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Environmental cues such as temperature induce macroscopic changes in the molting cycle of crustaceans, however, the physiological mechanisms behind these changes remain unclearWe aimed to investigate the regulatory mechanisms in the intermolt and premolt stages of the Callinectes sapidus molt cycle in response to thermal stimuli. The concentration of ecdysteroids and lipids in the hemolymph, and the expression of heat shock proteins (HSPs) and molt key genes were assessed at 19 °C, 24 °C and 29 °C. The premolt animals exhibited a much larger response to the colder temperature than intermolt animals. Ecdysteroids decreased drastically in premolt animals, whereas the expression of their hepatopancreas receptor (CasEcR) increased, possibly compensating for the low hemolymphatic levels at 19 °C. This decrease might be due to increased HSPs and inhibited ecdysteroidogenesis in the Y-organ. In addition, the molting-inhibiting hormone expression in the X-organ/sinus gland (XO/SG) remained constant between temperatures and stages, suggesting it is constitutive in this species. Lipid concentration in the hemolymph, and the expression of CasEcR and CasHSP90 in the XO/SG were influenced by the molting stage, not temperature. On the other hand, the expression of HSPs in the hepatopancreas is the result of the interaction between the two factors evaluated in the study. Our results demonstrated that temperature is an effective modulator of responses related to the molting cycle at the endocrine level and that temperature below the control condition caused a greater effect on the evaluated responses compared to the thermostable condition, especially when the animal was in the premolt stage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Molting enables growth and development across ecdysozoa. The molting process is strictly controlled by hormones - ecdysteroids. Ecdysteroidogenesis occurs in theprothoracic glands and stimulated by prothoracicotropic hormone in insects, while it ensues in the Y-organ and regulated by the molt inhibiting hormone in crustaceans. A peak in ecdysteroids in the hemolymph induces a cascade of multiple neuropeptides including Ecdysis Triggering Hormone (ETH) and Corazonin. The role of ETH is well defined in controlling the molt process in insects, but it is yet to be defined in crustaceans. In this study, we investigated the behavioral response of intermolt crayfish to ETH and Corazonin injections as well as the impact of ETH on the molt period using in vivo assays. Injection of Corazonin and ETH resulted in a clear and immediate eye twitching response to these two neuropeptides. The Corazonin injection induced eye twitching in slow and asynchronous manner, while ETH injection caused eye twitching in a relatively fast and synchronous way. A single injection of ETH to crayfish resulted in a remarkable prolong molt period, at twice the normal molting cycle, suggesting that ETH plays a key role in controlling the molt cycle in decapod crustaceans. Given the key significance of ETH in molt regulation and its plausible application in pest control, we characterized ETH across the pancrustacean orders. Bioinformatic analysis shows the mature ETH sequence is identical in all studied decapod species. ETH can be classified into specific groups based on the associated motif in each insect order and shows an insect motif -KxxPRx to be conserved in crustaceans., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The measurement of corticosterone levels in feathers (fCort) is gaining recognition as an effective means for describing links between stages of the annual cycle in birds. Many seabirds are especially good models for exploring these links, or carryover effects, due to their migratory behavior and reproductive investment in a single-egg clutch. Here, we measure fCort in Cassin's auklet (Ptychoramphus aleuticus) breast feathers at two colonies in British Columbia during 2011, a year of favorable oceanographic conditions, and examine its relationship with egg size. These feathers are grown at sea during the late winter period, 1-2 months prior to egg laying. Assuming that fCort provides some measure of nutritional stress and hypothalamic-pituitary-adrenal axis activity during feather growth, we predicted that fCort would be positively correlated with egg size via increased support for foraging activity during the nutritionally demanding molt stage. We also analyzed the δ 13 C and δ 15 N stable isotope content of breast feathers, measures commonly used to characterize seabird diet composition. Contrary to prediction, neither fCort nor stable isotope ratios were good predictors of egg size. Our results appear to conflict with two previous studies on alcids in which fCort and stable isotopes showed clear links with egg size; however, both studies were conducted in years when oceanographic and foraging conditions were poor. Under these conditions, upregulation of corticosterone may be needed to mediate the energetic tradeoffs between self-maintenance and reproduction, supporting increased foraging effort and thus increasing both the likelihood of reproduction and large egg size. However, when foraging conditions are favorable, we suggest that such tradeoffs and associated physiological constraints are minimized and regulation of egg size may be effectively independent of circulating corticosterone levels and diet type., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ecdysteroids play an essential role in the regulation of the molting processes of arthropods. Nuclear receptors of the spider Agelena silvatica that showed high homology with other arthropods especially in the functional domains were identified, two isoforms of ecdysone receptor (AsEcRA, AsEcRB), retinoid X receptor (AsRXR) and two isoforms of E75 (AsE75A, AsE75D). AsEcR and AsRXR mRNA did not show major changes in expression but occurred throughout the third instar nymphal stage. AsE75DBD was low or non-existent at first then showed a sudden increase from D7 to D10. On the other hand, AsE75D was expressed in the first half and decreased from D6 to D10. Ecdysteroid titers showed a peak on D6 in A. silvatica third instar nymphs. LC-MS/MS analysis of the ecdysteroid peak revealed only 20-hydroxyecdysone (20E) was present. The 20E peak on D6 and increase in AsE75DBD from D7 is likely a result of ecdysteroids binding to the heterodimer formed with constant expression of the AsEcR and AsRXR receptors. These findings indicate the mechanisms regulating molting widely conserved in insects and other arthropods also similarly function in spiders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cathepsins (CTSs) are peptidases that have biological roles in degrading extracellular matrix, catabolism of intracellular proteins, and processing of pro-hormones. Of these, cathepsin L (CTSL) is closely associated with morphological changes in reproductive organs required for proper function in mammals, including humans and mice, but little is known about CTSL in avian species. In the present study, the expression of CTSL was investigated in the oviduct of hens during regression and recrudescence in response to molting. Our results revealed that expression of CTSL mRNA increased (P<0.001) when the oviduct underwent regression during the molting period in hens. In situ hybridization and immunohistochemial analyses detected CTSL mRNA and protein predominantly in the luminal (LE) and glandular epithelia (GE) during regression of the oviduct, but not during regeneration of the oviduct. Expression of CTSL decreased in the oviduct of chicks treated with diethylstilbestrol (DES, a synthetic estrogen agonist). Furthermore, we discovered four miRNAs including miR-23b, miR-551, miR-1464 and miR-1803 that regulate expression of the CTSL gene at the post-transcriptional level, which suggests that CTSL mRNA can be regulated by specific miRNAs via 3'-UTR in chickens. Results of the present research suggest that estrogen regulates expression of CTSL during regression of the oviduct during molting and that down-regulation of CTSL is likely a prerequisite for the normal regeneration of oviductal tissues following molting in laying hens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In many male vertebrates, the secretion of reproductive (gonadal androgens) and adrenocortical (glucocorticoids) hormones varies seasonally and in response to environmental stimuli, and these hormones exert numerous behavioral and metabolic effects. We performed two field studies on adult male Rufous-winged Sparrows, Peucaea carpalis, a Sonoran Desert rain-dependent sedentary species, to (a) determine seasonal changes in initial (baseline) and acute stress-induced plasma testosterone (T), corticosterone (CORT), and two metabolites (uric acid and glucose) and (b) compare the effects of two types of social challenge (song playback or simulated territorial intrusion consisting of song playback plus exposure to a live decoy bird) on plasma T, CORT, these metabolites, and territorial behavior. Initial plasma T was higher during the summer breeding period than during post-breeding molt. Acute stress resulting from capture and restraint for 30 min decreased plasma T in breeding condition birds but not in the fall, revealing that this decrease is seasonally regulated. Initial plasma CORT did not change seasonally, but plasma CORT increased in response to acute stress. This increase was likewise seasonally regulated, being relatively smaller during autumnal molt than in the summer. We found no evidence that acute stress levels of CORT are functionally related to stress-depressed plasma T and, therefore, that plasma T decreases during stress as a result of elevated plasma CORT. Thirty minutes of exposure to simulated territorial intrusion resulted in different behavior than 30 min of exposure to song playback, with increased time spent near the decoy and decreased number of overhead flights. Neither type of social challenge influenced plasma T, thus offering no support for the hypothesis that plasma T either responds to or mediates the behavioral effects of social challenge. Exposure to both social challenges elevated plasma CORT, but simulated territorial intrusion was more effective in this respect than song playback. Plasma uric acid and glucose decreased during acute stress, but only plasma uric acid decreased during social challenge. Thus, an elevation in plasma CORT was consistently associated with a decrease in plasma uric acid, but not with a change in glycemia. These results enhance our understanding of the short-term relationships between T, CORT, and avian territorial behavior. They provide novel information on the endocrine effects of acute stress, in particular on plasma T, in free-ranging birds, and are among the first in these birds to link these effects to metabolic changes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Molt, a natural behavior that is initiated at the end of a lay cycle in birds, is implicated in the regression of the reproductive system in birds followed by a rejuvenation of egg-laying potential. The aim of the present study was to evaluate the physiological basis for the apparent rejuvenation of egg production that occurs following molting. Eighty-three-week-old Hy-line hens, were obtained and subjected to forced molting. Blood and tissue samples were obtained at the beginning of molt (at 83 weeks of age), during molt (at 85 weeks of age) and postmolt (at 89 weeks of age). The laying performance, egg quality, blood parameters and gene expression in the liver and the ovary were investigated before, during and after molt. There was an obvious increase in the postmolt laying rate from 70% premolt to 93% postmolt. Eggshell thickness, albumin height, Haugh unit and egg shape index were all significantly improved after molt. The circulating levels of estrogen and progesterone were lower in the postmolt hens, whereas the concentrations of luteinizing hormone and follicle stimulating hormone were not significantly affected by molt. These results indicate that enhanced hepatic yolk precursor synthesis and secretion contribute to increased postmolt laying performance. Molt enhanced the sensitivity of sex hormones in F1 follicles. Augmented gene expression in the ovary was involved in the rejuvenation of the reproductive performance of molted hens. These results suggest that facilitated yolk-precursor uptake by follicles is involved in the rejuvenation of the reproductive performance of molted hens., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Photoperiod is the major cue used by birds to time breeding seasons and molt. However, the annual cycle in photoperiod changes with latitude. Within species, for temperate and high latitude species, gonadal maturation and breeding start earlier at lower latitudes but regression and molt both occur at similar times at different latitudes. Earlier gonadal maturation can be explained simply by the fact that considerable maturation occurs before the equinox when photoperiod is longer at lower latitudes - genetic differences between populations are not necessary to explain earlier breeding at lower latitudes. Gonadal regression is caused either by absolute photorefractoriness or, in some species with long breeding seasons, relative photorefractoriness. In either case, the timing of regression and molt cannot be explained by absolute prevailing photoperiod or rate of change in photoperiod - birds appear to be using more subtle cues from the pattern of change in photoperiod. However, there may be no difference between absolute and relative photorefractory species in how they utilise the annual cycle in photoperiod to time regression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Very little is known regarding the release patterns of neuropeptides involved in ecdysis of crustaceans compared to insects. In particular, the dynamics of release of the insect cuticle hardening hormone bursicon, which has only recently been discovered in crustaceans, is unknown. Bursicon has not previously been identified as a circulating neurohormone in these animals. Since patterns of release were likely to be ephemeral, bursicon, as well as two other neurohormones involved in the ecdysis program in crustaceans, crustacean cardioactive peptide (CCAP) and crustacean hyperglycaemic hormone (CHH) were measured in single haemolymph samples in Carcinus maenas. For bursicon, an ultrasensitive time resolved-fluoroimmunoassay (TR-FIA) was developed, which firstly involved its characterisation by HPLC, bioassay and immunoassay. Simultaneous measurement of three neurohormones was performed at unparalleled levels of resolution, which has not previously been reported in any invertebrate. Additionally, expression patterns and architecture of neurones expressing both bursicon and CCAP were determined in the CNS during the moult cycle. Bursicon and CCAP are released in a massive surge, likely a single global exocytotic event on emergence, just after release of CHH. Despite co-localisation of CCAP and bursicon in neurones of the CNS, observations suggest that differential packaging of CCAP can occur in the pericardial organs in a small population of secretory boutons, thus accounting for observations showing release of some CCAP during the penultimate stages of the ecdysis program. The results obtained vividly illustrate the dynamism of neuropeptide cascades occurring during crustacean ecdysis, and also allow proposal of a hypothesis of its endocrine control., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In crustaceans, molting is known to be under the control of neuropeptide hormones synthesized and secreted from the eyestalk ganglia. While the role of molt-inhibiting hormone (MIH) in regulating molting has been described in several species using classical methods, an in vivo specific MIH targeted manipulation has not been described yet. In the present study, an MIH cDNA was isolated and sequenced from the eyestalk ganglia of the Australian freshwater red claw crayfish Cherax quadricarinatus (Cq) by 5' and 3' RACE. We analyzed the putative Cq-MIH based on sequence homology, a three dimensional structure model and transcript's tissue specificity. We further examined the involvement of Cq-MIH in the control of molt in the crayfish through RNAi by in vivo injections of Cq-MIH double-stranded RNA, which resulted in, similarly to eyestalk ablation, acceleration of molt cycles. This acceleration was reflected by a significant reduction (up to 32%) in molt interval and an increased rate in molt mineralization index (MMI), which correlated with the induction of ecdysteroid hormones compared to control. Altogether, this study provides a proof of function for the involvement of the Cq-MIH gene in molt regulation in the crayfish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In avian species that have evolved life-history strategies wherein molt and breeding overlap, there are potential conflicts between the regulatory roles of baseline prolactin and corticosterone in parental care (positive) and moult (negative). We describe seasonal patterns of hormonal secretion, moult, and parental behaviour in sibling species of giant petrels (Macronectes spp.) which begin moult during the incubation/early chick-rearing stage of reproduction. With the exception of male Southern giant petrels (Macronectes giganteus), prolactin secretion and moult in Northern (Macronectes halli) and female Southern giant petrels conformed to those observed in all other avian species, with the initiation of moult coincident with decreases from peak prolactin levels. However, male Southern giant petrels began moulting early in incubation when prolactin was increasing and had not yet peaked, which suggests a requirement of prolactin for incubation behaviour and a dissociation of prolactin from moult. Corticosterone showed little seasonal variation and no relationship with moult. When comparing prolactin, corticosterone, and moult in failed vs. active breeders, we found that failed breeding enabled a more rapid down-regulation of prolactin, thus facilitating a more rapid moult. We present specific examples of the behavioural ecology of giant petrels which we conclude help mediate any potential hormonal conflicts between parental care and moult., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

In arthropods, molting events are mediated by the binding of the ecdysone hormone to a heterodimer of two nuclear receptors: the ecdysone receptor (EcR) and the retinoid X receptor (RXR), a homolog of ultraspiracle (USP). We have cloned partial sequences of several isoforms for EcR and RXR genes from the centipede Lithobius peregrinus, and studied their expression profile during the second post-embryonic stage. LpEcR and LpRXR inferred amino acid sequences are very similar to other arthropod orthologs, especially to those of chelicerates and hemimetabolous insects, and their expression levels are significantly higher during the 48 h that precede the molt. Results obtained in this study represent the first data on the genetic basis of the ecdysone signal pathway for a myriapod, and in particular for an animal that, through a stereotyped developmental schedule paced by the molt cycle, completes trunk segmentation during post-embryonic life., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic hormone (CHH) family, inhibits the synthesis of ecdysteroid in Y-organ (YO) and plays a significant role in the regulation of molting and growth of crustaceans. A complete cDNA sequence encoding MIH (Ers-MIH, GenBank Accession No.: DQ341280) was cloned from eyestalk of Chinese mitten crab (Eriocheir sinensis) by 5' and 3' RACEs and PCR cloning. The full-length cDNA consists of 1457 bp with a 330 bp open reading frame, encoding 110 amino acids, containing a 75 amino acid mature peptide. The deduced amino acid sequence contains a typical CHH domain. Transcripts of Ers-MIH mRNA were detected in eyestalk by Northern blotting. The production of purified recombinant Ers-MIH (rErs-MIH) expressed in Escherichia coli was 0.3g/L. The LC-ESI-MS analysis showed that two peptide fragments of the recombinant protein were identical to the deduced amino acid sequence of Ers-MIH. By in vitro assay on E. sinensis YOs, a cGMP mediated suppression of rErs-MIH on ecdysteroidogenesis could be observed. Accumulation of cGMP in YOs showed a concentration-dependent manner within 0.01-1 nmol/mL of rErs-MIH; ecdysteroid secretion was inhibited significantly at the range of 0.01-100 nmol/mL rErs-MIH; furthermore, a significant inhibition effect on ecdysteroid releasing was shown when cGMP analog (8-Br-cGMP) concentration rose up to 100 nmol/mL. This study would facilitate to investigate the roles of MIH in molt cycle regulation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Molting is a highly complex process that requires precise coordination to be successful. We describe the early classical endocrinological experiments that elucidated the hormones and glands responsible for this process. We then describe the more recent experiments that have provided information on the cellular and molecular aspects of molting. In addition to providing a review of the scientific literature, we have also included our perspectives., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The hormonal factors that influence development from birth to weaning in otariid seals is still largely unknown. In the present study, a suite of thyroid hormones and cortisol were measured in Australian fur seal pups in order to determine baseline concentrations as well as to describe their endocrinology over this critical developmental period. A cross-section of newborn pups from a breeding colony located on Kanowna Island, Australia were sampled at six different times over the course of the 10 month lactation period. Sample times were designed to correspond to periods of heightened physiological change during pre-weaning development: post-natal, pre-molt, the initiation of molt, mid-molt, period of peak milk intake and weaning. Results indicate that the greatest hormonal changes were associated with the post-natal stage and molt, with molt showing the greatest changes, as has been reported for several species of pinnipeds. Two forms of thyroid hormones analyzed (Total T(4), and Free T(3)), increased with the initiation of the molt, and Free T(3) exhibited a second increase that was associated with the period of peak milk intake. The T(3):T(4) ratio was significantly lower during the initiation of molt than either pre- or mid-molt. The study was able to describe physiological change during the first year of life in Australian fur seals as well as document basal concentrations of thyroid hormones and cortisol in pups of this species., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Many birds face energetic trade-offs between different life history stages, such as reproductive effort, feather molt and the non-breeding period. Little is known about how physiological measures of condition (corticosterone, plasma metabolites) in free-living birds change from nesting stages to the post-breeding molt period or whether this is influenced by prior reproductive effort. We evaluated whether corticosterone (CORT) and plasma metabolite levels vary with date, nest stage and sex in a free-living migratory songbird, the wood thrush (Hylocichla mustelina). We also tested whether (1) baseline CORT levels early in the season were predictive of subsequent reproductive success and (2) whether prior reproductive effort influenced CORT levels and blood metabolites during molt. Baseline CORT levels decreased with date during both the incubation stage and nestling stage, but did not vary significantly across stage of breeding season. Stress-induced CORT declined with date during incubation and varied significantly across breeding stage, with lower levels during feather molt. Profiles of the metabolites of β-hydroxybutyrate, glycerol, and triglyceride did not vary significantly with date or breeding stage. Only triglycerides varied significantly with sex, with females having higher levels than males. Reproductive output was highly variable (0-10 fledglings per season) but baseline CORT levels in females during the first incubation period of the season was not related to subsequent reproductive output. Prior reproductive effort, measured as the cumulative number of young hatched during the breeding season, was positively related to stress-induced CORT during molt. High reproductive effort in wood thrush appears to have physiological carry-over effects into the molt period which could potentially affect rate of molt and preparation for fall migration., (Copyright © 2011. Published by Elsevier Inc.)

Fecal samples and behavioral data were collected at a fortnightly basis during 11 months period from free-living male American kestrels living in southeast Brazil (22 degrees S latitude). The aim was to investigate the seasonal changes in testicular and adrenal steroidogenic activity and their correlation to reproductive behaviors and environmental factors. The results revealed that monthly mean of fecal glucocorticoid metabolites in May and June were higher than those estimated in November. In parallel, monthly mean of androgen metabolites in September was higher than those from January to April and from October to November. Molt took place from January to March, whereas copulation was observed from June to October but peaked in September. Nest activity and food transfer to females occurred predominantly in October, and parental behavior was noticed only in November. Territorial aggressions were rare and scattered throughout the year. Multiple regression analysis revealed that fecal androgen levels are predicted by photoperiod and copulation, while fecal glucocorticoid levels are only predicted by photoperiod. Bivariate correlations showed that fecal androgen metabolites were positively correlated with fecal glucocorticoid metabolites and copulation, but negatively correlated with molt. Additionally, copulation was positively correlated with food transfer to females and nest activity, but negatively correlated with molt. These findings suggest that male American kestrels living in southeast Brazil exhibit significant seasonal changes in fecal androgen and glucocorticoid concentrations, which seem to be stimulated by decreasing daylength but not by rainfall or temperature., (Copyright 2009 Elsevier Inc. All rights reserved.)

Ecdysis, or the shedding of the old cuticle, depends on coordinated stereotyped behaviors, regulated by a number of neuropeptides. In the hornworm, Manduca sexta, two neuropeptides interact, namely ecdysis-triggering hormone (ETH) and eclosion hormone. We looked at the effects of ETH in vivo and in vitro, on the brain and the ventral nerve cord to determine the roles played by these hormones. We monitored ecdysis onset and the presence of cGMP and eclosion hormone immunoreactivity. In vivo, only a fraction of larvae lacking the cell bodies containing eclosion hormone, and injected with ETH, were able to undergo ecdysis, with a delayed response. These animals showed strongest cGMP immunoreactivity in the subesophageal and thoracic ganglia, with concomitant reductions in eclosion hormone immunoreactivity in descending axons in comparison with animals not undergoing ecdysis. Animals lacking the brain showed reduced to no cGMP levels in all ganglia. In vitro, isolated CNS preparations lacking the brain initiated ecdysis motor programs after incubation in ETH, with faster onset times than controls, and with reduced cGMP immunoreactivity. If ETH was applied only to the brain of the isolated CNS, cGMP immunoreactivity was noted primarily in the subesophageal and thoracic ganglia, with a decrease in eclosion hormone immunoreactivity in descending axons. ETH addition to the rest of the nerve cord showed reduced eclosion hormone immunoreactivity but little to no cGMP immunoreactivity in any ganglion. Controls showed strong cGMP immunoreactivity in all ganglia, and even greater reductions in eclosion hormone staining after ETH application. These results support previous suggestions that eclosion hormone is required for a positive feedback loop with ETH as well as onset of an inhibitory component, but also suggest that ETH stimulates eclosion hormone release at multiple spike initiation zones. The resultant up regulation of cGMP does not appear to be required for onset of ecdysis. A new model for ecdysis regulation is considered., (Copyright 2009 Elsevier Inc. All rights reserved.)

The aim of the study was to test the hypothesis that decreasing plasma prolactin stimulates or permits the initiation of avian molt. Changes in the concentration of plasma prolactin in Mute swans (Cygnus olor) were compared in non-breeding singletons and breeding pairs. In breeding swans, the onset of molt is delayed compared to non-breeders, and is delayed further in breeding males compared to their female partners. The seasonal decrease in prolactin in non-breeding birds of both sexes started at the end of May and was associated with the initiation of molt 4 weeks later. The decrease in plasma prolactin in incubating females was more pronounced, as a consequence of increased prolactin secretion associated with incubation behavior, but also started at end of May, and was associated the onset of molt 6 weeks later. In breeding males, plasma prolactin increased at the end of May when they started to care for their newly hatched cygnets. Correspondingly, prolactin began to decrease 3-5 weeks later in males than in females. These males started to molt in mid August, at least 4 weeks later than females. It is concluded that molt is related to decreasing plasma prolactin, and is inhibited when plasma prolactin is increasing or high.

Little is known about how frequent, acute stressors affect wild animals. We present two experiments conducted on captive, Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii) that explore how frequent, acute doses of corticosterone (CORT) affect condition and behavior. CORT was administered either once or three times a day to birds in pre-breeding, early-breeding, or late-breeding life-history stages. Two additional groups were included to control for the CORT delivery vehicle, DMSO, and the treatment process. Our results indicate that CORT treatment decreases condition, but that its effects are dependent on frequency and life stage. Specifically, CORT-treated birds delayed the onset of molt and had reduced body mass, flight muscle, and food consumption. CORT treatment did not affect fat stores, bile retention in the gallbladder, or the expression of migratory restlessness behavior. These results increase our understanding of the effects of frequent, acute stressors and the development of chronic stress states.

In temperate zone songbirds, such as canaries (Serinus canaria), seasonal variation in gonadal activity and behavior are associated with marked brain changes. These include gonadotropin-releasing hormone (GnRH) expression and the volume of brain areas controlling song production. Questions have been raised about the consistency of seasonal brain changes in canaries. Laboratory studies of the American singer strain raised doubts as to whether this strain exhibits a robust photoperiodic response along with changes in brain GnRH content, and studies of free-living canaries have failed to identify seasonal changes in volume of song control nuclei. We assessed differences in brain GnRH and the song control system associated with photoperiod-induced variation in reproductive state in Border canaries. We found that males and females maintained for 10 weeks on long days (14L:10D) regress their gonads, exhibit a decline in testosterone and initiate molt; a response consistent with the onset of absolute photorefractoriness (i.e., failed to respond to previously stimulating daylengths). All birds regained photosensitivity (i.e., exhibited gonadal response to stimulating daylengths) after experiencing short days (8L:16D) for 6 weeks. Furthermore, comparisons of birds in either a photosensitive, photostimulated, or photorefractory state revealed a marked increase in GnRH protein expression in the photosensitive and photostimulated birds over photorefractory birds. A similar variation was observed in the volume of key forebrain song nuclei. Thus, Border canaries demonstrate measurable neuroplasticity in response to photoperiodic manipulations. These data, along with previous work on other strains of canaries, indicate the presence of intra-specific variation in photoperiodically regulated neuroplasticity.

Bursicon is the ultimate hormone in insect ecdysis, which is involved in cuticle hardening. Here we show that mRNAs encoding the heterodimeric cystine knot protein bursicon (Burs alpha, beta), are present in crustaceans, suggesting ubiquity of this hormone in arthropods. We firstly report the cloning, sequencing of mRNAs encoding subunits from the water flea, Daphnia arenata and the CNS of the crab, Carcinus maenas, in comparison with insect bursicon subunits. Expression patterns of alpha and beta burs mRNAs were examined by in-situ hybridisation (ISH) and quantitative RT-PCR. In the thoracic ganglion, burs alpha and beta mRNAs were completely colocalised in neurones expressing crustacean cardioactive peptide (CCAP). However, in the brain and eyestalk, bursicon transcripts were never observed, despite a complex expression pattern of CCAP interneurones. Patterns of expression of burs alpha and beta mRNAs were constitutive during the moult cycle of adult crabs, in stark contrast to the situation in insects. Whilst copy numbers of burs beta transcripts closely matched those of CCAP, those of burs alpha mRNA were around 3-fold higher than burs beta. This pattern was apparent during embryogenesis, where bursicon transcripts were first observed at around 50% development-the same time as first expression of CCAP mRNA. Transcript ratios (burs alpha: beta) increased during development. Our studies have shown, for the first time, that bursicon mRNAs are expressed in identified neurones in the nervous system of crustaceans. These findings will now promote further investigation into the functions of bursicon during the moult cycle and development of crustaceans.

This study examines the possibility that metabolic or endocrinal factors initiate fledging in the king penguin, a semi-altricial seabird species breeding a single chick on the ground. Chick fledging (departure to sea) occurred 5d after completion of the molt. It was preceded by a 16d fasting period and by a 7-fold increase in locomotor activity. From the measurement of the plasma concentration of metabolites and of glucagon and insulin, pre-fledging king penguin chicks were found to adapt to fasting in a classical way, i.e. by sparing body protein and mobilizing fat stores. At fledging, chicks were in phase II of fasting and their departure to sea was not stimulated by reaching critical energy depletion (phase III), in contrast to that which has been reported in breeding-fasting adults. The plasma level of corticosterone remained unchanged throughout the whole pre-fledging period, providing no support for a role of this stress-hormone in the facilitation of fledging. Thus, king penguin fledglings did not appear to be environmentally or nutritionally stressed. The plasma levels of thyroid hormones were elevated during the pre-fledging molt, in accordance with their key role in molt control in adult penguins. These levels declined by the time of the molt end, the plasma level of T4 thereafter being directly related to the time left before fledging. These results do not support the view that chronically elevated levels of thyroid hormones are required for the energy-demanding transition between being ashore and in cold water, but they suggest that the maintenance of high T4 levels may delay fledging.

In the recent decade, tremendous progress has been realized in insect endocrinology as the result of the application of a variety of advanced methods in neuropeptidome- and receptor research. Hormones of which the existence had been shown by bioassays four decades ago, e.g. bursicon (a member of the glycoprotein hormone family) and pupariation factor (Neb-pyrokinin 2, a myotropin), could be identified, along with their respective receptors. In control of diurnal rhythms, clock genes got company from the neuropeptide Pigment Dispersing Factor (PDF), of which the receptor could also be identified. The discovery of Inka cells and their function in metamorphosis was a true hallmark. Analysis of the genomes of Caenorhabditis elegans, Drosophila melanogaster and Apis mellifera yielded about 75, 100 and 200 genes coding for putative signaling peptides, respectively, corresponding to approximately 57, 100 and 100 peptides of which the expression could already be proven by means of mass spectrometry. The comparative approach invertebrates-vertebrates recently yielded indications for the existence of counterparts in insects for prolactin, atrial natriuretic hormone and Growth Hormone Releasing Hormone (GRH). Substantial progress has been realized in identifying the Halloween genes, a membrane receptor(s) for ecdysteroids, a nuclear receptor for methylfarnesoate, and dozens of GPCRs for insect neuropeptides. The major remaining challenges concern the making match numerous orphan GPCRs with orphan peptidic ligands, and elucidating their functions. Furthermore, the endocrine control of growth, feeding-digestion, and of sexual differentiation, in particular of males, is still poorly understood. The finding that the prothoracic glands produce an autocrine factor with growth factor-like properties and secrete proteins necessitates a reevaluation of their role in development.

Insect ecdysis sequence is composed of pre-ecdysis, ecdysis and post-ecdysis behaviors controlled by a complex cascade of peptide hormones from endocrine Inka cells and neuropeptides in the central nervous system (CNS). Inka cells produce pre-ecdysis and ecdysis triggering hormones (ETH) which activate the ecdysis sequence through receptor-mediated actions on specific neurons in the CNS. Multiple experimental approaches have been used to determine mechanisms of ETH expression and release from Inka cells and its action on the CNS of moths and flies. During the preparatory phase 1-2 days prior to ecdysis, high ecdysteroid levels induce expression of ETH receptors in the CNS and increased ETH production in Inka cells, which coincides with expression of nuclear ecdysone receptor (EcR) and transcription factor cryptocephal (CRC). However, high ecdysteroid levels prevent ETH release from Inka cells. Acquisition of Inka cell competence to release ETH requires decline of ecdysteroid levels and beta-FTZ-F1 expression few hours prior to ecdysis. The behavioral phase is initiated by ETH secretion into the hemolymph, which is controlled by two brain neuropeptides-corazonin and eclosion hormone (EH). Corazonin acts on its receptor in Inka cells to elicit low level ETH secretion and initiation of pre-ecdysis, while EH induces cGMP-mediated ETH depletion and consequent activation of ecdysis. The activation of both behaviors is accomplished by ETH action on central neurons expressing ETH receptors A and B (ETHR-A and B). These neurons produce numerous excitatory or inhibitory neuropeptides which initiate or terminate different phases of the ecdysis sequence. Our data indicate that insect ecdysis is a very complex process characterized by two principal steps: (1) ecdysteroid-induced expression of receptors and transcription factors in the CNS and Inka cells. (2) Release and interaction of Inka cell peptide hormones and multiple central neuropeptides to control consecutive phases of the ecdysis sequence.

The molting cycle of the freshwater crayfish, Procambarus clarkii, has been used as a model to study the cellular physiology and molecular biology of Ca "supply" proteins that effect transcellular vectorial Ca(2+) movement to achieve organismal Ca homeostasis. Specifically, periods of net Ca(2+) influx (postmolt) have been compared with periods of net Ca(2+) balance (intermolt). The broader goal is to understand the paradox facing epithelial cells of maintaining low cytosolic Ca(2+)in the face of mass Ca(2+)transit across epithelial cells. This mini-review compares mRNA and protein expression profiles for a series of proteins that are of strategic importance in effecting transcellular Ca(2+) flux in a selected epithelium, the antennal gland (kidney analog) specifically during apical to basolateral Ca(2+) conveyance. Target proteins were selected as representative of key "stages" in the transcellular transfer of Ca(2+): import (epithelial Ca(2+) channel, ECaC); storage (sarco/endoplasmic reticulum Ca(2+) ATPase, SERCA); buffering (sarcoplasmic Ca(2+) binding protein, SCP); and export (plasma membrane Ca(2+) ATPase, PMCA and Na(+)/Ca(2+) exchanger, NCX). The purpose of this review is to assess coordination of expression of these target proteins at times of high Ca(2+) demand (premolt and postmolt) compared to low Ca demand (intermolt) as a function of cellular location (apical vs. basolateral; endomembranes vs. plasma membranes) and relative abundance within different regions of the antennal gland. Understanding the spatiotemporal regulation of Ca(2+) handling proteins involved in transcellular transport is fundamental to investigating their endocrine regulation.

Avian corticosterone (CORT) concentrations vary seasonally for many species, but most studies examined species from extreme environments or from only a few times of the year. It is unclear how general a phenomenon this is. We examined both baseline and stress-induced CORT from house sparrows (Passer domesticus) in two habitats-Massachusetts, similar to their ancestral habitat; and New Mexico, a semi-arid desert where the house sparrow only thrives as an obligate human commensal. We captured both males and females during four times of the year-Spring (when the male cloacal protuberance indicated birds were in breeding condition), Fall, Winter, and in the late summer when birds were undergoing a prebasic molt. Birds were heaviest and had the longest wing chord lengths in the Fall at both sites and Massachusetts birds were approximately 10% heavier than New Mexico birds. House sparrows also showed a seasonal variation in the amount of fat stores, but the seasonal pattern differed. Massachusetts birds were fatter overall and showed the most fat during Fall and Winter, whereas New Mexico birds showed the most fat in the Spring. Both baseline and stress-induced total CORT did not differ between sexes or sites, with the exceptions of baseline CORT in the Fall and stress-induced CORT in the Spring being elevated in Massachusetts males. There was a distinct seasonal pattern at both sites, however, with total CORT being highest in the Spring and Winter and lowest during Fall and molt. This seasonal pattern was mirrored in corticosterone binding globulin (CBG) capacities, and when free CORT was estimated, the seasonal pattern disappeared. Stress-induced free CORT, however, was higher in Massachusetts males and females during the Fall and Winter, suggesting a potential differential response to stress associated with commensalism.

Despite the importance of molt to birds, very little is known about its environmental or physiological control. In starlings Sturnus vulgaris, and other species, under both natural conditions and experimental regimes, gonadal regression coincides with peak prolactin secretion. The prebasic molt starts at the same time. The aim of this series of experiments was to keep starlings on photo-schedules that would challenge the normally close relationship between gonadal regression and molt, to determine how closely the start of molt is associated with gonadal regression and/or associated changes in prolactin concentrations. In one series of experiments, photosensitive starlings were moved from a short photoperiod, 8 h light per day (8L), to 13 or 18L, and from 13 to 18L or 13 to 8L during testicular maturation. Later, photorefractory birds under 13L that had finished molting were moved to 18L. In another series of experiments, photorefractory starlings were moved from 18 to 8L for 7 weeks, 4 weeks, 2 weeks, 1 week, 3 days, 1 day, or 0 days, before being returned to 18L. There was no consistent relationship between photoperiod, or the increase in photoperiod, and the timing of the start of molt. Nor was there a consistent relationship with gonadal regression and the start of molt-molt could be triggered in the absence of a gonadal cycle. However, there was always an association between the start of molt and prolactin. In all cases where molt was induced, there had been an earlier increase in prolactin. However, the timing of molt was related to the time of peak prolactin, not the magnitude of that peak. This relationship between peak prolactin and the start of molt could explain the normally close relationship between the end of breeding activity and the start of molt.

The measurement of hemolymph ecdysteroids using radioimmunoassay (RIA) indicates a biphasic increase during intermolt and premolt stages of the mole crab Emerita asiatica. The gradual rise during intermolt stage corresponds to vitellogenic activities in the ovary; whereas a distinctive premolt peak is characteristic of molting crustaceans. Injection experiments with 20-hydroxyecdysone (20E) during different molt cycle stages revealed the onset of precocious premolt changes, as determined by the epidermal retraction and setal development. Injection of 20E augmented protein synthesis in the ovary, hepatopancreas and integumentary tissues. Quantification of ecdysteroids in different developmental stages of ovary also indicated a progressive increase of ovarian ecdysteroids. Interestingly, the ovarian ecdysteroids after reaching a peak at C3 stage, start declining drastically to reach the lowest level at D(3-4) stage. This decline in the ovarian ecdysteroids is inversely related to rising hemolymph ecdysteroids during these active premolt stages. The hatching of the embryos, attached to the pleopods of the ovigerous females also occurs under a high titer of hemolymph ecdysteroids. In support, 20E injection at C3 stage crabs indicated a significant reduction in time duration of pleopodal embryonic development leading to hatching of zoea larvae. Understandably, the augmented hemolymph ecdysteroid titer helps in the synchronization of embryo hatching and the premolt changes, as occurring under the normal premolt conditions., (Copyright 2004 Elsevier Inc.)

In crustaceans, molt-inhibiting hormone (MIH) has been proposed to regulate molting by inhibiting the secretion of ecdysteroids from the Y-organ. Thus, MIH titer in the hemolymph should be inversely related to ecdysteroid titers during the molt cycle. However, it has not been demonstrated whether the MIH titer in the hemolymph changes during the molt cycle. The purpose of this study was to determine the changes in the MIH titers in the hemolymph during the molt cycle of the American crayfish, Procambarus clarkii, and to discuss the role of MIH in regulation of molting. As predicted by the hypothesis, the hemolymph MIH titer was high at the intermolt stage when the hemolymph ecdysteroid titer was low, and the MIH titer decreased to a basal level at the early premolt stage when the hemolymph ecdysteroid titer began to increase slightly. At the middle premolt stage when the hemolymph ecdysteroid titer increased, the MIH titer was restored to a level as high as that during the intermolt stage. This is in contradiction to the hypothesis. However, the Y-organs at this stage scarcely responded to MIH both in vitro and in vivo. The present findings suggest that ecdysteroid secretion from the Y-organ may be regulated not only by changes in the hemolymph MIH titer, but also by changes in the responsiveness of the Y-organ to MIH.

To understand the hormonal mechanism behind a unique strategy of breeding and molting in Humboldt penguins, six pairs of captive Humboldt penguins kept in an outdoor open display pen were observed and blood collected weekly for a year. They all molted between the middle of June and the middle of August within 10 days except one pair that molted about a month later. The late pair had been rearing a hatchling until July due to the successful second clutch after the first clutch failed. A peak of plasma levels of thyroxine and triiodothyronine, respectively, overlapped a period of molting in both sexes. Plasma testosterone concentrations in the males and females were lowest for two month during a period of pre-molt and molting. Plasma concentrations of estradiol were also lowest during the molt in both sexes. Except for the period of molting, sex steroid hormone concentrations were high although there was great individual variation. During the molt, the birds were forced to fast since they did not enter the pool in the display pen where they usually forage live fish. To compensate this forced fasting, they took more food than usual during pre-molting period and gained body mass to about 20% more than the baseline value. Increased flipper thickness was parallel to increased body mass indicating that the gained body mass attributed to fat reservoir. These data indicate that rapid molting in Humboldt penguins is correlated with a drastic increase and decrease of thyroid hormones during the period of lowest concentrations in sex steroid hormones.

The mountain white-crowned sparrow, Zonotrichia leucophrys oriantha, breeds in subalpine meadows throughout many mountainous regions of western North America. Mathematical analysis of 20 years of egg-laying dates at Tioga Pass, California (3030m elevation) indicated a highly predictable breeding season suggesting that precise environmental cues such as the annual change in day length were important for regulating reproductive function. Additionally, it appeared that there was sufficient yearly variation in the timing of breeding to suggest that other environmental cues may also be important for regulating adjustments in reproductive development and regression. Captive populations of Z. l. oriantha showed strong responses in gonadal development following transfer to longs days (15L 9D) and low temperature (5 degrees C) slowed down photoperiodically induced gonadal growth and subsequent regression, in both males and females. High temperature of 30 degrees C tended to accelerate gonadal development and regression whereas gonadal development was intermediate in a group exposed to 20 degrees C. Prior exposure to these temperature regimes while on short days (9L 15D) had no effect on body mass, fat, or plasma levels of luteinizing hormone (LH) and thyroid hormones. Curiously there was no effect of temperature on photoperiodically induced rises in LH in either sex despite marked effects on gonadal growth. Brood patch development was also enhanced in females exposed to 30 degrees C. Corticosterone levels measured in a subset of plasma samples from this experiment indicated no effect of temperature suggesting that the retarded gonadal development at 5 degrees C was not a result of thermal stress. Although there was a robust effect of photostimulation on thyroid hormone levels in blood of both sexes, temperature treatment had no effect on tri-iodothyronine (T3) concentrations. However, plasma levels of thyroxine (T4) were lower initially at 5 degrees C versus 20 and 30 degrees C treatments. This may be related to the protracted gonadal cycle at 5 degrees C versus the truncated gonadal cycle at 30 degrees C. Molt score, an indication of post-reproductive state and onset of photorefractoriness, was delayed in birds exposed to 5 degrees C. Body mass, and to a lesser extent fat score, tended to be lowest in birds exposed to 5 degrees C compared with those at 20 and 30 degrees C. These results demonstrate that ambient temperature significantly affected photoperiodically induced gonadal development and regression in these birds. The endocrine mechanisms underlying these effects require further study.

Captive starlings (Sturnus vulgaris) undergoing a prebasic molt were given exogenous insulin (INS) and corticosterone (CORT), to determine how these counterregulatory hormones would affect glucose and triglyceride concentrations during stress. Experiments were conducted at both morning (11:00) and night (23:00) to monitor daily variation in these responses. Concentrations of CORT, glucose, and triglycerides were measured in blood plasma within 3 min of disturbance (basal) and at 40, 70, and 150 min thereafter (stress-induced) to monitor the effect of injecting saline, INS, CORT, or INS+CORT. Saline injection (which included the stress of handling and restraint) increased CORT concentrations, decreased triglycerides, but had no effect on circulating glucose. Daily variation was not evident in CORT or glucose, but concentrations of triglycerides were higher at night than during the day. INS markedly suppressed glucose concentrations, but had no effect on plasma CORT or triglycerides. Glucose levels did not change in response to stress, but exogenous CORT elicited hyperglycemia during the day. Injected CORT also hastened the recovery of glucose concentrations from INS-induced hypoglycemia at night, and had no effect on circulating triglycerides. Basal concentrations of CORT, glucose, and triglycerides exhibited photoperiodic (mimicking seasonal) changes when combined with data from an earlier study in starlings held on long- and short-day photoperiods. During the prebasic molt, all three measurements were lower compared to other photoperiods. Together, these data suggest that glucose and triglycerides concentrations are regulated differently during molt, but INS and CORT maintain their traditional effects.

When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar., (Copyright 2002 Elsevier Science (USA))

Year-class differences in reproductive function were investigated in a free-living population of adult male Dark-eyed Juncos, Junco hyemalis, breeding in interior Alaska. Second-year males (SY, entering their first breeding season) were compared with after-second-year males (ASY, entering at least their second breeding season). We measured body mass, size of the cloacal protuberance (CP), testis mass, onset of prebasic molt, and concentrations of plasma luteinizing hormone (LH), testosterone (T), corticosterone (CORT), and prolactin (PRL) throughout the reproductive season (April to mid-July). There were no differences in SY and ASY body weights but SY males had smaller CPs and testis masses than ASY males during gonadal recrudescence and at the end of the breeding season. Plasma LH was elevated from April until mid-June and then decreased in the same way in both year classes. In contrast, plasma T was high from April until mid-May and was lower in SY than in ASY juncos shortly after they arrived on their breeding grounds at the end of April, but not at other times. In July, SY males started to molt earlier, suggesting that they became photorefractory earlier than ASY males. Plasma PRL increased progressively in both year classes between April and early June and decreased in early July. At this time, plasma PRL decreased earlier in SY than in ASY males. Plasma CORT changed seasonally, but did not differ between SY and ASY juncos. Thus, year-class differences in CP sizes and testis mass apparently did not result from SY males secreting less LH or more PRL or CORT than ASY males. It is suggested that differences in reproductive condition in SY and ASY juncos are mediated by interactions with conspecific birds and do not result from an intrinsic effect of age., (Copyright 2000 Academic Press.)

Harsh weather can have devastating effects on both the survival and the breeding success of wild animals. Corticosterone, released in response to the stress caused by harsh conditions, may trigger physiological and behavioral changes that help ameliorate these effects. We examined three species of Arctic-breeding passerine birds for correlations between circulating plasma corticosterone levels and weather conditions at the time of capture. Furthermore, because persistently poor weather conditions may be required to initiate a stress response, we also looked for a relationship between corticosterone levels and weather conditions over the 24 and 72 h preceding capture. None of the three species showed substantial effects of weather on unstressed corticosterone levels during the breeding season, although one species showed a significant relationship with stress-induced corticosterone levels. In two species, however, variations in weather during molt (an energetically costly period when birds replace their feathers) explained from 35 to 88% of the individual variation in corticosterone levels. In a third molting species, weather conditions in the preceding 24 and 72 h explained between 20 and 30% of the individual variation in corticosterone levels. It thus appears that adverse weather may be a potent stimulator of corticosterone release during molt, but not during the breeding season. Although extreme weather conditions (those experienced once every few years) can disrupt breeding, since none of the birds abandoned breeding we might conclude that the storms in this study were well within the ability of the birds to cope physiologically., (Copyright 2000 Academic Press.)

To study the in vitro release of PRL isoforms, anterior pituitary glands from medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries after collection and placed into separate perifusion chambers. Medium (M199) was continuously perifused through the system and pituitaries were stimulated with cVIP (10(-7) M). Total PRL content was monitored by RIA and, the ratio of immunoreactive PRL isoforms in the perifusate was estimated by Western blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg-laying and incubating hens and the band corresponding to the nonglycosylated isoform (24 kDa) was predominant in samples from out-of-lay and molting stages. No changes in the ratio of isoform released were detected during cVIP stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary gland in vitro in the same relative proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary gland in vitro., (Copyright 1999 Academic Press.)

In the fiddler crab, Uca pugilator, we have investigated the temporal expression of receptors in various tissues using probes that encode Uca ecdysteroid receptor (UpEcR) and retinoid-X-receptor (UpRXR) gene homologs. During molt stages C4 through D1-4, UpEcR and UpRXR transcripts are expressed in regenerating limb buds, gills, eyestalks, hypodermis, hepatopancreas, muscle from nonregenerating walking legs, and the large cheliped. Some of these tissues have not previously been recognized as ecdysteroid-target tissues. Levels of ecdysteroids in the hemolymph fluctuate significantly during the molt cycle of U. pugilator. The variation in steady-state concentrations of UpEcR transcripts in tissues from C4 to D1-4 implies molt cycle-related differences in the potential of these tissues to respond to changing titers of ecdysteroids in the hemolymph. In singly autotomized crabs, highest concentrations of UpEcR transcript in some tissues did not coincide with the highest levels of circulating ecdysteroids, suggesting that UpEcR expression in these tissues is not dependent on high ecdysteroid titers and may be induced by low or rising concentrations of ecdysteroids. UpEcR and UpRXR genes were expressed simultaneously in tissues, supporting the possibility of heterodimerization for EcR and RXR in vivo. In some tissues, however, levels of transcripts differed, suggesting other possible receptor interactions. Moreover, UpEcR expression in tissues from multiply autotomized crabs differed from the expression patterns in tissues from singly autotomized crabs., (Copyright 1998 Academic Press.)

Transfer of intact, photosensitive starlings from short to long days causes an increase in plasma thyroxine and gonadal maturation and later induces photorefractoriness. Thyroidectomy of starlings prevents the induction of photorefractoriness. This study investigated whether the long-day-induced increase in plasma thyroxine is necessary for the induction of photorefractoriness. Photosensitive starlings were thyroidectomised, given thyroxine in their drinking water at concentrations that result in plasma thyroxine at short-day physiological concentrations or lower, and transferred to long days. Plasma thyroxine and prolactin, gonadal size, and moult were monitored. The group with short-day concentrations of plasma thyroxine became photorefractory at the same time as intact controls transferred to long days. The other groups, with lower plasma thyroxine, also became photorefractory, but the onset of photorefractoriness was delayed. The increase in plasma prolactin following photostimulation was proportional to plasma thyroxine concentrations. The onset of moult was also related to plasma thyroxine. We conclude that the long-day concentrations of plasma thyroxine observed in the plasma of intact starlings are not necessary for the induction of photorefractoriness. This suggests that thyroxine acts as a permissive factor rather than actively driving the photorefractory process.

Temporal changes in DNA synthesis were examined in the corpora allata of the silkworm Bombyx mori during the penultimate larval stadium. 5'-Bromo-2'-deoxyuridine (BrdU) labeling revealed that allatal DNA synthesis rose early in the stadium, peaking at about 2% of corpus allatum (CA) cells 24 hr after ecdysis. This corresponded to a rising phase of the ecdysteroid titer reported previously. DNA synthesis declined to a nearly undetectable level 1 day later. In larvae fed on leaves coated with 20-hydroxyecdysone (20E), the number of BrdU-labeled cells increased continuously after the normal peak and reached about 8% of CA cells at the end of the stadium. However, total count of CA cells from whole-mount monolayers and colchicine treatment indicated that, regardless of DNA content, CA cells did not divide. To determine if the rise in 20E was responsible for the onset of allatal DNA synthesis, we have established an organ culture system to support de novo DNA synthesis in vitro. The number of BrdU-labeled cells increased with increasing 20E concentration between 0.1 and 0.5 microg ml-1 in the medium, corresponding well to the rising 20E titer in the hemolymph. The level of DNA synthesis in culture was further elevated when 20% hemolymph was added together with 20E, suggesting that other direct stimulatory factors for allatal DNA synthesis may also exist in the hemolymph.

Ovarian and hemolymph ecdysteroids in Armadillidium vulgare were analyzed at four stages of ovarian maturation through the reproductive molt cycle using high-performance liquid chromatography and radioimmunoassay. The major ecdysteroids in the ovaries and hemolymph of A. vulgare were 20-hydroxyecdysone and ecdysone in free and conjugated forms. The concentration of ovarian ecdysteroids reached a maximal level in maturing ovaries during stage D (premolt period) of the molt cycle. At the end of stage D, a high level of ecdysteroids was detected in fully matured ovaries. On the other hand, hemolymph ecdysteroid titers in reproductive females showed a peak during stage D and declined rapidly to a low level at the end of stage D. No appreciable differences in the amounts of hemolymph ecdysteroids and in molecular species of them were apparent in females in reproductive and nonreproductive molt cycles. The amounts of hemolymph ecdysteroids were about fivefold higher in females than those in males. These ecdysteroids may have a role in controlling ovarian development in female A. vulgare.

Effects of fenoxycarb on the secretory activity of the prothoracic glands (PGs) of the silkworm, Bombyx mori, were investigated. Fenoxycarb inhibited the secretory activity of PGs in vitro throughout the fifth instar. Incubation of PGs in the presence of 1 microgram of fenoxycarb resulted in a approximately 50% inhibition of their secretory activity. This inhibition of fenoxycarb was dose dependent. Stimulation of the PGs by recombinant prothoracicotropic hormone (rPTTH) reached a high activation ratio of 15.9. Simultaneous presence of fenoxycarb and 1 ng rPTTH significantly decreased the stimulatory effect of rPTTH on the PGs, and the activation ratio never exceeded 3.65. Early in the fifth instar, fenoxycarb strongly inhibited the secretory activity of the PGs and induced a developmental arrest as well. After the onset of pupal commitment, its action toward the PG secretory activity was inhibitory. However, fenoxycarb treatment was not able to induce a developmental arrest in intact animals after the pupal commitment although it could do so in head-ligated animals. On the contrary, application of fenoxycarb after the wandering behavior resulted in a significantly increased secretory activity by the PGs of both intact and head-ligated larvae.

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity of Carcinus maenas Y-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals. In vitro incubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (-cGMP/ +cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.

There is convincing evidence that euthyroid male American tree sparrows are already programmed for seasonal reproduction and postnuptial molt by Week 4 of photostimulation. To explore more precisely when, during early photostimulation, thyroid-dependent programming of seasonal events occurs, photosensitive male tree sparrows were radiothyroidectomized or sham thyroidectomized on the first day of photostimulation (Week 0) or at Weeks 1 or 3 thereafter. Birds were monitored for testicular growth and regression over 8 or 12 weeks and then tested for photosensitivity or photorefractoriness by exposing them to constant light and exogenous L-thyroxine for 4 weeks. Molt of the primary flight feathers was scored periodically, and at the end of the experiment, hypothalami were saved for cGnRH-I (chicken gonadotropin-releasing hormone I) assay. Because (1) the thyroid is already dysfunctional by Day 4 after radiothyroidectomy, (2) thyroid-dependent photoinduced gonadal growth is programmed growth, and as demonstrated here, (3) testis length at Week 6 on long days (i.e., maximum or near-maximum testis size) is independent of the time of thyroidectomy at or after the onset of photostimulation, we conclude that male American tree sparrows were programmed for photoperiodic testicular growth during the first week of photostimulation. Based on measurements of testis length and hypothalamic cGnRH-I content, only 2 of 11 birds thyroidectomized at Week 1 were already programmed for photorefractoriness by Week 1, whereas all birds thyroidectomized at Week 3 were so programmed by Week 3. Marked differences in molt scores between photorefractory (thyroidectomized and euthyroid) and photosensitive (thyroidectomized) birds argue that postnuptial molt also was programmed roughly between Weeks 1 and 3. To explain these results, we ascribe organizational-like actions directly or indirectly to endogenous thyroid hormones and argue that the onset of photostimulation opens a window, during which time euthyroid male American tree sparrows are programmed for seasonality. A clear dissociation of photorefractoriness from photoperiodic testicular growth in birds thyroidectomized at Week 1 suggests that separate control circuits mediate these two annually periodic events.

Prompted by evidence of a linkage between the thyroid gland and seasonal reproduction in obligately photoperiodic American tree sparrows (Spizella arborea), we measured circulating T4 (L-thyroxine), hypothalamic cGnRH-I (chicken gonadotropin-releasing hormone, variant I), pituitary and plasma LH (luteinizing hormone), and ovarian mass in euthyroid females moved from short to long days. Our purpose was to correlate temporal changes in T4 and cGnRH-I with each other and with pituitary and plasma LH and ovarian mass. T4 increased early during photostimulation and peaked at Week 1 or 2 on 20L:4D (20 hr light:4 hr dark per day). Hypothalamic cGnRH-I also increased during photostimulation and peaked between Weeks 2 and 4. Pituitary and plasma LH peaked at Weeks 4 and 1, respectively, and ovarian mass increased significantly by Week 6. In a separate study, we charted the progression of postnuptial molt. Molt of the primary remiges began at Week 9, proceeded rapidly through Week 15, and was complete by Week 18. In view of a demonstrated role for the thyroid gland in the control of seasonal reproduction in female tree sparrows and the time-dependent effects of thyroidectomy after photostimulation in male tree sparrows, the observation that circulating T4 increases early during photostimulation suggests the possibility of a causal relationship between the thyroid gland and the hypothalamus-pituitary-ovarian axis, hence a thyroid-hypothalamus-pituitary-ovarian axis.

The time course of thyroid dysfunction after injecting 0.30 mCi Na131I was charted in female American tree sparrows (Spizella arborea) transferred to long days on Day 0 and challenged with 30 micrograms bovine thyroid-stimulating hormone on Postinjection Days 4, 7, 46, and 105. Serum T4 (L-thyroxine) was not detectable in thyroidectomized birds, indicating complete thyroid dysfunction by Day 4 and no restoration of thyroid function by Day 105. By contrast, serum T4 concentrations in similarly challenged thyroid-intact controls greatly exceeded assay sensitivity. To determine whether a single injection of T4 can program thyroidectomized female tree sparrows for seasonal reproduction and postnuptial molt, as it can thyroidectomized male tree sparrows, thyroidectomized females were injected with T4 (100 micrograms) or alkaline vehicle (V) either on the first day of photostimulation or 1 week before photostimulation (during which time injected T4 likely was cleared metabolically). Females injected with T4 on the first day of photostimulation showed robust ovarian growth similar to that reported for photostimulated euthyroid females. Both groups of V-injected females, as well as females injected with T4 1 week before photostimulation, showed slow, but reliable, ovarian growth, indicating that thyroidectomized birds can detect an increase in day length. When birds were moved at Week 7.5 or 9 to constant light and given T4 in drinking water (a qualitative assay for absolute photorefractoriness), only one tested photorefractory and molted. The remaining birds, including those injected with T4 1 week before photostimulation and both groups of V-injected controls, tested photosensitive and did not molt. Taken together, these results indicate that T4, or one of its metabolites, programs thyroidectomized female tree sparrows for ovarian growth early during photostimulation. A single T4 injection (100 micrograms) on the first day of photostimulation usually also programs thyroidectomized male tree sparrows for photorefractoriness and molt. Such an injection is far less effective in programming females for these two late-season events.

Subspecies of the Houbara bustard are resident in North Africa (Chlamydotis undulata undulata) or are partial latitudinal migrants in Asia (Chlamydotis undulata macqueenii). Houbara breed in arid or semiarid zones, and the reproductive biology of wild Houbara is largely unknown. Body mass, molt, courtship display, laying, and plasma concentrations of LH, testosterone, and progesterone were measured in captive-bred birds of both subspecies, housed in outdoor cages under natural conditions, over a period of 1 year and 6 months, at Taif, Saudi Arabia (21 degrees 15' N). Captive Houbara bustards show a seasonal breeding pattern with a laying period from January to July. Males began displaying well in advance of any eggs laid and display long after the termination of laying. In females, gonadal activity and molt were mutually exclusive; however, in males, slight overlap between sexual display and molt was observed. In females, seasonal variation of LH and progesterone was synchronous with laying activity. In males both testosterone and LH plasma concentrations were highest in January (testosterone approximately 8 ng/ml; LH approximately 3 ng/ml) at the beginning display period. This could correspond, in the wild, to the establishment of territories. Levels dropped significantly before the females started laying but stayed at an intermediate level throughout the breeding season (testosterone approximately 3 ng/ml; LH approximately 1.5 ng/ml). In the middle of August, during molt, and following heavy rainfall, LH and testosterone peaked (testosterone approximately 2.3 ng/ml; LH approximately 1.8 ng/ml), suggesting a possible impact of rainfall on Houbara gonadal activity. Houbara stopped laying before the maximum ambient temperature reached 32 degrees, indicating that high temperature could be a factor terminating laying activity. No major differences were found between the two subspecies in the timing or duration of the reproductive period; only the annual variation of the body mass varied slightly between the subspecies.

Plasma levels of prolactin and luteinizing hormone have been examined in intact and pinealectomized European starling (Sturnus vulgaris) during termination and onset of photorefractoriness. Photorefractory starlings exposed to long (18L:6D) daily photoperiods for 10 weeks were pinealectomized. One week after pinealectomy these birds, along with the intact and sham-operated birds, were transferred to short (8L:16D) daily photoperiods. After 10 weeks on short day lengths the daily photoperiod was switched to long (18L:6D) and the birds remained on this regime for a further 10 weeks. Plasma prolactin and luteinizing hormone were determined prior to pinealectomy and then weekly throughout the experiment. Molt scores were also recorded. Profiles of both the hormones and the molt pattern were similar in pinealectomized, sham-operated, and intact control starlings. In European starlings the pineal gland does not seem to exert significant effects on the photoinduced rise in plasma prolactin and luteinizing hormone seen in photosensitive birds. It is also suggested that pinealectomy has no significant effect on the dissipation and onset of photorefractoriness assessed from plasma luteinizing hormone secretion in this bird.