Opsin-based photoreception in Crinoids: a molecular and behavioural study of Antedon bifida

Abstract

Opsins are essential photoreceptor proteins that enable both visual and nonvisual light perception in metazoans. Although extensively studied in eyed invertebrates, their role in eyeless organisms such as echinoderms remains underexplored. Within the echinoderm phylum, studies have primarily focused on sea stars, sea urchins, and brittle stars, leaving crinoids, the most basal echinoderm lineage, entirely unexplored. Currently, only a limited number of behavioural observations have suggested that crinoids may possess light sensitivity. This study investigated the behavioural, morphofunctional, and molecular foundations of opsin-based photoreception in Antedon bifida, a European crinoid species from the comatulid order. In this context, the behavioural response to different light wavelengths, characterisation of opsin genes in the recent chromosome-scale genome of this species, opsin immunolocalisation within the crinoid tissues, and in vitro functional characterisation of opsins have been investigated. In vivo tests indicated significant negative phototactic behaviour induced by a wide range of light wavelengths (463-630 nm) with maximum sensitivity to blue light ({lambda}max = 463 nm). In silico genome analyses revealed the presence of only three rhabdomeric opsin genes, located on chromosomes 4 (Abif-opsin 4.1) and 6 (Abif-opsin 4.2 and 4.3). All crinoid opsins were phylogenetically clustered as a sister group to all other echinoderm rhabdomeric opsins, supporting their evolution via duplication of an ancestral gene in the crinoid lineage. The low opsin diversity contrasts with that of other echinoderms, which are generally characterised by up to eight bilaterian opsin types. Interestingly, A. bifida opsin sequences present typical amino acid residues of rhabdomeric opsins of other bilaterians, including two conserved cysteines (C110 and C187), the probable ancestral E181 counterion, the typical G-protein signalling NPxxY(x)6F pattern, a highly conserved lysine potentially covalently bound to a chromophore, and the (D)RY motif, all of which support a photoreceptive function. Heterologous in vitro expression in HEK293T cell cultures subsequently confirmed the photoreceptive function of all three A. bifida opsins. Indeed, these three crinoid opsins formed active complexes with 11, cis-retinal association, and once purified, showed different absorbance peaks in the short wavelengths of the visible light spectrum ranging from 425 to 520 nm. Finally, immunoreactivity to newly generated antibodies against sea star opsins highlighted two potential crinoid opsins in several tissues associated with the ambulacral grooves of the calyx and pinnules. Within these tissues, one Abif-opsin is potentially expressed both in the ectoneural basiepithelial nerve plexus and in the hyponeural nerve plexus. However, a different opsin is also expressed in the sensory papillae of tube feet. Localisation of at least two opsins in different sensory structures suggests the presence of a complex extraocular photoreception system based exclusively on rhabdomeric opsins in this crinoid species.