Response of sea bream to handling

Understanding how gilthead sea bream, Sparus aurata L., an important Mediterranean Sea species for aquaculture, respond physiologically to stressors commonly encountered in intensive rearing is important for eiective production, as managing for stress is a major factor in maintaining healthy ¢sh stocks. Our objective was to determine whether holding juvenile gilthead sea bream at a high density (HD), as a chronic stressor, would aiect their physiological responses to a subsequent acute handling stressor. After acclimation at a low density (LD) of 6k g m 3 in 200-L circular tanks containing 33^ 36 g L 1 recirculating seawater at 191C under a normal photoperiod, juvenile 37-g gilthead sea bream were con¢ned for 14 days at a HD of 26 kg m 3 an

Functional evidence for the inflammatory reflex in teleosts: A novel alpha7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines

Modulation of innate immune-related genes and glucocorticoid synthesis in gnotobiotic full-sibling European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum

Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host-pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress-and innate immune-related genes in European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum. For this purpose, we challenged by bath full-sibling gnotobiotic sea bass larvae with 107 CFU mL(-1) of V. anguillarum strain HI 610 on day 5 post-hatching (dph). The mortality was monitored up to the end of the experiment [120 hours post-challenge (hpc)]. While no variations were registered in non-challenged larvae maintained under gnotobiotic conditions (93.20% survival at 120 hpc), in the challenged group a constant and sustained mortality was observed from 36 hpc onward, dropping to 18.31% survival at 120 hpc. Glucocorticoid quantification and expression analysis of stress-and innate immunity-related genes were carried out in single larvae. The increase of cortisol, cortisone and 20 beta-dihydrocortisone was observed at 120 hpc, although did not influence upon the modulation of stress-related genes (glucocorticoid receptor 1 [gr1], gr2, and heat shock protein 70 [hsp70]). On the other hand, the expression of lysozyme, transferrin, and il-10 differentially increased at 120 hpc together with a marked upregulation of the pro-inflammatory cytokines (il-1 beta and il-8) and hepcidin, suggesting a late activation of defense mechanisms against V. anguillarum. Importantly, this response coincided with the lowest survival observed in challenged groups. Therefore, the increase in markers associated with glucocorticoid synthesis together with the upregulation of genes associated with the anti-inflammatory response suggests that in larvae infected with V. anguillarum a pro-inflammatory response at systemic level takes place, which then leads to the participation of other physiological mechanisms at systemic level to counteract the effect and the consequences of such response. However, this late systemic response could be related to the previous high mortality observed in sea bass larvae challenged with V. anguillarum

Seasonal steroid variations in relation to maturity stages in the female Pacific red snapper Lutjanus peru in the Gulf of California, Mexico

Reproductive steroid concentration and gonadal development as well as plasma cortisol were investigated during the reproductive period (April to October) of the Pacific red snapper Lutjanus peru, a commercial and important local fishery species in the surrounding areas of Bahía La Paz, Gulf of California, Mexico. Gonads in April were immature and characterized by the presence of oocytes at the chromatin nucleolar and perinucleolar stages. In the next month, more than 50% of the fish showed gonads in vitellogenesis, characterized by the presence of cortical alveoli and early vitellogenic (V1) oocytes. Late vitellogenic and mature gonads were present in July, August and September. Postovulatory follicles were observed only in gonads collected in August, indicating spawning activity of these individuals. The gonadosomatic index (GSI) increased significantly (p <0.05) from April and May to July, when it reached its maximum value. A significant increase (p <0.05) in plasma testosterone (T) was also observed in July compared to previous months, and plasma estradiol (E2) concentration was significantly higher (p <0.05) in September than in April. Cortisol concentration was significantly higher (p <0.05) in September than in May. Significant differences (p <0.05) between gonadal stages were observed for GSI and E2 but not for T. We conclude that the Pacific red snapper, in the surrounding areas of Bahía de la Paz, showed an asynchronous ovarian growth starting in May when water temperature increased and that was characterized by several spawning events

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses

The present study examined the short and mid-term effects of a rise in temperature from 18 &ordm;C to 24 &ordm;C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6&nbsp;&deg;C, after 1 month of acclimation at 18 &ordm;C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1 hour, 24 hours, 3 days and 1 week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration.&nbsp; Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1 h as a short-term response, and a consecutive increase after one week, as a mid-term response.. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24 h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol

Unveiling the effect of dietary essential oils supplementation in Sparus aurata gills and its efficiency against the infestation by Sparicotyle chrysophrii

A microencapsulated feed additive composed by garlic, carvacrol and thymol essential oils (EOs) was evaluated regarding its protective effect in gills parasitized by Sparicotyle chrysophrii in Sparus aurata. A nutritional trial (65 days) followed by a cohabitation challenge with parasitized fish (39 days) were performed. Transcriptomic analysis by microarrays of gills of fish fed the EOs diet showed an up-regulation of genes related to biogenesis, vesicular transport and exocytosis, leukocyte-mediated immunity, oxidation–reduction and overall metabolism processes. The functional network obtained indicates a tissue-specific pro-inflammatory immune response arbitrated by degranulating acidophilic granulocytes, sustained by antioxidant and anti-inflammatory responses. The histochemical study of gills also showed an increase of carboxylate glycoproteins containing sialic acid in mucous and epithelial cells of fish fed the EOs diet, suggesting a mucosal defence mechanism through the modulation of mucin secretions. The outcomes of the in vivo challenge supported the transcriptomic results obtained from the nutritional trial, where a significant reduction of 78% in the abundance of S. chrysophrii total parasitation and a decrease in the prevalence of most parasitic developmental stages evaluated were observed in fish fed the EOs diet. These results suggest that the microencapsulation of garlic, carvacrol and thymol EOs could be considered an effective natural dietary strategy with antiparasitic properties against the ectoparasite S. chrysophrii.info:eu-repo/semantics/publishedVersio

The expression of TRPV channels, prostaglandin E2 and pro-inflammatory cytokines during behavioural fever in fish

A fever, or increased body temperature, is a symptom of inflammation, which is a complex defence reaction of the organism to pathogenic infections. After pathogens enter the body, immune cells secrete a number of agents, the functions of which stimulate the body to develop a functional immune and fever response. In mammals it is known that \{PGE2\} is the principal mediator of fever. The extent to which \{PGE2\} and other pro-inflammatory cytokines such as TNF-α, IL-6, or IL-1β could be involved in the induction of behavioural fever in fish remains to be clarified. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including \{TRPV1\} and TRPV2, which are activated by heat. Here we show that members of the \{TRP\} family, \{TRPV1\} and TRPV4, may participate in the coordination of temperature sensing during the behavioural fever. To examine the behavioral fever mechanism in Salmo salar an infection with IPNV, infectious pancreatic necrosis virus, was carried out by an immersion challenge with 10 ×105 PFU/mL-1 of IPNV. Behavioural fever impacted upon the expression levels of both \{TRPV1\} and \{TRPV4\} mRNAs after the viral challenge and revealed a juxtaposed regulation of \{TRPV\} channels. Our results suggest that an increase in the mRNA abundance of \{TRPV1\} is tightly correlated with a significant elevation in the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and PGE2) in the Pre-Optic Area (POA) and cytokine release in plasma. Together, these data indicate that the reduction of \{TRPV4\} expression during behavioural fever may contribute to the onset of behavioural fever influencing movement toward higher water temperatures. Our data also suggest an effect of \{TRPV\} channels in the regulation of behavioural fever through activation of \{EP3\} receptors in the central nervous system by \{PGE2\} induced by plasma-borne cytokines. These results highlight for first time in mobile ectotherms the key role of pro-inflammatory cytokines and \{TRPV\} channels in behavioural fever that likely involves a complex integration of prostaglandin induction, cytokine recognition and temperature sensing

Extending immunological profiling in the gilthead sea bream, sparus aurata, by enriched cDNA library analysis, microarray design and initial studies upon the inflammatory response to PAMPs

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata&nbsp;(SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata&nbsp;macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata&nbsp;with an emphasis upon immunity and the immune response