Laura Acerete

Effects of temperature decrease on feeding rates, immune indicators and histopathological changes of gilthead sea bream Sparus aurata fed with an experimental diet

Gilthead sea bream Sparus aurata were fed with either a commercial feed or a specifically prepared “winter” feed in order to assess whether fish showed signs similar to the so-called winter syndrome and whether the experimental diet showed protection against the syndrome. Fish were subjected to a ramp of temperature decrease and after 2 weeks, a further recovery of temperature. The temperature ramp produced changes on most immune indicators and feed intake was affected more severely than predicted by feeding tables. However, no generalised signs of the syndrome were detected. The experimental diet tested showed some degree of immune protection related to complement and phagocytosis activity and slight changes in the levels of leucocytic infiltration of the intestinal mucosa and submucosa.

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BackgroundIn 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.MethodsIn 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.ResultsLPS 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.ConclusionThese 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.

Stress-induced regulation of steroidogenic acute regulatory protein expression in head kidney of Gilthead seabream (Sparus aurata)

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane. In mammals, StAR controls this rate-limiting step of steroidogenesis, but its expression and regulation has not been well explored in fish. The present work investigates StAR mRNA expression in the head kidney of the gilthead seabream (Sparus aurata) under different stressors. We have cloned the StAR cDNA (1461 bp) in seabream (accession number EF640987), which has an open reading frame of 861 nucleotides encoding a polypeptide of 286 aa, and displays high sequence identity with StAR of other fish and mammalian counterparts. Seabream StAR transcripts were found to be expressed exclusively in head kidneys and gonads. In fish under acute stress (chased with a net), plasma cortisol levels peaked within 1 h, were still high after 6 h, and decreased after 16 h, although no increases in head kidney StAR expression were observed at any time post-stressor. Fish under chronic high-density stress showed cortisol levels 90-fold higher than controls and StAR mRNA levels increased threefold. Lipopolysaccharide (LPS) injection increased head kidney StAR mRNA levels after 6 h, reached a maximum at 12 h, and decreased until 72 h. When the head kidney cells were incubated in vitro and treated with ACTH or LPS, ACTH induced an increase in StAR expression as expected, but LPS induced a reduction in StAR expression. In conclusion, StAR expression in seabream head kidneys is highly regulated by different stressors.

Effects of Cortisol Administered through Slow-Release Implants on Innate Immune Responses in Rainbow Trout (Oncorhynchus mykiss).

Cortisol is a key hormone in the fish stress response with a well-known ability to regulate several physiological functions, including energy metabolism and the immune system. However, data concerning cortisol effects on fish innate immune system using a more controlled increase in cortisol levels isolated from any other stress related signaling is scarce. The present study describes the effect of doses of cortisol corresponding to acute and chronic levels on the complement and lysozyme activity in plasma of the rainbow trout (Oncorhynchus mykiss). We also evaluated the effects of these cortisol levels (from intraperitoneally implanted hydrocortisone) on the mRNA levels quantified by RT-qPCR of selected key immune-related genes in the liver, head kidney, and spleen. For that purpose, 60 specimens of rainbow trout were divided in to two groups: a control group injected with a coconut oil implant and another group injected with the same implant and cortisol (50 μg cortisol/g body weight). Our results demonstrate the role of cortisol as a modulator of the innate immune response without the direct contribution of other stress axes. Our results also show a relationship between the complement and lysozyme activity in plasma and mRNA levels in liver, supporting the important role of this organ in producing these immune system proteins after a rise of cortisol in the fish plasma.