Energy status of chironomid larvae (Diptera: Chironomidae) from high alpine rivers (Tyrol, Austria).

Chironomids (non-biting midges) inhabit almost every wet or semi-wet continental environment on Earth with probably 10,000 different species. Species occurrence and composition are undoubtedly limited by environmental harshness and food availability being reflected in their energy stores. Most animals store energy as glycogen and lipid. They enable the animals to survive adverse situations and to continue growth, development, and reproduction. This general statement is also true for insects and also particularly true for chironomid larvae. The rationale behind this research was, that probably any stress, any environmental burden, and any harmful influence increases the energy requirement of individual larvae depleting energy stores. We developed new methods to measure the glycogen and lipid content in small tissue samples. Here we show how to apply these methods to single chironomid larvae to demonstrate their energy stores. We compared different locations of the high Alpine rivers along harshness gradient densely populated and dominated by chironomid larvae. All samples show very low energy stores without any major differences. We found glycogen concentrations below 0.01% of dry weight (DW) and lipid concentrations below 5% of DW irrespective of the specific sampling point. These values are among the lowest ever observed in chironomid larvae. We demonstrate that individuals living in extreme environment are stressed leading to reduced energy stores in their bodies. This appears to be a general feature of high altitude regions. Our results provide new insights and a better understanding of population and ecological dynamics in harsh mountainous areas, also in view of a changing climate.

Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins.

Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains-α, ß1, ß2, ß3, γ, and δ-in a lineage-specific manner. We have functionally characterized the Neritimorpha ß3ß1 and the Patellogastropoda γß1 MTs, demonstrating the metal-binding capacity of the new γ domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding.

The Modular Architecture of Metallothioneins Facilitates Domain Rearrangements and Contributes to Their Evolvability in Metal-Accumulating Mollusks.

Protein domains are independent structural and functional modules that can rearrange to create new proteins. While the evolution of multidomain proteins through the shuffling of different preexisting domains has been well documented, the evolution of domain repeat proteins and the origin of new domains are less understood. Metallothioneins (MTs) provide a good case study considering that they consist of metal-binding domain repeats, some of them with a likely de novo origin. In mollusks, for instance, most MTs are bidomain proteins that arose by lineage-specific rearrangements between six putative domains: α, ß1, ß2, ß3, γ and δ. Some domains have been characterized in bivalves and gastropods, but nothing is known about the MTs and their domains of other Mollusca classes. To fill this gap, we investigated the metal-binding features of NpoMT1 of Nautilus pompilius (Cephalopoda class) and FcaMT1 of Falcidens caudatus (Caudofoveata class). Interestingly, whereas NpoMT1 consists of α and ß1 domains and has a prototypical Cd2+ preference, FcaMT1 has a singular preference for Zn2+ ions and a distinct domain composition, including a new Caudofoveata-specific δ domain. Overall, our results suggest that the modular architecture of MTs has contributed to MT evolution during mollusk diversification, and exemplify how modularity increases MT evolvability.

Metal-Specificity Divergence between Metallothioneins of Nerita peloronta (Neritimorpha, Gastropoda) Sets the Starting Point for a Novel Chemical MT Classification Proposal.

Metallothioneins' (MTs) biological function has been a matter of debate since their discovery. The importance to categorize these cysteine-rich proteins with high coordinating capacity into a specific group led to numerous classification proposals. We proposed a classification based on their metal-binding abilities, gradually sorting them from those with high selectivity towards Zn/Cd to those that are Cu-specific. However, the study of the NpeMT1 and NpeMT2isoforms of Nerita peloronta, has put a new perspective on this classification. N. peloronta has been chosen as a representative mollusk to elucidate the metal-binding abilities of Neritimorpha MTs, an order without any MTs characterized recently. Both isoforms have been recombinantly synthesized in cultures supplemented with ZnII, CdII, or CuII, and the purified metal-MT complexes have been thoroughly characterized by spectroscopic and spectrometric methods, leading to results that confirmed that Neritimorpha share Cd-selective MTs with Caenogastropoda and Heterobranchia, solving a so far unresolved question. NpeMTs show high coordinating preferences towards divalent metal ions, although one of them (NpeMT1) shares features with the so-called genuine Zn-thioneins, while the other (NpeMT2) exhibits a higher preference for Cd. The dissimilarities between the two isoforms let a window open to a new proposal of chemical MT classification.

Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae).

Terrestrial snails (Gastropoda) possess Cd-selective metallothioneins (CdMTs) that inactivate Cd2+ with high affinity. Most of these MTs are small Cysteine-rich proteins that bind 6 Cd2+ equivalents within two distinct metal-binding domains, with a binding stoichiometry of 3 Cd2+ ions per domain. Recently, unusually large, so-called multi-domain MTs (md-MTs) were discovered in the terrestrial door snail Alinda biplicata (A.b.). The aim of this study is to evaluate the ability of A.b. to cope with Cd stress and the potential involvement of md-MTs in its detoxification. Snails were exposed to increasing Cd concentrations, and Cd-tissue concentrations were quantified. The gene structure of two md-MTs (9md-MT and 10md-MT) was characterized, and the impact of Cd exposure on MT gene transcription was quantified via qRT PCR. A.b. efficiently accumulates Cd at moderately elevated concentrations in the feed, but avoids food uptake at excessively high Cd levels. The structure and expression of the long md-MT genes of A.b. were characterized. Although both genes are intronless, they are still transcribed, being significantly upregulated upon Cd exposure. Overall, our results contribute new knowledge regarding the metal handling of Alinda biplicata in particular, and the potential role of md-MTs in Cd detoxification of terrestrial snails, in general.

Hemocyanin genes as indicators of habitat shifts in Panpulmonata?

Hemocyanin is the primary respiratory protein for the majority of the Mollusca and therefore directly interfaces with the physiological requirements of each species and the environments to which they are adapted. Hemocyanin is therefore likely to have been evolutionarily imprinted by significant habitat shifts. In the gastropod clade Panpulmonata (>30,000 species) major realm transitions have occurred multiple times independently and may have contributed to the diversification of this group. Yet, little is known about the adaptive changes linked to these habitat shifts. In order to gain deeper insight into the evolution of panpulmonate hemocyanins and to infer possible impacts associated with those scenarios, we have assembled and analysed hemocyanin isoforms from 4 panpulmonate species: (i) Helix pomatia, (ii) Cantareus aspersus (both Helicidae, Stylommatophora), (iii) Arion vulgaris (Arionidae, Stylommatophora) and (iv) Lymnaea stagnalis (Lymnaeidae, Hygrophila). Additionally, we describe a new hemocyanin isoform within the genome of the euopisthobranch Aplysia californica. Using these newly acquired hemocyanin data, we performed a phylogenetic analysis that reveals independent duplication events of hemocyanin within lineages that correlate with significant habitat shifts.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

A de novo evolved domain improves the cadmium detoxification capacity of limpet metallothioneins.

Metallothioneins (MTs) constitute an important family of metal binding proteins. Mollusk MTs, in particular, have been used as model systems to better understand the evolution of their metal binding features and functional adaptation. In the present study two recombinantly produced MTs, LgiMT1 and LgiMT2, and their de novo evolved γ domain, of the marine limpet Lottia gigantea, were analyzed by electronic spectroscopy and mass spectrometry. Both MT proteins, as well as their γ domains, exhibit a strong binding specificity for Cd(II), but not for Zn(II) or Cu(I). The LgiMTs' γ domain renders an MII4(SCys)10 cluster with an increased Cd stoichiometry (binding 4 instead of 3 Cd2+ ions), representing a novel structural element in the world of MTs, probably featuring an adamantane 3D structure. This cluster significantly improves the Cd(II)-binding performance of the full length proteins and thus contributes to the particularly high Cd coping capacity observed in free-living limpets.

Sublethal cadmium exposure in the freshwater snail Lymnaea stagnalis meets a deficient, poorly responsive metallothionein system while evoking oxidative and cellular stress.

The Great Pond snail Lymnaea stagnalis (Gastropoda, Hygrophila) is a wide-spread freshwater gastropod, being considered as a model organism for research in many fields of biology, including ecotoxicology. The aim of the present study was to explore the Cd sensitivity of L. stagnalis through the measurement of a biomarker battery for oxidative, toxic and cellular stress. The interpretation of biomarker parameters occurred against the background of a truncated metallothionein protein with a limited Cd-binding capacity. Individuals of L. stagnalis were exposed through 14 days to uncontaminated water (controls) or to low (30 µg · L-1) or high (50 µg · L-1) Cd concentrations. The digestive gland of control and low-Cd exposed snails was processed for transcriptional analysis of the Metallothionein (MT) gene expression, and for determination of biomarkers for oxidative stress, toxicity and cellular stress. Digestive gland supernatants of high-Cd exposed snails were subjected to chromatography and subsequent analysis by spectrophotometry. It was shown that the MT system of L. stagnalis is functionally deficient, with a poor Cd responsiveness at both, the transcriptional and the protein expression levels. Instead, L. stagnalis appears to rely on alternative detoxification mechanisms such as Cd binding by phytochelatins and metal inactivation by compartmentalization within the lysosomal system. In spite of this, however, traces of Cd apparently leak out of the pre-determined detoxification pathways, leading to adverse effects, which is clearly indicated by biomarkers of oxidative and cellular stress.

Patterns of urban mercury contamination detected by bioindication with terrestrial isopods.

Mercury (Hg) is a trace element with high toxicological impact on potential receptors, including human beings. Global Hg emissions are predicted to increase significantly during the next 40 years. After emission, the metal is transported by air currents and precipitations, leading to increasing depositions even in areas far from emission sources. In the terrestrial environment, Hg is subjected to redistribution and transformation into different inorganic and metal-organic species that are taken up by vegetation and soil organisms. In the present study, the woodlouse (Porcellio scaber) was used as a biological indicator of total Hg pollution in the city of Dornbirn (province of Vorarlberg), Austria. Woodlice were collected from 30 sampling points scattered over the city area, 25 of them situated within a rectangular transect crossing the city area from west-northwest to east-southeast, starting near the Rheintal motorway and ending at the slopes of the Bregenzer Wald hills. In addition to woodlice, soil substrate samples were collected at nine of the selected sampling points. Total Hg concentrations were measured in isopod tissues and soil substrate samples by means of an Hg analyzer. Total Hg concentrations in isopod tissues were significantly correlated with Hg soil contents (P < 0.05). Moreover, a gradient of increasing Hg concentrations was observed in isopod samples along the transect across Dornbirn, with the lowest concentrations detected in woodlouse samples near the Rheintal motorway and the highest levels toward the ascending slopes of the Bregenzer Wald hills. This gradient of increasing Hg concentrations across the city matches a concomitant increase in wet precipitations along the same direction, indicating that deposition by wet precipitation may be an important source for Hg contamination in the city of Dornbirn. Overall, the degree of Hg contamination across the study area can be regarded as rather low, i.e., comparable with concentrations observed in other, unpolluted terrestrial habitats. It is concluded that bioindication by total Hg analysis in woodlice can be applied to distinguish between different levels and sources of contamination in urban areas.

Metal-dependent glycosylation in recombinant metallothioneins.

We show for the first time glycosylation of recombinant metallothioneins (MTs) produced in E. coli. Interestingly, our results show that the glycosylation level of the recombinant MTs is inversely proportional to the degree of protein structuration, and reflects their different metal preferences.

The evolution of hemocyanin genes in Tectipleura: a multitude of conserved introns in highly diverse gastropods.

BACKGROUND: Hemocyanin is the oxygen transporter of most molluscs. Since the oxygen affinity of hemocyanin is strongly temperature-dependent, this essential protein needs to be well-adapted to the environment. In Tectipleura, a very diverse group of gastropods with > 27,000 species living in all kinds of habitats, several hemocyanin genes have already been analyzed. Multiple independent duplications of this gene have been identified and may represent potential adaptations to different environments and lifestyles. The aim of this study is to further explore the evolution of these genes by analyzing their exon-intron architectures. RESULTS: We have reconstructed the gene architectures of ten hemocyanin genes from four Tectipleura species: Aplysia californica, Lymnaea stagnalis, Cornu aspersum and Helix pomatia. Their hemocyanin genes each contain 53 introns, significantly more than in the hemocyanin genes of Cephalopoda (9-11), Vetigastropoda (15) and Caenogastropoda (28-33). The gene structures of Tectipleura hemocyanins are identical in terms of intron number and location, with the exception of one out of two hemocyanin genes of L. stagnalis that comprises one additional intron. We found that gene structures that differ between molluscan lineages most probably evolved more recently through independent intron gains. CONCLUSIONS: The strict conservation of the large number of introns in Tectipleura hemocyanin genes over 200 million years suggests the influence of a selective pressure on this gene structure. While we could not identify conserved sequence motifs within these introns, it may be simply the great number of introns that offers increased possibilities of gene regulation relative to hemocyanin genes with less introns and thus may have facilitated habitat shifts and speciation events. This hypothesis is supported by the relatively high number of introns within the hemocyanin genes of Pomacea canaliculata that has evolved independently of the Tectipleura. Pomacea canaliculata belongs to the Caenogastropoda, the sister group of Heterobranchia (that encompass Tectipleura) which is also very diverse and comprises species living in different habitats. Our findings provide a hint to some of the molecular mechanisms that may have supported the spectacular radiation of one of Metazoa's most species rich groups.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

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.

Through evolution, marine snails have adapted several times independently to terrestrial life. A prime example for such transitions is the adaptation to terrestrial conditions in members of the gastropod clade of Littorinoidea (Caenogastropoda). Some species of this lineage like the periwinkle (Littorina littorea), live in intertidal habitats, where they are intermittently exposed to semi-terrestrial conditions. Pomatias elegans is a close relative of Littorina littorea that has successfully colonized terrestrial habitats. Evolutionary transitions from marine to terrestrial conditions have often been fostered in marine ancestors by acquisition of physiological pre-adaptations to terrestrial life. Such pre-adaptations are based, among others, on the optimization of a wide repertoire of stress resistance mechanisms, such as the expression of metal inactivating metallothioneins (MTs). The objective of our study was to explore the Cd handling strategy in the terrestrial snail Pomatias elegans in comparison to that observed previously in Littorina littorea. After Cd exposure, the metal is accumulated mainly in the midgut gland of Pomatias elegans, in a similar way as in its marine relative. Upon Cd exposure, Pomatias elegans expresses Cd-specific MTs, as also described from Littorina littorea. In contrast to the latter species, however, the detoxification of Cd in Pomatias elegans is mediated by two different MT isoforms, one two-domain and one three-domain MT. Although the MT proteins of both species are homologous and clearly originate from one common ancestor, the three-domain MT isoform of Pomatias elegans has evolved independently from the three-domain MT of its marine counterpart, probably by addition of a third domain to the pre-existing two-domain MT. Obviously, the occurrence of homologous MT structures in both species is a hereditary character, whereas the differentiation into two distinct MT isoforms with different upregulation capacities in Pomatias elegans is an adaptive feature that probably emerged upon transition to life on land.

Metal binding functions of metallothioneins in the slug Arion vulgaris differ from metal-specific isoforms of terrestrial snails.

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.

The physiological role and toxicological significance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails.

Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallothionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a major role in Cd2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first characterized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quantified in unexposed and Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This contrasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3'-UTR of the Cd/CuMT mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails.

Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails.

The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.