Mitochondrial bioenergetic processes are fundamental to development stress responses and health. Caenorhabditis elegans is widely used to study developmental biology mitochondrial disease and mitochondrial toxicity. Oxidative phosphorylation generally increases during development in many species and genetic and environmental factors may alter this normal trajectory. Altered mitochondrial function during development can lead to both drastic short-term responses including arrested development and death and subtle consequences that may persist throughout life and into subsequent generations. Understanding normal and altered developmental mitochondrial biology in C. elegans is currently constrained by incomplete and conflicting reports on how mitochondrial bioenergetic parameters change during development in this species. We used a Seahorse XFe24 Extracellular Flux (XF) Analyzer to carry out a comprehensive analysis of mitochondrial and non-mitochondrial oxygen consumption rates (OCR) throughout larval development in C. elegans. We optimized and describe conditions for analysis of basal OCR basal mitochondrial OCR ATP-linked OCR spare and maximal respiratory capacity proton leak and non-mitochondrial OCR. A key consideration is normalization and we present and discuss results as normalized per individual worm protein content worm volume mitochondrial DNA (mtDNA) count nuclear DNA (ncDNA) count and mtDNA:ncDNA ratio. Which normalization process is best depends on the question being asked and differences in normalization explain some of the discrepancies in previously reported developmental changes in OCR in C. elegans. Broadly when normalized to worm number our results agree with previous reports in showing dramatic increases in OCR throughout development. However when normalized to total protein worm volume or ncDNA or mtDNA count after a significant 2-3-fold increase from L1 to L2 stages we found small or no changes in most OCR parameters from the L2 to the L4 stage other than a marginal increase at L3 in spare and maximal respiratory capacity. Overall our results indicate an earlier cellular shift to oxidative metabolism than suggested in most previous literature.
Funding note: This work was funded by the National Institute of Health (R01ES028218 P42ES010356 R01ES034270). Some strains were provided by the Caenorhabditis Genetics Center which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript.
Funding note: This work was funded by the National Institute of Health (R01ES028218 P42ES010356 R01ES034270). Some strains were provided by the Caenorhabditis Genetics Center which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript.