Development 102, 377-385 (1988) 377 Printed in Great Britain © The Company of Biologists Limited 1988 Colour pattern regulation after surgery on the wing disks of Precis coenia (Lepidoptera: Nymphalidae) H. FREDERIK NIJHOUT and LAURA W. GRUNERT Department of Zoology, Duke University, Durham, North Carolina 27706, USA Summary Partial ablations were done in situ on the imaginal disk. When a cut was positioned near one of the dorsal disks of the hindwing in larvae of Precis coenia at ages eyespots, the outer rings of the eyspot opened up so between 2 and 9 days prior to pupation. While there that its central field became contiguous with the new was no regeneration of the wing lamina, the cut edge margin. The behaviour of the dorsal eyespots of the developed normal marginal scales and a marginal hindwing in response to ablation of the wing disk, as colour pattern if the ablation was done more than 3-5 well as to other developmental disturbances, appears days prior to pupation. The response of elements of to be the reverse of those on the forewing and ventral the marginal colour pattern to partial ablation of the hindwing. We conclude that the central field of a wing disk indicates that the wing margin has an dorsal eyespot and the wing margin share similar important role in colour pattern determination and controlling properties with respect to pattern, and that appears to act as a sink for a pattern-inducing signal. both appear to act as sinks or as the inverse of the While the elements of the marginal colour pattern sources of pattern-inducing signal found in the eye- regulate to the shape and position of the new wing spots of the forewing. margin, the eyespots changed their shape and size but Key words: butterfly, colour pattern, imaginal disk, not their position upon partial ablation of the wing pattern regulation, Precis coenia, regeneration. Introduction induced by their respective signalling sources during a 1- or 2-day period after pupation and in Precis coenia Colour pattern determination in Lepidoptera appears the large eyespots on the forewing are induced during to be a two-step process. The first step involves a a 48 h period beginning at or shortly before pupation process that establishes the positions of specialized (Kiihn & Von Engelhardt, 1936; Nijhout, 1978,1980). signalling sources on the wing surface. The second In Precis coenia, however, most of the other elements step is an activation of those sources, which then that made up the colour pattern (central symmetry induce certain pattern elements in the surrounding system, parafocal elements, submarginal bands) are wing epithelium. Border ocelli, the bands of the determined some time prior to pupation. This is central symmetry system and some of the bands at the evidenced by the fact that neither their position nor base of the wing have been shown to arise in this their shape can be altered by manipulation of the manner (Nijhout, 1978, 1985a). wing after pupation (Nijhout, 1980). Nothing is known at present about the mechanism Thus, to study the determination of many pattern or dynamics of the first step except that it must occur elements as well as the early processes responsible for on the wing imaginal disk some time before pupation. the positioning of signalling sources (Nijhout, 1985a), The second step in the process can occur at various it is necessary to be able to manipulate the wing times during development, depending on the species imaginal disk during the larval stage. It has been our and pattern element in question. In Ephestia kuh- experience, as well as that of others, that explanted niella, Malacosoma americana and Hyalophora cecro- fragments of disk remain alive and can be made to pia the bands of the central symmetry system are develop through metamorphosis, but develop few 378 H. F. Nijhout and L. W. Grunert scales and no detectable colour pattern. Thus, it is The principal limitation on the successful recovery from impossible, at this time, to determine the prospective surgery was the proximity of the pupal moult. Surgery fate for colour pattern of an imaginal disk explant. performed less than 24 h prior to pupation always led to We have developed a method that allows us to mechanical difficulties at ecdysis due to incomplete wound healing and thus animals older than one day prior to remove surgically small parts of wing imaginal disks in pupation could not be studied. We found that operated situ and to determine the regulative and regenerative animals experienced a delay in pupation of approximately capacities of the portion that retained its normal one day relative to unoperated controls, irrespective of attachment to the body. when during larval life the surgery was done. Thus, to The present paper will deal with our findings on the obtain the physiological age of the animal at the time of effects of partial ablation on the development of the surgery, we subtracted one day from the time that elapsed ocelli, parafocal elements and submarginal bands of between surgery and pupation. In the presentation that the hindwing colour pattern of Precis coenia. follows, all ages of experimental animals are given in terms of their physiological age relative to pupation. Animals of physiological ages between 2 and 6 days prior to pupation were obtained from surgery on last-instar larvae. Surgery Materials and methods was also successfully performed during the penultimate (fourth) larval instar and yielded the category of animals Larvae of Precis coenia were maintained at a constant ranging in age from 6 to 9 days prior to pupation. temperature of 27 °C under a 16L:8D photoperiod. In preparation for surgery, larvae were anaesthetized by submersion in distilled water for 15-20 min. Surgery was Results performed under saline. Imaginal disks were partially exposed by cutting off the lateral spine on either the Regeneration of the wing disk mesothorax or the metathorax, for the forewing and hindwing disks, respectively. With gentle pressure, the tip We observed, as have others (Henke, 1933; Magnus- of the imaginal disk was forced to protrude from this small sen, 1933), what appeared to be a modest but very opening and all or a portion of the protrusion was cut off erratic regeneration response after partial ablation of with iridectomy scissors. The principal limitation of this wing disks (Table 1). Large ablations always pro- method is that the initial incision has to be kept very small duced a defective wing, no matter how early in and the disk can never be laid out flat prior to ablation. development the operation was done. Small ab- Thus, we were limited to placing our cuts along only a single lations, however, occasionally resulted in the devel- plane (Fig. 1), controlled by the fact that the pointed tip on opment of normal-looking wings but these occurred the imaginal disk always protruded first. We have as yet no more frequently when surgery was done early in found no method that would allow us to extrude the entire disk through the incision, do a surgical manipulation and development (9 days prior to pupation) than when it then reinsert the disk without further damage. After was done late (2 days prior to pupation). Magnussen surgery larvae were placed on paper towels and stored in a (1933) reported that wing disks of Papilio machaon refrigerator for 6-12 h. During this cold incubation period, almost always failed to regenerate ablated parts even a clot formed over the wound which prevented blood loss after a 19-day period. This erratic and infrequent and extrusion of the viscera when the animals were brought regenerative response differs dramatically from the back to room temperature. Approximately 60 % of treated substantial and regular regenerative capacities of larvae survived this procedure and resumed feeding nor- wing disk fragments in insects like Drosophila mally upon return to room temperature. (Bryant, 1987). Table 1. Regeneration of wing disks in Precis coenia Day of Forewing Hindwing surgery (prior to surgery surgery surgery surgery pupation) operated normal operated normal 16 14 13 17 16 19 41 12 20 17 14 10 13 16 9 12 Normal animals are those that developed wings of normal size and shape after partial ablation of the wing disk. Others developed wings of reduced size and with obvious portions missing. Pattern regulation in wing disks 379 Magnussen (1933) proposed that this apparently erratic regenerative response has its basis in a peculiar feature of the wing disk in Lepidoptera. Unlike the well-known wing disk of Drosophila, which is compressed and invaginated, lepidopteran wing disks develop in a fully everted form and contain an accurate though miniature representation of the adult wing (Suffert, 1929; Nijhout, 1985a). During the last larval instar, the wing disk develops a system of radiating longitudinal lacunae most of which will become the future wing veins (Nijhout, 1985a). In addition, in each wing disk a 'bordering lacuna' develops that runs around the periphery of the disk but well within its outer edge. The bordering lacuna outlines the future margin of the wing (Fig. 1). After pupation all cells distal to the bordering lacuna undergo programmed cell death (C. Dohrmann & H. F. Nijhout, unpublished data) so that only the portion of the disk within the perimeter of the bordering lacuna develops into the adult wing. Species of 1A Lepidoptera differ greatly in the amount of periph- eral tissue in their wing imaginal disks. In some species, 20—30 % of the imaginal disk falls outside the bordering lacuna and is discarded (Suffert, 1929). Thus, ablations that only remove portions of the wing disk that fall outside the bordering lacuna will have no effect on the shape and size of the adult wing, even though a substantial amount of disk may have been removed.