Fig 1.
Representative trace of high-resolution respirometry using a multiple substrate—uncoupler-inhibitor titration protocol in the thoracic muscle of worker honeybees (Apis mellifera).
The oxygen consumption (y-axis) per mg of tissue mass is represented as a function of time (x-axis). Arrows indicate the timing of titrations of substrates, uncoupler and inhibitors. The protocol includes the following steps: pyruvate+malate+glutamate added before the homogenate (NADH-pathway, LEAK state), ADP (NADH-pathway, OXPHOS capacity); cytochrome c (Cyt. c; integrity of outer mitochondrial membrane), succinate (combined NADH and succinate pathways, NS-pathway, OXPHOS capacity), glycerophosphate (combined NADH, succinate, and Gp pathways, NSGp-pathway, OXPHOS capacity), uncoupler (dinitrophenol, DNP) titration (NSGp-pathway, maximal ETS capacity), rotenone (inhibition of Complex I, SGp-pathway, ETS capacity), antimycin A (inhibition of complex III, residual oxygen consumption, ROX), ascorbate (Asc.) and TMPD (Complex IV activity) and sodium azide (inhibition of Complex IV, chemical background).
Fig 2.
Tissue mass and biomarker of mitochondrial content in worker and queen bees across different ages and body regions.
Panels A–C report tissue mass (mg), panels D–F quantify citrate synthase (CS) activity as a mitochondrial content marker, and panels G–I present maximal electron transport system (ETS) capacity. All measures are shown for head (A, D, G), thoracic muscle (B, E, H), and abdominal fat body (C, F, I). CS activity and ETS capacity values are normalized to tissue mass (per mg). Box plots display the minimum, 25th percentile, median, 75th percentile, and maximum values. Data represent workers at 1 week (early-life, n = 8 and 4 weeks (late-life, n = 12 and queen at 1 week (early-life, n = 6) and 109 weeks (late-life, n = 4). Two-way ANOVA p-values for the effects of bee caste (workers vs. queens), age, and their interaction are indicated in each panel. Panel C, D, F and I did not meet the assumptions; therefore, Welch’s ANOVA with Games-Howell post-hoc test was performed instead. Significant differences between ages within a bee caste (shown below the boxes) and between bee castes at the same age (shown above the boxes) are denoted * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.001).
Fig 3.
Mitochondrial respiration pathway contributions in the head tissue of worker and queen bees across ages.
Respiration is expressed as the flux control ratio (FCR), normalized to the maximal electron transport system (ETS) capacity after uncoupling, based on the combined flux of the NADH, Succinate and glycerophosphate (Gp) pathways. Panel A: Relative contribution of the NADH-pathway to maximal flux. Panel B: Increase in respiration following the addition of succinate to mitochondria in the presence of NADH (NS-pathway – NADH-pathway). Panel C: Respiration with combined NS-pathway. Panel D: Effect of Gp addition to mitochondrial respiration in the presence of NS-pathway substrates (NSGp-pathway – NS-pathway). Panel E: Respiration after inhibition of the NADH-pathway with rotenone, with SGp-pathway remaining active. Panel F: Complex IV activity. Box plots display the minimum, 25th percentile, median, 75th percentile, and maximum values. Data represent workers at 1 week (early-life, n = 8) and 4 weeks (late-life, n = 12) and queen at 1 week (early-life, n = 6) and 109 weeks (late-life, n = 4). Two-way ANOVA p-values for the effects of bee caste (workers vs. queens), age, and their interaction are indicated in each panel. Panels A, C and D did not meet the assumptions; therefore, Welch’s ANOVA with Games-Howell post-hoc was performed for panel C and Kruskal-Wallis test with Dunn’s test was performed for panels A and D instead. Significant differences between ages within a bee caste (shown below the boxes) and between bee castes at the same age (shown above the boxes) are denoted * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.001).
Fig 4.
Mitochondrial respiration pathway contribution in the thoracic muscle of worker and queen bees across ages.
Respiration is expressed as the flux control ratio (FCR), normalized to the maximal electron transport system (ETS) capacity after decoupling, as described in Fig 3. Panels show: (A) NADH-pathway contribution, (B) increase in flux following succinate addition (NS-pathway – NADH-pathway), (C) combined NS-pathway respiration, (D) effect of glycerophosphate addition (NSGp – NS), (E) respiration after addition of rotenone (SGp-pathway), and (F) complex IV activity. Box plots display the minimum, 25th percentile, median, 75th percentile, and maximum values. Sample sizes; workers at 1 week (n = 8) and 4 weeks (n = 12); queen at 1 week (n = 6) and 109 weeks (n = 4). Two-way ANOVA p-values for caste, age, and their interaction are indicated. Significant differences within caste (below boxes) or between bee castes at a given age (above boxes) are denoted * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.001).
Fig 5.
Mitochondrial respiration pathway contributions in the abdominal fat tissue of worker and queen bees across ages.
Respiration is expressed as the flux control ratio (FCR), normalized to the maximal electron transport system (ETS) capacity after decoupling, as described in Fig 3. Panels show: (A) NADH-pathway contribution, (B) increase in flux following succinate addition (NS-pathway – NADH-pathway), (C) combined NS-pathway respication, (D) effect of glycerophosphate addition (NSGp – NS), (E) respiration after addition of rotenone (SGp-pathway), and (F) complex IV activity. Box plots display the minimum, 25th percentile, median, 75th percentile, and maximum values. Sample sizes; workers at 1 week (n = 8) and 4 weeks (n = 12); queen at 1 week (n = 6) and 109 weeks (n = 4). Two-way ANOVA p-values for caste, age, and their interaction are indicated. Significant differences within caste (below boxes) or between bee castes at a given age (above boxes) are denoted * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.001).
Fig 6.
Control of OXPHOS by the phosphorylation pathway in worker and queen bees across different ages and body regions.
Respiration with combined NSGp-pathways active, prior to decoupling with DNP, illustrating the limitation of OXPHOS by the phosphorylation pathway (1 indicates no limitation and 0 indicates 100% limitation), for the head tissue (A), thoracic muscle (B) and abdominal fat tissue (C). Box plots display the minimum, 25th percentile, median, 75th percentile, and maximum values. Data represent workers at 1 week (early-life, n = 8) and 4 weeks (late-life, n = 12) and queen at 1 week (early-life, n = 4) and 109 weeks (late-life, n = 4). Two-way ANOVA p-values for the effects of bee caste (workers vs. queens), age, and their interaction are indicated in each panel. Panel B did not meet the assumptions; therefore, Kruskal-Wallis test with Dunn’s test was performed instead. Significant differences between ages within a bee caste (shown below the boxes) and between bee castes at the same age (shown above the boxes) are denoted * (P ≤ 0.05), ** (P ≤ 0.01), and *** (P ≤ 0.001).