Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus)
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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Yanduo Wu and Yongshuang Xiao are the co-first authors of the article.