A Comparative Study of Endoparasites in Three Species of Sympatric Bufo (Anura: Bufonidae), from Texas

Home , Ascaridida, Cosmocercoides, Cyclophyllidea

Proc. Helminthol. Soc. Wash. 56(2), 1989, pp. 162-167

CHRIS T. McALLiSTER,1'2 STEVE J. UPTON,3 AND DAVID BRUCE CoNN4 1 Renal-Metabolic Lab (151-G), Veterans Administration Medical Center, 4500 S. Lancaster Road, Dallas, Texas 75216, 2 Department of Biological Sciences, University of North Texas, Denton, Texas 76203, 3 Division of Biology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506, and 4 Department of Biology, St. Lawrence University, Canton, New York 13617

ABSTRACT: Seventy immature and adult toads, Bufo spp., including 27 B. debilis debilis, 23 B. valliceps valliceps, and 20 B. woodhousii woodhousii from 4 counties of northcentral Texas, were examined for endoparasites. Forty-nine (70%) of the toads were infected with 1 or more species of protozoan or metazoan parasites. New host records are reported for Nyctotherus cordiformis Ehrenberg, 1838, Myxidium serotinum Kudo and Sprague, 1940, Distoichometra bufonis Dickey, 1921, and Cosmocercoides variabilis (Harwood, 1930) Travassos, 1931, in B. d. debilis; M. serotinum and Mesocestoides sp. Vaillant, 1863, in B. v. valliceps; and N. cordiformis and M. serotinum in B. w. woodhousii. A new geographic locality record is reported for D. bufonis. Prevalence of infection and parasite intensities varied among the 3 host taxa and differences were associated with breeding season, microhabitat selection, and climatological conditions. KEY WORDS: toads, Bufo spp., Bufo debilis debilis, Bufo woodhousii woodhousii, Bufo valliceps valliceps, Opalina sp., Nyctotherus cordiformis, Myxidium serotinum, Distoichometra bufonis, Adelina sp., Eimeria sp., Mesocestoides sp., metacestode, Cosmocercoides variabilis, prevalence, intensity, survey, anurans.

Toads of the genus Bufo are represented by 10 (x ± SE snout-vent length [SVL] = 33.9 ±1.1, range species distributed throughout various regions of 28-40 mm), 23 B. v. valliceps (85.1 ± 4.8, 35-122), Texas. These taxa inhabit diverse habitats, in- and 20 B. w. woodhousii (59.1 ± 3.3, 35-91), were cluding marshes, river bottoms, mountain can- collected in Denton, Hood, Johnson, and Somervell counties of northcentral Texas. Specimens were col- yons, desert streams, coastal prairies, irrigation lected by hand either by overturning rock shelters, ex- ditches, and upland cedar glades. Because bu- cavating shallow retreats, or by spotlighting during fonids are an integral part of the anuran com- rainfall at night on roadways. Toads were placed in munity in many areas, comparative study of their individually labeled plastic bags, transported to the laboratory, and examined within 24 hr for endopar- parasites may lead to better understanding of asites. These anurans were killed by pithing or by over- patterns of host specificity, prevalence of infec- dosing with sodium pentobarbital. Blood samples were tion, and possible effects of parasitism among obtained by heart puncture and smears stained with the different species. Giemsa and examined for hematozoa. The digestive The few previous studies on parasites of Bufo tract, lungs, heart, gall bladder, liver, coelomic cavity, and overlying muscle tissues of each individual were spp. have been conducted on single species in- examined for parasites. A portion of the large intestine, habiting widely separated geographic localities. along with feces, was placed in vials containing tap We chose to compare the endoparasites of 3 of water supplemented with antibiotic (100 I.U./ml pen- the more common bufonids of the state: the green icillin-G-100 Mg/nil streptomycin) and mailed to Kan- toad, B. debilis debilis Girard, 1854, Gulf Coast sas State University for examination of coccidia. Sam- ples were examined microscopically following notation toad, B. valliceps valliceps Wiegmann, 1833, and in a modified Sheather's sugar solution (sp. gr. 1.30). Woodhouse's toad, B. woodhousii woodhousii Gall bladder and intestinal contents were smeared on Girard, 1854. Only a few studies are available glass slides, placed in hot Schaudinn's fixative, and concerning the endoparasites of the latter 2 species stained with Gomori trichrome for examination of certain protozoans. Some tissues containing metacestodes (see Harwood, 1932; Trowbridge and Hefley, were removed from the hosts for parasite identification 1934; Walton, 1940, 1946; Kuntz, 1941; Kuntz and histopathological studies. This material was fixed and Self, 1944; Little, 1966); nothing is known in 10% formalin, embedded in Paraplast, sectioned at of the parasites of the former. 7 ^m, affixed to glass slides, stained with Harris' hematoxylin and eosin counterstain, and mounted in da- Materials and Methods mar. Non-encapsulated immature and adult cestodes were relaxed and killed by slowly warming them in Between May 1986 and September 1987, and again petri dishes containing 0.6% saline, fixed in alcohol- during June 1988, 70 toads, including 27 B. d. debilis formaldehyde-acetic acid (AFA) for 24 hr, and trans-

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Table 1. Endoparasites of 3 species of toads, Bufo spp., in Texas.

Host species and prevalence of infection Parasite B. d. debilis B. v. valliceps B. w. woodhousii

Protozoa Sarcomastigophora Opalina sp. 0/27 (0%) 2/23 (8.7%) 1/20 (5.0%) Ciliophora Nyctotherus cordiformis 2/27 (7.4%)* 0/23 (0%) 1/20(5.0%)* Apicomplexa Eucoccidiorida Adelina sp.-like coccidian 0/27 (0%) 2/23 (8.7%) 0/20 (0%) Eimeria sp. 0/27 (0%) 1/23 (4.3%) 0/20 (0%) Myxo/.oa Bivalvulida Myxidium serotinum 2/27 (7.4%)* 6/23(26.1%)* 7/20 (35.0%)* Cestoidea Cyclophyllidea Unidentified metacestode 0/27 (0%) 0/23 (0%) 1/20 (5.0%) Mesocestoides sp. 0/27 (0%) 3/23(13.0%)* 0/20 (0%) Distoichometra bufonis 4/27(14.8%)* 0/23 (0%) 10/20(50.0%) Nematoda Ascaridida Cosmocercoides variabilis 14/27(51.9%)* 16/23(69.6%) 0/20 (0%)

New host record. ferred to 70% ethanol. They were later stained in May- er's hematoxylin or Semichon's acetocarmine, dehy- (73 mm SVL) male B. w. woodhousii. Specific drated in a graded ethanol series, cleared in xylene, and identification was impossible because of a low mounted in permount. Nematodes were killed in hot prevalence of infection, an absence of a series of AFA, placed in 70% ethanol, and transferred to lac- infections, and a range of forms. Although we tophenol or glycerol for clearing and study. did not find opalinids in any of the 27 B. d. Voucher specimens are deposited in the U.S. Na- tional Museum Helminthological Collection, USDA, debilis, Metcalf (1923) noted "a very few cysts Beltsville, Maryland 20705 as follows: Opalina sp. of some species of Opalinidae" in 1/9 (11.1%) (USNM 80385); Nyctotherus cordiformis (USNM B. debilis from an unknown locality near the Tex- 80386); Myxidium serotinum (USNM 80382-80384); as-Mexico border. In addition, Walton (1946) Mesocestoides sp. tetrathyridia (USNM 80381); tetracetabulate anacanthocysticercus (USNM 80380); Dis- listed O. obtrigonoidea Metcalf, 1923, and O. toichometra bufonis (USNM 80387-80391); and Cos- triangularis Ghosh, 1918, in B. valliceps from mocercoides variabilis (USNM 80378-80379). Texas, and O. woodhousii Metcalf, 1923, and an unidentified Opalina sp. (of Trowbridge and Results and Discussion Hefley, 1934) in B. woodhousii from Utah, Ar- Forty-nine (70%) of the toads were infected izona, and Oklahoma. with 1 or more endoparasites, including 14 Walton (1946) reported Nyctotherus cordifor- (51.9%) B. d. debilis, 20 (87.0%) B. v. valliceps, mis Ehrenberg, 1838, in B. valliceps. However, and 15 (75.0%) B. w. woodhousii (Table 1). Blood we did not recover this endosymbiont from any samples were negative for apicomplexans or try- Gulf Coast toads; only 2 subadult (29 and 30 panosomes. New host records are reported for mm SVL) male B. d. debilis and an immature several endoparasites (see Table 1). (55 mm SVL) female B. w. woodhousii were in- An unidentified species of Opalina Purkinje fected. In addition, Frandsen and Grundmann and Valentin, 1840, was found in the rectum of (1960) reported TV. cordiformis in the boreal toad, an immature (35 mm SVL) and adult (83 mm B. boreas, from Utah. SVL) male B. v. valliceps and in a single adult In the gall bladder contents of all 3 species of

and, perhaps, as suggested by this observation, are also coprophagic. Two B. v. valliceps were found to be passing moderate numbers of what appeared to be adelid sporocysts (Apicomplexa: Adeleidae). Each sporocyst was spherical or subspherical, had a rel- atively thick wall, 2 sporozoites, and a coarse, granular residuum (Fig. 1). Because all known genera (Adelina Schneider, 1875) are found in invertebrates (annelids, chilopods, priapulids, and insects), we hypothesized that these coccidia were pseudoparasites. To test this hypothesis, we placed 2 wild-caught B. v. valliceps into 3 8-liter glass aquaria, examined feces for 3 wk to assure the absence of coccidia, and inoculated each toad orally with 2.0 x 104 3-wk-old sporocysts. Feces were collected continually for 2 wk after the in- oculations and microscopic examination revealed no additional coccidia. In addition, microscopic examination of the intestinal tract, liver, gall bladder, and kidneys from each of the toads Figure 1. Nomarski interference-contrast photo- micrograph of adelid-like sporocyst (Adelina sp.?) from at the end of the 2-wk period revealed no coc- Bufo valliceps valliceps. x 2,688. SR, sporocyst resid- cidial developmental stages, thus supporting our uum; SW, sporocyst wall; SZ, sporozoite. hypothesis. Three B. v. valliceps were infected with numerous tetrathyridia of Mesocestoides sp. Vail- toads, spores and trophozoites ofMyxidiwn se- lant, 1863. These occurred in the liver, intestines, rotinum Kudo and Sprague, 1940, were present. and musculature, where they frequently were lo- Only 2 adult (33 and 38 mm SVL) male B. d. calized under the peritoneal capsules and in ad- debilis were infected; however, prevalence was jacent mesenteries (Fig. 2). Presence of worms higher in B. v. valliceps and B. w. woodhousii was accompanied by a moderate amount of lo- (Table 1). This myxosporean has previously been calized inflammation. All the worms appeared reported in the bile of toads, Bufo sp. (Kudo, viable, but had been encapsulated by the host. 1943), and southern toads, B. terrestris (Kudo, Each tetrathyridium possessed a single deeply 1966) from Florida, and in frogs and salaman- invaginated scolex, and showed no sign of asex- ders (see McAllister, 1987; McAllister and Up- ual proliferation. ton, 1987a). Species of the genus Mesocestoides can be In the feces of a single B. v. valliceps, we ob- identified only from adult characteristics, but ge- served unsporulated coccidian oocysts. Upon neric identity of metacestodes can be determined sporulation in 2.5% (w/v) aqueous K2Cr2O7 for by their tetracetabulate scolex, lack of an apical 1 wk at ca. 23°C, oocysts were determined to be organ or armed rostellum, and solid cellular those of an Eimeria sp. Schneider, 1875. Oocysts hindbody that results from primitive rather than had a moderately pitted oocyst wall, measured neoteric development (Fig. 2). Thus, in terms of ca. 20 x 16 /im, and possessed a single globular comparative metacestode structure, tetrathyrid- oocyst residuum, polar granule, and ovoid spo- ia are invaginated anacantho-tetracetabuJo-ple- rocysts with Stieda bodies. Oocysts were of the rocercoids (Freeman, 1973). The possible taxo- "type A" morphology (see Reduker et al., 1987) nomic significance of asexual proliferation in characteristic of rodent coccidia. Since this toad tetrathyridia is unclear; however, worms report- was, at the time of capture, inhabiting an aban- ed here are of the far more common non-prolif- doned rodent's nest, these oocysts were judged erative type (Conn, 1988). Tetrathyridia have to be pseudoparasites of B. v. valliceps (Upton been reported previously from American toads, and McAllister, 1988). It is known that bufonid B. americanus, and Great Plains toads, B. cog- toads are quite opportunistic in their food habits natus, in Iowa and South Dakota (James and

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Figures 2, 3. Brightfield light micrographs of mid-longitudinal sections of metacestodes from Bufo spp. Each is surrounded by a fibrous host capsule (C) and possesses a deep invagination canal (I), distinct tegument (T), and well-developed tetracetabulate scolex (S). 2. Mesocestoides sp. tetrathyridium in the mesenteries of B. v. valliceps. Note the solid cellular hindbody (H). 3. Unidentified metacestode in the liver of B. w. woodhousii. The hindbody is occupied by a primary lacuna (L) filled with amorphous non-cellular material.

Ulmer, 1967). The only other anurans reported apparently belong to the order Cyclophyllidea. to serve as hosts in North America are green Cyclophyllidean, proteocephalidean, and pseu- frogs, Rana clamitans, leopard frogs, R. pipiens, dophyllidean metacestodes have been reported and Strecker's chorus frogs, Pseudacris streckeri from amphibians (Prudhoe and Bray, 1982). In streckeri (James and Ulmer, 1967; Williams and addition, Thomas et al. (1984) reported uniden- Taft, 1980; McAllister, 1987). Various mammals tifiable tetrathyridia in the gall bladder, mesen- and reptiles are also naturally infected (Mc- teries, heart, and liver of Houston toads, B. hous- Allister, 1988). tonensis, from southeastern Texas, and Brandt A single B. w. woodhousii harbored several (1936) found proteocephalid cysts in Fowler's unidentified metacestodes in the liver paren- toad, B. w. fowleri. Unfortunately, lack of ac- chyma (Fig. 3). Little inflammatory exudate sur- curate life cycle information on many amphibian rounded these worms, but most had been en- cestodes, and a paucity of detailed information capsulated by the host. Both worms and host on comparative metacestode structure, makes liver tissue appeared healthy. species identification impossible. The identity of these metacestodes could not Immature pre-stobilar metacestodes and adult be determined, but they clearly were not Meso- nematotaeniid tapeworms, Distoichometra bu- cestoides tetrathyridia. The most obvious distin- fonis Dickey, 1921, were found in the small in- guishing feature is the presence of a primary la- testine of 14 (20.6%) toads, including 3 male and cuna filled with amorphous non-cellular material 1 female B. d. debilis (adults, 37.0 ± 1.2 mm) in the unidentified worm (Fig. 3). According to and 6 male and 4 female B. w. woodhousii (ju- the scheme of Freeman (1973), these can be veniles and adults, 56.3 ± 4.0 mm). Mean in- described as invaginated anacantho-tetraceta- tensities were 5.5 ± 1.3 (range = 2-8) and 24.2 bulo-cysticerci or possibly -precysticerci, and ±9.5 (range = 4-105) worms in B. d. debilis and

B. w. woodhousii, respectively. Interestingly, none diversity of parasites), fluctuations in seasonal was found in any B. v. valliceps. This cestode has weather conditions may alter the pattern of par- been previously reported from southern toads, asitism as observed herein. Precipitation during B. lentiginosus (=terrestris) in Georgia (Dickey, the spring and early summer months initiates 1921); B. w.fowleri and eastern spadefoot toads, calling and male territoriality. The reproductive Scaphiopus holbrookii in North Carolina (Brandt, seasons of all 3 bufonids overlap, even to some 1936; Douglas, 1958); and Texas toads, B. spe- degree with other anurans in Texas (Wiest, 1982), ciosus, Great Plains toads, B. cognatus, Couch's and this would permit contact between larvae spadefoot toads, and S. couchii in Oklahoma and possible exchange of parasitic stages. (Kuntz, 1941); and B. w. woodhousii in Okla- Finally, our interpretation of the data was based homa (Kuntz, 1941) and Nebraska (Hardin and on information gathered over a period of several Janovy, 1988). months. In addition, the number of hosts ex- Since its original description as Oxysomatium amined was limited and the numbers of parasites variabilis from the intestine of B. valliceps, Cos- recovered are insufficient to permit statistical mocercoides variabilis (Harwood, 1930) Travas- comparisons between host species. We suggest sos, 1931, has been reported in numerous am- that comparative parasitological data must be phibians and reptiles of southern Texas and in accumulated for several years and additional wood frogs, R. sylvatica from New York (Baker, hosts should be examined before conclusive 1987). In our study, this nematode was the most statements may be made on parasitism among common parasite of both B. d. debilis and B. v. the species. valliceps (combined prevalence = 60%); mean intensity was 11.0 ± 1.8 (range = 3-25) and 13.9 Acknowledgments ± 3.6 (range 1-51), respectively. However, none We thank R. T. Howell for assistance with of the B. w. woodhousii was infected. collecting and K. L. Fry, G. Roberts, and the Recently, evidence provided by Vandenburgh Russells for allowing us to collect on their prop- and Anderson (1987) has shown that a related erties. We also thank 2 anonymous reviewers for species, C. dukae (Roll, 1928) Travassos, 1931, improving the manuscript. The senior author formerly thought to be a ubiquitous parasite of wishes to express his appreciation to the Texas amphibians (Baker, 1978; McAllister and Up- Parks and Wildlife Department for permission ton, 1987b), is primarily a roundworm parasite to collect toads via Scientific Collecting Permit of molluscs and represents an accidental parasite SPO44-1. of amphibians. In summary, differences in prevalence of in- Literature Cited fection and parasite intensities among the 3 Axtell, R. W. 1963. A reinterpretation of the distri- species of Bufo were evident in our study and bution of Bufo woodhousei woodhousei Girard, es- we offer the following observations which may pecially on the southeastern margin of its range. account for these findings. Of the 3 taxa, B. d. Herpetologica 19:115-122. debilis, a more arid-adapted toad, was seldom Baker, M. R. 1978. Transmission of Cosmocercoides dukae (Nematoda: Cosmocercoidea) to amphibi- seen in open areas except during and after pe- ans. Journal of Parasitology 64:765-766. riods of heavy rainfall; it most often remained . 1987. Synopsis of the Nematoda parasitic in inactive in burrows beneath limestone rocks. Its amphibians and reptiles. Memorial University of narrow temporal niche may explain the lower Newfoundland Occasional Papers in Biology 11: 1-325. prevalence and diversity of parasites in the Brandt, B. B. 1936. Parasites of certain North Car- species. Likewise, B. w. woodhousii, which was olina Salientia. Ecological Monographs 6:490-532. moderately parasitized, is not as widely distrib- Conn, D. B. 1988. Fine structure of the tegument of uted throughout the southwest and is highly re- Mesocestoides lineatus tetrathyridia (Cestoda: Cy- stricted to localized stream-edge habitat (Axtell, clophyllidea). International Journal for Parasitol- ogy 18:133-135. 1963; Wiest, 1982). However, the species show- Dickey, L. B. 1921. A new amphibian cestode. Jour- ing the highest prevalence and diversity of par- nal of Parasitology 7:129-136. asites, B. v. valliceps, occurs in a wide variety of Douglas, L.T. 1958. The taxonomy of nematotaeniid habitats, even in altered regions such as garbage cestodes. Journal of Parasitology 44:261-273. Frandsen, J. C., and A. W. Grundmann. 1960. The dumps and storm sewers. Since toads are re- parasites of some amphibians from Utah. Journal stricted to breeding in aquatic sites (i.e., sug- of Parasitology 46:678. gesting an increased opportunity for exposure to Freeman, R. S. 1973. Ontogeny of cestodes and its

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