Differential screening of cDNA libraries constructed from the foot muscle of marine snails Littorina littorea

of metabolic plasticity that enables them to tolerate the constantly changing environmental conditions imposed by the tidal cycle. One such species, the periwinkle snail Littorina littorea, is highly tolerant of oxygen deprivation and has also developed the ability to survive freezing. Many of the biochemical mechanisms that support freezing and/or anoxia survival in intertidal invertebrates have been identified. Anaerobiosis is supported by large stores of fermentable fuels (glycogen, aspartate) in all tissues and the use of modified pathways of substrate catabolism that link additional sites for the substrate-level phosphorylation of ADP with the production of end-products, including alanine and succinate (De Zwaan, 1983; Storey and Storey, 1990; Storey, 1992). Furthermore, as residual oxygen is depleted from tissues, anoxia-tolerant marine molluscs sharply lower their metabolic rate to <10% of the resting aerobic rate at the same temperature, thereby greatly extending the time that their fixed reserves of metabolic fuels can support anaerobiosis. Periwinkles also have a well-developed ability to endure the freezing of extracellular body fluids. In fact, survival after 8 days frozen at –8°C has been documented (Murphy, 1983). Freeze tolerance allows L. littorea to colonize the intertidal zone in temperate and subarctic regions and to endure low tide exposure to winter air temperatures that can fall well below the freezing point of body fluids (approximately –1.9°C for snails in full-strength seawater). In recent studies, our laboratory has begun to document the gene-expression responses involved in natural stress tolerance in L. littorea. Larade et al. (2001) reported transcriptional upregulation of the ribosomal protein L26 during anoxia, and proposed that upregulation of the protein might function to stabilize the existing mRNA pool during the anoxic period until normal oxygen levels are resumed. More recently, Larade and Storey (2002) described a novel cDNA that is both transcribed and translated during anoxia exposure, supporting a role in anoxia survival. In the present study, we report the isolation of a metallothionein (MT) cDNA that is upregulated in response to both anoxia and freezing exposures in L. littorea. MTs are a family of low molecular mass, cysteine-rich proteins that characteristically bind both essential and nonessential metal ions in a metal/sulfur complex. Although all 2517 The Journal of Experimental Biology 206, 2517-2524 © 2003 The Company of Biologists Ltd doi:10.1242/jeb.00465