Deep sequencing of transcriptomes from the nervous systems of two decapod crustaceans to characterize genes important for neural circuit function and modulation

Mixed nervous system transcriptome sequencing and de novo assembly

Constructing RNA-seq libraries from nervous tissues of adult crustaceans, a total of 414,978,768 and 452,237,240 raw reads were obtained from the paired - end sequencing of C. borealis and H. americanus, respectively. The average read length for both species was approximately 97 bp, as expected for 100 bp paired-end Illumina Sequencing. Following quality checks removing adaptors, contaminating sequences, and low-quality sequences, 391,060,790 (94.2 %) clean reads were found for C. borealis and 426,712,238 (94.4 %) for H. americanus. These high-quality cleaned reads were subsequently assembled de novo into contigs using two different assemblers: CLC Genomics and Seqman NGen. For C. borealis, CLC assembly resulted in 42,766 contigs with an average length of 1544 bp and an N50 length of 2178 bp, while SeqMan assembly resulted in 67,380 contigs with an average length of 1076 bp and N50 of 1239 (Table 1; Fig. 1a). For H. americanus, CLC assembly resulted in 60,273 contigs with an average length of 1657 bp and N50 length of 2357 bp, while SeqMan NGen resulted in 45,043 contigs with an average length of 1799 bp and N50 of 2258 (Table 1; Fig. 1a).

To compare the quality of our transcriptomes from multiple assemblies, a reference-based alignment was performed using BUSCO [23]. The arthropod BUSCO reference contains 2675 orthologous genes expected within most arthropod species that were compared against the gene content of our transcriptomes. The alignment of our transcriptomes against the arthropod reference resulted in similar percentages of the reference genes found as complete (C), fragmented (F), or missing (M) across our transcriptomes (Fig. 1b). The C. borealis metrics were C: 59.0 %, F: 13.5 %, and M: 27.5 % for the CLC Genomics assembly and C: 58.4 %, F: 20.1 %, and M: 21.5 % for the SeqMan NGen assembly. The H. americanus metrics were C: 56.1 %, F: 16.4 %, and M: 27.5 % for the CLC Genomics assembly and C: 66.5 %, F: 13.4 %, and M: 20.1 % for the SeqMan NGen assembly. These results were compared against the arthropod transcriptome reference scores provided in the BUSCO supplementary materials, a recently published Homarus americanus nervous system transcriptome assembled using Trinity [5], and a recent transcriptome of the freshwater crayfish Astacus astacus [29]. Our results are comparable with the Astacus transcriptome in completeness and an apparent extension of the published Homarus americanus transcriptome [5]. One possible explanation for the missing arthropod genes from our transcriptomes can be explained by the fact that our sequences were derived solely from nervous system tissue, while the references were built from arthropod genomic sequences.

Using the NCBI BLAST+ suite to perform a blastn of 28 Cancer borealis sequences already contained within GenBank against our assembled contigs, we found an average sequence identity from the CLC assembly of 99.2 %, with the lowest identity score 96 %. SeqMan NGen assembly for C. borealis transcripts had an average sequence identity of 99.03 %, and the lowest percent identity was 95 %. For H. americanus, 75 GenBank sequences were aligned against our transcriptome, resulting in an average sequence identity to CLC assembled sequences of 99.3 % with the lowest being 94.5 %. SeqMan NGen assembly for H. americanus transcripts had an average sequence identity of 98.97 %, and the lowest percent identity was 87 %.

Based on the relative similarity in many of the metrics for these two assembly methods, the somewhat better performance of CLC contigs when compared with Sanger sequencing generated orthologs, and the fact that portions of the H. americanus transcriptome based on the CLC assembly have previously been published [4], we chose to perform the remaining representative analysis of these sequence data based on the CLC assembled contigs. The H. americanus Transcriptome Shotgun Assembly (TSA) project has been deposited at GenBank under Accession No. GEBG00000000 (BioProject No. PRJNA300643; BioSample No. SAMN04230440). The C. borealis Transcriptome Shotgun Assembly (TSA) project has been deposited at GenBank under the Accession No. GEFB00000000 (BioProject No. PRJNA310325; BioSample No. SAMN04450329). The versions described in this paper represent the first versions, GEBG01000000 and GEFB01000000 respectively.

Annotation and gene ontology mapping

Entrez Gene IDs were obtained for both transcriptomes using blastx against the NCBI non-redundant (NR) protein database. These annotations consisted of 9489 unique proteins among C.borealis transcripts, and 11,061 among H, americanus transcripts. Mapping these gene IDs to Gene Ontology (GO) categories yielded 9351 (22 %) of the C. borealis and 6191 (10 %) of the H. americanus contigs successfully identified (Fig. 2a). Similar percentages have been observed in other de novo transcriptome analyses [27, 28]. From the functional annotation, transcripts were classified into three broad categories: cellular compartment (CC), molecular function (MF), and biological process (BP) [24]. Within these broad categorizations, the highest abundance GO terms of H. americanus and C. borealis were compared against each other, which included the top 9 CCs, 18 MFs, and 16 BPs for both species (Fig. 2b). The arrangement of GO terms was based on the highest abundance H. americanus terms, in descending order. The only notable exception to this order was the BP GO term “RNA-dependent DNA Replication” ontology due to its high abundance in C. borealis but relatively low abundance in H. americanus. These same GO terms were compared between the two species using the relative percentage of each GO term for its broad GO classification (CC, MF, BP) (Fig. 3). The most striking differences between the GO ontologies of each species include a much higher incidence of “protein binding” terms for C. borealis MF than that of H. americanus, a much higher incidence of “metabolic process” in H. americanus BP, and a prominent difference between the “RNA-dependent DNA Replication” term for BP. The source of these differences could be attributed to factors including, but not limited to, the variation in tissue types (abdominal nerve cord was used in H. americanus, but not C. borealis), depth of sequencing, or natural variation in transcript abundance.

Species comparisons: distribution and VennBLAST analysis

Using the Blast2GO software suite, the number of species that the C. borealis and H. americanus neural transcriptomes align with was determined from a blastx against the NCBI non-redundant database. The species distribution for both C. borealis and H. americanus gave similar top species, such as Tribolium castaneum, Daphnia pulex, and Strongylocentrotus purpuratus within the top 5 species hits (Fig. 4). The absence of termite (Zootermopsis nevadensis) from the C. borealis species distribution of blast hits is due to the fact that the Z. nevadensis protein sequences had yet to be uploaded to the NCBI non-redundant database at the time of the blast analysis of the C. borealis transcriptome, while the H. americanus analysis was performed after the Z. nevadensis reference became available.

Venn diagrams were generated (Fig. 5a) using the software VennBLAST [25] to compare the whole C. borealis and H. americanus transcriptomes against the Daphnia pulex (GCA_000187875.1) protein sequences from Ensembl Metazoa. The protein sequence database for D. pulex was chosen as a common subject to query the C. borealis and H. americanus transcriptomes against due to the mutual high top-hit species distribution (Fig. 4), as well as the well-annotated genome of the crustacean D. pulex [1]. Initially, a local blastx of C. borealis or H. americanus contigs against D. pulex protein sequences resulted in 17,343 and 14,818 hits, respectively. Upon overlapping these hits with the VennBLAST Merge tool, 11,258 hits from C. borealis and H. americanus were found to have the same top hit for D. pulex. A second analysis with increased stringency was performed using the VennBLAST Filter tool to retain only high-quality matches, leaving C. borealis with 7,460 and H. americanus with 7,268 hits to D. pulex. Subsequent merger of these filtered hits resulted in 6,226 common top-hits for D. pulex, resulting in an increased percentage (from 54 % overlap to 73 %) of common top-hits.

For the remainder of our transcriptome analysis, we identified and characterized sequences for 6 different Innexin proteins (gap junctions), 34 distinct ion channel types, 17 biogenic amine receptors, 5 GABA receptors, and 28 major transmitter receptor subtypes including glutamate and acetylcholine receptors. These are described in detail below. These receptor groups consisted of 27 different ligand-gated channel subunits (ionotropic receptors) and 23 metabotropic receptor types. To better quantify the similarity across lobster and crab, we performed analyses of percent amino acid identity and similarity between orthologs of a subset of genes (see Methods). Overall, the sequence similarity is very high between these species, as one might expect for members of the same Order (Fig. 5b); across 42 genes surveyed, there was a mean ± SD of 85.27 % ± 8.46 % amino acid identity between genes. However, we also noticed a significant trend across different types of gene products: there was significantly lower amino acid identity and similarity for metabotropic receptors than for the other classes of genes (Fig. 5b). In particular, the G-protein coupled (GPCRs) receptors that we analyzed were some of the most divergent between crab and lobster. For example, Octβ-R1, 3, and 4 shared 72, 72, and 74 % amino acid identity respectively. Conversely, the most highly conserved genes were in the Shaker family of voltage-gated K⁺ channels. Shaker, Shal, and Shaw1were 98, 98, and 96 % identical between crabs and lobsters. From these results we would predict more conservation in channel function and physiology across species than that of the GPCRs.

Ion channels

For our initial analysis of these crustacean transcriptomes, we decided to focus on some of the most critical proteins involved in nervous system function. We therefore first conducted an analysis of ion channel subtypes. Putative ion channels were identified based on tblastx searches utilizing the transcriptomes as a reference database and querying with known channel protein largely consisting of sequences from mouse (Mus musculus) and Drosophila melanogaster. A 100 % overlapping set of ion channels were found to be present in both C. borealis and H. americanus (Table 2; Fig. 6). We specifically hand-curated and annotated these channel sequences, and the full list is available in Table 2, including putative current types carried by each channel. We used the multiple sequence alignment (MSA) output from CLUSTALW [30] to develop a fairly comprehensive ion channel tree based on amino acid sequence similarity, allowing us to cluster channels by type to effectively interrogate the nervous system channel content of these crustaceans.

Our analysis of the crab and lobster transcriptomes led us to identify and characterize 34 distinct channel subtypes representing several gene families (Table 2; Fig. 6). Each identified channel transcript was found in both crab and lobster transcriptomes. Our analysis confirmed the presence of 3 major voltage-dependent calcium channel subtypes [31] corresponding one each to the L-type (CaV1), P/Q-type (CaV2), and T-type (CaV3) families of calcium channels. In addition, we identified a single member of the NaV-type voltage-gated Na⁺ channel representing the para type channel identified in other species. Finally, in both species we identified a non-selective sodium leak channel (NALCN) thought to underlie TTX-resistant Na⁺ conductance important in baseline neuronal excitability [32]. One other major family of non-selective cation channels we identified was the cyclic-nucleotide-gated channels of the HCN/CNG type. Both species contained one member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family, the channels that give rise to I_H type currents in crustacean neurons [33]. In addition, we identified 3 α -subunits and 1 β-subunit of the cyclic nucleotide gated ion channel types (CNG), which are activated by the binding of cAMP and cGMP to carry a non-selective cation current [34].

The pore-forming α-subunits of K⁺ channels can be sub-divided into voltage-dependent subunits (K_v), inward rectifiers (K_ir), two-pore subunits (K2P), and those activated by intracellular calcium (K_Ca) or sodium (K_Na) ions. We identified a diverse array of voltage-dependent potassium channel subtypes in the transcriptomes of both the crab and lobster (Table 2; Fig. 5). The best already characterized of these channels in crustaceans are the Shaker family of channels, having been identified in crab [35, 36] and spiny lobsters (Panulirus interruptus, [37, 38]), with the latter having an extensive characterization via expression in oocyte systems [39, 40]. Previously, 4 members of this family were already known from Cancer borealis: shaker, shal, shab, and shaw. We found orthologs to each of these in H. americanus as well. We further discovered that in both species there actually were 2 distinct shaw-related channel transcripts (Fig. 6). The newly re-named “Shaw1” transcript from this analysis is a perfect match with the previously identified shaw transcript from Cancer borealis (Accession #EF089569), while the newly identified shaw-like transcript is presented as Shaw2. We also identified two members of the KCNQ family of K⁺ channels. KCNQ genes encode a family of six transmembrane domain K⁺ channel alpha-subunits that have a wide range of physiological roles, including likely underlying the slow voltage-gated M-type currents [41]. Rounding out the voltage-dependent K⁺ channel subtypes are 3 members of the ether-a-go-go/KCNH family. In addition to voltage-dependent K⁺ channels, we also identified one K_ir channel (IRK), two members of the K2P family (KCNK), one sodium-activated K⁺ channel (KCNT), and two calcium-activated K⁺ channel types. These K_Ca channels had previously been identified in C. borealis [36] and correspond to one BK- and one SK- type channel.

Transient receptor potential (TRP) channels have been implicated as a primary channel for generation of sensations including temperature, taste, pain, pressure, and vision. In our analysis, we found various TRP subfamilies within both C. borealis and H. americanus (Table 2; Fig. 6). These subfamilies included TRPV (vanilloid), mediating odor and pain sensations; TRPA (ankyrin), associated with mechanical stress receptors; TRPM (melastatin), associated with magnesium reabsorption [42]; and TRP pyrexia, a thermal sensing receptor [43]. In crustaceans, TRP channels have been primarily studied for their role in olfactory reception [44] and stretch reception [45]. Not all orthologs of TRP channels were identified in both species. We did not identify in this data set orthologs of TRP-V6 and pyrexia from C. borealis and an ortholog of TRP-M1 was not identified in H. americanus. It is most likely that these “missing” orthologs are due to limitations in the sequence depth, although we cannot rule out the possibility that these two species have distinct complements of TRP channel genes. No sequences were found in either species that represent the TRPC, TRPP, TRPL, or TRPN subfamilies. These results are consistent with found in a previously published transcriptome of H. americanus that identified 2 TRPA, one pyrexia, and two TRPM type channels [5]. We extend these results to include identification of the TRPV family of channels in both H. americanus and C. borealis.

Biogenic amine receptors

Biogenic amine neuromodulators were some of the first modulatory compounds to be thoroughly studied in the crustacean nervous system [46–49], and specifically in the stomatogastric nervous system [47, 50–52] where some of the most comprehensive understanding of the multiple targets and modulatory impacts of these compounds on neural circuits has been described [53–59]. Therefore, we decided it would be valuable to provide a thorough characterization of these receptor subtypes as well to complement the extensive and elegant physiology work that has been going on for decades.

Dopamine has been perhaps the most extensively characterized biogenic amine from a functional and biochemical perspective in crustaceans. Previous work [60, 61] identified 3 subtypes of dopamine receptors in the nervous system of the spiny lobster, Panulirus interruptus: D1_αPan (Type 1A DAr), D1_βPan (Type 1B DAr), and D2_αPan (Type 2 DAr). We found clear orthologs to all three of these receptor subtypes in both C. borealis and H. americanus (Table 3; Fig. 7), and our transcriptome search protocol did not come up with any other putative DAr subunit transcripts. Therefore, it is likely that these three receptor subtypes represent the complete complement of dopamine receptors in these decapod crustaceans. In deference to the extensive characterization of these receptors in the closely related spiny lobster, we conform the naming of these channels to match with the Panulirus nomenclature: for example, Cb-D1αR, Cb-D1βR, and Cb-D2αR (Fig. 7).

Serotonin receptors are less described in crustaceans than the dopamine receptors. Previous reports describe two distinct subtypes of serotonin receptors in the P. interruptus [62] as well as the crayfish Procambarus clarkii [63]: one type-1 and one type-2 serotonin receptor. Our analysis of the transcriptomes of C. borealis and H. americanus found clear orthologs to both of these receptor subtypes, and based on homology with mouse and Drosophila sequences we identified two novel putative serotonin receptor subtypes as well. Figure 6 uses the existing P. interruptus and P. clarkii sequences to root the new sequences in a tree representing these crustacean serotonin receptors. Our analysis suggests that the previous type-1 5HTr subtypes identified are most similar to mammalian 5HT_1A (HTR1A) receptors, while the crustacean type-2 5HTr subunit is most similar to 5HT_2B (HTR2B) receptors. We also identified a putative type-1B receptor (HTR1B) as well as a putative type-7 receptor (HTR7). These identities are assigned based on mouse query sequences used in our tblastx protocols that generated the strongest hits (i.e. lowest e-values) when queried against the crustacean transcriptomes. We follow the mammalian classification and nomenclature guidelines for these 5HT receptors in assigning gene names (Table 3; Fig. 7), as these are well defined and organized relative to the invertebrate nomenclature: e.g. Cb-HTR1A, Cb-HTR1B, Cb-HTR2B, and Cb-HTR7.

Octopamine receptors are virtually undescribed in crustaceans, with the sole decapod receptor described as a tyramine/octopamine receptor from the freshwater prawn, Macrobrachium rosenbergii [64]. Thorough work with crustacean octopamine receptors is found in the barnacle, Balanus improvisus, where one alpha- and four beta-like receptor subtypes have been very nicely characterized [65]. Our analysis identified the same distribution of receptor types in C. borealis and H. americanus as was described in the barnacle – one alpha- and four beta-like subunits (Table 3; Fig. 7). However, there were no particularly conserved motifs that resulted in a clustering of decapod and barnacle receptor subtypes to converge on a common nomenclature for these receptors; the four beta-like receptors in barnacle most closely related one another rather than subtypes across species. As a result, we have simply named these β-like octopamine receptors subtypes with ascending numbers and in the style of the descriptions given to those identified in B. improvisus (Bi): e.g. Cb-Oct-αR, Cb-Octβ-R1, Cb-Octβ-R2, Cb-Octβ-R3, and Cb-Octβ-R4. However, we do not mean to imply direct orthology between BiOctβ-R1 and Cb-Octβ-R1, for example. Finally, we also identified a single putative tyramine receptor in both of our species (Cb-Tyr-R and Ha-Tyr-R), and these sequences are most similar to the tyramine/octopamine receptor reported from Macrobrachium rosenbergii, which is most effectively activated by tyramine [64, 66].

Histamine is also a major neurotransmitter in invertebrates, but sequence information for histamine receptors in crustaceans has yet to be described. In vertebrates, these receptors are part of a protein family of G-protein-coupled metabotropic receptors. In invertebrates, histamine acts exclusively through ionotropic histamine receptors, as no metabotropic histamine receptors appear to be present in invertebrates [67]. We discovered putative orthologs for 3 distinct ionotropic histamine receptors in both C. borealis as well as H. americanus (HisR1-3) and a fourth sequence was found in H. americanus that did not have an obvious match from C. borealis (Fig. 7). In sequence comparisons with Drosophila melanogaster, the closest orthologs are described histamine-gated chloride channels. This is consistent with the known physiological characterization of crustacean histamine gating of chloride channels [68, 69]. Because naming conventions are lacking for these receptors in crustaceans, we have simply assigned these transcripts ascending numbers (1–4) to distinguish between different putative histamine receptor subtypes (Fig. 7; Table 3).

Metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are seven-transmembrane domain proteins coupled to G-proteins capable of controlling many cellular processes through signaling cascades. mGluRs can be classified into three primary classes [70]: Class I consists of mGluR1 and mGluR5 and are associated with phospholipase-C and utilize intracellular calcium signaling cascades; Class II consists of mGluR2 and mGluR3; and Class III consists of mGluR4, mGluR6, mGluR7, and mGluR8 and are negatively coupled with adenylyl cyclase activity. These receptors exist as either homo- or heterodimers on the cell surface, and it is the associated G-protein alpha-subunit that determines which class the mGluR falls under (e.g. Class I associates with G_q and G₁₁). In crustaceans, mGluRs have a relatively short history of study, mostly owing to the fact that the metabotropic form of glutamate receptors had not been characterized until the late 1980s [71]. In crustacean preparations, it has been found that mGluRs play a role in rhythm generation in the stomatogastric ganglion [72, 73]. In our analysis of these transcriptomes, we found six mGluR sequences for each species, which covered all three primary classes of mGluRs (Table 4; Fig. 8). We did not find mGluR6 and mGluR8 orthologs in either species. It should also be noted that Ha-mGluR2 and Ha-mGluR4 aligned more closely to one another than to their C. borealis counterparts. This discrepancy could be due to the relatively short partial sequence found for Cb-mGluR2; that is, only the first 200 amino acids were found for Cb-mGluR2, while the Ha-mGluR2 sequence found is 1027 amino acids long.

Ionotropic glutamate receptors

The most common excitatory neurotransmitter found in crustaceans at the neuromuscular synapse is glutamate [74]. The three primary ionotropic glutamate receptors are NMDA, AMPA, and kainate receptors, named respectively after the agonists N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid that activate them. One of the initial characterizations of NMDA receptors from crustaceans was performed in the crayfish optic lobe [75]. Since then, NMDA receptors have been studied in crustaceans for their role in memory [76], axon-to-glial signaling [77], and central pattern generation [78]. We were able to identify 4 separate NMDA receptors from both C. borealis and H. americanus, which fell into two primary categories: 1-like and 2-like (Table 4; Fig. 8). We further identified these receptors into 1A, 1B, 2A, and 2B based on their pairing, but this is not meant to imply orthology with other naming schemes in other species. We also found one NMDA receptor in both C. borealis and H. americanus that we did not find a highly similar pair for, which we have included as Cb-NMDA-2-like and Ha-NMDA-2-like.

AMPA receptors have been virtually undescribed in crustacean preparations, which coincides with our results. In our analysis, we did not find any receptors that most closely resembled AMPA receptors. Known AMPA receptors blasted against our transcriptomes aligned best against the putative kainite-type receptors, which is unsurprising considering both AMPA and kainate are considered non-NMDA receptors. Kainate receptors have been implicated as modulators of synaptic transmission and excitability [79]. In crustacean systems, kainic acid historically has been shown to elicit depolarizations at the crab neuromuscular junction [80], as well as the crayfish neuromuscular junction [81]. Five pairs of kainite-like receptors were found between the C. borealis and H. americanus transcriptomes (Table 4; Fig. 8), falling into two discrete categories: kainate type-1-like and 2-like. Sequences were further subdivided into A, B, and C on the simple basis of pairing between the two species and not based on any specific orthology to other species.

Beyond the excitatory glutamate-gated cation channels (NMDA- and kainate-like), an inhibitory glutamate-gated chloride channel (GluCl) was also found for both C. borealis and H. americanus (Table 4). Soon after their discovery as extrajunctional receptors in locust muscles [82], GluCls were described as postsynaptic receptors in the crustacean stomatogastric ganglion [50, 83]. In our analysis, we found a single GluCl transcript for each species, named Cb-GluCl and Ha-GluCl. The finding of a single channel is consistent with some other invertebrates, with a single GluCl also found in most insects [84]. Because of their distinct characteristics, the GluCl channels we identified were not placed on any of the trees shown in the Figures.

GABA receptors

The neurotransmitter γ-amino butyric acid (GABA) has been studied in crustacean species for decades for its role in synaptic transmission and neural inhibition [85–88]. Interestingly, in invertebrates several different neural responses to GABA have been found that have distinct profiles from that of vertebrate GABA receptors [83, 89–91]. GABA receptors are classified into two major groups: GABA_A type receptors, comprising receptor complexes that are part of a ligand-gated ion channels, or GABA_B type receptors, G-protein-coupled receptors that act via metabotropic signaling systems. GABA_A receptors are pentameric transmembrane receptors responsible for fast, usually inhibitory synaptic currents, and heteromultimers of the individual subunit types can form distinct channel properties in invertebrates [92]. We identified orthologs of three GABA_A type receptor subunits from both H. americanus and C. borealis (Fig. 9; Table 3), including orthologs of Drosophila LCCH3-, RDL-, and GRD-like receptor subunits – and we have preserved naming conventions for these subtypes. Two GABA receptor subunits previously have been cloned from H. americanus [93], and very recently a GABA_A type receptor was identified in the crayfish, Procambarus clarkii [94]. Sequence comparison reveals these previously described sequences to be orthologous to the RDL-like receptor from our data. The metabotropic GABA_B type receptors are GPCRs responsible largely for slower inhibitory synaptic effects, and functional GABA_B receptors are heterodimers formed by GABA_B1 and GABA_B2 subunits [95]. We identified orthologs of both GABA_B1 and GABA_B2 subunits in both C. borealis and H. americanus (Table 3; Fig. 9).

Acetylcholine receptors

Acetylcholine receptors are classified into two family subtypes: nicotinic receptors (nAChRs), which are ligand-gated ion channels that are activated by nicotine; and muscarinic receptors (mAChRs), which are metabotropic GPCRs that respond to the agonist muscarine. Muscarinic acetylcholine receptors are further classified into subtypes based on the specific G-protein associated with the receptor [96]. In our analysis, we discovered two discrete subtypes of mAChRs (Table 4; Fig. 9) in both C. borealis and H. americanus, which is consistent with other arthropods [97]. The A- and B-type mAChR are defined based on their differential sensitivity to muscarine (A-type is 1000x more sensitive than B-type), as well as the antagonist binding properties (atropine, scopolamine, and QNB block A-type but not B-type) that each receptor exhibits [97].

Nicotinic acetylcholine receptors are common throughout the invertebrate central nervous system, mediating largely fast excitatory neurotransmission [98–101]. For nAChRs in nervous systems of C. borealis and H. americanus, each species was found to have 1 β-subtype and 8 α-subtypes (Fig. 9). A thorough characterization of invertebrate nAChRs has been performed for the snail, Lymnaea stagnalis [102]. Our analysis revealed that few direct orthologous sequences occurred from snail (Mollusca) to crustaceans that would allow us to adopt the nomenclature put forward in Lymnaea. Therefore, the crustacean receptor subtypes were named based on their most similar mammalian counterpart, with the exception of the α16 subunit found, named so due to its similarity to acr-16 found in C. elegans [103]. This subunit is most comparable to α7 in humans, but we found other sequences nAChRs more similar to human α7 than that of the putative α16.

Gap junction proteins (Innexins)

The proteins responsible for gap junctions in invertebrates are the Innexins [104]. A family of Innexins have previously been described for both C. borealis and H. americanus [105]. We include them here as characterized through the transcriptome analysis for completeness. Three full-length sequences were named Innexins 1–3 (Fig. 10; Table 5) based on significant sequence similarity to coding sequences of Innexins from multiple other organisms, including other decapod crustaceans [106]. Innexins 4–6 (Fig. 10; Table 5) were subsequently identified from our transcriptome analysis of C. borealis nervous system. We identified clear orthologs from lobster and crab for Innexins 1–4 and 6; but two Innexins showed enough dissimilarity to be classified separately, and these are classified as Innexin 5 in C. borealis and Innexin 7 in H. americanus (Fig. 10). All of these identified Innexin sequences have the signature motif YYQWV in the second TM domain as well as a series of other conserved amino acid residues considered hallmarks of Innexins [105].