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The Cellular and Physiological Mechanism of Wing-Body Scaling in Manduca sexta

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Science  17 Dec 2010:
Vol. 330, Issue 6011, pp. 1693-1695
DOI: 10.1126/science.1197292

Abstract

In animals, appendages develop in proportion to overall body size; when individual size varies, appendages covary proportionally. In insects with complete metamorphosis, adult appendages develop from precursor tissues called imaginal disks that grow after somatic growth has ceased. It is unclear, however, how the growth of these appendages is matched to the already established body size. We studied the pattern of cell division in the tobacco hornworm Manduca sexta and found that both the rate of cell division and the duration of growth of the wing imaginal disks depend on the size of the body in which they develop. Moreover, we found that both of these processes are controlled by the level and duration of secretion of the steroid hormone ecdysone. Thus, proportional growth is under hormonal control and indirectly regulated by the central nervous system.

The relative sizes of body parts are among the most characteristic traits of animals (13), and although methods for the analysis of scaling of appendages and other body parts have been described (36), the underlying developmental mechanisms that coordinate the relative sizes of body parts with overall body size remain largely unknown. Body size is determined by genetic and environmental factors. In a constant and optimal environment, insects grow to a characteristic species-specific size. Variation in nutrition and temperature can impose systematic deviations from this species-characteristic size. Increased rearing temperature typically results in smaller body size (79). In the tobacco hornworm Manduca sexta, the developmental mechanism underlying this temperature-size interaction is well understood (8). Nutrition, too, can have a profound effect on body size (7, 10, 11); in Manduca, variation in nutrition can result in variation in adult body mass by more than a factor of 2 (12).

Wings develop internally as imaginal disks. These disks undergo most of their growth after the larva has stopped feeding and growing (13, 14). At this stage, the forewing imaginal disk has a dry mass of 0.12 (±0.03) mg. The dry mass of an adult forewing is 26 (±0.6) mg, so >99% of a wing’s growth in mass occurs after the larva has stopped feeding. Small-bodied adults that develop from undernourished larvae have proportionally small wings, as expected. This raises an interesting problem, however, because the wings do not grow at the same time as the body. We sought to identify the mechanism that causes the wing disks to grow less when they develop in a small body than in a large body.

To investigate how control over the proportional growth of wings is exercised, we developed a system that reliably causes Manduca individuals to develop to half their normal adult size by taking advantage of the weight at which the endocrine events that culminate in the cessation of growth and metamorphosis are set in motion (the critical weight) in Manduca. Critical weight occurs in the middle of the last larval instar (15, 16). The last (fifth) larval instar is divided into the feeding phase, during which the larva grows from about 1.2 g to about 12.5 g, and the wandering phase, during which the larva purges its gut contents in preparation for pupation. In the strain used for this study, the critical weight is about 6.5 g, and larvae enter the wandering stage at about 12.5 g, 2.5 days after passing the critical weight.

When larvae were starved at a weight of 6.5 g, their development time to the wandering stage was identical to that of larvae that were allowed to continue feeding. Such starved larvae developed into normally proportioned adults of about half the mass of normally feeding controls, with wings half the mass of normally feeding controls and having about half the number of cells. Thus, the smaller wing size in adults that developed from undernourished larvae was due to a reduction in cell number in almost exact proportion to the diminution in wing size. Because the number of cells in the diminished wing was half of that in a normal wing, the two must differ by one cell division, on average. We thus sought to determine whether that missing cell division was averaged across the entire development of the wing or whether a cell division was missing at a particular time in development.

In normal development, there are 390,000 (±18,000) cells in the wing disk at the time a larva passes the critical weight, 1.64 million (±91,000) cells at the beginning of the pupal stage, and 8.59 million (±56,000) cells by the time cell division stops and the wing is fully developed. If we assume that all cells of the wing divide, then on average 2.06 cell divisions occur between the critical weight and pupation and 2.38 cell divisions occur between pupation and the fully developed wing.

We examined the pattern of growth of the forewing imaginal disks, beginning at the critical weight. Cell division in the wing disk stops during the fourth day of the wandering stage, about 24 hours before pupation; it resumes again 48 hours after pupation and stops permanently 6 days later. The growth trajectories of forewing disks of normally fed control and starved larvae (Fig. 1) show that the wing disks of starved larvae grew slower than those of fed larvae. The doubling time of cells in the wing disks during the first 3 days of growth in the wandering stage was calculated to be 1.96 days in control larvae and 2.12 days in starved larvae (see supporting online material).

Fig. 1

Growth of forewing imaginal disks during the wandering stage in normally feeding larvae and in larvae starved at 6.5 g (first time point). The wings of starved larvae stopped growing about 1 day before those of normally fed larvae. Each point is the mean of three to five individuals. Error bars show SEM.

In addition to having a lower growth rate, the disks of starved larvae stopped growing a full day before those of normally fed controls (Fig. 1). At the beginning of the wandering stage, the number of cells in the wing disks of starved larvae was 85% of that in controls, and when the disks stopped growing, those from starved larvae had 52% of the number of cells found in controls (Fig. 1). The temporal pattern of mitoses in wing disks during the wandering stage of normally fed and starved larvae (Fig. 2A) shows that cell proliferation was slower after the first day and terminated a day earlier in starved larvae.

Fig. 2

(A) Mitotic index (metaphases per 1000 cells) during the wandering stage of wing imaginal disks of normally fed control larvae and larvae starved at 6.5 g. Each point represents the mean of three to five individuals. (B) Ecdysone titers (in 20E equivalents) in the hemolymph during the wandering stage of normally fed control larvae and larvae starved at 6.5 g. Each point represents the mean of five to seven individuals assayed in triplicate. Error bars show SEM.

In the pupal stage, cell division resumed about 48 hours after pupation, continued for 5 days, and then stopped permanently in both feeding and starved individuals, with no differences in the duration of cell division. Pupae from feeding larvae had 1.64 million (±91,000) cells at the beginning and 8.59 million (±56,000) cells at the end of the growth phase. Pupae from starved larvae had 860,000 (±48,000) cells at the beginning and 4.73 million (±42,000) cells at the end of the growth phase. Thus, in both fed and starved larvae, the number of cells in the developing pupal wing increased by a factor of 5.35 (t test: Pfed = 0.197; Pstarved = 0.714). We conclude that the size difference between the wings of adults produced from fed and starved larvae was almost entirely due to differences in growth during the wandering phase. We estimate that the reduction of the growth rate alone, with an identical duration of growth period, would result in a 74% reduction in wing size. Similarly, a reduction of the growth period by 1 day would result in a 73% reduction in wing size. Therefore, reduced growth rate and development time contribute equally to the reduction of wing size at smaller body size.

We suspected that one or more insect developmental hormones might control the pattern of cellular growth. In Manduca and Precis, both ecdysone and bombyxin (an insect insulin-like growth factor) are required for growth of the wing imaginal disks in vitro (13, 14). Likewise, in Drosophila and other insects, ecdysone and insulin-like growth factors are known to regulate growth and size (1723). We examined the profile of ecdysteroid titers during the wandering stage of normally fed and starved Manduca larvae (Fig. 2B). In starved larvae, ecdysone secretion stopped earlier and its levels rose higher than in fed larvae.

These findings suggest that the duration of the wandering phase may be determined by the decline in ecdysone, and that high levels of ecdysone reduce the rate of cell division. Declining titers of ecdysone act as timers of developmental events during insect metamorphosis (2426), and we examined whether declining ecdysteroids were responsible for terminating the wandering phase. We injected fed larvae with 50 μg of 20-hydroxyecdysone (20E) on days 3 and 4 of the wandering stage, a time when their endogenous ecdysteroids are declining. These injections extended the wandering phase by an average of 18 hours (Fig. 3A).

Fig. 3

(A) Delay of pupation by injection of 20E (N = 20). (B) Reduction of adult forewing size by 20E injection. (C) Dose response of forewing imaginal disk growth by 20E injection during wandering stage. Dose was given three times at 24-hour intervals; percent growth indicates the percent increase in dry weight relative to saline-injected controls (each point is the mean of six to eight individuals). Error bars show SEM.

The resulting adults did not have larger wings than those of saline-injected controls; indeed, contrary to our expectation, their wings were significantly smaller (Fig. 3B), which suggests that the injected 20E inhibited wing growth. In comparison, injections of a human insulin at various times during the wandering stage had no effect on the duration of the wandering phase, nor on the size of the adult wings (t test, P = 0.323). In Drosophila, wing and body size are controlled by both ecdysone and insulin signaling (1721), and in Lepidoptera, in vitro culture has revealed that wing disks require both insulin and ecdysone for normal growth; the two act synergistically, and either factor by itself induces only minor growth (13, 14). The fact that insulin injection had no effect on wing size in wandering-stage larvae suggests that insulin was not limiting at this stage and that variation in ecdysone signaling is the only relevant factor. Insulin is typically involved in the regulation of nutrient-dependent growth (11, 27, 28), and because the wandering-stage larva does not feed, it is possible that insulin does not vary during this stage.

To investigate the possible inhibitory effect of 20E, we ligated larvae behind the head 6 hours after initiation of the wandering stage. Neck ligation isolates the body and wing disks from brain-derived neurosecretory hormones such as bombyxin (the lepidopteran insulin-like growth factor) and the prothoracicotropic hormone (PTTH), which stimulates secretion of ecdysteroids. After waiting 24 hours for endogenous hormones to decay, we injected these larvae with three doses of 20E at 24-hour intervals and compared the sizes of their wing disks to those of saline-injected controls (Fig. 3C). We observed an optimal dosage of about 15 to 20 μg that induced maximal wing growth. Doses below and above this optimum induced substantially less growth in the wing disks.

We conclude that the early termination of the wandering phase in starved larvae was caused by the early decline of ecdysteroid titers, which terminated the growth period of the wing disks. The decrease in the growth rate of the disks during the wandering phase appears to be caused by an elevated titer of ecdysteroids, surpassing the optimal level that promotes growth. This finding suggests that ecdysone levels can modulate tissue growth rates. Our conclusions are further supported by findings that growth rate and final body size in Drosophila are also influenced by ecdysone signaling (11, 19, 20, 29). Moreover, different levels of ecdysone signaling are known to have differential effects on tissue growth and differentiation during metamorphosis (14, 30). However, it is puzzling that decreased wing growth in starved larvae is regulated by an increase in ecdysone signaling. In principle, an increase or a decrease on either side of the optimal concentration should have equivalent effects (Fig. 3C). It may be that a decrease in ecdysone signaling cannot be tolerated because other developmental processes occurring during the larval-pupal transformation depend on ecdysone (30).

Finally, we examined whether wing disk growth was modulated to be intermediate at intermediate body sizes. We starved larvae at 9.5 g, an intermediate size between 6.5 g and the normal final weight of 12.5 g. Pupal mass, duration of the wandering stage, area of the pupal wing, and cell numbers of the wing at the beginning of the pupal stage were likewise intermediate (Fig. 4). Duration of the wandering stage was a nonlinear function of body size (Fig. 4A), which suggests that body size is the key determinant of the relative roles of development time and rate of cell division in adjusting wing size to body size.

Fig. 4

Larvae starved at 6.5 g, 9.5 g, and allowed to grow normally to 12.5 g produced pupae of 3.1 g, 4.7 g, and 5.9 g, respectively (abscissa). (A) Corresponding duration of wandering stage. (B) Surface area of pupal wing. (C) Cell number in pupal wing before cell division starts (N = 10 to 14). Error bars show SEM.

Our results show that the proportional regulation of wing growth to match body size occurs during the wandering stage of the larva, after somatic growth has stopped and before pupation. Furthermore, it depends on the timing and level of ecdysone secretion, which regulates both the duration and rate of growth. Because the brain controls the secretion of ecdysteroids via the production of a tropic neurosecretory hormone, PTTH (31), these findings imply that the regulation of proportional growth of wings is, in effect, controlled by the central nervous system. It is still unclear how the central nervous system becomes aware of body size and uses this information to time the pattern of ecdysteroid secretion in the wandering stage. In some insects, body size is measured by abdominal stretch receptors (32, 33), but there is no evidence that such a mechanism operates in Manduca (31, 34). Mechanisms that facilitate growth factor sensitivity to body size have been postulated (4, 34) but not yet demonstrated. Elucidating these mechanisms remains one of the great challenges in developmental biology.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1197292/DC1

Materials and Methods

References

References and Notes

  1. We thank C. Shreve, V. Callier, R. Dilling, K. Preuss, and I. Jalli for assistance and helpful discussions. Supported by NSF grants IOS-0641144 and IOS-0744952.
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