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Disseminated neoplasia (DN) is a form of cancer in bivalve molluscs that can be transmissible between individuals and in some cases across species. Neoplastic cells are highly proliferative, and infection is usually lethal. Commercially valuable bivalve species (mussels, cockles, softshell clams, and oysters) are affected by outbreaks of DN, making disease diagnosis and mitigation an important issue in ecological restoration efforts and aquaculture. Basket cockles (Clinocardium nuttallii) are native to the North American Pacific coast from California to Alaska. Recent concern from some Coast Salish Tribes regarding an observed long-term decline in basket cockle populations in Puget Sound, WA has increased interest in monitoring efforts and subsequent collection for aquarium-reared broodstock. Disseminated neoplasia was detected in Puget Sound basket cockle populations, delaying aquaculture efforts so that potential broodstock could be assessed for the presence of DN. This study details a minimally invasive, inexpensive, nonlethal method for high-throughput screening for DN in adult basket cockles. The hemolymph smear screening method to diagnose DN in C. nuttallii can be applied at field sites at low financial cost. Results of the hemolymph smear technique were validated against whole tissue histology, the standard method for DN diagnosis. Due to the similar cellular morphologies of DN in different bivalve species, it is proposed that hemolymph histology can likely be applied for diagnosing DN in other bivalves. 

Abstract The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes. Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa. To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophoreMnemiopsis leidyiand the oysterCrassostrea gigas. Here we report that ctenophores – thought to have diverged very early from the metazoan stem lineage – possess immune-like cells capable of phagocytosis and ETosis. We demonstrate that bothMnemiopsisandCrassostreaimmune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. We thus propose that ETosis is an evolutionarily conserved metazoan defense against pathogens. 

Abstract The cluster of differentiation 36 (CD36) domain defines the characteristic ectodomain associated with class B scavenger receptor (SR-B) proteins. In bilaterians, SR-Bs play critical roles in diverse biological processes including innate immunity functions such as pathogen recognition and apoptotic cell clearance, as well as metabolic sensing associated with fatty acid uptake and cholesterol transport. Although previous studies suggest this protein family is ancient, SR-B diversity across Eukarya has not been robustly characterized. We analyzed SR-B homologs identified from the genomes and transcriptomes of 165 diverse eukaryotic species. The presence of highly conserved amino acid motifs across major eukaryotic supergroups supports the presence of a SR-B homolog in the last eukaryotic common ancestor. Our comparative analyses of SR-B protein structure identify the retention of a canonical asymmetric beta barrel tertiary structure within the CD36 ectodomain across Eukarya. We also identify multiple instances of independent lineage-specific sequence expansions in the apex region of the CD36 ectodomain—a region functionally associated with ligand-sensing. We hypothesize that a combination of both sequence expansion and structural variation in the CD36 apex region may reflect the evolution of SR-B ligand-sensing specificity between diverse eukaryotic clades. 

Cell suspension fluidics, such as flow cytometry (FCS) and fluorescence-activated cell sorting (FACS), facilitates the identification and precise separation of individual cells based on phenotype. Since its introduction, flow cytometry has been used to analyze cell types and cellular processes in diverse non-vertebrate taxa, including cnidarians, molluscs, and arthropods. Ctenophores, which diverged very early from the metazoan stem lineage, have emerged as an informative clade for the study of metazoan cell type evolution. We present standardized methodologies for flow cytometry-mediated identification and analyses of cells from the model ctenophoreMnemiopsis leidyithat can also be applied to isolate targeted cell populations. Here we focus on the identification and isolation of ctenophore phagocytes. Implementing flow cytometry methods in ctenophores allows for fine scale analyses of fundamental cellular processes conserved broadly across animals, as well as potentially revealing novel cellular phenotypes and behaviors restricted to the ctenophore lineage. 

Abstract Innate immunity is an ancient physiological response critical for protecting metazoans from invading pathogens. It is the primary pathogen defense mechanism among invertebrates. While innate immunity has been studied extensively in diverse invertebrate taxa, including mollusks, crustaceans, and cnidarians, this system has not been well characterized in ctenophores. The ctenophores comprise an exclusively marine, non-bilaterian lineage that diverged early during metazoan diversification. The phylogenetic position of ctenophore lineage suggests that characterization of the ctenophore innate immune system will reveal important features associated with the early evolution of the metazoan innate immune system. Here, we review current understanding of the ctenophore immune repertoire and identify innate immunity genes recovered from three ctenophore species. We also isolate and characterize Mnemiopsis leidyi cells that display macrophage-like behavior when challenged with bacteria. Our results indicate that ctenophores possess cells capable of phagocytosing microbes and that two distantly related ctenophores, M. leidyi and Hormiphora californiensis, possess many candidate innate immunity proteins.