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.

Plasma biochemistry and hematologic values for wild-caught flying foxes (Pteropus giganteus) in India

Although bats of the genus Pteropus are important ecologically as pollinators and natural hosts for zoonotic pathogens, little is known about their basic physiology. Hematology and plasma biochemistries were determined from wild-caught flying foxes (Pteropus giganteus) in northern India (n = 41). Mean lymphocyte differential count was higher for juveniles than adults. Mean platelet count was lower than previously reported. No hemoparasites were observed. No differences were observed between plasma biochemistry values of male and female bats, juveniles and adults, or lactating and nonlactating females. Variation in aspartate aminotransferase (AST) was seen based on body condition score. Blood urea nitrogen and cholesterol concentrations were lower in P. giganteus than other mammalian groups, but were consistent with those reported from other Pteropus species. Alanine aminotransferase and AST concentrations were higher than those reported for Pteropus vampyrus, a closely related species. This study provides basic physiologic information that can be used in future health and disease studies of Indian flying foxes.

Major Cellular and Physiological Impacts of Ocean Acidification on a Reef Building Coral

As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surface temperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warming oceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use of physiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with two scenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression, respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparent at the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodinium populations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent with metabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrate upregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactions and cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studies of ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts on calcification.

Rhipicephalus microplus serine protease inhibitor family: annotation, expression and functional characterisation assessment

Background: Rhipicephalus (Boophilus) microplus evades the host's haemostatic system through a complex protein array secreted into tick saliva. Serine protease inhibitors (serpins) conform an important component of saliva which are represented by a large protease inhibitor family in Ixodidae. These secreted and non-secreted inhibitors modulate diverse and essential proteases involved in different physiological processes. Methods: The identification of R. microplus serpin sequences was performed through a web-based bioinformatics environment called Yabi. The database search was conducted on BmiGi V1, BmiGi V2.1, five SSH libraries, Australian tick transcriptome libraries and RmiTR V1 using bioinformatics methods. Semi quantitative PCR was carried out using different adult tissues and tick development stages. The cDNA of four identified R. microplus serpins were cloned and expressed in Pichia pastoris in order to determine biological targets of these serpins utilising protease inhibition assays. Results: A total of four out of twenty-two serpins identified in our analysis are new R. microplus serpins which were named as RmS-19 to RmS-22. The analyses of DNA and predicted amino acid sequences showed high conservation of the R. microplus serpin sequences. The expression data suggested ubiquitous expression of RmS except for RmS-6 and RmS-14 that were expressed only in nymphs and adult female ovaries, respectively. RmS-19, and -20 were expressed in all tissues samples analysed showing their important role in both parasitic and non-parasitic stages of R. microplus development. RmS-21 was not detected in ovaries and RmS-22 was not identified in ovary and nymph samples but were expressed in the rest of the samples analysed. A total of four expressed recombinant serpins showed protease specific inhibition for Chymotrypsin (RmS-1 and RmS-6), Chymotrypsin / Elastase (RmS-3) and Thrombin (RmS-15). Conclusion: This study constitutes an important contribution and improvement to the knowledge about the physiologic role of R. microplus serpins during the host-tick interaction.