Fig 1.
Schematic representation of the electron transport system, showing the sites of action of oxidative phosphorylation (OXPHOS) modulators (brown), the different substrates utilized throughout this study (pyruvate and proline, green; glycerol 3 phosphate, red; palmitoylcarnitine and malate, blue) and the known sites of superoxide (O2•¯) production (purple).
Table 1.
Mitochondrial oxygen consumption of insect flight muscle compared to vertebrate muscle induced by two distinct electron transport system sites.
Table 2.
Enzyme activities of A. aegypti flight muscle mitochondria.
Table 3.
Contribution of different substrates to respiration of isolated mitochondria from flight muscle of A. aegypti females.
Fig 2.
Complex I and G3PDH represent the major electron donor sites to support respiration in female A. aegypti flight muscle.
Oxygen consumption rates from female A. aegypti isolated mitochondria (A) and permeabilized flight muscle (B) were calculated from values shown in Tables 3 and 4. Data are expressed as mean ± SD of at least seven different experiments. Comparisons between groups were done by Kruskal-Wallis and a posteriori Dunn's tests. Figure (A): a p<0.05 relative to Complex I Pyr+pro; b p<0.05 relative to G3P; c p<0.01, relative to Complex I Pyr+pro; d p<0.001, relative to Complex I Pyr+pro and G3P. Figure (B): a p<0.001 relative to complex I; b p<0.001 relative to G3P.
Table 4.
Contribution of different substrates to sustain respiration in permeabilized flight muscle from A. aegypti females.
Table 5.
Mitochondrial bioenergetic efficiency and capacity in A. aegypti females flight muscle using different substrates.
Fig 3.
Comparative analyses of respiratory rates induced by different substrates among A. aegypti sexes.
Oxygen consumption rates from isolated mitochondria (A-C) and whole permeabilized flight muscle (D and E) from females (solid bars) and males (hatched bars) were plotted from values shown in Tables 3, 4, S2 and S3. Data are expressed as mean ± SD of at least six different experiments. Comparisons between groups were done by Student´s t- test. Figure (A): a p<0.005 and b p<0.05 relative to their equivalent metabolic state in female. Figure (D): a p<0.0001 and b p<0.05 relative to their equivalent metabolic state in female. Figure (E): a p<0.001 and b p<0.05 relative to their equivalent metabolic state in female.
Fig 4.
Preference towards proline oxidation in A. aegypti female mitochondria.
Oxygen consumption rates from isolated mitochondria (A-E) and whole permeabilized flight muscle (F-H) from females (solid bars) and males (hatched bars) were calculated from values shown in Fig. 3. Data are expressed as mean ± SD of at least seven different experiments. Comparisons between groups were done by Student´s t tests. Figure (B): b p<0.001 relative to female; Figure (G): a p<0.0001 relative to female; Figure (H): b p<0.038 relative to female.
Table 6.
Comparison of mitochondrial H2O2 production by insect flight muscle and vertebrate muscles.
Table 7.
Contribution of different substrates to mitochondrial H2O2 production in A. aegypti flight muscle.
Table 8.
Topology of H2O2 formation in A. aegypti flight muscle mitochondria.
Fig 5.
Contribution of different electron leak sites to H2O2 generation in isolated A. aegypti flight muscle mitochondria.
The contribution of site IF, ProDH+other dehydrogenases, G3PDH+other dehydrogenases and ETF:QOR+other dehydrogenases sites to H2O2 generation in A. aegypti mitochondria isolated from females (A, solid colors) and males (B, hatched bars) were calculated from data shown in Table 8. Data are expressed as mean ± SD of at least five different experiments. Comparisons between groups were done by ANOVA and a posteriori Tukey´s tests. Figure (A): a p<0.001 relative to IF (Pyr+pro); b p<0.001 relative to ETF:QOR+other dehydrogenases; c p<0.001 relative to IF (PC+Mal); d p<0.05 relative to G3PDH+other dehydrogenases; Figure (B): a p<0.001 relative to IF (Pyr+pro); b p<0.001 relative to ETF:QOR+other dehydrogenases; c p<0.001 relative to IF (PC+Mal); d p<0.05 relative to G3PDH+other dehydrogenases.
Fig 6.
Comparative analyses of H2O2 generation rates induced by different substrates among A. aegypti sexes. H2O2 formation rates of mitochondria isolated from females (solid bars) and males (hatched bars) were plotted using the values shown in Tables 7 and 8.
Data are expressed as mean ± SD of at least five different experiments. Comparisons between groups were done by Student´s t-test. Figure (B): a p<0.005, b p<0.0001 and c p<0.005 relative to their equivalent metabolic state in female. Figure (C): a p<0.05 relative to its equivalent metabolic state in female.
Fig 7.
Sexual differences in the contribution of different electron leak sites to H2O2 generation in isolated A. aegypti flight muscle mitochondria.
The contribution of site IF, ProDH+other dehydrogenases, G3PDH+other dehydrogenases and ETF:QOR+other dehydrogenases sites were calculated in A. aegypti mitochondria isolated from females (solid colors) and males (hatched bars) from data shown in Table 8. Data are expressed as mean ± SD of at least five different experiments. Comparisons between groups were done by Mann Whitney test. Figure (D): a p<0.01 relative to female IF (PC+Mal).
Fig 8.
Schematic representation of substrate utilization pathways driving respiration and O2•¯ formation in A. aegypti flight muscle mitochondria.
The dehydrogenases directly involved on mitochondrial electron transfer from nutrient oxidation to respiration are depicted in their respective colors utilized throughout this work, as following: complex I (light green), ProDH (dark green), G3PDH (red) and ETF:QOR (blue). The contribution of dehydrogenases to respiration are represented by their boxes, fonts, and lines sizes. Electron leak and O2•¯ formation induced by different substrates are represented by steam clouds, obeying the same color and size pattern described for dehydrogenases. Noteworthy, the steam cloud location in this scheme does not represent the exact site of O2•¯ production, since we were unable to precisely define these sites in this work. CACT, carnitine-acylcarnitine transferase; CPT2, carnitinepalmitoyl transferase 2; palm-CoA, palmitoyl-CoA; αKG, alpha-ketoglutarate; Δ1PC, Δ-1-pyrroline-5-carboxylate; DHAP, dihydroxyacetone phosphate; PDH, pyruvate dehydrogenase; IMS, intermembrane space; MM, mitochondrial matrix.