Insect Biochemistry and Molecular Biology Insect Biochemistry and Molecular Biology 37 (2007) 287–295 Differential gene expression profiles associated with heavy metal tolerance in the soil insect Orchesella cincta Dick Roelofs à , Janine Marie¨n, Nico M. van Straalen Department of Animal Ecology, Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Received 29 September 2006; received in revised form 28 November 2006; accepted 30 November 2006 Abstract Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola, springtails) populations occurring at metal-contaminated mining sites. Metal tolerance in O. cincta is correlated with heritable increase of excretion efficiency, decrease in cadmium-induced growth reduction, and over-expression of the single copy metallothionein (mt) gene. Although mt plays a major role in cadmium (Cd) detoxification an additional number of gene products may be altered in the tolerant phenotype that influence quantitative traits like excretion efficiency and growth. Using suppression subtractive hybridization analysis (SSH) genes were cloned that showed up- or down-regulation in response to Cd. This study reports about 192 SSH derived cDNA clones, of which 49 were confirmed to be differentially expressed after spot blot analysis. After sequence analysis 19 of the 49 differential clones could be annotated. Real-time RT-PCR of a reference culture and tolerant populations revealed significant gene responses upon Cd exposure. The reference culture showed stress induced gene expression, whereas the tolerant animals maintained normal gene expression or down regulation of cDNAs upon Cd exposure. Seven cDNAs with homology to genes involved in cell signaling, transport and programmed cell death showed an interaction between treatment and population. r 2007 Elsevier Ltd. All rights reserved. Keywords: Cadmium; Suppressive subtractive hybridization; Metallothionein; Orchesella cincta; Metal tolerance; Real-time RT-PCR 1. Introduction King and Wilson (1975) proposed variation in gene expression as a major driver of evolution. Recent studies indeed show that selection for altered transcriptional regulation can be a powerful mechanism for micro- evolution (Daborn et al., 2002; Gompel et al., 2005; Rockman et al., 2005), although Khaitovich et al. (2004) reported random neutral divergence of variation in gene expression within and among primate taxa. Earlier, we presented data that suggest the possibility for evolution of transcription regulation in a model for pollution-induced tolerance in the soil-living invertebrate Orchesella cincta (Arthropoda: Collembola, springtails). A tolerant popula- tion of springtails showed increased expression of the metal detoxifying protein metallothionein (mt) compared to a reference population (Hensbergen et al., 1999; Sterenborg and Roelofs, 2003). Moreover, in a reference population cadmium (Cd) induced mt over-expression contained a significant heritable component (Roelofs et al., 2006), suggesting that selection can act on elevated mt mRNAs in springtails at sites where Cd levels are a potential threat for the environment (Timmermans et al., 2005). The increase in transcriptional activation of mt may account for enhanced excretion efficiency that was observed in a tolerant population (Posthuma et al., 1993). However, it cannot explain other aspects of the metal-tolerant pheno- type, such as earlier reproduction (Posthuma et al., 1993). Differential gene expression patterns induced by expo- sure to heavy metals are poorly understood. Orchesella cincta is a soil dwelling arthropod that contains both sensitive as well as metal-tolerant populations and is thus an appropriate model to address the effect of heavy metals on the stress response system as well as for identifying genes that are possibly involved in metal tolerance. However, being a non-genomic model organism, DNA sequence information is lacking in O. cincta. In such a case ARTICLE IN PRESS www.elsevier.com/locate/ibmb 0965-1748/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2006.11.013 à Corresponding author. Tel.: +31 20 5987078; fax: +31 20 5987123. E-mail address: dick.roelofs@ecology.falw.vu.nl (D. Roelofs).