Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai)

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

NPF activates a specific NPF receptor and regulates food intake in Pacific abalone Haliotis discus hannai

Neuropeptides function through G protein-coupled receptors (GPCRs) with high specificity, implying a significant degree of neuropeptide-GPCR coevolution. However, potential neuropeptide signaling systems in non-chordates are relatively elusive. We determined the specificity of the neuropeptide F (Hdh-NPF) signaling system with a cognate receptor (Hdh-NPFR) in the Pacific abalone, Haliotis discus hannai. Phylogenetic and exon–intron arrangement analyses of bilaterian NPF and the chordate ortholog NPY with their receptor sequences revealed a likely common ancestor, and Hdh-NPFR was similar to the NPYR2 subtype among the NPYR1, NPYR2, and NPYR5 subtypes. Among four Hdh-NPFR-related receptors, Hdh-NPFR specifically responded to Hdh-NPF peptide, supported by the dose–response luciferase reporter curve, intracellular Ca2+ mobilization, and phosphorylation of ERK1/2 and its inhibition with a protein kinase C inhibitor. Peptide fragmentations and shuffling of Hdh-NPF with human NPY could not activate the cellular response of Hdh-NPFR. Three-dimensional in silico modeling suggested that interaction of Hdh-NPF C-terminal amino acids with the extracellular loops of Hdh-NPFR is critical for Hdh-NPFR activation. In vivo injection of Hdh-NPF peptide increased food consumption, and knockdown of Hdh-NPF expression decreased food consumption in Pacific abalone. These findings provide evidence for co-evolution of the NPF/Y ligand-receptor system, enabling further research on mollusk orexigenic neuropeptides.

Construction of a Genetic Linkage Map Based on SNP Markers, QTL Mapping and Detection of Candidate Genes of Growth-Related Traits in Pacific Abalone Using Genotyping-by-Sequencing

Pacific abalone (Haliotis discus hannai) is a commercially important high valued molluscan species. Its wild population has decreased in recent years. It is widely cultured in Korea. Traditional breeding programs have been implemented for hatchery production of abalone seeds. To obtain more genetic information for its molecular breeding program, a high-density linkage map and quantitative trait locus (QTL) for three growth-related traits was constructed for Pacific abalone. F1 cross population with two parents were sampled to construct the linkage map using genotyping by sequencing (GBS). A total of 664,630,534 clean reads and 56,686 SNPs were generated and 3,345 segregating SNPs were used to construct a consensus linkage map. The map spanned 1,747.023 cM with 18 linkage groups and an average interval of 0.55 cM. QTL analysis revealed two significant QTL in LG10 on the consensus linkage map of each growth-related trait. Both QTLs were located in the telomere region of the chromosome. Moreover, four potential candidate genes for growth-related traits were identified in the QTL region. Expression analysis revealed that these identified genes are involved in growth regulation of abalone. The newly constructed genetic linkage map, growth-related QTLs and potential candidate genes identified in the present study can be used as valuable genetic resources for marker-assisted selection (MAS) of Pacific abalone in molecular breeding program.

Comparative Transcriptome and DNA Methylation Analysis of Phenotypic Plasticity in the Pacific Abalone (Haliotis discus hannai)

Phenotypic plasticity is an adaptive mechanism used by organisms to cope with environmental fluctuations. Pacific abalone (Haliotis discus hannai) are large-scale farmed in the temperate area of northern China and in the warmer waters of southern China. RNA-seq and comparative transcriptomic analysis here were performed to determine if the northern and southern populations have evolved divergent plasticity and if functional differences are associated with protein synthesis and growth-related biological progress. The DNA methylation (5mC) landscape of H. discus hannai from the two populations using whole genomic bisulfite sequencing (WGBS), exhibited different epigenetic patterns. The southern population had significant genomic hypo-methylation that may have resulted from long-term acclimation to heat stress. Combining 790 differentially expressed genes (DEGs) and 7635 differentially methylated genes (DMGs), we found that methylation within the gene body might be important in predicting abalone gene expression. Genes related to growth, development, transduction, and apoptosis may be regulated by methylation and could explain the phenotypic divergence of H. discus hannai. Our findings not only emphasize the significant roles of adaptive plasticity in the acclimation of H. discus hannai to high temperatures but also provide a new understanding of the epigenetic mechanism underlying the phenotypic plasticity in adaptation to climate change for marine organisms.