ECTOSYMBIONTS 0F SELECYEEI LARVAL ANURANS AND AQUATIC URODELES FROM KALAMAZDO AND BARRY COUNTSES, MICHIGAN Thesis for the Degree of M. S. MICHIGAN STATE UMVERSITY Siguré Nelson, Jr. 1966 ‘i'HESIS LIBRARY Michigan Sum ' University .m.¢ W " W340 flit-:51 “fl-155' ECTOSYMBIONTS OF SELECTED LARVAL ANURANS AND AQUATIC URODELES FROM KALAMAZOO AND BARRY COUNTIES, MICHIGAN BY Sigurd Nelson, Jr. AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements . for the degree of MASTER OF SCIENCE Department of Zoology 1966 WES! ABSTRACT ECTOSYMBIONTS OF SELECTED LARVAL ANURANS AND AQUATIC URODELES FROM KALAMAZOO AND BARRY COUNTIES, MICHIGAN by Sigurd Nelson, Jr. Larval anurans and aquatic urodeles were collected from various habitats in Kalamazoo and Barry Counties. Michigan and examined for ectosymbiotic animals. An at- tempt was made to establish habitat preference and specific- ity of the various symbionts. Amphibians were collected by means of a dip net or by trapping, isolated in containers and removed to the laboratory. Prior to licrosc0pic examination, the amphibians were anesthetized with MS 222, which reduced undue activity without resulting in death to the animals. A record of the symbionts' attachment site, and relative abundance was made. Permanent slides, microphotographs and whole-mount preservations were prepared of ectosymbionts. The most abundant ectosymbiont recorded was the peritrichous ciliate Trichodina sp. Trichodina sp. oc- curred on all larval anurans except Bufo sp. Necturus maculosus was the only urodele found with Trichodina sp. Other peritrichous ciliates were less frequent. Sigurd Nelson, Jr. Testacean sarcodines, not previously reported in the literature, were found on larval anurans at three collection stations. Necturus maculosus served as the host for Trichodina sp., Sphyranura sp. (Monogenea), and clam glochidia. Leeches were found on both aquatic urodeles and larval anurans. No evidence was discovered indicating a specific ectosymbiont to be limited by habitat. Si ECTOSYMBIONTS OF SELECTED LARVAL ANURANS AND AQUATIC URODELES FROM KALAMAZOO AND BARRY COUNTIES, MICHIGAN BY Sigurd Nelson, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1966 ACKNOWL EDGMENTS I wish to express my appreciation to Dr. T. Wayne Porter for his encouragement, guidance, and critical re— view of this study. Special thanks are also extended to Drs. Roland L. Fischer and Ralph A. Pax for their suggestions and re- view of the manuscript. Drs. David T. Clark, Gerald W. Esch, M. M. Hensley, and William D. Lindquist were helpful in identifying speci- mens; from their respective disciplines. Graduate students J. Whitfield Gibbons, Martin Kopenski, Robert Lippson, Don L. McGregor and Peter Rich assisted in a variety of ways. Mrs. Bernadette Henderson has my sincere thanks for help in providing materials necessary for this study. Illustrations of ectosymbionts were made by Richard Burbidge. ii TABLE OF CONTENTS Introduction. . . . . . . . . . . . Review of the Literature. . . . . . Field Methods and Materials . . . . Laboratory Methods and Materials. . Description of the Collection Areas with the Host-Symbionts Found . . Tabulation of Results . . . . . . . Discussion Of Results . . . . . . . Conclusions . . . . . . . . . . . . Literature Cited. . . . . . . . . . Appendices . . . . . . . . . . . . iii Presented Page 10 13 28 32 38 39 43 Table LIST OF TABLES Page Percentage of Amphibia with the fol- lowing Ectosymbionts Related to Collection Station. . . . . . . . . . . . 29 Percentage of Amphibia with the fol— lowing Ectosymbionts. . . . . . . . . . . 30 Number of Amphibians Examined from Each Collection Station . . . . . . . . . . . 31 iv LIST OF APPENDICES Appendix Page I. II. III. IV. SPECIES OF ANURANS RECORDED FOR KALAMAZOO AND BARRY COUNTIES, MICHIGAN. . . . . . . 44 ENDOPARASITES FOUND DURING THIS STUDY . . 45 ECTOSYMBIOTIC MYCOPHYTA . . . . . . . . . 46 ILLUSTRATIONS OF COMMON ECTOSYMBIONTS FOUND. . C O O O O O O C O O C . . O O O O 47 ECTOSYMBIONTS OF SELECTED LARVAL ANURANS AND AQUATIC URODELES FROM KALAMAZOO AND BARRY COUNTIES, MICHIGAN This study is based on the ectosymbiotic animals found on larval anurans and aquatic urodeles. Adult anurans and terrestrial urodeles are excluded. Lentic habitats were selected in Barry and Kalamazoo Counties located in S.W. Michigan in an attempt to relate habitat preference as well as host specificity of the symbionts. Ecological notes related to habitat selections are in- corporated as well as some limnOIOgical data. The symbionts are presented qualitatively in a phylogenetic sequence with little attempt at a quantitative analysis. Symbiosis involves the interaction of two or more species, one usually larger than the other. A symbiotic relationship may be subdivided into such categories as parasitism, commensalism and mutualism. Arbitrary limits must be used to distinguish the above forms of symbiosis as no distinct line separates them. Parasitism is limited to those forms living on or within a host species. The host must be of a different species and larger than the parasite. In all instances. the parasite derives benefit at the host's expense. Rapid destruction of the host does not occur as in the l 2 case of the predator-prey relationship. Gradual degradation of the host, in part or as a whole, is the rule. Commensalism is limited to forms receiving benefit from a host species without causing harm, whereas mutal- ism involves the exchange of benefits between two or more symbionts. Unless direct evidence is presented, either by Observation of tissue damage or by literature citation, the symbiotic relationship is considered parasitic. Due to difficulty in ascertaining whether a host receives benefit, any mutualistic association may arbitrarily be considered as commensalistic. An ectosymbiont on larval anurans and aquatic urodeles is any symbiont located on the external sur— face of such amphibians. Those forms frequenting body orifices, but generally found on the body surface are included. Subcutaneous forms are deleted. A review of the literature indicated that a total of five phyla was represented as ectosymbionts of Amphibia. Included are Protozoa, Platyhelminthes, Annelida, Arthr0poda and Mollusca. 3 Phylum: Protozoa Classification after Kudo (1953) Class: MastigOphora Order: Polymastigina Family: Tetramitidae According to Wenrich (1924a), the ectozoic flagellate, Costia necatrix (Henneguy), occurred both on the skin and gills of Rana catesbeiana Shaw, 5; clamitans Latreille and gygpalustris Le Conte. The association did not ap- pear to cause any serious pathological results although they did attach themselves to the skin and gills. Class: Ciliata Order: Holotricha Family: Amphileptidae Wenrich (1924b) described the protozoan Amphileptus branchiarum on the gills of Rana catesbeiana, R; clamitans and g; palustris. The former two frogs were collected in the vicinity of Philadelphia, while 3:4palustris was reared in the laboratory. ‘A; branchiarum, according to Wenrich (1935), perhaps represents a transition between a predaceous status and a parasitic one. A. branchiarum has a free-swimming stage during which it roams about over the gills of the host tadpoles and if, while doing so, it meets an ectozoic Vorticella sp. or Trichodina sp. it may 4 indulge its predaceous tendencies by devouring one of them. At other times, and more commonly, it attaches itself to the gills by a thin membrane within which it gently swims in a rotating manner, pausing at approPriate times to engulf masses of gill cells to satisfy its food requirements. Order: Peritricha Family: Scyphidiidae Scyphidia sp. was reported by Wenrich (1924a) on the gills of Rana clamitans and Hyla versicolor Le Conte at Woods Hole, Massachusetts. Wenrich also reported a species which closely resembles Glossatella tintinnabulum (Kent) on R; catesbeiana, R; clamitans and §;_palustris tadpoles, which Kent ('80—82) found on the skin and gills of the larvae of the salamander Notophthalmus cristatus Laurenti. Family: Epistylidae Epistylis sp., Rhabdostyla sp. and Opercularia sp., according to Wenrich (1924a), have been found only on the skin, the tail region in the vicinity of the anus being the most favored location on tadpoles of Rana catesbeiana, _R; clamitans and R; palustris at Philadelphia. Family: Vorticellidae Vorticella sp. was reported by Wenrich (l924a)-— (see family Epistylidae for host and locality data). Family: Urceolariidae Fulton (1923) reported Trichodina pgdiculus (Muller) from the gills of Necturus sp. and Notophthalmussp. larvae, indicating that Trichodina sp. found on Hydra sp. are probably this same species. Wenrich (1924a) reported Trichodina sp. on the skin and gills of Rana catesbeiana, _R; clamitans and R; palustris at Philadelphia indicating a commensalistic association. Diller (1928) reported Trichodina sp. from tadpoles of Bufo sp., 3; clamitans, .R;gpalustris, R; sylvatica Le Conte and g; pipiens in Pennsylvania. Fulton (1923) reported one species of endozoic Trichodina sp. from the urinary bladder of amphibia. Wenrich (l924a) stated that the members of the family Urceolariidae, including the genus Trichodina sp. are all associated with other animals and for the most part lead commensalistic lives on the exterior of their hosts. Miscellaneous Protozoa Upon examination of mucus from the skin of tad- poles of Pelobates fuscus Laur., and Rana esculenta L. in EurOpe, Sassuchin (1928) found numerous ectosymbiotic 6 protozoans. Some of the tadpoles were found dead or were in the process of dying. Phylum: Platyhelminthes Classification after Yamaguti (1963) Class: Trematoda Order: Monogenea Family: Sphyranuridae Species of a single genus of monogenetic trematodes Sphyranura Wright, 1879, is ectosymbiotic on amphibians. The type species, g; osleri Wright was found on the skin and gills of Necturus maculosus in Canada. Price (1939) redescribed the original material and reported g; oligorchis Alvey from the skin of N;_maculosus in Pennsylvania. Alvey (1936) described S; polyorchis without giving data on host, location and distribution. Phylum: Annelida Classification after Pennak (1953) Class: Hirudinea Mann (1962) stated that it is very difficult to draw a sharp distinction between parasites and predators among leeches. Not only do their habits vary during the life history of an individual, but effect on the host may vary according to size. 7 Order: Rhynchobdellida Family: Glossiphoniidae Autrum (1953) reported Placobdella sp. from Rana esculenta and Pelobates fusca in EurOpe. Barrow (1958) exposed Batrachobdella picta (Verrill) to NotOphthalmus viridescens in an attempt to infect the newt with Trypanosoma diemyctyli Tobey. The transmission of the flagellate proved unsuccessful, but he concluded from the feeding experiments and from the presence of dead larvae in two ponds heavily infected with leeches that it appears that if a leech of any size feeds on the newt they may be killed by loss of blood. Mann (1962) stated that Theromyzon sp. and Oligobdella sp. attack Amphibia. Order: Arhynchobdellida Family: Hirudidae Macrobdella decora (Say), according to Moore (1923). attacks anuran eggs, tadpoles and adults. Blair (1927) reported Hirudo medicinalis L. feeding occasionally on frOgs and tadpoles. Phylum: Arthr0poda Class: Eucrustacea Classification after Pennak (1953) Order: EuCOpepoda Family: Lernaeidae Lernaea cyprinacea Linnaeus was reported by Okada (1927) on Notophthalmus pyrrhogaster Boie in Japan. He 8 indicated that the copepod was probably the same species as found by Watase on Rana catesbeiana near Tokyo. Stunkard and Cable (1931) described the COpepod, Lernaea ranae, on tadpoles of 3; clamitans in Ohio. Baldauf (1961) reported L; cyprinacea from tadpoles of g; catesbeiana kept in an aquarium for several months in Texas. Tidd (1962) introduced L; cyprinacea to tadpoles of R; pipiens and _R; sylvatica and found a parasitic interaction. Tidd and Shields (1963) described the tissue damage by L; gyprinacea on tadpoles of R; pipiens. Order: Branchiura Family: Argulidae Goin and Ogren (1956) reported Argulus americanus Wilson on the perennibranch salamander Pseudobranchus striatus axanthus Netting and Goin and the tadpole R. heckscheri Wright near Gainesville, Florida. Class: Arachnida Order: Acarina The first record of Acarina as ectOparasites of Amphibia was reported by Ewing (1926). Ewing 1926 described Hannemania hylae (Ewing) which parasitized the tree toad Hyla arenicolor COpe in southern California. The mite frequently penetrated the skin and assumed the role of an end0parasite. 9 Subsequently many acari have been reported as ect0parasites of adult anurans, however the literature does not reveal acari as ectoparasites of larval anurans or aquatic urodeles. Phylum: Mollusca Class: Pelecypoda Classification after Pennak (1953) Glochidia of Simpsoniconcha ambiqua Say were re- ported by Howard (1951) to parasitize Necturus maculosus in Illinois. The glochidia were found to be deeply embedded in the gill tissue. Glochidia of Megalonaias gigantea (Barnes) were also found but failed to embed in the gill tissue. Seshaiya (1941) reported relative degrees of encystment by glochidia of Lamellidens sp. after ex- perimentally infecting the tadpoles of Rana sp. FIELD METHODS AND MATERIALS Larval anurans were collected by means of a dip net. Generally the tadpoles were not observed and success in capturing was attained by randomly dipping in the aquatic vegetation. Aquatic salamanders were usually observed before capture and collected by means of a dip 10 net or, at one collection station, baitless minnow traps. If the salamanders were not visible, then the procedure paralleled that of the tadpole collecting. Animals, when collected, were isolated in containers at the collection site. This was done to prevent trans- fer of symbionts from one host to the other. Earlier collections were placed in distilled water while subsequent collections were placed in the water from the collection site. Field notes were recorded concerning water temper- ature, depth of capture, distance of capture from shore and bottom type. Aquatic plants, at the capture site were identified. A record of the time, date and location—- tier, range and section—-was taken. Selected weather data were recorded. LABORATORY METHODS AND MATERIALS Specimens were removed from the collection-site medium and anesthetized with MS 222 Sandoz at a con- centration of 1 x 10-3, an anesthetic suitable for cold- blooded vertebrates. It was found that many of the symbionts were anesthesized making protozoan identification difficult. In addition, anesthesized symbionts were often freed from attachment to the host, consequently settling to the bottom of the container. This problem was partially ll reduced by first observing the amphibians in distilled water, followed by anesthesia. The entire external body surface of each amphibian was examined with a minimum of 27X. An examination of the media was also made. The symbionts' attachment sites were recorded. The symbionts were identified by keys to the Protozoa: Jahn (1949), Kudo (1954) and Pennak (1953): Platyhelminthes: Yamaguti (1963); and Annelida: Pennak (1953). Amphibians were identified using Blair, Blair, Brodkorb, Cagle and Moore (1957), and Wright and Wright (1949). In addition, identifications were made by Dr. Gerald W. Esch (nematodes), Dr. M. M. Hensley (amphibians). Martin L. KOpenski (leeches and aquatic plants), Don L. McGregor (ostracodes) and Dr. T. Wayne Porter (other in— vertebrates). Symbionts were generally determined to genera. Anurans were identified to genera with the ex- ception of Acris crepitans blanchardi Harper, and urodeles to species. The anurans represent the species of gage. .Hyla and Rana found in Barry and Kalamazoo Counties. For a list of anurans representing the above genera see Appendix I. . Protozoan symbionts were preserved and stained with 10% nigrosin, and acidified methyl green (1% solution in 1% acetic acid). Platyhelminthes (Monogenea), nematodes, 12 arthrOpods and molluscs were preserved in 70% alcohol. Annelids (Hirudinea) were fixed in 5% formalin and stored in 70% alcohol. The Platyhelminthes were stained with paracarmine before mounting in balsam. Nematoda, Annelida, Arthr0poda and Mollusca were stored in vials. Due to difficulty in preparing protozoan slides, micrOphotographs were made of some protozoan ectosymbionts. A Spencer compound microsc0pe adapted with a 35 mm. camera was used. Illumination was made with a Spencer Ortho- Illuminator on to which the microscoPe and camera were mounted. Photographs were made using Kodak Panatomic—X (FX 135-20) film at a shutter speed of one-tenth to one- twenty-fifth of a second with high intensity light. Photographs were made from magnification of 200x to 430x. Total length of the amphibians was recorded along with snout—vent length of proteid salamanders. The amphibians were preserved in 5% formalin, placed in glass vials and jars, and deposited in the Michigan State University Museum. The 1965 collections of Necturus maculosus were examined, tagged and released at the capture site. DESCRIPTION OF THE COLLECTION AREAS PRESENTED WITH THE HOST-SYMBIONTS FOUND Collection Station 1 Kalamazoo Co., T 13. R 9W, S 28: Open alkaline system. _ I , - Amphibians were collected on two Occasions at a depth of (4 feet and within 12 feet of the shoreline among Chara sp. The substrate consisted of muck and marl. H25 and methane were detected at the collection sites. The first collection was made 31 October 1965 between 1500 and 1700 hours under 70% cloud cover. Water temperature was 9°C. Amphibians and ectosymbionts: Raga sp.——12 tad— poles examined; ectosymbionts, Difflugia sp.——two tadpoles, 13 14 each with one specimen, and one tadpole with two specimens (found in MS 222 examining media); Trichodina sp.-—few to abundant specimens on lateral body wall of ten tadpoles. The second collection was made 14 November 1965 between 1500 and 1600 hours under a cloudless sky with bright sun. Water temperature was 7°C. Amphibians and ectosymbionts: Rang sp.——eight tad- poles examined; ectosymbionts, Trichodina sp.——few specimens on the spiracle of one tadpole and abundant specimens on the entire body surface of a second tadpole. Collection Station 2 Kalamazoo Co., T 15, R 9W. S 21: Closed system with bog margin at south end. 15 Amphibians were collected on four occasions at a depth of < 3 feet and within 10 feet of the shoreline. Aquatic plants in the immediate vicinity included Sagittaria sp. (Arrowhead), Nuphar sp. (Yellow Water Lily), Nitella sp. (Characeae), and Utricularia sp. (Bladderwort). The substrate consisted of muck. The first collection was made 20 August 1965 be- tween 1800 and 2000 hours under a hazy sky. Water temper- ature was 24°C. Amphibians and ectosymbionts: Notophthalmus viridescens--seven efts examined; ectosymbionts, Scyphidiidae-- numerous specimens on the lateral body wall of one eft. N. viridescens--one adult examined: ectosymbionts, none. Acris crepitans--one tadpole examined; ectosymbionts, Vorticella sp.--few specimens attached to anal region. Rana sp.-—seven tadpoles examined; ectosymbionts, Vorticella sp.--few specimens attached to two tadpoles; Trichodina sp.--few specimens on entire body surface of one tadpole. The second collection was made 16 September 1965 between 1600 and 1800 hours under a hazy sky. Water temperature was 23°C. Amphibians and ectosymbionts: Notophthalmus viridescens-—seven efts examined; ectosymbionts, none. N. viridescens--two adults examined; ectosymbionts, none. l6 Rana sp.--fourteen tadpoles examined; ectosymbionts, Vorticella sp.—-few specimens found on body and in MS 222 of 10 tadpoles; Trichodina sp.--few specimens on entire body surface of one tadpole; Placobdella sp.--two specimens, one each on body wall of both tadpoles. The third collection was made 26 September 1965 between 1400 and 1600 hours under a cloudy sky. Water temperature was 13°C. Amphibians and ectosymbionts: .Rggg sp.-—five tad- poles examined; ectosymbionts, Arcella sp.--one specimen on lateral body wall of one tadpole; Trichodina sp.-- few to abundant specimens on three tadpoles. The fourth collection was made 10 October 1965 between 1500 and 1600 hours under a cloudy sky with inter- mittent sunshine. Water temperature was 12°C. Amphibians and ectosymbionts: .Rana sp.--ten tad- poles examined; ectosymbionts, Difflugia sp.--two specimens on two tadpoles (found in MS 222); Scyphidiidae--few to numerous specimens on lateral body wall of six tadpoles; Vorticella sp.—-five specimens on lateral body wall of one tadpole. ”"1 17 Collection Station 3 Kalamazoo Co., T 18, R 9W, SB: Breeding pond adjacent to Wintergreen Lake. Amphibians were collected at a depth of one foot and within 4 feet of the shoreline. Aquatic plants in the vicinity of the capture were LEEBE minor (Lesser Duckweed), Scirpus sp. (Bulrush) and Typhg sp. (Cattail). The substrate consisted of organic material in various stages of decomposition. The collection was made 21 July 1965 between 1800 and 2000 hours under a partly cloudy sky. Water temper- ature was 25°C. Amphibians and ectosymbionts: Raga sp.--seven tad— poles examined; ectosymbionts, Trichodina sp.—-moderate number of specimens on entire body surface of two tadpoles and confined to oral and anal region of one tadpole. 18 Collection Station 4 Gull Lake, Kalamazoo Co., T 18, R 9W, S 6 and 7. During the Spring Of 1965 a pilot study of Necturus maculOSus yielded the following ectosymbionts: Trichodina sp., Sphyranura sp. (Monogenea), and clam glochidia. During the spring of 1966 three collections were made. Amphibians were collected at a depth of . mm no on mm msmoasome 4% e mv 5 .mm mamm m VH m m 0 0m .Qm mcmm OQfi a mcmuflmmno .< m m 5H mcwommwaua> 4% om m ma .mm mcmm H , S G D 5 a w m. s m m a m I. m am 0 I e s q T J I. A o I. . X.d STL o D A 1 o I. o .4 s d .c 3 e H 3.I o I TI. 0 o 1 q T 3 q I I 3 m1. ea q an q o e o D T I n X I.q 33 Tm no 0. T. u D. a T. D. e 2 UT. .93. P e a e n T I I. I. T m a e o T I. S I J u T. S I. S e I pu UO 2 I. I s e e e s W C s w s u s e C s s s d e d o. S d d .d . . d a D I MCOQ c monucfls ,mowo on momsflaoz nonruua meflamc < usmcmumam u m coflumum COHDUOHHOO DAD Ou Counamm mOCOAQESmODOM mafizoaaom on» £ua3 manflsma< mo Ommucoouwm .H magma 3O w a he a; m A N v «Ha .mm mcmm mm m ma .mm mawm mm Ha m mcmuflmmno mfluo¢ m m ma .mm ousm n n ma escflumflu .d mm mm on mm mzmoasome mausuowz m om mcwomeHnH> .z ,b. D .a d .S . S G . I rA I I fd M M M. n. WW I W uuW w 1d e as q I. 11 I. M «o J . vxd D A.l o I K .3 4 s e T: q u. I I. I. o o J u. I. 4 u. I. T. 3 m I. Trm Pay m” no 2 W o r“ I. ?L n x Ina P ““0 T. u 9 TL P 2 Eu m u_E I. Did a e n I. T. I? T I. «we 2 s T. I u T. s T: .s e I. nvu I. .L s e e p. P d u s s e d s p. . s a o S S u s d a d p s 1a .d d . . 1.0 o o o m mm CHE momsaaoz Iommmud mpwamca< IHOQMwmwm MONOOOHm mDCOMQE>mODOm mcflzoAHOm OED nuwz mwnfinme< mo mmMDcouumm .N magma 31 a ma ma A 6 ON NHH .mm mcmm 4 ma .9. mg m H m mcmuflmmuo mfluofi ma ma .mm omsm Va ma Bazaumflu MEODmSQEE mm mm msmoasomfi mausuomz m ha om mcwomwpwuw> m5§HO£u£QOuOZ m- h- o m v m m H .02 :oHumOm aofluomaaoo Hmuoa swananmfi< OOADMDm COAOUOHHOO Comm Eoum COCHmem mcmwnflnmfi< mo Hwnfibz .m Canoe DISCUSSION OF RESULTS Protozoans were the most common ectosymbionts of amphibians examined. Ambystoma tigrinum was the only amphibian lacking protozoan ectosymbionts. The Ranidae had the most diversity of protozoan relationships with six taxa being represented from both sarcodine and ciliate Protozoa. The other anurans all possessed two genera of protozoans. No one protozoan ectosymbiotic genus was found on all the anuran genera examined. The urodeles, excluding A;_tigrinum, each possessed one genus or family of protozoan. Protozoan ectosymbionts were taken from all but Collection Station 5, where urodeles were taken exclusively. Protozoans were not found 0n.§; tigrinum from Collection Station 9, but were found on anurans taken from this same collection station. Protozoans were on urodeles from Collection Station 4 (Gull Lake) and Collection Station 2 where anurans were taken with protozoan ectosymbionts. Trichodina sp. was the most common protozoan found (Appendix IV, Plate 2). This peritrichous ciliate was on amphibians taken at all collection stations, except 5. Necturus maculosus was the only urodele with ectosym— biotic Trichodina sp. Only at Collection Station 2 32 33 was there a greater percentage of protozoan found than Trichodina sp., if Trichodina sp. occurred on an anuran genus at a particular collection station. Only three collection stations yielded other peritrichous ciliates. Eighty—four percent of the Hyla sp. examined possessed Trichodina sp., followed by 70% of the Necturus sp., 46.6% of the Rana sp., and 33.3% of the Acris crepitans. ‘N. viridescens, A; tigrinum and Bufo sp. lacked Trichodina sp. No other protozoan exceeded 11.6% infestation for Amphibia. The percentage (5.5) of Trichodina sp. given for Rana sp. at Collection Station 6 is extremely misleading. One specimen of Trichodina sp. was found on one tadpole. Trichodina sp., when present, generally exceeded 10 or more per individuals infested. Collection Station 6 was a closed marl ditch, and the Trichodina sp. present might have been introduced via adult anurans frequenting the habitat. The aloricate peritrichs lacking a stalk have been reported only as members of the family Scyphidiidae. At times, Scyphidia sp. (Appendix IV, Plate 1) and Glossatella sp. could readily be identified, while in other instances generic identification was not possible. Scyphidiidae were found on N; viridescens, Collection Station 2, and were the only ectosymbiotic protozoans 34 which occurred on urodeles other than Trichodina sp. Scyphidiidae were found on Raga sp. at this same col— lection station. An occurrence of testacean sarcodines Arcella sp. and Difflugia sp. had not previously been reported in the literature. According to Jepps (1956) testaceans are very widespread, and often abundant, in all habitats where protozoans occur, except in salt water, and that no parasitic forms are known. Arcella sp. were found on ‘Raga sp. exclusively. Difflugia sp. have not been ob- served on tadpoles but were always found in the MS 222 examining media. Heinis (1928) found one Difflugia constricta alive after washing the feet of a freshly killed Perdix cinerea and stated that it was probable that bird transportation was of greater significance than usually supposed. The writer felt that larval anurans may transport testaceans in much the same manner. No evidence has been observed in the present study which would indicate that the protozoans found were ectoparasitic. However, in the absence of histological studies, definite symbiotic levels could not be determined. Ninety-six percent of all Necturus maculosus were parasitized by the monogenetic trematode Sphyranura sp. (Appendix IV, Plate 3). Price (1939) separated the 35 Sphyranura sp. on the basis of the number of testes, .§; osleri 12-16, §;_oligorchis 5’7 and §;_polyorchis 20-23. Price further stated that should a re-examination of the hooks of S; polyorchis reveal lateral prominences, S, pplyorchis is possibly a synonym of g; osleri. An examination of specimens of Sphyranura sp. from Gull Lake, revealed that the testes varied from three to nine in number, thus not falling within the precepts of the three previously described species. It is the writer's Opinion that perhaps §;_osleri and S; oligorchis may represent the same species. Together with the doubt Price stated, this would place the three Sphyranura sp. into a single species, S; osleri. Leeches were found on Ambystoma tigrinum and Bag; sp. at three collection stations. Ratio of body size of the leech to body size of the amphibians indicated a parasitic rather than a predator-prey relationship. No amphibians were found with Macrobdella decora attached, as reported by Moore (1923). One tadpole of Rana was collected with a large triangulated lesion on the ventral surface which appeared to be the result of a dislodged leech. It would appear that if adult M;_decora attacked amphibians, such interactions would generally result in death to the amphibians and go unobserved during this study. 36 A single arthrOpod Cyprid0psis vidua (Ostracoda) was found about 1 mm. from the left rear leg of one Ambystoma tigrinum. This ostracode was a free—living form and may have been caught in the surface tension when transferring the salamander from distilled water to MS 222. Twenty—seven percent of all Necturus sp. were parasitized by clam glochidia which were partially em- bedded within the gill tissue. Without culturing glochidia to maturity, generic identification is impossible. No evidence was found which would indicate that an ectosymbiont was endemic to any particular habitat. ,Epistylis sp. and Vorticella sp. were found only on amphibians from bog areas, but both ciliates have been found in most aquatic habitats. Scyphidiidae were found in at least two diverse habitats. The multi-cellular ectosymbionts of N;_maculosus may have shown habitat pref- erence, but without collecting the salamander from other collection stations such habitat preferences could not be determined. Water temperature did not appear to be critical during this study. Trichodina sp. ectosymbionts were present on amphibians collected in habitats ranging from 4-35°C. Scyphidiidae were found on amphibians in areas ranging from 12—350C. Multicellular ectosymbionts were not influenced by water temperature. 37 With the exception of Amphileptus branchiarum, reported by Wenrich (1924b), little explanation can be made concerning the absence of previously reported ectosymbionts during this study. ‘A:branchiarum was commonly found swimming around the gills or embedded in the gill tissue of tadpoles. No tadpoles were examined during this study with gills exposed. The mastigOphoran Costia necatrix and the peritrichous ciliates Rhabdostyla sp. and Opercularia sp. reported by Wenrich (1924a) were not found. Both ciliates are members of the family Epistylidae. Trichodina sp. reported by Fulton (1923) on N; viridescens and by Diller (1928) on Bufo sp. ‘were not found on these anurans during this study. Per— haps too few Notophthalmus sp. and Bufg sp. were examined by the writer. The results are presented with no allowance being made for the periodic ecdyses which are characteristic of the Amphibia. Ectosymbionts attached by means of haptors, or embedded in the skins, are not influenced by this shedding. However, those ectosymbionts that were sessible or swimming freely on the surface of the amphibian would be dislodged and would have to re—establish their relationship. Generally, those amphibians examined im- mediately after capture had a greater number of ectosymbionts, while amphibians examined after some delay had fewer 38 ectosymbionts. Also, if an amphibian remained in the MS 222 examining media too long, ecdysis occurred. EndOparasites found during this study are reported in Appendix II. Ectosymbiotic myCOphyta are reported in Appendix III. CONCLUS IONS l. The peritrichous ciliate Trichodina sp. was ectosymbiotic on larval anurans at all collection stations where anurans were taken. Trichodina sp. occurred on all larval anurans examined with the exception of Bufo sp. The only urodele found with Trichodina sp. was Necturus maculosus. 2. Other peritrichous ciliates were less common and occurred on amphibians at three collection stations. Both anurans and urodeles had ectosymbiotic Scyphidiidae. 3. Testacean sarcodines, not previously described in the literature, were found on larval anurans at three collection stations. 4. Necturus maculosus served as the host for three phyla of ectosymbionts: Protozoa, Platyhelminthes, and Mollusca (Clamgfiochidia). Other urodeles were largely free of ectosymbionts. 5. Leeches were found on both aquatic urodeles and larval anurans. 6. Ectosymbionts do not seem to be limited by habitat. LI TERATURE CITED LITERATURE CITED Alvey, C. H. 1936. The morphology and development of monogenetic trematode Sphyranura oligorchis (Alvey, 1933) and description of g; polyorchis n sp. Parasitol., 28: 229-253. Autrum, H. J. 1932. Ein neuer Fundort von Haementeria costata (Fr. Muller) in Deutschland und Bemerkungen fiber den mOglichen Wirt des Egels. Zool. Anzeiger, 98 (1/2): 45-46. Baldauf, R. J. 1961. Another case of parasitic COpepods on amphibians. Jour. of Parasitol., 47 (2): 195. Barrow, J. H., Jr. 1958. The biology of Trypanosoma diemyctyli Tobey. Jour. of Prot., 5: 161-170. Blair, W. F., A. P. Blair, P. Brodkorb, F. R. Cagle and G. A. Moore. 1957. Vertebrates of the United States. New York: McGraw—Hill Book Co., 819 p. Blair, W. N. 1927. Notes on Hirudo medicinalis, the medicinal leech, as a British species. Proc. 2001. Soc. Lond., 999-1002. Chandler, A. C., and C. P. Read. 1961. Introduction to parasitology. New York: John Wiley and Sons, 822p. Diller, W. F. 1928. Binary fission and endoximis in the Trichodina from tadpoles. Jour. Morphol. PhYSiOlo: 46: 521. Ewing, H. E. 1926. The life history and biology of the tree—toed chigger, Trombicula hylae Ewing. Ann. Ent. Soc. Amer., 19(3): 261—267. Fassett, N. C. 1957. A manual of aquatic plants. The University of Wisconsin Press, Madison, 405 p. Fulton, J. F., Jr. 1923. Trichodina pediculus and a new closely related species. Proc. Boston SOC. Nat. Hist., 37: 1. Goin, C. J. and L. H. Ogren. 1956. Parasitic copepods (Argulidae) on amphibians. Jour. of Parasitol., 42: 72. 4O 41 Heinis, F. R. 1928. Betrag zur Moosfauna des Krakatau. Verh. Naturf. Ges. Basel, 39: 57—65. Howard, A. D. 1951. A river mussel parasitic on a salamander. Nat. Hist. Miscellanea, No. 77. Jahn, T. 1949. How to know the protozoa. Dubuque, Iowa: Wm. C. Brown Co., 234 p. Jepps. M. W. 1956. The protozoa, sarcodina. Edinburgh: Oliver and Boyd. Kudo, R. 1954. Protozoology. Springfield: Charles C. Thomas, 966 p. Mann, K. H. 1962. Leeches (Hirudinen) their structure, physiology, ecolOgy and embryology. New York: Pergamon Press. Moore, J. 1923. The control of blood-sucking leeches with an account of the leeches of palisades inter- state park. Roosevelt Wild Life Bulletin, Vol. 2, No. 1, 9—53. Okada, Y. K.‘ 1927. Copepode parasites des amphibians. Annotationes ZoolOgicae Japonenses. 11: 185-187. French trans. Pennak, R. W. 1953. Fresh—water invertebrates of the United States. New York: The Ronald Press Co., 769 p. Price, E. W. 1939. North American monOgenetic trematodes. Vol. IV. The family Polystomatidae. 6:80-92. Sassuchin, D. 1928. Zur Froge uber die ecto- und endo- parasitischen Protozoen der Froschkaulqueppen. (Ecto— and endOparasitic protozoa of tadpoles.) Arch. Protistenk. 64 (l/2):71-92. Seshaiya, R. V. 1941. Tadpoles as hosts for the glochidia of fresh—water mussels. Current Science, Bangalore, Oct. 12, 1941, 535—536. Stunkard, H. W. and R. M. Cable. 1931. Notes on a species of Lernaea parasitic in the larvae of Rana clamitans. Jour. of Parasit., 18: 92-97. Tidd, W. M. 1962. Experimental infestations of frog tadpoles by Lernaea cyprinacea. Jour. of Parasit., 48: 870. 42 Tidd, W. M. and R. J. Shields. 1963. Tissue damage in- flicted by Lernaea cyprinacea Linnaeus, a COpepod parasitic on tadpoles. Jour. of Parasit., 49: 693-696. Wenrich, D. H. 1924a. Protozoa on the skin and gills of tadpoles. Trans. Am. Microsc.£kxn,43: 200-202. Wenrich, D. H. 1924b. A new protozoan parasite,Amphi- leptus branchiarum, n. sp. on the gills of tadpoles. Trans. of the Amer. Microsc. Soc., 43: 191-199. Wenrich, D. H. 1935. Host—parasite relations between parasitic protozoa and their hosts. Proc. Am. Phil. Soc., 75: 605-50. Wright, A. H. and A. A. Wright. 1949. Handbook of frogs and toadsof the United States and Canada, 3d ed., Ithaca, New York: Comstock Publishing Associates, Inc. Yamaguti, S. 1963. Systema helminthum. Vol. IV monogenea and aspidocotylea. Interscience, New York, 699 p. APPENDICES APPENDIX I SPECIES LIST OF ANURANS RECORDED FOR KALAMAZOO AND BARRY COUNTIES, MICHIGAN Bufonidae: Bufo americanus Holbrook B. fowleri Hinckley Hylidae: Acris crepitans blanchardi Harper Pseudacris nigrita Schwartz Hyla versicolor LeConte g; crucifer Wied Ranidae: Rana clamitans Yarrow g; sylvatica Le Conte “R; pipiens Schreber '3; palustris Le Conte R. catesbeiana Shaw 44 APPENDIX II ENDOPARASITES FOUND DURING THIS STUDY EndOparasites found in the examining media following defecation by amphibians. . Collection End0para51te ' ’Host Station Opalinidae (Ciliate) Rana sp. 1 and 6 Nyctotherus sp. (Ciliate) Rana sp. 1 and 6 Cestoda §h_maculosus 4 Nematoda Rana sp. ‘ 1 and 2 Subcutaneous nematodes identified as Filariodea were found on 17 Necturus maculosus at Collection Station 4. 45 APPENDIX III ECTOSYMBIOTIC MYCOPHYTA Ectosymbiotic mycophyta of the family Saprolegni- aceae were found on the skin and gills of Necturus mac— ulosus from Gull Lake. Two of the 29 N;_maculosus examined were parasitized by the Saprolegniaceae. APPENDIX IV ILLUSTRATIONS OF COMMON ECTOSYMBIONTS FOUND 48 PLATE 1. Scyphidia sp. 49 i 1‘ )’ \ .,' ‘ ‘ ( I, s way.“ .u . \\\ _ ‘._._ -..-, . flu)” l .. é W .. é.“ / Kg . W/x 1, \ 3 \ . v' (OT) H "\i I? 25%. J‘ , _». ‘.‘ -.‘ «\ / ,l,‘-§‘:“-“.. v .' . w - .. m “T WWW ' 11H ‘ ( PLATE 2. Trichodina sp. PLATE 3. 50 Sghzranura sp. oral sucker pharynx intestine vitellaria testes caudal sucker large hook TTTTTT utflflflfyflfi‘m ”'Crflx'l‘fififlwijfflfi:EHJIIW;