Journal of Insect Physiology 50 (2004) 259–266 www.elsevier.com/locate/jinsphys Antifreeze proteins in Alaskan insects and spiders J.G. Duman a, , V. Bennett a , T. Sformo b , R. Hochstrasser c , B.M. Barnes b a Department of Biological Sciences, Box 369, University of Notre Dame, Notre Dame, IN 46556, USA b Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99775, USA c Sycamore Community High School, 7400 Cornell Road, Cincinnati, OH 45242, USA Received 18 September 2003; received in revised form 9 December 2003; accepted 9 December 2003 Abstract Prior to this study, antifreeze proteins (AFPs) had not been identified in terrestrial arthropods from the Arctic or anywhere in Alaska. The hemolymph of 75 species of insects and six spiders from interior and arctic Alaska were screened for thermal hyster- esis (a difference between the freezing and melting points), characteristic of the presence of AFPs. Eighteen species of insects and three spiders were shown to have AFPs. Ten of the insects with AFPs were beetles including the first species from the families Chrysomelidae, Pythidae, Silphidae and Carabidae. In addition, the first Neuropteran to have AFPs was identified, the lacewing Hemerobius simulans together with the second and third Diptera (the first Tipulids) and the second and third Hemiptera, the stinkbug Elasmostethus interstinctus (the first Pentatomid), and the water strider Limnoporus dissortis (the first Gerrid). Prior to this study, 33 species of insects and three spiders had been reported to have AFPs. Most AFP-producing terrestrial arthropods are freeze avoiding, and the AFPs function to prevent freezing. However, some of the AFP- producing insects identified in this study are known to be freeze tolerant (able to survive freezing) to very low temperatures (40 to 70 v C). # 2003 Elsevier Ltd. All rights reserved. Keywords: Antifreeze proteins; Insect cold tolerance; Spider cold tolerance; Arctic 1. Introduction Antifreeze proteins (AFPs), first discovered in the blood of Antarctic fish (DeVries, 1971), lower the freezing point of water in the presence of ice while not affecting the melting point. This produces a difference between the freezing and melting points which is termed thermal hysteresis (DeVries, 1986). AFPs are unique in their ability to produce thermal hysteresis and consequently it is diagnostic for their presence. The magnitude of thermal hysteresis is dependant on the specific activity and concentration of AFPs, and in some cases the presence of enhancers of AFP activity (Duman, 2001; Duman and Serianni, 2001). AFPs lower the freezing point by a non-colligative mech- anism whereby the AFPs adsorb onto preferred sur- faces of potential seed ice crystals (Raymond and DeVries, 1977; Raymond et al., 1989; Brown and So ¨nnichsen, 2002). Consequently, growth of the crystal (addition of water molecules to the crystal surfaces) can only occur between the adsorbed AFPs and in high radius of curvature fronts (high surface free energy), rather than the preferred low radius of curvature fronts (low surface free energy). Therefore, according to the Kelvin effect, the temperature must be lowered below the colligative melting point for growth to proceed. AFPs have been identified in many species of high latitude marine teleost fishes, where the AFPs have evolved independently multiple times (Cheng and De Vries, 2002; Brown and So ¨nnichsen, 2002). Five struc- turally different types of AFPs have been described in fish (Brown and So ¨nnichsen, 2002). AFPs are also present in numerous terrestrial arthropods including spiders (Duman, 1979a; Husby and Zachariassen, 1980), mites (Block and Duman, 1989; Sjursen and So ¨mme,2000), centipedes (Tursman et al., 1994; Tursman and Duman, 1995), and of course insects (Duman, 1977, 2001). Most terrestrial arthropods which produce AFPs are freeze avoiding. This occurs in overwintering larvae of the Pyrochroid beetle Dendroides canadensis  Corresponding author. Tel.: +1-219–631-7496; fax: +1-219-631- 7413. E-mail address: duman.1@nd.edu (J.G. Duman). 0022-1910/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2003.12.003