myo-inositol facilitates salinity tolerance by modulating multiple physiological functions in the turbot Scophthalmus maximus
Introduction
Euryhalinity in fish is modulated by diverse compatible organic osmolytes, such as myo-inositol. myo-inositol is synthesized by various animal tissues and commonly exists as a component of phosphatidylinositol in biological cell membranes of animals, where it acts on the reconciling of cellular responses to external stimuli (Mai et al., 2001). Therefore, myo-inositol is not only a growth factor in animals and microorganisms (Majumder and Biswas, 2006) but also mediates many physiological functions involving the regulation of hormonal signalling, protein stabilization, osmoregulation, and nerve transmission (Garcia-Perez and Burg, 1991; Di Paolo and De Camilli, 2006; Falkenburger et al., 2010; Goncalves et al., 2012). The effects of myo-inositol on cell proliferation, differentiation, oxidative status, antioxidant capacity, immunity, growth performance and structural integrity in teleost fish have been studied in detail (Shiau and Su, 2005; Lee et al., 2008; Jiang et al., 2011, Jiang et al., 2013, Jiang et al., 2015; Li et al., 2017).
Myo-inositol is a major intracellular osmolyte that can be accumulated to protect cells from a variety of stresses, including fluctuations in the osmolality of the survival environment (Michell, 2008). When osmotic stress persists for a long period of time, osmolytes that are more compatible than amino acids are required. During prolonged exposure of cells to hypertonicity, cell survival depends on the synthesis either of a compatible osmolyte or of intracellular accumulation of betaine, taurine and myo-inositol (Maurice et al., 1997). Kalujnaia et al. (2010) showed that in freshwater acclimated fish, myo-inositol levels were highest in osmoregulatory organs (gill and kidney), with very low levels in other fish tissues. The increases in myo-inositol concentrations resulting from increases in the intracellular production of the osmolyte could be responsible for the osmotic protection. Sakaguchi et al. (1993) reported that the chondrocytes of eel gill cartilage may function to produce and release inositol into the bronchial circulation, allowing the epithelial cells to take up the osmolyte via active membrane transport systems such as the sodium-dependent myo-inositol transporter. Although the effects of myo-inositol and its functions and production in salinity-stressed fish have been investigated, previous studies have focused on physiology, biochemistry, and individual gene action (Majumder and Biswas, 2006; Kalujnaia et al., 2010; Jiang et al., 2011). Information about the molecular regulatory mechanism of myo-inositol at the transcriptome level is lacking.
As a euryhaline flounder, turbot Scophthalmus maximus has the relatively remarkable ability to adapt to opposing osmotic challenges, which implies the regulation of a wide variety of biological functions (Fouchs et al., 2009). Several observations and studies have been conducted to explore the osmoregulatory mechanism of this species. The effects of salinity changes on seawater-adapted juvenile turbot were studied by examining their plasma osmolarity and ion concentrations, oxygen consumption, gill Na+-K+-ATPase activity, and growth parameters (Gaumet et al., 1995; Imsland et al., 2003). A genome scan for candidate genes induced by an abrupt change in salinity conditions was performed to identify potential adaptive variation in turbot (Vilas et al., 2015). Recently, a transcriptomic analysis was conducted to uncover putative osmoregulatory mechanisms in the kidney of S. maximus exposed to hyposaline seawater (Cui et al., 2019). Although many studies have examined osmotic regulation in turbot, few have focused on its regulation by anabolites, especially myo-inositol.
The goals of this study were to determine the distribution patterns of myo-inositol in turbot S. maximus under salinity stress from the physiological viewpoint, analyse the influence of myo-inositol on turbot survival time, and explore the effect of myo-inositol on the transcriptome. The accuracy of the conclusions was verified by qPCR analysis of functional genes related to steroid reductase and antioxidase, modulation of immune function, inhibition of the cell cycle, energy metabolism, and ion transport, which have been determined to affect the fish via myo-inositol. The results of this study also provide theoretical support for studies of the osmoregulatory mechanism in turbot and will be used to expand the ecological niche of this species and provide practical and technical support for the promotion of turbot culture in abnormal salinity areas.
Section snippets
Ethics statement
All experimental treatments for artificially cultivated fish were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health (Bethesda, MD, USA). The study protocol follows the recommendations of the Experimental Animal Ethics Committee, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, China.
Salinity stress experiment
Healthy juvenile turbot (total length 19.94 ± 1.27 cm) were obtained from Yantai Tianyuan
Characteristics of myo-inositol content under salinity stress
In seawater (salinity 30), the myo-inositol content was higher in the kidney (7.53 ± 0.11 ng g-1) than the gill (6.89 ± 0.29 ng g-1) of turbot, but this difference was not statistically significant. However, the myo-inositol concentration was significantly lower in plasma (4.34 ± 0.32 ng ml-1) than kidney and gill (p < .05). In all treatments, the content of myo-inositol was highest in the gill 5 min after salinity stress, reached the lowest level at 1 h and then increased over time (Fig. 1A).
Response of myo-inositol content to salinity transfer
Inositol, in particular myo-inositol, is an essential vitamin-like nutrient for most aquatic animals (Michael and Koshio, 2008). Many species of fish and their intestinal microbial flora can synthesize myo-inositol de novo in an amount sufficient to support normal physiological functions, such as tilapia (Oreochromis mossambicus) (Sacchi et al., 2014), channel catfish (Ictalurus punctatus) (Burtle and Lovell, 1989), and sunshine bass (Morone chrysops ♀ × Morone saxatilis ♂) (Deng et al., 2002).
Conclusion
In the present study, a significant increase in myo-inositol content was detected in the turbot gill during salinity stress. Additionally, myo-inositol supplementation via immersion or in the diet significantly increased the survival time of turbot under salinity stress. Transcriptome and qPCR data indicated that myo-inositol strengthened osmotic regulation, as represented by the expression of ion-channel genes, by stimulating the activities of steroid reductase and antioxidase, modulating
Availability of supporting data
The raw reads for the next generation sequencing data have been submitted to the NCBI Sequence Read Archive (SRA) under accession number PRJNA527368.
Submission declaration
The authors declare that the work described herein has not been published previously elsewhere in the same form, in English or in any other language.
Author contributions
Wenxiao Cui participated in the experimental design, investigations, data analyses and interpretation, and drafted the manuscript. Aijun Ma and Zhihui Huang participated in the experimental design and coordinated the study. Zhifeng Liu, Kai Yang and Wei Zhang helped to perform the fish exposure experiments and carried out sample preparation.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgments
This work was supported by the Earmarked Fund for Modern Agro-Industry Technology Research System [grant numbers CARS-47-G01]; and the AoShan Talents Cultivation Program supported by Qingdao National Laboratory for Marine Science and Technology [grant numbers 2017ASTCP-OS04]; the National Natural Science Foundation of China [grant numbers 41706168]; the Agricultural Fine Breed Project of Shandong [grant numbers 2016LZGC031]; the Key Research and Development Plan of Nation [grant numbers
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