Comparison of Calcium Balancing Strategies During Hypothermic Acclimation of Tilapia (Oreochromis mossambicus) and Goldfish (Carassius auratus)

Abstract

The body temperatures of teleost species fluctuate following changes in the aquatic environment. As such, decreased water temperature lowers the rates of biochemical reactions and affects many physiological processes, including active transport-dependent ion absorption. Previous studies have focused on the impacts of low temperature on the plasma ion concentrations or membrane transporters in fishes. However, very few in vivo or organism-level studies have been performed to more thoroughly elucidate the process of acclimation to low temperatures. In the present study, we compared the strategies for cold acclimation between stenothermic tilapia and eurythermic goldfish. Whole-body calcium content was more prominently diminished in tilapia than in goldfish after long-term cold exposure. This difference can be attributed to alterations in the transportation parameters for Ca²⁺ influx, i.e., maximum velocity (V_max ) and binding affinity (1/K_m ). There was also a significant difference in the regulation of Ca²⁺ efflux between the two fishes. Transcript levels for Ca²⁺ related transporters, including the Na⁺/Ca²⁺ exchanger and epithelial Ca²⁺ channel, were similarly regulated in both fishes. However, upregulation of plasma membrane Ca²⁺ATPase expression was more pronounced in goldfish than in tilapia. In addition, enhanced Na⁺/K⁺-ATPase abundance, which provides the major driving force for ion absorption, was only detected in tilapia, while upregulated Na⁺/K⁺-ATPase activity was only detected in goldfish. Based on the results of the present study, we have found that goldfish and tilapia differentially regulate gill epithelial plasma membrane Ca²⁺-ATPase (PMCA) expression and Na⁺/K⁺-ATPase activity in response to cold environments. These regulatory differences are potentially linked to more effective regulation of Ca²⁺ influx kinetics and better maintenance of whole body calcium content in goldfish than in tilapia.

Keywords: Ca2+ influx; Ca2+-ATPase; cold acclimation; gill; teleost.

FIGURE 1

Comparison of whole-body Ca²⁺ flux rates in (A) goldfish and (B) tilapia after short term (25°C→15°C) or long-term (15°C) cold acclimation. One-way ANOVA, Tukey’s multiple-comparison. ^ABp < 0.05 compared to short- and long-term acclimation Ca²⁺ influx, ^abp < 0.05 compared to short- and long-term Ca²⁺ net flux and ^∗p < 0.05 compared to short- and long-term Ca²⁺ efflux (n = 8) are significant different.

FIGURE 2

Comparison of Ca²⁺ influx kinetics after 2 weeks cold (15°C) acclimation or control (25°C) for (A) goldfish and (B) tilapia. Calcium influx was measured in tracer media over a range of calcium concentrations. The mean (closed squares for 15°C acclimated and open circles for 25°C acclimated) and deviation (bar) were obtained from four fish for each point (n = 4).

FIGURE 3

Effect of cold acclimation on the expression of Na⁺/K⁺-ATPase in tilapia or goldfish gill. (A) The abundance of Na⁺/K⁺-ATPase was determined by western blotting analysis. Different numbers (25 and 15) indicated acclimation temperature (°C). The immunoblot for actin served as an internal control. (B) The quantification of the Na⁺/K⁺-ATPase expression. The Na⁺/K⁺-ATPase expression was normalized to β-actin and was also relative to 25°C acclimated group (set as 1). Values shown are mean ± S.D. ^∗Significant difference between 15°C and 25°C acclimated groups in the same species (Student’s t-test, p ≤ 0.05, n = 4).

FIGURE 4

Effect of cold acclimation on the Na⁺/K⁺-ATPase activity in tilapia or goldfish gill. Na⁺/K⁺-ATPase activity was determined in 15°C and 25°C acclimated (A) goldfish or (B) tilapia at three reaction temperatures (15, 25, and 35°C). Asterisks indicate significant differences between the two acclimation temperature groups with Student’s t-test, p < 0.05, n = 4.

FIGURE 5

Effect of cold acclimation on the expression of calcium transporters in tilapia or goldfish gill. Relative mRNA expression of Na⁺/Ca²⁺ exchanger 1b (NCX1b), epithelial Ca²⁺ channel (ECaC), plasma membrane Ca²⁺ ATPase (PMCA) in (A) goldfish or (B) tilapia gill were measured by real-time quantitative PCR. The expression of 18S ribosomal RNA (18S rRNA) was used as an internal control for goldfish and ribosomal protein L7 (RPL7) was used for tilapia. Student’s t-test, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01 compared to 15°C acclimated fish. n = 6 per group.

FIGURE 6

Effect of cold acclimation on the Na⁺/K⁺-ATPase and PMCA-expressing ionocytes. Frozen sections of 25°C (A, first row) and 15°C (A, second row) acclimated tilapia, and 25°C (A, third row) and 15°C (A, fourth row) acclimated goldfish gills were stained with Na⁺/K⁺-ATPase (NKA) antibody and hybridized with PMCA RNA probes. Inset shows an enlarged picture of three co-stained cells. The numbers of PMCA only cells (arrow) or the double-stained (NKA/PMCA) cells (arrowhead) were averaged from three fish for each treatment (n = 3) (B). The cell numbers of three sections from different gill filaments of each fish are expressed as mean ± S.D. The means of control temperature (25°C) and cold-acclimated (15°C) groups from the same species were compared by Students’ t-test, ^∗p ≤ 0.05. Scale bar, 20 μm.