Metallothionein gene activation in the earthworm (Lumbricus rubellus).

In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs.

Coping with cadmium exposure in various ways: the two helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor-2, but differ in promoter organization and transcription of their Cd-metallothionein genes.

Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: II. AFLP analysis reflects human-aided local adaptation of a global pest species.

Originally resident in southeastern Europe, the codling moth (Cydia pomonella L.) (Tortricidae) has achieved a nearly global distribution, being one of the most successful pest insect species known today. As shown in our accompanying study, mitochondrial genetic markers suggest a Pleistocenic splitting of Cydia pomonella into two refugial clades which came into secondary contact after de-glaciation. The actual distribution pattern shows, however, that Central European codling moths have experienced a geographic splitting into many strains and locally adapted populations, which is not reflected by their mitochondrial haplotype distribution. We therefore have applied, in addition to mitochondrial markers, an approach with a higher resolution potential at the population level, based on the analysis of amplification fragment length polymorphisms (AFLPs). As shown in the present study, AFLP markers elucidate the genetic structure of codling moth strains and populations from different Central European apple orchard sites. While individual genetic diversity within codling moth strains and populations was small, a high degree of genetic differentiation was observed between the analyzed strains and populations, even at a small geographic scale. One of the main factors contributing to local differentiation may be limited gene flow among adjacent codling moth populations. In addition, microclimatic, ecological, and geographic constraints also may favour the splitting of Cydia pomonella into many local populations. Lastly, codling moths in Central European fruit orchards may experience considerable selective pressure due to pest control activities. As a consequence of all these selective forces, today in Central Europe we see a patchy distribution of many locally adapted codling moth populations, each of them having its own genetic fingerprint. Because of the complete absence of any correlation between insecticide resistance and geographic or genetic distances among populations, AFLP markers do not have a prognostic value for predicting an outbreak of pesticide resistance in the field. By combining mitochondrial genetic data and AFLP analysis it was possible, however, to track the recent evolutionary history of Cydia pomonella on three different time scales: from population splitting in Pleistocene, to interbreeding of mitochondrial haplotypes in Holocene, to human-aided complete intermixing and splitting into many locally adapted populations in very recent times. The case of Cydia pomonella is reminiscent of examples of sympatric speciation and another example of a human-induced globally successful pest species.

Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: I. Ancient clade splitting revealed by mitochondrial haplotype markers.

The codling moth (Cydia pomonella L., Tortricidae, Lepidoptera) is an important pest of pome fruit with global distribution. It has adapted successfully to different habitats by forming various ecotypes and populations, often termed strains, which differ among each other in several morphological, developmental, and physiological features. Many strains of Cydia pomonella have developed resistance against a broad range of chemically different pesticides. Obviously, pesticide-resistant strains must have a genetic basis inherent to the gene pool of codling moth populations, and this deserves our particular attention. The primary intention of the present study was to contribute novel information regarding the evolutionary phylogeny and phylogeography of codling moth populations in Central Europe. In addition, we aimed at testing the hypothesis that differential biological traits and response patterns towards pesticides in codling moth populations may be reflected at a mitochondrial DNA level. In particular, we wanted to test if pesticide resistance in codling moths is associated repeatedly and independently with more than one mitochondrial haplotype. To this end, we analyzed mitochondrial DNA and constructed phylogenetic trees based on three mitochondrial genes: cytochrome oxidase I (COI), the A+T-rich region of the control region (CR), and the nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5). The results indicate that Central European populations of Cydia pomonella are clearly divided in two ancient clades. As shown by means of a molecular clock approach, the splitting of the two clades can be dated to a time period between the lower and middle Pleistocene, about 1.29-0.20 million years ago. It is assumed that the cyclic changes of warm and cold periods during Pleistocene may have lead to the geographic separation of codling moth populations due to glaciation, giving rise to the formation of the two separate refugial clades, as already shown for many other European animal species. Due to their inclination towards developing novel detoxification gene variants, codling moth individuals from both clades independently and multifariously may have developed pesticide resistance, and this process may be ongoing. During their more recent evolutionary history, natural events such as the gradual disappearance of climate-specific geographic barriers, as well as human-aided dispersal in recent historic times, may have allowed codling moth haplotypes from the original clades to interbreed and completely merge again, creating a globally successful insect species with a gene pool capable of responding to novel selective challenges by rapid adaptation.

Environmental impacts of urban snow management--the alpine case study of Innsbruck.

In regions with colder climate, snow at roads can accumulate significant amounts of pollutant chemicals. In northern countries various efforts have been made to face this problem, but for the alpine region little is known about the pollution of urban snow. The present case study was carried out in the city of Innsbruck (Austria). It aimed at measuring pollution of roadside snow and estimating the impact of snow management practises on environmental quality. Concentrations of copper, zinc, lead, cadmium, suspended solids and chloride were determined during a series of sampling events. Various locations with low and high traffic densities and in different distances from a highway have been investigated. The concentrations of copper were generally higher at sites with high traffic density compared to locations with low traffic impact. In contrast to this, the concentrations of zinc and lead remained almost unvaried irrespective of traffic density at the different sampling sites. For cadmium, the picture was more diverse, showing moderately elevated concentrations of this metal also at the urban reference site not polluted by traffic. This indicates that there may be also other important sources for cadmium besides traffic. Suspended solids accumulated in the roadside snow, the highest concentrations were found at the sites with high traffic density. The chloride concentrations were considerable in the snow, especially at the highway. Based on the results of the present measurement campaign, the environmental impact of snow disposal in rivers was also estimated. A negative impact on rivers from snow disposal seems likely to occur, although the discharged loads could only be calculated with substantial uncertainty, considering the high variability of the measured pollutant concentrations. For a more accurate evaluation of this management practise on rivers, further investigations would be necessary.

Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia.

The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.

Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers.

During the last few years the subject of metallothioneins (MTs) in terrestrial invertebrates has gained increasing attention. One reason for this may be that terrestrial invertebrates provide new insights into the biological diversity of MTs, with the potential of discovering alternative models of structural and functional relationships. Four groups of terrestrial invertebrates have been studied in detail, namely nematodes, insects, snails and earthworms, with the present article focusing on MTs from the latter two groups. Snails are interesting because they possess distinct MT isoforms involved in different metal-specific tasks. In the Roman snail (Helix pomatia), for example, one isoform is predominantly expressed in the midgut gland, accounting for the accumulation, binding and detoxification of cadmium. The second isoform, which is present in the snail's mantle, is substantially different regarding its primary structure. Furthermore, it binds nearly exclusively copper, and thus is probably involved in the homeostatic regulation of essential trace elements. Earthworm MTs merit our attention because of another peculiarity: they seem to be much more unstable than snail MTs, particularly under conventional conditions of preparation. The cDNA of the brandling worm (Eisenia foetida), for instance, codes for a putative MT, which is about twice the size of the actual protein. The isolated MT peptide binds four Cd2+ ions and represents a one-domain MT entity that is stable and functional in vitro. This strongly suggests that earthworm MTs are either posttranslationally modified, or subjected to enzymatic cleavage during preparation. Both snail and earthworm MTs are inducible by metal exposure, especially by cadmium, thus supporting the idea of using them as potential biomarkers for environmental metal pollution. Whilst snail MTs have already been tested in this respect with some success, the use of earthworm MTs as biomarkers still remains to be evaluated, especially in the light of the unknown significance of their posttranslational instability.

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity.

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.

Isolation and characterization of a self-sufficient one-domain protein. (Cd)-metallothionein from Eisenia foetida.

Earthworms have been shown to accumulate trace elements in general, and particularly high amounts of metal ions such as cadmium, copper and zinc. The earthworm's response to metal contamination has been linked to the induction and expression of metallothionein (MT) proteins, a detoxification strategy analogous to that found in other biological systems. The present study focuses on an inducible Cd-MT isolated from the compost-dwelling brandling worm Eisenia foetida (Savigny). A full characterization of the protein (including protein induction, MT cDNA, amino-acid sequence and metal stoichiometry) revealed a new dimension of knowledge to the molecular genetic information available to date. Whereas the elucidated cDNA codes for a putative protein which possesses 80 amino-acid residues, the characterized protein bears only 41 amino acids. The isolated product has evidently attained its size and shape by cleavage near the N-terminal site and at the linker region between the two putative metal-binding domains of the translated product, yielding a small MT moiety which contains 12 Cys residues (including one triple Cys-motif) binding four cadmium ions. It can be shown that the isolated MT molecule represents a self-sufficient one-domain MT which is stable in vitro. The isolation of the single-domain MT peptide raises the question about the method of formation and significance in vivo of such small MT moieties from tissues of E. foetida and possibly other terrestrial invertebrates. In this respect, two hypotheses are discussed: firstly, the possibility of formation of small MT peptides due to enzymatic cleavage of the intact protein during the process of preparation and isolation; and secondly, the possibility of deliberate post-translational processing of the translated gene product to yield functional one-domain MT moieties.

Biomarkers in terrestrial invertebrates for ecotoxicological soil risk assessment.

This review has served to present the most recent information on a selected series of biomarker studies undertaken on soil invertebrates during two extensive European-funded scientific consortia, BIOPRINT and BIOPRINT-II. The goals were to develop and validate methods for the analysis of markers of stress in a range of soil-dwelling organisms. We have discussed the potential and limitations of the following invertebrate biomarkers for soil risk assessment purposes: heat shock proteins, histological and ultrastructural markers, metallothioneins and metal-binding proteins, esterases, lysosomal integrity, and the novel biomarker histidine. The hsp response in soil invertebrates is especially suitable to indicate the effects of exposure to comparatively low concentrations for a range of toxicants and can be regarded as a biomarker of general stress. The application of MTs and other metal-binding proteins as biomarkers for exposure in soil invertebrates has been well described, and new methods are being developed for analyzing MT induction both at the protein and molecular level, and reliable and reproducible methods are now available. (Cd)-MT is well characterized for the springtails and its MT concentration is a useful biomarker for exposure as well as for effect. For snails, (Cd)-MT can accumulate in the midgut gland over extended periods of time and therefore its concentration is a biomarker not only for recent intoxication but also for events of cadmium exposure that snails may have experienced a long time before the measurement took place. Cellular and histological alterations can be regarded as reflecting the "health" state of a cell, which may be a measure for the presence of toxicants. Histopathological work on terrestrial invertebrates, however, is still scarce. Isozymes have been poorly studied in soil invertebrates despite their promising role as potential biomarkers in aquatic organisms. Among the large diversity of isozymes, the most well studied are esterases that are frequently used a biomarkers of exposure to various classes of pesticides. Many other isozymes offer potentials for biomarker research, such as glucosephosphate isomerase and phosphoglucomutase, both enzymes necessary for the glycolytic pathway. The lysosomal system has been identified as a particular target for the toxic effects of xenobiotics, although it has yet a limited application in soil invertebrates. This marker is nonspecific, responding equally sensitively to organic or inorganic contamination; however, if used in combination with an earthworm immnunocompetence assay such as total immunoactivity of the coelomocytes, then it is possible to be more specific as to the likely nature of contamination. Free histidine was positively correlated with increasing copper exposure and total copper burden in earthworms from a semifield study. Histidine may thus act as a biomarker of exposure. The transient responses and confounding factors of biomarkers obscure a proper interpretation of biomarker responses under field conditions. These factors are still very poorly understood and require more study. For risk assessment purposes it is recommended that the aforementioned biomarkers may show promise when included in a suite of biomarkers among different soil invertebrate species. It is recommended that a risk assessment protocol draw upon ranking of biomarker responses on a defined scale. It is also hoped that the problems outlined in this review will aid the direction of future research on soil invertebrate biomarkers.

Mitochondrial DNA reveals cryptic oligochaete species differing in cadmium resistance.

Species of the family Tubificidae represent a major faunal element in benthic freshwater communities throughout the world. Some of them are considered particularly tolerant of the influence of toxicants such as cadmium. One of the most abundant species, "Tubifex tubifex," is frequently used as an indicator of environmental pollution, despite considerable taxonomic problems caused by phenotypic plasticity and genetic heterogeneity. Our study provides a phylogeny of "T. tubifex" based on a segment of the mitochondrial 16S rDNA and presents a rapid PCR-based method of genotype screening which was then applied in cadmium toxicity studies on natural populations. Phylogenetic analysis identified five major mitochondrial lineages, some of them separated by large genetic distances (up to 13%) but morphologically indistinguishable, thus highly suggestive of the existence of cryptic species. All lineages were present at different frequencies in the European river populations studied, with a tendency of the more resistant lineages to occur at higher frequencies in the more tolerant populations. In fact, lineage-specific toxicity experiments showed that individuals of different mitochondrial lineages consistently varied in cadmium resistance, suggesting that in benthic oligochaetes, evolution seems to proceed predominantly through natural selection acting on physiological, rather than morphological, characters. In consequence, toxicological studies involving "T. tubifex" as a monitoring or test organism should allow for the possibility of genetic inhomogeneity of this mudworm group by combining both toxicological and genetic methods.

Mitochondrial phylogeny of the genus Regulus and implications on the evolution of breeding behavior in sylvioid songbirds.

We tested four hypotheses about the relationships of the kinglets (genus Regulus)to seven closely related genera of the songbird superfamily Sylvioidea using mitochondrial DNA sequences. The kinglets were suggested to be closely related to the tits (Parus) or to the Old World Warblers (Phylloscopus) and were also suggested to constitute the, or at least one of the, most ancestral splits among the sylvioids. Our phylogenetic analysis grouped the kinglets as the sister group of a clade comprising Parus and Phylloscopus and including the genera Sylvia, Aegithalos, and Leptopoecile. Two of the taxa were placed more ancestral to the kinglets: Sitta and Certhia. We also identified the endemic kinglet species from the Canary Islands s the sister group of R. regulus. The superimposition of breeding behavior on the phylogeny suggests that hole nesting is ancestral and various other patterns of nest construction have evolved from it. The placement of Parus implies that hole nesting in the Paridae is likely to have originated secondarily. Further, Leptopoecile and Aegithalos, two genera for which a helper system of elder offspring in breeding was described, were resolved as a clade.

Primary structure of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod Helix pomatia L.

A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin.

Metallothionein research in terrestrial invertebrates: synopsis and perspectives.

While most of metallothionein research during the past years has been carried out on mammals or vertebrates, only relatively few studies have been directed towards invertebrates. Even fewer investigations have focussed on terrestrial invertebrates. The best studied metallothioneins and/or metallothionein genes among terrestrial invertebrates are those from an insect species (Drosophila melanogaster), a nematode (Caenorhabditis elegans) and some terrestrial gastropods (Helix pomatia, Arianta arbustorum). From these few examples it already appears that terrestrial invertebrate metallothioneins provide intriguing models to better understand the multiplicity of functions of these proteins and their evolution within the animal kingdom. Like in mammals, metallothioneins in terrestrial invertebrates seem to perform different functions simultaneously. This is exemplified by terrestrial gastropods, which are able to accumulate different metals in different tissues, in which metal-specific metallothionein isoforms or conformation forms are expressed, allowing these organisms to detoxify more efficiently nonessential trace elements such as cadmium, and at the same time to maintain the homeostasis of essential trace elements such as copper. A major proportion of metallothionein research in terrestrial invertebrates addresses the ecophysiological and ecotoxicological significance of these proteins with regard to the increasing risk due to chemical pollution. One promising aspect in this concern is the potential utilization of metallothioneins as biomarkers for risk assessment in terrestrial environments.

Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum.

1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.

Invertebrate organisms as biological indicators of heavy metal pollution.

Some species of invertebrate animals are known to be efficient accumulators of trace elements. Generally, metal accumulation by such organisms is based on efficient detoxification mechanisms, such as intracellular compartmentalization, or metal inactivation by binding to metallothioneins. Metal accumulators have often been used as accumulation indicators of environmental metal pollution. This means that, ideally, metal concentrations in the animal's body reflect quantitatively or semiquantitatively environmental pollution levels. In reality, however, many factors, such as the animal's weight and age, can disturb such quantitative relationships. These factors have, therefore, to be considered carefully before an invertebrate is utilized as accumulation indicator for metal pollution. Apart from accumulation, many invertebrates exposed to elevated metal concentrations respond to this stress by metal-induced synthesis of metallothioneins. Additionally, metallothionein in metal-loaded organisms can be present in different isoforms that are specifically synthesized in response to different metals. These facts make metallothionein a potential biomarker for metal stress in invertebrates. One possibility may be to assess parameters of metallothionein synthesis at the molecular or biochemical level. Moreover, metallothionein isoform patterns could provide information on different isoforms synthesized in response to different metals or chemicals. In any case, however, care must be taken to consider intrinsic physiological parameters, such as nutritional or developmental factors, which could also interfere with metallothionein synthesis.

Purification and primary structure of snail metallothionein. Similarity of the N-terminal sequence with histones H4 and H2A.

A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.

Terrestrial snails as quantitative indicators of environmental metal pollution.

Concentrations of cadmium, lead, copper and zinc were measured in individuals of Arianta arbustorum from different urban sampling sites. In comparison to snails from a reference site, the animals collected in the city showed higher concentrations of cadmium, lead, and copper, indicating elevated levels of metal pollution. The most pronounced difference in tissue concentrations between control animals and contaminated snails was observed for lead. Within the city, metal levels in snails differed significantly, even between adjacent populations. Arianta arbustorum is a suitable species for biomonitoring, because it is widespread, resident and easy to collect; it has a high capacity for metal accumulation and shows different concentrations depending on metal contamination of the sampling area. An interspecific comparison of metal concentrations in terrestrial gastropods was conducted to define background levels and classes of burden. Three pollution levels are distinguished on the basis of the snails' metal burden: no pollution (class 1: reference sites), moderate (class 2: traffic and other human activities in urban areas), and high pollution (class 3: mining and heavy industry).

Diversification of cadmium-binding proteins due to different levels of contamination in Arion lusitanicus.

Individuals of Arion lusitanicus were collected in the former mining area of Braubach (Federal Republic of Germany) which is highly polluted by various metals. The animals were transferred to the laboratory and fed contaminated litter from their original habitat. Groups of animals from a quarry near Heidelberg were reared in the laboratory and fed diets of different cadmium concentrations. Slugs fed uncontaminated food were used as controls. The concentration of cadmium in the midgut gland of cadmium-fed slugs increased in proportion with the metal concentration of the food. High amounts of zinc and copper were found in the midgut glands of slugs from Braubach. After centrifugation of homogenates, specific patterns of distribution between cytosolic components and pellets were observed for different metals. Cadmium and copper were predominantly bound to cytosolic components, whereas the main portion of zinc was associated with the pellet. In laboratory-fed slugs the increase of cadmium concentration in the food correlated with an increase of the metal content in the homogenate and in two components (supernatant, pellet) of the midgut gland. This correlation was most clearly expressed in the cytosolic components which contained 93-100% of total midgut gland cadmium. Total cadmium in control slugs was associated with components with a molecular weight of more than 15,000. In Braubach and in cadmium-loaded slugs, all the cadmium was bound to a protein with a molecular weight of 10,000, which also contained low amounts of zinc and copper. In highly contaminated individuals fed on the most concentrated cadmium diet, however, a spillover effect was observed, some cadmium being bound to an additional component with a molecular weight of more than 15,000.