Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a Painless homolog.

The transient receptor potential (TRP) family of cation channels are evolutionarily conserved proteins with critical roles in sensory physiology. Despite extensive studies in model species, knowledge of TRP channel functional diversity and physiological impact remains limited in many non-model insect species. To assess the TRP channel repertoire in a non-model agriculture pest species (Lygus hesperus), publicly available transcriptomic datasets were mined for potential homologs. Among the transcripts identified, 30 are predicted to encompass complete open reading frames that encode proteins representing each of the seven TRP channel subfamilies. Although no homologs were identified for the Pyrexia and Brivido channels, the TRP complement in L. hesperus exceeded the 13-16 channels reported in most insects. This diversity appears to be driven by a combination of alternative splicing, which impacted members of six subfamilies, and gene expansion of the TRPP subfamily. To validate the in silico data and provide more detailed analyses of L. hesperus TRP functionality, the putative Painless homolog was selected for more in depth analysis and its functional role in thermosensation examined in vitro. RT-PCR expression profiling revealed near ubiquitous expression of the Painless transcript throughout nymphal and adult development. Electrophysiological data generated using a Xenopus oocyte recombinant expression system indicated activation parameters for L. hesperus Painless homolog that are consistent with a role in noxious heat (40°-45 °C) thermosensation.

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.

Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning.

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.

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.

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.

Identification of putative neuropeptidergic signaling systems in the spiny lobster, Panulirus argus.

Members of the decapod infraorder Achelata, specifically species from the genus Panulirus, have storied histories as models for investigating the basic principles governing the generation, maintenance, and modulation of rhythmic motor behavior, including modulation by locally released and circulating peptides. Despite their contributions to our understanding of peptidergic neuromodulation, little is known about the identity of the native neuropeptides and neuronal peptide receptors present in these crustaceans. Here, a Panulirus argus nervous system-specific transcriptome was used to help fill this void, providing insight into the neuropeptidome and neuronal peptide receptome of this species. A neuropeptidome consisting of 266 distinct peptides was predicted using the P. argus assembly, 128 having structures placing them into a generally recognized arthropod peptide family: agatoxin-like peptide, allatostatin A (AST-A), allatostatin B, allatostatin C, bursicon, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31 (DH31), ecdysis-triggering hormone (ETH), FMRFamide-like peptide (FLP), glycoprotein hormone (GPH), GSEFLamide, inotocin, leucokinin, myosuppressin, natalisin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, periviscerokinin, pigment-dispersing hormone, pyrokinin, red pigment-concentrating hormone, RYamide, short neuropeptide F (sNPF), SIFamide, sulfakinin, tachykinin-related peptide (TRP), and trissin. Twenty-five putative neuronal receptors, encompassing 15 peptide groups, were also identified from the P. argus transcriptome: AST-A, bursicon, CCHamide, DH31, diuretic hormone 44, ETH, FLP, GPH, inotocin, insulin-like peptide, myosuppressin, natalisin, periviscerokinin, sNPF, and TRP. Collectively, the reported data provide a powerful resource for expanding studies of neuropeptidergic control of physiology and behavior in members of the genus Panulirus specifically, and decapods generally.

Assessment of midgut enteroendocrine peptide complement in the honey bee, Apis mellifera.

Peptides modulate physiological/behavioral control systems in all animals. In arthropods, midgut epithelial endocrine cells are one of the largest sources of these signaling agents. At present, little is known about the identity of the peptides that form arthropod midgut enteroendocrine peptidomes. While many techniques can be used for peptide structural identification, in silico transcriptome mining is one that has been used extensively for arthropod neuropeptidome prediction; this strategy has yet to be used for large-scale arthropod enteroendocrine peptide discovery. Here, a tissue-specific transcriptome was used to assess putative enteroendocrine peptide complement in the honey bee, Apis mellifera, midgut. Searches for transcripts encoding members of 42 peptide families were conducted, with evidence of expression for 15 groups found in the assembly: adipokinetic hormone, allatostatin A, allatostatin C, bursicon, CCHamide, CNMamide, diuretic hormone 31, diuretic hormone 44, insulin-like peptide, myosuppressin, neuropeptide F, pigment dispersing hormone, pyrokinin, short neuropeptide F, and tachykinin-related peptide. The proteins deduced from the midgut transcripts are identical in sequence, or nearly so, to those of Apis pre/preprohormones deposited previously into NCBI, providing increased confidence in the accuracy of the reported data. Seventy-five peptides were predicted from the deduced precursor proteins, 26 being members of known peptide families. Comparisons to previously published mass spectrometric data support the existence of many of the predicted Apis peptides. This study is the first prediction of an arthropod midgut peptidome using transcriptomics, and provides a powerful new resource for investigating enteroendocrine peptide signaling within/from the Apis midgut, a species of significant ecological/economic importance.

Identification of putative amine receptor complement in the eyestalk of the crayfish, Procambarus clarkii.

In decapod crustaceans, the amines dopamine, octopamine, serotonin, and histamine are known to serve as locally released and/or circulating neuromodulators. While many studies have focused on determining the modulatory actions of amines on decapod nervous systems, comparatively little is known about the identity of the receptors through which they exert their actions. Here, a crayfish, Procambarus clarkii, tissue-specific transcriptome was used to identify putative amine receptors in the eyestalk, a structure composed largely of the eyestalk ganglia, including the neuroendocrine X-organ-sinus gland system, and retina. Transcripts encoding 17 distinct putative amine receptors, three dopamine (one dopamine 1-like, one dopamine 2-like, and one dopamine/ecdysteroid-like), five octopamine (one alpha-like, three beta-like, and one octopamine/tyramine-like), three serotonin (two type-1-like and one type-7-like), and six histamine (five histamine-gated chloride channel A-like and one histamine-gated chloride channel B-like) were identified in the assembly. Comparison of the nucleotide sequence of the transcript encoding one predicted type-1-like serotonin receptor with that cloned previously from the P. clarkii nervous system shows the two sequences to be essentially identical, providing increased support for the validity of the transcripts used to deduce the proteins reported here. Reciprocal BLAST and structural/functional domain analyses support the protein family annotations ascribed to the putative P. clarkii receptors. These data represent the first large-scale description of amine receptors from P. clarkii, and as such provide a new resource for initiating gene-based studies of aminergic control of physiology/behavior at the level of receptors in this species.

What can transcriptomics reveal about the phylogenetic/structural conservation, tissue localization, and possible functions of CNMamide peptides in decapod crustaceans?

Over the past several years, in silico analyses of arthropod genomes/transcriptomes have led to the identification of several previously unknown peptide families. The CNMamides are one such peptide group, having been discovered via computational analyses of the fruit fly, Drosophila melanogaster, genome; both a CNMamide precursor and receptor were identified. Recently, a CNMamide family member, VMCHFKICNLamide (disulfide bridging between the cysteine residues), was predicted via in silico mining of a crayfish, Procambarus clarkii, transcriptome, suggesting the presence of this peptide group in members of the Decapoda. Here, using publically accessible transcriptomic data, the phylogenetic/structural conservation, tissue localization, and possible functions of the CNMamide family in decapods were explored. Evidence for CNMamide precursors was found for members of each decapod infraorder for which significant sequence data are available, suggesting a ubiquitous conservation of the CNMamide family in the Decapoda. For the Penaeoidea, Caridea, Astacidea and Achelata, the isoform of CNMamide originally identified from P. clarkii appears to be ubiquitously conserved; in members of the Brachyura, VMCHFKICNMamide (disulfide bridging between the cysteine residues) is the native isoform. Interestingly, the decapod CNMamide gene appears to also have a splice variant in which the carboxy-terminal portion of the preprohormone containing the CNMamide peptide is replaced by one containing a different disulfide bridged peptide that is structurally unrelated to it; this second peptide shows considerable conservation within, but variation among, decapod infraorders. A highly conserved putative CNMamide receptor was identified from members of the Penaeoidea, Astacidea and Brachyura. Phylogenetic analyses support the annotation of the decapod receptor as a true member of the CNMamide receptor family. The presence of precursor and receptor transcripts in both nervous system- and reproductive tissue-specific transcriptomes suggests CNMamides serve as modulators of decapod neural and reproductive control systems.

Identification of putative amine biosynthetic enzymes in the nervous system of the crab, Cancer borealis.

Amines function as neuromodulators throughout the animal kingdom. In decapod crustaceans, the amines serving neuromodulatory roles include dopamine, octopamine, serotonin and histamine. While much work has focused on examining the physiological effects of amines on decapod nervous systems, the identity of the native enzymes involved in their biosynthesis remains largely unknown. In an attempt to help fill this void, a transcriptome generated from multiple portions of the crab, Cancer borealis, nervous system, a species that has long served as a model species for investigating the neuromodulatory control of rhythmically active neural networks, was used to identify putative amine biosynthetic enzyme-encoding transcripts, and by proxy, proteins. Transcripts encoding full complements of the enzymes involved in the production of dopamine, octopamine, serotonin, and histamine were deduced from the C. borealis assembly, i.e., tryptophan-phenylalanine hydroxylase, tyrosine hydroxylase, DOPA decarboxylase, tyrosine decarboxylase, tyramine ß-hydroxylase, tryptophan hydroxylase, and histidine decarboxylase. All proteins deduced from the C. borealis transcripts appear to be full-length sequences, with reciprocal BLAST and structural domain analyses supporting the protein family annotations ascribed to them. These data provide the first descriptions of the native amine biosynthetic enzymes of C. borealis, and as such, serve as a resource for initiating gene-based studies of aminergic control of physiology and behavior at the level of biosynthesis in this important biomedical model.

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.