Short-Term Exposure of Mytilus coruscus to Decreased pH and Salinity Change Impacts Immune Parameters of Their Haemocytes

Abstract

With the release of large amounts of CO₂, ocean acidification is intensifying and affecting aquatic organisms. In addition, salinity also plays an important role for marine organisms and fluctuates greatly in estuarine and coastal ecosystem, where ocean acidification frequently occurs. In present study, flow cytometry was used to investigate immune parameters of haemocytes in the thick shell mussel Mytilus coruscus exposed to different salinities (15, 25, and 35‰) and two pH levels (7.3 and 8.1). A 7-day in vivo and a 5-h in vitro experiments were performed. In both experiments, low pH had significant effects on all tested immune parameters. When exposed to decreased pH, total haemocyte count (THC), phagocytosis (Pha), esterase (Est), and lysosomal content (Lyso) were significantly decreased, whereas haemocyte mortality (HM) and reactive oxygen species (ROS) were increased. High salinity had no significant effects on the immune parameters of haemocytes as compared with low salinity. However, an interaction between pH and salinity was observed in both experiments for most tested haemocyte parameters. This study showed that high salinity, low salinity and low pH have negative and interactive effects on haemocytes of mussels. As a consequence, it can be expected that the combined effect of low pH and changed salinity will have more severe effects on mussel health than predicted by single exposure.

Keywords: Mytilus coruscus; acidification; flow cytometry; haemocyte; immune response; salinity.

Figure 1

(A) Haemocyte mortality (HM), (B) Phagocytosis (Pha), (C) Esterase (Est), (D) Reactive oxygen species (ROS), (E) Lysosomal content (Lyso) of M. coruscus exposed to six combinations of salinity (15, 25, and 35‰) and pH (7.3 and 8.1) at 2 and 5 h for in vitro experiment. Different capital letters indicate significant differences among time points within each salinity level in pH 7.3 level or control group (p < 0.05). Different small letters indicate significant differences between salinity within each time point in pH 7.3 level or control group (p < 0.05). Asterisk indicates significant differences between pH within each time point and fixed salinity treatment (p < 0.05).

Figure 2

(A) Haemocyte mortality (HM), (B) Phagocytosis (Pha), (C) Esterase (Est), (D) Reactive oxygen species (ROS), (E) Lysosomal content (Lyso), (F) Total haemocyte count (THC) of M. coruscus exposed to six combinations of salinity (15, 25, and 35‰) and pH (7.3 and 8.1) at 1, 2, 4, and 7 days for in vivo experiment. Different capital letters indicate significant differences among time points within each salinity level in pH 7.3 level or control group (p < 0.05). Different small letters indicate significant differences between salinity within each time point in pH 7.3 level or control group (p < 0.05). Asterisk indicates significant differences between pH within each time point and fixed salinity treatment (p < 0.05).

Figure 3

(A) Biplot originating from PCA integrating all measured variables (HM, Pha, Est, ROS, and Lyso) and two time points (hours: 2 and 5) at six different treatments (■−15‰ × pH 8.1, □−15‰ × pH 7.3, ▴−25‰ × pH 8.1, Δ-25‰ × pH 7.3, ♦−35‰ × pH 8.1, ♢−35‰ × pH 7.3) for in vitro experiment. (B) Biplot originating from principal component analysis integrating all measured variables (HM, Pha, Est, ROS, Lyso, and THC) and four time points (days: 1, 2, 4, and 7) at six different treatments (■−15‰ × pH 8.1, □−15‰ × pH 7.3, ▴−25‰ × pH 8.1, Δ-25‰ × pH 7.3, ♦−35‰ × pH 8.1, ♢−35‰ × pH 7.3) for in vivo experiment. Both the loadings of the variables (•) and the scores of the experimental conditions were shown.