Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.

Differential expression profiling of components associated with exoskeletal hardening in crustaceans

Background: Exoskeletal 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. Results: Two 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. Conclusion: Cuticle 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.

Gene Expression Profiling of the Cephalothorax and Eyestalk in Penaeus Monodon during Ovarian Maturation.

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-beta-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters

Carotenoids are associated with various health benefits, such as prevention of age-related macular degeneration, cataract, certain cancers, rheumatoid arthritis, muscular dystrophy and cardiovascular problems. As microalgae contain considerable amounts of carotenoids, there is a need to find species with high carotenoid content. Out of hundreds of Australian isolates, twelve microalgal species were screened for carotenoid profiles, carotenoid productivity, and in vitro antioxidant capacity (total phenolic content (TPC) and ORAC). The top four carotenoid producers at 4.68-6.88 mg/g dry weight (DW) were Dunaliella salina, Tetraselmis suecica, Isochrysis galbana, and Pavlova salina. TPC was low, with D. salina possessing the highest TPC (1.54 mg Gallic Acid Equivalents/g DW) and ORAC (577 μmol Trolox Equivalents/g DW). Results indicate that T. suecica, D. salina, P. salina and I. galbana could be further developed for commercial carotenoid production.

Profiling ellagic acid content: The importance of form and ascorbic acid levels

As the importance of plant-based antioxidants to human health becomes clearer there is a rapidly expanding search for rich sources of these compounds. Much attention is currently focussed on the antioxidant potential of ellagic acid (EA). Making assessment difficult is that EA occurs in different forms: free EA, EA glycosides and polymeric ellagitannins. The overall structure of these forms has a pronounced effect on their antioxidant efficiency and is responsible for widely differing reactivity, solubility and hence bioavailability properties. Often associated with EA is vitamin C which also contributes to the plant foods total antioxidant activity. Previous studies have suggested that ascorbic acid may have protective effects on the polyphenol content of plants. With a view to gaining evidence that the bioactive forms of vitamin C influence EA content, several fruits with a range of EA and vitamin C contents were examined. To facilitate a more detailed assessment of the selected fruits antioxidant potential the relative proportions of EA forms were also determined. In strawberries and boysenberries EA content was predominantly in the polymeric form (21% and 12% free EA plus EA glycoside vs total EA levels for strawberry and boysenberry respectively), while in Kakadu plum it was mainly in the free form (70% of total EA). An increasing percentage of dehydroascorbic acid (9 to 14% of total vitamin C) indicating enhanced transformation of ascorbic acid to its oxidative degradation product together with stable free EA levels (≈ 950 mg/100 g DW) over the 4 month frozen storage period for the Kakadu plum samples are consistent with a possible protective effect of EA by ascorbic acid.