After Experiment selleck chem inhibitor 2, we decided to test the three groups as pools, and chose growth neurotrophic genes. A separate experi ment was carried out with embryonic treatments identi cal to those used in Experiment 1. Whole embryos were homogenized in TRIzol using a Mini Bead Beater 8, and total RNA isolation was as described above. Two differ ent pools were created for each condition, Control1, ALC NTC1, ALC NTO1, Control2, ALC NTC2, ALC NTO2. The relative quantification of expression of each RNA pool was performed using the ABI Prism 7700 Sequence Detection System and calculated using the standard curve method. In each experiment, a relative expression level was determined for the two pools from each group in triplicate, 3 4 repeat experiments were performed, resulting in 18 24 values from each group.

The treatment groups were compared with one way ANOVA followed by Students t test. Moulting is a cyclic process that occurs in all arthro pods, from insects to crustaceans, and is essential for growth, reproduction and metamorphosis. The crusta cean moult cycle encompasses the period between two successive moults and has been subdivided into 4 major stages, intermoult, pre moult, ecdysis, and post moult. The intermoult period is the longest stage of the moult cycle, during which muscle regeneration and the accumulation of energy reserves such as glycogen and lipids occurs. Pre moult sees the atrophy of somatic muscle, the resorption of the old exoskeleton, and the formation of a new exoskeleton in preparation for the onset of ecdysis.

Ecdysis, or the moult itself, involves the shedding of the exoskeleton through a rapid uptake of water from the environment, causing the exoskeleton to rupture. Further water uptake occurs during post moult facilitating the expansion of the new, still soft, exoskeleton, this expansion is essential for the growth of the animal. Exoskeletal hardening, via scleroti zation and mineralisation, then takes place. Moulting is regulated by an elaborate interplay of hormones, including those which promote, and those which negatively regulate moulting. Among the hor mones involved in the induction of moulting are two families of nonpeptidergic hormones, the steroids, and the sesquiterpenoids and crustacean methyl farnesoate. Ecdysteroids initiate and coordinate each moult, and are synthesised and secreted by the Y organs.

MF is synthesised by the mandibu GSK-3 lar organs, and has been implicated in the regulation of crustacean morphogenesis, metamorphosis, reproduction and moulting. MF has been shown to directly stimulate the secretion of ecdysteroids in Cancer magister Y organs. Additionally, the duration of premoult was significantly reduced in the prawn Penaeus setiferus that had been implanted with mandibular organs from C. magister. The negative regulatory centre in crustaceans is the sinus gland X organ complex, a neurohaemal organ located in the eyestalk.