N-acetyl-ß-d-glucosaminidase activity in Palaemon serratus - Methodological optimisation and intrinsic variability.

Chitinolytic enzymes fulfil a key role in the moulting process of crustaceans, in degrading the endocuticle during apolysis. Measuring the enzyme activity is an interesting manner to monitor the moult process at sub-individual level, complementary to the classical observation of the integument morphogenesis, ecdysis success, or moult cycle duration. The present study aimed to optimise the methodology of using N-acetyl-ß-D-glucosaminidase (NAGase) activity to monitor moulting in the marine prawn Palaemon serratus, and to compare NAGase activity levels along the moult cycle of both male and female specimens. First, to optimise protocols for five different organs, different reaction medium compositions were tested, considering the type buffer, concentration of the substrate, and the load in enzymatic extract. Second, levels of NAGase activity were closely monitored during eight moulting stages in male prawns. Variations in NAGase activity were observed during the moult cycle, with an increase in activity in the late premoult phase of approximately 2.4-fold the level of the intermoult phase. This response profile was observed for each tested organ. The levels of NAGase activity of male and female specimens were compared during three stages of the premoult phase. The patterns observed for both sexes were similar for all the tested organs.

Additive effect of calcium depletion and low resource quality on Gammarus fossarum (Crustacea, Amphipoda) life history traits.

Gammarus fossarum is an often-abundant crustacean detritivore that contributes importantly to leaf litter breakdown in oligotrophic, mainly heterotrophic, headwater streams. This species requires large amounts of Ca to moult, thus allowing growth and reproduction. Because resource quality is tightly coupled to the organism's growth and physiological status, we hypothesised that low Ca concentration [Ca] and low food resource quality (low phosphorus [P] and/or reduced highly unsaturated fatty acid [HUFA] contents) would interactively impair molecular responses (gene expression) and reproduction of G. fossarum. To investigate the effects of food resources quality, we experimentally manipulated the P content of sycamore leaves and also used diatoms because they contain high amounts of HUFAs. Three resource quality treatments were tested: low quality (LQ, unmanipulated leaves: low P content), high quality 1 (HQ1; P-manipulated leaves: high P content), and high quality 2 (unmanipulated leaves supplemented with a pellet containing diatoms: high P and HUFA content). Naturally, demineralised stream water was supplemented with CaSO4 to obtain three Ca concentrations (2, 3.5, and 10.5 mg Ca L-1). For 21 days, pairs of G. fossarum were individually exposed to one of the nine treatments (3 [Ca] × 3 resource qualities). At the individual level, strong and significant delays in moult stage were observed in gammarids exposed to lower [Ca] and to lower resource quality, with additive effects lengthening the duration of the reproductive cycle. Effects at the molecular level were investigated by measuring expression of 12 genes involved in energy production, translation, or Ca or P homeostasis. Expression of ATP synthase beta (higher in HQ2), calcified cuticle protein (higher in HQ1 and HQ2), and tropomyosin (higher in HQ2 compared to HQ1) was significantly affected by resource quality, and significant additive effects on Ca transporting ATPase expression were induced by [Ca] and resource quality (higher for low [Ca] and higher resource quality). These results highlight the potential drastic deleterious effects of water [Ca] depletion on G. fossarum physiology, populations, and ecosystem functioning, especially in oligotrophic environments.