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.

Earthworm genomes, genes and proteins: the (re)discovery of Darwin's worms.

Small incremental biological change, winnowed by natural selection over geological time scales to produce large consequences, was Darwin's singular insight that revolutionized the life sciences. His publications after 1859, including the 'earthworm book', were all written to amplify and support the evolutionary theory presented in the Origin. Darwin was unable to provide a physical basis for the inheritance of favoured traits because of the absence of genetic knowledge that much later led to the 'modern synthesis'. Mistaken though he was in advocating systemic 'gemmules' as agents of inheritance, Darwin was perceptive in seeking to underpin his core vision with concrete factors that both determine the nature of a trait in one generation and convey it to subsequent generations. This brief review evaluates the molecular genetic literature on earthworms published during the last decade, and casts light on the specific aspects of earthworm evolutionary biology that more or less engaged Darwin: (i) biogeography, (ii) species diversity, (iii) local adaptations and (iv) sensitivity. We predict that the current understanding will deepen with the announcement of a draft earthworm genome in Darwin's bicentenary year, 2009. Subsequently, the earthworm may be elevated from the status of a soil sentinel to that elusive entity, an ecologically relevant genetic model organism.

Identification of heavy metal induced changes in the expression patterns of the translationally controlled tumour protein (TCTP) in the earthworm Lumbricus rubellus1.

Heavy metal contaminated soils are assessed for specific human health and ecological risk by governmental regulatory agencies utilizing the abundant soil invertebrate, the earthworm, in a biomonitoring process. Fingerprinting the molecular genetic responses resulting from heavy metal exposure facilitates the identification of biomarkers for assessing the impact of such pollution on individual organisms. This paper reports the identification of a novel translationally controlled tumour protein (TCTP) in the earthworm Lumbricus rubellus. In addition to the standard molecular biological technique of differential Southern blotting, a fully quantitative approach (fluorescent microvolume PCR) was performed to assess the specific expression profiles of TCTP in earthworms exposed to different heavy metal regimes. After normalizing with actin as an invariant control, the results showed that TCTP was upregulated by at least a factor of 4 in the population originating from a Pb/Zn/Cd polluted mine, compared to an unpolluted control population. An even more pronounced increase was identified in earthworms native to a Cu polluted mine, where TCTP increased 335-fold. TCTP copies in earthworms exposed to artificial soil with a single stressor (Cd) were 14 times higher than in the appropriate control earthworms (maintained on artificial soil without Cd). The data presented are novel in two ways: first, they provide evidence for an upregulation that is induced by heavy metals (especially copper); second, they show that TCTP can also be under transcriptional control, therefore upregulation is not limited to translational modifications as TCTP's nomenclature suggests.

Characterisation and quantification of earthworm cyclophilins: identification of invariant and heavy metal responsive isoforms.

We report the identification of two earthworm cyclophilin genes, which resemble the cytosolic cyclophilin-A and the signal sequence containing cyclophilin-B. Using fully quantitative PCR we were able to assess the transcript regulation of both cyclophilin isoforms, as well as a further independent control gene (actin), during exposure to heavy metals. Whilst the expression of cyclophilin-B and actin remained exceptionally constant, cyclophilin-A was up-regulated 38-fold. This intriguing observation has profound implications regarding cyclophilin's use as an invariant control and highlights the fact that it is essential to treat cyclophilin isoforms as separate entities, rather than one functional unit.

C. elegans metallothioneins: new insights into the phenotypic effects of cadmium toxicosis.

Metallothioneins are considered to be the primary player in the detoxification of and protection from cadmium, a teratogen, mutagen and potentially lethal heavy metal. The nematode Caenorhabditis elegans has only two metallothioneins, mtl-i and mtIl-2, thus making it an ideal organism to investigate the phenotypic effects of cadmium toxicosis. The functional importance of metallothioneins in cadmium trafficking was highlighted through the generation of viable green fluorescent protein (GFP) expressing transgenes, a metallothionein null allele, as well as RNAi mediated metallothionein knock-downs. A highly sensitive dose and temporal transcriptional response to cadmium, but not copper or zinc, was shown to be equally prevalent in both isoforms. No measurable compensatory up-regulation of mtl-l could be observed in the null allele of mtl-2, suggesting that both isoforms are independent and not synergistic in their mode of action. Exposure to cadmium affected all demographic indices measured, manifested by a reduction in body size, generation time, brood size and lifespan. These effects were magnified in the knock-out or wild-type subjected to a knock down by RNAi, however, only in the presence of cadmium. This substantiates the notion that metallothioneins play a pivotal role in the protection from cadmium toxicosis. Finally, an earthworm metallothionein-GFP construct could be activated in C. elegans upon exposure to cadmium, the results providing further evidence that the transcriptional control of metallothioneins is fundamentally divergent in lower invertebrates and not mediated via MTF-1 as in more complex organisms.

Molecular cloning and characterization of Cpn60 in the free-living nematode Plectus acuminatus.

Heat shock proteins (Hsps) have provoked interest not only because of their involvement in human diseases but also for their potential as biomarkers of environmental pollution. Whereas the former interest is covered by numerous reports, the latter is an exciting new field of research. We report the isolation of the full-length cpn60 messenger ribonucleic acid (mRNA) and partial genomic deoxyribonucleic acid from the free-living, environmental sentinel nematode Plectus acuminatus, a species used in classical ecotoxicity tests. Although the primary sequence displays high identity scores to other nematodes and human Cpn60 (75% and 70%, respectively), the intron-exon structure differs markedly. Furthermore, although mRNA levels remained constant after exposure to ZnCl2 (0-330 microM) under laboratory conditions, protein levels increased significantly in a dose-dependent manner. In conclusion, this first account of molecular genetic similarities and differences of Cpn60 in a neglected nematode taxon provides a valuable insight into its potential uses in gene-based ecotoxicological risk assessment exercises.

The identification, cloning and characterization of earthworm metallothionein.

Combining standard gel chromatographic techniques and novel molecular methodologies (Directed Differential Display and quantitative PCR), it has been possible to isolate and sequence two isoforms of the first true earthworm metallothionein. Both proteins are characteristically high in cysteine residues and possess no significant aromatic residues. Metal responsiveness was confirmed by determining metallothionein specific expression profiles in earthworms exposed to soils of differing heavy metal concentrations. Analysis of the derived amino acid sequence of isoform 2 identified two putative N-glycosylation signal sequences, suggesting that the two isoforms may have different subcellular distributions and functions. Possible implications for intracellular metal trafficking are discussed.

Control genes in quantitative molecular biological techniques: the variability of invariance.

The measurement of transcript levels constitutes the foundation of today's molecular genetics. Independent of the techniques used, quantifications are generally normalised using invariant control genes to account for sample handling, loading and experimental variation. All of the widely used control genes are evaluated, dissecting different methodological approaches and issues regarding the experimental context (e.g. development and tissue type). Furthermore, the major sources of error are highlighted when applying these techniques. Finally, different approaches undertaken to assess the invariance of control genes are critically analysed to generate a procedure that will help to discern the best control for novel experiments.

Cu-Cd interactions in earthworms maintained in laboratory microcosms: the examination of a putative copper paradox.

Earthworms (Lumbricus rubellus) from Ecton (predominantly Cu-contaminated), Shipham (Cd-contaminated) and Dinas Powys (uncontaminated, reference) were maintained in the laboratory on soil from the sampling sites. Two principle exposure protocols were used: (1) a 4-week 'no pre-exposure experiment', where batches of earthworms were maintained on soils from each habitat and (2) a 'pre-exposure experiment' where uncontaminated control worms were maintained on Shipham soil for 4 weeks (the pre-exposure period), and then transferred to Ecton soil for a further 4 weeks, and vice versa. In summary, there were two primary findings. Firstly, exposure of worms to Cd-rich soil (Shipham) before exposure to Cu-rich soil (Ecton) increased the Cu burden and secondly, biochemical analysis of metal binding proteins in analogous earthworm populations indicated that Cu and Cd, but not Zn, are bound to a metallothionein homologue. These observations are consistent with the hypothesis that Cu does not induce thionein synthesis efficiently in earthworm tissues, but may bind avidly to Cd-induced thionein by substituting for Cd in the molecule because Cu-thionein is a more stable complex.

Biosynthesis of luminescent quantum dots in an earthworm.

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworm's metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape.

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in EST(Pb) libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Comparative transcriptomic responses to chronic cadmium, fluoranthene, and atrazine exposure in Lumbricus rubellus.

Transcriptional responses of a soil-dwelling organism (the earthworm Lumbricus rubellus) to three chemicals, cadmium (Cd), fluoranthene (FA), and atrazine (AZ), were measured following chronic exposure, with the aim of identifying the nature of any shared transcriptional response. Principal component analysis indicated full or partial separation of control and exposed samples for each compound but not for the composite set of all control and exposed samples. Partial least-squares discriminant analysis allowed separation of the control and exposed samples for each chemical and also for the composite data set, suggesting a common transcriptional response to exposure. Genes identified as changing in expression level (by the least stringent test for significance) following exposure to two chemicals indicated a substantial number of common genes (> 127). The three compound overlapping gene set, however, comprised only 25 genes. We suggest that the low commonality in transcriptional response may be linked to the chronic concentrations (approximately 10% EC50) and chronic duration (28 days) used. Annotations of the three compound overlapping gene set indicated that genes from pathways most often associated with responses to environmental stress, such as heat shock, phase I and II metabolism, antioxidant defense, and cation balance, were not represented. The strongest annotation signature was for genes important in mitochondrial function and energy metabolism.

Ligand arsenic complexation and immunoperoxidase detection of metallothionein in the earthworm Lumbricus rubellus inhabiting arsenic-rich soil.

Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is sequestered by the metalloprotein in certain target cells and tissues.

Metal ion trafficking in earthworms. Identification of a cadmium-specific metallothionein.

Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situ using polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.

Earthworm pre-procarboxypeptidase: a copper responsive enzyme.

This paper describes the identification and molecular cloning of the earthworm metalloenzyme preprocarboxypeptidase. It was possible to approximate the putative pre-pro cleavage sites, which after removal activates a 35.7 kDa mature peptide with a length of 317 amino acids. The primary, secondary and predicted tertiary structure of the mature chain is highly homologous to other carboxypeptidases from diverse phylogenetic origin. Using a fully quantitative PCR, we were able to assess relative expression of this gene in earthworms exposed to different heavy metal contaminated substrates. In summary, cadmium on its own or in combination with lead and zinc, did not trigger transcriptional up-regulation of carboxypeptidase. In contrast, copper exposed earthworms were assessed to have a 4-fold increase in carboxypeptidase transcript numbers. A hypothetical model is presented to explain how the exposure to heavy metals may influence transcriptional control and/or function of this enzyme. Finally, the significance of these observations in terms of risk assessment and biomonitoring of contaminated soils is discussed.

Differential metallothionein expression in earthworm (Lumbricus rubellus) tissues.

This article describes the immunoperoxidase localization of metallothionein (MT) in the major organs and tissues of the earthworm Lumbricus rubellus sampled from a mine soil heavily polluted with Pb, Zn, and Cd. The polyclonal antiserum used was raised against the MT isoform (wMT2), the molecular characteristics and focal subcellular distribution of which indicate a primary role for it in the sequestration of certain nonessential metals, such as Cd. Intense MT immunostaining was detectable in chloragogenous tissue throughout the body: around the intestine, in the typhlosolar infolding, around blood vessels anterior and posterior to the crop/gizzard, and around the calciferous gland. Electron probe X-ray microanalysis of neutral red-labeled vesicular structures in the chloragogenous tissue indicated that this acidic compartment, probably lysosomal, yielded the strong Cd and S signals associated with Cd-MT. MT expression was also detected in the apical cytoplasm of intestinal epithelial cells; in coelomocytes contiguous with chloragocytes attached to the gut; within the narrow tubular region of nephridia, in the secretory epithelia of the calciferous gland, but not anywhere in the body wall. We concluded that (a) the main route of Cd uptake is probably via absorptive alimentary surfaces, and not across the external epidermal layer; (b) nephridia are involved with Cd excretion and/or are a major target of Cd-induced pathological damage; (c) tentatively, a combination of immunohistochemistry and proton-induced X-ray emission analysis indicated that the calciferous gland is probably not a major "heavy metal" excretory route.

Developing a new method for soil pollution monitoring using molecular genetic biomarkers.

Physiological responses to environmental stressors may induce changes in gene expression as part of an organism's homeostatic mechanisms. Thus molecular genetic biomarkers have the potential to be used for monitoring sublethal chemical exposure in ecosystems. This paper describes a methodological assessment of the suitability of a protocol to monitor selected biomarkers. The TaqMan real-time quantitative polymerase chain reaction was used to measure gene transcription in earthworms (Lumbricus rubellus) maintained on control or cadmium- or copper-spiked soil. Changes in the expression of two target genes, that encoding metallothionein isoform 2 (MT-2) and that encoding the mitochondrial large ribosomal subunit (MLRS), were quantified against the internal control gene beta-actin. The protocol used produced reliable and reproducible results. Transcript levels displayed qualitative and quantitative differences in the responses to the two metal ions. MLRS gene levels were unaffected by exposure to cadmium but displayed a response to high levels of copper. Conversely, cadmium greatly induced MT-2 gene expression, but copper only altered transcription of this gene at high exposure concentrations. This study demonstrates that it is now technically feasible to use gene expression as an index of pollution exposure in environmentally relevant organisms.