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Lepidosaurian reptiles, particularly snakes, periodically shed the outer epidermal layers of their skin (ecdysis) to restore or enhance vital functions such as regulating water and gaseous exchange, growth, and protection against insult, infection or physical injury. Although many studies have focused on the nature and mechanisms of skin shedding, little attention has been paid to the timing of the first ecdysis in neonates following birth or hatching. A recent study investigated patterns of the time to first postnatal ecdysis in snakes based on a large dataset taken from the literature. The analysis demonstrated patterns in the time to first postnatal ecdysis related to phylogeny as well as several life history traits. While this assessment provides important advances in our knowledge of this topic, data on known biophysical drivers of ecdysis - temperature and humidity - were largely unavailable and were not evaluated. The first postnatal ecdysis of neonatal snakes can be viewed as an adaptive adjustment to the transition from the aqueous environment of the embryo to the aerial environment of the newborn. Hence, the timing of the first postnatal ecdysis is logically influenced by the aerial environment into which a newborn snake or hatchling finds itself. Therefore, in this Commentary, we first emphasize the putative plasticity of ecdysis with respect to epidermal lipids that structure the water permeability barrier and are established or renewed during ecdysis to reduce transepidermal evaporative water loss. We then discuss the likely importance of biophysical variables as influential covariates that need future investigation as potential co-determinants of the timing of first postnatal ecdysis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Once a year, penguins undergo a catastrophic moult, replacing their entire plumage during a fasting period on land or on sea-ice during which time individuals can lose 45% of their body mass. In penguins, new feather synthesis precedes the loss of old feathers, leading to an accumulation of two feather layers (double coat) before the old plumage is shed. We hypothesized that the combination of the high metabolism required for new feather synthesis and the potentially high thermal insulation linked to the double coat could lead to a thermal challenge requiring additional peripheral circulation to thermal windows to dissipate the extra heat. To test this hypothesis, we measured the surface temperature of different body regions of captive gentoo penguins (Pygoscelis papua) throughout the moult under constant environmental conditions. The surface temperature of the main body trunk decreased during the initial stages of the moult, suggesting greater thermal insulation. In contrast, the periorbital region, a potential proxy of core temperature in birds, increased during these same early moulting stages. The surface temperature of the bill, flipper and foot (thermal windows) tended to initially increase during the moult, highlighting the likely need for extra heat dissipation in moulting penguins. These results raise questions regarding the thermoregulatory capacities of penguins in the wild during the challenging period of moulting on land in the current context of global warming., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Seals haul out of water for extended periods during the annual molt, when they shed and regrow their pelage. This behavior is believed to limit heat loss to the environment given increased peripheral blood flow to support tissue regeneration. The degree to which time in water, particularly during the molt, may affect thermoregulatory costs is poorly understood. We measured the resting metabolism of three spotted seals (Phoca largha), one ringed seal (Pusa hispida) and one bearded seal (Erignathus barbatus) during and outside the molting period, while resting in water and when hauled out. Metabolic rates were elevated in spotted and ringed seals during molt, but comparable in water and air for individuals of all species, regardless of molt status. Our data indicate that elevated metabolism during molt primarily reflects the cost of tissue regeneration, while increased haul out behavior is driven by the need to maintain elevated skin temperatures to support tissue regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In ecdysozoan animals, moulting entails the production of a new exoskeleton and shedding of the old one during ecdysis. It is induced by a pulse of ecdysone that regulates the expression of different hormonal receptors and activates a peptide-mediated signalling cascade. In Holometabola, the peptidergic cascade regulating ecdysis has been well described. However, very little functional information regarding the neuroendocrine regulation of ecdysis is available for Hemimetabola, which display an incomplete metamorphosis. We use Rhodnius prolixus as a convenient experimental model to test two hypotheses: (1) the role of neuropeptides that regulate ecdysis in Holometabola is conserved in hemimetabolous insects; and (2) the neuropeptides regulating ecdysis play a role in the regulation of female reproduction during the adult stage. The RNA interference-mediated reduction of ecdysis triggering hormone (ETH) mRNA levels in fourth-instar nymphs resulted in lethality at the expected time of ecdysis. Unlike in holometabolous insects, knockdown of eth and orcokinin isoform A (oka) did not affect oviposition in adult females, pointing to a different endocrine regulation of ovary maturation. However, eth knockdown prevented egg hatching. The blockage of egg hatching appears to be a consequence of embryonic ecdysis failure. Most of the first-instar nymphs hatched from the eggs laid by females injected with dsRNA for eclosion hormone (dsEH), crustacean cardioactive peptide (dsCCAP) and dsOKA died at the expected time of ecdysis, indicating the crucial involvement of these genes in post-embryonic development. No phenotypes were observed upon corazonin (cz) knockdown in nymphs or adult females. The results are relevant for evolutionary entomology and could reveal targets for neuropeptide-based pest control tools., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Like all birds, penguins undergo periodic molt, during which they replace old feathers. However, unlike other birds, penguins replace their entire plumage within a short period while fasting ashore. During molt, king penguins ( Aptenodytes patagonicus ) lose half of their initial body mass, most importantly their insulating subcutaneous fat and half of their pectoral muscle mass. The latter might challenge their capacity to generate and sustain a sufficient mechanical power output to swim to distant food sources and propel themselves to great depth for successful prey capture. To investigate the effects of the annual molt fast on their dive/foraging performance, we studied various dive/foraging parameters and peripheral temperature patterns in immature king penguins across two molt cycles, after birds had spent their first and second year at sea, using implanted data-loggers. We found that the dive/foraging performance of immature king penguins was significantly reduced during post-molt foraging trips. Dive and bottom duration for a given depth were shorter during post-molt and post-dive surface interval duration was longer, reducing overall dive efficiency and underwater foraging time. We attribute this decline to the severe physiological changes that birds undergo during their annual molt. Peripheral temperature patterns differed greatly between pre- and post-molt trips, indicating the loss of the insulating subcutaneous fat layer during molt. Peripheral perfusion, as inferred from peripheral temperature, was restricted to short periods at night during pre-molt but occurred throughout extended periods during post-molt, reflecting the need to rapidly deposit an insulating fat layer during the latter period., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Intraspecific discrete polymorphism is associated with the use of alternative life-history strategies, reflected by distinct reproductive or copying strategies in individuals of different morphs. Yet, morph-specific costs and benefits related to different life-history strategies remain unclear. Here, we examined in the polymorphic Gouldian finch ( Erythrura gouldiae ) whether markers of somatic maintenance (body mass, oxidative status and telomere length) differed between red- and black-headed birds under energetically demanding conditions (during heatwaves of different intensity, and during moult or breeding following heatwaves). During heatwaves, red-headed birds showed a homogeneous response, as males and females initially tended to gain mass and had reduced plasma hydroperoxide levels (a marker of oxidative damage) irrespective of heatwave intensity. In contrast, black-headed birds showed a stronger and more heterogeneous response, as black-headed males gained mass at the beginning of the thermoneutral heatwave and showed stable oxidative status, whereas black-headed females lost mass and tended to show higher hydroperoxide levels at the end of the thermocritical heatwave. Following heatwaves, we found morph-specific oxidative costs owing to moult or reproduction, with oxidative markers varying only in black-headed birds. Again, oxidative markers varied differently in black-headed males and females, as plasma antioxidant capacity decreased in moulting or breeding females, whereas males showed higher hydroperoxide levels with larger broods. For the first time, our study highlights that within polymorphic species, some individuals appear more vulnerable than others when coping with energetically demanding conditions. In the context of climate change, such differential effects may ultimately alter the currently observed balance between morphs and sexes within natural populations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Mechanistic target of rapamymcin (mTOR) is a highly conserved protein kinase that controls cellular protein synthesis and energy homeostasis. We hypothesize that mTOR integrates intrinsic signals (moulting hormones) and extrinsic signals (thermal stress) to regulate moulting and growth in decapod crustaceans. The effects of temperature on survival, moulting and mRNA levels of mTOR signalling genes ( Mm-Rheb , Mm-mTOR , Mm-AMPKα , Mm-S6K and Mm-AKT ) and neuropeptides ( Mm-CHH and Mm-MIH ) were quantified in juvenile Metacarcinus magister Crabs at different moult stages (12, 19 or 26 days postmoult) were transferred from ambient temperature (∼15°C) to temperatures between 5 and 30°C for up to 14 days. Survival was 97-100% from 5 to 20°C, but none survived at 25 or 30°C. Moult stage progression accelerated from 5 to 15°C, but did not accelerate further at 20°C. In eyestalk ganglia, Mm-Rheb , Mm-AMPKα and Mm-AKT mRNA levels decreased with increasing temperatures. Mm-MIH and Mm-CHH mRNA levels were lowest in the eyestalk ganglia of mid-premoult animals at 20°C. In the Y-organ, Mm-Rheb mRNA levels decreased with increasing temperature and increased during premoult, and were positively correlated with haemolymph ecdysteroid titre. In the heart, moult stage had no effect on mTOR signalling gene mRNA levels; only Mm-Rheb , Mm-S6K and Mm-mTOR mRNA levels were higher in intermoult animals at 10°C. These data suggest that temperature compensation of neuropeptide and mTOR signalling gene expression in the eyestalk ganglia and Y-organ contributes to regulate moulting in the 10 to 20°C range. The limited warm compensation in the heart may contribute to mortality at temperatures above 20°C., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The transition from aquatic to terrestrial environments places significant mechanical challenges on skeletal support systems. Crabs have made this transition multiple times and are the largest arthropods to inhabit both environments. Furthermore, they alternate between rigid and hydrostatic skeletons, making them an interesting system to examine mechanical adaptations in skeletal support systems. I hypothesized that terrestrial crabs have modified morphology to enhance mechanical stiffness and that rigid and hydrostatic skeletons scale differently from each other, with stronger allometric relationships on land. Using the aquatic blue crab, Callinectes sapidus , and the terrestrial blackback land crab, Gecarcinus lateralis , I measured and compared body mass, merus morphology (dimensions, cuticle thickness and the second moment of area I ) and mechanics (flexural stiffness EI , elastic modulus E , critical stress and hydrostatic pressure) of rigid and hydrostatic stage crabs encompassing a range of sizes ( C. sapidus : 1.5-133 g, N ≤24; G. lateralis : 22-70 g, N ≤15). The results revealed that rigid G. lateralis has similar morphology (limb length to diameter L / D and cuticle thickness to limb diameter T / D ratio) to C. sapidus , and the mechanics and most scaling relationships are the same. Hydrostatic land crabs differ from aquatic crabs by having different morphology (thinner cuticle), mechanics (greater internal pressures) and scaling relationship (cuticle thickness). These results suggest that the rigid crab body plan is inherently overbuilt and sufficient to deal with the greater gravitational loading that occurs on land, while mechanical adaptations are important for hydrostatically supported crabs. Compared with other arthropods and hydrostatic animals, crabs possess distinct strategies for adapting mechanically to life on land., Competing Interests: Competing interestsThe author declares no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Body size is a key life history trait, and knowledge of its mechanistic basis is crucial in life history biology. Such knowledge is accumulating for holometabolous insects, whose growth is characterised and body size affected by moulting. According to the oxygen-dependent induction of moulting (ODIM) hypothesis, moult is induced at a critical mass at which oxygen demand of growing tissues overrides the supply from the tracheal respiratory system, which principally grows only at moults. Support for the ODIM hypothesis is controversial, partly because of a lack of proper data to explicitly test the hypothesis. The ODIM hypothesis predicts that the critical mass is positively correlated with oxygen partial pressure ( P O 2 ) and negatively with temperature. To resolve the controversy that surrounds the ODIM hypothesis, we rigorously test these predictions by exposing penultimate-instar Orthosia gothica (Lepidoptera: Noctuidae) larvae to temperature and moderate P O 2  manipulations in a factorial experiment. The relative mass increment in the focal instar increased along with increasing P O 2 , as predicted, but there was only weak suggestive evidence of the temperature effect. Probably owing to a high measurement error in the trait, the effect of P O 2  on the critical mass was sex specific; high P O 2  had a positive effect only in females, whereas low P O 2  had a negative effect only in males. Critical mass was independent of temperature. Support for the ODIM hypothesis is partial because of only suggestive evidence of a temperature effect on moulting, but the role of oxygen in moult induction seems unambiguous. The ODIM mechanism thus seems worth considering in body size analyses., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Amphibian skin is a multifunctional organ providing protection from the external environment and facilitating the physiological exchange of gases, water and salts with the environment. In order to maintain these functions, the outer layer of skin is regularly replaced in a process called sloughing. During sloughing, the outermost layer of the skin is removed in its entirety, which has the potential to interfere with skin permeability and ion transport, disrupting homeostasis. In this study, we measured, in vivo , the effects of sloughing on the cutaneous efflux of ions in toads Rhinella marina kept in freshwater conditions. We also measured transepithelial potential, cutaneous resistance, active ion transport and the distribution, abundance and gene expression of the key ion transport proteins sodium-potassium ATPase (NKA) and epithelial sodium channel (ENaC) during sloughing. We hypothesised that the increase in transepithelial efflux of ions during sloughing is a consequence of increased permeability and/or a reduction in the abundance or expression of cutaneous ion transport proteins, resulting in disruption of internal ion homeostasis. There was a significant increase in sodium and chloride efflux during sloughing in R. marina However, although in vitro skin resistance decreased after sloughing, active sodium transport increased commensurate with an increase in NKA and ENaC protein abundance in the skin. These changes in skin function associated with sloughing did not affect the maintenance of internal electrolyte homeostasis. These results suggest that during sloughing, amphibians actively maintain internal homeostasis by increasing cutaneous rates of ion uptake., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Recent data suggest that oxygen limitation may induce moulting in larval insects. This oxygen-dependent induction of moulting (ODIM) hypothesis stems from the fact that the tracheal respiratory system of insects grows primarily at moults, whereas tissue mass increases massively between moults. This may result in a mismatch between oxygen supply and demand at the end of each larval instar because oxygen demand of growing tissues exceeds the relatively fixed supply capacity of the respiratory system. The ODIM hypothesis predicts that, within larval instars, respiration and metabolic rates of an individual larva first increase with increasing body mass but eventually level off once the supply capacity of the tracheal system starts to constrain metabolism. Here, we provide the first individual-level test of this key prediction of the ODIM hypothesis. We use a novel methodology where we repeatedly measure respiration and metabolic rates throughout the penultimate- and final-instar larvae in the butterfly Pieris napi In the penultimate instar, respiration and metabolic rates gradually decelerated along with growth, supporting the ODIM hypothesis. However, respiration and metabolic rates increased linearly during growth in the final instar, contradicting the prediction. Moreover, our data suggest considerable variation among individuals in the association between respiration rate and mass in the final instar. Overall, the results provide partial support for the ODIM hypothesis and suggest that oxygen limitation may emerge gradually within a larval instar. The results also suggest that there may be different moult induction mechanisms in larva-to-larva moults compared with the final metamorphic moult., (© 2016. Published by The Company of Biologists Ltd.)

Locusts jump by using a catapult mechanism in which energy produced by slow contractions of the extensor tibiae muscles of the hind legs is stored in distortions of the exoskeleton, most notably (1) the two semi-lunar processes at each knee joint and (2) the tendons of the extensor muscles themselves. The energy is then suddenly released from these stores to power the rapid, propulsive movements of the hind legs. The reliance on the mechanical storage of energy is likely to impact on jumping because growth occurs by a series of five moults, at each of which the exoskeleton is replaced by a new one. All developmental stages (instars) nevertheless jump as a means of forward locomotion, or as an escape movement. Here, I show that in each instar, resilin is added to the semi-lunar processes and to the core of the extensor tendons so that their thickness increases. As the next moult approaches, a new exoskeleton forms within the old one, with resilin already present in the new semi-lunar processes. The old exoskeleton, the tendons and their resilin are discarded at moulting. The resilin of the semi-lunar processes and tendons of the new instar is initially thin, but a similar pattern of deposition results in an increase of their thickness. In adults, resilin continues to be deposited so that at 4 weeks old the thickness in the semi-lunar processes has increased fourfold. These changes in the energy stores accompany changes in jumping ability and performance during each moulting cycle., (© 2016. Published by The Company of Biologists Ltd.)

Molting in decapod crustaceans is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ (XO)/sinus gland (SG) complex of the eyestalk ganglia (ESG). Pulsatile release of MIH from the SG suppresses ecdysteroidogenesis by the molting gland or Y-organ (YO). The hypothesis is that nitric oxide (NO), a neuromodulator that controls neurotransmitter release at presynaptic membranes, depresses the frequency and/or amount of MIH pulses to induce molting. NO synthase (NOS) mRNA was present in Carcinus maenas ESG and other tissues and NOS protein was present in the SG. A copper based ligand (CuFL), which reacts with NO to form a highly fluorescent product (NO-FL), was used to image NO in the ESG and SG and quantify the effects of NO scavenger (cPTIO), NOS inhibitor (l-NAME), and sodium azide (NaN3) on NO production in the SG. Pre-incubation with cPTIO prior to CuFL loading decreased NO-FL fluorescence ~30%; including l-NAME had no additional effect. Incubating SG with l-NAME during pre-incubation and loading decreased NO-FL fluorescence ~40%, indicating that over half of the NO release was not directly dependent on NOS activity. Azide, which reacts with NO-binding metal groups in proteins, reduced NO-FL fluorescence to near background levels without extensive cell death. Spectral shift analysis showed that azide displaced NO from a soluble protein in SG extract. These data suggest that the SG contains NO-binding protein(s) that sequester NO and releases it over a prolonged period. This NO release may modulate neuropeptide secretion from the axon termini in the SG., (© 2015. Published by The Company of Biologists Ltd.)

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure., (© 2014. Published by The Company of Biologists Ltd.)

Circannual rhythms regulate seasonal reproduction in many vertebrates. The present study investigated whether circannual reproductive phenotypes (rhythms in growth of gonads and molt) were generated independently of the circadian clocks in the subtropical non-photoperiodic spotted munia (Lonchura punctulata). Birds were subjected to light:dark (LD) cycles with identical light but varying dark hours, such that the period of LD cycle (T) equaled 16 h (T16; 12 h L:4 h D), 21 h (T21; 12 h L:9 h D), 24 h (T24; 12 h L:12 h D) and 27 h (T27; 12 h L:15 h D), or to continuous light (LL, 24 h L:0 h D) at ~18°C. During the ~21 month exposure, munia underwent at least two cycles of gonadal development and molt; changes in body mass were not rhythmic. This was similar to the occurrence of annual cycles in reproduction and molt observed in wild birds. A greater asynchrony between circannual cycles of gonad development and molt indicated their independent regulation. Females showed reproductive rhythms with similar circannual periods, whilst in males, circannual periods measured between peak gonadal size were longer in T21 and T24 than in T16 or T27. This suggested a sex-dependent timing of annual reproduction in the spotted munia. Also, food availability periods may not influence the circannual timing of reproduction, as shown by the results on the rhythm in gonadal growth and regression in munia under T-photocycles and LL that provided differential light (feeding) hours. Further, a short-term experiment revealed that activity-rest patterns in munia were synchronized with T-photocycles, but were arrhythmic under LL. We conclude that circadian rhythms are not involved in the timing of the annual reproductive cycle in the spotted munia., (© 2014. Published by The Company of Biologists Ltd.)

Female blue crabs (Callinectes sapidus) in their pubertal moult stage release unidentified sex pheromone molecules in their urine, causing males to respond with courtship behaviours including a display called courtship stationary paddling and a form of precopulatory guarding called cradle carry. We hypothesized that pheromones are mixtures of molecules and are more concentrated in urine of pubertal premoult females compared with other moulting stages and thus that these molecules are biomarkers (i.e. metabolites that can be used as an indicator of some biological state or condition) of pubertal premoult females. We tested this hypothesis by combining bioassay-guided fractionation and biomarker targeting. To evaluate the molecular mass of the putative pheromone by bioassay-guided fractionation, we separated urine from pubertal premoult females and intermoult males by ultrafiltration into three molecular mass fractions. The <500 Da fraction and the 500-1000 Da fraction but not the >1000 Da fraction of female urine induced male courtship stationary paddling, but none of the fractions of male urine did. Thus, female urine contains molecules of <1000 Da that stimulate courtship behaviours in males. Biomarker targeting using nuclear magnetic resonance (NMR) spectral analysis of the 500-1000 Da fraction of urine from premoult and postmoult males and females revealed a premoult biomarker. Purification, nuclear magnetic resonance, mass spectrometry and high pressure liquid chromatography analysis of this premoult biomarker identified it as N-acetylglucosamino-1,5-lactone (NAGL) and showed that it is more abundant in urine of premoult females and males than in urine of either postmoult or juvenile females and males. NAGL has not been reported before as a natural product or as a molecule of the chitin metabolic pathway. Physiological and behavioural experiments demonstrated that blue crabs can detect NAGL through their olfactory pathway. Thus, we hypothesize that NAGL is a component of the sex pheromone and that it acts in conjunction with other yet unidentified components.

Animal behavior is often organized into stereotyped sequences that promote the goals of reproduction, development and survival. However, for most behaviors, the neural mechanisms that govern the order of execution of the motor programs within a sequence are poorly understood. An important model in understanding the hormonal determinants of behavioral sequencing is the ecdysis sequence, which is performed by insects at each developmental transition, or molt. The adult ecdysis sequence in Drosophila includes the emergence of the insect from the pupal case followed by expansion and hardening of the wings. Wing expansion is governed by the hormone bursicon, and stimulation of the bursicon-expressing neurons in newly eclosed flies induces rapid wing expansion. Here we show that that such stimulation delivered prior to eclosion has no immediate effect, but does cause rapid wing expansion after eclosion if the stimulus is delivered within 40 min of that event. We observe a similar delayed effect upon stimulation of a single pair of bursicon-expressing neurons previously identified as command neurons for wing expansion. We conclude that command neuron stimulation enables the motor output pathway for wing expansion, but that this pathway is blocked prior to eclosion. By manipulating the time of eclosion, we demonstrate that some physiological process tightly coupled to adult ecdysis releases the block on wing expansion. Eclosion thus serves as a behavioral checkpoint and complements hormonal mechanisms to ensure that wing expansion strictly follows eclosion in the ecdysis sequence.

Habitat heterogeneity that occurs within an individual's lifetime may favour the evolution of reversible plasticity. Colour reversibility has many different functions in animals, such as thermoregulation, crypsis through background matching and social interactions. However, the mechanisms underlying reversible colour changes are yet to be thoroughly investigated. This study aims to determine the environmental and hormonal factors underlying morphological colour changes in Thomisus onustus crab spiders and the biochemical metabolites produced during these changes. We quantified the dynamics of colour changes over time: spiders were kept in yellow and white containers under natural light conditions and their colour was measured over 15 days using a spectrophotometer. We also characterised the chemical metabolites of spiders changing to a yellow colour using HPLC. Hormonal control of colour change was investigated by injecting 20-hydroxyecdysone (20E) into spiders. We found that background colouration was a major environmental factor responsible for colour change in crab spiders: individuals presented with white and yellow backgrounds changed to white and yellow colours, respectively. An ommochrome precursor, 3-OH-kynurenine, was the main pigment responsible for yellow colour. Spiders injected with 20E displayed a similar rate of change towards yellow colouration as spiders kept in yellow containers and exposed to natural sunlight. This study demonstrates novel hormonal manipulations that are capable of inducing reversible colour change.

The timing and duration of gonadal phases in the year indicates that breeding cycles are regulated by endogenous mechanisms. The present study on tropical spotted munia (Lonchura punctulata) investigates whether such mechanisms are based on circannual rhythms, and whether circannual rhythms between sexes differ in their relationship with the light environment. Birds were subjected to 12 h light per day (12L:12D), alternate days of light and darkness (24L:24D, LL/DD) and continuous light (LL), with L=22 lx and D≤1 lx, for 28 months at constant temperature (18±1°C). Groups kept on natural day lengths (NDL) served as controls. Measurement of body mass, gonads and molts of the primary wing feathers and body plumage at regular intervals showed that birds underwent repeated cycles in gonads and molt, but not in body mass. Under NDL, gonadal phases in both sexes cycled with 12 month periods. Under other conditions, males cycled with similar periods of ~11 months, but females cycled with relatively large period variations, ~10-13 months. Gonadal recrudescence-regression phase was longer in males than in females and, in both sexes, longer in the second year compared with the first year. The molt of wing primaries was more closely coupled to gonadal maturation in groups on NDL and 12L:12D than in groups on LL and LL/DD, but this relationship drifted apart in the second year. Body plumage molts were relatively more highly variable in both frequency and pattern in females than in males. It is suggested that annual breeding cycle in spotted munia is regulated by the self-sustained circannual rhythms, which probably interact with the annual photoperiodic cycle to synchronize breeding cycles to calendar year. Both sexes seem to have independent timing strategies, but females appear to share a greater role in defining the reproductive season in relation with the environment.

Costly events in the life history cycle of organisms such as reproduction, migration and pelage/plumage replacement are typically separated in time to maximize their outcome. Such temporal separation is thought to be necessitated by energetical trade-offs, and mediated through physiological processes. However, certain species, such as tropical birds, are able to overlap two costly life history stages: reproduction and feather replacement. It has remained unclear how both events progress when they co-occur over extended periods of time. Here we determined the consequences and potential costs of such overlap by comparing molt and behavioral patterns in both sexes of captive zebra finches (Taeniopygia guttata castanotis) that were solely molting or were overlapping breeding and molt. Individuals overlapping the early stages of breeding with molt showed a roughly 40% decrease in the growth rate of individual feathers compared with birds that were molting but not breeding. Further, individuals that overlapped breeding and molt tended to molt fewer feathers simultaneously and exhibited longer intervals between shedding consecutive feathers on the tail or the same wing as well as delays in shedding corresponding flight feathers on opposite sides. Overlapping individuals also altered their time budgets: they devoted more than twice the time to feeding while halving the time spent for feather care in comparison to molt-only individuals. These data provide experimental support for the previously untested hypothesis that when molt and reproduction overlap in time, feather replacement will occur at a slower and less intense rate. There were no sex differences in any of the variables assessed, except for a tendency in females to decline body condition more strongly over time during the overlap than males. Our data indicate the existence of major consequences of overlapping breeding and molt, manifested in changes in both molt dynamics and time budgets of both sexes. It is likely that under harsher conditions in natural environments such consequences will be more severe and may result in fitness consequences.

Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.

In crustaceans with compound eyes, the corneal lens of each facet is part of the exoskeleton and thus shed during molting. Here we used an optomotor assay to evaluate the impact of molting on visual acuity (as measured by the minimum resolvable angle, α(min)) in the female blue crab, Callinectes sapidus. We found that visual acuity decreases substantially in the days prior to molting and is gradually recovered after molting. Four days prior to molting, α(min) was 1.8 deg (N=5), a value approximating the best possible acuity in this species. In the 24 h before ecdysis occurred, α(min) increased to 15.0 deg (N=12), corresponding to an eightfold drop in visual acuity. Within 6 days after molting, α(min) returned to the pre-molting value. Micrographs of C. sapidus eyes showed that a gap between the corneal lens and the crystalline cone first appeared approximately 5 days prior to shedding and increased in width as the process progressed. This separation was likely responsible for the loss of visual acuity observed in behavioral tests. In blue crabs, mating is limited to the period of the female's pubertal molt, and a reduction in acuity during this time may have an effect on the sensory cues used in female mate choice. The results described here may be broadly applicable to all arthropods that molt and have particular importance for crustaceans that molt multiple times in their lifetime or have mating cycles that are paired with molting.

In Caenorhabditis elegans, a well-defined pathway of heterochronic genes ensures the proper timing of stage-specific developmental events. During the final larval stage, an upregulation of the let-7 microRNA indirectly activates the terminal differentiation factor and central regulator of the larval-to-adult transition, LIN-29, via the downregulation of the let-7 target genes lin-41 and hbl-1. Here, we identify a new heterochronic gene, mab-10, and show that mab-10 encodes a NAB (NGFI-A-binding protein) transcriptional co-factor. MAB-10 acts with LIN-29 to control the expression of genes required to regulate a subset of differentiation events during the larval-to-adult transition, and we show that the NAB-interaction domain of LIN-29 is conserved in Kruppel-family EGR (early growth response) proteins. In mammals, EGR proteins control the differentiation of multiple cell lineages, and EGR-1 acts with NAB proteins to initiate menarche by regulating the transcription of the luteinizing hormone β subunit. Genome-wide association studies of humans and various studies of mouse recently have implicated the mammalian homologs of the C. elegans heterochronic gene lin-28 in regulating cellular differentiation and the timing of menarche. Our work suggests that human homologs of multiple C. elegans heterochronic genes might act in an evolutionarily conserved pathway to promote cellular differentiation and the onset of puberty.

Trade-offs between self-maintenance processes can affect life-history evolution. Integument replacement and the stress response both promote self-maintenance and affect survival in vertebrates. Relationships between the two processes have been studied most extensively in birds, where hormonal stress suppression is down regulated during molt in seasonal species, suggesting a resource-based trade-off between the two processes. The only species found to differ are the rock dove and Eurasian tree sparrow, at least one of which performs a very slow molt that may reduce resource demands during feather growth, permitting investment in the stress response. To test for the presence of a molt-stress response trade-off, we measured hormonal stress responsiveness during and outside molt in two additional species with extended molts, red crossbills (Loxia curvirostra) and zebra finches (Taeniopygia guttata). We found that both species maintain hormonal stress responsiveness during molt. Further, a comparative analysis of all available species revealed a strong relationship between molt duration and degree of hormonal suppression. Though our results support trade-off hypotheses, these data can also be explained by alternative hypotheses that have not been formally addressed in the literature. We found a strong relationship between stress suppression and seasonality of breeding and evidence suggesting that the degree of suppression may be either locally adaptable or plastic and responsive to local environmental conditions. We hypothesize that environmental unpredictability favors extended molt duration, which in turn allows for maintenance of the hormonal stress response, and discuss implications of a possible trade-off for the evolution of molt schedules.

Retrograde BMP signaling in neurons plays conserved roles in synaptic efficacy and subtype-specific gene expression. However, a role for retrograde BMP signaling in the behavioral output of neuronal networks has not been established. Insect development proceeds through a series of stages punctuated by ecdysis, a complex patterned behavior coordinated by a dedicated neuronal network. In Drosophila, larval ecdysis sheds the old cuticle between larval stages, and pupal ecdysis everts the head and appendages to their adult external position during metamorphosis. Here, we found that mutants of the type II BMP receptor wit exhibited a defect in the timing of larval ecdysis and in the completion of pupal ecdysis. These phenotypes largely recapitulate those previously observed upon ablation of CCAP neurons, an integral subset of the ecdysis neuronal network. Here, we establish that retrograde BMP signaling in only the efferent subset of CCAP neurons (CCAP-ENs) is required to cell-autonomously upregulate expression of the peptide hormones CCAP, Mip and Bursicon β. In wit mutants, restoration of wit exclusively in CCAP neurons significantly rescued peptide hormone expression and ecdysis phenotypes. Moreover, combinatorial restoration of peptide hormone expression in CCAP neurons in wit mutants also significantly rescued wit ecdysis phenotypes. Collectively, our data demonstrate a novel role for retrograde BMP signaling in maintaining the behavioral output of a neuronal network and uncover the underlying cellular and gene regulatory substrates.

The transition from juvenile to adult in the Australian western rock lobster, Panulirus cygnus (George), is preceded by a mass migration from inshore nursery reefs to offshore breeding grounds. Associated with this migration is a moult which results in the animals that are due to migrate undergoing characteristic colour change from deep red to pale pink, known as the ;white' phase, which is believed to be triggered by environmental factors. To investigate this phenomenon, the colour change of wild-caught animals was measured over two separate years in response to two important modifiers of crustacean shell colour, dietary carotenoid and background substrate colour. Changes in shell colour during this colour transition period were influenced more greatly by other factors independent of diet or background substrate and no mass colour change was induced during this time. Shell colour measurement and carotenoid quantification confirmed the presence of animals similar to wild-caught ;whites', regardless of the treatment. From these experimental observations we infer that the ;white' phase of the western rock lobster is not triggered by dietary modification or in response to background substrate. We propose that this transition is under the regulation of an ontogenetic program activated at a specific moult, which induces presently unidentified molecular changes linked to shell colour production. This unique colour transition may have evolved to provide protective camouflage during migration, and serves as an excellent model to study the genetic mechanisms underlying crustacean shell colouration. These data also provide insight into the changes in carotenoid levels induced by environmental factors, and the ability to modify crustacean shell colour in aquaculture.

Insect ecdysis is a precisely coordinated series of behavioral and hormonal events that occur at the end of each molt. A great deal is known about the hormonal events that underlie this process, although less is known about the neuronal circuitry involved. In this study we identified two populations of neurons that are required for larval and adult ecdyses in the fruit fly, Drosophila melanogaster (Meigen). These neurons were identified by using the upstream region of two genes that code for atypical soluble guanylyl cyclases to drive tetanus toxin in the neurons that express these cyclases to block their synaptic activity. Expression of tetanus toxin in neurons that express Gyc-89Da blocked adult eclosion whereas expression of tetanus toxin in neurons that express Gyc-89Db prevented the initiation of the first larval ecdysis. Expression of tetanus toxin in the Gyc-89Da neurons also resulted in about 50% lethality just prior to pupariation; however, this was probably due to suffocation in the food as lethality was prevented by stopping the larvae from burrowing deep within the food. This result is consistent with our model that the atypical soluble guanylyl cyclases can act as molecular oxygen detectors. The expression pattern of these cyclases did not overlap with any of the neurons containing peptides known to regulate ecdysis and eclosion behaviors. By using the conditional expression of tetanus toxin we were also able to demonstrate that synaptic activity in the Gyc-89Da and Gyc-89Db neurons is required during early adult development for adult eclosion.

Molting in crustaceans involves significant changes in the structure and function of the exoskeleton as the old cuticle is shed and a new one is secreted. The flimsy new cuticle takes several days to harden and during this time crabs rely on a hydrostatic skeletal support system for support and movement. This change from a rigid to a hydrostatic skeletal support mechanism implies correlated changes in the function, and thus mechanical properties, of the cuticle. In particular, it must change from primarily resisting compression, bending and torsional forces to resisting tension. This study was designed to explore the changes in the mechanical properties of the crustacean cuticle as the animals switch between two distinct skeletal support mechanisms. Samples of cuticle were removed from blue crabs, Callinectes sapidus, at 1 h (soft-shell stage), 12 h (paper-shell stage), and 7 days (hard-shell stage) following molting. We measured and compared the flexural stiffness, Young's modulus of elasticity (in tension), and tensile strength for each postmolt stage. We found that the hard-shell cuticle has a flexural stiffness fully four orders of magnitude greater than the soft-shell and paper-shell cuticle. Although the soft-shell cuticle has a Young's modulus significantly lower than that of the paper-shell and hard-shell cuticle, it has the same tensile strength. Thus, the soft-shell and paper-shell cuticles are unable to resist the significant bending forces associated with a rigid skeletal support system, but can resist the tensile forces that characterize hydrostatic support systems. The mechanical properties of the cuticle thus change dramatically during molting in association with the change in function of the cuticle. These results emphasize the significant role that mechanics plays in the evolution of the molting process in arthropods, and possibly other ecdysozoans.

Two eyestalk (ES) neuropeptides, molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), increase intracellular cGMP levels in target tissues. Both MIH and CHH inhibit ecdysteroid secretion by the molting gland or Y-organ (YO), but apparently through different guanylyl cyclase (GC)-dependent pathways. MIH signaling may be mediated by nitric oxide synthase (NOS) and NO-sensitive GC. CHH binds to a membrane receptor GC. As molting affects neuropeptide signaling, the effects of ecdysteroid on the expression of the land crab Gecarcinus lateralis beta subunit of a NO-sensitive GC (Gl-GC-Ibeta), a membrane receptor GC (Gl-GC-II) and a NO-insensitive soluble GC (Gl-GC-III) were determined. Gl-GC-Ibeta isoforms differing in the absence or presence of an N-terminal 32-amino acid sequence and Gl-GC-III were expressed at higher mRNA levels in ES ganglia, gill, hepatopancreas, ovary and testis, and at lower levels in YO, heart and skeletal muscle. Three Gl-GC-II isoforms, which vary in the length of insertions (+18, +9 and +0 amino acids) within the N-terminal ligand-binding domain, differed in tissue distribution. Gl-GC-II(+18) was expressed highly in striated muscle (skeletal and cardiac muscles); Gl-GC-II(+9) was expressed in all tissues examined (ES ganglia, YO, gill, hepatopancreas, striated muscles and gonads); and Gl-GC-II(+0) was expressed in most tissues and was the dominant isoform in ES and thoracic ganglia. ES ablation, which increased hemolymph ecdysteroid, increased Gl-GC-II(+18) mRNA level in claw muscle. Using real-time RT-PCR, ES ablation increased Gl-GC-Ibeta, Gl-GC-III and ecdysone receptor mRNA levels in the YOs approximately ten-, approximately four- and approximately twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A single injection of 20-hydroxyecdysone into intact animals transiently lowered Gl-GC-Ibeta in hepatopancreas, testis and skeletal muscle, and certain Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other tissues can modulate responses to neuropeptides by altering GC expression.

Many different physiological changes have been observed in wild waterfowl during the flightless stage of wing moult, including a loss of body mass. We aimed to determine whether captive barnacle geese (Branta leucopsis) would show the characteristic decrease in body mass during their wing moult, even though they had unlimited and unrestricted access to food. Fourteen captive geese were weighed at 1-2-week intervals for two complete years. During the flightless period of the moult, body mass decreased by approximately 25% from the pre-moult value. To understand the basis of this change, the rate of oxygen consumption was measured during daytime and nighttime at six points in the second year, and at three points (before, during and after wing moult) behavioural observations were made. Measurements of the rate of oxygen consumption showed an 80% increase above that of the nonmoulting periods of the year. We propose that metabolism was increased during moult because of the cost of feather synthesis. Although food was available, the captive birds chose not to forage and instead increased the proportion of time spent resting. It is likely that this behaviour in response to wing moult is a strategy to avoid predation in the wild. Thus, the innate nature of this behaviour has potential survival value for wild birds of this species. We conclude that the increase in metabolism led to the use of endogenous energy reserves because the birds reduced rather than increased their food intake rates, and as a result, the barnacle geese lost body mass during wing moult.

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. Here, we show that the paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family.

In the moth, Manduca sexta, anterior foregut motility is modulated during the larval-larval molts in order to control the timing of molting fluid (MF) ingestion. MF is the enzymatic mixture that destroys the outer cuticle so that it can be shed at the end of the molt. The onset of the larval-larval molt is characterized by a dramatic decline in the amplitude of the anterior foregut contractions so that MF is not prematurely ingested. As the end of the molt approaches, the robust contractions of the anterior foregut return and the MF is ingested, enabling the larva to free itself from its old cuticle. In the present study we examine possible mechanisms involved in modulating anterior foregut motility during a larval-larval molt. Our results reveal that the release of a blood-borne factor plays a role in the decline in anterior foregut peristaltic activity during the molt. This blood-borne factor reduces the efficacy of the presynaptic endings of the motorneurons, resulting in a reduction in the amplitude of the excitatory junctional potential (EJP) recorded from the anterior foregut musculature. We also present evidence that crustacean cardioactive peptide (CCAP) targets the motorneuron terminals and its actions are sufficient to trigger the dramatic increase in EJP amplitude and anterior foregut contractions. Finally, the surgical ablation of the subesophageal ganglion, which has been previously described to be a source of CCAP neurons and the CCAP projections to the anterior foregut region, blocks both the increase in anterior foregut motility and the ingestion of MF that normally occur at the end of a larval-larval molt.

Species with different reproductive schedules may differ in how they respond to the annual change in photoperiod (the adaptive specialization hypothesis). Seasonally breeding species are predicted to use photorefractoriness to terminate reproduction prior to inclement conditions in autumn and winter, whereas opportunistically breeding species may not exhibit photorefractoriness. We tested for absolute photorefractoriness in four species of cardueline finch that differ in their reproductive schedules: opportunistically breeding red crossbills, flexibly breeding pine siskins, and seasonally breeding Cassin's finches and gray-crowned rosy-finches. Field observations indicated that all four species regress their gonads and begin prebasic feather molt in late summer or autumn. However, exposure to a long day photoperiod in autumn (24 h:0 h L:D) resulted in elevation of gonadotropins and testicular recrudescence in all species except Cassin's finches. Thus, by this criterion, some of the seasonally breeding species tested here did not exhibit absolute photorefractoriness. These results indicate that phylogenetic history needs to be taken into account when considering the adaptive nature of photoperiod response systems.

In insects, continuous growth requires the periodic replacement of the exoskeleton during the moult. A moulting insect displays a stereotypical set of behaviours that culminate in the shedding of the old cuticle at ecdysis. Moulting is an intricate process requiring tightly regulated physiological changes and behaviours to allow integration of environmental cues and to ensure the proper timing and sequence of its components. This is under complex hormonal regulation, and is an important point of interaction between endocrine and neural control. Here, we focus on the locust frontal ganglion (FG), an important player in moulting behaviour, as a previously unexplored target for ecdysis peptides. We show that application of 10(-7) mol l(-1) ecdysis-triggering hormone (ETH) or 10(-7) mol l(-1) and 10(-6) mol l(-1) Pre-ecdysis-triggering hormone (PETH) to an isolated FG preparation caused an increase in bursting frequency in the FG, whereas application of 10(-6) mol l(-1) eclosion hormone (EH) caused an instantaneous, though temporary, total inhibition of all FG rhythmic activity. Crustacean cardioactive peptide (CCAP), an important peptide believed to turn on ecdysis behaviour, caused a dose-dependent increase of FG burst frequency. Our results imply a novel role for this peptide in generating air-swallowing behaviour during the early stages of ecdysis. Furthermore, we show that the modulatory effects of CCAP on the FG motor circuits are dependent on behavioural state and physiological context. Thus, we report that pre-treatment with ETH caused CCAP-induced effects similar to those induced by CCAP alone during pre-ecdysis. Thus, the action of CCAP seems to depend on pre-exposure to ETH, which is thought to be released before CCAP in vivo.

C. elegans develops through four larval stages (L1 to L4) separated by molts. The identity of larval stages is mostly determined by stage-specific expression of heterochronic genes, which constitute an intrinsic genetic timer. However, extrinsic cues such as food availability or population density also modulate the developmental timing of C. elegans by mechanisms that remain largely unknown. To investigate a potential role of the nervous system in the temporal regulation of C. elegans development, we pharmacologically manipulated nicotinic neurotransmission, which represents a prominent signaling component in C. elegans nervous system. Exposure to the nicotinic agonist DMPP during post-embryonic development is lethal at the L2/L3 molt. Specifically, it delays cell divisions and differentiation during the L2 stage but does not affect the timing of the molt cycle, hence causing exposure of a defective L3 cuticle to the environment after the L2/L3 molt. Forcing development through a previously uncharacterized L2 diapause resynchronizes these events and suppresses DMPP-induced lethality. Nicotinic acetylcholine receptors (nAChRs) containing the UNC-63 subunit are required, probably in neurons, to trigger the action of DMPP. Using a forward genetic screen, we further demonstrated that the nuclear hormone receptor (NHR) DAF-12 is necessary to implement the developmental effects of DMPP. Therefore, a novel neuroendocrine pathway involving nAChRs and the NHR DAF-12 can control the speed of stage-specific developmental events in C. elegans. Activation of DMPP-sensitive nAChRs during the second larval stage uncouples a molting timer and a developmental timer, thus causing a heterochronic phenotype that is lethal at the subsequent molt.

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.

Hemocyanin, the blue blood protein of many arthropods and molluscs, reversibly binds oxygen at its highly conserved copper-oxygen-binding sites and supplies tissues with oxygen. Cryptocyanin, closely related structurally and phylogenetically to arthropod hemocyanin, lacks several of the six critical copper-binding histidines, however, and has lost the ability to bind oxygen. Despite this loss of function, cryptocyanin continues to be synthesized, an indication that it has been exploited to carry out new functions. Here, we show that cryptocyanin is present in extremely high concentrations in the hemolymph of the crab during the premolt portion of the molt cycle. Both proteins are specifically expressed in the same type of cell in the hepatopancreas and secreted into the hemolymph, but cryptocyanin plays a major role in forming the new exoskeleton, while hemocyanin functions in oxygen transport. A cessation in cryptocyanin, but not hemocyanin, synthesis after eyestalk ablation supports our hypothesis that cryptocyanin is closely regulated by molting hormones. The contrasts between the two gene products illustrate how a gene duplication of a copper-oxygen protein and its subsequent mutation may work in concert with the evolution of new regulatory mechanisms, leading to the assumption of new functions.

The uterine (shell gland) epithelium from the domestic chicken was mounted in Ussing chambers, bathed in symmetric avian saline solution on both apical and basolateral aspects and voltage clamped at 0 mV. The epithelium exhibited a basal short circuit current (I(sc)) that was partially inhibited by the epithelial Na(+) channel (ENaC) blockers, amiloride and benzamil (IC(50) values of 0.8 and 0.12 micromol l(-1), respectively). Inhibition of basal Na(+) absorption by 10 micromol l(-1) amiloride was confirmed by measurements of transepithelial Na(+) and Cl(-) fluxes, where inhibition of the apical-to-basolateral and net Na(+) flux occurred, but no significant effects on Cl(-) fluxes were detected. The amiloride-insensitive portion of the basal I(sc) was both Cl(-) and HCO(3)(-) dependent and was inhibited by the Cl(-) channel blocker, diphenyl-2-carboxylate (DPC; 100 micromol l(-1)). Stimulation with 8-(4-chlorophenylthio)-cyclic 3'-5', adenosine monophosphate (8-cpt cAMP) produced a sustained increase in I(sc) that was dependent on both Cl(-) and HCO(3)(-). The magnitude of the amiloride-sensitive I(sc) was approximately twofold greater in birds where shell formation was complete, but oviposition had not yet occurred. In addition, the amiloride-sensitive I(sc) was greater in hens over the age of 55 weeks and in molting birds. The anion-dependent component of the basal I(sc) was reduced in older birds, and electrogenic HCO(3)(-) transport was nearly absent in molting birds. These results demonstrated that electrogenic Na(+) transport in avian shell gland was similar to the mammalian uterine epithelium and increased with age and during molting. Electrogenic Cl(-) and HCO(3)(-) transport were coupled under basal and cAMP stimulated conditions and basal anion transport decreased with age and during molting.

Molting is required for progression between larval stages in the life cycle of nematodes. We have identified four mutant alleles of a Caenorhabditis elegans metalloprotease gene, nas-37, that cause incomplete ecdysis. At each molt the cuticle fails to open sufficiently at the anterior end and the partially shed cuticle is dragged behind the animal. The gene is expressed in hypodermal cells 4 hours before ecdysis during all larval stages. The NAS-37 protein accumulates in the anterior cuticle and is shed in the cuticle after ecdysis. This pattern of protein accumulation places NAS-37 in the right place and at the right time to degrade the cuticle to facilitate ecdysis. The nas-37 gene has orthologs in other nematode species, including parasitic nematodes, and they undergo a similar shedding process. For example, Haemonchus contortus molts by digesting a ring of cuticle at the tip of the nose. Incubating Haemonchus larvae in extracted exsheathing fluids causes a refractile ring of digested cuticle to form at the tip of the nose. When Haemonchus cuticles are incubated with purified NAS-37, a similar refractile ring forms. NAS-37 degradation of the Haemonchus cuticle suggests that the metalloproteases and the cuticle substrates involved in exsheathment of parasitic nematodes are conserved in free-living nematodes.

The discontinuous pattern of crustacean cuticular mineralization (the molting cycle) has emerged as a model system to study the spatial and temporal regulation of genes that code for Ca2+-transporting proteins including pumps, channels and exchangers. The plasma membrane Ca2+-ATPase (PMCA) is potentially of significant interest due to its role in the active transport of Ca2+ across the basolateral membrane, which is required for routine maintenance of intracellular Ca2+ as well as unidirectional Ca2+ influx. Prior research has suggested that PMCA expression is upregulated during periods of elevated Ca2+ influx associated with postmolt cuticular mineralization. This paper describes the cloning, sequencing and functional characterization of a novel PMCA3 gene from the antennal gland (kidney) of the crayfish Procambarus clarkii. The complete sequence, the first obtained from a non-genetic invertebrate species, was obtained through reverse transcription-polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) techniques. Crayfish PMCA3 consists of 4148 bp with a 3546 bp open reading frame coding for 1182 amino acid residues with a molecular mass of 130 kDa. It exhibits 77.5-80.9% identity at the mRNA level and 85.3-86.9% identity at the protein level with PMCA3 from human, mouse and rat. Membrane topography was typical of published mammalian PMCAs. Northern blot analysis of total RNA from crayfish gill, antennal gland, cardiac muscle and axial abdominal muscle revealed that a 7.5 kb species was ubiquitous. The level of PMCA3 mRNA expression in all tissues (transporting epithelia and muscle) increased significantly in pre/postmolt stages compared with relatively low abundance in intermolt. Western analysis confirmed corresponding changes in PMCA protein expression (130 kDa).

Large and medium-sized alcids have a very intense wing molt wherein many flight feathers are shed in rapid succession and wing surface area is reduced by as much as 40%. Although these birds are rendered flightless during wing molt, they must still use their wings to propel themselves underwater. A molt-induced loss of wing area could simply reduce wing propulsion such that more muscular work would be required to maintain a given speed. Alternatively, molt could reduce drag on the wings, making a bird more penguin-like and actually enhancing diving ability. I addressed this issue by filming captive common guillemots Uria aalge and tufted puffins Fratercula cirrhata using an array of video cameras to plot the birds' movements in three dimensions. From these coordinate data I calculated swimming velocities, angles of descent and absolute depths. These values allowed me to estimate the forces due to drag and buoyancy that must be counteracted by flapping, which in turn yielded estimates of the amount of work generated during each flap as well as the average power and cost of transport. Within-bird comparisons of diving performance when wings were intact and during several stages of wing molt indicated that molt is associated with more frequent flapping, reduced displacement during the flap cycle, and possibly reduced work per flap. These negative effects on diving may explain why primary and secondary molts were offset in the birds I studied such that the period during which all of the flight feathers are effectively missing is minimized.

NO signaling is involved in many physiological processes in invertebrates. In crustaceans, it plays a role in the regulation of the nervous system and muscle contraction. Nested reverse transcription-polymerase chain reaction (RT-PCR) and 5' and 3' rapid amplification of cDNA ends (RACE) PCR generated a full-length cDNA sequence (3982 bp) of land crab NO synthase (Gl-NOS) from molting gland (Y-organ) and thoracic ganglion mRNA. The open reading frame encoded a protein of 1199 amino acids with an estimated mass of 135 624 Da. Gl-NOS had the highest sequence identity with insect NOS. The amino acid sequences for binding heme and tetrahydrobiopterin in the oxygenase domain, binding calmodulin and binding FMN, FAD and NADPH in the reductase domain were highly conserved. Gl-NOS had single amino acid differences in all three highly conserved FAD-binding sequences, which distinguished it from other NOS sequences. RT-PCR showed that the Gl-NOS mRNA was present in testis, ovary, gill, eyestalk neural ganglia, thoracic ganglion and Y-organ. NOS mRNA varied between preparations of Y-organ, thoracic ganglion and gill, while NOS mRNA was at consistently high levels in the ovary, testis and eyestalk ganglia. Immunohistochemistry confirmed that the Gl-NOS protein was expressed in Y-organ, ovary and gill. These results suggest that NOS has functions in addition to neuromodulation in adults, such as regulating or modulating ecdysteroid synthesis in the Y-organ.

A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.

The formation and resorption of CaCO(3) by epithelial cell layers require epithelial transport of protons. We used the anterior sternal epithelium of the terrestrial isopod Porcellio scaber as a model to study the expression pattern and immunolocalization of a V-type H(+)-ATPase during the mineralization and demineralization of intermittent CaCO(3) deposits. Semiquantitative RT-PCR revealed that the expression of the V-type H(+)-ATPase increases from non Ca(2+)-transporting control stages to the stages of CaCO(3) deposit formation and resorption. In the Ca(2+)-transporting stages the expression was larger in the anterior than in the posterior sternal epithelium, which is not involved in deposit formation and transports just moderate amounts of CaCO(3). Immunocytochemistry of the B-subunit of the V-type H(+)-ATPase in the anterior sternal epithelium reveals an increase in the abundance of the protein within the basolateral membrane, from undetectable to strong signals in the control stage to the stages of CaCO(3) deposit formation, respectively. From the stage of CaCO(3) deposit formation to that of CaCO(3) resorption the signal decreased within the basolateral plasma membrane and increased within the apical plasma membrane. For the first time the results indicate a contribution of a V-type H(+)-ATPase to CaCO(3) deposition and a reversal of its polarity from the basolateral to the apical plasma membrane compartment within the same cells.

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing spreading behavior after eclosion. The cell death was accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy revealed that this cell death exhibited extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibited the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrated that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This was supported by evidence that wing cell death was inhibited by a mutation of rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeant analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induced precocious death. Conversely, cell death was inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion.

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.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

We examined the role of the foregut in the resorption of molting fluid (MF) from the exuvial space during the last larval-larval molt of the moth Manduca sexta. In intermolt larvae, the activity of the foregut is characterized by robust peristaltic contractions. With the onset of the molt, MF is secreted into the exuvial space where it digests and weakens the old cuticle. The appearance of MF in the exuvial space is accompanied by a dramatic reduction in the amplitude of the foregut contractions. Foregut peristalsis returned about halfway through the molt, followed shortly by the appearance of MF in the gut. These observations suggested that larvae use their foreguts to remove MF from the exuvial space. Animals whose foreguts were surgically inactivated did not resorb their MF and most failed to successfully shed their old cuticles. The reduction in foregut motility at the onset of the molt was correlated with a sharp decline in the amplitude of the excitatory junctional potentials. With the onset of the molt there was also a decline in the number of presynaptic terminals on the foregut that loaded with the activity-dependent dye FM1-43. In the second half of the molt, the appearance of MF in the foregut and the return of foregut motility was correlated with an increase in FM1-43 loading. These data reveal that during a larval-larval molt, vesicle release and/or recycling of the presynaptic endings on the foregut muscles is modulated to assure the proper timing of MF resorption.