Diet-induced developmental acceleration independent of TOR and insulin in C. elegans.

Dietary composition has major effects on physiology. Here, we show that developmental rate, reproduction, and lifespan are altered in C. elegans fed Comamonas DA1877 relative to those fed a standard E. coli OP50 diet. We identify a set of genes that change in expression in response to this diet and use the promoter of one of these (acdh-1) as a dietary sensor. Remarkably, the effects on transcription and development occur even when Comamonas DA1877 is diluted with another diet, suggesting that Comamonas DA1877 generates a signal that is sensed by the nematode. Surprisingly, the developmental effect is independent from TOR and insulin signaling. Rather, Comamonas DA1877 affects cyclic gene expression during molting, likely through the nuclear hormone receptor NHR-23. Altogether, our findings indicate that different bacteria elicit various responses via distinct mechanisms, which has implications for diseases such as obesity and the interactions between the human microbiome and intestinal cells.

The pupal moulting fluid has evolved social functions in ants.

Insect societies are tightly integrated, complex biological systems in which group-level properties arise from the interactions between individuals1-4. However, these interactions have not been studied systematically and therefore remain incompletely known. Here, using a reverse engineering approach, we reveal that unlike solitary insects, ant pupae extrude a secretion derived from the moulting fluid that is rich in nutrients, hormones and neuroactive substances. This secretion elicits parental care behaviour and is rapidly removed and consumed by the adults. This behaviour is crucial for pupal survival; if the secretion is not removed, pupae develop fungal infections and die. Analogous to mammalian milk, the secretion is also an important source of early larval nutrition, and young larvae exhibit stunted growth and decreased survival without access to the fluid. We show that this derived social function of the moulting fluid generalizes across the ants. This secretion thus forms the basis of a central and hitherto overlooked interaction network in ant societies, and constitutes a rare example of how a conserved developmental process can be co-opted to provide the mechanistic basis of social interactions. These results implicate moulting fluids in having a major role in the evolution of ant eusociality.

C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1.

C. elegans develops through four larval stages that are rhythmically terminated by molts, that is, the synthesis and shedding of a cuticular exoskeleton. Each larval cycle involves rhythmic accumulation of thousands of transcripts, which we show here relies on rhythmic transcription. To uncover the responsible gene regulatory networks (GRNs), we screened for transcription factors that promote progression through the larval stages and identified GRH-1, BLMP-1, NHR-23, NHR-25, MYRF-1, and BED-3. We further characterize GRH-1, a Grainyhead/LSF transcription factor, whose orthologues in other animals are key epithelial cell-fate regulators. We find that GRH-1 depletion extends molt durations, impairs cuticle integrity and shedding, and causes larval death. GRH-1 is required for, and accumulates prior to, each molt, and preferentially binds to the promoters of genes expressed during this time window. Binding to the promoters of additional genes identified in our screen furthermore suggests that we have identified components of a core molting-clock GRN. Since the mammalian orthologues of GRH-1, BLMP-1 and NHR-23, have been implicated in rhythmic homeostatic skin regeneration in mouse, the mechanisms underlying rhythmic C. elegans molting may apply beyond nematodes.

Sea ice concentration decline in an important Adélie penguin molt area.

Unlike in many polar regions, the spatial extent and duration of the sea ice season have increased in the Ross Sea sector of the Southern Ocean during the satellite era. Simultaneously, populations of Adélie penguins, a sea ice obligate, have been stable or increasing in the region. Relationships between Adélie penguin population growth and sea ice concentration (SIC) are complex, with sea ice driving different, sometimes contrasting, demographic patterns. Adélie penguins undergo a complete molt annually, replacing all their feathers while fasting shortly after the breeding season. Unlike most penguin species, a majority of Adélies are thought to molt on sea ice, away from the breeding colonies, which makes this period particularly difficult to study. Here, we evaluate the hypothesis that persistent areas of high SIC provide an important molting habitat for Adélie penguins. We analyzed data from geolocating dive recorders deployed year-round on 195 adult penguins at two colonies in the Ross Sea from 2017 to 2019. We identified molt by detecting extended gaps in postbreeding diving activity and used associated locations to define two key molting areas. Remotely sensed data indicated that SIC during molt was anomalously low during the study and has declined in the primary molt area since 1980. Further, annual return rates of penguins to breeding colonies were positively correlated with SIC in the molt areas over 20 y. Together these results suggest that sea ice conditions during Adélie penguin molt may represent a previously underappreciated annual bottleneck for adult survival.

The sea spider Pycnogonum litorale overturns the paradigm of the absence of axial regeneration in molting animals.

Regenerative abilities and their evolution in the different animal lineages have fascinated generations of biologists. While some taxa are capable of restoring entire individuals from small body fragments, others can regrow only specific structures or lack structural regeneration completely. In contrast to many other protostomes, including the segmented annelids, molting animals (Ecdysozoa) are commonly considered incapable of primary body axis regeneration, which has been hypothesized to be linked to the evolution of their protective cuticular exoskeleton. This holds also for the extraordinarily diverse, segmented arthropods. Contradicting this long-standing paradigm, we here show that immatures of the sea spider Pycnogonum litorale reestablish the posterior body pole after transverse amputation and can regrow almost complete segments and the terminal body region, including the hindgut, anus, and musculature. Depending on the amputation level, normal phenotypes or hypomeric six-legged forms develop. Remarkably, also the hypomeric animals regain reproductive functionality by ectopic formation of gonoducts and gonopores. The discovery of such complex regenerative patterns in an extant arthropod challenges the hitherto widely assumed evolutionary loss of axial regeneration during ecdysozoan evolution. Rather, the branching of sea spiders at the base of Chelicerata and their likely ancestral anamorphic development suggests that the arthropod stem species may have featured similar regenerative capabilities. Accordingly, our results provide an incentive for renewed comparative regeneration studies across ecdysozoans, with the aim to resolve whether this trait was potentially even inherited from the protostome ancestor.

Specification of the endocrine primordia controlling insect moulting and metamorphosis by the JAK/STAT signalling pathway.

The corpora allata and the prothoracic glands control moulting and metamorphosis in insects. These endocrine glands are specified in the maxillary and labial segments at positions homologous to those forming the trachea in more posterior segments. Glands and trachea can be homeotically transformed into each other suggesting that all three evolved from a metamerically repeated organ that diverged to form glands in the head and respiratory organs in the trunk. While much is known about tracheal specification, there is limited information about corpora allata and prothorathic gland specification. Here we show that the expression of a key regulator of early gland development, the snail gene, is controlled by the Dfd and Scr Hox genes and by the Hedgehog and Wnt signalling pathways that induce localised transcription of upd, the ligand of the JAK/STAT signalling pathway, which lies at the heart of gland specification. Our results show that the same upstream regulators are required for the early gland and tracheal primordia specification, reinforcing the hypothesis that they originated from a segmentally repeated organ present in an ancient arthropod.

An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting.

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a mediator of molting. Loss of adm-2, including mutations that disrupt the metalloprotease domain, led to the strong suppression of molting defects in partial loss-of-function nekl mutants. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of apical epidermal LRP-1, ADM-2 overexpression caused a reduction in LRP-1 levels. Consistent with this, several mammalian ADAMs, including the meltrin ADAM12, have been shown to regulate mammalian LRP1 via proteolysis. In contrast to mammalian homologs, however, the regulation of LRP-1 by ADM-2 does not appear to involve the metalloprotease function of ADM-2, nor is proteolytic processing of LRP-1 strongly affected in adm-2 mutants. Our findings suggest a noncanonical role for an ADAM family member in the regulation of a lipoprotein-like receptor and lead us to propose that endocytic trafficking may be important for both the internalization of factors that promote molting as well as the removal of proteins that can inhibit the process.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

Individual variation in life-history timing: synchronous presence, asynchronous events and phenological compensation in a wild mammal.

Many animals and plants have species-typical annual cycles, but individuals vary in their timing of life-history events. Individual variation in fur replacement (moult) timing is poorly understood in mammals due to the challenge of repeated observations and longitudinal sampling. We examined factors that influence variation in moult duration and timing among elephant seals (Mirounga angustirostris). We quantified the onset and progression of fur loss in 1178 individuals. We found that an exceptionally rapid visible moult (7 days, the shortest of any mammals or birds), and a wide range of moult start dates (spanning 6-10× the event duration) facilitated high asynchrony across individuals (only 20% of individuals in the population moulting at the same time). Some of the variation was due to reproductive state, as reproductively mature females that skipped a breeding season moulted a week earlier than reproductive females. Moreover, individual variation in timing and duration within age-sex categories far outweighed (76-80%) variation among age-sex categories. Individuals arriving at the end of the moult season spent 50% less time on the beach, which allowed them to catch up in their annual cycles and reduce population-level variance during breeding. These findings underscore the importance of individual variation in annual cycles.

Changes in body surface temperature reveal the thermal challenge associated with catastrophic moult in captive gentoo penguins.

Once a year, penguins undergo a catastrophic moult, replacing their entire plumage during a fasting period on land or on sea-ice during which time individuals can lose 45% of their body mass. In penguins, new feather synthesis precedes the loss of old feathers, leading to an accumulation of two feather layers (double coat) before the old plumage is shed. We hypothesized that the combination of the high metabolism required for new feather synthesis and the potentially high thermal insulation linked to the double coat could lead to a thermal challenge requiring additional peripheral circulation to thermal windows to dissipate the extra heat. To test this hypothesis, we measured the surface temperature of different body regions of captive gentoo penguins (Pygoscelis papua) throughout the moult under constant environmental conditions. The surface temperature of the main body trunk decreased during the initial stages of the moult, suggesting greater thermal insulation. In contrast, the periorbital region, a potential proxy of core temperature in birds, increased during these same early moulting stages. The surface temperature of the bill, flipper and foot (thermal windows) tended to initially increase during the moult, highlighting the likely need for extra heat dissipation in moulting penguins. These results raise questions regarding the thermoregulatory capacities of penguins in the wild during the challenging period of moulting on land in the current context of global warming.

Complexity in the timing of the first postnatal ecdysis in snakes.

Lepidosaurian reptiles, particularly snakes, periodically shed the outer epidermal layers of their skin (ecdysis) to restore or enhance vital functions such as regulating water and gaseous exchange, growth, and protection against insult, infection or physical injury. Although many studies have focused on the nature and mechanisms of skin shedding, little attention has been paid to the timing of the first ecdysis in neonates following birth or hatching. A recent study investigated patterns of the time to first postnatal ecdysis in snakes based on a large dataset taken from the literature. The analysis demonstrated patterns in the time to first postnatal ecdysis related to phylogeny as well as several life history traits. While this assessment provides important advances in our knowledge of this topic, data on known biophysical drivers of ecdysis - temperature and humidity - were largely unavailable and were not evaluated. The first postnatal ecdysis of neonatal snakes can be viewed as an adaptive adjustment to the transition from the aqueous environment of the embryo to the aerial environment of the newborn. Hence, the timing of the first postnatal ecdysis is logically influenced by the aerial environment into which a newborn snake or hatchling finds itself. Therefore, in this Commentary, we first emphasize the putative plasticity of ecdysis with respect to epidermal lipids that structure the water permeability barrier and are established or renewed during ecdysis to reduce transepidermal evaporative water loss. We then discuss the likely importance of biophysical variables as influential covariates that need future investigation as potential co-determinants of the timing of first postnatal ecdysis.

Effects of sublethal methylmercury and food stress on songbird energetic performance: metabolic rates, molt and feather quality.

Organisms regularly adjust their physiology and energy balance in response to predictable seasonal environmental changes. Stressors and contaminants have the potential to disrupt these critical seasonal transitions. No studies have investigated how simultaneous exposure to the ubiquitous toxin methylmercury (MeHg) and food stress affects birds' physiological performance across seasons. We quantified several aspects of energetic performance in song sparrows, Melospiza melodia, exposed or not to unpredictable food stress and MeHg in a 2×2 experimental design, over 3 months during the breeding season, followed by 3 months post-exposure. Birds exposed to food stress had reduced basal metabolic rate and non-significant higher factorial metabolic scope during the exposure period, and had a greater increase in lean mass throughout most of the experimental period. Birds exposed to MeHg had increased molt duration, and increased mass:length ratio of some of their primary feathers. Birds exposed to the combined food stress and MeHg treatment often had responses similar to the stress-only or MeHg-only exposure groups, suggesting these treatments affected physiological performance through different mechanisms and resulted in compensatory or independent effects. Because the MeHg and stress variables were selected in candidate models with a ΔAICc lower than 2 but the 95% confidence interval of these variables overlapped zero, we found weak support for MeHg effects on all measures except basal metabolic rate, and for food stress effects on maximum metabolic rate, factorial metabolic scope and feather mass:length ratio. This suggests that MeHg and food stress effects on these measures are statistically identified but not simple and/or were too weak to be detected via linear regression. Overall, combined exposure to ecologically relevant MeHg and unpredictable food stress during the breeding season does not appear to induce extra energetic costs for songbirds in the post-exposure period. However, MeHg effects on molt duration could carry over across multiple annual cycle stages.

Earliest evidence of avian primary feather moult.

Feather moulting is a crucial process in the avian life cycle, which evolved to maintain plumage functionality. However, moulting involves both energetic and functional costs. During moulting, plumage function temporarily decreases between the shedding of old feathers and the full growth of new ones. In flying taxa, a gradual and sequential replacement of flight feathers evolved to maintain aerodynamic capabilities during the moulting period. Little is known about the moult strategies of non-avian pennaraptoran dinosaurs and stem birds, before the emergence of crown lineage. Here, we report on two Early Cretaceous pygostylian birds from the Yixian Formation (125 mya), probably referable to Confuciusornithiformes, exhibiting morphological characteristics that suggest a gradual and sequential moult of wing flight feathers. Short primary feathers interpreted as immature are symmetrically present on both wings, as is typical among extant flying birds. Our survey of the enormous collection of the Tianyu Museum confirms previous findings that evidence of active moult in non-neornithine pennaraptorans is rare and likely indicates a moult cycle greater than one year. Documenting moult in Mesozoic feathered dinosaurs is critical for understanding their ecology, locomotor ability and the evolution of this important life-history process in birds.

Evidence for cryptic molting behavior in the trilobite Toxochasmops vormsiensis from the Upper Ordovician Katian Kõrgessaare Formation, Estonia.

Documentation of cryptic trilobite behavior has presented important insights into the paleoecology of this fully extinct arthropod group. One such example is the preservation of trilobites inside the remains of larger animals. To date, evidence for trilobites within cephalopods, gastropods, hyoliths, and other trilobites has been presented. Importantly, most of these interactions show trilobite molts, suggesting that trilobites used larger animals for protection during molting. To expand the record of molted trilobites within cephalopods, we present a unique case of a Toxochasmops vormsiensis trilobite within the body chamber of a Gorbyoceras textumaraneum nautiloid from the Upper Ordovician Kõrgessaare Formation of Estonia. By examining this material, we present new insights into the ecology of pterygometopid trilobites, highlighting how these forms used large cephalopods as areas to successfully molt.

The association between haemosporidian infection and non-breeding moult location in great reed warblers revisited by combining feather stable isotope profiles and geolocator data.

Stable isotope analysis provides valuable insights into the ecology of long-distance migratory birds during periods spent away from a specific study site. In a previous study, Swedish great reed warblers (Acrocephalus arundinaceus) infected with haemosporidian parasites differed in feather isotope ratios compared to non-infected birds, suggesting that infected and non-infected birds spent the non-breeding season in different locations or habitats. Here, we use a novel dataset comprising geolocator data, isotopes, and haemosporidian infection status of 92 individuals from four Eurasian populations to investigate whether parasite transmission varies with geography or habitats. We found that the probability of harbouring Plasmodium and Leucocytozoon parasites was higher in birds moulting in the eastern region of the non-breeding grounds. However, no geographic pattern occurred for Haemoproteus infections or overall infection status. In contrast to the previous study, we did not find any relationship between feather isotope ratios and overall haemosporidian infection for the entire current dataset. Plasmodium-infected birds had lower feather δ15N values indicating that they occupied more mesic habitats. Leucocytozoon-infected birds had higher feather δ34S values suggesting more coastal sites or wetlands with anoxic sulphate reduction. As the composition and prevalence of haemosporidian parasites differed between the old and the current dataset, we suggest that the differences might be a consequence of temporal dynamics of haemosporidian parasites. Our results emphasize the importance of replicating studies conducted on a single population over a restricted time period, as the patterns can become more complex for data from wider geographical areas and different time periods.

Local age-dependent neuromodulation in Rhodnius prolixus antennae.

Kissing bugs do not respond to host cues when recently molted and only exhibit robust host-seeking several days after ecdysis. Behavioral plasticity has peripheral correlates in antennal gene expression changes through the week after ecdysis. The mechanisms regulating these peripheral changes are still unknown, but neuropeptide, G-protein coupled receptor, nuclear receptor, and takeout genes likely modulate peripheral sensory physiology. We evaluated their expression in antennal transcriptomes along the first week postecdysis of Rhodnius prolixus 5th instar larvae. Besides, we performed clustering and co-expression analyses to reveal relationships between neuromodulatory (NM) and sensory genes. Significant changes in transcript abundance were detected for 50 NM genes. We identified 73 sensory-related and NM genes that were assigned to nine clusters. According to their expression patterns, clusters were classified into four groups: two including genes up or downregulated immediately after ecdysis; and two with genes with expression altered at day 2. Several NM genes together with sensory genes belong to the first group, suggesting functional interactions. Co-expression network analysis revealed a set of genes that seem to connect with sensory system maturation. Significant expression changes in NM components were described in the antennae of R. prolixus after ecdysis, suggesting that a local NM system acts on antennal physiology. These changes may modify the sensitivity of kissing bugs to host cues during this maturation interval.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ).

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

Neochloris aquatica induces larval mortality, molting defects, and unstable flightless adults in the Asian tiger mosquito.

The Asian tiger mosquito, Aedes albopictus, is a highly invasive and aggressive species capable of transmitting a large number of etiological agents of medical and veterinary importance, posing a high risk for the transmission of emerging viruses between animals and humans. In this work, we evaluated the mosquitocidal activity of Neochloris aquatica against A. albopictus throughout its development and analyzed whether this effect was potentiated when the microalga was cultivated under stress conditions due to nutrient deprivation. Our results suggest that N. aquatica produces metabolites that have negative effects on these insects, including larval mortality, interruption of pupal development, and incomplete emergence of adults when fed on microalgae in the larval stages. When microalgae were cultured under stress conditions, an increase in molting defects was recorded, and the number of healthy adults emerged drastically decreased. Histological studies revealed severe signs of total disintegration of different tissues and organs in the thorax and abdomen regions. The muscles and fat bodies in the midgut and foregut were severely distorted. In particular, larval intestinal tissue damage included vacuolization of the cytoplasm, destruction of brush border microvilli, and dilation of the intercellular space, which are distinctive morphological characteristics of apoptotic cells. Evidence suggests that N. aquatica produces metabolites with mosquitocidal effects that affect development and, therefore, the ability to vector etiological agents of medical and veterinary importance.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis.

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera).

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.