period that is dependent upon appropriate water and nutrient balance, and likewise, elevated cardiac activity is a hallmark response to a variety of physiological stresses in a variety of organisms as is a circadian regulation of stress hormones [15]. Indeed, during the revision of this manuscript, two review papers hypothesized a similar role of corazonin functioning within the physiology of stress responses [16–17]. Additionally, the parallels between mammalian GnRH and corazonin, suggest that corazo- nin may be modulated by stress hormones and consequently, may represent an important substrate in the behavioral and physiolog- ical responses to stress. We report herein that different manipulations of corazonin neuronal function in Drosophila are manifest as differences in lifespan under different stresses. We find these differences are sex dependent and that a component of the dimorphic aspects is localized to corazonin neurons. Materials and Methods Drosophila Husbandry All Drosophila stocks were reared on a standard cornmeal-malt- agar-molasses medium that was supplemented with proprionic acid. Stocks were housed in uncrowded conditions at 25 C on a 12:12 LD cycle. The following stocks were used in the study: pdf-GAL4 kindly donated by Paul Taghert, Crz-GAL4 (on chromosome II) kindly donated by Jae Park, UAS-ork, UAS-rpr, UAS-NaChBac (on chromosome III), UAS-CD8-GFP (on chromosome II), UAS-traF (on chromosome II), and w1118 from the Bloomington stock center. All transgenes were backcrossed in the w1118 background. Lifespan Measurements Measurements of lifespan have been widely employed in Drosophila as a metric of stress sensitivity [e.g, 18]. We placed 30 three to ten day old flies (males and females housed separately) in vials with a solution containing the source of the stress. We had previously determined that survivorship under the physiological stressors employed in this study was similar across this age transect (Figure S1). Animals were collected under mild CO2 anesthesia and placed in a vial containing the stress at ZT0 (lights on) following three days of entrainment to a 12:12 LD cycle. A two percent agar solution was used to starve the animals, food containing 0.6 M NaCl was used to induce osmotic stress, and food at a final concentration of 20 mM paraquat was used for assessment of responses to oxidative stress. For each vial, we assessed median survival (the time of death for 50% of the population) employing non-linear regression analysis (GraphPad Prism) to calculate a mean median survival and then employed a one-way ANOVA with post-hoc Tukey’s comparison for differences between genotypes/treatments. Mated males and females were housed independently and mortality was assessed every twelve hours. Locomotor Measurements Locomotor activity was monitored with a TriKinetics Locomo- tor Monitor (Waltham, MA) on the aggregate population of thirty 3210 day old flies. Flies were housed in a 12:12 LD cycle for three days prior to the experiments. Flies were transferred to a vial containing the appropriate medium at ZT0. Total beam breaks were monitored continuously through an automated system and for duration of 48 hours, to avoid skewed measurements of dwindling populations. We subtracted out baseline locomotion from locomotion during stress for each genotype and then assessed the percent change in locomotion to isolate the amount of activity associated with the stress. We employed a one-way ANOVA followed by post-hoc Tukey comparisons for differences in this activity parameter between genotypes. Dimorphic Stress Hormones Immunocytochemistry Larval and adult progeny from flies carrying the Crz-GAL4 transgene crossed to the UAS-CD8-GFP lines were dissected. Brains were fixed in a 4% Paraformaldehyde 7% picric acid fixative for one hour at room temperature and washed six times with phosphate buffered saline (PBS) containing Triton-X 100. A 1:1000 dilution of anti-rabbit corazonin, a generous gift from Jan Veenstra, was incubated overnight at 4 C. Brains were washed and a Cy-3 conjugated anti-rabbit secondary antibody was applied overnight at 1:1000 dilution. Tissues were then mounted and viewed on a Zeiss confocal microscope. All images were collected using the same settings and confocal protocol. Images were imported into Image ProPlus for quantification of immunosignals. This was done by counting the pixel intensity of corazonin immunosignals and expressing this as a ratio of autofluorescence of a similar size region of the brain using a different channel. Triglyceride Measurements Total triglyceride levels were assessed through use of a Serum Total Triglyceride Kit (Sigma, St. Louis) according to manufac- turer’s instructions. Thirty flies were weighed, and homogenized completely in 1 mL 100 mM Tris-HCl (pH 7.4), 1 NaCl, 1% TritonX-100 buffer. Homogenates were centrifuged for 2 minutes at room temperature at 130006g to collect particulates. One hundred microliters of supernatant was transferred to a 96 well plate and an equal volume of working triglyceride reagent was added to each well. Triplicate wells were performed for each sample and three replicate samples of flies were run for each treatment condition. Absorbance at 510 nm wavelength was measured on a Perkin Elmer Victor III multilabel microplate reader. Absorbance values were compared to standards and normalized by fly weight. A homogeneity of slopes test was performed on linear regression to compare triglyceride levels of different genotypes following starvation conditions. Dopamine Measurements Dopamine levels were measured via HPLC as previously described [19] and through a receptor bioassay. For HPLC analysis, five brains from adults aged 3210 days old were dissected in calcium-free ringers and placed directly into 100 mL of ice-cold 50 mM citrate acetate buffer, and homogenized completely. Extraction of adult hemolymph was done by piercing the cuticle on the thorax and abdomen and spinning for thirty seconds at 20006G in a tube with holes to allow passage of liquid components and collection of particulates. This procedure was carried out on ninety individual flies per sample, and three samples for each sex and each genotype was collected. Two microliters of hemolymph was diluted in 100 uL of citrate buffer. Homogenates were then spun through a 0.22 micron spin filter (Millipore Corporation, Bedford, MA) prior to analysis. Samples were loaded onto a HPLC system. Dopamine levels were quantified and analyzed using Jasco ChromPass chromatography software and compared to standards. Dopamine appeared as a unitary and well- resolved peak when detected at an oxidation potential of +500 mv relative to an Ag+/AgCl reference electrode. This peak was confirmed as dopamine by co-migration with an authentic DA standard, and by electrochemical analysis of the half-oxidation potential. Potential statistical differences between genotypes were assessed by employing ANOVA (GraphPad, Prism). Corazonin Transcript Analysis Three replicate vials of thirty flies were subjected to stress as just described. Flies were then quickly frozen and were homogenized PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9141