Showing 1 - 6 of 6 Items

Date: 2017-03-01

Creator: Andrew E. Christie, Vittoria Roncalli, Matthew C. Cieslak, Micah G. Pascual, Andy, Yu, Tess J. Lameyer, Meredith E. Stanhope, Patsy S. Dickinson

Access: Open access

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 β5, 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.

Date: 2018-12-01

Creator: Andrew E. Christie, Meredith E. Stanhope, Helen I. Gandler, Tess J. Lameyer, Micah G., Pascual, Devlin N. Shea, Andy Yu, Patsy S. Dickinson, J. Joe Hull

Access: Open access

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 β-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.

Date: 2018-03-01

Creator: Andrew E. Christie, Alexandra Miller, Rebecca Fernandez, Evyn S. Dickinson, Audrey, Jordan, Jessica Kohn, Mina C. Youn, Patsy S. Dickinson

Access: Open access

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.

Date: 2020-12-01

Creator: J. Joe Hull, Melissa A. Stefanek, Patsy S. Dickinson, Andrew E. Christie

Access: Open access

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.

Date: 2020-06-01

Creator: Andrew E. Christie, J. Joe Hull, Patsy S. Dickinson

Access: Open access

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.

Date: 2018-07-01

Creator: Andrew E. Christie, Andy Yu, Micah G. Pascual, Vittoria Roncalli, Matthew C., Cieslak, Amanda N. Warner, Tess J. Lameyer, Meredith E. Stanhope, Patsy S. Dickinson, J. Joe Hull

Access: Open access

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.