European Union and American legislations require that contaminated soils are tested for the presence of toxic compounds to assess human health risks. Earthworms of the Lumbricus genus are ideal bio-monitors for heavy metal toxicity testing of soil. However, until recently, no information has been available concerning the molecular genetic responses of earthworms to heavy metals. Furthermore, although it is well established that earthworms can adapt to toxic heavy metal polluted environments, no information concerning this adaptation process has been forthcoming. Therefore, the underlying rationale of this longstanding project is to understand not only the molecular responses to acute heavy metal exposure but also to understand the molecular mechanisms invoked that allow earthworms to adapt to heavy metal contaminated environments.
Numerous novel techniques including several differential and subtractive approaches as well as fully quantitative PCR were applied to identify and evaluate metal responsive genes in the earthworm. Highest priority has been given to the identification and functional characterization of major metal binding proteins such as metallothionein (MT). Earthworm metallothioneins 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. These findings have now been substantiated by the production of recombinant MT (both isoforms), which has since allowed detailed studies on metal stoichiometry, metal stability as well as MT localization. The latter analyses were performed using polyclonal antibodies raised against the recombinant proteins and subsequently used in Light- and Electron-Microscopy. Overall, the results provide concrete evidence that the two isoforms have different sub-cellular distributions and functions, namely the homeostasis and detoxification of essential and non-essential metals, respectively.
The earthworm Expressed Sequence Tag (EST) project was initiated in the year 2000 and now contains over 17000 sequences from libraries synthesised from dissected late cocoon embryos, juveniles, adults, anterior sections, and adult earthworms exposed to atrazine, cadmium, copper, fluoranthene or lead. In total, over 7500 unique gene objects were identified and have allowed the creation of earthworm micro-arrays, a resource that has recently been exploited in numerous gene expression experiments. A draft genome has been assembled using Roche 454, Illumina SOLEXA Bacterial Artificial Chromosome (BAC) library sequencing.
We were able to demonstrate that the earthworm, when exposed to cadmium and tellurium salts, biosynthesizes high quality, luminescent quantum dots via their innate metal transport and storage mechanism. This pathway requires no solvents other than water, no specialist glassware, no heating and no extraneous reagents (such as inert gases). To exploit this biosynthesis route further, we are improving the biosynthetic efficiency of the nanomaterial production and in doing so aim to advance the field of the next generation of biosynthesized QDs, namely biotechnology which is less toxic, clean, biocompatible, versatile, tunable and cost effective.
The earthworm is capable of fully regenerating its brain within weeks of surgically removal. We are studying the molecular genetic basis and the mechanistic drivers of this biological phenomenon in fine detail. By identifying and characterizing key molecular transcripts and proteins involved in brain regeneration we are working towards the recreation of a 3D earthworm brain ex vivo.
Transcriptomics, qPCR, metabolomics, life cycle analysis, X-ray absorption fine structure (XAFS), X-ray fluorescence imaging (XFI), Coherent anti-Stokes Raman spectroscopy (CARS), microsurgery, Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS), etc.