Amino Acids and TOR Signaling Promote Prothoracic Gland Growth and the Initiation of Larval Molts in the Tobacco Hornworm <em>Manduca sexta</em>

<div><p>Molting in arthropods is orchestrated by a series of endocrine changes that occur towards the end of an instar. However, little is understood about the mechanisms that trigger these endocrine changes. Here, nutritional inputs were manipulated to investigate the minimal nutritional inputs required for a <em>Manduca sexta</em> larva to initiate a molt. Amino acids were found to be necessary for a larva to molt, indicating the involvement of an amino acid sensitive pathway. Feeding rapamycin, an inhibitor of the target of rapamycin (TOR) signaling, delayed the onset of a molt and resulted in abnormally larger larvae. Rapamycin also suppressed the growth of the prothoracic glands relative to the whole body growth, and this was accompanied by suppression of ecdysone production and secretion. Higher doses of rapamycin also slowed the growth rate, indicating that TOR signaling also plays a role in systemic growth. TOR signaling therefore couples the nutritional status of the larva to the endocrine system to regulate the timing of a molt.</p> </div

Growth of fourth instar <i>M. sexta</i> larvae transferred from sucrose to casein diets and vice versa.

<p>(A–E) Growth trajectory of fourth instar larvae transferred from sucrose to casein diets and vice versa. Day 0 larvae were fed 5.8% casein and 7% sucrose in the specified order, and their growth trajectory was monitored. The red dots indicate the timing of head capsule slippage (n = 8–20 for each treatment). Black circles indicate the animals that molted whereas grey circles represent the animals that ultimately died without molting. (F) The time taken for larvae to initiate a molt. Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p<0.05; n = 4–20). p<0.0001 for all statistically different pairwise comparisons except between larvae fed on casein for either one or two days and transferred to sucrose (p = 0.0002), and between those fed on normal diet and those fed on sucrose for one day followed by casein (p = 0.0008). Error bars represent standard errors.</p

Effect of rapamycin and various nutrients on prothoracic gland growth.

<p>(A) Representative prothoracic glands, two days post feeding diets containing various nutrients. (B,C) Effect of rapamycin (B) and various nutrients (C) on prothoracic gland growth relative to body size. Prothoracic glands were dissected out from larvae at various weights. The size of prothoracic glands was determined by measuring the cross-sectional area of the glands. Larvae were transferred to the treated diets after being fed a non-nutritive diet for two days post head capsule slip at the end of the third instar. The glands were dissected after the larvae were fed the respective diets for at least one day.</p

Effects of rapamycin and nutrients on ecdysone levels and 4E-BP phosphorylation states, respectively.

<p>(A) Effects of rapamycin treatment on ecdysone levels. Prothoracic glands and hemolymph from larvae fed 0.1 mg/ml rapamycin- or control DMSO-treated diets were isolated at average weights of 0.59±0.04 g and 0.61±0.02 g, respectively (Student’s t-test, <i>p</i> = 0.70, n = 8 larvae per group). Significant differences were observed between control and rapamyin-fed groups in hemolymph ecdysteroid levels, and in ecdysone secretion by isolated prothoracic glands (Student’s t-test, <i>p</i> = 0.014 and 0.009, respectively). Error bars indicate standard error for ecdysone secretion. (B) Effect of nutrients on 4E-BP phosphorylation states in the prothoracic glands and the fat body. In both the prothoracic glands and the fat body, the presence of amino acids led to elevated phosphorylated 4E-BP levels relative to sucrose fed larvae or starved larvae. In the prothoracic glands, phospho-4E-BP levels were also elevated in the starved larva relative to the sucrose fed larva.</p

Effect of diet dilution on the timing of a molt and the growth of prothoracic glands.

<p>(A) Size of larvae at the time of head capsule slippage in larvae fed 40%, 60% or 100% diet from the first instar. (B) The relationship between body size and prothoracic gland size in larvae fed diluted diet from the first instar.</p

Model of how environmental cues might interact with TOR signaling to influence the timing of a molt.

<p>Model of how environmental cues might interact with TOR signaling to influence the timing of a molt.</p

Effect of various nutrients on the survival and time to molt.

<p>(A) Time to molt (black bars) or death (open bars) in 4^th instar larvae fed various concentrations of casein or sucrose only diets (n = 8–12 for each treatment). (B) Final size at the time of the molt for larvae fed normal or casein-only diets. Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p<0.0001). The normal diet fed larvae were significantly larger than those fed 4% (p<0.0001), 5.8% (p = 0.0001) or 8% (p = 0.0001) casein only diets. The various casein diets did not produce significantly different effects on the final size at the time of molt (n = 8–12 for each treatment). Error bars represent standard errors. (C) Time to molt (black bars) or death (open bars) in 4^th instar larvae fed casein diets supplemented with sucrose (n = 10–11 for each treatment). Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p<0.0001). Molting time of larvae fed a 2% casein diet supplemented with 6% sucrose was significantly delayed relative to the 8% casein only, 5.8% casein/2.2% sucrose and 4% casein/4% sucrose diets (p<0.0001, p<0.0001 and p = 0.001, respectively). (D) Final size at the time of the molt for larvae fed casein diets supplemented with sucrose. Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p = 0.0007). Larvae fed a 2% casein diet supplemented with 6% sucrose molted at a significantly smaller size relative to the 8% casein only, 5.8% casein/2.2% sucrose and 4% casein/4% sucrose diets (p = 0.0352, p = 0.0003 and p = 0.0247, respectively).</p

The effect of 20-hydroxyecdysone injection on starved 4^th instar larvae.

<p>The effect of 20-hydroxyecdysone injection on starved 4^th instar larvae.</p

Effect of rapamycin on larval molt and time to molt.

<p>(A) Growth trajectories of larvae fed different concentrations of rapamycin. Differently colored lines represent different concentrations of rapamycin. (B) Time taken for larvae at various body sizes to initiate a molt. (C) Total time taken for larvae to initiate a molt. Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p<0.0001). All pairwise comparisons are significantly different from each other (p<0.0001) except between those treated with 1 mg/g and 10 mg/g rapamycin (p = 0.961). (D) Final body size at time of head capsule slip (n = 19–24 for each treatment). Different letters represent statistically significant differences (ANOVA with Tukey-Kramer HSD test; p<0.0001). All rapamycin fed larvae molted at a significantly larger size relative to the DMSO treated control larvae (p<0.0001). Larvae fed 1 mg/g rapamycin molted at a significantly larger size compared to those fed with 0.1 mg/g rapamycin (p = 0.0171). Error bars represent standard errors.</p