Camp. Biochem. Physiol. Vol. 83A, No. 2, pp. 353-358, 1986 0300-9629186 $3.00 + 0.00 Printed in Great Britain ,i‘ 1986 Pergamon Press Ltd HEMOLYMPH FERRITIN AND RADULA STRUCTURE IN THE LIMPETS PAZ?XLOIDA ALTICOSTA 7-A AND PA TELLA PERONII (MOLLUSCA : GASTROPODA) M. A. BURFORD, D. J. MACEY and J. WEBB* School of Environmental and Life Sciences, and *School of Mathematical and Physical Sciences, Murdoch University, Murdoch, Western Australia 6150. Telephone: (09) 332-2211 (Received 7 June 1985) Abstract-l. The mean concentration of total hemolymph iron was 3060 pg/IOO ml in Putella peronii and 2950 pg/lOO ml in Patelloida alticostata. 2. Ferritin was found to act as a major iron-binding protein in the hemolymph of both P. peronii and P. alticosiata. 3. P. alticostata ferritin has a molecular weight of approximately 505,000, while that of P. peronii has a mol wt. of approximately 520,000. 4. The lateral radula teeth of both species are mineralized by deposits of silica (SiO,) and iron in the form of goethite (a-FeOOH). 5. Hemolvmoh ferritin is sueeested to act as a high capacity transport system to supply iron to the mineralizing- front of the radul~~ INTRODUCTION Limpets (Mollusca: Gastropoda) together with chit- ons, are common members of the intertidal and shallow water molluscan community which occur on rocky shores in most temperate parts of the world (Barnes, 1980). Limpets are microphagous, feeding on fine algae, sponges and other encrusting or- ganisms which they obtain using a specially devel- oped tongue or radula which is extruded from the mouth and rasped along the substratum. The struc- ture of the molluscan radula and the teeth which are arranged in a series of transverse rows upon it, have been shown to vary both with the type and more particularly with the hardness of the food material consumed (Steneck, 1982; Steneck and Watling, 1982). In several species of limpet the radula teeth are mineralized and are thus able to excavate, to a limited extent, the rocks over which they browse (Jones et al., 1935; Lowenstam, 1962, 1967, 1971, 1981; Runham ef al., 1969; Steneck and Watling, 1982). Quantitative analysis of the radula teeth of a northern hemisphere limpet species Patellu athletica, has shown that they possess an iron content equiv- alent to 54% Fe,O, (Jones et al., 1935). The crys- tallographic form of the iron present in the limpet radula has been identified by X-ray diffraction and infrared spectroscopy as goethite, cc-FeOOH (Low- enstam, 1962). These goethite crystals co-exist with mineralized, but amorphous silica, SiO, in the or- ganic matrix of the teeth (Runham et al., 1969; Lowenstam, 1971; Grime et al., 1985). This parallels the situation found in the mature teeth of chitons which also show extensive bio- mineralization of iron in the form of ferrihydrite (5 Fe,0,.9H,O), lepidocrocite (y-FeOOH) and mag- netite (Fe,O,) (Carefoot, 1965; Lowenstam, 1962; Kirschvink and Lowenstam, 1979). Recent work in our laboratory has shown that in addition to miner- alized teeth, one of the local chiton species Cluvari- zona hirtosa, has hemolymph that contains particu- larly high concentrations of iron (Webb and Macey, 1983). Most (ca 70%) of this iron is bound to the protein ferritin which contains, in general, micelles (ca 70A) of ferrihydrite (Towe ahd Bradley, 1967; Harrison et al., 1980). The hemolymph concentration of ferritin, up to 400~g/lOOml is the highest yet reported for any hemolymph or plasma (Kim et al., in preparation). The high blood ferritin levels in this species thus presumably act as a delivery system for transporting iron to the radula where mineralization takes place (Webb and Macey, 1983; Kim et al., in preparation). Two limpet species, Patelloida alticostata and Pa- tellu peronii occur in close sympatric association with this chiton species in the south-west of Western Australia, all three species being found browsing mainly on the crustose coralline algae which cover the limestone rocks found in this area (Hodgkin, 1959). This study was therefore undertaken to determine whether the two limpet species P. alticostata and P. peronii also contained high hemolymph iron concen- trations, the nature of the major hemolymph iron- binding protein present and the degree and form of mineralization of the radula. A preliminary account of some of the data reported here has appeared (Burford et al., 1983). MATERIALS AND METHODS Limpets (Patelloida alticostata and Patella peronii) were obtained from intertidal reefs near the Perth metropolitan area (latitude 32’S, longitude 116 ‘E). All chemicals used were of the highest grade commercially available. The Sephacryl S-300 and Sepharose 6B used for protein sep- arations were obtained from Pharmacia Fine Chemicals. Proteins used as molecular weight markers for gel electro- phoresis in sodium dodecyl sulphate (SDS) were purchased 353