The main objective of this study was to investigate differences in the expression of putative chemosensory receptors in male and female Crown-of-thorns starfish (COTS; Acanthaster cf. solaris) tube feet (TF) and sensory tentacles (ST). We identify several receptor genes which display either sex- or tissue-biased expression based on their log2FC values, making them ideal candidates for further investigation in terms of functional validation and biological significance. Our results indicate significant differences in the expression of numerous 7TM receptors between the ST and TF of COTS (TFALL vs. STALL). There is also evidence of male-biased expression of three receptors within the ST (STM vs. STF). In contrast, there is relatively little difference in receptor expression between male and female TF (TFM vs. TFF). Overall, we reveal a clear difference in putative chemosensory function between the COTS TF and STs (TFALL vs. STALL), and ST of both sexes (STM vs. STF). The TFALL vs. STALL group contained the highest number of DEGs, including 295 receptor-type genes. Of these, 24 were previously identified COTS ApORs. The majority of ApORs were significantly overexpressed in the STALL when compared to TFALL. These results confirm previous research which suggested a predominantly chemosensory role for the STs in COTS .
Many types of receptor genes are differentially expressed in the olfactory organs of male and female COTS. In particular, 11 putative chemoreceptor genes were targeted for further analysis due to their significant differential expression pattern. Three receptor genes showed male-biased expression within the sensory tentacles of COTS: one adrenergic receptor (ADRA1A), a G protein-coupled receptor 52 (GPCR52) and a metabotropic glutamate receptor (mGluR3). Eight receptor genes displayed ST-biased expression but no sex-biased expression: two adrenergic receptors (ADRA1D-like isoform X1 and ADRA1D-like isoform X2), two ionotropic glutamate receptors (gKAR2 and GluR2), a metabotropic glutamate receptor (mGluR7), a trace amine associated receptor 13c (TAAR13c), a cholecystokinin receptor type A (CCKRa) and G protein-coupled receptor (GRL101). While not all of these 11 genes are known to have chemosensory functions in other species, their presence within COTS olfactory organs implicates them with a likely role in chemosensation for this species.
COTS display significant overexpression of two iGluRs in their STs (STALL), gKaR2 and Glu2. Both genes contain three TM domains and several conserved regions with bilaterian homologs. Both receptor genes are expressed broadly in COTS tissues. Most notably, gKAR2 was expressed in several female tissues, but was only expressed in male TF and STs. The gKAR2 protein contains the ANF receptor domain of iGluRs, which is the extracellular ligand-binding domain , however, its variability in this region from the M. musculus homolog may be indicative of functional difference. In many invertebrates, variant iGluRs have been identified as having a chemosensory function, including Drosophila [1, 36], Lepidoptera spp. , Danio rerio , the water flea Daphnia pulex  and marine crustaceans such as the spiny lobster Panulirus argus  and the hermit crab Coenobita clypeatus . More recently, glutamate receptor-like genes have been found to be critical for reproduction in mosses through sperm chemotaxis and transcriptional regulation . These examples provide further support for a chemosensory role for these receptors in COTS. The COTS sequences show some conservation with both mammalian iGluRs and Drosophila IRs, however, they display variability in the three key amino acids which are known to bind the glutamate ligand. Phylogenetic analysis positions COTS gKAR2 separately from the gKAR2 group, however, while it bears some similarity to the IR8a and IR25a groups, it appears to have diverged after the separation of the conserved IRs from the iGluRs. Based on these results, it is likely this gene represents a possible variant IR which may be involved in chemosensation in COTS, as they are in Drosophila and other invertebrates.
mGluRs are class C GPCRs, originally characterised in mammalian nervous systems and having a role in neurotransmission . Two types of mGluRs, mGluR3 and mGluR7, were found to be differentially expressed in our comparisons. mGluR3 shows male-biased expression within the ST, being significantly overexpressed in male STs compared to female STs, while mGluR7 shows no sex-biased expression, being significantly overexpressed in COTS TF when compared to STs. RT-PCR demonstrated that both of these genes are also expressed in several other tissues in male and female COTS. Both are also expressed in the male spine (SPM) and male and female radial nerves (RNM and RNF). Despite their original characterisation within the central nervous system of vertebrates, mGluRs have also been detected in the main olfactory bulb of M. musculus  and the olfactory organ of the sea lamprey, Petromyzon marinus . They have also more recently been discovered to act as taste receptors in rat . COTS mGluR3 and mGluR7 proteins both display conservation within the N-terminus ANF domain, which is the putative ligand-binding region, and contain all of the conserved cysteine residues within the NCD3G domain, which are known to form disulphide bridges. Given the variety of chemosensory functions of this type of receptor in other organisms, it is also likely they are involved in chemosensation in COTS.
The ADRA1A receptor displays male-biased expression within COTS ST, while two isoforms of the ADRA1D-like receptor are significantly overexpressed within the STs of COTS (STALL) compared to TF (TFALL), indicating a possible olfactory role. ADRs belong to a large family that are known to control cardiovascular, respiratory and neuronal functions in humans and other vertebrates . It has previously been suggested that this gene family evolved via several ancient gene duplication events in the mammalian lineage . They remain relatively unexplored in invertebrates, making comparisons challenging. RT-PCR results show that they are expressed in multiple COTS tissues between males and females. Phylogenetic analysis shows distinct clustering of COTS ADRs in a manner that suggests lineage-specific expansions. Many of these were previously characterised as ApORs and cluster within the COTS genome . These results confirm that COTS have a considerable expansion of ADR genes and that many of them may be involved in chemosensation.
TAARs are known to be involved in olfaction in humans, mice and other vertebrates [45, 46]. The TAAR13c receptor binds specific ligands emitted from carrion, producing an attraction/aversion response in many vertebrate species, including the zebrafish D. rerio . In COTS, this gene shows a substantial increase in expression in STs compared to TF, the highest of any receptor investigated, however, RT-PCR shows that this gene is also expressed in several other tissues. COTS and zebrafish TAAR13c protein sequences have several conserved cysteine residues, however, there are also many regions of variability, including the characteristic DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3. In COTS TAAR13c this motif is DRF (Asp-Arg-Phe). While this entire motif was once thought to be critical for the interaction between GPCRs and their corresponding G-proteins, it has now been established that this is not always the case . The Tyr residue is the least conserved and functional studies have demonstrated that it is not involved in receptor activation . If TAAR13c has a similar role in COTS as it does in zebrafish, it may be a target for biological control through interfering with COTS attraction to food stimulus such as coral.
CCKRs have been well characterised in vertebrates where they have important roles in the regulation of feeding behaviour and energy homeostasis . In COTS, CCKRa is overexpressed in STs (STALL). CCKRa has also been identified in invertebrates such as Caenorhabditis elegans. C elegans shares significant similarity to those found in vertebrates, however, their function in invertebrates has not been confirmed . The COTS CCKRa protein lacks the characteristic N-terminal domain that, in other CCKRa proteins, adopts a tertiary structure of helical turns and a disulphide cross-linked loop, and is essential for the interaction with its ligand. We speculate that its ligand-binding specificity in COTS may be different than in vertebrates, but that it likely remains involved in the regulation of feeding behaviours. Therefore, it may be an interesting target for disrupting COTS behaviours such as foraging and feeding.
GPCR52 shows male-biased expression within the COTS ST and is an orphan receptor belonging to the rhodopsin-like family of GPCRs. In mammals it is highly expressed in the brain and inhibits dopamine signalling . It has been implicated in psychosis and neurodegenerative diseases in humans and as such is a valuable target for the treatment of these conditions. In COTS, it is overexpressed in STM (ie. underexpressed in STF) and RT-PCR shows expression of this gene in multiple tissues in males and females. Multiple sequence alignment between M. musculus and COTS GPCR52 proteins shows variability in the DRY motif at the intracellular end of transmembrane helix 3. In COTS, the Asp residue is substituted for Glu, resulting in ERY (Glu-Arg-Tyr). The Asp residue is typically conserved and forms an acidic side chain. This component is critical for regulating the activation of GPCRs and their interaction with associated G proteins. Glutamic acid is also able to form an acidic side chain however, and GPCRs with ERY motifs are still able to activate and couple to G proteins . Interestingly, despite being typically highly expressed in the brains of mammals, GPCR52 showed low expression in the radial nerve of male and female COTS (RNM and RNF) as compared to TF and ST. This gene may have varying function between invertebrate and vertebrate phyla but its expression in COTS, particularly the male ST, implicates it in chemosensation. It may be the case that it this is a male-specific receptor which detects biological cues released from female COTS.
GRL101 is also differentially expressed within the COTS olfactory organs, and RT-PCR shows it is expressed more so in female tissues than male tissues. This gene belongs to a family of leucine-rich repeat containing GPCRs or LGRs, including the relaxin and glycoprotein receptors originally characterised in mammals . LGRs are present in a wide range of animal phyla, and a subtype of group C LGRs have been recently discovered in the purple sea urchin S. purpuratus, and several other invertebrates, including decapod crustaceans . Based on the number of LDLa and LRR motifs, the COTS GRL101-like protein sequence bears the most homology to the type C2 LGRs, which are non-classical relaxin receptors. It has been suggested that type C2 LGRs bind insulin-like peptides (ILPs), which belong to the larger insulin superfamily of peptides . Their precise function has not yet been described in non-vertebrate species, however they appear to be involved in metabolism, growth, reproduction and aging in other animals . The overexpression of GRL101 within COTS ST (STALL) suggests it may have a role in chemosensation. Based on its function in other species, it could also influence growth or reproduction.
While olfactory chemoreceptors are expressed within the sensory epithelia of specialised organs, it has been established that this is not always the case. In Homo sapiens, ORs are expressed in the olfactory epithelium of the vomeronasal organ, yet many of these ORs are also involved in chemosensation in tissues as diverse as muscle, sperm, kidney and the cardiovascular system (Reviewed in ). For example, Olfr78 binds short chain fatty acids and is expressed in the kidney where it mediates the secretion of renin . Likewise, GRs that bind bitter molecules in the human tongue are also expressed in the ciliated epithelium of the lungs, where they mediate bronchodilation in response to inhaled ligands . In addition, for Drosophila the main taste organ is the labellum, yet GRs that respond to sweet and bitter taste molecules are also found in several other tissues, including the proboscis, legs, abdomen and wings .These GRs are not only functional, as determined by gene knockout studies, but may be associated with specialised behaviours, including the exploration of ecological niches . Based on these findings, it is not surprising that many COTS putative chemosensory receptors, such as those presented in this study, are expressed in tissues other than the TF and STs. It is also possible that the variation in expression indicates diversification of functions of these receptors.
While there is extensive literature available describing the function of ORs in terrestrial insects and vertebrates, particularly model species such as Drosophila and M. musculus, there are very few studies describing ORs in aquatic invertebrates, and even fewer in Echinoderms. This presents a considerable challenge in determining function for the number of interesting genes identified in this study. Indeed, even within vertebrate and insect lineages, ORs have a wide range of functions, many of which are unknown or not yet fully understood. Discovering which ligands bind to the ORs described in this study is essential if this is to be an avenue for the development of a biological control for COTS. Whilst homology provides a starting point, inferring the function of a differentially expressed receptor in this species based on the available literature requires further in-depth investigation that is outside the scope of this study. Thus, a significant challenge moving forward will be to elucidate the function of these receptors.