Intestinal ion regulation exhibits a daily rhythm in Gymnocypris przewalskii exposed to high saline and alkaline water

Experimental animals

Gymnocypris przewalskii used in this study were obtained from the Rescue Center for Naked Carp of Lake Qinghai in Xining. Only healthy fish without visible body damage were used. Wet mass and body length were recorded before the fish were sampled. All fish were collected under permits issued by local and national authorities, and experimental procedures were in accordance with national animal care regulations. Experimental waters were prepared daily, and water qualities were measured before each experiment. The water temperature and salinity were measured using an YSI6600 multiprobe sensor (YSI Incorporated, Ohio, USA), and the carbonate alkalinity was determined by titration³⁹. All fish (average body weight: 33.21 ± 2.74 g; average body length:14.81 ± 0.35 cm) were held in an indoor RAS system at a density of approximately 6.5 kg m⁻³. The holding and experimental water were filtered tap water (Canature/AC/KDF150-1–300) (salinity 0.16, pH 7.56, carbonate alkalinity 2.7 mmol L⁻¹, temperature 17.1 ± 0.61 °C). Fish were fed daily with commercial feed. Fish husbandry and experimental procedures were approved by the Second Scientific Research Ethics Committee of East China Sea Fisheries Research Institute.

Experimental design

Fish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx; 19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. To examine the effect of rhythm on osmoregulation and acid–base balance, this study measured four endpoints: drinking rate, self-feeding intake, mRNA expression and the single cell expression level of osmoregulation and acid–base regulation relevant proteins. Fish held in filtered tap water were transferred directly to saline-alkaline lake water with salinities of 15 (L15, salinity 14.83, pH 8.65, carbonate alkalinity 30.54 mmol L⁻¹) and 17 (L17, salinity 16.80, pH 9.02, carbonate alkalinity 34.61 mmol L⁻¹), which was prepared by adding the same ratio of NaCl, MgCl₂.6H₂O, Na₂SO_4, CaCl₂, KCl, NaHCO₃ and Na₂CO₃ as in Qinghai Lake. The experimental period was 4–5 days.

Drinking rate during high saline-alkaline transfer

Fish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, feeding was stopped 48 h prior to the experiments. Fresh water fish were transferred directly to L15 and L17 PEG-4000 free water for up to 4 days. For drinking rate analysis, new tanks were prepared which contained 50L saline-alkaline lake water with salinity 15 or 17 and with PEG-4000 (final concentration was 1.00 g L⁻¹) during the day (10:00–16:00) and night (4:00–22:00) on the fourth day respectively. Nine fish per treatment were individually transferred from PEG-4000 free water to each tank which contained 1.00 g L⁻¹ PEG-4000 at 10:00 or 4:00. Water samples were collected at 15 min after the fish were transferred to each treatment group for the determination of PEG-4000 concentration. The fish were terminally anesthetized with MS-222 (0.40 g L⁻¹) after 6 h. The intestines were then quickly dissected out from nine individual fish per treatment group and the intestinal fluid were collected and stored at 4 °C. All fish were weighed before sampling.

Self-feeding intake during high saline-alkaline transfer

Fish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, fish were kept in freshwater (FW) or acclimated to L15 for more than 15 days before the experiment started. Six RAS glass tanks (95 cm × 60 cm × 60 cm), which belong to two circulatory systems (3 tanks for FW and 3 for L15), were used for self-feeding experiment. Each tank had 15 individuals.

Before the experiment, fish were trained by a custom-made self-feeding system (Fig. S2). Trained fish triggered the self-feeder when they want to feed. In the self-feeding system, the photoelectric sensor converts the change of optical signal into the change of electrical signal, and the feeder release feed by recognizing level fluctuation.

During the formal experiment, we collected feed data at 5:00 and 19:00, which were the time points of the light and dark transition. Feed intakes of naked carp were calculated by weighing the feed quantities at two time points. The experiment lasted 5 days.

mRNA expression of osmoregulation and acid–base regulation relevant proteins during high saline-alkaline transfer

Single cell positive rate of osmoregulation and acid–base regulation relevant proteins

Fish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, feeding was stopped 48 h prior to the experiments. To analyze the single cell positive rate of acid–base relevant proteins, a separate experiment was conducted. Fresh water fish were transferred directly to L17 for up to 4 days. There were 3 tanks (6 fish per tank) in this experimental group. At the fourth day, three fish per tank were individually removed and terminally anesthetized with MS-222 (0.40 g L⁻¹) at 16:00 and 22:00, respectively. The mid-intestine was quickly dissected out from nine individual fish and immediately prepared for single-cell suspensions.

Analytical techniques

Drinking rate analysis

The measurement of drinking rate was performed according to the study of Buxton et al.⁴⁰. After weighing the collected intestinal fluid, it was centrifuged at 13,000g for 1 min, and 50 μL of the supernatant was taken, added dropwise to 350 μL of 72% pre-cooled (4 °C) acetone, and vortexed to mix. Samples were then centrifuged at 2000g for 10 min at 4 °C, the supernatant filtered with 0.45 μm filter paper, followed by addition of 100 μL of filtrate to 175 μL 25 mg L⁻¹ gum arabic and vortexed to mix. Finally, 200 μL of TCA-CaCl₂ (trichloroacetic acid-calcium chloride, 30% and 5% by mass) was added to the mixture and the reaction allowed to proceed at room temperature for 20 min. An Epoch microplate (Bio Tek) spectrophotometry unit was used to measure the absorbance at 650 nm. The remaining solution was weighed again after drying at 60 °C for 48 h, and the volume of intestinal fluid was determined (quantity of collected intestinal fluid-mass after drying). The same method as above was used to process the standard solution. Solute concentrations for standard curve were prepared as 0.00 g L⁻¹, 0.10 g L⁻¹, 0.20 g L⁻¹, 0.40 g L⁻¹, 0.60 g/L⁻¹, 0.80 g L^–1, 1 g L⁻¹, and 2 g L⁻¹ PEG-4000. The PEG-4000 concentration of intestinal fluid was calculated based on the standard curve. Drinking rate (μLg^-1h^-1) = 1000 × (C_I × V_I)/(C_W × W × t), where C_I is the concentration of PEG-4000 in the intestinal fluid (gL^-1), V_I is the volume of intestinal fluid (mL), C_W is the concentration of PEG-4000 in experimental water (gL^-1),W is the body weight of the fish (g), t is the duration of the experiment (h).

Molecular biology

The known sequences of the NKA-α gene of naked carp were compared with the corresponding genes of other species in GenBank, and highly conserved regions were selected for primer design (Table 1). The reference gene EF1α was used according to Yao et al.³. Previously published primers were used for SLC26A6 and SLC4A4 genes⁶. After extracting total RNA with Trizol (Invitrogen), the integrity of RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of total RNA were determined by a Bio Tek Epoch microplate spectrophotometer. The Rever Tra Ace-α (TOYOBO) kit was used to reverse transcribe mRNA to cDNA. Fluorescence quantitative PCR analysis was performed using a QuantStudio™ Real-Time PCR (Thermo life) with the SYBR Premix Ex TaqIII (TaKaRa) kit: total reaction volume of 10 μL, including 5 μL SYBR Premix Ex Taq, 2 μL upstream primers, 2 μL downstream Primers, and 1 μL cDNA template. The amplification procedure was as follows: 95 °C 30 s, 1 cycle; 95 °C 5 s, 60 °C 20 s, 40 cycles. Three replicates were included for each sample, with EF1α as the internal reference gene. The relative expression of each gene was calculated using the 2^−ΔΔCt method⁴¹. Melting curve analysis was performed following each reaction to confirm that there was only a single product and no primer-dimer artifacts. In addition, representative samples were electrophoresed to verify that only a single product was present. Negative control reactions were performed for representative samples using RNA that had not been reverse transcribed to control for the possible presence of genomic DNA contamination. No-template control reactions were also performed to verify the absence of contaminating DNA or primer-dimer amplification in the reactions.

Table 1 Nucleotide sequences of the primers used for amplification.

Full size table

Single cell staining analysis

The naked carp mid-intestine was isolated and transferred to HBSS on ice. The mid-intestine was washed by HBSS (Corning, 21-022-CV) and transferred to pre-warmed digestion medium containing 0.2 mg·mL⁻¹ Collagenase I (Gibco, 17100-017), 0.06 mg mL⁻¹ Collagenase II (Gibco, 17101-015) and 0.2 mg mL⁻¹ Collagenase IV (Gibco, 17104-019), which was shaken vigorously for 30 s and further incubated at 37 °C for about 30 min in incubator with gentle shaking every 5 min to release cells. Cells were then collected by centrifuging at 300 × g for 5 min, and resuspended in D-PBS (BBI, E607009-0500). Then taken an appropriate amount of single cell suspension and dropped it on poly-L-lysine-coated slides where the experimental area was drawn with a hydrophobic marker to allow the single cells to settle freely. When the cell sedimentation density was moderate, aspirated the excess cell suspension, slides were fixed with 4% paraformaldehyde fix solution (BBI, E672002-0500) for 10 min, and blocked with 3% BSA (Sigma, B2064) for 1 h, three washes in D-PBS. Subsequently, slides were incubated in NKA-α or SLC26A6 (antibody dilution ratio was 1:100) for overnight at 4 °C. The NKA-α antibody was a commercial polyclonal rabbit Na⁺/K⁺-ATPase α antibody (Santa Cruz Biotechnology, sc-28800). The SLC26A6 antibody was a commercial polyclonal rabbit SLC26A6 antibody (Abcam, ab-172684). After the incubation, three washes in D-PBS. The secondary antibodies consisted of Alexa flour 568 goat anti-rabbit IgG (Thermo Fisher Scientific, A11036) (antibody dilution ratio was 1:400). Slides were incubated in room temperature for 1 h, followed by three washes in D-PBS. Finally, incubate with Hochest for 30 min. Cells were then photographed with a fluorescence microscope. For every fish, positive protein expression was counted using at least three pictures. Image J was used to analyze the fluorescence intensity and record the positivity rate.

Statistical analysis

The data was expressed as mean ± standard error (SE). Two-way ANOVA and One-way ANOVA with LSD multiple comparison were employed to compare drinking rate, food intake and relative gene expression among different treatments and time courses respectively. Differences in single cell positive rate between 16:00 and 22:00 in L17 were evaluated by chi-square test. Assumptions for all parametric models (normality and equal residuals) were assessed via diagnostic plots. Means were considered significantly different when P < 0.05. All statistical analyses were conducted with SPSS 11. The bar charts were created using Origin 8.6.

Source: Ecology - nature.com