A switch in the control of growth of the wing imaginal disks of Manduca sexta

Abstract

Background: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs.

Methodology: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment.

Principal findings: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth.

Conclusions: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.

Figure 1. Somatic and wing imaginal disk growth during the last larval instar of Manduca sexta.

Vertical dashed line between days 5–6 indicates the transition from the feeding to the wandering phase. (A) Body growth; feeding stops and mass declines during the wandering phase. Each point is the mean of >50 individuals; bars are standard deviations. (B) Semilogarithmic plot of growth in dry mass of the fore wing imaginal disks. The growth rate of the wing disk increases after larvae enter the wandering stage. Lines are exponential regressions. During the feeding phase the growth is given by the exponential equation mass = 0.011e^0.45, r² = 0.93; during the wandering phase growth is given by the exponential equation mass = 0.007e^0.55, r² = 0.97. Each point is the mean of 20–22 individuals; bars are standard deviations. (C) Semilogarithmic plot of the increase in cell number of the fore wing imaginal disk. Cell division stops after day 8. Each point represents the mean of 12 individuals; bars are standard deviations.

Figure 2. Effect of starvation before and after critical weight on wing imaginal disk growth.

The grey bar represents the mass interval at which 95% of larvae attain the critical weight. Before the critical weight starvation (open triangles) stopped growth. After the critical weight starvation did not stop growth but reduced the growth rate to approximately 66% of control (black circles).

Figure 3. Effect of juvenile hormone on wing disk growth.

Graph shows the size of the disks after 4 days for JH-treated larvae and acetone-treated controls. Each bar represents the mean of 6–8 larvae. Error bars SEM.

Figure 4. Effect of the brain removal and abdominal ligation on wing imaginal disk growth.

Dry weight gain after 24h of brain removal (p<0.001) or abdominal ligation (p<0.001) for the forewing disk of first day wandering phase (day 6). Each bar represents the mean in forewing disk size of 18–20 disks. Error bars SEM.

Figure 5. Quantification of cell proliferation in the forewing imaginal disk.

Cell proliferation was measured as the average number of PH3 positive cells in an area of 0.0875mm² of the wing imaginal disk. Effect of starvation: larvae were starved on day 4 of the feeding phase and PH3 cells in the wing disk were counted 48h later (p = 0.021). Effect of brain removal: the brain was removed the first day of the wandering phase (day 6) and PH3 cells were counted 24h later (p<0.001). Effect of abdominal ligation: a ligation was placed between the thorax and the abdomen on day 6 larvae, and PH3 cells were counted 24h later (p<0.001). Each bar represents the mean of 10 disks. Error bars SEM.

Figure 6. Effect of mid-thoracic ligation on fore- and hindwing imaginal disk growth.

A ligation was placed between the second and third thoracic segment on the first day of the wandering phase (day 6). The ligation prevented the forewing from receiving most factors coming from the abdominal region, and also prevented the hindwing from receiving brain factors. The relationship between fore- and hindwing sizes for controls fall on a common regression. In ligated larvae (open triangles) the forewing disks grew little in 24 hours, and the hindwings grew about half as much as controls (black circles) The regression lines for control and ligated larvae for fore- vs hindwing disk sizes are statistically different (p<0.001).

Figure 7. Hemolymph glucose and trehalose concentration during the last larval instar.

Values are means of 10–16 individuals ±SD.

Figure 8. Effect of starvation on hemolymph glucose and trehalose concentration.

(A) Glucose concentration in larvae starved before (BCW) and after critical weight (ACW). (B) Trehalose concentration in larvae starved before and after critical weight. Values are means of 8–16 independent samples ±SD.

Figure 9. Effect of glucose and trehalose injection on wing disk growth.

Glucose and trehalose were injected 3 times at 12h interval during 48 h. Each bar represents the mean of 6–10 disks. Error bars SEM.

Figure 10. Bbx and InR mRNA transcript levels.

(A) Last larval instar. Effect of food deprivation at day 2 (B), and at day 4 (C) of the feeding phase. Transcript levels were measured 24h after starvation. Each point represents the mean of 3–5 biological replicates measured in triplicate. Error bars SD.