Crustacean Cardioactive Peptide Research Articles

Modulation by Neuropeptides with Overlapping Targets Results in Functional Overlap in Oscillatory Circuit Activation.

Neuromodulation lends flexibility to neural circuit operation but the general notion that different neuromodulators sculpt neural circuit activity into distinct and characteristic patterns is complicated by interindividual variability. In addition, some neuromodulators converge onto the same signaling pathways, with similar effects on neurons and synapses. We compared the effects of three neuropeptides on the rhythmic pyloric circuit in the stomatogastric ganglion of male crabs, Cancer borealis Proctolin (PROC), crustacean cardioactive peptide (CCAP), and red pigment concentrating hormone (RPCH) activate the same modulatory inward current, I MI, and have convergent actions on synapses. However, while PROC targets all four neuron types in the core pyloric circuit, CCAP and RPCH target the same subset of only two neurons. After removal of spontaneous neuromodulator release, none of the neuropeptides restored the control cycle frequency, but all restored the relative timing between neuron types. Consequently, differences between neuropeptide effects were mainly found in the spiking activity of different neuron types. We performed statistical comparisons using the Euclidean distance in the multidimensional space of normalized output attributes to obtain a single measure of difference between modulatory states. Across preparations, the circuit output in PROC was distinguishable from CCAP and RPCH, but CCAP and RPCH were not distinguishable from each other. However, we argue that even between PROC and the other two neuropeptides, population data overlapped enough to prevent reliable identification of individual output patterns as characteristic for a specific neuropeptide. We confirmed this notion by showing that blind classifications by machine learning algorithms were only moderately successful.Significance Statement It is commonly assumed that distinct behaviors or circuit activities can be elicited by different neuromodulators. Yet it is unknown to what extent these characteristic actions remain distinct across individuals. We use a well-studied circuit model of neuromodulation to examine the effects of three neuropeptides, each known to produce a distinct activity pattern in controlled studies. We find that, when compared across individuals, the three peptides elicit activity patterns that are either statistically indistinguishable or show too much overlap to be labeled characteristic. We ascribe this to interindividual variability and overlapping subcellular actions of the modulators. Because both factors are common in all neural circuits, these findings have broad significance for understanding chemical neuromodulatory actions while considering interindividual variability.

Crustacean cardioactive peptide signaling system in the gastropod mollusk Pacific abalone

Crustacean cardioactive peptide (CCAP) signaling systems have been characterized in a diverse range of protostomes, representatively in arthropods. The cyclic CX5C-type CCAP regulates various biological activities through CCAP receptors (CCAPRs), which are orthologous to neuropeptide S receptors (NPSRs) in deuterostomes. However, the CCAPRs of the lophotrochozoa remain poorly characterized; therefore, the relationship between the CCAP, NPS, and CX4C-type oxytocin/vasopressin (OT/VP) signaling systems is unclear. In this study, we identified a CCAP precursor and two CCAPR isoforms in the Pacific abalone (Haliotis discus hannai; Hdh). The Hdh-CCAP precursor was found to harbor three CX5C-type and one CX4C-type CCAPs. The Hdh-CCAPRs displayed homology with protostome CCAPRs and deuterostome NPSRs, having characteristics of the rhodopsin-type G protein-coupled receptors. Phylogenetic analysis showed that lophotrochozoan CCAPRs, including Hdh-CCAPRs, form a monophyletic group distinct from arthropod CCAPRs. Reporter assays demonstrated that all examined Hdh-CCAPs and insect CCAP-induced intracellular Ca2+ mobilization and cAMP accumulation in Hdh-CCAPR-expressing HEK293 cells, whereas none of the CCAP peptides inhibited the forskolin-stimulated cAMP signaling pathway even at micromolar concentrations. In silico ligand-receptor docking models showed that the N-terminal FCN motifs of Hdh-CCAPs are deeply inserted inside the binding pocket of Hdh-CCAPR, forming extensive hydrophobic interactions. In mature Pacific abalone, the transcripts for Hdh-CCAP precursor and Hdh-CCAPR were highly expressed in the neural ganglia compared to the peripheral tissues. Collectively, this study characterized the first CCAP signaling system linked to both Ca2+/PKC and cAMP/PKA signal transduction pathways in gastropod mollusks and gives insights into the evolutional origins of deuterostomian NPS and OT/VP signaling systems.

Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion.

Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the interoceptive subesophageal zone neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. Here, we investigate the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, we find that the ISNs synapse with a novel cell-type bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPCs), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion.

Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion

Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the interoceptive subesophageal zone neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. Here, we investigate the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, we find that the ISNs synapse with a novel cell-type bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPCs), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion.

Cloning and deorphanization of three inotocin (insect oxytocin/vasopressin-like) receptors and their ligand from the tick Ixodes scapularis

Many insects produce the cyclic neuropeptide inotocin (CLITNCPRGamide), which is the insect orthologue of the mammalian neuropeptides oxytocin and vasopressin. These insects also have one inotocin G protein-coupled receptor (GPCR), which is the orthologue of the mammalian oxytocin and vasopressin receptors. The tick Ixodes scapularis belongs to the subphylum Chelicerata, an arthropod taxon different from insects, to which also spiders, scorpions, and mites belong. I. scapularis is an ectoparasite and a health risk for humans, because it transfers pathogenic microorganisms to its human host during a blood meal, thereby causing serious neurological diseases, among them Lyme disease and tick-borne encephalitis (TBE). By annotating the genomic sequence of I. scapularis, we previously found one presumed tick inotocin preprohormone gene and, in contrast to insects, three genes coding for presumed inotocin GPCRs. We now find that these GPCR genes cluster on one genomic contig, suggesting that they originated by recent gene duplications. Closely located on the same contig are also four adipokinetic hormone/corazonin-related peptide (ACP) GPCR genes, and one crustacean cardioactive peptide (CCAP) GPCR gene, suggesting evolutionary relationships. These evolutionary relationships are confirmed by phylogenetic tree analyses of their gene products. We also cloned the tick inotocin preprohormone, which has a structural organization closely resembling mammalian oxytocin and vasopressin preprohormones, including the presence of a conserved neurophysin sequence, having seven cystine bridges. This neurophysin sequence has two cystine-knot domains, but in contrast to mammalian neurophysins, the tick neurophysin contains a canonical prohormone convertase cleavage signal and a peptide C-terminal amidation sequence (GKR), suggesting cleavage into two biologically active cystine-knot peptides. This cleavage/amidation sequence occurs in neurophysins from most hard tick species, but not in other chelicerates. Mature tick inotocin is different from insect inotocin and has the sequence CFITNCPPGamide. Finally, we cloned and stably expressed the three tick inotocin receptors in Chinese Hamster Ovary cells and found that each of them was activated by nanomolar concentrations of tick inotocin (EC50 for ITR1 = 1.6 × 10−8 M; EC50 for ITR2 = 5.8 × 10−9 M; EC50 for ITR3 = 9.3 × 10−9 M), thereby establishing that they are genuine tick inotocin receptors.

Functional Analysis of a Multiple-Domain CTL15 in the Innate Immunity, Eclosion, and Reproduction of Tribolium castaneum.

C-type lectin X (CTL-X) plays critical roles in immune defense, cell adhesion, and developmental regulation. Here, a transmembrane CTL-X of Tribolium castaneum, TcCTL15, with multiple domains was characterized. It was highly expressed in the early and late pupae and early adults and was distributed in all examined tissues. In addition, its expression levels were significantly induced after being challenged with pathogen-associated molecular patterns (PAMPs) and bacteria. In vitro, the recombinant TcCTL15 could recognize bacteria through binding PAMPs and exhibit agglutinating activity against a narrow range of bacteria in the presence of Ca2+. RNAi-mediated TcCTL15-knockdown-larvae infected with Escherichia coli and Staphylococcus aureus showed less survival, had activated immune signaling pathways, and induced the expression of antimicrobial peptide genes. Moreover, silencing TcCTL15 caused eclosion defects by impairing ecdysone and crustacean cardioactive peptide receptors (CCAPRs). Suppression of TcCTL15 in female adults led to defects in ovary development and fecundity, accompanied by concomitant reductions in the mRNA levels of vitellogenin (TcVg) and farnesol dehydrogenase (TcFDH). These findings imply that TcCTL15 has extensive functions in developmental regulation and antibacterial immunity. Uncovering the function of TcCTL15 will enrich the understanding of CTL-X in invertebrates. Its multiple biological functions endow the potential to be an attractive target for pest control.

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.

SoxC is Required for Ecdysteroid Induction of Neuropeptide Genes During Insect Eclosion.

In insects, the shedding of the old exoskeleton is accomplished through ecdysis which is typically followed by the expansion and tanning of the new cuticle. Four neuropeptides, eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon (Bur) are known to control ecdysis. However, the regulation of these neuropeptide genes is still poorly understood. Here, we report that in the red flour beetle (RFB) Tribolium castaneum and the fall armyworm (FAW) Spodoptera frugiperda, knockdown or knockout of the SoxC gene caused eclosion defects. The expansion and tanning of wings were not complete. In both RFB and FAW, the knockdown or knockout of SoxC resulted in a decrease in the expression of EH gene. Electrophoretic mobility shift assays revealed that the SfSoxC protein directly binds to a motif present in the promoter of SfEH. The luciferase reporter assays in Sf9 cells confirmed these results. These data suggest that transcription factor SoxC plays a key role in ecdysteroid induction of genes coding for neuropeptides such as EH involved in the regulation of insect eclosion.

Early-Stage Defense Mechanism of the Cotton Aphid Aphis gossypii Against Infection With the Insect-Killing Fungus Beauveria bassiana JEF-544.

Aphis gossypii, commonly known as the cotton aphid, is a widely distributed pest of agricultural crops and acts as a vector for many serious plant viruses. Cotton aphid shows high resistance to chemical insecticides due to rapid rates of genetic diversity as a result of its short life cycle, seasonal migration, and host alteration. As an alternative, entomopathogenic fungi can be used to control cotton aphids in an environmentally sound manner. However, little is known about how cotton aphids respond to fungal infection. In this work, a new Beauveria bassiana strain JEF-544 (Bb JEF-544) was selected and isolated through bioassays with high virulence against cotton aphid. Early response of cotton aphid to Bb JEF-544 infection was analyzed at the transcriptome level. Infected aphids were collected two days after treatment at 25% lethal time (LT25), and total RNA of non-infected and Bb JEF-544-infected aphids was independently subjected to sequencing. Infected aphids showed significant up-regulation of the insect hormone biosynthesis pathway. Bursicon (Burs) and crustacean cardioactive peptide (CCAP) receptors involved in molting along with ecdysone synthesis were also strongly up-regulated in the aphid response to the fungal infection. In the immune response, melanization in the hemocoel was significantly up-regulated, while phagocytosis was less actively transcribed. In conclusion, cotton aphids protect themselves from Bb JEF-544 infection by activating the immune response including melanization and insect molting hormones to shed infected cuticles. In addition to describing the initial stages of Bb JEF-544 infection at the transcriptome level, this work provides potential treatment targets and insight into how fungal isolates can effectively be used to control this serious aphid species.

Modulatory capacity correlates with dietary diversity in three species of decapod crustaceans

The central nervous systems of decapod crustaceans contains numerous amines, amino acids, and peptides that modulate the central pattern generating (CPG) circuits found within its stomatogastric nervous system (STNS) and cardiac ganglion (CG); over 30 modulators have been identified in one species of crab (C. borealis). CPGs in the STNS are responsible for internally chewing (Gastric Mill CPG) and filtering food (Pyloric CPG) into the midgut, while the CG drives the heartbeat. Neuromodulation allows these CPGs to flexibly produce a variety of output patterns; however, the variety of modulators exceeds the variety of induced output, leading us to ask why so many modulators are present. Although modulatory capacity, a measure of the extent to which a neuronal circuit can be modulated, may be solely an inherited trait, we hypothesize a functional correlation: decapods that need more behavioral flexibility utilize a broader range of modulators than those with more stereotyped behaviors. We thus predicted that decapods with broad diets would have a larger modulatory capacity in the STNS, but not the CG, than those with limited diets. To test this, we compared responses of the STNS and CG CPGs in three species of majoid crabs (Pugettia producta, Libinia emarginata, and Chionoecetes opilio) to the same neuromodulators. L. emarginata and C. opilio both have broad diets, while P. producta primarily eats kelp. With one exception, each species’ CG exhibited similar responses to all modulators. The STNS of crabs with broader diets responded similarly to all modulators, whereas the P. producta STNS showed limited responses to the same set of neuromodulators. CG responses did not follow this trend, as all crabs showed varied CG response to modulation. Modulators: Cancer borealisTachykinin Related Peptide (CabTRP), Crustacean Cardioactive Peptide (CCAP), Red Pigment Concentrating Hormone (RPCH), Myosuppressin (Myo), Oxotremorine (Oxo), Dopamine (DA), Proctolin (Proc).

Nutrients and pheromones promote insulin release to inhibit courtship drive.

Food and reproduction are the fundamental needs for all animals. However, the neural mechanisms that orchestrate nutrient intake and sexual behaviors are not well understood. Here, we find that sugar feeding immediately suppresses sexual drive of male Drosophila, a regulation mediated by insulin that acts on insulin receptors on the courtship-promoting P1 neurons. The same pathway was co-opted by anaphrodisiac pheromones to suppress sexual hyperactivity to suboptimal mates. Activated by repulsive pheromones, male-specific PPK23 neurons on the leg tarsus release crustacean cardioactive peptide (CCAP) that acts on CCAP receptor on the insulin-producing cells in the brain to trigger insulin release, which then inhibits P1 neurons. Our results reveal how male flies avoid promiscuity by balancing the weight between aphrodisiac and anaphrodisiac inputs from multiple peripheral sensory pathways and nutritional states. Such a regulation enables male animals to make an appropriate mating decision under fluctuating feeding conditions.

Ecdysis Triggering Hormone, Eclosion Hormone, and Crustacean Cardioactive Peptide Play Essential but Different Roles in the Molting Process of Mud Crab, Scylla paramamosain

Molting behavior in insects is controlled by the ecdysis triggering hormone (ETH), eclosion hormone (EH) and the crustacean cardioactive peptide (CCAP). At present, the regulation of molting behavior in crustaceans remains unclear. Here, we studied the roles of ETH, EH, and CCAP in the molt regulation of the crab, Scylla paramamosain from their expression pattern and in vivo assays. The results showed that transcripts of ETH, EH, and CCAP were mainly localized in thoracic ganglia and fluctuated periodically with the molting cycle. When ETH or CCAP was knockdown at early premolt stage (D0), molting of crabs was interrupted and all animals died at late premolt stage (D2). While the EH gene was knock-down, most crabs were dead before D2. Injection of synthetic peptide for ETH or CCAP rescued ETH- or CCAP-gene knock-down crabs separately. However, none of peptides could rescue dsEH-injected crabs. At D0 stage, knockdown of ETH down-regulated the transcriptions of EH and CCAP; while ETH was up-regulated when EH was knockdown. At D2 stage, ETH transcripts levels were reduced with the injection of dsEH but increased with the same dose of dsETH as crabs at D0 stage. Co-injection of dsETH and dsEH down-regulated ETH at D2 stage. Results showed that ETH, EH, and CCAP play essential but different roles in molt regulation in mud crab. In summary, the result of this study contributes to the discovery of different molecular mechanisms between Insecta and Crustacea and may provide insight to develop fishery drugs that helps aquacultured crustaceans to molt successfully.

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.

Immunomodulatory role of crustacean cardioactive peptide in the mud crab Scylla paramamosain

Crustacean cardioactive peptide (CCAP) is a pleiotropic neuropeptide, but its immunomodulatory role is not clear. Herein, the mud crab Scylla paramamosain provides a primitive model to study crosstalk between the neuroendocrine and immune systems. In this study, in situ hybridization showed that Sp-CCAP positive signal localized in multiple cells in the nervous tissue, while its conjugate receptor (Sp-CCAPR) positive signal mainly localized in the semigranular cells of hemocytes. The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide (LPS) stimulation, but the Sp-CCAP mRNA expression level was up-regulated firstly and then down-regulated after the stimulation of polyriboinosinic polyribocytidylic acid [Poly (I:C)]. After the injection of Sp-CCAP synthesis peptide, the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group. Simultaneously, the mRNA expression of phagocytosis related gene (Sp-Rab5), nuclear transcription factor NF-κB homologues (Sp-Relish), C-type lectin (Sp-CTL-B), prophenoloxidase (Sp-proPO), pro-inflammatory cytokines factor (Sp-TNFSF, Sp-IL16) and antimicrobial peptides (Sp-ALF1 and Sp-ALF5) in the hemocytes were also significantly up-regulated at different time points after the injection of Sp-CCAP synthetic peptide, but Sp-TNFSF, Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h. In addition, RNA interference of Sp-CCAP suppressed the phagocytic activity of hemocytes and inhibited the mRNA expression of Sp-Rab5, Sp-Relish, Sp-CTL-B, Sp-TNFSF, Sp-IL16 and Sp-ALF5 in the hemocytes, and ultimately weakened the ability of hemolymph bacteria clearance of mud crab. Taken together, these results revealed that CCAP induced innate immune and increased the anti-infection ability in the mud crab.

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.

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.

Neonicotinoids can cause arrested pupal ecdysis in Lepidoptera

Recently, we reported a novel mode of action in monarch butterfly (Danaus plexippus) larvae exposed to neonicotinoid insecticides: arrest in pupal ecdysis following successful larval ecdysis. In this paper, we explore arrested pupal ecdysis in greater detail and propose adverse outcome pathways to explain how neonicotinoids cause this effect. Using imidacloprid as a model compound, we determined that final-instar monarchs, corn earworms (Helicoverpa zea), and wax moths (Galleria mellonella) showed high susceptibility to arrested pupal ecdysis while painted ladies (Vanessa cardui) and red admirals (Vanessa atalanta) showed low susceptibility. Fall armyworms (Spodoptera frugiperda) and European corn borers (Ostrinia nubilalis) were recalcitrant. All larvae with arrested ecdysis developed pupal cuticle, but with incomplete shedding of larval cuticle and unexpanded pupal appendages; corn earworm larvae successfully developed into adults with unexpanded appendages. Delayed initiation of pupal ecdysis was also observed with treated larvae. Imidacloprid exposure was required at least 26 h prior to pupal ecdysis to disrupt the molt. These observations suggest neonicotinoids may disrupt the function of crustacean cardioactive peptide (CCAP) neurons, either by directly acting on their nicotinic acetylcholine receptors or by acting on receptors of inhibitory neurons that regulate CCAP activity.

Ecdysis-related neuropeptide expression and metamorphosis in a non-ecdysozoan bilaterian.

Ecdysis-related neuropeptides (ERNs), including eclosion hormone, crustacean cardioactive peptide, myoinhibitory peptide, bursicon alpha, and bursicon beta regulate molting in insects and crustaceans. Recent evidence further revealed that ERNs likely play an ancestral role in invertebrate life cycle transitions, but their tempo-spatial expression patterns have not been investigated outside Arthropoda. Using RNA-seq and in situ hybridization, we show that ERNs are broadly expressed in the developing nervous system of a mollusk, the polyplacophoran Acanthochitona fascicularis. While some ERN-expressing neurons persist from larval to juvenile stages, others are only present during settlement and metamorphosis. These transient neurons belong to the "ampullary system," a polyplacophoran-specific larval sensory structure. Surprisingly, however, ERN expression is absent from the apical organ, another larval sensory structure that degenerates before settlement is completed in A. fascicularis. Our findings thus support a role of ERNs in A. fascicularis metamorphosis but contradict the common notion that the apical organ-like structures shared by various aquatic invertebrates (i.e., cnidarians, annelids, mollusks, echinoderms) are of general importance for this process.

Molecular and physiological characterization of a crustacean cardioactive signaling system in a lophotrochozoan - the Pacific oyster (Crassostrea gigas): a role in reproduction and salinity acclimation.

The crustacean cardioactive peptide (CCAP) is an important neuropeptide involved in the regulation of a variety of physiological processes in arthropods. Although this family of peptides has an ancestral origin, its function remains poorly understood among protostome species - apart from arthropods. We functionally characterized three G protein-coupled receptors (GPCRs) in the oyster Crassostrea gigas, phylogenetically related to ecdysozoan CCAP receptors (CCAPRs) and to chordate neuropeptide S receptors (NPSRs). Cragi-CCAPR1 and Cragi-CCAPR2 were specifically activated by the Cragi-CCAP1 and Cragi-CCAP2 peptides, respectively, both derived from the same CCAP precursor. In contrast, Cragi-CCAPR3 was only partially activated by CCAP1 and CCAP2 at high concentrations. The Cragi-CCAPR1 and Cragi-CCAPR2 genes were expressed in various adult tissues. They are both most expressed in the gills, while Cragi-CCAPR3 is mainly expressed in the visceral ganglia (VG). Cragi-CCAP precursor transcripts are higher in the VG, the labial palps and the gills. Receptor and ligand-encoding transcripts are more abundantly expressed in the gonads in the first stages of gametogenesis, while the Cragi-CCAP precursor is upregulated in the VG in the last stages of gametogenesis. This suggests a role of the CCAP signaling system in the regulation of reproductive processes. A role in water and ionic regulation is also supported considering the differential expression of the CCAP signaling components in oysters exposed to brackish water.

Crustacean cardioactive peptide as a stimulator of feeding and a regulator of ecdysis in Leptinotarsa decemlineata

Crustacean cardioactive peptide (CCAP), a highly conserved amidated neuropeptide, stimulates feeding in Drosophila melanogaster and Periplaneta americana, and regulates pupa-adult transition in Tribolium castaneum and Manduca sexta. In the present paper, we intended to address whether CCAP plays the dual roles in the Colorado potato beetle Leptinotarsa decemlineata. We found that the levels of Ldccap were high in the dissected samples of brain-corpora cardiaca-corpora allata complex and ventral nerve cord, midgut and hindgut in the final (fourth)-instar larvae. A pulse of 20-hydroxyecdysone triggered the expression of Ldccap in the central nervous system but decreased the transcription in the midgut. In contrast, juvenile hormone intensified the expression of Ldccap in the midgut. RNA interference (RNAi)-aided knockdown of Ldccap at the penultimate instar stage inhibited foliage consumption, reduced the contents of trehalose and chitin, and lowered the mRNA levels of two chitin biosynthesis genes (LdUAP1 and LdChSAb). Moreover, around 70% of the Ldccap RNAi larvae remained as prepupae, completely wrapped in the old larval exuviae, and finally died. The remaining RNAi beetles continually developed to severely-deformed adults: most having wrinkled and smaller elytra and hindwings, and shortened legs. Therefore, CCAP plays three distinct roles, stimulating feeding in foraging larval stage, regulating ecdysis, and facilitating wing expansion and appendage elongation in a coleopteran. In addition, Ldccap can be used as a potential target gene for developing novel management strategies against this coleopteran pest.