Identification of a putative antifreeze protein gene that is highly expressed during preparation for winter in the sunn pest, Eurygaster maura Nurper Guz a,⇑ , Umut Toprak a , Asli Dageri a , M. Oktay Gurkan a , David L. Denlinger b a Department of Plant Protection, Faculty of Agriculture, University of Ankara, Ankara, Turkey b Departments of Entomology and Evolution, Ecology and Organismal Biology, Ohio State University, USA article info Article history: Received 11 February 2014 Received in revised form 19 June 2014 Accepted 30 June 2014 Available online 7 July 2014 Keywords: Sunn pest Antifreeze protein Cold tolerance Diapause abstract A cDNA library generated from the fat body of field-collected, diapausing adults of the sunn pest, Eurygaster maura revealed the presence of a transcript that encodes a protein that shares the distinct physiochemical and structural features of an insect antifreeze protein. The transcript, which is most abundant in the midgut, accumulates in adults as they leave the fields in late summer and migrate to sur- rounding mountainous areas to overwinter. Transcript abundance again declines when adults return to the fields the following spring. This winter pattern of abundance suggests that this protein may be critical for winter survival in the cold regions where the bug enters its obligatory diapause. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Several recent transcriptome studies reveal dramatic differ- ences in gene expression between diapausing and nondiapausing insects (e.g. Baker and Russell, 2009; Ragland et al., 2010, 2011; Emerson et al., 2010; Poelchau et al. 2013). Prominent among such distinctions are genes that are up-regulated during diapause to enhance resistance to low temperature, desiccation, microbial attack, and other forms of environmental stress (Denlinger, 2002). The identification of such genes offers insights on specific mechanisms that may be used by the diapausing insect to success- fully overwinter and may provide valuable biomarkers for identify- ing the insect’s diapause status. It was the goal of this project to identify key features of diapause and potential diapause-specific markers in the sunn pest, Eurygaster maura (Hemiptera: Scutelleridae), the dominant pest of cereal crops in the Near- and Middle-East, West and Central Asia, North Africa, and Eastern and Southern Europe (Brown, 1965; Critchley, 1998; Parker et al., 2002). This univoltine species feeds and completes its development in grain fields during spring and summer and then migrates as an adult to the mountains in late summer where it enters an obligatory diapause. Diapause consists of two phases: an early period of aestivation during the hot, dry months of late summer and autumn and hibernation during the cold winter months (Critchley, 1998). In the spring, the adults ter- minate diapause and fly back to the fields where they mate and lay eggs. It is not at all clear how this insect survives in the mountains where prevailing temperatures in the overwintering areas can be as low as À30 °C. In this study we used a targeted approach to identify genes that may contribute to the overwintering success of E. maura. Among genes of potential interest were those encoding antifreeze proteins (AFPs), a group of proteins once thought to be limited to Antarctic fish (DeVries, 1971) and a few insects (Duman, 1977; Duman et al., 1998), but now appear to be more widely distributed than origi- nally assumed (Duman, 2001; Duman et al., 2010). A scan of the cDNA library for proteins with possible antifreeze protein domains revealed one conspicuous cDNA that reflects a pronounced winter pattern of expression, suggesting a prominent role for this gene in the insect’s overwintering biology. The transcript is particularly abundant in the adult midgut. The fact that this predicted structure suggests that this cDNA may encode an antifreeze protein is con- sistent with the abundance of this transcript in overwintering adults. http://dx.doi.org/10.1016/j.jinsphys.2014.06.021 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +90 312 596 1524; fax: +90 312 318 7029. E-mail address: nurperguz@agri.ankara.edu.tr (N. Guz). Journal of Insect Physiology 68 (2014) 30–35 Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys