THE BLOOD PARASITES OF ANURANS FROM COSTA RICA WITH REFLECTIONS ON THE TAXONOMY OF THEIR TRYPANOSOMES

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1 J. Parasitol., 87(1), 2001, p American Society of Parasitologists 2001 THE BLOOD PARASITES OF ANURANS FROM COSTA RICA WITH REFLECTIONS ON THE TAXONOMY OF THEIR TRYPANOSOMES Sherwin S. Desser Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 3G5 ABSTRACT: During May 1997, specimens of 7 species of anurans, that included 5 Phrynohyas venulosa Laurenti, 5 Rana forreri Boulenger, 7 Rana vaillanti Brucchi, 6 Eleutherodactylus fitzingeri Schimdt, 4 Smilisca baudinii Duméril and Bibron, 1 Leptodactylus melanonotus, and 3 Bufo marinus Linneaus, from the Guanacaste Conservation Area, Costa Rica were examined for blood parasites. Their hematozoan fauna included intraerythrocytic and intraleukocytic icosahedral viruses, a rickettsia (Aegyptianella sp.), 2 species of Hepatozoon, Lankesterella minima, 2 unknown species of apicomplexans, 9 morphologically distinct types of trypanosomes, and 2 species of microfilariae. Rana vaillanti, the most aquatic species of frog, harbored the most species of parasites. Recent evidence indicates that morphological changes in the highly pleomorphic trypanosomes of anurans from different geographical regions have not kept pace with biochemical (isozyme) and molecular (DNA sequence) changes. Describing new species based solely on bloodstream trypomastigotes is discouraged. Additional criteria described herein should be applied when naming new species of anuran trypanosomes. Anurans are exposed to several hematophagous vectors in their aquatic and terrrestrial habitats and are infected with a wide variety of intra- and extracellular blood parasites, including viruses, rickettsiae, species of several genera of protozoa, a yeast, and microfilariae. Surveys of the blood parasites of anurans have been conducted in several geographical regions and include among others, those of Sergent and Sergent (1905), Dutton et al. (1907), Nöller (1913), Cleland (1914), Kudo (1922), Tanabe (1931), Fantham et al. (1942), Mohammed and Mansour (1959), Mackerras and Mackerras (1961), Walton (1964a, 1964b), Barta and Desser (1984), Miyata (1987), Desser and Yekutiel (1987), and Barta et al. (1989). An All Taxon Biodiversity Inventory being conducted in Costa Rica afforded the opportunity to study the hematozoan parasites of 7 species of anurans from the Guanacaste Conservation Area. In the present paper, their rich parasite fauna is described, illustrated, and discussed in relation to similar parasites recorded from other areas, and the current status of the taxonomy and biology of anuran trypanosomes is critically evaluated. MATERIALS AND METHODS Anurans from several locations in the Guanacaste Conservation Area (10 55 N, W), Costa Rica, were captured during May The captive specimens consisted of 5 veined treefrogs Phrynohyas venulosa Laurenti, 1768, 5 Forrer s grass frogs Rana forreri Boulenger, 1883, 7 Vaillant s frogs Rana vaillanti Brucchi, 1877, 6 common rain frogs Eleutherodactylus fitzingeri Schmidt, 1857, 4 Mexican treefrogs Smilisca baudinii Duméril and Bibron, 1841, 1 black-backed frog Leptodactylus melanonotus Hallowell, 1861, and 3 cane toads Bufo marinus Linneaus, Blood from a cut digit of each animal was smeared directly onto a glass slide. Thin films were air-dried, fixed, and stained with Diff-Quik (Baxter Healthcare Corporation, McGaw Park, Illinois). Parasites were measured with an ocular micrometer and photographed using a Zeiss photomicroscope I with Kodak TMAX 100 film. When possible, measurements of parasites were made from 12 specimens and are given in m, followed by the standard deviation and sample size. Some trypanosome species were so scanty that their measurements were based on as few as 2 specimens. Voucher specimens have been deposited in the Canadian Museum of Nature, Invertebrate Zoology Collection, Ottawa, Canada (catalogue numbers CMNPA , CMNPA , CMNPA , CMNPA ). Received 25 February 2000; revised 13 June 2000; accepted 13 June RESULTS A variety of blood parasites, including viruses, bacteria, protozoa, and microfilariae, were observed in the frogs and toads (see Table I). The parasites are described by their individual hosts. Bufo marinus Erythrocytes of 1 of the 3 cane toads were infected with what appeared to be an apicomplexan parasite. These small, elongated organisms measuring about m had a lightly stained cytoplasm and a densely stained, terminally positioned nucleus (Fig. 1). Sheathed microfilariae that measured (n 12) were seen in the blood of 2 of the 3 toads (Fig. 2). Eleutherodactylus fitzingeri One of the 6 common rain frogs was infected with 2 viruses. Frog erythrocytic virus (FEV) with its characteristic spherical pink-staining viroplasm, measuring 2 4 m in diameter, was seen in the cytoplasm of many erythrocytes. The nucleus of infected cells was enlarged and diffusely stained, and the cytoplasm often appeared vacuolated (Fig. 3). The peripheral cytoplasm of many infected erythrocytes appeared distorted and unstained (Fig. 4). A second virus, frog leukocytic virus (FLV), was observed in mononuclear leukocytes. Like FEV, the pinkstaining, roughly spherical viroplasm measured 2 4 m and was usually surrounded by lighter staining, irregularly shaped inclusions (Fig. 5). A slender trypanosome was observed in 1 of the 6 common rain frogs. This parasite had an elongated, centrally positioned nucleus and a vacuolated cytoplasm. The kinetoplast was situated near the pointed posterior end, and a long flagellum extended from the tapered anterior end (Fig. 6). The dimensions of this trypanosome, designated sp. (a), and of all the other trypanosomes with the exception of the amastigote types, sp. (b) and chattoni, are given in Table II. Phrynohyas venulosa Two species of were observed in 1 of the 5 veined tree frogs. One designated sp. (b) was 152

2 DESSER ANURAN BLOOD PARASITES OF COSTA RICA 153 TABLE I. Blood parasites in anurans from the Guanacaste Conservation Area, Costa Rica. Host n spp.* Hepatozoon sp. Lankesterella sp.? Aegyptianella sp. FEV FLV Microfilariae Bufo marinus 3 Eleutherodactylus 6 fitzingeri (1 sp.) Leptodactylus 1 melanonotus Phrynohyas 5 venulosa (2 spp.) Rana forreri 5 (1 sp.) Rana vaillanti 7 (5 spp.) Smilisca baudinii 4 * Nine morphologically distinct trypanosomes. Unknown apicomplexans. Frog erythrocytic virus. Frog leukocytic virus. roughly ovoid in shape and did not have a flagellum or undulating membrane (Fig. 7). It measured (n 3). A second species designated sp. (c) was elongate and slender with a centrally positioned spherical nucleus, a well developed undulating membrane, and long free flagellum. The kinetoplast was situated near the tapered and pointed posterior end (Fig. 8). Rana forreri Gamonts, typical of ranid Hepatozoon species, were seen in erythrocytes of 3 of the 5 Forrer s grass frogs. The gamonts were sausage-shaped with a tapering posterior region that folded back along the body of the gamont. The nucleus of infected erythrocytes was often enlarged and displaced either laterally or to 1 end of the cell (Fig. 9). Intracellular gamonts measured (n 12). Many free gamonts (Fig. 10) that measured (n 12) occurred in 2 heavily infected frogs. Two of the 5 frogs were infected with a species of Lankesterella. Intraerythrocytic sporozoites were rare and were sharply reflexed, lying in small pink-staining vacuoles in the cytoplasm. Many free sporozoites, measuring (n 12), were observed. They contained a centrally positioned nucleus, flanked by 2 light-staining inclusions (Fig. 11). The broader anterior end was stained pink. A single species of trypanosome seen in the blood of 1 of the 5 Forrer s grass frogs was designated sp. (d). It was large and broad-bodied with tapered pointed ends, a well developed undulating membrane, and a long free flagellum (Fig. 12). Sheathed microfilariae were observed in the blood of 2 of the 5 R. forreri (Fig. 13). They measured (n 12). Rana vaillanti Vaillant s frogs harbored the most species of parasites and the highest parasitemias among the anurans examined. Four of 7 frogs were infected with a species of Hepatozoon, intraerythrocytic gamonts of which were reflexed sharply, with both ends approximately equal in length (Fig. 14). The densely stained nucleus usually lay in the broader anterior region. The gamonts were enclosed within a capsule that appeared as a narrow ( 1 m) halo surrounding the parasite. The host-cell nucleus was deeply stained and displaced either laterally or to 1 end of the cell. Intracellular gamonts measured (n 12). Free gamonts (Fig. 15) appeared longer and more slender than their intracellular counterparts, measuring (n 12). Four of the 7 R. vaillanti were infected with a species of Lankesterella. Most of the sporozoites in the blood film were extracellular. They were slender, with a centrally positioned nucleus, flanked by 2 lightly stained bodies (Fig. 16). The blunter anterior end stained pink. Extracellular sporozoites measured (n 12). In 1 of the heavily infected frogs, mononuclear leukocytes containing up to 12 sporozoites were seen (Fig. 17). A novel apicomplexan parasite was seen in thrombocytes of 3 Vaillant s frogs. Immature stages of the parasite were sausageshaped and distorted the thrombocyte nucleus and cytoplasm (Fig. 18). The large granular nucleus of the parasite lay in a vacuolated cytoplasm. Immature stages measured (n 12). The parasites transformed into large, beanshaped forms measuring (n 12). These apparently mature stages, possibly gamonts, had a large diffuse, granular nucleus and vacuolated cytoplasm (Fig. 19). A capsule like that enclosing mature gamonts of Hepatozoon sp. was not observed. Intraerythrocytic rickettsiae, Aegyptianella sp., were seen in 3 Vaillant s frogs. The rickettsiae, enclosed in a spherical vacuole in the erythrocyte cytoplasm, were barely perceptible by light microscopy (Fig. 20). In most cases, the wall of the vacuole was densely stained (Fig. 21). Five morphologically distinct types of trypanosomes were observed in the blood of R. vaillanti. All 7 frogs harbored 2 or more species of trypanosomes; however, only 1 frog was infected with all 5 species. Descriptions and dimensions of these trypanosomes are given in Table II.

3 154 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 1, FEBRUARY 2001 FIGURES Photomicrographs of hematozoan parasites of Bufo marinus, Eleutherodactylus fitzingeri, Phrynohyas venulosa, and Rana forreri. Scale bar 10 m. 1. Unknown intraerythrocytic apicomplexan parasite in B. marinus. 2. Microfilaria in B. marinus. 3, 4. Frog erythrocytic virus (FEV) in E. fitzingeri. Arrow indicates viroplasm. Note disrupted appearance of erythrocyte cytoplasm and lightly stained peripheral cytoplasm of host cell in Figure Frog leukocytic virus (FLV) in mononuclear leukocyte of E. fitzingeri. Arrow indicates viroplasm. Note associated lightly stained bodies, a common cytopathological feature associated with this parasite. 6. sp. (a) in E. fitzingeri. 7. chattoni-like trypanosome, sp. (b) in P. venulosa. 8. sp. (c) in P. venulosa. 9. Mature gamont of Hepatozoon sp. in erythrocyte of R. forreri. 10. Free gamont of Hepatozoon sp. in R. forreri. 11. Free sporozoite of Lankesterella minima in R. forreri. chattoni Mathis and Leger, 1911, was large, flattened, and circular in outline, and measured (n 12). A centrally positioned, spherical nucleus and adjacent kinetoplast lay in a dense, patterned cytoplasm (Fig. 22). These trypanosomes occurred in all 7 frogs. loricatum (Mayer, 1843) França and Athias, 1906, was large with a broad, costate body, usually about ½ of its length or greater in width, with a well-developed undulating membrane and no free flagellum. The anterior end was usually pointed and slightly recurved. The spherical nucleus lay in the midbody region, and the kinetoplast, roughly half way between the nucleus and usually blunt posterior end (Fig. 23). loricatum was observed in 5 of the 7 frogs. sp. (e) had a broad midbody with sharply tapering ends and was observed in 2 of the 7 frogs. The body of this trypanosome exhibited a spiral pattern that was most obvious in the posterior region (Fig. 24). The kinetoplast lay adjacent to the ovoid nucleus in the midbody region. This trypanosome had a well developed undulating membrane and a long free flagellum. sp. (f) was long and slender, with sharply pointed anterior and posterior ends, and a prominent undulating membrane and short free flagellum (Fig. 25). The kinetoplast lay near the pointed posterior end. This trypanosome was observed in 2 frogs. sp. (g) was seen in only 1 Vaillant s frog. The trypanosome had a prominent ovoid nucleus and a vacuolated cytoplasm. The kinetoplast was situated near the pointed posterior end. The parasite had a narrow undulating membrane and a long free flagellum (Fig. 26).

4 DESSER ANURAN BLOOD PARASITES OF COSTA RICA 155 TABLE II. Dimensions of trypanosomes observed in frogs from the Guanacaste Conservation Area, Costa Rica.* Host E. fitzingeri P. venulosa R. forreri R. vaillanti R. vaillanti R. vaillanti R. vaillanti species sp. (a) n 4 sp. (c) n 2 sp. (d) n 7 T. loricatum n 12 sp. (e) n 5 sp. (f) n 7 sp. (g) n 2 PF PA FF BW LN WN PK KN NA PN PK/PN PK/PA PN/PA BW/PA FF/PA ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) n/a ( ) ( ) ( ) ( ) ( ) ( ) ( ) n/a ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) * Excluding the T. chattoni-like trypanosome in R. venulosa and T. chattoni in R. vaillanti, the dimensions of which are given in the text. PF total length including free flagellum, PA body length excluding free flagellum, FF length of free flagellum (n/a no free flagellum), BW maximum body width excluding undulating membrane, LN length of nucleus, WN width of nucleus, PK distance from posterior to kinetoplast, KN distance from kinetoplast to center of nucleus, NA distance from center of nucleus to anterior end, PN distance from posterior end to center of nucleus. Parasites were not seen in the blood of S. baudinii or of L. melanonotus. DISCUSSION Among the many pro- and eukaryotic parasites recorded in Costa Rican anurans, the most prevalent were trypanosomes. The parasite fauna was similar to that described from anurans in Ontario, Canada (Barta and Desser, 1984) and in Corsica (Barta et al., 1989). Few species of parasites were shared by more than 1 host species. These observations will be discussed with respect to the individual parasites and with reference to similar studies elsewhere. Viruses FEV and FLV recorded from E. fitzingeri and R. vaillanti were morphologically similar to viruses in ranids from Ontario (Desser and Barta, 1984; Desser, 1992). FEV was shown by in situ hybridization to be a cytoplasmic poxlike DNA virus (Gruia-Gray et al., 1992). Electron microscopy revealed that the red-staining spherical inclusions corresponded to viroplasms that were surrounded by mature icosahedral viral particles (Gruia-Gray et al., 1989). Gruia-Gray and Desser (1989) demonstrated that FEV may be transmitted mechanically among ranids by the interrupted feeding of the amphibian-feeding ceratopogonid, Forcipomyia (Lasiohelia) fairfaxensis. Rana catesbeiana, with up to 90% parasitemias, exhibited little evidence of pathogenicity. The spherical red-staining bodies and associated cytopathology seen in lymphocytes of E. fitzingeri and R. vaillanti were similar to those observed in mononuclear leukocytes and heterophils of Rana clamitans and Rana septentrionalis in Ontario. Like FEV, the prominent red-staining cytoplasmic inclusions of FLV corresponded to viral assembly centers containing nucleocapsids in various stages of development. Disrupted areas in the cytoplasm, like those seen in lymphocytes of R. vaillanti, contained viral inclusions with assembled viral particles, varying from loosely arranged icosahedral virions to closely packed linearly arranged groupings (Desser, 1992). Bacteria Intraerythrocytic bacteria like those seen in R. vaillanti were shown by electron microscopy to be rickettsiae in ranids from Ontario (Desser and Barta, 1984; Desser, 1987) and from Corsica (Desser and Barta, 1989). These rickettsiae were designated Aegyptianella ranarum and Aegyptianella bacterifera, respectively. The former species was smaller, like the organisms in R. vaillanti, and could not be resolved clearly by light microscopy. The rickettsiae in Costa Rican frogs, like those in

5 156 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 1, FEBRUARY 2001 FIGURES Photomicrographs of hematozoan parasites of R. forreri and R. vaillanti. Scale bar 10 m. 12. sp. (d) in R. forreri. 13. Microfilaria in R. forreri. 14. Mature gamont of Hepatozoon sp. in erythrocyte of R. vaillanti. 15. Free gamont of Hepatozoon sp. in R. vaillanti. 16. Intraerythrocytic, and free sporozoite of L. minima. Note lightly stained paranuclear bodies (arrows). 17. Monocyte containing several sporozoites of L. minima. 18. Young stage of unknown apicomplexan parasite (arrow) in thrombocyte of R. vaillanti. Note 3 adjacent uninfected thrombocytes. 19. Various stages of development of an unknown apicomplexan parasite from immature to large, mature, bean-shaped stages Aegyptianella sp. in erythrocytes of R. vaillanti. Note barely resolvable rickettsiae within vacuoles (arrows) and darkly stained vacuolar border in Figure Trypanosomes in R. vaillanti. 22. chattoni. 23. loricatum. 24. sp. (e). Note spiral pattern. 25. sp. (f). 26. sp. (g).

6 DESSER ANURAN BLOOD PARASITES OF COSTA RICA 157 Ontario, were restricted to ranids. Aegyptianella species appear to be cosmopolitan parasites of erythrocytes of ranids having been reported from North America, Europe, and Africa (Dutton et al., 1907; Brygoo, 1963). Preliminary evidence suggested that the intermediate host and vector of A. ranarum in Ontario is the common amphibian-feeding leech, Desserobdella picta, in which the rickettsiae undergo prolific development (Desser, 1987). Apicomplexa Hepatozoon species: Gamonts of 2 species of Hepatozoon were observed in the Costa Rican ranids. Gamonts of the species in R. forreri were smaller than those in R. vaillanti and assumed a different position within the parasitophorous vacuole. Two species of Hepatozoon were also recorded from ranids in Ontario (Barta and Desser, 1984). Although the gamonts of these species were morphologically indistinguishable, the cytopathology differed markedly. Rana catesbeiana and R. clamitans were susceptible to experimental infection with both species of Hepatozoon that exhibited significant differences in their ITS-1 nucleotide sequences (Kim et al., 1998). Intraerythrocytic apicomplexan gamonts recorded from frogs in many localities have until recently been described as species of Haemogregarina. Desser et al. (1995) demonstrated that Culex territans, a mosquito that feeds on amphibians in Ontario, serves as the definitive host and vector of Hepatozoon catesbianae in bullfrogs and described its sporogonic development in the Malpighian tubules of the fly. This development revealed that the parasite was a species of Hepatozoon, not Haemogregarina. On the basis of this new information, Smith (1996) transferred all the species of anuran Haemogregarina to Hepatozoon. Subsequently, Kim et al. (1998) demonstrated a similar type of development for Hepatozoon clamitae. Presumably, a local species of mosquito serves as the vector for Hepatozoon species in Costa Rican ranids. Although the gamonts of both species of Hepatozoon resemble those described from ranids elsewhere, I prefer to describe them without assigning specific epithets until their life cycles are elucidated. Lankesterella: Sporozoites of a species of Lankesterella were seen in many of the Costa Rican ranids, several of which were heavily infected, with free sporozoites in their peripheral circulation. Sporozoites observed in R. forreri and R. vaillanti were similar in size and morphology and probably belong to the cosmopolitan species Lankesterella minima, which has been described from several localities in North America (Desser and Barta, 1984) and Europe (Nöller, 1913; Barta et al., 1989). Lankesterella minima is transmitted through the bite of its leech vector in which the parasite does not replicate. Tse et al. (1986) documented ultrastructural differences between sporozoites in the gut ceca and those in the salivary glands of the leech vector D. picta. The latter sporozoites were infective as Desser et al. (1990) were able to transmit L. minima to laboratory-reared tadpoles through the bite of experimentally infected, laboratoryreared leeches. Monocytes containing several sporozoites of L. minima in heavily infected R. vaillanti probably reflected the overwhelming infections in which many free sporozoites were observed. Previously undescribed apicomplexans The small intraerythrocytic parasites seen in B. marinus were unlike any apicomplexan parasites described thus far from toads but did resemble merozoites and young trophozoites of Babesiosoma stableri observed in frogs from Ontario (Barta and Desser, 1986). Unfortunately, it was not possible to identify the parasite on the basis of this stage alone. The unusual intrathrombocytic parasites observed in R. vaillanti resembled stages of development of hemogregarine gamonts. On the basis of these stages alone, it was impossible to determine whether this parasite was a species of Hepatozoon, Hemolivia, or possibly Haemogregarina. Intrathrombocytic parasites are unusual. The only other parasite of anuran thrombocytes recorded was the yeast Thrombocytozoons ranarum that was reported in Ontario ranids (Desser and Barta, 1988). Microfilariae On the basis of the adult worms, microfilariae in R. forreri were identified as Foleyella sp., and the shorter, stouter microfilariae in B. marinus, as a species of Ochotenerella. Filarial nematodes of anurans are cosmopolitan (Walton, 1964a, 1964b; Schacher and Crans, 1973; Barta and Desser, 1984; Miyata, 1987). The biology of Icosiella neglecta in European frogs and its transmission by the ceratopogonid Lasiohelia (syn. Forcipomyia) velox was described by Desportes (1941, 1942). Schacher and Crans (1973) described the transmission of North American species of Foleyella by mosquitoes, mostly species of Culex. A comparison of the parasite fauna of anurans in Costa Rica with that of anurans from Ontario and Corsica revealed that in all 3 localities, the presence of leech-vectored parasites including trypanosomes, L. minima and Aegyptianella sp., was highest in the most aquatic anurans, e.g., R. vaillanti in Costa Rica, R. catesbeiana and R. clamitans in Canada, and Rana esculenta in Corsica. Leech-transmitted dactylosomatids, B. stableri in North American ranids (Barta and Desser, 1989) and Dactylosoma ranarum in Corsican R. esculenta (see Barta et al., 1989), were not seen in Costa Rican ranids nor was the diplomonad flagellate B. algonquinensis (see Desser et al., 1992). This may have been due to the relatively small sample size. Trypanosomes Trypanosomes, the most prevalent hematozoan parasites in the Costa Rican anurans, were recorded in 4 of the 7 species sampled. Nine morphologically distinct trypanosomes were observed, with R. vaillanti, the most aquatic species, serving as the host for 5 of these. Amphibians from Ontario, Canada showed a similar pattern with the more aquatic Rana species harboring the most species of trypanosomes as well as the apicomplexan parasites L. minima and B. stableri, all of which were shown to be transmitted by the leech, D. picta (Barta and Desser, 1984; Desser et al., 1990). Surprisingly, none of the trypanosome types was shared by different hosts, with the possible exception of the morphologically similar sp. (a) in E. fitzingeri and sp. (g) in R. vaillanti. Although similar in size, sp. (a) had a much broader undulating membrane than the latter. In his survey of trypanosomes of amphibians from

7 158 THE JOURNAL OF PARASITOLOGY, VOL. 87, NO. 1, FEBRUARY 2001 the Pacific coast of Colombia, Carvajal (1982) described 2 types of trypanosomes from E. fitzingeri, neither of which resembled sp. (a) from the same host in Costa Rica. Of the 8 types of trypanosomes observed in Costa Rican anurans, only 2 were sufficiently distinctive to be assigned to species, i.e., the large, flattened amastigote stages of T. chattoni, and the broad-bodied, costate forms lacking a free flagellum of T. loricatum. Although both these species are cosmopolitan in distribution, morphometric and other differences have been recorded among forms in geographically isolated populations of the same species of frogs and between different host species (Diamond, 1965; Woo, 1969; Bardsley and Harmsen, 1973; Miyata, 1978; Barta and Desser, 1984). This phenomenon is further exemplified in the present study in which the amastigote form of the T. chattoni-like parasite observed in P. venulosa differed morphologically and morphometrically from that seen in R. vaillanti. I have refrained from naming the other trypanosomes because most trypanosomes of anurans are highly pleomorphic, and frequently members of the same species have been assigned separate species status, leading to a profusion of species and considerable confusion. Of the more than 70 named species of anuran trypanosomes, Diamond (1965), in his comprehensive review, recognized only 26 species. The task of compiling an accurate list of valid species is presently impossible, as most species descriptions were based solely on the pleomorphic bloodstream trypomastigotes. An example of this type of problem is the confusion surrounding the most frequently named trypanosome rotatorium (see Bardsley and Harmsen, 1973; Barta et al., 1989). A second reason for caution in naming new species concerns the various stages of development of the parasites in blood films from wild-caught animals. Infective metacyclic forms in the leech vectors are tiny and morphologically distinct from the usually much larger, mature bloodstream trypomastigotes, and growth to the mature forms may require several days (Diamond, 1965; Martin and Desser, 1991a, 1991b). Unless development of these parasites has been followed in experimentally infected anurans, the smaller forms may be mistaken for distinct species. This problem has undoubtedly contributed to the incorrect naming of several anuran trypanosome species. Which criteria should be satisfied when naming species of anuran trypanosomes? I suggest that, in addition to the morphological and morphometric features of the parasite, and the host and its locality, stages in the vector and in culture should be described. Also, changes in the flagellates as they mature in the blood of experimentally infected hosts should be documented. Other than pipientis (see Diamond, 1950) and fallisi (see Martin and Desser, 1991a, 1991b), few species of trypanosomes have been characterized using these criteria. Isozyme and DNA sequence data have provided further insights into the status of species of anuran trypanosomes (Martin, Desser, and Hong, 1992; Martin, Desser, and Werner, 1992; Lun and Desser, 1995, 1996). Using random amplified polymorphic DNA and karyotype analysis by pulsedfield gradient gel electrophoresis, Lun and Desser (1995, 1996) showed significant variations in morphologically similar strains and species of anuran trypanosomes. These studies indicate that morphological changes in anuran trypanosomes from different geographic regions did not keep pace with molecular and biochemical changes, further complicating the taxonomic conundrum. Another feature of anuran trypanosomes that has received little attention is their host specificity, which appears quite strict from the limited information available. In their study of the host range for T. fallisi of the American toad in Ontario, Martin and Desser (1991b) were able to experimentally transmit only transient infections to another toad Bufo valiceps, and a tree frog Hyla versicolor. Rana species were not susceptible to infections with T. fallisi. This observation is important because in an earlier study, on the basis of morphological similarity, Barta and Desser (1984) misidentified T. fallisi for T. ranarum, a common parasite of ranids in Ontario. A brief consideration of the status of our understanding of the vectors of anuran trypanosomes is warranted. In addition to leeches that are generally acknowledged to be the primary vectors, there have been reports of phlebotomine flies serving as vectors of trypanosomes of toads, i.e., Lutzomyia vecatrix occidentalis for bufophlebotomi in Bufo boreas in California (Ayala, 1971), and Phlebotomus squamirostris for bocagei in Bufo B. gargarizans in China (Feng and Chung, 1940). In both cases, replication of the trypanosomes and colonization of the hindgut and rectum of the flies by epimastigotes were observed. Unfortunately, in neither study was the experimental transmission of parasites to toads achieved. Desser et al. (1973), likewise recorded the prolific development of T. rotatorium from R. catesbeiana in laboratory-reared amphibian-feeding mosquitoes Culex territans. The terminal epimastigote stages in the rectum of the mosquitoes were, however, not infective to the normal ranid hosts (Desser et al., 1975). Despite the fact that amphibian-feeding flies may provide a suitable habitat for replication, the parasites do not develop to the infective metacyclic trypomastigote stage seen in the leech vector. Even the brief aquatic sojourn of more terrestrial anurans, like the American toad, was shown to be sufficient for transmission of T. fallisi by the leech vectors (Martin and Desser, 1991a). The Costa Rican counterparts of these vectors of anuran trypanosomes and L. minima, as well as the dipteran vectors of the other hematozoan parasites described, await discovery. Despite Bardsley and Harmsen s (1973) caution concerning the naming of anuran trypanosomes solely on the basis of specimens in blood films, some authors (e.g., Miyata, 1978) have persisted in doing so. Others (e.g., Carvajal, 1982) have prudently designated the parasites as types of species. This approach should be encouraged until a better understanding of the biology of anuran trypanosomes is achieved. ACKNOWLEDGMENTS The author is grateful to Dan Janzen for his kind hospitality and for providing laboratory facilities at the Area de Conservacion Guanacaste (ACG) in northwestern Costa Rica. This study is part of an inventory of eukaryotic parasites inhabiting the 940 species of vertebrates living in the ACG. I am grateful to the scientific and technical staff of the ACG for their help in capturing anurans, and in particular to Sigifredo Marin, Roger Blanco, Alejandro Masis, Maria Marta Chavarria, Felipe Chavarria, Guillermo Jiminez, Calixto Moraga, Carolina Cano, Elda Araya, and Fredy Queseda. I am also grateful to Daniel R. Brooks for identifying the filarial worms, to Henry Hong for his superb technical assistance, to Henry, Don Martin, Todd Smith, Kowthar Salim, Janet Koprivnikar, Anne Koehler, and Amanda Martyn for their critical review of the manuscript,

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