The interplay between prior selection, mild intermittent exposure, and acute severe exposure in phenotypic and transcriptional response to hypoxia

Abstract

Hypoxia has profound and diverse effects on aerobic organisms, disrupting oxidative phosphorylation and activating several protective pathways. Predictions have been made that exposure to mild intermittent hypoxia may be protective against more severe exposure and may extend lifespan. Here we report the lifespan effects of chronic, mild, intermittent hypoxia, and short-term survival in acute severe hypoxia in four clones of Daphnia magna originating from either permanent or intermittent habitats. We test the hypothesis that acclimation to chronic mild intermittent hypoxia can extend lifespan through activation of antioxidant and stress-tolerance pathways and increase survival in acute severe hypoxia through activation of oxygen transport and storage proteins and adjustment to carbohydrate metabolism. Unexpectedly, we show that chronic hypoxia extended the lifespan in the two clones originating from intermittent habitats but had the opposite effect in the two clones from permanent habitats, which also showed lower tolerance to acute hypoxia. Exposure to chronic hypoxia did not protect against acute hypoxia; to the contrary, Daphnia from the chronic hypoxia treatment had lower acute hypoxia tolerance than normoxic controls. Few transcripts changed their abundance in response to the chronic hypoxia treatment in any of the clones. After 12 h of acute hypoxia treatment, the transcriptional response was more pronounced, with numerous protein-coding genes with functionality in oxygen transport, mitochondrial and respiratory metabolism, and gluconeogenesis, showing upregulation. While clones from intermittent habitats showed somewhat stronger differential expression in response to acute hypoxia than those from permanent habitats, contrary to predictions, there were no significant hypoxia-by-habitat of origin or chronic-by-acute treatment interactions. GO enrichment analysis revealed a possible hypoxia tolerance role by accelerating the molting cycle and regulating neuron survival through upregulation of cuticular proteins and neurotrophins, respectively.

Keywords: Daphnia; differential gene expression; hypoxia; lifespan; local adaptation; mitochondrial membrane potential; tolerance.

FIGURE 1

Survival curves of Daphnia from intermittent habitats (a, dashed lines) and permanent habitats (b, solid lines) in normoxic conditions (8 mgO₂/L, green), chronic mild intermittent hypoxia (4 mg O₂ /L twice daily; orange), or after switch from 4 to 8 mgO₂/L at day 30 (blue). Clones within habitat types were not a significant factor in the proportional hazards analysis and are polled together here, as are replicate tanks within each treatment. P values for Log‐rank test for survival differences between groups and cohort sizes (N) are shown. See Table 2 for detailed survival analysis. See Figure S4 for the same data grouped by hypoxia conditions rather than by habitats of origin.

FIGURE 2

Body length at age 18 days (a), feeding rate (b), respiration rate (c, d) and fecundity (e) in four Daphnia from intermittent (FI, IL) and permanent (GB, HU) habitats reared either in normoxic conditions (8 mgO₂/L, green) or chronic mild intermittent hypoxia (CMIH, 4 mg O₂/L twice daily; orange). Respiration rate is measured either at an initial O₂ concentration of 8 mg/L (c) or at an initial O₂ concentration equal to that in the rearing conditions (d). The bars here and below are standard errors. Letters on bars indicate significant differences among groups in Tukey test, p < .05: groups sharing a letter are not different. Not shown when no differences are significant. See Table 1 for clones' provenance and Table 3 for full statistics.

FIGURE 3

Survival in acute severe hypoxia (ASH; <1 mg/L O₂) of Daphnia from intermittent habitats (dotted lines) and permanent habitats (solid lines) reared in normoxia (green) or in chronic mild intermittent hypoxia (CMIH; 4 mg O₂ /L; orange). See Table 4 for detailed survival analysis.

FIGURE 4

Whole body lactate/pyruvate ratio (a) and protein‐normalized lactate and pyruvate concentrations (b, c) in young (15–20 days) and moderately aged (55–60 days) Daphnia from the two clones studied reared at either normoxic control (green) or CMIH (orange) conditions. See Table 5 for statistics.

FIGURE 5

Median rhodamin‐123 fluorescence used as a measure of mitochondrial membrane potential in antenna‐driving muscle (m), epipodite (osmoregulation/gas exchange organ, e), brain (b), and optical lobe (ol) in Daphnia from either intermittent or permanent habitats reared either in normoxic (8 mg O₂/L, green) or CMIH (4 mg O₂/L twice daily, orange) conditions. Letters on the bars are the results of Tukey test conducted for each tissue separately. See Table 6 for statistical analysis.

FIGURE 6

PCA of 48 samples (2CMIH × 2ASH × 4clones/libraries × three biological replicates) in the space of 587 transcripts with at least one uncorrected p < .01. Colors as on previous figures, namely green: CMIH control, orange: CMIH treatment. Circles: ASH control, triangles: ASH treatment. Ellipses encircling clone/library blocks drawn by hand. Clones from intermittent habitats: FI, IL; from permanent habitats: GB, HU. See Table 1 for clones' provenance and Table 7 for statistics.

FIGURE 7

Read abundance (RPKM) in select transcripts with a significant (p _adj < .1) effect of CMIH or ASH in the 3‐way LRT analysis. *: p _adj < .1; **: p _adj < .01; ***: p _adj < .001; ****: p _adj < .0001. (a–e) hemoglobins, cytoglobins, and heme synthesis‐related genes. (f) HiF prolyl hydrolase. (g–i) pyruvate metabolism and gluconeogenesis‐related genes. (j) choriolytic enzyme.

FIGURE 8

RPKM values of transcripts whose GOs are significantly enriched in the gene set with possible ASH × habitat type interactions. (a) 14 paralogs of cuticulum proteins; (b) six paralogs of neurotrophins. Both groups of transcripts show upregulation in Daphnia from intermittent habitats and downregulation in those from permanent habitats.