Comparative proteomic profiling represents an inhibition of protein synthesis to regulate osmotic stress in Nile tilapia (Oreochromis niloticus) embryos.

Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, can tolerate up to 30 g.L-1 SW but rarely produce offspring. The embryos of SW-acclimated O. niloticus survived equally well from 0- to 10-g.L-1 environment but not under 20-g. L-1. However, when the embryos were incubated under 10 g.L-1 during days 0-3, and then the salinity was suddenly shifted to and maintained at 20 g.L-1 during days 4-6, their survival rate was comparable to those incubated under 0 and 10 g.L-1. To elucidate a molecular adaptation of the embryos that survived different salinity environments, the proteomic profiles of the newly hatched embryos, or early larvae, hatched under 0 g.L-1, 10 g.L-1, and those being incubated at 10 g.L-1 during days 0-3 followed at 20 g.L-1 during days 4-6 were compared. Total proteins extracted from the samples were identified with a gel-free shot-gun proteomics approach using the Nile tilapia protein database. The early larvae from the three groups expressed 2295 proteins, and 279 proteins showed statistically different expressions among groups. Downregulation of the 182 proteins in the larvae hatched under 10 and 20 g.L-1 was found to include 22 proteins that are responsible for cellular responses to osmotic stress. This adaptation may be a crucial factor in reducing cellular metabolism and ion transport between the intra- and extra-cellular environment to stabilize cellular osmolality. In addition, some of these proteins suppress cellular damage from oxygen free radicals generated from the osmotic stress. Eighty-seven proteins significantly changed in the larvae hatched under 20 g.L-1 were clustered. Nineteen of the cellular stress response proteins, which were considered to be mortality induction, were described.

Development of polymorphic microsatellites for genetic studies of white scar oyster (Crassostrea belcheri) using paired-end shotgun sequencing.

White scar oyster Crassostrea belcheri is a commercially important bivalve species in Thailand. Appropriate genetic markers are needed for effective management to elevate its production efficiency. Type II microsatellites of C. belcheri were identified and characterized using an Illumina paired-end shotgun sequencing. A total of 14,743,710 reads were generated for which 198,849 reads containing microsatellites and 217,998 microsatellite loci were found. Twenty out of 60 microsatellite loci (33.33%) were polymorphic and these microsatellites were further tested against DNA bulks (N = 10 each) originating from 7 different geographic locations in Thai waters. Results indicated that newly developed microsatellites can be used for genetic diversity analysis of C. belcheri. Genotyping of C. belcheri collected from Surat Thani (Gulf of Thailand; N = 50) were performed. The number of alleles per locus ranged from 2 to 12 (average = 4.95). Observed and expected heterozygosities ranged from 0.0000 to 0.9400 (average = 0.3419) and 0.1139 to 0.8190 (average = 0.5844), respectively. Genome information and 20 newly isolated microsatellites will facilitate further studies in population genetics, stock management, and genetic improvement of C. belcheri in Thailand.

A novel beta-defensin antimicrobial peptide in Atlantic cod with stimulatory effect on phagocytic activity.

A novel defensin antimicrobial peptide gene was identified in Atlantic cod, Gadus morhua. This three exon/two intron defensin gene codes for a peptide precursor consisting of two domains: a signal peptide of 26 amino acids and a mature peptide of 40 residues. The mature cod defensin has six conserved cysteine residues that form 1-5, 2-4 and 3-6 disulphide bridges. This pattern is typical of beta-defensins and this gene was therefore named cod beta-defensin (defb). The tertiary structure of Defb exhibits an α/ß fold with one α helix and ß1ß2ß3 sheets. RT-PCR analysis indicated that defb transcripts were present mainly in the swim bladder and peritoneum wall but could also be detected at moderate to low levels in skin, head- and excretory kidneys. In situ hybridisation revealed that defb was specifically expressed by cells located in the swim bladder submucosa and the oocytes. During embryonic development, defb gene transcripts were detectable from the golden eye stage onwards and their expression was restricted to the swim bladder and retina. Defb was differentially expressed in several tissues following antigenic challenge with Vibrio anguillarum, being up-regulated up to 25-fold in head kidney. Recombinant Defb displayed antibacterial activity, with a minimal inhibitory concentration of 0.4-0.8 µM and 25-50 µM against the Gram-(+) bacteria Planococcus citreus and Micrococcus luteus, respectively. In addition, Defb stimulated phagocytic activity of cod head kidney leucocytes in vitro. These findings imply that beta-defensins may play an important role in the innate immune response of Atlantic cod.

Ubiquitous presence of piscidin-1 in Atlantic cod as evidenced by immunolocalisation.

BACKGROUND: Antimicrobial peptides (AMPs), the natural antibiotics bestowed upon all forms of life, consist of small molecular weight proteins with a broad spectrum antimicrobial activity against a variety of pathogenic microorganisms. Piscidins are one of the AMP families that are imperative for the innate defence mechanisms of teleosts. Atlantic cod, a basal fish belonging to the superorder Paracanthopterygii also possesses multiple piscidin peptides. Two piscidin paralogues (pis1 and pis2) and a novel alternative splice variant of pis2 of this fish were previously described by us. To shed light on other potent roles of these molecules, now we have mapped the distribution of piscidin 1 (Pis1), in different tissues and organs of cod through immunohistochemistry (IHC) employing an affinity purified polyclonal antibody specific to Pis1. RESULTS: Various cell types and tissues of Atlantic cod including those from the immune organs of naïve fish are armed with Pis1 peptide. Different types of the blood leucocytes and phagocytic cells among the leucocytes examined gave a relatively strong indication of Pis1 immunopositivity. In addition, other cell types such as hematopoietic cells, epithelial cells and multi-granular cells located in the mucosal and hematopoietic tissues were also Pis1-immunoreactive. More interestingly, chondrocytes appear to produce Pis1 and this is the first report on the presence of an AMP in cartilage tissue of fish. Furthermore, Pis1 immunopositivity was detected in other tissues and organs of naïve fish including neural tissues, exocrine and endocrine glands, compound gland cells, excretory kidney, intestinal and respiratory epithelial cells, swim bladder, skin and hypodermis layer, myosepta, liver, heart, eye and oocytes. CONCLUSIONS: Pis1 peptide is produced by various cell types located in different tissues and organs of Atlantic cod. It is present in all immune-related organs of naïve fish and the elevated peptide expression following phagocytosis strongly suggest their involvement in innate defence. Further, its widespread occurrence in non-immune tissues and organs of apparently healthy fish implies that piscidin may have other functions in addition to its role as an immune effector molecule.

Differential expression and biological activity of two piscidin paralogues and a novel splice variant in Atlantic cod (Gadus morhua L.).

The piscidin (pis) family of potent antimicrobial peptides with broad-spectrum activity has an important role in innate host defence. We have identified and characterized two pis paralogues in Atlantic cod (pis1 and pis2), as well as a novel splice variant of pis2, termed pis2-ß. Pis1 and pis2 genes have most likely originated from a recent duplication event, since they share the same four-exon structure with up to 91% identity at the intron level. The alternative transcript pis2-ß is derived from intron retention and even if not translated it may regulate pis expression through nonsense mediated decay. In spite of their overall conservation, pis genes are being shaped by positive selection and pis1, pis2 and pis2-ß code for structurally diverse mature peptides, which have different functional properties. Synthetic Pis1 displays antibacterial activity in the micromolar range against Gram-(+) and Gram-(-) bacteria, including the fish pathogens Vibrio anguillarum and Yersinia ruckeri. In contrast, synthetic Pis2 and Pis2-ß have limited or no antibacterial activity, respectively, but exhibit more potent antiparasitic activity against Tetrahymena pyriformis. In adult cod, pis1 and pis2-ß are constitutively expressed in immune-related organs, whereas pis2 is constitutively expressed in all tissues examined. Differential expression is also observed during embryonic development. In particular, pis2 and pis2-ß are maternally inherited but pis1 transcripts are only present from gastrulation onwards. It was found that antigenic challenge with attenuated V. anguillarum induces a general down-regulation of all pis in head kidney, spleen and distal intestine, suggesting that they may be used as health indicators. Taken together, our data indicate that pis is an important component of the cod innate immune system. Moreover, the two pis paralogues have undergone structural diversification and it is likely that they play multifunctional roles in Atlantic cod.

Atlantic cod piscidin and its diversification through positive selection.

Piscidins constitute a family of cationic antimicrobial peptides that are thought to play an important role in the innate immune response of teleosts. On the one hand they show a remarkable diversity, which indicates that they are shaped by positive selection, but on the other hand they are ancient and have specific targets, suggesting that they are constrained by purifying selection. Until now piscidins had only been found in fish species from the superorder Acanthopterygii but we have recently identified a piscidin gene in Atlantic cod (Gadus morhua), thus showing that these antimicrobial peptides are not restricted to evolutionarily modern teleosts. Nucleotide diversity was much higher in the regions of the piscidin gene that code for the mature peptide and its pro domain than in the signal peptide. Maximum likelihood analyses with different evolution models revealed that the piscidin gene is under positive selection. Charge or hydrophobicity-changing amino acid substitutions observed in positively selected sites within the mature peptide influence its amphipathic structure and can have a marked effect on its function. This diversification might be associated with adaptation to new habitats or rapidly evolving pathogens.

Antimicrobial activity in the tissues of Atlantic cod (Gadus morhua L.).

Antimicrobial peptides (AMP) are important components of the innate immune system in metazoans. They have been studied widely in several fishes, but little is known about these defence factors in Atlantic cod, which is thought to have a less sophisticated adaptive immune system compared to other teleosts. The aim of the present study was to screen for potential AMPs in various tissues of Atlantic cod and to examine their spectra of activity. Acidic crude extracts were prepared from thirteen tissues (i.e. mucus, gills, skin, intestine, rectum, head kidney, spleen, blood, gall bladder, liver, ovary, muscle and peritoneal wall). Following partial purification by solid-phase extraction, 78 fractions were obtained and these were assayed for antimicrobial activity using a two-layer radial diffusion assay. Some of the fractions prepared from several tissues examined had potent activity against the test bacteria. In general, acetonitrile rich fractions displayed higher antibacterial activity than the aqueous ones. The most potent fractions were obtained from the gall bladder and they exhibited potent antimicrobial activity against 8 of the 9 test bacteria, including the cod pathogen Vibrio anguillarum. Antibacterial activity was completely eliminated or reduced upon treatment with proteinase K in most fractions. Protein profiles obtained by SDS-PAGE and two-dimensional gel electrophoresis showed that antimicrobial activity of the partially purified tissue extracts might be due to cationic, low molecular weight peptides.