Optimization and establishment of laboratory rearing conditions for Cimex lectularius L. against variable temperature and relative humidity.

Emerging infestations of bed bugs are affecting normal human lifestyle globally. This study has been designed to optimize the rearing conditions for Cimex lectularius L. (Hemiptera), to support the scientific research on them. Bed bugs have been projected onto three different temperature (20 °C, 25 °C, and 30 °C) and relative humidity (50%, 70%, and 90%) conditions to check their overall growth and survival rate. Adult mortality, weight loss, egg laying, percentage hatching, hatching initiation and completion, nymph mortality, and molting have been evaluated to optimize the best conditions. The temperature at 25 °C with 90% RH showed minimum mortality for adults (female 13.33 ± 3.33% and male 6.67 ± 3.33%) and nymphs (13.33 ± 3.33%), while maximum egg laying (40.33 ± 1.86), with highest percentage hatching (98.23 ± 0.58%). At 30 °C with 90% RH, hatching initiation and completion (5.19 ± 0.12 days and 7.23 ± 0.16 days) as well as molting initiation and completion (3.73 ± 0.12 days and 7.00 ± 0.24 days) were found to be fastest. Thus, it can be concluded that 25 °C with 90% RH is ideal for rearing of adults and 30 °C with 90% RH is appropriate for rapid growth of nymphs.

Differential fluorescence features and recovery speeds of different scorpion exoskeleton parts during the molting process.

Scorpion fluorescence under ultraviolet light is a well-known phenomenon, but its features under excitation in the UVA, UVB and UVC bands have not been characterized. Systematic fluorescence characterization revealed indistinguishable fluorescence spectra with a peak wavelength of 475 nm for whole exuviae from second-, third- and fifth-instar scorpions under different ultraviolet light ranges. In-depth investigations of the chelae, mesosoma, metasoma and telson of adult scorpions further indicated heterogeneity in the typical fluorescence spectrum within the visible light range and in the newly reported fluorescence spectrum with a peak wavelength of 320 nm within the ultraviolet light range, which both showed excitation wavelength-independent features. Dynamic fluorescence changes during the molting process of third-instar scorpions revealed the fluorescence heterogeneity-dependent recovery speed of scorpion exoskeletons. The typical fluorescence spectra of the molted chelae and telson rapidly recovered approximately 6 h after ecdysis under UVA light and approximately 36 h after ecdysis under UVB and UVC light. However, it took approximately 12 h and 24 h to obtain the typical fluorescence spectra of the molted metasoma and mesosoma, respectively, under UVA irradiation and 72 h to obtain the typical fluorescence spectra under UVB and UVC irradiation. The fluorescence heterogeneity-dependent fluorescence recovery of the scorpion exoskeleton was further confirmed by tissue section analysis of different segments from molting third-instar scorpions. These findings reveal novel scorpion fluorescence features and provide potential clues on the biological function of scorpion fluorescence.

Inter-organ steroid hormone signaling promotes myoblast fusion via direct transcriptional regulation of a single key effector gene.

Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a single non-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as "ecdysone") secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of ecdysone receptor (EcR) and ultraspiracle (USP) via ecdysone-response elements but also more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific TF Twist synergistically activate ants expression in vitro and in vivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.

Snow cover-related camouflage mismatch increases detection by predators.

Camouflage expressed by animals is an adaptation to local environments that certain animals express to maximize survival and fitness. Animals at higher latitudes change their coat color according to a seasonally changing environment, expressing a white coat in winter and a darker coat in summer. The timing of molting is tightly linked to the appearance and disappearance of snow and is mainly regulated by photoperiod. However, due to climate change, an increasing mismatch is observed between the coat color of these species and their environment. Here, we conducted an experiment in northern Sweden, with white and brown decoys to study how camouflage (mis)-match influenced (1) predator attraction to decoys, and (2) predation events. Using camera trap data, we showed that mismatching decoys attracted more predators and experienced a higher likelihood of predation events in comparison to matching decoys, suggesting that camouflage mismatched animals experience increased detection by predators. These results provide insight into the function of a seasonal color coat and the need for this adaptation to maximize fitness in an environment that is exposed to high seasonality. Thus, our results suggest that, with increasing climate change and reduced snow cover, animals expressing a seasonal color coat will experience a decrease in survival.

Reduction in metabolic noise reveals rejuvenation following transient severe caloric restriction.

Among land vertebrates, the laying hen stands out due to its great reproductive efficiency: producing an egg daily all year long. This production rate makes the laying hen a special model animal to study the general process of reproduction and aging. One unique aspect of hens is their ability to undergo reproductive plasticity and to rejuvenate their reproductive tract during molting, a standard industrial feed restriction protocol for transiently pausing reproduction, followed by improved laying efficiency almost to peak production. Here we use longitudinal metabolomics, immunology, and physiological assays to show that molting promotes reproduction, compresses morbidity, and restores youthfulness when applied to old hens. We identified circulating metabolic biomarkers that quantitatively predict the reproduction and age of individuals. Lastly, we introduce metabolic noise, a robust, unitless, and quantifiable measure for heterogeneity of the complete metabolome as a general marker that can indicate the rate of aging of a population. Indeed, metabolic noise increased with age in control hens, whereas molted hens exhibited reduced noise following molting, indicating systemic rejuvenation. Our results suggest that metabolic noise can be used as a quick and universal proxy for assessing successful aging treatments, accelerating the timeline for drug development.

Adenosine Monophosphate-Activated Protein Kinase (AMPK) Phosphorylation Is Required for 20-Hydroxyecdysone Regulates Ecdysis in Apolygus lucorum.

The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.

Application of Natural Bioresources to Sustainable Agriculture: A C-Glycoside Insecticide Based on N-Acetyl-glucosamine for Regulating Insect Molting of Ostrinia furnacalis.

In order to increase the application of natural bioresources in drug discovery and development, a study on N-acetyl-glucosamine (GlcNAc) derivatives of chitin as green pesticides was necessary. In this study, we designed and synthesized a series of novel C-glycoside naphthalimides using GlcNAc as a starting material. Compound 10l showed high inhibitory activity against OfHex1 (IC50 = 1.77 µM), with a nearly 30-fold increase in activity over our previously reported C-glycoside CAUZL-A (IC50 = 47.47 µM). By observing the morphology of the Ostrinia furnacalis, we found that the synthesized compounds significantly inhibited the molting process. In addition, we further explored the morphological changes of the inhibitor-treated O. furnacalis cuticle using scanning electron microscopy. This is the first study to validate the insecticidal mechanism of OfHex1 inhibitors at the microscale level. Several compounds also exhibited excellent larvicidal activity against Plutella xylostella. Moreover, the toxicity measurements and predictions indicated that the C-glycoside naphthalimides have little effect on the natural enemy Trichogramma ostriniae and rats. Together, our results highlight an approach for the design of green pesticides, taking advantage of natural bioresources to control pests in agriculture.

Reproductive status of a female white cockatoo (Cacatua alba) based on relationships among urofecal steroid hormone dynamics, molting, and body weight.

The detailed reproductive physiology of cockatoos based on gonadal hormone dynamics is unclear. In this study, we aimed to investigate ovarian activity by monitoring urofecal sex steroid hormone profiles in a captive female white cockatoo (Cacatua alba) and to noninvasively reveal basic reproductive physiology by comparing the hormone profiles with the laying dates, body mass changes, and molt progress. Urofeces were collected regularly for approximately 4 years from one female that frequently laid unfertilized eggs under single-rearing conditions. Urofecal progesterone (P4) and estradiol-17ß (E2) were measured by enzyme immunoassay. In addition, body mass and the number of fallen feathers were measured periodically. The urofecal P4 concentration peaked at an average of 17.7 days after the start of the rise in urofecal E2 concentration, and egg laying began on the day after the peak urofecal P4 concentration. The clutch size was usually two eggs, with an average interval of 4.5 days between eggs in each egg-laying cycle. There was a significant correlation between the dynamics of E2 concentration in urofeces and body mass. The results strongly suggest that E2 and P4 reflect the follicle growth and ovulation status, respectively, and that noninvasive monitoring of hormone dynamics using urofeces can accurately capture ovarian activity in the white cockatoo. Furthermore, changes in body mass can predict follicular growth, and reproduction and molt are antagonistic.

Molecular characterization of TRPA1 and its function in temperature preference in Eriocheir sinensis.

Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species, and its growth and development are regulated by temperature, but the molecular mechanisms of the responses to temperature remain unclear. Herein, we identified TRPA1 from E. sinensis, a member of the TRP family of heat receptor potential channels, performed RACE cloning and bioinformatics analysis, and investigated the effect of TRPA1 on temperature responses and molting by real-time PCR and RNA interference (RNAi). The open reading frame of Es-TRPA1 is 3660 bp, and the encoded protein has a molecular weight of 136.91 kDa, and is expressed in embryos and juveniles. RNAi-mediated silencing decreased Es-TRPA1 expression in juvenile crabs, molting rate was decreased, mortality was increased, and crabs avoided cold areas (4 °C) much less than control juvenile crabs. The results suggest that Es-TRPA1 is involved in regulating temperature adaptation and molting processes in E. sinensis. The findings lay a foundation for further exploration of temperature regulation mechanisms in E. sinensis and other crustaceans.

What is the optimal fertigation start time and frequency in lettuce seedlings?

Although lettuce is one of the most important vegetable crops cultivated in Brazil, producers conduct seedling production empirically, as there are no published reports on the optimal start time and management strategy for seedling fertigation. The present aimed to assess the influence of fertigation management on the growth, physiological aspects and nutritional status of lettuce seedlings and to determine the optimal fertigation start time and frequency. Two experiments were conducted, each with a randomized block design and six repetitions. The first consisted of six treatments, namely six fertigation start times at 0, 3, 6, 9, 12, and 15 d after emergence (DAE), and the second consisted of five treatments, representing different application frequencies at 3, 4, 5, 6, and 7 d intervals. The assessment of nutrient accumulation levels and biometric and physiological characteristics of the seedlings were performed after transplanting. Fertigation start times significantly affected 14 of the 18 variables assessed, particularly the number of leaves, shoot dry weight, leaf area, initial chlorophyll fluorescence, and P, K, Ca, Mg, and S accumulation. The best results for ten variables were obtained when fertigation began at emergence, with values 17.77 - 35.63% higher than those at fertigation onset at 15 DAE. Application frequency only influenced chlorophyll content and N, P, K, and S accumulation, with optimal results obtained at 3 - 6 d intervals. Beginning fertigation at plant emergence favors dry weight production, nutrition and photosynthesis and shortens the production time of lettuce seedlings. The optimal start time for lettuce seedling fertigation is at emergence, with application performed every 6 d.

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.

Proteomic analysis of pharate pupal molting fluid from the tobacco hornworm, Manduca sexta.

The insect cuticle is a key component of their success, being important for protection, communication, locomotion, and support. Conversely, as an exoskeleton, it also limits the size of the insect and must be periodically molted and a new one synthesized, to permit growth. To achieve this, the insect secretes a solution of chitinases, proteases and other proteins, known collectively as molting fluid, during each molting process to break down and recycle components of the old cuticle. Previous research has focused on the degradative enzymes in molting fluid and offered some characterization of their biochemical properties. However, identification of the specific proteins involved remained to be determined. We have used 2D SDS-PAGE and LC/MS-based proteomic analysis to identify proteins in the molting fluid of the tobacco hornworm, Manduca sexta, undergoing the larval to pupal molt. We categorized these proteins based on their proposed functions including chitin metabolism, proteases, peptidases, and immunity. This analysis complements previous reported work on M. sexta molting fluid and identifies candidate genes for enzymes involved in cuticle remodeling. Proteins classified as having an immune function highlight potential for molting fluid to act as an immune barrier to prevent infections during the cuticle degradation and ecdysis processes. Several proteins known to function in melanin synthesis as an immune response in hemolymph were present in molting fluid. We demonstrated that the bacterium Micrococcus luteus and the entomopathogenic fungus Beauveria bassiana can stimulate activation of phenoloxidase in molting fluid, indicating that the recognition proteins, protease cascade, and prophenoloxidase needed for melanin synthesis are present as a defense against infection during cuticle degradation. This analysis offers insights for proteins that may be important not only for molting in M. sexta but for insects in general.

Bacterial ectosymbionts in cuticular organs chemically protect a beetle during molting stages.

In invertebrates, the cuticle is the first and major protective barrier against predators and pathogen infections. While immune responses and behavioral defenses are also known to be important for insect protection, the potential of cuticle-associated microbial symbionts to aid in preventing pathogen entry during molting and throughout larval development remains unexplored. Here, we show that bacterial symbionts of the beetle Lagria villosa inhabit unusual dorsal invaginations of the insect cuticle, which remain open to the outer surface and persist throughout larval development. This specialized location enables the release of several symbiont cells and the associated protective compounds during molting. This facilitates ectosymbiont maintenance and extended defense during larval development against antagonistic fungi. One Burkholderia strain, which produces the antifungal compound lagriamide, dominates the community across all life stages, and removal of the community significantly impairs the survival probability of young larvae when exposed to different pathogenic fungi. We localize both the dominant bacterial strain and lagriamide on the surface of eggs, larvae, pupae, and on the inner surface of the molted cuticle (exuvia), supporting extended protection. These results highlight adaptations for effective defense of immature insects by cuticle-associated ectosymbionts, a potentially key advantage for a ground-dwelling insect when confronting pathogenic microbes.

The role of neuropeptides in regulating ecdysis and reproduction in the hemimetabolous insect Rhodnius prolixus.

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

Deviations from temporal scaling support a stage-specific regulation for C. elegans postembryonic development.

BACKGROUND: After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. RESULTS: We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. CONCLUSIONS: Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

Practices and issues of moulting programs for laying hens: a review.

1. Moulting is a natural physiological process in birds when they shed their old feathers and replace them with new ones, and it is followed by reproductive quiescence resulting in reduced egg production. Different birds undergo moulting at different points in their life. Some birds have seasonal moulting while some moult at the end of their breeding cycle. This review will mainly focus on moulting practices associated with commercial layer birds because, in all other bird types, this is not managed.2. Commercial farms commonly analyse the cost-benefit ratio to decide the time and method to adopt for moulting. Commercial layer farms adopt different practices to force birds out of moult and restart the production cycle. Animal welfare groups consider this as stressful and against animal welfare, raising questions about the ethics of this practice.3. Many studies have been conducted using complete or partial feed withdrawal and non-feed withdrawal programs to measure their effectiveness in maintaining animal welfare, economy, and post-moult performance in mind.4. Animal welfare should not be compromised during moulting. The United States Egg Producers and other such groups from the United Kingdom and Europe have decided to sell eggs produced only through a non-feed withdrawal moulting programs.

Crustacean cardioactive peptide and its receptor modulate the ecdysis behavior in the pea aphid, Acyrthosiphon pisum.

Insects must undergo ecdysis for successful development and growth, in which crustacean cardioactive peptide (CCAP) is a master hormone. However, the function of CCAP signaling in pea aphid, Acyrthosiphon pisum, remains unclear. In this study, we determined the sequence of the CCAP precursor and its receptor in A. pisum. We identified the functional receptor ApCCAPR, and then expressed this receptor in Chinese hamster ovary (CHO) cells, which in consequence exhibited high sensitivity to the ApCCAP mature peptide. The ApCCAP transcript was detected in the central nervous system of A. pisum. Neurons containing CCAP were also identified by immunohistochemical staining against insect CCAP. RNAi silencing of ApCCAP or ApCCAP-R signals caused developmental failure during nymph-adult ecdysis. The dsRNA-treated fourth-instar nymphs could not shed their old cuticle and died. Taking these findings together, we conclude that ApCCAP, via the activation of ApCCAP-R, plays an essential role in regulating the process of nymph-adult ecdysis in A. pisum. Our results deepen our understanding of the regulation of early ecdysis in A. pisum.

Metabolic heat loss in southern elephant seals (Mirounga leonina) differs with stage of moult and between habitats.

The moult in southern elephant seals (Mirounga leonina) represents an especially energetically demanding period during which seals must maintain high skin temperature to facilitate complete replacement of body fur and upper dermis. In this study, heat flux from the body surface was measured on 18 moulting southern elephant seals to estimate metabolic heat loss in three different habitats (beach, wallow and vegetation). Temperature data loggers were also deployed on 10 southern elephant seals to monitor skin surface temperature. On average, heat loss of animals on the beach was greater than in wallows or vegetation, and greater in wallows than in vegetation. Heat loss across all habitats during the moult equated to 1.8 x resting metabolic rate (RMR). The greatest heat loss of animals was recorded in the beach habitat during the late moult, that represented 2.3 x RMR. Mass loss was 3.6 ± 0.3 kg day-1, resulting in changes in body condition as the moult progressed. As body condition declined, skin surface temperature also decreased, suggesting that as animals approached the end of the moult blood flow to the skin surface was no longer required for hair growth.

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior.

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.