I29 The Structure, Development, and Operation of the Hinge Ligament of Ostrea edulis By E. R. TRUEMAN (From the Department of Zoology, University College, Hull) SUMMARY 1. The ligamental structure of Ostrea edulis is briefly described. The ligament is situated between the valves of the shell immediately below the urhbo and may be divided into two main layers, the outer and the inner, the principal features of which appear to correspond with those of other bivalves. The outer layer is divided into anterior and posterior halves by the inner layer, which occupies a central position. 2. The axis (hinge or pivotal axis) about which the valves open is situated in the adult oyster along a line drawn through the outer layer and the upper part of the inner layer. When the valves are closed the ligament above this axis is subjected to a tensile strain and that portion below to compression, the forces so produced causing the open- ing of the valves. An attempt has been made to measure this force in relation to the surface area of the valves and it is found to be approximately 4/5 gm. mm./mm.z This figure is comparable to those obtained when certain other species of bivalves are used. 3. The ligament is shown to develop from the simple outer layer of the early larval stages, first by the addition of an inner layer and then by growth of this structure chiefly in a ventral direction. The initial dorsal region of the ligament soon degenerates and at the same time the pivotal axis moves ventrally between the valves, thus increas- ing in length. This method of growth is compared with that of an elongate external ligament and the mechanical implications are suggested. INTRODUCTION EVERAL papers on various aspects of the hinge ligament of certain S lamellibranchs have recently been published (Trueman, 1949. 1950a and b). This paper deals with the structure of the hinge ligament of Ostrea edulis and makes reference to its development and method of operation. A brief comparison is made between it and those ligaments previously described (of Mytilus edulis and Tellina tenuis). It is hoped shortly to extend this survey to other forms. Some work on the hinge ligament of the oyster was carried out many years ago, notably by Horst (1882), Jackson (1888), and Bernard (1896). Mention should be made of recent observations by Rees (1950) on the spat of certain bivalves including that of O. edulis. The methods used in this investigation follow those previously described (Trueman, 1949). Decalcified specimens of all stages up to an overall length of 30 mm. (i.e. about 12 months after spatfall) were sectioned in ester wax and stained in Mallory's triple stain to facilitate easy and rapid differentiation of the layers of the ligament. Sections of the adult ligament were obtained by [Quarterly Journal of Microscopical Science, Vol. 92, part 2, June 1951.] 2421.18 K i3o Trueman—Hinge Ligament of Ostrea edulis sawing through the shell and ligament or by cutting frozen sections of the isolated ligament. The author is indebted to Professors P. G. 'Espinasse and C. M. Yonge, F.R.S., for reading this paper in manuscript. THE STRUCTURE OF THE ADULT LIGAMENT The hinge ligament of the adult oyster is situated between the valves in the umbonal region of the shell. It is completely internal and no part can be easily degenerate part of inner layer (2) tenor part of anterior part of the the outer layer (1) outer layer (1) pivotal axis pivotal axis anterior margin ventral surface of right valve of inner layer 10 mm. TEXT-FIG, I. Diagram of the right valve of Ostrea edulis, with the ligament cut in longi- tudinal section. The position of the pivotal axis is indicated. seen from the exterior. The location of the ligament is shown in Text-fig, i, which represents a median longitudinal section of Ostrea edulis. The term 'alivincular' has been suggested (Dall, 1895) ^or tn's tvPe °f ligament and describes its general appearance in longitudinal section. The term is not restricted to the Ostreidae but is used to describe the similar ligaments of, for example, Nucula or Pecten. The ligament may be divided into three parts which are most clearly seen in longitudinal section. The most anterior and posterior portions are both dark brown in colour and they cover the central part antero- and postero- dorsally, thus representing the outer layer (1) of the ligament (Text-fig. 1). In the young stages the outer layer is continuous between the dorsal margins of the valves but in the adult oyster it has become divided into anterior and posterior parts. The central part or inner layer (2) (Text-fig. 1) is generally whitish-grey in colour with some light-brown striations running parallel to the ventral border. Striations may also be seen in the outer layers, again running parallel to the ventral border, probably representing growth stages of the ligament. In the inner layer only, fine fibres may be observed with their long axes in the plane normal to the ventral margin of the ligament (Text- fig. 1). The structure of the inner layer is thus not very dissimilar from that of the inner layer of the ligaments' of Tellina or Mytilus previously described Trueman—Hinge Ligament of Ostrea edulis 131 (Trueman, 1949, 1950&). The striations of the outer layer give it a laminated appearance which is more marked than the laminae described in the same layer (1) of the ligament of Tellina. Rapid differentiation between the outer and inner layers may be obtained by the use of Mallory's triple stain in which the outer layer stains red and the inner blue. This staining reaction is similar to that observed in the ligament of other bivalves (Trueman, 19506, p. 227) and emphasizes the homologies of the outer and inner layers of the ligament. Work on the nature of the liga- ment of lamellibranchs (Trueman, 1950a) shows that the two layers are different chemically. The outer layers consist principally of a quinone-tanned protein, whereas the inner layer is composed of a calcified protein, which, however, appears to be subjected to varying amounts of tanning in different bivalves. This latter fact may be the explanation of the light-brown striations of the inner layer of the ligament of Ostrea. THE DEVELOPMENT OF THE LIGAMENT Any study of the morphology of the hinge ligament would be incomplete without some consideration of its development. With the kind co-operation of Dr. H. A. Cole of the Fisheries Experiment Station, Conway, it was possible to collect specimens of most stages of the development of Ostrea edulis, so that changes in the form of the ligament could be studied. The shell of lamellibranchs is secreted by the shell gland of the young larva, as a single dome-shaped membranous structure which soon becomes slightly calcified in areas on each side of the midline. The central region is not calcified and becomes the first part of the hinge ligament. Thus as the shell grows it forms the characteristic pair of valves joined together initially by a thin membrane and subsequently by a much thicker hinge ligament. Ransom (1939) has described stages in the development of the shell of O. edulis and observed that the valves of an early prodissoconch stage (Jackson, 1888) of o-i2 mm. length, were attached to each other by a thin transparent pellicle which he considers to be part of the primitive unpaired shell. The shell of the blacksick stage of 0-19 mm. length taken from the gill filaments of the oyster, has a hinge ligament consisting of the thin outer membrane, which is probably equivalent to that described by Ransom and a thin layer of ligament (i'5/J- thick) which is similar to the outer layer of most hinge liga- ments. This simple ligament joins the dorsal margins of the valves for a distance of about 70/x and is about 7/4 wide. The function of this early ligament is probably twofold. First, it must hold the valves together along their dorsal margin and secondly it must cause them to open on relaxation of the adductor muscles. The second function may be assisted by any other outward pressure from the animal. The force required to open the valves at this early stage of development (blacksick) is in any case small, owing to the small size of the valves. The next stage examined was the free-swimming spat of 0-20 and 0-22 mm. overall length. In these specimens the differentiation of the ligament into outer 132 Trueman—Hinge Ligament of Ostrea edulis and inner layers can be observed (Text-fig. 2). The outer layer still extends rather like the 'covers' of Tellina (Trueman, 1949) along most of the dorsal margin of the shell, and below this a small blue-staining (in Mallory's triple stain) inner layer is present. At this stage the latter is about 5^. long, 2/x thick, and 5 ft wide. This is considerably smaller in thickness and in width than the inner layer observed in similar stages in the development of Mytilus. Rees (1950) in his extensive work on bivalve larvae has shown that in many species there occurs a well-marked hinge ligament which appears rectangular in shape when viewed from the side (Rees, 1950, fig. 3). Such a hinge liga- ment has been described by the present author in the young stages of Mytilus periosfcracum outer layer (1) ,valve inner layer (2) TEXT-FIG. 2. Diagram of a transverse section through the ligament of an oyster spat 0-20 mm.