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

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

European Sea Bass (Dicentrarchus labrax) immune status and disease resistance are impaired by arginine dietary supplementation

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.European Union's Seventh Framework Programme AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research) [262336]; AQUAIMPROV [NORTE-07-0124-FEDER-000038]; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte) , under the National Strategic Reference Framework, through the European Regional Development Fund; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte), under the National Strategic Reference Framework through the COMPETE - Operational Competitiveness Programme; Fundacao para a Ciencia e Tecnologia; Fundacao para a Ciencia e Tecnologia [SFRH/BD/89457/2012, SFRH/BPD/77210/2011]; Generalitat Valenciana through the project REVIDPAQUA [ISIC/2012/003]; [PEst-C/MAR/LA0015/2013]; [UID/Multi/04423/2013]info:eu-repo/semantics/publishedVersio

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

Transport and recovery of gilthead sea bream (Sparus aurata L.) sedated with clove oil and MS222: Effects on oxidative stress status

The use of anesthesia is a common practice in aquaculture to sedate fish and mitigate handling stress. Although the employ of anesthesia is considered beneficial for fish, as it reduces stress and improves welfare, at the same time it may induce hazardous side-effects. The aim of the present study was to investigate the effects of clove oil (CO) and tricaine methanesulfonate (MS222), two of the most used anesthetics, on several oxidative stress related parameters in gilthead sea bream (Sparus aurata), as these types of effects of anesthetics have been seldom investigated. To assess these effects, S. aurata juveniles were placed in a setup of mobile water tanks and were transported during 6 h with either 2.5 mg/L CO or 5 mg/L MS222. After transport, half of the fish were sampled, whereas the remaining fish were transferred to tanks without anesthetics where they were allowed to recover for 18 h before sampling. Changes in the expression levels of several target genes related with the antioxidant response and cell-tissue repair were evaluated in the gills, liver and brain. Those transcripts included glutathione peroxidase 1 (gpx1), catalase (cat), glutathione S-transferase 3 (gst3), glutathione reductase (gr), superoxide dismutase [Zn] (sod2), heat shock protein-70 (hsp70), and metallothionein (mt). Antioxidant enzymatic activities glutathione S-transferase, GST; catalase, CAT; and glutathione reductase, GR, levels of non-enzymatic antioxidants (non-protein thiols – NPT), and pro-oxidative damage, assessed as lipid peroxidation (LPO), were determined in gills, liver and brain. Acetylcholinesterase activity (AChE) was determined in plasma, gills, brain, muscle and heart as an indicator of neuro-muscular alterations. In plasma, the total antioxidant capacity (TAC) and total oxidative status (TOS) were also measured. Results showed that the use of both anesthetic agents, CO and MS222, interferes with fish antioxidant status. All tested biological matrices displayed alterations in antioxidant endpoints, confirming that these substances, although minimizing the effects of transport stress, may have long term effects on fish defenses. This result is of high relevance to aquaculture considering that the oxidative stress, may increase the susceptibility to different environmental or biotic stress and different types of pathologies

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

Thymol-carvacrol essential oil extract mitigates the mucosal stress-immune response induced by intraperitoneal immunization in sea bass (Dicentrarchus labrax)

The present study investigates the mitigation of the stress response induced by a bivalent vaccine against vibriosis and pasteurellosis in sea bass through treatment with thymol-carvacrol oil extracts, known as FishEase- C (FEC) and FishEase-L (FEL). Plasma and skin mucus cortisol as well as mucosal transcript stress-immune response were measured at 1 and 24 h post-vaccination (hpv). Our results indicate that FEL but not FEC was capable of reducing plasmatic cortisol secretion by 24 hpv, in contrast to vaccinated and vaccinated plus treated with FEC groups. A similar pattern was noted in the skin mucus though secretion of cortisol was prevented by the FEL. The expression of stress-immune related gene transcripts (gr1, gr2, crh, hsp70, cox2, il1β, il6, il8, il10, tgfβ1, lysozyme, igm, igt and igd/igt) revealed that stress-immune gene regulation was not homogenous among mucosae. Gills were more responsive to the stress/inflammation induced by FEC, as shown by crh, hsp70, cox2, il1β and il6 enhanced expression, suggesting higher absorption of FEC via this mucosae. The gills also appeared to be more resilient to the stressful effect of the vaccine, while higher regulation was observed in skin and intestine. In summary, administration of FEL was able to mitigate the stress response at physological (e.g., cortisol) and the stress-immune genes activation in skin and intestine(hsp70, il1β, il6), mainly at 24 hpv. We recommend FEL as a customized formulation to farmers for use on sea bass prior to vaccination, in order to mitigate negative effect of vaccination procedure

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