The formation of pattern on the wing of the moth, Ephestia kuhniella

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1 Development 13, (1988) Printed in Great Britain The Company of Biologists Limited The formation of pattern on the wing of the moth, Ephestia kuhniella NEIL TOUSSAINT and VERNON FRENCH Department of Zoology, Kings Buildings, West Mains Road, Edinburgh EH9 3JT, UK Summary Transverse bands of coloured scales are a common feature of lepidopteran wing patterns and it has been suggested that their positions are specified by a propagated 'Determination Wave' (Kuhn & von Englehardt), or a 'Gradient' of diffusible morphogen (Nijhout). We have assessed these models in an experimental study of the formation of bands in the moth, Ephestia kuhniella. The banding pattern can be altered by microcautery of the pupal wing between 1 h and 48 h postpupation (at 2 C) and effects are of two types: Local modifications follow early (1-3 h postpupation) cautery located on or between the presumptive sites of the bands; operations more proximally or distally on the wing have no effect. Patterns consist of a loop deflecting the nearest band medially, around the site of cautery; or an isolated ring of band scales surrounding the operation site, with the two bands in their normal positions. The type of pattern formed depends only on the location of the cautery, with rings following medial cautery (midway between the bands). Global modifications follow a h cautery anywhere on the wing surface and they consist of a medial displacement of both bands. The degree of displacement is very variable (with band separation reduced to % of that on the contralateral control wing) but this does not depend on location of the cautery. Furthermore, the degree of modification does not depend on the precise age at cautery, as an equivalent range of effects is produced at 36 h, at 42 h and at 48 h. The Determination Wave model is disproved since it explicitly assumes that the different degrees of global modification form a temporal sequence (from severe early to mild late) and they do not. Furthermore, this model cannot explain adequately the formation of loop or ring local modifications. The Gradient model can account for many features of the results, but it does not readily explain the ring local modifications or the effect of (36-48 h) cautery in causing a global change in the banding pattern, independent of the site or precise time of operation. Key words: pattern formation, insect wing, Ephestia kuhniella. Introduction The colour patterns on butterfly and moth wings are particularly spectacular and convenient experimental systems for investigating position-dependent differentiation. In the late pupa, epidermal scale cells in different positions on the wing synthesize different pigments and deposit them in the scale cuticle. Lepidopteran wings show an enormous diversity of colour patterns, but common elements are transverse bands and eyespots (concentric rings). The development of bands has been studied experimentally in two closely related moth species, Ephestia kuhniella and Plodia interpunctella. Ephestia has a simple banding pattern and microcautery of the early pupal wing results in a local pattern modification (a medial deflection of the band close to the site of operation), while a later cautery anywhere on the wing surface results in a global pattern modification in which both proximal and distal bands are displaced medially to a variable extent (Kuhn & von Englehardt, 1933). The Plodia wing has a more complicated pattern, but here also cautery results in early local and later global changes (Wehrmaker, 1959; Schwartz, 1962; Wilnecker, 198). From their results, Kuhn & von Englehardt (1933) postulated that the position of the bands is defined by a 'Determination Wave' which originates at sites midway along the anterior and posterior margins and travels out over the wing surface. They suggested no detailed mechanism, but the wave is best considered as a propagated signal which temporarily switches cells from 'field' to 'band' state (see Wilnecker, 198). Recently, Nijhout (1978, 1985ft) has reinterpreted

2 78 N. Toussaint and V. French the Ephestia results and proposed that the two major elements of lepidopteran wing patterns (bands and eyespots) are formed by the same 'Gradient' mechanism. He showed that the development of the eyespot of the butterfly, Precis coenia, depends on the presence of a small central region (the focus), and suggested that this may be the source of a gradient of diffusible morphogen whose different concentrations direct pigment synthesis (Nijhout, 198). A single focus would produce a conical gradient and an eyespot pattern, while a line of foci across the wing could produce a ridge and transverse banding pattern (Nijhout, 1978, 19856). The Determination Wave and Gradient models lead to different interpretations of the modified patterns resulting from cautery, and make some different predictions. A critical feature of the former model was the proposal that global modifications result from stopping the wave and, therefore, that cautery early in the sensitive period should result in the most extreme modifications. This was assumed by Kiihn & von Englehardt (and in most subsequent reviews, e.g. Sondhi, 1963), but they used coarsely timed animals and could not demonstrate the relationship, and no further work has been done on the simple banding pattern of Ephestia. A temporal sequence in the severity of global effects in Plodia was denied by Wehrmaker (1959) but supported by Schwartz (1962), but their animals were probably not adequately timed. More recently, Wilnecker (198) re-examined the effects of cautery on accurately timed Plodia pupae and obtained a wide variety of complex pattern modifications, but these did not show a clear relationship between severity of effect and time of operation. In the present experiments, we cauterized precisely timed Ephestia pupae, studied the range of modifications induced and, in particular, examined whether the degree of global modification depends on the time of the operation. Materials and methods Cultures of Ephestia kuhniella were kept in a 16:8 h light: dark cycle at 25 C C and fed on a mixture of 1 parts heat-sterilized wholemeal flour: 2 parts glycerol:l part yeast. The melanic mutant (b~) was used because the banding is simpler and clearer than on the wild-type moth, and alteration in pattern can be scored more accurately (see Kiihn & von Englehardt, 1933). Late final instar larvae were isolated and examined at regular intervals to determine the time of pupation (average accuracy = ±2min, range = -±85min). Pupae were placed in a 2 ± -5 C incubator and cauterized at approximately 1, 24,26, 31,42, 48, 6 and 72 h postpupation. There was no possible overlap between the age classes (see Table 1A). The left pupal forewing was cauterized with a sharpened tungsten needle attached to the heating element of a variable power source. The temperature of the tip of the needle was adjusted to 7 C using histological waxes. 'Sham-cautery' experiments were also performed on 24 h pupae, using an unheated needle. Pupae were secured on plasticine with the dorsal surface of the forewing upper- Table 1. Pattern modifications caused by cautery of the pupal wing Age class (h) sham con (A) Frequency No. Unmod. Mod. ND (B) Nature Loops Rings Global (A) The total number (no.) of animals cauterized in each age class, and the percentage with experimental wing patterns similar to (unmod.) and differing from the control (mod.) or nondeveloping (ND). sham refers to 24h pupae pierced with an unheated needle and con are control animals. Each age class (e.g. 26h) contained animals cauterized at approximately the same age. Operations at 25,56h (25h 56min) after a pupation timed to ±,7 h and at 26,35 h after pupation timed to ±,15 h must be between 25 h and 27 h, while an operation 26,38 h after pupation timed to ±,46 h could be 1 h older (+1 h). Examined in this way, maximum variability within age classes was: 1 h - 99/1 were -2h (1, +lh); 24h - 48/48 were 23-25h; 26h - 6/69 were 25-27h (9, +lh); 31 h - 45/87 were 3O-32h (37, -lh, 5, +1 h); 36 h- 7/73 were h (2, +lh, 1, -lh); 42h - 51/68 were h (1, +lh,3, +2h, 4, +3 h); 48h - 63/75 were h (11, +lh, 1, -1 h); 6h- 5/67 were h (7, +lh, 1, +2h), and 72h - 75/78 were 71-73h (2, +lh, 1, +2h). There was no possible overlap between age classes. (B) The number of animals with local (loop and ring) and global pattern modifications on the experimental wing.

3 Pattern formation on moth wings 79 most. With the needle supported in a micromanipulator, the cuticle was pierced at a site located with reference to the pattern of veins, the needle held in place for 1-2 s and withdrawn. In some animals, the extent of epidermal cell death was examined immediately or 24 h after cautery. Wings were dissected in insect saline, covered with a 4% trypan blue solution for 2-5 min and examined under phase contrast. Cauterized pupae were returned to 2 C until adult emergence (approximately 16 days). The adult moths were placed into a 2 C freezer immediately following wing extension, to ensure that as few scales as possible were lost from the surface of the wing. Wings were removed, glued onto microscope slides, illuminated from the distal end and viewed through a polarizing filter to reduce reflection from the scales. Camera-lucida drawings were made of each experimental and contralateral control wing, showing banding patterns and the extent of any damage caused by the operation. Results The normal wing pattern Most of the dorsal surface of the forewing of the melanic mutant Ephestia is covered with dark pigmented scales, but two irregular bands of whitetipped scales extend from its anterior to posterior margin (Fig. 1A). The wings of different animals vary in size and details of pattern, but the right and left wings were always highly symmetrical (Fig. 2), so the contralateral wing of an experimental animal can be used as a precise control for the effect of cautery. Damage caused by microcautery The area of dead cells present around a site of cautery immediately after the operation usually corresponded to 1-3cells. 24h after the operation, the area of trypan blue staining was smaller but still variable in size. Cautery of the pupal wing at various times during early development almost always resulted in damage to the adult wing, with the formation of a lesion (a patch of cuticle lacking scales and sockets) and sometimes also a hole. Although cautery was performed at a standard temperature for a constant duration, the area of the lesion on the adult wing was variable and seemingly unrelated to the age of the pupa. Lesions did not develop following 'shamcautery' with an unheated needle. By relating the location at which the pupal wing was cauterized to the site of the resulting lesion on the adult wing, a correspondence map was constructed (Fig. 3). Lesions resulting from cautery at a particular pupal location were restricted to a fairly specific region on the wing, especially following cautery at a readily defined position such as the branch point of pupal veins. The effect of cautery on the banding pattern Following cautery performed between 1 and 48 h after pupation, the banding pattern on the experimental wing frequently differed from that on the contralateral control wing, although the frequency of modifications was rather low on wings cauterized at lh (Table 1A). Effects were rare (<5 %) following cautery at or after 6 h, indicating that the future position of the bands becomes determined between 48 and 6 h postpupation. Depending on the exact timing and location of cautery, the banding pattern was altered in one of two characteristic ways (Table IB): (1) Local pattern modifications Only part of the banding pattern was affected and the effect depended on the location of the lesion. These patterns were produced mainly after early cautery (1 31 h postpupation) and there were two types: loops (116 cases) and rings (75 cases). (a) Loops. The transverse band nearest the site of the lesion was deflected medially, isolating the lesion from the central field (Figs IB, 4A). Loop patterns were rather variable in nature, involving only a localized region of the band (Fig. 4Ai) or sometimes displacing most or all of the band (Fig. 4Aiv). This variability is not clearly correlated with age at cautery (e.g. Fig. 4Ai & ii show wings with similar loops following cautery at different ages) or with severity of the operation (as judged by the extent of damage on the adult wing). Cautery in a medial position gave a more extensive loop than cautery close to the normal band site, but did not necessarily involve the whole band (compare Figs IB and 4Aiii). (b) Rings. The position of both transverse bands was unaltered with respect to the control pattern, but an ectopic ring (often incomplete) of white band scales surrounded the lesion (Figs 1C, 4B). Rings produced by cautery towards the proximal or distal edge of the central field were usually fused with the nearest transverse band (Fig. 4Biii), and those formed close to the anterior or posterior wing margin were incomplete (Fig. 4Biv). Rings were rather variable in diameter, but this was not correlated with age at cautery or the severity of the operation (as judged by the extent of damage). However, a few rings formed following 'sham-cautery' (see Table IB) and these were very small. The location of the site of cautery determined which type of local pattern modification was formed. Operations in the proximal and distal regions of the central field formed proximal and distal loops, respectively, while cautery in the medial part of the wing tended to result in the formation of a ring

4 71 N. Toussaint and V. French Fig. 1. Normal and altered banding patterns on the dorsal surface of the forewing of Ephestia. (A) Control wing showing the proximal (P) and distal (Z)) bands of white-tipped scales and a distal fringe of marginal scales (m). Bar, 1 mm. (B) Local pattern modification in which the proximal band loops around the lesion (arrowed). (C) Ring of band scales (R) surrounding medial lesion, while the position of proximal and distal transverse bands is unaltered. (D) Global pattern modification in which both transverse bands are located more medially than on the control wing. (Fig. 4C). Local modifications were not observed when the lesion was located outside the prospective central field. (2) Global pattern modifications The whole banding pattern on the experimental wing was altered with respect to the control wing; the

5 Pattern formation on moth wings 711 post ant proximal band (often enlarged) occupied a more distal position and the distal band was displaced proximally (Figs ID, 5A). Global pattern modifications were produced mainly after cautery at h after pupation (Table IB) and, unlike the local effects, occurred even when the site of cautery was outside the prospective central field (Figs ID, 5Aiv, 5B). The size of the central field was measured along the medial axis of the experimental and control wings (Fig. 2C), and the degree of pattern modification was found to be variable (central field reduced to % of its control). There was no indication that the degree of pattern modification was related to the size of lesion on the wing, suggesting that differences in the severity of the operation do not account for the variability in result. Fig. 2. (A) Camera lucida drawings of left and right forewings of a control animal to show proximal (P) and distal (D) bands, the region they enclose (the central field, CF), the proximal and distal fields (PF and DF) and the vein pattern. The distal margin of the wing has a fringe of long marginal scales (m), the maximum extent of which is illustrated by the dotted line. Bar, lmm. (B) The similarity of banding pattern of right and left wings. Wings were drawn at constant magnification (xl8) onto tracing paper and the image of the left wing reversed and superimposed onto the right (bands shown by dotted lines) such : that the anterior (ant), posterior (post) and distal (dist) margins of the two coincide. (C) Separation of proximal and distal bands. The measurement was taken along the medial axis of the wing. Relative size of the central fields (left wing with respect to right) for the wing pair shown in B is 98-5%. Furthermore, there were no significant differences (P>-5) in the mean reduction in the size of the central field between animals cauterized in the presumptive proximal, central and distal fields (Table 2B), showing that the response was independent of the site of cautery. It has previously been assumed that the degree of global pattern modification is directly related to age at cautery (see Introduction). In the present experiment, most modifications occurred following cautery at 36, 42 and 48h postpupation (Table IB). Each of the three age classes contained the full range of modified patterns (Fig. 6A), and there were no significant differences (P> -5) in the degree to which the two bands were displaced (Table 2C). Furthermore, there was no indication that the few global

6 712 N. Toussaint and V. French \ Fig. 3. Defect map relating the site of cautery on the pupal wing to the location of the lesion on the adult wing. Central figure shows the left pupal wing (nomenclature for venation follows Kuhn & von Englehardt, 1933) and the four outer figures shown left adult wings with lines linking sites of pupal cautery to the centres of the resulting lesions. The numbers on the outer figures indicate the number of adults with lesions at that site. modifications produced by early (31 h) or late (6, 72 h) cautery differed from the rest (Fig. 6B). These results clearly demonstrate that the degree of global modification is not related to the time of cautery and the patterns do not constitute a temporal sequence. Discussion Cautery of the forewing of the early pupa can alter the banding pattern which appears much later, as the adult scales become pigmented. Analysis of these modifications eliminates one of the mechanisms that have been proposed for specifying the location of the bands. Effects of cautery Trypan blue staining demonstrates directly that the cautery kills cells and that these are replaced, presumably by migration of surrounding epidermal cells into the wounded area and perhaps also by local stimulation of division to restore cell density (see Wright & Lawrence, 1981; Anderson & French, 1985; Campbell, 1987). There was considerable variability in the initial area of cell death, perhaps resulting from slight differences in the duration and the depth of cautery. Since most operations result in a lesion (a patch of cuticle lacking scales and sockets), it seems likely that new scale mother cells (whose granddaughters will form scale and socket; Stossberg, 1938) are already determined and new ones cannot be regenerated after cautery. However, the size of the lesion will depend on the degree of scale mother cell migration during healing, so it may not be a reliable measure of the severity of cautery and initial damage. Pattern modifications and the mechanism of band formation Cautery kills a small region of cells and directly

7 Pattern formation on moth wings 713 Fig. 4. Local pattern modifications. Black area shows the lesion or hole in the wing. (A) Four loop patterns resulting from distal (j,ii) and proximal (iii.iv) cautery at (i) 1,5 ±,11 h, (it) 24,7 ±,12 h, (Hi) 26,35 ±,15 h and (iv) 3,56 ±,5h postpupation. (B) Four ring patterns following cautery in a medial location at (i) 25,56 ±,7 h, (u) at 24,4 ±,9 h, (Hi) 24,32 ±,h and (iv) 26,2 ±,15 h postpupation. (C) The location of lesions (shaded or stippled regions) on the wings which formed (i) distal (D,n = 39) and proximal loops (P, n = 34) and (it) rings (R, n = 63). Not all patterns are represented in this figure because, in some cases, there was no detectable lesion. affects nearby survivors, and an early cautery alters the pattern locally, in the region of the lesion. However, a later cautery has a global effect, changing the location of both the proximal and distal bands, regardless of the position of the cautery on the wing surface. The global response is not systemic, however, since only the cauterized wing is modified, while the contralateral wing forms a normal pattern.

8 714 N. Toussaint and V. French \ iv) Fig. 5. Global pattern modifications. (A) Patterns following cautery in different locations at (1) 48,1 ±,15 h, causing a reduction of the central field to 61-7 %, (u) 36,23 ±,3h (central field 54-1 %), («i) 48,5 ±,25h (central field 71-1 %) and (iv) 36,5 ±,22 h (central field 74-5 %). (B) The location of lesions (stippled) on wings with global pattern modifications (n = 113). Not all patterns are represented in this figure because, in some cases, there was no detectable lesion. Table 2. Global pattern modifications: effect of time and location of cautery Central field mean size (mm)' Central field modification n right left left/right % ±1-2 (A) Controls (B) Position of lesion Proximal Medial Distal (C) Age at cautery 36h 42 h 48h ± ± ± ±16 41 ± ±1 4-1 ± ± ± ± ± ± ± ± ± ± ± ± ± ±4-5 The mean size of the central field (* with 95 % confidence limits) of the right and left wings of (A) controls and (B,C) animals showing global modifications after cautery of the left wing. Operated animals were classified as cauterized (B) in the proximal field (proximal), the central field (medial) or the distal field (distal), and (C) at 36, 42 or 48h after pupation. The table also shows the mean degree of pattern modification (* with 95 % confidence limits), with the central field of the left wing expressed as a percentage of that on its control right wing. Not all wing patterns are represented (see Table 1) as sometimes there was no detectable lesion, or the central field could not be accurately measured. There is no significant difference (t test, / > >5) in the degree of global modification between any pair of position classes (B), or between any pair of age classes (C), although all differ (P<5) from the control (A).

9 Pattern formation on moth wings 715 Fig. 6. Lack of effect of age at cautery on degree of global pattern modification. (A) Different degrees of modification following cautery of pupae of the 42h age class, with central field reduced to (/') 86-5 %, (ii) 8- %, (Hi) 73-1 % and (iv) 55-9 % of the size of the control. (B) Similar modifications resulting from cautery (i) early, at 3,5 ±,2 h (central field 72-2 %) and (ii) late, at 6,8 ±,15 h (central field 86-3 %). In many lepidopteran species, abnormal wing patterns ('phenocopies') can be induced by heat or cold shocks at pupal stage (Goldschmidt, 1938, Nijhout, 1984). In this way, a small but variable reduction in the separation of the bands can be produced on the Ephestia wing (Kiihn & Henke, 1936) and Nijhout (1985a) has suggested that global modifications are, in fact, phenocopies restricted to the cauterized, heated wing. A heat shock response to cautery could interfere with pattern formation by stopping gene transcription (see Mitchell & Lipps, 1978), but it is difficult to understand why this should be uniform over the wing surface, and generate similar effects on bands close to and far from the heat source (e.g. Fig. 5). In almost all cases, the response of the banding pattern to cautery can be readily classified as 'local' or 'global', as was found by Kiihn & von Englehardt (1933). In the present experiments, the pattern formed on each cauterized wing was compared precisely with its appropriate control (Fig. 2) and, with this technique and much more precise timing of operated animals, we have been able to extend Kiihn & von Englehardt's (1933) results in several ways. (i) A local pattern modification may be either a medial loop in one of the bands or a ring, formed in addition to the normally positioned bands. As the site of cautery is moved medially within the presumptive central field, the resulting pattern changes smoothly from small loop (Fig. 4Ai), to large loop (Fig. IB), to ring merging with a band (Fig. 4Biii) to an isolated ring plus normal bands (Fig. 4Bi,ii). (ii) As shown in Table IB, the response changes abruptly from local (91 % of interpretable modifications following cautery at 31 h postpupation) to global (76% at 36 h), indicating a rapid change of state within the epidermis. Kuhn & von Englehardt (1933), working with pupae timed only to ±6h, found a more gradual change in response. (iii) Kiihn & von Englehardt (1933) assumed, but did not establish, that the global modifications form a temporal sequence (from early severe to late mild), but the present results show clearly that this is not the case. Direct measurement of the central fields of experimental and contralateral control wings quantifies the degree of modification, and there is no

10 716 N. Toussaint and V. French ant post Fig. 7. Models of the formation of the banding pattern on Ephestia wings. (A) Determination wave model (adapted from Kuhn & von Englehardt, 1933). (i) The normal pattern of proximal (P) and distal (D) bands formed at the front of the 'determination wave' (arrows) emanating from anterior (ant) and posterior (post) edges of the wing and spreading nonuniformly across the surface. Early cautery (it) inside the prospective bands causes an area of dead or damaged cells (black) which locally block subsequent wave propagation, preventing more marginal cells being switched to 'band' state. It is unclear why this gives a continuous loop with medial cells (nonstippled) not reverting from 'band' to 'field' state. Cautery during the period of propagation (Hi) freezes the wave, even if it is inflicted outside the band positions (star). Early cautery gives an extreme modification, while stopping the wave later gives bands (dashed lines) close to their normal positions. (B) Gradient model (from Nijhout, 1978, 19856). (i) Three foci (F) act as sources of diffusible morphogen which forms a transverse ridge gradient (concentration contours 9-1 shown by dashed lines). Band scales are formed at gradient levels 2-5. Early cautery (if) forms a local morphogen sink (open circle, concentration O) distorting the gradient profile and causing the distal bands (D) to loop around the site of cautery. In some way, later cautery (Hi) may lower the gradient concentration and thereby cause the bands to form in a more medial position. difference between the patterns produced by cautery at 36, 42 and 48 h (Table 2B). A dependence of the pattern on age would produce a difference in the average degree of modification after early and late cautery, even if the animals varied in the time of onset or the rate of determination of band position, or if the response also depended upon the severity of the operation. The Ephestia global modifications, however, show no indication of a dependence on age at cautery. Two models have been proposed for the formation of the wing pattern in Ephestia and they can be reassessed in the light of these new results. (1) The Determination Wave model Kuhn & von Englehardt (1933) proposed that the location of the bands was defined by the position of a propagating wave on the wing epidermis at the time of scale colour determination (Fig. 7Ai). They suggested local (loop) modifications occur because early cautery, inflicted before the wave is generated, creates a local barrier to wave propagation. This could explain the failure of cells beyond (marginal to) a lesion to form white band scales, but not the formation of a continuous loop pattern involving the cells medial to the lesion (Fig. 7Aii). Similarly, a propagated wave could not explain the isolated medial ring of bands scales accompanying complete bands in their normal positions (Fig. 4B). Also, cautery on the wing margins (at or close to the postulated origins of the wave) should produce major disruptions of proximal and distal bands, rather than the isolated half-rings that result from this operation (Fig. 4Biv). The global pattern modifications were interpreted

11 Pattern formation on moth wings 111 by Kiihn & von Engelhardt (1933) as evidence for the spread of the 'determination wave'. They assumed that wave propagation across the wing blade occurs during the period of sensitivity and is completely arrested by cautery. They arranged the global patterns into what they assumed was a temporal sequence (figs 12,13 of Kiihn & von Englehardt, 1933). Early cautery would stop the wave close to its origin and produce a severe modification with bands close together, while late cautery would stop the wave close to its normal terminus and produce a mild effect with bands only slightly closer than normal (Fig. 7Aiii). The present results show clearly that there is no temporal sequence underlying the variability in the degree of global modification and hence disprove the Determination Wave model. (2) The Gradient model Nijhout (1978, 1985a) suggested that the Ephestia bands are formed in response to levels of a gradient of diffusible morphogen produced by a line of three sources situated on the anterior and posterior margins and in the centre of the wing (Fig. 7B). In computer simulations, cautery within the presumptive central field can cause a loop to form in the nearby band, but only if it locally destroys morphogen, rather than merely creates a barrier to diffusion (fig. 5 of Nijhout, 1985a - see also Murray, 1981). It is also possible to simulate the formation of more-or-less normal bands plus a small ring surrounding the site of cautery (fig. 5D of Nijhout, 1985a). However, it is difficult to explain many of the present local modifications where a large ring is formed within the central field but the positions of both bands are unaffected. In many cases, the large ring clearly encloses the postulated central source (e.g. Fig. 5Bi,ii) and the central region of both bands would be expected to be displaced medially. Similarly, a lesion close to the margin, which produces a large arc enclosing the postulated anterior or posterior source (Fig. 5Biv), would be expected to reduce the width of the central field at that edge. Nijhout (1978, 1985a, >) does not discuss Ephestia global pattern modifications in detail, but the medial displacement of both bands would correspond to a lower gradient profile (Fig. 7Biii), which could depend on the time of cautery. If the cautery had a permanent but gradual effect on the gradient (e.g. by stopping or reducing morphogen synthesis), an early operation would produce a severe change and a late cautery only a mild disturbance. Alternatively, if cautery had only a temporary effect from which the system could recover, a late operation just before pattern determination would cause the most extreme modification. The lack of any temporal relationship suggests that cautery within the h time period has a rapid and permanent effect on the gradient (or the way in which it is interpreted). Conclusion The Determination Wave model must be rejected as it is unable to explain the range of local pattern modifications induced by cautery. Furthermore, it explicitly predicts that the global modifications will form a temporal sequence, and they do not. The Gradient model remains plausible for band formation, but it is difficult to explain the ring class of local modifications which follow early cautery, or to interpret the effect of later (36-48 h) cautery in causing a global change independent of the site or precise time of operation. This work was supported by a Sir David Baxter Memorial Scholarship (to N.T.) and the stock of Ephestia was kindly provided by Professor W. B. Cotter, We thank Jonathan Bard, Paul Brakefield, Jim Murray and, especially, Brian Goodwin for helpful and enjoyable arguments. References ANDERSON, H. J. & FRENCH, V. (1985). Cell division during intercalary regeneration in the cockroach leg. /. Embryol. exp. Morph. 9, CAMPBELL, G. L. (1987). Cell behaviour during postembryonic pattern regulation in the insect abdomen (Oncopeltus fasciatus). II. Intrasegmental regulation. Development 11, GOLDSCHMIDT, R. B. (1938). Physiological Genetics. New York: McGraw-Hill. KUHN, A. & HENKE, K. (1936). Genetische und Entwicklungsphysiologische Untersuchungen an der Mehlmotte Ephestia kuhniella Zeller. Abh. Ges. Wiss. Gott. Math.-Phys. Kl N.F. 15, KUHN, A. & VON ENGLEHARDT, M. (1933). Uber die Determination des Symmetriesystems auf dem Vorderfliigel von Ephestia kuhniella. Wilhelm Roux' Arch. EntwMech. 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