Vision is a two-step process, including phototransduction initiated by a retinal-bound opsin photopigment, and subsequent photopigment regeneration wherein the inactive state is restored. Because retinal isomerization, or changing of the molecular structural conformation, drives the activation of opsin photopigments, the reverse process is a necessary part of regeneration. In some animal lineages, a second opsin functions as a photoisomerase that converts retinal back to the isomer needed for the visual opsin, but very little is known about their spectral properties. We investigated spectral variation and spatial expression of scallop retinochrome, an opsin used for light-driven regeneration of molluscan visual photopigments. We show that retinochrome is expressed in scallop retina, along with visual opsins, indicating a conserved role in supporting vision. We compared retinochrome spectral absorption across six species with different ecologies, revealing considerable functional variation possibly associated with photic environments. Finally, we characterized multiple amino acid sites contributing to spectral tuning-shifts in the specific maximally sensitive wavelength of light-of this opsin. These results reveal retinochrome as a functionally dynamic component of the scallop visual cycle that may be adapted to different photic environments across species and highlights spectral tuning in an ocular but otherwise non-visual photopigment, which has been largely overlooked in studying the evolution of visual cycles.

Opsin proteins are central to photoreception and have diversified extensively to support both visual and non-visual functions in animals. While the molecular conservation is striking, the evolutionary pathways that shaped opsin diversity, particularly in relation to visual and extraocular roles, remain poorly understood. To address this gap, we examined opsin expression in Pteriomorphia, a diverse group of mostly sessile marine bivalves with multiple photoreceptor systems and ecologies. We generated transcriptomes for 12 species, including eyed and eyeless adults, and analysed published RNA-seq data from five additional species covering all taxonomic orders and eye types. We identified and classified opsins expressed in the photosensitive mantle margin and eyes using phylogenetically informed annotation. We tested whether opsin diversity is associated with eye presence or mobility and determined which opsins are widely used. Our results suggest that bivalve eyes generally express more opsins than extraocular tissues. Additionally, convergent expansions and reductions of opsin diversity were estimated, likely derived from an ancestral repertoire in the mantle margin. Curiously, differences in mobility among species do not influence opsin repertoire. Our findings further support that the rhodopsin-retinochrome system is pervasive across bivalves and identify visual and non-visual opsins used in both eyes and dermal photoreception.

IntroductionMany marine organisms have a biphasic life cycle that transitions between a swimming larva with a more sedentary adult form. At the end of the first phase, larvae must identify suitable sites to settle and undergo a dramatic morphological change. Environmental factors, including photic and chemical cues, appear to influence settlement, but the sensory receptors involved are largely unknown. We targeted the protein receptor, opsin, which belongs to large superfamily of transmembrane receptors that detects environmental stimuli, hormones, and neurotransmitters. While opsins are well-known for light-sensing, including vision, a growing number of studies have demonstrated light-independent functions. We therefore examined opsin expression in the Pteriomorphia, a large, diverse clade of marine bivalves, that includes commercially important species, such as oysters, mussels, and scallops.MethodsGenomic annotations combined with phylogenetic analysis show great variation of opsin abundance among pteriomorphian bivalves, including surprisingly high genomic abundance in many species that are eyeless as adults, such as mussels. Therefore, we investigated the diversity of opsin expression from the perspective of larval development. We collected opsin gene expression in four families of Pteriomorphia, across three distinct larval stages, i.e., trochophore, veliger, and pediveliger, and compared those to adult tissues.ResultsWe found larvae express all opsin types in these bivalves, but opsin expression patterns are largely species-specific across development. Few opsins are expressed in the adult mantle, but many are highly expressed in adult eyes. Intriguingly, opsin genes such as retinochrome, xenopsins, and Go-opsins have higher levels of expression in the later larval stages when substrates for settlement are being tested, such as the pediveliger.ConclusionInvestigating opsin gene expression during larval development provides crucial insights into their intricate interactions with the surroundings, which may shed light on how opsin receptors of these organisms respond to various environmental cues that play a pivotal role in their settlement process.

ABSTRACTThe bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present the first limid genome assembly, the disco clam,Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTMproximity-ligation sequencing. The final assembly is ∼2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a completely annotated mitochondrial genome, also a first for Limida. The ∼20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. TheC. alesnuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops,C. aleslacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact,C. aleshas uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids.SignificanceThis high-quality chromosome-level genome assembly forCtenoides ales, the disco clam, is the first genome sequenced from the bivalve order Limida, the only group of Pteriomorphia – a highly diverse and ecologically important clade – lacking genomic resources. The sequence and annotation of theC. alesgenome will be a useful resource for molluscan phylogenetics and comparative genomics.

Animals have evolved numerous mechanisms to perceive and interact with the environment that can be translated into different sensory modalities. However, the genomic and phenotypic features that support sensory functions remain enigmatic for many invertebrates, such as bivalves, an ecologically and economically important taxonomic group. No repertoire of sensory genes has been characterized in bivalves, representing a significant knowledge gap in molluscan sensory biology. Here, we gather multiple lines of evidence to explore the specialized sensory function of bivalve tentacles in the common jingle shell, Anomia simplex. In addition to applying microscopy techniques, we performed transcriptome sequencing of dissected tentacles using phylogenetically-informed annotation to identify candidate receptors. Our results demonstrate the expression of candidate GPCRs, including one opsin type, five small-molecule receptors, and 11 chemosensory-related receptors, supporting the involvement of sensory neurons in the organ, likely in association with the ciliated receptor cells observed along the tentacle surface. In addition, we identified seven ionotropic receptors as putative chemosensory receptors and one member of the Piezo mechanosensitive ion channel, which might be involved in touch sensation by ciliated sensory receptors. Our results provide the first evidence of putative sensory genes expressed in a bivalve sensory organ, representing an important starting point to investigate chemosensation in this class.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

ABSTRACT Left–right asymmetries are consistent differences between the left and right sides and represent an intriguing feature of molluscan morphology. Interestingly, external asymmetries, such as inequivalve shells, are often coupled with lateralization in the nervous system, which often includes functional and structural specializations of the left or right sides. In the case of visual asymmetries, lateralized phenotypes frequently include left–right differences in eye position, structure and use. To investigate lateralization and visual asymmetries among bivalves, we examined the visual and nervous systems of the scallop Nodipecten nodosus. We characterized different eye morphologies in the species and determined eye-side frequencies. We also studied the anatomy of the nervous system, focusing on the parietovisceral ganglion (PVG). Our results reveal that the visual and nervous systems of the scallop N. nodosus comprise consistent left–right asymmetries: (1) a greater number of eyes on the left side, (2) increased size of the left PVG lateral lobe, (3) larger glomeruli in the left PVG lateral lobe and (4) radial pallial nerves associated with the left lateral lobe spaced more widely than the more compactly arranged pallial nerves associated with the right lateral lobe. In addition, eyes with a distinctive morphology, where the optic vesicle is rotated, are more frequent on the left side. Considering the habit of this species to rest on the right valve, we hypothesize that curved eyes might contribute to expanding the overall visual field. Even though bivalves are not typically considered in the literature on lateralization, accumulating evidence for scallops, as suggested by our results, indicates their potential to reveal novel patterns of visual asymmetry in benthic invertebrates.

Eyes are remarkable systems to investigate the complex interaction between ecological drivers and phenotypic outcomes. Some animals, such as scallops, have many eyes for visual perception, but to date, the evolution of multiple-eye systems remains obscure. For instance, it is unclear whether eye number changes over a lifetime or varies among species. Scallops are a suitable model group to investigate these questions considering the interspecific variation of adult size and ecological diversity. We tested whether eye abundance scales with body size among individuals and species and whether it varies with life habits. We performed comparative analyses, including a phylogenetic ANCOVA and evolutionary model comparisons, based on eye count and shell height (as a proxy of body size) across 31 scallop species. Our analyses reveal that patterns of increasing relationship with body size are not concordant among taxa and suggest ontogenetic convergence caused by similar ecologies. Accordingly, selective optima in eye numbers are associated with shifts in life habits. For instance, species with increased mobility have significantly more eyes than less mobile species. The convergent evolution of greater eye abundance in more mobile scallops likely indicates a visual improvement based on increased levels of oversampling of the surrounding environment.

The relationship between genotype and phenotype is nontrivial due to often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket are responsible for fine spectral tuning, or small changes in the λmax of these light sensitive proteins Most mutations caused a blue shift regardless of the retinochrome background, with shifts ranging from a 12 nm blue shift to a 5 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggests the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis. Author summary Site-directed mutagenesis determined that spectral tuning in retinochrome is not solely additive, but is influenced by intra-molecular epistasis.

Tentacles are fascinating, multifunctional organs found in many aquatic invertebrate groups. In bivalves, tentacles are morphologically diverse, performing protective and sensory roles in taxa from different ecological niches. Such diversity is particularly accentuated in Pteriomorphia, a clade comprising scallops, oysters, file clams, and relatives. However, little is known about the evolution of these organs and their role in bivalve radiation. To test hypotheses of convergent tentacular evolution and a possible association between tentacles and body orientation on the substrate, we first examined tentacle morphology in 108 preserved species representing 15 families across Pteriomorphia. Morphological descriptions of tentacle type (inner mantle fold tentacles, IFT; middle mantle fold tentacles, MFT) and position (marginal and submarginal) are provided, expanding the knowledge of less studied bivalve taxa. Then, we placed the morphological dataset under a molecular phylogenetic framework to estimate ancestral states. IFT had likely four independent origins, while MFT emerged twice independently. After being gained, tentacles have not been lost. In addition, evolution of MFT coincides with transitions in body position with the midsagittal plane parallel to the substrate in the clades of scallops (Pectinida) and oysters (Ostreida). Such a shift could be related to the increase of mantle exposure, favoring the emergence of serially repeated organs, such as tentacles. Altogether, our results support the convergent evolution of tentacles across different taxonomic levels, corroborating the plasticity of the molluscan body and the relevance of evolutionary convergences in the radiation of bivalves.