Abalone Haliotis Discus Hannai Ino Research Articles

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Effects of dietary eicosapentaenoic acid on the growth performance, fatty acid profile, immunity and heat tolerance of juvenile abalone Haliotis discus hannai Ino

A 93-day growth trial was performed to evaluate the influences of graded dietary EPA levels (0.03% (control group), 0.24%, 0.63%, 1.07%, 1.72% and 2.25%) on the growth performance, immunity and heat tolerance of juvenile abalone Haliotis discus hannai with a mean initial weight of 4.15 ± 0.18 g. After the feeding trial, the findings found that the highest weight gain rate (WGR) belonged to the groups fed with 0.63% ∼ 1.72% of dietary EPA (P < 0.05). The lowest feed conversion ratio was distinguished in the groups with 0.63% ∼ 1.07% of dietary EPA (P < 0.05). Also, EPA supplementation in abalone feed increased the content of crude lipid in the soft body and the activity of lipase in the intestine (P < 0.05). The total antioxidative capacity increased when dietary EPA levels rose from 0.03% to 1.07%, and then decreased (P < 0.05). Furthermore, compared to the control group, the 0.24% and 0.24% ∼ 0.63% of dietary EPA significantly down-regulated the mRNA levels of IκB kinase and kelch-like ECH-associated protein 1, respectively (P < 0.05). However, nuclear factor-κB and interleukin 1 receptor-associated kinase 4 genes expression in the digestive gland were significantly up-regulated by the use of 1.72% and 2.25% of EPA in diet (P < 0.05). The mRNA levels of tumor necrosis factor α and interleukin 16 were significantly up-regulated by the 2.25% of dietary EPA (P < 0.05). The 1.72% of dietary EPA significantly increased the falling rate of abalone after the heat stress compared with the control (P < 0.05). In conclusion, the optimal dietary EPA requirement of juvenile abalone was determined as 1.36% based on quadratic regression analysis against WGR. Moderate dietary EPA content (0.63% ∼ 1.07%) improved the immunity and heat resistance of abalone. Excessive dietary EPA content (1.72% ∼ 2.25%) decreased the growth, meanwhile, increasing the oxidative stress, inflammation and susceptibility of abalone to heat stress.

An efficient approach for extraction of polysaccharide from abalone (Haliotis Discus Hannai Ino) viscera by natural deep eutectic solvent

In this study, a natural deep eutectic solvent (NADES) was proposed for the ultrasonic-assisted extraction of polysaccharides from abalone (Haliotis Discus Hannai Ino) viscera. Eleven NADESs were employed for abalone viscera polysaccharide (AVP) extraction. NADES, composed of choline chloride and ethylene glycol in a molar ratio 1: 3 had the highest extraction efficiency. The optimal extraction conditions were obtained using a four-factor, three-level Box-Behnken design and specific response surface methodology. The maximum predicted polysaccharide yield was 17.32 %. Fick's second law was fitted to the extraction process of AVP by ultrasonic-assisted NADES based on a high linear correlation (R2 ≥ 0.9). The extraction rate constants (k), diffusion coefficients (Du) and half-lives (t1/2) were calculated. Compared to the polysaccharides prepared by the conventional method, the polysaccharides extracted by NADES had a higher sugar content, lower molecular weight, more glucuronic acid, and stronger antioxidant capacity. Overall, the NADES extraction method established in this research can become a strategy for the preparation of high-purity and highly bioactive abalone viscera polysaccharides, which has implications for the exploitation and application of marine food byproduct resources.

Dietary arachidonic acid affects the growth performance, fatty acid profile, immunity and resistance to heat stress in juvenile abalone Haliotis discus hannai Ino

A 93-day feeding trial was performed to evaluate the effects of dietary arachidonic acid (ARA) on the growth performance, antioxidative capacity, immunity and anti-stress capacity of juvenile abalone Haliotis discus hannai Ino with an initial mean body weight of 4.26 ± 0.11 g. Six practical diets with 0.04% (the control), 0.31%, 0.44%, 0.68%, 0.94% and 1.34% of ARA were designed. After the feeding trial, abalones were subjected to a heat stress test. The findings showed that the survival of abalone (91.7–96.7%) after the feeding trial was not significantly influenced by dietary ARA. The 0.31–0.44% of dietary ARA significantly increased the weight gain rate (WGR). The feed conversion ratio (FCR) was decreased by 0.31–0.68% of dietary ARA. The 0.44% of dietary ARA significantly enhanced the activity of trypsin, lipase and α-amylase in the intestine. Besides, dietary ARA supplementation significantly elevated the total antioxidative capacity and activity of superoxide dismutase, catalase and lysozyme in the cell-free hemolymph. Higher levels of dietary ARA (≥0.94%) significantly up-regulated the mRNA levels of nuclear factor κB, inhibitor of κBα and transcription factor AP-1 in the digestive gland. The 0.68% of dietary ARA significantly decreased the falling rate of abalone after heat stress. In conclusion, appropriate dietary ARA levels improved the growth performance and feed utilization of abalone. The optimal dietary ARA content for juvenile abalone was 0.52% based on the quadratic regression analysis of WGR. The 0.44–0.68% of dietary ARA improved the antioxidative capacity, immunity and anti-heat stress of abalone.

Interaction between dietary lipid and bile acids on the growth performance and lipid metabolism in abalone Haliotis discus hannai Ino

A 105-day feeding trail was conducted to evaluate the effects of dietary lipid and bile acids levels as well as their interactions on the growth, digestion and lipid metabolism in abalone Haliotis discus hannai (initial weight: 5.29 ± 0.02 g). Eight isonitrogenous diets with two levels of lipid (4.3% and 6.3%) and four levels of bile acids (0, 15, 60 and 240 mg/kg) were formulated in a 2 × 4 two-factor design. The results showed that 6.3% of dietary lipid decreased the weight gain rate (WGR) of abalone, which increased as the dietary bile acids level increasing from 0 mg/kg to 60 mg/kg. The treatment with the highest WGR contained 4.3% of dietary lipid and 60 mg/kg of dietary bile acids. In the groups with 4.3% of dietary lipid, the expression of genes involved in lipid transport and lipolysis was increased (P < 0.05). And dietary bile acids increased the expression of genes involved in lipid transport and lipolysis while decreased the expression of genes involved in lipogenesis (P < 0.05). The expression of genes involved in lipid metabolism, such as cluster of differentiation, fatty acid transport protein, microsomal triglyceride transfer protein and peroxisome proliferator-activated receptor α, revealed the interactions between dietary lipid and bile acids (P < 0.05). In conclusion, the growth and digestion of abalone were negatively impacted by an excessively high dietary lipid content (6.3%). Appropriate supplemented level of dietary bile acids (60 mg/kg) significantly enhanced the growth and digestion of abalone. Dietary lipid and bile acids had an interactive effect on the lipid metabolism in abalone, including lipid transport, lipolysis and lipogenesis.

Interactions between dietary protein level and water temperature on the growth performance, innate immunity and disease resistance of juvenile abalone Haliotis discus hannai Ino

A 100-day feeding trial was performed to examine the effects of dietary protein level, water temperature and their interactions on the growth performance, innate immunity and disease resistance of abalone Haliotis discus hannai with an initial body weight of 12.20 ± 0.1 g and an initial shell length of 42.64 ± 0.29 mm. With graded levels of dietary protein (9.33%, 27.53% and 40.42%, respectively), three isolipidic and isoenergetic diets were made and used in two water temperatures (17 °C and 23 °C) in a form of a 3 × 2 factorial design. The results indicated that the weight gain rate (WGR) and daily increment in shell length of abalones (DISL) were significantly higher in 27.53% of dietary protein level at both 17 °C and 23 °C water temperatures. At 17 °C and 23 °C of water temperatures, 27.53% of dietary protein significantly increased the trypsin and α- amylase as well as decreased the malondialdehyde content and glutamate dehydrogenase activities of abalone. Dietary protein and water temperature interact significantly with WGR and α-amylase. The histological analysis of abalone’s digestive gland showed that 27.53% of dietary protein caused normal development at both water temperatures. The mRNA levels of tumor necrosis factor alpha, toll-like receptor 2 and 4, inhibitor κBα, iκb kinase α, myeloid differentiation primary response gene 88, interleukin1 receptor-associated kinase 4, caspase 3 and B-cell lymphoma protein-2-associated X protein were significantly down-regulated by 27.53% of dietary protein at 17 °C of water temperature. The total hemocyte counts (THC), respiratory burst (RB) and B-cell lymphoma 2 (Bcl-2) were significantly increased by 27.53% of dietary protein at 17 °C water temperature. Dietary protein and water temperature interact significantly with THC, RB and Bcl-2. The use of 27.53% of dietary protein at 17 °C of water temperature showed no mortality when challenged with V. parahaemolyticus. In conclusion, 27.53% of dietary protein level at 17 °C of water temperature activated the innate immunity by reducing inflammation and apoptosis response, hence increasing disease resistance in abalones after the Vibrio challenge test.

Development of organic-inorganic hybrid antimicrobial materials by mechanical force and application for active packaging

The loading and delivery of nanomaterials have attracted considerable interest. In this study, the organic-inorganic hybrid antimicrobial materials of clove essential oil (CEO) intercalated organic montmorillonite (MMT) were prepared using a facile and novel cryogenic ball milling technology and characterized. Fourier transform infrared spectroscopy, powder X-ray diffraction, and transmission electron micrograph analysis confirmed the successful preparation of the hybrid antimicrobial materials, and thermogravimetric analysis further demonstrated the thermal stability. In a novel application, hybrid antimicrobial materials were extruded with thermoplastic starch (TPS), polylactic acid (PLA), and poly (butylene adipate-co-terephthalate) (PBAT) to prepare active packaging films. The film release experiments showed that the CEO in the film, containing hybrid antimicrobial materials, was in a sustained release state, and its equilibrium release was higher than that of other films. The prepared films were used to preserve fresh abalone （Haliotis discus hannai Ino）, physicochemical changes and microbiological counts were determined. The results showed that the shelf life of fresh abalone packed in films containing hybrid antimicrobial materials was extended by 3–5 days, indicating that cryogenic ball milling is a simple and effective method for preparing organic-inorganic hybrid antimicrobial materials for active packaging.

Comparative evaluation on the effects of dietary docosahexaenoic acid on growth performance, fatty acid profile and lipid metabolism in two sizes of abalone Haliotis discus hannai Ino

A 93-day feeding trial was conducted to comparatively evaluate the effects of graded levels of dietary docosahexaenoic acid (DHA) (0.06%, 0.46%, 0.99%, 1.57%, 1.95% and 2.25%) on growth performance, fatty acid profile and lipid metabolism in two different sizes of abalone Haliotis discus hannai (initial body weight: 2.83 ± 0.03 g and 26.81 ± 0.17 g, respectively). Results showed that 0.99%–1.95% of dietary DHA contents significantly increased the weight gain rate (WGR) in large size of abalone (P < 0.05), while the WGR in small size of abalone was significantly increased by the 0.46%–0.99% of dietary DHA contents (P < 0.05). There was no significant difference in the survival of abalone among all the six groups (P > 0.05). Based on the WGR, the optimal dietary DHA content for large and small size abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA supplementation significantly increased the activities of trypsin and lipase in the intestine, the content of ∑n-3 polyunsaturated fatty acids (PUFA) and ∑n-3/n-6 PUFA ratio in digestive gland in both sizes of abalone (P < 0.05). The total glyceride concentration in the cell-free hemolymph in two sizes of abalone was decreased. In large size of abalone, 1.95%–2.25% of dietary DHA reduced the lipid content in digestive gland. Lipogenic genes in the digestive gland, including sterol regulatory element binding protein-1 (srebp-1) and diacylglycerol acyltransferase, were up-regulated in the 0.46%–1.57% DHA group (P > 0.05). The mRNA expression levels of hormone sensitive lipase (hsl) and carnitine palmitoyl transferase-1 (cpt-1) of lipolytic genes in the digestive gland were significantly increased in the 0.46% and 0.99% DHA groups, respectively (P < 0.05). In the small size of abalone, the lipid content in digestive gland and soft body decreased significantly in the 1.95% and 2.25% DHA groups, respectively (P < 0.05). Lipogenic genes in the digestive gland, including srebp-1 and acetyl-CoA carboxylase, were significantly down-regulated in the 0.99%–1.95% DHA group (P < 0.05). Lipolytic genes in the digestive gland, including hsl and cpt-1, were not significantly different from the control group (P > 0.05). In conclusion, based on the WGR, the dietary DHA requirements for large and small size of abalone were estimated to be 1.32% and 0.88%, respectively. Dietary DHA reduced lipid deposition in both sizes of abalone by promoting lipid decomposition and inhibiting lipid synthesis.

Structural characterization and immunostimulatory activity of a water-soluble polysaccharide from abalone (Haliotis discus hannai Ino) muscle

A water-soluble polysaccharide from abalone muscle (AMPP) was isolated. The contents of carbohydrate, protein, uronic acid, and sulfate in AMPP were 83.5 %, 0.5 %, 2.7 %, and 2.6 %, respectively. High-performance liquid chromatography analysis indicated that AMPP was homogeneous and had an average molecular weight of approximately 3.2 kDa. The main monosaccharides of AMPP were glucose (Glc) and mannose with a molar ratio of 99.7:0.3. The structural characteristics of AMPP were elucidated through methylation analysis, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The linkages of AMPP consisted of terminal, 1,4-linked, 1,6-linked, and 1,4,6-linked Glcp with a molar ratio of 3.1:7.2:1.0:2.5. In one repeat unit of the proposed AMPP structure, the backbone chain was composed of eight 1→4 glycosidic bonds and one 1→6 glycosidic bond, with three branch chains linked by 1→6 glycosidic bond. In addition, AMPP was found to possess potent immunostimulatory activity via rising phagocytosis of RAW264.7 cells and promoting secretion of TNF-α.

Transcriptome study on immune response against Vibrio parahaemolyticus challenge in gill of abalone Haliotis discus hannai Ino

Abalone (Haliotis discus hannai Ino) is one of the most vital economic shellfish cultured in China. However, bacterial and viral infection often leads to its high mortality and serious economic losses. In this study, transcriptome sequencing was performed on gill of abalone challenged by Vibrio parahaemolyticus for 0 h (control group, A0h), 8 h (A8h), and 48 h (A48h), respectively. Each library has an average of 53,361,675 clean reads. Approximately 85.71% reads were mapped to the reference genome of Haliotis discus hannai Ino. In comparison with A0h group, 7,656 and 2,789 genes were differentially expressed in the A8h and A48h groups, respectively. The differentially expressed genes (DEGs) were mostly enriched in oxidation–reduction process and oxidoreductase activity from GO analysis as well as immunity and metabolism-related pathways from Kyoto Encyclopedia of Genes and Genomes analysis. Cluster analysis demonstrated that all DEGs were categorized into eight subclusters by adopting the gene expression model. Specifically, a total of 1,070 significantly downregulated genes from two clusters were related to some metabolic pathways, ABC transporters, and cell adhesion. Moreover, 212 upregulated genes in one subcluster were enriched in several immune-related pathways, including nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway and apoptosis. Some members of the pattern recognition receptors family, which can initiate inflammatory signaling and induce apoptosis, were stimulated to upregulate in gill after challenge including C1q, perlucin, and low-density lipoprotein receptor–related protein. In addition, caspase-dependent apoptosis pathway was inactive, whereas caspase-independent apoptosis pathway was stimulating in response to Vibrio challenge. To eliminate invading pathogen, the immune effectors such as interleukin-17, matrix metalloproteinase, lysozyme, superoxide, and ferritin were also stimulated in gill. Moreover, this study also provides more information for comprehending the immunity and defense mechanisms against V. parahaemolyticus infection in abalone.

Interactions of dietary eicosapentaenoic acid and vitamin C on growth performance, anti-oxidation and muscle quality of abalone Haliotis discus hannai Ino

A 93-day feeding trial was conducted to investigate the effects of dietary eicosapentaenoic acid (EPA) and vitamin C (VC) on the growth performance, anti-oxidation and muscle quality of abalone Haliotis discus hannai (initial body weight: 26.89 ± 0.26 g; initial shell length: 60.36 ± 0.25 mm). Six experimental diets in a 3 × 2 two-factor design were formulated with graded levels of dietary EPA (0.25%, 0.75% and 1.25%) and dietary VC (0 and 1260 mg/kg). Results showed that the weight gain rate (WGR) of abalone was significantly increased with the increasing dietary EPA levels (P < 0.05), but there was no significant effect of dietary VC on the WGR. The significant highest superoxide dismutase (SOD) activity was observed in the group with 1.25% of dietary EPA and 1260 mg/kg of dietary VC, and there was a significant interaction between dietary EPA and VC (P < 0.05). Dietary EPA supplementation significantly improved muscle hardness and the content of ∑n-3 polyunsaturated fatty acids, volatile compounds, adenosine-5′-monophosphate (AMP) and inosine-5′-monophosphate (IMP) in muscle. Dietary VC supplementation significantly improved the muscle hardness, springiness, chewiness, and the contents of volatile compounds, hydroxyproline and IMP in muscle (P < 0.05). Significant interactions between dietary EPA and VC were observed on volatile compounds, AMP and IMP (P < 0.05). In conclusion, dietary EPA and VC interactively influence the anti-oxidative capability and muscle quality of abalone.

Replacement of dietary fish meal by soybean meal on growth performance, immunity, anti-oxidative capacity and mTOR pathways in juvenile abalone Haliotis discus hannai Ino

This study aimed to evaluate the effects of replacing fish meal with soybean meal (SBM) on growth performance, immunity, anti-oxidative capacity and mTOR pathway in juvenile abalone Haliotis discus hannai Ino. Five isonitrogenous (about 29% of crude protein) and isolipidic (about 4% of crude lipid) experimental diets with graded levels of fish meal replaced by SBM (0, 25, 50, 75 and 100%, respectively) were fed to abalone (initial body weight: 21.87 ± 0.07 g, initial shell length: 53.18 ± 0.18 mm) for 16 weeks. These experimental diets were named as SBM0, SBM25, SBM50, SBM75 and SBM100, respectively. The results showed that: 1) There was no significant difference in survival of abalone among all the treatments. The highest weight gain rate was found in the SBM50 group, and the group did not significantly differ from those in the SBM75 and SBM100 groups. 2) Replacement of fish meal by SBM linearly and quadratically increased the crude lipid content in muscle of abalone, which reached its highest level in the SBM50 group. 3) Trypsin activity in the digestive gland of abalone fed SBM25 diet was significantly higher than that of other groups. 4) The highest total anti-oxidative capacity and superoxidase activity in digestive gland were found in the SBM50 group. 5) The highest mRNA levels of pi3k and eif-4e, and the lowest level of 4e-bp in muscle were found in the SBM50 group. There was no significant difference in the mRNA level of mtor in the muscle among all the groups. 6). The lowest expression level of nf-κb and the highest expression level of mytimacin 6 in digestive gland of abalone were observed fed SBM50 diet. A significant quadratic trend was found in the mRNA levels of il-16 with increasing levels of dietary SBM. Meanwhile, the mRNA level of β-defensin was linearly decreased after total replacement of fish meal by SBM. Take the growth, antioxidation, immune and mTOR pathway into account, 50% replacement of dietary fish meal by SBM was recommended for abalone.

Substitution effect of Saccharina japonica with beet leaf by-product in feed on the growth and chemical composition of the soft body of juvenile abalone (Haliotis discus hannai Ino 1952) subjected to air exposure

Dietary substitution effect of beet leaf by-product (BLB) for Saccharina japonica on the growth and soft body composition of abalone (Haliotis discus hannai) subjected to air exposure was determined. A total of 1260 juvenile abalone was randomly distributed into 18 containers. Five experimental diets were prepared. The control (Con) diet contained 20% S. japonica. Twenty five percent, 50%, 75% and 100% S. japonica were substituted with equal amount of BLB, referred to as the BLB25, BLB50, BLB75 and BLB100 diets, respectively. The dry S. japonica was also prepared to compare effect of formulated diets on growth of abalone. The experimental diets were fed to triplicate groups of abalone for 16 weeks, and then exposed to air for 24 h. Survival of abalone was monitored for the next 6-day post observation period after the 24-h air exposure. Weight gain and specific growth rate (SGR) of abalone fed with formulated diets were greater than those of abalone fed with the S. japonica. The greatest weight gain and SGR were obtained in abalone fed the BLB25 diet, followed by the BLB50, BLB75, BLB100 and Con diets, in that order. The maximum values of weight gain, SGR, shell length and shell width of abalone were estimated to be approximately 42.3%, 43.5%, 39.8% and 33.7% of substitution level of BLB for S. japonica in abalone feed. The chemical composition of abalone soft body was not affected by the experimental diets. Survival of abalone fed with formulated diets was higher than that of abalone fed with the S. japonica at the end of the 6-day post observation period. In conclusion, S. japonica could be completely replaced with BLB in abalone feed without retardation of growth and survival of abalone subjected to the 24-h air exposure. The greatest weight gain and SGR were obtained in abalone fed the BLB25 diet.

High dietary lipid level decreases the immunity and disease resistance of abalone Haliotis discus hannai and affects the perilipin‐2/TLR4, JNK and Keap1/Nrf2 pathways

A 100-day feeding trial was conducted to investigate the influences of dietary lipid levels on the immunity and mechanism in abalone Haliotis discus hannai Ino. Abalones (initial weight: 10.98 ± 0.05 g) were fed with graded levels of dietary lipid, which were 15.73, 23.41, 31.72, 38.25, 46.35, 55.63, 61.70 and 67.19 g/kg. Results showed that compared with the treatment with 38.25 g/kg of dietary lipid, 67.19 g/kg of dietary lipid increased the cumulative mortality of abalone after a challenge with Vibrio parahaemolyticus. Meanwhile, 67.19 g/kg of dietary lipid decreased the activities of lysozyme (LZ) and acid phosphatase (ACP) in serum, and the mRNA levels of β-defensin and mytimacin 6 in hepatopancreas of abalone. The mRNA levels of tumour necrosis factor α (tnfα), activator protein 1 (ap-1), nuclear factor κB (nf-κb), interleukin 1 receptor-associated kinase-4 (irak4), myeloid differentiation primary response protein MyD88 (myd88), toll-like receptor 2 (tlr2), tlr4 and perilipin-2, and the protein level of interleukin-1β (IL-1β), TNFα, NF-κBp65, AP-1, MyD88 and Perilipin-2 in hepatopancreas were up-regulated by 67.19 g/kg of dietary lipid. However, the mRNA levels of arginase and inhibitor of κBα (iκbα) were down-regulated. Besides, 67.19 g/kg of dietary lipid up-regulated the mRNA levels of executor apoptosis-related cysteine peptidase 3 (caspase-3), caspase-7 and B-cell lymphoma protein-2-associated X protein (bax), and the protein levels of caspase-3 and c-Jun N-terminal kinases (JNK) in hepatopancreas. Moreover, high dietary lipid level down-regulated the mRNA levels of copper/zinc superoxide dismutase (cuznsod), manganese superoxide dismutase (mnsod), catalase (cat), glutathione peroxidase (gpx), glutathione-S-transferase (gst) and NF-E2-related factor 2 (nrf2), while up-regulated the mRNA and protein levels of Kelch-like ECH-associated protein 1 (Keap1) in hepatopancreas. It was concluded that a high level (67.19 g/kg) of dietary lipid decreased the immunity of abalone, which might be related to the aggravated inflammation and apoptosis, and decreased anti-oxidative capacity under Perilipin-2/TLRs/MyD88/IRAK4/(NF-κB or AP-1), JNK/Bcl-2/Bax and Keap1/Nrf2 signalling.

Ascorbic Acid Regulates the Immunity, Anti-Oxidation and Apoptosis in Abalone Haliotis discus hannai Ino.

The present study was conducted to investigate the roles of ascorbic acid (AA) in immune response, anti-oxidation and apoptosis in abalone (Haliotis discus hannai Ino). Seven semi-purified diets with graded levels of AA (0, 50, 100, 200, 500, 1000 and 5000 mg/kg) were fed to abalone (initial weight: 12.01 ± 0.001 g, initial shell length: 48.44 ± 0.069 mm) for 100 days. The survival, weight gain rate and daily increment in shell length were not affected by dietary AA. The AA content in the gill, muscle and digestive glands of abalone was significantly increased by dietary AA. In terms of immunity, dietary AA significantly improved the total hemocyte count, respiratory burst and phagocytic activity in hemolymph, and lysozyme activity in cell-free hemolymph (CFH). In the digestive gland, the TLR-MyD88-dependent and TLR-MyD88-independent signaling pathways were suppressed by dietary AA supplementation. The mRNA levels of β-defensin and arginase-I in the digestive gland were significantly increased by dietary AA. In the gill, only the TLR-MyD88-dependent signaling pathway was depressed by dietary AA to reduce inflammation in abalone. The level of mytimacin 6 in the gill was significantly upregulated by dietary AA. After Vibrio parahaemolyticus infection, the TLR signaling pathway in the digestive gland was suppressed by dietary AA, which reduced inflammation in the abalone. In terms of anti-oxidation, superoxide dismutase, glutathione peroxidase and catalase activities, as well as total anti-oxidative capacity and reduced glutathione content in CFH, were all significantly upregulated. The malondialdehyde content was significantly downregulated by dietary AA. The anti-oxidative capacity was improved by triggering the Keap1-Nrf2 pathway in abalone. In terms of apoptosis, dietary AA could enhance the anti-apoptosis ability via the JNK-Bcl-2/Bax signaling cascade in abalone. To conclude, dietary AA was involved in regulating immunity, anti-oxidation and apoptosis in abalone.

Lipid deposition in abalone Haliotis discus hannai affected by dietary lipid levels through AMPKα2/PPARα and JNK/mTOR/SREBP-1c pathway

A 100-day feeding trial was conducted to investigate the effects of dietary lipid on growth performance and lipid deposition as well as the regulation mechanism in abalone Haliotis discus hannai Ino. Abalones (initial weight: 10.98 ± 0.05 g) were fed with graded levels of dietary lipid (1.57%, 2.34%, 3.17%, 3.82%, 4.63%, 5.56%, 6.17% and 6.72%). Results showed that weight gain rate (WGR) and increment in shell length of abalone were highest in 3.82% dietary lipid level. Based on the quadratic regression analysis for WGR, the optimal dietary lipid level for abalone was estimated to be 3.87%. Concentrations of the total triacylglycerol, low-density lipoprotein cholesterol and non-esterified fatty acids in serum as well as lipid contents in hepatopancreas and muscle were positively related to dietary lipid levels. Compared to the group with 3.82% of dietary lipid, higher (6.72%) and lower (1.57%) dietary lipid down regulated the expression of genes involved in fatty acids transport and lipolysis in hepatopancreas and muscle, which might be related to AMP-activated protein kinase α2 (AMPKα2)/peroxisome proliferator-activated receptor α (PPARα) signaling. Higher dietary lipid (6.72%) down regulated, while lower dietary lipid (1.57%) up-regulated the expressions of genes involved in de-novo lipogenesis in hepatopancreas. Both higher (6.72%) and lower (1.57%) dietary lipid down regulated these genes' expression in muscle. It was found that c-Jun N-terminal kinases (JNK)/mammalian target of rapamycin (mTOR)/sterol regulatory element-binding protein 1c (SREBP-1c) signaling participate in the regulation of de-novo lipogenesis. In conclusion, lipid deposition in abalone was affected by dietary lipid levels through AMPKα2/PPARα and JNK/mTOR/SREBP-1c pathway.

Mapping and marker identification for sex-determining in the Pacific abalone, Haliotis discus hannai Ino

Pacific abalone (Haliotis discus hannai Ino) is an economically important mollusk. It has a male heterogametic (XX/XY) sex determination system. However, little is currently known on the molecular basis of sex determination in the species. Therefore, we identified the sex-linked genomic segment and markers with genome re-sequencing in Pacific abalone. A total of 8,473,763 high quality SNPs and 6,332,868 indels were obtained with resequencing and further processing. Among them, 2397 SNPs and 98 indels were detected as sexually dimorphic. According to distribution of sexually dimorphic SNPs, a major segment on Chr. 9 (~2.59 Mb) and a minor one on Chr. 2 (~50.02 Kb) were identified as sex-linked, consistent to the results of genome-wide association analysis (GWAS). In addition, a diagnostic marker (MSP-2) for genetic sex of Pacific abalone was developed according to the male-specific deletion rs4391834 (12 bp). These results were useful in further researches on the molecular mechanism of sex determination and sex-control breeding in Pacific abalone.

Molecular characterization, purification, and antioxidant activity of recombinant superoxide dismutase from the Pacific abalone Haliotis discus hannai Ino

Superoxide dismutase (SOD) is an acidic metalloenzyme that scavenges free radicals produced by endogenous and exogenous substances. In the present study, the tissue distribution of the superoxide dismutase HdhCu/Zn-SOD was investigated in Haliotis discus hannai Ino. The expression profile after lipopolysaccharide (LPS) challenge was determined using quantitative real-time polymerase chain reaction (qPCR). To study the antioxidant activity of a recombinant HdhCu/Zn-SOD protein, the HdhCu/Zn-SOD gene was cloned into the pPIC9K vector and transformed into the Pichia pastoris GS115 strain by electroporation. After induction by methanol, the recombinant product was purified using immobilized metal affinity chromatography and confirmed using mass spectrometry. The optimal expression conditions were determined to be incubation with 0.5% methanol at pH 6.0, resulting in a stable expressed product with the molecular weight of approximately 17 kDa and 21 kDa. The enzymatic activity of HdhCu/Zn-SOD consistently increased with increasing Cu2+ concentrations and showed good thermal stability. Recombinant HdhCu/Zn-SOD showed a strong ability to scavenge superoxide anions and hydroxyl radicals and protected L929 cells against the toxicity caused by H2O2 through its in vitro antioxidant activity. The heterologous expression of HdhCu/Zn-SOD in P. pastoris and the antioxidant activity of this enzyme are reported for the first time.Graphic abstract

Shelter selection by juvenile Pacific abalone ( Haliotis discus hannai Ino) as a function of food distribution and water flow velocity

Juvenile Pacific abalone (Haliotis discus hannai Ino) are currently reared in land-based aquaculture systems until they reach the necessary size for seeding in the sea. One problem that this industry faces is that an uneven distribution of juveniles in tanks can lead to variations in the growth rate. Understanding the cues that affect the sheltering behaviours exhibited by juvenile Pacific abalone—namely, the food distribution and the water flow velocity—will help optimize abalone culture settings. In this study, a group of 1,000 juvenile Pacific abalone (distributed across three tanks) was visually observed and enumerated during six experiments that were conducted over a 5-month study period. It was found that juvenile Pacific abalone preferred to shelter close to food sources when the food was unevenly distributed. When the food was evenly distributed, the juveniles tended to avoid areas of rapid water flow and distributed evenly across the sheltered areas receiving the equal water velocity. This distribution might be the confounding effects between water flow velocity and food stimulus. Based on these findings, it was recommend that the food and water velocity be evenly distributed in an abalone aquaculture system.

CHARACTERISATION OF PATHOGENIC VIBRIO SPP. ISOLATED FROM LIVE PACIFIC ABALONE (HALIOTIS DISCUS HANNAI INO, 1953) MARKETED IN SOUTH KOREA

This study aimed to evaluate the profile of virulence factors and antimicrobial resistance in Vibrio spp. isolated from live Pacific abalone (Haliotis discus hannai Ino, 1953) marketed in South Korea. A total of 32 Vibrio isolates comprising V. alginolyticus (n=15), V. diabolicus (n=14), V. antiquarius (n=2) and V. parahaemolyticus (n=1) were analysed. All isolates demonstrated DNase, lipase, phospholipase and amylase activities. Additionally, production of slime (97%), gelatinase (94%), α-haemolysin (22%), β-haemolysin (78%) and protease (53%) were detected. Virulence genes viz., toxR, tlh, tdh, VAC, VPI, ctxAB and hupO were recorded in 56, 59, 13, 100, 41, 9 and 9% of the isolates, respectively. All isolates were resistant to ampicillin and 88% of the isolates were resistant to cephalothin and colistin sulphate. Twentyone isolates (66%) showed multiple antimicrobial resistance (MAR) index ≥0.2. Antimicrobial resistance genes blaCTX (85%), blaTEM (10%), blaSHV (10%), strAB (13%) and aphA-IAB (22%) and class 1 integrons (19%) were detected. V. diabolicus and V. antiquarius were identified and characterised for the first time in the Pacific abalone. Our findings imply the significance of integrated monitoring and surveillance programmes for the occurrence, virulence, and antimicrobial resistance patterns of vibrios in Pacific abalone.