Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).

The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene ß-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of ß-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of ß-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out ß-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.

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.

Divergences of the RLR Gene Families across Lophotrochozoans: Domain Grafting, Exon-Intron Structure, Expression, and Positive Selection.

Invertebrates do not possess adaptive immunity but have evolved a variety of unique repertoires of innate immune sensors. In this study, we explored the immune diversity and specificity of invertebrates based on the lophotrochozoan RLRs, a major component in antiviral immune recognition. By annotating RLRs in the genomes of 58 representative species across metazoan evolution, we explored the gene expansion of RLRs in Lophotrochozoa. Of note, the N-terminal domains of lophotrochozoan RLRs showed the most striking diversity which evolved independently by domain grafting. Exon-intron structures were revealed to be prevalent in the domain grafting of lophotrochozoan RLRs based on an analysis of sibling paralogs and orthologs. In more than half of the cases, the mechanism of 'exonization/pseudoexonization' led to the generation of non-canonical N-terminal domains. Transcriptomic studies revealed that many non-canonical RLRs display immune-related expression patterns. Two of these RLRs showed obvious evidence of positive selection, which may be the result of host defense selection pressure. Overall, our study suggests that the complex and unique domain arrangement of lophotrochozoan RLRs might result from domain grafting, exon-intron divergence, expression diversification, and positive selection, which may have led to functionally distinct lophotrochozoan RLRs.

Gut Microbial Divergence Between Three Hadal Amphipod Species from the Isolated Hadal Trenches.

Amphipods are the dominant scavenging metazoan species in the hadal trenches at water depths below 6,000 m. The gut microbiota have been considered to be contribution to the adaptation of deep-sea organisms; however, few comparative analyses of animal gut microbiota between different isolated hadal environments have been done so far. Here, we employed high-throughput 16S rRNA sequencing to compare the gut microbial taxonomic composition and functional potential diversity of three hadal amphipod species, Hirondellea gigas, Bathycallisoma schellenbergi, and Alicella gigantea, collected from the Mariana Trench, Marceau Trench, and New Britain Trench in the Pacific Ocean, respectively. Results showed that different community compositions were detected across all the amphipod specimens based on the analyses of alpha-diversity, hierarchical cluster tree, and PCoA (principal coordinate analysis). Moreover, almost no correlation was observed between genera overrepresented in different amphipods by microbe-microbe correlations analysis, which suggested that the colonization of symbionts were host-specific. At genus level, Psychromonas was dominant in H. gigas, and Candidatus Hepatoplasma was overall dominant in A. gigantea and B. schellenbergi. Comparison of the functional potential showed that, though three hadal amphipod species shared the same predominant functional pathways, the abundances of those most shared pathways showed distinct differences across all the specimens. These findings pointed to the enrichment of particular functional pathways in the gut microbiota of the different isolated trench amphipods. Moreover, in terms of species relative abundance, alpha-diversity and beta-diversity, there was high similarity of gut microbiota between the two A. gigantea populations, which dwelled in two different localities of the same hadal trench. Altogether, this study provides an initial investigation into the gut-microbial interactions and evolution at the hadal depths within amphipod. Each of these three amphipod species would be a model taxa for future studies investigating the influence habitat difference and geography on gut-microbial communities.

Transcriptomic Response to Perkinsus marinus in Two Crassostrea Oysters Reveals Evolutionary Dynamics of Host-Parasite Interactions.

Infectious disease outbreaks are causing widespread declines of marine invertebrates including corals, sea stars, shrimps, and molluscs. Dermo is a lethal infectious disease of the eastern oyster Crassostrea virginica caused by the protist Perkinsus marinus. The Pacific oyster Crassostrea gigas is resistant to Dermo due to differences in the host-parasite interaction that is not well understood. We compared transcriptomic responses to P. marinus challenge in the two oysters at early and late infection stages. Dynamic and orchestrated regulation of large sets of innate immune response genes were observed in both species with remarkably similar patterns for most orthologs, although responses in C. virginica were stronger, suggesting strong or over-reacting immune response could be a cause of host mortality. Between the two species, several key immune response gene families differed in their expansion, sequence variation and/or transcriptional response to P. marinus, reflecting evolutionary divergence in host-parasite interaction. Of note, significant upregulation of inhibitors of apoptosis (IAPs) was observed in resistant C. gigas but not in susceptible C. virginica, suggesting upregulation of IAPs is an active defense mechanism, not a passive response orchestrated by P. marinus. Compared with C. gigas, C. virginica exhibited greater expansion of toll-like receptors (TLRs) and positive selection in P. marinus responsive TLRs. The C1q domain containing proteins (C1qDCs) with the galactose-binding lectin domain that is involved in P. marinus recognition, were only present and significantly upregulated in C. virginica. These results point to previously undescribed differences in host defense genes between the two oyster species that may account for the difference in susceptibility, providing an expanded portrait of the evolutionary dynamics of host-parasite interaction in lophotrochozoans that lack adaptive immunity. Our findings suggest that C. virginica and P. marinus have a history of coevolution and the recent outbreaks may be due to increased virulence of the parasite.

Metagenomic Insights Into the Structure and Function of Intestinal Microbiota of the Hadal Amphipods.

Hadal trenches are the deepest known areas of the ocean. Amphipods are considered to be the dominant scavengers in the hadal food webs. The studies on the structure and function of the hadal intestinal microbiotas are largely lacking. Here, the intestinal microbiotas of three hadal amphipods, Hirondellea gigas, Scopelocheirus schellenbergi, and Alicella gigantea, from Mariana Trench, Marceau Trench, and New Britain Trench, respectively, were investigated. The taxonomic analysis identified 358 microbial genera commonly shared within the three amphipods. Different amphipod species possessed their own characteristic dominant microbial component, Psychromonas in H. gigas and Candidatus Hepatoplasma in A. gigantea and S. schellenbergi. Functional composition analysis showed that "Carbohydrate Metabolism," "Lipid Metabolism," "Cell Motility," "Replication and Repair," and "Membrane Transport" were among the most represented Gene Ontology (GO) Categories in the gut microbiotas. To test the possible functions of "Bacterial Chemotaxis" within the "Cell Motility" category, the methyl-accepting chemotaxis protein (MCP) gene involved in the "Bacterial Chemotaxis" pathway was obtained and used for swarming motility assays. Results showed that bacteria transformed with the gut bacterial MCP gene showed significantly faster growths compared with the control group, suggesting MCP promoted the bacterial swimming capability and nutrient utilization ability. This result suggested that hadal gut microbes could promote their survival in poor nutrient conditions by enhancing chemotaxis and motility. In addition, large quantities of probiotic genera were detected in the hadal amphipod gut microbiotas, which indicated that those probiotics would be possible contributors for promoting the host's growth and development, which could facilitate adaptation of hadal amphipods to the extreme environment.

Genetic Diversity and Population Structure Analysis of Three Deep-Sea Amphipod Species from Geographically Isolated Hadal Trenches in the Pacific Ocean.

Amphipods of the superfamily Lysianassoidea that inhabit the hadal zone ( > 6000 m) have large bathymetric ranges and play a key role in deep ocean ecosystems. The endemism of these amphipod species makes them a good model for investigating potent natural selection and restricted dispersal in deep ocean trenches. Here, we describe genetic diversity and intraspecific population differentiation among three amphipod species from four Pacific trenches based on a mtDNA concatenated dataset (CO Ι and 16S rRNA genes) from 150 amphipod individuals. All amphipod populations had low genetic diversity, as indicated by haplotype and nucleotide diversity values. Population geographic relationship analysis of two Alicella gigantea populations revealed no genetic differentiation between these two localities (pairwise genetic differentiation coefficient = 0.00032, gene flow = 784.58), and the major variation (99.97%) was derived from variation within the populations. Historical demographic events were investigated using Tajima's D and Fu's F neutrality tests and analysis of mismatch distribution. Consistent results provided strong evidence to support the premise that demographic expansion occurred only for the Mariana population of Hirondellea gigas, possibly within the last 2.1-3.4 million years. These findings suggest that the formation of amphipod population structure might be the result of multiple factors including high hydrostatic pressure, food distribution, trench topographic forcing and potential ecological interactions.

miRNA-152 targets GATA1 to regulate erythropoiesis in Chionodraco hamatus.

Chionodraco hamatus is a teleost within the suborder Notothenioidei, the members of which are known to lack functional erythrocytes with modified hematopoiesis. Hematopoiesis is an essential process during the development of animals, where it is tightly regulated by many different transcription factors, signaling proteins, chromatin modifications, and microRNAs (miRNAs). The miRNAs are known to regulate the expression of their target genes at the post-transcriptional level. However, little is known about the miRNA-mediated regulation of hematopoiesis. In this study, we confirmed that miR-152 plays a crucial role in hematopoiesis during the development of C. hamatus. The overexpression of miR-152 reduced hematopoiesis according to the decreased expression of GATA1 and reduced o-dianisidine staining of hemoglobin. Mechanistically, reduced hematopoiesis was regulated by the miR-152-mediated down-regulated expression of GATA1. Bioinformatics analysis was used to predict the target gene of miR-152. Western blotting as well as dual luciferase and EGFP reporter assays were employed to investigate the expression of GATA1 mediated by miR-152. Finally, verification experiments in the zebrafish autologous model strongly supported the effect of miR-152 on hematopoiesis. In conclusion, we suggest that miR-152 is a novel molecular factor that regulates hematopoiesis during the development of C. hamatus by down-regulating the expression of GATA1.

Neofunctionalization of zona pellucida proteins enhances freeze-prevention in the eggs of Antarctic notothenioids.

The mechanisms by which the eggs of the Antarctic notothenioid fishes avoid freezing are not fully understood. Zona pellucida proteins (ZPs) are constituents of the chorion which forms a protective matrix surrounding the egg. Here we report occurrence of freezing temperature-related gene expansion and acquisition of unusual ice melting-promoting (IMP) activity in a family of Antarctic notothenioid ZPs (AnnotoZPs). Members of AnnotoZPs are shown to bind with ice and non-colligatively depress the melting point of a solution in a range of 0.26 to 0.65 °C at a moderate concentration. Eggs of zebrafishes expressing an AnnotoZP transgene show improved melting point depression and enhanced survival in freezing conditions. Mutational analyses in a representative AnnotoZP indicate the ZP domain and patches of acidic residues are essential structures for the IMP activity. AnnotoZPs, therefore, represent a group of macromolecules that prevent freezing by a unique ZP-ice interaction mechanism distinct from the known antifreeze proteins.

Evolutionary suppression of erythropoiesis via the modulation of TGF-ß signalling in an Antarctic icefish.

The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-ß signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-ß superfamily revealed signs of positive selection in TGF-ß1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-ß signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.