2.4. Digestive enzyme activities
Enzymatic activities were determined on digestive organs (hepatopancreas and intestine) for each individual. Single tissues were homogenized in 350–460 μl distilled water and sonicated in ice bath until total tissue disintegration (Ultrasonic Dismembrator, Fisher Scientific, USA). The homogenates were then centrifuged for 20 min at 16900 × g at 4 °C and the extracts used for the analysis. Fluorometric analyses were performed to determine the digestive activities except for neutral lipase, which was assayed as shown by Iijima et al. (1998). Trypsin and pancreatic lipase were measured according to Rotllant et al. (2008), modified by Toledo-Cuevas et al. (2011). Total alkaline proteases were performed based on Twining (1984) and amylase activity was recorded by the EnzChek Ultra AmylaseAssay kit (E33651 Molecular Probes, Invitrogen). The fluorometric measurements were carried out using a Fluoroskan Ascent equipment (Thermo Fisher Scientific, Vantaa, Finland). The activities were reported as total activity [Fluorescence Units (FU) per segment (FU segment-1)] and specific activity (FU mg protein-1). For neutral lipase, 1 U of enzyme activity was defined as 1 mmol of hydrolyzed substrate released per min. Each assay was performed in duplicate. Protein concentration was measured in homogenates as described by Bradford (1976).