Cryptobia Cataractae Sp. N. (Kinetoplastida: Cryptobiidae), a Hemoflagellate of Some Cyprinid Fishes of West Virginia1

18 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY

tode egg counts in feedlot cattle. Am. J. bendazole-tolerant strain of Haemonchus con- Vet. Res. 31: 1203-1215. tortus in lambs. Am. J. Vet. Res. 30: 81-84. Nelson, D. L., R. G. White, J. O. Mozier, and Stober, M., and H. Ende. 1964. Behandlungs- A. D. Allen. 1970. Toxicity of larger-than- versuche mit Maretin beim Magendarm- recommended doses of naphthalophos to cattle, wurmbefall des Rindes. Berl. Miinch. tieiiirztl. sheep and goats. Am. J. Vet. Res. 31: 199- Wchnschr. 77: 100-105. 201. Train, C. T., R. G. White, and M. F. Hansen. Partosoedjono, S., J. H. Drudge, E. T. Lyons, 1968. Efficacy of coumaphos and naph- and F. W. Knapp. 1969. Evaluation of thalophos against nematodes of lambs. Am. J. naphthalophos against Oestrus ovis and a thia- Vet. Res. 29: 2331-2335.

Cryptobia cataractae sp. n. (Kinetoplastida: Cryptobiidae), a Hemoflagellate of some Cyprinid Fishes of West Virginia1

ROBERT E. PUTZ Bureau of Sport Fisheries and Wildlife, Eastern Fish Disease Laboratory, Kearneysville, West Virginia 25430

ABSTRACT: Cnjptobia cataractae sp. n. is described from the blood of some cyprinid fishes of West Virginia. The leech vector is Cystobranchus virginicus. It is compared with the Cryptobia species found in the blood of North American freshwater fishes.

Cnjptobia (Leidy, 1846) (Synonym: Trypano- Virginia, latitude 39°22'30" N, longitude plasma) (Laveran and Mesnil, 1901) is a 77°57'30"W. genus of biflagellated protozoan (Kineto- Fishes from nature were collected by seining plastida: Cryptobiidae). It has been recorded or electrofishing using a 115-volt alternating in. both marine and freshwater fishes of North current system. America; from marine fishes as parasites of the The leech vector of Cnjptobia cataractae, blood (Strout, 1965) and digestive tract Cystobranchus virginicus (Hoffman, 1964), (Noble, 1968); from freshwater fishes as para- was collected manually with small forceps sites of the skin (Swezy, 1919), gills (Wen- from the host fishes, Rhinichthijs cataractae, rich, 1931), and blood. after anesthetization with MS-222 (tricaine The following are reports of Cryptobia methanesulfonate) at a concentration of 1 found in North American freshwater fishes: C. part to 15,000 parts water. Piscicola salmo- borelli (Mavor, 1915), C. gurneyomm (Laird, sitica leeches used for comparative studies were 1961), and C. salmositica (Katz, 1951; Davi- collected and sent by Dr. Dale C. Becker, son et al., 1954; Wales and Wolf, 1955; Becker Biology Department, Battelle-Northwest, Rich- and Katz, 1965a, b, c, 1966, 1969; Katz et al., land, Washington 99352. Prior to examina- 1966). Except for C. salmositica, the orga- tion, leeches were put in 500 ml of fresh nisms have not been extensively studied. spring water and placed into a 4 C refrigerator Materials and Methods for 4 to 6 days. This permitted much of the blood within the leech to be digested. The geographical area of natural study and After refrigeration, fasted leeches were specimen collection was the Opequon Creek placed on tissue paper to remove excess water, at Dandridge Ford, Jefferson County, West macerated in a mortar with Hanks' balanced salt solution, lightly centrifuged, let stand for 1 A portion of a dissertation submitted in partial fulfill- ment of the requirements for the degree of Doctor of 10 minutes, and the saline with contained Philosophy in the Department of Biological Sciences, Ford- ham University, The Bronx, New York 10458. Cnjptobia decanted.

Figure 1. Freehand sketch of Cryptobia cataractae sp. n. showing organelles: Abbreviations: AF, anterior flagella; B, blepharoplast; K, kinetoplast; N, nucleus; UM, undulating membrane; PF, posterior flagella.

Fish used for transmission and host range days, anesthetized with MS-222, and examined. studies were laboratory-reared or collected Blood was obtained by cardiac puncture using from streams. Those collected from streams a heparinized 25-gauge needle and syringe, were tested for the presence or absence of and diluted with equal part of BSS and ex- Cryptobia by withdrawal and microscopic amined microscopically. examination of blood. Blood containing C. cataractae was obtained Results from host fishes after anesthetization with Cryptobia cataractae sp. n. (Fig. 1) is a MS-222 by cardiac puncture, puncture of biflagellated protozoan (Kinetoplastida: Crypto- vessels under the haemal arch in the caudal biidae) which is in constant motion, with peduncle region, and severing of the tail at movements due to flagellar, undulating mem- the posterior end of the caudal peduncle brane, and entire body movements. region. MORPHOMETRICS (in microns): From 50 To prevent coagulation of the blood, a drop of 2% heparinized saline was placed into con- formalin-fixed specimens measured with an tainers receiving blood, including syringes and ocular micrometer. Body length: 17 (14.7— needles. 18.9); body width: 2 (1.54-2.2); length of For transmission and host range studies, anterior flagellum: 11 (9.6-13.2); length of approximately 100 Cryptobia in 0.05 ml of posterior flagellum (free part): 14 (11-16.4); Hank's balanced salt solution were injected nucleus diameter: 1—1.5; kinetoplast length: into fishes intramuscularly near the base of 2.6-3.1. the dorsal fin. HOSTS, NATURAL: Longnose dace, Rhinichthys To determine transmission and host sus- cataractae (primary host); blacknose dace, ceptibility, inoculated fishes were held for 10 Rhinichthys atratulus; cutlips minnow, Exo-

Table 1. Comparison of Cryptobia cataractae with other North American Cryptobia from freshwater fishes. Measurements in microns.

Length Length Body- Body anterior posterior Kinetoplast Species Vector length width flagella flagella Nucleus length Cryptobia 17 2 11 14 1-1.5 2.6-3.1 cataractae1 C. Virginians (14.7-18.9) (1.54-2.2) (9.6-13.2) (11-16.4)

C. salmositica2 P. salmositica 14.94 2.46 16.05 8.96 1.5-3.5 4.58 X 0.78 C. gurnet/orum* None given 25.1 6.7 19 10 None given None given (19.2-28.2) (3.1-7.1) Scattered giants 38.8 9.1 C. borreli* None given 20—25 3—4 None given. None given None given None given Natural hosts: 1 R. cataractae, R. atratulus, E. maxillingtia, C. anomalurn. - O. kisutch, C. rhoiheus, C. alenticus, S. gairdneri, O. tshawytscha, S. trutta, S. gairdneri gairdneri, C. snyderi, O. keta, O. gorbuscha, Prosopium williamsoni, C. bairdi, C. gubsiis, C. beldingi, C. perplexus, C. asper, R. cataractae, Gasterosteus aculeatus. A C. clupeaformis, S. namaycush, E. Indus. •l C. commersonii. glossum maxillingua; stoneroller, Campostoma the flagella. The writer therefore provisionally anomalum. identifies it with this species." HOSTS, EXPERIMENTAL: Creek chub, Semo- Swezy (1919) discussed T. carassii, which tilus atromaculatiis; banded killifish, Fundulus he named in 1915, as an ectocommensal para- diaphaniis. site of goldfish, Carassius auratus, in aquaria SITE OF INFECTION: Blood and body fluids. at the University of California. He reports, VECTOR: Cystobranchus virginicus Hoffman, "A comparison of T. ca.ra.ssii with the blood 1964. inhabiting species of the genus shows no im- TYPE LOCALITY: Opequoii Creek at Dan- portant differences." He does not indicate dridge Ford, Jefferson County, West Virginia, why he considered it a separate species. latitude 39°22'30" N, longitude 77°57'30" W. Wenrich (1931) found a Cryptobia on the TYPE SPECIMENS: USNM Helm. Coll. (No. gills of the carp, Cyprinus carpio, from the 63198). Schuylkill River in Philadelphia. He stated, Discussion "The structure of this trypanoplasm is some- what different from that attributed to T. Previous descriptions of Cryptobia species carassii described by Swezy from the skin of from North American freshwater teleosts have goldfish and different from T. cyprimift from been based on. morphometrics and hosts they the blood of European, carp." He doesn't say were parasitizing. Experiments to elucidate how it is different and does not elaborate as to the biology of the organism, i.e., life cycle, its taxonomic status. vector, experimental transmission, and possible Katz (1951) described two new species of physiological processes, were not used for de- Cryptobia: C. salmositica from silver salmon, termining species. Oncorhynchus kisutch, and C. lynchi from two Mavor (1915), the first to report a Crypto- cottids, Cottus rhotheus and C. aleuticus, from bia from North America, found the organism the same geographical area in the state of in a moribund sucker, Catostomus commersoni, Washington. He separated them on the basis from Go Home Bay of Lake Huron. He states, of their morphometrics and host difference. "The trypanoplasm found in the sucker has Wales and Wolf (1955) found Cryptobia all the morphological characters described and from rainbow trout, Salmo gairdneri, king sal- figured for Tnjpanoplasma borreli by Lavern mon, O. tshawytscha, steelhead rainbow trout, and Mesnil (1902), size and shape of the body, Salmo gairdneri gairdneri, brown trout, S. position and shape of the nuclei, and length of trutta, coho salmon, O. kisutch, Klamath

Table 2. Natural host range of Cryptobia cataractae over a 3-year period, 1967-69.

Fish Genera and Number Number Per cent family species examined infected infection Cyprinidae Rhinichthtjs cataractae 111 108 97 Rhinichthys atratulus 84 25 30 Exoglossum maxillingua 77 14 18 Campostoma anomalum 69 13 19 Semotilus corporalis 75 20 27 Semotilus atromaculatus 57 0 0 Notropis cornutus 72 0 0 Cyprinodontidae Ftmdulus diaphanus 53 0 0 Cottidae Cottus sp. 100 0 0 Centrarchidae Ambloplites rupestris 65 0 0 Microptenis dolomieui 48 0 0 Ictaluridae Nottirus sp. 61 0 0 large-scaled sucker, Catostomus snyderi, scul- within the family Cyprinidae. Natural infec- pin, Cottus sp., and the leech, P. salmositica, tions were not observed in the families in the state of California. They designated Cyprinidontidae, Cottidae, Centrarchidae, and the Cryptobia as C. borreli without giving any Ictaluridae (Table 2). This natural host range reasons for doing so. was checked for 3 consecutive years to de- Laird (1961) recorded a hemoflagellate termine if any fluctuation occurred. It re- from northern pike, Esox Indus, the salmonids mained the same which indicated a similar Coregomis clupeaformis and Salvelinus namay- constant host range for the vector, C. virginicus. cush, calling it C. gurneyorum (Minchin). He Since the incidence of infection was the highest based speciation on morphometrics and the (97%) among the longnose dace, R. cataractae, presence of cytoplasmic chromophilic granules. and subsequent investigation showed this fish The procedure of naming a new species of during its spawning season to be the only Cryptobia when infections are found in new species in the endemic geographical area to hosts, and on minor morphometric differences harbor the vector, it is considered to be the alone, is questionable since the polymorphic reservoir host of C. cataractae. diversity of Cryptobia species is well known (Table 1). This is further substantiated by the work of Becker and Katz (1965b). Becker Table 3. Comparative experimental host range of (pers. comm.) believes C. salmositica from the Cryptobia salmositica from the Pacific northwest- Pacific Northwest of the United States to be a ern U. S. and Cryptobia cataractae from the eastern valid species primarily on the basis of the dis- U.S. tribution and association with its common C. salmositica C. cataractae endemic leech vector, P. salmositica. C. Western U. S. Eastern U. S. cataractae is declared a new species after com- Number Number Number Number paring its morphometrics with the other three Fish injected infected injected infected species noted from North America (Table 1), Salmonidae Salmo gairdneri 10 10 10 0 natural and experimental host range with that Salmo fontinalis 10 10 10 0 of C. salmositica from the western United Salmo trutta 10 10 10 0 Oncorhynchns States (Tables 2, 3), and other biological tschawytscha 10 10 10 0 parameters,1 i.e., vectors, cryogenic preser- Oncorhynchus kisutch 10 10 10 0 vation, histopathology, maintenance, and cul- Cyprinidae Rhinichthys atratulus 10 10 10 10 ture attempts. The specific name for this Semotilus atromaculatus 5 5 10 7 Cryptobia is taken from the primary host fish, Cyprinodontidae Fundulus diaphanus 0 0 10 10 Rhinichthys cataractae. Cottidae Cottus sp. 10 10 10 0 The natural host range of C. cataractae is Centrarchidae Lepomis macrochirus 10 0 10 0 Lepomis cyanellus 10 0 10 0 1 To be published by U. S. Department of the Interior, Microptenis dolomieui 5 0 5 0 Bureau of Sport Fisheries and Wildlife as a Technical Ambloplites rupestris 10 0 0 0 Paper.

C. cataractae showed a wider experimental 1951, in freshwater teleosts of the Pacific host range (Table 3) than was found in nature Coast. Tr. Am. Fish. Soc. 94: 327-333. (Table 2). An additional fish species of , and . 1965c. Distribution, ecol- the Cyprinidae family, the creek chub, S. ogy, and biology of the salmonid leech, Pisci- atromaculatus, when experimentally injected cola salmositica (Rhynchobdellae: Pisci- colidae). J. Fish. Res. Bd. Can. 22: 1175- with C. cataractae, became infected as did the 1195. banded killifish, F. diaphanus, a member of , and . 1966. Host relationships another fish family, Cyprinodontidae. of Cryptobia salmositica (Protozoa: Mastigoph- When C. salmositica and C. cataractae were ora) in a western Washington hatchery each injected into the same species of fishes, stream. Tr. Am. Fish. Soc. 95: 196-202. C. salmositica showed the widest experimental , and . 1969. Refractive gran- host range infecting eight species of fishes from ules in Cryptobia salmositica, a hemoflagellate three different families, while C. cataractae was of freshwater teleosts. Tr. Am. Micr. Soc. 88: infective to only three species of two families 300-304. Davison, R. C., W. Breese, and M. Katz. 1954. (TableS). The hemoflagellate, Cryptobia salmositica, in Only two fish species, R. atratulus and S. Oregon salmon. J. Parasit. 40: 703-704. atromaculatus, both of the family Cyprinidae, Hoffman, R. L. 1964. A new species of Cysto- showed susceptibility to both Cryptobia species. branchus from southwestern Virginia (Hir- The leech vector, Cystobranchus virginicus, udinea: Piscicolidae). Am. Midi. Nat. 72: was found only on the longnose dace, R. 390-395. cataractae. The probability of the natural host Katz, M. 1951. Two new hemoflagellates range of Cryptobia cataractae being a direct (genus Cryptobia) from some western Wash- biological function of leech preference or spec- ington teleosts. J. Parasit. 32: 245-250. ificity existed. This apparently is the case as , J. C. Woodey, C. D. Becker, P. T. K. Woo, and J. R. Adams. 1966. Records of shown by the natural and experimental host Cnjptobia salmositica from sockeye salmon range of C. cataractae. from the Fraser River drainage and from the Acknowledgments State of Washington. J. Fish. Res. Bd. Can. 23: 1965-1966. The author wishes to thank Mr. Charles P. Laird, M. 1961. Parasites from northern Can- Carlson, Hatchery Biologist, Experimental Fish ada. II. Haematozoa of fishes. Can. J. Zool. Farming Station, Stuttgart, Arkansas, for noting 39: 541-548. Mavor, J. W. 1915. On the occurrence of a the presence of Cryptobia cataractae sp. n. in trypanoplasm, probably Tnjpano plasma bor- the blood of longnose dace, Rhinichthys cata- reli Laveran et Mesnil, in the blood of the ractae, while a trainee in fish diseases at this common sucker, Catostomus commersonii. J. laboratory; Dr. John J. A. McLaughlin, chair- Parasit. 2: 1-6. man of the Department of Biological Sciences, Noble, E. R. 1968. The flagellate Cryptobia Fordham University, and Director of The in two species of deep sea fishes from the Louis Calder Conservation and Ecology Study eastern Pacific J. Parasit. 54: 720-724. Center for his mentorship during the study; Strout, R. G. 1965. A new hemoflagellate Drs. S. F. Snieszko and G. L. Hoffman of the (genus Cryptobia) from marine fishes of Eastern Fish Disease Laboratory for their northern New England. J. Parasit. 51: 654- assistance and critical review of the total 659. dissertation. Swezy, O. 1919. The occurrence of Trypano- plasma as an ectoparasite. Tr. Am. Micr. Soc. Literature Cited 38: 20-24. Becker, C. D., and M. Katz. 1965a. Trans- Wales, J. H., and H. Wolf. 1955. Three mission of the hemoflagellate, Cryptobia sal- protozoan diseases of trout in California. mositica Katz, 1951, by a Rhynchobdellid vec- Calif. Fish and Game 41: 183-187. tor. J. Parasit. 51: 95-99. Wenrich, D. H. 1931. A trypanoplasm on the , and . 1965b. Infections of the gills of carp from the Schuylkill River. J. hemoflagellate, Cryptobia salmositica Katz, Parasit. 38: 133.