Differential response to Cadmium exposure by expression of a two and a three-domain metallothionein isoform in the land winkle Pomatias elegans: Valuating the marine heritage of a land snail
Graphical abstract
Introduction
Through the history of evolution, marine snails from different clades of Gastropoda have adapted several times independently to life on land (Rosenberg, 1996; Barker, 2001). Occasionally, such transitions may have been fostered in marine ancestors by acquisition of physiological pre-adaptations to intertidal and terrestrial conditions (Little, 1989; Marshall and McQuaid, 1991).
Pomatias elegans (the mouth-rounded snail) is a little South and Western European land snail living in moist terrestrial habitats of mainly calcareous soils (Kerney and Cameron, 1979; Pfenninger, 2002). In contrast to most other terrestrial snails, Pomatias elegans belongs to the gastropod class of Caenogastropoda and specifically, to the clade of Littorinoidea (Barker, 2001). It is considered as a land winkle closely related to the North Atlantic littoral periwinkle Littorina littorea, both of them being included into the superfamily of Littorinacea (Jordaens et al., 2001). Littorina littorea is optimally adapted to life in the intertidal zone, being alternately exposed to marine and terrestrial conditions. In contrast, Pomatias elegans and its close relatives of the Pomatiidae family have succeeded to become true terrestrial snails. Their emergence goes probably back to the cretaceous age, when they originated from an ancestral clade that gave rise to both, the modern periwinkles and the terrestrial Pomatiidae (Barker, 2001). Like in other gastropod lineages that made the transition from marine to terrestrial life (Marshall and McQuaid, 1991), specific physiological pre-adaptations may have facilitated the colonization of terrestrial habitats by ancestral Littorinoidea. Such pre-adaptations may include metabolic depression and high anaerobic capacity during exposure to air and under freezing conditions (Churchill and Storey, 1996; Storey et al., 2013), or increased capacity for osmoregulation (Klekowski, 1963; Taylor and Andrews, 1988). Consistently, adaptations of gastropods to life on land have also required the acquisition of particular adjustments at the biochemical and molecular levels. In Littorina littorea, hypoxic conditions due to the interruption of oxygen uptake by gills during aerial exposure can trigger transcriptional arrest (Larade and Storey, 2007) and regulation of gene expression by involvement of microRNAs (Biggar et al., 2012). At the same time, activation of protective mechanisms may prevent or mitigate adverse effects during intertidal phases. The expression of a metallothionein (MT) in the foot and the midgut gland due to freezing and anoxic impact, for example, may increase the stress resistance of air-exposed periwinkles (English and Storey, 2003). The respective MT has been shown, moreover, to be a Cd-selective protein (Palacios et al., 2017). Its three-dimensional structure was recently disclosed by solution NMR, revealing a novel three-domain structure, by which this protein is able to bind 9 Cd2+ ions per MT molecule (Baumann et al., 2017). Hence, apart from possible, other functions (English and Storey, 2003), the MT of Littorina littorea seems to be primarily involved in Cd detoxification. In fact, this metal is strongly accumulated in the snail midgut gland, where the MT gene is readily induced due to metal exposure in a concentration- and time dependent manner (Benito et al., 2017). As a consequence, most of the Cd accumulated in this organ is bound to the expressed MT protein (Bebianno et al., 1992). At the same time, virtually no Cd excretion can be detected, which may be attributed to the extremely low half time of MT degradation in the periwinkle's midgut gland (Bebianno and Langston, 1998). Apparently, the strategy of Littorina littorea with respect to the non-essential trace element Cd is a process of macro-concentration, a phenomenon that was previously described for some other terrestrial gastropods, too (Dallinger, 1993).
Against this background, the central objective of our study was to explore the Cd handling strategy of Pomatias elegans, a close relative of Littorina littorea, but with an exclusive terrestrial lifestyle. First, we were interested in the question whether, after Cd exposure, the toxic metal would be accumulated and retained mainly in the midgut gland of Pomatias elegans in a similar way as previously observed in its marine relative, Littorina littorea. Second, we wanted to explore if the handling and detoxification of Cd in Pomatias elegans is predominantly mediated by MT or MT isoforms, as in Littorina littorea. Thirdly, we wanted to find out whether the MT system of Pomatias elegans was homologous to that of the intertidal periwinkle, in terms of primary and tertiary structure and gene expression capacity in response to environmental Cd exposure. Fourthly, we wanted to understand if potential ecophysiological similarities and/or differences in the functioning of the MT system between the two closely related species could rather be explained by their common ancestry or, instead, by their adaptation to strongly different lifestyles (marine versus terrestrial). Finally, we tried to evaluate the ecotoxicological implications and biomarker potential of the MT system of Pomatias elegans.
Section snippets
Animals and rearing conditions
Individuals of Pomatias elegans were collected in summer 2014 on the island of Brač (Croatia), in a field site near Sumartin (43.2906522N, 16.8740650E). They were kindly provided to our lab by Dr. Lucie Juřičková (Department of Zoology, Charles University Praha, Czech Republic). During the winter season of 2014/15, the snails hibernated buried in dry soil substrate within a covered glass terrarium in our laboratory at room temperature (18–23 °C) and an average ambient air humidity of 36%. The
The midgut gland of Pomatias elegans as a central organ of Cd accumulation
As shown in Fig. 1, Cd accumulation by Pomatias elegans occurred in a tissue-specific manner similar to findings in other mollusc species (Boshoff et al., 2013; Gundacker, 1999; Sokolova et al., 2005). Clearly, the organ reaching the highest accumulation levels was the midgut gland, with an increase from 28.48 μg Cd/g (dry wt.) at the start of the exposure experiment to a maximal concentration of 119.77 μg Cd/g at day 14 (Fig. 1A). In contrast, Cd concentrations in the midgut gland of control
Acknowledgements
This study was supported by a grant of the Austrian Science Foundation (FWF, DACH proj. No. I 1482-N28) to R.D.
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Both authors contributed equally to this study.