Dimorphic Stress Hormones Figure 6. Corazonin neuronal alterations impact the levels of dopamine. Dopamine concentrations as determined by HPLC measurements on hemolymph derived from males and females with varying corazonin phenotypes. For females, only the Crz-rpr lines significantly differed in dopamine levels compared to all other control genotypes (P,0.05, Tukey’s Post Test for means). In males, both the Crz-rpr and Crz-ork lines were significantly different than control parental genotypes. doi:10.1371/journal.pone.0009141.g006 are events that preceded these observations [38]. Additionally, we report similar patterns of altered dopamine levels in two different life stages, those of wandering third instar larva and adults, suggesting that altered dopamine levels are a specific consequence of corazonin neuronal manipulations. Corazonin Expression Decreases during Stress The above results demonstrate a requirement of corazonin neurons within multiple behavioral and physiological responses accompanying different stresses. However, a possible explanation for these phenotypic differences is that corazonin neurons participate in the developmental organization of responsible central and/or peripheral targets as opposed to acute actions of these neurons during stress. To begin to differentiate between these possibilities, we measured corazonin transcript expression during periods of stress. To gauge relative expression of the corazonin transcript, we normalized levels to those ascertained for RP49. These experiments show a decrease in corazonin transcript expression following starvation and osmotic stress, and such decreases were more pronounced in males (Figure 7). This rapid decline of expression levels gradually returns to normal levels as a function of starvation stress duration in males. Corazonin transcript levels were largely unaffected by oxidative stress. Discussion Our observations implicate corazonin neurons as a critical anatomical locus that participates in stress response, however, we cannot rule out the possibility that the phenotypes associated with corazonin neuronal manipulations reflect changes in an unknown co-transmitter. However, there is no other known transmitter that is co-expressed with corazonin, and previous functional descrip- tions of the corazonin peptide are consistent with a role in mediating stress responses. Specifically, previous analysis of the actions associated with corazonin, include acceleration of heart rate [5], association with the ecdysial behavioral program and the circadian system [7,8], and corazonin levels are correlated with gregarious phase pigmentation in Locusta [6]. We suggest that these disparate phenotypes might reflect a general involvement of corazonin neurons functioning within stress responses for the reasons given previously (Figure 8). We also note that the demonstration of corazonin neuron involvement within stress response behaviors is paralleled by previous observations describ- ing suppression of GnRH (suspected corazonin homolog, by virtue of receptor similarities) release as part of the stress response in mammals, by virtue of its modulation of stress hormones [4]. The observations that manipulations of crz neuronal physiology manifest as alterations in lifespan and other behavioral and physiological parameters during heterotypic stresses suggest a general contribution of crz neurons to stress responses. In general, stress responses are thought to reflect a shift away from physiologies less critical for the maintenance of homeostasis during adverse environmental changes [39]. Throughout the mammals, responses to stress include inhibition of urinary, digestive, immune, and reproductive physiologies and simulta- neous activation of cardiac, muscle, respiratory, and energy mobilization processes. The enhanced levels of activity are of significant selective advantage, as this would facilitate the removal PLoS ONE | www.plosone.org 10 February 2010 | Volume 5 | Issue 2 | e9141