7922 Nucleic Acids Research, 2012, Vol. 40, No. 16 Figure 4. Serial UVC exposure results in mtDNA damage accumula- tion. (a) Schematic of serial UVC protocol. L1 nematodes on unseeded plates are dosed with UVC at 254 nm every 24 h for a total of three doses. Following the third dose, nematodes are provided with food and developmental stage recorded at 48, 72 and 96 h post UVC exposure. (b) mtDNA damage accumulates over serial UVC exposure and persists 24 h after the last UVC exposure and addition of food. Following 24 h recovery periods, nDNA damage was repaired. Bars ± SEM. To address speciﬁcally the effects of mtDNA damage and not nDNA damage, we developed an experimental protocol (Figure 4a) that results in accumulation of mtDNA damage in early larval development but permits repair of nDNA damage. In this protocol, age-synchronized L1 nematodes are dosed three times with UVC over the course of 48 h, with 24 h recovery periods to allow nDNA damage repair. This larval arrest protocol results in time-dependent accumulation of mtDNA damage to a level similar to that induced in adult experiments given earlier in the text, whereas nDNA damage levels were reduced to close to the limit of detec- tion (0.1 lesions/10 kb; (56)) (Figure 4b). Following the third exposure to UVC, L1 nematodes were provided with food and screened at 48, 72 and 96 h to assess the frequency and duration of L3 arrest. A UVC dose-dependent increase in L3 arrest was observed with signiﬁcant inhibition starting at 7.5 J/m2 (Figure 5a). Steady state ATP levels, quantiﬁed 24 h after the third dose and prior to detectable larval arrest, were signiﬁcantly lower at all UVC exposures (Figure 5b). As a more speciﬁc measure of mitochondrial respir- ation, oxygen consumption was assessed at 0, 24 and 48 h after exposure to 10 J/m2 UVC and addition of food. UVC-exposed nematodes displayed signiﬁcantly lower oxygen consumption by 24 h (Figure 5c) with a more signiﬁcant effect in treated nematodes at 48 h. At 48 h when larval arrest is ﬁrst quantiﬁed, control nema- todes are at young adult stage while 40% of nematodes in the UVC treated group are L2 and L3. We separated Figure 5. Serial UVC exposure results in dose-dependent L3 arrest, lower steady state ATP level and reduced O2 consumption. (a) Larval arrest increased in a dose-dependent manner following serial UVC exposure. (b) Steady state ATP levels were signiﬁcantly lower by 24 h after UVC exposure and addition of food. Asterisks denote a signiﬁ- cant treatment effect compared with untreated control (Fisher’s PLSD, P < 0.05). (c) Nematodes exposed to serial UVC 10 J/m2 had signiﬁ- cantly lower O2 consumption compared with untreated nematodes at 0, 24 and 48 h post exposure and addition of food. At 48 h, O2 consump- tion was further decreased in arrested nematodes compared with non-arrested nematodes and both were decreased compared with the untreated group. Two-way ANOVA indicated a signiﬁcant treat- ment recovery interaction (P < 0.0001) and asterisks denote signiﬁcant differences at each time point (Fisher’s PLSD, P < 0.05). Bars ± SEM. these two populations based on size in the UVC exposed group using a COPAS Biosort, deﬁning large or non-arrested nematodes as those which were the same size as untreated nematodes and smaller or ‘arrested’ nematodes as those smaller than untreated nematodes. Both the arrested and non-arrested nematodes had signiﬁ- cantly lower oxygen consumption compared with controls, but oxygen consumption in arrested nematodes was signiﬁcantly lower than non-arrested UVC-treated nematodes. Thus, accumulated mtDNA damage resulted in an overall decrease in mitochondrial function as indicated by increased L3 arrest, lower steady state ATP level and reduced oxygen consumption. Although nDNA damage is repaired throughout larval exposure and the recovery period, we cannot entirely rule out the possibility that