The Structure, Development, and Operation of the Hinge Ligament of Ostrea Edulis by E

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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 opening 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 longitudinal 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 ligament 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 ligaments. 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 ligament 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. in size immediately following the secretion of the first part of the inner layer (2). The position of the valves is indicated in this figure and in Text-figs. 3 and 4 by the stippled area, the extent of which has been determined as closely as practicable from sections of decalcified specimens. edulis and it seems probable that the main part of the ligament observed by Rees is composed of the typical inner layer (2). He has not observed such a well-marked structure in the spat of Ostrea, but this may well be due to the relatively small inner layer found at this stage. The general similarity in the form of the bivalve spat and of their ligaments shown by Rees, even in species which have very different hinge ligaments in the adult, should be noted. It seems probable that the larval ligament is more closely related to the larval shell and larval mode of life than to the various adult structures. The stages immediately following spatfall (specimens of o-6 to 2 mm. in size), when the metamorphosis of the larva takes place (Cole, 1938), were examined. The importance of this stage is due to the change of mode of life and to the subsequent increase in size and weight of the valves. Jackson (1888) observed that at the stage immediately following spatfall the shell secreted changed in structure to that typical of an adult oyster, having outer prismatic and inner nacreous layers. The form of the ligament in the stages just after spatfall is shown in Text- figs. 3 and 6a. In a typical transverse section (Text-fig. 3c) its appearance is not dissimilar from that of a transverse section of the ligament of Mytilus edulis (Text-fig. 7a). In front of the former section the inner layer becomes much thinner and ultimately disappears, leaving only the outer layer (Text- fig. 3d). This layer connects the dorsal margins of the valves together in much the same way as the anterior and posterior covers of the hinge ligament of Trueman—Hinge Ligament of Ostrea edulis 133 Tellina tenuis (Trueman, 1949, figs. 1 and 2). The anterior and posterior covers of the oyster spat both subsequently become incorporated in the operational part of the ligament, whereas in Tellina only the posterior cover is involved. The posterior cover (Text-fig. 3a) is frequently differentiated into two parts; the inner stains less intensely than the outer and must obviously be a subsequent secretion. These layers may be equivalent to those observed

outer part of periostracum outer layer (I) outer layer (I) inner layer (2) valve

inner part of ventral process outer layer (1) of layer 1

outer layer (1) slightly fractured dorsally outer layer (1)

inner layer

0-OSmm. TEXT-FIG. 3. Semi-diagrammatic representation of a series of transverse sections of the ligament of an oyster of about two weeks after spatfall. Section a is the most posterior, showing the outer layer (i) divided into two parts which may be homologous with layers IA and IB of the ligament of some other bivalves. In section b (8 p anterior) both outer (i) and inner (2) layers are present. The latter has become split in the midline and enclosed by the outer layer. Section e (16/1 in front of b) shows the normal appearance of the ligament at this stage with a well-developed inner layer. Anterior to this, section d, only the outer layer extends forward. and described (named IA and IB) in the hinge ligaments of Mytilus and Tellina. Text-fig. 36 shows an interesting phenomenon which is also seen in a more advanced condition in older oysters (Text-fig. 46). The inner layer (2) has become constricted and eventually split in the midline and below this a layer of the same type as the outer is secreted to fill the gap. It is significant that the fracture is filled by a secretion similar to the outer layer (tanned protein) rather than that of the calcified inner layer. This is similar to what has been observed in the ligament of Mytilus edulis. The split observed in the inner layer of the ligament and shown in transverse section in Text-fig. 4b and c, is characteristic of this layer in Ostrea edulis. The fractured nature of the inner layer is most apparent in the posterior region of the ligament of one-year-old oysters, for at this stage the secretion 134 Truetnan—Hinge Ligament of Ostrea edulis of the inner layer is anteriorly directed below the anterior portion of the outer layer (i) (Text-figs. 5 and 6). Growth of the ligament anterior to the umbo is not found in many species

inner layer {I), fractured outer layer (1' fractured dorsally

ventral part of outer layer ,(l) below the inner(2)

outer layer (1) fractured dorsaUy inner lajer (2) fractured "~ dorsal ly

O-Smm. TEXT-FIG. 4. A series of transverse sections of the ligament of the oyster, about one year after spatfall. The most posterior (a) and anterior (rf) show a well-developed outer layer which is torn dorsally along the greater part of the length of the ligament. The fracture is greater near the posterior end. Section b (200 ft anterior to a) shows the dorsal part of the outer layer completely separated into two halves, the inner layer (2) in a similar condition and below this a process of the outer layer (1). Section c (450/1 anterior to b) shows the typical appearance of the ligament at this stage of its development; both outer (1) and inner (2) layers are however split in the midline. of bivalves, where extension generally proceeds posteriorly to the umbo. A possible explanation of this phenomenon in the oyster is that at this stage the prodissoconch becomes posteriorly directed so that the axis of the ligament is turned into a different plane and anterior prolongation of the ligament is possible, at least to a limited extent. Ransom (1939 and 1948) has observed that in the prodissoconch of Gryphaea angulata the ligament has become established anteriorly. He considers that this displacement of the ligament Trueman—Hinge Ligament of Ostrea edulis 135

inner layer degenerate part

anterior part of outer layer posterior part of outer layer

prodissoconchal ""'••••.. shell ' •. •••" 2mm.

TEXT-FIG. 5. Drawing of Ostrea edulis showing the interior of the right valve with a longitudinal section of the ligament, at approximately one year after spatfall. The shape of the prodissoconchal shell is indicated. The umbo is turned posteriorly and the tendency towards growth of the ligament in an anterior direction may be observed.

one year after spatfall

TEXT-FIG. 6. Representations of longitudinal sections of the ligaments of two oysters, approximately two weeks and one year after spatfall, the anterior part of the ligament being on the left. The outer layer is shown in black where intact and by dots where torn or fractured; the inner layer is indicated by vertical lines where intact and by dashes where split. The anteriorly directed growth of the inner layer and the process of the posterior part of the outer layer (1) under layer 2 can be clearly seen. Only the intact part of the ligament can be functional so that very little of the ligament at stage a remains operational at stage b. The probable position of the pivotal axis is indicated by a straight line; this obviously moves ventrally during growth of the ligament. which is median in other Ostreidae is due to the extraordinary asymmetry of the prodissoconch in this species. In the typical adult condition (Text-fig. 1) the early parts of the ligament, as described above, have been lost. 136 Trueman—Hinge Ligament of Ostrea edulis

OBSERVATIONS ON THE OPERATION OF THE LIGAMENT To observe the operation of the hinge ligament of the oyster the adductor muscle was cut and the tissues of the animal removed, the valves being allowed to open only slightly during this procedure. The ligament then caused the valves to open more or less widely according to the external pressure. When investigating bivalves with an external ligament it has been possible to remove portions of the ligament in order to observe their effect. This investigation of the hinge ligament of the oyster required the precise removal of the anterior

Mybilus edulis Ostrea edulis Tellina benuis («0 (b) (c) TEXT-FIG. 7. Transverse sections through the centre of the functional part of the ligament of (a) Mytilus edulis, (b) Ostrea edulis, and.(c) Tellina tenuis. The outer and inner layers are shown black and by vertical lines respectively. The probable position of the pivotal axis is indicated by means of a cross. or posterior portions of the outer layer (Text-fig. 1). Owing to the sub- umbonal position of the ligament and the thick nacreous adult shell this procedure is difficult, but portions of the shell and ligament were removed by use of a small circular saw. From previous work (Trueman, 1949 and 1950&) on the external hinge ligament of Tellina tenuis and that of Mytilus edulis it is clear that the two main layers of the ligament have a specific function in relation to one another (Text-fig. 7). The outer layer (1) is normally subjected to a tensile strain and the inner (2) to compression. If the latter is stretched it readily fractures. The ligament of both these bivalves may be taken to open about an axis (hinge or pivotal axis) drawn in the midline between the main layers (Text- fig. 7a and c). Both Tellina and Mytilus have elongate ligaments situated along the straight dorsal margin of the shell. Growth of such a ligament is by means of posterior prolongation, first of the outer layers, then of the inner layer. As the mussel grows older the anterior region of the ligament ceases to cause the valves to open and the new secretion at the posterior end gradually takes over this function of the ligament. In the adult oyster the conditions of operation of the ligament are more complex. Transverse sections of the ligament and valves were cut in different regions. Measurements of the ligament were made in the closed and open positions of the valves, and these showed clearly that the hinge or pivotal axis is situated near the upper region of the inner layer (2) (Text-figs. 1 and yb) Trueman—Hinge Ligament of Ostrea edulis 137 arid not along the boundaries between layers 1 and 2 as in certain other bivalves (Text-fig. 7a and c). In such a transverse section as Text-fig. 76 through the centre of the ligament of Ostrea there is no outer layer directly above the inner. The pivotal axis is situated within the inner layer (2) so that when the valves are closed the part of this layer above this axis is subjected to a tensile strain and the region below to compression. In the ligaments of other bivalves layer 2 is normally subjected only to compression and the region of this layer below the pivotal axis in the ligament of the oyster is directly comparable in this respect. The portion of the inner layer above the axis is

counter balance arm to centroid of valve I

weight pan

/ iiiilllllllTTJi TEXT-FIG. 8. Diagram of the apparatus used to determine the opening moment or thrust of a hinge ligament. A full description is given in the text. unusual. It has been noted above that this layer readily fractures under conditions of tensile strain. This is probably the reason for the beginning of the degeneration of layer 2 (Text-figs. 1 and 7) and for the fractured appearance of this layer in the sections shown in Text-figs. 3 and 4, though the possibility that this fracturing may have resulted from the decalcification necessary for the preparation of the section cannot wholly be excluded. The position of the pivotal axis in relation to the outer layer of the ligament may be observed in Text-figs. 1 and 6. Not all of layer 1 is dorsal to the axis. The portion below must be under compression when the shell is closed; although this is not the usual condition for the outer layer it may well be elastic to compression and operate similarly to layer 2. In oysters in which the tissues had been removed it was possible to measure the opening moment or thrust of the ligament as follows. The left valve was firmly embedded in plasticine and weights were applied to the centroid of the right valve. The apparatus used consists of a counterbalanced beam with an arm to the centroid of the valve and a weight pan (Text-fig. 8). These are arranged so that the weight (w) applied to the valve is twice that in the pan. The weights were gradually increased until the valves were just closed so that the opening moment, M, was exactly counterbalanced. Thus the ratio d(2w+v) (= M) the surface area of the valve 138 Trueman—Hinge Ligament of Ostrea edulis could be determined, where d represents the distance from the point of application of the weight, w, to the pivotal axis, and v, the weight of the upper valve. In many bivalves the centre of gravity of the valve is very near the centroid. This ratio produced a result of approximately 4-5 gm. mm./mm.2 for the adult ligament of Ostrea edulis. Results of work on the elasticity of the ligaments of other bivalves together with a more complete discussion of this problem will be published shortly. After completion of these experiments an attempt was made to remove portions of the ligament in the same individuals and to assess the results. Removal of either the anterior or the posterior halves of the ligament considerably reduces the figure obtained (to approximately one quarter). Removal of either the anterior or posterior portions of the outer layer produces a similar reduction. Removal of both parts of layer 1 causes the loss of function of the ligament, the easy splitting of layer 2 and consequent separation of the valves. The importance of the outer layer in holding the valves together can thus be easily demonstrated. From these observations it would appear that the general conditions of operation and function of layers 1 and 2 of the ligament of Ostrea are similar to those of certain other bivalves. The outer layer, while not situated along the whole of the dorsal margin of the ligament (Text-fig. 1), is clearly essential. The part of layer 1 together with the very much less significant part of layer 2 situated above the pivotal axis, must be considered to be under tension when the valves are closed. At the same time the parts of both layers (1 and 2) below the pivotal axis are under compression. These two forces combine to open the shell. The exact method of operation of the inner layer in relation to the incomplete 'roof of the outer layer is difficult to interpret. In the free-swimming larva and early spat the latter condition is not found. The outer layer extends over most of the dorsal surface of the inner. At this stage the ligament probably operates rather similarly to that of Mytilus edulis (Trueman, 1950J). In the initial shelled stages (p. 131) it has been observed that the ligament consists only of an outer layer. There is no reason why this structure should not efficiently open the valves even although it is not differentiated into inner and outer layers. Indeed, all hinge ligaments composed of two layers must pass through a stage in development in which only the outer layer is present. When layer 2 is secreted the compressive function of the outer layer is probably gradually taken over. As this happens the hinge or pivotal axis moves ventrally to approximately the boundary of the two layers. In the ligament of the oyster this ventral movement is carried on throughout life and the upper part of layer 2 becomes split when the pivotal axis has passed below. By observation of the extent of the fracture it is possible to ascertain the functional portion of the ligament and probable position of the pivotal axis. This has been attempted and the results incorporated in Text-fig. 6, which represents longitudinal sections of the ligament of specimens a, 2 weeks, and b, Trueman—Hinge Ligament of Ostrea edulis 139 12 months, after spatfall. In Text-fig. 6a very little of the outer or inner layers has become split so that the greater part of layer 1 is above the pivotal axis and forms a complete 'roof over the inner layer. In Text-fig. 66 both layers have become extensively fractured in the dorsal region, the pivotal axis has moved ventrally and is increased in length.

DISCUSSION In any consideration of the function of the different layers of the ligament attention should be paid to their composition. Brief reference has already been made to the nature of layers 1 and 2. More detailed work has been carried out on the hinge ligament of Anodonta cygnea and it is hoped to publish this shortly. A few preliminary observations are included in this paper. The outer layers of ligaments seem to be generally composed of a tanned protein complex and are not calcified. This layer is therefore probably much more resistant and less permeable than the calcified inner layer. The periostracum has generally similar properties to the outer layer of the ligament and both appear to have a protective function. The upper surface of the ligament of the adult oyster which is exposed to the exterior, shows signs of degeneration. It may be noted that in Mytilus edulis (Trueman, 19506, p. 228) both the initial outer and the inner layers of the young ligament become fractured and into the gap so formed a layer similar in appearance to the outer layer is secreted. A somewhat similar phenomenon has been observed in the oyster (see above, p. 133). The most important property of the outer layer is its elasticity to tensile strains. The tensile strength of the protein of layer 1 is probably increased by tanning in the same way as it is increased in rubber by means of vulcanization (see Meyer, 1942, p. 176). Layer 2 is typically a calcified protein complex the exact nature of which is not yet clear. This layer has a characteristic fibrous appearance with the main fibre axis normal to the ventral surface and if this layer is subjected to tension, splitting takes place between the fibres. The hinge ligament of adult bivalves develops in many species in a larva, e.g. that of Mytilus, Cardium, or Macoma, which in the early prodissoconch stage ('phylembryo' of Jackson, 1890, p. 312) has a characteristically straight hinge line. This condition has been described above (p. 131) for the oyster larva, in which only the outer layer is present. Newell (1942, p. 28) observes that the alivincular type of ligament commonly appears early in the ontogeny of many genera, e.g. Nucula, Mytilus, Venus, and Mactra, and that it is reasonable to suspect that this was the primitive ligament from which other types are derived. The form of any ligament probably bears a close relation- ship to the shell shape. It is dangerous to assume that the larval ligament of any particular species is necessarily a primitive form. The hinge ligament normally has a straight hinge or pivotal axis. For obvious reasons a ligament forming a curve or an arc of a circle is mechani- cally much more complex if not impossible. In the early stages of a shelled larva this condition is fulfilled by the straight dorsal margin of the shell, 140 Trueman—Hinge Ligament of Ostrea edulis which is long in relation to the overall size of the valves. As the shell grows this ratio is reduced but a straight pivotal axis is maintained. Dall (1895) observed that the longer the line covered by a functional ligament the more rigidly will the opening and closing of the valves be controlled. To obtain a long straight line on the border of a rounded body, such as a developing bivalve, either the pivotal axis must descend or the outline of the body must be modified. The former, which is possibly the easier process, is the method used by the oyster and those bivalves with similar internal ligaments. Thus during growth of Ostrea the pivotal axis is moved ventrally, this being the only possible change in position in the oyster shell which will allow of the elongation of the ligament. Posterior elongation of the ligament is commonly employed by those bivalves with straight dorsal margins to the shell. From results of work on the opening moment and on the elasticity of the ligament of other species of bivalves, which it is hoped to publish shortly, the ligament of Ostrea appears to be no less efficient than the external type. The internal ligament of the oyster has, however, certain marked disadvantages, such as the exposure of the inner layer. This cannot be repaired by the secretion of the typical outer layer (as in the elongate ligament of Mytilus edulis), since the ligament is situated between the fracture and the secretory epithelial tissue of the pallial suture. The valves of Ostrea edulis are relatively flat in transverse section. If they were more curved like those of Cardium, there. would be a greater angular change at the ligament during the growth of the shell (see Trueman, 19506, for fuller discussion). This would probably cause a greater fracture, possibly too large for the ventral growth of the ligament fully to compensate. The internal ligament of the oyster may possibly limit the tumidity of the shell and the angle to which the valves may open. It would be of interest to observe the conditions of operation and growth of the hinge ligament of Gryphaea in this respect.

REFERENCES TO LITERATURE BERNARD, F., 1896. Bull. Soc. geol. Fr., 24, 54 and 412. COLE, H. A., 1938. J. mar. biol. Ass. U.K., 22, 469. DALL, W. H., 189s- Trans. Wagner Inst. Sci. Philad., 3, 486. HORST, R-, 1882. Quart. J. micr. Sci., 22, 341. JACKSON, R. T., 1888. Proc. Boston. Soc. nat. Hist., 23, 531. 1890. Mem. Boston Soc. nat. Hist., 4, 277. MEYER, K. H., 1942. Natural and Synthetic High Polymers. New York (Interscience). NEWELL, N. D., 1942. Univ. Kansas Publ., 10, pt. 2, 1. RANSOM, G., 1939. Bull. Mus. Hist. nat. Paris, 11, 318. 1948. Bull. Mus. Hist. nat. Belg., 24, no. 42, 1. REES, C. B., 1950. Hull Bull. Mar. Ecol., 3, 19. TRUEMAN, E. R., 1949. Proc. zool. Soc. Lond., 119, 717. 1950a. Nature, Lond., 165, 397. 19506. Quart. J. micr. Sci., 91, 225.