LIFE-HISTORY OF BUCEPHALUS HAIMEANUS. 635 A Study of the Life-history of Bucephalus Haimeanus; a Parasite of the Oyster. By David Hilt Tcimeiit. With Plates 39—42. INTRODUCTION. AMONG a number of oysters which I procured in February, 1902, for the purpose of a demonstration, were several which were badly infected with the cercaria Bucephalus. At the suggestion of Professor Brooks I undertook a study of the origin of the germ-cells, from which the cercarias arise, the material at my command promising to be favourable for such an investigation. NOTE.—Since I began my studies on the origin of the germ- cells in Bucephalus haimeanus two papers on the process in related forms have appeared. I refer to those of Reuss (41, 1903) on Distomum duplicatum, and of Haswell (23, 1903) on Echinostomum sp. Further mention of these papers will be made at another place in this paper. Economic considerations have also made a study of the life history of the parasite of importance. Exact data as to the extent of infection, its means of spreading, the effect of infection upon the oyster, etc., have been desired. In my investigations I have endeavoured to determine not only facts of a purely scientific interest, but also those which will be of use to the oyster planter. 636 DAVJD HILT TENNENT. From this rather limited field my research gradually broadened until it included not only the study of the germ- cells, but also that of the older cercaria stages, as well as investigations along various lines for the purpose of deter- mining the complete life history of the form under considera- tion. This work, of which the present paper is an account, has been carried on in the Zoological Laboratory of the Johns Hopkins University, and in the Fisheries Laboratory at Beaufort, North Carolina. It is with pleasure that I acknowledge my indebtedness to the Honourable George M, Bowers, United States Commis- sioner of Fish and Fisheries, for the opportunity of enjoying the advantages of the Beaufort Laboratory, and to Dr. Caswell Grave, Director of the Laboratory, by whom many courtesies and privileges have been extended. To Mr. R. E. Coker I am indebted for material from Pamlico Sound, and to Mr. 0. C, .Glascr for infected oysters from Cameron, Louisiana. To Professor Brooks I express my most hearty appreciation of the unfailing interest, and of the many kindnesses which have characterised his direction of my work. MATERIAL AND METHODS. The abundance of infected oysters at Beaufort provided the opportunity of making extended observations on living material. That this has been done will be apparent from the account of my work. For the continuation of my work in Baltimore, recourse was had to preserved material. In my work on ' Bucephalus' I made use of many fixing agents. Corrosive sublimate solution, either with or without the addition of acetic acid, picric acid liquids, and Perenyi's fluid gave extremely poor results. Tissues placed in Gilson's mixture for two hours were excellently fixed and gave very useful preparations. By far the best fixation, however, was obtained by the use of LIFE-HISTORY OP BUCEPHALUS HA1MEAHUS. 637 Flemming's chromo-aceto-osmic mixture (weaker formula). Tissues were allowed to remain in this reagent for twenty- four hours and were then removed and washed in running water for twenty-four hours. In my study of Gasterostomum I have tried but three fixing agents : acetic sublimate solution, Gilson's mixture, and cold saturated aqueous solution of corrosive sublimate warmed to 35° C. The material preserved by the use of the last-named reagent has been satisfactory. For staining I have used borax carmine and acid borax carmine, both of which gave fairly good results; Delafield's haamatoxylin and eosin, which were useful; gentian violet, which was unsatisfactory; Flemniing's triple stain (safranin, gentian violet and orange G.), which was excellent in the study of the germ-cells, and Heidenhairi's iron hfematoxyiin, with eosin as a counter stain. By far the best results were obtained by the use of this method. The material was imbedded in paraffin and cut in one, two, and three micron sections. In studying the nervous system I used methylene blue as an intra-vitam stain, by means of which I was able to make out the topography of the nervous system as well as the scattered ganglion cells. For the study of the nervous system in preserved material I found Heidenhain's iron hasmatoxylin with a counter stain of eosin in 95 per cent, alcohol most useful. Several chloride of gold methods were tried, none of which proved satis- factory. HISTORICAL ACCOUNT or GASTBROSTOMUH. Von Siebold (1, 1848) discovered and named Gastero- stomum fimbriatum, which he found in the intestine of Perca and Lucioperca. Wagener (2, 1852) described this species, giving also descriptions and figures of two new species, G. minimum, from Trigla microlepidota, and G. gracilescens = 638 DAYID HILT TENNENT. Distomum gracilescens, Rud. (Synopsis, p. 409), from Lophius piscatorius. (Gasterostomum rather than Dis- tomum because the mouth is situated in the ventral sucking disc.) In 1857 Wagener (3) gave figures of an old and two young specimens of G. fimbriatum. Wedl (4, 1857) redescribed and figured Distoma cam- panula, Dujardin ('Histoire nafcurelle des Helminthes/ p. 435), from intestinal slime of Esox. Wagener (5, 1858) recognised the identity of this form with G. fimbriatum, v. Siebold. In this paper (1858) he again discusses the three species then known. 1. G. minimum, Wagener,from intestine Triglse rnicro- lipidotEe. 2. G. gracilescens, Wagener, from intestine Lophii piscatorii = Distoma gracilescens, Rud. 3. G. fimbriatum, v. Siebold = Distoma campanula, Dujardin, from intestine Esocis lucii, Percje fluviatilis, Luciopercse Sandras; encystedforms on gills of Cyprinus, showing the points in which they agree, and calling attention to the fact that the first two are from the Mediterranean where B. haimeanus is found, while the last is a fresh-water form, as is B. polymorphus. As a means of distinguishing the species he proposes making use of the difference in the direction of the gut, and the relative size of the sucking disc, and of the eggs.. Diesing (6, 1858) in a systematic paper separates the three forms above mentioned, retaining the genus Gasterostomum for the species fimbriatum, and creating a new genus, Rhipi- docotyle, for the species gracilescens and minimum. 1. Rhipidocotyle gracilescens = Distoma graci- lescens, Rud. = G. gracilescens, Wag. 2. R. minimum = G. minimum. Wag. The complex and peculiar structure of the anterior end of G. fimbriatum, which was described by Wagener (1857), LIFE-HISTORY OF BUCEPHALUS HAIMEANUS. 639 seemed to him sufficient reason for the formation of a new genus. Ziegler (7, 1883, p. 549) shows the error in Wagener's interpretation. Diesing also confuses the posterior with the anterior end in the three species named. Molin (8,1858) describes anew species, G. armatum from Conger conger. Gervais and Beneden, 1858 ('Zool. Med. 11/ p. 207), describe G. crucibulum = Monostomum crucibulum, Eud. Cobbold (9, 1858) gives a careful description and figures of G. gracilescens, which add little to the knowledge of that form. Diesing (10,1859), iu a revision of his classification, accepts three species of G-asterostomum : 1. Gr. fimbriatum, v. Siebold. 2. G. armatum, Molin. 3. G. crucibulum, Gerv. and Ben. = M. crucibulum, Rud. Molin (11, 1861) gives a complete description and figures of his G. armatum, describing acornucopiaeform acetabulum and distinct genital apertures. Olsson (12, 1867) redescribes G. armatum, Mol., and G. gracilescens, Eud. Maddox (46, 1867) found what seems to have been young G. gracilescens encysted in the nerves of the haddock. He describes the vitellaria (sixteen on each side) and flame-cells, which he calls pulsating bodies, connected with the water vascular system. Van Beneden (13, 1871) gives as new species: Gr. viperse—intestine of Trachinus vipera, Cuv. „ triglse— „ Trigla gurnardis, L. „ clupeas— „ Clupea sprattus. „ vivee— „ Trachinus draco, L. „ nova, sp. ,, Cyclopterus lumpus, L. Of these new species he gives figures of two, G. triglee 640 DAVID HILT TENNENT. and G. viperse, as well as giving new figures of G. gracilescens and G. crucibulum. Olsson (14, 1875) redescribes: . : 1. G. gracilescens, Rud.; embryonic forms from brain of Gadus minutus, adnlt from Lophius piscatorius. 2. G. armatnm, Molin, correcting Molin's error with, regard to the genital aperture. 3. G. crucibulum, Rud. = Monostomum crucibulum, Rud. = Distoma crucibulum, Dujardin. = Gasterostom a crucibulum, Diesing. and gives a figure of the last-named form. Rud. v. Willemoes Suhm (15, 1873) expresses the opinion that G. crucibulum = G. armatnm. He gives figures of the embryos of these forms, and thinks that Bucephalus haimeanus is a stage in the life history. Levinsen (16, 1881) redescribes G. armatum, confirming Olsson (13) in his observation as to the single genital aperture. Linton (17, 1899) describes and figures: 1. G. ovatum, Lt. ("Monostomum orbiculare," Rud. Lt. in 'Proc. U.S. Nat. Mus./ Vol. xx, p. 541) from Lobotes surinamensis. 2. G. arcuatum, Lt., from Sarda sarda and Carcha- rinus obscurus. 3. Gasterostomum sp. from Scomberomorus maculatus. 4. Gasterostomum sp. from Tylosurus marinus. The form which is to be considered in the present paper is that designated by Linton (17) as Gasterostomum sp. from Tylosurus marinus. For reasons which can best be discussed in a later section of this paper, I shall tentatively consider the form as G. gracilescens, Wagener. LIFE-HISTORY OP BUCEPHALUS HAIMEANUS. 641 HISTORICAL ACCOUNT OF BUCEPHALUS. Several varieties of Bucephalus, the larval form of species of Gasterostoma, are known. B. polyrnorphus, von Baer (18, 1827), from Anodonta mutabilis, var. Anatina and Oelleusis, and from Unto pictorum. B. intermedius, Ulicny (19, 1878), from Anodonta cellensis. B.