Oxygen sensing in crustaceans: functions and mechanisms.

Animals that live in changing environments need to adjust their metabolism to maintain body functions, and sensing these changing conditions is essential for mediating the short- and long-term physiological and behavioral responses that make these adjustments. Previous research on nematodes and insects facing changing oxygen levels has shown that these animals rapidly respond using atypical soluble guanylyl cyclases (sGCs) as oxygen sensors connected to downstream cGMP pathways, and they respond more slowly using hypoxia-inducible transcription factors (HIFs) that are further modulated by oxygen-sensing prolyl hydroxylases (PHs). Crustaceans are known to respond in different ways to hypoxia, but the mechanisms responsible for sensing oxygen levels are more poorly understood than in nematodes and insects. Our paper reviews the functions of and mechanisms underlying oxygen sensing in crustaceans. Furthermore, using the oxygen sensing abilities of nematodes and insects as guides in analyzing available crustacean transcriptomes, we identified orthologues of atypical sGCs, HIFs, and PHs in crustaceans, including in their chemosensory organs and neurons. These molecules include atypical sGCs activated by hypoxia (Gyc-88E/GCY-31 and Gyc-89D/GCY-33) but not those activated by hyperoxia (GCY-35, GCY-36), as well as orthologues of HIF-α, HIF-ß, and PH. We offer possible directions for future research on oxygen sensing by crustaceans.

Single cell transcriptomes reveal expression patterns of chemoreceptor genes in olfactory sensory neurons of the Caribbean spiny lobster, Panulirus argus.

BACKGROUND: Crustaceans express several classes of receptor genes in their antennules, which house olfactory sensory neurons (OSNs) and non-olfactory chemosensory neurons. Transcriptomics studies reveal that candidate chemoreceptor proteins include variant Ionotropic Receptors (IRs) including both co-receptor IRs and tuning IRs, Transient Receptor Potential (TRP) channels, Gustatory Receptors, epithelial sodium channels, and class A G-protein coupled receptors (GPCRs). The Caribbean spiny lobster, Panulirus argus, expresses in its antennules nearly 600 IRs, 17 TRP channels, 1 Gustatory Receptor, 7 epithelial sodium channels, 81 GPCRs, 6 G proteins, and dozens of enzymes in signaling pathways. However, the specific combinatorial expression patterns of these proteins in single sensory neurons are not known for any crustacean, limiting our understanding of how their chemosensory systems encode chemical quality. RESULTS: The goal of this study was to use transcriptomics to describe expression patterns of chemoreceptor genes in OSNs of P. argus. We generated and analyzed transcriptomes from 7 single OSNs, some of which were shown to respond to a food odor, as well as an additional 7 multicell transcriptomes from preparations containing few (2-4), several (ca. 15), or many (ca. 400) OSNs. We found that each OSN expressed the same 2 co-receptor IRs (IR25a, IR93a) but not the other 2 antennular coIRs (IR8a, IR76b), 9-53 tuning IRs but only one to a few in high abundance, the same 5 TRP channels plus up to 5 additional TRPs, 12-17 GPCRs including the same 5 expressed in every single cell transcriptome, the same 3 G proteins plus others, many enzymes in the signaling pathways, but no Gustatory Receptors or epithelial sodium channels. The greatest difference in receptor expression among the OSNs was the identity of the tuning IRs. CONCLUSIONS: Our results provide an initial view of the combinatorial expression patterns of receptor molecules in single OSNs in one species of decapod crustacean, including receptors directly involved in olfactory transduction and others likely involved in modulation. Our results also suggest differences in receptor expression in OSNs vs. other chemosensory neurons.

Molecular Mechanisms of Reception and Perireception in Crustacean Chemoreception: A Comparative Review.

This review summarizes our present knowledge of chemoreceptor proteins in crustaceans, using a comparative perspective to review these molecules in crustaceans relative to other metazoan models of chemoreception including mammals, insects, nematodes, and molluscs. Evolution has resulted in unique expansions of specific gene families and repurposing of them for chemosensation in various clades, including crustaceans. A major class of chemoreceptor proteins across crustaceans is the Ionotropic Receptors, which diversified from ionotropic glutamate receptors in ancient protostomes but which are not present in deuterostomes. Representatives of another major class of chemoreceptor proteins-the Grl/GR/OR family of ionotropic 7-transmembrane receptors-are diversified in insects but to date have been reported in only one crustacean species, Daphnia pulex So far, canonic 7-transmembrane G-protein coupled receptors, the principal chemoreceptors in vertebrates and reported in a few protostome clades, have not been identified in crustaceans. More types of chemoreceptors are known throughout the metazoans and might well be expected to be discovered in crustaceans. Our review also provides a comparative coverage of perireceptor events in crustacean chemoreception, including molecules involved in stimulus acquisition, stimulus delivery, and stimulus removal, though much less is known about these events in crustaceans, particularly at the molecular level.

CrusTome: a transcriptome database resource for large-scale analyses across Crustacea.

Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans.

In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates.

Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab, Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth, Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designated CHH Family Receptor Candidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. In G. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24ß1, -A24ß2, -A34α2, -A34ß1, and -A34ß2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded ß-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34ß1/ß2 had an additional two-stranded ß-sheet. We hypothesize that this second ß-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays.

G protein-coupled receptors as candidates for modulation and activation of the chemical senses in decapod crustaceans.

Many studies have characterized class A GPCRs in crustaceans; however, their expression in crustacean chemosensory organs has yet to be detailed. Class A GPCRs comprise several subclasses mediating diverse functions. In this study, using sequence homology, we classified all putative class A GPCRs in two chemosensory organs (antennular lateral flagellum [LF] and walking leg dactyls) and brain of four species of decapod crustaceans (Caribbean spiny lobster Panulirus argus, American lobster Homarus americanus, red-swamp crayfish Procambarus clarkii, and blue crab Callinectes sapidus). We identified 333 putative class A GPCRs- 83 from P. argus, 81 from H. americanus, 102 from P. clarkii, and 67 from C. sapidus-which belong to five distinct subclasses. The numbers of sequences for each subclass in the four decapod species are (in parentheses): opsins (19), small-molecule receptors including biogenic amine receptors (83), neuropeptide receptors (90), leucine-rich repeat-containing GPCRs (LGRs) (24), orphan receptors (117). Most class A GPCRs are predominately expressed in the brain; however, we identified multiple transcripts enriched in the LF and several in the dactyl. In total, we found 55 sequences with higher expression in the chemosensory organs relative to the brain across three decapod species. We also identified novel transcripts enriched in the LF including a metabotropic histamine receptor and numerous orphan receptors. Our work establishes expression patterns for class A GPCRs in the chemosensory organs of crustaceans, providing insight into molecular mechanisms mediating neurotransmission, neuromodulation, and possibly chemoreception.

Comparison of transcriptomes from two chemosensory organs in four decapod crustaceans reveals hundreds of candidate chemoreceptor proteins.

Crustaceans express genes for at least three classes of putative chemosensory proteins. These are: Ionotropic Receptors (IRs), derived from the heterotetrameric ionotropic glutamate receptors (iGluRs); Transient Receptor Potential (TRP) channels, a diverse set of sensor-channels that include several families of chemoreceptor channels; and Gustatory Receptor Like receptors (GRLs), ionotropic receptors that are homologues of Gustatory Receptors (GRs) of insects and are expressed sparingly in most crustaceans so far studied. IRs are typically numerically the most dominant of these receptor proteins in crustaceans and include two classes: co-receptor IRs, which are necessary for making a functional receptor-channel; and tuning IRs, whose specific combination in the IR subunits in the heterotetramer confers chemical specificity. Previous work showed that the transcriptomes from two major chemosensory organs-the lateral flagellum of the antennule (LF) and the tips of the legs (dactyls)-of the Caribbean spiny lobster Panulirus argus express four co-receptor IRs and over 100 tuning IRs. In this paper, we examined and compared the transcriptomes from the LF and dactyls of P. argus and three other decapod crustaceans-the clawed lobster Homarus americanus, red swamp crayfish Procambarus clarkii, and the blue crab Callinectes sapidus. Each species has at least ca. 100 to 250 IRs, 1 to 4 GRLs, and ca. 15 TRP channels including those shown to be involved in chemoreception in other species. The IRs show different degrees of phylogenetic conservation: some are arthropod-conserved, others are pancrustacean-conserved, others appear to be crustacean-conserved, and some appear to be species-specific. Many IRs appear to be more highly expressed in the LF than dactyl. Our results show that decapod crustaceans express an abundance of genes for chemoreceptor proteins of different types, phylogenetic conservation, and expression patterns. An understanding of their functional roles awaits determining their expression patterns in individual chemosensory neurons and the central projections of those neurons.

Chemoreceptor proteins in the Caribbean spiny lobster, Panulirus argus: Expression of Ionotropic Receptors, Gustatory Receptors, and TRP channels in two chemosensory organs and brain.

The spiny lobster, Panulirus argus, has two classes of chemosensilla representing "olfaction" and "distributed chemoreception," as is typical for decapod crustaceans. Olfactory sensilla are found exclusively on antennular lateral flagella and are innervated only by olfactory receptor neurons (ORNs) that project into olfactory lobes organized into glomeruli in the brain. Distributed chemoreceptor sensilla are found on all body surfaces including the antennular lateral flagella (LF) and walking leg dactyls (dactyls), and are innervated by both chemoreceptor neurons (CRNs) and mechanoreceptor neurons that project into somatotopically organized neuropils. Here, we examined expression of three classes of chemosensory genes in transcriptomes of the LF (with ORNs and CRNs), dactyls (with only CRNs), and brain of P. argus: Ionotropic Receptors (IRs), which are related to ionotropic glutamate receptors and found in all protostomes including crustaceans; Gustatory Receptors (GRs), which are ionotropic receptors that are abundantly expressed in insects but more restricted in crustaceans; and Transient Receptor Potential (TRP) channels, a diverse set of sensor-channels that include several chemosensors in diverse animals. We identified 108 IRs, one GR, and 18 homologues representing all seven subfamilies of TRP channels. The number of IRs expressed in the LF is far greater than in dactyls, possibly reflecting the contribution of receptor proteins associated with the ORNs beyond those associated with CRNs. We found co-receptor IRs (IR8a, IR25a, IR76b, IR93a) and conserved IRs (IR21a, IR40a) in addition to the numerous divergent IRs in the LF, dactyl, and brain. Immunocytochemistry showed that IR25a is expressed in ORNs, CRNs, and a specific type of cell located in the brain near the olfactory lobes. While the function of IRs, TRP channels, and the GR was not explored, our results suggest that P. argus has an abundance of diverse putative chemoreceptor proteins that it may use in chemoreception.