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ABSTRACT Over the course of hundreds of millions of years, biomineralization has evolved independently many times across all kingdoms of life. Among animals, the phylum Mollusca displays a remarkable diversity in biomineral structures, particularly the molluscan shell, which varies greatly in shape, size, pigmentation, and patterning. Shell matrix proteins (SMPs) are key components of these shells, and are thought to drive the precipitation of calcium carbonate minerals and influence shell morphology. However, this structure‐function relationship has rarely been studied directly because tools for knocking out genes did not exist in molluscs until recently. In this study, we report the first successful use of CRISPR/Cas9 gene editing to target an SMP in gastropod molluscs. Using the emerging model gastropodCrepidula atrasolea, we generated knockouts of theSMP1gene. Successful gene editing was confirmed by Sanger and MiSeq sequencing, and loss ofSMP1expression was validated through high‐content imaging of crispant embryos. This study establishesC. atrasoleaas a valuable model for investigating the genetic basis of shell formation and provides a framework for applying CRISPR/Cas9 technology in other molluscan species. Our approach will enable future studies to thoroughly test the role of SMPs in shaping the diverse array of molluscan shell structures. 

Sea urchins are basal deuterostomes that share key molecular components of innate immunity with vertebrates. They are a powerful model for the study of innate immune system evolution and function, especially during early development. Here we characterize the morphology and associated molecular markers of larval immune cell types in a newly developed model sea urchin, Lytechinus pictus. We then challenge larvae through infection with an established pathogenic Vibrio and characterize phenotypic and molecular responses. We contrast these to the previously described immune responses of the purple sea urchin Strongylocentrotus purpuratus . The results revealed shared cellular morphologies and homologs of known pigment cell immunocyte markers ( PKS, srcr142 ) but a striking absence of subsets of perforin‐like macpf genes in blastocoelar cell immunocytes. We also identified novel patterning of cells expressing a scavenger receptor cysteine rich (SRCR) gene in the coelomic pouches of the larva (the embryonic stem cell niche). The SRCR signal becomes further enriched in both pouches in response to bacterial infection. Collectively, these results provide a foundation for the study of immune responses in L. pictus. The characterization of the larval immune system of this rapidly developing and genetically enabled sea urchin species will facilitate more sophisticated studies of innate immunity and the crosstalk between the immune system and development. 

ABSTRACT Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources. 

ABSTRACT The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host–microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment. 

Abstract BackgroundSea urchin embryos have been used for more than a century in the study of fertilization and early development. However, several of the species used, such asStrongylocentrotus purpuratus, have long generation times making them suboptimal for transgenerational studies. ResultsHere, we present an overview of the development of a rapidly developing echinoderm species,Lytechinus pictus, from fertilization through sexual maturation. When grown at room temperature (20°C) embryos complete the first cell cycle in 90 minutes, followed by subsequent cleavages every 45 minutes, leading to hatching at 9 hours postfertilization (hpf). The swimming embryos gastrulate from 12 to 36 hpf and produce the cells which subsequently give rise to the larval skeleton and immunocytes. Larvae begin to feed at 2 days and metamorphose by 3 weeks. Juveniles reach sexual maturity at 4 to 6 months of age, depending on individual growth rate. ConclusionsThis staging scheme lays a foundation for future studies inL. pictus, which share many of the attractive features of other urchins but have the key advantage of rapid development to sexual maturation. This is significant for multigenerational and genetic studies newly enabled by CRISPR‐CAS mediated gene editing. 

Although persistent, bioaccumulative and toxic pollutants (PBTs) are well-studied individually, their distribution and variability on a global scale are largely unknown, particularly in marine fish. Using 2,662 measurements collected from peer-reviewed literature spanning 1969–2012, we examined variability of five classes of PBTs, considering effects of geography, habitat, and trophic level on observed concentrations. While we see large-scale spatial patterning in some PBTs (chlordanes, polychlorinated biphenyls), habitat type and trophic level did not contribute to significant patterning, with the exception of mercury. We further examined patterns of change in PBT concentration as a function of sampling year. All PBTs showed significant declines in concentration levels through time, ranging from 15–30% reduction per decade across PBT groups. Despite consistent evidence of reductions, variation in pollutant concentration remains high, indicating ongoing consumer risk of exposure to fish with pollutant levels exceeding EPA screening values. The temporal trends indicate that mitigation programs are effective, but that global levels decline slowly. In order for monitoring efforts to provide more targeted assessments of risk to PBT exposure, these data highlight an urgent need for improved replication and standardization of pollutant monitoring protocols for marine finfish.