Manduca sexta larvae are a model for growth control in insects, particularly for the demonstration of critical weight, a threshold weight that the larva must surpass before it can enter metamorphosis on a normal schedule, and the inhibitory action of juvenile hormone on this checkpoint. We examined the effects of nutrition on allatectomized (CAX) larvae that lack juvenile hormone to impose the critical weight checkpoint. Normal larvae respond to prolonged starvation at the start of the last larval stage, by extending their subsequent feeding period to ensure that they begin metamorphosis above critical weight. CAX larvae, by contrast, show no homeostatic adjustment to starvation but start metamorphosis 4 d after feeding onset, regardless of larval size or the state of development of their imaginal discs. By feeding starved CAX larvae for various durations, we found that feeding for only 12-24 h was sufficient to result in metamorphosis on day 4, regardless of further feeding or body size. Manipulation of diet composition showed that protein was the critical macronutrient to initiate this timing. This constant period between the start of feeding and the onset of metamorphosis suggests that larvae possess a molt timer that establishes a minimal time to metamorphosis. Ligation experiments indicate that a portion of the timing may occur in the prothoracic glands. This positive system that promotes molting and the negative control via the critical weight checkpoint provide antagonistic pathways that evolution can modify to adapt growth to the ecological needs of different insects.

To elucidate the role of juvenile hormone (JH) in metamorphosis of Drosophila melanogaster, the corpora allata cells, which produce JH, were killed using the cell death gene grim. These allatectomized (CAX) larvae were smaller at pupariation and died at head eversion. They showed premature ecdysone receptor B1 (EcR-B1) in the photoreceptors and in the optic lobe, downregulation of proliferation in the optic lobe, and separation of R7 from R8 in the medulla during the prepupal period. All of these effects of allatectomy were reversed by feeding third instar larvae on a diet containing the JH mimic (JHM) pyriproxifen or by application of JH III or JHM at the onset of wandering. Eye and optic lobe development in the Methoprene-tolerant (Met)-null mutant mimicked that of CAX prepupae, but the mutant formed viable adults, which had marked abnormalities in the organization of their optic lobe neuropils. Feeding Met(27) larvae on the JHM diet did not rescue the premature EcR-B1 expression or the downregulation of proliferation but did partially rescue the premature separation of R7, suggesting that other pathways besides Met might be involved in mediating the response to JH. Selective expression of Met RNAi in the photoreceptors caused their premature expression of EcR-B1 and the separation of R7 and R8, but driving Met RNAi in lamina neurons led only to the precocious appearance of EcR-B1 in the lamina. Thus, the lack of JH and its receptor Met causes a heterochronic shift in the development of the visual system that is likely to result from some cells ’misinterpreting’ the ecdysteroid peaks that drive metamorphosis.

The regulation of static allometry is a fundamental developmental process, yet little is understood of the mechanisms that ensure organs scale correctly across a range of body sizes. Recent studies have revealed the physiological and genetic mechanisms that control nutritional variation in the final body and organ size in holometabolous insects. The implications these mechanisms have for the regulation of static allometry is, however, unknown. Here, we formulate a mathematical description of the nutritional control of body and organ size in Drosophila melanogaster and use it to explore how the developmental regulators of size influence static allometry. The model suggests that the slope of nutritional static allometries, the ’allometric coefficient’, is controlled by the relative sensitivity of an organ’s growth rate to changes in nutrition, and the relative duration of development when nutrition affects an organ’s final size. The model also predicts that, in order to maintain correct scaling, sensitivity to changes in nutrition varies among organs, and within organs through time. We present experimental data that support these predictions. By revealing how specific physiological and genetic regulators of size influence allometry, the model serves to identify developmental processes upon which evolution may act to alter scaling relationships.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

The transcription factor E74 is one of the early genes induced by ecdysteroids during metamorphosis of Drosophila melanogaster. Here, we report the cloning and hormonal regulation of E74 from the tobacco hornworm, Manduca sexta (MsE74). MsE74 is 98% identical to that of D. melanogaster within the DNA-binding ETS domain of the protein. The 5’-isoform-specific regions of MsE74A and MsE74B share significantly lower sequence similarity (30-40%). Developmental expression by Northern blot analysis reveals that, during the 5th larval instar, MsE74B expression correlates with pupal commitment on day 3 and is induced to maximal levels within 12h by low levels of 20-hydroxyecdysone (20E) and repressed by physiologically relevant levels of juvenile hormone I (JH I). Immunocytochemical analysis shows that MsE74B appears in the epidermis before the 20E-induced Broad transcription factor that is correlated with pupal commitment (Zhou and Riddiford, 2001). In contrast, MsE74A is expressed late in the larval and the pupal molts when the ecdysteroid titer has declined to low levels and in the adult molt just as the ecdysteroid titer begins to decline. This change in timing during the adult molt appears not to be due to the absence of JH as there was no change during the pupal molt of allatectomized animals. When either 4th or 5th instar larval epidermis was explanted and subjected to hormonal manipulations, MsE74A induction occurred only after exposure to 20E followed by its removal. Thus, MsE74B appears to have a similar role at the onset of metamorphosis in Manduca as it does in Drosophila, whereas MsE74A is regulated differently at pupation in Manduca than at pupariation in Drosophila.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Metand gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

In starved larvae of the tobacco hornworm moth Manduca sexta, larval and imaginal tissues stop growing, the former because they lack nutrient-dependent signals but the latter because of suppression by juvenile hormone. Without juvenile hormone, imaginal discs form and grow despite severe starvation. This hormone inhibits the intrinsic signaling needed for disc morphogenesis and does so independently of ecdysteroid action. Starvation and juvenile hormone treatments allowed the separation of intrinsic and nutrient-dependent aspects of disc growth and showed that both aspects must occur during the early phases of disc morphogenesis to ensure normal growth leading to typical-sized adults.

The development of the adult optic lobe (OL) of is directed by a wave of ingrowth of the photoreceptors over a two day period at the outset of metamorphosis which is accompanied by the appearance of the pupal-specific transcription factor Broad-Z3 (Br-Z3) and expression of early drivers in OL neurons. During this time, there are pulses of ecdysteroids that time the metamorphic events. At the outset, the transient appearance of juvenile hormone (JH) prevents precocious development of the OL caused by the ecdysteroid peak that initiates pupariation, but the artificial maintenance of JH after this time misdirects subsequent development. Axon ingrowth, Br-Z3 appearance and the expression of early drivers were unaffected, but aspects of later development such as the dendritic expansion of the lamina monopolar neurons and the expression of late drivers were suppressed. This effect of the exogenous JH mimic (JHM) pyriproxifen is lost by 24 hr after pupariation. Part of this effect of JHM is due to its suppression of the appearance of ecdysone receptor EcR-B1 that occurs after pupation and during early adult development.

An adult moth sheds its pupal skin only during a specific period of the day. The brain is necessary for the synchronization of this behavior with the environmental photoperiod. This function is fully preserved when all the brain’s nervous connections are severed or when a "loose" brain is transplanted into the tip of the abdomen. By appropriate experiments it was possible to show that the entire mechanism is brain-centered. The components include a photoreceptor mechanism, a clock, and a neuroendocrine output. The clock-controlled release of the hormone acts on the central nervous system to trigger a species-specific behavior pattern which culminates in ecdysis.

Insect molting is triggered by ecdysteroids, which are produced in the prothoracic glands (PG). The broad (br) gene is one of the ’early genes’ directly regulated by ecdysteroids. Ectopic expression of the BR-Z3 isoform in early second instar Drosophila larvae (L2) before the rise of the ecdysteroid titer prevented molting to the third instar, but the larvae subsequently formed L2 prepupae after prolonged feeding. When these larvae were fed on diet containing 20-hydroxyecdysone (20E), they formed pharate third instar larvae. The critical weight for normal L3 pupariation of w(1118) larvae was found to be 0.8 mg and that for L2 pupariation was 0.45 mg. We also defined a threshold weight for metamorphosis of 0.3 mg, above which L2 larvae will metamorphose when provided with 20E. BR-Z3 apparently works through the PG cells of the ring gland but not the putative neurosecretory cells that drive ecdysone secretion, because ectopic expression of BR-Z3 specifically in the ring gland caused 53% of the larvae to become permanent first instar larvae. Driving other BR isoforms in the ring gland prevented larval molting or pupariation to varying degrees. These molting defects were rescued by feeding 20E. Overexpression of each of the BR isoforms caused degeneration of the PG cells but on different time courses, indicating that BR is a signal for the degeneration of the PG cells that normally occurs during the pupal-adult transition.