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).

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.

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.

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.

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.

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.

Identification of the molecular components of a putative Jasus edwardsii (Crustacea; Decapoda; Achelata) circadian signaling system.

Like all organisms, members of the crustacean order Decapoda must coordinate their physiology and behavior to accommodate recurring patterns of environmental change. Genetically encoded biological clocks are responsible, at least in part, for the proper timing of these organism-environment patternings. While biological clocks cycling on a wide range of timescales have been identified, the circadian signaling system, which serves to coordinate physiological/behavioral events to the solar day, is perhaps the best known and most thoroughly investigated. While many circadian patterns of physiology/behavior have been documented in decapods, few data exist concerning the identity of circadian genes/proteins in members of this taxon. In fact, large collections of circadian genes/proteins have been described from just a handful of decapod species. Here, a publicly accessible transcriptome, produced from tissues that included the nervous system (brain and eyestalk ganglia), was used to identify the molecular components of a circadian signaling system for rock lobster, Jasus edwardsii, a member of the decapod infraorder Achelata. Complete sets of core clock (those involved in the establishment of the molecular feedback loop that allows for ~ 24-h cyclical timing), clock-associated (those involved in modulation of core clock output), and clock input pathway (those that allow for synchronization of the core clock to the solar day) genes/proteins are reported. This is the first description of a putative circadian signaling system from any member of the infraorder Achelata, and as such, expands the decapod taxa for which complete complements of putative circadian genes/proteins have been identified.

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.

Non-amidated and amidated members of the C-type allatostatin (AST-C) family are differentially distributed in the stomatogastric nervous system of the American lobster, Homarus americanus.

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~ 14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~ 42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.

Prediction of a neuropeptidome for the eyestalk ganglia of the lobster Homarus americanus using a tissue-specific de novo assembled transcriptome.

In silico transcriptome mining is a powerful tool for crustacean peptidome prediction. Using homology-based BLAST searches and a simple bioinformatics workflow, large peptidomes have recently been predicted for a variety of crustaceans, including the lobster, Homarus americanus. Interestingly, no in silico studies have been conducted on the eyestalk ganglia (lamina ganglionaris, medulla externa, medulla interna and medulla terminalis) of the lobster, although the eyestalk is the location of a major neuroendocrine complex, i.e., the X-organ-sinus gland system. Here, an H. americanus eyestalk ganglia-specific transcriptome was produced using the de novo assembler Trinity. This transcriptome was generated from 130,973,220 Illumina reads and consists of 147,542 unique contigs. Eighty-nine neuropeptide-encoding transcripts were identified from this dataset, allowing for the deduction of 62 distinct pre/preprohormones. Two hundred sixty-two neuropeptides were predicted from this set of precursors; the peptides include members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon α, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, glycoprotein hormone ß5, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families. The predicted peptides expand the H. americanus eyestalk ganglia neuropeptidome approximately 7-fold, and include 78 peptides new to the lobster. The transcriptome and predicted neuropeptidome described here provide new resources for investigating peptidergic signaling within/from the lobster eyestalk ganglia.

Neuropeptidergic Signaling in the American Lobster Homarus americanus: New Insights from High-Throughput Nucleotide Sequencing.

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system.

Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family.

Distinct or shared actions of peptide family isoforms: I. Peptide-specific actions of pyrokinins in the lobster cardiac neuromuscular system.

Although the crustacean heart is modulated by a large number of peptides and amines, few of these molecules have been localized to the cardiac ganglion itself; most appear to reach the cardiac ganglion only by hormonal routes. Immunohistochemistry in the American lobster Homarus americanus indicates that pyrokinins are present not only in neuroendocrine organs (pericardial organ and sinus gland), but also in the cardiac ganglion itself, where pyrokinin-positive terminals were found in the pacemaker cell region, as well as surrounding the motor neurons. Surprisingly, the single pyrokinin peptide identified from H. americanus, FSPRLamide, which consists solely of the conserved FXPRLamide residues that characterize pyrokinins, did not alter the activity of the cardiac neuromuscular system. However, a pyrokinin from the shrimp Litopenaeus vannamei [ADFAFNPRLamide, also known as Penaeus vannamei pyrokinin 2 (PevPK2)] increased both the frequency and amplitude of heart contractions when perfused through the isolated whole heart. None of the other crustacean pyrokinins tested (another from L. vannamei and two from the crab Cancer borealis) had any effect on the lobster heart. Similarly, altering the PevPK2 sequence either by truncation or by the substitution of single amino acids resulted in much lower or no activity in all cases; only the conservative substitution of serine for alanine at position 1 resulted in any activity on the heart. Thus, in contrast to other systems (cockroach and crab) in which all tested pyrokinins elicit similar bioactivities, activation of the pyrokinin receptor in the lobster heart appears to be highly isoform specific.

Neuropeptide discovery in the Araneae (Arthropoda, Chelicerata, Arachnida): elucidation of true spider peptidomes using that of the Western black widow as a reference.

The public deposition of large transcriptome shotgun assembly (TSA) datasets for the Araneae (true spiders) provides a resource for determining the structures of the native neuropeptides present in members of this chelicerate order. Here, the Araneae TSA data were mined for putative peptide-encoding transcripts using the recently deduced neuropeptide precursors from the Western black widow Latrodectus hesperus as query templates. Neuropeptide-encoding transcripts from five spiders, Latrodectus tredecimguttatus, Stegodyphus mimosarum, Stegodyphus lineatus, Stegodyphus tentoriicola and Acanthoscurria geniculata, were identified, including ones encoding members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, CAPA/periviscerokinin/pyrokinin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. A total of 156 distinct peptides were predicted from the precursor proteins deduced from the S. mimosarum transcripts, with 65, 26, 21 and 12 peptides predicted from those deduced from the A. geniculata, L. tredecimguttatus, S. lineatus and S. tentoriicola sequences, respectively. Among the peptides identified were variant isoforms of FLP, orcokinin and TRP, peptides whose structures are similar to ones previously identified from L. hesperus. The prediction of these atypical peptides from multiple spiders suggests that they may be broadly conserved within the Araneae rather than being species-specific variants. Taken collectively, the data described here greatly expand the number of known Araneae neuropeptides, providing a foundation for future functional studies of peptidergic signaling in this important Chelicerate order.

In silico characterization of the neuropeptidome of the Western black widow spider Latrodectus hesperus.

Technological advancements in high-throughput sequencing have resulted in the production/public deposition of an ever-growing number of arthropod transcriptomes. While most sequencing projects have focused on hexapods, transcriptomes have also been generated for members of the Chelicerata. One chelicerate for which a large transcriptome has recently been released is the Western black widow Latrodectus hesperus, a member of the Araneae (true spiders). Here, a neuropeptidome for L. hesperus was predicted using this resource. Thirty-eight peptide-encoding transcripts were mined from the L. hesperus transcriptome, with 216 distinct peptides predicted from the deduced pre/preprohormones. The identified peptides included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon ß, CAPA/periviscerokinin/pyrokinin, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, neuropeptide F (NPF), orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. Of particular note were the identifications of a carboxyl (C)-terminally extended corazonin, FLPs possessing -IMRFamide, -MMYFamide, and -MIHFamide C-termini, a NPF and a sulfakinin each ending in -RYamide rather than -RFamide, a precursor whose orcokinins include C-terminally amidated isoforms, and a collection of TRPs possessing -FXPXLamide rather than the stereotypical -FXGXLamide C-termini. The L. hesperus peptidome is by far the largest thus far published for any member of the Chelicerata. Taken collectively, these data serve as a reference for future neuropeptide discovery in the Araneae and provide a foundation for future studies of peptidergic control in L. hesperus and other spiders.