Identification of genes expressed in juvenile Haliotis rufescens in response to different copper concentrations in the north of Chile under controlled conditions Fernando Silva-Aciares a,⇑ , Manuel Zapata b , Jennifer Tournois c , Dario Moraga c , Carlos Riquelme a,b a Laboratorio de Ecología Microbiana, Universidad de Antofagasta 170, Antofagasta, Chile b Centro Investigación Científica y Tecnológica para la Minería CICITEM, Avenida Jose Miguel Carrera 1701 4° piso, Antofagasta, Chile c Laboratoire des Sciences de l’Environnement Marin, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Place Nicolas Copernic, 29280 Plouzané, France article info Keywords: Haliotis rufescens Copper stress Suppression subtractive hybridization (SSH) Gene expression Real time PCR abstract This study reports molecular markers potentially associated with resistance or sensitivity to the impact of copper in juvenile red abalone, Haliotis rufescens, in the north of Chile under experimental conditions. Genomic analysis was made applying subtractive hybridization libraries (SSH) to identify genes up- and down regulated during cooper exposure in abalone over periods of 12 and 168 h exposed to 2.5 and 10 lg/L of Cu +2 . Results obtained from the SSH library revealed 368 different sequences regulated by copper, that correspond to eight major physiological functions. The validation of these sequences obtained by SSH as well as their expression kinetics were made by PCR in real time on 14 potential genes regulated by metal stress. This study provides information for the characterization of potential genomic markers that may be used in future environmental monitoring and to investigate new mechanisms of stress to copper in this commercially important marine species. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. 1. Introduction The abalone is a slow-growing marine herbivore gastropod mol- lusk that lives in rocky substrates in temperate seas associated with brown algae prairies which are its food (Viana, 2002). There are more than 100 species of the genus Haliotis around the world, but less than 10% of them are commercially important. Its larval development under cultivation conditions lasts approximately 4–5 days at a temperature of 16 °C. In the settling period the larvae start feeding on microalgal films, and after 8 months, when they reach a size of 20 mm, their diet is supplemented with macroalgae plantlets in the order of 10–30% of their body weight every day (Leighton, 2000). The cultivation and marketing of abalone is not devoid of problems in aspects such as: seed supply (insufficient, irregular, and not always of good quality), instability in the provi- sion of natural food, and the high cost of artificial food. One of the critical stages is the one called ‘‘weaning,’’ which consists in the transition from food based on diatomaceous biofilms to macroal- gae, and it is the stage that has the highest mortality, which ex- ceeds 30–40% of the organisms (Flores-Aguilar et al., 2007). On the other hand, over the last years abalone populations have de- creased considerably all over the world. Overexploitation, habitat destruction, increasing environmental pollution, and diseases threaten the populations of these mollusks (Davis et al., 1994; DFG, 2003). Because of the reduction of the natural populations, abalone aquaculture became public domain with the purpose of guarantee- ing a stable contribution to the marketing of this mollusk. Because of the high commercial value of abalone in the international mar- ket, its aquaculture has developed around the world and continues to grow, with a 50% increase in recent years, with China holding first place in abalone production in 2006, with 5000 tons (Flores- Aguilar et al., 2007). Of the 100 abalone species that have been identified so far, the red abalone, Haliotis rufescens, of Southern California, was intro- duced in Chile in 1977, but its cultivation started increasing only recently (Viana, 2005; Enriquez and Villagrán, 2008). In fact, it held fifth place in abalone production with 304 tons in 2006 (Flores- Aguilar et al., 2007). During the last few years the red abalone has been cultivated on land, in tanks, in hatcheries in the northern coasts of Chile. Due to the high financial costs of this cultivation system, it culture has now started in maritime concessions, espe- cially in the south of Chile (Flores-Aguilar et al., 2007). The introduction of abalone in the coast of the South Pacific may be subject to high anthropogenic pressure that can have an impact on the development, growth and survival of this mollusk. There is no doubt that pollution by heavy metals is one of the highest risk factors for coastal organisms because they are exposed to indus- trial and urban effluents from this mining region (Salamanca et al., 2004). These human activities along the coast, together with 0025-326X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.09.023 ⇑ Corresponding author. Tel.: +56 55 637537; fax: +56 55 637804. E-mail address: fsaciares@uantof.cl (F. Silva-Aciares). Marine Pollution Bulletin 62 (2011) 2671–2680 Contents lists available at SciVerse ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul