stress. Likewise, co-introduction of the traF with the NaChBac element lengthened survival times of the NaChBac alone (35.260.4 hrs as compared to 26.061.2 hrs for starvation, 81.761.1 hrs as compared to 21.960.6 hrs for osmotic stress, and 120.2620.1 hrs as compared to 24.06 hrs for oxidative stress). While we acknowledge that feminization in conjunction with other corazonin neuronal manipulations do not strictly lead to the female phenotype likely reflects that feminization was limited to corazonin neurons and other dimorphic aspects of the circuitry are still male. However, the finding that any significant change is a consequence of feminization on a complex trait such as lifespan indicates an inherent dimorphism that maps to these cells, which is clear by examination of locomotor phenotypes. Corazonin Neurons Are Essential for Normal Stress-Induced Behavior How does the loss of a neuronal population lead to increased resistance to stress? We reasoned that one possible mechanism is that the loss of corazonin neurons might lead to critical failures in the formation of stress behaviors, thereby increasing lifespan under stress. For example, one of the many behavioral responses to starvation (and other physiological stress) is elevated activity levels [29–30] which ultimately causes the further loss of energy reserves and exacerbates the deleterious effects of the stress. Animals defective in producing such a behavioral response, as has recently been shown in animals deficient in the cells producing the Adipokinetic hormone, have enhanced longevity during starvation [29–30]. Thus, this behavioral response is counter-productive in the long-term as it leads to the further exhaustion of energetic stores, but in the short term would facilitate enhanced foraging opportunities. An alternative explanation for the changes in lifespan under stress in animals with altered corazonin neuronal function may be that secretion and/or expression of corazonin might be normally inhibited by stress, and the manipulations of corazonin neuronal excitability may either mimic or impede these normal actions of stress. To distinguish between these and other possible mechanisms, we initiated studies to evaluate specific aspects of stress response behaviors and utilized locomotor activity as a marker for such behaviors. We observed a period of hyperactivity in locomotion in w1118 flies with the presentation of starvation (Figure 4A and 4B) and osmotic stress (Figure 4B), but decreased activity in flies challenged with oxidative stress (Figure 4B). In order to evaluate any potential effects of altered corazonin neuronal functions on stress-induced locomotion, we calculated the percent change of locomotion by normalizing to unstressed locomotor activity levels. Again, a strong dimorphism in this parameter was observed. Males with either silenced or ablated corazonin neurons show a diminution of stress-induced activity for starvation and osmotic stress, and are resistant to the decreases in locomotor activity caused by oxidative stress (Figure 4B). In contrast, activation of male corazonin neurons leads to enhanced activity under osmotic and starvation challenges (P,0.05, ANOVA). In females, there was also a lack of consistent effects from manipulations of corazonin neuronal excitability, although interestingly, ablation of female corazonin neurons leads to enhanced stress responses (Figure 4B), despite the observations that this alteration increases stress resistance as determined by lifespan measurements (P,0.05, ANOVA). Corazonin Neuronal Manipulations Alter Energy Stores Given the behavioral phenotypes associated with manipulated corazonin neuronal function, the next pertinent question is: are energy stores and/or utilization altered in these backgrounds or under stress conditions? To assess whether corazonin differentially Dimorphic Stress Hormones impacted either the capacity of energy stores or its utilization, we measured total triglyceride levels in animals with different corazonin genotypes. Under normal (unstressed) conditions, levels of triglyc- erides vary between genotypes, but within a given genotype, there appear to be no differences between males and females. Specifically, for both sexes, ablation of corazonin neurons produced flies with the largest amount of triglycerides, followed by corazonin silenced animals, then w1118 flies, and lastly flies expressing the NaChBac channel in corazonin neurons (P,0.02, ANOVA). To evaluate whether corazonin neurons alter the profile of how energy is utilized during stress, we subjected animals to starvation conditions and measured triglyceride levels. We chose to limit this analysis to starvation conditions to circumvent potential confounds of altered dietary intakes with other stresses, as dietary introduction was the method of stress-induction. In these experiments, the total amount of triglycerides decreased as a function of starvation duration for all genotypes. For comparisons of genotypes, a homogeneity of slopes test was performed following a linear regression transform. For males, this analysis revealed no differences in the slopes of the lines (F = 0.20, P = 0.81) (Figure 5A), but did reveal differences in intercept (F = 17.5, P,0.0001). This suggests that there are no differences in how triglyceride levels are mobilized or utilized during starvation, but rather the effects of genotype are limited on total triglyceride levels prior to starvation. However, for females expressing different transgenes in corazonin neurons, the slopes were significantly impacted (F = 13.8, P,0.0001). Comparing these differences in triglycerides to the changes in stress-induced lifespan suggests that these changes in energy balance may underlie the differences exhibited in stress survivorship (Figure 5B). Specifically, for males, the order of triglyceride levels matches the order of lifespan: ablated, silenced, w1118, and activated corazonin neurons. The profile of energy utilization exhibited in females with altered corazonin neuronal function also predicts stress sensitivities. Specifically, while female flies with activated corazonin neurons (NaChBac-expressing) have lower triglyceride levels, the slower turnover or utilization of these stores correlates with the longer lifespan under starvation stress. Levels of Dopamine Differ in Corazonin Deficient Flies Given the observation that flies with altered corazonin neuronal function differ in their hyperactivity and normal locomotor activity profiles, we investigated the possibility that corazonin may be acting in concert with other hormones to shape these behavioral responses. One candidate transmitter that may be interacting with corazonin neurons is dopamine, based on previous descriptions of altered dopamine synthesis during stress and phenotypes associated with genetic differences in dopaminergic signaling [31–35]. We tested the possibility that dopamine signaling may be altered in the flies with genetically altered corazonin neuronal functions. We first tested this hypothesis through employing a receptor bioassay. Larval hemo- lymph derived from flies with specific corazonin neuronal alterations was applied to HEK-293 cells expressing the dopamine receptor encoded by CG9761 [36] and a luciferase reporter for cAMP [37] the second messenger through which this receptor signals. The amount of luminescence detected was greatest from hemolymph derived from flies lacking (rpr) followed by silenced (ork), w1118 and activated (NaChBac) corazonin neurons (Figure S4). While such results are indicative of an interaction between corazonin function and dopamine content, this measure only provides a relative measure of dopamine, as reporter-only transfected cells exhibited greater luminescence than vehicle treated cells, and for technical reasons only allowed investigation of larval stages. Therefore, we employed HPLC measurements on adult flies with altered corazonin neuronal function to quantify the impact of corazonin on dopamine levels PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9141