Discussion

We have used genetic alterations of OA and TA action to elucidate the role of these amines in survival and sugar responsiveness of fruit flies. Our data suggest complex, central and peripheral actions of these amines on physiology and behavior.
We have shown that the tßh gene is involved in starvation-induced survival and an increase in sugar response. The phenotype was reported in females (Fig. 1) and males (Fig. 6), in three different genetic backgrounds (w+ and w-,tßhnM18; hs-tßh and w-,tßhnM18, UAS-tßh) and is independent of the egg-retention phenotype Partridge 1987, which is rescued in w-;tßhnM18;UAS-tßh control mutant flies. It is interesting to see that the sugar response phenotype appears to vanish with longer starvation periods Yang 2015. The phenotype  was not found in previous reports focused on the learning phenotype of these flies Schwaerzel 2003, possibly because the assay used was dependent on locomotion, which is also affected in tßhnM18 mutants Koon 2010Fox 2006Saraswati 2004. Complementary results were obtained using a different approach in Drosophila Scheiner 2014 and also in Apis melifera (companion paper).
OA/TA and metabolism
Since sugar response is dependent on starvation Colomb 2009, a decreased sugar response as found in tßhnM18 mutants can be understood as resistance to the starvation treatment, an hypothesis that our results appeared to confirm. Indeed, we found that the levels of carbohydrates in the hemolymph of tßhnM18 mutant flies are higher after starvation than in control flies (Fig. 2). Since trehalose constitutes the energy store of a fly and its hemolymph concentration reflects starvation level Thompson 2003 Isabel 2004, it is reasonable to argue that the mutant flies were affected less by the starvation treatment than the controls, even though they were deprived of food for the same amount of time. This interpretation is also supported by longer survival of tßhnM18 mutants under starvation conditions (Fig. 3, a result which we independently replicated Li 2016Scheiner 2014: our experiments were carried out before the ones cited). Complementing our analysis in flies, injection of the OA-receptor antagonist epinastine in honey bees also prolonged survival (companion paper). Taken together, these results suggest that the absence of OA-signaling saves the mutant animal’s energy, making them less sensitive to starvation, a conclusion in line with previous reports on the role of OA in trigylceride Erion 2012Woodring 1989 and carbohydrate Park 1998Blau 1994 metabolism.
OA/TA and sugar responsiveness.
While tßh is affecting starvation resistance, we asked whether it could also have a role on the neuronal modification due to starvation, and our results could separate the two phenotypes. The mutant phenotype is partially rescued by acute tßh expression, while expression during the starvation period had no effect (Fig. 4). This suggests that the decrease of carbohydrate levels is not the only tßh-dependent starvation-induced alteration that leads to a normal sugar response. Indeed, the sensitivity of the sugar sensing neurons is affected by TA/OA imbalance (Fig. 5), but only after starvation. Interestingly, the control w+ strain did not show the expected Nishimura 2012Meunier 2007 increase in sensitivity after starvation (Fig. 5A), while more common wild type strains showed the increase in the same experiment (Fig. 5B). Since the w+ control flies do show an increase in their proboscis extension response to sugar (Fig. 1), there must be a modulatory mechanism downstream of taste receptor activity. Taken together, these data suggest that in addition to the internal state that is altered by starvation, both the sensory input and the likelihood to extend the proboscis for the same input are modified.
Where is the site of OA/TA-action?
In order to identify the cells contributing to the behavioral response to sucrose and the metabolic response to starvation, we expressed tßh in different cells inside or outside the nervous system in the mutant flies, using the UAS/Gal4 system (Fig. 6). The predicted effect of this manipulation is a production of OA and a decrease in the concentration of TA in the affected cells. Ubiquitous expression of tßh with the actin-Gal4 driver does increase the PER of starved flies. The non-neuronal Tdc1-GAL4-driver drives expression in crop and hind gut tyraminergic cells Blumenthal 2009Chintapalli 2007Cole 2005, that normally do not produce OA, but only TA Monastirioti 1995. Ectopic production of OA in these cells rescues the sugar responsiveness phenotypes (Fig. 6). Because ectopic OA would lack necessary receptors, we tentatively interpret this result as an effect of presumably reduced TA levels. However, the OA produced might also be released into the heamolymph and taken up by neurons, as is proposed to happen when feeding OA Schwaerzel 2003Scheiner 2014. Interestingly, pan-neuronal tßh expression with nsyb-Gal4, but not expression with drivers specifically labeling OA/TA neurons (tdc2-Gal4 and NP7088-Gal4) rescues the phenotype. These results suggest that both neuronal and non-neuronal tissues are affecting the starvation-induced increase in sugar responsiveness (and that the two most commonly used OA/TA drivers remain suboptimal tools to study OA action).
OA and TA specificity
The TßH enzyme converts TA into OA such that tßhnM18 mutants not only lack OA but also accumulate TA. To disentangle the roles of the two amines, we tested OA- or TA-receptor mutants in two experiments, starvation survival and starvation-induced sugar response (Fig. 7). We decided not to test TDC2 mutants, since the mutant also affect both OA and TA levels and both similar and dissimilar phenotypes could be explained by the effect on TA actions only. Perhaps not surprisingly, given that several processes appear to mediate both starvation-induced effects, we also found the sugar responsiveness and the starvation resistance phenotypes of the tested mutants to be separable as well: some mutants exhibit a phenotype in none (oamb286, oamb584), both (tßhnM18, honoka), or in individual assays: only in starvation resistance (TyrII-TyrR∆124) or only in sugar responsiveness (TyrR∆124). These results reinforce our previous conclusion that  starvation resistance and sugar response are not mediated by the same OA/TA-cells and receptors, but by different sub-populations. In addition, the data indicate that both OA and TA play a role in starvation-induced sugar response. OA- and TA-receptor mutants tend to perform similarly, suggesting they may not be counteracting each other in this behavior, as previously suggested for crawling behavior Saraswati 2004 or for flight Brembs 2007.

Conclusions

Taken together with the experiments from our accompanying paper(Buckemüller et al.,), our results suggest that the
OA/TA-system is involved in both the physiological and the behavioral
changes that follow starvation, and that these changes are regulated
independently. They also show that the behavioral change is due not
only to a modulation of the taste neuron activity and to action of
TA-specific cells in peripheral, non-neuronal organs, but that a more central
effect seems additionally to be at play.