Changes in complement responses in Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) under crowding stress, plus viral and bacterial challenges

Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) were subjected to either experimental infection with Photobacterium damselae subsp. piscicida or Nodavirus after a period of 2 weeks of crowding in which fish were subjected to a 5-fold increase in density (10-50 kg/m(3)). Samples were obtained before the crowding period (0 h or control) and at 24h and 72 h after crowding from both groups of infected fish. The Complement haemolytic activity and the expression of the C3 gene were evaluated in blood and liver samples respectively. The bacteriolytic and lysozyme activities were also assessed. The results showed that Complement haemolytic activity was reduced at 72 h with both bacteria and virus in high density Gilthead seabream, and a similar increase was observed at low density. Bacteriolytic activity under both bacterial and viral challenges for both species was increased at 24h, under low density. At high density, the bacterial challenge did not induce significant changes. C3 mRNA abundance was substantially increased after pathogen treatments in low density groups at 24h but no significant changes were detected at high densities. These results support the idea of the suppressor effect of stressors on the immune system since a reduction of Complement activity under virus and high density, or lack of response in C3 expression under high density were observed.     

Morphological differences between wild and farmed Mediterranean fish

Gilthead seabream (Sparus aurata L.) and European seabass (Dicentrarchus labrax L.) are important commercial marine fish species both for aquaculture and fisheries in the Mediterranean. It is known that farmed individuals escape from farm facilities, but the extent of escape events is not easy to report and estimate because of the difficulty to distinguish between wild and farmed individuals. In this study, significant differences provided through morphometry evidence that the cranial and body regions of seabream and seabass are different regarding their farm or wild origin at different scales. Morphological variations have been shown to be a valuable tool for describing changes in shape features. Therefore, the biomass contribution of escapees to local habitats could be determined by identifying escaped individuals from fisheries landings as a first step to assess the potential negative effects of fish farm escapees on the environment, and their influence on wild stocks and local fisheries.     

Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the ‘MedFish’ SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4–99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.     

Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation

Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the alpha-glycoprotein subunit (alphaGSU) and gonadotropin beta subunits (Ibeta and IIbeta) of red seabream using the 5'- and 3'-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of alphaGSU, Ibeta, and IIbeta are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Ibeta mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexual maturation, indicating sexual dimorphism in the expression pattern of Ibeta. In contrast, IIbeta mRNA levels of both sexes are maintained at high levels from the beginning of gametogenesis to spawning season. These results are different than those of salmonids and suggest that GTH-I may have important roles in male, but not female, gametogenesis. GTH-II may be involved in regulation of early and late gametogenesis in both male and female red seabream.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Molecular characterization of the aryl hydrocarbon receptors (AHR1 and AHR2) from red seabream (Pagrus major)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs). Bony fishes exposed to PHAHs exhibit a wide range of developmental defects. However, functional roles of fish AHR are not yet fully understood, compared with those of mammalian AHRs. To investigate the potential sensitivity to PHAHs toxic effects, an AHR cDNA was initially cloned and sequenced from red seabream (Pagrus major), an important fishery resource in Japan. The present study succeeded in identifying two highly divergent red seabream AHR cDNA clones, which shared only 32% identity in full-length amino acid sequence. The phylogenetic analysis revealed that one belonged to AHR1 clade (rsAHR1) and another to AHR2 clade (rsAHR2). The rsAHR1 encoded a 846-residue protein with a predicted molecular mass of 93.2 kDa, and 990 amino acids and 108.9 kDa encoded rsAHR2. In the N-terminal half, both rsAHR genes included bHLH and PAS domains, which participate in ligand binding, AHR/ARNT dimerization and DNA binding. The C-terminal half, which is responsible for transactivation, was poorly conserved between rsAHRs. Quantitative analyses of both rsAHRs mRNAs revealed that their tissue expression profiles were isoform-specific; rsAHR1 mRNA expressed primarily in brain, heart, ovary and spleen, while rsAHR2 mRNA was observed in all tissues examined, indicating distinct roles of each rsAHR. Furthermore, there appeared to be species-differences in the tissue expression profiles of AHR isoforms between red seabream and other fish. These results suggest that there are isoform- and species-specific functions in piscine AHRs.     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.     

Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European seabass (Dicentrarchus labrax)

The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), β-actin (two regions of β-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), β2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization.     

Conservation of RET proto-oncogene splicing variants and implications for RET isoform function

The RET proto-oncogene encodes a receptor tyrosine kinase required for development of the kidney and neural crest-derived cell types. Alternative splicing of the 3' exons of human RET results in three protein isoforms with distinct C-termini: RET9, RET51, and RET43. These RET isoforms show differential binding to downstream adapter molecules, suggesting they may have distinct signaling functions. We have characterized Ret 3' sequences in mouse and investigated alternative splicing of this region. We found that the organization of Ret 3' sequences is very similar to human RET. The mouse locus also has alternatively spliced C-terminal coding regions, and the sequences corresponding to RET9 and RET51 are highly conserved in both position and sequence with the human locus. Further, we compared the predicted C-terminal amino acids of RET9 and RET51 in seven vertebrate species, and found that they are well conserved. We have identified sequence encoding a putative ret43 isoform in mouse, however the predicted amino acid sequence showed low homology to human RET43. Our data suggest that RET isoforms are evolutionarily highly conserved over a broad range of species, which may indicate that each isoform has a distinct role in normal RET function.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Molecular characterization and sex-specific tissue expression of estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a) and ovarian aromatase (cyp19a1a) in yellow perch (Perca flavescens)

Yellow perch (Perca flavescens) exhibits an estrogen-stimulated sexual size dimorphism (SSD) wherein females grow faster and larger than males. To aid in the examination of this phenomenon, the cDNA sequences encoding estrogen receptor-alpha (esr1), estrogen receptor-betaa (esr2a) and ovarian aromatase (cyp19a1a) for the teleost yellow perch were obtained. Several tissues were analyzed from both male and female adult yellow perch for sex-specific tissue expression. The full length cDNAs of yellow perch esr1, esr2a and cyp19a1a consist of 3052 bp, 2462 bp and 1859 bp with open reading frames encoding putative proteins of 576 amino acids, 555 amino acids and 518 amino acids, respectively. Esr1 and esr2a expression was highest in female ovary and liver tissues with low to moderate expression in other tissues. Esr2a showed a more global tissue expression pattern than esr1, particularly in males but also in females. Cyp19a1a expression was highest in both male and female spleen tissue and oocytes with moderate expression in male pituitary and gill tissue. Cyp19a1a expression was moderately high in female liver tissue with undetectable expression in male liver tissue, suggesting its involvement in sexually dimorphic growth. These sequences are valuable molecular tools that can be used in future studies investigating estrogen mechanisms and actions, such as SSD, in yellow perch.     

A peroxidase gene family and gene trees in Heterobasidion and related genera

Four putative peroxidase-encoding gene fragments, named mnp1a, mnp1b, mnp2 and mnp3, were amplified with degenerative primers from the white-rot basidiomycete genus Heterobasidion. The fragments were cloned and sequenced. Similar fragments were produced and analyzed from the related genera Amylostereum, Bondarzewia and Echinodontium. Each amplified fragment contains three identically positioned introns. According to the predicted amino acid sequence, these fragments are most similar to two Mn peroxidase-encoding genes (MPGI and mnp2) and gene pgv of Trametes versicolor. Conserved residues thought to be essential for peroxidase function were identified. All four MnP gene loci of Heterobasidion were detected only in H. parviporum. Variation occurred in the predicted amino-acid sequences (131-132 amino acids) of all four fragments originating from the 47 Heterobasidion isolates tested. Amino acid variation in fragments of mnp2 and mnp3 separated European Heterobasidion parviporum ("S-type") and H. abietinum ("F-type"), known to have identical rDNA sequences. Asian and western North American isolates from fir, spruce and other hosts had the peroxidase amino acid sequences of European H. parviporum. American and European H. annosum ("P-type") isolates had different amino acid sequences and might be cryptic species.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

Cloning of a neonatal calcium atpase isoform (SERCA 1B) from extraocular muscle of adult blue marlin (Makaira nigricans)

Complete cDNAs for the fast-twitch Ca2+ -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin (Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa. Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2+ -ATPase normally expressed in early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source of functional differences between fish and mammalian SERCAs.     

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.