High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins Giancarlo Lopez-Martinez a,Ã , Michael A. Elnitsky b , Joshua B. Benoit a , Richard E. Lee Jr. b , David L. Denlinger a a Department of Entomology, Ohio State University, Columbus, OH 43210, USA b Department of Zoology, Miami University, Oxford, OH 45056, USA article info Article history: Received 21 December 2007 Received in revised form 13 May 2008 Accepted 18 May 2008 Keywords: Antarctic midge SOD Catalase Oxidative stress ROS Ultraviolet radiation Heat shock proteins abstract Intense ultraviolet radiation, coupled with frequent bouts of freezing–thawing and anoxia, have the potential to generate high levels of oxidative stress in Antarctic organisms. In this study, we examined mechanisms used by the Antarctic midge, Belgica antarctica, to counter oxidative stress. We cloned genes encoding two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), and showed that SOD mRNA was expressed continuously and at very high levels in larvae, but not in adults, while Cat mRNA was expressed in both larvae and adults but at a somewhat reduced level. SOD mRNA was expressed at even higher levels in larvae that were exposed to direct sunlight. Catalase, a small heat shock protein, Hsp70 and Hsp90 mRNAs were also strongly upregulated in response to sunlight. Total antioxidant capacity of the adults was higher than that of the larvae, but levels in both stages of the midge were much higher than observed in a freeze-tolerant, temperate zone insect, the gall fly Eurosta solidaginis. Assays to measure oxidative damage (lipid peroxidation TBARS and carbonyl proteins) demonstrated that the Antarctic midge is highly resistant to oxidative stress. & 2008 Elsevier Ltd. All rights reserved. 1. Introduction In addition to the conspicuous challenges of low temperature and desiccation, organisms living in Antarctica are bombarded with especially high levels of ultraviolet radiation during the summer, an effect that has been exacerbated recently due to openings in the ozone layer (Solomon, 1990; Liao and Frederick, 2005; Weatherhead and Andersen, 2006). Adding to the oxidative stress caused by ultraviolet radiation is the potential for oxygen radical generation by frequent freeze–thaw and anoxia cycles (Joanisse and Storey, 1998; Hermes-Lima and Zenteno-Savin, 2002). Oxygen radicals and non-radicals, like hydrogen peroxide, are known collectively as reactive oxygen species (ROS). ROS can cause lipid peroxidation which disrupts membrane fluidity, and the degradation products can initiate apoptosis in the mitochon- dria (Halliwell and Gutteridge, 1999; Green and Reed, 1998). Oxidative damage to proteins can range from specific amino acid modifications and fragmentation of the peptide chain to total enzyme inactivation by superoxide anions (Stadtman, 1986). ROS can also lead to DNA deletions, mutations, base degradation, single-strand breakage and cross-linkage of proteins (Imlay and Linn, 1988; Imlay, 2003). Superoxide radicals generated by oxidative stress act as oxidants or reductants that lead to the production of hydroxyl radicals (Fridovich, 1995). The hydroxyl radicals, though short-lived, are highly reactive and readily damage DNA by denaturing nucleic acids (Lesser, 2006). Two of the enzymes most crucial for inactivating these potentially damaging oxygen agents are superoxide dismutase (SOD) and catalase (Orr and Sohal, 1994). SOD converts superoxide into oxygen and hydrogen peroxide, while catalase then converts hydrogen peroxide into oxygen and water. In this study, we examine the response of an Antarctic insect, the midge Belgica antarctica Jacobs (Diptera, Chironomidae), to oxidative stress. This insect, which is endemic to maritime Antarctic, has a patchy distribution on the Antarctic Peninsula and its nearby islands (Gressitt, 1967; Convey and Block, 1996). The midge is freeze-tolerant and spends much of its 2-yr life cycle as a larva encased in a matrix of ice and substrate, but during the brief (approximately 2 months) austral summer the larvae feed on algae and bacteria located in the substrate near penguin rookeries (Sugg et al., 1983). The wingless adults emerge late December to early January. They can be found in aggregations on the surfaces of rocks located near the larvae, but they never stray far from the ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ibmb Insect Biochemistry and Molecular Biology 0965-1748/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2008.05.006 Ã Corresponding author. Tel.: +1614 2924477; fax: +1614 292 2180. E-mail address: lopez-martinez.1@osu.edu (G. Lopez-Martinez). Insect Biochemistry and Molecular Biology 38 (2008) 796– 804