J. Insect Physiol. Vol. 35, No. 4, pp. 277-281, 1989 Printed in Great Britain. All rights reserved 0022-1910/89 $3.00 + 0.00 Copyright 0 1989 Pergamon Press plc EFFECTS OF LOW TEMPERATURE ON DIAPAUSING AGLAIS URTICAE AND INACHIS IO (LEPIDOPTERA: NYMPHALIDAE): COLD HARDINESS AND OVERWINTERING SURVIVAL A. S. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED FULLIN and J. S. BALE Department of Pure and Applied Biology, Agricultural Sciences Building, University of Leeds, Leeds LS2 9JT, England (Received 5 August 1988; reked 16 September 1988) Abstraract-Two species of nymphalid butterflies, Agluis urticae and Inachis io were exposed to four different temperature regimes (10, 2, - Y’C, and cycling - S/lWC) during diapause to determine patterns of cold hardiness and overwintering survival. Supercooling ability increases in all groups from cessation of feeding to 100 days in diapause, but the lowest temperature regime does not produce the lowest mean crytallisation temperature. Pre and post-diapause feeding reduce supercooling as does surface water. Fresh weight loss follows a temperature-dependent pattern with greatest weight loss at 10” and least at - 5°C in both species. Mortality is highest at lo” accounting for 80% of I. io after 100 days and 75% of A. urticae after 170 days. Mortality at - 5 and - S/lo”C differs between species; in A. urticue only 30% had died after 170 days in both groups but in I. io a sharp rise in mortality after 100 days resulted in 65% mortality after 155 days in both regimes. There was no evidence of individuals freezing in the subzero regimes. Initial wet weights of survivors are significantly higher than those of non-survivors at all temperatures. Implications for assessment of cold hardiness in insects are discussed. Key Word Index: Overwintering, cold hardiness, Agfais, Znachis, supercooling, butterfly INTRODU(XON Insects employ a number of strategies to increase their chances of survival during winter. The accumu- lation of energy reserves in the form of lipids and the induction of a state or reduced metabolic activity are the most common adaptations in response to such a long period without food. Additionally, most insects increase their winter cold hardiness as an aid to survival at subzero temperatures. Insects are classified as freeze tolerant or intolerant based on the ability of the species to survive freezing of their extracellular fluids (Salt, 1961). Freeze-tolerant spe- cies are able to survive extracellular ice formation within the body tissues, whereas freezing-intolerant insects are killed by freezing and rely on supercooling to avoid this lethal event. More recently, Knight et al. (1986) found that some aphids, although able to supercool extensively, die after a brief exposure to sub-zero temperatures above their crystallisation temperature (often referred to as the supercooling point). Assessment of cold-hardiness is complicated in freezing-intolerant insects by variations in super- cooling during diapause (Mansingh and Smallman, 1972). This may reflect changes in the levels of cryoprotective substances in response to the nutri- tional and diapause status of the population, or to variation in preceding weather conditions. In addi- tion, supercooling may be reduced by the presence of internal ice nucleators such as food in the gut (Salt, 1953), or by surface moisture causing inoculative freezing through the cuticle (Salt, 1956). Most research on insect cold hardiness has centred on short-term exposures to low temperatures and few studies have attempted to assess the effects of pro- longed exposure throughout the winter period. In one study, Turnock et al. (1983) found that exposure of diapausing pupae of Mamestra ConJigurata to tem- peratures between -5 and -20°C over 140 days reduced survival in the post-diapause stages. If re- duced survival is common after longer term exposure to low temperature then previous assumptions about levels of mortality in overwintering populations based on short-term exposures and supercooling data may be erroneous (Bale, 1987). The temperate nymphalid buttedies, Agluis urticue and In&is io are widely distributed on the European continent but A. urticae extends to higher latitudes and altitudes. Both species overwinter in imaginal diapause after seeking out semi-sheltered sites amongst vegetation, or in tree-holes and buildings. The survival of these species at moderate temperature (4°C) during this period has already been studied (Pullin, 1987). The aims of the research described in this paper are 2-fold. Firstly to determine the cold hardiness of adults at specific stages before, during and after the overwintering process, and to assess mortality during long-term exposure to temperatures above the crystallisation temperature; and secondly to investigate the influence of interactions between diapause, nutritional status and abiotic factors on overwintering. MATERIALS AND METHODS Rearing Both species of butterfly were reared in the labora- tory following the method of Pullin (1987) over one 277