THE UNIVERSITY OF ILLINOIS LIBRARY S 10.5 i L L -- IRF Ps The person charging this material is re- sponsible for its return to the library from which it was withdrawn on or before the Latest Date stamped below. Theft, mutilation, and underlining of books are reasons for disciplinary action and may result in dismissal from the University. UNIVERSITY OF ILLINOIS LIBRARY AT URBANA-CHAMPAIGN AUG 2 Au’ 4| ‘i NOV 29 98I.. rane 4 4/1983 APR 6 (i983 ILLINOIS BIOLOGICAL MONOGRAPHS PUBLISHED QUARTERLY UNDER THE AUSPICES OF THE GRADUATE SCHOOL BY THE UNIVERSITY OF ILLINOIS VOLUME xX Urbana, Illinois 1926 EpItoRIAL COMMITTEE STEPHEN ALFRED FORBES WILLIAM TRELEASE HENRY BALDWIN WARD TABLE OF CONTENTS VOLUME X NUMBERS 1. Studies on the Avian Species of the Cestode Family Hymenolepididae. 7 By R. L. Mayhew. With 9 plates and 2 text figures. . 2. Some North American Fish Trematodes. By H. W. Manter. With 6 plates, 2 charts, and 1 text figure 3. Comparative Studies on Furcocercous Cercariae. By H. M. Miller, Jr. With 8 plates and 2 text figures ; 4. A Comparison of the Animal Communities of Coniferous and Deciduous Forests. By I. H. Blake, With 16 plates and 25 tables 1-126 127-264 265-370 371-520 Digitized by the Internet Archive in 2011 with funding from University of Illinois Urbana-Champaign http://www.archive.org/details/somenorthamerical1Omant SOME NORTH AMERICAN FISH TREMATODES WITH 6 PLATES, 2 CHARTS AND 1 TEXTFIGURE BY HAROLD WINFRED MANTER Contributions from the ee Laboratory of the paiveraty of inate under the direction ot Henry B. War THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ZOOLOGY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS 1925 TABLE OF CONTENTS Introduction. . ssibdetiayd divas egsile) Poe aith dha ateustiite Suseta shane aye e-ce enero te, Pea db aie rele oes Material and Methods...........0.cccceccceccceeeucceecccsecceeunee. General Distribution of Entozoa in Hosts Examined. Historical Survey. . Ses caren einai ets The Morphology of Otodistomum cestoides. Edi diGuveeoentsgalscamenecev ens ee Thickness of Regions of Body Wall. . Tae srae Pata Roses cite ae ernie maton Qbodastoragn Vela POT stata see: sce:0e scares scuah oa auhy sks daemon edooaisl dual dibntcbavaiquyace avace.ik,@las aces Olods stom COSLOTE ESS os c.508 wae im seo 0ie,a(s wae o 6 Reise wv edie 6 Ale 6 ee DeLee Oe cee Variation in Posterior Extent of Vitellaria in O. cestoides. . Deviate oe emia Growth Changes in Otodistomum cestoides Within the Final Host..............--- Chart I—Showing Relations Between Body Length and Distance from the Anterior End to Ventral Sucker in O. cesfoides.... 2... cece eee ee eee COLD ES sitar orcs cia Gisinrs Soloists S visleeiaie aie oisicss\pibie Sins Bore, SF eae ae wie eres Comparison of Uterus Region with Posterior Body Region in Young Specimens Of Olodistonttii CeStOId eS <5 oc ee signe 6 dae es sos Cease os e805 8b ee88 Comparison of Uterus Region with Posterior Body Region in Adult Specimen OL OL0dtSLOMITA.COSLOMLOS = = sina, dioreiess diatazs seis 6: cers iol evaveie- tis skelecn.esiaiete e838 Comparison of Neck Region with Posterior Body Region in Young Specimens OL OLOdtSLO DUMMY COSLOUDES oo. oxc: ect nseserereleie ciate as Wio16s0 8 46 oe a taleCorosecs & Comparison of Neck Region with Posterior Body Region in Adult Specimens Of Ofodistomune Cestoides... o.oo ccicneessceccteeticee tse ssvectoe Diagram to Show Growth Changes in Otodistomum cestoides................ Comparison of Ofodistomum cestoides and O. veli, eure Rae eater a alate mete nee ares e The Miracidium of Otodistomum cestoides. . ee Notes on the Life History of Otodistomum cestoides.. A Systematic Review of the Family Azygiidae. . Pe thatele tas teenth Ca Gvee renee Sas Azygia angusticauda (Stafford ron bial gia Sr atesa sav hier 4 ol ieee a wresdsl cera ee Redvers Scene Azygia acuminata Goldberger 1911. . si gvtiaee ar eirolaus's ovaliera see e-ayGiai dis Guanes Azygialonga (Leidy 1851)....... e aay bare rtsus helena oc Meu taaeeta reer a cue afhcith cuemeea ASV OIG SEDELO sooo swresss.noe tinea oes Op etle Gale Hale emee eS Ee bent ALLY BUD DULOOSE aici uatate le wiviviche oa1s'cixiereidiel orale sie latelare atens ansie aradaig year Cooper’s Asygialuciit from L. Ronen) eRe Si oie Mee ea tet ear ete pecber) Tee in fom peaks Beant atataeknic’s ae oN os wien ors Azygia robusta Odhner 1911. vette Azygia perryii Fujita 1918. . Monet ee eree ye Mee area cne Azygia volgensis (v. Linstow) Odhner 1911............... 0.2. s seers ee eee Synopsis of the Genus Azygia. . ficeiate eyclerd Aiaas os Merieete ets A General Study of Some Marine Fish Trematodes. . Sa, Micke ius, Sue. daria eared The: Genus Podocotyles acc vsa0 os te cecds mecca Sicha ee neue been sade ete Podocotyle atomon (Rudolphi 1802)... 2.2... 06. Podocotyle olssoni Odhner 1905..... Stephaochasmus baccatus Nicoll 1907. . Lepidapedon rachion (Cobbold) Stafford 1904. Lepidapedon elongatum (Lebour 1908)... eerie eerie emer es Homalometron pallidum Stafford 1904... 00006 cece eee Steganoderma formosum Stafford 1904.2... 600 e eee eee MhePlemiuridaes.:.,. o.siecsisavareeai sities @ sie. snes aun eee Dede ee meee Hemiuris levinsent Odbner 1905.20... oc. cs cece nn cseeeecea ceaetacesese Brachylhallus crenatus (Rudolphi 1802)... 2. 0.0.6 eee Lecithaster gibbosus (Rudolphi 1802).......000 00 0c cee eee eee A ponurus sphaerolecithus Manter 1925........000 00 eee ee eee Genolinea laticauda Manter 1925. ...ic.06 2 ccs oe cee tones tesa beens s Gonocerca phycidis Manter 1925......... 2.000. - cece cece ect e ee en tenes Derogenes varicus (Miiller 1784).....0 00000 ec ce eee e eens Hirudinella fusca (Poirier 1885). . etapa shvavanese) Hier aera srearary aero ete Siphodera vinaledwardsii (Linton 1899). jase ifindutautaalanpetneenirees.e Deropristis inflata (Molin 1859)... 2.2.0. cece eee ee nee ence nen eeeeens Acanthocotyle verrilli Goto 1899... cece eet e en eee Dactylocotyle minor (Olsson 1868)... . Reet are ene Saran Summary and Conclusions.... 2.0... 0606 ce ence cece teen aces Bibliography. . eee Explanation of Plates. . Index to Scientific Names. . 133] SOME NORTH AMERICAN FISH TREMATODES—MANTER 7 INTRODUCTION The study of the parasites of North American fish, especially marine species, offers a comparatively new field to the investigator. Linton has opened this field in a broad sense by his papers on fish parasites of the Woods Hole region and of the Atlantic waters of Southern United States, This pioneer work has been necessarily incomplete in some respects. On the parasites of fishes inhabiting the colder waters of the northern Atlantic, almost no work has been done in America. Stafford and Cooper have made small collections in these regions. On European shores considerably more research has been done, and here the early work of Van Beneden, Olsson, and Levinsen has been followed by valuable contributions from Looss, Odhner, Lebour, Nicoll, and others. The present paper is based very largely upon studies of marine fish parasites from the Maine coast. These studies were first undertaken at the Mount Desert Island Biological Laboratory during the summer of 1924, although some material was also collected in the same region in 1923, Early in the work of collection, attention was directed to a common trematode (Ofodistomum cestoides) of the barn-door skate. Considerable time was devoted to the structure of this form and its life history. Later, a large series of fresh-water forms related to this species were also studied and compared. A brief report of the trematodes collected from marine fish has already been published (Manter, 1925). Sincere appreciation is here extended to Professor Ulric Dahlgren for the use of the laboratory facilities at the Mount Desert Island Biological Station. Acknowledgment is also rendered to the Hygienic Laboratory of the U. S. Public Health Service and to the U. S. National Museum for the loan of valuable material. Above all, is the writer indebted to Dr. Henry B. Ward, under whose direction these studies were undertaken. To him appreciation is extended, not only for his constant interest, but also for the loan of material from his personal collection of parasites, and for the use of his extensive library. To others, who, like fellow-students, have been less intimately associated with this work, but who have gladly cooperated in many ways, gratitude is likewise expressed. MATERIAL AND METHODS The collection of parasites was not limited to any particular group. In general, a broad collection of metazoan fish parasites was attempted. The final collection included trematodes, cestodes, nematodes, acantho- 8 ILLINOIS BIOLOGICAL MONOGRAPHS (134 cephala, copepods, leeches, an ectoparasitic turbellarian, and an ecto- parasitic isopod. From this collection the trematodes were chosen for special study. The most common shore fish such as sculpin, flounder, herring, and skates were obtained from Frenchman’s Bay in the immediate vicinity of the Laboratory. Most of the fish examined, however, were obtained at the small fishing village of Manset on the south side of Mount Desert Island. Here specimens of the larger food fish such as cod, haddock, and hake were available in large numbers. Identification of the host was usually simple as most of the fish examined were common and well known species. Some uncertainty was unavoidable in the correct identification of a few forms, such as the sculpins. The following table (Table 1) shows the general occurrence of the different groups of parasites according to hosts. TABLE 1 GENERAL DISTRIBUTION OF ENTOZOA IN Hosts EXAMINED Name of Host Number of Hosts With With ee With With Scientific Common Examined | Trema- | Nema- Ces- | Acantho- . todes todes todes | cephala Acanthias vulgaris | Common dog fish 12 2 12 Raia erinacea Bonnet skate 8 1 1 8 Raia diaphanes Big skate 2 2 1 Raia stabuliforis Barn-door skate 20 18 7 20 Raia scabrata (?) Skate 1 1 1 Anguilla chrysypa | Common eel 2 2 Clupea harengus Herring 28 9 6 Osmerus mordax Smelt 5 3 2 Scomberscombrus | Mackerel 5 5 Fundulus Killifish, heteroclitus minnow 25 9 6 23 Tautogolabrus adspersus Cunner 4 3 1 135] SOME NORTH AMERICAN FISH TREMATODES—MANTER 9 TaBLe 1 (continued) Name of Host Number of Hosts ee With With With With Scientific Common Examined | Trema- | Nema- Ces- |Acantho- todes todes todes cephala Myxocephalus octodecimspinosus | Sculpin 11 3 7 4 9 Pholis gunnellus Butterfish 9 w Anarrhichaslupus | Wolf-fish 2 1 1 Zoarces anguillaris | Eel pout 1 1 Pollachius virens Pollack 4 2 3 2 Gadus callarias Cod 10 8 9 2 10 Melanogrammus aeglifinus Haddock 17 4 9 5 17 Urophycis tenuis Hake 3 3 1 1 2 Urophycis chuss Squirrel hake 6 5 6 1 4 Hippoglossus hippoglossus Halibut ?} 1 Z 2 Pseudopleuronectes americanus Flounder 19 3 3 2 15 Limanda ferruginea (?) Sand dab 2 2 2 1 Total 198 72 70 66 90 The barn-door skate (Raia stabuliforis) was found to furnish the most varied and interesting parasites. Ecto-parasitic on this skate were found; Micropharynx parasitica (a tri-clad turbellarian), Aega psora (an isopod), and a large leech, Oxyostoma typica.* In the nasal cavity was found Charopinus dalmanni (Retz.) a large parasitic copepod. The stomach was usually heavily infected with the trematode, Otodistomum cestoides. Large numbers of cestodes occurred in the spiral valve. Although only a part * For the identification I am indebted to Prof. J. Percy Moore who reports that the leeches “‘are representatives of Oxyostoma typica Malm or possibly a closely related species of the same genus.” 10 ILLINOIS BIOLOGICAL MONOGRAPHS [136 of this cestode material was examined, two forms were identifiéd as Rhynchobothrius erinaceus (van Ben.) and Acanthobothrium coronatum (Rud.). One interesting case of Acanthocephalan infection was found in Fundu- lus heteroclitus. The livers of these minnows were almost invariably heavily infected with a juvenile form of Neoechinorhynchus. These parasites were sometimes free in the liver tissue but usually were coiled in a very thin- walled cyst. In one liver 25 or 26 such cysts could be counted. Only two of about 25 specimens failed to show the cysts macroscopically. Inonecase, this same form of juvenile Acanthocephalan was found in the intestine. This possible occurrence of the parasite in the intestine led to feeding experiments. A Fundulus was isolated and fed liver containing cysts from other minnows. As some of the material was disgorged, it could not be ascertained exactly how many cysts were ingested. The fish was fed again on the following day, and killed and examined on the third day. Three of the Acanthocephala were found in the intestine. This experiment was repeated by again feeding an isolated Fundulus the cysts on two days, and examining it on the third day. This second fish contained six of the Acanthocephala in its intestine. It is evident that the parasite can be transmitted from the liver to the intestine of the same species host. A possible explanation of the rare occurrence of this type of transfer in nature is found in the fact that the viscera of dead minnows in the aquaria are readily devoured by the other minnows. No evidence was found that the parasite ever reaches sexual maturity in this host. The final host of the parasite was found to be the common eel, Anguilla chrysypa. The locality from which the minnows were caught harbored numerous eels. Two of these fish were caught and examined for parasites. The first contained large numbers (about 50) of a Neoechinorhynchus in the lower part of the intestine. The parasites were apparently identical with the young form taken from the minnow, differing only in beingsexually mature. The second eel contained three minnows in the stomach. One of these minnows was practically digested. From the stomach content in this region one of the Acanthocephalan cysts was recovered. Furthermore, there was found free in the upper part of the intestine one of the juvenile Acanthocephalans without the cyst. Lower down in the intestine occurred large numbers of the adult parasite attached to the intestine wall. The species of Acanthocephala was kindly identified by Dr. H. J. Van Cleave as Neoechinornynchus cylindratus (Van Cleave). The demonstration seems to be quite complete that the eel (at least in this particular locality) acquires (at least in part) its infection through the minnow, Fundulus heteroclitus. Whether the minnow is a necessary link in the life-history of the parasite is very doubtful. Van Cleave (1920) in explaining juvenile forms of Acanthocephala encysted in various fish, 135] SOME NORTH AMERICAN FISH TREMATODES—MANTER 11 suggests that they may result from ingestion of the larva when it is too young to maintain itself in the intestine, the immature larva developing to an infective juvenile form in some tissue outside the digestive tract. In the course of the present work, no new methods of technique were discovered. The finding of the larger parasites such as most cestodes, acanthocephala, and nematodes is simple. To collect the smaller forms such as many trematodes, the content of the digestive tract of the host must be minutely examined. Best results were obtained by diluting a small amount of material with considerable water in a large glass dish. A chang- ing from light to dark of the background below the dish is often helpful. Careful scraping of the wall of the digestive tract is usually necessary to remove many of the smaller parasites. Cestodes were killed in HgCl: solution. Agitation by shaking or the actual stretching by hand of cestodes is necessary to prevent excessive contraction. Trematodes were killed according to the Looss method (Looss 1901) by first shaking in water and then in a 50% solution of HgCl. A modified Gilson’s solution (Petrunkevitch solution) was also found very satisfactory for trematodes. Nematodes were killed by placing in hot 70% alcohol or hot Petrunkevitch solution. Acanthocephala from the marine fish were first placed in fresh water which causes complete extension of the proboscis. When the Acanthocephala no longer respond to stimuli they are removed to the killing solution. Some difficulty was experienced by some forms, especially trematodes, later becoming quite black, due apparently to a precipitation of metallic mercury. This condition might be due to insufficient or delayed treatment with iodized alcohol. A semisatisfactory method of reclaiming such specimens was found to consist of treating them with a weak solution of nitric acid. The acid removes the mercury but slightly stains the tissues. In staining trematodes for total mounts, Ehrlich’s or Delafield’s hematoxylin or a mixture of the two was found to be very satisfactory. Alum cochineal also gave good results. Sections were stained with the ordinary reagents such as hematoxylin, iron hematoxylin, safranin, eosin, orange G, and Lyon’s blue. The present studies can be divided into three rather distinct parts as follows. First, an intensive study of Otodistomum cestoides, its first larval form, its growth changes within the final host, and its morphology. Some data was also obtained from an attempt to trace the life history of this form. Second, a comparative study of the entire family of the Azygiidae, and a revision of the American representatives of the genus Azygia. Third, a briefer account of each of the other forms of marine trematodes in the collection. This last account also includes data on several forms from the collection of Dr. H. B. Ward. These latter were collected from the Woods Hole region. 12 ILLINOIS BIOLOGICAL MONOGRAPHS {138 HISTORICAL SURVEY Van Beneden (1858 and 1871) was one of the earliest workers to deal particularly with fish parasites and many of the more common species were first described by him. His collections were made along the coast of Belgium. Olsson in 1868 records 32 different trematode parasites from Scandinavian fish. Twenty-seven of these parasites were distomes, and eight were reported as new. Olsson’s work covered a wide host range, the trematodes being collected from 42 different species of fish. Levinsen published in 1881 results of his studies on trematodes of arctic fish. This work is the product of two years spent on the west coast of Greenland and constitutes the first comprehensive record of trematodes from strictly arctic marine fish. The number of host species examined was small (six), but the trematode fauna of a few fish such as the sculpin was thoroughly studied. Nine different species of trematodes were recorded from this host. Levinsen lists thirteen different sexually mature forms, most of which are distomes. Seven species are described as new. Early work on the fish trematodes of the Mediterranean was done by Rudolphi, Monticelli, Stossich, and Looss. The detailed and accurate morphological observations of Looss have been of special service, and his ° (1899) conception of the genus among distomes has resulted in a complete systematic reorganization of that group. Probably the most important paper on trematodes of arctic fish is Odhner’s contribution (1905) to the Fauna Arctica, Trematoden des arktischen Gebietes. This critical monograph of the arctic trematode fauna is not limited to fish parasites but includes a few forms from avian and mammalian hosts. Odhner also restudied Levinsen’s material. As the paper deals with considerable change in the ‘‘natural system” of trema- todes, short descriptions are given of some forms outside the arctic regions. Twenty-two different trematodes, 19 of which are Digenea, are recorded from fish. Among these fish trematodes, four new genera and three new species are described. Several previously known species are redescribed, and some important systematic reorganizations are made. In a series of later short papers under the general title, Zum naturlichen System der digenen Trematoden, Odhner has made numerous subsequent additions to our knowledge of relationships among the trematodes, and his conclusions have been largely accepted by Nicoll (1915) and others. The trematodes of the British marine fish are probably better known than those of any other particular region. This is largely due to the re- searches of Lebour, Johnstone, T. Scott, A. Scott, and Nicoll. According 139] SOME NORTH AMERICAN FISH TREMATODES—MANTER 13 to Nicoll, up to 1915 nearly 100 species of trematodes had been recorded from British marine fish. Nicoll’s(1915)list of trematodes from marine fish contains 241 different species of trematodes belonging to 119 genera. Of these trematodes, 157 species and 73 genera are distomes, In America the work of Linton at Woods Hole, Beaufort, N. C., and the Tortugas stands almost alone. The last named region is especially interesting in revealing a very rich and varied trematode fauna (Linton 1911) with forms differing considerably from most of the more northern forms. Stafford (1904) gives a list of 37 trematodes from 32 different Canadian marine fish. Cooper (1915) gives more complete data on a few forms from the same region. A large amount of the literature is, of course, in the nature of special studies on individual or few forms. Examples of papers of this type are found in the works of Poirier (1885), Villot (1879), Pratt (1898), Darr (1902), Buttel-Reepen (1903), and Miihlschlag (1914). 14 ILLINOIS BIOLOGICAL MONOGRAPHS {140 THE MORPHOLOGY OF OTODISTOMUM CESTOIDES OTODISTOMUM CESTOIDES (VAN BENEDEN 1871) Otodistomum cestoides (van Ben.) Syns.: Distomum cestoides van Ben. 1871 Otiodistomum veliporum of Stafford 1904 Otiodistomum veliporum of Lebour 1908 Otiodistomum veliporum of Lénnberg 1891 From stomach, Raia stabuliforis (=R. laevis) Reported hosts: Raia batis Raia laevis Raia fullonica Raia lintea Raia clavata Raia radiata Raia macrorhyncha Chlamydoselache anguinea Dist. cestoides was first obtained by van Beneden (1871) from Raia batis. The genus Otodistomum was named by Stafford (1904) for a form he obtained from the stomach of Raia stabuliforis (=R. laevis). This form he called Otodistomum veliporum, identifying it as the Dist. veliporum of of Creplin. The close resemblance between the two species cestoides and veliporum has been a cause of general confusion, and, indeed, the two have been considered synonymous. Odhner (1911b) first showed that O. cestoides (van Ben.) had been incorrectly identified as O. veliporum by previous workers including Stafford, Lebour, and Lénnberg. In the course of the present studies both species were available for comparison. Specimens of O. veliporum were obtained from the collection of Dr. H. B. Ward* and were collected by him from Raia binoculata in Alaska in 1909. Further material of this same form was collected by a fellow-student from the same host at Friday Harbor, Washington, in 1924. Frequent reference will be made to this species in connection with the following discussion of the morphology of O. cestoides. The host records of the two species are doubtless somewhat confused. According to the literature, O. veliporum appears to have a much wider host range among the Selachians, O. cestoides being almost entirely re- stricted to Raia species. In the region of Mount Desert Island O. cestoides occurs abundantly in Raia stabuliforis, but was never found in any other species of skate. Both Stafford and Cooper report it from Canadian waters. * Collection of Dr. H. B. Ward, vial No. 48a from Excursion Inlet, Alaska, July 22, 1909. 141] SOME NORTH AMERICAN FISH TREMATODES—MANTER 15 Linton also records it from Woods Hole but there it seems to be a rare parasite. The trematode was found in the lower part of the pyloric stomach of Raia stabuliforis. It is one of the most common fish trematodes in the region of Mount Desert Island as almost all the skates of this species we re- infected, sometimes heavily. Only two individuals among twenty examined in 1924 were uninfected, while all of the several examined in 1923 contained the trematode. The average degree of infection is about 21, although the number varies from only 2 or 3 to about 150. In a number of cases only young immature forms were found. It is also common, especially if the infection is light, to find only mature forms or at least no very young specimens, Several instances were found wherein all stages were present. Table 2 shows a record of the collection of this parasite. The trematode is one of the largest known. Stafford records worms as long as 80 mm. when extended. There is a remarkable size variation. The longest specimen in the present collection measured about 65 mm., while several were found only 2 or 3 mm. in length. Sexual maturity is reached when the worm has a length of about 11 mm. The body form is elongate and somewhat flattened dorso-ventrally especially in the posterior region. The anterior end tapers slightly in front of the ventral sucker and is bluntly pointed. Behind the ventral sucker the body tapers only very gradually and the posterior end is usually blunt. In some cases, however, when the worm is extended the posterior tip is sharply pointed. In this case, the body form is spindle-like. The two suckers are close together near the anterior end. The ventral sucker is the larger and very powerful. When in use this ventral sucker may be protruded from the body very prominently. The worms cling tenaciously by means of the ventral sucker and may extend the anterior end of the body for some distance, feeling about in a leech-like manner. When removed from their host the worms cling to each other by means of their ventral suckers, and it is often difficult to separate individuals after they have become attached in this manner. There is a marked tendency for the worms to bend slightly inward, (i.e., ventrad), so that in profile they assume a curved or crescentic shape. This shape is especially marked when the trematodes are killed unless they are prevented by some mechanical means from so curling. The color of the worms is a translucent white when alive, becoming opaque white when killed. The region of the uterus just posterior to the ventral sucker is dark brown in color, due to the presence of many eggs which possess a light brown shell. The oral sucker is smaller than the ventral sucker and the size ratio of the two does not show any progressive change with growth, averaging the same in very small individuals as it does in the largest. The anterior sucker averages slightly over .6 the size of the ventral one, or a proportion of 16 ILLINOIS BIOLOGICAL MONOGRAPHS [142 TABLE 2 InpivipvaL INFECTION OF Raia stabuliforis witTH Otodistomum cestoides Date July 16 527. August 5 “ 28 Content of Stomach | Number of parasites Empty Sculpin Empty Empty “ a“ a a “a Crab, shrimp Remains of flounder 3 herring, part of lobster Empty “ “a 8-10 small crabs, shrimp Empty a Remains of fish, Buccinum Flounder Several 4-5 Many None 12-15 160-170 Several Z Condition Mostly mature Mature Small, immature Mature 3 Small All sizes, 10 or more mature Mostly mature Heaviest infection. 100 immature All sizes Mature about 3:5. Odhner (1911b), however, gives a ratio of 3 : 4 for O. cestoides and 3:5 for O. veliporum. Miss Lebour’s single specimen with a ratio of 1:2 must have been (as Odhner suggests) an abnormal condition. For the measurements of the suckers see Table 4. The circular opening of the oral sucker may be directed anteriorly, but is usually ventral. The ventral sucker is very deep, extending nearly to the dorsal surface of the body when the worm is extended. The cavity of this sucker extends posteriorly as well as dorsally, a condition due to the 143] SOME NORTH AMERICAN FISH TREMATODES—MANTER 17 greater development of its muscles in the posterior region. While interest- ing because of the high degree of muscular development, the structure of the two suckers is like that already described for similar forms by Poirier (1885) and for O. veliporum by Miihlschlag (1914). From the ventral anterior rim of the ventral sucker two muscle bands pass dorsally and can be traced nearly to the longitudinal body muscles of the dorsal wall. Crossing them obliquely a strong band of muscles can be seen to extend ventrally and anteriorly from the central dorsal border of the sucker to the longitudinal muscles of the ventral body wall. There are also muscle fibers extending laterally from this anterior ventral border of the sucker. Muscle bands at the posterior border of the sucker extend laterally on each side, and other bands extend dorsally to the dorsal body wall. : Short muscles are attached to the anterior margin of the oral sucker and course anteriorly and dorsally where they seem to join longitudinal body muscles of the dorsal side. Just posterior to these occur lateral oblique muscles running dorsally and laterally from each side of the sucker. They also are closely related with the dorsal longitudinal body muscles. Some of these fibers extend to the outer edge of the sucker, Finally, from the posterior edge of the sucker oblique muscle bands run dorsally and pos- teriorly and attach themselves to the pharynx. The body wall consists of the cuticula, a layer of circular muscles, and a layer of longitudinal muscles (Fig.12). The cuticula is a thick structureless layer surrounding the entire body. In a specimen about 25 mm. in length, this cuticula was about 17y thick on the ventral surface of the neck region and about 28 thick on the ventral surface of the posterior region. The thicknesses on the dorsal surfaces of the same regions were 34 and 37y, Hence, the same relative thicknesses are found that Mihlschlag noted for O. veliporum. For the latter species, Miihlschlag’s measurements are all much smaller than were found in the present species, but the size of the specimen was not given in the former case. The cuticula is much thinner over the inner surfaces of the two suckers. Here it measures only 5 to 8y in thickness. The body wall is thrown into circular folds which are more prominent with increased degree of contraction. These folds give the ringed appear- ance characteristic of this group of trematodes. Sections show that these folds involve the cuticula, and the layer of circular muscles, but not the layer of longitudinal muscles. The thickness of the circular layer, therefore, varies greatly. The layer is very thick in the center of the folds, and is greatly reduced in the furrows between the folds. In the following table a medium condition is represented as far as possible. 18 ILLINOIS BIOLOGICAL MONOGRAPHS [144 THICKNESS OF REGIONS OF Bopy WALL (Specimen about 25 mm. long) Ventral Surface Neck Region Testis Region Posterior Region Cuticula 17 og 28-30u 28-30 Circular muscles 40-50 50-55 50-55 Longitudinal muscles 34 17-20 17 Dorsal Surface Cuticula 340 40 wu 35-45 Circular muscles 50 50 38 Longitudinal muscles 22 20 16 The parenchyma presents no unusual features. It consists of a spongy tissue, filling in the regions between organs. Small nuclei are common, and also larger cells which are possibly nervous in function. The parenchyma is set through with muscle bands. Many of these, especially near the body wall, and within the neck region are oblique or diagonal, but the majority run in longitudinal bands throughout the length of the body. In species of Azygia (a related genus) a very definite and relatively narrow band of these longitudinal muscles occurs. In cross-section, the body is separated by them into medullary and cortical regions. Various Azygia species were available for comparison with O. cestoides in this respect. The localization of the muscles is much more pronounced and definite in Azygia where they form a rather compact layer. Here also the follicles of the vitellaria are outside this layer, that is, in the cortical region. In Otodistomum, the longitudinal parenchyma muscles are not limited to such a narrow layer and their formation is much less compact (Fig. 23). The region of their occurrence is, however, definite enough so that it can be noted that the follicles of the vitellaria lie largely internal to them, a condition to be contrasted with that occurring in Azygia species. The nervous system could not be worked out in detail, only its more prominent features being noted. These agreed with data already known for similar forms. The pharynx is ovoid or egg-shaped. It is located just posterior and slightly dorsal to the oral sucker. Its size is about 0.5 mm. by 0.3 to 0.4 mm. in average sized specimens. The largest examples measured about 0.75 mm. in length. Ina specimen 24.5 mm. in length the pharynx measured 0.56 by 0.37 mm., and the thickness of its walls was about 0.18 mm. There is no pre-pharynx and the pharynx itself may protrude slightly into the cavity of the oral sucker. The pharynx usually extends obliquely dorsally and posteriorly. The opening between pharynx and oral sucker is therefore facing obliquely between a dorso-ventral and a posterio-anterior direction. The walls of the pharynx are very muscular and show the same sets of muscles found in the suckers. Circular or equatorial muscles are especially 145] SOME NORTH AMERICAN FISH TREMATODES—MANTER 19 powerful in the posterior region where the esophagus joins the pharynx and these may act as a sphincter muscle between the two (Fig. 10). Special muscle bands run obliquely forward from the dorsal and ventral sides of the pharynx to the oral sucker. These bands are much more prominent on the ventral side. The degree of contraction of these muscles no doubt accounts for the varying position of the pharynx especially its tip in the dorso-ventral direction in relation to the oral sucker. Sometimes the pharynx entirely overlaps the sucker dorsally and rarely it extends almost directly posteriorly. The pharynx leads directly into the esophagus. In a specimen about 25 mm. long the anterio-posterior length of the unpaired region of the esophagus adjacent to the pharynx is only about 57, but the organ divides immediately into two lateral stems and is actually a tube running laterally and perpendicular to the long axis of the pharynx. The length of each lateral stem is about 0.4 mm. Each stem bends toward the anterior and extends in that direction about 0.3 mm. before opening into the intestine proper (Fig. 10). The esophagus lies dorsal and partly lateral to the pharynx. Outside the cuticula layer of the esophagus occurs a layer of circular muscles covered in turn by a thin coat of longitudinal muscles. The circular muscles are more prominent in the region where the esophagus joins the intestine, and serve here as a sphincter muscle usually giving rise to a slight constriction in this region. Usually at about the level of the middle of the pharynx the esophageal stems on each side open into the ceca of the intestine proper. The internal cuticular layer of the esophagus ends abruptly at this point (Fig. 10). The intestinal ceca continue forward for a short distance, then bend abruptly and lead posteriorly. The intestine shows no special regions. The two branches stretch nearly to the posterior tip of the body. They spread apart in the region of the ventral sucker, but approach each other closely immediately posterior to the sucker. They are also forced somewhat laterally by the ovary and testes. Just behind the posterior testis they again approach each other slightly. As in other related trematodes (Azygia and Leuceruthrus), the ceca are thrown into small folds throughout their length, the folds being more pronounced the higher the degree of body contraction. Each branch of the intestine ends blindly. There may be a slight difference in their length. That no significance can be attached to the frequently unequal length of the ceca is shown by the fact that the right is sometimes longer and sometimes shorter than the left. The intestine is lined internally by a layer of cuboid or low cylindrical cells from which long wavy protoplasmic processes stream out into the lumen usually nearly filling it (Fig. 10). The internal boundary of these intestinal cells is very indefinite. The size of the cells is the same in very 20 ILLINOIS BIOLOGICAL MONOGRAPHS [140 small specimens and in the largest. A thin membrane surrounds the intestine which is also provided with a thin circular (internal) and longi- tudinal (external) muscle layers. The excretory system is like that of similar forms. It consists posteriorly of a large single median tube which may be much swollen, lying between the branches of the intestine, and opening by means of a short duct at the posterior tip of the body. This tube branches a short distance behind the posterior testis, its two branches crossing the intestinal ceca ventrally and proceeding anteriorly lateral to these ceca. The two branches are con- tinuous anterior to the oral sucker.. The main excretory system consists then, of a Y-shaped tube with the two forks of the Y continuous. This tube may be thrown into folds comparable with those described for the intestine. Its outline in cross-section is very irregular and it can be seen to be con- tinually receiving minute lateral branches. Its thin membraneous lining is obscured by many deeply staining spherical granules which thickly adhere to the wall of the main tube throughout its length. These con- cretions are generally considered as waste products, and are commonly known in many trematodes. , Flame cells are numerous. They are small cells oval in shape, with prominent nuclei. Favorable sections show that they occupy ends of minute excretory ducts and that their tips are prolonged into a tuft of cilia about equal to the cell body in length. The cells measure about 14 by 8p. The genital pore is ventral, median, between the two suckers and much closer te the oral sucker. It leads into a roomy genital atrium within which usually projects the nipple-shaped genital cone or papilla (Fig. 5). Both the genital atrium and the papilla are lined with cuticula which, however, becomes very thin at the tip of the cone. Just beneath this cuticula the wall of the atrium is strengthened by a thick layer of circular muscles. These muscles continue about the base of the papilla but gradually dis- appear toward its tip. Outside these circular muscles is a layer of longi- tudinal muscles, that is, muscles running in the direction of the long axis of the papilla. These muscles are continuous from the wall of the atrium into the papilla where they are internal to the circular muscles. Oblique muscles run off from the region of the longitudinal muscle layer of the atrium. In the solid-appearing tissue of the cone occur numerous nuclei which are more numerous near the tip of the papilla. What at first appeared to be a marked difference in the size and occur- rence of this papilla in the two species of Otodistomum led to a study of the permanency and variability of this organ. In the related genus Azygia, the genital atrium is usually without a papilla, the common sex duct opening at its base. This base of the atrium is, however, protrusible so that it can be thrust out papilla-like. Thus, here the genital papilla is only a momentary or temporary structure. Odhner (1911b: 518) says in ~ 147) SOME NORTH AMERICAN FISH TREMATODES—MANTER 21 regard to this condition; ‘‘Dies konnte den Verdacht erwecken, dass es sich bei Otodistomum am Ende in derselben Weise verhielte; doch scheint mir dort die Papille einen mehr ‘soliden’ Eindruck zu machen.” Sections through O. veliporum from the Pacific showed the genital papilla almost always entirely absent or quite rudimentary in size. In O. cestoides the papilla was usually of robust form filling most of the atrium, or even entirely protruded from it (Fig. 6). In the latter case, the atrium itself is practically eliminated. In order to determine the constancy of the papilla, specimens of various sizes and degrees of body contraction were sectioned. Specimens collected in different years and killed in different solutions were also compared. The usual and contrasting conditions found are represented in Fig. 8 (O. cestoides) and Fig. 7 (O. veliporum). Of the specimens of O. veliporum sectioned, six showed the papilla absent, or a condition as in Azygia, three showed a small papilla (Fig. 7), while one showed a prominent papilla partially protruded from the pore (Fig. 29). Among eight specimens of O. cestoides sectioned, one showed the papilla entirely protruded (Fig. 6), six showed a robust papilla largely filling the genital atrium (Figs. 5 and 8), while one showed the papilla entirely absent (Fig. 28). Odhner’s suspicion is therefore correct. Although the genital papilla has always been described and figured as a prominent feature in Otodistomum species it is capable of being entirely withdrawn as is normally the condition in Azygia. What appeared, then, to be a clear distinction between the two forms studied becomes a weak taxonomic character since it is variable and inconstant. The atrium in O. veliporum from the Pacific almost constantly led very sharply posteriad and only slightly dorsad, while in the Atlantic form (O. cestoides) the slope of the atrium was almost directly dorsad and only slightly posteriad. This degree of slope of the atrium is probably associated with the condition of the papilla as when this structure was completely withdrawn in O. cestoides the atrium led sharply posteriad (Fig. 28). Measurements on the size of the atrium and papilla in different speci- mens are as follows: Otodistomum veliporum Genital atrium Genital cone 1. 0.845 0.093 mm. 0.102 by 0.08 mm. 2. 0.9 by 0.06 0.075 0.035 3. 0.935 0.112 absent 4. 0.935 0.168 0.056 0.093 5. 0.935 0.149 absent 6. 2.04 0.32 absent de Andi] 0.3 absent 8. 1.57 0.28 absent 9. 1.25 0.23 absent 10. 0.9 0.5 1.25 0.43 22 ILLINOIS BIOLOGICAL MONOGRAPHS [148 Olodistomum cestoides Genital atrium Genital cone 1. 0.243 X& 0.187 mm. 0.168 X& 0.149 mm. 2. absent 0.71 0.52 35/0233 0.28 0.28 0.2 4. 0.617 0.355 0.317 0.317 5. 0.37 0.187 0.187 0.187 6. 0.6 0.6 0.39 0.54 7. 0.94 0.62 0.39 0.54 8; 1.25 O15 absent It is certain that the size of the papilla is independent of general body contraction. Measurements No. 4 in the above table represent a strongly contracted specimen of O. veliporwm and an extended specimen of O. cestoides. The size of this structure is also independent of the killing fluid. Sometimes the body wall projects lip-like about the genital pore. This condition also is not associated with the size of the papilla. A definite system of muscles about the papilla and in the neck region seems to be responsible for the protrusion and withdrawal of the papilla. The exact mechanism for the protruding of the papilla is difficult to determine and probably depends upon a rather complex system of muscles. The longitudinal muscles about the wall of the atrium have processes which attach themselves to the anterior (or ventral) part of the atrium (Fig. 28). At the other end these muscles are continuous with the longi- tudinal body muscles. Contraction of these muscles of the atrium would have a tendency to widen and to pull forward the anterior part of the atrium. The actual pushing out of the base of the atrium to form the papilla is probably brought about by the numerous diagonal and dorso- ventral muscles of the neck region. Contraction of these muscles causing a compression of body tissue in that region would provide a pushing force at the base of the atrium. Once the movement of the extrusion has started, it would be aided by the contraction of the circular muscles in the wall of the atrium and base of the papilla. Of the two specimens with papilla protruded O. cestoides showed the ejaculatory duct somewhat coiled even in the papilla itself, while this duct was straight in the papilla of O. veli- porum. The duct is always coiled between the cirrus sac and the papilla, and in unprotruded papillae. The sudden projection of sperm and seminal fluids through this muscular duct would have a tendency to straighten its coiled condition just as such an effect is brought about by sudden pressure of water in a coiled hose. This influence is, of course, an uncertain one in this case and even if present would probably have a negligible effect in elongating the papilla. The retraction of the papilla doubtless results from the contraction of the longitudinal muscles which it possesses. The papilla probably functions as a copulatory organ. In the specimen of O. veliporum with protruded papilla (Fig. 29), a large mass of sperma- 149] SOME NORTH AMERICAN FISH TREMATODES—MANTER 23 tozoa was emerging from the genital duct at the tip of the papilla. Sperma- tozoa were also found crowding the distal tip of the vagina for a short distance, as well as in the genital atrium outside the papilla. None were found in the vagina posterior to the papilla. Eggs were frequently found in the atrium but only when the papilla was completely withdrawn, a fact which indicates that egg laying occurs while the papilla is completely subsided. The two testes lie one immediately behind the other in the median line at about the center of the body. They are close together and very com- monly are in contact with each other. This condition depends, however, upon the state of body contraction. The posterior testis is always slightly larger than the anterior testis. Each testis is surrounded by a fibrous-like membrane containing a few flattened nuclei. In adult forms from the Atlantic collection (O. cestoides) the size of the anterior testis was 0.62 to 1.25 mm. by 0.8 to 1.12 mm., while the posterior testis measured 0.8 to 1.37 mm. by 0.8 to 1.2 mm. The organs seemed to be rather consistently somewhat larger in O. veliporum. The duct from the anterior testis leads from the ventral side of that organ near its anterior end, and extends anteriorly in the right half of the body. The other male duct leads from the ventral side of the posterior testis about 1/4 the length of the organ from its anterior end. Thus, in the specimen studied, the posterior testis measured 1.235 mm. and the duct opened 0.365 mm. from the anterior end. This condition may be at least ’ partially due to the angle at which the sections were cut. The duct from the posterior testis leads anteriorly in the left half of the body. Both ducts at first lie ventral to the uterus but like the uterus pass dorsal to the ventral sucker, and in this region they also gradually become dorsal to the uterus. Slightly anterior to the posterior margin of the cirrus sac both ducts swing ventrally. The two do not unite until just before they empty into the seminal vesicle. Relations of the ducts were found to be the same in both O. cestoides and O. veliporum. Miihlschlag, however, gives the ducts as arising from the median anterior borders of the testes and uniting into a common duct dorsal to the cirrus sac. The diameter of the vas deferens varies but when expanded measures 26 to 39u with a very thin mem- braneous wall except in regions where the wall expands into prominent cells. (Fig. 25.) The cirrus sac is large and elongate-ovoid in shape. It lies between the two suckers somewhat nearer the oral. In average sized specimens (23 to 35 mm.) it measures 0.84 to 1.02 mm. by 0.65 to 0.84 mm. It may attain a length of 1.3 mm. It is somewhat larger in O. veliporum where it reaches a length of 1.4 mm. The sac is definitely bounded by a thin membrane about which is an inconspicuous coat of circular muscles. Both seminal vesicle and prostate gland are contained entirely within the sac. 24 ILLINOIS BIOLOGICAL MONOGRAPHS {150 The seminal vesicle is a large swollen tube almost filling the posterior half of the cirrus sac. It is somewhat curved in shape and always packed with sperm cells. The sperm cells are thread-like in form with minute round heads. The wall of the vesicle is made up of flattened cells with large nuclei. The pars prostatica of the male duct leads from the anterior end of the seminal vesicle, proceeds posteriorly and dorsally, then bends anteriorly and runs diagonally forward to the anterior end of the cirrus sac. It is surrounded by the large prostate gland which fills the remainder of the cirrus sac. Just before the duct leaves the cirrus sac its character changes very abruptly into that of the ejaculatory duct. The ejaculatory duct follows a much winding course leading to the tip of the genital papilla. Distally, it is joined ventrally by the vagina. The coiling of the duct in O. cestoides continues in the genital cone itself even when the latter is fully extended. The duct is lined by a thick layer of cuticula-like material continuous with the body cuticula. This layer attains a thickness of 10 to 134 and shows a folded irregular outline in cross-section. It is surrounded by a thick coat of circular muscles which becomes 20 to 25u in thickness near the cirrus sac. No longitudinal muscles could be noted. In development the male reproductive system precedes that of the female. The seminal vesicle is filled with sperm cells some time before eggs appear in the uterus and before the vitellaria appear at all. The ovary lies immediately in front of the anterior testis and usually in contact with it. In both organs the surface of contact is somewhat flattened. The ovary is therefore flattened on its posterior surface and its longest dimension is in the right and left direction. It may lie a little to one side of the testis, either to the right or to the left. (Figs. 13 to 18.) Its position in this respect is very inconstant and the various descriptions of the position of the ovary in this and similar forms is probably without significance. It is normally almost directly in front of the anterior testis. The size of the ovary in fully mature forms is 0.43 to 0.6 mm. by 0.78 to 0.9 mm. It is slightly larger in O. veliporum where it reaches 0.8 by 1 mm. The ootype lies immediately anterior and slightly dorsal to the ovary with which it is in close contact. Both organs are surrounded by a common fibrous tissue which also separates the two. The ootype is somewhat smaller than the ovary. In an average sized specimen where the ovary measured 0.5 by 0.84 mm., the ootype measured 0.35 by 0.53 mm. The oviduct leads from the middle anterior aspect of the ovary. It projects into the ovary in the form of a funnel-like structure with thick walls. This condition was found in both species (Fig. 11). The walls are composed of fibrous tissue which is continuous with the tissue covering the ovary and the ootype. This tissue is quite thick between ovary and ootype, and in it occur the two lateral vitelline ducts. As these ducts 151] SOME NORTH AMERICAN FISH TREMATODES—MANTER 25 approach each other they also tend to encroach upon the ootype so that they give the impression of pushing into it from the rear. Since the fibrous lining tissue becomes very thin between the yolk duct and the cells of the ootype, it is difficult to determine the exact point of penetration of the ootype by the yolk duct on either side. Frontal sections of O. cestoides indicate that the lateral ducts may penetrate the ootype before uniting to form the common yolk duct. More commonly, however, the two lateral ducts unite while still within the fibrous tissue close to the ootype and only the common duct actually penetrates into that organ. Almost immediately after entering the ootype proper the oviduct is joined by the common vitelline duct and by Laurer’s canal. The common vitelline duct is very short. A rather unusual condition was found in at least two cases where Laurer’s canal instead of joining the oviduct opened at the point of union of the two yolk ducts, so that the three canals opened together into the oviduct (Fig. 24). In other cases noted, Laurer’s canal was slightly to the right and appeared to join the right vitelline duct. Again, Laurer’s canal may join the oviduct at about the same point as does the common yolk duct. Laurer’s canal is very well developed. It is lined with cuticula 4 to 64 in thickness. In medium-sized specimens the duct is about 25y in diameter. A circular layer of muscles surrounds it but no special longitudinal muscles could be made out. The tube is much coiled and leads dorsally and either anteriorly or posteriorly as will be shown later. Within the lumen of the canal are many sperm cells. In some cases, in the region adjacent the oviduct a few yolk cells were noted. A seminal receptacle is absent. After receiving the yolk duct and Laurer’s canal, the oviduct, now the uterus, becomes a small tube only about 26y in diameter with a thick wall made up of very definite cells with large nuclei. Into the lumen of this tube project cilia-like processes. The tube very shortly enlarges and the nature of its wall becomes one of large cuboidal and heavily granular cells (13 to 23u in thickness). This part of the tube coils about more or less within the ootype for a short time and continues also outside of that organ. All stages in formation of the egg shell can be traced from the point where the yolk duct joins the oviduct. The shell material can be first seen as a shapeless irregular mass surrounding an egg cell and a group of yolk cells. Within the cellular-walled uterus region the eggs still lack their characteristic shape. The inner surface of the forming shell often shows vacuole-like spaces (Fig. 26). The cellular region of the uterus as it coils anteriorly soon passes over into a region characterized by a thin simple membraneous wall. By the time the eggs have reached this region they possess hardened and fully formed shells. The uterus contains thousands of eggs and extends anteriorly in many transverse coils. It passes dorsal to the ventral sucker and ventral to the 26 ILLINOIS BIOLOGICAL MONOGRAPHS [152 seminal vesicle. At about the level of the ventral sucker it becomes less coiled and the nature of its wall changes to produce a region which may be called the vagina. The vagina like the male ejaculatory duct is lined internally by cuticula. It possesses two layers of conspicuous muscles, an inner circular layer and an outer longitudinal layer. The vagina contains few eggs and above the ventral sucker is almost a straight tube. It leads to near the tip of the genital cone where it unites with the male ejaculatory duct. There is a common duct for a distance of about 25. In development the uterus first appears as a solid string of cells without a lumen. Sections of a very young specimen seem to show a lumen appear- ing first within the ootype the start of which appears early with the beginning of the ovary. It is an interesting fact that in slightly older specimens when the uterus can first be made out in toto-mounts but before eggs are being produced, this string-like beginning of the uterus assumes the coiling which characterizes the organ when filled with eggs. It winds transversely back and forth between the ovary and ventral sucker, crossing the body as many as 40 to 50 times, approximately the same es ceetce of folding that is found in the adult. The first eggs to be produced are abortive. A specimen 11.5 mm. in length showed the earliest egg still in the region of the ventral sucker. The most anterior eggs (15 to 20) were only about half the size of the others and were almost spherical with very thin shells. They measured from 22.8 to about 30u, the smallest being most anterior. The eggs nearer the ovary were larger but still showed a very thin shell, in striking contrast to the thick shell found in the adult egg. No eggs in this specimen measured over 68, a measurement slightly below the average adult size. The vitellaria are interesting because of the variation of their extent and arrangement. They are of the follicular type and consist of separate spherical bodies grouped in two longitudinal rows along the sides of the body. The follicles lie mostly ventral to the digestive system. They do not appear until relatively late in development, or just before eggs begin to be formed. At this time the follicles are very small, approximately the size of the eggs (about 50u in diameter), but in the adult they reach a diameter of 150 to 190u. Several hundred of these follicles are connected by ducts which unite into a common lateral duct on each side at the level of the ootype, and these two lateral ducts unite in the ootype as described. The rows of follicles are usually narrow, but the width of the rows often increases to such an extent that the two approach each other medianly. Thus, Miss Lebour (1908) figures the vitellaria massed together posterior to the testes. Such variations are not common, however, the two rows being usually distinctly separate. Irregular breaks or spaces free of follicles are common, and certainly can have no specific significance within the 153] SOME NORTH AMERICAN FISH TREMATODES—MANTER 27 genus Otodistomum. Even the differences in the vitellaria given by Odhner (1911b) to distinguish O. cestoides from O. veliporum are quite useless. He gives the vitellaria in O. veliporum as beginning behind the middle of the uterus and as being compressed into narrow rows, while in TABLE 3 VARIATION IN POSTERIOR EXTENT OF VITELLARIA IN O. cestoides Body length | Distance from Extent of right Extent of left posterior testis to} vitellaria beyond | vitellaria beyond b b posterior tip posterior testis posterior testis C ei a b G d 31. mm. 16. mm. 6.4 mm. 7.6 mm. i) ae ae mee: BRE S S Ss 1) OL Z| Ss = Pe iesen ees Ost 0s - ee ee ee | tL Reese z isk), | ua} jo sojdnjnu uw $19] OUNITTUA-J]eY 9}LOIPUT SOUT] [eJWOZIIOY OY], “SPU [LOIOA oATSssadNs ueVMj}9q PayUasaidal Ie S[ENPIAIPUI US], *9AINO JaMOy ay} Aq poyuasaidar are suatmpeds Surpuodsosi09 Jo syypIM ayy, ‘suouTdads jo spud so110}sod oy} aarnd saddn ay} ‘spue Jolzeyue 94} S}Uasaidal (() OUT] aseq oy], "sap10jsa9 wnuojsrpojsQ JO suoTAIDads QOZ UT TIPIM Apoq pur y}Sue] Apoq [e}0} UseMJaq suOIOdold jo uoTeyUaseIder STydvINH *7Z LAVHD a = x a _ ia a i ie 159] SOME NORTH AMERICAN FISH TREMATODES—MANTER 33 Thus, in individuals 2 or 3 mm. in length the ventral sucker is located just anterior to the center of the body, the proportion of the region in advance of the posterior border of the sucker to the entire body length being 1: 2.3, 1:2.5, 1:3, etc. (see Chart 1). As the trematodes increase in size, the region posterior to the ventral sucker gradually and constantly gains in size in proportion to the anterior region, which grows relatively little. This change can be followed in the accompanying chart (Chart 1) on which the body lengths and positions of the ventral suckers have been plotted. The total length in half millimeters is plotted on the vertical lines as is also the position of the ventral sucker. Each vertical line, then, repre- sents one trematode so that if the base line be considered as the anterior ends of the worms, the points where the lower curve intersects the vertical lines represent the positions of the ventral suckers, while the upper curve represents the posterior ends of the trematodes. Ten specimens are repre- sented between two successive verical lines. The change in proportion and the constancy of the change can be seen at a glance. The fluctuations in the lower curve are due to differences in contraction of individual specimens. Because of such differences the chart can only be considered as approximate. Quite a number of the specimens were stretched slightly by hand when killed in order to prevent contraction. Separate tables and charts were prepared, however, for individuals not so treated, and in every case results were the same as in the complete table of all individuals. In the collection of material numerous of the more mature trematodes were destroyed for the purpose of obtaining eggs. Without doubt, the only change which would be caused in the chart by such loss would be to reduce the degree of incline or steepness of the upper curve in the region above horizontal line 50 or 60. Study of the data on length and width of the trematode at different stages showed corresponding results. The measurements are plotted in Chart 2 where it can be seen that increase in width is very little in com- parison with increase in length. The elongate form which gives the adult worm a cestode-like appearance is only gradually assumed with age, and the youngest individuals are very characteristically trematode-shaped. In Chart 2 the distance on any vertical line from the point of intersection of the lower curve to the base line represents the width of an individual specimen whose length is represented by the distance from the upper curve to the base line. As the specimens are arranged in order of length, here again all fluctuations due to varying degrees of contraction appear in the lower curve only. In spite of these radical] changes in body proportions, the ratio between the sizes of the two suckers remains constant. That is, the two suckers grow equally evenly, although the body is growing much more rapidly posteriorly. 34 ILLINOIS BIOLOGICAL MONOGRAPHS {160 This constancy of the sucker ratio is somewhat unexpected since other trematodes, especially when body proportions are altered, show an increase of the ventral sucker over the oral. Most conspicuous is the actual reversal in sucker proportion in Schistosoma japonicum where, as already given, the ventral sucker is smaller than the oral in the cercaria, but later assumes equality with it, and in the adult is definitely larger. A similar tendency in F. hepatica has also been noted. The following table (Table 4, Column b/ c) shows very clearly how all sizes of O. cestoides vary closely about the average ratio (oral sucker .66 of the ventral sucker). It is true that in the four smallest specimens is found the largest ratio (up to 0.82), but specimens almost as small show a ratio slightly below (0.64 and 0.65) the average, and some of the largest individuals show a ratio of 0.7 or more. TABLE 4 SUCKER SIZE IN RELATION TO GROWTH IN O. cestoides Body length Body width Diameter of Diameter of anterior sucker ventral sucker = pis (a) ) () ‘ 2 39. mm. 2.87 mm. 1.43 mm. 2.18 mm. -65 17.8 38. 2.06 1.25 1.81 atl 21; 38. 2.25 1.312 2s -66 19. 32° 23 1.06 1.62 -65 19. Shs 3.5 1.56 2.18 71 14.2 31. 253 133 1.8 BY 17.2 29. Di5 1.43 2.31 -62 12.5 29. 2:3 1; 1.56 -63 18.5 28. 2.18 1212 5) 64 16. 28. 123 1.37 Ze .68 14. 28. 2AS ie 1.68 .58 16.6 28. 2.87 1618 1.68 Wf 16.6 26. 1.6 93 1.37 .67 18.9 26. 1.87 1.06 1537 whe 18.9 24.5 2.37 1525 1.87 61 13. 24. 2.8 1.37 2.18 -62 11, 21. oot. 1.25 175) Ae) 125 20. 1.68 0.878 1.327 -66 15: 19. 2.12 Ai: 1.437 71 13.9 18. 2:5 1.14 1.77 64 10.1 17, 225 1.028 1.77 -07 9.6 16. 2.06 0.916 1.34 .68 11.9 16. 255, 1.02 1253 66 10.4 14. 1.9 1.06 1.5 : 64 9.3 13. 2 15 175 .66 8.6 12. 1.8 1; 1.37 .73 8.7 1155 Pat 0.93 155 -62 7.6 11, 1.5 0.75 1,12 .66 9.8 6 I 2.1 0.93 155 62, Wsd 161] SOME NORTH AMERICAN FISH TREMATODES—MANTER 35 Tas_e 4 (continued) Body length Body width Diameter of Diameter of anterior sucker ventral sucker b c (a) (0) (c) c a 10.8 mm. 1.75 mm. 0.75 mm. 1.25 mm. .6 8.6 10.5 1.8 0.68 1.31 .52 8. 10.5 123 0.68 1.06 64 9.9 6.5 At 0.54 0.785 .68 8.3 6.06 L212 0.525 0.748 ay A 8. 3s 0.93 0.448 0.654 .67 7.6 4.5 0.56 0.448 0.654 .67 6.9 4.37 0.9 0.414 0.673 .62 6.5 4.31 1.047 0.504 0.766 65 5.6 4.18 0.937 0.467 0.635 .73 6.5 4.125 0.991 0.397 0.617 64 6.6 3.6 0.75 0.414 0.635 65 5.6 2.37 0.562 0.317 0.43 Rif} 5.5 De25 0.748 0.414 0.504 .82 4.2 2.18 0.71 0.355 0.43 .82 5, 1.9 0.56 0.28 0.39 74 4.8 Average .66 Another interesting point is the relatively much larger size of the suckers in comparison with body size in the young. This observation would follow from the fact that the diameters of the suckers keep pace with the width of the worm. This marked change in proportion between sucker diameter and body length can be followed in Table 4, column c/a. In the smallest specimens, the body length was 4.8 times the diameter of the ventral sucker, while in all the largest specimens measured the body length was about 20 times the diameter of the ventral sucker. The change between these two extremes is seen to be consistent and gradual exactly as was the change body length and width. Young forms in general show the body length to be about 5 times the diameter of the ventral sucker, medium sized specimens have a length about 10-11 times this diameter, while a specimen 38 mm. long was 21 times longer than the diameter of the ventral sucker. It is interesting to note what effect, if any, the arrival at sexual maturity has upon these growth rates. The uterus is located posterior to the ventral sucker, and it might be expected that the relatively sudden filling of the uterus with eggs would affect growth in this region. The body size at which the trematode attains sexual maturity is quite constant. Almost invariably eggs begin to be produced when the worm has a length of about 11 mm. A few eggs have been found in specimens 10.5 mm. in length. It can be stated quite certainly that sexual maturity is attained between the body lengths of 10 and 15 mm. 36 ILLINOIS BIOLOGICAL MONOGRAPHS [162 Reference to the charts shows 49 specimens between 5 and 10 mm. in length, 30 between 10 and 15 mm. in length, and 22 between 15 and 20 mm. in length. All specimens of these sizes were preserved. As fewer individuals were found intergrading between the sizes 10 and 15 mm. than between 5 and 10 mm., it might be inferred that growth is more rapid in the former case. The indication can only be considered as a slight one, however, as the difference is not large, the numbers are somewhat few, and there is a possibility of various unknown factors such as infection rate. Further data on the effect of sexual maturity on growth can be obtained by comparing distances from the posterior testis to the ventral sucker (region of uterus) and from this same point to the posterior end of the worm (tail region). Ata certain period (11 to 15 mm.) the uterus becomes quite suddenly crowded with eggs. Whether this change alters the pro- portions of the length of the uterus region to the length of the posterior region where no organs of importance are located, and where no important change is occurring, can be shown approximately by comparing the lengths of these regions in mature and immature specimens. In 21 wholly mature TABLE 5 CoMPARISON OF Uterus REGION WITH PosTERIOR Bopy REGIONS IN YOUNG SPECIMENS oF Otodistomum cestoides Body length Length of tail region Length of uterus region (a) (b) ae a 14. mm. 6.5 mm. 5. mm a 13. Ue 3.56 Pro 12'. 6. 3.9 -58 11.5 5.3 3: .56 11), 53 3. 58 11. 5.25 2.37 45 10.8 5. 3525 -65 10.5 4.8 2.87 6 10.5 5. 2.4 -58 10. SES 2.18 37 6.5 3,29 1.43 44 6.06 3: 1.47 .49 5 233 1.12 48 4.5 1.68 1. 6 4,187 1.68 0.937 55 4.37 1.5 al -66 4.31 1.5 0.75 5 4.125 1.9 0.687 35 3.6 * iy | 0.57 34 2.37 Ls 0.525 52 2.18 0.74 0.37 5 Average -493 163] SOME NOR17H AMERICAN FISH TREMATODES—MANTER 37 individuals the length of the uterus region averaged 0.67 the length of the tail region. In 21 immature individuals including a few very young forms, this uterus region averaged 0.493 the length of the posterior region. That is, the uterus region upon becoming filled with eggs increases on the average its ratio to the tail region by about 0.2. While this increase is small, it is very definite. Reference to Tables 5 and 6 shows that the proportion of the TABLE 6 CoMPARISON OF UTERUS REGION WITH POSTERIOR Bopy REGION IN ADULT SPECIMENS OF Otodistomum cestoides Body length Length of tail region Length of uterus region b (a) () 38. mm. 21. mm. 12.5 mm. 6 38. 18. 14. a7 32% 16. 11. 67 31. 11.5 9. 78 31. iy 9. .53 29. 14. 9.75 .69 29. 15: 8.5 .56 28. 14. 10. will 28. 15. 9. 6 28. 18. Ts A 28. 12.5 10. 8 26. 11.06 11.06 1 26. 15: 8. .56 24.5 13:2 dec .53 2h. 12.5 6.25 25 20. 8. 8. 1 Us 19, 10. 6.5 65 18. het ks 5.93 ai) 17. (aS) 5.8 .78 16. 6.12 6.12 1. 16. 7.9 4.6 58 Average .67 uterus to the tail region in the young forms measured attained only once a point as high as 0.7 and went as low as 0.34. Moreover, even in these forms the uterus region tended to be slightly greater in individuals with a few eggs (those specimens 10 to 14 mm. in length). Of the mature forms, however, the uterus region was never less than 0.4 the tail region and several times attained equal length with it. The increase of the length of the body posterior to the ventral sucker over the length anterior to this sucker is by no means due entirely, however, to this seeming increase in the uterus region. To show this fact, measure- ments of the neck region (that region from the anterior end to the ventral sucker) were compared with measurements of the tail region. The neck 38 ILLINOIS BIOLOGICAL MONOGRAPHS (164 region in 18 immature or recently mature forms averaged 0.77 the length of the tail region, while in 12 mature forms the neck region averaged only 0.39 of the tail region. That is, while the uterus region is gaining on the tail region the latter is also gaining even more rapidly on the neck region (Tables 7 and 8). TABLE 7 COMPARISON OF NECK REGION WITH POSTERIOR Bopy REGION IN YOUNG SPECIMENS OF Otodistomum cestoides Anterior end to Posterior testis to P Body length ventral sucker posterior end = (a (0) : 13. mm. 3. mm 7. mm. 43 11.5 335 S23 66 11. 3. 5.3 56 11. 3: 5.25 Ay / 10.8 2.56 5. 751 10.5 2.68 4.8 .56 10.5 215 5: .55 6.5 2.12 azo 65 6.06 125) 3. 35) 5: 1.87 253 81 4.5 1.8 1.6 1.1 4.187 1.8 1.68 1.05 4.37 1.87 1.5 1.24 4.31 1.37 175) 91 4.125 1.4 1.9 74 3.6 1.25 1.7 AY fs} 2.3 0.93 1; 93 2.18 - 1.04 0.74 1.4 Average | .77 In stating that the uterus region increases its proportion to the tail region, it has been assumed that the position of the posterior testis is relatively stationary. If, however, the uterus, in filling with eggs, pushes the reproductive organs backward, then the increased growth in the uterus region is only apparent, since the position of the hind testis was taken as a point of measurement. Similarly, illusions would follow from any local movement of the gonads either forward or backward within the body. In general, the gonad group appears to occupy a fixed position. Commonly, especially in extended specimens, the two testes are separate from each other by a short space. When the two organs are in contact with each other the surface of contact becomes flattened. Thus, if the uterus did have a tendency to force the gonads backward, some leeway would be allowed this movement before it exerted an influence on the position of the hind testis, upon which present measurements were based. 165] SOME NORTH AMERICAN FISH TREMATODES—MANTER 39 TABLE 8 CoMPARISON OF NECK REGION witH PosTERIOR Bopy REGION IN ADULT SPECIMENS OF Otodistomum cestoides Body length Anterior end to Posterior testis to P ventral sucker posterior end = (a) (6) : 38. mm. 5. mm. 21. mm. 24 Sie 6. 17. <3 38. 6. 18. 34 29. 5 14. «35 28. 4. 14. 28 24.5 4, 13.2 3 20. 4, 8. ‘5 19. Sie) 10. =35 18. 3. hielo .38 ily fe 4. Ups) 53 16. 4. 6.12 65 16. ee) 7.9 44 Average 39 Some slight evidence that the crowded uterus may cause a forcing back of the ovary against the anterior testis is found in certain variations in the relative position of the pore of Laurer’s canal. This pore furnishes a fixed point and if it retains a constant position in relation to the ovary in young and in mature specimens, it can be inferred that the position of the ovary is also constant. Such a relation does not exist. The pore was found to be sometimes anterior to the anterior end of ovary, sometimes almost exactly dorsal to it, and sometimes slightly posterior to it. As serial sections were necessary to determine this point, it was ascertained in relatively few cases. In O. cestoides the pore was always found to be posterior to the anterior border of the ovary in young or recently mature specimens, and usually anterior to it in mature forms. One exception in both O. cestoides and O. veliporum shows that the pore may be posterior to the anterior edge of the ovary even in mature individuals. In all young forms measured the pore was posterior to this point. A table follows showing the position of the pore in specimens of different ages: O. cestoides Length Maturity . Pore in relation to Distance anterior end of ovary 10 mm. uterus with few eggs Posterior 0.56mm. Small immature “ 0.18 Small only a few eggs in uterus id 0.23 14mm. Recently mature Anterior 0.14 25mm. Mature 0.18 Large Mature . 0.18 Large Mature Posterior Slight 40 ILLINOIS BIOLOGICAL MONOGRAPHS {166 O. veliporum 23 mm. Mature Anterior 0.6 mm. 25 mm. Mature Posterior 0.285 In any case, the extent of this shifting as far as it is indicated by relative pore position is not sufficient to affect materially the position of the hind testis. Conclusions in regard to growth changes can be more graphically expressed by means of a diagram (Diagram 1). Dracraqm. 1. Diagram to show growth changes in Olodistomum cestoides. Distance B constantly grows faster than Distance A, even before sexual maturity. Distance C increases slightly its proportion to D after sexual maturity. Distance D increases in length more rapidly than A, even after sexual maturity. Body length increases much more rapidly than body width. Size ratio of the two suckers does not change with size of the worm. Suckers are proportionally much larger in young. 167] SOME NORTH AMERICAN FISH TREMATODES—MANTER 41 COMPARISON OF OTODISTOMUM CESTOIDES AND O. VELIPORUM The close resemblance of these two species has already been noted. Odhner (1911b) gives the following differences: O. veliporum O. cestoides 1. Size: 50 mm. by 5 to 6 mm. Size: to 65 mm. by 3 to 5 mm. 2. Ratio of suckers: 3:5 Ratio of suckers: 3 : 4 3. Vitellaria beginning behind the middle of Vitellaria usually beginning in front of uterus field, compressed into narrow middle of uterus field, not so narrowly bands. compressed. 4, Egg: 0.86 by 0.06 to 0.063 mm. Egg: 0.065 to 0.072 by 0.043 mm. 5. Thickness of egg shell 0.006 mm. Thickness of egg shell 0.003 mm. Of these differences, numbers 2 and 3 are certainly useless as all con- ditions involved were commonly found in the present studies of the single species, O. cestoides. Odhner placed importance on the thickness of the egg shell. Miihlschlag (1914) worked on O. veliporum and concluded that the following features separate it from O. cestoides: (1) its body is less in length, greater in width; (2) its eggs are larger, and (3) have a much thicker shell. He found that the thickness of the egg shell of O. veliporum to reach 7p or more than twice as thick as commonly reported for O. cestoides. The trematode from Raia binoculata from the Pacific has already been frequently compared with O. cestoides from the Atlantic ‘‘barn-door’”’ skate. It has been seen that the Pacific form presents very few significant differences. Chief among these differences is the egg size. The average egg size from over 50 measurements in the Pacific form gives 85.5 by 57.8u as compared with 69.4 by 46.2u in O. cestoides. This difference is real, constant, and significant. In no case did the egg size overlap between the two species. Thus, the eggs in the Pacific form agree almost exactly with the reported egg size for O. veliporum. One of the other reported differences between these two species is a slightly greater body width in O. veliporum. The average width of 75 Atlantic specimens (QO. cestoides) between 23 and 50 mm. in length was 3.22 mm. The average width of 65 Pacific specimens (O. veliporum) between the same length limits was 3.81 mm. Thus, again the Pacific form agrees with descriptions of O. veliporum. The Pacific specimens were quite constantly of greater thickness, however, and their slightly greater width might be due to an average higher degree of body contraction. The only other specific criterion to be applied is the thickness of the egg shell. O. veliporum supposedly possesses a much thicker egg shell than 42 ILLINOIS BIOLOGICAL MONOGRAPHS. [168 does O. cestoides. In this particular, the Pacific form studied does not agree with the published records for O. veliporum. An average of numerous measurements on the egg shell gave a thickness of about 4y. For O. cestoides an egg shell thickness of about 4.54 was found. Thus, instead of having an egg shell twice as thick as does O. cestoides, the Pacific form actually has a slightly thinner egg shell. Measurements were taken on mature eggs in distal regions of the uterus. The eggs nearer the ovary had a slightly thicker shell. Even though this discrepancy in egg shell thickness appears to be a constant feature in material studied, it hardly seems to be in itself a specific character. In the first place, the absolute difference in egg shell thickness between the Atlantic form and records for O. veliporum is slight (about 2) since the Atlantic material showed an egg shell somewhat thicker than recorded for O. cestoides. Again, such minute measurements are difficult to determine exactly, especially as slightly oblique sections through the egg exaggerate the apparent thickness of its shell. Finally, the constanty of this feature is not well established, as is, for example, egg size in this genus. In respect to other differences noted between the two forms, the con- dition of the genital atrium and papilla has already been discussed and showed to be unreliable as a specific character. It can only be said that, in general, the genital papilla was either absent or small in size in O. veliporum, and usually prominent and robust in O. cestoides. It may, however, be flattened out entirely in O. cestoides and entirely protruded in O. veliporum. The genital organs seemed proportionally somewhat larger in O. veliporum. Actual measurements (in millimeters) on specimens of approx- imately similar size are as follows: O. cestoides O. veliporum Length Length 32mm. | Testis (ant.) 0.9 by 0.87 32mm. | Testis (ant.) 1.25 by 1.25 “ (post.) 1. 0.81 “ (post.) 1.5 1.25 Ovary 0.43 0.81 Ovary 1 0.75 31 Testis (ant.) 0.68 by 0.9 30 Testis (ant.) 1. by 0.9 “(post.) 0.8 0.9 “ (post.) 1.27 1 Ovary 0.5 0.68 Ovary 0.8 1 24 Testis (ant.) 0.89 by 0.37 25 Testis (ant.) 1 by 0.89 “ (post.) 0.78 0.86 “ (post.) 1.3 0.89 Ovary 0.46 0.7 Ovary 0.8 0.6 A 39 mm. specimen of O. cestoides agreed more nearly with the 32 mm. specimen of O. veliporum in gonad size, as follows: 39 mm. Testis (ant.) 1.25 by 0.87 mm. Testis (post.) 1.37 0.9 Ovary 0.6 0.9 169] SOME NORTH AMERICAN FISH TREMATODES—MANTER 43 These differences in gonad size are not, however, sufficient to be sig- nificant. Miss Lebour (1908) figures very large gonads for her species which was clearly O. cestoides. From this study, it has been concluded that the Pacific material repre- sents Otodistomum veliporum. Furthermore O. veliporum and O, cestoides are shown to be even more similar than has hitherto been pointed out. The most certain distinction is found in egg size. Raia binoculata (= R. cooperi) is a new host for O. veliporum. O. veliporum (Creplin) has the following synonyms: Dist. veliporum Creplin Dist. insigne Dies. Dist. microcephalum Baird } according to Ariola 1899 Dist. scymni Risso Fasc. squali grisei Risso Dist. veliporum of Johnstone 1902 It has been recorded from the following hosts: WNotidanus griseus, Notidanus cinereus, Echinorhinus spinosus, Carcharias milbertii, Raia batis, R. clavata, R. fullonica, R. radiata, R. lintea, R. binoculata, R. stabuliforis (=R. laevis), Scymnus nicaeensis, Acanthias vulgaris, Carcharias sp., Laemargus melanostoma, Scyllium canicula, Chimaera monstrosa, Carcharias rondeletti, Centrolophus pompilus. 44 ILLINOIS BIOLOGICAL MONOGRAPHS [170 THE MIRACIDIUM OF OTODISTOMUM CESTOIDES [FIGS. 31-43] The first larval form or miracidium of O. cestoides was studied from live material and in toto-mounts. As the larva is well developed and ready to hatch when the eggs are laid, sections of eggs in the vagina formed the basis for study from sections. Many thousands of the eggs can be obtained by cutting the adult worm just beneath the ventral sucker and pressing out the eggs in the distal portion of the uterus. This method gives a mixed culture of eggs of various degrees of maturity. A more uniformly mature collection results from saving only the eggs from the vagina region. Un- successful attempts were made to secure eggs that had been normally laid. Adults were kept both in sea-water and in sea-water with mucous and fragments from the stomach of the host. In the latter case, some specimens were kept alive two or three days, but in no case were any eggs obtained. There is no reason to believe, however, that the eggs in the vagina do not represent the normal condition of the eggs when laid. The eggs can be readily hatched. They were kept in sea-water in vials surrounded by running sea-water. The water in the vials was changed twice daily. The eggs were found to be very hardy. In mixed cultures containing many immature egg stages, hatching larvae appeared most frequently only after 7 to 10 days. In the case of cultures of only the most mature eggs, several miracidia were observed only 5 hours after the eggs had been removed from the worm. This very early hatching shows that the larva is fully developed in the egg at the time of oviposition. In general, even when the eggs in a vial seemed quite uniform, no uniform period of hatching was observed. Only a small proportion of the eggs hatched, and these hatchings, few at a time, extended over periods of some days (up to 15). The large number of cultures undertaken gave ample opportunity of studying the rather peculiar miracidium. The youngest immature eggs are pale yellow in color and quite trans- parent. They average only slightly smaller than mature eggs and the thickness of the shell seems to be about the same at all stages (in O. ces- toides). In the early stages the embryo consists of only a few cells collected at the opercular end of the shell, while the rest of the space is occupied by numerous yolk cells. These latter gradually disappear as the embryo grows, until the egg shell seems to contain a uniform mass of cells. The more mature eggs while alive have a characteristic appearance. The shell is brownish-yellow in color. A few small, globular bodies are 171] SOME NORTH AMERICAN FISH TREMATODES—MANTER 45 scattered about within the shell while at one end, the anterior, a dark opaque granulated area appears, concealing the anterior tip of the embryo. This area thins out posteriorly so that a dark crescent-shape appearance is given (Fig. 31). The same condition is described by Leuckart (1886:380) for D. lanceolatum. Just posterior to this area and within the body of the embryo are. two conspicuous, hump-like structures continuous anteriorly, but of two parts (resembling lobes) posteriorly. What was probably a similar structure was seen and pictured by von Nordmann (1832) for the eggs of D. rosaceum (= Azygia lucit). It represents a conspicuous organ of the embryo. Nordman noted the bifid character of the organ, describing it as “eine gleichsam zusammengekrummte, dunkler begrenzte Figur, welche an einem Ende wie mehr oder weniger gespalten ershien.’”’ All subsequent descriptions of the larva of A. /ucii consider the organ as simple, sac-like in structure and it has been interpreted as representing a simple type of intestine. In one case, in eggs of O. cestoides the body appeared to be split into three rounded lobes (Fig. 32). Even while the embryo is still in the egg, a more or less circular lighter area can be made out within the “‘lobes”’ of this organ, an appearance suggesting the presence of a lumen in each. The Hiillmembran of Schauinsland (1883) can be found in stained material. It is a thin membrane containing a few flattened cells. It is left behind in the egg shell at time of hatching. The miracidium is non-ciliated and normally pear-shaped, with a narrow anterior region. Its shape is continually changing, however, as the worm-like movement of the larva results in alternate expansion and con- traction of the body. When at full length the shape is narrow and elongate, reaching a maximum of 90 to 100u; when fully contracted the body becomes spherical and about 45 to 50u in diameter. The posterior end is sometimes pointed. The anterior end of the larva is pulled in and pushed out simul- taneously with the contraction and extension of the body. The action of this narrowed anterior region is exactly like that of a proboscis. The earliest movement noted within the egg can only be described as an indefinite “squirming.” At no time is there any indication of ciliary movement. The process of hatching was observed several times. At this period, the movement of the larva has become very definite and like that of the free miracidium, consisting of the alternate pulling in and pushing out of the somewhat pointed anterior end. The movement results in a series of pushes or blows directed at the operculum which sooner or later opens and the worm slides out. The ease of hatching seemed to vary considerably. Thus, one case was observed when the entire process of hatching required only about 30 seconds and almost without effort on the part of the embryo. Another case studied showed a very active embryo which did not succeed in hatching even after several hours and finally its movements slowed down and ceased. Focusing showed that this animal did not seem to be accurately 46 ILLINOIS BIOLOGICAL MONOGRAPHS (172 oriented in the shell and the force of the propulsion of the proboscis was directed a little to one side of the operculum. Although there was ample space for the worm to change its position, it seemed to persist in directing its “blows” at one spot. With the escape of the embryo from the shell, a small stream of minute granules is also given out. These particles attracted small infusorians in the culture and sometimes an empty egg shell would be well filled with small ciliated protozoa. The body of the miracidium is covered by a very thin cuticula-like layer which is non-ciliated. About the anterior end is a region of bristles or spines. Such an occurrence of spines is common in non-ciliated miracidia, In Azygia lucii they are described as occurring on four plates (Borsten- platten) surrounding the anterior end of the larva. This Azygia larva also has a posterior series of four bristle plates. Although Odhner (1911b) states that ‘‘Borstenplatten” are not present in the genus Otodistomum, the spiny areas were very conspicuous in living material. They were also discernible in toto-mounts and could occasionally be definitely made out in sections of embryos within the eggs. Unlike their condition in A. lucii, these bristles occur on five strips or areas radiating from the anterior tip. Each strip is tapering in form, being broadest posteriorly. The bristles are longer near the tip and gradually become shorter posteriorly. When the larva first hatches these strips or plates of bristles lie quite flat. Very soon, however, they become loosened at the anterior end and peel off backwards. This shedding of the bristle-plates often gives to the miracidium a very peculiar appearance, as the strips remaining attached posteriorly resemble appendages. The strips are of a very thin epiderm-like material, and when free from the larva tend to curl slightly (Figs. 34 and 43). The process of shedding the strips can be compared in a homely way with the peeling of a banana. Creutzburg (1890) in his work on the life history of Dist. ovocaudatum (=Halipegus ovocaudatus) which has a larva very much like that of O. cestoides, found that the miracidium of that species lost its coat of bristles only after it had pierced the intestinal wall of a snail. While he does not mention any localized areas of the bristles he describes the shedding of the epiderm as proceeding from the head end. He says (p. 22) the embryo ‘‘verwandelt er sich,....durch Abwerfen der fusseren stacheltragenden Bedeckung, deren Loslésung nach meinen Beobachtungen zunichst am Kopfpol vor sich geht..... ” Tt is therefore quite possible that the spiny region normally serves to aid the miracidium in piercing the tissues of the host. In my material, only one live individual was seen which seemed to have completely shed the bristle plates. Larvae were usually closely associated with the empty egg shell from which they emerged. The posterior end of the larva seemed to show a tendency to adhere to the egg shell. Only a few larvae were seen at any 173] SOME NORTH AMERICAN FISH TREMATODES—MANTER 47 great distance from their egg shells. Locomotion was certainly very limited and even the most active larvae progressed very little, if any, in the watchglasses wherein they were studied. The anterior end seemed to adhere rather tenaciously to any debris with which it came in contact (Fig. 34). The only conspicuous organ of the larva is the structure already mentioned as visible in later egg stages. It extends to about the middle of the body and has a paired sac-like appearance. In one favorable specimen it was observed that leading forward from this organ is a minute duct which opens at the extreme anterior tip of the larva. This anterior tip when free from the bristle-plates is rounded and knob-like in shape. The rounded lighter areas in the central part of each of the lobes of the organ have the appearance of lumens, and the impression gained from study of live material is that the entire organ represents a bi-lobed or bifid intestine. Such a conclusion is supported by the fact that many miracidia have been described as possessing a simple type of intestine. Furthermore, what is certainly the same structure in similar larvae (miracidia of A. lucii, H. ovocaudatus), has been commonly interpreted by Looss, Leuckart, Creutzburg, Schauinsland, and others as an intestine. Stained material, and especially sections through mature eggs, present evidence which seems to warrant questioning very seriously the conclusion that this organ represents any form of intestine. In the first place, the organ is found to consist not of two parts but of four. The outline of these “lobes” or parts stains with hematoxylin clearly. The bifid appearance is explained by the fact that the four parts are arranged in pairs (Figs. 36 and 38). The elements of a pair are closely associated, but the pairs may be wide apart (Fig. 38). Furthermore, the central region (of each lobe) which had been inter- preted as a lumen, invariably stains darkly like a nucleus. The granular appearance of the organ in life disappears in sections and in its place is seen a clear transparent area. Safranin staining gave results similar to those obtained with various hematoxylin stains. In fact, safranin stains brought out the four-partite and four-nucleate condition even more clearly than hematoxylin. The fact that the organ is made up of four similar parts, and that the central regions stain like nuclei make it seem more probable that the organ is not an intestine but a group of unicellular glands. One bit of evidence in favor of viewing the organ as an intestine has been the reported occurrence of a minute pharynx or muscular region about the duct. Only the slightest suggestion of such a structure was detected in the present studies although no special staining of live material was attempted. Von Linstow (1890) reports what appears to be a similar structure in D. cylindraceum as a “Stutsapparatus.” Schauinsland (1883), however, describes and pictures 48 ILLINOIS BIOLOGICAL MONOGRAPHS [174 a minute pharynx-like structure in the larvae of Azygia lucit and other trematodes. He does not hesitate to consider such a structure as a true pharynx and the sac-like organ as an intestine. It is interesting to note that in no case does he show any form of cellular structure around the so-called intestine, nor does he show that any lumen is present. On the contrary, he does represent nuclei-like bodies within the intestine in A. /ucii larvae. It is quite evident that these are the same type of bodies which stain like nuclei in my sections. Schauinsland refers to them as ‘‘Kerne im Darmin- halt.”’ In one case, he reports “Im Darm bemerkt man 3 Kerne.’”’ Both for the larva of A. Jucii and for the larvae of the other forms he studied, Schauinsland shows from one to four nuclei within the contents of the intestine. Looss (1894) shows the same condition in his figures. Particularly interesting is his figure of the larva of A. lucit. Here three rounded bodies are clearly present within the organ interpreted as an intestine. In the present studies Azygia material was found to be most favorable in sections of A. acuminata. Here were found four apparent nuclei within the organ in question (Fig. 44). The organ seemed to be simple and sac-like in shape. Because of the much smaller size of the larvae in Azygia species it would be very difficult if not impossible to make out the four-partite condition which the larger larvae of Otodistomum reveal. The miracidium of Schistosoma japonicum possesses in addition to a sac-like intestine with four nuclei, a pair of large ‘‘cephalic glands,”’ one gland being located on each side of the intestine. The glands are nearly as large as the intestine. The four-partite condition found in O. cestoides showed, however, no such differentiation between the parts. Creutzburg (1890) has given not only a description of the appearance of this organ in the similar larva of Halipegus ovocaudatus (=D. ovocauda- tum), but also a history of its fate in later development. In the larva he studied, the organ seemed to be simple and sac-like in form. He says (p. 21): “Dieser kérnige Inhalt zeigt oft blischenihnliche Einschliisse, die auf das Vorhandensein eines Lumens schliessen lassen.’’ This larva (which does not hatch until the egg has been eaten by a snail) penetrates the intestinal wall of the snail. Soon after it has passed into the body cavity of the snail the larval “intestine” or organ in question begins to diminish in size and finally disappears. Creutzburg says (p. 21): ‘Die weitere Entwickelung des schlauchformigen Organs lisst sich noch bei den Sporo- cysten verfolgen, wo es, seine urspriingliche Lage am vordern Kérperende beibehaltend, spaterhin an Grésse allmihlich abnimmt, und schliesslich ganz verschwindet.” Creutzburg himself was somewhat uncertain as to the correct interpretation of the organ, but in view of its similarity to so-called intestines in other miracidia he concluded it represented a rudi- mentary intestine. 175] SOME NORTH AMERICAN FISH TREMATODES—MANTER 49 The conception of an intestine implies the presence of a cellular wall and a lumen. In none of the above instances has either of these conditions been demonstrated. That the organ in question might be interpreted as a group of uni-cellular glands seems more justified. This latter view would be more in accord with the nuclear-like content noted in present material and also described by Schauinsland and others. It would also explain the four-partite condition shown in Figures 36-38 and 40-41. These divisions of the organ into more than two parts seem to offer the strongest evidence against its interpretation as an intestine. Furthermore, if the possible glandular secretion served to aid penetration into the intermediate host, the gradual disappearance of the organ after this act was accomplished in the case of H. ovocaudatus would be explained. . The tendency showed by the larvae of O. cestoides to cling to debris at their anterior ends will be recalled. While it is true that the miracidia of many trematodes require no glandular secretion in order to penetrate their host, yet it should also be remembered that most miracidia are strongly ciliated and an effective boring force is attained by the action of the cilia which supply a constant forward pressure. On the other hand, the propulsive force of the miracidia- form under consideration is quite different in that when the force is directed against an object the entire body of the larva tends to be pushed backward. As the contents of the intestine of a snail are probabily less resistant than the intestinal wall, the physical power of unciliated miracidia would seem to be less effective in this first tissue penetration than is such power in ciliated miracidia. Unciliated miracidia of digenetic trematodes are very rare. Except among the Azygiidae they are definitely known only in Halipegus ovo- caudatus. Von Siebold as early as 1837 described an unciliated miracidium for Dist. variegatum (later Dist. cylindraceum=Haplometra cylindracea) but later workers disagreed with this conclusion. Schauinsland (1883) found the larva to possess a coat of cilia which it shed at time of hatching. Von Linstow (1890) found that the shedding of the ciliated coat was due to premature hatching, and that the larva is normally free-swimming. Willemoes-Suhm (1871) described the miracidium of Ptychogonimus megastomus and showed it to be very similar to Otodistomum larvae, possessing bristle plates and no cilia. The non-ciliated larvae of Azygia lucti and Halipegus ovocaudatus have been best known. Leuceruthrus now remains the only genus of the Azygiidae whose miracidium has not been studied. 50 ILLINOIS BIOLOGICAL MONOGRAPHS [176 NOTES ON THE LIFE HISTORY OF OTODISTOMUM CESTOIDES The very thick shell of the egg in O. cestoides indicates that these eggs possess the capacity for waiting a long period of time before hatching. On the other hand, the mature larva within the egg at the time of ovi- position shows that under favorable conditions the egg may hatch im- mediately. The further fact that the miracidium lacks the power of locomotion indicates that the eggs do not normally hatch until eaten by a snail. Schauinsland (1883) believes that such is normally the case for Azygia lucii, the eggs of which he found hatching in the intestine of a snail. Leuckart (1886:66-67) says that this condition is probably true for all unciliated miracidia. He states: “unter den Arten mit glatter Embryonal- haut scheint es doch manche zu geben, die im Wasser entweder gar nicht oder doch nur selten ausschliipfen, vielmehr solches vermuthlich erst dann thun, wenn sie, noch umschlossen von der Eischale, in den Darm eines geeigneten Trigers gerathen sind. So sah ich die Embryonen des Distomum ovocaudatum niemals im Freien ausschliipfen, obwohl ich die schon im Mutterleibe vollstindig sich entwickelnden Eier wochenlang, bis zum Absterben, im Wasser cultivirte. Eine ahnliche Beobachtung machte Schauinsland an Dist. tereticolle, dessen Embryonen im Darme von Lymna- eus palustris und L. stagnalis noch nach 24-36 Stunden lebhaft umher- kriechen, aber im Wasser nur selten frei werden.” The larvae of Halipegus ovocaudatus after hatching in the intestine of the intermediate snail host develop into sporocysts as shown by Leuckart and Creutzburg. The explanation of the ready hatching of the eggs of O. cestoides in sea water is found in the effect of the changes in concentration of the sea water due to evaporation. This effect’ was accidentally dis- covered when a culture of mixed eggs was left exposed in a watch glass for several hours. Evaporation of the sea water caused the opercula of the eggs to open whereupon the larvae, even though immature, partially escaped. The same result was repeatedly noted when sea water containing eggs was allowed to evaporate. The more mature eggs hatch more readily and the larvae appear active and healthy. When the evaporation is pro- nounced the less mature eggs open and the embryos partially slide out from the shell. Embryos thus prematurely hatched show no movement. Young eggs wherein the body form of the embryo is not definitely estab- lished do not open. In regular cultures the evaporation was slight and gradual, so that only mature eggs were stimulated to hatch. . 177] SOME NORTH AMERICAN FISH TREMATODES—MANTER 51 Still further evidence that the eggs do not normally hatch in a free state is furnished by the following data: Three vials of fresh eggs were covered with an animal parchment membrane and immersed in a large aquarium of running sea water. Here any change of concentration due to evaporation would be negligible. Although all of these cultures were examined at intervals for about a week, no hatched eggs or larvae were found, while eggs collected at the same time but kept in open vials hatched with customary regularity. This experiment to eliminate evaporation was tried with the purpose and hope of increasing the hatching of the eggs. The fact, however, that this more normal environment of the eggs reduced or eliminated hatching is in full accordance with the later discovered cause of hatchings by evaporation of the sea water. The opening of the operculum of the egg shell therefore seems to be induced directly or indirectly by an increase in density or osmotic pressure in the surrounding medium. The indication is that the stimulus is primarily a physical one rather than a chemical one. That is, increased density in the content of the snail’s intestine as compared with the density of sea water, rather than any chemical peculiarity of the location within the snail, induces the hatching of the eggs. This conclusion receives support from Schauinsland’s experiments in hatching the eggs of A. lucii. He obtained normally laid eggs, and found that the speed of their hatching could be greatly increased by placing them in a solution of common salt. In this solution the eggs hatched in about 15 minutes or practically im- mediately. It seems clear that no particular chemical substance is necessary for the stimulation to hatch, but merely an increase in osmotic pressure in the surrounding medium. Whether such a stimulus to hatch is direct or indirect is, of course, unknown. It is possible that the discrepancy in densities within and without the egg shell stimulates a secretion from the gland cells of the embryo, and that this secretion is the direct cause of the opening of the operculum. Some evidence in favor of this possibility is seen in the fact that even excessive evaporation does not cause the younger eggs to open although the operculum is well defined in the one-celled egg stage. Various molluscs common in the vicinity of Frenchman’s Bay were kept in vials of egg cultures. After periods varying from about 12 hours up to several days, these molluscs were removed and examined microscopically by means of smears. In general, all of this work was unsatisfactory. Eggs retained between the shell and body of a snail showed a tendency to hatch after the snail had been out of water for some time. This result would be expected, being probably due to sea water evaporation. There was no evidence that larvae so hatched penetrated the tissues of the snail. Un- fortunately, during most of the work, no special examination was made for the presence of eggs in the intestine. One very conspicuous case was ILLINOIS BIOLOGICAL MONOGRAPHS {178 on to discovered, however, when it was found that Littorina litorea ate these eggs very readily. Pellets of fecal or food matter in the intestine showed eggs present in large numbers. The snail had been kept for four days in a vial containing eggs. One pellet from the intestine contained over 50 eggs, another 25, 15, 12, etc., down to 4 or 5. A live larva was found by lightly crushing one of these pellets under a cover glass. The location and con- dition of this larva leaves no doubt that it had recently hatched from one of the eggs in the intestine of the snail. The snail had been dry and out of water for about a day and a half. Two out of six other specimens of Littorina examined by smears showed eggs in the intestine. This condition was further investigated by examination of serial sections of snails exposed to eggs. All of four specimens of Littorina so sectioned showed numerous eggs in the intestine. These eggs sometimes occurred in large numbers (several hundred). One case of a partly hatched larva was discovered in these sections. Careful examination revealed no certain case wherein such Jarvae had penetrated the wall of the intestine. Serial sections of two specimens of Thais lapillus, one of which had been in a very vigorous culture for over a week, gave practically negative results. Occasionally, isolated eggs were found in the digestive tract of one of these specimens, while the other contained no eggs. Sections of Buccinum undatum (which had, however, been in the egg culture only one night) showed no eggs. An encysted distome was not uncommon in both Littorina and Thais, but it could not be determined as Otodistomum. It occurred in thin walled cysts near the digestive tract. Redia and cercaria were also found in these snails, but probably those discovered did not belong to Otodistomum. No definite conclusions can therefore be drawn in regard to the first inter- mediate host, except that some species of snails (especially Littorina) readily devour the trematode eggs, and that these eggs can hatch in the intestine of the snail. Other species of snails under similar conditions seem to eat very few or none of the eggs. In the meantime, rather conclusive evidence has been found in regard to the last intermediate host of this trematode, at least in European waters. Scott (1909), in his report on fish parasites of Scotland waters, states in his discussion of Dist. cestoides: ‘“‘Several young specimens of a Distomum, which closely resembles the immature D. cestoides from the skate, were found encysted on the walls of the stomach of a Witch Sole, Pleuronectes microcephalus, captured in Moray Firth. There were several cysts observed, and all those examined contained only young Distomids—in some cases one, in others two examples. “Fishes form a considerable proportion of the food of large skates, and probably the Witch Sole, which lives in moderately deep water, sometimes becomes the prey of these large Plagiostomes.” 179] SOME NORTH AMERICAN FISH TREMATODES—MANTER 53 Nicoll (1913a) records Otodistomum cestoides (van Ben.) from Raia macrorhynca and adds the following: “In regard to the life history of this species, it is probable that the cercaria discovered by Scott, encysted in the stomach wall of the witch, Plewronectes cynoglossus (in Scott’s paper this species is inadvertently named P. microcephalus) represents the larva of O. cestoides. In the same fish I have found encysted in the wall of the stomach a cercaria, which from its large size and general structure, cannot be referred to any other species. It occurred as a large orange-yellow globular cyst 1.95 mm. in diameter, firmly attached to the outer wall of the stomach by a short pedicle. The wall of the cyst consisted of a thick, tough outer coat within which was a large amount of viscous yellow material. The cercaria measured 2.4 mm. in length, with a maximum breadth across the ventral sucker of 0.66 mm. Both suckers were trans- versely elongated, the oral measuring 0.29 by 0.35 mm. and the ventral 0.43 by 0.59 mm. The latter was situated at a distance of 0.96 mm. from the anterior end. The pharynx was small and situated close to the oral sucker. There was a very short esophagus and wide diverticula extending in a slightly sinuous manner to the posterior end of the body. The excretory vesicle was Y-shaped and consisted of a median stem extending a quarter of the length of the body, and two lateral branches reaching forward as far as the pharynx. No other organs were visible.” This cercaria described by Nicoll resembles almost exactly my youngest forms from the skate. A copy of Nicoll’s figure is given for comparison (Figures 2 and 3). As Nicoll points out, no other trematode is known to which this cercaria could belong except O. cestoides. Reference to my records shows that the sand dab and flounder are among the most common fish food of the big skate. No cysts were found containing cercaria, but relatively few of these flat fish were examined. It is very probable that the sand dab or flounder conveys the trematode to the big skate. The degree of infection and range of size of the parasite in the skate indicating a con- tinuous infection is in accordance with this conclusion. The immature trematode found encysted in the stomach wall of Lophius piscatorius and named ‘‘Xenodistomum melanocystis” by Stafford appears to be an immature O. cestoides. It is so identified by Odhner (1911b), who concludes that the goose-fish does not represent a true intermediate host, but that the cysts in that host represent an accidental infection. 54 ILLINOIS BIOLOGICAL MONOGRAPHS [180 SYSTEMATIC REVIEW OF THE FAMILY AZYGIIDAE The characteristics of the family Azygiidae as given by Odhner (1911b: 513-14) are as follows: Mehr oder weniger langgestreckte und abgeplattete “‘Distomen” mit einem derben, muskelkriftigen Kérper von 5-75 mm. Linge. Saugnipfe sehr kriftig entwickelt, einander genihert. Haut unbewaffnet, mit dicker Cuticula, die sich beider Kontraktion in unregel- missige Querfalten legt. Darm mit kriftigem Pharynx, dusserst kurzem Oesophagus und bis ins Hinterende reichenden Darmschenkeln; ein Pripharynx fehlt. Excretionsblase Y-férmig mit sehr langen, bis ins Kopfende reichenden (und sich dort mitunter vor dem Mundsaugnapf vereinigenden) Schenkeln. (Bei Ptychogonimus ist der Hauptstamm sehr verkurzt.) Genitalporus median, zwischen den Saugnipfen. Die charakteristische Ent- wicklung der Endteile der Geschlechtsweg biétet die beste Biirgschaft fiir die nahe Ver- wandtschaft der in diese Familie zusammengestellen Gattungen: der Genitalsinus ist sehr geriumig entwickelt; die minnlichen Leitungsweg bestehen aus Ductus ejaculatorius, Pars prostatica und Samenblase, die beiden letzteren schlauchférmig und ausser bei Ptychogonimus von einem Cirrusbeutel umhiillt; die ganze Komplex liegt unmittelbar vor oder iiber dem Bauchsaugnapf. Ovar und Hoden median, unmittelbar hintereinander in Hinterkérper gelegen. (Ausnahme: Leuceruthrus.) Uterus von Ovar aus nach vorn ziehend (Da er bei Ptychogonimus vor dem Ovar zuwenig Platz fiir seine Entfaltung findet, sendet er jederseits zwischen den Geschlechtsdriisen und Dotterstécken eine lange Schlinge nach hinten bis in die Nihe des Hinterendes). Laurerscher Kanal vorhanden, Receptaculum seminis fehlt. Dottersticke follikulir entwickelt, in den Seiten des Hinterkérpers, nicht bis ins dusserste Hinterende reichend. Vagina vorhanden. Fier etwa 0.045-0.085 mm. lang, gedeckelt; sie enthalten bei der Ablage ein reifes, anscheinend immer unbewimpertes Miracidium.— Magenparasiten bei Fischen. Odhner included the following genera in this family: Otodistomum, Azygia, Leuceruthrus, and Ptychogonimus. Azygia and Otodistomum are the most nearly related genera. Leuceruthrus has a very different arrange- ment of gonads, and Goldberger (1911:7) suggested that it might represent the type of a new family. In this genus the testes are lateral and anterior, being far removed from the median and more posterior ovary. Odhner, however, shows that this forward migration of the testes is of secondary importance, compared with the similarity which the genus shows to Azygia in the character of the genital atrium. Ptychogonimus shows the most marked deviations from the family type. These deviations lead toward certain characteristics of another group of marine trematodes known as the Distomum-clavatum group. Consideration of this relationship might be of value in determining the systematic position of the family Azygiidae. Trematodes of the Dist. clavatum group are also of large size with powerful, muscular bodies, and are found in the stomachs of marine fish. The group, containing a number of species, has been an isolated one 181] SOME NORTH AMERICAN FISH TREMATODES—MANTER 55 taxonomically. In 1911, Odhner placed the group as a sub-family of the Hemiuridae. Although designating these forms as of sub-family rank, he did not give a sub-family name. Nicoll (1915) in his list of trematodes of British fish, classified them under the Accacoeliinae. The term “Dist. clavatum group,” although awkward, is curiously persistent. The species in question (Dist. clavata (Menz.) Rud.) is actually Hirudinella clavata as designated by Blainville in 1824. Blainville later included also in this genus the former Fasciola ventricosa. The forms have been usually referred to the genus Distomum. All species in the group probably belong to the genus Hirudinella. Similarities of this group to the Azygiidae in general are: large size, muscular bodies, no pre-pharynx, very short esophagus, similar excretory system, and similar genital atrium. This latter condition forms a very important and striking resemblance. Jigerskiold (1900) describes the genital sucker of Distomum megastomum (= Ptychogonimus megastomus) as “ein neuer Typus von Kopulationsorgenen.’’ He compares the terminal genital regions of D. veliporum, D. clavatum, D. verrucosum, and D. megasto- mum, and offers the suggestion that the latter (which now represents the genus Ptychogonimus) is phylogenetically derived from a form resembling D. veliporum through a series now represented by members of the Dist. clavatum group. ‘This suggestion is based solely on comparison of the “Kopulationsorganen.’”’ The comparison does show that so far as the distal genital apparatus is concerned, Ptychogonimus is more similar to the Hirudinella group than to the genera of the Azygiidae. This similarity is marked not only by the more conspicuous folds and muscle rings in the wall of the atrium, but also by the fact that both sex ducts open separately on the genital papilla in Ptychogonimus and in Hirudinella. The lack of a cirrus sac in Ptychogonimus is another important feature in which that genus is like Hirudinella and different from other Azygiidae. Furthermore, the uterus in Ptychogonimus sends two coils posterior to the ovary and extending nearly to the posterior end of the body. This dis- tribution of the uterus is unlike anything found in other Azygiidae but very similar to the condition in Hirudinella. Odhner’s distinction between the Azygiidae and the Hirudinella group is that in the latter not only have the testes migrated forward (as in Leucerthrus) but the ovary has followed also. The ovary is then located directly behind the testes instead of in front of them as is the normal condition in the Azygiidae. He continues (p. 524): ‘‘—dadurch werden also die Lagebeziehungen zwischen Ovar und der Hauptmasse des Uterus die umgekehrten zu denen der Azygiiden. Nach diesem zweifellos recht schwerwiegenden Merkmal habe ich in erster Linie die Familiengrenze gezogen.” 56 ILLINOIS BIOLOGICAL MONOGRAPHS [182 However, as two coils of the uterus extend posteriad in Ptychogonimus, in this genus also the chief mass of the uterus is posterior to the ovary as can readily be seen from the figures of Jacoby (1899). One of the chief distinctions, then, between this genus and the Hirudinella group seems to be the position of the ovary in relation to the testes. Yet the significance of gonad location was discounted by Odhner himself when he included Leuceruthrus in the Azygiidae. Ptychogonimus, however, possesses follicular vitellaria as in Azygia, and not tubular as in Hirudinella. Again, its body is flattened and less muscular than Hirudinella. Considering these latter features, the genus is probably more appropriately considered as a member of the Azygiidae. Yet evidently it shows a relationship between the two groups and through it the family Azygiidae leads to the Hemiuridae and to a sub-family near the Accacoeliinae. The miracidium of Ptychogonimus is non-ciliated and bears a spiny anterior region as does the miracidium of Azygia. Odhner considers Ptychogonimus to be more closely related to Azygia than to Otodistomum “namentlich auf Grund des Baues der Miracidien.” It has, however, already been shown that Otodistomum larvae are also equipped with bristle-plates. This inference of relationship between genera on the basis of larval forms is interesting. No description of the miracidia of Hirudinella species could be found, but, judging from sectioned material of Hirudinella fusca, the mature eggs in this species do not contain larvae at all similar to those contained in mature eggs of Otodistomum. Hence, the first larval form of Ptychogonimus probably relates that genus more closely to the Azygiidae than to Hirudinella, 7f similarity of larval forms is reliable evidence. But there is reason to believe that similarity in miracidia of different species does not necessarily indicate close specific or even generic relationship of the adult trematodes. Thus, the miracidium of Halipegius ovocaudatus, a parasite of the frog, has the very same morphological features that characterize the miracidia of Azygia and Otodistomum. It is of the same shape and movement, is unciliated, possesses bristle plates, and has the so-called “intestine” (Creutzberg, 1890). Yet the adult form of Halipegus is not closely related to Azygia. The natural explanation is that the adults have undergone evolution independently of their larval forms. The non-ciliated miracidium is probably always associated with the fact that the egg normally hatches only after eaten by a snail. The retention of this feature in developmental history results in the retention of a certain type of larva. In the meantime, the adults (in the two cases cited) seem to have evolved along different lines without change in their miracidia. That is, one finds here the very common condition of constancy of larval forms and divergence of adults. The other possibility of con- vergence of larval forms is not probable in this case because the miracidia resemble each other in such great detail. 183] SOME NORTH AMERICAN FISH TREMATODES—MANTER 57 The genera of the Azygiidae can be separated by the following key: Uterus sending coils posterior to ovary................-.0005 Ptychogonimus Uterus entirely anterior to ovary Ovary and testes more or less directly behind one another in median line Genital pore close to acetabulum, branches of excretory SYSLEMUSEPATALG aaa ace cereieul esa eis teicieme re sdiete tiaceue ine ... Azygia Genital pore nearer oral sucker, branches of excretory vesicle united anterior to oralsucker............ Otodistomum Testes lateral and anterior to ovary.............00 0000 eee cece Leuceruthrus Ptychogonimus has the single species megastomus. Leuceruthrus micro pteri is the only representative of the genus Leuceruthrus. The two species of Otodistomum have already been considered at length. The egg size is the most certain distinction between them. Eggs averaging about 69 by 46u................ ......0. cestotdes Eggs averaging about 86 by 58u................-..055 O. veliporum A key to the species of Azygia will follow later. Azygia is the only genus of the family showing taxonomic confusion in its species. As it is morphologically very similar to Otodistomum, some of the difficulties leading to confusion were encountered in the present studies, and led to further comparative study of different species of Azygia as well as of the two genera themselves. These difficulties involved the question of relative significance of such factors as: body size and shape; extent and arrangement of the vitellaria; relative size and position of the suckers; shape of pharynx; position of gonads; and size of eggs. In the first place it is important to note that these forms are all very muscular and highly contractile. Their nature in this respect can be contrasted with such forms as Dicrocoelium and Opisthorchis, so that even though the internal arrangement of organs may,be similar, the heavy muscular body sharply distinguishes the Azygia group from them. Looss noted this fact in 1899 when he pointed out that the common descriptions of A. lucii (= Dist. tereticolle) hardly separated it from the genus Opis- thorchis. Yet Opisthorchis is a delicate, quite muscularless form, and not closely related to Azygia. Body contraction in the Azygia group where the worms are elongate in form not only alters the general shape, but it also changes the relation of the ovary and testes so that the former may lie almost lateral to the anterior testis, instead of directly anterior to it. In spite of the fact that Goldberger (1911) noted this variation in his A. acuminata, he nevertheless separated his genus “‘Hassalius’’upon this condition. The genus Hassalius has already been rightly reduced to synonymy with Azygia by Odhner (1911b) and by Ward (1917). Moreover, body contraction results in throwing the intestinal ceca into folds, giving them a zig-zag appearance. 58 ILLINOIS BIOLOGICAL MONOGRAPHS [184 Such an appearance is entirely without significance except as it indicates to some extent the degree of contraction. It should be eliminated as a distinction between species. A pointed or blunt caudal region is also a character depending largely upon temporary body contraction. Some confusion among genera and species is also due to a rather extreme range in size exhibited by the trematodes of this group. The conspicuous growth of Otodistomum cestoides even after arrival at sexual maturity, has already been considered. While this feature is common among trematodes, it seems to be particularly prominent in Azygia species. Odhner is therefore justified in considering Stafford’s genera, Megadistomum and Mimo- distomum (which were largely based on body size) as synonymous with Azygia. ; The extent and arrangement of the vitellaria are often very constant in trematodes. In the Azygiidae, the vitellaria are less reliable for specific diagnosis than might be expected. The variability of these organs in O. cestoides has been noted in detail. Odhner allows them almost unlimited range in Azygia species also. Certainly the importance given to details of these organs by Goldberger has been unwarranted. In the present studies, the constancy of the vitellaria in the various forms was critically studied, and will be considered in connection with the different species. One histological feature of special interest in highly muscular trema- todes such as the Azygiidae, is the arrangement of body muscles. Body muscles in trematodes usually consist of certain layers (circular, longi- tudinal, and diagonal) immediately beneath the cuticula. In addition to these muscle layers of the body wall, there occurs in the present group a well developed layer of longitudinal muscles within the parenchyma. These muscles running parallel with the longitudinal muscles of the body wall occur in bundles forming a more or less compact layer, so that in cross-section there is separated a central region containing most of the organs from an outer cortical region. Ward (1910) called particular attention to these muscles in Azygia sebago. They were also described and figured by Leuckart (1886) for Azygia tereticolle. Concerning them Leuckart (p. 18-19) says: ‘‘Unterhalb des Hautmuskelschlauches ordnen sich diese Parenchymfasern hier und da wieder in formliche Schichten, wie z.B. bei Distomum tereticolle, bei dem sich in einiger Entfernung von den Diagonal- muskeln der Rinde eine scharf begrenzte zweite Lingsfaserschicht bildet, die aus kriftigen Spindelzellen besteht und mit Ausschluss der Dotterstécke die in dem hellen, von Bindegewebe erfiillten Zwischenraum zwischen ihr und dem Hautmuskelschlauche zu liegen kommen, simmtliche Eingeweide in sich einschliesst.’’ Thus, the vitellaria occurred outside the muscles, as Ward found for A. sebago. In the present study of different American forms of Azygia, the vitellaria always occurred outside this muscle layer. The 185] SOME NORTH AMERICAN FISH TREMATODES—MANTER 59 available sections of Goldberger’s A. acuminata showed the muscles less developed and more widely scattered. The occurrence of similar body muscles in Otodistomum serves as an additional histological link of relationship between these forms and Azygia. Villot (1879:7) describes the grouping of such muscles for D. insigne (=Otodistomum veliporum). Ue says: ‘Une derniére couche de fibres longitudinales disséminées dans la zone limite du parenchyme. Ces fibres sont trés-fortes.’’ Villot’s figures clearly show these muscles as a definite layer. They represent a condition similar to that found in Azygia species, but their position is distinctly different in Otodistomum where they com- pletely enclose the vitellaria. My own studies on Ofodistomum cestoides and O. veliporum show the muscles present in a discernible layer but more scattered than in Azygia species, and external to the vitellaria. What may represent a homologous arrangement of longitudinal muscles has also been described for trematodes of the Hirudinella clavata (= Disto- mum clavatum) group. These forms are extremely muscular and contractile. In the neck region of H. clavata the following muscle layers occur: circular layer, longitudinal layer, oblique layer, and finally another internal zone of longitudinal muscles. Concerning this latter, Poirier (1885:483) says: “Celle-ci se compose d’un grand nombre de faisceaux musculairessouvent trés gros, et formés de fibres longitudinales d’un fort diamétre.” Poirier describes a rather peculiar condition posterior to the ventral sucker. In this region, he says (p. 484): ‘‘toutes les zones de fibres musculaires a Vexception de celle des faisceaux longitudinaux internes, ont complétement disparu. Cette derniére, par contre, a pris un développement considérable. Elle est formée de faisceaux composés d’un grand nombre de fibres, trés serrés les uns contre les autres, de facon 4 former une gaine épaisse, 4 peu prés continue, 4 lantérieur de laquelle se trouvent le parenchyme du corps et les différents organs de l’animal.”’ I have found (in agreement with Miihlschlag, 1914) a similar condition in Hirudinella fusca. The other body muscles, however, do not here com- pletely disappear as recorded by Poirier. Throughout the body length in this form there occur from the cuticula inward the following muscle layers: (1) circular, (2) longitudinal, (3) circular, (4) longitudinal. Layer (2) is always very weak with only scattered fibers. In mid-body region, layer (4) is very powerful with heavy fibers grouped in large bundles. It is this inner layer which is possibly homologous with the internal parenchyma muscles of Azygia and Otodistomum. It differs from them in being immediately surrounded externally by a ring of circular muscles. Granting that these muscles are present in all three groups, they differ in Azygia species by being located internal to the vitellaria, whereas in Otodistomum and Hirudinella they are found external to the vitellaria. 60 ILLINOIS BIOLOGICAL MONOGRAPHS [186 The writer has been fortunate in obtaining for study type or original material of all the different Azygia forms described from America with the exception of those recorded by Stafford. Co-type material of Azygia perryit Fujita from Japan was also studied. The conclusions reached will be included under the following discussions of accepted species. AZYGIA ANGUSTICAUDA! (STAFFORD 1904) [Fig. 27] Synonyms: Mimodistomum angusticaudum Stafford 1904 Azygia loossii Marshall and Gilbert 1905 Azygia loossi Odhner 1911 The descriptions of the two above mentioned forms appeared at about the same time. Since in the present work material was obtained only of A. loossii, the distinctive specific characters will be taken from that material. Goldberger (1911) has redescribed this species at some length. Reference to Stafford’s description will be made a little later. The position of the gonads in the extreme posterior region of the body seems to be a point in which A. Joossii is distinct from all other species. In this form the testes are located about 1/7 to 1/8 the body length from the posterior tip. Furthermore, the ventral sucker in this species is only slightly anterior to the middle of the body, whereas in other species it is distinctly more anterior. Such marked differences could possibly be explained by a highly extended anterior region and a highly contracted posterior region. But in the few specimens available, it was very clear that the entire body was extended. The uterus in A. loossii appears to be tubular with eggs in linear order, but this condition may be due to the possibility that the specimen had but recently arrived at sexual maturity. The musculature of the genital atrium seems to be particularly well developed in this species, so that the genital pore appears to be surrounded by a small sucker. The eggs average about 52 by 28y which is intermediate between the recorded sizes of eggs in A. sebago and A. acuminata. What seems to be an additional important feature of this species is the extent of the vitellaria almost to the region of the ventral sucker. Although the vitellaria actually begin in the mid-body region instead of anterior to it as is common in other species, the fact that the ventral sucker itself is equatorial in position explains the relative proximity of the vitellaria to it. The proportion of body length to distance from ventral sucker to most anterior vitellaria was found to be 27.2 and 26.2, a ratio reached only by A. acuminata. (See Table 18.) A. loossii was collected 1 Due to an obvious misprint the name of this species was spelled “augusticaudum” in Stafford’s original paper. 187] SOME NORTH AMERICAN FISH TREMATODES—MANTER 61 from Micropterus salmoides, Lucius lucius, and Amia calva from Wisconsin lakes. The above characters do not seem to warrant a new genus for this species, as in every important respect it agrees with the genus Azygia. It seems quite certain, however, that it does represent the same form described by Stafford as ““Mimodistomum augusticaudum’” from the mouth, pharynx, esophagus, and stomach of Lota maculosa and Stizostedion vitreum. Stafford describes the ventral sucker ‘‘as situated in the middle of the length of the worm in the most normal cases. The genital glands are flattened against each other and crowded backwards near to the ends of the caeca.”” Comparing it with Azygia, he says: “In the 12 mm. Azygia I selected as example, the ventral sucker is 2 mm. from the anterior end. In the 7 mm. Mimodistomum it is 3.5 mm. from the end, while the relatively long distance between sucker and ovary is in marked contrast to the short distance between the latter and the posterior end of the worm.” Thus, although Stafford’s description is brief, the differences he points out are exactly those between A. Joossii and other American species. That is, there is no point in Stafford’s description of ‘‘Mimodistomum angusti- caudatum”’ that does not agree with A. Joossii and practically every point mentioned is characteristic for that species. Hf, therefore, Stafford’s description is to be accepted there seems to be no escape from considering the two species synonymous. AZYGIA ACUMINATA GOLDBERGER 1911 [Figs. 21, 22] Goldberger unfortunately emphasizes unimportant points (e.g., vitel- laria unbroken, ceca zigzag, tail pointed, constricted neck region) in distinguishing this species. A study of his material, together with specimens of the same species collected and identified by Cooper, has led to the belief that this form represents a true species capable of distinction by definite features. A few specimens* from Dr. Ward’s collection from the same host (Amiatus calvus) apparently also belong to this species. Certain similarities in all of the material could be demonstrated. The constriction of the neck just anterior to the ventral sucker appears to be fairly constant and noticeable. This character should not be emphasized, however, as Goldberger’s own figure of his Azygia bulbosa shows that some slight localized and temporary constriction may occur in this region in other species. The most distinguishing specific characters were found to be: relatively wide body, anterior extent of the vitellaria, egg size, and poorly developed condition of the internal parenchyma muscles. It should be realized that the nature of all of these features is of somewhat precarious * Collection of Dr. H. B. Ward, vials Nos, 25.27 and 25.28. 62 ILLINOIS BIOLOGICAL MONOGRAPHS [188 standing in this genus. Probably no one of them, unless very marked, would justify a recognition of a separate species. Only because of the general association of all of these characters can the forms be separated from the other common American species. It may also be of some sig- nificance that all of the material is from the same host, Amiatus calvus. The proportion of body width to body length can be seen in Table 15. This point cannot be considered a reliable specific character. Generally speaking, A. acuminata usually shows a wider body than most of the other forms. In nearly every case, the vitellaria began at about the level of the posterior edge of the ventral sucker, rarely appreciably posterior to it. In none of the other American species was this general condition found except in A. angusticauda (Staff.) wherein this matter has already been discussed. Table 18 compares A. acuminata with the other forms in this respect. The average egg size of twenty measurements from Cooper’s specimens was 64.8 by 28u. The average egg size of about fifty measurements from Goldberger’s specimens was 63.4 by 34u. The egg size in Ward’s material was 63 by 29u. All of these averages are considerably above the egg size of the most nearly related Azygia species (Table 14). This large size is attained in the largest specimens of a few forms (like Leidy’s Dist. longum), but was never found in specimens of sizes similar to A. acuminata. Four sectioned specimens (three from Goldberger’s material, and one from Cooper’s) agreed in showing a much more scattered and less compact TABLE 9. Asygia acuminata Measurements in millimeters gfele? lee. | 28 | 32 | 23 5 Za »|BelBos/ Ba ESse] BS2 lag] ag bo S| aed |e AS seas i elas ol mete o§ SES 8 Me) a3 aS gad Pn eC 2h a sp 4H S&S |/Oo8g/>2 [ae 8)a 5 sass as SS O60 |\\ieea 8.* 1.68: | 1. 0.75 2.6 343 0.4 1.37 0.31 0.31, 11.3* 225 133 1. 3.9 0.65 2.56 1.56 0.96 10.9* 2:5 £3 0.93 3.4 3.87 0.37 2t5 0.63 1.01 O275* | 153 iI 0.68 3.3 3.1 0.5 2.4 0.5 1.5 e285 1255/18 3.87 0.74 968% [2255 1S 1. 3 3.75 0.3 9.87* | 2.6 1.25 | 1.06 3.56 3.62 0.35 1.4 0.65 1.15 6.87 1.8 1.18 | 0.9 1.9 200 0. 1.6 0.78 0.84 6.56 1.75 | 1.06 | 0.74 223 1.75 0.5 156 0.95 0.76 6.3 135 0.93 | 0.78 2. 2.18 0. 1.4 0.84 0.13 a5 2° 1.18 | 1. 2.68 2375 0. 1.68 0.88 1.03 8.877 | 1.4 0.87 | 0.812 2.93 Rigs) 0:3 2218 1.5 1.56 9.377.) 1:3 0.87 | 0.812 3.75 320 0.75 2.18 0.87 1.18 1 oF 0.93 | 0.75 3.87 4.12 0.5 2.8 1.87 1.9 189] SOME NORTH AMERICAN FISH TREMATODES—MANTER 63 arrangement of internal, longitudinal parenchyma muscles than was found in the other Azygia species. Table 9 (page 60) gives measurements of the available A. acuminata specimens. Those marked * were collected by Cooper; those marked + are from the Ward collection and were obtained at Fairport, Iowa, in 1916 by T. B. Magath. In each case the host was Amiatus calvus. Pearse (1924) records A. acuminata from the ‘‘wall-eyed pike” in Lake Pepin, Wisconsin, but gives no further data. AZYGIA LONGA (LEIDY 1851) [Figs. 19, 20 and 30] Synonyms: Dist omum longum Leidy 1851 Dist omum tereticolle of Leidy 1851 Megadistomum longum (Leidy) Stafford 1904 Azygia tereticolle of Stafford 1904 zygia sebago Ward 1910 Azygia bulbosa Goldberger 1911 Hassallius hassalli Goldberger 1911 Azygia lucii of Cooper 1915 Authentic material of all these forms (with the exception of Stafford’s) was obtained. A careful comparative study of this material has led to the acceptance of the above list of synonyms. The occurrence of intermediate conditions between somewhat extreme types, and the high degree of variability of some factors, necessitates the extension of the limits of the species as will be shown. In view of the high degree of contractility and marked growth changes of trematodes in this group, all comparisons were made relative to body length and on the basis of body proportions. Even on this basis consideration must be given to relative changes in proportions with growth. Azygia sebago Ward (1910) from the Sebago salmon is the best described of American species. Leidy’s Dist. longum reported in 1851 and the various other species recorded from America since 1910 have appeared to differ more or less markedly from A. sebago. These differences will now be critically examined. In separating A. sebago from the European species, A. lucii (=A. tereti- colle) Ward emphasized the posterior extent of the vitellaria. Although Odhner (1911b) is partially justified in discounting dependence upon vitellaria in this genus, the distinction seems to be a valid one. Not only in A. sebago, but in all American forms of Azygia examined, the vitellaria always extend appreciably beyond the hind testis, usually at least half way from this point to the posterior end, and may even reach to the extreme posterior tip as was observed in one case. According to descriptions and figures of A. Jucii, the vitellaria end in the region of the hind testis and 64 ILLINOIS BIOLOGICAL MONOGRAPHS [190 rarely extend appreciably posterior to it, although van Beneden (1858) says the vitellaria reach ‘‘depuis la ventouse posterior jusqu’en dessous du second testicule.” His figures show the vitellaria extending beyond the hind testis to a point about 1/4 the distance between that organ and the posterior end. Looss (1894:16) describes their extent for the European species as follows: “‘Die Dotterstécke erstrecken sich in den Seitentheilen des Leibes ausserhalb der Darmschenkel nach vorn hin nicht bis an den Bauchsaugnapfi—sie endigen ungefiihr die Linge seines Durchmessers vorher-, nach hinten zu kaum jemals bis iiber hintere Grenze des zweiten Hodens hinaus.” Further distinction between American species and A. lucii can probably be found in pharynx shape, a point upon which Odhner separates a new species, A. robusta. The pharynx of American species is more globose than the elongate pharynx of A. lucii. This point will be considered in more detail under A. robusta. A. sebago averages about 6 to 8 mm. in length. Specimens were found as small as 1 mm. and no ova were present in forms 2.85 mm. long. In regard to sucker proportions: ‘“The ventral sucker or acetabulum is usually distinctly smaller than the oral. In the extreme case it appears about equal in size or, on the other hand, only about half as large’ (Ward, 1910:1177). The questions of body size, size at sexual maturity, egg size, and sucker proportion are important points of comparison in American Azygia species. Of the other Azygia species, A. bulbosa Goldberger is most evidently identical with A. sebago. The descriptions of the two forms show no important differences. Type material of both species was studied. Refer- ence to comparative tables (Tables 15 to 18) shows almost identical body size and proportions, as well as similar position and arrangement of organs. Goldberger gives the egg size of A. bulbosa as 56 by 25u. Although the eggs may reach a length of 57y, the average of my 110 measurements gave the egg size as 48.3 by 28.7yu. Ward gives the egg size of A. sebago as 48 by 27x. The original type material of Hassallius hassalli Goldberger was also examined for comparison. That all of this material was strongly contracted was made very evident by the ring-like foldings of the cuticula and the excessive foldings of the intestine. Goldberger himself explained the “numerous transverse sulci’ of the cuticula as “apparently due to con- traction of the worm.” The high degree of body contraction is also evi- denced by the longitudinal compression of the organs, especially the uterus, gonads, and vitellaria. The pharynx is brought down closer to the ventral sucker than is normal. Yet, in spite of the evident body contraction, Goldberger separates his genus from Azygia on a thicker, shorter body form, and a lateral position of the ovary in relation to the anterior testis, both of which characters would be caused by body contraction. The only 191] SOME NORTH AMERICAN FISH TREMATODES—MANTER 65 comparison Goldberger makes between the two genera is in these words: “This new genus, aside from its external characters, differs from Azygia in the position of the ovary, which here is by the side of, that is in the same transverse plane as the cephalic testis, instead of cephalad of the latter as in Azygia.” The shorter and thicker body shape is directly due to con- traction. In regard to the position of the ovary, reference need only be made to Goldberger’s figure of Azygia acuminata (his Fig. 8) and to the following sentence from his description of this Azygia species: ‘‘In one of four press preparations, the ovary bears exactly the same relation to the testes as that in Hassallius hassalli; that is, it is within the zone of the cephalic testis.” In Azygia species as in Otodistomum (see Figs. 13-18), the ovary may be located slightly to the right or to the left of the anterior testis, and is consequently forced into the “‘zone’’of this testis when the body is contracted. Until the ovary can be shown to be lateral to the testis in uncontracted specimens, this character cannot be given even specific significance. In fact, after allowance is made for body contraction, this form cannot be distinguished from the other common American forms as represented by A. sebago and A. bulbosa. Goldberger’s own description and figures show the terminal genital apparatus in his A. bulbosa and Hassallius hassalli to be exactly similar. Study of his publication and original material reveals not a single point of difference between the two forms, except differences that might be due directly to body contraction. Comparison of sagittal sections of this species with sagittal sections of A. sebago and the A. lucii of Cooper shows no differences that can in any sense be considered specific. Goldberger gives an egg size of 48 by 26u measured from sections. My measurements of eggs from toto mount gave about 49 by 24y, but the larger eggs measured from sections were from 54 to 57y in length by about 24u in width, or practically exactly the measurements for A. bulbosa. The internal parenchyma muscles in Hassallius hassalli are prominent and are exactly as in A. sebago and the A. lucii of Cooper. Leidy in 1851 described Distomum longum from the mouth of Esox estor (=E. lucius). Stafford (1904) designates as ‘‘Megadistomum longum Leidy” forms which he collected from the mouth, esophagus, and stomach of Esox masquinongy. These trematodes are very long, Leidy’s speci- mens being up to 76. mm. (3 inches) in length, while Stafford reports living worms as extending to 127. mm. (Sinches), a truly prodigious length. Stafford gives a normal length of 75 mm. Both Leidy and Stafford give the oral sucker as slightly larger than the acetabulum. Odhner (1911b) expresses the view that this trematode should be considered as a large Azygia species. Cooper (1915) collecting material from L. masquinongy considers his form the same as that reported by Stafford and identifies it as Azygia lucit. 66 ILLINOIS BIOLOGICAL MONOGRAPHS [192 Study of Leidy’s original Distomum longum and Cooper’s material clearly shows that they represent members of the genus Azygia. Their large size is certainly not sufficient basis to warrant Stafford in forming a genus, Megadistomum. Leidy’s Dist. tereticolle (from Esox reticulatus) also was compared with them, and in the single specimen available in the Leidy collection, the oral sucker, contrary to Leidy’s description, was found to be slightly larger than the acetabulum. In Dist. longum, Leidy describes the genital opening as “‘just anterior to the middle of the neck, or nearer the oral acetabulum.”’ Such was not the condition in the two specimens of this form available. In each case the genital aperture was close in front of the ventral sucker, as is, indeed, characteristic of the genus Azygia. In the most elongate specimen the esophagus was unusually extended longitudinally and at its base a swollen region showed a marked resemblance to the cirrus sac. It is possible that Leidy mistook this appear- ance for the end-apparatus of the genital system. The true position of the terminal genital-apparatus in Dist. longum is seen in Figure 30. Cooper also collected Azygia species from the pike (L. luctus). These latter trematodes were much smaller in size, and apparently a different species, but Cooper, after a careful comparison of his smallest examples from the maskinonge with those from the pike, concludes that they repre- sent a single species. He also points out the highly variable size at which egg production begins. A 14. mm. trematode showed less mature eggs than one 6. mm. long from the same host. ‘On the other hand,” Cooper adds, “examples of intervening lengths may have their uteri distended with ripe eggs!’ (Cooper 1915:192). Forms from the trout were all immature although they reached a length of 11 mm. This variable size at which eggs are first produced in Azygia species is in contrast with the fairly constant size at sexual maturity in Otodistomum species. The large number of different hosts in which Azygia species occur may be associated with this variability. Another possible factor is the season of the year, but the influence of this factor would be difficult to determine. Eleven different fish hosts have been reported for the present Azygia species. Ward collected his material in July and August and found that those forms from the smelt although attaining a length up to 11 mm., were always immature. Cooper does not indicate the season of his collection but found all forms from the trout and from the small-mouthed black bass immature. Stafford examined his fresh water fish in the spring and fall. One specimen of his ““Megadistomum longum”’ measured 18. mm. long but contained no eggs; and the largest individuals of his ‘‘A. tereticolle’’ were smaller than immature individuals of Megadistomum. He found that most of his “Mimodistomum angusticaudum” were immature, but “towards the end of October” found a few sexually mature. 193] SOME NORTH AMERICAN FISH TREMATODES—MANTER 67 Whatever effect the season of collection may have, it is certain that what is evidently the same Azygia species does not attain sexual maturity at the same time in the different hosts in which it occurs. Thus, while average sized forms are producing eggs in such hosts as pike, pickerel, and salmon, specimens fully as large are still immature in such hosts as smelt, trout, small-mouthed black bass, and perch. That.variation in body size at sexual maturity may occur within a single host is shown by Cooper’s report of a 14 mm. trematode with less mature eggs than were commonly found in specimens 6 to 14 mm. in length. Further data is needed, however, to show the extent of this variation. It has not been noted by other workers, and certainly is not so conspicuous as the marked differences found in specimens from different hosts. The maximum body size which the trematode attains also seems associated with the host. In those cases wherein the greatest size is reached (as in the maskinonge and pike) there also seems to be considerable growth before sexual maturity (e.g. Stafford’s immature 18 mm. specimen), and it is very possible that the ultimate size which the trematode may reach is associated with the amount of growth before eggs are produced, and this latter condition may, in turn, depend upon the fish host. The following tables (Tables 10-13) show absolute measurements on these different forms. The 66 mm. specimen of Dist. longum was very highly extended and had apparently been stretched in killing. Moreover, the body was not equally extended but was more elongate in the uterus region just posterior to the acetabulum. The 37 mm. specimen therefore probably furnishes the more normal proportions. TABLE 10. Azygia sebago Measurements in millimeters 2 8 8 35.34 g a) Plo r) cs 2 oa 8 % 3 3 o of o ov 39 “| Be au = has Boies eee loo eles lg] eat} ee | 8s S |B \68| 2 |eee 28s 883) 28 | 22 | 22 5.9 0.9 0.65 | 0.59 2 1.4 0.31 2.12 0.72 0.72 5.9 0.9 0.65 | 0.59 23 1.4 0.31 212 0.72 0.72 10. 0.93 | 0.65 | 0.58 4.37 1.9 0.65 2215 Ze 2: 6.7 1.06 | 0.65 | 0.56 2525 1:5 0.41 2.18 1.31 1.18 11.5 0.75 | 0.67 | 0.56 4.06 2 0.5 3.75 2.3 2.4 13%, 0.5 0.74 | 0.56 5.3 2.5 0.9 3.25 1.9 1.9 9. 1.5 0.68 | 0.62 259. 1.8 0.4 2.8 1.12 0.55 12. nie | 0.8 0.5 4.8 3. 23412 3.43 1.9 1.9 10. 0.81 | 0.71 | 0.52 4.18 2.56 1.3 3.18 1.93 2.28 10.5 0.81 | 0.65 | 0.56 3.6 2.4 1.06 3.37 212; 1.87 68 ILLINOIS BIOLOGICAL MONOGRAPHS [194 TABLE 11. Azygia bulbosa Measurements in millimeters g z| 2.3 | 23 ; a |, 2/22 38.ea $3 | 22) 23 2 = | se [oe el Se ee sal) ee eae eee a SZ |SS] BS |Bss/ Bs gasses] ee] ss] 2S 4 Fe 168| « ISS Bla “ISSAs AS | FB | Fs 5,68 | 1. 0.81 | 0.62 2.43 1.68 0.65 1.49 0.75 0.75 6.3 1.37 | 0.87 | 0.62 Qed, 1.8 0.65 1.5 0.75 0.75 6.2 1.25 | 0.81 | 0.68 2.4 2.1 0.58 1.6 0.7 0.7 8.4 2.9 | 1.25.1 1: 2.6 2:5 0.84 2.5 1732, 125 3.9 0.9 | 0.66 | 0.54 1525 125 0.37 0.78 0.28 0.28 5.6 0.9 | 0.69 | 0.54 2: 1.6 0.58 1.6 0.73 0.92 7.1 1.1 | 0.87 | 0.63 2. 2.5 0.69 2.1 ake 1.2 5.1 1. 0.75 | 0.56 1.68 1.62 0.9 1.3 0.74 0.74 4.18 | 0.9 | 0.56 | 0.35 1.3 1.49 0.5 1512 0.55 0.55 6.9 1.1 | 0.81 | 0.62 1.9 2: 0.8 2.3 1.43 1.43 5.9 1.1. | 0.75 | 0,63 1.9 1.8 1.56 1.6 0.85 0.85 TABLE 12. Cooper’s Azygia lucii from L. masquinongy Measurements in millimeters » n n 25) .8|e2/| 33 2 8, 818,~(g22| $2 | 22) 22 S | Sel es (ee ore ae eee le eee ae FI ZS (BS) B [Bss[essless| BS] e258] 2B 4 Se |/Oa] < |@eki[Raesi(aaes| GS | FB | FB 29. 2.06 | 1.06 | 1.06 15.3 4. 4.8 6.87 S21 4.07 29. 2a 1d 1.1 12.3 4.4 decd: 9.6 4. 6.1 39. 2.18 | 1.25 | 1.25 17. 4.3 8.2 9. 5.6 4.4 31 1.8 | 1. 1 14. 3.68 5.3 9.1 5.23 323. 2.3 1.25. ||, 1:37 5.12 9.7 1.87 8.4 6.22 529 1.68 9.6 A) 6.8 1.18 | 1.18 0.87 | 0. 3.4 0.5 | 0.48] 0.5 * The large size of Dist. longum and the form from the maskinonge seems at first to separate them from most of the other forms. Leidy’s Disé. terelicolle, however, somewhat bridges the gap. The equal size of the suckers in the form from the maskinonge is distinct in these tables, but Cooper himself reports the oral sucker as larger than the acetabulum. In Dist. longum which this form most closely resembles, the oral sucker is larger. Moreover, Ward reports occasional equality of sucker-size in A. sebago. In view of these facts, the apparently constant equality of the size of the suckers in the above form is probably a coin- cidence, and at any rate could not alone justify a new species. 195] SOME NORTH AMERICAN FISH TREMATODES—MANTER 69 TABLE 13. Cooper’s Azygia lucii from the pike Measurements in millimeters ww un wn Oe no] os r=] = 2 8,218, ~/$22| 82 | 22] 22 g 3 ~| 8 oS S/SSs/oSs} of gy gy a) oO eens . 4 fe Nae 3 2 ° q Ss |8a Br) apmdiasgeslags!] as Ssuiss 3 < 4 Se |oa| « jagsdlass(as8| o8 | FB | FB 142 1.6 | 0.93 | 0.75 4.18 Zed 0.25 5. 2.8 2.5 14. 1.12 | 0.78 | 0.56 6.3 2.9 0.89 5: 251 2.3 8. 1 0.5 | 0.43 3.75 1.8 0.48 1.75 0.75 0.68 11. 0.75 | 0.68 | 0.56 4.75 2.5 15 2.68 1.18 1.37 7.5 0.87 | 0.67 | 0.52 2.9 1.6 0.28 2% 0.9 0.87 8.5 0.68 0:5 3.4 1.8 0.43 1.4 0.8 0.8 12. 0.93 | 0.75 | 0.62 5.5 3.9 0.9 2.18 1:5 fy, Leidy’s Distomum tereticolle 20.3 | 0.97 | Ors 0.71 «| 9:3. | 23.9 2.5 | 732 1 36 3.6 Distomum longum 1.3 38. | 6.8 18.7 | 12.1 | el RA 1.131917 5.68 9. 4, 4. 66. ea 1.42 37. 12565 J 037 The egg size is larger in the larger forms, but intermediate sizes were found in some of the material from the pike as shown by the following measurements. TABLE 14 Average egg sizes in American forms of Azygia ASV E10 SCDEZO S oisssieicccs ave ciadtececnneecss 48 by 27 Asy eta bulbosa: coe s