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 (: )

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: , 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 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 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 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. Magnussen (1933) attributed the er- ratic regeneration observed in her study and in those of others to a failure to recognize this structural feature of the wing imaginal disk, and concluded that in Papilio machaon at least, wing disks did not regenerate ablated parts. Thus, the rare and erratic regeneration behaviour shown in Table 1 is most- likely explained by Magnussen's (1933) hypothesis. We are as yet unable to control the site of our ablations with sufficient precision to test this hypoth- esis rigorously, but we conclude provisionally that the B wing disks of Precis exhibited no significant ability to regenerate during the 9-day period prior to pupation. Work is presently under way to study cellular behav- Fig. 1. Imaginal disks of the hindwing of Precis coenia iour at the cut edge to determine the degree and taken from larvae at 3 days (A) and 7 days (B) prior to pupation. The lacunae (/) that will become the wing veins pattern of cell proliferation after partial ablation. are evident at both stages. Tracheae (r) have begun to invade the lacunae at 3 days prior to pupation. Arrows Regeneration of the marginal colour pattern indicate the bordering lacuna. All cells distal to the The marginal pattern of the hindwing consists of an bordering lacuna will undergo programmed cell death alternation of dark (brown) and light (yellow) bands. during the pupal stage. The adult wing is clearly outlined The dark bands constitute the pattern elements and within the imaginal disk. Since the disk contains a nearly are named the edge band (eb), the submarginal band perfect miniature of the adult wing, the fate map for (smb) and the parafocal element (pfe) (Fig.. 2). The colour pattern maps directly onto the wing disk (distorted edge band and submarginal band always run perfectly only by a slight Cartesian transformation that maps the disk onto the wing proper). The line indicates the parallel to the wing margin while -the parafocal orientation of the cuts we made. All cuts were made element has a pronounced proximally directed peak parallel to this line at various positions within the disk. or projection along the midline of each wing cell. This Scale bar is 0-5 mm, both disks are at the same feature is particularly pronounced on the ventral magnification. 380 H. F. Nijhout and L. W. Grunert

Fig. 3. Close-ups of wing edge showing the long marginal scales. (A) Normal wing edge; (B) regenerated edge after removal of approximately one third of wing disk at 7 days prior to pupation. Bar, 0-5 mm.

100

Fig. 2. Normal colour pattern of the dorsal (A) and ventral (B) hindwing of Precis coenia. eb, edge band; smb, submarginal band; pfe, parafocal element. Veins M,, M2, Q. 15 60 Cui and Cu2 are indicated (see test). Scale bar is 5 mm.

E 40 surface (Fig. 2B). All pattern elements show disconti- nuities in shape or colour at the wing veins. In addition, the margin bears a fringe of specialized 20 marginal scales that differ in shape and are more than three times as long as the normal wing scales 987654321 (Fig. 3A). Physiological age at time of surgery When a portion of the wing imaginal disk was (days prior to pupation) surgically removed, many animals developed normal specialized marginal scales and a normal or nearly Fig. 4. Graph of the percentage of wings that developed normal marginal colour pattern along the new cut a marginal pattern after partial ablation of the wing disk edge. These 'regenerated' marginal scales and colour at various ages prior to pupation. The number of patterns were always smoothly continuous with their individuals represented by each point can be found in Table 1 (number operated minus number normal). homologues on the undisturbed portion of the wing Normal marginal pattern developed in more than 50 % of margin. There was a nearly perfect correspondence individuals when surgery was done at a physiological age between individuals that regenerated a normal mar- younger than 3 days prior to pupation for the forewing ginal colour pattern and those that developed normal (black circles), and 5 days prior to pupation for the marginal scales on the new wing margin. Of the more hindwing (white circles). The lines are linear regressions. ft/

5A

S3

B s a. o Fig. 5. Effects of partial ablation of a hindwing disk on the morphology of wing and colour pattern. (A,B) Dorsal and ventral view, respectively, of an animal that had only the edge of an imaginal disk removed 5 days prior to pupation. Bracket indicates the extent of the cut. Eyespots near the cut on dorsal and ventral side are reduced in size. Marginal patterns are slightly I narrower than on the unoperated side and the parafocal elements (pfe) along the cut edge do not have their distinctive peaked shape. (CD). Dorsal and ventral view, respectively, of an animal that had three quarters of an imaginal disk removed 7 days prior to pupation. Marginal pattern elements formed on both dorsal and ventral sides. Pigmentation of the marginal pattern identities each element as shown: pfe, parafocal element; smb, submarginal band. For reasons that are unclear at present, there were no wing veins present in the small wing, and the apparent fragmentation of the pfe on the ventral side is not associated with CO any visible structure in the wing. Bar, 5 mm; all specimens same magnification. 382 H. F. Nijhout and L. W. Grunert

Fig. 6. Hindwing that developed after removal of two thirds of the wing imaginal disk 6 days prior to pupation illustrating smb continuity of marginal colour pattern elements, uninterrupted by wing veins. (A) Ventral surface; (B) dorsal surface, pfe, parafocal element; smb, submarginal band; eb, edge band. The smb is absent on ventral side and along most of the dorsal side in this specimen. Bar, lmm. than 150 animals that developed a marginal colour Pattern regulation in eyespots on the hindwing pattern only 7 did not have marginal scales, and we Eyespots are a common feature of butterfly colour never obtained an animal with normal marginal scales patterns. They are induced by discrete signalling that did not also have a marginal colour pattern. sources that are always located on the midline of a The percentage of animals that developed normal wing cell (a wing cell is defined as the area on a wing marginal scales and wing pattern depended on the demarcated by wing veins). Potentially, each wing time that elapsed between surgery and pupation. cell can bear one eyespot (Nijhout, 1980, 1985a). The dorsal surface of the hindwing of Precis coenia bears Approximately a 3-day period was required for 50 % two large eyespots, centred in wing cells M]—M and of the animals to regenerate pattern on the hindwing 2 Cu!-Cu2 (Fig. 2A). The ventral surface bears two and a 5-day period for the forewing (Fig. 4). The smaller eyespots in homologous locations and, in ability to regenerate a marginal pattern and marginal addition, small dot-like patterns, homologous to the scales depended only on the timing of the ablation, centres of eyespots (which in some individuals in our not on its location, nor on the amount of tissue population develop into very small ocelli), on the removed from the disk (e.g. Figs 5B, 6). midlines of three additional wing cells (Fig. 2B). While the regenerated marginal patterns always The eyespots of the dorsal and ventral wing sur- had the same sequence of pattern elements and faces responded very differently to ablation of part of colours as normal marginal patterns, they differed in the wing disk. The eyespots on the ventral surface three respects from a perfectly normal colour pattern. were invariably diminished in size and often reduced First, the bands were almost always narrower than in to dots after ablations distal to their position the normal pattern (Figs 5,6). Second, the submar- (Fig. 5B). When an ablation removed the portion ginal band was absent in about one third of the of the wing disk containing the presumptive location animals we operated on (Fig. 6). We did not find its of the eyspots, they were always absent from the presence or absence to correlate with any parameter adult wing, even though normal marginal patterns of the experimental procedure. Third, the parafocal developed. elements often formed smooth continuous lines, par- The eyespots on the dorsal wing surface, by con- allel to the wing margin, instead of being indented trast, had two modes of response. If the cut was made into chevron-like segments by the wing veins as in the at a short distance from a presumptive eyespot the normal pattern (compare Figs 2 and 6). Smooth latter became diminished in size. However, if the cut was made through the field of a presumptive eyespot, parafocal element bands, which showed no interrup- it became enlarged and highly distorted. This distor- tions where they crossed a wing vein, occurred only tion had the characteristic of an 'opening up' of the after ablation of more than one third of the wing outer black ring of the eyespot into a horseshoe- or surface. This observation suggests that the wing veins omega-shaped figure with its opening towards the cut were able to affect the shape of the parafocal el- edge (Fig. 7A,B). These long eyespot-derived arcs ements only in the distal portions of the wing disk. By (Fig. 7C) could be easily distinguished from contrast, if a parafocal element is induced in the elongated parafocal elements (e.g. Fig. 6B) by differ- proximal portion of a wing disk, the veins appear to ences in their coloration. The outer rings of an have no effect on its morphology. eyespot are deep black, while the parafocal elements Pattern regulation in wing disks 383

are brown. In the most extreme cases, the outer black ring formed into a long black arc parallel to the wing margin (Fig. 7C). The fate of the marginal patterns along the wing edge next to such a distorted eyespot was variable. In the 34 cases of eyespot distortion that we produced in the course of this study, five devel- oped a normal marginal pattern along the cut edge. In the rest, the margin had yellow scales identical to those of the light ring immediately inside the black outer ring of the eyespot (Fig. 7). Thus, in the majority of cases, the margin took on the character- istic of the central field of the eyespot. These responses of the hindwing eyespots were found to depend only on the site of the cut, not on its timing. Ablations done at a physiological age of one day prior to pupation (the latest developmental time for which we were able to obtain data) induced 7A identical distortions to those done 9 days prior to pupation. We did not find a single instance in which the position of an eyespot was altered from its expected location in response to partial ablation of the wing disk. This observation suggests that the positions of the signalling sources that induce eyespots (Nijhout, 1980, 1985a) are already fixed by 9 days prior to pupation, even though the shape of the eyespots they will induce is not. We have also found that the eyespots on the dorsal and ventral side of the forewing responded to partial ablation in a manner identical to those on the ventral hindwing, as will be detailed elsewhere. Thus the eyespots on the dorsal hindwing of Precis coenia behave very differently from those on other wing surfaces.

Discussion

The wing disks of Precis coenia exhibited no ability to regenerate ablated portions, even in individuals that had a 9-day period between ablation and pupation. During the last 9 days of larval life, the wing disks undergo extensive mitoses and increase more than fourfold in size and cell number (Kremen, 1987). The failure to undergo compensatory growth in the face of an ample capacity for cell division suggests that there exists no mechanism for size and shape regulation in Fig. 7. Effects of partial ablation on the shape of the dorsal eyespots. (A,B) Cuts very near or just within the field of the eyespot cause a break or opening to develop in the outer black (b) and yellow (y) rings that faces the cut edge. (C) An extreme case of eyespot distortion in which the outer black (b) and yellow (y) rings have opened to form a long arc parallel to the wing margin. Note absence of marginal pattern elements in areas where eyespot central field (yellow ring and black central disk) approaches the margin closely. Bar, 5 mm. 384 H. F. Nijhout and L. W. Grunert the wing disk during the last 9 days of larval life. It is known from previous studies that the eyespots Similar failures of wing disks to regenerate have been on the dorsal hindwing differ morphologically and reported for Philosamia cynthia (Henke, 1933) and developmentally from those on the other wing sur- Papilio machaon (Magnussen, 1933). These findings faces (Nijhout, 1984, 19856). Developmentally, the stand in contrast to those of Meisenheimer (1908, eyespots on the dorsal hindwing and their surround- 1909), Von Ubisch (1911) and Bodenstein (1936) who ing epidermis have been shown to respond very have reported wing disk regeneration after complete differently to cautery during the pupal stage than do extirpation in Lymantria dispar and Vanessa urticae. homologous areas on the other wing surfaces (Nij- Von Ubisch (1911) has presented a well-documented hout, 19856). Cautery of the central region of a dorsal study, including drawings from histological sections, hindwing eyespot has no effect whatever on its showing that Lymantria can regenerate wings from development. By contrast, cautery of the cells at the undifferentiated thoracic epidermis, but neither he centre of a forewing eyespot early in the pupal stage nor Bodenstein appear to have investigated the completely abolishes its development, because these potential for regeneration after only partial ablation cells serve as the inductive centres for eyespot forma- of a wing disk. We are presently attempting to tion. Furthermore, cautery of the wing surface out- replicate the Von Ubisch experiments, to find histo- side an eyespot on the hindwing induces a small logical confirmation of this extraordinary regenerat- supernumerary eyespot around the site of injury ive response, and to determine whether and how this (Nijhout, 19856). This stands in contrast to the response differs in fully and partially ablated disks. findings that cauteries of the extraocellar areas of the When a portion of the wing imaginal disk was forewing or ventral hindwing have no effect on removed more than 3-5 days prior to pupation pattern (Nijhout, 1980). Nijhout (19856) has shown (Fig. 4), the new margin developed a normal fringe of by computer simulation that the unusual response of distinctive marginal scales and a normal or nearly the hindwing to cautery can be understood if it is normal colour pattern parallel to the new margin. assumed, first, that injury causes a local destruction The finding that the marginal patterns were always of the morphogenetic signal responsible for eyespot parallel to the new wing margin, irrespective of its induction and, second, that the normal eyespots on shape, together with the observation that homolo- the dorsal hindwing developed around relative sinks gous pattern elements in species with a highly scal- of that morphogenetic signal, not around sources as loped wing margin (e.g. Baeotus baeotus, Cethosia do those on the forewing. chrysippe) are strongly arched and also run parallel to If we now add to this the observations presented the normal outline of the margin, suggest that the above that the central field of the eyespots on the margin, perhaps through some special function of the dorsal hindwing and the wing margin share similar bordering lacuna, acts as an organizing centre for that properties and that the margin has an inductive role portion of the colour pattern. in colour pattern formation, we may provisionally There appears to be no regulation of the position of conclude that both the centre of the dorsal eyespot eyespots on a partial wing disk. This finding suggests and the wing margin have similar and compatible that the position of the eyespot-inducing centres inductive properties and that these are in some sense (foci, Nijhout, 1980) was already fixed at the time our the inverse (or opposite) of the inductive signal that is ablations were done and that an ablated disk was produced by the foci on the forewing (Nijhout, 1980). incapable of reinitiating the processes that lead to eyespot localization. By contrast, the shape and size We wish to thank Claire Kremen for critical comments on of eyespots could be altered dramatically, depending the manuscript. This work was supported by grants PCM- on the proximity of the cut to the field of the 8214535 and DCB-8517210 from the National Science Foun- presumptive ocellus and depending on the wing dation. surface in question. Eyespots on the ventral side were invariably diminished in size and reduced to dots. The large eyespots on the dorsal surface, by contrast, References were increased in size and highly distorted in shape when a cut was made within their presumptive field. BODENSTEIN, D. (1936). Das Determinationsgeschehen In the more extreme cases, the dorsal eyespots were bei Insekten mit Ausschluss der fruembryonalen modified into a broad open arc facing the cut edge. Determination. Ergebn. Biol. 13P, 174-234. Thus, the new cut edge in effect became part of the BRYANT, P. J. (1987). Experimental and genetic analysis of growth and cell proliferation in Drosophila imaginal central field of the eyespot. This observation suggests discs. In Genetic Regulation of Development (ed. W. F. that the central field of a dorsal eyespot and the wing Loomis), pp. 339-372. New York: A. R. Liss. margin share an important common developmental HENKE, K. (1936). Untersuchungen an Philosamia cynthia determinant. Drury zur Entwicklungsphysiologie des Pattern regulation in wing disks 385

Zeichnungsmusters auf dem Schmetterlingsfliigel. NIJHOUT, H. F. (1980). Pattern formation on lepidopteran Wilhelm Roux' Arch. EntwMech. Org. 128, 15-107. wings: Determination of an eyespot. Devi Biol. 80, KREMEN, C. (1987). Metamorphosis of the imaginal disks 267-274. and epidermis in the Buckeye butterfly, Precis coenia. NIJHOUT, H. F. (1984). Colour pattern modification by coldshock in Lepidoptera. J. Embryol. exp. Morph. 81, Thesis, Duke University. 287-305. KUHN, VON ENGELHARDT, A. & M. (1936). Uber die NIJHOUT, H. F. (1985a). The developmental physiology of Determination des Symmetriesystems auf dem color patterns in Lepidoptera. Adv. . Physiol. 18, Vorderfliigel von Ephestia kilhniella. Wilhelm Roux' 181-247. Archiv. EntwMech. Org. 130, 660-703. NIJHOUT, H. F. (1985b). Cautery-induced colour patterns MAGNUSSEN, K. (1933). Untersuchungen zur in Precis coenia (Lepidoptera: Nymphalidae). J. Entwicklungsphysiologie des Schmetterlingsfliigels. Embryol. exp. Morph. 86, 191-203. Wilhelm Roux' Archiv. EntwMech. Org. 128, 447-479. SUFFERT, F. (1929). Die Ausbildung des imaginalen MEISENHEIMER, J. (1908). Uber Flugelregeneration bei Fliigelschnittes in der Schmetterlingspuppe. Z. Morph. Schmetterlingen. Zool. Anz. 33, 689-698. Okol. Tiere 14, 338-359. VON UBISCH, L. (1911). Uber Flugelregeneration beim MEISENHEIMER, J. (1909). Die Flugelregeneration bei Schwammspinner, Lymantria dispar. Wilhelm Roux' Schmetterlingen. Verh. dt. zool. Ges. 19, 174-182. Archiv. EntwMech. Org. 31, 637-653. NIJHOUT, H. F. (1978). Pattern formation on lepidopteran wings: A model. J. exp. Zool. 206, 119-136. (Accepted 20 October 1987)