Sea Bass Head Kidney Research Articles

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Sensitive periods for 17β-estradiol exposure during immune system development in sea bass head kidney.

An increasing body of evidence suggests that sex steroids play an important role in the development and regulation of vertebrate immune defense. Therefore, compounds with estrogenic activity may influence the immune system via receptor-mediated pathways. The presence of estrogen receptors in immune cells and organs during the early stages of development may indicate that female steroid hormones are involved in the maturation of the fish immune system. This is of particular importance, as some marine fish are probably exposed to sources of exogenous estrogens while they reside in their estuarine nursery grounds. In this study, the influence of 17β-estradiol (E2) on estrogen receptor and cytokine gene expression was assessed in juvenile sea bass (Dicentrarchus labrax) together with characterization of the head kidney leukocyte populations and corresponding phagocytic activity during organ regionalization from 98 to 239 dph. E2 exposure, beginning at 90 dph resulted in indirect and delayed modifications of interleukin 1β and estrogen receptor α gene expression, which may affect B-lymphocyte proliferation in the sea bass head kidney. The E2 treatment of 120 dph fish led to an increase in estrogen receptor β2 and a decrease in transforming growth factor β1 gene expression, which coincided with decreased phagocytic activity of head kidney lymphocytes and monocytes/macrophages. Additionally, these changes were observed during developmental periods described as critical phases for B-lymphocyte development in mammals. Consequently, exogenous estrogens have the potential to modify the innate immune response in juvenile sea bass and to exert detrimental effects on head kidney development. Copyright © 2015 John Wiley & Sons, Ltd.

Elevated cortisol modulates Hsp70 and Hsp90 gene expression and protein in sea bass head kidney and isolated leukocytes

In fish, interactions between Hsps and cortisol are involved in stress modulated physiological processes including innate immune responses. Cortisol exerts a role in the regulation of Hsps synthesis. Fish head kidney is a lymphomieloid and endocrine organ releasing cortisol, and it is the central organ for immune-endocrine interactions. In sea bass, cortisol intraperitoneal injection and in vitro treatment of head kidney cells show that inducible Hsp70 and Hsp90 are modulated by this hormone. However, an inverse relationship between mRNA expression (real-time PCR) and Hsp70 and Hsp90 protein levels (densitometric band analysis) was found. Time-course assays indicate a cortisol-mediated regulation. Furthermore, Hsp70 gene modulation appears to be more susceptible to the cortisol action and the mRNA was transcribed within 3 h post-injection. The restoration of the homeostatic conditions was observed at a week p.i., when plasma cortisol baseline was reached. Although fish manipulation and injection exerted stressing effects as indicated by serological parameters, differences between cortisol treated specimens compared to untreated or sham fish are statistically significant. Similar results were found by examining in vitro total cells and isolated leukocytes from head kidney cultured for 3 h with increasing cortisol concentration. Finally, MTT test and DNA fragmentation experiments showed that the apoptotic effect expected in cortisol-treated cells could be counteracted by high Hsp70 intracellular levels.

Searching for immunomodulatory sequences in sea bass ( Dicentrarchus labrax L.): Transcripts analysis from thymus

The thymus is a key organ of the immune system in most vertebrates and, for this reason, it has been used in this paper for the generation of a normalized cDNA library from sea bass ( Dicentrarchus labrax), one of the most extensively cultured species in South Mediterranean aquaculture. A total of 1632 ESTs from this library were initially analysed for sequence quality and vector sequences and, after this control, 1264 (77% of total clones sequenced) high-quality ESTs were further processed. The total collection of D. labrax thymus ESTs has been deposited in the EBI-GenBank-DBJ database (GenBank accession numbers from FN565576 to FN566839). The functional classification of ESTs was performed by Gene Ontology and KEGG annotation and, successively, the sequences were analysed using the ImmunomeBase software to identify potentially immuno-related genes. Using this approach, we found about 100 putative genes involved in immune system responses, most new in sea bass, that were analysed more in detail. Some of the potentially interesting genes identified by these in silico analyses were studied by real-time PCR to verify their expression both at basal level and after in vitro stimulation of sea bass head kidney leukocytes. The used strategy has been confirmed as a good approach to discover new immuno-related genes and to improve the knowledge of specific markers that could help the discrimination of T-cell subpopulations in sea bass and, in general, in Teleosts.

Differential expression of two glucocorticoid receptors in seabass (teleost fish) head kidney after exogeneous cortisol inoculation

Stressful conditions include a prompt release of corticosteroid hormones which can mediate gene expression through glucocorticoid receptors (GR). Since two seabass ( Dicentrarchus labrax) GRs have been cloned and sequenced from peritoneal cavity cells (DlGR1) and liver (DlGR2), a comparative amino acid sequence analysis that included Haplochromis burtoni HbGRs, was carried out and homologies disclosed. The DlGR1 and DlGR2 deduced aminoacid sequences showed 61% identity (I) and 70% similarity (S). Moreover, DlGR2 was similar to HbGR2b (69% I, 73% S), and the DlGR1 to HbGR1 (72% I, 78% S). In addition, we examined the expression of the DlGRs after exogeneous cortisol inoculation into the peritoneal cavity, mimicking stress effects. At various times after the administration (3 h, 24 h, 1 week), gene expressions was evaluated in head kidney by real-time PCR. In addition, immunoblotting and densitometry analyses were performed with anti-DlGR1 antibodies. Although sea bass head kidney expressed both DlGR1 and DlGR2 they were differentially modulated by intraperitoneal implant of exogeneous cortisol.

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1beta, TNF-alpha, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924 bp cDNA of sea bass caspase-9 was obtained, consisting of 1308 bp open reading frame coding for 435 amino acids, 199 bp of the 5′-UTR and 417 bp of the 3′-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp 121, Asp 325 and Asp 343. The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida ( Phdp) strain PP3, showed increased expression from 0 to 12 h returning to control levels at 24 h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48 h post-infection, when the fish were with advanced septicaemia.

Biological Activity of Sea Bass (Dicentrarchus labrax L.) Recombinant Interleukin-1β

Biological activities of a putative mature sea bass interleukin-1beta peptide, produced as a recombinant protein (rIL-1beta) in Escherichia coli, have been investigated. The rIL-1beta contains a 6-histidine tag at the N-terminus, and protein purification has been achieved through this tag by affinity chromatography. Biological activities have been investigated both at the cellular and gene expression levels. In in vitro assays sea bass rIL-1beta induced the proliferation of murine D10.G4.1 cells and increased yeast phagocytosis by sea bass head kidney leukocytes. The purified cytokine was also tested in a lymphocyte-activation factor assay, where it induced the proliferation of sea bass thymocytes. Finally, in an in vivo assay, rIL-1beta administered intraperitoneally increased expression levels of the IL-1beta gene and activated macrophages to produce a cyclooxygenase 2 homologue (COX-2) gene in the head kidney.

Mercury chloride effects on the function and cellular integrity of sea bass ( Dicentrarchus labrax) head kidney macrophages

The objective of this work was to study mercury chloride effects on the function and integrity of sea bass ( Dicentrarchus labrax) head kidney macrophages (S-HKM), and to evaluate the response of HgCl 2-exposed cells to macrophage activating factor(s) (MAF) produced by sea bass head kidney leukocytes. There was considerable variability in the effects of HgCl 2 on the production of reactive oxygen species (ROS) by S-HKM. When incubated with HgCl 2, cells from five out of nine fish tested showed a decrease in ROS production as compared to cells incubated with medium alone. In those cultures, MAF addition prevented the mercury chloride-induced decrease in ROS production. In other S-HKM cultures isolated from different fish, mercury chloride abrogated the up-regulating effect of MAF on the respiratory burst. MAF activation of the phagocytic activity of S-HKM was also impaired by HgCl 2 addition. Mercury chloride induced apoptosis in S-HKM cultures and MAF addition prevented this effect.

Effects of temperature on chemiluminescence of phagocytes from sea bass, Dicentrarchus labrax L.

Abstract Sea bass head kidney cells were incubated at several temperatures with or without opsonized zymosan, Luminol was added to amplify light emission. When stimulated by zymosan cells emitted higher chemiluminescence (CL) at low temperatures (5–20°C) than at high temperatures (25–40°C). CL reached its peak earlier at high temperatures than at low temperatures. Unstimulated cells showed a low level CL response which rose with increasing temperature. Sodium azide inhibited CL by 97%.