Table 1.
Mean (± SD) shell length and wet weight of the Ruditapes philippinarum selected for the two hypoxia experiments.
Fig 1.
Design of the automated hypoxia simulation device.
(A) The sketch showed the main structure of the device. The structure labeled with ‘a’ was the environmental simulation system; the structure labeled with ‘b’ was the water-exchange system; the structure labeled with ‘c’ was the central control system. (B) A logic diagram indicated how the device was running, including the water flow and gas flow.
Table 2.
Dissolved oxygen (DO) concentrations used in Experiment 2.
Fig 2.
Dissolved oxygen (DO) treatments used in Experiments 1 and 2; the control group in each experiment was maintained at 6.0 mg L-1 DO.
(A) Experiment 1 consisted of three treatment groups, set at 0.5, 1.0 and 2.0 mg L-1 DO, and a control group. (B) Experiment 2 comprised four independent experiments with different sets of treatments: Experiment 2.1 –treatments 0.40, 0.60 and 0.80 mg L-1 DO, and a control group; Experiment 2.2 –treatments 0.45, 0.65 and 0.85 mg L-1 DO, and a control group; Experiment 2.3 –treatments 0.50, 0.70 and 0.90 mg L-1, and a control group; Experiment 2.4 –treatments 0.55, 0.75 and 0.95 mg L-1, and a control group.
Fig 3.
Changes in the number of surviving and buried Ruditapes philippinarum in Experiment 1.
(A) Treatment group exposed to 0.5 mg L-1 DO. The dashed lines denote the estimated critical-time thresholds dividing the curve into different phases; the first dashed line marks the beginning of the mortalities, and the second cuts the rest of the curve into two parts that could each be linearly fitted with best fitness (the largest sum of R values in Excel). (B) Treatment group exposed to 1.0 mg L-1 DO. (C) Treatment group exposed to 2.0 mg L-1 DO. (D) Control group maintained at 6.0 mg L-1 DO. N = 72 per treatment.
Fig 4.
Survival rates of the Ruditapes philippinarum in Experiment 2.
Each circle represents one replicate in one specific experiment. The 20-day LC50 for DO was estimated by probit analysis, based on the combined results of Experiments 2.1–2.4. N = 96 per treatment.
Table 3.
Calibrated values (CV) for survival rates and burial rates of the Ruditapes philippinarum (* indicates p < 0.05).
Fig 5.
Metabolic response to hypoxia by Ruditapes philippinarum on the 10th day and at the end of the experiment, under different DO concentrations.
(A) Oxygen consumption rate on the tenth day; (B) ammonia-N excretion rate on the tenth day; (C) O:N ratio on the tenth day. (D) oxygen consumption rate at the end of experiment; (E) ammonia-N excretion rate at the end of experiment; (F) O:N ratio at the end of experiment. Error bars indicate mean ± SE (** indicates p < 0.01, * indicates p < 0.05).
Fig 6.
Biochemical responses to hypoxia measured in adductor muscle of Ruditapes philippinarum on the 10th day and at the end of the experiment, under different DO concentrations.
(A) Lactate dehydrogenase activity on the tenth day; (B) phosphofructokinase activity on the tenth day; (C) pyruvate kinase activity on the tenth day. (D) lactate dehydrogenase activity at the end of experiment; (E) phosphofructokinase activity at the end of experiment; (F) pyruvate kinase activity at the end of experiment. Error bars indicate mean ± SE (** indicates p < 0.01, * indicates p < 0.05).
Fig 7.
Transmission electron microscope images of the foot muscle of Ruditapes philippinarum.
(A), (B), (C) and (D) represent the DO groups of 6.0, 2.0, 1.0 and 0.5 mg L-1, respectively. White arrows point to normal mitochondria while black arrows point to mitochondria with collapsed cristae (judged by sparse cristae) and vacuolization (judged by shallower staining). Cell vacuolization was apparent according to the shallower cell-staining. Some other cellular structures for point of reference are listed as follows: ‘a’ refer to glycogen granules; ‘b’ refer to myofilaments; ‘c’ refer to cell membrane.