Aquatic Toxicology 93 (2009) 116–124 Contents lists available at ScienceDirect Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox Gene expression profiling in the deep water horse mussel Modiolus modiolus (L.) located near a marine municipal wastewater outfall Nik Veldhoen a , Christopher J. Lowe b,c , Céline Davis c , Asit Mazumder b , Caren C. Helbing a,∗ a Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, British Columbia, Canada V8W 3P6 b Department of Biology, University of Victoria, P.O. Box 3020, STN CSC, Victoria, British Columbia, Canada V8W 3N5 c Environmental Services Department, Capital Regional District, 625 Fisgard Street, P.O. Box 1000, Victoria, British Columbia, Canada V8W 2S6 article info Article history: Received 24 January 2009 Received in revised form 7 April 2009 Accepted 11 April 2009 Keywords: Gene expression mRNA Wastewater Modiolus Environmental contaminant Real-time PCR Bivalve abstract The marine bivalve Modiolus modiolus (L.) is a sentinel species used for the assessment of potential bio- logical exposure to anthropogenic contaminants in benthic environments. Herein, we have developed real-time quantitative polymerase chain reaction assays for 12 specific mRNAs. The levels of each mRNA transcript were evaluated in adductor muscle, gonad, and hepatopancreas tissue collected from animals located at a reference site and a site near a preliminary municipal wastewater treatment outfall. Signifi- cant differences in mRNA abundance in animals located at the wastewater outfall site were observed for CAT and NET/SCF6 in all three tissues examined, ABCA4 and HSP70 transcript abundance were increased in the adductor muscle and hepatopancreas, respectively. Transcript levels for MDR, CYP4, rpS4, rpS9, and Ca 2+ -ATPase were not different between sites in the three tissues examined. This study sets the foun- dation for further evaluation of these transcripts’ utility in the evaluation of effluent effects within the marine environment. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Bivalves have been used extensively in ecotoxicological biomon- itoring programs (Acosta and Lodeiros, 2004; Yusof et al., 2004; De Luca-Abbott et al., 2005), with Mytilus spp. representing the pre- dominant genus (Adami et al., 2002; St-Jean et al., 2003). In spite of their widespread distribution, Modiolus species are less used as a sentinel, due in part to their lack of commercial value and coloniza- tion depth. However, horse mussels (Modiolus modiolus [L.]) are one of the most populous benthic species in the vicinity of the Capital Regional District (CRD) Clover Point municipal wastewater outfall located in Victoria, British Columbia and, as such, have provided the CRD with information to help assess the potential spatial and temporal effects of the outfall on seafloor organisms (CRD, 2008). The effluent is comprised of complex mixture that includes met- als, various organic substances and emerging chemicals of concern, such as pharmaceuticals and personal care products (CRD, 2008). Currently few toxicity reference values are available for the growing list of emerging substances. The emerging application of ecotoxicogenomic endpoints in the evaluation of sentinel organisms provides for an additional highly sensitive evaluation criterion in wastewater management and has been applied to a limited extent to bivalve species. Sev- ∗ Corresponding author. Tel.: +1 250 721 6146; fax: +1 250 721 8855. E-mail address: chelbing@uvic.ca (C.C. Helbing). eral gene transcripts within the mussel transcriptome exhibited altered steady-state levels following exposure to pollutants that include components of personal care products, pesticides, crude petroleum, metals, and different bacterial species (Franzellitti and Fabbri, 2005; La Porte, 2005; Luckenbach and Epel, 2005; Tanguy et al., 2005; Dondero et al., 2006a,b; Feldstein et al., 2006; Franzellitti and Fabbri, 2006; Song et al., 2006; Venier et al., 2006; Cellura et al., 2007). These mRNA transcripts encode proteins involved in such diverse metabolic pathways as xenobiotic detoxification, immune system response, protection against oxidative and heat stress, and regulation of gene expression. Such recent work indicates the potential to determine the presence and nature of pollutant expo- sure through changes in mRNA transcript profiles within affected mussel populations. Current monitoring endpoints for M. modiolus include size and growth metrics, age, stage of gonadal development, histopatho- logical assessments and tissue chemistry (CRD, 2006, 2008). The present study augments this ongoing monitoring program and involves the isolation of 12 gene-specific cDNA sequences rep- resentative of expressed mRNA and their utilization to form an informative gene expression profile that can differentiate between organisms exposed to municipal wastewater effluent and those residing at reference locations (Fig. 1). A quantitative real-time polymerase chain reaction (QPCR) method that employs these species-specific molecular tools was carried out and differential expression of a number of gene transcripts was observed to signif- icantly associate with proximity to the effluent outfall site. Several 0166-445X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2009.04.002