Knock down of dilp2 did, however, lead to an increase in total RAD001 mTOR inhibitor trehalose content of the same magnitude as that resulting from mNSC ablation. dilp2 expression is therefore not limiting for lifespan, and it plays an individual role only in the increase in trehalose storage among the phenotypes affected by mNSC ablation. Furthermore, our data show that increased trehalose storage alone is not sufficient to extend lifespan. It should be noted that the increase in lipid observed previously due to mNSC-ablation was not observed in the current study in the mNSC-ablated or dilp2RNAi flies. In addition, the increased hemolymph sugar phenotype of the mNSC-ablated flies was slightly different to that seen previously. The alteration of these metabolic phenotypes may be due to the yeast used or the sucrose content in the food for the current experiments, both of which have been optimised for VE-822 lifespan measurements. The yeast used in this study is from a different supplier than that used previously, and the nutritional make-up of yeasts from different suppliers varies greatly resulting in different effects on lifespan and fecundity. The metabolic phenotypes in the ablated flies appear to be sensitive to the nutrient content of the food, and the same may account for the loss of the paraquat resistance phenotype in the mNSC-ablated flies, where differences in the total antioxidant activity of the yeasts may have resulted in a difference in paraquat tolerance. dilp2 is the most highly expressed of the mNSC-derived dilps, and several studies have indicated its functional significance and the prominent role it may play in mediating lifespan-extension due to reduced IIS, prompting us to investigate the role of DILP2 directly. We were successful in specifically reducing both the levels of RNA and protein, and found that DILP2 levels were limiting for only one of the phenotypes resulting from mNSC ablation, increased whole body trehalose content. It remains quite possible that the lifespan extensions due to the FOXO, JNK or p53 manipulations were mediated by reductions in one or more of DILPs 2, 3 and 5, because the context in which the level of dilp2 was lowered was different in each case. This could, for instance, have resulted in different levels of transcript for other dilps or different states of the intracellular IIS pathway. Our findings do show clearly, however, that neither reduction in dilp2 RNA and DILP2 protein expression alone nor increased trehalose storage alone is sufficient to extend lifespan. Of the phenotypes observed on mNSC-ablation, we found that reduction in DILP2 had an effect only on whole body trehalose levels. This result could indicate that reduction in DILP2 per se is required for increased stored tehalose upon mNSC-ablation. Alternatively, if there is functional redundancy between the 3 DILPs produced in the mNSCs, it is possible that trehalose storage is the most sensitive of the phenotypes to a reduction in the overall expression of DILPs in the mNSCs.