Limited genomic signatures of population collapse in the critically endangered black abalone (Haliotis cracherodii).

The black abalone, Haliotis cracherodii, is a large, long-lived marine mollusc that inhabits rocky intertidal habitats along the coast of California and Mexico. In 1985, populations were impacted by a bacterial disease known as withering syndrome (WS) that wiped out >90% of individuals, leading to the closure of all U.S. black abalone fisheries since 1993. Current conservation strategies include restoring diminished populations by translocating healthy individuals. However, population collapse on this scale may have dramatically lowered genetic diversity and strengthened geographic differentiation, making translocation-based recovery contentious. Additionally, the current prevalence of WS remains unknown. To address these uncertainties, we sequenced and analysed the genomes of 133 black abalone individuals from across their present range. We observed no spatial genetic structure among black abalone, with the exception of a single chromosomal inversion that increases in frequency with latitude. Outside the inversion, genetic differentiation between sites is minimal and does not scale with either geographic distance or environmental dissimilarity. Genetic diversity appears uniformly high across the range. Demographic inference does indicate a severe population bottleneck beginning just 15 generations in the past, but this decline is short lived, with present-day size far exceeding the pre-bottleneck status quo. Finally, we find the bacterial agent of WS is equally present across the sampled range, but only in 10% of individuals. The lack of population genetic structure, uniform diversity and prevalence of WS bacteria indicates that translocation could be a valid and low-risk means of population restoration for black abalone species' recovery.

Within- and transgenerational stress legacy effects of ocean acidification on red abalone (Haliotis rufescens) growth and survival.

Understanding the mechanisms by which individual organisms respond and populations adapt to global climate change is a critical challenge. The role of plasticity and acclimation, within and across generations, may be essential given the pace of change. We investigated plasticity across generations and life stages in response to ocean acidification (OA), which poses a growing threat to both wild populations and the sustainable aquaculture of shellfish. Most studies of OA on shellfish focus on acute effects, and less is known regarding the longer term carryover effects that may manifest within or across generations. We assessed these longer term effects in red abalone (Haliotis rufescens) using a multi-generational split-brood experiment. We spawned adults raised in ambient conditions to create offspring that we then exposed to high pCO2 (1180 µatm; simulating OA) or low pCO2 (450 µatm; control or ambient conditions) during the first 3 months of life. We then allowed these animals to reach maturity in ambient common garden conditions for 4 years before returning the adults into high or low pCO2 treatments for 11 months and measuring growth and reproductive potential. Early-life exposure to OA in the F1 generation decreased adult growth rate even after 5 years especially when abalone were re-exposed to OA as adults. Adult but not early-life exposure to OA negatively impacted fecundity. We then exposed the F2 offspring to high or low pCO2 treatments for the first 3 months of life in a fully factorial, split-brood design. We found negative transgenerational effects of parental OA exposure on survival and growth of F2 offspring, in addition to significant direct effects of OA on F2 survival. These results show that the negative impacts of OA can last within and across generations, but that buffering against OA conditions at critical life-history windows can mitigate these effects.

Functional Characterization of Gonadotropin-Releasing Hormone and Corazonin Signaling Systems in Pacific Abalone: Toward Reclassification of Invertebrate Neuropeptides.

INTRODUCTION: The proposed evolutionary origins and corresponding nomenclature of bilaterian gonadotropin-releasing hormone (GnRH)-related neuropeptides have changed tremendously with the aid of receptor deorphanization. However, the reclassification of the GnRH and corazonin (CRZ) signaling systems in Lophotrochozoa remains unclear. METHODS: We characterized GnRH and CRZ receptors in the mollusk Pacific abalone, Haliotis discus hannai (Hdh), by phylogenetic and gene expression analyses, bioluminescence-based reporter, Western blotting, substitution of peptide amino acids, in vivo neuropeptide injection, and RNA interference assays. RESULTS: Two Hdh CRZ-like receptors (Hdh-CRZR-A and Hdh-CRZR-B) and three Hdh GnRH-like receptors (Hdh-GnRHR1-A, Hdh-GnRHR1-B, and Hdh-GnRHR2) were identified. In phylogenetic analysis, Hdh-CRZR-A and -B grouped within the CRZ-type receptors, whereas Hdh-GnRHR1-A/-B and Hdh-GnRHR2 clustered within the GnRH/adipokinetic hormone (AKH)/CRZ-related peptide-type receptors. Hdh-CRZR-A/-B and Hdh-GnRHR1-A were activated by Hdh-CRZ (pQNYHFSNGWHA-NH2) and Hdh-GnRH (pQISFSPNWGT-NH2), respectively. Hdh-CRZR-A/-B dually coupled with the Gαq and Gαs signaling pathways, whereas Hdh-GnRHR1-A was linked only with Gαq signaling. Analysis of substituted peptides, [I2S3]Hdh-CRZ and [N2Y3H4]Hdh-GnRH, and in silico docking models revealed that the N-terminal amino acids of the peptides are critical for the selectivity of Hdh-CRZR and Hdh-GnRHR. Two precursor transcripts for Hdh-CRZ and Hdh-GnRH peptides and their receptors were mainly expressed in the neural ganglia, and their levels increased in starved abalones. Injection of Hdh-CRZ peptide into abalones decreased food consumption, whereas Hdh-CRZR knockdown increased food consumption. Moreover, Hdh-CRZ induced germinal vesicle breakdown in mature oocytes. CONCLUSION: Characterization of Hdh-CRZRs and Hdh-GnRHRs and their cognate peptides provides new insight into the evolutionary route of GnRH-related signaling systems in bilaterians.

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone.

INTRODUCTION: Neuropeptides regulate vital physiological processes in multicellular organisms, including growth, reproduction, metamorphosis, and feeding. Recent transcriptome analyses have revealed neuropeptide genes with potential roles in vertebrate and invertebrate growth and reproduction. Among these genes, haliotid growth-associated peptide (HGAP) was identified as a novel gene in abalone. METHODS: This study focused on HGAP in Pacific abalone (Haliotis discus hannai), where the complete cDNA sequence named Hdh-HGAP was identified and characterized. Samples from different experiments, such as metamorphosis, juvenile abalone growth, gonad development stages, muscle remodeling, and starvation, were collected for mRNA expression analysis. RESULTS: The sequence spans 552 bp, encoding 96 amino acids with a molecular weight of 10.96 kDa. Expression analysis revealed that Hdh-HGAP exhibited higher levels in muscle tissue. Notably, during metamorphosis, Hdh-HGAP exhibited greater expression in the trochophore, veliger, and juvenile stages than in the cell division stages. Regarding growth patterns, Hdh-HGAP was highly expressed during rapid growth compared to stunted, minimal, and normal growth. In gonadal development, Hdh-HGAP mRNA reached its highest expression level during the ripening stage, indicating a potential role in gonadal cell proliferation and maturation. The in vivo effects of GnRH on gonad development and the expression of the Hdh-HGAP neuropeptide indicate its involvement in regulating reproduction in Pacific abalone. While tissue remodeling is primarily governed by immune genes, Hdh-HGAP was also upregulated during muscle tissue remodeling. Conversely, Hdh-HGAP was downregulated during prolonged starvation. CONCLUSION: This study marks the first comprehensive exploration of the Hdh-HGAP neuropeptide gene in Pacific abalone, shedding light on its involvement in growth, reproduction, metamorphosis, tissue remodeling, and response to starvation, although regulatory mechanisms are mostly unknown.

Spirochaete genome identified in red abalone sample represents a novel genus Candidatus Haliotispira gen. nov. within the order Spirochaetales.

A fully assembled spirochaete genome was identified as a contaminating scaffold in our red abalone (Haliotis rufescens) genome assembly. In this paper, we describe the analysis of this bacterial genome. The assembled spirochaete genome is 3.25 Mb in size with 48.5 mol% G+C content. The proteomes of 38 species were compared with the spirochaete genome and it was discovered to form an independent branch within the family Spirochaetaceae on the phylogenetic tree. The comparison of 16S rRNA sequences and average nucleotide identity scores between the spirochaete genome with known species of different families in Spirochaetia indicate that it is an unknown species. Further, the percentage of conserved proteins compared to neighbouring taxa confirm that it does not belong to a known genus within Spirochaetaceae. We propose the name Candidatus Haliotispira prima gen. nov., sp. nov. based on its taxonomic placement and origin. We also tested for the presence of this species in different species of abalone and found that it is also present in white abalone (Haliotis sorenseni). In addition, we highlight the need for better classification of taxa within the class Spirochaetia.

Growth performance and feed utilisation of Australian hybrid abalone (Haliotis rubra × Haliotis laevigata) fed increasing dietary protein levels at three water temperatures.

Determining the macronutrient requirements for commercially valuable aquaculture species remains crucial for maximising production efficiency. Yet, such information is lacking for Australian hybrid abalone (Haliotis rubra × Haliotis laevigata), particularly with respect to life stage and water temperatures. The present study aimed to evaluate the effect of dietary protein inclusion level on the growth performance, nutrient utilisation and nutritional quality of juvenile (3·3 g) Australian hybrid abalone reared at three different temperatures representative of winter (12°C), average annual (17°C) and summer (22°C) grow-out periods and fed five diets containing graded dietary protein levels of 35, 38, 41, 44 and 47 %. Abalone growth increased with increasing water temperature with weight gains of approximately 100, 280 and 380 % of their initial weight at 12, 17 and 22°C, respectively. Furthermore, the present study clearly demonstrated that higher dietary protein inclusion levels (41 %) than those currently used commercially (35 %) would significantly improve the growth performance when water temperatures are ≥17°C without any adverse impacts on nutrient utilisation, nutrient deposition or nutritional quality of the abalone soft tissue. For example, at 22°C abalone fed a diet containing 41 % protein obtained a significantly higher weight gain percentage (421 %) compared with those fed a diet containing 35 % protein (356 %). Lastly, it is suggested that maintaining a dietary protein inclusion level of 35 % or implementing a 'least cost' feeding approach during cooler seasons, or where water temperatures are ∼12°C, may be beneficial, considering only marginal growth improvements were observed during these periods of slow growth.

Exposure to bisphenol A and fiber-type microplastics induce oxidative stress and cell damage in disk abalone Haliotis discus hannai: Bioaccumulation and toxicity.

Along with environmental pollution caused by rapid economic development and industrialization, plastic waste is emerging as a global concern in relation to marine ecosystems and human health. Among the microplastics, fiber-type microfibers (MF) and bisphenol A (BPA), which are widely used as plasticizers, do not decompose well in the ocean, and tend to accumulate in organisms, generating an increased oxidative stress response. This study investigated the abalones' antioxidant and cell death responses following exposure to the environmental pollutants MF and BPA. Levels of malondialdehyde (MDA) and DNA damage increased over time, demonstrating the degree of lipid peroxidation and DNA damage in abalones exposed to individual and combined environmental conditions of MF and BPA. Compared to the single MF and BPA exposure groups, the combined exposure group showed a higher expression of antioxidant enzymes. A similar pattern was seen in the expression of the apoptosis enzyme caspase-3. Both MF and BPA caused oxidative stress and antioxidant enzymes were expressed to alleviate it, but it is believed that cell damage occurred because the stress level exceeded the allowed range.

Dietary Clostridium autoethanogenum protein has dose-dependent influence on the gut microbiota, immunity, inflammation and disease resistance of abalone Haliotis discus hannai.

Clostridium autoethanogenum protein (CAP) is an eco-friendly protein source and has great application potential in aquafeeds. The present study aimed to investigate the effects of dietary CAP inclusion on the anti-oxidation, immunity, inflammation, disease resistance and gut microbiota of abalone Haliotis discus hannai after a 110-day feeding trial. Three isonitrogenous and isolipidic diets were formulated by adding 0 % (control), 4.10 % (CAP4.10) and 16.25 % (CAP16.25) of CAP, respectively. A total of 540 abalones with an initial mean body weight of 22.05 ± 0.19 g were randomly distributed in three groups with three replicates per group and 60 abalones per replicate. Results showed that the activities of superoxide dismutase and glutathione peroxidase in the cell-free hemolymph (CFH) were significantly decreased and the content of malondialdehyde in CFH was significantly increased in the CAP16.25 group. The diet with 4.1 % of CAP significantly increased the activities of lysozyme and acid phosphatase in CFH. The expressions of pro-inflammatory genes such as tumor necrosis factor-α (tnf-α), nuclear factor-κb (nf-κb) and toll-like receptor 4 (tlr4) in digestive gland were downregulated, and the expressions of anti-inflammatory genes such as ß-defensin and mytimacin 6 in digestive gland were upregulated in the CAP4.10 group. Dietary CAP inclusion significantly decreased the cumulative mortality of abalone after the challenge test with Vibrio parahaemolyticus for 7 days. Dietary CAP inclusion changed the composition of gut microbiota of abalone. Besides, the balance of the ecological interaction network of bacterial genera in the intestine of abalone was enhanced by dietary CAP. The association analysis showed that two bacterial genera Ruegeria and Bacteroides were closely correlated with the inflammatory genes. In conclusion, the 4.10 % of dietary CAP enhanced the immunity and disease resistance as well as inhibited the inflammation of abalone. The 16.25 % of dietary CAP decreased the anti-oxidative capacity of abalone. The structure of the gut microbiota of abalone changed with dietary CAP levels.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Structural and functional characterization of Hdh-HSBP1 and its involvement in heat stress and early development in Pacific abalone, Haliotis discus hannai.

Heat shock factor binding protein 1 (HSBP1) is known to regulate the activity of heat shock factor 1 (HSF1) and the early development of organisms. To understand the involvement of HSBP1 in the heat shock response and embryonic and larval development of Pacific abalone (Haliotis discus hannai), the Hdh-HSBP1 gene was sequenced from the digestive gland (DG) tissue. The full-length sequence of Hdh-HSBP1 encompassed 738 nucleotides, encoding an 8.42 kDa protein consisting of 75 deduced amino acids. The protein contains an HSBP1 domain and a coiled-coil domain, which are conserved features in the HSBP1 protein family. Protein-protein molecular docking revealed that the coiled-coil region of Hdh-HSBP1 binds to the coiled-coil region of Hdh-HSF1. Tissue expression analysis demonstrated that the highest Hdh-HSBP1 expression occurred in the DG, whereas seasonal expression analysis revealed that this gene was most highly expressed in summer. In heat-stressed abalone, the highest expression of Hdh-HSBP1 occurred at 30 °C. Moreover, time-series analysis revealed that the expression of this gene began to increase significantly at 6 h post-heat stress, with higher expression observed at 12 h and 24 h post-heat stress. Furthermore, Hdh-HSBP1 mRNA expression showed a link to ROS production. Additionally, the expression of Hdh-HSBP1 showed significantly higher expression in the early stages of embryonic development in Pacific abalone. These results suggest that Hdh-HSBP1 plays a crucial role in the stress physiology of Pacific abalone by interacting with Hdh-HSF1, as well as its embryonic development.

Carryover effects of embryonic hypoxia exposure on adult fitness of the Pacific abalone.

The widespread and severe drop in dissolved oxygen concentration in the open ocean and coastal waters has attracted much attention, but assessments of the impacts of environmental hypoxia on aquatic organisms have focused primarily on responses to current exposure. Past stress exposure might also affect the performance of aquatic organisms through carryover effects, and whether these effects scale from positive to negative based on exposure degree is unknown. We investigated the carryover effects of varying embryonic hypoxia levels (mediate hypoxia: 3.0-3.1 mg O2/L; severe hypoxia: 2.0-2.1 mg O2/L) on the fitness traits of adult Pacific abalone (Haliotis discus hannai), including growth, hypoxia tolerance, oxygen consumption, ammonia excretion rate, and biochemical responses to acute hypoxia. Moderate embryonic hypoxia exposure significantly improved the hypoxia tolerance of adult Pacific abalone without sacrificing growth and survival. Adult abalone exposed to embryonic hypoxia exhibited physiological plasticity, including decreased oxygen consumption rates under environmental stress, increased basal methylation levels, and a more active response to acute hypoxia, which might support their higher hypoxia tolerance. Thus, moderate oxygen declines in early life have persistent effects on the fitness of abalone even two years later, further affecting population dynamics. The results suggested that incorporating the carryover effects of embryonic hypoxia exposure into genetic breeding programs would be an important step toward rapidly improving the hypoxia tolerance of aquatic animals. The study also inspires the protection of endangered wild animals and other vulnerable species under global climate change.

Histopathological investigation of four populations of abalone (Haliotis iris) exhibiting divergent growth performance.

The black-foot abalone (paua), Haliotis iris, is a unique and valuable species to New Zealand with cultural importance for Maori. Abalone are marine gastropods that can display a high level of phenotypic variation, including slow-growing or 'stunted' variants. This investigation focused on identifying factors that are associated with growth performance, with particular interest in the slow-growing variants. Tissue alterations in H. iris were examined using histopathological techniques, in relation to growth performance, contrasting populations classified by commercial harvesters as 'stunted' (i.e., slow-growing) and 'non-stunted' (i.e., fast-growing) from four sites around the Chatham Islands (New Zealand). Ten adults and 10 sub-adults were collected from each of the four sites and prepared for histological assessment of condition, tissue alterations, presence of food and presence of parasites. The gut epithelium connective tissue, digestive gland, gill lamellae and right kidney tissues all displayed signs of structural differences between the slow-growing and fast-growing populations. Overall, several factors appear to be correlated to growth performance. The individuals from slow-growing populations were observed to have more degraded macroalgal fragments in the midgut, increased numbers of ceroid granules in multiple tissues, as well as increased prevalence of birefringent mineral crystals and haplosporidian-like parasites in the right kidney. The histopathological approaches presented here complement anecdotal field observations of reduced seaweed availability and increased sand incursion at slow-growing sites, while providing an insight into the health of individual abalone and sub-populations. The approaches described here will ultimately help elucidate the drivers behind variable growth performance which, in turn, supports fisheries management decisions and future surveillance programs.

Toxic effects of combined exposure to homoyessotoxin and nitrite on the survival, antioxidative responses, and apoptosis of the abalone Haliotis discus hannai.

Homoyessotoxin (homo-YTX) and nitrite (NO2-N), released during harmful dinoflagellate cell lysis adversely affect abalones. However, their toxicity mechanisms in shellfish remain unclear. This study investigated the economic abalone species Haliotis discus hannai exposed to varying concentrations of homo-YTX (0, 2, 5, and 10 µg L-1) and NO2-N (0, 3, and 6 mg L-1) on the basis of their 12 h LC50 values (5.05 µg L-1 and 4.25 mg L-1, respectively) and the environmentally relevant dissolved concentrations during severe dinoflagellate blooms, including mixtures. The test abalones were exposed to homo-YTX and NO2-N for 12 h. The mortality rate (D), reactive oxygen species (ROS) levels, antioxidant defense capabilities, and expression levels of antioxidant-related, Hsp-related, and apoptosis-related genes in abalone gills were assessed. Results showed that the combined exposure to homo-YTX and NO2-N increased the D and ROS levels and upregulated B-cell lymphoma-2 (BCL2)-associated X (BAX) and caspase3 (CASP3) expression levels while reducing glutathione peroxidase (GPx) activity and GPx, CuZnSOD, and BCL2 expression levels. High concentrations of homo-YTX (10 µg L-1) and NO2-N (6 mg L-1) solutions and the combinations of these toxicants inhibited the activities of superoxide dismutase (SOD) and catalase (CAT) and downregulated the expression levels of MnSOD, CAT, Hsp70, and Hsp90. The ROS levels were negatively correlated with the activities of SOD, CAT, and GPx and the expression levels of MnSOD, CuZnSOD, CAT, GPx, Hsp70, Hsp90, and BCL2. These results suggest that homo-YTX, in conjunction with NO2-N, induces oxidative stress, disrupts antioxidant defense systems, and triggers caspase-dependent apoptosis in the gills of abalone. ROS-mediated antioxidative and heat-shock responses and apoptosis emerge as potential toxicity mechanisms affecting the survival of H. discus hannai due to homo-YTX and NO2-N exposure.

EF-Hand-Binding Secreted Protein Hdh-SMP5 Regulates Shell Biomineralization and Responses to Stress in Pacific Abalone, Haliotis discus hannai.

The development of a shell is a complex calcium metabolic process involving shell matrix proteins (SMPs). In this study, we describe the isolation, characterization, and expression of SMP5 and investigate its potential regulatory role in the shell biomineralization of Pacific abalone Haliotis discus hannai. The full-length Hdh-SMP5 cDNA contains 685 bp and encodes a polypeptide of 134 amino acids. Structurally, the Hdh-SMP5 protein belongs to the EF-hand-binding superfamily, which possesses three EF-hand Ca2+-binding regions and is rich in aspartic acid. The distinct clustering patterns in the phylogenetic tree indicate that the amino acid composition and structure of this protein may vary among different SMPs. During early development, significantly higher expression was observed in the trochophore and veliger stages. Hdh-SMP5 was also upregulated during shell biomineralization in Pacific abalone. Long periods of starvation cause Hdh-SMP5 expression to decrease. Furthermore, Hdh-SMP5 expression was observed to be significantly higher under thermal stress at temperatures of 15, 30, and 25 °C for durations of 6 h, 12 h, and 48 h, respectively. Our study is the first to characterize Hdh-SMP5 comprehensively and analyze its expression to elucidate its dynamic roles in ontogenetic development, shell biomineralization, and the response to starvation and thermal stress.

Pathogenicity, genomic analysis and structure of abalone asfa-like virus: evidence for classification in the family Asfarviridae.

This paper presents the rationale for classifying abalone asfa-like virus (AbALV) in the family Asfarviridae based on analyses of the host, whole genome and electron microscopic observations. AbALV caused >80 % cumulative mortality in an experimentally infected mollusc, Haliotis madaka. The AbALV genome was found to be linear, approximately 281 kb in length, with a G+C content of 31.32 %. Of the 309 predicted ORFs, 48 of the top hits with African swine fever virus (ASFV) genes in homology analysis were found to be in the central region of the genome. Synteny in the central region of the genome was conserved with ASFV. Similar to ASFV, paralogous genes were present at both ends of the genome. The pairwise average amino acid identity (AAI) between the AbALV and ASFV genomes was 33.97 %, within the range of intra-family AAI values for Nucleocytoviricota. Electron microscopy analysis of the gills revealed ~200 nm icosahedral virus particles in the cytoplasm of epithelial cells, and the size and morphology resembled ASFV. In addition to swine, ASFV also infects ticks, which are protostomes like abalone. The overall genome structure and virion morphology of AbALV and ASFV are similar, and both viruses infect protostomes, suggesting that AbALV is a new member of the family Asfarviridae.

Effects of dietary bile acids levels on growth performance, anti-oxidative capacity, immunity and intestinal microbiota of abalone Haliotis discus hannai.

Abalone Haliotis discus hannai (initial weight: 38.79 ± 0.70 g) was used as the experimental animal in a 105-day feeding trial to investigate the influence of dietary bile acids levels on the growth, anti-oxidation, immune response and intestinal microbiota. Six isonitrogenous and isolipidic diets were prepared by adding 0 (control group), 15, 30, 60, 120 and 240 mg/kg of bile acids, respectively (named BA0, BA15, BA30, BA60, BA120 and BA240, respectively). It was found that survival of abalone between groups had no significant difference (P > 0.05). Compared to the control, significant improvements in weight gain rate (WGR) were observed in the groups of BA30 and BA60 (P < 0.05). Based on WGR, the broken line regression model analysis showed that the optimum demand for dietary bile acids for abalone was 35.47 mg/kg. Dietary bile acids increased the total anti-oxidative capacity and activities of catalase, superoxide dismutase, lysozyme and alkaline phosphatase, meanwhile decreased the content of malondialdehyde, alanine aminotransferase and aspartate aminotransferase activities in the cell-free hemolymph (P < 0.05). When bile acids were added to the diets, mRNA levels of genes related to pro-inflammatory factors and apoptosis in the digestive gland were down-regulated (P < 0.05). In contrast, the expression of genes related to anti-oxidation was significantly up-regulated (P < 0.05). The Firmicutes, Actinobacteriota and Proteobacteria were the most abundant phyla in intestine. And dietary bile acids significantly decreased the abundance of Actinobacteria and increased the abundance of Firmicutes (P < 0.05). In conclusion, supplementation of dietary bile acids within 120 mg/kg significantly increased the growth of abalone. The 34.62 mg/kg of dietary bile acids significantly increased the anti-oxidative capacity of abalone. Appropriate levels of dietary bile acids (34.62-61.75 mg/kg) promote the immunity of abalone. Application of appropriate levels of bile acids in diets (34.62 mg/kg) changed the intestinal microbiota and promoted the intestinal health of abalone.

From waste to feed: Microbial fermented abalone waste improves the digestibility, gut health, and immunity in marron, Cherax cainii.

New aquafeed ingredients produced by a circular economy approach are the opportunity for sustainable and resilient aquaculture. In the light of this approach, the mixture of abalone waste and Sargassum spp (9:1) fermented by Saccharomyces cereviceae and Lactobacillus casei (Yakult®) (FMAS) were used to replace 0% (FMAS0), 25% (FMAS-25), 50% (FMAS-50), 75% (FMAS-75), and 100% (FMAS-100) of fishmeal (FM) protein in marron, Cherax cainii diet. The marron was fed these diets in triplicate for 90 days. Growth, feed utilization and protein efficiency ratio were unchanged in marron-fed all test diets. Improvement in apparent protein digestibility was aligned with an increase in the size and number of B-cells in the hepatopancreas. Most of the immune responses, except for haemocyte clotting time, hyaline cells and neutral red retention time (NRR time) were unchanged by 42- and 90-days feeding trials compared to those of the control group. 90 days post-feeding marron with FMAS25 showed a lower haemocyte clotting time than the post 42 days feeding marron with the same diet. Hyaline cells increased in marron fed FMAS75 for 90 days compared to marron fed the same diet for 42 days. The challenge test involved injecting marron with Vibrio mimicus resulted in a 100% survival rate after 96 h of exposure. During the challenge test, phagocytosis activity in 24 and 48-h post-challenged marron fed FMAS75 decreased which recovered after 96 h post-challenge. Marron fed FMAS50 also recorded a significantly higher proportion of granular cells after 24 h and NRR time at 96 h compared with that of other treatments. Given the above indicators of bio-growth, feed efficiency and immune responses, total replacement of FM protein of marron practical feed with FMAS are considered feasible and optimum to maintain health status and resistance to disease.

Resolving the identity of commercially cultivated Ulva (Ulvaceae, Chlorophyta) in integrated seaweed-abalone aquaculture farms in South Africa.

Species of Ulva have a wide range of commercial applications and are increasingly being recognized as promising candidates for integrated aquaculture. In South Africa, Ulva has been commercially cultivated in integrated seaweed-abalone aquaculture farms since 2002, with more than 2000 tonnes of biomass cultivated per annum in land-based paddle raceways. However, the identity of the species of Ulva grown on these farms remains uncertain. We therefore characterized samples of Ulva cultivated in five integrated multi-trophic aquaculture farms (IMTA) across a wide geographical range and compared them with foliose Ulva specimens from neighboring seashores. The molecular markers employed for this study were the chloroplast-encoded Ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL), the Internal Transcribed Spacer (ITS) of the nuclear, and the chloroplast elongation factor tufA. All currently cultivated specimens of Ulva were molecularly resolved as a single species, U. lacinulata. The same species has been cultivated for over a decade, although a few specimens of two other species were also present in early South African IMTA systems. The name Ulva uncialis is adopted for the Ulva "Species A" by Fort et al. (2021), Molecular Ecology Resources, 22, 86) significantly extending the distribution range for this species. A comparison with wild Ulva on seashores close to the farms resulted in five new distribution records for South Africa (U. lacinulata, U. ohnoi, U. australis, U. stenophylloides, and U. aragoënsis), the first report of a foliose form of U. compressa in the region, and one new distribution record for Namibia (U. australis). This study reiterates the need for DNA confirmation, especially when identifying morphologically simple macroalgae with potential commercial applications.

Impact assessment of metals realeased by aluminium-based galvanic anode on the physiology of the abalone Haliotis tuberculata in controlled conditions.

To protect metal structures immersed in the sea from corrosion, the galvanic anode cathodic protection system (GACP) is often applied. However, this association leads to continuous oxidation of the galvanic anode and therefore to a release of a metal cocktail in the forms of ions or oxy-hydroxides. Therefore, the main objective of our study was to investigate the toxicity of elements released from the dissolution of an aluminium-based galvanic anode (∼95% Al, ∼5% Zn, <0.1% for In, Cu, Cd, Mn, Fe) on a grazing gastropod, the abalone Haliotis tuberculata. The present study was carried out in complement to other research currently in submission. Gastropods were exposed for 16 weeks (12 weeks of exposure and 4 weeks of decontamination phase) to 6 conditions including a control, 4 concentrations based on total aluminium level (86, 425, 1096 and 3549 µg L-1) and a trophic control, corresponding to abalones placed in non-contaminated natural seawater but fed with contaminated algae. The effects of metals on growth, glycogen levels, brix index of hemolymph, MDA levels in digestive gland and gills, hemocyte phagocytic activity, ROS production, lysosomal system and the progress of gametogenesis were investigated throughout the entire exposure allowing the realization of kinetics. The results revealed that the aluminium-based anode does not seem to have an effect on the health status of the individuals for environmentally realistic concentrations. However, in extreme conditions strong effects were reported on the growth, immune system and reproduction of abalone.

Computer-Aided Virtual Screening and In Vitro Validation of Biomimetic Tyrosinase Inhibitory Peptides from Abalone Peptidome.

Hyperpigmentation is a medical and cosmetic problem caused by an excess accumulation of melanin or the overexpression of the enzyme tyrosinase, leading to several skin disorders, i.e., freckles, melasma, and skin cancer. Tyrosinase is a key enzyme in melanogenesis and thus a target for reducing melanin production. Although abalone is a good source of bioactive peptides that have been used for several properties including depigmentation, the available information on the anti-tyrosinase property of abalone peptides remains insufficient. This study investigated the anti-tyrosinase properties of Haliotis diversicolor tyrosinase inhibitory peptides (hdTIPs) based on mushroom tyrosinase, cellular tyrosinase, and melanin content assays. The binding conformation between peptides and tyrosinase was also examined by molecular docking and dynamics study. KNN1 showed a high potent inhibitory effect on mushroom tyrosinase with an IC50 of 70.83 µM. Moreover, our selected hdTIPs could inhibit melanin production through the reductions in tyrosinase activity and reactive oxygen species (ROS) levels by enhancing the antioxidative enzymes. RF1 showed the highest activity on both cellular tyrosinase inhibition and ROS reduction. leading to the lower melanin content in B16F10 murine melanoma cells. Accordingly, it can be assumed that our selected peptides exhibited high potential in medical cosmetology applications.