Tjalf E. de Boer

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.

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.

The effect of soil pH and temperature on Folsomia candida transcriptional regulation.

Differences in abiotic factors like temperature and soil pH can have a significant physiological impact on soil dwelling invertebrates and may confound results in ecotoxicological testing. In this study we exposed Folsomia candida to a range of two abiotic stress treatments (pH and temperature) for 3 days and measured gene expression of a panel of nine stress response genes with real-time Q-PCR. The exposure to different pH values had a minimal effect on the expression of the nine selected genes: only V-ATPase expression was significantly increased due to decreasing pH. ATPase expression was up-regulated, possibly due to increased proton trafficking across the cell membrane, at a lower pH. HSP70 was up-regulated in collembolans exposed to 30 degrees C, and along with HSP40 at 0 degrees C. We speculate that the minor pH effect on gene expression, compared to the temperature treatment, can be explained by the spatial restricted exposure to the external pH in the gut. Our data showed that only 1 or 2 stress response genes were transcriptionally affected by pH and temperature thus exerting minimal effects. The physiological effects of these treatments on F. candida might indicate interesting novel molecular mechanisms.

Exposure to mercury reduces heat tolerance and heat hardening ability of the springtail Folsomia candida

We investigated the combined effects of mercury (HgCl(2)) and acute heat on survival of the springtail Folsomia candida. The springtails were exposed to a range of aqueous concentrations (0-48 mg Hg(2+)/L) of HgCl(2) for 24 h. Subsequently, the same individuals were exposed to a range of high temperatures, from 20 to 35.5 degrees C. We found a highly significant synergistic interaction between effects of mercury and heat, with a reduced tolerance to heat after exposure to sublethal concentrations of mercury. Further, the heat hardening ability of F. candida was studied at sublethal concentrations of mercury. F. candida was able to heat harden (exposure to a mild heat treatment increasing survival of subsequent severe heat); however, when the springtails experienced a previous exposure to as little as 1 mg Hg(2+)/L, heat hardening failed to improve survival of heat shock at 34.5 degrees C, even though this was much lower than concentrations affecting survival without heat stress. Mild heat stress is known to induce the heat shock protein, HSP70, and real-time quantitative PCR confirmed that pre-acclimation to 32 degrees C did indeed cause >5-fold up-regulation of HSP70 expression. This up-regulation was not affected by previous exposure to 1 mg Hg(2+)/L.

A Functional Isopenicillin N Synthase in an Animal Genome

Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the β-lactam antibiotics biosynthesis pathway, in the genome of the soil-living collembolan species, Folsomia candida (FcIPNS). At present, this gene is only known from bacteria and fungi, as is the capacity to produce β-lactam antibiotics. The FcIPNS gene was located on two genomic contigs, was physically linked to a predicted insect ATP-binding cassette transporter gene, and contained three introns each flanked by eukaryotic splicing recognition sites (GT/AG). Homology searches revealed no similarity between these introns and the FcIPNS regions of bacteria or fungi. All amino acids conserved across bacteria and fungi were also conserved in F. candida. Recombinant FcIPNS was able to convert its substrate amino δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine into isopenicillin N, providing strong evidence that FcIPNS is functional. Phylogenetic analysis clustered FcIPNS outside the bacterial IPNS clade, and also outside the fungal IPNS clade, suggesting an ancient gene transfer followed by divergence in the F. candida genome. In conclusion, the data suggest that the soil-living collembolan F. candida has assimilated the capacity for antibacterial activity by horizontal gene transfer, which may be an important adaptive trait in the microbe-dominated soil ecosystem.

Comment on "Ecotoxicogenomics: bridging the gap between genes and populations".

Fedorenkova et al. (1) developed a species-sensitivity distribution (SSD) for cadmium-induced gene expression and compared it to SSDs for cadmium toxicity evaluated at the level of the no-observed effect (NOEC) and median lethality (LC50). They concluded that the SSD for gene expression is shifted about four times above the NOEC, therefore, gene expression is a less sensitive end point than the classical sublethal end points, growth and reproduction. We argue that these conclusions are premature and biased, for the following three reasons.

Integrating transcriptomics into triad-based soil-quality assessment

The present study examined how transcriptomics tools can be included in a triad-based soil-quality assessment to assess the toxicity of soils from riverbanks polluted by metals. To that end, the authors measured chemical soil properties and used the International Organization for Standardization guideline for ecotoxicological tests and a newly developed microarray for gene expression in the indicator soil arthropod Folsomia candida. Microarray analysis revealed that the oxidative stress response pathway was significantly affected in all soils except one. The data indicate that changes in cell redox homeostasis are a significant signature of metal stress. Finally, 32 genes showed significant dose-dependent expression with metal concentrations. They are promising genetic markers providing an early indication of the need for higher-tier testing of soil quality. During the bioassay, the toxicity of the least polluted soils could be removed by sterilization. The gene expression profile for this soil did not show a metal-related signature, confirming that a factor other than metals (most likely of biological origin) caused the toxicity. The present study demonstrates the feasibility and advantages of integrating transcriptomics into triad-based soil-quality assessment. Combining molecular and organismal life-history trait stress responses helps to identify causes of adverse effects in bioassays. Further validation is needed for verifying the set of genes with dose-dependent expression patterns linked with toxic stress.

Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening

Environmental risk assessment relies on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic end point in bioassays and effect-based screening. Data analysis and its results, however, are complex and often not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expression data set assembled from multiple studies and experiments in the springtail Folsomia candida, with the aim of selecting a set of genes that can be trained to classify general toxic stress. By performing differential expression analysis prior to classifier training, we were able to select a set of 135 genes which was enriched in stress related processes. Classifier models from this set were used to classify two test sets comprised of chemical spiked, polluted, and clean soils and compared to another, more traditional classifier feature selection. The gene set presented here outperformed the more traditionally selected gene set. This gene set has the potential to be used as a biomarker to test for adverse effects caused by chemicals in springtails to provide end points in environmental risk assessment.

Population-specific transcriptional differences associated with freeze tolerance in a terrestrial worm

Abstract Enchytraeus albidus is a terrestrial earthworm widespread along the coasts of northern Europe and the Arctic. This species tolerates freezing of body fluids and survives winters in a frozen state. Their acclimatory physiological mechanisms behind freeze tolerance involve increased fluidity of membrane lipids during cold exposure and accumulation of cryoprotectants (glucose) during the freezing process. Gene regulatory processes of these physiological responses have not been studied, partly because no gene expression tools were developed. The main aim of this study was to understand whether the freeze tolerance mechanisms have a transcriptomic basis in E. albidus. For that purpose, first the transcriptome of E. albidus was assembled with RNAseq data. Second, two strains from contrasting thermal environments (Germany and Greenland) were compared by mapping barcoded RNAseq data onto the assembled transcriptome. Both of these strains are freeze tolerant, but Greenland is extremely freeze tolerant. Results showed more plastic responses in the Greenland strain as well as higher constitutive expression of particular stress response genes. These altered transcriptional networks are associated with an adapted homeostasis coping with prolonged freezing conditions in Greenland animals. Previously identified physiological alterations in freeze‐tolerant strains of E. albidus are underpinned at the transcriptome level. These processes involve anion transport in the hemolymph, fatty acid metabolism, metabolism, and transport of cryoprotective sugars as well as protection against oxidative stress. Pathway analysis supported most of these processes, and identified additional differentially expressed pathways such as peroxisome and Toll‐like receptor signaling. We propose that the freeze‐tolerant phenotype is the consequence of genetic adaptation to cold stress and may have driven evolutionary divergence of the two strains.

Mechanisms of phenanthrene toxicity in the soil invertebrate, Enchytraeus crypticus

Polycyclic aromatic hydrocarbons (PAHs) continue to cause environmental challenges as a result of their release into the environment by a great variety of anthropogenic activities and their accumulation in soils. Studies were conducted on the toxicological effect of the model PAH phenanthrene using the soil invertebrate model Enchytraeus crypticus at the individual, tissue, and molecular levels. Animals were exposed for 2 d and 21 d to phenanthrene concentrations corresponding to the (previously estimated) 3-wk effective concentration, 10% (EC10) and EC50 for effects on reproduction. Gene expression profiling did not reveal a typical phenanthrene-induced biotransformation signature, as it usually does in arthropods and vertebrates. Instead, after 2 d of exposure, only general metabolic processes were affected, such as translation and adenosine triphosphate synthesis-coupled electron transport. Histological sections of tissues of 2-d exposed animals showed no deviations from control. In contrast, after prolonged exposure of up to 21 d, histopathological effects were found: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. Environ Toxicol Chem 2016;35:2713-2720.