Reference: Biol. Bull. 167: 704-712. (December, 1984) RELATIONSHIPS BETWEEN FREE CUPRIC ION CONCENTRATIONS IN SEA WATER AND COPPER METABOLISM AND GROWTH IN CRAB LARVAE BRENDA M. SANDERS'^ AND KENNETH D. JENKINS'^ ^Molecular Ecology Institute, California State University, Long Beach, California, 90840: ^Duke University Marine Laboratory, Beaufort, North Carolina, 28516; and ^Department of Biology, California State University, Long Beach, California, 90840 ^ Abstract Crab larvae {Rhithropanopeus harrisii) were exposed to a range of free cupric ion concentrations, [Cu^^], regulated in sea water by a metal chelate buffer system. We found a biphasic relationship between intracellular copper distribution and [Cu^^] in sea water. At [Cu"^] within the ambient range (10 '"^'* to 10 '°^ M), cytosolic copper was associated with both metallothionein (MT) and high molecular weight (HMW) ligands, and was independent of external [Cu*^^]. At higher [Cu^^], copper was also associated with very low molecular weight (VLMW) ligands, and accumulated in this ligand pool and the MT pool as external [Cu*^^] increased. In marked contrast, copper in the HMW ligand pool did not correlate with [Cu'^] in sea water over the entire range of exposures. Reductions in larval growth occurred at greater than es-timated ambient [Cu^^] and correlated with copper accumulation in the MT and VLMW pools. Introduction The concentrations and subcellular distributions of metals can provide valuable information on an organism's capacity to adapt to accumulated metals. As a con-sequence, it has been suggested that the more subtle aspects of metal toxicity in aquatic organisms can be more accurately estimated by examining the distribution of metals among the various intracellular ligand pools (Bayne et ai, 1980). However, to understand the ecological significance of these data we must also be able to relate this information on metal metabolism to effects on the organism, the population, and the community (Sanders et al, 1983). In a previous study on larvae of the crab Rhithropanopeus harrisii (Sanders et al, 1983) we used metal-chelate buffer systems to control Cu speciation since the biological availability of Cu is related to the concentration of the free cupric ion, [Cu^^], rather than to the total or chelated Cu concentration (Sunda and Guillard, 1976; Anderson and Morel, 1978; Jackson and Morgan, 1978; Zamuda and Sunda, 1982). These buffers enabled us to expose larvae throughout zoeal development to a range of calculated [Cu^^] while the concentrations of potentially competitive metals were kept constant. In that study we found that most cytosolic Cu was associated with metallothionein, a cysteine-rich metal-binding protein whose synthesis is induced by metals (Hildebrand et al, 1979). This ubiquitous protein has been implicated in metal uptake, metabolism, and detoxication in vertebrates and invertebrates (Richards and Cousins, 1976; Brown et ai, 1977; Li et ai, 1980; Roesijadi, 1980; Jenkins et ai, 1982). We found that the concentration of Cu-thionein in crab larvae was related Received 16 April 1984; accepted 18 September 1984. 704
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