Endogenous PolyP levels were measured as described (Ruiz et al., 2001). Briefly, 24-h-old eggs were homogenized in distilled water at 4 °C. For long-chain PolyP, egg homogenate was added to lysis buffer (6 M Guanidine Thiocyanate, 50 mM Tris–HCl pH 7.0) and powdered glass was used to bind mTOR inhibitor PolyP. After DNase and RNase treatment, bound PolyP was eluted at 95 °C in 50 mM Tris–HCl pH 8.0. For short chain PolyP, egg homogenate was

added to 0.5 N HClO4 followed by neutralization using KOH and KHCO3. Both long chain and short chain PolyP levels were determined as the amount of Pi released upon treatment with an excess of recombinant exopolyphosphatase from Saccharomyces cerevisiae (scPPX). Aliquots of short chain or long chain polyP were incubated for 15 min at 37 °C in reaction medium (60 mM Tris–HCl pH 7.5, 6 mM MgCl2) containing purified scPPX and the malachite green assay was used for Pi quantification

( Gomes et al., 2010). Following, the ability of agAP to hydrolyze endogenous PolyP was evaluated by addition of 1.5 μg of agAP in a reaction medium (10 mM DTT, 10 mM EDTA, 0.1 M sodium acetate buffer pH 4.0) containing either short chain or long chain PolyP obtained from A. gemmatalis eggs. Incubation was held for 60 min at 37 °C. After that, PolyP levels were determined by the scPPX assay. Freshly-laid eggs were collected and left to develop for different times before homogenization in 20 mM Hepes pH 7.5 supplemented with P8340 protease inhibitor cocktail. After centrifugation (10,000g, 10 min, 4 °C), supernatants were submitted to 12.5% SDS–PAGE Fossariinae and stained check details with Coomassie blue. Egg homogenates were prepared using 24- and 48-h-old egg extracts in 50 mM sodium acetate pH 5.0 followed by two washing steps (10,000g, 10 min, 4 °C). Protease assays were performed at room temperature in a reaction medium (0.2 M NaCl, 5 mM EDTA, 2.5 mM DTT, sodium acetate buffer 50 mM pH

5.0) containing 5 μM z-Phe-Arg-AMC. Steady-state velocities were obtained by linear regression of the hydrolysis curve ( Lima et al., 2001) as followed in an Fmax fluorescence microplate reader (molecular Devices) using 380/440 nm as excitation/emission wavelengths for 30 min. When expressed, the influence of PolyP was determined by the addition of PolyP-3, -25 or -75 into the reaction medium. As yolk granule hydrolases are strongly associated with yolk mobilization, we wondered whether yolk mobilization was correlated with hydrolase activity during Anticarsia development. From homogenates prepared from eggs at different times after oviposition, we observed a smooth mobilization of major storage proteins around 80, 30, and 10 kDa ( Fig. 1A). The first evidences of yolk mobilization were observed 20–40 h after oviposition – the same period where an increase of acid phosphatase activity was also detected in egg extracts ( Fig.