Dick Roelofs

Development and application of the adverse outcome pathway framework for understanding and predicting chronic toxicity: I. Challenges and research needs in ecotoxicology

To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of toxicity events across scales of biological organization that lead to adverse outcomes relevant for risk assessment. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. This will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species in the future.

Transcriptional and posttranscriptional gene silencing are mechanistically related

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.

Cre recombinase expression can result in phenotypic aberrations in plants

AbstractThe cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression, aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severity of the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing of cre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severe phenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype was observed when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for the application of recombinases in plants, expression is best limited to specific tissues and a short time frame.[12pt] Abbreviations: bar, the phosphinotricin acetyltransferase gene; CAM, chloramphenicol resistance gene; Ds 5′ & Ds 3′, borders of the Ds transposable element from maize forming a functional transposable element that embodies the interjacent DNA; gus, the β-glucoronidase gene; gus-int, the gus gene interrupted by a plant intron; hpt, the hygromycin phosphotransferase gene; nptII, the neomycin phosphotransferase gene; ORI, bacterial origin for plasmid replication in Escherichia coli of plasmid p15A

Molecular mechanisms of heavy metal tolerance and evolution in invertebrates.

Following the genomics revolution, our knowledge of the molecular mechanisms underlying defenses against stress has been greatly expanded. Under strong selective pressure many animals may evolve an enhanced stress tolerance. This can be achieved by altering the structure of proteins (through mutations in the coding regions of genes) or by altering the amount of protein (through changes in transcriptional regulation). The latter type of evolution can be achieved by substitutions in the promoter of the gene of interest (cis‐regulatory change) or by altering the structure or amount of transcriptional regulator proteins (trans‐regulatory change). The metallothionein system is one of the best studied stress response systems in the context of heavy metals. Metallothionein expression is assumed to be regulated by metal transcription factor 1 (MTF‐1); however, up to now the involvement of MTF‐1 has only been proven for some vertebrates and Drosophila. Data on invertebrates such as nematodes and earthworms suggest that other mechanisms of metallothionein induction may be present. A detailed study of Cd tolerance was done for a species of soil‐living springtail, Orchesella cincta. The metallothionein gene of this species is overexpressed in metal‐exposed field populations. Analysis of the metallothionein promoter has demonstrated extensive polymorphisms that have a functional significance, as shown in bioreporter assays. In a study comparing 20 different populations, the frequency of a high‐expresser promoter allele was positively correlated with the concentration of metals in soil, especially Cd. The springtail study shows that cis‐regulatory change of genes involved in the cellular stress response may contribute to evolution of metal tolerance.

Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

BackgroundGenomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.ResultsEight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable.ConclusionWe present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.

Adaptive differences in gene expression associated with heavy metal tolerance in the soil arthropod Orchesella cincta

Field‐selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal‐contaminated mining sites. This tolerance correlated with heritable increase in metal excretion efficiency, less pronounced cadmium (Cd)‐induced growth reduction and overexpression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and Cd‐tolerant populations. Many cDNAs responded to Cd exposure in the reference population. Significantly fewer clones were Cd responsive in tolerant animals. Analysis of variance revealed transcripts that interact between Cd exposure and population. Hierarchical cluster analysis of these clones identified two major groups. The first one contained cDNAs that were up‐regulated by Cd in the reference culture but non‐responsive or down‐regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti‐microbial defence, calcium channel‐blocking, sulphur assimilation and chromatin remodelling. The second group consisted of cDNAs down‐regulated in reference animals but not responding or slightly up‐regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+‐dependent stress signalling, redox state, proteolysis and digestion. The reference population showed a strong signature of stress‐induced genome‐wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon Cd exposure. We confirmed the micro‐evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress‐adapted phenotype.

Collembase: a repository for springtail genomics and soil quality assessment.

BackgroundEnvironmental quality assessment is traditionally based on responses of reproduction and survival of indicator organisms. For soil assessment the springtail Folsomia candida (Collembola) is an accepted standard test organism. We argue that environmental quality assessment using gene expression profiles of indicator organisms exposed to test substrates is more sensitive, more toxicant specific and significantly faster than current risk assessment methods. To apply this species as a genomic model for soil quality testing we conducted an EST sequencing project and developed an online database.DescriptionCollembase is a web-accessible database comprising springtail (F. candida) genomic data. Presently, the database contains information on 8686 ESTs that are assembled into 5952 unique gene objects. Of those gene objects ~40% showed homology to other protein sequences available in GenBank (blastx analysis; non-redundant (nr) database; expect-value < 10-5). Software was applied to infer protein sequences. The putative peptides, which had an average length of 115 amino-acids (ranging between 23 and 440) were annotated with Gene Ontology (GO) terms. In total 1025 peptides (~17% of the gene objects) were assigned at least one GO term (expect-value < 10-25). Within Collembase searches can be conducted based on BLAST and GO annotation, cluster name or using a BLAST server. The system furthermore enables easy sequence retrieval for functional genomic and Quantitative-PCR experiments. Sequences are submitted to GenBank (Accession numbers: EV473060 – EV481745).ConclusionCollembase http://www.collembase.org is a resource of sequence data on the springtail F. candida. The information within the database will be linked to a custom made microarray, based on the Agilent platform, which can be applied for soil quality testing. In addition, Collembase supplies information that is valuable for related scientific disciplines such as molecular ecology, ecogenomics, molecular evolution and phylogenetics.

Molecular evidence for an extinct parent of the tetraploid species Microseris acuminata and M. campestris (Asteraceae, Lactuceae)

To determine the origin of the tetraploid annuals Microseris campestris and M. acuminata, chloroplast RFLP, RAPD and ITS sequence variability among nine populations of the two polyploids and 14 populations of the diploid annuals M. elegans and M. douglasii have been surveyed. Previously described variable chloroplast restriction sites infer M. douglasii as the possible maternal parent of both tetraploid species. However, the chloroplast genome typical for M. douglasii has now also been found in some plants of M. elegans. RAPD analysis revealed 172 polymorphic DNA markers that defined all four species as genetically distinct groups, but demonstrated closer associations between M. douglasii and M. acuminata, and between M. elegans and M. campestris. Sequencing of the ITS‐1 and ITS‐2 region yielded 73 phylogenetically informative sites. Thirty base‐pair mutations separated the annual Microseris species from the outgroup, Uropappus lindleyi. The putative interspecific allotetraploid M. campestris contained only one type ITS sequence that, on the basis of eight synapomorphic substitutions was derived from M. elegans. The single ITS of M. acuminata shares six common sites with M. douglasii. Surprisingly, six sites were synapomorphic for the two tetraploids, M. campestris and M. acuminata, suggesting recombination within the ITS of both species with that of a common, now extinct, parental taxon, possibly the donor of the M. douglasii type chloroplasts found in both tetraploids. These results confirm the interpretation of M. campestris as derived from M. douglasii (extinct population) and M. elegans, and resolve the unknown origin of M. acuminata as an intraspecific hybrid between two very distinct populations of M. douglasii, one of them the same extinct M. douglasii form that contributes to M. campestris.

De novo transcriptome assembly and SNP discovery in the wing polymorphic salt marsh beetle Pogonus chalceus (Coleoptera, Carabidae).

Background The salt marsh beetle Pogonus chalceus represents a unique opportunity to understand and study the origin and evolution of dispersal polymorphisms as remarkable inter-population divergence in dispersal related traits (e.g. wing development, body size and metabolism) has been shown to persist in face of strong homogenizing gene flow. Sequencing and assembling the transcriptome of P. chalceus is a first step in developing large scale genetic information that will allow us to further study the recurrent phenotypic evolution in dispersal traits in these natural populations. Methodology/Results We used the Illumina HiSeq2000 to sequence 37 Gbases of the transcriptome and performed de novo transcriptome assembly with the Trinity short read assembler. This resulted in 65,766 contigs, clustering into 39,393 unique transcripts (unigenes). A subset of 12,987 show similarity (BLAST) to known proteins in the NCBI database and 7,589 are assigned Gene Ontology (GO). Using homology searches we identified all reported genes involved in wing development, juvenile- and ecdysteroid hormone pathways in Tribolium castaneum. About half (56.7%) of the unique assembled genes are shared among three life stages (third-instar larva, pupa, and imago). We identified 38,141 single nucleotide polymorphisms (SNPs) in these unigenes. Of these SNPs, 26,823 (70.3%) were found in a predicted open reading frame (ORF) and 6,998 (18.3%) were nonsynonymous. Conclusions The assembled transcriptome and SNP data are essential genomic resources for further study of the developmental pathways, genetic mechanisms and metabolic consequences of adaptive divergence in dispersal power in natural populations.

Transcriptomics reveals extensive inducible biotransformation in the soil-dwelling invertebrate Folsomia candida exposed to phenanthrene

BackgroundPolycyclic aromatic hydrocarbons are common pollutants in soil, have negative effects on soil ecosystems, and are potentially carcinogenic. The Springtail (Collembola) Folsomia candida is often used as an indicator species for soil toxicity. Here we report a toxicogenomic study that translates the ecological effects of the polycyclic aromatic hydrocarbon phenanthrene in soil to the early transcriptomic responses in Folsomia candida.ResultsMicroarrays were used to examine two different exposure concentrations of phenanthrene, namely the EC10 (24.95 mg kg-1 soil) and EC50 (45.80 mg kg-1 soil) on reproduction of this springtail, which evoked 405 and 251 differentially expressed transcripts, respectively. Fifty transcripts were differential in response to either concentration. Many transcripts encoding xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) were upregulated in response to either concentration. Furthermore, indications of general and oxidative stress were found in response to phenanthrene. Chitin metabolism appeared to be disrupted particularly at the low concentration, and protein translation appeared suppressed at the high concentration of phenanthrene; most likely in order to reallocate energy budgets for the detoxification process. Finally, an immune response was evoked especially in response to the high effect concentration, which was also described in a previous transcriptomic study using the same effect concentration (EC50) of cadmium.ConclusionOur study provides new insights in the molecular mode of action of the important polluting class of polycyclic aromatic hydrocarbons in soil animals. Furthermore, we present a fast, sensitive, and specific soil toxicity test which enhances traditional tests and may help to improve current environmental risk assessments and monitoring of potentially polluted sites.