Survival, immune response and growth of Penaeid shrimp as affected by immunostimulants: A meta-analysis.

Immunostimulants represent the most innovative approach for combating shrimp diseases. They are molecules that effectively enhance the host's nonspecific defenses against invading microorganisms. However, methodological differences exist among immunostimulants based on the same source. Therefore, conducting a meta-analysis is essential to derive valid conclusions. The effect size value utilized in this study was Hedges' d. Heterogeneity among studies was assessed using the DerSimonian and Laird tests (Q-statistic). Meta-regression analysis was conducted to explore the sources of heterogeneity in treatment effects. In this study, dose served as a covariate because it was the only continuous variable that significantly contributed to the observed heterogeneity. Funnel plots and the fail-safe number were employed to assess publication bias within the datasets. The article collection process followed the PRISMA methodology. Based on the results of the meta-analysis and meta-regression conducted with 83 articles, it can be concluded that immunostimulants have a significant effect, characterized by high category standard mean difference (SMD) values, on the survival, growth, and immune response of Penaeid family shrimp. Among potential immunostimulants options, algae ingredients exhibited the most favorable effects on the survival, growth, and immune response of Penaeid family shrimp. Subgroup analysis outcomes revealed that various extraction methods significantly impacted the efficacy of immunostimulants, with the ethanol solvent method proving to be the most effective. Among different administration methods, no significant effect was observed on immunostimulant efficacy across all parameters, with positive SMD values for all administration methods. Regarding challenged test pathogens, immunostimulants were observed to enhance immune response, survival, and weight gain against various pathogens. Meta-regression results indicated that algal treatments had a lower optimal dose point, leading to decreased efficacy as the dose increased. In contrast, fungi exhibited a higher optimum dose point, resulting in increased efficacy at higher doses.

Effects of dietary astaxanthin on immune status and lipid metabolism in gilthead seabream (Sparus aurata).

Astaxanthin (AX) is a carotenoid known to have one of the highest documented antioxidant capacities and has attracted considerable scientific and commercial interest. The incorporation of AX into aquaculture practices has been associated with improved pigmentation, modulation of the immune and endocrine systems, stress reduction, reproductive efficiency and general fish health. This study describes the effects of dietary AX (0, control, 20, 100 and 500 mg kg-1 AX per kg of diet) for 15 and 30 days on growth performance, immune and antioxidant status, histology and gene expression in gilthead seabream (Sparus aurata). Fish fed diets enriched with 500 mg kg-1 of AX for 15 days decreased in skin mucus peroxidase activity while at 30 days of trial, fish fed a diet supplemented with 20 mg kg-1 AX increased the peroxidase activity in serum. In addition, bactericidal activity against Vibrio harveyi increased in the skin mucus of fish fed any of the AX supplemented diets. Regarding antioxidant activities in the liver, catalase and glutathione reductase were decreased and increased, respectively, in fish fed a diet supplemented with 500 mg kg-1 of AX. Finally, although the expression of up to 21 inflammatory and lipid metabolism-related genes was analysed in visceral adipose tissue, only the expression of the interleukin 6 (il6) gene was up-regulated in fish fed a diet supplemented with 20 mg kg-1 of AX. The present results provide a detailed insight into the potent antioxidant properties of AX and its possible modulatory effects on the immune status and lipid metabolism of seabream, which may be of interest to the aquaculture sector.

Dietary sulfated polysaccharides extracted from Caulerpa sp. and Padina sp. modulated physiological performance, antibacterial activity and ammonia challenge test in juvenile rainbow trout (Oncorhynchus mykiss).

Nowadays, the use of seaweed derivatives in aquaculture has drawn attention for their potential as an immunostimulant and growth promotor. The sulfated polysaccharide extracted (SPE ) from green (Caulerpa sp.; SPC) and brown (Padina sp.; SPP) seaweeds with two concentrations (0.05% and 0.1%); nominated in four groups: SPC0.05 , SPC0.1 , SPP0.05 , SPP0.1 and control group (free of SPE ) were used for juvenile rainbow trout (Oncorhynchus mykiss) diet. Fish (N: 150; 8.5 ± 0.2 g) were selected aleatory distributed in 15 circular tanks (triplicate for the group) and fed test diets for 56 days. The outcomes revealed that the supplementation of SPE up to 1 g kg-1 failed to show significant differences in the organosomatic indices as compared to the control group. The most inferior protein value of dress-out fish composition was observed in the fish fed the control diet, which was statistically lower than the SCP0.1 group (p < 0.05), while no significant difference was observed in other macronutrient composition among the treatments. Total monounsaturated fatty acid (MUFA) had lower trend in the carcass of fish fed SPE supplemented diets, so that lowest MUFA were observed in SPC0.05 group (p < 0.05; 25.22 ± 4.29%). The lowest value of docosahexaenoic acid was observed in the control diet compared to the SPE -supplemented diets (p < 0.05). The serum alternative complement pathway levels in all treatments tend to promote compared to the control treatment. A similar trend was observed for lysozyme activity. According to the results, the superoxide dismutase (SOD) value were highest in SPC0.05 and SPC0.1 compared to the other treatments (p < 0.05), while a further elevation of the SPE Padina sp. extracted level (SPP0.1 ) leads to a decrease in SOD value. Thiobarbituric acid reactive substances of plasma was indicated not to influence by sulfated polysaccharide extracts in the refrigerated storage. The lowest serum stress indicators were observed in fish fed SPP0.05 group postchallenge test. Taken together, our outcomes revealed that SPE of two species of seaweeds bestows benefits in some of the immunity and antioxidant system. Also, notable elevations in HUFA were observed in juvenile rainbow trout fed supplemented with SPE .

Synergetic response on herbal and probiotic applications: a review.

Herbs and their by-products are important traditional medicines and food supplements; they provide numerous beneficial effects for animals. Consequently, probiotics are living cell organisms, nontoxic, and friendly microbes. Probiotics have numerous beneficial activities such as inhibition of pathogens, enhancement of the immune system, growth, disease resistance, improving water quality, reducing toxic effects, synthesis of vitamins, prevention of cancer, reduction of irritable bowel syndrome, and more positive responses in animals. Herbal and probiotic combinations have more active responses and produce new substances to enhance beneficial responses in animals. Herbal and probiotic mixture report is still limited applications for animals. However, the mechanisms by which they interact with the immune system and gut microbiota in animals are largely unclear. This review provides some information on the effect of herbal and probiotic blend on animals. This review discusses current research advancements to fulfill research gaps and promote effective and healthy animal production.

A review of immune modulators and immunotherapy in infectious diseases.

The human immune system responds to harmful foreign invaders frequently encountered by the body and employs defense mechanisms to counteract such assaults. Various exogenous and endogenous factors play a prominent role in maintaining the balanced functioning of the immune system, which can result in immune suppression or immune stimulation. With the advent of different immune-modulatory agents, immune responses can be modulated or regulated to control infections and other health effects. Literature provides evidence on various immunomodulators from different sources and their role in modulating immune responses. Due to the limited efficacy of current drugs and the rise in drug resistance, there is a growing need for new therapies for infectious diseases. In this review, we aim to provide a comprehensive overview of different immune-modulating agents and immune therapies specifically focused on viral infectious diseases.

Mechanistic insight into immunomodulatory effects of food-functioned plant secondary metabolites.

Medicinally important plant-foods offer a balanced immune function, which is essential for protecting the body against antigenic invasion, mainly by microorganisms. Immunomodulators play pivotal roles in supporting immune function either suppressing or stimulating the immune system's response to invading pathogens. Among different immunomodulators, plant-based secondary metabolites have emerged as high potential not only for immune defense but also for cellular immunoresponsiveness. These natural immunomodulators can be developed into safer alternatives to the clinically used immunosuppressants and immunostimulant cytotoxic drugs which possess serious side effects. Many plants of different species have been reported to possess strong immunomodulating properties. The immunomodulatory effects of plant extracts and their bioactive metabolites have been suggested due to their diverse mechanisms of modulation of the complex immune system and their multifarious molecular targets. Phytochemicals such as alkaloids, flavonoids, terpenoids, carbohydrates and polyphenols have been reported as responsible for the immunomodulatory effects of several medicinal plants. This review illustrates the potent immunomodulatory effects of 65 plant secondary metabolites, including dietary compounds and their underlying mechanisms of action on cellular and humoral immune functions in in vitro and in vivo studies. The clinical potential of some of the compounds to be used for various immune-related disorders is highlighted.

Generation and structure-activity relationships of novel imidazo-thienopyridine based TLR7 agonists: application as payloads for immunostimulatory antibody drug-conjugates.

Pairing immunostimulatory small molecules with the targeting capability of an antibody has emerged as a novel therapeutic modality with the potential to treat a variety of solid tumors. A series of compounds based on an imidazo-thienopyridine scaffold were synthesized and tested for their ability to agonize the innate immune sensors toll-like receptor 7 and 8 (TLR7/8). Structure-activity relationship (SAR) studies revealed that certain simple amino-substituents could enable TLR7 agonism at low nanomolar concentrations. Drug-linkers containing either payload 1 or 20h were conjugated to the HER2-targeting antibody trastuzumab at the interchain disulfide cysteine residues using a cleavable valine-citrulline dipeptide linker and stochastic thiol-maleimide chemistry. In vitro, these immune-stimulating antibody drug-conjugates (ADCs) were found to induce cytokine release in a murine splenocyte assay when co-cultured with the HER2-high NCI-N87 cancer cell line. In vivo, tumor regression was observed with a single dose in an NCI-N87 gastric carcinoma xenograft model in BALB/c nude mice.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.

Use of marine microorganisms in designing anti-infective strategies for sustainable aquaculture production.

Aquaculture, a noteworthy food production sector, is confronted with disease occurrences. Treatment of aquaculture pathogens with antibiotics is often rendered ineffective due to biofilm formation and the development of resistant strains. Marine ecosystems encompass unusual microorganisms that produce novel bioactive compounds, including agents that could be used as alternatives to antibiotics. Moreover, biomass and/or biomolecules associated with these microorganisms could act as feed supplements to enhance the overall health of aquaculture species' and improve water quality parameters. The present review summarizes the contents of studies on such marine microorganisms with the potential to be developed as agents for tackling bacterial diseases in the aquaculture segment. Bioactive compounds produced by marine bacteria are known to inhibit biofilm-associated infections mediated by their bactericidal properties (produced by Bacillus, Vibrio, Photobacterium, and Pseudoalteromonas species), surfactant activity (obtained from different species of Bacillus and Staphylococcus lentus), anti-adhesive activity (derived from Bacillus sp. and Brevibacterium sp.), and quorum sensing inhibition. Several marine fungal isolates capable of producing antibacterial agents have also been effective in inhibiting aquaculture-associated pathogens. Another strategy followed by investigators to reduce the severity of infections is the use of bacterial, yeast, and microalgae biomass as feed supplements, probiotics, and immunostimulants. In some cases, marine microalgae have been employed as sustainable alternatives to fish oil and fish meal without compromising on nutritional quality. Their inclusion in aquaculture feed has enhanced growth, favored better survival of cultured species, and improved water quality parameters. Marine microorganisms (by providing effective bioactive compounds and being used as feed supplements) could enable aquaculture practices to be more sustainable in the future.

Synthesis, evaluation and molecular dynamics study of human toll-like receptor 2/6 specific monoacyl lipopeptides as candidate immunostimulants.

TLR2 agonists typified by the S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (Pam2CS) motif have exhibited powerful immunostimulatory activities. Based on simplified monoacyl lipopeptide (Carbamate-linked N-Ac PamCS), we describe interesting SAR investigations where modifications are done to alter the size of substituents on the cysteine amine, introduce ionizable groups to the terminal and insert aromatic substitutions to the aliphatic chain. Our structural modifications have led to a highly specific human TLR2/6 agonist 14a (EC50 = 0.424 nM), which behaves like Pam2CSK4 by inducing NF-κB activation to trigger downstream signaling pathways, such as subsequent phosphorylation of related proteins (p65, p38) and production of key inflammatory cytokines (IL-6, IL-1ß, TNF-α). Importantly, the ability to stimulate enhanced T cell response compared to Carbamate-linked N-Ac PamCS makes compound 14a a further potential candidate immunostimulant.

The immunomodulatory and antioxidant effects of ß-glucans in invertebrates.

ß-glucans (ßGs) are carbohydrate polymers linked by ß-1,3, 1,4 or 1,6 bonds, they have been used to protect against potential pathogens and prevent lethal diseases. The immune system possesses several receptors that identify a wide range of structures and trigger cellular and humoral mechanisms. However, the mechanisms by which ßGs activate the immune system of invertebrate organisms have not been fully clarified. This review is focused on evaluating the effect of ßGs on innate immune system in invertebrates. ßGs stimulate different cellular and humoral mechanisms, such as phagocytosis, oxygen species production, extracellular trap formation, proPO system, and antimicrobial peptide synthesis, moreover, ßGs increase survival rate and decrease pathogen load in several species.

Aeromonas veronii infection remarkably increases expression of lysozymes in grass carp (Ctenopharyngodon idellus) and injection of lysozyme expression cassette along with QCDC adjuvant significantly upregulates immune factors and decreases cumulative mortality.

Aeromonas veronii AvX005 is a pathogenic bacterium with high toxicity to grass carp (Ctenopharyngodon idellus). The expression levels of g-type (goose-type lysozyme, Lys-g) and c-type lysozyme (chicken-type lysozyme, Lys-c) in the spleen of grass carp infected with AvX005 were significantly increased by approximately 4.5 times and 27 times, respectively. The recombinant proteins rLys-g and rLys-c produced in a recombinant expression system of Escherichia coli showed significant antibacterial activity against the pathogenic bacteria AvX005. A challenge test was conducted after rLys-g and rLys-c were expressed in grass carp L8824 liver cells, and compared with the survival rate of the control cells (46.3%), the survival rate of the experimental cells (77.6% for rLys-g and 68.6% for rLys-c) was significantly increased. Grass carp were infected with AvX005 on the second day after delivering pcDNA3.1-lys-g and pcDNA-lys-c with the Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant, and both pcDNA3.1-lys-g and pcDNA-lys-c provided 70% relative protection for grass carp. The activity of lysozyme and alkaline phosphatase in the serum of grass carp was significantly increased after injection of DNA. The expression of the immune factors IgM, C3 and IL8 in the kidney was upregulated to varying degrees for pcDNA3.1-lys-g and immune factors C3 and IgM was upregulated for pcDNA-lys-c. The results indicated that pcDNA3.1-lys-g and pcDNA-lys-c may be used as immunostimulants to protect grass carp from the pathogenic bacterium AvX005.

Immune responses to prebiotics in farmed salmonid fish: How transcriptomic approaches help interpret responses.

Within aquaculture, prebiotics are composed of complex carbohydrate molecules that cannot be digested by the fish directly but are metabolised by the microbial communities within the host gut, with the desire that "healthy" bacterial species are promoted with subsequently improved performance of the fish, there are likely some direct responses of intestinal cells to these dietary components. The sources and processing of prebiotics, which fall under the overarching theme of "functional feeds" are highly varied between species and types of prebiotics administered. How these feeds exert their effect, and the host responses are hard to determine, but new technologies and the development of high-throughput technologies (omics) are enabling the mechanisms and methods of action to be further understood. The recent advances in the availability of 'omics' technologies with the transition from single gene assays to microarray and RNA-seq in fish health have enabled novel functional ingredients to be analysed. This review will focus on recent studies on targeted gene expression and 'omics' technologies to characterize immune responses. Comparisons between the immunomodulatory effect of different prebiotics have been made and specific examples of how transcriptomics techniques have been used to identify immune responses to prebiotics are given.

Combined dynamic transcriptomics and metabolomics analyses revealed the effects of trans-vp28 gene Synechocystis sp. PCC6803 on the hepatopancreas of Litopenaeus vannamei.

Litopenaeus vannamei is the most important shrimp species throughout the world. However, diseases are increasing with the development of the industry, so enhancing the immunity of shrimp is of great significance. In this study, 1800 shrimp were divided into two groups randomly: the control group (N, feed with brine shrimp flake) and the experimental group (M, feed with mutant of Synechocystis sp. cells) (300 shrimp/group/replication) and each trial was conducted in triplicates. After immunization, sixty shrimp (with three replicates of twenty) were collected at 0 h in group N and 24, 72, and 144 h in group M, respectively, and the hepatopancreas were isolated for transcriptomic and metabolomic analysis. Transcriptome data revealed that compared with group N, genes related to antimicrobial peptides, cytoskeleton remodeling, detoxification, apoptosis, blood coagulation, immune defense, and antioxidant systems were differentially expressed in group M. In addition, combined transcriptomic and metabolomic analysis revealed that some immune-related differential genes or differential metabolites were consistently expressed in both omics. All the above results indicated that trans-vp28 gene Synechocystis sp. PCC6803 could improve the immunity of L. vannamei. This is the first report of the integration of dynamic transcriptomics combined with metabolomics to study the effect of trans-vp28 gene Synechocystis sp. PCC6803 in the hepatopancreas of L. vannamei and provided important information about the defense and immune mechanisms used by invertebrates against pathogens.

Bioactive immunostimulants as health-promoting feed additives in aquaculture: A review.

Bioactive immunostimulants could be derived from different sources like plants, animals, microbes, algae, yeast, etc. Bioactive immunostimulants are the most significant role to enhance aquatic production, as well as the cost of this method, which is effective, non-toxic, and environment-friendly. These immunostimulants are supportive to increase the immune system, growth, antioxidant, anti-inflammatory, and disease resistance of aquatic animals' health and also improve aquatic animal feed. Diseases are mainly targeted to the immune system of aquatic organisms in such a way that different processes of bioactive immunostimulants progress are considered imperative techniques for the development of aquaculture production. Communicable infections are the main problem for aquaculture, while the mortality and morbidity connected with some outbreaks significantly limit the productivity of some sectors. Aquaculture is considered the mainly developing food production sector globally. Protein insists is an important issue in human nutrition. Aquaculture has been an exercise for thousands of years, and it has now surpassed capture fisheries as the most vital source of seafood in the world. Limited study reports are available to focal point on bioactive immunostimulants in aquaculture applications. This review report provides information on the nutritional administration of bioactive immunostimulants, their types, functions, and beneficial impacts on aquatic animals' health as well as for the feed quality development in the aquaculture industry. The scope of this review combined to afford various kinds of natural derived bioactive molecules utilization and their beneficial effects in aquaculture applications.

Antioxidative and immunostimulant potential of fruit derived biomolecules in aquaculture.

Huge amounts of non-edible by-products could be generated from fruit industrial processes. They consist mainly of peels together with low amounts of pulp and seeds. These by-products pose an environmental hazard due to soil, air, and water pollution. Moreover, treating these by-products is very expensive and under strict governmental regulations. Nevertheless, they are an excellent source of bioactive constituents, such as phenols, flavonoids, terpenes, and glucans. Based on their constituents, these by-products can significantly enhance the antioxidant defense, immune response, and modulation of gut microbiota and host resistance against various diseases. Therefore, sustainable valorization of fruits by-products can efficiently obtain value-added products that improve the well-being of organisms and reduce environmental stress, in addition to earning an additional industrial income. Since aquaculture is a vital economic sector, there is urgent to look for inexpensive natural food additives that improve health and maintain high nutritional quality for farming organisms without harming the environment and human health. Therefore, using fruit wastes as feed additives represents a striking alternative for fruitful aquaculture. In order to make use of these value-added products, it is a dire need to determine their biological effects on aquaculture organisms by understanding their mechanism of action. In this context, this review will holistically address a comprehensive focus on utilizing fruits by-products and their immunostimulant and antioxidative action.

The effects of dietary immunostimulants on the innate immune response of Indian major carp: A review.

Immunostimulants, as feed additives, play an important role in maintaining fish health and enhancing their overall growth by providing resistance against diseases in cultured fish. At the initial stages of life of fish, innate immunity is the essential mechanism in their survival. Later, innate immunity has an instructive role in adapting acquired immune response and homeostasis through different receptor proteins. Several studies have been conducted to analyze the effect of dietary immunostimulants like algae, plant extracts, vitamins, herbs, probiotics, and prebiotics-containing diets in Indian major carps. Many bacterial, fungal and viral pathogens are responsible for high death rates in both wild and cultured fish. It's a major limiting factor for world aquaculture industries. Recognition of invading pathogens by different pathogen recognition receptor plays an important role for the activation of different pathways to initiate protective immune responses. Hence, there is a growing need to control the devastating effects of diseases without recourse to toxic chemicals or antibiotics. Keeping with alternative approaches without using toxic chemicals to control fish diseases in mind, many immunostimulants are used, which enhance immune responses along with their gene expression level through different signaling pathway. The objective of this review is to summarize and evaluate the current knowledge of various immunostimulants and their immune responses in three Indian major carps namely Catla catla, Labeo rohita and Cirrhinus mrigala, which are preferred by the people.

In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon bacterial challenge.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Immunostimulant plant proteins: Potential candidates as vaccine adjuvants.

The COVID-19 pandemic is shaking up global scientific structures toward addressing antibiotic resistance threats and indicates an urgent need to develop more cost-effective vaccines. Vaccine adjuvants play a crucial role in boosting immunogenicity and improving vaccine efficacy. The toxicity and adversity of most adjuvant formulations are the major human immunization problems, especially in routine pediatric and immunocompromised patients. The present review focused on preclinical studies of immunoadjuvant plant proteins in use with antiparasitic, antifungal, and antiviral vaccines. Moreover, this report outlines the current perspective of immunostimulant plant protein candidates that can be used by researchers in developing new generations of vaccine-adjuvants. Future clinical studies are required to substantiate the plant proteins' safety and applicability as a vaccine adjuvant in pharmaceutical manufacturing.

Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbß) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbß in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbß mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.