CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, Heliocidaris crassispina.

Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. Heliocidaris crassispina is an important marine fishery resource with edible gonads. Although H. crassispina has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to H. crassispina. In this study, we targeted genes encoding ETS transcription factor (HcEts) and pigmentation-related polyketide synthase (HcPks1). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When Ets gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the Pks1 gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the Pks1 gene in Hemicentrotus pulcherrimus, mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, Pks1 knockout H. crassispina exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin H. crassispina.

Local Genomic Instability of the SpTransformer Gene Family in the Purple Sea Urchin Inferred from BAC Insert Deletions.

The SpTransformer (SpTrf) gene family in the purple sea urchin, Strongylocentrotus purpuratus, encodes immune response proteins. The genes are clustered, surrounded by short tandem repeats, and some are present in genomic segmental duplications. The genes share regions of sequence and include repeats in the coding exon. This complex structure is consistent with putative local genomic instability. Instability of the SpTrf gene cluster was tested by 10 days of growth of Escherichia coli harboring bacterial artificial chromosome (BAC) clones of sea urchin genomic DNA with inserts containing SpTrf genes. After the growth period, the BAC DNA inserts were analyzed for size and SpTrf gene content. Clones with multiple SpTrf genes showed a variety of deletions, including loss of one, most, or all genes from the cluster. Alternatively, a BAC insert with a single SpTrf gene was stable. BAC insert instability is consistent with variations in the gene family composition among sea urchins, the types of SpTrf genes in the family, and a reduction in the gene copy number in single coelomocytes. Based on the sequence variability among SpTrf genes within and among sea urchins, local genomic instability of the family may be important for driving sequence diversity in this gene family that would be of benefit to sea urchins in their arms race with marine microbes.

Transglobal spread of an ecologically relevant sea urchin parasite.

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s contributed to a persistent phase shift from coral- to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, though the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of Diadema setosum occurred along the Sultanate of Oman's coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman, that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in Diadema setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

Genome-Wide Comparative Analysis of SRCR Gene Superfamily in Invertebrates Reveals Massive and Independent Gene Expansions in the Sponge and Sea Urchin.

Without general adaptative immunity, invertebrates evolved a vast number of heterogeneous non-self recognition strategies. One of those well-known adaptations is the expansion of the immune receptor gene superfamily coding for scavenger receptor cysteine-rich domain containing proteins (SRCR) in a few invertebrates. Here, we investigated the evolutionary history of the SRCR gene superfamily (SRCR-SF) across 29 metazoan species with an emphasis on invertebrates. We analyzed their domain architectures, genome locations and phylogenetic distribution. Our analysis shows extensive genome-wide duplications of the SRCR-SFs in Amphimedon queenslandica and Strongylocentrotus purpuratus. Further molecular evolution study reveals various patterns of conserved cysteines in the sponge and sea urchin SRCR-SFs, indicating independent and convergent evolution of SRCR-SF expansion during invertebrate evolution. In the case of the sponge SRCR-SFs, a novel motif with seven conserved cysteines was identified. Exon-intron structure analysis suggests the rapid evolution of SRCR-SFs during gene duplications in both the sponge and the sea urchin. Our findings across nine representative metazoans also underscore a heightened expression of SRCR-SFs in immune-related tissues, notably the digestive glands. This observation indicates the potential role of SRCR-SFs in reinforcing distinct immune functions in these invertebrates. Collectively, our results reveal that gene duplication, motif structure variation, and exon-intron divergence might lead to the convergent evolution of SRCR-SF expansions in the genomes of the sponge and sea urchin. Our study also suggests that the utilization of SRCR-SF receptor duplication may be a general and basal strategy to increase immune diversity and tissue specificity for the invertebrates.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

Spotting disease disrupts the microbiome of infected purple sea urchins, Strongylocentrotus purpuratus.

BACKGROUND: Spotting disease infects a variety of sea urchin species across many different marine locations. The disease is characterized by discrete lesions on the body surface composed of discolored necrotic tissue that cause the loss of all surface appendages within the lesioned area. A similar, but separate disease of sea urchins called bald sea urchin disease (BSUD) has overlapping symptoms with spotting disease, resulting in confusions in distinguishing the two diseases. Previous studies have focus on identifying the underlying causative agent of spotting disease, which has resulted in the identification of a wide array of pathogenic bacteria that vary based on location and sea urchin species. Our aim was to investigate the spotting disease infection by characterizing the microbiomes of the animal surface and various tissues. RESULTS: We collected samples of the global body surface, the lesion surface, lesioned and non-lesioned body wall, and coelomic fluid, in addition to samples from healthy sea urchins. 16S rRNA gene was amplified and sequenced from the genomic DNA. Results show that the lesions are composed mainly of Cyclobacteriaceae, Cryomorphaceae, and a few other taxa, and that the microbial composition of lesions is the same for all infected sea urchins. Spotting disease also alters the microbial composition of the non-lesioned body wall and coelomic fluid of infected sea urchins. In our closed aquarium systems, sea urchins contracted spotting disease and BSUD separately and therefore direct comparisons could be made between the microbiomes from diseased and healthy sea urchins. CONCLUSION: Results show that spotting disease and BSUD are separate diseases with distinct symptoms and distinct microbial compositions.

Characterization of p53/p63/p73 and Myc expressions during embryogenesis of the sea urchin.

BACKGROUND: Some marine invertebrate organisms are considered not to develop tumors due to unknown mechanisms. To gain an initial insight into how tumor-related genes may be expressed and function during marine invertebrate development, we here leverage sea urchin embryos as a model system and characterize the expressions of Myc and p53/p63/p73 which are reported to function synergistically in mammalian models as an oncogene and tumor suppressor, respectively. RESULTS: During sea urchin embryogenesis, a combo gene of p53/p63/p73 is found to be maternally loaded and decrease after fertilization both in transcript and protein, while Myc transcript and protein are zygotically expressed. p53/p63/p73 and Myc proteins are observed in the cytoplasm and nucleus of every blastomere, respectively, throughout embryogenesis. Both p53/p63/p73 and Myc overexpression results in compromised development with increased DNA damage after the blastula stage. p53/p63/p73 increases the expression of parp1, a DNA repair/cell death marker gene, and suppresses endomesoderm gene expressions. In contrast, Myc does not alter the expression of specification genes or oncogenes yet induces disorganized morphology. CONCLUSIONS: p53/p63/p73 appears to be important for controlling cell differentiation, while Myc induces disorganized morphology yet not through conventional oncogene regulations or apoptotic pathways during embryogenesis of the sea urchin.

Parentage influence on gene expression under acidification revealed through single-embryo sequencing.

The dissolution of anthropogenic carbon dioxide (CO2 ) in seawater has altered its carbonate chemistry in the process of ocean acidification (OA). OA affects the viability of marine species. In particular, calcifying organisms and their early planktonic larval stages are considered vulnerable. These organisms often utilize energy reserves for metabolism rather than growth and calcification as supported by bulk RNA-sequencing (RNA-seq) experiments. Yet, transcriptomic profiling of a bulk sample reflects the average gene expression of the population, neglecting the variations between individuals, which forms the basis for natural selection. Here, we used single-embryo RNA-seq on larval sea urchin Heliocidaris crassispina, which is a commercially and ecologically valuable species in East Asia, to document gene expression changes to OA at an individual and family level. Three paternal half-sibs groups were fertilized and exposed to 3 pH conditions (ambient pH 8.0, 7.7 and 7.4) for 12 h prior to sequencing and oxygen consumption assay. The resulting transcriptomic profile of all embryos can be distinguished into four clusters, with differences in gene expressions that govern biomineralization, cell differentiation and patterning, as well as metabolism. While these responses were influenced by pH conditions, the male identities also had an effect. Specifically, a regression model and goodness of fit tests indicated a significant interaction between sire and pH on the probability of embryo membership in different clusters of gene expression. The single-embryo RNA-seq approach is promising in climate stressor research because not only does it highlight potential impacts before phenotypic changes were observed, but it also highlights variations between individuals and lineages, thus enabling a better determination of evolutionary potential.

Whole-Genome Sequencing Reveals That Regulatory and Low Pleiotropy Variants Underlie Local Adaptation to Environmental Variability in Purple Sea Urchins.

AbstractOrganisms experience environments that vary across both space and time. Such environmental heterogeneity shapes standing genetic variation and may influence species' capacity to adapt to rapid environmental change. However, we know little about the kind of genetic variation that is involved in local adaptation to environmental variability. To address this gap, we sequenced the whole genomes of 140 purple sea urchins (Strongylocentrotus purpuratus) from seven populations that vary in their degree of pH variability. Despite no evidence of global population structure, we found a suite of single-nucleotide polymorphisms (SNPs) tightly correlated with local pH variability (outlier SNPs), which were overrepresented in regions putatively involved in gene regulation (long noncoding RNA and enhancers), supporting the idea that variation in regulatory regions is important for local adaptation to variability. In addition, outliers in genes were found to be (i) enriched for biomineralization and ion homeostasis functions related to low pH response, (ii) less central to the protein-protein interaction network, and (iii) underrepresented among genes highly expressed during early development. Taken together, these results suggest that loci that underlie local adaptation to pH variability in purple sea urchins fall in regions with potentially low pleiotropic effects (based on analyses involving regulatory regions, network centrality, and expression time) involved in low pH response (based on functional enrichment).

Hybrid Epigenomes Reveal Extensive Local Genetic Changes to Chromatin Accessibility Contribute to Divergence in Embryonic Gene Expression Between Species.

Chromatin accessibility plays an important role in shaping gene expression, yet little is known about the genetic and molecular mechanisms that influence the evolution of chromatin configuration. Both local (cis) and distant (trans) genetic influences can in principle influence chromatin accessibility and are based on distinct molecular mechanisms. We, therefore, sought to characterize the role that each of these plays in altering chromatin accessibility in 2 closely related sea urchin species. Using hybrids of Heliocidaris erythrogramma and Heliocidaris tuberculata, and adapting a statistical framework previously developed for the analysis of cis and trans influences on the transcriptome, we examined how these mechanisms shape the regulatory landscape at 3 important developmental stages, and compared our results to similar analyses of the transcriptome. We found extensive cis- and trans-based influences on evolutionary changes in chromatin, with cis effects generally larger in effect. Evolutionary changes in accessibility and gene expression are correlated, especially when expression has a local genetic basis. Maternal influences appear to have more of an effect on chromatin accessibility than on gene expression, persisting well past the maternal-to-zygotic transition. Chromatin accessibility near gene regulatory network genes appears to be distinctly regulated, with trans factors appearing to play an outsized role in the configuration of chromatin near these genes. Together, our results represent the first attempt to quantify cis and trans influences on evolutionary divergence in chromatin configuration in an outbred natural study system and suggest that chromatin regulation is more genetically complex than was previously appreciated.

Single-Cell Transcriptomic Analysis Reveals the Molecular Profile of Go-Opsin Photoreceptor Cells in Sea Urchin Larvae.

The ability to perceive and respond to light stimuli is fundamental not only for spatial vision but also to many other light-mediated interactions with the environment. In animals, light perception is performed by specific cells known as photoreceptors and, at molecular level, by a group of GPCRs known as opsins. Sea urchin larvae possess a group of photoreceptor cells (PRCs) deploying a Go-Opsin (Opsin3.2) which have been shown to share transcription factors and morphology with PRCs of the ciliary type, raising new questions related to how this sea urchin larva PRC is specified and whether it shares a common ancestor with ciliary PRCs or it if evolved independently through convergent evolution. To answer these questions, we combined immunohistochemistry and fluorescent in situ hybridization to investigate how the Opsin3.2 PRCs develop in the sea urchin Strongylocentrotus purpuratus larva. Subsequently, we applied single-cell transcriptomics to investigate the molecular signature of the Sp-Opsin3.2-expressing cells and show that they deploy an ancient regulatory program responsible for photoreceptors specification. Finally, we also discuss the possible functions of the Opsin3.2-positive cells based on their molecular fingerprint, and we suggest that they are involved in a variety of signaling pathways, including those entailing the thyrotropin-releasing hormone.

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

The crucial role of CTCF in mitotic progression during early development of sea urchin.

CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.

Characterization of digestive proteases in the gut of a basal deuterostome.

Digestive systems are complex organs that allow organisms to absorb energy from their environment to fuel vital processes such as growth, development and the maintenance of homeostasis. A comprehensive understanding of digestive physiology is therefore essential to fully understand the energetics of an organism. The digestion of proteins is of particular importance because most heterotrophic organisms are not able to synthesize all essential amino acids. While Echinoderms are basal deuterostomes that share a large genetic similarity with vertebrates, their digestion physiology remains largely unexplored. Using a genetic approach, this work demonstrated that several protease genes including an enteropeptidase, aminopeptidase, carboxypeptidase and trypsin involved in mammalian digestive networks are also found in sea urchin larvae. Through characterization including perturbation experiments with different food treatments and pharmacological inhibition of proteases using specific inhibitors, as well as transcriptomic analysis, we conclude that the trypsin-2 gene codes for a crucial enzyme for protein digestion in Strongylocentrotus purpuratus. Measurements of in vivo digestion rates in the transparent sea urchin larva were not altered by pharmacological inhibition of trypsin (using soybean trypsin inhibitor) or serine proteases (aprotinin), suggesting that proteases are not critically involved in the initial step of microalgal breakdown. This work provides new insights into the digestive physiology of a basal deuterostome and allows comparisons from the molecular to the functional level in the digestive systems of vertebrates and mammals. This knowledge will contribute to a better understanding for conserved digestive mechanisms that evolved in close interaction with their biotic and abiotic environment.

New hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinoderms.

Cell types are the building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos has contributed significantly to understanding the evolution of novel cell types, through the examination of skeletogenic mesenchyme and pigment cells, which are found in sea urchin larvae, but not sea star larvae. This paper outlines the development of a gene expression atlas for the bat sea star, Patiria miniata, using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 23 cell clusters covering all expected cell types from the endoderm, mesoderm, and ectoderm germ layers. In particular, four distinct neural clusters, an immune-like cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single-cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. The equivalent of S. purpuratus piwil3+ Cells were not detected in P. miniata, while the Left Coelom of P. miniata has no equivalent cell cluster in S. purpuratus. These differences may reflect changes in developmental timing between these species. While considered novel morphologically, the Pigment Cells of S. purpuratus map to clusters containing Immune-like Mesenchyme and Neural cells of P. miniata, while the Skeletogenic Mesenchyme of S. purpuratus are revealed as orthologous to the Right Coelom cluster of P. miniata. These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types.

Natural antisense transcription of presenilin in sea urchin reveals a possible role for natural antisense transcription in the general control of gene expression during development

One presenilin gene (PSEN) is expressed in the sea urchin embryo, in the vegetal pole of the gastrula and then mainly in cilia cells located around the digestive system of the pluteus, as we recently have reported. PSEN expression must be accurately regulated for correct execution of these two steps of development. While investigating PSEN expression changes in embryos after expansion of endoderm with LiCl or of ectoderm with Zn2+ by whole-mount in situ hybridization (WISH) and quantitative PCR (qPCR), we detected natural antisense transcription of PSEN. We then found that Endo16 and Wnt5, markers of endo-mesoderm, and of Hnf6 and Gsc, markers of ectoderm, are also sense and antisense transcribed. We discuss that general gene expression could depend on both sense and antisense transcription. This mechanism, together with the PSEN gene, should be included in gene regulatory networks (GRNs) that theorize diverse processes in this species. We suggest that it would also be relevant to investigate natural antisense transcription of PSEN in the field of Alzheimer's disease (AD) where the role of human PSEN1 and PSEN2 is well known.

Near-Chromosomal-Level Genome Assembly of the Sea Urchin Echinometra lucunter, a Model for Speciation in the Sea.

Echinometra lucunter, the rock-boring sea urchin, is a widely distributed echinoid and a model for ecological studies of reproduction, responses to climate change, and speciation. We present a near chromosome-level genome assembly of E. lucunter, including 21 scaffolds larger than 10 Mb predicted to represent each of the chromosomes of the species. The 760.4 Mb assembly includes a scaffold N50 of 30.0 Mb and BUSCO (benchmarking universal single-copy orthologue) single copy and a duplicated score of 95.8% and 1.4%, respectively. Ab-initio gene model prediction and annotation with transcriptomic data constructed 33,989 gene models composing 50.4% of the assembly, including 37,036 transcripts. Repetitive elements make up approximately 39.6% of the assembly, and unresolved gap sequences are estimated to be 0.65%. Whole genome alignment with Echinometra sp. EZ revealed high synteny and conservation between the two species, further bolstering Echinometra as an emerging genus for comparative genomics studies. This genome assembly represents a high-quality genomic resource for future evolutionary and developmental studies of this species and more broadly of echinoderms.

The lysosome-phagosome pathway mediates immune regulatory mechanisms in Mesocentrotus nudus against Vibrio coralliilyticus infection.

Sea urchins are a popular model species for studying invertebrate diseases. The immune regulatory mechanisms of the sea urchin Mesocentrotus nudus during pathogenic infection are currently unknown. This study aimed to reveal the potential molecular mechanisms of M. nudus during resistance to Vibrio coralliilyticus infection by integrative transcriptomic and proteomic analyses. Here, we identified a total of 135,868 unigenes and 4,351 proteins in the four infection periods of 0 h, 20 h, 60 h and 100 h in M. nudus. In the I20, I60 and I100 infection comparison groups, 10,861, 15,201 and 8,809 differentially expressed genes (DEGs) and 2,188, 2,386 and 2,516 differentially expressed proteins (DEPs) were identified, respectively. We performed an integrated comparative analysis of the transcriptome and proteome throughout the infection phase and found very a low correlation between transcriptome and proteome changes. KEGG pathway analysis revealed that most upregulated DEGs and DEPs were involved in immune strategies. Notably, "lysosome" and "phagosome" activated throughout the infection process, could be considered the two most important enrichment pathways at the mRNA and protein levels. The significant increase in phagocytosis of infected M. nudus coelomocytes further demonstrated that the lysosome-phagosome pathway played an important immunological role in M. nudus resistance to pathogenic infection. Key gene expression profiles and protein‒protein interaction analysis revealed that cathepsin family and V-ATPase family genes might be key bridges in the lysosome-phagosome pathway. In addition, the expression patterns of key immune genes were verified using qRT‒PCR, and the different expression trends of candidate genes reflected, to some extent, the regulatory mechanism of immune homeostasis mediated by the lysosome-phagosome pathway in M. nudus against pathogenic infection. This work will provide new insights into the immune regulatory mechanisms of sea urchins under pathogenic stress and help identify key potential genes/proteins for sea urchin immune responses.

Phylogeny, ancestral ranges and reclassification of sand dollars.

Classification of the Class Echinoidea is under significant revision in light of emerging molecular phylogenetic evidence. In particular, the sister-group relationships within the superorder Luminacea (Echinoidea: Irregularia) have been considerably updated. However, the placement of many families remains largely unresolved due to a series of incongruent evidence obtained from morphological, paleontological, and genetic data for the majority of extant representatives. In this study, we investigated the phylogenetic relationships of 25 taxa, belonging to eleven luminacean families. We proposed three new superfamilies: Astriclypeoidea, Mellitoidea, and Taiwanasteroidea (including Dendrasteridae, Taiwanasteridae, Scutellidae, and Echinarachniidae), instead of the currently recognized superfamily Scutelloidea Gray, 1825. In light of the new data obtained from ten additional species, the historical biogeography reconstructed shows that the tropical western Pacific and eastern Indian Oceans are the cradle for early sand dollar diversification. Hothouse conditions during the late Cretaceous and early Paleogene were coupled with diversification events of major clades of sand dollars. We also demonstrate that Taiwan fauna can play a key role in terms of understanding the major Cenozoic migration and dispersal events in the evolutionary history of Luminacea.

Functional annotation of a hugely expanded nanos repertoire in Lytechinus variegatus, the green sea urchin.

Nanos genes encode essential RNA-binding proteins involved in germline determination and germline stem cell maintenance. When examining diverse classes of echinoderms, typically three, sometimes four, nanos genes are present. In this analysis, we identify and annotate nine nanos orthologs in the green sea urchin, Lytechinus variegatus (Lv). All nine genes are transcribed and grouped into three distinct classes. Class one includes the germline Nanos, with one member: Nanos2. Class two includes Nanos3-like genes, with significant sequence similarity to Nanos3 in the purple sea urchin, Strongylocentrotus purpuratus (Sp), but with wildly variable expression patterns. The third class includes several previously undescribed nanos zinc-finger genes that may be the result of duplications of Nanos2. All nine nanos transcripts occupy unique genomic loci and are expressed with unique temporal profiles during development. Importantly, here we describe and characterize the unique genomic location, conservation, and phylogeny of the Lv ortholog of the well-studied Sp Nanos2. However, in addition to the conserved germline functioning Nanos2, the green sea urchin appears to be an outlier in the echinoderm phyla with eight additional nanos genes. We hypothesize that this expansion of nanos gene members may be the result of a previously uncharacterized L1-class transposon encoded on the opposite strand of a nanos2 pseudogene present on chromosome 12 in this species. The expansion of nanos genes described here represents intriguing insights into germline specification and nanos evolution in this species of sea urchin.