Diversity of neuropeptidergic modulation in decapod crustacean cardiac and feeding systems.

All nervous systems are multiply modulated by polypeptides. However, a bulk of transmitter and modulation research has historically focused on small molecule transmitters released at synaptic sites. The stomatogastric nervous system (controls digestive movements of the foregut) and cardiac nervous system of decapod crustaceans have long been used to understand the processes that underlie neuromodulation. The circuits governing the rhythmic output from these nervous systems are comprised of a relatively small number of identified neurons, and the details of these nervous systems are well-defined. Here we discuss recent research highlighting advances in our understanding of peptidergic modulation in these systems. In particular, we focus on our ability to identify specific signaling peptide sequences and relate their expression patterns to their physiological effects, as well as on the multiple sites within a pattern generator-effector system at which modulation takes place. Recent efforts have enabled us to understand how co-modulation by two or more peptides can generate surprising effects on circuit physiology and that modulation at different receptor sites can produce supra-additive effects. Finally, we examine the protective role modulation plays in making circuits robust to perturbations, in this case, changes in temperature.

The role of feedback and modulation in determining temperature resiliency in the lobster cardiac nervous system.

Changes in ambient temperature affect all biological processes. However, these effects are process specific and often vary non-linearly. It is thus a non-trivial problem for neuronal circuits to maintain coordinated, functional output across a range of temperatures. The cardiac nervous systems in two species of decapod crustaceans, Homarus americanus and Cancer borealis, can maintain function across a wide but physiologically relevant temperature range. However, the processes that underlie temperature resilience in neuronal circuits and muscle systems are not fully understood. Here, we demonstrate that the non-isolated cardiac nervous system (i.e., the whole heart: neurons, effector organs, intrinsic feedback systems) in the American lobster, H. americanus, is more sensitive to warm temperatures than the isolated cardiac ganglion (CG) that controls the heartbeat. This was surprising as modulatory processes known to stabilize the output from the CG are absent when the ganglion is isolated. One source of inhibitory feedback in the intact cardiac neuromuscular system is nitric oxide (NO), which is released in response to heart contractions. We hypothesized that the greater temperature tolerance observed in the isolated CG is due to the absence of NO feedback. Here, we demonstrate that applying an NO donor to the isolated CG reduces its temperature tolerance. Similarly, we show that the NO synthase inhibitor L-nitroarginine (LNA) increases the temperature tolerance of the non-isolated nervous system. This is sufficient to explain differences in temperature tolerance between the isolated CG and the whole heart. However, in an intact lobster, the heart and CG are modulated by an array of endogenous peptides and hormones, many of which are positive regulators of the heartbeat. Many studies have demonstrated that excitatory modulators increase temperature resilience. However, this neuromuscular system is regulated by both excitatory and inhibitory peptide modulators. Perfusing SGRNFLRFamide, a FLRFamide-like peptide, through the heart increases the non-isolated nervous system's tolerance to high temperatures. In contrast, perfusing myosuppressin, a peptide that negatively regulates the heartbeat frequency, decreases the temperature tolerance. Our data suggest that, in this nervous system, positive regulators of neural output increase temperature tolerance of the neuromuscular system, while modulators that decrease neural output decrease temperature tolerance.

Structural variation between neuropeptide isoforms affects function in the lobster cardiac system.

Neuronal responses to peptide signaling are determined by the specific binding of a peptide to its receptor(s). For example, isoforms of the same peptide family can drive distinct responses in the same circuit by having different affinities for the same receptor, by having each isoform bind to a different receptor, or by a combination of these scenarios. Small changes in peptide composition can alter the binding kinetics and overall physiological response to a given peptide. In the American lobster (Homarus americanus), native isoforms of C-type allatostatins (AST-Cs) usually decrease heartbeat frequency and alter contraction force. However, one of the three AST-C isoforms, AST-C II, drives a cardiac response distinct from the response elicited by the other two. To investigate the aspects of the peptide that might be responsible for these differential responses, we altered various features of each peptide sequence. Although the presence of an amide group at the end of a peptide sequence (amidation) is often essential for determining physiological function, we demonstrate that C-terminal amidation does not dictate the AST-C response in the lobster cardiac system. However, single amino acid substitution within the consensus sequence did account for many of the differences in specific response characteristics (e.g. contraction frequency or force).

Isoforms of the neuropeptide myosuppressin differentially modulate the cardiac neuromuscular system of the American lobster, Homarus americanus.

Post-translational modifications (PTMs) diversify peptide structure and allow for greater flexibility within signaling networks. The cardiac neuromuscular system of the American lobster, Homarus americanus, is made up of a central pattern generator, the cardiac ganglion (CG), and peripheral cardiac muscle. Together, these components produce flexible output in response to peptidergic modulation. Here, we examined the role of PTMs in determining the effects of a cardioactive neuropeptide, myosuppressin (pQDLDHVFLRFamide), on the whole heart, the neuromuscular junction/muscle, the isolated CG, and the neurons of the CG. Mature myosuppressin and noncyclized myosuppressin (QDLDHVFLRFamide) elicited similar and significant changes in whole heart contraction amplitude and frequency, stimulated muscle contraction amplitude and the bursting pattern of the intact and ligatured neurons of the ganglion. In the whole heart, nonamidated myosuppressin (pQDLDHVFLRFG) elicited only a small decrease in frequency and amplitude. In the absence of motor neuron input, nonamidated myosuppressin did not cause any significant changes in the amplitude of stimulated contractions. In the intact CG, nonamidated myosuppressin elicited a small but significant decrease in burst duration. Further analysis revealed a correlation between the extent of modulation elicited by nonamidated myosuppressin in the whole heart and the isolated, intact CG. When the neurons of the CG were physically decoupled, nonamidated myosuppressin elicited highly variable responses. Taken together, these data suggest that amidation, but not cyclization, is critical in enabling this peptide to exert its effects on the cardiac neuromuscular system.NEW & NOTEWORTHY Myosuppressin (pQDLDHVFLRFamide), a well-characterized crustacean neuropeptide, and its noncyclized (QDLDHVFLRFamide) and nonamidated (pQDLDHVFLRFG) isoforms alter the output of the cardiac neuromuscular system of the American lobster, Homarus americanus. Mature myosuppressin and noncyclized myosuppressin elicited similar and significant changes across all levels of the isolated system, whereas responses to nonamidated myosuppressin were significantly different from other isoforms and were highly variable. These data support the diversity of peptide action as a function of peptide structure.

Does Differential Receptor Distribution Underlie Variable Responses to a Neuropeptide in the Lobster Cardiac System?

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

Mass Spectrometry Quantification, Localization, and Discovery of Feeding-Related Neuropeptides in Cancer borealis.

The crab Cancer borealis nervous system is an important model for understanding neural circuit dynamics and modulation, but the identity of neuromodulatory substances and their influence on circuit dynamics in this system remains incomplete, particularly with respect to behavioral state-dependent modulation. Therefore, we used a multifaceted mass spectrometry (MS) method to identify neuropeptides that differentiate the unfed and fed states. Duplex stable isotope labeling revealed that the abundance of 80 of 278 identified neuropeptides was distinct in ganglia and/or neurohemal tissue from fed vs unfed animals. MS imaging revealed that an additional 7 and 11 neuropeptides exhibited altered spatial distributions in the brain and the neuroendocrine pericardial organs (POs), respectively, during these two feeding states. Furthermore, de novo sequencing yielded 69 newly identified putative neuropeptides that may influence feeding state-related neuromodulation. Two of these latter neuropeptides were determined to be upregulated in PO tissue from fed crabs, and one of these two peptides influenced heartbeat in ex vivo preparations. Overall, the results presented here identify a cohort of neuropeptides that are poised to influence feeding-related behaviors, providing valuable opportunities for future functional studies.

Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus.

Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5' terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans.

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification.

Differential neuropeptide modulation of premotor and motor neurons in the lobster cardiac ganglion.

The American lobster, Homarus americanus, cardiac neuromuscular system is controlled by the cardiac ganglion (CG), a central pattern generator consisting of four premotor and five motor neurons. Here, we show that the premotor and motor neurons can establish independent bursting patterns when decoupled by a physical ligature. We also show that mRNA encoding myosuppressin, a cardioactive neuropeptide, is produced within the CG. We thus asked whether myosuppressin modulates the decoupled premotor and motor neurons, and if so, how this modulation might underlie the role(s) that these neurons play in myosuppressin's effects on ganglionic output. Although myosuppressin exerted dose-dependent effects on burst frequency and duration in both premotor and motor neurons in the intact CG, its effects on the ligatured ganglion were more complex, with different effects and thresholds on the two types of neurons. These data suggest that the motor neurons are more important in determining the changes in frequency of the CG elicited by low concentrations of myosuppressin, whereas the premotor neurons have a greater impact on changes elicited in burst duration. A single putative myosuppressin receptor (MSR-I) was previously described from the Homarus nervous system. We identified four additional putative MSRs (MSR-II-V) and investigated their individual distributions in the CG premotor and motor neurons using RT-PCR. Transcripts for only three receptors (MSR-II-IV) were amplified from the CG. Potential differential distributions of the receptors were observed between the premotor and motor neurons; these differences may contribute to the distinct physiological responses of the two neuron types to myosuppressin.NEW & NOTEWORTHY Premotor and motor neurons of the Homarus americanus cardiac ganglion (CG) are normally electrically and chemically coupled, and generate rhythmic bursting that drives cardiac contractions; we show that they can establish independent bursting patterns when physically decoupled by a ligature. The neuropeptide myosuppressin modulates different aspects of the bursting pattern in these neuron types to determine the overall modulation of the intact CG. Differential distribution of myosuppressin receptors may underlie the observed responses to myosuppressin.

Assessment and comparison of putative amine receptor complement/diversity in the brain and eyestalk ganglia of the lobster, Homarus americanus.

In decapods, dopamine, octopamine, serotonin, and histamine function as locally released/hormonally delivered modulators of physiology/behavior. Although the functional roles played by amines in decapods have been examined extensively, little is known about the identity/diversity of their amine receptors. Recently, a Homarus americanus mixed nervous system transcriptome was used to identify putative neuronal amine receptors in this species. While many receptors were identified, some were fragmentary, and no evidence of splice/other variants was found. Here, the previously predicted proteins were used to search brain- and eyestalk ganglia-specific transcriptomes to assess/compare amine receptor complements in these portions of the lobster nervous system. All previously identified receptors were reidentified from the brain and/or eyestalk ganglia transcriptomes, i.e., dopamine alpha-1, beta-1, and alpha-2 (Homam-DAα2R) receptors, octopamine alpha (Homam-OctαR), beta-1, beta-2, beta-3, beta-4, and octopamine-tyramine (Homam-OTR-I) receptors, serotonin type-1A, type-1B (Homam-5HTR1B), type-2B, and type-7 receptors; and histamine type-1 (Homam-HA1R), type-2, type-3, and type-4 receptors. For many previously partial proteins, full-length receptors were deduced from brain and/or eyestalk ganglia transcripts, i.e., Homam-DAα2R, Homam-OctαR, Homam-OTR-I, and Homam-5HTR1B. In addition, novel dopamine/ecdysteroid, octopamine alpha-2, and OTR receptors were discovered, the latter, Homam-OTR-II, being a putative paralog of Homam-OTR-I. Finally, evidence for splice/other variants was found for many receptors, including evidence for some being assembly-specific, e.g., a brain-specific Homam-OTR-I variant and an eyestalk ganglia-specific Homam-HA1R variant. To increase confidence in the transcriptome-derived sequences, a subset of receptors was cloned using RT-PCR. These data complement/augment those reported previously, providing a more complete picture of amine receptor complement/diversity in the lobster nervous system.

In silico analyses suggest the cardiac ganglion of the lobster, Homarus americanus, contains a diverse array of putative innexin/innexin-like proteins, including both known and novel members of this protein family.

Gap junctions are physical channels that connect adjacent cells, permitting the flow of small molecules/ions between the cytoplasms of the coupled units. Innexin/innexin-like proteins are responsible for the formation of invertebrate gap junctions. Within the nervous system, gap junctions often function as electrical synapses, providing a means for coordinating activity among electrically coupled neurons. While some gap junctions allow the bidirectional flow of small molecules/ions between coupled cells, others permit flow in one direction only or preferentially. The complement of innexins present in a gap junction determines its specific properties. Thus, understanding innexin diversity is key for understanding the full potential of electrical coupling in a species/system. The decapod crustacean cardiac ganglion (CG), which controls cardiac muscle contractions, is a simple pattern-generating neural network with extensive electrical coupling among its circuit elements. In the lobster, Homarus americanus, prior work suggested that the adult neuronal innexin complement consists of six innexins (Homam-Inx1-4 and Homam-Inx6-7). Here, using a H. americanus CG-specific transcriptome, we explored innexin complement in this portion of the lobster nervous system. With the exception of Homam-Inx4, all of the previously described innexins appear to be expressed in the H. americanus CG. In addition, transcripts encoding seven novel putative innexins (Homam-Inx8-14) were identified, four (Homam-Inx8-11) having multiple splice variants, e.g., six for Homam-Inx8. Collectively, these data indicate that the innexin complement of the lobster nervous system in general, and the CG specifically, is likely significantly greater than previously reported, suggesting the possibility of expanded gap junction diversity and function in H. americanus.

SIFamide peptides modulate cardiac activity differently in two species of Cancer crab.

The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif -SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly1-SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val1-SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly1- and Val1-SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10-9 to 10-8 M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion.

To what extent may peptide receptor gene diversity/complement contribute to functional flexibility in a simple pattern-generating neural network?

Peptides are known to contribute to central pattern generator (CPG) flexibility throughout the animal kingdom. However, the role played by receptor diversity/complement in determining this functional flexibility is not clear. The stomatogastric ganglion (STG) of the crab, Cancer borealis, contains CPGs that are models for investigating peptidergic control of rhythmic behavior. Although many Cancer peptides have been identified, their peptide receptors are largely unknown. Thus, the extent to which receptor diversity/complement contributes to modulatory flexibility in this system remains unresolved. Here, a Cancer mixed nervous system transcriptome was used to determine the peptide receptor complement for the crab nervous system as a whole. Receptors for 27 peptide families, including multiple receptors for some groups, were identified. To increase confidence in the predicted sequences, receptors for allatostatin-A, allatostatin-B, and allatostatin-C were cloned, sequenced, and expressed in an insect cell line; as expected, all three receptors trafficked to the cell membrane. RT-PCR was used to determine whether each receptor was expressed in the Cancer STG. Transcripts for 36 of the 46 identified receptors were amplified; these included at least one for each peptide family except RYamide. Finally, two peptides untested on the crab STG were assessed for their influence on its motor outputs. Myosuppressin, for which STG receptors were identified, exhibited clear modulatory effects on the motor patterns of the ganglion, while a native RYamide, for which no STG receptors were found, elicited no consistent modulatory effects. These data support receptor diversity/complement as a major contributor to the functional flexibility of CPGs.

Similarities and differences in circuit responses to applied Gly1-SIFamide and peptidergic (Gly1-SIFamide) neuron stimulation.

Microcircuit modulation by peptides is well established, but the cellular/synaptic mechanisms whereby identified neurons with identified peptide transmitters modulate microcircuits remain unknown for most systems. Here, we describe the distribution of GYRKPPFNGSIFamide (Gly1-SIFamide) immunoreactivity (Gly1-SIFamide-IR) in the stomatogastric nervous system (STNS) of the crab Cancer borealis and the Gly1-SIFamide actions on the two feeding-related circuits in the stomatogastric ganglion (STG). Gly1-SIFamide-IR localized to somata in the paired commissural ganglia (CoGs), two axons in the nerves connecting each CoG with the STG, and the CoG and STG neuropil. We identified one Gly1-SIFamide-IR projection neuron innervating the STG as the previously identified modulatory commissural neuron 5 (MCN5). Brief (~10 s) MCN5 stimulation excites some pyloric circuit neurons. We now find that bath applying Gly1-SIFamide to the isolated STG also enhanced pyloric rhythm activity and activated an imperfectly coordinated gastric mill rhythm that included unusually prolonged bursts in two circuit neurons [inferior cardiac (IC), lateral posterior gastric (LPG)]. Furthermore, longer duration (>30 s) MCN5 stimulation activated a Gly1-SIFamide-like gastric mill rhythm, including prolonged IC and LPG bursting. The prolonged LPG bursting decreased the coincidence of its activity with neurons to which it is electrically coupled. We also identified local circuit feedback onto the MCN5 axon terminals, which may contribute to some distinctions between the responses to MCN5 stimulation and Gly1-SIFamide application. Thus, MCN5 adds to the few identified projection neurons that modulate a well-defined circuit at least partly via an identified neuropeptide transmitter and provides an opportunity to study peptide regulation of electrical coupled neurons in a functional context. NEW & NOTEWORTHY Limited insight exists regarding how identified peptidergic neurons modulate microcircuits. We show that the modulatory projection neuron modulatory commissural neuron 5 (MCN5) is peptidergic, containing Gly1-SIFamide. MCN5 and Gly1-SIFamide elicit similar output from two well-defined motor circuits. Their distinct actions may result partly from circuit feedback onto the MCN5 axon terminals. Their similar actions include eliciting divergent activity patterns in normally coactive, electrically coupled neurons, providing an opportunity to examine peptide modulation of electrically coupled neurons in a functional context.

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus.

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods.

Molecular characterization of putative neuropeptide, amine, diffusible gas and small molecule transmitter biosynthetic enzymes in the eyestalk ganglia of the American lobster, Homarus americanus.

The American lobster, Homarus americanus, is a model for investigating the neuromodulatory control of physiology and behavior. Prior studies have shown that multiple classes of chemicals serve as locally released/circulating neuromodulators/neurotransmitters in this species. Interestingly, while many neuroactive compounds are known from Homarus, little work has focused on identifying/characterizing the enzymes responsible for their biosynthesis, despite the fact that these enzymes are key components for regulating neuromodulation/neurotransmission. Here, an eyestalk ganglia-specific transcriptome was mined for transcripts encoding enzymes involved in neuropeptide, amine, diffusible gas and small molecule transmitter biosynthesis. Using known Drosophila melanogaster proteins as templates, transcripts encoding putative Homarus homologs of peptide precursor processing (signal peptide peptidase, prohormone processing protease and carboxypeptidase) and immature peptide modifying (glutaminyl cyclase, tyrosylprotein sulfotransferase, protein disulfide isomerase, peptidylglycine-α-hydroxylating monooxygenase and peptidyl-α-hydroxyglycine-α-amidating lyase) enzymes were identified in the eyestalk assembly. Similarly, transcripts encoding full complements of the enzymes responsible for dopamine [tryptophan-phenylalanine hydroxylase (TPH), tyrosine hydroxylase and DOPA decarboxylase (DDC)], octopamine (TPH, tyrosine decarboxylase and tyramine ß-hydroxylase), serotonin (TPH or tryptophan hydroxylase and DDC) and histamine (histidine decarboxylase) biosynthesis were identified from the eyestalk ganglia, as were those responsible for the generation of the gases nitric oxide (nitric oxide synthase) and carbon monoxide (heme oxygenase), and the small molecule transmitters acetylcholine (choline acetyltransferase), glutamate (glutaminase) and GABA (glutamic acid decarboxylase). The presence and identity of the transcriptome-derived transcripts were confirmed using RT-PCR. The data presented here provide a foundation for future gene-based studies of neuromodulatory control at the level of neurotransmitter/modulator biosynthesis in Homarus.

Molecular evidence for an intrinsic circadian pacemaker in the cardiac ganglion of the American lobster, Homarus americanus - Is diel cycling of heartbeat frequency controlled by a peripheral clock system?

Whether cardiac output in decapod crustaceans is under circadian control has long been debated, with mixed evidence for and against the hypothesis. Moreover, the locus of the clock system controlling cardiac activity, if it is under circadian control, is unknown. However, a report that the crayfish heart in organ culture maintains a circadian oscillation in heartbeat frequency suggests the presence of a peripheral pacemaker within the cardiac neuromuscular system itself. Because the decapod heart is neurogenic, with contractions controlled by the five motor and four premotor neurons that make up the cardiac ganglion (CG), a likely locus for a circadian clock is the CG itself. Here, a CG-specific transcriptome was generated for the lobster, Homarus americanus, and was used to assess the presence/absence of transcripts encoding putative clock-related proteins in the ganglion. Using known Homarus brain/eyestalk ganglia clock-related proteins as queries, BLAST searches of the CG transcriptome were conducted for the five proteins that form the core clock, i.e., clock, cryptochrome 2, cycle, period and timeless, as well as for a variety of clock-associated, clock input pathway and clock output pathway proteins. With the exception of pigment dispersing hormone receptor [PDHR], a putative clock output pathway protein, one or more transcripts encoding each of the proteins searched for were identified from the CG assembly; no PDHR-encoding transcripts were found. RT-PCR confirmed the expression of all core clock transcripts in multiple independent CG cDNAs; RNA-Seq data suggest that both the motor and premotor neurons could contribute to the cellular locus of a pacemaker. These data provide support for the possible existence of an intrinsic circadian clock in the H. americanus CG, and form a foundation for guiding future anatomical, molecular and physiological investigations of circadian signaling in the lobster cardiac neuromuscular system.

Characterization of the mature form of a ß-defensin-like peptide, Hoa-D1, in the lobster Homarus americanus.

We report on the characterization of the native form of an American lobster, Homarus americanus, ß-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to ß-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system.

Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system.

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.