Elsevier

Fish & Shellfish Immunology

Volume 121, February 2022, Pages 183-196
Fish & Shellfish Immunology

Molecular interaction analysis of β-1, 3 glucan binding protein with Bacillus licheniformis and evaluation of its immunostimulant property in Oreochromis mossambicus

https://doi.org/10.1016/j.fsi.2021.12.044Get rights and content

Highlights

  • A synbiotic diet was prepared and nourished to the experimental fish Oreochromis mossambicus.

  • The growth rate was significantly increased in the fish fed with a synbiotic supplemented diet.

  • Immune responses and disease resistance were elevated in fish fed with a synbiotic enriched diet.

  • Molecular docking studies proved that β-glucan specifically binds to the active site of β-GBP.

Abstract

Analyzing the health benefits of any two immunostimulants (synbiotics) in combined form and information on their interactions gain more visibility in the usage of synbiotics in aquafarms. With this intention, the current work explores the immunostimulant effect and structural interaction of synbiotic (β-1, 3 glucan binding protein from marine crab, Portunus pelagicus (Ppβ-GBP) and Bacillus licheniformis) on Oreochromis mossambicus. The experimental diet was prepared with Ppβ-GBP and B. licheniformis, and nourished to the fingerlings of O. mossambicus for 30 days. After the experimental trial, a higher growth rate and immune reactions (lysozyme, protease, myeloperoxidase and alkaline phosphatase activity) were noticed in the fish nourished with synbiotic (B. licheniformis and Ppβ-GBP) enriched diet. Moreover, the synbiotic enriched diet elevated the antioxidant responses like glutathione peroxidase (GSH-Px) and catalase (CAT) activity in the experimental diet-nurtured fish. At the end of the feed trial, synbiotic diet nourished fish shows an increased survival rate during Aeromonas hydrophila infection, reflecting the disease resistance potential of experimental fish. Also, the interaction between Ppβ-GBP and Bacillus licheniformis was analyzed through computational approaches. The results evidenced that, Ppβ-GBP interacts with the B. licheniformis through sugar-based ligand, β-glucan through a hydrogen bond with a good docking score. Thus, the synbiotic diet would be an effective immunostimulant to strengthen the fish immune system for better productivity.

Introduction

Currently, disease appearance in aquaculture and the development of antibiotic-resistant genes in pathogenic microorganisms become a significant hindrance towards aquaculture production. Fish culture under a restricted environment (intensive farming) creates stress conditions that weaken the immune system and makes the animal more prone to infectious disease-causing microbes. An innovative feeding method is one of the left practices employed in aqua farming by feed manufacturers and farmers to attain better growth in cultured fish [1]. Immunostimulants are harmless and beneficial bio-agents administered to farming animals (intensive or extensive) to prevent the entry of infectious microbes and improve the growth rate of the cultured organisms. Supplementation of immunostimulants such as probiotics, prebiotics, and synbiotics in the fish diet is a new approach to strengthening fish's defense mechanism against infectious pathogens [2]. Recently, lactoferrin [3,4], emodin [5], fucoidan [6], β-glucan [7], prebiotics [8], probiotics [9], and vitamin C [10,11] were applied as immune-boosting agent (immunostimulants) in the aquafarming industry to reduce disease outbreaks.

Probiotics and prebiotics receive much attention due to their beneficial effects on cultured animals and the environment. Probiotics administration in the aquaculture industry is an inventive approach due to its positive impacts on aquatic organisms and the environment. The administration of probiotics has become widespread in aquaculture to solve and prevent infective bacterial pathogens. Gram-positive Bacillus sp, Lactobacillus sp, Bifidobacterium sp, Micrococcus sp, Enterococcus sp, and Gram-negative Alteromonas sp, Pseudomonas sp, Photorhodobacterium sp. and Vibrios sp were used as probiotics [12]. Bacillus sp is a broadly used probiotic that shows practical immunomodulatory effects [13,14]. However, due to their limited viability in the intestinal tract, researchers focused on the concept of prebiotic to create competitive potential in the probiotic among commensal microbiota to increase stable longevity. Prebiotics are a non-digestible food ingredient that shows resistance to gastric acidity of the intestinal microbiota of the host and increases the beneficial bacteria in the digestive zone [15,16]. Various prebiotics includes inulin, oligofructose, xylooligosaccharides, galactooligosaccharides, and fructooligosaccharides, were used as immunostimulants in the aquaculture industry. These boost up the defense response of cultured animals, reflecting an adequate growth rate, good microbial colonies in the intestinal tract, and disease resistance potential [17]. The administration of prebiotics may alter the gastrointestinal tract's microbial abundance by elevating immune responses [18].

Synbiotics, the name itself denotes the combination of probiotics and prebiotics in defined concentration. The supplementation of synbiotics in cultured animals enhances the immune responses and elevates mucus production [19]. Moreover, synbiotics act as an excellent antioxidant that prevents cell damage due to excess reactive oxygen species (ROS) formed in the cultured aquatic animals [[20], [21], [22]]. Synbiotics beneficially modulate the intestinal microflora of the host and develop synergistic relations between microbial colonies in the gut and host environment [17]. So far, the literature has focused on analyzing the immunostimulant effect of synbiotics, but there is no report on its molecular interaction. Hence, this study aimed to assess the immunostimulant effect of synbiotic in Tilapia O. mossambicus and analyze the structural interaction between B. licheniformis and Ppβ-GBP through molecular docking studies. The molecular interaction analysis provides more insight into the relationship of synbiotics for the immunostimulant effect.

Section snippets

Experimental animal collection and purification of Ppβ-GBP

Collection of healthy live crabs and purification of β-GBP from the heamolymph of P. pelagicus was carried out as per the method described in our previous publication [23]. For the experimental analysis, fresh and live freshwater Mozambique Tilapia Oreochromis mossambicus (7.5 ± 0.5 g weight and 8 ± 0.5 cm length) were gathered safely from the local fish farm carried to the laboratory. Fish were placed in 300 L fiber-reinforced plastic (FRP) tanks with an optimum temperature of 29 ± 2.0 °C, pH

Growth performance

The influence of an experimental diet on fish growth is depicted in Table 1. Growth rate remained to be increased in experimental diet nourished fish than control fish. Though the growth rate was improved in the fish nourished with a prebiotic and probiotic enriched diet, a significant improvement in growth was noticed in the fish nourished with a synbiotic enriched diet after 30 days of the feeding trial. Synbiotic diet nourished fish shows a two-fold increase in weight than basal diet

Discussion

Recently, immunostimulants have been applied in the aquafarms to expand the growth rate in fish and boost up the immune response of aquatic animals against infectious pathogens. In particular, prebiotics and probiotics are widely applied as a substitute to the conventional treatment in aquaculture to enhance disease resistance by improving the humoral and cellular mediated immune responses in cultured fishes [15,16]. Henceforward, the present work was focused on investigating the synergistic

Conclusion

In conclusion, the experimental diet nourished fish exhibits improved growth parameters, such as lysozyme and peroxidase, protease and ALP activity in serum and mucus during the 30-day feeding trial. Besides, the antioxidant response (GSH-Px and CAT) was also increased in the experimental diet of nourished fish. The experimental diet increases the disease resistance capacity in the fish against A. hydrophila and thus increases the relative percentage of survival. In particular, adequate growth,

CRediT authorship contribution statement

Mahalingam Anjugam: Conceptualization, Methodology, Software. Arokiadhas Iswarya: Formal analysis. Ashokkumar Sibiya: Formal analysis. Chandrabose Selvaraj: Visualization, Validation. Sanjeev Kumar Singh: Formal analysis. Marimuthu Govindarajan: Writing – review & editing. Naiyf S. Alharbi: Resources, Validation. Shine Kadaikunnan: Visualization, Validation. Jamal M. Khaled: Formal analysis. Jeyachandran Sivakamavalli: Formal analysis. Baskaralingam Vaseeharan: Conceptualization, Visualization,

Acknowledgement

The authors thank RUSA phase.2.0 grant (Refer. No.24-51/2014-U, policy) TN. Multi-Gen, Department of Education, Government of India. The authors express their sincere appreciation to the Researchers Supporting Project Number (RSP-2021/70), King Saud University, Riyadh, Saudi Arabia.

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