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The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes ( CdMT ,  CuMT  and  Cd/CuMT ), already known from adults, in the Early Life Stage (ELS) of  Cantareus aspersus . This was accomplished by detection of the  MT  isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three  MT  genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three  MT  isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the  CdMT  and  Cd/CuMT  genes, but not the  CuMT  gene, are expressed in embryos at the first day of development. The transcription of the 3  MT  genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed  MT  gene was that of the  Cd/CuMT  isoform, whose transcription levels greatly exceeded those of the other two  MT  genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the  CdMT  gene in a concentration-dependent manner, whereas the expression of the  Cd/CuMT  gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the  CdMT  gene.

Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress