Ciliated trochophore-type larvae are widespread among protostome animals with spiral cleavage. The respective phyla are often united into the superclade Spiralia or Lophotrochozoa that includes, for example, mollusks, annelids, and platyhelminths. Mollusks (bivalves, gastropods, cephalopods, polyplacophorans, and their kin) in particular are known for their morphological innovations and lineage-specific plasticity of homologous characters (e.g., radula, shell, foot, neuromuscular systems), raising questions concerning the cell types and the molecular toolkit that underlie this variation. Here, we report on the gene expression profile of individual cells of the trochophore larva of the invasive freshwater bivalve Dreissena rostriformis as inferred from single cell RNA sequencing. We generated transcriptomes of 632 individual cells and identified seven transcriptionally distinct cell populations. Developmental trajectory analyses identify cell populations that, for example, share an ectodermal origin such as the nervous system, the shell field, and the prototroch. To annotate these cell populations, we examined ontology terms from the gene sets that characterize each individual cluster. These were compared to gene expression data previously reported from other lophotrochozoans. Genes expected to be specific to certain tissues, such as Hox1 (in the shell field), Caveolin (in prototrochal cells), or FoxJ (in other cillia-bearing cells) provide evidence that the recovered cell populations contribute to various distinct tissues and organs known from morphological studies. This dataset provides the first molecular atlas of gene expression underlying bivalve organogenesis and generates an important framework for future comparative studies into cell and tissue type development in Mollusca and Metazoa as a whole.

The mesoderm gives rise to several key morphological features of bilaterian animals including endoskeletal elements and the musculature. A number of regulatory genes involved in mesoderm and/or muscle formation (e.g., Brachyury (Bra), even-skipped (eve), Mox, myosin II heavy chain (mhc)) have been identified chiefly from chordates and the ecdysozoans Drosophila and Caenorhabditis elegans, but data for non-model protostomes, especially those belonging to the ecdysozoan sister clade, Lophotrochozoa (e.g., flatworms, annelids, mollusks), are only beginning to emerge. Within the lophotrochozoans, Mollusca constitutes the most speciose and diverse phylum. Interestingly, however, information on the morphological and molecular underpinnings of key ontogenetic processes such as mesoderm formation and myogenesis remains scarce even for prominent molluscan sublineages such as the bivalves. Here, we investigated myogenesis and developmental expression of Bra, eve, Mox, and mhc in the quagga mussel Dreissena rostriformis, an invasive freshwater bivalve and an emerging model in invertebrate evodevo. We found that all four genes are expressed during mesoderm formation, but some show additional, individual sites of expression during ontogeny. While Mox and mhc are involved in early myogenesis, eve is also expressed in the embryonic shell field and Bra is additionally present in the foregut. Comparative analysis suggests that Mox has an ancestral role in mesoderm and possibly muscle formation in bilaterians, while Bra and eve are conserved regulators of mesoderm development of nephrozoans (protostomes and deuterostomes). The fully developed Dreissena veliger larva shows a highly complex muscular architecture, supporting a muscular ground pattern of autobranch bivalve larvae that includes at least a velum muscle ring, three or four pairs of velum retractors, one or two pairs of larval retractors, two pairs of foot retractors, a pedal plexus, possibly two pairs of mantle retractors, and the muscles of the pallial line, as well as an anterior and a posterior adductor. As is typical for their molluscan kin, remodelling and loss of prominent larval features such as the velum musculature and various retractor systems appear to be also common in bivalves.