Long-Term survival of anoxia despite rapid ATP decline in embryos of the annual killifish Austrofundulus limnaeus

Abstract

Embryos of the annual killifish Austrofundulus limnaeus can survive for months in the complete absence of oxygen. Survival of anoxia is associated with entry into a state of metabolic dormancy known as diapause. However, extreme tolerance of anoxia is retained for several days of post-diapause development. Rates of heat dissipation in diapause II and 4 days post-diapause II embryos were measured under aerobic conditions and during the transition into anoxia. Phosphorylated adenylate compounds were quantified in embryos during entry into anoxia and after 12 hr of aerobic recovery. Rates of heat dissipation were not affected by exposure to anoxia in diapause II embryos, while post-diapause II embryos experienced a profound decrease in heat dissipation. ATP decreased substantially in both developmental stages upon exposure to anoxia, and all indicators of cellular energetic status indicated energetic stress, at least based on the mammalian paradigm. The rate of decline in ATP is the most acute reported for any vertebrate. The mechanisms responsible for cellular survival despite a clear dysregulation between energy production and energy consumption remain to be identified. Necrotic and apoptotic cell death in response to hypoxia contribute to poor survival during many diseases and pathological conditions in mammals. Understanding the mechanisms that are in place to prevent maladaptive cell death in embryos of A. limnaeus may greatly improve treatment strategies in diseases that involve hypoxia and reperfusion injuries.

Fig. 1

Representative heat dissipation data for aerobic and anoxic embryos of A. limnaeus. The dashed line indicates the initiation of anoxic conditions, and the gap in the tracing is due to removal and re-equilibration of the ampule containing the embryos following establishment of anoxic conditions. The last 15 min of each aerobic and anoxic run were used to extract data for the heat dissipation data presented in Fig. 2.

Fig. 2

Rate of heat dissipation in diapause II and post-diapause II embryos under aerobic and anoxic conditions. Bars represent the mean ± s.e.m. (n = 4). Bars with different letters are statistically different (ANOVA, Tukey’s post hoc, p < 0.001).

Fig. 3

Phosphorylated adenylate compounds in embryos of A. limnaeus exposed to 48 h of anoxia followed by 12 h of aerobic recovery. Symbols represent the mean ± s.e.m. (n = 3). Statistical differences from control values (t = 0) are indicated by a † for total adenylates, a ‡ for ATP, and a * for AMP (ANOVA, Dunnett’s post hoc test, p < 0.01). Total adenylates and ATP levels are statistically different in the two developmental stages at t = 0 (ANOVA, Tukey’s post hoc, p < 0.002), while ADP and AMP levels are not.

Fig. 4

Measures of energetic status for embryos of A. limnaeus exposed to anoxia followed by 12 h of aerobic recovery. Symbols are means ± s.e.m. (n = 3). Statistically significant differences from values at t = 0 are indicated by a * for post-diapause II, and a † for diapause II embryos (ANOVA, Dunnett’s post hoc, p < 0.01).

Fig. 5

The relationship between depletion of ATP levels and survival of anoxia in animals. (A) Invertebrate species with high tolerances of anoxia, Artemia fransciscana (brine shrimp, early anoxia ATP levels from Anchordoguy and Hand, ‘94; survival time and long-term anoxia ATP levels from Warner and Clegg, 2001), Hirudo medicinalis (leech, Zebe et al, ‘81) Nephelopsis obscura (leech, Reddy and Davies, ‘93 for ATP levels, survival time from Davies et al, ‘87), Mytilus galloprovincialis (mussel, Isani et al, ‘95). (B) Tissues of anoxic turtles (Chrysemys picta) exposed to anoxia at 3–5°C (Jackson et al, ‘95; Stecyk et al, 2009 for heart). Survival time estimated at 180 d at 3°C (Ultsch and Jackson, ‘82). (C) Levels of ATP in brain tissue of goldfish Carassius auratus (van den Thillart, ‘82), frog Rana pipiens (Lutz and Reiners, ‘97), 10 d old rat Rattus norvegicus (Lolley and Samson, ‘62 for ATP levels; Adolph, ‘69 for survival), and post-diapause II embryos of A. limnaeus (this study).