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Body composition with respect to white adipose tissue and lean body mass is of central importance and a determining factor of adiposity-associated perturbations, such as diabetes type 2, cardiovascular disease, and cancer. Therefore, modulation of body composition and especially the reduction of body fat are the main foci of dietary and lifestyle interventions. In the last few years, Caenorhabditis elegans became a model organism for investigating fat storage and metabolism, as reviewed by different authors ( 1-3 ). Based on forward genetics ( 4-6 ), functional genomics, and candidate gene approaches ( 7,8 ), more than 400 genes possibly involved in fat storage were described in C. elegans . Most of these genes are evolutionarily conserved and allocated in common pathways and are of relevance for the study of human adiposity.Although C. elegans was extensively used as a model organism to study the genetic and functional basis of fat storage, robust lipid droplet markers such as perilipin are not available at the moment ( 2 ). Appropriate methods concerning the distribution behavior of dyes in the worm or the reproducibility of staining procedures are still under discussion ( 3 ). The classical fi xative Sudan black dye ( 9 ) is shown to be very error prone in C. elegans because of the last ethanol-based washing steps. Recently O' Rourke et al. ( 10 ) demonstrated that the vital dyes Nile red and C1-C12-BODIPY-labeled fatty acid, which were used in more than 75 reports, stain lysosome-like granules rather than lipid droplets within the C. elegans intestine. These dyes were shown not to localize with the fi xative neutral fat fl uorophore LipidTox ( 10 ) 17 March 2011. Published, JLR Papers in Press, March 18, 2011 DOI 10.1194 Fluorescence-based fi xative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and fl ow cytometry approaches Abbreviations: BP, bandpass; CARS, coherent anti-Stokes Raman scattering; COPAS, cytometry-based object parametric analysis and sorting system; LRO, lysosome-related organelle; OPO, optical parametric oscillator; TOF, time of fl ight; TAG, triacylglycerol. Manuscript received 8 October 2010 and in revised form

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Dietary restriction (DR) extends lifespan in man species and modulates evolutionary conserved signalling and metabolic pathways. Most of these studies were done in adult animals. Here we investigated fat phenotypes of C. elegans larvae and adults which were exposed to DR during development. This approach was named “developmental-DR” (dDR). Moderate as well as stringent dDR increased the triglyceride to protein ratio in L4 larvae and adult worms. This alteration was accompanied by a marked expansion of intestinal and hypodermal lipid droplets. In comparison to ad libitum condition, the relative proportion of fat stored in large lipid droplets (>50 µm3) was increased by a factor of about 5 to 6 in larvae exposed to dDR. Microarray-based expression profiling identified several dDR-regulated genes of lipolysis and lipogenesis which may contribute to the observed fat phenotypes. In conclusion, dDR increases the triglyceride to protein ratio, enlarges lipid droplets and alters the expression of genes functioning in lipid metabolism in C. elegans. These changes might be an effective adaptation to conserve fat stores in animals subjected to limiting food supply during development.

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