RNA-seq analyses of Marine Medaka (Oryzias melastigma) reveals salinity responsive transcriptomes in the gills and livers

Highlights

• Salinity induced adaptive mechanisms and histochemical responses were evaluated in O. melastigma.

• 1664 genes exhibited expression responses under extreme hypertonic environmental stress in livers.

• 609 genes showed expression responses in gills under extreme hypotonic environmental stress.

• The qRT-PCR analysis validated the results of transcriptomic profiling.

Abstract

Increasing salinity levels in marine and estuarine ecosystems greatly influence developmental, physiological and molecular activities of inhabiting fauna. Marine medaka (Oryzias melastigma), a euryhaline research model, has extraordinary abilities to survive in a wide range of aquatic salinity. To elucidate how marine medaka copes with salinity differences, the responses of Oryzias melastigma after being transferred to different salt concentrations [0 practical salinity units (psu), 15 psu, 30 psu (control), 45 psu] were studied at developmental, histochemical and transcriptome levels in the gill and liver tissues. A greater number of gills differentially expressed genes (DEG) under 0 psu (609) than 15 psu (157) and 45 psu (312), indicating transcriptomic adjustments in gills were more sensitive to the extreme hypotonic environment. A greater number of livers DEGs were observed in 45 psu (1,664) than 0 psu (87) and L15 psu (512), suggesting that liver was more susceptible to hypertonic environment. Further functional analyses of DEGs showed that gills have a more immediate response, mainly in adjusting ion balance, immune and signal transduction. In contrast, DEGs in livers were involved in protein synthesis and processing. We also identified common DEGs in both gill and liver and found they were mostly involved in osmotic regulation of amino sugar and nucleotide sugar metabolism and steroid biosynthesis. Additionally, salinity stresses showed no significant effects on most developmental and histochemical parameters except increased heartbeat with increasing salinity and decreased glycogen after transferred from stable conditions (30 psu) to other salinity environments. These findings suggested that salinity-stress induced changes in gene expressions could reduce the effects on developmental and histochemical parameters. Overall, this study provides a useful resource for understanding the molecular mechanisms of fish responses to salinity stresses.

Introduction

Salinity has been long recognized as one of the critical environmental stresses threatening marine and estuarine organisms worldwide (Solan et al., 2016). In recent years, changes in the salinity levels have changed many molecular and physiological mechanisms of aquatic species, such as reproduction, survival, distribution, osmoregulation, and gene expression patterns (Su et al., 2020; Zhang et al., 2017). The extent of these effects varies according to the intensity of stress and the adaptability of the organism (Chasiotis and Kelly, 2011; McCormick et al., 2013). For example, most stenohaline fish species can only tolerate a limited amount of salinity thus restricting their ability to move between different salinity environments (Kultz, 2015). While euryhaline fish acquired the capability to rapidly adjust, reproduce and move between hypo- and hyper-tonic environments (Kultz, 2015), they were recognized as valuable organisms for studying the salinity-induced molecular and physiological changes. Euryhaline fish species have acquired the ability to adapt to various salinity environments during evolution, contributing to fish evolution and diversity (R et al., 2015). Osmotic regulation is a critical adaptive mechanism commonly found in aquatic organisms living in different salinity environments (Kultz, 2015). Many studies investigated the developmental aspects of salt stress in teleost fish species (Laiz-Carrión et al., 2005; Li et al., 2020). Still, little is known about how salinity fluctuations affect the acquisition of adaptations at the molecular level in euryhaline fish species. Therefore, elucidating the mechanisms underlying the physiological capacity to tolerate osmotic challenges will help to better understand the evolutionary processes associated with habitat alteration in euryhaline species.

The marine medaka is a euryhaline teleost that can survive in a wide range of salinities (Inoue and Takei, 2002). More than 30 species are known in the genus Oryzias, but their tolerance capabilities to different salinity environments are diverse (Matsuda and Sakaizumi, 2016). For example, a study by Inoue and Takei (Inoue and Takei, 2002) showed that Oryzias melastigma and Oryzias javanica were able to survive under different salinities environments. On the contrary, both Oryzias marmoratus and Oryzias latipes died within 2 h and 9.5 h, respectively, after transferred from freshwater to seawater. Likewise, Oryzias marmoratus did not even survive in 50% seawater (all died within 25 h). Moreover, Inoue and Takei also treated the eggs laid by the four species under the same salinity gradients and found that all four fish species could lay eggs and fertilize in freshwater. However, only Oryzias melastigma and Oryzias javanicus can spawn and fertilize in both seawater and freshwater. These critical biological characteristics, as well as the adapting capability to diverse environments make Oryzias melastigma a promising model organism for marine ecotoxicological studies.

The gill is a complex organ composed of cells performing multiple physiological and metabolic functions (Evans et al., 2005). Due to its direct contact with the surrounding environment, the gill plays an essential role in exchanging gasses between water and blood and in osmoregulation, ion regulation, acid-base regulation, and nitrogen waste excretion (Evans et al., 2005). The cell signal sensing system stimulates multiple responses under salinity stresses in gills, such as gill epithelial functional transitions to regulate cell volume and ion transport across the membrane (D. Kültz, 2012; D. Kültz, 2012). Previous studies showed that the primary response mechanisms for maintaining plasma ion and osmolarity homeostasis involve the gill Na⁺/K⁺-ATPase (NKA) (Stewart et al., 2016). The NKA is located primarily in chloride cells and the enzyme generates a chemical gradient that eliminates excess intracellular and extracellular Na⁺ and Cl⁻ in a hypertonic environment and absorbs Cl⁻ in a hypotonic environment (Wood, 2011). In most teleost, changes in the NKA activity correlate with adaptive responses against changes in the salinity levels (Zhang et al., 2019; Teng et al., 2020).

The liver is considered a key source of energy-related metabolic responses under salinity stresses, such as carbohydrates in osmoregulation (Zhang et al., 2017; Tseng and Hwang, 2008). Osmoregulation is one of the most energy-intensive metabolic activities in teleost (Boeuf and Payan, 2001). Therefore, when under hypo- or hyper-tonic conditions, fish require a large amount of energy to maintain osmotic balance (Zhang et al., 2017). Several studies have shown that stress or environmental fluctuations can lead to changes in the blood glucose levels and energy metabolism levels (Huang et al., 2015,; Lin et al., 2011). The metabolic demands for energy during environmental fluctuations are mainly met by many energy-rich compounds, such as carbohydrates, lipids and proteins, which can either be used directly as fuel (respiratory substrate) or stored in the body. The production of these compounds in different tissues is well linked with the expression patterns of different genes, such as apolipoprotein, GLUT family (Zhang et al., 2017; Sun et al., 2020; Chang et al., 2007). Altogether, changes in the salinity levels affect chemical and developmental features, and influence the expression patterns of different genes of aquatic organisms, and these expression patterns even vary among tissues. Therefore, it is necessary to uncover the tissue-specific gene expression response of marine fish.

In this study, developmental and histochemical parameters and RNA-Seq was used to study the responses of O. melastigma under chronic salinity stress to evaluate the osmoregulatory mechanisms in O. melastigma. To our knowledge, this is the first complete salinity responsive transcriptomes study of O. melastigma. It will provide vital information for enriching its genetic resources.