Mitochondrial function and energy levels are compromised in rps-10(0)/+ mutants. (A) Mitochondrial activity, quantified via the uptake of fluorescent MitoTracker Red CMXRos dye in each RP mutant compared to its wild-type counterpart. MitoTracker intensity per area was measured, and the values for RP animals were normalized to the median of stage-matched wild-type controls and plotted. P values represent the results of a two-tailed Student’s t test with Bonferroni correction comparing each RP haploinsufficient mutant sample to its stage-matched wild-type control. Data distribution was assumed to be normal but this was not formally tested. (B) The ADP/ATP ratio, reflecting overall energy levels of RP haploinsufficient animals normalized to stage-matched controls is shown for each mutant. Measurements were taken from ∼50 RP mutant and wild-type animals per biological replicate, with each replicate represented as a single dot on the plot. (C) The relative oxygen consumption of rps-23(0)/+ and rps-10(0)/+ mutants compared to wild type was plotted. Animals were stage-matched to ensure similar body sizes, and oxygen consumption rates were normalized to the number of animals utilized in each assay. In B and C each point reflects the ratio of RP haploinsufficient animals to respective wild-type controls per biological replicate. The bold line represents the median. P values represent the results of a paired, two-tailed Student’s t test with Bonferroni correction comparing each RP haploinsufficient mutant to its stage-matched wild-type control. Data distribution was assumed to be normal but this was not formally tested. (D) The genetic modification strategy for the rps-10 gene is illustrated. The wild-type rps-10 gene with the PAM sequence highlighted in purple is shown on top; the introduction of tandem early stop codons, generating the rps-10(0) variant with a new PAM sequence in purple is indicated in the middle; the restoration of rps-10(0) to its wild-type sequence marked in green is demonstrated at bottom. (E) The normalized MitoTracker intensity was compared in wild type and two [rps-10(0) to wild-type] rescued strains (rescue-1, rescue-2). P values represent the results of a two-tailed Student’s t test with Bonferroni correction comparing each rescue strain to the stage-matched wild-type controls. Data distribution was assumed to be normal but this was not formally tested. (F) The normalized oxygen consumption was compared in wild type and two [rps-10(0) to wild-type] rescued strains (rescue-1, rescue-2). P values represent the results of a paired, two-tailed Student’s t test with Bonferroni correction comparing each rescue strain to the stage-matched wild-type control. (G) The relative intensity distribution of TOMM-20::TagRFP and COX-4::GFP levels in rps-10 (0)/+ mutants and stage-matched wild-type controls are shown. Fluorescence intensities rps-10 (0)/+ mutant animals were normalized to the median of stage matched wild-type intensities. P values represent a two-tailed Student’s t test. Data distribution was assumed to be normal but this was not formally tested. The bar represents interquartile distribution and the bold horizontal line represents the median. (H) Normalized oxygen consumption rates were plotted for gcn-1(n4827) mutants and gcn-1 RNAi compared to their controls (wild type and non-target RNAi) (left). The effects of rps-10 and rps-23 RNAi in a wild-type background (middle), alongside the rps-10 and gas-1 RNAi effects in gcn-1(n4827) mutants (right), with respective controls for each group are shown. Each point represents the ratio of gene-specific RNAi to the respective non-target RNAi for each biological replicate. The bold line indicates the median. P values represent the results of a paired, two-tailed Student’s t test with Bonferroni correction, comparing each gene-specific RNAi to the non-target RNAi control. Data distribution was assumed to be normal but this was not formally tested. All experiments were performed in at least three biological replicates, and the animals were grown at 16°C. Superscript numbers denote the specific balancers compared between an RP mutant and its wild type counterpart. Balancer chromosomes are denoted as follows: +¹ = tmC20, +² = tmC5, +³ = mIn1, +⁴ = nT1. “n” at the top of each graph represents the total number of animals when all biological replicates combined. For comparisons, roughly equal number of animals were used.