Genomic approach to the study of moulting and cuticle formation in crustaceans

Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles across all moult cycle stages was used in this study. Custom cDNA microarrays were constructed for P. pelagicus. The chips contained 5000 transcripts derived from both the whole organism, and from individual tissues such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages. Thus the arrays were designed to study global gene expression profiles of transcripts relevant to the moulting process, across the entire moult cycle. A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 217 singlets and 62 contigs, resulting in 279 unique putative genes. The isolated transcripts were classified into the following biological functions: cuticular proteins associated with calcified and/or uncalcified arthropod exoskeletons, FaMeT, proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators, ribosomal RNA relevant to protein biosynthesis, other sequences which did not fall into these groups, and those transcripts which displayed no significant sequence homology with sequences deposited in the NCBI database. Partial characterisation of the unannotated transcripts was achieved via conceptual domain analysis. Moult cycle-related differential expression profiles were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, which were shown to be up-regulated at key stages of moulting, and the gene for putative FaMeT which was up-regulated only in intermoult. The moult cycle-related differential expression profiles of genes involved in exoskeletal formation were analysed and described in detail. Additional characterisation was carried out in the form of conceptual domain analysis, uncovering many types of cuticle proteins. Twenty-one distinct transcripts representing crustacean cuticular proteins were isolated. Thirteen contain copies of the cuticle_1 domain, previously isolated only from calcified regions of the crustacean exoskeleton. Four transcripts contained a chitin_bind_4 domain, associated with both the calcified and un-calcified cuticle of crustaceans. Another four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally cryptocyanin, a haemolymph protein, involved in cuticle synthesis and structural integrity, also displayed moult cycle-related differential expression. Moult stage-specific expression analysis of these transcripts revealed that differential expression occurs both within transcripts containing the same domain and between transcripts containing different domains. Exoskeletal hardening in crustaceans occurs through the mineralisation and sclerotization of the organic matrix. Glycosylation levels of proteins in an organic matrix have been attributed to the regulation of calcification. Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display moult cyclerelated differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the postmoult stage, during which calcification takes place, implicating both in the regulation of calcification. Genes known to be involved in the phenoloxidase activation pathway, which facilitates sclerotization, also displayed moult cycle-related differential expression profiles. Trypsin and chymotrypsin were up-regulated in the intermoult stage when compared to postmoult, while trypsin was also up-regulated in premoult compared to ecdysis. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and postmoult. FaMeT is the enzyme responsible for the conversion of FA to MF in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three isoforms. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis, and the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present in only the long isoform of putative FaMeT