Development of a microarray for Enchytraeus albidus (Oligochaeta): preliminary tool with diverse applications

Abstract

Standard bioassays allow hazard assessment at the population level, but much remains to be learned about the molecular level response of organisms to stressors. The main aim of this study was the development of a DNA microarray for Enchytraeus albidus, a common soil worm species. Further, this microarray was tested using worms exposed to Cu, phenmedipham, and different soil types. Hybridization onto the developed microarray revealed several genes with homology to known sequences. Genes of interest were confirmed through real-time polymerase chain reaction. It was possible to discriminate between natural and chemical stressors and chemical concentrations. Gene responses were detected under conditions known to have effects in the reproduction of individuals. It was confirmed that the integration of different endpoints improves the assessment process and enhances the understanding of the modes of action of stressors. The chemical stress-induced genes were related to factors such as immune response, stress response, metabolic processes, and/or signal transduction. The present study represents the first step of a gene-level study in the ecologically relevant and standard test species E. albidus. It demonstrates the usefulness of cDNA normalization in the production of cDNA libraries of ecotoxicological standard organisms that are not genome models like E. albidus.

Gene Expression Responses Linked to Reproduction Effect Concentrations (EC10,20,50,90) of Dimethoate, Atrazine and Carbendazim, in Enchytraeus albidus

“…The development of a cDNA microarray for E. albidus [23], [24] provided a new tool to assess molecular mechanisms of pesticide toxicity. This microarray was enhanced (Agilent oligonucleotide microarray) with the development of two new cDNA libraries enriched with metal and pesticide responsive genes, and all the sequence information was made available in EnchyBASE (http://bioinformatics.ua.pt/enchybase/) [25].…”

Differential gene expression analysis in Enchytraeus albidus exposed to natural and chemical stressors at different exposure periods

“…Therefore, in the present study, organisms were exposed to the same stressors (chemical and natural) used by Amorim et al (2011) but with two more exposure periods, 4 and 21 days. The gene expression analysis was performed using the existing cDNA library for Enchytraeus albidus and the results of different exposure times were compared with the purpose to obtain information on the evolution of gene expression in E. albidus over time and covering periods that could produce effects at higher levels of biological organization.…”

“…Similar approaches can be used in the case of standard organisms whose genomes are still poorly characterized, such as E. albidus, by developing the necessary molecular tools. In this context, Amorim et al (2011) have designed a Electronic supplementary material The online version of this article (doi:10.1007/s10646-011-0780-4) contains supplementary material, which is available to authorized users. custom-made DNA microarray for E. albidus based on a normalised cDNA library, and were able to identify differences in gene expression that were specific to the chemical and natural stresses tested.…”

Enchytraeus albidus Microarray: Enrichment, Design, Annotation and Database (EnchyBASE)

“…Regarding E. albidus , Amorim and co-authors started the EST sequencing project with a normalized cDNA library [16]. A cDNA microarray was developed based on this normalized library and has been used to study the effects of phenmedipham, copper, different soil properties or exposure duration [16], [21]–[23].…”

“…A cDNA microarray was developed based on this normalized library and has been used to study the effects of phenmedipham, copper, different soil properties or exposure duration [16], [21]–[23]. The existing cDNA library was improved using suppression subtractive hybridization-PCR (SSH-PCR), a technique that combines high subtraction efficiency with a normalization step to generate differentially expressed sequences equally represented in the library [24], [25].…”