BACKGROUND
ever since charles darwin's classic work  <cit> , earthworms  have been renowned as 'ecosystem engineers' in recognition of the direct and indirect effects they have on water, nutrient and carbon cycling in temperate and tropical soils  <cit> .
earthworms have therefore been widely adopted by international and national agencies for the diagnosis of soil ecosystem health, and for predicting the potential environmental impact of xenobiotics, such as industrial chemicals, pesticides and medicines, from anthropogenic sources  <cit> .
quantification of chemical toxicity to earthworms currently relies on measuring the effects of exposure on key life-history traits  in standardised laboratory bioassays conducted with certain test species .
these bioassays can produce sensitive estimates of population effects, but are not suited to elucidation of mechanisms of action, and thus may be difficult to generalise from.
for example, xenobiotic exposure may affect individual physiology, cocoon-lay rates, cocoon viability and juvenile growth rates in a specific manner, resulting in different outcomes for population growth rate and age structure.
complementing measurements of gross toxicity with molecular profiling and genomic studies can make plain the modes of action of specific xenobiotics and identify the generality of biological process affected as well as the molecular response pathways invoked  <cit> .
taking a specific example, it has been observed that l. rubellus is able to colonise highly metal-contaminated environments  <cit> .
detailed work identified that the primary molecules responsible for this metal tolerance were earthworm metallothioneins  <cit> .
though successful, the progress of these mechanistic studies has been handicapped by the lack of available sequence data for earthworms in public databases and the finding that very few  of the few available earthworm gene fragments could be identified by sequence similarity to previously sequenced genes.
to help bridge this gap in available sequence information, a previously described small-scale  expressed sequence tag  dataset for l. rubellus was generated  <cit> .
this has prompted the extended survey of the l. rubellus transcribed genome through extensive sampling of additional cdna libraries from earthworms at defined lifecycle stages , specific tissues  and following acute exposure to model chemicals representing three different contaminant classes: inorganic , organic , and agrochemical  that is reported here.
from this transcriptome resource, we defined ~ <dig> genes, and have fabricated a cdna microarray to investigate the transcriptome responses of l. rubellus through normal growth and following sub-lethal exposure to a series of sub-lethal concentrations of cd, fla, and atz.
from the derived transcript profiles we identify the key molecular responses and biochemical pathways associated with each life-stage treatment, and propose mechanisms of action for the three chemicals.
RESULTS
generation of ests from cdna libraries spanning the l. rubellus lifecycle and following exposure to target chemicals
we generated  <dig>  high-quality ests from nine different cdna libraries constructed from l. rubellus raised under defined conditions .
we maximised the gene discovery rate by screening out, from some libraries, a small number of highly-expressed mrnas .
the ests were clustered using the partigene suite of tools  <cit>  and  <dig>  clusters  were defined.
this number of putative genes is likely to be an overestimate because  alternative splice forms may be assembled into different clusters,  the partial nature of ests may result in non-overlap between sequences from the same mrna, and  our earthworms were drawn from an outbred source and allelic variation is likely.
within-dataset analyses suggested that the effects of these factors are minor, and that the true diversity in the dataset is ~ <dig>  different loci.
if, like other non-vertebrates, l. rubellus has between  <dig>  and  <dig>  protein-coding genes, the sequencing effort has generated sequence tags for ~40–50% of all genes in this key species.
<dig>  see additional file  <dig> for more detailed descriptions of the libraries used.
<dig>  a cluster is counted as derived from a library if it contains at least one est from that library.
thus the longest cluster  contains ests from five of the nine libraries and counts as a member of all five.
clusters  with one est member accounted for 53%  of all clusters .
there were  <dig> clusters containing ten or fewer ests , and only  <dig> clusters had over  <dig> ests.
of the clusters with more than one est, approximately 50% were library-specific.
such specificity may simply reflect the low density of sampling rather than real expression differences between libraries.
for each cluster, we derived a consensus cdna sequence, and from this derived a putative protein translation using prot4est  <cit> .
some clusters  yielded more than one consensus, possibly due to alternative splice forms or divergent alleles.
across all the l. rubellus libraries, the  <dig>  consensus sequences had a mean length of  <dig> bases  .
the mean length of l. rubellus putative proteins was  <dig> amino acids .
variation in mean protein length between libraries was directly related to the variation in consensus length.
the sequence and annotation data  are available in a relational database, lumbribase.
the database has been used as a workbench for our analyses , and is presented on the web through a php-scripted interface  <cit> .
functional annotation of the l. rubellus gene set
gene ontology  terms  <cit>  were assigned to l. rubellus predicted proteins using gotcha  <cit>  and the analysis summarised by assessing representation of higher-level  terms .
less than half  of the putative protein translations were assigned a go term at a confidence greater than  <dig>  this apparently high proportion of unannotated genes in l. rubellus can be explained by our application of the conservative gotcha algorithm and by the paucity of genomic information from other annelid species.
for those goslim "function" and "process" categories with a mean representation across all libraries of ≥  <dig>  we examined under- and over-representation in each library .
the developing cocoon library had the highest representation of nucleic acid binding, nucleotide binding, chaperone, and protein binding, likely derived from the presence of actively developing embryos in these animals, while the juvenile library had an over representation of lipid-binding annotations .
as expected, the control adult library was lacking in terms associated with heavy metal binding compared to libraries derived from cu- or cd-treated earthworms.
however, unexpectedly, the library from fla-exposed animals also had increased representation of this annotation.
the fla library also exhibited overrepresentation of carbohydrate-binding and fk506-sensitive peptidyl-prolyl isomerase annotations.
in the library from cd-treated animals both receptor signalling and globin classes were abundant compared to other libraries, perhaps reflecting the interference of cd with the functions of other, essential metals.
in the less-closely defined "process" categories, cd-treated and juvenile worm-derived libraries had more annotations of 'cell death' than other libraries .
many of these general observations were confirmed through the use of microarrays .
the only other "soil" animal for which extensive gene annotation data exist is the rhabditid nematode caenorhabditis elegans, though ecotoxicology analyses focussed on this species are limited  <cit> .
identification of possible orthologues between l. rubellus and c. elegans using blast similarity identified  <dig>  l. rubellus sequences with a blast hit in the c. elegans proteome  with an e-value of less than 1e- <dig>   <dig> of which were reciprocal top hits between the species .
of these,  <dig> had annotations relating to c. elegans rnai experiments  but these were largely uninformative as to biological function.
fabrication and validation of a l. rubellus cdna microarray
to profile life-cycle and xenobiotic transcriptome responses, a custom cdna microarray was fabricated using representative reporters from each cluster.
each spotted cdna originated from a clone processed for sequencing.
the hybridisation was performed using a nested reference design.
in detail, the reference sample  was derived from two 65–70-mer oligonucleotides designed against vector sequence between the amplification primer and the inserted cdna and co-amplified during insert preparation .
probes derived from mrna preparations were labelled with cy <dig>  this nested reference design permitted direct comparison between samples both within and between experiments.
four experiments were undertaken, comparing juvenile and adult stages and comparing transcriptional responses following exposure to cd, fla and atz.
all exposures were generated using a common experimental set-up designed to generate a set of biologically replicated samples for a series of exposure concentrations .
after hybridisation, array scanning, and pre-processing, the initial technical validation included visual inspection of images to identify gross abnormalities or background.
prior to normalisation the sensitivity of the array and relationship between rna concentration and fluorescent signal was assessed by calculating the signal intensity generated by reporters complementary to  <dig> "alien" rna spikes introduced at known concentrations, from  <dig> pmole to  <dig> nmole, prior to labelling .
following normalisation the distribution of data responses was examined and any samples showing abnormal distribution were discarded from the analysis .
array data were further validated by generating ma-plots, graphical representations of log ratio of the average normalised data from control samples compared to xenobiotic-exposed counterparts against the fluorescence intensity  <cit>  .
the control samples were of high quality by this measure.
for example, when compared to the cd exposure controls, >99% of the genes exhibiting an acceptable signal had < <dig> -fold variance in expression .
in contrast, for hybridisations involving mrna from animals exposed to  <dig> μg/kg cd, ~5% of genes showed an expression change > <dig> -fold with respect to the controls  .
further, a group of reporters showed very significant up-regulation in response to cd.
annotations for these reporters indicated that they included clusters encoding various metallothionein isoforms, a gene set previously shown to be highly cd responsive   <cit> .
gene expression changes through the l. rubellus lifecycle
transcript profiles were compared for  <dig> control groups of juveniles and biological replicate groups of adults .
these groups were derived from a common stock and selected to minimise the possible confounding effects of seasonal variation .
after initial pre-processing of the data, including normalisation relative to the median gene expression in juveniles and adults combined, both principal component analysis and hierarchical condition clustering  indicated a primary separation of samples based on developmental stage.
intriguingly, a further separation was observed within the adult samples.
this separated the samples into two groups that accorded with the time of year the adults were harvested from culture for experimental use in the laboratory experiment .
earthworms for two experiments  were harvested in november and those for the third  were harvested in late december.
transcript profiling may therefore also be able to provide molecular signatures relating to seasonally-responsive biology and behaviour.
substantive differences were evident between the juvenile and adult groups, with ~45% of the reporters that consistently generated features passing automated and manual quality criteria yielding measurements showing > <dig> -fold expression change between the two conditions.
a t-test identified  <dig> genes with significantly altered transcript levels .
using only these significantly changed genes, hcc separated all samples by developmental stage .
annotation of genes with changed expression levels between juveniles and adults indicated that transcripts associated with macromolecular biosynthesis, energy production, and connective tissue synthesis  were over-expressed in juveniles .
also over-expressed in juveniles was an invertebrate oxygen-carrier, erythrocruorin, which showed up to 5-fold bias.
genes with higher expression in adults reflected activities associated with turnover of cellular components, including biopolymer metabolism, catabolism, and hydrolase activities.
multiple isoforms of ferritin heavy chain  were over-expressed 50- to 100-fold in adults.
although a library was constructed from the anterior segments  to sample from reproductive tissues  we were not able to identify many transcripts associated with the biological process of sexual reproduction.
a notable and intriguing exception is an homologue of the estrogen receptor co-activator  which, although originally identified from the juvenile library , did show a mean >3-fold, and statistically significant, increase in expression level in the adult samples.
while assessment of goslim annotations of individual clusters by library yielded similar patterns of inference as to biology , the granularity of the est dataset made it a far less sensitive probe of animal physiology than the microarray analyses.
importantly, the microarrays permitted assessment of expression of genes sampled only a few times in the est strategy.
transcript responses of l. rubellus to xenobiotic exposure
dose-response transcription profiles were determined for three xenobiotics from different chemical classes: inorganic , organic , and agrochemical  .
those data passing quality control criteria were analysed as independent compound exposures employing a standard normalisation, followed by filtering to select those reporters that passed automated and manual quality criteria and showed a > <dig> -fold expression change in all conditions.
this dataset was scrutinised further to identify statistically significant expression changes.
gene lists for each compound exposure are given in additional files  <dig>   <dig> and  <dig>  the resultant transcript profiles for replicate samples for each exposure concentration for each compound were hierarchically clustered , the average transcript profiles computed for each exposure condition , and key genes providing functional insights into the response profile identified  .
for each compound, samples from the same exposure condition were more closely related to each other than to other doses .
the only exception was seen in fla, where the groups exposed to the two highest concentrations  are inter-mixed, but clustered separately from the remaining exposure conditions .
for all compounds, hcc analysis indicated a second tier of clustering, separating controls from the two lower doses and both of these groups from all replicates for the two higher doses.
since all exposure experiments were designed with a logarithmic dose series across the full sub-lethal exposure range , this similarity in clustering pattern may reflect similar patterns in transcript change associated with the extent of toxic effects.
molecular insights into xenobiotic response pathways
in our study, earthworms were maintained in the presence of each chemical for a relatively long period  compared to more standard, acute assays .
this extended exposure was intended to permit the measurement of transcriptome responses at a physiological acclimated plateau rather than in a dynamic flux state associated with the initial exposure stress.
we have mined these transcriptional changes to provide insights, some established and some novel, into the mode of action of each xenobiotic.
since our array only reports on a subset of the complete l. rubellus transcriptome, and does not address alternative splicing or posttranscriptional modifications, the insights derived are by default fragmented.
inevitably, our analysis exploits primarily a small proportion of well annotated genes, including invariant controls  and compound responsive transcripts .
however, the majority of genes that show statistically significant relationships between xenobiotic dose and transcript levels do not have informative similarity to known proteins .
while we can annotate these genes as 'regulated by xenobiotic exposure', we are obliged to leave assignment of their molecular functions and relationships as key avenues for future work on the molecular physiology of l. rubellus.
cadmium: a response to inorganic exposure
cadmium is a recognised carcinogen with a well described underlying molecular aetiology .
it has been shown to induce oxidative damage, modulate dna repair and interfere with metabolism of essential metal ions, including fe, ca, and zn, in yeast and cultured cells.
for l. rubellus the annotated cdna microarray identified dose dependent transcript profiles congruent with these molecular mechanisms, together with tantalising novel insights into the secondary impacts of chronic cd exposure on the earthworm.
the cluster of transcripts exhibiting the most significant dose dependent induction in response to cadmium were members of the key protective and detoxification pathway for cd, namely, isoforms of the small, cysteine-rich, metal-binding protein, metallothionein .
the role of transcriptional up-regulation of metallothionein isoforms in cellular detoxification of cadmium in earthworms has been well characterised  <cit> .
the induction profile was confirmed by qpcr analysis on independently transcribed samples of the total rna used for microarray analysis .
the highly significant correlation  of the microarray and qpcr data within the experiment, and the agreement with the previous independent studies cited above, provides confirmation of the validity of our microarray findings.
cd also inhibits the electron transport chain, and causes the generation of reactive oxygen species .
it has been proposed that the specific sites of impact of cd are in complexes ii  and iii , with the majority of ros production being associated with the inhibition of complex iii  <cit> .
the array results supported this model, showing a significant dose-dependent reduction of a cytochrome c oxidase from complex iii, and a modulation of the expression of nadh-ubiquinone oxidoreductase ii and cytochrome b  .
the observed inductions of a cytochrome p <dig> and glutathione-s transferase  alpha by cd  are perhaps a response to ros-induced damage, and have previously been observed in yeast and rat liver, respectively  <cit> .
intriguingly, the induction of the cellular response cascade to cd-induced oxidative damage has been linked to increased levels of the transcription factor nrf <dig> through direct cd interference with ubiquitin-mediated proteolysis  <cit> .
the earthworm array indicates a transcriptional down-regulation of an ubiquitin specific protease , and it will be important to establish whether this inhibition is linked to modulation of nrf <dig> degradation.
although at high concentrations cd may impact on dna directly, causing dna conformational alterations, at lower levels it causes cytotoxic effects through inhibition of dna repair  <cit> .
cd affects both early and late steps of nucleotide excision repair, and also inhibits repair of dna strand breaks and post-replication mismatch repair.
however, the exact mechanisms underlying these effects have not been described.
our data may provide a novel insight into this process, as l. rubellus genes with significant similarity to rad <dig>  <cit>  and xpa  <cit> , key components of repair pathways for double stranded dna breaks and excision repair respectively, are down-regulated in response to cd .
since both rad <dig> and xpa associate with zn it is conceivable that cd may disrupt or inhibit these key components of the dna repair pathway through molecular mimicry.
it will be important to confirm these observations within other species.
as established in model species, molecular mimicry arguably provides the most wide-ranging vehicle through which cd can elicit toxic effects on a biological system.
ca, zn and fe are all employed as key cofactors in proteins that function as structural components, second messengers, transcriptional regulators and redox centres.
the binding of cd to these metallo-proteins will have wide-ranging effects.
the established link between cd toxicity and disruption of fe metabolism leading to anaemia was reflected within the array data through the significant down-regulation of heavy chain ferritin .
whether this is a direct effect of cd or a result of disruption of general fe homeostatic sensing  is unclear.
the impact of cd on ca metabolism was evident on the arrays as a negative transcriptional influence on the ca-binding proteins calcineurin, calexcitin and sarcoplasmic ca-binding protein  .
furthermore, the long-term effects of cd on the cytoskeleton  <cit>  were also reflected in the cd-specific up-regulation of tropomyosin  <dig> .
fluoranthene: a representative polycyclic aromatic hydrocarbon toxin
pahs are lipophilic and so can incorporate into and disrupt the functions of biological membranes  <cit> .
low-level exposure to pah has been linked to several adverse effects, including carcinogenesis  <cit> , teratogenesis  <cit> , and induction of cardiovascular disease  <cit> .
in mammals the molecular trigger coordinating cellular responses to certain pahs is the upregulation of aryl hydrocarbon receptor  activity.
this activation is not mediated transcriptionally, but results from reduction of the proteolytic turnover of ahr by the ubiquitin-26s proteasome pathway  <cit> .
in l. rubellus, exposure to our model pah, fla, caused the down-regulation of two members of this proteolytic pathway, proteasome 26s sub-unit atpase  <dig> and 26s proteasome regulatory sub-unit p <dig> .
a second regulatory mechanism is moderation of an ahr – heat shock protein  <dig> complex by fk506-binding proteins   <cit> .
in l. rubellus, fla exposure caused a >30-fold down-regulation of one fkbp isoform .
these observations suggest that l. rubellus orchestrates its response to fla through these established mechanisms.
ahr is a transcriptional regulator.
specific inhibition of degradation increases the cellular concentration of ahr, and thus causes transcriptional up-regulation of a set of target metabolic and detoxification mechanisms.
classically these responses have been associated by the induction of cytochrome p <dig> reductases, but recently alternative metabolic pathways have been identified, including gsts and nadh:quinone oxidoreductases  <cit> .
in l. rubellus exposed to fla, this detoxification response is exemplified by the up-regulation of a homologue to a pi class gst, a cytosolic isoform that has previously been linked to pah metabolism  <cit>  .
ahr independent pathways activated by pah include those involving early growth response factor  <dig> and peroxisome proliferation-activated receptor alpha  <cit> .
biotransformation of pah leads to the generation of reactive oxygenated metabolites  and rom-mediated oxidative stress  <cit> .
this process activates a broad spectrum of pathways associated with inflammation and hypoxia.
this is reflected through the up-regulation of factors involved in the positive regulation of global transcription, including homologues of habt <dig>  churchill protein and cgi- <dig> .
a specific link has been established between the activation of hypoxia-inducible erythropoietin and the activation pathways under the control of nfkb  <cit> .
nfkb serves as a second messenger to induce a series of cellular cytokines in the response to cellular damage invoked by, amongst others, reactive oxygen species generated by pah exposure.
the observed up-regulation of a cgi- <dig> protein homolog, a putative nfkb activating protein, in response to fla exposure supports the assertion that the earthworm is responding to rom-induced stress .
the direct cellular impacts of pahs have been associated with:  mitochondrial dysfunction or decay leading to the uncoupling of mitochondrial respiration, and inhibiting electron transport;  direct genotoxic damage through dna adduct formation; and,  hypoxia and ros and rom generation  <cit> .
all three processes are clearly evident in the earthworm transcript response profiles.
the mitochondrial-encoded genes nadh dehydrogenase subunits  <dig>  &  <dig>  cytochrome b, and cytochrome c oxidase subunit ii, showed significant down-regulation, with those involved in dna damage repair, such as btbd <dig> protein and apex nuclease   <dig>  were significantly up-regulated .
overwhelmingly, the responses observed within the earthworm are comparable with studies performed in mammalian systems.
however, one area where these annelids show a distinct difference is in the down-regulation of microsomal-based metabolic machinery, including phase i  enzymes) and phase ii  detoxification reactions.
significantly, homologues of two microsomal gsts show significant down-regulation .
furthermore, no homologue of cytochrome p <dig> isoform 1a  has so far been identified in l. rubellus.
this finding is supported by a number of studies in earthworms  <cit>  which did not observe an increase in enzyme activity using assays which target microsomal cytochrome p <dig> enzymes involved in biotransformation of organic xenobiotics .
however, other studies have shown induction of cyp1a  in invertebrates, such as molluscs, following exposure to organic chemicals  <cit> .
while the role of the cytochrome p <dig> mfo system is well understood in vertebrates, the data presented here suggest that there is a subtly distinct, hitherto undescribed variant mechanism active in l. rubellus.
atrazine: an s-triazine-ring herbicide
compared to the significant body of research relating to the gross toxicology of atz , very little is known of the molecular pathways underlying these physiological effects.
it has been proposed that atz breakdown is linked to standard organic phase i and phase ii metabolic pathways, implying that cytochrome p <dig> reductases and gst may be invoked as the primary detoxification mechanisms.
this proposition is supported by the l. rubellus data, which indicated transcriptional up-regulation of representative p <dig> and gst isoforms .
atz is classed as a potential carcinogen with teratogenic activity  <cit> .
on exposure to atz, l. rubellus showed up-regulation of a number of genes that are associated with dna damage, such as uv excision repair protein rad <dig> homologue b and hus <dig> like protein .
furthermore, enzymes directly involved specifically in uv-responsive excision repair are also up-regulated, along with genes involved with chromatin remodelling .
an impact on the transcript levels of genes involved in controlling the cell cycle was also observed.
this may indicate a link to mechanisms that control cell cycle progression in response to genetic integrity of the cell .
as an herbicide, the mode of action of atz is the disruption of electron transport by targeting chloroplast photosystem ii.
inhibition occurs at the level of protein-bound plastoquinone b  <cit> .
the response of l. rubellus to atz measured on the arrays indicated a significant up-regulation of several members of the oxidative phosphorylation pathway and of the tricarboxylic acid cycle .
this may indicate that, at high doses, atz elevates mitochondrial electron transport and alternative routes of atp generation to compensate for a partial uncoupling of oxidative phosphorylation.
however, in the l. rubellus response to atz, the largest and most significantly overrepresented group of genes were associated with protein synthesis and catabolism .
the up-regulation by atz of the ubiquitin-conjugating enzyme e <dig> which is central for the targeting of protein for degradation in concert with ubiquitin-protein ligases  via the hect pathway is indicative of this raised catabolic state  <cit> .
this was counterbalanced by observation of increases in a large number of genes associated with up-regulated protein synthesis such as ribosomal proteins and amino acid transporters.
a possible explanation for these observations might be an increase in the generation of incorrectly folded proteins.
degradation of these non-functional proteins and their re-synthesis would account for the transcript changes observed.
CONCLUSIONS
a thirty-fold accretion in the genetic knowledge-base for any species belonging to an acknowledged keystone taxon that has been previously neglected in sequencing studies might reasonably be expected to yield general and specific insights into individual species and group systematics.
indeed, a recent phylogenetic revision of the animal kingdom, partly based on the l. rubellus ests, defining  <dig>  gene objects generated as a component of this work, clearly grouped earthworms with flatworms and molluscs in a superphylum, the lophotrochozoa, alongside the superphylum ecdysozoa, to form one of the two major animal divisions, the protostomia  <cit> .
the sequencing work described here has, thus, already made a valued contribution to understanding the evolutionary relationships between animal phyla.
the availability and functional annotation of the est resource, and the subsequent clustering to identify a set of gene objects has allowed the first high density microarray for l. rubellus to be fabricated.
the development of this  <dig> -feature array provides a valuable resource to the earthworm community for future studies in areas such as tissue regeneration and immune system function, for which earthworms are already in use as model systems  <cit> .
through experimental studies, we have clearly demonstrated the capacity of the microarray to provide novel insights by investigating the molecular basis of the responses of the species to environmental perturbations.
all our data and analyses have been collated and presented on a web-available database, lumbribase  <cit> , that permits querying of sequence, annotation and microarray data through an easy-to navigate interface.
these analyses promise radical changes in the use of earthworms for the biological assessment of soil contamination.
thus, not only have we revealed differences in the qualitative and quantitative expression patterns of a high proportion  of all l. rubellus genes following exposure to three chemicals of contrasting modes of action, but it has also provided novel molecular insights that complement the outcomes of previous, targeted functional biochemical and metabolomics studies on this species  <cit> .
with this expanding set of tools now available, it can only be assumed that l. rubellus will increasingly be viewed as a powerful tool through which to probe, with an evolutionary perspective, the biochemical, physiological and evolutionary responses of a 'soil engineering' species to environmental perturbations and changes.

 ever since charles darwin's classic work, earthworms have been renowned as'ecosystem engineers' in recognition of the direct and indirect effects they have on water, nutrient and carbon cycling in temperate and tropical soils. earthworms have therefore been widely adopted by international and national agencies for the diagnosis of soil ecosystem health, and for predicting the potential environmental impact of xenobiotics, such as industrial chemicals, pesticides and medicines, from anthropogenic sources. quantification of chemical toxicity to earthworms currently relies on measuring the effects of exposure on key life-history traits in standardised laboratory bioassays conducted with certain test species. these bioassays can produce sensitive estimates of population effects, but are not suited to elucidation of mechanisms of action, and thus may be difficult to generalise from. for example, xenobiotic exposure may affect individual physiology, cocoon-lay rates, cocoon viability and juvenile growth rates in a specific manner, resulting in different outcomes for population growth rate and age structure. complementing measurements of gross toxicity with molecular profiling and genomic studies can make plain the modes of action of specific xenobiotics and identify the generality of biological process affected as well as the molecular response pathways invoked. taking a specific example, it has been observed that l. rubellus is able to colonise highly metal-contaminated environments. detailed work identified that the primary molecules responsible for this metal tolerance were earthworm metallothioneins. though successful, the progress of these mechanistic studies has been handicapped by the lack of available sequence data for earthworms in public databases and the finding that very few of the few available earthworm gene fragments could be identified by sequence similarity to previously sequenced genes. to help bridge this gap in available sequence information, a previously described small-scale expressed sequence tag dataset for l. rubellus was generated. this has prompted the extended survey of the l. rubellus transcribed genome through extensive sampling of additional cdna libraries from earthworms at defined lifecycle stages, specific tissues and following acute exposure to model chemicals representing three different contaminant classes: inorganic, organic, and agrochemical that is reported here. from this transcriptome resource, we defined ~ genes, and have fabricated a cdna microarray to investigate the transcriptome responses of l. rubellus through normal growth and following sub-lethal exposure to a series of sub-lethal concentrations of cd, fla, and atz. from the derived transcript profiles we identify the key molecular responses and biochemical pathways associated with each life-stage treatment, and propose mechanisms of action for the three chemicals.  generation of ests from cdna libraries spanning the l. rubellus lifecycle and following exposure to target chemicals we generated high-quality ests from nine different cdna libraries constructed from l. rubellus raised under defined conditions. we maximised the gene discovery rate by screening out, from some libraries, a small number of highly-expressed mrnas. the ests were clustered using the partigene suite of tools and clusters were defined. this number of putative genes is likely to be an overestimate because alternative splice forms may be assembled into different clusters, the partial nature of ests may result in non-overlap between sequences from the same mrna, and our earthworms were drawn from an outbred source and allelic variation is likely. within-dataset analyses suggested that the effects of these factors are minor, and that the true diversity in the dataset is ~ different loci. if, like other non-vertebrates, l. rubellus has between and protein-coding genes, the sequencing effort has generated sequence tags for ~40–50% of all genes in this key species. see additional file for more detailed descriptions of the libraries used. a cluster is counted as derived from a library if it contains at least one est from that library. thus the longest cluster contains ests from five of the nine libraries and counts as a member of all five. clusters with one est member accounted for 53% of all clusters. there were clusters containing ten or fewer ests, and only clusters had over ests. of the clusters with more than one est, approximately 50% were library-specific. such specificity may simply reflect the low density of sampling rather than real expression differences between libraries. for each cluster, we derived a consensus cdna sequence, and from this derived a putative protein translation using prot4est. some clusters yielded more than one consensus, possibly due to alternative splice forms or divergent alleles. across all the l. rubellus libraries, the consensus sequences had a mean length of bases. the mean length of l. rubellus putative proteins was amino acids. variation in mean protein length between libraries was directly related to the variation in consensus length. the sequence and annotation data are available in a relational database, lumbribase. the database has been used as a workbench for our analyses, and is presented on the web through a php-scripted interface. functional annotation of the l. rubellus gene set gene ontology terms were assigned to l. rubellus predicted proteins using gotcha and the analysis summarised by assessing representation of higher-level terms. less than half of the putative protein translations were assigned a go term at a confidence greater than this apparently high proportion of unannotated genes in l. rubellus can be explained by our application of the conservative gotcha algorithm and by the paucity of genomic information from other annelid species. for those goslim "function" and "process" categories with a mean representation across all libraries of ≥ we examined under- and over-representation in each library. the developing cocoon library had the highest representation of nucleic acid binding, nucleotide binding, chaperone, and protein binding, likely derived from the presence of actively developing embryos in these animals, while the juvenile library had an over representation of lipid-binding annotations. as expected, the control adult library was lacking in terms associated with heavy metal binding compared to libraries derived from cu- or cd-treated earthworms. however, unexpectedly, the library from fla-exposed animals also had increased representation of this annotation. the fla library also exhibited overrepresentation of carbohydrate-binding and fk506-sensitive peptidyl-prolyl isomerase annotations. in the library from cd-treated animals both receptor signalling and globin classes were abundant compared to other libraries, perhaps reflecting the interference of cd with the functions of other, essential metals. in the less-closely defined "process" categories, cd-treated and juvenile worm-derived libraries had more annotations of'cell death' than other libraries. many of these general observations were confirmed through the use of microarrays. the only other "soil" animal for which extensive gene annotation data exist is the rhabditid nematode caenorhabditis elegans, though ecotoxicology analyses focussed on this species are limited. identification of possible orthologues between l. rubellus and c. elegans using blast similarity identified l. rubellus sequences with a blast hit in the c. elegans proteome with an e-value of less than 1e- of which were reciprocal top hits between the species. of these, had annotations relating to c. elegans rnai experiments but these were largely uninformative as to biological function. fabrication and validation of a l. rubellus cdna microarray to profile life-cycle and xenobiotic transcriptome responses, a custom cdna microarray was fabricated using representative reporters from each cluster. each spotted cdna originated from a clone processed for sequencing. the hybridisation was performed using a nested reference design. in detail, the reference sample was derived from two 65–70-mer oligonucleotides designed against vector sequence between the amplification primer and the inserted cdna and co-amplified during insert preparation. probes derived from mrna preparations were labelled with cy this nested reference design permitted direct comparison between samples both within and between experiments. four experiments were undertaken, comparing juvenile and adult stages and comparing transcriptional responses following exposure to cd, fla and atz. all exposures were generated using a common experimental set-up designed to generate a set of biologically replicated samples for a series of exposure concentrations. after hybridisation, array scanning, and pre-processing, the initial technical validation included visual inspection of images to identify gross abnormalities or background. prior to normalisation the sensitivity of the array and relationship between rna concentration and fluorescent signal was assessed by calculating the signal intensity generated by reporters complementary to "alien" rna spikes introduced at known concentrations, from pmole to nmole, prior to labelling. following normalisation the distribution of data responses was examined and any samples showing abnormal distribution were discarded from the analysis. array data were further validated by generating ma-plots, graphical representations of log ratio of the average normalised data from control samples compared to xenobiotic-exposed counterparts against the fluorescence intensity. the control samples were of high quality by this measure. for example, when compared to the cd exposure controls, >99% of the genes exhibiting an acceptable signal had < -fold variance in expression. in contrast, for hybridisations involving mrna from animals exposed to μg/kg cd, ~5% of genes showed an expression change > -fold with respect to the controls. further, a group of reporters showed very significant up-regulation in response to cd. annotations for these reporters indicated that they included clusters encoding various metallothionein isoforms, a gene set previously shown to be highly cd responsive. gene expression changes through the l. rubellus lifecycle transcript profiles were compared for control groups of juveniles and biological replicate groups of adults. these groups were derived from a common stock and selected to minimise the possible confounding effects of seasonal variation. after initial pre-processing of the data, including normalisation relative to the median gene expression in juveniles and adults combined, both principal component analysis and hierarchical condition clustering indicated a primary separation of samples based on developmental stage. intriguingly, a further separation was observed within the adult samples. this separated the samples into two groups that accorded with the time of year the adults were harvested from culture for experimental use in the laboratory experiment. earthworms for two experiments were harvested in november and those for the third were harvested in late december. transcript profiling may therefore also be able to provide molecular signatures relating to seasonally-responsive biology and behaviour. substantive differences were evident between the juvenile and adult groups, with ~45% of the reporters that consistently generated features passing automated and manual quality criteria yielding measurements showing > -fold expression change between the two conditions. a t-test identified genes with significantly altered transcript levels. using only these significantly changed genes, hcc separated all samples by developmental stage. annotation of genes with changed expression levels between juveniles and adults indicated that transcripts associated with macromolecular biosynthesis, energy production, and connective tissue synthesis were over-expressed in juveniles. also over-expressed in juveniles was an invertebrate oxygen-carrier, erythrocruorin, which showed up to 5-fold bias. genes with higher expression in adults reflected activities associated with turnover of cellular components, including biopolymer metabolism, catabolism, and hydrolase activities. multiple isoforms of ferritin heavy chain were over-expressed 50- to 100-fold in adults. although a library was constructed from the anterior segments to sample from reproductive tissues we were not able to identify many transcripts associated with the biological process of sexual reproduction. a notable and intriguing exception is an homologue of the estrogen receptor co-activator which, although originally identified from the juvenile library, did show a mean >3-fold, and statistically significant, increase in expression level in the adult samples. while assessment of goslim annotations of individual clusters by library yielded similar patterns of inference as to biology, the granularity of the est dataset made it a far less sensitive probe of animal physiology than the microarray analyses. importantly, the microarrays permitted assessment of expression of genes sampled only a few times in the est strategy. transcript responses of l. rubellus to xenobiotic exposure dose-response transcription profiles were determined for three xenobiotics from different chemical classes: inorganic, organic, and agrochemical. those data passing quality control criteria were analysed as independent compound exposures employing a standard normalisation, followed by filtering to select those reporters that passed automated and manual quality criteria and showed a > -fold expression change in all conditions. this dataset was scrutinised further to identify statistically significant expression changes. gene lists for each compound exposure are given in additional files and the resultant transcript profiles for replicate samples for each exposure concentration for each compound were hierarchically clustered, the average transcript profiles computed for each exposure condition, and key genes providing functional insights into the response profile identified. for each compound, samples from the same exposure condition were more closely related to each other than to other doses. the only exception was seen in fla, where the groups exposed to the two highest concentrations are inter-mixed, but clustered separately from the remaining exposure conditions. for all compounds, hcc analysis indicated a second tier of clustering, separating controls from the two lower doses and both of these groups from all replicates for the two higher doses. since all exposure experiments were designed with a logarithmic dose series across the full sub-lethal exposure range, this similarity in clustering pattern may reflect similar patterns in transcript change associated with the extent of toxic effects. molecular insights into xenobiotic response pathways in our study, earthworms were maintained in the presence of each chemical for a relatively long period compared to more standard, acute assays. this extended exposure was intended to permit the measurement of transcriptome responses at a physiological acclimated plateau rather than in a dynamic flux state associated with the initial exposure stress. we have mined these transcriptional changes to provide insights, some established and some novel, into the mode of action of each xenobiotic. since our array only reports on a subset of the complete l. rubellus transcriptome, and does not address alternative splicing or posttranscriptional modifications, the insights derived are by default fragmented. inevitably, our analysis exploits primarily a small proportion of well annotated genes, including invariant controls and compound responsive transcripts. however, the majority of genes that show statistically significant relationships between xenobiotic dose and transcript levels do not have informative similarity to known proteins. while we can annotate these genes as'regulated by xenobiotic exposure', we are obliged to leave assignment of their molecular functions and relationships as key avenues for future work on the molecular physiology of l. rubellus. cadmium: a response to inorganic exposure cadmium is a recognised carcinogen with a well described underlying molecular aetiology. it has been shown to induce oxidative damage, modulate dna repair and interfere with metabolism of essential metal ions, including fe, ca, and zn, in yeast and cultured cells. for l. rubellus the annotated cdna microarray identified dose dependent transcript profiles congruent with these molecular mechanisms, together with tantalising novel insights into the secondary impacts of chronic cd exposure on the earthworm. the cluster of transcripts exhibiting the most significant dose dependent induction in response to cadmium were members of the key protective and detoxification pathway for cd, namely, isoforms of the small, cysteine-rich, metal-binding protein, metallothionein. the role of transcriptional up-regulation of metallothionein isoforms in cellular detoxification of cadmium in earthworms has been well characterised. the induction profile was confirmed by qpcr analysis on independently transcribed samples of the total rna used for microarray analysis. the highly significant correlation of the microarray and qpcr data within the experiment, and the agreement with the previous independent studies cited above, provides confirmation of the validity of our microarray findings. cd also inhibits the electron transport chain, and causes the generation of reactive oxygen species. it has been proposed that the specific sites of impact of cd are in complexes ii and iii, with the majority of ros production being associated with the inhibition of complex iii. the array results supported this model, showing a significant dose-dependent reduction of a cytochrome c oxidase from complex iii, and a modulation of the expression of nadh-ubiquinone oxidoreductase ii and cytochrome b. the observed inductions of a cytochrome p and glutathione-s transferase alpha by cd are perhaps a response to ros-induced damage, and have previously been observed in yeast and rat liver, respectively. intriguingly, the induction of the cellular response cascade to cd-induced oxidative damage has been linked to increased levels of the transcription factor nrf through direct cd interference with ubiquitin-mediated proteolysis. the earthworm array indicates a transcriptional down-regulation of an ubiquitin specific protease, and it will be important to establish whether this inhibition is linked to modulation of nrf degradation. although at high concentrations cd may impact on dna directly, causing dna conformational alterations, at lower levels it causes cytotoxic effects through inhibition of dna repair. cd affects both early and late steps of nucleotide excision repair, and also inhibits repair of dna strand breaks and post-replication mismatch repair. however, the exact mechanisms underlying these effects have not been described. our data may provide a novel insight into this process, as l. rubellus genes with significant similarity to rad and xpa, key components of repair pathways for double stranded dna breaks and excision repair respectively, are down-regulated in response to cd. since both rad and xpa associate with zn it is conceivable that cd may disrupt or inhibit these key components of the dna repair pathway through molecular mimicry. it will be important to confirm these observations within other species. as established in model species, molecular mimicry arguably provides the most wide-ranging vehicle through which cd can elicit toxic effects on a biological system. ca, zn and fe are all employed as key cofactors in proteins that function as structural components, second messengers, transcriptional regulators and redox centres. the binding of cd to these metallo-proteins will have wide-ranging effects. the established link between cd toxicity and disruption of fe metabolism leading to anaemia was reflected within the array data through the significant down-regulation of heavy chain ferritin. whether this is a direct effect of cd or a result of disruption of general fe homeostatic sensing is unclear. the impact of cd on ca metabolism was evident on the arrays as a negative transcriptional influence on the ca-binding proteins calcineurin, calexcitin and sarcoplasmic ca-binding protein. furthermore, the long-term effects of cd on the cytoskeleton were also reflected in the cd-specific up-regulation of tropomyosin. fluoranthene: a representative polycyclic aromatic hydrocarbon toxin pahs are lipophilic and so can incorporate into and disrupt the functions of biological membranes. low-level exposure to pah has been linked to several adverse effects, including carcinogenesis, teratogenesis, and induction of cardiovascular disease. in mammals the molecular trigger coordinating cellular responses to certain pahs is the upregulation of aryl hydrocarbon receptor activity. this activation is not mediated transcriptionally, but results from reduction of the proteolytic turnover of ahr by the ubiquitin-26s proteasome pathway. in l. rubellus, exposure to our model pah, fla, caused the down-regulation of two members of this proteolytic pathway, proteasome 26s sub-unit atpase and 26s proteasome regulatory sub-unit p. a second regulatory mechanism is moderation of an ahr – heat shock protein complex by fk506-binding proteins. in l. rubellus, fla exposure caused a >30-fold down-regulation of one fkbp isoform. these observations suggest that l. rubellus orchestrates its response to fla through these established mechanisms. ahr is a transcriptional regulator. specific inhibition of degradation increases the cellular concentration of ahr, and thus causes transcriptional up-regulation of a set of target metabolic and detoxification mechanisms. classically these responses have been associated by the induction of cytochrome p reductases, but recently alternative metabolic pathways have been identified, including gsts and nadh:quinone oxidoreductases. in l. rubellus exposed to fla, this detoxification response is exemplified by the up-regulation of a homologue to a pi class gst, a cytosolic isoform that has previously been linked to pah metabolism. ahr independent pathways activated by pah include those involving early growth response factor and peroxisome proliferation-activated receptor alpha. biotransformation of pah leads to the generation of reactive oxygenated metabolites and rom-mediated oxidative stress. this process activates a broad spectrum of pathways associated with inflammation and hypoxia. this is reflected through the up-regulation of factors involved in the positive regulation of global transcription, including homologues of habt churchill protein and cgi-. a specific link has been established between the activation of hypoxia-inducible erythropoietin and the activation pathways under the control of nfkb. nfkb serves as a second messenger to induce a series of cellular cytokines in the response to cellular damage invoked by, amongst others, reactive oxygen species generated by pah exposure. the observed up-regulation of a cgi- protein homolog, a putative nfkb activating protein, in response to fla exposure supports the assertion that the earthworm is responding to rom-induced stress. the direct cellular impacts of pahs have been associated with: mitochondrial dysfunction or decay leading to the uncoupling of mitochondrial respiration, and inhibiting electron transport; direct genotoxic damage through dna adduct formation; and, hypoxia and ros and rom generation. all three processes are clearly evident in the earthworm transcript response profiles. the mitochondrial-encoded genes nadh dehydrogenase subunits & cytochrome b, and cytochrome c oxidase subunit ii, showed significant down-regulation, with those involved in dna damage repair, such as btbd protein and apex nuclease were significantly up-regulated. overwhelmingly, the responses observed within the earthworm are comparable with studies performed in mammalian systems. however, one area where these annelids show a distinct difference is in the down-regulation of microsomal-based metabolic machinery, including phase i enzymes) and phase ii detoxification reactions. significantly, homologues of two microsomal gsts show significant down-regulation. furthermore, no homologue of cytochrome p isoform 1a has so far been identified in l. rubellus. this finding is supported by a number of studies in earthworms which did not observe an increase in enzyme activity using assays which target microsomal cytochrome p enzymes involved in biotransformation of organic xenobiotics. however, other studies have shown induction of cyp1a in invertebrates, such as molluscs, following exposure to organic chemicals. while the role of the cytochrome p mfo system is well understood in vertebrates, the data presented here suggest that there is a subtly distinct, hitherto undescribed variant mechanism active in l. rubellus. atrazine: an s-triazine-ring herbicide compared to the significant body of research relating to the gross toxicology of atz, very little is known of the molecular pathways underlying these physiological effects. it has been proposed that atz breakdown is linked to standard organic phase i and phase ii metabolic pathways, implying that cytochrome p reductases and gst may be invoked as the primary detoxification mechanisms. this proposition is supported by the l. rubellus data, which indicated transcriptional up-regulation of representative p and gst isoforms. atz is classed as a potential carcinogen with teratogenic activity. on exposure to atz, l. rubellus showed up-regulation of a number of genes that are associated with dna damage, such as uv excision repair protein rad homologue b and hus like protein. furthermore, enzymes directly involved specifically in uv-responsive excision repair are also up-regulated, along with genes involved with chromatin remodelling. an impact on the transcript levels of genes involved in controlling the cell cycle was also observed. this may indicate a link to mechanisms that control cell cycle progression in response to genetic integrity of the cell. as an herbicide, the mode of action of atz is the disruption of electron transport by targeting chloroplast photosystem ii. inhibition occurs at the level of protein-bound plastoquinone b. the response of l. rubellus to atz measured on the arrays indicated a significant up-regulation of several members of the oxidative phosphorylation pathway and of the tricarboxylic acid cycle. this may indicate that, at high doses, atz elevates mitochondrial electron transport and alternative routes of atp generation to compensate for a partial uncoupling of oxidative phosphorylation. however, in the l. rubellus response to atz, the largest and most significantly overrepresented group of genes were associated with protein synthesis and catabolism. the up-regulation by atz of the ubiquitin-conjugating enzyme e which is central for the targeting of protein for degradation in concert with ubiquitin-protein ligases via the hect pathway is indicative of this raised catabolic state. this was counterbalanced by observation of increases in a large number of genes associated with up-regulated protein synthesis such as ribosomal proteins and amino acid transporters. a possible explanation for these observations might be an increase in the generation of incorrectly folded proteins. degradation of these non-functional proteins and their re-synthesis would account for the transcript changes observed.  a thirty-fold accretion in the genetic knowledge-base for any species belonging to an acknowledged keystone taxon that has been previously neglected in sequencing studies might reasonably be expected to yield general and specific insights into individual species and group systematics. indeed, a recent phylogenetic revision of the animal kingdom, partly based on the l. rubellus ests, defining gene objects generated as a component of this work, clearly grouped earthworms with flatworms and molluscs in a superphylum, the lophotrochozoa, alongside the superphylum ecdysozoa, to form one of the two major animal divisions, the protostomia. the sequencing work described here has, thus, already made a valued contribution to understanding the evolutionary relationships between animal phyla. the availability and functional annotation of the est resource, and the subsequent clustering to identify a set of gene objects has allowed the first high density microarray for l. rubellus to be fabricated. the development of this -feature array provides a valuable resource to the earthworm community for future studies in areas such as tissue regeneration and immune system function, for which earthworms are already in use as model systems. through experimental studies, we have clearly demonstrated the capacity of the microarray to provide novel insights by investigating the molecular basis of the responses of the species to environmental perturbations. all our data and analyses have been collated and presented on a web-available database, lumbribase, that permits querying of sequence, annotation and microarray data through an easy-to navigate interface. these analyses promise radical changes in the use of earthworms for the biological assessment of soil contamination. thus, not only have we revealed differences in the qualitative and quantitative expression patterns of a high proportion of all l. rubellus genes following exposure to three chemicals of contrasting modes of action, but it has also provided novel molecular insights that complement the outcomes of previous, targeted functional biochemical and metabolomics studies on this species. with this expanding set of tools now available, it can only be assumed that l. rubellus will increasingly be viewed as a powerful tool through which to probe, with an evolutionary perspective, the biochemical, physiological and evolutionary responses of a'soil engineering' species to environmental perturbations and changes. 
we generated a sequence expressed sequence tag dataset, defining ~ different putative genes, and built an -element transcriptome microarray for l. rubellus. strikingly, less than half the putative genes were assigned annotations from the gene ontology system; this reflects the phylogenetic uniqueness of earthworms compared to the well-annotated model animals. l. rubellus has a complex response to contaminant exposure, but this can be efficiently analysed using molecular methods, revealing unique response profiles for different classes of effector. the microarray was used to identify adult- and juvenile-specific transcript profiles in untreated animals and to determine dose-response transcription profiles following exposure to three xenobiotics from different chemical classes: inorganic, organic, and agrochemical.
 natural contamination and anthropogenic pollution of soils are likely to be major determinants of functioning and survival of keystone invertebrate taxa. soil animals will have both evolutionary adaptation and genetically programmed responses to these toxic chemicals, but mechanistic understanding of such is sparse. the clitellate annelid lumbricus rubellus is a model organism for soil health testing, but genetic data have been lacking.  we generated a sequence expressed sequence tag dataset, defining ~ different putative genes, and built an -element transcriptome microarray for l. rubellus. strikingly, less than half the putative genes were assigned annotations from the gene ontology system; this reflects the phylogenetic uniqueness of earthworms compared to the well-annotated model animals. the microarray was used to identify adult- and juvenile-specific transcript profiles in untreated animals and to determine dose-response transcription profiles following exposure to three xenobiotics from different chemical classes: inorganic, organic, and agrochemical. analysis of these profiles revealed compound-specific fingerprints which identify the molecular responses of this annelid to each contaminant. the data and analyses are available in an integrated database, lumbribase.  l. rubellus has a complex response to contaminant exposure, but this can be efficiently analysed using molecular methods, revealing unique response profiles for different classes of effector. these profiles may assist in the development of novel monitoring or bioremediation protocols, as well as in understanding the ecosystem effects of exposure. 