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Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae)
Journal of Insect Physiology 46 (2000) 251–258 www.elsevier.com/locate/jinsphys
Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae) Andrew L. Miner, Allison J. Rosenberg, H. Frederik Nijhout
*
Department of Zoology, Duke University, Durham, NC 27708-0325, USA Received 25 May 1999; accepted 3 August 1999
Abstract During the last larval instar, the wing imaginal disks of Precis coenia grow continuously. The rate of disk growth is not diskautonomous but closely matches the rate of somatic growth of the larva, so that the size of the disks is a function of the size of the body, irrespective of the growth rate of the larva. When larvae are starved, their wing disks cease growth within 4 h, which indicates the existence of an efficient coupling mechanism between the growth of the soma and growth of the imaginal disks. Disk growth is inhibited by juvenile hormone in a dose-dependent manner. In the presence of the hormone the wing disks stop growing even while the larva continues to grow normally. During the last larval instar the wing imaginal disks also undergo a complex differentiation, consisting of the development of the lacunae and tracheation that define the future adult wing venation system. In normally growing larvae, differentiation of the wing disk is tightly correlated with wing size. Differentiation and size can be dissociated by starvation. If larvae are starved at any time after differentiation has begun, differentiation continues at a normal rate, even though the wing disk does not grow. Differentiation does not begin spontaneously in larvae that are starved before differentiation has begun. These findings indicate that the initiation of differentiation and its continuation are controlled independently. Juvenile hormone inhibits differentiation in a dose-dependent manner. Upon treatment with juvenile hormone, the stage of differentiation becomes fixed. These findings indicate that continued differentiation of the wing disk can only occur in the absence of juvenile hormone. Although the circulating level of juvenile hormone may be elevated during starvation, it is unlikely that this elevation is responsible for the observed effect of starvation on growth and differentiation of the disk. 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Precis coenia; Imaginal disk; Growth; Methoprene; Juvenile hormone; Nutrition
1. Introduction The adult appendages of holometabolous insects begin their development within the larva as imaginal disks. Under normal conditions the imaginal disks grow continuously and roughly exponentially, at least during the later larval stages and prepupal stage (Sehnal, 1985; Kremen and Nijhout, 1998). At some time during metamorphosis the disks stop growing and then proceed to differentiate into appendages that are correctly proportioned to the adult body. Little is known about the mechanisms that control the growth rate of imaginal disks, nor about the mechanisms
* Corresponding author. Tel.: +1-919-684-2793; fax: +1-919-6846168. E-mail address: [email protected] (H. Frederik Nijhout)
that control the final size to which a particular disk will grow. Evidence from Drosophila suggests that disks control their own growth through an intrinsic mechanism. Numerous experiments have demonstrated that when disks, or fragments of disks, are transplanted into an adult abdomen they grow to about their normal final size, in spite of the fact that the adult environment is vastly different from that of the larva in which disks normally grow and develop (Bryant and Simpson, 1984; Wilder and Perrimon, 1996). Hence, in Drosophila, the differences in the larval and adult environment appear to be irrelevant to the normal control of disk growth. In Precis coenia, by contrast, disk growth is quite sensitive to the developmental environment. Surgical removal of one or two wing disks, for instance, enhances the growth of the remaining wing disks, which then grow much larger than they otherwise would (Nijhout and Emlen, 1998; Klingenberg and Nijhout, 1998). This finding indi-
0022-1910/00/$ - see front matter 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 9 ) 0 0 1 7 7 - 8
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cates that the growth of wing disks is somehow limited by the presence of other disks, possibly through competition for a limiting resource required for growth. The final size of the disk thus appears to be affected by factors in the environment in which the disk develops. The regulation of the final size of a disk could occur through two mechanisms. The disk could always remain a function of the body size of the larva as it grows, irrespective of the vagaries of nutrition, so that it always produces a final disk that is correctly proportioned to the final size of the larva. Alternatively, a disk could have a growth trajectory that is largely independent of the growth of the larva, in which case final size regulation must take place sometime during metamorphosis. In either case, there must exist some kind of interaction between disks and body by which the proportionality of disk to body size is either maintained throughout growth or achieved during metamorphosis. In the present paper we investigate the physiological control of wing disk growth in Precis coenia, during normal growth and during starvation of the larva, in order to determine whether wing disks can grow independently of body growth. We also examine the ability of juvenile hormone to dissociate disk and body growth.
2. Materials and methods Larvae of Precis coenia were reared on an artificial diet at 27°C and a long-day (16L:8D) photoperiod. All larvae used in these experiments were selected and weighed within 1 h of eclosion to the 5th (final) larval instar, after which they were placed in individual cups and their growth was monitored by daily weighing. Prior to surgery larvae were anaesthetized by submersion in water for 10 min. Wing disks were removed from larvae submerged in saline, through a small incision in the lateral thoracic wall immediately overlying the disk. More than 90% of the larvae survived this operation and continued to grow normally at a rate indistinguishable from that of sham operated larvae. The size of wing disks was determined by means of a bicinchoninic acid (BCA) protein assay (Pierce), using the following protocol. Each disk was sonicated three times for 5 s in 200 µl of saline using a Fisher Dismembranator with a microprobe attachment. The homogenate was then placed on ice and 1 ml of test reagent was added. The mixture was incubated in a water bath at 36°C for exactly 30 min and then returned to ice. After cooling the mixture was centrifuged at 14,000g for 2 min and the absorbance of the supernatant was read in a spectrophotometer at 562 nm. A bovine serum albumin (Sigma) solution was used as a standard, and a dilution series of standards was processed with each set of samples. The juvenile hormone analog Methoprene was dissolved in acetone to a concentration that allowed the appropriate dose to be applied
in a volume of either 5 or 10 µl, and applied with a micropipette to the dorsum of the larva. Control larvae received either 5 or 10 µl of acetone. In the course of the final larval instar the wing imaginal disks undergo a complex differentiation, consisting of the development of an extensive pattern of lacunae and tracheae that define the future venation system of the wing. A scoring system was developed for measuring the degree of vein differentiation of the wing disk. This scoring system is described in Fig. 4 and its caption.
3. Results 3.1. The relation between body size and wing disk size In order to study the control of growth and differentiation of the wing disks it is necessary to have a method for obtaining standardized larvae whose stage of wing disk development can be predicted. Chronological age since the last molt and weight are typically used for staging lepidopteran larvae (Nijhout and Williams, 1974; Goodman et al., 1985). Larvae of Precis are quite variable in their growth rate and also vary in the duration of the 5th instar, which ranges from 5 to 9 days. Furthermore, the size of larvae at the beginning of the 5th instar is highly variable and can range from 0.09 to 0.22 g (live weight). Larvae with large body sizes at the beginning of the 5th instar grow faster than larvae with initially small body sizes (Kremen and Nijhout, 1998). Consequently, the chronological age and the body weight of a 5th instar larva cannot, by themselves, provide an accurate measure of physiological or developmental stage of the larva. We found that larvae of the same chronological age can have imaginal disks at very different stages of development (r2=0.48). Likewise, larvae of the same body weight can have imaginal disks at very different stages in development (r2=0.69). Thus neither chronological age nor body size of the larva can be used by itself to predict the stage of development of its imaginal disks precisely. We tested whether a combination of both measures provided greater predictive ability by measuring the wing disks of a cohort of larvae of the same weight and age. Such selected larvae also had wing disks in different stages of development, and this combination of measures actually provided a poorer predictor than either weight or age alone (r2=0.41). We suspected that the variation in initial size of the 5th instar could be responsible for the poor correlation between larval age, size, and the degree of wing disk development, because larvae that start the 5th instar at different initial sizes have different growth trajectories. We found that wing disk size and the stage of development were, in fact, most highly correlated with the percentage of weight gained since the beginning of the 5th
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instar (r2=0.76). Accordingly, all our size measures will be given as the ratio between the weight at the time of observation and the initial weight of the 5th instar larva. We’ll call this measure of size the growth ratio (a growth ratio of 2.0 means that at the time of observation the larva was twice the weight it had at the beginning of the instar). The relationship between the growth ratio and the size of the fore wing imaginal disk is shown in Fig. 1. A linear and an exponential regression fit the data equally well. In view of the fact that an exponential relationship most accurately describes other features of imaginal disk growth (see below, and Nijhout and Wheeler, 1995; Kremen and Nijhout, 1989), we present the exponential regression in Fig. 1. The accuracy of our method for determining disk size was tested by measuring sets of fore and hind wings of the same individual (Fig. 2). The fore wing imaginal disk of Precis is about 1.4× the size of the hind wing disk over a broad range of disk sizes (Fig. 2). These measures were highly correlated (r2=0.92), which suggests that the scatter observed in Fig. 1 represents real individual variation rather than measurement error. 3.2. The growth of disks during starvation In the growing 5th instar larva the sizes of the wing disks are a function of the growth ratio (Fig. 1). Because the growth rate of the larvae is inherently variable in this species, this observation implies that disks must grow more slowly in slow growing larvae than they do in fast growing larvae. A mechanism must therefore exist that coordinates the rate of disk growth with overall growth of the body.
Fig. 1. Relationship between body size and wing imaginal disk size during the final larval instar. Body size is measured as the growth ratio, the ratio of the weight of the larva at the time of measurement and the initial weight of the larva at the molt to the final instar. An exponential regression is fitted to the data (r2=0.76).
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Fig. 2. Relationship between the size of fore and hind wing imaginal disks during the last larval instar (r2=0.92). Fore and hind wing maintain the same relative sizes throughout their growth period.
We determined whether the growth rate of the disk is directly responsive to alterations of the growth rate of the body by starving larvae at various body weights and noting the effect of starvation on the subsequent growth of the wing disks. Experimental larvae were weighed, and one fore wing disk was removed to determine the size of the disks at the beginning of the experiment. Larvae were then starved for either 24 or 48 h, after which the other fore wing disk was removed. All experimental larvae were provided with a small block of 4% agar in distilled water to prevent dehydration. Control larvae were weighed and also had one fore wing disk removed, but were then allowed to feed normally for 24 or 48 h before the other fore wing disk was removed. The results of this experiment are illustrated in Fig. 3, which plots the percent growth of wing disks after 24 and 48 h for
Fig. 3. Growth of the fore wing imaginal disk in starved larvae and normally growing control larvae. Growth is measured as the percentage increase of disk size after 24 and 48 h. Bars indicate standard errors of the means. The imaginal disks appear to stop growing within hours after food is removed.
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both experimental and control larvae. The wing disks of control larvae grew normally during the experimental period and maintained the relationship to body size (as measured by the growth ratio) shown in Fig. 1. Control disks grew by 65% during the 24 h feeding period and by 177% during the 48 h period, which is consistent with the exponential growth trajectory of disks shown in Fig. 1. The disks of starved larvae grew 4% during the 24 h starvation period and 15% during the 48 h starvation period. The amounts of disk growth during 24 and 48 h of starvation were not significantly different form each other (t-test, P=0.075). It is possible to obtain an estimate of the time required for the disks to stop growing after the initiation of starvation from the data in Fig. 3. The averaged disk growth rates for control animals were 2.8 and 3.6% per hour for the 24 and 48 h periods, respectively. If we assume the 4 and 15% growth increments of the disks in starved larvae are accurate measures of how much the disks continued to grow after starvation began, then the disks of starved animals stopped growing after 1.4 and 4.2 h, respectively. Taking the average of these figures, the data suggest that in starved animals the wing disk continued to grow about 2.8 h after starvation began. The data available do not allow us to determine whether the disks grew normally for several hours and then stopped abruptly, or whether their growth rate gradually diminished over a longer period of time.
significantly from that in feeding larvae. This relationship is shown in Fig. 5 (curve B). In starved larvae, the wing venation was more differentiated for disks of a particular size than was the case in control larvae. This observation implies that differentiation of the venation system must continue for some time after disk growth stops. By only considering the relationship between disk size and its state of differentiation, the data presented in Fig. 5 conceal an interesting feature about the dynamics of vein differentiation. The effect of starvation on the dynamics of differentiation is revealed when we consider how much the state of differentiation of a disk changed during a 48 h period of feeding or starvation. Fig. 6 shows that the progression of disk differentiation during starvation depends on the degree of differentiation the disk had attained at the beginning of starvation. Disks that had not yet begun to differentiate did not initiate differentiation, whereas disks that had already initiated differentiation progressed about as far during the next 48 h of starvation as did disks in normally feeding larvae. Analysis of the data in Fig. 6 indicates that the initiation of differentiation of the wing disk appears to be coupled to the growth (or size) of the disk, but the continuation of differentiation is uncoupled from disk growth and is only marginally inhibited by starvation.
3.3. The relationship between disk growth and differentiation
Kremen and Nijhout (1998) presented evidence that mitosis in wing imaginal disks of 5th instar larvae of Precis can be inhibited by JH. The functional significance of this inhibitory effect is not clear at present, but it appears to be a special feature of the physiology of the last larval instar, because such inhibition does not occur in earlier instars (Kremen and Nijhout, 1998). In Manduca sexta, titers of JH gradually become elevated during starvation, and if this is the case in Precis also, then an increase in JH titer in response to starvation could account for the inhibition of mitosis and disk growth during starvation. We examined the effect of exogenous applications of the JH analog methoprene (JHA) on the growth of wing imaginal disks. Larvae for this experiment were synchronized as follows: freshly molted 5th instar larvae weighing between 0.14 and 0.18 g were selected and allowed to grow in individual containers for 48 h. At that time, only larvae weighing between 0.27 and 0.34 g were selected for treatment with JHA. The appropriate dose of JHA, dissolved in 5 µl of acetone, was applied to the dorsum of each larva once a day for three consecutive days. Control animals received daily applications of acetone alone. On the 5th day the fore wing disks were removed, their degree of differentiation was scored, and they were then homogenized for protein analysis.
The wing imaginal disks of Precis undergo considerable differentiation in the course of the 5th larval instar. During the first third of the instar a system of lacunae forms in the wing disk that outline the future venation of the adult wing. During the middle and later portion of the instar these lacunae are invaded by the tracheal system so that at the end of the 5th instar the wing disk is fully tracheated and the complete venation pattern of the adult is evident. In order to measure the timing and rate of vein differentiation and tracheation we used the numerical scoring system illustrated in Fig. 4. The differentiation of the venation system is not well correlated with the chronological age of the larvae, nor with its weight. Rather differentiation is correlated with the size of the wing disk, as illustrated in Fig. 5 (curve A). We noted that in starved larvae the tracheal differentiation of the wing disk was also inhibited, and we investigated whether growth and differentiation of the disk remained tightly coupled or whether they could be uncoupled by starvation. Uncoupling of growth and differentiation can be measured by determining whether the relationship between disk size and differentiation in larvae starved for 48 hours at a range of body sizes deviates
3.4. The effect of juvenile hormone (JH) on disk growth and differentiation
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Fig. 4. Differentiation of the fore wing imaginal disk during the middle portion of the last larval instar. Tracheae are air-filled and show up black in these transmitted-light photographs. Scoring system for differentiation is indicated at the bottom of each panel: (a) score 0: tight tracheal bundle at base of wing disk; (b) score 1.0: blade of wing has begun to grow, early traces of lacunae for venation system visible, one or two tracheae have emerged from the basal tracheal bundle; (c) score 1.5: many tracheae have emerged from the basal bundle and are beginning to enter the lacunae; (d) score 2.0: trachea fill most lacunae and many trachea extend almost to the end of the lacunae; (e) score 2.5: all lacunae are filled with tracheae, and tracheae begin to branch at their tips; (f) score 3.0: tracheae are extensively branched at their tips, and many lateral branches have developed. Disks that were intermediate between any two of these nominal categories were given an intermediate score (e.g. 2.25 or 2.75). For the scoring system used in this paper we also identified a score 4.0 (not shown here) characterized by the development of a brown pigment over the entire surface of the disk. This pigmentation develops about 24 h before entry into the prepupal stage.
Fig. 5. Relationship between size and differentiation of the fore wing disk. Scoring of differentiation is according to Fig. 4. Black circles (curve A), control larvae; white circles (curve B), starved larvae. Larvae with differentiation scores of 0 (zero) are not shown (see Fig. 6). Curves are logarithmic regressions.
Treatment with JHA inhibited disk growth and differentiation in a dose-dependent manner. Dose–response curves for disk growth and vein differentiation are shown in Fig. 7(A) and (B), respectively. Doses smaller than 10⫺3 µg had no effect on either process, while doses
Fig. 6. Progress of differentiation of the fore wing disk during normal growth (䊊,쐌) and starvation (䊐,䊏). Progress is measured as the change in differentiation score over 48 h. Open symbols are data points, filled symbols are means (and standard errors). Starvation had no effect on differentiation of disks that were initially larger than 13 µg (to the right of the dashed line), but almost completely inhibited differentiation of disks smaller than 13 µg. By contrast, in feeding (control) larvae, disks initially smaller than 13 µg differentiated to the same degree as larger disks. Starvation is thus able to uncouple differentiation from growth. Once differentiation has begun, it continues normally, even under conditions of starvation when disk growth is inhibited.
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Fig. 8. Relationship between size and differentiation of the fore wing disk in methoprene-treated (䊊) and control (쐌) larvae. Larvae with differentiation scores of 0 (zero) are not plotted. Curves are logarithmic regressions. Unlike starvation, methoprene does not appear to be able to dissociate growth from differentiation of the wing.
Fig. 7. Effect of the JHA methoprene on growth (A) and differentiation (B) of the fore wing disk. Both growth and differentiation are inhibited in a dose-dependent manner: 50% inhibition of growth occurs at a dose of 700 ng, and 50% inhibition of differentiation occurs at a dose of 800 ng per larva. Open circles at left are mean values for acetone-treated controls. Bars are standard errors of the means.
larger than 1 µg had a maximal effect in inhibiting both growth and differentiation of the disks. The relationship between disk size and stage of vein differentiation in JHA treated animals was identical to that of controls (Fig. 8), indicating that the JHA did not have a differential effect on the growth and differentiation of the wing disks. Wing disks that were maximally inhibited had an average size of 35.5 µg protein, which was not significantly different from the disk size of larvae at the beginning of the 3-day JH treatment.
4. Discussion A summary diagram of the principal findings an conclusions of the work described above is given in Fig. 9. The wing disks of Precis appear to undergo a constant rate of cell division and, in normal-growing larvae, the
disks grow continuously and exponentially, at least during the last two larval instars (Kremen and Nijhout, 1998). Our results show that during the growth period of the last larval instar, the size of the wing disks maintains a constant relation to body size (Fig. 1). Because the growth rate of these larvae is highly variable, this implies that the disks do not grow at a constant rate, but that their growth rate must be a function of that of the larva: the disks of slow-growing larvae must grow more slowly than those of fast-growing larvae. Moreover, larvae do not all metamorphose at the same body size, and that means that in order to develop wings that are correctly proportioned to body size there must exist a mechanism for coordinating the growth of disks and the growth of the body. Although the need for proportionality in the growth of the body and its various parts is self-evident, the mechanism that controls proportional growth is still unknown. Experiments on disk transplantation and disk regeneration in Drosophila suggest that size regulation of imaginal disks is autonomous and that they require no regulatory input from their developmental environment (Bryant and Simpson, 1984; Wilder and Perrimon, 1996). Other lines of evidence suggest, however, that the growth of imaginal disks is not only responsive to the internal environment of the larva, but must be exquisitely so. It is well known that when larvae of Drosophila and other insects are starved or malnourished, they develop into miniature adults that nevertheless have normally proportioned (but in effect miniature) wings and appendages. Disks must therefore somehow receive information about the size of the body in which they develop. One simple possibility is that all parts of the body of a growing larva share the available nutrient resources in
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Fig. 9. Summary diagram of the control of growth and differentiation of the wing disks of Precis coenia. Growth of the body and the wing disks is stimulated by feeding, and vein differentiation begins when the growing wing disks surpasses a critical size. The continuation of vein differentiation does not require continued growth of the wing disk, because in starved animals disk growth stops and differentiation continues normally. Both growth of the disk and vein differentiation are inhibited by the JHA, methoprene in a dose-dependent manner. Imaginal disks interfere with each other’s growth, possibly through competition for limiting growth resources (Nijhout and Emlen, 1998). The coordination between disk growth and somatic growth is such that the disks always remain well-proportioned to the body. Whether this is due to an independent joint response to circulating nutrients or to a special interaction between disks and soma (indicated by a question mark) is unknown. The exact mechanisms through which the various stimulations and inhibitions depicted in this diagram take place are still unknown.
a specific manner, so that they are all equally affected by the feeding rate and nutritional status of the larva. Several lines of evidence suggest that the situation is not as simple as this. The wing disks of Precis, for instance, continue to grow at an exponential rate after the larva stops feeding at the end of the 5th instar (Kremen and Nijhout, 1998). In addition, there appears to be a competitive interaction among disks, so that the growth of some disks is enhanced when the growth of others is inhibited (Nijhout and Emlen, 1998; Klingenberg and Nijhout, 1998). The imaginal disks of Precis thus grow as a function of body size at some stages of development but not at others, and their growth is affected by the presence of other disks. When a larva is starved, the wing disks stop growing. The lag time between the removal of food from the larvae and the cessation of disk growth is on the order of a few hours. Considering the error of our estimates of disk size, the lag time may not be significantly different from zero hours and probably lies somewhere between 0 and 4 h. This finding implies that the growth of wing disks is extremely sensitive to the nutritional status of the larva. Something in the environment of the disk, presumably a hemolymph-borne factor, must somehow track the nutritive intake of the larva. This factor must be modulated to allow the rate of disk growth to match that of the body over a large range of growth rates, and it must fall rapidly below some minimal value when larvae are starved. This factor could be a critical nutrient (such as an amino acid or a vitamin), a specific growth factor (analogous to those found in vertebrates), a growth inhibitor (whose level rises during starvation), or it could
be a hormone. Whatever the factor is, it must vary as a function of the growth rate of the larva. That disks must be exquisitely sensitive to this regulatory mechanism is suggested by the highly localized nature of the interaction among imaginal disks (Klingenberg and Nijhout, 1998). We also investigated the relationship between disk growth and differentiation. In normally growing larvae, differentiation of the venation system is tightly coupled to disk size and not to the age of the larva (Fig. 5). However, if larvae are starved after vein differentiation has begun, the disk ceases growth almost immediately but vein differentiation continues at a rate indistinguishable from that of normally growing larvae. By contrast, if larvae are starved before the venation system has begun to differentiate, it does not initiate differentiation but remains undifferentiated.(Fig. 6). Thus, there appears to be independent control over the initiation and over the progression of vein differentiation. It is possible that initiation is triggered only when the disk reaches a critical size. Once initiated, differentiation is actually independent of the growth of the disk and of the nutritional status of the larva. Thus, although differentiation is normally highly correlated with disk growth, these two processes are actually controlled independently. Kremen and Nijhout (1998) showed that growth of the wing disks in 5th instar larvae of Precis can be inhibited by large doses of the JHA methoprene, whereas growth of the wing disks of 4th instar larvae is insensitive to this hormone analog. In order to find the minimal dose of JH that can inhibit growth of the wing disks we determined the dose–response relationship for the inhibi-
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tory effect of JHA in 5th instar disks. The growth of the wing disks was inhibited in a dose-dependent manner, as was the degree of differentiation of the wing venation. There was no dissociation between disk size and the degree of disk differentiation, which suggests that, unlike starvation, JHA inhibited disk growth and differentiation equally. It is possible, therefore, that inhibition of disk growth by starvation is mediated by an elevation in the titer of JH during starvation. Indeed, in 5th instar larvae of Manduca that are starved, the JH titer rises to about fivefold, to 3 ng/ml, and remains elevated for several days before gradually declining to a level typical of that found in feeding larvae (Cymborowski et al., 1982). The native JH titer during the first three days of the 5th instar of Precis has been estimated at between 1 and 2 ng/ml (Kremen and Nijhout, 1989). The lowest acute dose of JHA that inhibited disk development was just under 30 ng, which, in a 0.5 g larva, would produce a hemolymph concentration in excess of 120 ng/ml. This is substantially above the level to which the endogenous JH titer is likely to rise, even without accounting for the fact that, in Precis, methoprene is about five times more active than the native JH (JH-I; Kremen and Nijhout, unpublished). It is unlikely, therefore, that fluctuation of the JH titer is the mechanism that modulates the rate of disk growth in normally-growing or in starved larvae. The recent discovery of circulating growth factors that stimulate mitosis and cellular growth of internal tissues and imaginal disks of Drosophila (Britton and Edgar, 1998; Kawamura et al., 1999) suggests an alternative mechanism for the regulation of disk growth. If these factors stimulate the rate of mitosis and cytoplasmic growth in a concentration-dependent manner, they could provide the required mechanism that modulates disks growth and matches disk growth to the somatic growth of the larva. For this to be the case, these factors must also vary with the nutritional status of the larva, that is, they must decline rapidly under conditions of starvation. If these factors are somehow sequestered or inactivated by growing wing disks, then they may also account for the apparent competitive interaction among growing disks described by Nijhout and Emlen (1998) and Klingenberg and Nijhout (1998). Acknowledgements We thank Chris Klingenberg, Armin Moczek, and Lou D’Amico for critical comments on the manuscript. This
work was supported in part by a grant (IBN-9728727) from the National Science Foundation.
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Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae) Andrew L. Miner, Allison J. Rosenberg, H. Frederik Nijhout
*
Department of Zoology, Duke University, Durham, NC 27708-0325, USA Received 25 May 1999; accepted 3 August 1999
Abstract During the last larval instar, the wing imaginal disks of Precis coenia grow continuously. The rate of disk growth is not diskautonomous but closely matches the rate of somatic growth of the larva, so that the size of the disks is a function of the size of the body, irrespective of the growth rate of the larva. When larvae are starved, their wing disks cease growth within 4 h, which indicates the existence of an efficient coupling mechanism between the growth of the soma and growth of the imaginal disks. Disk growth is inhibited by juvenile hormone in a dose-dependent manner. In the presence of the hormone the wing disks stop growing even while the larva continues to grow normally. During the last larval instar the wing imaginal disks also undergo a complex differentiation, consisting of the development of the lacunae and tracheation that define the future adult wing venation system. In normally growing larvae, differentiation of the wing disk is tightly correlated with wing size. Differentiation and size can be dissociated by starvation. If larvae are starved at any time after differentiation has begun, differentiation continues at a normal rate, even though the wing disk does not grow. Differentiation does not begin spontaneously in larvae that are starved before differentiation has begun. These findings indicate that the initiation of differentiation and its continuation are controlled independently. Juvenile hormone inhibits differentiation in a dose-dependent manner. Upon treatment with juvenile hormone, the stage of differentiation becomes fixed. These findings indicate that continued differentiation of the wing disk can only occur in the absence of juvenile hormone. Although the circulating level of juvenile hormone may be elevated during starvation, it is unlikely that this elevation is responsible for the observed effect of starvation on growth and differentiation of the disk. 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Precis coenia; Imaginal disk; Growth; Methoprene; Juvenile hormone; Nutrition
1. Introduction The adult appendages of holometabolous insects begin their development within the larva as imaginal disks. Under normal conditions the imaginal disks grow continuously and roughly exponentially, at least during the later larval stages and prepupal stage (Sehnal, 1985; Kremen and Nijhout, 1998). At some time during metamorphosis the disks stop growing and then proceed to differentiate into appendages that are correctly proportioned to the adult body. Little is known about the mechanisms that control the growth rate of imaginal disks, nor about the mechanisms
* Corresponding author. Tel.: +1-919-684-2793; fax: +1-919-6846168. E-mail address: [email protected] (H. Frederik Nijhout)
that control the final size to which a particular disk will grow. Evidence from Drosophila suggests that disks control their own growth through an intrinsic mechanism. Numerous experiments have demonstrated that when disks, or fragments of disks, are transplanted into an adult abdomen they grow to about their normal final size, in spite of the fact that the adult environment is vastly different from that of the larva in which disks normally grow and develop (Bryant and Simpson, 1984; Wilder and Perrimon, 1996). Hence, in Drosophila, the differences in the larval and adult environment appear to be irrelevant to the normal control of disk growth. In Precis coenia, by contrast, disk growth is quite sensitive to the developmental environment. Surgical removal of one or two wing disks, for instance, enhances the growth of the remaining wing disks, which then grow much larger than they otherwise would (Nijhout and Emlen, 1998; Klingenberg and Nijhout, 1998). This finding indi-
0022-1910/00/$ - see front matter 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 9 ) 0 0 1 7 7 - 8
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cates that the growth of wing disks is somehow limited by the presence of other disks, possibly through competition for a limiting resource required for growth. The final size of the disk thus appears to be affected by factors in the environment in which the disk develops. The regulation of the final size of a disk could occur through two mechanisms. The disk could always remain a function of the body size of the larva as it grows, irrespective of the vagaries of nutrition, so that it always produces a final disk that is correctly proportioned to the final size of the larva. Alternatively, a disk could have a growth trajectory that is largely independent of the growth of the larva, in which case final size regulation must take place sometime during metamorphosis. In either case, there must exist some kind of interaction between disks and body by which the proportionality of disk to body size is either maintained throughout growth or achieved during metamorphosis. In the present paper we investigate the physiological control of wing disk growth in Precis coenia, during normal growth and during starvation of the larva, in order to determine whether wing disks can grow independently of body growth. We also examine the ability of juvenile hormone to dissociate disk and body growth.
2. Materials and methods Larvae of Precis coenia were reared on an artificial diet at 27°C and a long-day (16L:8D) photoperiod. All larvae used in these experiments were selected and weighed within 1 h of eclosion to the 5th (final) larval instar, after which they were placed in individual cups and their growth was monitored by daily weighing. Prior to surgery larvae were anaesthetized by submersion in water for 10 min. Wing disks were removed from larvae submerged in saline, through a small incision in the lateral thoracic wall immediately overlying the disk. More than 90% of the larvae survived this operation and continued to grow normally at a rate indistinguishable from that of sham operated larvae. The size of wing disks was determined by means of a bicinchoninic acid (BCA) protein assay (Pierce), using the following protocol. Each disk was sonicated three times for 5 s in 200 µl of saline using a Fisher Dismembranator with a microprobe attachment. The homogenate was then placed on ice and 1 ml of test reagent was added. The mixture was incubated in a water bath at 36°C for exactly 30 min and then returned to ice. After cooling the mixture was centrifuged at 14,000g for 2 min and the absorbance of the supernatant was read in a spectrophotometer at 562 nm. A bovine serum albumin (Sigma) solution was used as a standard, and a dilution series of standards was processed with each set of samples. The juvenile hormone analog Methoprene was dissolved in acetone to a concentration that allowed the appropriate dose to be applied
in a volume of either 5 or 10 µl, and applied with a micropipette to the dorsum of the larva. Control larvae received either 5 or 10 µl of acetone. In the course of the final larval instar the wing imaginal disks undergo a complex differentiation, consisting of the development of an extensive pattern of lacunae and tracheae that define the future venation system of the wing. A scoring system was developed for measuring the degree of vein differentiation of the wing disk. This scoring system is described in Fig. 4 and its caption.
3. Results 3.1. The relation between body size and wing disk size In order to study the control of growth and differentiation of the wing disks it is necessary to have a method for obtaining standardized larvae whose stage of wing disk development can be predicted. Chronological age since the last molt and weight are typically used for staging lepidopteran larvae (Nijhout and Williams, 1974; Goodman et al., 1985). Larvae of Precis are quite variable in their growth rate and also vary in the duration of the 5th instar, which ranges from 5 to 9 days. Furthermore, the size of larvae at the beginning of the 5th instar is highly variable and can range from 0.09 to 0.22 g (live weight). Larvae with large body sizes at the beginning of the 5th instar grow faster than larvae with initially small body sizes (Kremen and Nijhout, 1998). Consequently, the chronological age and the body weight of a 5th instar larva cannot, by themselves, provide an accurate measure of physiological or developmental stage of the larva. We found that larvae of the same chronological age can have imaginal disks at very different stages of development (r2=0.48). Likewise, larvae of the same body weight can have imaginal disks at very different stages in development (r2=0.69). Thus neither chronological age nor body size of the larva can be used by itself to predict the stage of development of its imaginal disks precisely. We tested whether a combination of both measures provided greater predictive ability by measuring the wing disks of a cohort of larvae of the same weight and age. Such selected larvae also had wing disks in different stages of development, and this combination of measures actually provided a poorer predictor than either weight or age alone (r2=0.41). We suspected that the variation in initial size of the 5th instar could be responsible for the poor correlation between larval age, size, and the degree of wing disk development, because larvae that start the 5th instar at different initial sizes have different growth trajectories. We found that wing disk size and the stage of development were, in fact, most highly correlated with the percentage of weight gained since the beginning of the 5th
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instar (r2=0.76). Accordingly, all our size measures will be given as the ratio between the weight at the time of observation and the initial weight of the 5th instar larva. We’ll call this measure of size the growth ratio (a growth ratio of 2.0 means that at the time of observation the larva was twice the weight it had at the beginning of the instar). The relationship between the growth ratio and the size of the fore wing imaginal disk is shown in Fig. 1. A linear and an exponential regression fit the data equally well. In view of the fact that an exponential relationship most accurately describes other features of imaginal disk growth (see below, and Nijhout and Wheeler, 1995; Kremen and Nijhout, 1989), we present the exponential regression in Fig. 1. The accuracy of our method for determining disk size was tested by measuring sets of fore and hind wings of the same individual (Fig. 2). The fore wing imaginal disk of Precis is about 1.4× the size of the hind wing disk over a broad range of disk sizes (Fig. 2). These measures were highly correlated (r2=0.92), which suggests that the scatter observed in Fig. 1 represents real individual variation rather than measurement error. 3.2. The growth of disks during starvation In the growing 5th instar larva the sizes of the wing disks are a function of the growth ratio (Fig. 1). Because the growth rate of the larvae is inherently variable in this species, this observation implies that disks must grow more slowly in slow growing larvae than they do in fast growing larvae. A mechanism must therefore exist that coordinates the rate of disk growth with overall growth of the body.
Fig. 1. Relationship between body size and wing imaginal disk size during the final larval instar. Body size is measured as the growth ratio, the ratio of the weight of the larva at the time of measurement and the initial weight of the larva at the molt to the final instar. An exponential regression is fitted to the data (r2=0.76).
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Fig. 2. Relationship between the size of fore and hind wing imaginal disks during the last larval instar (r2=0.92). Fore and hind wing maintain the same relative sizes throughout their growth period.
We determined whether the growth rate of the disk is directly responsive to alterations of the growth rate of the body by starving larvae at various body weights and noting the effect of starvation on the subsequent growth of the wing disks. Experimental larvae were weighed, and one fore wing disk was removed to determine the size of the disks at the beginning of the experiment. Larvae were then starved for either 24 or 48 h, after which the other fore wing disk was removed. All experimental larvae were provided with a small block of 4% agar in distilled water to prevent dehydration. Control larvae were weighed and also had one fore wing disk removed, but were then allowed to feed normally for 24 or 48 h before the other fore wing disk was removed. The results of this experiment are illustrated in Fig. 3, which plots the percent growth of wing disks after 24 and 48 h for
Fig. 3. Growth of the fore wing imaginal disk in starved larvae and normally growing control larvae. Growth is measured as the percentage increase of disk size after 24 and 48 h. Bars indicate standard errors of the means. The imaginal disks appear to stop growing within hours after food is removed.
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both experimental and control larvae. The wing disks of control larvae grew normally during the experimental period and maintained the relationship to body size (as measured by the growth ratio) shown in Fig. 1. Control disks grew by 65% during the 24 h feeding period and by 177% during the 48 h period, which is consistent with the exponential growth trajectory of disks shown in Fig. 1. The disks of starved larvae grew 4% during the 24 h starvation period and 15% during the 48 h starvation period. The amounts of disk growth during 24 and 48 h of starvation were not significantly different form each other (t-test, P=0.075). It is possible to obtain an estimate of the time required for the disks to stop growing after the initiation of starvation from the data in Fig. 3. The averaged disk growth rates for control animals were 2.8 and 3.6% per hour for the 24 and 48 h periods, respectively. If we assume the 4 and 15% growth increments of the disks in starved larvae are accurate measures of how much the disks continued to grow after starvation began, then the disks of starved animals stopped growing after 1.4 and 4.2 h, respectively. Taking the average of these figures, the data suggest that in starved animals the wing disk continued to grow about 2.8 h after starvation began. The data available do not allow us to determine whether the disks grew normally for several hours and then stopped abruptly, or whether their growth rate gradually diminished over a longer period of time.
significantly from that in feeding larvae. This relationship is shown in Fig. 5 (curve B). In starved larvae, the wing venation was more differentiated for disks of a particular size than was the case in control larvae. This observation implies that differentiation of the venation system must continue for some time after disk growth stops. By only considering the relationship between disk size and its state of differentiation, the data presented in Fig. 5 conceal an interesting feature about the dynamics of vein differentiation. The effect of starvation on the dynamics of differentiation is revealed when we consider how much the state of differentiation of a disk changed during a 48 h period of feeding or starvation. Fig. 6 shows that the progression of disk differentiation during starvation depends on the degree of differentiation the disk had attained at the beginning of starvation. Disks that had not yet begun to differentiate did not initiate differentiation, whereas disks that had already initiated differentiation progressed about as far during the next 48 h of starvation as did disks in normally feeding larvae. Analysis of the data in Fig. 6 indicates that the initiation of differentiation of the wing disk appears to be coupled to the growth (or size) of the disk, but the continuation of differentiation is uncoupled from disk growth and is only marginally inhibited by starvation.
3.3. The relationship between disk growth and differentiation
Kremen and Nijhout (1998) presented evidence that mitosis in wing imaginal disks of 5th instar larvae of Precis can be inhibited by JH. The functional significance of this inhibitory effect is not clear at present, but it appears to be a special feature of the physiology of the last larval instar, because such inhibition does not occur in earlier instars (Kremen and Nijhout, 1998). In Manduca sexta, titers of JH gradually become elevated during starvation, and if this is the case in Precis also, then an increase in JH titer in response to starvation could account for the inhibition of mitosis and disk growth during starvation. We examined the effect of exogenous applications of the JH analog methoprene (JHA) on the growth of wing imaginal disks. Larvae for this experiment were synchronized as follows: freshly molted 5th instar larvae weighing between 0.14 and 0.18 g were selected and allowed to grow in individual containers for 48 h. At that time, only larvae weighing between 0.27 and 0.34 g were selected for treatment with JHA. The appropriate dose of JHA, dissolved in 5 µl of acetone, was applied to the dorsum of each larva once a day for three consecutive days. Control animals received daily applications of acetone alone. On the 5th day the fore wing disks were removed, their degree of differentiation was scored, and they were then homogenized for protein analysis.
The wing imaginal disks of Precis undergo considerable differentiation in the course of the 5th larval instar. During the first third of the instar a system of lacunae forms in the wing disk that outline the future venation of the adult wing. During the middle and later portion of the instar these lacunae are invaded by the tracheal system so that at the end of the 5th instar the wing disk is fully tracheated and the complete venation pattern of the adult is evident. In order to measure the timing and rate of vein differentiation and tracheation we used the numerical scoring system illustrated in Fig. 4. The differentiation of the venation system is not well correlated with the chronological age of the larvae, nor with its weight. Rather differentiation is correlated with the size of the wing disk, as illustrated in Fig. 5 (curve A). We noted that in starved larvae the tracheal differentiation of the wing disk was also inhibited, and we investigated whether growth and differentiation of the disk remained tightly coupled or whether they could be uncoupled by starvation. Uncoupling of growth and differentiation can be measured by determining whether the relationship between disk size and differentiation in larvae starved for 48 hours at a range of body sizes deviates
3.4. The effect of juvenile hormone (JH) on disk growth and differentiation
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Fig. 4. Differentiation of the fore wing imaginal disk during the middle portion of the last larval instar. Tracheae are air-filled and show up black in these transmitted-light photographs. Scoring system for differentiation is indicated at the bottom of each panel: (a) score 0: tight tracheal bundle at base of wing disk; (b) score 1.0: blade of wing has begun to grow, early traces of lacunae for venation system visible, one or two tracheae have emerged from the basal tracheal bundle; (c) score 1.5: many tracheae have emerged from the basal bundle and are beginning to enter the lacunae; (d) score 2.0: trachea fill most lacunae and many trachea extend almost to the end of the lacunae; (e) score 2.5: all lacunae are filled with tracheae, and tracheae begin to branch at their tips; (f) score 3.0: tracheae are extensively branched at their tips, and many lateral branches have developed. Disks that were intermediate between any two of these nominal categories were given an intermediate score (e.g. 2.25 or 2.75). For the scoring system used in this paper we also identified a score 4.0 (not shown here) characterized by the development of a brown pigment over the entire surface of the disk. This pigmentation develops about 24 h before entry into the prepupal stage.
Fig. 5. Relationship between size and differentiation of the fore wing disk. Scoring of differentiation is according to Fig. 4. Black circles (curve A), control larvae; white circles (curve B), starved larvae. Larvae with differentiation scores of 0 (zero) are not shown (see Fig. 6). Curves are logarithmic regressions.
Treatment with JHA inhibited disk growth and differentiation in a dose-dependent manner. Dose–response curves for disk growth and vein differentiation are shown in Fig. 7(A) and (B), respectively. Doses smaller than 10⫺3 µg had no effect on either process, while doses
Fig. 6. Progress of differentiation of the fore wing disk during normal growth (䊊,쐌) and starvation (䊐,䊏). Progress is measured as the change in differentiation score over 48 h. Open symbols are data points, filled symbols are means (and standard errors). Starvation had no effect on differentiation of disks that were initially larger than 13 µg (to the right of the dashed line), but almost completely inhibited differentiation of disks smaller than 13 µg. By contrast, in feeding (control) larvae, disks initially smaller than 13 µg differentiated to the same degree as larger disks. Starvation is thus able to uncouple differentiation from growth. Once differentiation has begun, it continues normally, even under conditions of starvation when disk growth is inhibited.
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Fig. 8. Relationship between size and differentiation of the fore wing disk in methoprene-treated (䊊) and control (쐌) larvae. Larvae with differentiation scores of 0 (zero) are not plotted. Curves are logarithmic regressions. Unlike starvation, methoprene does not appear to be able to dissociate growth from differentiation of the wing.
Fig. 7. Effect of the JHA methoprene on growth (A) and differentiation (B) of the fore wing disk. Both growth and differentiation are inhibited in a dose-dependent manner: 50% inhibition of growth occurs at a dose of 700 ng, and 50% inhibition of differentiation occurs at a dose of 800 ng per larva. Open circles at left are mean values for acetone-treated controls. Bars are standard errors of the means.
larger than 1 µg had a maximal effect in inhibiting both growth and differentiation of the disks. The relationship between disk size and stage of vein differentiation in JHA treated animals was identical to that of controls (Fig. 8), indicating that the JHA did not have a differential effect on the growth and differentiation of the wing disks. Wing disks that were maximally inhibited had an average size of 35.5 µg protein, which was not significantly different from the disk size of larvae at the beginning of the 3-day JH treatment.
4. Discussion A summary diagram of the principal findings an conclusions of the work described above is given in Fig. 9. The wing disks of Precis appear to undergo a constant rate of cell division and, in normal-growing larvae, the
disks grow continuously and exponentially, at least during the last two larval instars (Kremen and Nijhout, 1998). Our results show that during the growth period of the last larval instar, the size of the wing disks maintains a constant relation to body size (Fig. 1). Because the growth rate of these larvae is highly variable, this implies that the disks do not grow at a constant rate, but that their growth rate must be a function of that of the larva: the disks of slow-growing larvae must grow more slowly than those of fast-growing larvae. Moreover, larvae do not all metamorphose at the same body size, and that means that in order to develop wings that are correctly proportioned to body size there must exist a mechanism for coordinating the growth of disks and the growth of the body. Although the need for proportionality in the growth of the body and its various parts is self-evident, the mechanism that controls proportional growth is still unknown. Experiments on disk transplantation and disk regeneration in Drosophila suggest that size regulation of imaginal disks is autonomous and that they require no regulatory input from their developmental environment (Bryant and Simpson, 1984; Wilder and Perrimon, 1996). Other lines of evidence suggest, however, that the growth of imaginal disks is not only responsive to the internal environment of the larva, but must be exquisitely so. It is well known that when larvae of Drosophila and other insects are starved or malnourished, they develop into miniature adults that nevertheless have normally proportioned (but in effect miniature) wings and appendages. Disks must therefore somehow receive information about the size of the body in which they develop. One simple possibility is that all parts of the body of a growing larva share the available nutrient resources in
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Fig. 9. Summary diagram of the control of growth and differentiation of the wing disks of Precis coenia. Growth of the body and the wing disks is stimulated by feeding, and vein differentiation begins when the growing wing disks surpasses a critical size. The continuation of vein differentiation does not require continued growth of the wing disk, because in starved animals disk growth stops and differentiation continues normally. Both growth of the disk and vein differentiation are inhibited by the JHA, methoprene in a dose-dependent manner. Imaginal disks interfere with each other’s growth, possibly through competition for limiting growth resources (Nijhout and Emlen, 1998). The coordination between disk growth and somatic growth is such that the disks always remain well-proportioned to the body. Whether this is due to an independent joint response to circulating nutrients or to a special interaction between disks and soma (indicated by a question mark) is unknown. The exact mechanisms through which the various stimulations and inhibitions depicted in this diagram take place are still unknown.
a specific manner, so that they are all equally affected by the feeding rate and nutritional status of the larva. Several lines of evidence suggest that the situation is not as simple as this. The wing disks of Precis, for instance, continue to grow at an exponential rate after the larva stops feeding at the end of the 5th instar (Kremen and Nijhout, 1998). In addition, there appears to be a competitive interaction among disks, so that the growth of some disks is enhanced when the growth of others is inhibited (Nijhout and Emlen, 1998; Klingenberg and Nijhout, 1998). The imaginal disks of Precis thus grow as a function of body size at some stages of development but not at others, and their growth is affected by the presence of other disks. When a larva is starved, the wing disks stop growing. The lag time between the removal of food from the larvae and the cessation of disk growth is on the order of a few hours. Considering the error of our estimates of disk size, the lag time may not be significantly different from zero hours and probably lies somewhere between 0 and 4 h. This finding implies that the growth of wing disks is extremely sensitive to the nutritional status of the larva. Something in the environment of the disk, presumably a hemolymph-borne factor, must somehow track the nutritive intake of the larva. This factor must be modulated to allow the rate of disk growth to match that of the body over a large range of growth rates, and it must fall rapidly below some minimal value when larvae are starved. This factor could be a critical nutrient (such as an amino acid or a vitamin), a specific growth factor (analogous to those found in vertebrates), a growth inhibitor (whose level rises during starvation), or it could
be a hormone. Whatever the factor is, it must vary as a function of the growth rate of the larva. That disks must be exquisitely sensitive to this regulatory mechanism is suggested by the highly localized nature of the interaction among imaginal disks (Klingenberg and Nijhout, 1998). We also investigated the relationship between disk growth and differentiation. In normally growing larvae, differentiation of the venation system is tightly coupled to disk size and not to the age of the larva (Fig. 5). However, if larvae are starved after vein differentiation has begun, the disk ceases growth almost immediately but vein differentiation continues at a rate indistinguishable from that of normally growing larvae. By contrast, if larvae are starved before the venation system has begun to differentiate, it does not initiate differentiation but remains undifferentiated.(Fig. 6). Thus, there appears to be independent control over the initiation and over the progression of vein differentiation. It is possible that initiation is triggered only when the disk reaches a critical size. Once initiated, differentiation is actually independent of the growth of the disk and of the nutritional status of the larva. Thus, although differentiation is normally highly correlated with disk growth, these two processes are actually controlled independently. Kremen and Nijhout (1998) showed that growth of the wing disks in 5th instar larvae of Precis can be inhibited by large doses of the JHA methoprene, whereas growth of the wing disks of 4th instar larvae is insensitive to this hormone analog. In order to find the minimal dose of JH that can inhibit growth of the wing disks we determined the dose–response relationship for the inhibi-
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tory effect of JHA in 5th instar disks. The growth of the wing disks was inhibited in a dose-dependent manner, as was the degree of differentiation of the wing venation. There was no dissociation between disk size and the degree of disk differentiation, which suggests that, unlike starvation, JHA inhibited disk growth and differentiation equally. It is possible, therefore, that inhibition of disk growth by starvation is mediated by an elevation in the titer of JH during starvation. Indeed, in 5th instar larvae of Manduca that are starved, the JH titer rises to about fivefold, to 3 ng/ml, and remains elevated for several days before gradually declining to a level typical of that found in feeding larvae (Cymborowski et al., 1982). The native JH titer during the first three days of the 5th instar of Precis has been estimated at between 1 and 2 ng/ml (Kremen and Nijhout, 1989). The lowest acute dose of JHA that inhibited disk development was just under 30 ng, which, in a 0.5 g larva, would produce a hemolymph concentration in excess of 120 ng/ml. This is substantially above the level to which the endogenous JH titer is likely to rise, even without accounting for the fact that, in Precis, methoprene is about five times more active than the native JH (JH-I; Kremen and Nijhout, unpublished). It is unlikely, therefore, that fluctuation of the JH titer is the mechanism that modulates the rate of disk growth in normally-growing or in starved larvae. The recent discovery of circulating growth factors that stimulate mitosis and cellular growth of internal tissues and imaginal disks of Drosophila (Britton and Edgar, 1998; Kawamura et al., 1999) suggests an alternative mechanism for the regulation of disk growth. If these factors stimulate the rate of mitosis and cytoplasmic growth in a concentration-dependent manner, they could provide the required mechanism that modulates disks growth and matches disk growth to the somatic growth of the larva. For this to be the case, these factors must also vary with the nutritional status of the larva, that is, they must decline rapidly under conditions of starvation. If these factors are somehow sequestered or inactivated by growing wing disks, then they may also account for the apparent competitive interaction among growing disks described by Nijhout and Emlen (1998) and Klingenberg and Nijhout (1998). Acknowledgements We thank Chris Klingenberg, Armin Moczek, and Lou D’Amico for critical comments on the manuscript. This
work was supported in part by a grant (IBN-9728727) from the National Science Foundation.
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