High throughput nano-liter RT-qPCR to classify soil contamination using a soil arthropod

Abstract

BackgroundTo incorporate genomics data into environmental assessments a mechanistic perspective of interactions between chemicals and induced biological processes needs to be developed. Since chemical compounds with structural similarity often induce comparable biological responses in exposed animals, gene expression signatures can serve as a starting point for the assessment of chemicals and their toxicity, but only when relevant and stable gene panels are available. To design such a panel, we isolated differentially expressed gene fragments from the soil arthropod Folsomia candida, a species often used for ecotoxicological testing. Animals were exposed to two chemically distinct compounds, being a metal (cadmium) and a polycyclic aromatic hydrocarbon (phenanthrene). We investigated the affected molecular responses resulting from either treatment and developed and validated 44 qPCR assays for their responses using a high throughput nano-liter RT-qPCR platform for the analysis of the samples.ResultsSuppressive subtractive hybridization (SSH) was used to retrieve stress-related gene fragments. SSH libraries revealed pathways involved in mitochondrial dysfunction and protein degradation for cadmium and biotransformation for phenanthrene to be overrepresented. Amongst a small cluster of SSH-derived cadmium responsive markers were an inflammatory response protein and an endo-glucanase. Conversely, cytochrome P450 family 6 or 9 was specifically induced by phenanthrene. Differential expressions of these candidate biomarkers were also highly significant in the independently generated test sample set. Toxicity levels in different training samples were not reflected by any of the markers' intensity of expressions. Though, a model based on partial least squares differential analysis (PLS-DA) (with RMSEPs between 9 and 22% and R2s between 0.82 and 0.97) using gene expressions of 25 important qPCR assays correctly predicted the nature of exposures of test samples.ConclusionsFor the application of molecular bio-indication in environmental assessments, multivariate analyses obviously have an added value over univariate methods. Our results suggest that compound discrimination can be achieved by PLS-DA, based on a hard classification of the within-class rankings of samples from a test set. This study clearly shows that the use of high throughput RT-qPCR could be a valuable tool in ecotoxicology combining high throughput with analytical sensitivity.

Phenotypic and transcriptional responses associated with multi-generation exposure of Folsomia candida to engineered nanomaterials

“…16 A study by de Boer et al (2011) showed that multiple ABC transporters and copper pumps (copper-transporting ATPase 1, 2) were upregulated in response to cadmium toxicity. 43 In the present study, significantly induced metallothionein expression was found in F. candida exposed to either CuO or WCCo NMs and induction of the ABC transporter gene even persisted in animals after 2 recovery generations while in clean soil when previously exposed to WCCo-NM; all indicating a function in maintaining metal or redox homeostasis.…”

“…A previous study showed that metal stress affects cell differentiation and adhesion in the basal lamina in human fibroblasts, where collagen and laminin production decreased upon low-dose exposure to silver and gold nanoparticles. 47 Laminin A gene expression was also affected in cadmium and phenanthrene exposed F. candida, irrespective of compound (De Boer et al 2011); 43 suggesting persistent differential gene expression as a result of metal stress. In the present study, a significant effect of the interaction History*Generation on gene expression was found in WCCo-NM exposed F. candida.…”

Functional Environmental Genomics of a Municipal Landfill Soil

“…Quantitative PCR (QPCR) was performed using a selected group of genes according to de Boer et al ( 2011b ) on a Biomark HD system (Fluidigm). Information on gene description and PCR primer sequence can be found in Table 2 of the results section.…”

Transgenerational and multigenerational stress gene responses to the insecticide etofenprox in Folsomia candida (Collembola)

“…Environmental stressors, such as insecticides, can activate many genes but down-regulate others in the collembolan F. candida (de Boer et al, 2011). In fact, differential regulation of genes is considered as an early warning sign of homeostasis disruption due to stress, since it precedes protein translation and metabolite biosynthesis in response to a changing environment.…”