Diego Crespo

Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): Potential effects on the immune response

Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole (Solea senegalensis) juveniles, a 2 x 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m(-2)) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m(-2)) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray

BackgroundSenegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution.ResultsA comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB.ConclusionsTranscriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout

BackgroundThe relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta).MethodsTo determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.ResultsLPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha.ConclusionsIn view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Evolutionary structural and functional conservation of an ortholog of the GLUT2 glucose transporter gene (SLC2A2) in zebrafish

In mammals, GLUT2 plays an essential role in glucose homeostasis. From an evolutionary perspective, relatively little is known about the biology of GLUT2, or other GLUTs, in nonmammalian vertebrates. Here, we have conducted studies to functionally characterize GLUT2 in zebrafish. First, we cloned the zebrafish ortholog of GLUT2 (zfGLUT2) encoding a protein of 504 amino acids with high-sequence identity to other known vertebrate GLUT2 proteins. The zfGLUT2 gene consists of 11 exons and 10 introns, spanning 20 kb and mapping to a region of chromosome 2 that exhibits conserved synteny with human chromosome 3. When expressed in Xenopus oocytes, zfGLUT2 transported 2-deoxyglucose (2-DG) with similar affinity than mammalian GLUT2 (K(m) of 11 mM). Transport of 2-DG was competed mostly by D-fructose and D-mannose and was inhibited by cytochalasin B. During early development, zfGLUT2 expression was detected already at 10 h postfertilization and remained elevated in 5-day larvae, when it was clearly localized to the liver and intestinal bulb. In the adult, zfGLUT2 expression was highest in testis, brain, skin, kidney, and intestine, followed by liver and muscle. In the intestine, zfGLUT2 transcripts were detected in absorptive enterocytes, and its mRNA levels were altered by fasting and refeeding, suggesting that its expression in the intestine may be regulated by the nutritional status. These results indicate that the structure and function of GLUT2 has been remarkably well conserved during vertebrate evolution and open the way for the use of zebrafish as a model species in which to study the biology and pathophysiology of GLUT2.

Tumor Necrosis Factor Alpha May Act as an Intraovarian Mediator of Luteinizing Hormone-Induced Oocyte Maturation in Trout

ABSTRACT Infish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.

Saving energy to fuel exercise: swimming suppresses oocyte development and downregulates ovarian transcriptomic response of rainbow trout Oncorhynchus mykiss

Metabolic processes and sexual maturation closely interact during the long-distance reproductive migration of many fish species to their spawning grounds. In the present study, we have used exercise experimentally to investigate the effects on sexual maturation in rainbow trout. Pubertal autumn-spawning seawater-raised female rainbow trout Oncorhynchus mykiss (n = 26; 50 cm, 1.5 kg) were rested or swum at a near optimal speed of 0.75 body lengths per second in a 6,000-liter swim flume under natural reproductive conditions (16 degrees C fresh-water, starvation, 8:16-h light-dark photoperiod). Fish were sampled after arrival and subsequently after 10 days (resting or swimming 307 km) and 20 days (resting or swimming 636 km). Ovarian development was significantly reduced in the swimmers. Analysis of the expression of key factors in the reproductive axis included pituitary kiss1-receptor, lh, and fsh and ovarian lh-receptor, fsh-receptor, aromatase, and vitellogenin-receptor (vtgr). Swimmers had lower pituitary lh and ovarian vtgr expression than resters. Furthermore, the number of late vitellogenic oocytes was lower in swimmers than in resters, probably resulting from the lower vtgr expression, and vitellogenin plasma levels were higher. Therefore, swimming exercise suppresses oocyte development possibly by inhibiting vitellogenin uptake. Transcriptomic changes that occurred in the ovary of exercised fish were investigated using a salmonid cDNA microarray platform. Protein biosynthesis and energy provision were among the 16 functional categories that were all downregulated in the ovary. Downregulation of the transcriptomic response in the ovary illustrates the priority of energy reallocation and will save energy to fuel exercise. A swimming-induced ovarian developmental suppression at the start of vitellogenesis during long-term reproductive migration may be a strategy to avoid precocious muscle atrophy.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Luteinizing hormone stimulation of in vitro ovulation in brook trout (Salvelinus fontinalis) involves follicle contraction and activation of proteolytic genes

Luteinizing hormone (LH) is an essential hormone for the stimulation of the ovulatory process in vertebrates. However, little is known in fish regarding the different mechanisms induced by LH during ovulation that facilitate the rupture of the follicle wall and the subsequent expulsion of the mature oocyte. In this study, the effects of salmon LH (sLH) on in vitro ovulation were investigated in brook trout (Salvelinus fontinalis) isolated follicles. sLH significantly stimulated in vitro ovulation and contraction of brook trout preovulatory follicles. In order to investigate the possible involvement of proteolytic events in the ovulatory action of LH, the expression of genes known to have a crucial role in the degradation of follicle wall structure was examined. Our results show that sLH clearly stimulated the mRNA expression levels of matrix metalloproteinases (MMPs; including mmp2 and mmp19) and other enzymes with proteolytic action during ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs 1 (adamts1) and plasminogen (plg), in brook trout preovulatory follicles. In addition, the expression of mmp2, adamts1 and plg increased in brook trout follicles during the progression of LH-induced ovulation. Interestingly, the expression of tissue inhibitor of matrix metalloproteinase 2 (timp2), a known regulator of MMP2 activity, paralleled that of mmp2, suggesting the existence of a controlled mechanism of MMP2 action. Therefore, the known increase in proteolytic activity during ovulation in fish could be the result of the stimulation of the expression of proteolytic enzymes by LH in preovulatory follicles. We propose that LH may stimulate ovulation in brook trout follicles by stimulating proteolysis of the follicle wall and by stimulating follicle contraction.

Luteinizing hormone induces ovulation via tumor necrosis factor α-dependent increases in prostaglandin F2α in a nonmammalian vertebrate.

Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin (PG) F2α (PGF2α) are involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH is not well established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Recombinant trout Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and all actions of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfα–dependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility.

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss)

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout.