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Oxygen, hydrogen, carbon and nitrogen stable isotopes were measured on a comprehensive sampling of feathers from two spring Hooded Warblers (Setophaga citrina) in Texas to evaluate isotopic variability between feathers and during molt. Isotopic homogeneity within each bird was found across all four isotopic systems, supporting the hypothesis that molt in these neotropical migrants is fully completed on the breeding grounds. This homogeneity suggests that the isotopic composition of a single feather is may be representative of the whole songbird. However, each bird was found to have one or two outlier feathers, which could signify regrowth of lost feathers after prebasic molt., Competing Interests: The authors have declared that no competing interests exist.

Introduced bird species can become invasive in agroecosystems and their management is inhibited if their origin and movements are not well understood. Stable isotope measurements of feathers can be used to infer molt origins and interstate movements in North America. We analyzed stable-hydrogen (δ2H), carbon (δ13C) and nitrogen (δ 15N) isotope ratios in feathers to better understand the molt origin of European starlings (Sturnus vulgaris) collected at dairies and feedlots throughout the United States. Primary feathers were used from 596 adult and 90 juvenile starlings collected during winter at dairies and feedlots that experience starling damages in Arizona, California, Colorado, Idaho, Illinois, Iowa, Kansas, Minnesota, Missouri, Nebraska, Nevada, New Hampshire, New Mexico, New York, North Carolina, Oregon, Texas, Vermont, Washington and Wisconsin. The best-fit model indicated that the combination of feather δ2H, δ13C and δ15N values best predicted the state where samples were collected and thus supported use of this approach for tracing molt origins in European starlings. Interestingly, molt origins of starlings collected at dairies and feedlots generally west of -90° longitude (i.e. 11 of 15 states west of the Mississippi River, including Wisconsin) were assigned to the collection state and/or the state adjacent to the collection state. In contrast, molt origins of starlings collected generally east of -90° longitude (four of five eastern states) were not assigned to the collection state and/or the state adjacent to the collection state. Among all starlings (N = 686), 23% were assigned to the collection state and 19% were assigned to the state adjacent to the collection state. Among all males (N = 489) and all females (N = 197), 23% and 26% were assigned to the collection state and 19% and 13% were assigned to the state adjacent to the collection state, respectively. We observed a greater proportion (88%) of juvenile starlings assigned to states other than their collection state (i.e. potentially a result of natal dispersal) than that proportion (76%) in adult starlings. This study included an unprecedented sample of feather isotopes from European starlings throughout the United States. As a novel contribution to the ecology and management of invasive and migratory passerines, we demonstrate how such feather isoscapes can be used to predict molt origin and, potentially, interstate movements of European starlings for subsequent ecological and management investigations., Competing Interests: The authors have declared that no competing interests exist.

Biological rhythms regulate innumerable physiological processes, yet little is known of factors that regulate many of these rhythms. Disruption in the timing of these rhythms can have devastating impacts on population sustainability. We hypothesized that the timing of the molt infradian rhythm in the crustacean Daphnia magna is regulated by the joint action of the protein E75 and nitric oxide. Further, we hypothesized that disruption of the function of E75 would adversely impact several physiological processes related to growth and reproduction. Analysis of mRNA levels of several genes, involved in regulating the molt cycle in insects, revealed the sequential accumulation of E75, its dimer partner HR3, FTZ-F1, and CYP18a1 during the molt cycle. Exposure to the nitric oxide donor sodium nitroprusside early in the molt cycle had no effect on E75 or HR3 mRNA levels, but delayed the peak accumulation of FTZ-F1 and CYP18a1 mRNA. The subsequent exuviation was also delayed consistent with the delay in peak accumulation of FTZ-F1 and CYP18a1. These results supported our assertion that nitric oxide binds E75 rendering it incapable of binding HR3. Excess HR3 protein then enhanced the accumulation of the downstream products FTZ-F1 and CYP18a1. Similarly, suppression of E75 mRNA levels, using siRNA, had no effect on mRNA levels of HR3 but elevated mRNA levels of FTZ-F1. Consistent with these molecular responses, the suppression of E75 using siRNA increased the duration of the molt cycle and reduced the number of offspring produced. We conclude that the molt cycle of daphnids is regulated in a manner similar to insects and disruption of E75 results in a lengthening of the molt cycle and a reduction the release of viable offspring., Competing Interests: The authors have declared that no competing interests exist.

In many seabirds, individuals abstain from eating during the moult period. Penguins have an intense moult that lasts for weeks, during which they are confined to land. Despite the importance for survival, it is still unclear how the faecal microbiota of Antarctic penguins changes in response to the moult fast. Here, we investigated the faecal microbiota of chinstrap (Pygoscelis antarcticus) and gentoo penguins (Pygoscelis papua) on King George Island, Antarctica. The bacterial community compositions during the feeding and moulting stages were compared for both species using bacterial 16S rRNA gene amplicon on an Illumina MiSeq platform. Our results showed that the moult fast altered the bacterial community structures in both penguin species. Interestingly, the bacterial community composition shifted in the same direction in response to the moult fast but formed two distinct clusters that were specific to each penguin species. A significant increase in bacterial diversity was observed in gentoo penguins, whereas no such change was observed for chinstrap penguins. By analysing the contribution of the ecological processes that determine bacterial community assembly, we observed that processes regulating community turnover were considerably different between the feeding and moulting stages for each penguin. At the phylum level, the relative abundances of Fusobacteria, Firmicutes and Proteobacteria were dominant in chinstrap penguins, and no significant changes were detected in these phyla between the feeding and moulting periods. Our results suggest that moult fast-induced changes in the faecal microbiota occur in both species., Competing Interests: The authors have declared that no competing interests exist.

In several countries in northwestern Europe, the number of resident greylag geese (Anser anser) has increased by up to 20% a year over the last few decades. The increasing numbers of geese are causing problems in tourism and agriculture. The number of resident greylag geese visiting the Altmuehlsee lake, a recreational area in the south of Germany, has also increased, especially in June and July. However, the geese leave the lake again at the beginning of August. 30 adult greylag geese were banded with telemetry transmitters during molting at the Altmuehlsee lake to determine when the geese leave the lake in August and what structures they need in their winter habitat. The distances the greylag geese flew from their resting sites to foraging sites were calculated, as well as the size of their home ranges and the proportions of each habitat type in the home range. It was found that the greylag geese used small home ranges while rearing their young, and fed close to the water. After molting, the size of their home ranges increased. They fed on fields and meadows around the lake. In the winter habitat, the geese also had a small home range, and they preferred eating grain and corn and the availability of an open body of water. In addition, one group of geese remained in the city of Munich during the winter, benefiting from supplementary food from people. This information helps us to understand the different requirements of resident greylag geese. It can be used to inform management decisions aimed at preventing agricultural damage. Special goose areas can for example be designated for the different periods in a goose's life cycle., Competing Interests: The authors have declared that no competing interests exist.

Migratory species often roam vast distances bringing them into contact with diverse conditions and threats that could play significant roles in their population dynamics. This is especially true if long-range travels occur within crucial stages of a species' annual life-cycle. Crested penguins, for example, usually disperse over several hundreds of kilometres after completing the energetically demanding breeding season and in preparation for the costly annual moult. A basic understanding of crested penguins' pre-moult dispersal is therefore paramount in order to be able to assess factors affecting individual survival. The Fiordland penguin, or Tawaki, the only crested penguin species breeding on the New Zealand mainland, is currently one of the least studied and rarest penguin species in the world. We successfully satellite tracked the pre-moult dispersal of 17 adult Tawaki from a single colony located in the species' northern breeding distribution. Over the course of 8-10 weeks the penguins travelled up to 2,500 km away from their breeding colony, covering total swimming distances of up to 6,800 km. During outbound travels all penguins headed south-west within a well-defined corridor before branching out towards two general trip destinations. Birds leaving in late November travelled towards the Subtropical Front some 800 km south of Tasmania, whereas penguins that left in December headed further towards the subantarctic front. Using K-select analysis we examined the influence of oceanographic factors on the penguins' dispersal. Water depth, surface current velocity and sea level anomalies had the greatest influence on penguin movements at the subantarctic Front, while sea surface temperature and chlorophyll a concentration were key for birds travelling to the subtropical front. We discuss our findings in the light of anthropogenic activities (or lack thereof) in the regions visited by the penguins as well as the potential consequences of Tawaki pre-moult dispersal for the species' breeding distribution on the New Zealand mainland., Competing Interests: The authors have declared that no competing interests exist.

The oriental river prawn, Macrobrachium nipponense, is an important commercial aquaculture resource in China. In order to overwinter, M. nipponense displays decreased physiological activity and less consumption of energy. Sudden warming would trigger molting and cause an extensive death, resulting in huge economic losses. Therefore, it is of great practical significance to study the molting mechanism of oriental river prawns. Molt-inhibiting hormone gene (MIH) plays a major role in regulating molting in crustaceans. In this study, a full length MIH cDNA of M. nipponense (Mn-MIH) was cloned from the eyestalk. The total length of the Mn-MIH was 925 bp, encoding a protein of 119 amino acids. Tissue distribution analysis showed that Mn-MIH was highly expressed in the eyestalk, and that it had relatively low expression in gill, ovary, and abdominal ganglion. Mn-MIH was detected in all developmental stages, and changed regularly in line with the molting cycle of the embryo and larva. Mn-MIH varied in response to the molting cycle, suggesting that Mn-MIH negatively regulates ecdysteroidogenesis. Mn-MIH inhibition by RNAi resulted in a significant acceleration of molting cycles in both males and females, confirming the inhibitory role of MIH in molting. After long-term RNAi males, but not females had significant weight gain, confirming that Mn-MIH plays an important role in growth of M. nipponense. Our work contributes to a better understanding of the role of Mn-MIH in crustacean molting and growth., Competing Interests: The authors have declared that no competing interests exist.

Bowhead whales (Balaena mysticetus) have a nearly circumpolar distribution, and occasionally occupy warmer shallow coastal areas during summertime that may facilitate molting. However, relatively little is known about the occurrence of molting and associated behaviors in bowhead whales. We opportunistically observed whales in Cumberland Sound, Nunavut, Canada with skin irregularities consistent with molting during August 2014, and collected a skin sample from a biopsied whale that revealed loose epidermis and sloughing. During August 2016, we flew a small unmanned aerial system (sUAS) over whales to take video and still images to: 1) determine unique individuals; 2) estimate the proportion of the body of unique individuals that exhibited sloughing skin; 3) determine the presence or absence of superficial lines representative of rock-rubbing behavior; and 4) measure body lengths to infer age-class. The still images revealed that all individuals (n = 81 whales) were sloughing skin, and that nearly 40% of them had mottled skin over more than two-thirds of their bodies. The video images captured bowhead whales rubbing on large rocks in shallow, coastal areas-likely to facilitate molting. Molting and rock rubbing appears to be pervasive during late summer for whales in the eastern Canadian Arctic.

Wing morphology strongly affects flight performance which may consequently decline during feather moult due to the creation of feather gaps in the wing. Hence, the size and shape of moult-related wing gap may directly affect flight capacity. Here I examined the rare divergent primary moult sequence compared to the more common descendant moult sequence. In the divergent moult, the focus of primary moult is shifted from P1 (primary feather numbered descendantly) to another primary between P2 and P5, and then primaries are moulted in two concurrent waves, one descendant and the other ascendant. The result of this rare moult sequence is the splitting of the wing gap to two smaller gaps. Using a large moult database including 6,763 individuals of 32 Western Palaearctic passerine species, I found evidence of divergent moult only among 27 individuals of 12 species. I examined the speed of wing-feather moult for each individual that moulted divergently compared to a control group of individuals at the same moult stage which moulted following the common descending sequence. The results indicate that the sequence of primary moult and moult speed are correlated. Individuals which moulted divergently moulted their primaries with higher moult speed than descendant moulters. The applicability of this study is weakened by the dearth of moult data, thus making it difficult to draw conclusions for a large range of species. Ornithologists and bird ringers are therefore encouraged to collect more basic moult data during their field study.

Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems. We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.

Background: Molting is an essential biological process throughout the life history of crustaceans, which is regulated by many neuropeptide hormones expressed in the eyestalk. To better understand the molting mechanism in Portunus trituberculatus, we used digital gene expression (DGE) to analyze single eyestalk samples during the molting cycle by high-throughput sequencing., Results: We obtained 14,387,942, 12,631,508 and 13,060,062 clean sequence reads from inter-molt (InM), pre-molt (PrM) and post-molt (PoM) cDNA libraries, respectively. A total of 1,394 molt-related differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analysis identified some important processes and pathways with key roles in molting regulation, such as chitin metabolism, peptidase inhibitor activity, and the ribosome. We first observed a pattern associated with the neuromodulator-related pathways during the molting cycle, which were up-regulated in PrM and down-regulated in PoM. Four categories of important molting-related transcripts were clustered and most of them had similar expression patterns, which suggests that there is a connection between these genes throughout the molt cycle., Conclusion: Our work is the first molt-related investigation of P. trituberculatus focusing on the eyestalk at the whole transcriptome level. Together, our results, including DEGs, identification of molting-related biological processes and pathways, and observed expression patterns of important genes, provide a novel insight into the function of the eyestalk in molting regulation.

Increased shipping in British Columbia (BC) waters poses risks for marine birds from marine oil spills. Ancient Murrelets (Synthliboramphus antiquus), small marine diving birds of which half of the world's population breeds in BC, are especially susceptible to oiling immediately after departing from their breeding colonies, as their offspring are flightless, constraining their parents to remain with them. In 2014 we deployed geolocator loggers on Ancient Murrelets at four breeding colonies, two on the east and two on the west coast of Haida Gwaii to investigate patterns of post-breeding dispersal and subsequent migratory movements. Birds from east coast colonies moved south and east after leaving their colonies, remaining in Queen Charlotte Sound and adjacent waters for 4-6 weeks, whereas those from west coast colonies moved steadily north and west, so that they left BC waters earlier than those from east coast colonies. These movements were consistent with being driven by surface currents. In June, all birds moved rapidly to the eastern Aleutians, SE Bering Sea, and waters off Kamchatka, where they probably moulted. In August, most moved north, some passing through Bering Straits into the Chukchi Sea. In October-November some birds returned to waters off western N America (33%) and the remainder carried on westwards to waters off Japan, Korea and NE China. For the former group the movement to the Bering Sea in June constituted a moult migration and, as such, is the first described for an auk. Those birds wintering in Asia began moving east in February and arrived off BC in March, when observations at colonies show that burrow visits begin in Haida Gwaii. Our data suggest that, immediately after colony departure, birds from the east coast colonies (about half the population of Haida Gwaii) are at higher risk from potential oil spills in northern British Columbia waters than those breeding on the west coast.

In insects, extra-molting has been viewed as a compensatory mechanism for nymphal growth that contributes to optimize body weight for successful reproduction. However, little is known on the capacity of extra-molting to evolve in natural populations, which limits our understanding of how selection acts on nymphal growth. We used a multi-generational pedigree, individual monitoring and quantitative genetics models to investigate the evolution of extra-molting and its impact on nymphal growth in a solitarious population of the desert locust, Schistocerca gregaria. Growth compensation via extra-molting was observed for 46% of the females, whose adult weight exceeded by 4% that of other females, at a cost of a 22% longer development time. We found a null heritability for body weight threshold only, and the highest and a strongly female-biased heritability for extra molting. Our genetic estimates show that (1) directional selection can act on growth rate, development time and extra-molting to optimize body weight threshold, the target of stabilizing selection, (2) extra-molting can evolve in natural populations, and (3) a genetic conflict, due to sexually antagonistic selection on extra-molting, might prevent its fixation. Finally, we discuss how antagonistic selection between solitarious and gregarious environments and/or genetic correlations between growth and phase traits might also impact the evolution of extra-molting in locusts.

Aquatic birds have high cost of thermoregulation, especially during the moulting period, yet the effect of water temperature on the moulting strategy of aquatic birds has rarely been studied. Our general hypothesis is that energy savings associated with lower thermoregulation costs would be allocated to moulting processes. We predicted that aquatic birds moulting in warm water would have a higher level of body reserves, a faster growth rate of feathers, and an earlier remigial moult onset compared with birds moulting in cold water. We used the common eider (Somateria mollissima dresseri), a large sea duck, as the model species. Captive individuals were experimentally exposed to warm (18°C) and cold (8°C) water treatments during a three year period with individuals swapped between treatments. We found a similar feather growth rate for the two water temperature treatments and in contrast to our predictions, eiders exposed to warm water had a lower body mass and showed a delayed onset of remigial moult of approximately 7 days compared with those exposed to cold water. Our data indicate that body mass variations influence the timing of moult in unexpected ways and we suggest that it likely controls the occurrence of wing moult through a hormonal cascade. This study emphasizes the importance of improving our knowledge of the effects of water temperature on remigial moult of aquatic birds, to better assert the potential effects of global warming on their survival.

Adult passerines renew their flight feathers at least once every year. This complete moult occurs either in the breeding areas, just after breeding (summer moult), or, in some long-distance migratory species, at the non-breeding areas, after arrival to the southern wintering area at the end of autumn migration (winter moult). The aim of this study was to relate moult strategies with the DMD, the difference in median migration date, through Israel, between juveniles and adults. Our data on autumn migration timing in juveniles and adults was based on ringing data of 49,125 individuals belonging to 23 passerine species that breed in Europe and Western Asia and migrate through Israel. We found that DMD was associated with moult timing. In all species that perform a winter moult, adults preceded juveniles during autumn. Among migrants who perform a summer moult, we found evidence of both migration timing patterns: juveniles preceding adults or adults preceding juveniles. In addition, in summer moulters, we found a significant, positive correlation between mean breeding latitude and DMD. Although previous studies described that moult duration and extent can be affected by migration, we suggest that moult strategies affect both migration timing and migration strategy. These two moult strategies (summer or winter moult) also represent two unique migration strategies. Our findings highlight the evolutionary interplay between moult and migration strategies.

Cuticular structures of arthropods undergo dramatic molt-related changes from being soft to becoming hard. The shell-hardening process of decapod crustaceans includes sclerotization and mineralization. Hemocyte PPO plays a central role in melanization and sclerotization particularly in wound healing in crustaceans. However, little is known about its role in the crustacean initial shell-hardening process. The earlier findings of the aggregation of heavily granulated hemocytes beneath the hypodermis during ecdysis imply that the hemocytes may be involved in the shell-hardening process. In order to determine if hemocytes and hemocyte PPO have a role in the shell-hardening of crustaceans, a knockdown study using specific CasPPO-hemo-dsRNA was carried out with juvenile blue crabs, Callinectes sapidus. Multiple injections of CasPPO-hemo-dsRNA reduce specifically the levels of CasPPO-hemo expression by 57% and PO activity by 54% in hemocyte lysate at the postmolt, while they have no effect on the total hemocyte numbers. Immunocytochemistry and flow cytometry analysis using a specific antiserum generated against CasPPO show granulocytes, semigranulocytes and hyaline cells as the cellular sources for PPO at the postmolt. Interestingly, the type of hemocytes, as the cellular sources of PPO, varies by molt stage. The granulocytes always contain PPO throughout the molt cycle. However, semigranulocytes and hyaline cells become CasPPO immune-positive only at early premolt and postmolt, indicating that PPO expression in these cells may be involved in the shell-hardening process of C. sapidus.

The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.

Physiological trade-offs mediated by limiting energy, resources or time constrain the simultaneous expression of major functions and can lead to the evolution of temporal separation between demanding activities. In birds, plumage renewal is a demanding activity, which accomplishes fundamental functions, such as allowing thermal insulation, aerodynamics and socio-sexual signaling. Feather renewal is a very expensive and disabling process, and molt is often partitioned from breeding and migration. However, trade-offs between feather renewal and breeding have been only sparsely studied. In barn swallows (Hirundo rustica) breeding in Italy and undergoing molt during wintering in sub-Saharan Africa, we studied this trade-off by removing a tail feather from a large sample of individuals and analyzing growth bar width, reflecting feather growth rate, and length of the growing replacement feather in relation to the stage in the breeding cycle at removal and clutch size. Growth bar width of females and length of the growing replacement feather of both sexes were smaller when the original feather had been removed after clutch initiation. Importantly, in females both growth bar width and replacement feather length were negatively predicted by clutch size, and more strongly so for large clutches and when feather removal occurred immediately after clutch completion. Hence, we found strong, coherent evidence for a trade-off between reproduction, and laying effort in particular, and the ability to generate new feathers. These results support the hypothesis that the derived condition of molting during wintering in long-distance migrants is maintained by the costs of overlapping breeding and molt.

The effects of environmental perturbations or stressors on individual states can be carried over to subsequent life stages and ultimately affect survival and reproduction. The concentration of corticosterone (CORT) in feathers is an integrated measure of hypothalamic-pituitary-adrenal activity during the molting period, providing information on the total baseline and stress-induced CORT secreted during the period of feather growth. Common eiders and greater snow geese replace all flight feathers once a year during the pre-basic molt, which occurs following breeding. Thus, CORT contained in feathers of pre-breeding individuals sampled in spring reflects the total CORT secreted during the previous molting event, which may provide insight into the magnitude or extent of stress experienced during this time period. We used data from multiple recaptures to disentangle the contribution of individual quality vs. external factors (i.e., breeding investment or environmental conditions) on feather CORT in arctic-nesting waterfowl. Our results revealed no repeatability of feather CORT within individuals of either species. In common eiders, feather CORT was not affected by prior reproductive investment, nor by pre-breeding (spring) body condition prior to the molting period. Individual feather CORT greatly varied according to the year, and August-September temperatures explained most of the annual variation in feather CORT. Understanding mechanisms that affect energetic costs and stress responses during molting will require further studies either using long-term data or experiments. Although our study period encompassed only five years, it nonetheless provides evidence that CORT measured in feathers likely reflects responses to environmental conditions experienced by birds during molt, and could be used as a metric to study carry-over effects.

Light pollution is known to affect important biological functions of wild animals, including daily and annual cycles. However, knowledge about long-term effects of chronic exposure to artificial light at night is still very limited. Here we present data on reproductive physiology, molt and locomotor activity during two-year cycles of European blackbirds (Turdus merula) exposed to either dark nights or 0.3 lux at night. As expected, control birds kept under dark nights exhibited two regular testicular and testosterone cycles during the two-year experiment. Control urban birds developed testes faster than their control rural conspecifics. Conversely, while in the first year blackbirds exposed to light at night showed a normal but earlier gonadal cycle compared to control birds, during the second year the reproductive system did not develop at all: both testicular size and testosterone concentration were at baseline levels in all birds. In addition, molt sequence in light-treated birds was more irregular than in control birds in both years. Analysis of locomotor activity showed that birds were still synchronized to the underlying light-dark cycle. We suggest that the lack of reproductive activity and irregular molt progression were possibly the results of i) birds being stuck in a photorefractory state and/or ii) chronic stress. Our data show that chronic low intensities of light at night can dramatically affect the reproductive system. Future studies are needed in order to investigate if and how urban animals avoid such negative impact and to elucidate the physiological mechanisms behind these profound long-term effects of artificial light at night. Finally we call for collaboration between scientists and policy makers to limit the impact of light pollution on animals and ecosystems.

Insects require molting fluids to shed the old cuticle during molting. β-N-acetyl-D-hexosaminidase, known as Hex1, together with various chitinases, is responsible for degrading the chitin component of the old cuticle. This study showed that another β-N-acetyl-D-hexosaminidase, termed OfHex3, interacted with Hex1 and functioned in the molting fluid, although the homolog of OfHex3 was known as a sperm-plasma enzyme functioning in egg-sperm recognition. OfHex3 is an enzyme cloned from the insect Asian corn borer, Ostrinia furnacalis, which is one of the most destructive pests of maize. The enzymatic activity analysis indicated that OfHex3 was able to degrade chitooligosaccharides, but at a lower rate than that of OfHex1. Because OfHex3 did not have substrate inhibition, we deduced that the presence of OfHex3 might help OfHex1 relieve substrate inhibition during chitin degradation during molting. The expression patterns of OfHex3 during O. furnacalis development were studied by real-time PCR as well as western blot. The results showed that both gene transcription and protein translation levels of OfHex3 were up-regulated during larval-larval molting. The tissue-specific expression pattern analysis indicated that OfHex3 was mostly localized in the fat body and testis. All these data further supported that Hex3 was involved in molting as well as in fertilization. This study may help to understand the complexity of cuticle degradation during insect molting, and may provide a possible target for pest control.

A temporal separation of energetically costly life history events like reproduction and maintenance of the integumentary system is thought to be promoted by selection to avoid trade-offs and maximize fitness. It has therefore remained somewhat of a paradox that certain vertebrate species can undergo both events simultaneously. Identifying potential costs of overlapping two demanding life history stages will further our understanding of the selection pressures that shape the temporal regulation of life history events in vertebrates. We studied free-living tropical Slaty brush-finches (Atlapetes schistaceus), in which individuals spontaneously overlap reproduction and moult or undergo both events in separation. To assess possible costs of such an overlap we quantified feather quality and flight performance of individuals in different states. We determined individual's life history state by measuring gonad size and scoring moult stage, and collected a newly grown 7(th) primary wing feather for later analysis of feather quality. Finally, we quantified flight performance for each individual in the wild. Overlapping individuals produced lighter and shorter wing feathers than individuals just moulting, with females decreasing feather quality more strongly during the overlap than males. Moreover, overlapping individuals had a reduced flight speed during escape flights, while their foraging flight speed was unaffected. Despite overlappers being larger and having a smaller wing area, their lower body mass resulted in a similar wing load as in breeders or moulters. Individuals measured repeatedly in different states also showed significant decreases in feather quality and escape flight speed during the overlap. Reduced escape flight speed may represent a major consequence of the overlap by increasing predation risk. Our data document costs to undergoing two life history stages simultaneously, which likely arise from energetic trade-offs. Impairments in individual quality and performance may represent important factors that select for temporal separation of life history stages in other species.

The failure of animals to fit all life-cycle stages into an annual cycle could reduce the chances of successful breeding. In some cases, non-optimal strategies will be adopted in order to maintain the life-cycle within the scope of one year. We studied trade-offs made by a High Arctic migrant shorebird, the red knot Calidris canutus islandica, between reproduction and wing feather molt carried out in the non-breeding period in the Dutch Wadden Sea. We compared primary molt duration between birds undertaking the full migratory and breeding schedule with birds that forego breeding because they are young or are maintained in captivity. Molt duration was ca. 71 days in breeding adults, which was achieved by an accelerated feather replacement strategy. Second-year birds and captive adults took ca. 22% and 27% longer, respectively. Second-year birds start molt in late June, more than four weeks before captive adults, and almost seven weeks before adults that return from breeding in late July-August. Adults finish molt in October when steeply increasing thermostatic costs and reductions in food availability occur. Primary molt duration was longer in female than in male knots (all ages), which was accordance with the somewhat larger body size of females. Since fast growth leads to lower quality feathers, the speedy wing molt shown by Arctic-breeding birds may represent a time constraint that is an unavoidable and routine cost of reproduction. So far it was hypothesized that only birds over 1 kg would have difficulty fitting molt within a year. Here we show that in birds an order of magnitude smaller, temporal imperatives may impose the adoption of non-optimal life-cycle routines in the entire actively breeding population.

The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal 'stiff-jointed' gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species.

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

Background: The trade-off between current and residual reproductive values is central to life history theory, although the possible mechanisms underlying this trade-off are largely unknown. The 'molt constraint' hypothesis suggests that molt and plumage functionality are compromised by the preceding breeding event, yet this candidate mechanism remains insufficiently explored., Methodology/principal Findings: The seasonal change in photoperiod was manipulated to accelerate the molt rate. This treatment simulates the case of naturally late-breeding birds. House sparrows Passer domesticus experiencing accelerated molt developed shorter flight feathers with more fault bars and body feathers with supposedly lower insulation capacity (i.e. shorter, smaller, with a higher barbule density and fewer plumulaceous barbs). However, the wing, tail and primary feather lengths were shorter in fast-molting birds if they had an inferior body condition, which has been largely overlooked in previous studies. The rachis width of flight feathers was not affected by the treatment, but it was still condition-dependent., Conclusions/significance: This study shows that sedentary birds might face evolutionary costs because of the molt rate-feather quality conflict. This is the first study to experimentally demonstrate that (1) molt rate affects several aspects of body feathers as well as flight feathers and (2) the costly effects of rapid molt are condition-specific. We conclude that molt rate and its association with feather quality might be a major mediator of life history trade-offs. Our findings also suggest a novel advantage of early breeding, i.e. the facilitation of slower molt and the condition-dependent regulation of feather growth.

Explanations for the hypoallometric scaling of metabolic rate through ontogeny generally fall into two categories: supply-side constraints on delivery of oxygen, or decreased mass-specific intrinsic demand for oxygen. In many animals, supply and demand increase together as the body grows, thus making it impossible to tease apart the relative contributions of changing supply and demand to the observed scaling of metabolic rate. In larval insects, the large components of the tracheal system are set in size at each molt, but then remain constant in size until the next molt. Larvae of Manduca sexta increase up to ten-fold in mass between molts, leading to increased oxygen need without a concomitant increase in supply. At the molt, the tracheal system is shed and replaced with a new, larger one. Due to this discontinuous growth of the tracheal system, insect larvae present an ideal system in which to examine the relative contributions of supply and demand of oxygen to the ontogenetic scaling of metabolic rate. We observed that the metabolic rate at the beginning of successive instars scales hypoallometrically. This decrease in specific intrinsic demand could be due to a decrease in the proportion of highly metabolically active tissues (the midgut) or to a decrease in mitochondrial activity in individual cells. We found that decreased intrinsic demand, mediated by a decrease in the proportion of highly metabolically active tissues in the fifth instar, along with a decrease in the specific mitochondrial activity, contribute to the hypoallometric scaling of metabolic rate.

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.

Food limitation greatly affects bird breeding performance, but the effect of nutritive stress on molt has barely been investigated outside of laboratory settings. Here we show changes in molting patterns for an entire native Hawaiian bird community at 1650-1900 m elevation on the Island of Hawaii between 1989-1999 and 2000-2006, associated with severe food limitation throughout the year beginning in 2000. Young birds and adults of all species took longer to complete their molt, including months never or rarely used during the 1989-1999 decade. These included the cold winter months and even the early months of the following breeding season. In addition, more adults of most species initiated their molt one to two months earlier, during the breeding season. Suspended molt, indicated by birds temporarily not molting primary flight feathers during the months of peak primary molt, increased in prevalence. Food limitation reached the point where individuals of all species had asymmetric molt, with different primary flight feathers molted on each wing. These multiple changes in molt, unprecedented in birds, had survival consequences. Adult birds captured during January to March, 2000-2004, had lower survival in four of five species with little effect of extended molt. Extended molt may be adaptive for a nutrient stressed bird to survive warm temperatures but not cool winter temperatures that may obliterate the energy savings. The changing molt of Hawaiian birds has many implications for conservation and for understanding life history aspects of molt of tropical birds.

It is often assumed that larval food stress reduces lifetime fitness regardless of the conditions subsequently faced by adults. However, according to the environment-matching hypothesis, a plastic developmental response to poor nutrition results in an adult phenotype that is better adapted to restricted food conditions than one having developed in high food conditions. Such a strategy might evolve when current conditions are a reliable predictor of future conditions. To test this hypothesis, we assessed the effects of larval food conditions (low, improving and high food) on reproductive fitness in both low and high food adults environments. Contrary to this hypothesis, we found no evidence that food restriction in larval ladybird beetles produced adults that were better suited to continuing food stress. In fact, reproductive rate was invariably lower in females that were reared at low food, regardless of whether adults were well fed or food stressed. Juveniles that encountered improving conditions during the larval stage compensated for delayed growth by accelerating subsequent growth, and thus showed no evidence of a reduced reproductive rate. However, these same individuals lost more mass during the period of starvation in adults, which indicates that accelerated growth results in an increased risk of starvation during subsequent periods of food stress.

The majority of bird species studied to date have molt schedules that are not concurrent with other energy demanding life history stages, an outcome assumed to arise from energetic trade-offs. Empirical studies reveal that molt is one of the most energetically demanding and perplexingly inefficient growth processes measured. Furthermore, small birds, which have the highest mass-specific basal metabolic rates (BMR(m)), have the highest costs of molt per gram of feathers produced. However, many small passerines, including white-plumed honeyeaters (WPHE; Lichenostomus penicillatus), breed in response to resource availability at any time of year, and do so without interrupting their annual molt. We examined the energetic cost of molt in WPHE by quantifying weekly changes in minimum resting metabolic rate (RMR(min)) during a natural-molt period in 7 wild-caught birds. We also measured the energetic cost of feather replacement in a second group of WPHEs that we forced to replace an additional 25% of their plumage at the start of their natural molt period. Energy expenditure during natural molt revealed an energy conversion efficiency of just 6.9% (±0.57) close to values reported for similar-sized birds from more predictable north-temperate environments. Maximum increases in RMR(min) during the molt of WPHE, at 82% (±5.59) above individual pre-molt levels, were some of the highest yet reported. Yet RMR(min) maxima during molt were not coincident with the peak period of feather replacement in naturally molting or plucked birds. Given the tight relationship between molt efficiency and mass-specific metabolic rate in all species studied to date, regardless of life-history pattern (Efficiency (%)  = 35.720 x 10(-0.494BMRm); r² = 0.944; p  =  or < 0.0001), there appears to be concomitant physiological costs entrained in the molt period that is not directly due to feather replacement. Despite these high total expenditures, the protracted molt period of WPHE significantly reduces these added costs on a daily basis.

Background: Litopenaeus Vannamei, well known as pacific white shrimp, is the most popular shrimp in the world shrimp market. Identification and characterization of shrimp muscle regulatory genes are not only important for shrimp genetic improvement, but also facilitate comparative genomic tools for understanding of muscle development and regeneration., Methodology/principal Findings: A novel mRNA encoding for a putative DNA-binding protein LvDBP23 was identified from Litopenaeus vannamei abdominal muscle cDNA library. The LvDBP23 cDNA contains 639 nucleotides of protein-coding sequence with deduced 212 amino acids of predicted molecular mass 23.32 kDa with glycine-rich domain at amino acid position 94-130. The mRNA sequence is successfully used for producing LvDBP23 recombinant protein in sf9 insect cell expression system. The expression of LvDBP23 mRNA is presented in abdominal muscle and swimming leg muscle, as well as other tissues including intestine, lymphoid and gill. The mRNA expression has the highest level in abdominal muscle in all tested tissues. LVDBP23 transcript during the molt cycle is highly expressed in the intermolt stage. In vitro nucleic acid-binding assays reveal that LvDBP23 protein can bind to both ssDNA and dsDNA, indicating its possible role of regulation of gene transcription., Conclusions/significance: We are the first to report a DNA-binding protein identified from the abdominal muscle tissue of marine shrimp L. Vannamei. Its high-level specific expression during the intermot stage suggests its role in the regulation of muscle buildup during the growth phase of shrimp molt cycle.

In this study we describe a distinctive pigment ring that appears in spider eyes after ecdysis and successively decreases in size in the days thereafter. Although pigment stops in spider eyes are well known, size variability is, to our knowledge, reported here for the first time. Representative species from three families (Ctenidae, Sparassidae and Lycosidae) are investigated and, for one of these species (Cupiennius salei, Ctenidae), the progressive increase in pupil diameter is monitored. In this species the pupil occupies only a fourth of the total projected lens surface after ecdysis and reaches its final size after approximately ten days. MicroCT images suggest that the decrease of the pigment ring is linked to the growth of the corneal lens after ecdysis. The pigment rings might improve vision in the immature eye by shielding light rays that would otherwise enter the eye via peripheral regions of the cornea, beside the growing crystalline lens.

Background: Many animals display colorful signals in their integument which convey information about the quality of their bearer. Theoretically, these ornaments incur differential production and/or maintenance costs that enforce their honesty. However, the proximate mechanisms of production costs are poorly understood and contentious in cases of non-carotenoid-based plumage ornaments like the melanin-based badge and depigmented white wing-bar in house sparrows Passer domesticus. Costly life-history events are adaptively separated in time, thus, when reproduction is extended, the time available for molt is curtailed and, in turn, molt rate is accelerated., Methodology/principal Findings: We experimentally accelerated the molt rate by shortening the photoperiod in order to test whether this environmental constraint is mirrored in the expression of plumage ornaments. Sparrows which had undergone an accelerated molt developed smaller badges and less bright wing-bars compared to conspecifics that molted at a natural rate being held at natural-like photoperiod. There was no difference in the brightness of the badge or the size of the wing-bar., Conclusions/significance: These results indicate that the time available for molt and thus the rate at which molt occurs may constrain the expression of melanin-based and depigmented plumage advertisements. This mechanism may lead to the evolution of honest signaling if the onset of molt is condition-dependent through the timing of and/or trade-off between breeding and molt.

The functional life span of feathers is always much less than the potential life span of birds, so feathers must be renewed regularly. But feather renewal entails important energetic, time and performance costs that must be integrated into the annual cycle. Across species the time required to replace flight feather increases disproportionately with body size, resulting in complex, multiple waves of feather replacement in the primaries of many large birds. We describe the rules of flight feather replacement for Hemiprocne mystacea, a small, 60 g tree swift from the New Guinea region. This species breeds and molts in all months of the year, and flight feather molt occurs during breeding in some individuals. H. mystacea is one to be the smallest species for which stepwise replacement of the primaries and secondaries has been documented; yet, primary replacement is extremely slow in this aerial forager, requiring more than 300 days if molt is not interrupted. We used growth bands to show that primaries grow at an average rate of 2.86 mm/d. The 10 primaries are a single molt series, while the 11 secondaries and five rectrices are each broken into two molt series. In large birds stepwise replacement of the primaries serves to increase the rate of primary replacement while minimizing gaps in the wing. But stepwise replacement of the wing quills in H. mystacea proceeds so slowly that it may be a consequence of the ontogeny of stepwise molting, rather than an adaptation, because the average number of growing primaries is probably lower than 1.14 feathers per wing.