Additionally, transcripts poten tially involved in the before deposition of lipids in the newly forming cuticle of crustaceans, were up regulated in the pre moult stage of P. pelagicus. A large diversity of genes representing many impor tant biological functions related to moulting in crusta ceans were able to annotated, and their expression profiles mapped across consecutive stages of the moult cycle of P. pelagicus in a time series manner. This approach aims to enhance the knowledge of the molecu lar mechanisms and regulating factors involved in the moult cycle, and allows the identification of target genes which may control important aspects of various stages of the moult cycle. Methods Animal selection P. pelagicus crabs were supplied by staff at the Depart ment of Employment, Economic Development and Inno vation, Bribie Island Research Centre.
The crabs were individually housed in a flowthrough system at an ambient water temperature of 24 C, and fed a commer cial diet twice daily. Two size groups of crabs were used, small crabs of an average carapace width of 4 cm, and larger crabs of an average carapace width of 11 cm. All crabs were moult staged by examination of pleopod paddles for epidermal retraction and grouped into the following moult stages, moult, post moult, intermoult early and late stage pre moult. cDNA library construction Two cDNA libraries were constructed using various source tissues, selected in order to provide a diverse col lection of transcripts, and representing a broad range of tissue functions and physiological states in all moult stages.
One of the cDNA libraries was synthesised from whole animals in order to obtain transcripts from each tissue type. For this library, six small crabs, from each of the following five moult stages, moult, post moult, inter moult, early and late pre moult stages, were selected, snap frozen and individually ground under liquid nitro gen. The second cDNA library was derived from organs previously identified as being important to the moult cycle of crustaceans and served to enrich the array with sequences particularly relevant to crustacean moulting. The tissues represented in the P. pelagicus organ library were brain, eyestalk, mandibular organ and Y organ. These tissues were obtained from six anaesthe tised large P. pelagicus crabs from each of moult, post moult, intermoult, and early and late pre moult stages, and stored in RNA later.
Total RNA was purified from each sample using TRI ZOL reagent as recommended by the manufacturer. Con centration and purity of the RNA were determined using a spectrophotometer with 260 and 280 nm readings. RNA quality was assessed for all sam ples by visualisation on Cilengitide a denaturing formaldehyde RNA gel and ethidium bromide staining. Each cDNA library was constructed by pooling equal amounts of total RNA from all moult cycle stages.