Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus)

https://doi.org/10.1016/j.fsi.2023.108817Get rights and content

Highlights

  • To investigate whether the addition of immune enhancers to bait can stimulate the immunity of the spotted knifejaw.

  • Immune enhancers can significantly improve the growth performance.

  • Immune enhancers can significantly stimulate the immune response ability.

  • Different immune enhancers stimulate different TLR genes immune responses.

  • Point out the direction for the healthy development of breeding industry.

Abstract

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

Introduction

The spotted knifejaw (Oplegnathus punctatus) is a new explored species in China's marine fish farming industry with high commercial value (35 dollar/kg), ornamental beauty, and notable ecological benefits [[1], [2], [3]]. However, frequent disease outbreaks pose a challenge to the sustainable development of spotted knifejaw breeding and cause significant losses for farmers [4]. To promote the healthy breeding of this species and advance the aquaculture industry, it is crucial to investigate the effects of exogenous immune enhancers on immune regulation in spotted knifejaw.

Immune enhancers are substances that stimulate the host defense response and enhance an organism's ability to resist disease [5]. In recent years, the addition of immune enhancers to baits has been partially reported. A study on Wuchang bream (Megalobrama amblycephala) found that adding 0.5 mg/kg selenium yeast or tea polyphenols (TP) to the basic diet effectively promoted growth, inhibited the rise of serum cortisol levels, and improved the activity of antioxidant enzymes [6]. Astaxanthin (AS) is widely used as a feed additive and dietary supplement in the aquaculture industry due to its positive impact on fish coloration, immune system enhancement, growth performance, and survival [7,8]. Melittin (ME), a transparent liquid secreted by the venom glands and accessory glands of bee workers, is the primary bioactive substance in bee venom [9]. Adding melittin to animal diets has been shown to enhance production performance, immunity, and antioxidant capacity, effectively preventing diseases [[10], [11], [12]]. Nevertheless, little research has been conducted on the role of melittins in fish immunity.

As one of the most crucial pattern recognition receptors (PRRs), Toll-like receptors (TLR) are ancient and highly conserved innate PRRs in the immune system. They play a crucial role in recognizing pathogen-associated microbial patterns (PAMPs) and danger-associated molecular patterns (DAMPs) [13]. In teleost fish, 21 tlrs genes, including tlr1-5, 5s, tlr7-9, tlr13, tlr14, tlr18-23, and tlr25-28, have been identified. Notably, the tlr4 gene is absent in most teleost fish, and teleost-specific tlrs are tlr5s, 18–20, 23, tlr25-28. However, the tlr gene family has significantly contracted in O. punctatus according to recent research[14,15], with only five tlr genes (namely tlr1, tlr2, tlr5, tlr14, and tlr23) left for the species. It remains unclear whether the contraction of the associated immune gene family is related to poor immunity in O. punctatus, and further research on the immune response between the tlr gene and immune enhancers is necessary to explore this topic thoroughly.

In this work, we fed O. punctatus with three different types of immune enhancers (tea polyphenols, astaxanthin, and melittin) at different levels (0, 200, 400, 600 and 800 mg/kg) in the basic feed to study the immune response of tlr gene and the effects of growth in O. punctatus. Our work will lay a solid foundation for subsequent immune research of O. punctatus.

Section snippets

Fish and experimental preparation

The breeding experiment was conducted at Laizhou Mingbo Aquatic Products Co., Ltd. (Yantai, Shandong Province). We selected a batch of juvenile fish with identical health, body shape, and specifications, and temporarily raised them in the breeding workshop. A total of 351 juvenile fish were randomly assigned to four groups and placed into experimental ponds with a volume of 0.2 m3. The juvenile fish had an average body weight of 36.02 ± 6.57 g and an average body length of 8.99 ± 0.82 cm. The

Effect of different immune enhancers on the mRNA expression level of tlr genes

The expression of different tlr genes in O. punctatus tissues demonstrated significant differences (p < 0.05). The TLR1 family of O. punctatus consists of tlr1, tlr2, and tlr14, which demonstrates the expression pattern of each of these three genes after the addition of immune enhancers. tlr1 gene was mainly expressed in immune tissues, such as the spleen, and head kidney; In the TP group, tlr1 gene expression was lowest in the spleen when the additive dose was 400 mg/kg, and the rest of the

Discussion

A large number of studies have shown that adding different levels of TP and AS to feed can significantly improve the immune capacity of organisms [18,19]. Fish rely on their innate immune system for effective defense against invading microbial pathogens, and Toll-like receptors (TLR), an important protein molecule in innate immunity, trigger an immune response due to pathogen invasion [20]. Recently, our team conducted experiments to verify the relationship between tlr genes and immune

Conclusion

In conclusion, our results have demonstrated that adding varying doses of TP, AS, and ME to the diets of O. punctatus promotes growth performance and induces an immune response. The most effective dosage was 400 mg/kg for TP, and 200 mg/kg for both AS and ME. TP, AS, and ME act as immunostimulants, and tlr genes are stimulated differently by each immune enhancer. tlr1, tlr14 and tlr23 respond to TP, tlr14 responds to AS, and all tlr genes, except for tlr5, are stimulated by ME. The best

Financial support

This work was supported by grants from the National Key Research and Development Program (2022YFC3103600, 2022YFC3102004), National Natural Science Foundation of China (No. 42276107); CARs for Marine Fish Culture Industry (CARS-47), Key Deployment Projects of Center for Ocean Mega-Science, Chinese Academy of Sciences (Frontier Cross-category, COMS2020Q05).

CRediT authorship contribution statement

Yanduo Wu: Conceptualization, performed the experiments, Formal analysis, Writing – original draft. Yongshuang Xiao: Conceptualization, Formal analysis, Writing – original draft, Supervision. Wensheng Li: performed the experiments. Chuanjun Yang: performed the experiments. Wenhui Ma: performed the experiments. Zunfang Pang: performed the experiments. Jiawei Zhang: performed the experiments. Zhizhong Xiao: Conceptualization, Provision of experimental materials, Writing – review & editing. Jun Li:

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgments

We would like to thank Laizhou Mingbo Aquatic Products Co., Ltd. The company for providing materials for conducting this study. We also like to thank all the fish who participated in this study.

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