There have been reports on artificially induced shell internalization before. Removal of distinct micromeres during early cleavage in Ilyanassa obsoleta can lead to veliger larvae that do not develop an exterior shell but in which an internal precipitate of calcium carbonate can be found [18, 19]. McCain [19] suggested that the reason for the development of an internal shell in Ilyanassa embryos is a disruption of normal inductive interactions between the calcium carbonate precursor cells (cells that regulate shell and statocyst formation) in addition to the interactions between the 3D macromere and the micromeres. Dictus and Damen [20] showed that the progeny of several micromeres contribute to the shell-secreting tissue of the larva in Patella vulgata. For Marisa cornuarietis, however, it is not known which micromeres contribute to which tissue [7] and, therefore, any speculation about a possible connection between a tissue’s sensitivity to platinum and its progenitor cells is difficult. Besides, the induction of shell internalization by deletion of micromeres happens very early in embryonic development, whereas the platinum-induced shell internalization becomes feasible only in later stages of development, caused by a different mechanism that affects tissue growth and not tissue formation.
In normally developing Marisa embryos two different directions of differential growth can be observed: first, the mantle anlage, the shell gland and the mantle edge overgrow the visceral sac in a way that leads to a horizontal rotation of the visceral sac by 180° [7]. Subsequently, differential growth of the dorsal part of mantle, shell gland, and mantle edge, results in shell coiling [21]. Torsion and coiling are independent from each other [21, 22]. Marschner et al. [3] showed that ontogenetic torsion does not take place in “sluggish” Marisa snails, however, it is not clear whether it is also absent in the “partly-shelled” individuals described here. A horizontal rotation cannot be observed in “partly-shelled” snails, although, in some specimens the ctenidium was positioned above the head which is a position which is supposed to be caused by ontogenetic torsion. In the “partly-shelled” snails the mantle anlage, the shell gland, and the mantle edge rotate vertically by 90° just like in the “sluggish” conspecifics. This rotation is unrelated to torsion and its cause is unknown. However, in contrast to the “sluggish” snails, the mantle tissue, the shell gland and the mantle edge resume growth in the “partly-shelled” animals. These tissues overgrow the visceral sac from a position on the ventral side of the visceral sac and thus the growth vector is not angular to the longitudinal body axis and no rotation of the visceral sac occurs. During this process, the tissue on the dorsal side of the visceral sac is pushed craniad, including the ctenidium which is drawn closer to its “normal” position above the snails’ head. Due to the vertical rotation by 90°, the ctenidium starts its movement from the left side of the snail’s body, and since the growth vector is parallel to the longitudinal body axis, the ctenidium ends up above the snail’s head but is located still more to the left side instead of right as it is in controls. This way, a criterion that is usually attributed to torsion, the anterior ctenidium, can be formed without any horizontal rotation of the visceral sac. Comparing normally developing and exposed embryos, it can be concluded that platinum inhibits the differential growth of both shell gland and mantle edge. In the case of the Pt-exposed “sluggish” animals these tissues remain inactive and neither angular nor straight differential growth takes place. In contrast, the combination of platinum and heat first leads to the described Pt-induced arrest of tissue growth but, some time later, heat reverses the effect. However, an angular growth does not take place, and straight differential growth leads to coiling. The platinum effect on the embryonic development of Marisa cornuarietis is obviously tissue-specific in the way that platinum specifically inhibits the growth of these tissues.
Growth of tissues and organs often occurs through cell proliferation which is usually, but not always, induced by growth factors [23]. Grande and Patel [24] investigated a signalling molecule of the growth factor β superfamily named Nodal. Nodal is involved in the development of chirality in snails and its inhibition by the chemical SB-431542 can lead to a loss of chirality that results in uncoiled shells. Disruption of the Nodal pathway obviously disrupts the differential growth that leads to coiling. The morphology of the resulting phenotype differs from the one induced by platinum, but is another example for body plan modification resulting from the inhibition of differential tissue growth by a single agent. Gene expression in the shell-secreting tissue has been investigated in Haliotis asinina [25] and Patella vulgata [26]. Although many of these upregulated genes are probably involved in shell formation, the gene dpp-BMP2/4, that was traced by Nederbragt et al. [26] in the ectodermal tissue surrounding the mantle edge, is known for its role in the specification of the dorsoventral axis in vertebrates and insects [27]. Nederbragt et al. [26] hypothesized that engrailed, which is expressed in the shell-secreting tissue, and dpp-BMP2/4 together set up a compartment boundary between shell-secreting and non-secreting tissue. This hypothesis was supported by Baratte et al. [28] who found engrailed proteins at the border of the mantle edge in the shell sac of Sepia officinalis, a cephalopod with an internal shell. These findings are especially interesting since dpp-BMP2/4, like Nodal, also belongs to the transforming growth factor β superfamily [29]. Both engrailed and dpp-BMP2/4 expression should therefore be investigated in control and platinum-exposed Marisa cornuarietis in the future, in order to find out, whether the inhibition of the differential growth in platinum-exposed snails is also due to a disruption of a growth factor as it is in the case of inhibited shell coiling.
There are also several accounts on how heavy metals can influence tissue growth and development. Hanna et al. [30] investigated several heavy metals and their impact on cell proliferation in embryos. They found that heavy metals could significantly reduce the number of cells in developing mouse embryos. Heavy metals can also act as carcinogens on different stages of carcinogenesis, including mutagenesis and altering gene expression [31].
Since some organoplatinum compounds are potent anticancer drugs, the genotoxicity of these chemicals has received reasonable attention. The genotoxicity of the different organoplatinum agents varies with their valency, structure, and conformation [32]. While Gebel et al. [32] did not observe any induction of micronuclei in human peripheral lymphocytes by PtCl2, Migliore et al. [33] found a significant increase of micronuclei in human lmyphocytes. According to Nordlind [34], PtCl2 inhibits the DNA synthesis of human lymphoid cells. Also, Osterauer et al. [35] observed a significant increase in DNA damage induced by PtCl2 in Marisa cornuarietis. It is, however, unlikely that genotoxicity alone can explain the highly tissue specific action of PtCl2 observed in the present study.
It is striking that, while exposure to platinum alone results only in a single type of “sluggish” individuals, the combination of platinum and elevated temperature generates phenotypes displaying a whole continuum of shell forms ranging from completely internal shells to “partly-shelled” animals of different morphology, and normally shelled animals. Consequently, the higher temperature must be assigned to be responsible for the re-start of the differential growth of the mantle edge and the shell gland. The higher the temperature the faster is embryonic development in the exotherm species Marisa cornuarietis [7] - this effect could also be observed in our experiments. Actually, the animals exposed to platinum at higher temperatures (28°C - 30°C) exhibited a lower mortality rate than at the lower temperature (26°C).
Heat stress, but also heavy metal exposure and other kinds of proteotoxic stress, induce stress protein synthesis (reviewed in [36]). However, constitutive levels of stress proteins are also important in stabilizing normal embryonic development [37, 38] but apparently need to be elevated under proteotoxic condition to enable a proper embryogenesis. Gunter and Degnan [39] exposed embryos of Haliotis asinina to slightly elevated temperatures and observed larvae with abnormally located tissues, although the tissues themselves kept their unique stress protein expression patterns.
There are several examples showing that an induction of stress proteins during embryonic development can reduce the disrupting effect of teratogenic agents. In Drosophila melanogaster an elevated Hsp70 level either caused by a preceding heat shock or by genetic manipulation, mitigates the toxic effects of the mitotic poisons vinblastine and colchicine [40]. Hsp70 and Hsp90, and probably other stress-induced proteins as well, are involved in the cell cycle and in proliferating cells higher Hsp levels can be detected than in cells at another stage of the cell cycle (reviewed in [41]). Hunter and Dix [42] found that in mouse embryos stress-induced Hsp70 was able to decrease the incidence of neural-tube malformations induced by the metalloid arsenite. A further study on human cells by Barnes et al. [43] revealed protection from arsenite-induced genotoxic effects by elevated Hsp70 levels. Osterauer et al. [13] investigated Hsp70-levels in juvenile platinum-exposed Marisa cornuarietis individuals. The highest concentration tested was 100 μg/L PtCl2, half of the concentration that was used in this study and, although histological investigations showed histopathological tissue alterations, the Hsp70 level was not significantly elevated. Also own preliminary studies on the induction of stress protein in platinum- and platinum-plus-heat-exposed Marisa embryos did not reveal a consistend induction pattern so far which could have been indicative for a protective role of chaperones in the context of shell gland and mantle edge outgrowth. Thus, for the moment, the role of Hsps in the signalling cascade triggering differential growth in Marisa embryos remains unclear.
In Marisa, the evidently different phenotypes belong in fact to a continuum of gradual morphological variation and result from a more or less intense phenotypic expression of a single developmental trait: the formation and differential growth of an exterior mantle, and, consequently, an exterior shell. The further the exterior mantle is developed, the more “normal” is the development of the snail. This means, that, in this study, elevated temperatures, which are usually considered stressful, actually lead to a more normal development than lower temperatures when interacting with a developmental disruptor, platinum. Together with the observation that mortality in the platinum expositions was lower at higher temperatures than at 26°C this leads to the conclusion that the increase in temperature in this case was not posing additional stress but actually protected development to some extent. There is also another group of stress-induced proteins that should be discussed regarding their possible interaction with platinum: metallothioneins, proteins that are involved in metal trafficking and detoxification. Serafim et al. [44] observed that heavy metal-induced metallothionein levels were higher at higher temperatures in Mytilus galloprovincialis, and Piano et al. [45] even found a temperature-induced increase of metallothionein levels in the absence of heavy metal exposure in the oyster Ostrea edulis. Metallothioneins have also received attention as possible cytosolic sinks for platinum-containing anticancer drugs [46]. Possibly, heat-induced metallothioneins may scavenge platinum in Marisa and thus decrease the internal concentration of “active” platinum. It is, however, unlikely that solely a quantitative reduction of “free” platinum is responsible for the formation of various “partial” shells, because these phenotypes never occurred in exposure experiments with lower platinum concentrations in the medium [10].
Internal shells have evolved in a number of gastropod and other molluscan clades independently from one another. So the question arises, whether there are any similarities between Marisa’s platinum-induced shell internalization and the formation of internal shells which have evolved in some molluscan taxa. In some groups (e.g. Naticidae), developing individuals start with an external shell which is then overgrown by extensions of the foot, the mantle, or both [47]. In Opisthobranchia, shell-internalization and reduction is very common and is theorized to have arisen independently in the different subgroups [48]. In Stylocheilus longicauda, for example, the juvenile shell is overgrown by the parapodia and then shed [49]. The Nudibranchia all lose their juvenile shells and the visceral sac is overgrown by the mantle [50], whereas the Saccoglossa are a heterogenous group with shell-bearing and non-shelled groups [48]. However, in non-shelled Saccoglossa it is not the the mantle that overgrows the visceral sac, it is material of the foot that covers the naked visceral sac [50]. Another gastropod group without external shells is the group of terrestrial pulmonate slugs. In the slugs Arion subfuscens [51] and Limax maximus [52] the shell develops inside a shell sac which is located directly under the dorsal mantle region where it lies horizontally [53]. Slugs, with occasional exceptions like the jumping-slugs of the genus Hemphillia, have a completely internal shell, but they have an external mantle unlike the “sluggish” phenotype of Marisa cornuarietis. Apart from pulmonate slugs, all gastropods have, at least at one time of their lives, external shells which may then be internalized during further development. In “sluggish” Marisa this is similar but less obvious: the first part of their shells, in this study called “plate”, is secreted on the ventral part of the visceral sac and is never covered directly by any tissue but is only hidden from sight by the foot. Only the later formed part of the internal shell is covered by the lobe and calcium carbonate is secreted internally.
Gastropods are not the only molluscan group in which shell reduction and internalization have evolved convergently. Kröger et al. [54] described several different reduction mechanisms in cephalopods, e.g. demineralisation of the shell or reduction of organic compounds. All these examples show that there is a phylogenetic continuum running between the two extrema - shell-bearing and non-shelled - and that different groups have evolved different mechanisms to reach their respective stages in this continuum independently from each other. While the presence or absence of an external shell is a very obvious trait in molluscs, it does not seem to be, mechanistically, a very conserved one. However, there is also a similarity between the platinum-induced shell internalization in Marisa and the internal shell of cephalopods. In some coleoids a second layer of prismatic material is deposited on top of the original one [55]. According to Bandel [56] mineralization of the primary shell occurs both inside and outside the shell and the calcium carbonate is attached to the periostracum from the exterior and the interior. This means that in these animals a part of the shell-producing tissue faces inwards just like it does in “sluggish” and “partly-shelled” Marisa individuals in which the shell gland is located on a lobe. Assuming, that the tissue of this lobe is in fact the tissue that would normally form the mantle, the shell in “sluggish”İ and “partly-shelled” Marisa individuals lies both on the mantle and is, at least partly, covered by it. This condition is similar to the one found in molluscs in which the mantle overgrows the shell, resulting in an inwardly facing shell-secreting tissue, like in almost all extant cephalopods.