Body size profoundly affects many aspects of animal biology, including metamorphosis, allometry, size-dependent alternative pathways of gene expression, and the social and ecological roles of individuals. However, regulation of body size is one of the fundamental unsolved problems in developmental biology. The control of body size requires a mechanism that assesses size and stops growth within a characteristic range of sizes. Under normal growth conditions in Manduca sexta , the endocrine cascade that causes the brain to initiate metamorphosis starts when the larva reaches a critical weight. Metamorphosis is initiated by a size-sensing mechanism, but the nature of this mechanism has remained elusive. Here we show that this size-sensing mechanism depends on the limited ability of a fixed tracheal system to sustain the oxygen supply to a growing individual. As body mass increases, the demand for oxygen also increases, but the fixed tracheal system does not allow a corresponding increase in oxygen supply. We show that interinstar molting has the same size-related oxygen-dependent mechanism of regulation as metamorphosis. We show that low oxygen tension induces molting at smaller body size, consistent with the hypothesis that under normal growth conditions, body size is regulated by a mechanism that senses oxygen limitation. We also found that under poor growth conditions, larvae may never attain the critical weight but eventually molt regardless. We show that under these conditions, larvae do not use the critical weight mechanism, but instead use a size-independent mechanism that is independent of the brain.

A mathematical model is developed for the folate cycle based on standard biochemical kinetics. We use the model to provide new insights into several different mechanisms of folate homeostasis. The model reproduces the known pool sizes of folate substrates and the fluxes through each of the loops of the folate cycle and has the qualitative behavior observed in a variety of experimental studies. Vitamin B(12) deficiency, modeled as a reduction in the V(max) of the methionine synthase reaction, results in a secondary folate deficiency via the accumulation of folate as 5-methyltetrahydrofolate (the "methyl trap"). One form of homeostasis is revealed by the fact that a 100-fold up-regulation of thymidylate synthase and dihydrofolate reductase (known to occur at the G(1)/S transition) dramatically increases pyrimidine production without affecting the other reactions of the folate cycle. The model also predicts that an almost total inhibition of dihydrofolate reductase is required to significantly inhibit the thymidylate synthase reaction, consistent with experimental and clinical studies on the effects of methotrexate. Sensitivity to variation in enzymatic parameters tends to be local in the cycle and inversely proportional to the number of reactions that interconvert two folate substrates. Another form of homeostasis is a consequence of the nonenzymatic binding of folate substrates to folate enzymes. Without folate binding, the velocities of the reactions decrease approximately linearly as total folate is decreased. In the presence of folate binding and allosteric inhibition, the velocities show a remarkable constancy as total folate is decreased.

The mechanisms that control the growth rate of internal tissues during postembryonic development are poorly understood. In insects, the growth rate of imaginal disks varies with nutrition and keeps pace with variation in somatic growth. We describe here a mechanism by which the growth of wing imaginal disks is controlled. When wing imaginal disks of the butterflyPrecis coeniaare removed from the larva and placed in a standard nutrient-rich tissue culture medium they stop growing, suggesting that nutrients alone are not sufficient to support normal growth. Such disks can be made to grow at a normal rate by supplementing the culture medium with an optimal concentration of the steroid hormone 20-hydroxyecdysone and with hemolymph taken from growing larvae. The growth-promoting activity of the hemolymph is caused by a heat-stable factor that can be extracted from the CNS and appears to be identical to the neurohormone bombyxin, a member of the insulin family of proteins. Synthetic bombyxin stimulates growth at concentrations as low as 30 ng/ml, and specific antibodies to bombyxin completely remove growth-promoting activity from the hemolymph. Bombyxin evidently acts together with 20-hydroxyecdysone to stimulate cell division and growth of wing imaginal disks. It appears that the level of bombyxin in the hemolymph is modulated by the brain in response to variation in nutrition and is part of the mechanism that coordinates the growth of internal organs with overall somatic growth.