EVOLUTION & DEVELOPMENT 5:3, 231–239 (2003) © BLACKWELL PUBLISHING, INC. 231 Heat shock in the developmentally sensitive period of butterfly eyespots fails to increase fluctuating asymmetry Casper J. Breuker* and Paul M. Brakefield Evolutionary Biology, Institute of Evolutionary and Ecological Sciences, Leiden University, 2300 RA Leiden, The Netherlands *Author for correspondence (e-mail: breuker@rulsfb.leidenuniv.nl) SUMMARY Fluctuating asymmetry (FA) is considered to provide a means of evaluating developmental stability and to reflect an individual’s quality or the stress experienced dur- ing development. Stress is predicted to increase the pheno- typic variation of both FA and trait size. In this study we examined the effect of a particular heat shock on both FA and size of eyespots in the butterfly, Bicyclus anynana. We also examined whether those eyespots thought to be involved in partner choice and sexual selection were particularly sensi- tive to stress. We applied a heat shock of 39.5 ° C for 3 h before, during, and after a sensitive period in eyespot development. We examined the FA, variation in FA, size, and variation in size of five eyespots, two on the dorsal forewing (sexually se- lected traits), two on the ventral forewing, and one on the ventral hindwing (nonsexually selected traits). For each sex and treatment, the heat shock did not result in significant changes in mean trait size and FA nor in the variation of size and FA. There were no differences in the response to the heat shock between sexually and nonsexually selected traits. We discuss how the increased production of heat shock pro- teins, including HSP60, may have stabilized development and how this might explain the results. Introduction A developing organism is likely to encounter forms of stress of environmental and genetic origin (Parsons 1990). Fluctu- ating asymmetry (FA) describes small nondirectional devia- tions from bilateral symmetry. FA is thought to provide a means of evaluating developmental stability or the capacity of individuals to buffer development against the disruptive ef- fects of developmental stress (Van Valen 1962; Palmer and Strobeck 1986, 1992; Palmer 1994; Markow 1995). If there are no a priori reasons to assume significant differences be- tween individuals in developmental stability, then FA may be used as a reliable indicator of stress (Leary and Allendorf 1989). Results from several studies have been inconsistent, however. FA does not always increase under stress, and if so, it seems to do so in a trait-specific way (Bjorksten et al. 2000; Clarke et al. 2000). In this study we examined the ef- fect of a particular heat shock on both FA and size of eye- spots in the butterfly, Bicyclus anynana. We also examined whether those eyespots thought to be involved in partner choice and sexual selection were particularly sensitive to the shock. It is hypothesized that the development of sexual orna- ments is weakly stabilized due to their presumed recent his- tory of directional selection (experimental evidence reviewed by Møller and Swaddle 1997). Furthermore, The develop- ment of these traits are proposed to be highly condition de- pendent and thereby more likely to show higher levels of FA and a greater variability in trait size and FA (Møller 1990). It thus follows that sexual traits are more likely to respond to stress than homologous nonsexual traits (Hunt and Simmons 1997; Woods et al. 1999; Bjorksten et al. 2000). However, the results of six studies reported by Bjorksten et al. (2000) testing these hypotheses are also inconsistent. The wings of the tropical butterfly B. anynana have eye- spots on both their ventral and dorsal surfaces. Each eyespot consists of concentric rings of different color centered around a white “pupil.” The development of these eyespots is progressively specified in four stages (Brakefield et al. 1996; Brunetti et al. 2001) (Fig. 1A). In the first stage, during the last larval instar, the transcription factor Distal-less (Dll) is expressed in a prepattern in the developing wing disks. In the second stage, organizing groups of cells called foci are established in the particular areas of the larval wing disk des- tined to become the pupil areas of each adult eyespot. The expression of Dll and other developmental genes is up- regulated in the cells of an eyespot focus. In the third stage, covering the first 24 h after pupation, an information gradient is established around each focus apparently by focal signaling. This is then interpreted by surrounding cells to determine which color pigments will be produced later, just before adult eclosion (fourth stage). Brunetti et al. (2001) showed