In this study, we investigated the histology and ultrastructure of the RNC in A. cf. solaris and revealed the presence of unique bulbous structures in adult A. cf. solaris. There is no evidence of similar structures in similar studies of the RNCs of other phylogenetically diverse seastars including, Meridiastra gunnii [22], Echinaster sepositus [23], Echinaster echinophorus [24], Patiria pectinifera [25], Asterias rubens [26], Marthasterias glacialis [27], Patiriella regularis and Parvulastra exigua [28]; to name a few species that all have the smooth epithelial surface, typical of the asteroid RNC. These comparisons provided strong evidence that the neural bulbs of A. cf. solaris are a novel innovation of this seastar. Meanwhile, neural bulbs were absent from juvenile A. cf. solaris RNC, suggesting that the neural bulbs were only developed when A. cf. solaris become an adult. Interestingly, we found no evidence for the presence of the bulbous structures in the closely related adult A. brevispinus which inhabits soft sediment substrates in deeper waters adjacent to reefs and feeds on molluscs and detritus [29]. The evolutionary divergence between these two Acanthaster species has been established by molecular data (approximate similarity of 89% from mitochondrial genomic data) [30, 31], as well as their disparate morphology [29]. In spite of that, they do retain the capacity for interbreeding, which suggests they have undergone recent speciation [29].
Acanthaster cf. solaris is unusual in comparison to most seastars in that it is a specialised corallivore, being able to overcome the waxy lipid coral tissue due to a specialised stomach enzyme system [32]. Since the neural bulbs extending form the RNC were absent in both the early juvenile stage of A. cf. solaris as well as the adult A. brevispinus, neither of which feed on coral, we could therefore speculate that the possession of the neural bulbs may be related to diet and habitat. A. cf. solaris starts its benthic life as a herbivore with a preference for coralline algae [33, 34], while A. brevispinus feeds on molluscs and detritus [29]. Juvenile A. cf. solaris transition from herbivory into coral-feeding “sub-adults” at ~ 6 months of age [35], although this perilous transition can be delayed for years when coral diet is limited [36]. Corals are a hostile, highly defended food source, enveloped in allelochemicals and stinging cnidae, with many species also possessing sweeper tentacles and/or mesenterial filaments [32]. Corals often injure juvenile A. cf. solaris as they make the dietary switch, including central disc lesions and loss of arms that may lead to death [33]. We hypothesised that the RNC bulbs may be neural adaptations to traversing and feeding on coral. They may function as counter-defences that enable the successful predation and co-habitation of A. cf. solaris with corals. Thus, it is plausible that the contents of the neural bulb cells could have defensive and/or offensive roles, thereby protecting the RNCs from coral-derived neurotoxins and/or releasing neurochemicals that inhibit or suppress the coral defence mechanisms. [29].
Aside from the neural bulbs, the histology and ultrastructure of the RNC of A. cf. solaris is similar to that of other seastars. The hyponeural region of the RNC contains motor neurons [15, 26, 37] and for the seastar A. rubens it has been shown that these express a variety of neuropeptides [38]. The underlying ectoneural portion of the RNC is by far the largest of the three regions in seastars. In A. cf. solaris and A. rubens [39], its border with the connective tissue layer is quite level, in stark contrast to the vacillating boundary observed in the seastar P. pectinifera [40] and the sea cucumber Eupentacta fraudatrix [41]. The fibre bundles have been identified as bipolar glial cells [39, 42], although the presence of glia in echinoderm nervous tissue was repeatedly dismissed by prominent researchers in the field as recently as the late 1990s [2, 43]. Within the ectoneural neuropile was a heavy meshwork of smaller neurons, dendrites, vacuoles, glial fibre bundles and electron dense vesicles. Surrounding the uppermost portion of these fibre bundles were sensory knobs comprising of relatively enlarged nerve fibres that contain an abundance of synaptic vesicles [40]. The non-neuronal bipolar cells within the ectoneural layer are secretory and show immunoreactivity to Reissner’s substance, distinguishing them as a distant relative of the radial glial cells found in chordates [39, 42]. The lowermost region of the ectoneural layer is covered by a thick, microvilli-edged neuroepithelium occupying more space than the hyponeural plexus and connective tissue layer combined. The goblet cells interspersed within the epithelial portion of this layer potentially function to secrete mucous for microvilli on the epithelial surface. Importantly, the bulbous structures project from the dorsal side of the ectoneural tissue and may serve to increase the regions surface area, a common biological trope in structuring tissue.
The neural bulbs appeared to be dominated by secretory cells with potentially two types present, the cells with clear vesicles whose contents may have been lost in processing and cells with large vesicles containing protein-like secretory material destined for exocytosis at the surface. The large vesicles may indicate the presence of goblet-like cells which are known to secrete mucus, while the vesicles with dense protein-like inclusions indicated the presence of secretory cells with material in various stages of maturation [44, 45]. The juxtaposition of these cells is reminiscent of a duo-glandular system [45]. Cells within the epithelial portion of the neural bulbs were known to produce neurotransmitters such as serotonin and GABA (gamma-aminobutyric acid) [14].
Our proteomic analysis of the adult A. cf. solaris RNC, including the neural bulbs, identified almost 900 proteins, 16% of which were predicted to be secreted, with a significant number known to be involved in cellular and metabolic processes, including ependymin-related proteins (EPDRs). In A. cf. solaris, the EPDRs have expanded significantly through tandem duplication [46], and due to their presence in A. cf. solaris-conditioned seawater, it was hypothesised that they could play a role in conspecific communication [19]. Several of these A. cf. solaris EPDRs were also found in our RNC proteome, suggesting that the RNC could be a potential source. In the vertebrates, EPDRs appear to function in hydrophobic molecule binding, based on structural and in silico interaction analysis [47]. A previous study by Smith et al [13] analysed neuropeptides in the adult A. cf. solaris RNC based on a molecular weight cut-off prior to MS analysis. Our RP-HPLC and LC-MS/MS workflow yielded similarities to some of these neuropeptides, although some additional neuropeptides were also found, including ApNp11, ApNp22-like, bombyxin-type, calcitonin-type, CRH-type, orexin type-1, and RGP.
Providing a lower signal-to-biological-noise ratio caused by sample complexity, the more targeted analysis of isolated neural bulbs allowed for the identification of proteins that were specific or relatively abundant in the neural bulbs. These included several neuropeptides such as ApNP15a, calcitonin-type, F-type SALMFamide, myorelaxant peptide, TRH-type, and bombyxin. The latter was exclusively detected in neural bulbs, suggesting that the neural bulbs may be a major production and/or secretion site of this neuropeptide. Interestingly, a few neuropeptides detected in the neural bulbs were functionally annotated as myorelaxant agents (e.g., myorelaxant peptide, SALMFamide, and calcitonin-type peptides). A myorelaxant peptide has been found in the RNC and neuromuscular tissues of the P. pectinifera, where it stimulates muscle relaxation [48]. Similarly, SALMFamide peptides, which were the first echinoderm neuropeptides to be fully sequenced [49] and are derived from L-type (LxFamide motif) or F-type (FxFamide motif) precursor proteins, also act as myorelaxants [50]. Calcitonin is traditionally known for its role in vertebrate calcium metabolism [51] and calcitonin-type peptides have been identified in several echinoderm species [52–54]. Furthermore, it was recently discovered that calcitonin-type neuropeptides act as muscle relaxants in the seastars A. rubens and P. pectinifera [26]. Therefore, we hypothesised that the neural bulbs might associate with a regulation of muscle function in A. cf. solaris.
Four DMBT1-like proteins were elucidated from the adult A. cf. solaris RNC, two of which were present in neural bulb preparations. Although DMBT1-like proteins have not been adequately investigated in echinoderms, it is known from other eumetazoans that the Dmbt gene encodes alternatively spliced glycoproteins associated with the membrane or products of epithelial secretions [55]. These glycoproteins have been individually identified in both terrestrial and aquatic animal species [named DMBT1, salivary agglutinin (SAG), crp-ductin, gp-340, ebnerin, vomeroglandin, hensin, and muclin], where they have a variety of functions [55]. Dmbt1 tissue expression levels are highly species and organ dependent [55]. For example, in mice, the Dmbt1 (known as vomeroglandin) is actively expressed within the vomeronasal glands, important for pheromone perception among vertebrates [56, 57]. In humans, SAGs aggregate and bind to a large variety of microorganisms such as viruses, bacteria and fungi [58]. In adult A. cf. solaris, it is possible that DMBT1-like proteins provide defence against foreign microbes or upon exposure to coral. The A. cf. solaris genome contains a total of 56 genes that encode proteins annotated as DMBT-like, and 124 proteins that contain one or more SRCR domains [19]. By comparison, humans contain 22 SRCR-containing proteins, while the purple sea urchin S. purpuratus has 407 [19]. This suggested an expansion of DMBT/DMBT-like proteins in the echinoderm lineage.