Journal of Insect Physiology 49 (2003) 999–1004 www.elsevier.com/locate/jinsphys Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes Shu-Xia Yi, Richard E. Lee Jr. * Department of Zoology, Miami University, Oxford, OH 45056, USA Received 8 May 2003; received in revised form 27 June 2003; accepted 27 June 2003 Abstract This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 °C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY- 14) and propidium iodide. Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20–30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.  2003 Elsevier Ltd. All rights reserved. Keywords: Freeze tolerance; Cryoprotection; Cold-hardening; Vital fluorescent dyes 1. Introduction Winter cold poses a great challenge to the survival of many insects and other ectotherms. For temperate spec- ies, seasonal cold-hardening is a prerequisite to avoid chilling and freezing injury (Lee and Denlinger, 1991). An array of physiological and biochemical responses, including the accumulation of glycerol, sorbitol and other low molecular mass cryoprotectants, antifreeze proteins and ice nucleating proteins are associated with increases in insect cold tolerance (Duman, 2001; Denlinger and Lee, 1998). Although we understand, in part, the underlying mechanisms by which cold tolerance is enhanced sea- sonally, it is also clear that much is not known concern- * Corresponding author. Tel.: +1-513-529-3141; fax: +1-513-529- 6900. E-mail address: leere@muohio.edu (R.E. Lee, Jr). 0022-1910/$ - see front matter  2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-1910(03)00168-9 ing the nature of cold-hardening, particularly at the cellu- lar level (Collins et al., 1997; Orvar et al., 2000). Using logistic regression modeling to identify factors influenc- ing cold-hardening of fat body cells, Bennett and Lee (1997) found that seasonal increases in low molecular mass cryoprotectants were insufficient to fully explain cold-hardening. Instead, unidentified changes intrinsic to the cell had occurred also. First, second, and early third instar larvae of the gold- enrod gall fly, Eurosta solidaginis, are not tolerant of freezing. However, in late summer and early autumn third instar larvae gradually acquire freeze tolerance. One of the first indications of larval cold-hardening is the synthesis of glycerol, triggered by the drying of sur- rounding gall tissue as the plant senesces (Rojas et al., 1986). A few weeks later, when environmental tempera- tures cool to 5 °C or below, larvae respond by producing large amounts of sorbitol (Baust and Lee, 1982). Cold tolerance is also promoted by a reduction in supercooling capacity, caused by the appearance in the Malpighian