Construction of plasmid pSLGCV
The backbone for pSLGCV was plasmid pPD95.79 (Firelab, Carnegie Institution for Science), which contains gfp. To this backbone we added sequentially the sur-5 promoter , PCR-amplified from plasmid pTG96 and then the luciferase gene PCR-amplified from vector pSP-luc+ (Promega). A 3.7 kb fragment upstream of the sur-5 gene containing the promoter, was amplified using primers 5'- ATAAGCTTGCATGCCTGCATTGC-3' and 5'-AGACACCCCGGGCTTTCTGAAAAC-3' (this introduces a Sma I site and removes the start codon for the sur-5 gene). The sur-5 promoter was inserted in the pPD95.79 backbone using appropriate restriction sites (Sph I and Sma I).
The luc+ gene was amplified using primers 5'-TCCCGGGAAGCTTTCCATGGAAGAC-3' and 5'-CTAGGGTACCACGGCGATCTTTCC-3'. Sma I and Kpn I were used to insert luc+ downstream of the sur-5 promoter and upstream of and in frame with gfp. The peroxisome tagging sequence is absent from luc+ and the NLS for sur-5 was excluded from pSLGCV, so that LUC+::GFP was expressed in the cytoplasm.
Microinjection of the gonad syncytium was carried out as standard . The plasmid pSLGCV was co-injected with the marker pRF4 at 250 ng/ml each. A transgenic line, feEx44 [sur-5::luc+::gfp; rol-6(su1006)], was isolated and the array was integrated by EMS mutagenesis , followed by selection of F2 animals that gave rise to 100% roller progeny. We obtained two independently integrated lines, the first into the X chromosome and the second where the transgene appears to be in chromosome V. The first line was out-crossed 8 times with the wild-type N2 strain to generate PE255 (feIs5), and the second was out-crossed 10 times to generate PE254 (feIs4). The data shown were obtained with feIs5, except where otherwise stated.
Measurement of luminescence
Luminescence was measured in a Clarity microplate luminometer (Biotek) in the visible spectral range between 300 and 600 nm, (firefly luciferase typically emits at 550–570 nm). White microplates were used (Greiner) with approximately 100 worms per well (in 100 μl). An automated dispenser delivered 50 μl of luminescence buffer to each well, consisting of citrate phosphate buffer pH 6.5, 0.1 mM D-luciferin, 1% DMSO and 0.05 % triton-X (all final concentrations). Luminescence of the PE255 strain increases steeply in the first minute after adding luciferin, followed by a slower increase to its maximum levels observed within the second minute. The light levels remain fairly stable during the first 5 minutes, followed by a gradual decrease to 60–80% of the maximum luminescence values in the first half hour (Additional file 1). Luminescence was read for 1 sec, 5 min after adding luciferin, except for the azide recovery experiments, where incubation with luciferin was only 3 min. These incubation periods allowed for maximum luminescence to be reached. During incubation with luciferin plates were shaken at 160 rpm. Except where otherwise mentioned, all luminescence measurements were carried out in vivo.
Measurement of fluorescence
Fluorescence was quantified in a FLUOstar OPTIMA microplate reader (BMG labtech) using 485 nm excitation filter and a 520 nm emission filter. Background measurements were subtracted from readings.
Sodium azide (NaN3) exposure bioassays
Worm cultures were synchronised by bleaching and overnight hatching in M9 . L1 stage nematodes were washed and incubated (20°C, 160 rpm) for 45 h in S medium supplemented with 15 g/L E. coli OP50, at a density of 20 to 30 worms per 10 μl. 50 μl of the worm culture was aliquoted to each well of 96-well plates, white for luminescence or black for fluorescence. 50 μl of sodium azide (SIGMA) was added per well to final concentrations of 0, 1, 2.5, 5, 10 and 15 mM. Each concentration was tested on 8 replicate wells. The plates were covered with air permeable seals (ABgene), and incubated at 20°C, 160 rpm, for 30 min prior to measurement of light output or fluorescence. To assess survival worms exposed to each NaN3 concentration were pooled and washed 3 times with S basal , supplemented with 0.01% Tween-20 to prevent worms from sticking to plasticware. Triplicate 10 μl samples were plated on to NGM plates  and viability assessed by motility within 4 h.
Pharyngeal pumping assay
Worms were treated as described in NaN3 exposure bioassays. An adaptation of the method of Mörck et al.  was used for assessing pharyngeal pumping. Briefly, after exposure to the various azide concentrations, Fluoresbrite™ Polychromatic red microspheres, 0.5 μm diameter (Polyscience, Inc), suspended in luminescence buffer, were added to each well, to a final dilution of 50 times. After 10 min contact time, worms were washed with M9 plus 1 mM levamisole (600 × g, 2 min), transferred to 2% agarose (w/v in M9) pads and mounted for observation under fluorescent microscopy (Texas red filters). A minimum of 30 worms were scored for the presence of microspheres in their pharynx and gut, for each of the azide concentrations tested.
Recovery from azide
Synchronised worms were exposed to 10 mM NaN3 for 30 min, in S complete supplemented with E. coli OP50. Luminescence was read just prior to washing worms from NaN3, and this reading was adopted as the zero time point for recovery (t = 0). Recovery was timed from removal of azide solution. Worms were then washed once, centrifuged (1 min, 600 × g) resuspended in S complete with 7.5 g/L E. coli OP50, and aliquoted to 96-well plates. Luminescence was measured at different time points, as quickly as possible, during the first half hour of recovery. At each time point, a different set of samples was read, so that the incubation time with luciferin was the same (3 min) each time. Each luminescence time point is the average of 24 replicate wells. Luminescence of worms not exposed to azide was taken as the guideline for full recovery.
In vitro luciferase assay
Exposure to 10 mM azide was carried out as described above. Worms were then washed 3 times (1 min, 600 × g) in S basal, and approximately 1000 worms in 100 μl of S basal were frozen in liquid nitrogen and stored at -80°C until analysis. Four replicate samples for each azide concentration were thawed on ice and 200 mg glass beads (212–300 μm) and CCLR reagent were added (Promega). To obtain worm lysates samples were put through a Fastprep homogeniser 8 times (5000 rev/min, 30 s) with incubation on ice in between runs. Lysates were analysed using Luciferase Assay system (Promega), according to manufacturer's instructions. The in vitro luminescence data were normalised to protein content of samples.
In vitro ATP determination
A luminometric method adapted from Braeckman et al.  (Braeckman pers. comm.) was used. Briefly, four replicate samples of washed worms were collected in 100 μl of S basal, quickly frozen in liquid nitrogen and stored at -80°C. Worms were broken up by disruption with glass beads (212–300 μm) in a Fastprep homogeniser after addition of 8 % (v/v) HClO4. Extracts were neutralised with 1.3 M KHCO3, and buffered with 1 M K phosphate buffer (pH 7.6) prior to measurement of ATP concentrations with the ATP bioluminescence CLS II kit (Roche), in a Clarity luminometer (Biotek). ATP concentrations were normalised to protein content of samples.
BCA kit (Pierce) was used in all protein determinations according to manufacturer's instructions, with incubation at 60°C.
RNAi Knockdown of respiratory chain genes
The Rivers et al. protocol for RNAi feeding in liquid culture was used . Approximately 15 synchronised L1 nematodes were placed in each well of 96 -well microplates and cultivated with bacteria expressing dsRNA for respiratory chain genes (MRC GeneService clones), or control clones (GFP clone: pPD128.110; empty vector control: pPD129.36; both Firelab vectors), for 4 days at 25°C without shaking, prior to measurement of in vivo luminescence (as above) and length. To control for reduced body size, luminescence readings were normalised to average worm length. A microscope equipped with an ocular micrometer was used to measure worms after immobilisation with levamisole (200 μM). At least 30 measurements were taken for each experimental condition.
Imaging of worms
A Photometrics CASCADE II 512 Air cooled (-70°C) CCD sensor and a Deltavision microscope were used to capture images of the glp-4(bn2); feIs5 strain. Worms were picked into 5 μl of luminescence buffer plus 200 μM levamisole (to immobilise worms) and mounted in 2% agarose pads. Luminescence was captured with no incident light, in a dark chamber, with 10 sec integration, 18 min (± 2 min) after treatment with the luminescence buffer plus 200 μM levamisole. As a control, conditions for imaging luminescence were tested on worms that carry the plasmid PTG96  and display expression of GFP on its own. No luminescence was captured for the control. For imaging fluorescence illumination through FITC filters was used with an exposure time of 0.2 seconds. A Zeiss Axioplan2 microscope with a HAMAMATSU ORCA-ER CA742–80 digital camera was used for additional photographs of worms, including those of the feIs5 strain and the pharyngeal pumping assay.
Statistical analysis was performed with SPSS 15.0 and Microsoft Office Excel 2003. Least significant differences (LSD)  were calculated for statistically significant ANOVA results.