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Alignment of Homarus americanus and related signal peptide peptidases. (A) MAFFT alignment of H. americanus signal peptide peptidase (Homam-SPP) and Drosophila melanogaster signal peptide peptidase (Drome-SPP; Accession No. AAF51486; Adams et al., 2000). (B) MAFFT alignment of Homam-SPP and Daphnia pulex signal peptide peptidase (Dappu-SPP; Accession No. EFX80723; Colbourne et al., 2011). In the line immediately below each sequence grouping, "*" indicates identical amino acid residues, while ":" and "." denote amino acids that are similar in structure between sequences. In this figure, signal peptide peptidase domains identified by Pfam analyses are highlighted in yellow.
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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, li...
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... protein listed as "full-length" exhibits a "start" methionine and is flanked on its C-terminus by a stop codon. Proteins described here as "partial" lack either a start methionine (referred to as C-terminal partial proteins) or a stop codon (referred to as N-terminal partial proteins); the amino acid sequences of all proteins deduced from the lobster eyestalk transcriptome can be found in Supplemental Figure 1. To confirm that each of the proteins identified here is most similar to the D. melanogaster sequence used to identify the transcript encoding it, each Homarus protein was used as the input query in a BLAST search of the annotated Drosophila protein dataset present in FlyBase (version FB2016_05; Gramates et al., 2017). ...
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... searches of the eyestalk ganglia transcriptome using known D. melanogaster proteins as the query sequences identified transcripts encoding putative homologs of each of the abovementioned neuropeptide precursor processing and immature peptide modifying enzymes ( Table 1). Translation of these sequences revealed one SPP (Homam-SPP; Fig. 1), one PPP (Homam-PPP), one CP (Homam-CP), one QC (Homam-QC), one TPST (Homam-TPST), two PDIs (Homam-PDI-v1 and v2), one PHM (Homam-PHM; Fig. 2) and three PALs (Homam-PAL-I-v1 and v2 and Homam-PAL-II; Fig. 3). All of the proteins deduced from the eyestalk ganglia transcriptome appear to be full-length sequences, with the exception of ...
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... which they were named, each lobster protein was analyzed for structural domains using Pfam, and the identified domains were compared to those predicted by the program for the protein's top FlyBase and NCBI arthropod non-redundant protein hits (see above). As expected, the domain complements predicted by Pfam for each Homarus sequence (Table 4) (Fig. 1). Similarly, one copper type II ascorbate-dependent monooxygenase N-terminal domain and one copper type II ascorbate-dependent monooxygenase C-terminal domain were predicted by Pfam in Homam-PHM (Fig. 2), which is the same domain complement predicted by the program for both the D. melanogaster (Accession No. AAF47127; ; Fig 2012; Bucher ...
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... isoforms of the abovementioned proteins as the query sequence identified transcripts encoding putative homologs of each enzyme in the H. americanus eyestalk ganglia transcriptome ( Table 1). Translation of the identified lobster transcripts revealed one CHAT (Homam-CHAT; Fig. 9), six GLSs (Homam-GLS-I-v1 through v4 and Homam-GLS-II-v1 and v2; Fig. 10), which are likely the products of two different genes, both showing probable alternative splicing, and two GADs (Homam-GAD-I and Homam-GAD-II; Fig. 11), which are, in all likelihood, the products of different genes. With the exception of Homam-GLS-I-v4, which is a C-terminal partial protein, the deduced H. americanus small molecule ...
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... eyestalk ganglia transcriptome ( Table 1). Translation of the identified lobster transcripts revealed one CHAT (Homam-CHAT; Fig. 9), six GLSs (Homam-GLS-I-v1 through v4 and Homam-GLS-II-v1 and v2; Fig. 10), which are likely the products of two different genes, both showing probable alternative splicing, and two GADs (Homam-GAD-I and Homam-GAD-II; Fig. 11), which are, in all likelihood, the products of different genes. With the exception of Homam-GLS-I-v4, which is a C-terminal partial protein, the deduced H. americanus small molecule transmitter biosynthetic enzymes all appear to be full-length ...
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... above are expressed in the eyestalk ganglia, fragments (~ 500 bp) of the respective transcripts were amplified using RT-PCR and compared against the transcriptome sequences. Each of the 23 transcripts assessed was consistently amplified with varying degrees of efficiency from each of the three independent eyestalk ganglia cDNA replicates ( Fig. 12; representative image). Demonstration that the PCR products exhibited >99% nucleotide identity with their respective transcriptome-derived sequences further supports eyestalk expression and accuracy of the de novo ...
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... of neuromodulator-associated hydroxylase families (four classes of substrate) and decarboxylase families (five classes of substrate). The respective maximum-likelihood cladograms revealed high bootstrap support for clade-specific clustering of the enzyme families, with each of the Homarus protein sequences sorting as initially annotated (Figs. 13 and ...
Citations
... Accession numbers for the sequences used to construct the tree are available in Supplementary Table S1 and nucleotide and peptide sequences of P. ornatus sequences for the processing enzymes are available in Supplementary Table S3. For other processing enzymes involved with neuropeptide maturation, homologue nucleotide sequences from Homarus americanus (Christie et al., 2018) were obtained from NCBI, translated using Expasy Translate (https://web.expasy.org/translate/) and submitted to a tBLASTN search of P. ornatus adult tissues using CrustyBase as mentioned previously. ...
... Studies on decapod proprotein convertases (PCs) have identified the presence of PC2 in the eyestalk ganglia of the crayfish O. limosus (Toullec et al., 2002) and American lobster H. americanus (Christie et al., 2018), as a major site for the production of neuropeptides in decapods. Studies on the substrates of PC2 have been carried out in the model fly D. melanogaster, where PC2 (encoded by the amontillado gene in this species) is likely to be involved with the processing of multiple neuropeptide precursors including corazonin and AKH (Rhea et al., 2010). ...
Neuropeptides are commonly produced in the neural tissues yet can have effects on far-reaching targets, with varied biological responses. We describe here the neuropeptidome of the ornate spiny lobster, Panulirus ornatus, a species of emerging importance to closed-system aquaculture, with a focus on peptide hormones produced by the reproductive tissues.
Transcripts for a precursor to one neuropeptide, adipokinetic hormone/corazonin-related peptide (ACP) were identified in high numbers in the sperm duct of adult spiny lobsters suggesting a role for ACP in the reproduction of this species. Neuropeptide production in the sperm duct may be linked with physiological control of spermatophore production in the male, or alternatively may function in signalling to the female.
The enzymes which process nascent neuropeptide precursors into their mature, active forms have seldom been studied in decapods, and never before at the multi-tissue level. We have identified transcripts for multiple members of the proprotein convertase subtisilin/kexin family in the ornate spiny lobster, with some enzymes showing specificity to certain tissues. In addition, other enzyme transcripts involved with neuropeptide processing are identified along with their tissue and life stage expression patterns.
... Many species of crustacean decapods have been utilized over the past decades as biomedical models to understand neural network and physiology due to neurons of large size and relatively simple nervous circuits. Recent high-throughput approaches, as new generation genoma and transcriptoma analysis, made it possible to identify molecules that are involved in the neuromodulator/neurotransmitter pathways and in their control [17]. ...
Invertebrates represent about 95% of existing species, and most of them belong to aquatic ecosystems. Marine invertebrates are found at intermediate levels of the food chain and, therefore, they play a central role in the biodiversity of ecosystems. Furthermore, these organisms have a short life cycle, easy laboratory manipulation, and high sensitivity to marine pollution and, therefore, they are considered to be optimal bioindicators for assessing detrimental chemical agents that are related to the marine environment and with potential toxicity to human health, including neurotoxicity. In general, albeit simple, the nervous system of marine invertebrates is composed of neuronal and glial cells, and it exhibits biochemical and functional similarities with the vertebrate nervous system, including humans. In recent decades, new genetic and transcriptomic technologies have made the identification of many neural genes and transcription factors homologous to those in humans possible. Neuroinflammation, oxidative stress, and altered levels of neurotransmitters are some of the aspects of neurotoxic effects that can also occur in marine invertebrate organisms. The purpose of this review is to provide an overview of major marine pollutants, such as heavy metals, pesticides, and micro and nano-plastics, with a focus on their neurotoxic effects in marine invertebrate organisms. This review could be a stimulus to bio-research towards the use of invertebrate model systems other than traditional, ethically questionable, time-consuming, and highly expensive mammalian models.
... Although these datasets were initially developed to serve a variety of functions, they have proven to be powerful resources for a wide array of gene and, by proxy, protein discoveries. Crustacean genomes and transcriptomes have been extensively exploited to facilitate the identification of the molecular components (genes and proteins) of peptidergic signaling systems, including peptide precursors (e.g., Bao et al., 2015;Christie and Hull, 2019;Christie and Pascual, 2016;Christie and Yu, 2019;Christie et al., 2015;Nguyen et al., 2016;Oliphant et al., 2018;Veenstra, 2015Veenstra, , 2016, peptide processing enzymes (Christie et al., 2018c), and peptide receptors (e.g., Bao et al., 2018;Buckley et al., 2016;Christie and Hull, 2019;Christie and Yu, 2019;Christie et al., 2015;Dickinson et al., 2019;Oliphant et al., 2018;Tran et al., 2019;Veenstra, 2016). ...
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.
... A publicly accessible mixed nervous system (supraoesophageal ganglion [brain], abdominal nerve cord, cardiac ganglion, and stomatogastric nervous system) assembly exists for H. americanus (BioProject No. PRJNA300643;Northcutt et al. 2016), as do several nervous system regionspecific ones, e.g., those for the brain (BioProject No. PRJNA379629; Christie et al. 2018a) and eyestalk ganglia (BioProject No. PRJNA338672;Christie et al. 2017). These and other Homarus assemblies have proven to be powerful resources from which to identify genes/proteins in specific areas of interest, e.g., those involved in neuropeptidergic signaling (e.g., Christie et al. 2015Christie et al. , 2017Christie et al. , 2018c and those involved in the establishment of neuronal circadian signaling systems (Christie et al. 2018a, b), providing substrates for initiating molecular and physiological studies of neural control in H. americanus, and by proxy, other members of the Decapoda. ...
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.
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.
The biogenic amines, tyramine and octopamine, in the octopaminergic synthesis pathway play critical roles in regulating physiological and immunological homeostasis in Litopenaeus vannamei. Tyrosine decarboxylase (TDC) is an enzyme catalyzing the first decarboxylation step in the biosynthesis of tyramine and octopamine. The full-length gene sequence of TDC cloned from the brain of L. vannamei (LvTDC) was predicted to encode a 779-amino acid protein with a pyridoxal-dependent decarboxylase-conserved domain in close phylogenetic relationship with arthropod TDCs. LvTDC gene expression was found to be abundant in nervous thoracic ganglia. RNA interference was used to assess the immune and physiological function of LvTDC. The LvTDC knockdown shrimp revealed significant decreases in the total haemocyte count, hyaline cells, antimicrobial peptides, respiratory bursts, gene expression, respiratory bursts of haemocytes per unit of haemolymph, and phagocytic activity and clearance efficiency toward Vibrio alginolyticus. Furthermore, LvTDC knockdown was accompanied by decreases in octopamine deficiency. In the V. alginolyticus challenge test, the survival rate of LvTDC knockdown shrimp was lower than the shrimp injected with DEPC-water or GAPDH-dsRNA. In conclusion, the cloned LvTDC was responsible for octopaminergic synthesis, which then regulated physiological and immune responses in L. vannamei.
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.
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 β-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.
