Abstract
BackgroundExoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from Portunus pelagicus were used to identify genes possibly associated with the activation pathways involved in these processes.ResultsTwo genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related 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 post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt.ConclusionCuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.
Highlights
Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix
The temporal differential expression patterns for genes relevant to cuticle hardening are summarised in Tables 1, 2, 3, 4, comparing post-molt and intermolt, intermolt and early premolt, and late pre-molt and ecdysis
A graphic representation of the log2 fold change in gene expression across molt stage comparisons is depicted in Figure 1. 280 unique transcripts were identified within the scope of the microarray experiments described, only those potentially associated with cuticle hardening will be discussed in this paper
Summary
Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Arthropods periodically undergo cyclic molting (shedding of the exoskeleton), a process that is essential for growth, metamorphosis and reproduction. At ecdysis the old exoskeleton is shed, followed by the expansion, continued deposition, and hardening of the new cuticle in post-molt, until the crab enters intermolt [2]. The crustacean intermolt cuticle is divided into four layers; the outermost is the epicuticle, beneath which lies the exocuticle (both are deposited pre-ecdysially), and below these are the principle and membranous layers of the endocuticle (deposited post-ecdysially) [3,4,5]. The crustacean cuticle is biphasic and is composed of an intertwining organic phase, consisting of chitin rods embedded in a protein matrix, and a mineral phase mainly comprised of calcium salts (in the calcified regions of the cuticle) [5]. The exocuticle, and principle layer of the endocuticle, contain both calcium salts and a matrix of chitin and protein, whereas the membranous layer contains the chitin-protein matrix but remains uncalcified [4,5]
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