Inducing the Alternative Oxidase Forms Part of the Molecular Strategy of Anoxic Survival in Freshwater Bivalves

Abstract

Hypoxia in freshwater ecosystems is spreading as a consequence of global change, including pollution and eutrophication. In the Patagonian Andes, a decline in precipitation causes reduced lake water volumes and stagnant conditions that limit oxygen transport and exacerbate hypoxia below the upper mixed layer. We analyzed the molecular and biochemical response of the North Patagonian bivalve Diplodon chilensis after 10 days of experimental anoxia (<0.2 mg O₂/L), hypoxia (2 mg O₂/L), and normoxia (9 mg O₂/L). Specifically, we investigated the expression of an alternative oxidase (AOX) pathway assumed to shortcut the regular mitochondrial electron transport system (ETS) during metabolic rate depression (MRD) in hypoxia-tolerant invertebrates. Whereas, the AOX system was strongly upregulated during anoxia in gills, ETS activities and energy mobilization decreased [less transcription of glycogen phosphorylase (GlyP) and succinate dehydrogenase (SDH) in gills and mantle]. Accumulation of succinate and induction of malate dehydrogenase (MDH) activity could indicate activation of anaerobic mitochondrial pathways to support anoxic survival in D. chilensis. Oxidative stress [protein carbonylation, glutathione peroxidase (GPx) expression] and apoptotic intensity (caspase 3/7 activity) decreased, whereas an unfolded protein response (HSP90) was induced under anoxia. This is the first clear evidence of the concerted regulation of the AOX and ETS genes in a hypoxia-tolerant freshwater bivalve and yet another example that exposure to hypoxia and anoxia is not necessarily accompanied by oxidative stress in hypoxia-tolerant mollusks.

Keywords: Diplodon chilensis; alternative oxidase; anaerobiosis; hypoxia; mitochondrial electron transport; oxidative stress.

Figure 1

Gene expression levels of (A) PK (pyruvate kinase), GlyP (glycogen phosphorylase), SDH (succinate dehydrogenase), CS (citrate synthetase), and AOX (alternative oxidase); (B) GSS (glutathione synthetase) and GPx (glutathione peroxidase); and (C) HSP-70, HSP-90, and LR (laminin receptor) in gill (white bars) and mantle (black bars) of D. chilensis exposed to anoxia (<0.2 mg O₂/L), hypoxia (2 mg O₂/L), and normoxia (9 mg O₂/L, control group), (means ± SD, n = 5, except for normoxia-exposed bivalves in mantle n = 4). Letters a and b indicate significant differences between tissues, c and d among oxygen concentration conditions (p < 0.05). For AOX different letters indicate significant differences (p < 0.05).

Figure 2

Enzyme activities of (A) PK (pyruvate kinase), PEPCK (phosphoenol-pyruvate-carboxykinase), MDH (malate dehydrogenase), LDH (lactate dehydrogenase) in gill (white bars) and mantle (black bars), (B) SOD (superoxide dismutase), CAT (catalase), and GPx (glutathione peroxidase) in mantle; and (C) carbonyl levels in gill and caspase 3/7 activity in mantle of Diplodon chilensis exposed to anoxia (<0.2 mg O₂/L), hypoxia (2 mg O₂/L), and normoxia (9 mg O₂/L, control group). Different letters indicate significant differences (p < 0.05) (means ± SD).

Figure 3

Schematic diagram summarizing the major physiological mechanisms of adaptation to different oxygenation scenarios. Scenarios “normoxic,” “hypoxic,” and “anoxic” are based on evidence from the present study. AOX, alternative oxidase; ETS, electron transport system of complex I and III; unfolded PR, unfolded protein response; MRD, metabolic rate depression.