Current address: Department of Biology, Colorado State University, Ft. Collins, Colorado, United States of America
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
All data are freely available in open access data repositories. Raw reads are available under BioProject accession PRJNA596786 on NCBI (
The nervous system comprises a network of neurons that regulate the physiology and behavior of animals [
Class A GPCR subclasses in crustaceans include opsins and receptors for small-molecule neurotransmitters, neuropeptides, and hormones. Most crustacean GPCRs that have been functionally characterized are involved in neurotransmission and neuromodulation rather than sensory transduction [
Thus, our goal was to explore whether class A GPCRs are candidate chemoreceptors in crustaceans. To do this, we analyzed the transcriptomes of two chemosensory organs in four decapod crustacean species for all class A GPCRs. We also analyzed the brain transcriptomes from three of the four decapod crustacean species. The four species were Caribbean spiny lobster
We identified class A GPCRs in the LF, dactyl, and brain transcriptomes, and we analyzed expression levels in all three organs. Among the GPCRs that we identified in our study, many have homologues that have been functionally characterized in other species including numerous small-molecule receptors, neuropeptide receptors, and hormone receptors. We believe that these receptors expressed in the chemosensory organs may be important for neurotransmission and neuromodulation involved with chemical sensing, especially GPCRs unique to the LF since it is the olfactory organ. In addition to the known types, we also identified several novel receptor types and multiple orphan receptors expressed in the chemosensory organs, which may also be involved in neurotransmission, neuromodulation, or the detection of external chemical cues.
Formal approval from the Institutional Animal Care and Use Committee of Georgia State University or other ethics committees was not required since our work did not involve vertebrate animals. Nonetheless, our protocols complied with standard practices including collecting organs and sacrificing animals using cold anesthesia.
All analyses were performed on
For
Quality assessment on Agilent Bioanalyzer2000 and TapeStation of total RNA extracted, mRNA specific cDNA synthesis, and cDNA paired-end sequencing on the Illumina HiSeq 2500 high-throughput sequencer were performed by Beckman Coulter Genomics (now part of GENEWIZ, South Plainfield, New Jersey) as previously described in Kozma et al. 2018 and 2020a [
Individual reference transcriptomes for
Transcript expression was determined by quantifying overall transcript abundance in each organ transcriptome and by comparing transcript expression levels between organ transcriptomes to find differentially expressed genes (DEGs). Transcript abundance was estimated using RSEM [
High performance computing systems at Georgia State University were used for this analysis [
Sequences (and their corresponding transcripts) were named based on a two-part naming system comprising a prefix and suffix. The prefix abbreviation represents the respective decapod genus and species: Parg (
For analyses yielding more than one homologue for a given receptor type, a number was assigned to each respective homologue (e.g., R1, R2) and a letter was assigned for multiple transcript variants (e.g., R1a, R1b). For example, three homologues were identified for the CCHamide receptor (e.g., CCHa-R1, CCHa-R2, CCHa-R3) and two variants for CCHaR1 in
Sequences were also classified more generally according to the putative receptor subclass to which they belong. These categories include opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs (LGRs, which are glycoprotein hormone receptors), and orphan receptors.
The total number of putative class A GPCR sequences containing the PF00001 domain and the number of sequences that met inclusion criteria for all four decapod species are shown in
Species | # of seqs with PF0001 domain | # of selected seqs |
---|---|---|
120 | 83 | |
122 | 81 | |
140 | 102 | |
105 | 67 |
The numbers of sequences in each of the five subclasses of class A GPCRs–opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs, and orphan receptors–are listed for each of the four decapod crustaceans in
Class A GPCR subclasses | Species | |||
---|---|---|---|---|
Parg | Hame | Pcla | Csap | |
Opsins | 5 | 5 | 3 | 6 |
Small-molecule | 22 | 22 | 23 | 16 |
Neuropeptide | 23 | 19 | 34 | 14 |
LGRs | 6 | 5 | 10 | 3 |
Orphans | 27 | 30 | 32 | 28 |
Phylogenetic analyses of the sequences expressed in the transcriptomes of the four decapod species–
Colors represents five clades of visual and non-visual opsins. Maroon: Long wavelength-sensitive (LWS) opsins. Dark green: Medium wavelength-sensitive (MWS) opsins. Purple:
Colors represent clades of sequences. Dark green: Dopamine and Dopamine/Ecdysteroid receptors; Orange: 5HT receptors; Teal: Acetylcholine receptors; Yellow: Tyramine receptors; Dark blue: Histamine receptors; Brown: Eicosanoid (prostaglandin) receptors (PGE); Light purple: Adenosine (Ado) receptors; Dark purple: Octopamine (Oct) receptors; Gray: Adrenergic receptors. Colors representing decapod crustacean species:
Colors representing receptor types are neuropeptide receptors (dark green), leucine-rich repeat-containing GPCRs (brown), and orphan receptors (purple). Colors representing decapod crustacean species:
Colors representing the receptor clades are: Small-molecule receptors (blue), Neuropeptide receptors (green), Opsins (orange), LGRs (brown), Orphan receptors (gray). Colors representing decapod crustacean species are:
We discovered 19 putative opsin sequences encoding eight different receptor types (
There are two major neurotransmitter classes comprising peptides and small-molecule neurotransmitters. Small-molecule neurotransmitters comprise low molecular weight neurotransmitters such as biogenic amines, amino acids, purines, eicosanoids, and acetylcholine. Twenty-six putative small-molecule receptor subtypes were identified in the four decapods (
Neuropeptide receptors were the most diverse receptor subclass in this study. We identified 44 subtypes with putative homologues for the following receptors: sulfakinin (SKR), natalisin (NTLR), tachykinin (TKR), RYamide (RYa-R), leucokinin (LKR), neuropeptide F (NPFR), long neuropeptide F (LNPFR), short neuropeptide F (SNPFR), SIFamide (SIFa-R), allatostatin A (AstA-R), allatostatin C (AstC-R), CCHamide (CCHa-R), proctolin (ProcR), myosuppressin (MSR), FMRFamide (FMRFa-R), CNMamide (CNMa-R), pyrokinin (PKR), capability (CapaR), ecdysis triggering hormone (ETH-R), trissin (TrisR), elevenin (ElevR), crustacean hyperglycemic hormone (CHH-R), adipokinetic hormone/corazonin-related peptide (ACP-R), red pigment concentrating hormone (RPCH-R), corazonin (CrzR), inotocin (InotR), and crustacean cardioactive peptide (CCAP-R) (
LGRs are defined by the large leucine-rich ectodomain, which serves as the ligand-binding region for glycoprotein hormones [
We identified 117 sequences encoding putative orphan receptor in the four decapod transcriptomes, including homologues for ten orphan receptors identified in prior studies in other species, though their ligands and/or functions are unclear. These include homologues for seven putative
From the three decapods with transcriptomes from the brain and chemosensory organs (
Species | Transcripts of GPCRs enriched in LF | |||
---|---|---|---|---|
Opsins | Small-molecule | Neuropeptide | Orphan | |
Onychopsin-like | TyrR1, H1R | CCHa-R1b | HP1R, GG13995, GPCR_E1, GPCR_G1, GPCR_I1, GPCR_K1 | |
- | DopEcR, OctαR, TyrR1, H1R, PGE2R2b | ProcR1 | HP1R, CG13995, GPCR_C2, GPCR_E1, GPCR_G1 | |
Neuropsin | PGE2R1, PGE2R2a | ACP-R1, RPCH-R2, CHH-Rb | MoodyR, Tre1R, CG12290, GPR150, GPCR_A3, GPCR_C3, GPCR_D2y, GPCR_E1, GPCR_F1, GPCR_F2y, GPCR_G1, GPCR_K1, GPCR_L9 |
Class A GPCR transcripts with higher relative expression (fold-change > 2.82) in LF based on DESeq2 analysis of the organ comparisons, LF vs. Brain.
Species | Transcripts of GPCRs enriched in Dactyl | |||
---|---|---|---|---|
Opsins | Small-molecule | Neuropeptide | Orphan | |
- | TyR2 | CCHa-R1b, ACP-R1, RPCH-R2 | GPCR_A1, GPCR_C2x, GPCR_E1, GPCR_G1, GPCR_K1 | |
LWS_Opsin1, LWS_Opsin3 | 5HT4R, PGE2R2a, PGE2R2b, H1R | FMRFa-R | CG12290, GPCR_L4, GPCR_L5, GPCR_E1 | |
- | - | CHH-Rb | GPCR_A3 |
Class A GPCR transcripts with higher relative expression (fold-change > 2.82) in dactyls based on DESeq2 analysis of the organ comparisons, Dactyl vs. Brain.
There were eight transcripts expressed at higher levels (fold-change > 2.82) in both the LF and dactyl relative to the brain including CCHa-R1b, GPCR_E1, and GPCR_G1 in
Expression patterns between LF and dactyl varied between species. The number of transcripts expressed at higher levels (fold-change > 2.82) in the LF than dactyl are: 17 in
Our study identified dozens of class A GPCRs in two chemosensory organs (antennular lateral flagellum [LF] and walking leg dactyls, see
Opsins are used as visual pigments in ocular and extra-ocular photoreception in many animals, including crustaceans [
We also found an onychopsin-like sequence in
We also identified putative homologues for four non-visual opsins (neuropsin, arthropsin, peropsin, pteropsin) all of which are expressed in the decapod chemosensory organs. Functional characterization of non-visual opsins is lacking in crustaceans; however, there is evidence for their function in mammals. For example, neuropsin is a UV-light sensor in mammals [
Small-molecule receptors are highly conserved and expressed throughout the brain and chemosensory organs of the four decapods. Most of these small-molecule receptors are homologues to sequences from
Another novel receptor type was found in
We also found expression of a metabotropic H1 histamine receptor (H1R) conserved in three decapod crustaceans. Ionotropic histamine-gated chloride channels have been physiologically characterized in the OSNs of decapod crustaceans [
Several other small-molecule receptors are more highly expressed in the chemosensory organs than the brain, including the E2 prostaglandin receptor and receptors for tyramine and octopamine. Both tyramine and octopamine modulate olfactory processes, including pheromone sensitivity in arthropods [
The dopamine/ecdysteroid receptor is also expressed at higher levels in the LF compared the brain. DopEcR can bind 20-hydroxyecdysone (20E) and dopamine, but it more readily binds 20E [
Two serotonin receptors were detected in the OSNs of
Several small-molecule receptors are highly expressed in OSNs of
Many (44) neuropeptide receptors were identified in our study, most having higher expression in the brain, though several have higher expression in the chemosensory organs. For example, ACP-R and the RPCH-R have varied expression in the chemosensory organs between species. ACP-R1 and RPCH-R2 are more highly expressed in the LF of
Overall, expression of neuropeptide receptors is lower in the LF compared to other subclasses, with the exception of the proctolin receptor in
Neuropeptide receptor expression in the OSNs of the LF is sparse, with only the allatostatin C receptor (AstCR2) expressed in the OSNs of
Based on these studies, we hypothesize that in the LF of decapod crustaceans, several neuropeptide receptors, including ACP-R, RPCH-R, and the proctolin receptor, may support homeostatic functions, and AstCR2 may modulate the sensitivity of OSNs. CHH-R and FMRFa-R may support homeostatic function or modulate chemoreception in the dactyl. While there are several neuropeptide receptors enriched in the chemosensory organs; expression patterns suggest that neuropeptide receptors dominate the brain and are likely more pertinent to CNS function.
LGRs are found throughout the central and peripheral nervous system of decapod crustaceans. LGRs act as receptors for glycoprotein hormones and carry out numerous functions. LGR3, LGR4, and LGR5 serve as receptors for gonadulin, relaxin, and arthropod insulin-like growth factor respectively [
In summary, in decapod crustaceans, LGR1 and LGR2 are more evenly expressed throughout the LF, dactyl, and brain, while LGR3, LGR4, and LGR5 are more highly expressed in the brain compared to the chemosensory organs. Of the five LGRs, only LGR1 is expressed in the OSNs of
Homologues to seven
We also identified one crustacean orphan receptor known as HP1R. HP1R was originally discovered in the hepatopancreas of
Two other putative orphan receptors identified in this study–GPR150 and GPR161 –share homology with orphan receptors classified in vertebrates. Little is known about their phylogeny or function. In the phylogenetic tree, GPR150 clusters closely with inotocin receptors indicating that these transcripts have some shared homology. GPR161 clusters with the
Several other orphan receptors are expressed at higher levels in the chemosensory organs, mainly in the LF. GPCR_E1 and GPCR_G1 were more highly expressed in the LF than brain of
Class A GPCRs are expressed in the chemosensory organs and brain of decapod crustaceans. We identified a subset of class A GPCRs that are enriched in the LF and dactyl. These receptors may mediate chemical sensing, possibly olfaction, though their roles remain undetermined. We can only speculate the function and the expression of these putative receptors. mRNA can be used as a measure to predict protein expression; however, mRNA may not directly translate to protein expression. We can only infer that transcripts expressed more highly in a particular organ may be important for its function. Analysis of the proteome and functional characterization of these transcripts will be essential in deciphering the role of these putative receptors in decapod crustaceans.
(TXT)
Click here for additional data file.
(TIF)
Click here for additional data file.
MA-plots showing distribution of transcripts from DESeq2 analyses between organ types. Each grey dot represents a transcript from the transcriptomes of Parg (
(PDF)
Click here for additional data file.
Receptors are organized by subclass: opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs, and orphan receptors. Values are (from left to right) number of transcript expected counts from RSEM for each transcriptome, and the log2 fold change and actual fold change for LF vs. brain, LF vs. dactyl, and dactyl vs brain. Green box (RSEM expected count > 1000). Orange box (1000 > RSEM expected count > 100). Blue box = log2 FC > 1.5 between the two organs. Pink box = log2 FC < -1.5 between the two organs. Yellow box = log2 FC between –1.5 and 1.5.
(XLSX)
Click here for additional data file.
Receptors are organized by type: opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs, and orphan receptors. Values are (from left to right) number of transcript expected counts from RSEM for each transcriptome, and the log2 fold change and actual fold change for LF vs. brain, LF vs. dactyl, and dactyl vs brain. Green box (RSEM expected count > 1000). Orange box (1000 > RSEM expected count > 100). Blue box = log2 FC > 1.5 between the two organs. Pink box = log2 FC < -1.5 between the two organs. Yellow box = log2 FC between –1.5 and 1.5.
(XLSX)
Click here for additional data file.
Receptors are organized by type: opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs, and orphan receptors. Values are (from left to right) number of transcript expected counts from RSEM for each transcriptome, and the log2 fold change and actual fold change for LF vs. brain, LF vs. dactyl, and dactyl vs brain. Green box (RSEM expected count > 1000). Orange box (1000 > RSEM expected count > 100). Blue box = log2 FC > 1.5 between the two organs. Pink box = log2 FC < -1.5 between the two organs. Yellow box = log2 FC between –1.5 and 1.5.
(XLSX)
Click here for additional data file.
Receptors are organized by type: opsins, small-molecule receptors, neuropeptide receptors, leucine-rich repeat-containing GPCRs, and orphan receptors. Values are (from left to right) number of transcript expected counts from RSEM for each transcriptome, and the log2 fold change and actual fold change for LF vs. brain, LF vs. dactyl, and dactyl vs brain. Green box (RSEM expected count > 1000). Orange box (1000 > RSEM expected count > 100). Blue box = log2 FC > 1.5 between the two organs. Pink box = log2 FC < -1.5 between the two organs. Yellow box = log2 FC between –1.5 and 1.5.
(XLSX)
Click here for additional data file.
GPCR homologues are listed along with the species (
(XLSX)
Click here for additional data file.
Listed are the original names in Kozma et al. (2020b) [Ref
(XLSX)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
(FA)
Click here for additional data file.
We thank Dr. Adriano Senatore and Yeun Yan Wong for their support in developing the EVG transcriptomes of the four decapod crustaceans. We thank Vivian Ngo-Vu and Drs. Manfred Schmidt and Dan Cox for many helpful discussions and suggestions.
G protein-coupled receptor
variant Ionotropic Receptor
lateral flagella of the antennule
olfactory sensory neuron