in 1 mL of TriZol (Invitrogen, Ca). RNA was isolated according to manufacturer’s recommendations. Total RNA was extracted and used as template for cDNA synthesis using a SuperScript III Reverse Transcription Kit (Invitrogen). Specific primers for the corazonin ORF were designed to flank intervening sequences to readily distinguish if amplicons originated from cDNA versus contaminating genomic DNA. The primers for corazonin were: Forward: 59- 39 ACT CTA ATG GCG AGA ATG TTT TGT and Reverse: ATT GAA CGG ATA GTG GCT AAT GTT. Specific primers were also designed to amplify the housekeeping gene, RP49. Intensity of EtBR fluorescence was determined using a Hitachi Gene Systems equipped with a CCD camera and PCR conditions were determined to be in the linear range of amplification. Values were normalized to RP49 across samples and expressed as % of baseline, i.e., normal conditions. Results Manipulations of Corazonin Neurons Alter Stress Sensitivity We tested the possibility that corazonin neurons were part of the central circuitry mediating responses to various stressors by employing the GAL4-UAS binary system [20] to alter corazonin neuronal function and evaluate stress responses. We introduced the pro-apoptotic gene reaper to ablate corazonin cells using a specific corazonin-GAL4 line, thereby producing a corazonin deficient animal [9,21]. We also introduced modified ion channel constructs, which have been used to silence and activate (Dork and NaChBac, respectively) discrete neuronal populations in Drosophila [22–24]. We used these constructs to alter excitability with the expectation of modulating subsequent corazonin peptide release. To evaluate the impact of transgene expression within corazonin neurons with respect to expected neuronal alterations, we employed an immunocytochemical evaluation of peptide quantity using a specific antibody against the corazonin neuropeptide (Figure 1A). We observed a complete loss of corazonin immunolabeling in flies expressing the reaper transgene (Figure 1C), a depletion of corazonin immunolabeling in NaChBac expressing (Figure 1D), and a slight increase in corazonin immunolabeling in ork expressing flies (Figure 1B) compared to flies expressing a GFP reporter (P,0.0001, Two-way ANOVA) (Figure 1E). Therefore, we conclude that these transgenes are producing phenotypes consistent with heightened secretion (NaChBac expressing), silenced secretion (ork-expressing), and ablated (reaper) corazonin neurons. However, we note that immunosignals derived from adult female brains expressing the ork channel in corazonin neurons did not significantly differ from females expressing GFP in corazonin neurons, which may reflect saturation of immunosignals in these two backgrounds. To evaluate the potential roles of corazonin neurons in mediating stress response, we first measured lifespan of these different genotypes under three different physiological stress conditions: starvation, osmotic, and oxidative. Corazonin cell ablation consistently extended lifespan during these stresses in males and females, as compared to the parental stocks or w1118 genetic background controls, although the degree to which lifespan was extended varied with both gender and the nature of the stress (Figure 2). Introduction of a modified ion channel construct (Dork) to electrically silence corazonin neurons, also resulted in a lengthened lifespan under these stresses, with these effects most pronounced in males and dependent upon the nature of the stress in females. In contrast, activation of these neurons produced differential stress sensitivities in males and females. Predictably, expression of the NaChBac channel in males, results to increased Dimorphic Stress Hormones stress sensitivity for all three stresses tested, as measured by lifespan. However, in females there is a strong stress by gender interaction. Notably, both the introduction of a reporter gene (UAS-CD8-GFP) to corazonin cells and the ablation of a group of well-defined neurons that express a different neuropeptide, Pigment Dispersing Factor (PDF) [25], showed stress sensitivities that were generally comparable to the w1118 background controls under these heterotypic stresses (Figure 2). This indicates that changes in lifespan are likely the consequence of manipulations to corazonin neuronal populations and not solely caused by increased hybrid vigor or differences in genetic backgrounds. Although there are strong differences in quantitative traits such as lifespan [26], and some of the results we present here may be attributed to genetic background effects, our control genotypes neither showed the same large effects of the corazonin manipulations nor in many cases were background effect in the same direction. Additionally, evaluation of lifespan of genotypes with altered corazonin neuronal function indicates that the majority of these changes are a specific consequence of physiological stress, as for almost all genotypes, aging was not significantly impacted (Figure S2). Corazonin Neuronal Manipulations Produce Sex-Specific Effects on Locomotor Activity We reasoned that the alterations in lifespan exhibited in flies differing in corazonin neuronal function may, in part, stem from changes in activity levels as activity may correlate with stress- sensitivity. We measured activity levels in flies that varied in corazonin neuronal function under a normal 12:12 LD lighting regime. We found that the overall amount of activity levels of w1118 males and females were different from each other, consistent with previous observations [27]. However, there was a significant impact on activity levels that were influenced by sex and the specific alteration to corazonin neuronal function. Specifically, males expressing the Dork channel in corazonin neurons displayed greater activity levels than w1118 males. Furthermore, the opposite manipulation (NaChBac introduction) produced hypoactive males relative to w1118 (Figure 3A). Both of these phenotypes were not evident in females, where both manipulations produced activity levels indistinguishable from parental genotypes (Figure S3). Ablation of corazonin neurons produced activity levels slightly lower than w1118 levels for both males and females (Figure 3A). While we predicted that the ablation and silencing phenotypes would be similar for males, this inconsistent finding may be attributable to either differences in transgene impact (silencing is incomplete as compared to ablation) or developmental or physiological compen- sation in animals lacking corazonin neurons. Sexual Dimorphisms Map to Corazonin Neuronal Functions To evaluate whether these dimorphisms were autonomous to corazonin neurons, as opposed to dimorphic downstream elements of the circuitry, we introduced the transformer element, which feminizes male cells [28], to corazonin neurons. Transformer expression in male corazonin cells produced intermediate amounts of locomotion between male and females, indicating a contribution of corazonin cells in the inherent dimorphisms in locomotor behaviors (Figure 3B). Based on the observations that silencing and activation of corazonin neurons produced sex-specific effects, we investigated the consequences of silencing/activation in conjunc- tion with feminization of corazonin cells. We recombined the ork and NaChBac elements with the transformer element and monitored activity levels and found significant differences in the amount of locomotion that were dependent upon sex and genotype PLoS ONE | www.plosone.org 3 February 2010 | Volume 5 | Issue 2 | e9141