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The innervation of the antennal heart of the cockroach Periplaneta americana was studied with immunocytochemical techniques on both the light and electron microscopic levels. The antennal heart is innervated by two efferent systems, both using one biogenic amine in combination with neuropeptides. In one, we found co-localization of serotonin with proctolin and allatostatin. These fibers most likely originate from paired neurons located in the suboesophageal ganglion. In the second system, we found octopamine co-localized with the short neuropeptide F. The source of this second system is dorsal unpaired median (DUM) neurons, also located in the suboesophageal ganglion. The possible effects of these neuromediators on different targets are discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: The kissing bug, Rhodnius prolixus, is a blood-gorging insect that is medically important, being a principal vector of Trypanosoma cruzi, which causes Chagas disease. Understanding the reproductive biology of R. prolixus is, therefore, of some importance from a scientific perspective, but also in the medical context in order to control the spread of the disease. Here we show that the male reproductive system of R. prolixus is composed of muscular tissues performing contractions that aid in the transfer of sperm and other fluids into the female during mating. These contractions are further shown to be controlled by neuropeptides released by the nervous system that act on the receptors to either increase or inhibit the contractions. The male reproductive structures are, therefore, coordinated for successful copulation, whereby the transfer of sperm and fluid occurring during copulation is facilitated through a coordination of the contraction of the muscles within the male reproductive system. This study is critically important to provide novel options for pest management. The gross anatomy of the male reproductive structures and their associated musculature are described in the blood-gorging vector of Chagas disease, Rhodnius prolixus. The male reproductive system is composed of muscular tissues each performing contractions that aid in the movement of sperm out of the testis into the vas deferens, seminal vesicle and then into the ejaculatory duct, along with proteins and lipids from the transparent and opaque accessory glands. Phalloidin staining shows the various patterns of muscle fiber layers, from thin circular to more complex crisscross patterns, implying subtle differences in the form of the contractions and movement of each of the structures, allowing for waves of contractions or twisting patterns. The transcripts for the receptors for proctolin, myosuppressin (RhoprMS) and for the extended FMRFamides are expressed in the various regions of the reproductive system, and the nerve processes extending over the reproductive structures are positive for FMRFamide-like immunoreactivity, as are neurosecretory cells lying on the nerves. Proctolin and AKDNFIRFamide are strong stimulators for the frequency of the contractions, and RhoprMS can inhibit the proctolin-induced contractions. Taken together, this work implicates these two families of peptides in coordinating the male reproductive structures for the successful transfer of sperm and the associated accessory gland fluid to the female during copulation. [ABSTRACT FROM AUTHOR]

The crustacean cardiac ganglion (CG) comprises nine neurons that provide rhythmic drive to the heart. The CG is the direct target of multiple modulators. Synapsin‐like immunoreactivity was found clustered around the somata of the large cells (LC) and in a neuropil at the anterior branch of the CG trunk of Cancer borealis. This implicates the soma as a key site of synaptic integration, an unusual configuration in invertebrates. Proctolin is an excitatory neuromodulator of the CG, and proctolin‐like immunoreactivity exhibited partial overlap with putative chemical synapses near the LCs and at the neuropil. A proctolin‐like projection was also found in a pair of excitatory nerves entering the CG. GABA‐like immunoreactivity was nearly completely colocalized with chemical synapses near the LCs but absent at the anterior branch neuropil. GABA‐like projections were found in a pair of inhibitory nerves entering the CG. C. borealis Allatostatin B1 (CbASTB), red pigment concentrating hormone, and FLRFamide‐like immunoreactivity each had a unique pattern of staining and co‐localization with putative chemical synapses. These results provide morphological evidence that synaptic input is integrated at LC somata in the CG. Our findings provide a topographical organization for some of the multiple inhibitory and excitatory modulators that alter the rhythmic output of this semi‐autonomous motor circuit. [ABSTRACT FROM AUTHOR]

The ability of insects to tolerate low temperatures, known as chill tolerance, contributes to their global distribution. However, the mechanisms underlying insect chill tolerance remain poorly understood. At low temperatures, insects enter chill coma, a reversible state of paralysis, owing to disrupted ion and water homeostasis. Upon returning to normal temperatures, insects reestablish ion and water homeostasis and recover the ability to move. In this study, we used the two-spotted cricket, Gryllus bimaculatus, as an experimental model and unveiled the roles of neuropeptides in regulating chill tolerance, typically evaluated by the time taken to recover from chill coma. Screening of 37 neuropeptides revealed that Allatotropin, DH31, and Proctolin inhibited chill coma recovery and decreased the survival rate under cold stress. RT-qPCR analyses revealed that the receptors for Allatotropin and DH31 were predominantly expressed in the hindgut. Injection of the three neuropeptides decreased both hemolymph mass and gut water content at low temperatures, most likely by increasing water excretion from the hindgut due to their effects on the rectum contraction. Additionally, Allatotropin and DH31 were produced by the terminal abdominal ganglion (TAG) innervating the hindgut since they were partly co-localized in the TAG, and their mature peptides were detected in the TAG-hindgut nerves. Moreover, the transcriptional levels of the neuropeptides in the TAG and receptors in the hindgut changed with cold exposure and rewarming. Based on these findings, we propose that Allatotropin, DH31, and Proctolin affect the physiological activities of the gut, probably the hindgut, to disrupt water homeostasis at low temperatures, thereby reducing chill tolerance in crickets., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024. Published by Elsevier Ltd.)

The gross anatomy of the male reproductive structures and their associated musculature are described in the blood-gorging vector of Chagas disease, Rhodnius prolixus. The male reproductive system is composed of muscular tissues each performing contractions that aid in the movement of sperm out of the testis into the vas deferens, seminal vesicle and then into the ejaculatory duct, along with proteins and lipids from the transparent and opaque accessory glands. Phalloidin staining shows the various patterns of muscle fiber layers, from thin circular to more complex crisscross patterns, implying subtle differences in the form of the contractions and movement of each of the structures, allowing for waves of contractions or twisting patterns. The transcripts for the receptors for proctolin, myosuppressin (RhoprMS) and for the extended FMRFamides are expressed in the various regions of the reproductive system, and the nerve processes extending over the reproductive structures are positive for FMRFamide-like immunoreactivity, as are neurosecretory cells lying on the nerves. Proctolin and AKDNFIRFamide are strong stimulators for the frequency of the contractions, and RhoprMS can inhibit the proctolin-induced contractions. Taken together, this work implicates these two families of peptides in coordinating the male reproductive structures for the successful transfer of sperm and the associated accessory gland fluid to the female during copulation.

Proctolin (RYLPT) has been the first invertebrate neuropeptide to be fully sequenced (already in 1975). Its structure is identical in both crustaceans and insects, where it has myo- and neurostimulatory actions. The Drosophila receptor has been the first invertebrate proctolin receptor to be identified. Over the years, a large number of agonists and antagonists have been synthesized to find the structure-activity relationship. Computer methods now enable the quick analyses of various analogs. The aim of the present study is to model the proctolin receptor and to perform docking with known analogues, both agonist and antagonist. The new model of a proctolin receptor is modeled and a significant correlation between the results of docking and in vitro experiments (Pearson's r = 0.8627 and P value = 0.0270) is found. The results obtained are promising and open an area of a further profound investigation aiming to optimize the receptor structure and to design new agonists and antagonists of proctolin. [ABSTRACT FROM AUTHOR]

Neurons in the central pattern-generating circuits in the crustacean stornatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide la (CabTRPla), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (lMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRPla and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRPla in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP lain the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na4 current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na + channels, and one that can persist in their absence. [ABSTRACT FROM AUTHOR]

Over the past four decades, Drosophila melanogaster has become an increasingly important model system for studying the modulation of chemical synapses and muscle contraction by cotransmitters and neurohormones. This review describes how advantages provided by Drosophila have been utilized to investigate synaptic modulation, and it discusses key findings from investigations of cotransmitters and neurohormones that act on body wall muscles of 3rd instar Drosophila larvae. These studies have contributed much to our understanding of how neuromuscular systems are modulated by neuropeptides and biogenic amines, but there are still gaps in relating these peripheral modulatory effects to behavior. [ABSTRACT FROM AUTHOR]

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg 1 ]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg 1 ]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg 1 ]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg 1 ]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg 1 ]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys. [ABSTRACT FROM AUTHOR]

The neuropeptide proctolin (RYLPT) plays important roles as both a neurohormone and a cotransmitter in arthropod neuromuscular systems. We used third-instar Drosophila larvae as a model system to differentiate synaptic effects of this peptide from its direct effects on muscle contractility and to determine whether proctolin can work in a cell-selective manner on muscle fibers. Proctolin did not appear to alter the amplitude of excitatory junctional potentials but did induce sustained muscle contractions in preparations where the CNS had been removed and no stimuli were applied to the remaining nerves. Proctolin-induced contractions were dose-dependent, were reduced by knocking down expression of the Drosophila proctolin receptor in muscle tissue, and were larger in some muscle cells than others (i.e., larger in fibers 4, 12, and 13 than in 6 and 7). Proctolin also increased the amplitude of nerve-evoked contractions in a dose-dependent manner, and the magnitude of this effect was also larger in some muscle cells than others (again, larger in fibers 4, 12, and 13 than in 6 and 7). Increasing the intraburst impulse frequency and number of impulses per burst increased the magnitude of proctolin’s enhancement of nerve-evoked contractions and decreased the threshold and EC50 concentrations for proctolin to enhance nerve-evoked contractions. Reducing proctolin receptor expression decreased the velocity of larval crawling at higher temperatures, and thermal preference in these larvae. Our results suggest that proctolin acts directly on body-wall muscles to elicit slow, sustained contractions and to enhance nerve-evoked contractions, and that proctolin affects muscle fibers in a cell-selective manner. [ABSTRACT FROM AUTHOR]

Heartbeat reversal patterns have been monitored in the body of diapausing pupae of M. sexta 2 h before and 3 h after the injections of [Arg7]-corazonin, using noninvasive thermographic and optocardiographic methods. Large dosages (10-6 M final concentrations of corazonin in the body) caused almost immediate, adrenaline-like enhancement of the anterograde heartbeat. During the relatively short, acute phase of the tachycardia induced by corazonin, the systolic anterograde contractions of the heart increased in average from 10.5 to 24 pulses per min, culminating at 2.5 min after the injections. Duration of the acute period of tachycardia was only 7 to 20 min, which was followed by a period of slightly elevated, residual anterograde heartbeat which persisted occasionally for 1 to 3 h. Smaller dosages of corazonin (10-7M concentrations in the body) occasionally also produced a less intensive cardiotropic effect, while the more diluted samples were completely inactive. In pupae of the beetle T. molitor, injections of corazonin (10-6 M in the body) had no effect on the rate of in vivo heartbeat at all. Pharmacological analysis of the effects of corazonin in M. sexta indicated that the cardiostimulating effects of corazonin did not conform with the expected action of a peptidic neurohormone. A possibility that these effects might be artifacts produced by the low molecular breakdown products of corazonin has been discussed.

We studied the relationship between neuropeptide receptor transcript expression and current responses in the stomatogastric ganglion (STG) of the crab, Cancer borealis. We identified a transcript with high sequence similarity to crustacean cardioactive peptide (CCAP) receptors in insects and mammalian neuropeptide S receptors. This transcript was expressed throughout the nervous system, consistent with the role of CCAP in a range of different behaviors. In the STG, single-cell qPCR showed expression in only a subset of neurons. This subset had previously been shown to respond to CCAP with the activation of a modulator-activated inward current (IMI), with one exception. In the one cell type that showed expression but no IMI responses, we found CCAP modulation of synaptic currents. Expression levels within STG neuron types were fairly variable, but significantly different between some neuron types. We tested the magnitude and concentration dependence of IMI responses to CCAP application in two identified neurons, the lateral pyloric (LP) and the inferior cardiac (IC) neurons. LP had several-fold higher expression and showed larger current responses. It also was more sensitive to low CCAP concentrations and showed saturation at lower concentrations, as sigmoid fits showed smaller EC50 values and steeper slopes. In addition, occlusion experiments with proctolin, a different neuropeptide converging onto IMI, showed that saturating concentrations of CCAP activated all available IMI in LP, but only approximately two-thirds in IC, the neuron with lower receptor transcript expression. The implications of these findings for comodulation are discussed. [ABSTRACT FROM AUTHOR]

A variety of neurons and synapses shows a maximal response at a preferred frequency, generally considered to be important in shaping network activity. We are interested in whether all neurons and synapses in a recurrent oscillatory network can have preferred frequencies and, if so, whether these frequencies are the same or correlated, and whether they influence the network activity. We address this question using identified neurons in the pyloric network of the crab Cancer borealis. Previous work has shown that the pyloric pacemaker neurons exhibit membrane potential resonance whose resonance frequency is correlated with the network frequency. The follower lateral pyloric (LP) neuron makes reciprocally inhibitory synapses with the pacemakers. We find that LP shows resonance at a higher frequency than the pacemakers and the network frequency falls between the two. We also find that the reciprocal synapses between the pacemakers and LP have preferred frequencies but at significantly lower values. The preferred frequency of the LP to pacemaker synapse is correlated with the presynaptic preferred frequency, which is most pronounced when the peak voltage of the LP waveform is within the dynamic range of the synaptic activation curve and a shift in the activation curve by the modulatory neuropeptide proctolin shifts the frequency preference. Proctolin also changes the power of the LP neuron resonance without significantly changing the resonance frequency. These results indicate that different neuron types and synapses in a network may have distinct preferred frequencies, which are subject to neuromodulation and may interact to shape network oscillations. [ABSTRACT FROM AUTHOR]

Linear leak currents have been implicated in the regulation of neuronal excitability, generation of neuronal and network oscillations, and network state transitions. Yet, few studies have directly tested the dependence of network oscillations on leak currents or explored the role of leak currents on network activity. In the oscillatory pyloric network of decapod crustaceans neuromodulatory inputs are necessary for pacemaker activity. A large subset of neuromodulators is known to activate a single voltage-gated inward current IMI, which has been shown to regulate the rhythmic activity of the network and its pacemaker neurons. Using the dynamic clamp technique, we show that the crucial component of IMI for the generation of oscillatory activity is only a close-to-linear portion of the current-voltage relationship. The nature of this conductance is such that the presence or the absence of neuromodulators effectively regulates the amount of leak current and the input resistance in the pacemaker neurons. When deprived of neuromodulatory inputs, pyloric oscillations are disrupted; yet, a linear reduction of the total conductance in a single neuron within the pacemaker group recovers not only the pacemaker activity in that neuron, but also leads to a recovery of oscillations in the entire pyloric network. The recovered activity produces proper frequency and phasing that is similar to that induced by neuromodulators. These results show that the passive properties of pacemaker neurons can significantly affect their capacity to generate and regulate the oscillatory activity of an entire network, and that this feature is exploited by neuromodulatory inputs.

Abstract: MALDI-TOF MS and MS/MS techniques were used for the isolation and identification of neuropeptides from the ventral nerve cord (VNC) of two beetle species Tenebrio molitor and Zophobas atratus. Two peptides, proctolin and myosuppressin (Zopat-MS), with well-established myotropic properties were identified as well as Trica-NVPL-4trunc. The presence of proctolin and myosuppressin was confirmed by immunocytochemical studies in adults and larvae of both beetles. In addition, the myosuppressin gene in Z. atratus was sequenced and expression analyses showed that it is present in all parts of the beetle central nervous system. Results suggest that the identified peptides act as neurotransmitters/neuromodulators in beetles, regulate visceral muscle contractions and indirectly influence important physiological processes such as feeding and reproduction. [Copyright &y& Elsevier]

Although synaptic output is known to be modulated by changes in presynaptic calcium channels, additional pathways for calcium entry into the presynaptic terminal, such as non-selective channels, could contribute to modulation of short term synaptic dynamics. We address this issue using computational modeling. The neuropeptide proctolin modulates the inhibitory synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron, two slow-wave bursting neurons in the pyloric network of the crab Cancer borealis. Proctolin enhances the strength of this synapse and also changes its dynamics. Whereas in control saline the synapse shows depression independent of the amplitude of the presynaptic LP signal, in proctolin, with high-amplitude presynaptic LP stimulation the synapse remains depressing while low-amplitude stimulation causes facilitation. We use simple calcium-dependent release models to explore two alternative mechanisms underlying these modulatory effects. In the first model, proctolin directly targets calcium channels by changing their activation kinetics which results in gradual accumulation of calcium with low-amplitude presynaptic stimulation, leading to facilitation. The second model uses the fact that proctolin is known to activate a non-specific cation current I. In this model, we assume that the MI channels have some permeability to calcium, modeled to be a result of slow conformation change after binding calcium. This generates a gradual increase in calcium influx into the presynaptic terminals through the modulatory channel similar to that described in the first model. Each of these models can explain the modulation of the synapse by proctolin but with different consequences for network activity. [ABSTRACT FROM AUTHOR]

The regulation of insect gut physiology is complex and involves the interactions of a number of mechanisms, including the neural regulation of gut contraction by altering neural input and the modulation of gut contractions by neuropeptides directly affecting the muscle. The FGLa-type allatostatins (FGLa/ASTs) are known brain/gut peptides with numerous physiological roles, including modulation of gut contraction and neural input. To further investigate the pleiotropic roles of FGLa/AST peptides in Locusta migratoria, we have examined the role of a locust FGLa/AST (Scg-AST-6) In the gut. Proctolin and Scg-AST-6 have opposing effects on gut contraction, where proctolin dose-dependently increases gut muscle tension, while Scg-AST-6 inhibits both muscle tension and spontaneous and neurogenic contractions in a dose-dependent manner. Results from neurophysiological recordings indicate that there may be a central pattern generator (CPG) within the ventricular ganglia regulated by descending inhibition, and the addition of Scg-AST-6 dose-dependently modulates this ventricular ganglion CPG. This work provides a comprehensive picture of how FGLa/ASTs may modulate and coordinate each region of the locust gut, and shows that FGLa/ASTs have both central effects, on the ventricular ganglion CPG, and peripheral effects on the gut muscle. Overall, this study shows how FGLa/ASTs contribute to the complex regulation and fine tuning of gut contraction. [ABSTRACT FROM AUTHOR]

Neuropeptides are important with respect to almost all physiological processes and behavioural patterns in an organism. In the present study, muscle bioassays, immunohistochemistry and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry are used to investigate the distribution and efficacy of proctolin in the antennal heart of 36 species of lower Neoptera. In total, 20 species of Dictyoptera (cockroaches, termites, praying mantids), eight species of Saltatoria (crickets and grasshoppers) and eight species of Phasmatodea (stick insects) are investigated. The antennal heart of all tested Blattoidea, including the termite Mastotermes darwiniensis Froggatt, exhibit a strong proctolin-like immunoreactivity, as well as a high sensitivity to proctolin, whereas members of the second clade of cockroaches (Blaberoidea) are largely insensitive to proctolin and show only a weak proctolin-like immunoreactivity. The antennal heart of praying mantids (Mantodea) also contains only few proctolin-like immunoreactive fibres but is highly sensitive to proctolin. Such a high sensitivity is found also in Phasmatodea, although the antennal heart of these insects does not have proctolin-like immunoreactive fibres. These findings are supported by MALDI-TOF mass spectrometry; no trace of proctolin is detected in antennal heart preparations of Phasmatodea. In Saltatoria, only weak (or no) effects of proctolin are observed and, in most subtaxa, no proctolin-like immunoreactivity is visible in the antennal heart preparations. Only in Grylloidea is strong proctolin-like immunostaining found in processes of the antennal heart. In these species, weak or moderate effects of proctolin are observed in the antennal heart bioassay. In general, the differences in distribution of proctolin and effects on the antennal heart within the basal Neoptera cannot be deduced from their phylogenetic position, although they show conformity within each (sub)taxon. [ABSTRACT FROM AUTHOR]

Abstract: The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identifieid, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown. [Copyright &y& Elsevier]

The peptides proctolin, crustacean cardioactive peptide (CCAP) and FMRFamide, which are known to modulate insect muscle contractions, were assayed for their action on oviduct contractions in Hylobius abietis. A video microscopy technique and computer-based method of data acquisition and analysis were used to investigate the effects of theses peptides on spontaneous contractions of continuously perfused oviducts. All three peptides tested stimulate spontaneous contraction activity of the pine weevil oviduct, increasing the frequency and amplitude of phasic contractions in a dose-dependent manner. Proctolin is more potent as a stimulator than CCAP. For proctolin a threshold response of oviduct muscles is at concentration of peptide 10-10 mol/L and for CCAP at concentration range 10-10 mol/L. FMRFamide exerts a weak stimulatory effect on the oviduct, and at higher concentrations of the peptide (above 10 mol/L). The peptides exert different responses on oviduct contractions and they may play a role as functional regulators in such processes as egg movement and oviposition. [ABSTRACT FROM AUTHOR]

Abstract: Genome mining has provided a valuable tool for peptide discovery in many species, yet no crustacean has undergone this analysis. Currently, the only crustacean with a sequenced genome is the cladoceran Daphnia pulex, a model organism in many fields of biology. Here, we have mined the D. pulex genome for peptide-encoding genes. For each gene identified, the encoded precursor protein was deduced, and its mature peptides predicted. Twenty-four peptide-encoding genes were identified, including ones predicted to produce members of the A-type allatostatin, B-type allatostatin, C-type allatostatin, allatotropin (ATR), bursicon α, bursicon β, calcitonin-like diuretic hormone, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, ecdysis-triggering hormone, eclosion hormone (EH), insulin-like peptide (ILP), molt-inhibiting hormone, neuropeptide F, orcokinin (two genes), pigment-dispersing hormone, proctolin, red pigment concentrating hormone/adipokinetic hormone (RPCH/AKH), short neuropeptide F, SIFamide, sulfakinin, and tachykinin-related peptide (TRP) families/subfamilies. In total, 96 peptides were predicted from these genes. Our identification of isoforms of corazonin, EH, ILP, proctolin, RPCH/AKH, sulfakinin and TRP are the first for D. pulex, while our prediction of ATR from this species is the first from any crustacean. The number of peptides predicted in our study shows the power of genome mining for peptide discovery, and provides a model for future genomic analyses of the peptidomes of other crustaceans. In addition, the data presented in our study provide foundations for future molecular, biochemical, anatomical, and physiological investigation of peptidergic signaling in D. pulex and other cladoceran species. [Copyright &y& Elsevier]

We examined if the pentapeptide proctolin, a well-investigated myotropic neutrotrasmitter/neuromodulator for which a possible role in insect control has been suggested, affected the gastrointestinal function in lepidopteran larvae. We verified in the model species Bombyx mori (Lepidoptera, Bombicidae) if the visceral muscles of the larval gut expressed receptors for the peptide, by monitoring the spontaneous contractions of the hindgut in the absence or in the presence of proctolin. The frequency of the peristaltic contractions increased only at a very high concentration of the peptide, suggesting that proctolin could effectively bind with low affinity to non-specific receptors. The outcome on larval fitness of increasing doses of proctolin orally administered to fourth instar larvae were investigated by recording the standard nutritional indices. A significant decrease of the Efficiency of Conversion of Ingested and Digested food to body substance with a concomitant reduction of the Relative Growth Rate (RGR) was observed at the lowest dose tested (0.18 mg/g larva). The amount of peptide administered appeared critical, because a 10-fold higher dose of proctolin abolished the negative effect on RGR by a stimulation of the feeding behaviour, possibly as a result of the activation of compensatory mechanisms. A similar decrease of larval RGR with a low concentration of proctolin was observed also in the polyphagous pest Spodoptera littoralis (Lepidoptera, Noctuidae). [ABSTRACT FROM AUTHOR]

Proctolin is a neuroactive pentapeptide first isolated from the cockroach Periplaneta americana in which it has an excitatory effect on contractions on visceral muscles of the hindgut. Subsequently, proctolin is reported in a wide variety of invertebrates, and considerable efforts have been made to determine its mode of action. Its primary role appears to be that of a neuromodulator rather than a classical neurotransmitter, and it may also serve as a neurohormone, depending on the muscles examined. The present study identifies the vagina muscles of the blood-sucking insect Rhodnius prolixus (Stål) as a proctolinergic system. Physiological doses of proctolin generate prolonged contractions that closely mimic the effects of motor nerve stimulation. This preparation is convenient and robust, warranting its use as an experimental system to further understand the role of proctolin in the regulation of muscle contractions in insects. Moreover, these muscles are innervated by an identifiable inhibitory component providing a means to investigate the interaction between proctolin excitation and neural inhibition. [ABSTRACT FROM AUTHOR]

Abstract: The Lottia gigantea genome was prospected for the presence of genes coding neuropeptides and neurohormones. Four genes code insulin-related peptides: two genes code molluscan insulin-like growth hormones, one gene an insulin very similar to vertebrate insulin, and the fourth a peptide related to drosophila insulin-like peptide 7. Four other genes encode the cysteine-knot proteins GPA2/GPB5 and bursicon/parabursicon. Another 37 genes code for precursors of the following neuropeptides: achatin, APGWamide, allatostatin C, allatotropin, buccalin (perhaps an allatostatin A homolog), cerebrin, CCAP, conopressin, elevenin (the predicted neuropeptide made by abdominal neuron 11 in Aplysia), egg laying hormone (two genes), enterin, feeding circuit activating neuropeptide (FCAP), FFamide, FMRFamide, GGNG, a GnRH-like peptide, the newly discovered LASGLVamide, LFRFamide, LFRYamide, LRNFVamide, luqin, lymnokinin, myomodulin (two genes), the newly discovered NKY, NPY, pedal peptide (three genes), PKYMDT, pleurin, PXFVamide, small cardioactive peptides, tachykinins (two genes) and WWamide (an allatostatin B homolog). One gene was found to encode FWISamide, while about 20 closely related genes were found to encode WWFamide. These small neuropeptides appear homologous to the NdWFamide, which contains d-Trp; these genes are similar to the Aplysia gene encoding NWFamide. Some of these peptides had not been previously identified from mollusks, such as the predicted hormones similar to Drosophila and vertebrate insulins, bursicon, the putative proctolin homolog PKYMDT and allatostatin C. Together with neuropeptides which are likely homologs of other insect neuropeptides, such as cerebrin and WWamide, this shows that despite significant differences the molluscan and arthropod neuropeptidomes are more similar than generally recognized. [Copyright &y& Elsevier]

Abstract: Acoustic communication in male Chorthippus biguttulus is a suitable behavioral model to explore the physiological effects and underlying molecular mechanisms of identified neuropeptides. Proctolin plays a modulatory role in a neuronal circuit controlling sexual behavior in these insects. Activation of proctolin receptors triggers courtship singing (the second level of excitation in sexual behavior) that is preceded by call singing triggered by muscarinic acetylcholine receptor activation. The pharmacological studies presented here revealed that the Phospholipase C pathway was involved in courtship singing since neomycin and Li+ strongly inhibited proctolin-stimulated singing. In addition, injecting phorbol ester into proctolin-sensitive neural sites only elicited stridulation. This confirms that proteinkinase C can mediate the effects of Phospholipase C activation in this model. These results partially clarify the molecular mechanisms in the decision making brain center that controls sexual behavior in these insects. [Copyright &y& Elsevier]

Investigations into cardiac physiology in Myriapoda are rare, but heart beat generation is not considered to be uniform throughout this taxon. Although cardiac automatism in Chilopoda is neurogenic, superimposed onto a myogenic automatism, the present study reveals, on the basis of electrophysiological experiments including electrocardiograms and the first intracellular recordings from dorsal vessel muscle cells of Archispirostreptus gigas Peters, 1855 (Diplopoda: Spirostreptida, Spirostreptidae), that heartbeat generation in Diplopoda is clearly myogenic. Experiments with tetrodotoxin confirm this result, and also show that proctolin, acetylcholine and octopamine have no effect. The results are discussed from the perspective of comparative cardiac physiology in arthropods. [ABSTRACT FROM AUTHOR]

Abstract: The cladoceran crustacean Daphnia pulex has emerged as a model species for many biological fields, in particular environmental toxicology and toxicogenomics. Recently, this species has been the subject of an extensive transcriptome project, resulting in the generation and public deposition of over 150,000 expressed sequence tags (ESTs). This resource makes D. pulex an excellent model for protein discovery using bioinformatics. Here, in silico searches of the D. pulex EST database were conducted to identify transcripts encoding putative peptide precursors. Moreover, the mature peptides contained within the deduced prepro-hormones were predicted using online peptide processing programs and homology to known arthropod isoforms. In total, 63 putative peptide-encoding ESTs were identified encompassing 14 distinct peptide families/subfamilies: A-type allatostatin, B-type allatostatin, C-type allatostatin, bursicon (both α and β subunit peptides), crustacean cardioactive peptide (CCAP), crustacean hyperglycemic hormone (CHH)/ion transport peptide (both CHH- and moult-inhibiting hormone-like subfamilies), diuretic hormone (calcitonin-like), ecdysis-triggering hormone (ETH), FMRFamide (both neuropeptide F and short neuropeptide F subfamilies), orcokinin and pigment dispersing hormone. From these transcripts, the structures of 76 full-length/partial peptides were predicted, which included the first C-type allatostatin-like peptide identified from a crustacean, the first crustacean calcitonin-like diuretic hormone, an undescribed CCAP isoform, two hitherto unknown ETH variants, and two new orcokinins. Neuronal localization of several of the identified peptide families was confirmed using immunohistochemitry (i.e. A-type allatostatin, CCAP, FMRFamide and PDH). In addition, immunohistochemical analyses identified other putative neuropeptides for which no ESTs had been found (i.e. corazonin, insect kinin, proctolin, red pigment concentrating hormone, SIFamide, sulfakinin and tachykinin-related peptide). Collectively, the data presented here not only catalog an extensive array of putative D. pulex peptide paracrines/hormones, but also provide a strong foundation for future investigations of the effects of environmental/anthropogenic stressors on peptidergic control in this model organism. [Copyright &y& Elsevier]

Abstract: In earwigs, the male reproductive system is complex, comprising accessory glands and long dual intromittent organs for transfer of materials to the female and for removal of rival sperm. We investigated potential factors altering contractions of the male reproductive tracts in vitro. Tracts from 0-day (newly emerged) males displayed relatively little motility in vitro; however, those from 5-day (intermediate stage of sexual maturity) and 8-day (fully mature) males pulsed vigorously. Both 1 and 100 nM proctolin (RYLPT-OH) stimulated the rate of contraction of reproductive tracts from both 5-day and 8-day males. In contrast, 1 nM and 100 nM FGLa AST (cockroach allatostatin) did not affect pulsations. However, 10 µM FGLa AST decreased activity of reproductive tracts. Mating decreased motility of tracts from 5-day old males, but did not alter motility of tracts from 8-day old males. Castration of larvae significantly suppressed reproductive tract motility in subsequent 8-day old adults compared with those of intact or sham-operated adults. Castration also suppressed seminal vesicle size. Lastly, we assessed the presence and distribution of proctolin-like and allatostatin-like immunoreactivity in tissues. Immunoreactivity to FGLa AST and proctolin was widespread, occurring in the brain and ventral ganglia. Surprisingly, we did not detect immunoreactivity to either FGLa AST or proctolin within the reproductive system; however, proctolin immunoreactivity was evident in nerves extending from the terminal ganglion of 8-day, but not 0-day, males. Collectively, these experiments demonstrate that the male earwig reproductive system is an appropriate model for use in addressing sexual maturation and activities in male insects. [Copyright &y& Elsevier]

Abstract: The Aphidoidea is an insect superfamily comprising most of the known aphid species. While small in size, these animals are of considerable economic importance as many members of this taxon are serious agricultural pests, inflicting physical damage upon crop plants and serving as vectors in the transmission of viral plant diseases. In terms of identifying the paracrines/hormones used to modulate behavior, particularly peptides, members of the Aphidoidea have largely been ignored, as it is not tractable to isolate the large pools of tissue needed for standard biochemical investigations. Here, a bioinformatics approach to peptide discovery has been used to overcome this limitation of scale. Specifically, in silico searches of publicly accessible aphidoidean ESTs were conducted to identify transcripts encoding putative peptides precursors, with the mature peptides contained within them deduced using peptide processing software and homology to known arthropod sequences. In total, 39 ESTs encoding putative peptides precursors were identified from four aphid species: Acyrthosiphon pisum (14 ESTs), Aphis gossypii (four ESTs), Myzus persicae (20 ESTs) and Toxoptera citricida (one EST). These precursors included ones predicted to encode isoforms of B-type allatostatin, crustacean cardioactive peptide, FMRFamide-related peptide (both myosuppressin and short neuropeptide F subfamilies), insect kinin, orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide and tachykinin-related peptide. In total, 83 peptides were characterized from the identified precursors, most novel, including two B-type allatostatins possessing the variant −WX 7Wamide motif, two N-terminally extended proctolin isoforms and an N-terminally truncated and substituted SIFamide. Collectively, these results expand greatly the number of known/predicted aphid peptide paracrines/hormones, and provide a strong foundation for future molecular and physiological investigations of peptidergic control in this insect group. [Copyright &y& Elsevier]

Abstract: The Ixodoidea (ticks) are important vectors in the transmission of many human diseases; for example, the blacklegged tick Ixodes scapularis is the major vector in the transmission of Lyme disease, the most frequently reported vector-borne illness in the United States. The development of expressed sequence tags (ESTs) for ixodoidean cDNA libraries, and their public deposition, has generated a rich resource for protein discovery in members of this taxon, thereby providing an opportunity for better understanding the physiology and behavior of these disease vectors. Here, in silico searches of publicly accessible ESTs were conducted to identify transcripts encoding putative ixodoidean neuropeptide precursors, with the mature peptides contained within them predicted using online peptide processing programs and homology to known arthropod sequences. In total, 37 putative neuropeptide-encoding ESTs were identified from three ixodoidean species: I. scapularis (29 ESTs), Rhipicephalus microplus (seven ESTs) and Amblyomma americanum (one EST). Among those identified from I. scapularis were ones predicted to encode isoforms of corazonin, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone (both calcitonin- and corticotropin-releasing factor-like), FMRFamide-related peptide (both short neuropeptide F and sulfakinin subfamilies) orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide, and tachykinin-related peptide. Collectively, 80 distinct ixodoidean neuropeptides were characterized from the identified precursors. These results not only expand greatly the number of known/predicted ixodoidean neuropeptides, but also provide a strong foundation for future molecular and physiological investigations of peptidergic control in this important group of disease-transmitting arthropods. [Copyright &y& Elsevier]

Abstract: Dippu-allatostatins (ASTs) have pleiotropic effects in Locusta migratoria. Dippu-ASTs act as releasing factors for adipokinetic hormone I (AKH I) from the corpus cardiacum (CC) and also alter juvenile hormone (JH) biosynthesis and release from the corpus allatum (CA). Dippu-AST-like immunoreactivity is found within lateral neurosecretory cells (LNCs) of the brain and axons within the paired nervi corporis cardiaci II (NCC II) to the CC and the CA, where there are extensive processes and nerve endings over both of these neuroendocrine organs. There was co-localization of Dippu-AST-like and proctolin-like immunoreactivity within these regions. Dippu-ASTs increase the release of AKH I in a dose-dependent manner, with thresholds below 10−11 M (Dippu-AST 7) and between 10−13 and 10−12 M (Dippu-AST 2). Both proctolin and Dippu-AST 2 caused an increase in the cAMP content of the glandular lobe of the CC. Dippu-AST 2 also altered the release of JH from the locust CA, but this effect depended on the concentration of peptide and the basal release rates of the CA. These physiological effects for Dippu-ASTs in Locusta have not been shown previously. [Copyright &y& Elsevier]

Abstract: The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP. [Copyright &y& Elsevier]

Network plasticity arises in large part due to the effects of exogenous neuromodulators. We investigate the neuromodulatory effects on short-term synaptic dynamics. The synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron in the pyloric network of the crab C. borealis has both spike-mediated and non-spike-mediated (graded) components. Previous studies have shown that the graded component of this synapse exhibits short-term depression. Recent results from our lab indicate that in the presence of the neuromodulatory peptide proctolin, low-amplitude presynaptic stimuli switch the short-term dynamics of this graded component from depression to facilitation. In this study, we show that this facilitation is correlated with the activation of a presynaptic inward current that is blocked by Mn2+, suggesting that it is a slowly accumulating Ca2+ current. We modify a mechanistic model of synaptic release by assuming that the low-voltage-activating Ca2+ current in our system is composed of two currents with fast (I CaF) and slow (I CaS) kinetics. We show that if proctolin adjusts the activation rate of I CaS, this leads to accumulation of local intracellular Ca2+ in response to multiple presynaptic voltage stimuli, which, in turn, results in synaptic facilitation. Additionally, we assume that proctolin increases the maximal conductances of Ca2+ currents in the model, consistent with the increased synaptic release found in the experiments. We find that these two presynaptic actions of proctolin in the model are sufficient to describe its actions on the short-term dynamics of the LP to PD synapse. [Copyright &y& Elsevier]

Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming ≈ 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at ≈ 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin ( Km≈ 4 µm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts. [ABSTRACT FROM AUTHOR]

Studies of release under physiological conditions provide more direct data about the identity of neuromodulatory signaling molecules than studies of tissue localization that cannot distinguish between processing precursors and biologically active neuropeptides. We have identified neuropeptides released by electrical stimulation of nerves that contain the axons of the modulatory projection neurons to the stomatogastric ganglion of the crab, Cancer borealis. Preparations were bathed in saline containing a cocktail of peptidase inhibitors to minimize peptide degradation. Both electrical stimulation of projection nerves and depolarization with high K+ saline were used to evoke release. Releasates were desalted and then identified by mass using MALDI–TOF (matrix-assisted laser desorption/ionization–time-of-flight) mass spectrometry. Both previously known and novel peptides were detected. Subsequent to electrical stimulation proctolin, Cancer borealis tachykinin-related peptide (CabTRP), FVNSRYa, carcinustatin-8, allatostatin-3 (AST-3), red pigment concentrating hormone, NRNFLRFa, AST-5, SGFYANRYa, TNRNFLRFa, AST-9, orcomyotropin-related peptide, corazonin, Ala13-orcokinin, and Ser9-Val13-orcokinin were detected. Some of these were also detected after high K+ depolarization. Release was calcium dependent. In summary, we have shown release of the neuropeptides thought to play an important neuromodulatory role in the stomatogastric ganglion, as well as numerous other candidate neuromodulators that remain to be identified. [ABSTRACT FROM AUTHOR]

The effectsin vivoof cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae ofManduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10−6 M (calculated fromµg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10-9 − 10-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10-8 − 10-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10−6 M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptidesin vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditionsin vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae ofM. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions. [ABSTRACT FROM AUTHOR]

At the threshold concentration (1–10 pmol l-1), the neuropeptide hormones proctolin (PR) and the FLRFamide-like peptide (FLP) F2 cause an increase in amplitude of electrically evoked contractions (each contraction is a brief tetanus) of lobster heart ostial muscle. At higher concentrations each peptide also induces an increase in tonus (contracture). The PR-induced contracture and augmentation of tetani are proportional to increases in [Ca2+]i. The rate of onset and recovery of peptide-induced effects on both tetani and contracture appeared to reduced by Ca2+ storage by the sarcoplasmic reticulum (SR). Enhanced tetani following a contracture may be due to enhanced voltage-gated Ca2+ current and sarcoplasmic reticular (SR) Ca2+ loading. The SR Ca2+ loading appears to be specific for PR and F2, since glutamic-acid-induced contractures are not followed by increased tetani. The prolonged elevation of [Ca2+]i during contracture causes a right-ward shift in the force-pCa curve indicating a decrease in myofibrillar sensitivity to Ca2+. Blocking voltage-gated Ca2+ channels with Cd2+, nifedipine or verapamil, while reducing tetani, does not prevent peptide-induced contracture and enhanced tetani. Opening SR Ca2+ channels and depleting SR Ca2+ with either caffeine or ryanodine blocked tetani but permitted accelerated peptide-induced contractures. We conclude that PR and F2 at low concentration enhance voltage-dependent Ca2+ induced Ca2+ release from the SR, while higher hormone levels directly gate Ca2+ entry across the sarcolemma. [ABSTRACT FROM AUTHOR]

The present investigation studied the influence of a number of neuropeptides on semi-open preparations of the isolated and perfused anterior stomach of larval Aedes aegypti. Effects of peptides were observed on the lumen negative transepithelial voltage (Vte) that is present with serotonin in the bath; this voltage most likely reflects active HCO3- secretion involved in alkalization of the larval anterior stomach. The five different A. aegypti allatostatins (allatostatin A 1–5) all affected Vte in almost identical ways, causing a 10–15% reduction of the voltage at 10-7 moll-1. A. aegypti neuropeptide F and proctolin reduced Vte at submicromolar concentrations. At 10-6 moll-1, neuropeptide F reduced Vte by 30% and proctolin reduced Vte by 50%. In contrast, A. aegypti allatotropin, A. aegypti head peptides I and III and A. aegypti short neuropeptide F were without effect on Vte. During the investigation it was observed that the peristaltic contractions of the preparations caused a dynamic component of Vte. Peristaltic contractions and the correlated voltage fluctuations depended on the presence of serotonin. Peristaltic activity and Vie deflections were progressively inhibited by A. aegypti head peptides I and III by A. aegypti short neuropeptide F and by A. aegypti neuropeptide F when the peptide concentrations were increased from 10-8 to 10-6 moll-1. These observations show that physiological concentrations of some of the tested neuropeptides affect two processes that require coordination: ion transport and motility of the larval anterior stomach. [ABSTRACT FROM AUTHOR]

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (K m ≈ 4 µm). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 ± 0.15 µm). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation. [ABSTRACT FROM AUTHOR]

In Jasus edwardsii (Hutton) the vascular resistance of each of the seven major arterial systems leaving the heart was increased in response to several of the following neurotransmitters and neurohormones: acetylcholine, adrenalin, serotonin, dopamine, octopamine and peptides proctolin and FLRFamide-related peptide F1. The resistance to flow through the infrabranchial sinus (IBS), part of the venous system, was also sensitive to these drugs. Unexpectedly, the responses of the IBS continued after removal of the gills. Differences in the profiles of responses of the arteries to individual hormones and in the magnitudes and the time courses of back pressure changes, eliminate a common downstream location such as the venous sinuses or gills, as the source of the arterial responses. Vasoactive drugs were effective when applied either via the lumen or, with longer delay, to the basal side of an artery via the IBS. It is concluded that the resistance of each of these sections of the vascular system is independently controllable by hormones. [Copyright &y& Elsevier]