The Utilization of I¹³¹ by Certain Lamellibranchs, with Particular Reference to Shell Secretion

In contrast to the skeletons of certain sponges and corals which contain iodinated tyrosine and also traces of thyroxine (Roche, 1952), the structural proteins of molluscan shells appear to contain little or no natural iodine. Cameron (1914, 1915), for example, could not detect iodine in the shell pf My a arenaria although he found 0-042% in the byssus of Mytilus. However, in the course of recent work on the utilization of radioactive iodine by inverte-brates, Gorbman, Clements, and O’Brien (1954) showed that molluscs are in fact able to accumulate the isotope into the body and that protein-bound radioiodine can subsequently be located both in the shell and in the underlying mantle. They found, moreover, that in the bivalve Musculiumpartumeium, the isotope can be identified in hydrolysates of the body tissues in the form of monoiodotyrosine, diiodotyrosine, and thyroxine.

These observations are of particular interest in view of the fact that tyrosine is an important constituent of the scleroproteins in the periostracum and the outer shell layers in Anodonta cygnea and other lamellibranchs (Roche, Ranson, and Eysseric-Lafon, 1951; Beedham 19586). If tyrosine compounds labelled with radioiodine are utilized in shell formation, it is reasonable to suppose that these particular regions of the valves and ligament will specifically accumulate the isotope. Consequently, it seemed worth while to investigate in greater detail the metabolism of radioactive iodine in the Lamellibranchia with a view to tracing the secretion of artificially iodinated tyrosine from the mantle, in particular from the outer mantle fold, which forms the periostracum and the outer layers of valves and ligament.

The general procedures followed in the present investigation are the same as those adopted by Gorbman, Clements, and O’Brien (1954), Animals were exposed to the radioactive iodine by immersion in water containing the isotope I¹³¹ (carrier-free, as iodide), and the precise localization of iodine in the tissues was subsequently determined by autoradiography.

The lamellibranchs used were A. cygnea (small specimens, 4 to 5 cm long), Sphaerium corneum, and Mytilus edulis. Anodonta was employed mainly for studying the general distribution of iodine in the body after relatively short periods of exposure. Specimens were immersed in water containing an initial concentration of 1 mC of I¹³¹ per litre for periods ranging from 24 h to 14 days. Small specimens of Mytilus, which were primarily used for investigating the metabolism of iodine in relation to the secretion of byssus threads, were exposed to a similar concentration of the isotope for 1 week.

In addition, long-term experiments were conducted with Sphaerium to observe the localization of iodine after appreciable shell formation has been allowed to take place. Sphaerium is particularly useful for experiments of this kind since it readily secretes new shell material even under laboratory conditions. The animals were given exposure periods of up to 10 weeks in water containing 200 μ C of I¹³¹ per litre, this concentration being maintained as nearly as possible at a constant level.

After exposure to iodine, the animals were fixed in absolute alcohol or Bouin’s fluid and embedded in paraffin wax or ester wax (Steedman, 1947). Decalcification was carried out in 30% formic acid, which is less liable to leach out the radioactive material than the inorganic acids, nitric and hydrochloric (Siffert, 1948). Autoradiographs were prepared by the stripping film technique of Pele as described by Pearse (1953). They were developed in Ilford D 19B or amidol and the sections counterstained with haemalum. According to Gorbman, Clements, and O’Brien (1954), only the proteinbound form of radioiodine would be expected to remain in the sections after the procedures involved in the preparation of material for autoradiography.

The most noticeable feature shown on autoradiographs of the body of Anodonta is the accumulation of iodine along the surfaces of the organs within the mantle cavity. The isotope is localized on the inner epithelium of the mantle, and on the epithelia of the gills, labial palps, and much of the foot. The concentration of iodine on the inner surfaces of the palps is particularly intense (figs. 1, A, B). The radioiodine associated with the gills occurs mainly on the outer surfaces of the filaments, especially in the region of the lateral and latero-frontal cilia, although moderately strong autoradiographs are also given by the remainder of the filament surfaces and by-the epithelia lining the interlamellar spaces and the exhalant chamber. It is interesting to observe that the areas of the mantle and foot which are usually in direct contact with the external medium show much less tendency to concentrate iodine than the regions facing the mantle cavity. Negligible amounts of I¹³¹ occur on the middle and inner marginal folds of the mantle (i.e. the pallial curtain) and on the tip of the foot which normally protrudes between the ventral margins of the valves.

This distribution of the isotope suggests that a positive mechanism exists for the direct uptake of iodine by the epithelia lining the mantle cavity. Autoradiographs show that the iodine is mainly localized on the external borders of these epithelia, although it probably also occurs to a lesser extent in the cytoplasm in the outermost part of the epithelial cells. It has already been shown that radioactive calcium can be absorbed directly from the surrounding medium by the mantle, foot, and gills of A. cygnea (Schoffeniels, 1951), and by the mantle in mantle shell preparations of Crassostrea virginica (Jodfrey, 1953). Pomeroy and Haskin (1954) found that labelled phosphate ions enter the oyster principally by way of the gills, and they observed that this may also be the main source of calcium for shell desposition. However, there is little doubt that radioiodine also enters the body from the digestive system. Appreciable concentrations of the isotope can be detected on autoradiographs of the digestive gland. These deposits are often diffuse but in some specimens the iodine is found to be concentrated both in the lumen and in the large vacuolated cells of the tubules of the digestive diverticula. The isotope can also be located in the lumen of the stomach and intestine but it is not known whether iodine passes through the epithelia lining these parts of the gut.

Except for the accumulation in the digestive gland, the amount of iodine demonstrable by autoradiography within the body is small. After absorption, the iodine is probably dispersed throughout the tissues by the haemocoel, but it does not appear to be stored in any part of the body. Little or no iodine could be detected in the mantle, apart from the deposits occurring on its inner surface. The epithelia concerned with the deposition of the periostracum and the different layers of the valves and ligament consistently give negative autoradiographs.

The relationship of radioiodine to the non-calcareous material (conchiolin) in the shell is particularly interesting. Autoradiographs show that iodine becomes deposited over the whole outer surface of the shell. Large concentrations of the isotope occur both on the periostracum which superficially covers the valves and on the fusion layer which, because the periostracum becomes worn away in the hinge region, extends over much of the external surface of the ligament (Beedham, 1958a). It should be noted, however, that the iodine is only associated directly with these layers where they are in actual contact with the surrounding medium. As illustrated in fig. 1, c, the isotope accumulates equally well on the detritus which tends to form an irregular layer over large areas of the shell. If the shell is coated with celloidin before exposure to the isotope, the iodine is similarly deposited on this artificial covering and does not affect the periostracum. Although it is possible that the I¹³¹ occurring on the periostracum and fusion layer may be chemically combined with the protein at the surfaces of these layers, it seems unlikely that the radioiodine associated with the extraneous matter can be in a protein-bound form. Since carrier-free isotopes tend to be strongly adsorbed on any solids with which they come into contact (Francis, Mulligan, and Wormall, 1954), it may be suggested that a similar factor is at least partly responsible for the accumulation of I¹³¹ on the shell. This adsorption may well be sufficient to withstand the techniques employed in the preparation of autoradiographs.

In general the iodine associated with the shell is localized only on the surfaces which have direct access to the isotope. With the exception of one specimen, in which small deposits of iodine could be detected on the under surface of the outer calcareous layer near the margin of the valves, the conchiolin of the outer and inner layers of both valves and ligament was found to give negative autoradiographs. Owing to interference from the radioiodine deposited on the outer surface of the shell, it is difficult to assess the precise localization of the isotope on autoradiographs of the margins of the valves and ligament. However, there is no positive evidence from these sections that I¹³¹ becomes actively incorporated either into the periostracum or into the fusion layer. The amount of iodine occurring on the periostracum certainly diminishes in extent towards the extreme mantle edge, especially in the region of the periostracal groove where this layer is secreted.

The results of the experiments on Sphaerium are similar in many respects to those obtained for Anodonta. Despite the fact that shell formation occurred in the presence of radioiodine, the isotope could not be detected either in the non-calcareous matrix of the shell or in the underlying mantle tissues. Apart from small local concentrations of iodine in the lumen of the alimentary canal, the isotope is scantily distributed throughout the body. The strongest autoradiographs of specimens examined after either 2 or 10 weeks are invariably given by the external surfaces of the valves and ligament. However, although the iodine becomes deposited over much of the periostracum it does not appear to accumulate in the portion most recently secreted (fig. 1, D). There is a sharp demarcation line on autoradiographs between the youngest part of the periostracum which lies protected within the folds of the mantle edge and the remainder of the periostracum which is exposed to the surrounding medium (fig. 1, D).

As in Sphaerium, the radioiodine distributed on the shell occurs only on the external surface of the periostracum which is in direct contact with the water containing the isotope. At its origin, the periostracum gives negative autoradiographs, as does the remainder of the shell conchiolin and also the mantle. It is noticeable that the localization of iodine in relation to the byssus threads is essentially the same as that associated with the shell. As found by Gorbman, Clements, and O’Brien (1954), newly formed byssus threads autoradiograph strongly. Nevertheless, the threads only begin to accumulate iodine as they are secreted into the posterior groove of the foot and become fully exposed to the radioactive sea-water. No iodine was detected in the byssus threads whilst they were being formed within the foot, nor in any of the tissues responsible for the deposition of byssal material.

The experiments described here indicate that the main distribution of iodine in bivalves exposed to the isotope, I¹³¹, is on surfaces of the shell or body in contact with the radioactive medium. Although there is a mechanism for the uptake of iodine into the tissues where it can subsequently be recognized in the form of tyrosine-rich compounds labelled with the isotope (Gorbman, Clements, and O’Brien, 1954), the iodine does not appear to be concentrated in specific loci in the body. As also found by Fretter (1952) in experiments with I¹³¹ on species of Helix, autoradiographs show that iodine is scantily dispersed throughout the tissues, apart from deposits in the digestive gland. Where iodine accumulates on the mantle, as occurs in Anodonta, it is largely confined to the inner surface facing the mantle cavity.

The principal concentration of iodine occurs in association with the shell. Gorbman, Clements, and O’Brien (1954) considered that the shell and periostracum of molluscs and the byssus threads of Mytilus, in common with horny structures in other invertebrates (e.g. the exoskeleton of arthropods and the setae of polychaete annelids) can accumulate protein-bound iodine. However, whilst these authors clearly showed by radiochromatography the distribution of iodine in the tissues of Musculium in the same chemical forms, monoiodotyrosine and diiodotyrosine, as are found in the thyroid gland of vertebrates as precursors of thyroxine, the present investigation found no evidence for the secretion of these substances into the shell. The accumulated iodine which we have found in the shell and byssus of lamellibranchs must have been acquired almost entirely from the surrounding medium and not by way of the tissues of the body.

These deposits of iodine are located mainly on surfaces of the shell and byssus which have immediate access to the isotope, whereas the remainder of these structures show little or no tendency to concentrate the radioactive material. Moreover, there is some evidence to suggest that the I¹³¹ associated with the shell has accumulated at least partially as a result of adsorption.

Only in that part of the shell secreted during or subsequent to treatment with radioactive iodine could one possibly expect to find protein-bound iodine which had been deposited by the activity of the mantle. The molluscs used by Gorbman, Clements, and O’Brien (1954) were exposed to I¹³¹ for only 24 to 48 h, during which time shell formation cannot have occurred to any appreciable extent. Even after the much longer exposure periods used in the present investigation, which ensured that the shell material was formed in the presence of radioiodine, the newly secreted parts of the shell gave weak or negative autoradiographs.

These features, coupled with the fact that iodine could not be detected in the regions of the mantle which secrete the shell, nor in the tissues forming the byssal threads in Mytilus, indicate that caution should be attached to the view that the isotope, I¹³¹, can be utilized in shell and byssus secretion. While it is possible that the failure to demonstrate iodine in the secretory tissues is due to there being a rapid turnover of very small amounts of the isotope in these regions of the mantle and foot (Jodfrey, 1953, for instance, has shown that such a condition occurs in connexion with the calcium involved in shell deposition in the oyster), there appears at present to be insufficient evidence to show that I¹³¹ is actively incorporated in a protein-bound form into the shell and byssus in the Lamellibranchia.

This investigation was carried out in the Department of Zoology, The University, Hull. The authors wish to acknowledge the support and encouragement received from Professor P. G. ‘Espinasse.