Transcriptional up-regulation of the myo-inositol biosynthesis pathway is enhanced by NFAT5 in hyperosmotically stressed tilapia cells.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyper-osmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (p < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (p<0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO induced activation of the MIB pathway.

Ancient and Recent Hybridization in the Oreochromis Cichlid Fishes.

Cichlid fishes of the genus Oreochromis (tilapia) are among the most important fish for inland capture fisheries and global aquaculture. Deliberate introductions of non-native species for fisheries improvement and accidental escapees from farms have resulted in admixture with indigenous species. Such hybridization may be detrimental to native biodiversity, potentially leading to genomic homogenization of populations and the loss of important genetic material associated with local adaptation. By contrast, introgression may fuel diversification when combined with ecological opportunity, by supplying novel genetic combinations. To date, the role of introgression in the evolutionary history of tilapia has not been explored. Here we studied both ancient and recent hybridization in tilapia, using whole genome resequencing of 575 individuals from 23 species. We focused on Tanzania, a natural hotspot of tilapia diversity, and a country where hybridization between exotic and native species in the natural environment has been previously reported. We reconstruct the first genome-scale phylogeny of the genus and reveal prevalent ancient gene flow across the Oreochromis phylogeny. This has likely resulted in the hybrid speciation of one species, O. chungruruensis. We identify multiple cases of recent hybridization between native and introduced species in the wild, linked to the use of non-native species in both capture fisheries improvement and aquaculture. This has potential implications for both conservation of wild populations and the development of the global tilapia aquaculture industry.

Effects of CRISPR/Cas9 targeting of the myo-inositol biosynthesis pathway on hyper-osmotic tolerance of tilapia cells.

Myo-inositol is an important compatible osmolyte in vertebrates. This osmolyte is produced by the myo-inositol biosynthesis (MIB) pathway composed of myo-inositol phosphate synthase and inositol monophosphatase. These enzymes are among the highest upregulated proteins in tissues and cell cultures from teleost fish exposed to hyperosmotic conditions indicating high importance of this pathway for tolerating this type of stress. CRISPR/Cas9 gene editing of tilapia cells produced knockout lines of MIB enzymes and control genes. Metabolic activity decreased significantly for MIB KO lines in hyperosmotic media. Trends of faster growth of the MIB knockout lines in isosmotic media and faster decline of MIB knockout lines in hyperosmotic media were also observed. These results indicate a decline in metabolic fitness but only moderate effects on cell survival when tilapia cells with disrupted MIB genes are exposed to hyperosmolality. Therefore MIB genes are required for full osmotolerance of tilapia cells.

Cell-based homologous expression system for in-vitro characterization of environmental effects on transmembrane peptide transport in fish.

All organisms encounter environmental changes that lead to physiological adjustments that could drive evolutionary adaptations. The ability to adjust performance in order to cope with environmental changes depends on the organism's physiological plasticity. These adjustments can be reflected in behavioral, physiological, and molecular changes, which interact and affect each other. Deciphering the role of molecular adjustments in physiological changes will help to understand how multiple levels of biological organization are synchronized during adaptations. Transmembrane transporters, which facilitate a cell's interaction with its surroundings, are prime targets for molecular studies of the environmental effects on an organism's physiology. Fish are subjected to environmental fluctuations and exhibit different coping mechanisms. To study the molecular adjustments of fish transporters to their external surrounding, suitable experimental systems must be established. The Mozambique tilapia (Oreochromis mossambicus) is an excellent model for environmental stress studies, due to its extreme salinity tolerance. We established a homologous cellular-based expression system and uptake assay that allowed us to study the effects of environmental conditions on transmembrane transport. We applied our expression system to investigate the effects of environmental conditions on the activity of PepT2, a transmembrane transporter critical in the absorption of dietary peptides and drugs. We created a stable, modified fish cell-line, in which we exogenously expressed the tilapia PepT2, and tested the effects of water temperature and salinity on the uptake of a fluorescent di-peptide, ß-Ala-Lys-AMCA. While temperature affected only Vmax, medium salinity had a bi-directional effect, with significantly reduced Vmax in hyposaline conditions and significantly increased Km in hypersaline conditions. These assays demonstrate the importance of suitable experimental systems for fish ecophysiology studies. Furthermore, our in-vitro results show how the effect of hypersaline conditions on the transporter activity can explain expression shifts seen in the intestine of saltwater-acclimated fish, emphasizing the importance of complimentary studies in better understanding environmental physiology. This research highlights the advantages of using homologous expression systems to study environmental effects encountered by fish, in a relevant cellular context. The presented tools and methods can be adapted to study other transporters in-vitro.

Removal of evolutionarily conserved functional MYC domains in a tilapia cell line using a vector-based CRISPR/Cas9 system.

MYC transcription factors have critical roles in facilitating a variety of cellular functions that have been highly conserved among species during evolution. However, despite circumstantial evidence for an involvement of MYC in animal osmoregulation, mechanistic links between MYC function and osmoregulation are missing. Mozambique tilapia (Oreochromis mossambicus) represents an excellent model system to study these links because it is highly euryhaline and highly tolerant to osmotic (salinity) stress at both the whole organism and cellular levels of biological organization. Here, we utilize an O. mossambicus brain cell line and an optimized vector-based CRISPR/Cas9 system to functionally disrupt MYC in the tilapia genome and to establish causal links between MYC and cell functions, including cellular osmoregulation. A cell isolation and dilution strategy yielded polyclonal myca (a gene encoding MYC) knockout (ko) cell pools with low genetic variability and high gene editing efficiencies (as high as 98.2%). Subsequent isolation and dilution of cells from these pools produced a myca ko cell line harboring a 1-bp deletion that caused a frameshift mutation. This frameshift functionally inactivated the transcriptional regulatory and DNA-binding domains predicted by bioinformatics and structural analyses. Both the polyclonal and monoclonal myca ko cell lines were viable, propagated well in standard medium, and differed from wild-type cells in morphology. As such, they represent a new tool for causally linking myca to cellular osmoregulation and other cell functions.

Different transcriptomic architecture of the gill epithelia in Nile and Mozambique tilapia after salinity challenge.

Tilapiine fishes of the genus Oreochromis vary in their euryhaline capabilities, therefore inhabiting aquatic environments of different salinities across the African continent. We analyzed the differential gene expression in the gills before and after 6 weeks salinity challenge between the highly tolerant Mozambique tilapia (Oreochromis mossambicus) and the less tolerant Nile tilapia (O. niloticus). The pathways triggered by salinity in both tilapia species reveal immune and cell stress responses as well as turnover of ionocytes. Nevertheless, the actual differential expressed genes vary between these two species, pointing at differential transcriptomic architecture, which likely contribute to the species osmoregulation capabilities in elevated salinities.

Genome Editing Using the CRISPR-Cas9 System to Generate a Solid-Red Germline of Nile Tilapia (Oreochromis niloticus).

In recent years, there has been increasing demand for red tilapia, which are commercial strains of hybrids of different tilapiine species or red variants of highly inbred Nile tilapia. However, red tilapia phenotypes are genetically unstable and affected by environmental factors, resulting in nonuniform coloration with black or dark-red color blotches that reduce their market value. Solute carrier family 45 member 2 (SLC45A2) is a membrane transporter that mediates melanin biosynthesis and is evolutionarily conserved from fish to humans. In the present study, we describe the generation of a stable and heritable red tilapia phenotype by inducing loss-of-function mutations in the slc45a2 gene. For this purpose, we identified the slc45a2 gene in Nile tilapia and designed highly specific guide RNAs (gRNA) for its genomic sequence. Multiplex microinjection of slc45a2-specific ribonucleoproteins to Nile tilapia zygotes induced up to 97-99% albinism, including loss of melanin in the eye. Next-generation sequencing of the injected zygotes demonstrated that all injected fish carried mutant alleles with variable mutagenesis efficiencies. Sanger sequencing of the genomic target region in the slc45a2 gene from fin clips, sperm, and F1 offspring of a highly mutant male identified various genomic indels and germline transmission of the sperm-identified indels. Overall, this work demonstrates the generation of somatic and germline slc45a2 mutant alleles, which leads to complete albinism in Nile tilapia.

Nonlinear effects of environmental salinity on the gill transcriptome versus proteome of Oreochromis niloticus modulate epithelial cell turnover.

A data-independent acquisition (DIA) assay library for targeted quantitation of thousands of Oreochromis niloticus gill proteins using a label- and gel-free workflow was generated and used to compare protein and mRNA abundances. This approach generated complimentary rather than redundant data for 1899 unique genes in gills of tilapia acclimated to freshwater and brackish water. Functional enrichment analyses identified mitochondrial energy metabolism, serine protease and immunity-related functions, and cytoskeleton/ extracellular matrix organization as major processes controlled by salinity in O. niloticus gills. Non-linearity in salinity-dependent transcriptome versus proteome regulation was revealed for specific functional groups of genes. The relationship was more linear for other molecular functions/ cellular processes, suggesting that the salinity-dependent regulation of O. niloticus gill function relies on post-transcriptional mechanisms for some functions/ processes more than others. This integrative systems biology approach can be adopted for other tissues and organisms to study cellular dynamics for many changing ecological contexts.

A data-independent acquisition (DIA) assay library for quantitation of environmental effects on the kidney proteome of Oreochromis niloticus.

Interactions of organisms with their environment are complex and environmental regulation at different levels of biological organization is often nonlinear. Therefore, the genotype to phenotype continuum requires study at multiple levels of organization. While studies of transcriptome regulation are now common for many species, quantitative studies of environmental effects on proteomes are needed. Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of thousands of Oreochromis niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. We demonstrate the usefulness of this DIA assay library by discerning environmental effects on the kidney proteome of O. niloticus. Moreover, we demonstrate that the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomic data. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms. The application of functional enrichment analyses using STRING and KEGG to DIA assay data sets is demonstrated by identifying myo-inositol metabolism, antioxidant and xenobiotic functions, and signalling mechanisms as key elements controlled by salinity in tilapia kidneys. The DIA assay library resource presented here can be adopted for other tissues and other organisms to study proteome dynamics during changing ecological contexts.

Antibiotic effect and microbiome persistence vary along the European seabass gut.

The constant increase in aquaculture production has led to extensive use of antibiotics as a means to prevent and treat diseases, with adverse implications on the environment, animal health and commensal microbes. Gut microbes are important for the host proper functioning, thus evaluating such impacts is highly crucial. Examining the antibiotic impact on gut segments with different physiological roles may provide insight into their effects on these microhabitats. Hence, we evaluated the effect of feed-administrated antibiotics on the composition and metabolic potential of the gut microbiome in the European seabass, an economically important aquaculture species. We used quantitative PCR to measure bacterial copy numbers, and amplicon sequencing of the 16S rRNA gene to describe the composition along the gut, after 7-days administration of two broad-range antibiotic mixtures at two concentrations. While positive correlation was found between antibiotic concentration and bacterial abundance, we showed a differential effect of antibiotics on the composition along the gut, highlighting distinct impacts on these microbial niches. Moreover, we found an increase in abundance of predicted pathways related to antibiotic-resistance. Overall, we show that a high portion of the European seabass gut microbiome persisted, despite the examined antibiotic intake, indicating high stability to perturbations.

Corrigendum: Salinity-Dependent Shift in the Localization of Three Peptide Transporters along the Intestine of the Mozambique Tilapia (Oreochromis mossambicus).

[This corrects the article DOI: 10.3389/fphys.2017.00008.].

Core gut microbial communities are maintained by beneficial interactions and strain variability in fish.

The term core microbiome describes microbes that are consistently present in a particular habitat. If the conditions in that habitat are highly variable, core microbes may also be considered to be ecological generalists. However, little is known about whether metabolic competition and microbial interactions influence the ability of some microbes to persist in the core microbiome while others cannot. We investigated microbial communities at three sites in the guts of European seabass under four dietary conditions. We identified generalist core microbial populations in each gut site that are shared across fish, present under multiple diets and persistent over time. We found that core microbes tend to show synergistic growth in co-culture, and low levels of predicted and validated metabolic competition. Within core microbial species, we found high levels of intraspecific variability and strain-specific habitat specialization. Thus, both intraspecific variability and interspecific facilitation may contribute to the ecological stability of the animal core microbiome.

Peptide Transporters in the Primary Gastrointestinal Tract of Pre-Feeding Mozambique Tilapia Larva.

Fish larvae differ greatly from the adult form in their morphology and organ functionality. The functionality of the gastrointestinal tract depends on the expression of various pumps, transporters, and channels responsible for feed digestion and nutrients absorption. During the larval period, the gastrointestinal tract develops from a simple closed tube, into its complex form with differentiated segments, crypts and villi, as found in the adult. In this study, we characterized the expression of three peptide transporters (PepT1a, PepT1b, and PepT2) in the gastrointestinal tract of Mozambique tilapia (Oreochromis mossambicus) larvae along 12 days of development, from pre-hatching to the completion of yolk sac absorption. Gene expression analysis revealed differential and complimentary time-dependent expression of the PepT1 variants and PepT2 along the larval development period. Immunofluorescence analysis showed differential protein localization of the three peptide transporters (PepTs) along the gastrointestinal tract, in a similar pattern to the adult. In addition, PepT1a was localized in mucosal cells in the larvae esophagus, in much higher abundance than in the adults. The results of this study demonstrate specialization of intestinal sections and absorbance potential of the enterocytes prior to the onset of active exogenous feeding, thus pointing to an uncharacterized function and role of the gastrointestinal tract and its transporters during the larval period.

Transcriptome Analysis Reveals Common and Differential Response to Low Temperature Exposure Between Tolerant and Sensitive Blue Tilapia (Oreochromis aureus).

Tilapias are very important to the world's aquaculture. As befitting fish of their tropical origin, their distribution, and culture practices are highly affected by low temperatures. In this study, we used genetic and genomic methodologies to reveal pathways involved in the response and tolerance of blue tilapia (Oreochromis aureus) to low temperature stress. Cold tolerance was characterized in 66 families of blue tilapia. Fish from cold-tolerant and cold-sensitive families were sampled at 24 and 12°C, and the transcriptional responses to low-temperature exposure were measured in the gills and liver by high-throughput mRNA sequencing. Four hundred and ninety four genes displayed a similar temperature-dependent expression in both tolerant and sensitive fish and in the two tissues, representing the core molecular response to low temperature exposure. KEGG pathway analysis of these genes revealed down-regulation of focal-adhesion and other cell-extracellular matrix (ECM) interactions, and up-regulation of proteasome and various intra-cellular proteolytic activities. Differential responses between cold-tolerant and cold-sensitive fish were found with genes and pathways that were up-regulated in one group and down-regulated in the other. This reverse response was characterized by genes involved in metabolic pathways such as glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, with low temperature down-regulation in tolerant fish and up-regulation in sensitive fish.

Short- and long-term low-salinity acclimation effects on the branchial and intestinal gene expression in the European seabass (Dicentrarchus labrax).

The European seabass (Dicentrarchus labrax) is a teleost remarkably adapted to a wide range of water salinity, through osmoregulatory mechanisms, mainly operating in the gills and the intestine. As an important aquaculture species, its rearing in low-salinity conditions offers benefits for its inland culture. However, this demands a full comprehension of the European seabass osmoregulatory mechanisms and its response to acclimation protocols. The purpose of this study was to evaluate different acclimation protocols in terms of osmoregularity and stress response, following transferring of European seabass juveniles from seawater to freshwater. In addition, nutrient absorption was also examined since drinking rates are sensitive to salinity. The acclimation challenge was applied through three protocols: direct transfer (0 h) to freshwater, gradual transfer during 3 h and during 72 h. The short- (1 h after complete change to freshwater) and long-term effects (after 2 months) of each acclimation protocol were evaluated by assessing the expression of 1. The osmoregulatory genes: Na+/K+-ATPase α1, Na+/K+/2Cl- 1 co-transporter, aquaporins 1 and 3, and the cystic fibrosis transmembrane conductance regulator; 2. The heat shock protein 70 gene; 3. The peptide transporter genes corresponding to PepT1a, PepT1b and PepT2. The short-term acclimation response was pronounced in both gills and the intestine affecting stress-, osmoregulatory- and nutrient-related gene expression. Long-term effects were only evident in the intestine. Direct transfer in freshwater mainly induced a long-term stress response, while the short-term effect was more pronounced in the 3 h-transfer, potentially due to handling. Our results suggest that although the European seabass can withstand direct transfer to low-salinity conditions, a gradual transfer is recommended to prevent long-term stress effects.

Host genetic selection for cold tolerance shapes microbiome composition and modulates its response to temperature.

The hologenome concept proposes that microbes and their host organism are an independent unit of selection. Motivated by this concept, we hypothesized that thermal acclimation in poikilothermic organisms, owing to their inability to maintain their body temperature, is connected to their microbiome composition. To test this hypothesis, we used a unique experimental setup with a transgenerational selective breeding scheme for cold tolerance in tropical tilapias. We tested the effects of the selection on the gut microbiome and on host transcriptomic response. Interestingly, we found that host genetic selection for thermal tolerance shapes the microbiome composition and its response to cold. The microbiomes of cold-resistant fish showed higher resilience to temperature changes, indicating that the microbiome is shaped by its host's selection. These findings are consistent with the hologenome concept and highlight the connection between the host and its microbiome's response to the environment.

Dietary salt levels affect digestibility, intestinal gene expression, and the microbiome, in Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is the world's most widely cultured fish species. Therefore, its nutritional physiology is of great interest from an aquaculture perspective. Studies conducted on several fish species, including tilapia, demonstrated the beneficial effects of dietary salt supplementation on growth; however, the mechanism behind these beneficial effects is still not fully understood. The fish intestine is a complex system, with functions, such as nutrient absorption, ion equilibrium and acid-base balance that are tightly linked and dependent on each other's activities and products. Ions are the driving force in the absorption of feed components through pumps, transporters and protein channels. In this study, we examined the impact of 5% increase in dietary NaCl on protein, lipid, ash and dry matter digestibility, as well as on the expression of intestinal peptide transporters (PepTs) and ion pumps (Na+/K+-ATPase, V-H+-ATPase, N+/H+-Exchanger) in Nile tilapia. In addition, effects on the gut microbiome were evaluated. Our results show that dietary salt supplementation significantly increased digestibility of all measured nutritional components, peptide transporters expression and ion pumps activity. Moreover, changes in the gut microbial diversity were observed, and were associated with lipid digestibility and Na+/K+-ATPase expression.

Effects of the acclimation to high salinity on intestinal ion and peptide transporters in two tilapia species that differ in their salinity tolerance.

Tilapiine species, widely distributed across habitats with diverse water salinities, are important to aquaculture as well as a laboratory model. The effects of water salinity on two tilapia species, that differ in their salinity tolerance, was evaluated. Oreochromis niloticus reared in brackish-water, showed a significant decrease in growth and feed efficiency, whereas O. mossambicus reared in seawater did not show any significant changes. The expression and activity of Na+/K+-ATPase (NKA), V-type H+-ATPase (VHA) and carbonic anhydrase (CA), as well as expression levels of genes encoding two HCO3- and three peptide transporters (nbc1, slc26a6, slc15a1a, slc15a1b and slc15a2) were measured in three intestinal sections of these two species, grown in freshwater and brackish/sea-water. Overall, the spatial distribution along the intestine of the genes examined in this study was similar between the two species, with the exception of tcaIV. The salinity response, on the other hand, varied greatly between these species. In O. mossambicus, there was a salinity-dependent increased expression of most of the examined genes (except slc26a6 and slc15a2), while in O. niloticus the expression of most genes did not change, or even decreased (tcaIV, nbc1 and slc15a1b). This study highlighted differences in the intestinal response to salinity acclimation between closely- related species that differ in their salinity tolerance. O. mossambicus, which has a high salinity tolerance, showed expression patterns and responses similar to marine species, and differed from the low-salinity-tolerance O. niloticus, which showed a response that differed from the accepted models, that are based on marine and diadromous fishes.

Salinity-Dependent Shift in the Localization of Three Peptide Transporters along the Intestine of the Mozambique Tilapia (Oreochromis mossambicus).

The peptide transporter (PepT) systems are well-known for their importance to protein absorption in all vertebrate species. These symporters use H+ gradient at the apical membrane of the intestinal epithelial cells to mediate the absorption of small peptides. In fish, the intestine is a multifunctional organ, involved in osmoregulation, acid-base regulation, and nutrient absorption. Therefore, we expected environmental stimuli to affect peptide absorption. We examined the effect of three environmental factors; salinity, pH and feeding, on the expression, activity and localization of three PepT transporters (PepT1a, PepT1b, PepT2) along the intestine of the Mozambique tilapia (Oreochromis mossambicus). Quantitative real time PCR (qPCR) analysis demonstrated that the two PepT1 variants are typical to the proximal intestinal section while PepT2 is typical to the distal intestinal sections. Immunofluorescence analysis with custom-made antibodies supported the qPCR results, localized both transporters on the apical membrane of enterocytes and provided the first evidence for the participation of PepT2 in nutrient absorption. This first description of segment-specific expression and localization points to a complementary role of the different peptide transporters, corresponding to the changes in nutrient availability along the intestine. Both gene expression and absorption activity assays showed that an increase in water salinity shifted the localization of the PepT genes transcription and activity down along the intestinal tract. Additionally, an unexpected pH effect was found on the absorption of small peptides, with increased activity at higher pH levels. This work emphasizes the relationships between different functions of the fish intestine and how they are affected by environmental conditions.

Intestinal transcriptome analysis revealed differential salinity adaptation between two tilapiine species.

Tilapias are a group of freshwater species, which vary in their ability to adapt to high salinity water. Osmotic regulation in fish is conducted mainly in the gills, kidney, and gastrointestinal tract (GIT). The mechanisms involved in ion and water transport through the GIT is not well-characterized, with only a few described complexes. Comparing the transcriptome of the anterior and posterior intestinal sections of a freshwater and saltwater adapted fish by deep-sequencing, we examined the salinity adaptation of two tilapia species: the high salinity-tolerant Oreochromis mossambicus (Mozambique tilapia), and the less salinity-tolerant Oreochromis niloticus (Nile tilapia). This comparative analysis revealed high similarity in gene expression response to salinity change between species in the posterior intestine and large differences in the anterior intestine. Furthermore, in the anterior intestine 68 genes were saltwater up-regulated in one species and down-regulated in the other species (47 genes up-regulated in O. niloticus and down-regulated in O. mossambicus, with 21 genes showing the reverse pattern). Gene ontology (GO) analysis showed a high proportion of transporter and ion channel function among these genes. The results of this study point to a group of genes that differed in their salinity-dependent regulation pattern in the anterior intestine as potentially having a role in the differential salinity tolerance of these two closely related species.