Elsevier

Fish & Shellfish Immunology

Volume 64, May 2017, Pages 457-468
Fish & Shellfish Immunology

Full length article
The hot-water extract of leaves of noni, Morinda citrifolia, promotes the immunocompetence of giant freshwater prawn, Macrobrachium rosenbergii

https://doi.org/10.1016/j.fsi.2017.03.045Get rights and content

Highlights

  • HMLE triggers phenoloxidase and phagocytotic activities, and respiratory bursts of hemocytes.

  • Prawn fed the HMLE-containing diet at 0.6 g kg−1 enhance the immunocompentence.

  • The deduced mortality against infection reveal in prawns fed the HMLE-containing diet at 0.6 g kg−1.

Abstract

The hot-water Morinda citrifolia leaf extract (HMLE) was prepared for in vitro assessment on phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity (PA). Furthermore, the HMLE was administrated in the diet at 0.6, 3, and 6 g (kg diet)−1 for Macrobrachium rosenbergii, and the potential effects on the immunocompetence of prawns were evaluated. PO activity, RBs, and PA in hemocytes incubated with the HMLE at 140, 20, 20, and 140 mg l−1 significantly increased. The immune parameters of the total hemocyte count (THC), differential hemocyte count (DHC), RBs, PO activity, superoxide dismutase (SOD) activity, PA, transglutaminase (TG) activity and hemolymph clotting time were evaluated before and after 1, 3, 5, 7, and 9 weeks of the feeding trial. During 9 weeks of the feeding trial, higher THCs, DHCs, RBs, PO, and TG as well as accelerated clotting times were observed in prawns fed HMLE-containing diets at 0.6 g kg−1. The mRNA expressions of prophenoloxidase, TG, crustin, and lysozyme of prawns fed HMLE-containing diets at 0.6 g kg−1 for 9 weeks of the feeding trial significantly increased. The susceptibility of prawns fed the HMLE at 0.6 g kg−1 to Lactococcus garvieae infection significantly decreased, and the relative survival percentage was 23.1%. We therefore found that HMLE administrated through the diet at 0.6 g kg−1 was capable of enhancing the immunity and resistance against L. garvieae in M. rosenbergii.

Introduction

The giant freshwater prawn, Macrobrachium rosenbergii, is an important freshwater farmed crustacean species in many countries because of its high commercial value. However, in Taiwan, serious economic losses have occurred due to infection by yeasts in the cool season [1] and bacteria in the hot season [2]. To avoid economic losses, veterinary drugs are commonly used in aquaculture to prevent or treat disease outbreaks, and the regular administration as additives in fish food or in baths and injections is a commonly used strategy as prophylactics, therapeutics, or growth promoters [3]. However, the side effects of veterinary drugs in terms of both environmental and health safety have become a major concern. Vaccinations are used as potential treatments against disease outbreaks in aquaculture; nevertheless, the cost and multivalent vaccine development are main issues in the spectrum of vaccine use in aquaculture [4]. The prophylactic administration of immunostimulants is considered an alternative treatment to chemotherapy and vaccines, and is applied in aquaculture to control disease because of their broad-spectrum activity, cost effectiveness, and eco-friendly disease preventative effects through strengthening defense mechanisms [5]. Immunostimulants are effective means of increasing the immunocompetency and disease resistance by enhancing both specific and non-specific defense mechanisms of fish and shellfish [6], [7].

Invertebrates lack an adaptive immune system and rely on their effective cellular and humoral innate immune responses to combat invading microbes [8]. In recent years, even though some evidence of specific immune priming revealed the protective specificity upon a second exposure to a pathogen in shrimp, it is still not generally accepted that this implies the presence of immunological memory [9]. Plants and their byproducts, such as alkaloids, terpenoids, tannins, saponins, glycosides, flavonoids, phenolics, steroids, and essential oils, in plant extracts were reported to have such properties as growth-promoting ability, immune system improvement, antimicrobial capability, and stimulating the appetite and anti-stress characteristics in aquaculture species [7], [10]. In addition, the reduced costs of treatment, biodegradable characteristics, and less drug resistance are advantages of using plant extracts [4]. Therefore, plant extracts as sustainable and effective substitutes for veterinary drugs and vaccines in aquaculture was widely reviewed by Reverter et al. [4], Harikrishnan et al. [7], Citarasu [10], and Van Hai [11]. As interest has grown in prawns, the enhanced immunological responses, resistance to pathogen infection, and better growth performance were reported in Mac. rosenbergii fed diets containing extracts of banana (Musa acuminata) peels [12], Eichhornia crassipes [13], Rheum officinale Bail [14], or Withania somnifera [15].

Morinda citrifolia, known commercially as noni, grows widely throughout the Pacific and is the most significant source of traditional medicines among Pacific Island societies. This small evergreen tree or shrub is native to areas from Southeastern Asia (Indonesia) to Australia, and now has a pantropical distribution. Extracts of various parts of the noni plant were reported to have many significant effects, such as antibacterial, antifungal, and tumor suppressive activities of the fruit [16]; antioxidative activities of the root [17]; inhibitor of elastase and tyrosinase of seeds [18]; and treatment of ulcerations and minor infections of the leaves [19]. In aquaculture, Kumaran et al. [20] reported that methanol extract of Morinda tinctoria leaf enhanced the resistance to Vibrio parahaemolyticus in the freshwater crab, Oziotelphusa senex senex and also promoted non-specific and specific defense mechanisms.

Alcoholic and organic solvents can more highly efficiently extract secondary bioactive metabolites (polar and non-polar) with antimicrobial and immunostimulant activities, compared to water-based methods [21], [22], [23]. However, during processing with organosolvent extraction, the material must be further concentrated in a rotary evaporator under reduced pressure to remove the solvent. Preparing plant extracts with water can be easily conducted, and the byproducts obtained in this series of processes can readily be directly administered as a dietary supplement. Therefore, to develop a potential immunostimulant for Mac. rosenbergii to promote the benefits in aquaculture industry, hot water Mor. citrifolia leaf extract (HMLE) was used to supplement diets of prawns for 9 weeks of a feeding trial, and the immunological responses, immune-related gene expressions, and susceptibility of Mac. rosenbergii to Lactococcus garvieae infection were investigated in the present study.

Section snippets

Process of obtaining the HMLE

Green leaves of Mor. citrifolia were collected from Noni Farm, Pingtung, southern Taiwan. Fresh leaves of Mor. citrifolia were cleaned with tap water and rinsed with distilled water to remove contaminants and debris. After the leaves were chopped into small pieces and dried in an oven at 63 °C for 12 h, the dried material was ground into a powder with a grinder. Known weights of Mor. citrifolia leaf powder of 2, 6, and 10 g were boiled in 200 ml of distilled water (1%, 3%, and 5%) at 100 ± 4 °C

In vitro assessment of the HMLE

PO activity of hemocytes incubated with the HMLE at 140 mg l−1 significantly increased by 150.92%, and meanwhile, no significant difference was observed in those at 20 and 220 mg l−1, compared to the control (Fig. 1A). After incubating with the HMLE at 20 mg l−1, RBs of hemocytes were significantly higher by 133.45% than the control. However, no significant differences were observed among treatments of the control, 140, and 220 mg l−1 HMLE (Fig. 1B). PAs of hemocytes incubated with the HMLE at

Discussion

In a review, Bulfon et al. [23] stated that the solvent used for plant extraction can influence the spectrum of antibacterial and immunomodulatory properties in aquaculture, and in consideration of the effectiveness of extraction, a higher efficiency of extracting secondary bioactive metabolites (polar and non-polar) with antimicrobial and immunostimulant activities using inorganic or organic solvents was mentioned compared to those extracted with water-based methods. The same phenomenon was

Acknowledgements

This research was supported by a grant from the Ministry of Science and Technology (102-2313-B-020 -006 -MY3), Taiwan, ROC.

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