ABSTRACT
Although the morphology of the radulae of molluscs have been extensively studied, but little is known of their composition ; it is generally stated to be of a chitinous nature. There are two exceptions to this statement: Troschel (1856) and Sollas (1907). The former reported that calcium, phosphorus and iron were present in the enamel which covered the teeth and that the organ contained both organic and inorganic constituents. Sollas found that the radulae of the Docoglossa contained up to 27 per cent, of ash of which silica was the chief constituent: from microdeterminations of specific gravity she suggested that the silica was in the form of a hydrate or opal ; the marginal teeth were found to contain no silica. She recognised the presence of iron and stated that “the ash contained (in addition to silica) a noticeable quantity of calcium, iron, magnesium and phosphoric acid.” No other groups of molluscs were found to have siliceous teeth in their radulae, but to have chitinous teeth hardened by deposits of iron, calcium and phosphorus. The Chitonidae were somewhat exceptional, for the most important constituent of their teeth was ferric oxide.
The work of Sollas does not seem to have received sufficient recognition, for Bowell (1928) refers only to Troschel’s work when referring to the chemical composition of radulae.
The present study was commenced on a small number of radulae of Patella vulgata obtained from Plymouth and was continued on a small number from Hastings. Finally over 3000 P. athletica were collected at Broadstairs and their radulae dissected out. The latter were washed with a stream of distilled water on a piece of muslin and dried to constant weight. A detailed chemical study has been made of this material ; the analyses carried out on the small samples from Plymouth and Hastings gave essentially similar results. A qualitative examination of a number of other radulae has also been made for the presence of large quantities of iron and silica. Most of these radulae have been obtained from Plymouth, but Miss M. Rothschild very kindly obtained a number of species for us at Naples.
METHODS
Qualitative and microchemical tests for iron have been carried out on microscope slides, (a) by dilute hydrochloric acid followed by potassium ferrocyanide, (b) by ammonium sulphide. Qualitative tests for silica have been made by heating the radulae in concentrated aqua regia at 100 ° C. Siliceous teeth retain their shape and often their colour for an hour or so under this treatment, and can be identified under a microscope.
Sodium, potassium, calcium, magnesium and iron were determined as described by McCance and Shipp (1933). Iron was also determined by a thioglycollic acid method (Lyons, 1927). Copper was determined by sodium diethyldithio-carbamate reagent (Callan and Henderson, 1929). Aluminium was determined by aluminon (Myers, Mull and Morrison, 1928). Silicon was determined by the loss of weight on heating with hydrofluoric acid. The results agreed very closely with determinations made on the assumption that the residue which remained after ashing the radulae at 400 ° C. to constant weight, extracting the ash for eight hours with hydrochloric acid and washing the residue repeatedly, was silica. Chloride was determined by heating with concentrated nitric acid and excess of standard silver nitrate for some hours, with the precautions, described by McCance and Shipp (1933), for the subsequent titration of the excess of silver. Phosphorus was estimated by Briggs’ (1922) method, using the extract employed for the determination of metals.
PATELLA ATHLETIC A
Microscopically, the iron was not found to be uniformly distributed along the radula. The portion nearest the radular sac showed no reaction for iron. Proceeding distally, a reaction for iron gradually appeared in the radular membrane at the bases of the pigmented teeth. Near the tip of the radula, all the teeth (marginals, laterals and centrals) showed the presence of the metal (Plate I, fig. 1), but the brown pigmented teeth took longer to stain than the rest probably owing to the delay produced by their resistant shell on the rate of entry of the reagents. That most of the iron was in the pigmented teeth was suggested by the results of X-ray photography. Plate I, figs. 2 and 3 are enlargements of surface lateral and X-ray photographs of a radula. It is clear that the central and lateral teeth are sharply defined while the rest of the radula is hardly visible. The iron was present in the ferric state, but was non-magnetic.
When dried radulae were ashed at 400-500 ° C. the general form and colour of the central and lateral teeth remained unaltered, while the rest of the radulae was destroyed. On extracting the ash on a water-bath with concentrated hydrochloric acid or aqua regia the pigment was gradually removed from the bases of the teeth towards the tips, leaving a colourless shell which had the same shape as that of the original teeth. Similar results were obtained after heating samples of radulae to about 1000 ° C. These results, which agreed with those of Sollas, suggested that the skeleton of the teeth consisted of silica. This was confirmed by obtaining complete solution of the decolorised teeth in hydrofluoric acid. Further, when teeth were heated with fusion mixture in a nickel crucible, the cooled melt extracted with hot water, filtered and acidified, a residue was obtained (on evaporating to dryness) which was insoluble in hot aqua regia.
Table I gives the total inorganic constitution of the radulae as determined by the methods already described. The percentage of ash varied between 25.96 and 27.00 in the four samples which have been analysed. The figures given refer to a sample which contained 26.50 per cent. ash. It will be noticed that calcium and phosphorus are present in insignificant amounts and that there is nearly twice as much iron as silica.
The proportions of the elements found were so unusual in a substance of biological origin that when Dr R. Lessing very kindly offered to make an independent analysis, we gladly accepted his proposal.
He obtained the following results :
Through the kindness of Dr Sheldon, a spectrographic analysis was made of the radulae of P. athletica by Dr H. Ramage. This revealed, in addition to the above substances, the presence of small amounts of strontium, silver, manganese, lithium and lead.
The other organs of Patella were found to contain relatively little iron, and there is little distinctive or worthy of comment in their inorganic constitution (see Table II).
Less attention has been given to the organic substances. 1.75 per cent, of the dried radulae were found to be soluble in ether. Proteins were present as shown by the biuret test, but reactions for tryptophane and tyrosine were not obtained. Sulphur tests were positive. After hydrolysis at 95 ° C. in 80 per cent, (by volume) hydrochloric acid, reducing properties appeared equivalent to about 23 per cent, of the original (as glucose), and crystals of glucosazone were readily obtained. These results suggest that the body of the radula consists partly of a protein of the connective tissue type, and partly of a polysaccharide, probably chitin. The dried material contained 6.8 per cent, of nitrogen (Kjeldahl) which is also in harmony with this view.
Since sea water contains so little iron it is probable that Patella relies mainly upon its food for the iron required for the radular teeth. Patella feeds mainly on the green seaweed Enteromorpha, but to some extent also upon the brown seaweeds Fucus platycarpus and Dictyota dichotoma. Analyses of these seaweeds were made and the results are shown in Table III. The large amount of iron in Enteromorpha, which has Keen found in both the specimens analysed, would be enough to provide Patella with all its requirements. The mechanism of storage in the radula is at present quite unknown.
OTHER GASTROPODS
Qualitative and microchemical tests for iron and silica have been carried out on a number of British and Mediterranean species with the following results :
DISCUSSION
It is not possible to state definitely that iron and silica are only found together in the Patellidae, but the evidence suggests that such a combination is a characteristic of this family.
It would appear that iron is not present as a silicate but that the skeleton of the teeth consists of the silica and that iron is present as the oxide or carbonate, or possibly as an organic compound. The iron/silicate ratios (as determined by Dr R. Lessing and ourselves) would not conform to any simple relationship. According to Dr R. Lessing they would best be satisfied by some such formula as [(FeAl)2O3]3[SiO2]4 but this appears to be improbable. The microscopic evidence indicates that the percentage of iron increases progressively from the proximal to the distal segments of the radulae. Silica may increase pari passu, but there is no evidence that the iron which collects in the small lateral teeth is associated with silica. The presence of iron without silica in the Chitonidae and the presence of silica without iron in Dentalium suggests that the two constituents are independent of each other and possibly only occur together in the Patellidae by chance.
SUMMARY
The radular teeth of the Patellidae consist largely of silica and iron.
The teeth of the Chitonidae contain large amounts of iron and little or no silica.
The teeth of Dentalium contain small amounts of silica but no iron.
Iron and silica have not been found in other radular teeth.
ACKNOWLEDGEMENTS
We are indebted to Miss M. Rothschild for Neapolitan specimens, and for the photographs from which the figures were made, to Dr Ramage for the spectrographic analyses, and to Dr R. Lessing for his chemical assistance. We have to thank Miss Franklin for taking the X-ray photographs.
