Remnants of ancestral larval eyes in an eyeless mollusk? Molecular characterization of photoreceptors in the scaphopod Antalis entalis

Background Eyes have evolved and been lost multiple times during animal evolution, however, the process of eye loss has only been reconstructed in a few cases. Mollusks exhibit eyes as varied as the octopod camera eye or the gastropod cup eye and are ideal systems for studying the evolution of eyes, photoreceptors, and opsins. Results Here, we identify genes related to photoreceptor formation and function in an eyeless conchiferan mollusk, the scaphopod Antalis entalis, and investigate their spatial and temporal expression patterns during development. Our study reveals that the scaphopod early mid-stage trochophore larva has putative photoreceptors in a similar location and with a similar gene expression profile as the trochophore of polyplacophoran mollusks. The apical and post-trochal putative photoreceptors appear to co-express go-opsin, six1/2, myoV, and eya, while expression domains in the posterior foot and pavilion (posterior mantle opening) show co-expression of several other candidate genes but not go-opsin. Sequence analysis reveals that the scaphopod Go-opsin amino acid sequence lacks the functionally important lysine (K296; Schiff base) in the retinal-binding domain, but has not accumulated nonsense mutations and still exhibits the canonical G-protein activation domain. Conclusions The scaphopod Go-opsin sequence reported here is the only known example of a bilaterian opsin that lacks lysine K296 in the retinal-binding domain. Although this may render the Go-opsin unable to detect light, the protein may still perform sensory functions. The location, innervation, development, and gene expression profiles of the scaphopod and polyplacophoran apical and post-trochal photoreceptors suggest that they are homologous, even though the scaphopod post-trochal photoreceptors have degenerated. This indicates that post-trochal eyes are not a polyplacophoran apomorphy but likely a molluscan synapomorphy lost in other mollusks. Scaphopod eye degeneration is probably a result of the transition to an infaunal life history and is reflected in the likely functional degeneration of Go-opsin, the loss of photoreceptor shielding pigments, and the scarce expression of genes involved in phototransduction and eye development. Our results emphasize the importance of studying a phylogenetically broad range of taxa to infer the mechanisms and direction of body plan evolution.


Background
Vision is among the most important sensory modalities for bilaterian animals and it has been suggested that eyes have been independently gained and lost several times [1][2][3][4]. Simple cup-shaped eyes composed of photoreceptor cells and shading pigments probably already existed in the last common bilaterian ancestor [5]. Photoreceptor cells possess expanded surface areas to store photopigments (opsins), and while rhabdomeric photoreceptors possess microvilli on their apical surface for this purpose, ciliary photoreceptors possess surface extended cilia [5]. Similar gene expression profiles and comparisons of molecular and morphological characteristics of photoreceptors have shaped inferences of the putative ancestral organization of shared receptor cells [6,7]. For example, recent studies suggest that the last common bilaterian ancestor possessed several opsins, including a canonical R-opsin, a non-canonical R-opsin, a C-opsin, a Go-opsin, a retinal pigment epithelium-retinal G-protein-coupled receptor/peropsin/retinochrome, and a neuropsin [8]. For the majority of bilaterians, it remains, however, unclear where these different opsins are expressed and whether given photoreceptor cells in different taxa are homologous or originated via evolutionary convergence (see [5,7] for detailed studies).
Among bilaterians mollusks are textbook examples for eye evolution with designs as varied as the octopod camera eye, the nautiloid pinhole eye, the gastropod cup eye, or the camera-type eyes of strombid conchs [9]. Although most adult bivalves lack eyes, ark clams possess sophisticated compound eyes and scallops exhibit mirror-based eyes. Adult polyplacophorans lack cerebral eyes but certain species possess image-forming eyes embedded in their outermost tegmental shell valve layer (esthetes) [10]. Within earlier developmental stages, post-trochal eye spots are known from polyplacophoran trochophore larvae [10], and cerebrally innervated eyespots occur in gastropod and bivalve larvae [11]. Notably, there are also molluscan clades that neither possess eyes as adults nor during earlier developmental stages, such as the wormshaped aplacophorans or the tusk-shelled scaphopods (Fig. 1). These enigmatic animals live an infaunal, cryptic lifestyle as adults but possess free-swimming trochophore-like larvae, in which phototactic behavior has not been reported [12][13][14][15][16].
Reconstructing the evolutionary history of photoreceptors is challenging within the Mollusca. A recent study showed that the polyplacophoran mollusk Leptochiton asellus possesses three clusters of photoreceptors located in the apical, post-trochal, and the most posterior region of its trochophore larva [17,18]. The topography and cell lineage of the post-trochal eyes were used as arguments against their homology with cerebral eyes of other mollusks and other bilaterians [19]. Despite this, all three types of polyplacophoran photoreceptors share gene expression profiles of cerebral eyes and express photoreceptor genes such as r-opsin and xenopsin, as well as genes implicated in the development of cerebral eyes in other taxa [17,18]. The latter genes include members of the Pax-Six-Eya-Dach network [paired box protein 6 (pax6), sine oculis homeobox gene 1/2 (six1/2), eyes absent (eya), and dachshund (dach)], transcription factors that are involved in the development of eyes, other sensory organs, and the brain [20,21]. Transient receptor potential cation channel (trpC) is involved in phototransduction, myosinV (myoV) in intracellular r-opsin transport, while retinitis pigmentosa GTPase regulator (rpgr) is implicated in ciliary opsin targeting [22][23][24]. The 'cerebral' molecular fingerprint of polyplacophoran photoreceptors was interpreted as a heterotopic replication of the 'cerebral eyes' in the post-trochal region without a change in their underlying genetic circuitry [17]. According to this hypothesis, the cerebral eyes of polyplacophoran ancestors were replicated by a single saltatory event; this could be, for example, a single change in expression Fig. 1 The presence and absence of cerebral eyes in adult recent mollusks. The last common cephalopod and gastropod ancestors possessed probably a pair of adult cerebral eyes (+), while the ancestors of all other molluscan clades most likely did not exhibit cerebral eyes (−). Adult polyplacophorans possess a sensory system with lenses in the tegmental layer of the shell valves, so-called esthetes. Certain bivalves evolved eyes associated with their mantle tissue. Phylogenetic analysis based on Smith et al. [28] Sketch drawing modified from Wollesen et al. [44] of a regulatory gene with concomitant changes of co-regulated downstream target genes. In the developmentally distinct post-trochal region, this would result in a pair of novel post-trochal eyes with underlying identical gene inventory to cerebral eyes. Caveats of this hypothesis are, however, that the underlying gene regulatory network is not known for polyplacophorans and that comparable data from closely related molluscan species are lacking.
In the present study, we took advantage of the case of an eyeless conchiferan mollusk, the scaphopod Antalis entalis, to investigate the molecular signature of eye, photoreceptor, and opsin functional degeneration. To this end, we searched for homologues of opsins and other eye or photoreceptor-related genes that have been described previously for the polyplacophoran mollusk L. asellus [17,18]. Two opsin genes, go-opsin and xenopsin, as well as pax6, six1/2, eya, dach, trpC, rpgr, and myos-inV have been identified to provide further insights into possible phototransduction pathways. We show that the scaphopod A. entalis and the polyplacophoran L. asellus express opsins in similar body regions and propose an evolutionary scenario of molluscan eye and photoreceptor evolution.

Phylogenetic and sequence analysis
We detected putative sequences of go-opsin, xenopsin, dach, rpgr, six1/2, myosinV (myoV), pax6, eya, and trpC within the Antalis entalis (aen) transcriptome, and the predicted protein sequences of each of these genes cluster with their bilaterian orthologs in phylogenetic analyses (Additional file 1: Figure S1). Two partial aen transcripts were found that encode peptides which fall within the xenopsin clade (Additional file 1: Figure S1a). The two peptide fragments do not overlap in the alignment and it is possible that they represent parts of the same gene, however, attempts to join the two fragments by PCR were unsuccessful (data not shown). We note that the Xenopsin clade is unsupported in our phylogenetic analysis, however, both partial sequences align well with other Xenopsins, and cluster with well-supported Xenopsin sequences from the more extensive analysis performed by Ramirez et al. [8]. The c-terminal Xenopsin sequence a2932192_2 was used for aen-xenopsin riboprobe synthesis for in situ hybridization. Both the c-terminal Xenopsin and the Go-opsin (aen transcript-60_140421) contain the characteristic 'NPXXY' motif and tripeptide for G-protein activation ( Fig. 2; [18]). In the Xenopsin, the tripeptide is 'NKQ' (found in C-opsins and some other Xenopsins), while in the Go-opsin the tripeptide is 'HMK' (Fig. 2). The predicted amino acid sequence of go-opsin lacks the highly conserved lysine ('K296') in the retinal-binding domain (Fig. 2), which is completely conserved in all other opsins other than placopsins [25]. Every raw transcriptome read spanning this motif contained the same sequence, suggesting that the lack of a predicted lysine is not the result of a sequencing or assembly error (data not shown). In addition, the sequence spanning this motif has been amplified and Sanger sequenced, confirming the lack of the predicted lysine (Additional file 1). Structural prediction of aen-Go-opsin and comparison to bovine rhodopsin (PDB ID code 1U19) demonstrated the absence Fig. 2 The retinal-binding domain of scaphopod Go-opsin lacks the highly conserved lysine K296. Alignment of representative sequences from Ramirez et al. [8] highlight conserved motifs as per Vöcking et al. [18]. The highly conserved lysine (K) at position 296 is absent from placozoan opsins (='placopsins') and from aen-Go-opsin of any additional lysine residues within the retinal-binding pocket that may be able to compensate for the loss of K296 (as has been observed for Rhodopsin mutants in vitro, [26] (Fig. 3).
Clear single-copy orthologs were found for all other genes investigated, except six1/2. Three distinct A. entalis transcripts encoded proteins that fell within the six1/2 clade (Additional file 1: Figure S1g), suggesting that gene duplication has occurred in this lineage. The six1/2 sequence most similar to the previously investigated L. asellus six1/2 sequence was used for in situ hybridization.

Gene expression analyses
In situ hybridization experiments with riboprobes against trpC and xenopsin did not yield labeling of transcripts in any of the developmental stages examined (data not shown). PCR products were amplified from pooled larval cDNA, confirming that the genes are expressed during development, but perhaps at a concentration too low to be detected by hybridization techniques.
The earliest expression patterns detected in developmental stages of the scaphopod Antalis entalis are found in early trochophore larvae (Fig. 4a-c; Additional file 1: Figures S2-S8a-c). In the episphere, pax6+ cells are located below the cerebral pits, i.e., the invaginations of the cerebral ganglia placodes (Additional file 1: Figure S6a-c; [29][30][31]). Close to the pax6+ cells, six1/2+ cells are situated that may also be associated with the nervous system ( Fig. 4c; Additional file 1: Figure S8a-c). More posteriorly dach is expressed in two cells close to the foregut (Additional file 1: Figure  S3a Early mid-stage trochophores express go-opsin in two apical cells located in the lateral episphere adjacent to the trochoblasts, and in two cells of the anterior inner mantle posterior to the prototroch (Figs. 4d-f; 6a; Additional file 1: Figure S2d-f ). No shading pigments are associated with these cells. The apical go-opsin+ cells are flask-shaped, send processes to the apical surface (Fig. 4f ), and connect via axons to the neuropilar plexus underlying the apical organ (data not shown). These cells appear to co-express go-opsin, eya and myoV, however, we could not unequivocally determine that expression was located within the same cells (Figs. 4g, h; 6a). Pax6 and six1/2 are expressed in other flask-shaped apical organ cells, i.e., are not coexpressed with go-opsin, eya, and myoV (Figs. 4i; 6a; Additional file 1: Figure S6d At later stages, the episphere invaginates and accordingly the prototroch is situated more anteriorly [32]. Consequently, both apical go-opsin+ cells that appear to co-express eya and myoV are located in the interior of mid-stage trochophores (Fig. 4j-l; Additional file 1: Figures S4h, S5i). Other apical cells also express pax6 and six1/2, while six1/2+ cells are also present in the region of the cerebral and pedal ganglia ( Fig. 4m; Additional file 1: Figures S6h, j, S8h, j). Eya is expressed in the region connecting the hyposphere and episphere (Fig. 4k). Both post-trochal go-opsin+ cells are still visible in the mantle and still appear to co-express eya, six1/2, and myoV ( Fig. 4j-m; Additional file 1: Figures S2g-j). Dach, eya, myoV, pax6 and six1/2 are expressed in diverse regions of the mantle (Fig. 4j- Figures S5h, S6h). Rpgr+ cells were not detected in the mid-stage trochophore larva (Additional file 1: Figure S7g-j).

Are scaphopod go-opsins functional?
Go-opsins are a poorly characterized, but evolutionarily ancient, group of opsin proteins that have been lost in ecdysozoans and many vertebrates [18,25]. They were first discovered in the ciliary receptors of the distal retina in the mantle eyes of scallops [33]. In annelids, go-opsin1 exhibits a lambda absorption maximum of 488 nm, and these photoreceptors are involved in the phototactic response to light in trochophores and in the mediation of the shadow reflex in adults [34,35]. Therefore, Go-opsins function in the mediation of light responses in both annelids and mollusks.
In all neuralians studied so far, all opsins are linked via a highly conserved lysine (Schiff base) to a chromophore to form a visual pigment [25]. A covalent interaction between the Schiff base in the seventh transmembrane helix and the retinal chromophore leads to photosensation [36,37]. In the scaphopod Antalis entalis, the predicted Go-opsin amino acid sequence does not contain this lysine (K296, named after the position of the residue in bovine Rhodopsin) (Fig. 2). Absence of the Schiff base is so far only known from fungal, haloarchael, and placozoan opsin-like receptors [25,38,39], where it was hypothesized that they may be unable to detect light [25]. However, in vitro experiments indicate that this may not necessarily be the case. Mutation experiments on bovine  Rhodopsin revealed that proteins in which K296 had been substituted with another amino acid were constitutively active, i.e., were able to activate signaling via the G-protein transducin in the absence of a chromophore [40]. Further experiments also showed that light-dependent activation for these K296 mutants could be rescued by modification of another residue within the active site to a lysine [26]. In these cases, the protein regains the ability to form a pigment with 11-cis-retinal and to activate G-proteins in response to light, although the spectral properties are slightly altered [26,40]. Prediction of the secondary structure of A. entalis Go-opsin revealed that there are no other lysine residues within the retinal-binding pocket (Fig. 3), therefore rescue of responsiveness to light is unlikely to have occurred via this mechanism. Given this, and that the aen-go-opsin sequence contains a domain for G-protein activation (NPIIY motif and tripeptide in Fig. 2), we speculate that the scaphopod Go-opsin may still be functional as a sensory receptor of unknown modality.

Homology of polyplacophoran and remnant scaphopod apical and post-trochal photoreceptors
Aen-go-opsin+ cells are located in the vicinity of the trochoblasts in the episphere, i.e., a region that may be part of the apical organ (Fig. 4a-c; [32]. Both latter cells are flask shaped, their dendritic processes penetrate the epidermis and their axons run in the direction of the neural plexus underlying the apical organ (data not shown). Therefore, they resemble apical chemoreceptors [32,41]. The other pair of aen-go-opsin+ cells is present in the inner anterolateral mantle margin posterior to the prototroch in the early mid-stage trochophore (Fig. 4d-f ). None of the aen-go-opsin+ cells are accompanied by cells with discrete shading pigments which are required for directional photoreception. Recent studies have demonstrated positive photoresponse behavior without any discrete shading pigment in brachiopods [42], indicating that the opaqueness of the larval body may be used for shielding. Photoreceptors of other mollusks are arranged in a strikingly similar fashion as those of the scaphopod A. entalis (Fig. 6a, b). The trochophore of the polyplacophoran Leptochiton asellus also possesses a pair of photoreceptor cells in the apical organ or close to it, and the veliger larvae of gastropods and bivalves exhibit cerebrally innervated eyes close to the apical organ (Fig. 6b, d; [17,18]). The polyplacophoran trochophore additionally possesses a pair of post-trochal larval eyes reminiscent of the scaphopod condition, as well as another pair of photoreceptors at the posteriormost end of the trochophore ( Fig. 6b; [17,18]). Like scaphopod and polyplacophoran trochophore larvae, adult scallops possess photoreceptors within the mantle, albeit within mirror eyes located on the tips of tentacles extending from the middle mantle fold [33]. In contrast to scaphopods (this study), bivalves [33], and gastropods [8], go-opsin has probably been secondarily lost during evolution in polyplacophorans and cephalopods (go-opsin appears to be absent in the genome of Octopus bimaculoides and no polyplacophoran genome has been published so far). In polyplacophorans, go-opsin has been functionally replaced by r-opsin and xenopsin within photoreceptors ( Fig. 6b; [8,17,18,35]).

Crucial phototransduction machinery genes are not expressed in scaphopod photoreceptors
To infer whether the scaphopod go-opsin+ cells possess the genetic inventory for phototransduction, we carried out in situ hybridization experiments on genes involved in phototransduction, ciliary opsin targeting, intracellular R-opsin transport, and eye development as previously reported for the polyplacophoran L. asellus [17,18]. While in L. asellus all three groups of photoreceptors co-express xenopsin, r-opsin, eya, dach, six1/2, myoV, trypC, and rpgr [17,18], only few of these genes are potentially co-expressed in the scaphopod go-opsin+ cells (Fig. 6a, b). Aen-go-opsin appears to be co-expressed with myoV and eya in the apical cells and six1/2, myoV, and eya in the post-trochal cells, but no co-expression was observed with trpC, rpgr, pax6, or dach (Fig. 6a). In contrast to apical and post-trochal go-opsin+ cells which do not express a number of crucial genes implicated in phototransduction or eye development, numerous important phototransduction machinery genes (but not opsins) appear to be co-expressed in few cells of the posterior ventral foot (dach, six1/2, pax6, eya) and the pavilion (dach, six1/2, rpgr, eya, myoV) (Fig. 6a). This resembles the condition found in the polyplacophoran posterior most photoreceptors that co-express dach, six1/2, eya, pax6, rpgr, and myoV in r-opsin+/xenopsin+ cells ( Fig. 6b; [17,18]). While no orthologs of r-opsin or c-opsin were found within publicly accessible scaphopod transcriptomic resources, we cannot discount that some of these genes may be co-expressed with xenopsin, for which a partial sequence was discovered within the Antalis entalis developmental transcriptome. We were unable to amplify this gene from larval cDNA, therefore Aen-xenopsin may be lowly expressed, or may only be expressed during a very short developmental time frame that has not been considered in our study. The latter condition has been found in cave fish species and their closely related surface-dwelling species that exhibit significantly different opsin expression levels depending on the need for short-or long-wavelength sensitivity [43]. Interestingly, only one of the genes identified here (six1/2) could be found by BLAST within an additional Antalis entalis transcriptome dataset [28], (NCBI Bio-Project PRJNA72139), presumably generated from adult tissue, indicating that expression of the majority of these genes may be restricted to larval stages.

An evolutionary scenario of molluscan photoreceptor evolution
Based on their 'cerebral' expression profile, their topological location, and their distinct cell lineage from other mollusks or annelids, the polyplacophoran post-trochal eyes are argued to have arisen by heterotopic replication from ancestral cerebral eyes under retention of transcriptional activity of genes involved in phototransduction and eye development [17]. Our study highlights a similar location of apical and post-trochal photoreceptors in polyplacophoran and scaphopod trochophores, respectively (Additional file 1: Table S1). The polyplacophoran condition is most similar to the scaphopod early midstage trochophore, considered to be the 'phylotypic stage' in which staggered Hox gene expression bears most resemblance to the ancestral bilaterian condition (Fig. 6a, b; [44]).
We propose homology of the apical photoreceptor cells of scaphopods, polyplacophorans, and annelids (Fig. 6ac; [35]). Spatial expression of opsin genes has not been investigated in gastropod and bivalve larvae, however, based on their proximity to the apical organ and their cerebral innervation their larval eyes may be homologous to the scaphopod and polyplacophoran apical photoreceptors ( Fig. 6d; Additional file 1: Table S1). Due to a similar location in the trochophore larva and a similar gene expression profile, we propose homology of the post-trochal (mantle) photoreceptors of scaphopods and polyplacophorans. Although adult scallops also express go-opsin in their photoreceptors within their mantlebased mirror eyes, we hesitate to consider this as support for our hypothesis, since bivalve adult eyes have evolved several times independently and were probably not present in the last common bivalve ancestor ( Fig. 1; [33]). Several genes co-expressed in the posterior most polyplacophoran photoreceptors are also expressed in a domain in the posterior scaphopod foot. However, the expression of an opsin in this region could not been proven for scaphopods and therefore the homology of these regions remains unclear.
Given the similarity between polyplacophoran and remnant scaphopod post-trochal photoreceptors, we argue that the evolution of these photoreceptors via heterotopic replication from cerebral eyes may have occurred in the molluscan stem lineage and not only in the polyplacophoran stem lineage. If correct, larval post-trochal/mantle edge-associated eyes could therefore be considered a molluscan (not a polyplacophoran) synapomorphy, with loss of post-trochal (mantle margin) photoreceptors in other mollusks. Alternatively, the evolution of post-trochal eyes may have occurred via the gradual co-option of photoreceptor gene expression to the post-trochal region in both polyplacophorans and scaphopods. Although additional data are needed, we consider the first evolutionary scenario more parsimonious as it could have occurred via the change of expression of a single regulatory gene, while stepwise co-option of single genes of a gene regulatory network requires several evolutionary events.

Conclusions
Our results indicate that the scaphopod go-opsin+ cells represent former photoreceptors which have probably evolved into receptors of another sensory modality. This degeneration can be seen in the light-insensitive Goopsin, the loss of photoreceptor shielding pigments, and the loss of expression of several genes involved in phototransduction and eye development. The similar topographical constellation of remnant photoreceptor cells with functional photoreceptors in polyplacophorans suggests that the last common molluscan ancestor also possessed apical, post-trochal, and probably posterior photoreceptors, which represent previously unrecognized molluscan synapomorphies.

Ethics, collection and culture of animals
Adults of the scaphopod Antalis entalis Jeffreys 1869 were collected from approximately 25 m depth by the staff of the research vessel Neomys off the coast of Roscoff (France) in the summer of 2014 and 2017. Animals spawned and developmental stages were reared at 18-20 °C as described previously [16].

RNA extraction and fixation of animals for in situ hybridization experiments
Several hundred individuals of early cleavage stages to settled metamorphosed individuals were investigated. All developmental stages were carefully anesthetized in 7.14% MgCl 2 before fixation for in situ hybridization experiments as previously described [16].

Alignment, phylogenetic analysis and secondary structure prediction
Candidate genes were identified by BLAST searches against the published transcriptomes of the scaphopod A. entalis ( [16,27]; NCBI bioproject PRJNA357466; assembly available at https ://zoolo gy.univi e.ac.at/opendata/) were derived from pooled RNA from several hundred specimens of early embryos to postmetamorphic individuals. Phylogenetic analyses were performed for