The Enhanced Immune Protection in Small Abalone Haliotis diversicolor Against a Secondary Infection With Vibrio harveyi

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

K-Nearest Neighbor and Random Forest-Based Prediction of Putative Tyrosinase Inhibitory Peptides of Abalone Haliotis diversicolor

Skin pigment disorders are common cosmetic and medical problems. Many known compounds inhibit the key melanin-producing enzyme, tyrosinase, but their use is limited due to side effects. Natural-derived peptides also display tyrosinase inhibition. Abalone is a good source of peptides, and the abalone proteins have been used widely in pharmaceutical and cosmetic products, but not for melanin inhibition. This study aimed to predict putative tyrosinase inhibitory peptides (TIPs) from abalone, Haliotis diversicolor, using k-nearest neighbor (kNN) and random forest (RF) algorithms. The kNN and RF predictors were trained and tested against 133 peptides with known anti-tyrosinase properties with 97% and 99% accuracy. The kNN predictor suggested 1075 putative TIPs and six TIPs from the RF predictor. Two helical peptides were predicted by both methods and showed possible interaction with the predicted structure of mushroom tyrosinase, similar to those of the known TIPs. These two peptides had arginine and aromatic amino acids, which were common to the known TIPs, suggesting non-competitive inhibition on the tyrosinase. Therefore, the first version of the TIP predictors could suggest a reasonable number of the TIP candidates for further experiments. More experimental data will be important for improving the performance of these predictors, and they can be extended to discover more TIPs from other organisms. The confirmation of TIPs in abalone will be a new commercial opportunity for abalone farmers and industry.

Integrated Analysis of mRNA and miRNA Changes in Two Haliotis diversicolor Genotypes and Their Hybrid

Heterosis is a widely distributed phenomenon in mollusks. It is vital in aquaculture by bringing beneficial traits into hybrids. People have utilized the heterosis theory in aquaculture for years. However, the molecular basis of heterosis remains elusive. Evident growth and survival heterosis were shown in the hybrid (“Dongyou-1”) of two Haliotis diversicolor geographic genotypes (Japan and Taiwan). To explore the molecular basis underlying the hybrid abalone’s heterosis, we conducted comparative mRNA and miRNA transcriptional analysis in the hybrid and parental genotypes. Differentially expression analysis identified 5,562 differentially expressed genes (DEGs) and 102 differentially expressed miRNAs (DEMs) between the three genotypes. 1,789 DEGs and 71 DEMs were found to be non-additively expressed in the hybrid. Meanwhile, both the expression level dominance pattern (ELD) and expression level overdominance pattern (ELOD) were found in the DEGs and DEMs, showing the existence of dominance and overdominance models in the hybrid’s transcriptome and post-transcriptional regulation. Functional analysis showed the non-additively expressed genes, ELD genes, and ELOD genes were significantly enriched in growth, immunity, and stress response related pathways, while some of the pathways were regulated by the mRNA-miRNA interactions. The expression levels of FGF, C1Q, HC, CAT, SEGPX, and MGST were significantly up-regulated in the hybrid compared to the middle parent value. In conclusion, we identified the existence of non-additivity, dominance, and overdominance models in the transcriptome and miRNAome of the H. diversicolor hybrid; these models facilitate the advantageous parental alleles’ integration into the hybrid, contributing to the hybrid’s growth and survival heterosis.

Calcineurin subunit B is involved in shell regeneration in Haliotis diversicolor

Abalone shells are mainly composed of two major polymorphs of CaCO3 that are distributed in different layers of the shell. The process of shell biomineralization is controlled by genes and proteins expressed within the mantle epithelium. In this present paper, we conducted a shell regeneration experiment to study the role of HcCNA and HcCNB (individual subunits of calcineurin) in shell biomineralization in H. diversicolor. The results of qPCR showed that HcCNB is upregulated to a greater extent than HcCNA in the mantle after shell notching. In vivo study of the effects of rHcCNB injection showed a significantly higher percentage of regenerated shell length, but not area, in the injected group compared to the control group. In addition, SEM observation of the inner surface of the regenerated shells revealed three different zones including prismatic, nacreous, and a distinct transition zone. Changes in the crystal organization and ultrastructure are clearly evident in these three zones, particularly after 3 weeks of rHcCNB administration. We hypothesize that this is due to faster biomineralization rates in the rHcCNB treated group. Taken together, our results demonstrate that HcCNB participates in shell regeneration in H. diversicolor. As calcineurin subunits have also been implicated in shell formation in bivalves, these findings suggest that calcineurin subunits may play important roles in biomineralization in all conchiferans.