Anguillid Eels as a Model Species for Understanding Endocrinological Influences on the Onset of Spawning Migration of Fishes.

Anguillid eels are the iconic example of catadromous fishes, because of their long-distance offshore spawning migrations. They are also a good model for research on the onset mechanisms of migrations to breeding areas, because the migrations begin in inland waters. When eels transform from yellow eels to silver eels, it is called silvering. Silver eels show various synchronous external and internal changes during silvering, that include coloration changes, eye-size increases, and gonadal development, which appear to be pre-adaptations to the oceanic environment and for reproductive maturation. A strong gonadotropic axis activation occurs during silvering, whereas somatotropic and thyrotropic axes are not activated. Among various hormones, 11-ketotestosterone (11-KT) drastically increases during spawning migration onset. Gradual water temperature decreases simulating the autumn migratory season, inducing 11-KT increases. Administration of 11-KT appeared to cause changes related to silvering, such as early-stage oocyte growth and eye enlargement. Moreover, 11-KT may be an endogenous factor that elevates the migratory drive needed for the spawning migration onset. These findings suggested that water temperature decreases cause 11-KT to increase in autumn and this induces silvering and increases migratory drive. In addition, we newly report that 11-KT is associated with a corticotropin-releasing hormone that influences migratory behavior of salmonids. This evidence that 11-KT might be among the most important factors in the spawning migration onset of anguillid eels can help provide useful knowledge for understanding endocrinological mechanisms of the initiation of spawning migrations.

Slower growth of farmed eels stocked into rivers with higher wild eel density.

Farmed anguillid eels are frequently stocked into natural fresh waters to enhance eel resources, but little is known about what happens to these eels or their interactions with wild eels after stocking. A recent study observed a depressed survival and growth rate of farmed Japanese eels when they were reared with wild eels, which indicated that wild eels might interfere with the survival and growth of farmed-and-stocked eels through intraspecific competition. To contribute to improving eel stocking efficiency, the growth of farmed-and-stocked Japanese eels was compared among four rivers with different wild eel densities using mark-and-recapture studies. Based on the 2-year recapture survey after stocking, it was found that the density of the farmed-and-stocked eels was not significantly different among rivers. The daily growth rates of farmed-and-stocked eels in the rivers with lower wild eel density were significantly higher than those of the eels stocked into the rivers with higher wild eel density. The farmed-and-stocked eels moved significantly greater distances downstream than wild eels that showed sedentary behaviour. This and previous studies indicate that significant questions remain about the effectiveness of stocking farmed eels into water bodies where naturally recruited wild eels are present.

Ghrelin and food acquisition in wild and cultured Japanese eel (Anguilla japonica).

To clarify the relationships between growth, endocrine status and habitat characteristics in Japanese eel (Anguilla japonica), plasma and stomach mRNA levels of ghrelin were examined in wild eels captured in the river and the bay, and in cultured eels during and after experimental fasting. Wild juvenile eels captured in freshwater habitats within the river showed significantly higher plasma and stomach mRNA levels of ghrelin than did fish obtained from brackish-water habitats within the bay. In cultured eels experimentally fasted for 4 weeks, plasma and stomach mRNA levels of ghrelin increased. After refeeding, the both parameters returned to the levels observed in continuously feeding control fish. In pigmented elvers, 2 months of feed restriction resulted in a significant increase in whole-body ghrelin mRNA. It is suggested that interaction between ghrelin and feeding is related to their habitats through differential food acquisition in fresh and brackish water environments.

Changes in PRL Gene Expression During Upstream Movement of the Japanese Eel, Anguilla japonica.

Changes in mRNA levels of prolactin (PRL) during upstream movement were examined in juvenile Japanese eels, Anguilla japonica. Glass eels and elvers were collected from 2007 to 2009 near the entrance of Hamana Lake, and in a small inflowing stream, the Egawa River. Quantification of mRNA was performed by real-time PCR and expressed as whole-body content. PRL mRNA levels of glass eels caught in the coastal zone and tidal area were low. Eels that moved downward in the tidal zone and migrated upstream to enter into freshwater showed increased levels of PRL mRNA. These changes suggest the importance of up-regulation of PRL gene expression in juvenile eels during their upstream movement from seawater to fresh water, particularly in relation to hyperosmoregulation.

Down-regulation of corticosteroid receptor in leucocytes of stressed rainbow trout.

The relationship between stress and immunosuppression was investigated in peripheral blood leucocytes (PBL) in rainbow trout, with reference to corticosteroid receptor (CR) expression and responses to cortisol- and/or lipopolysaccharide (LPS)-administration. Confinement stress in shallow water resulted in a sustained elevation of plasma cortisol, whereas lysozyme and immunoglobin levels were suppressed. Significant increases in mRNA levels of caspase-6 and insulin-like growth factor (IGF)-I were observed in PBL isolated from stressed fish. Confinement stress also suppressed proinflammatory cytokine, interleukin (IL)-1ß, expression in PBL. There were decreasing tendencies for the mRNA levels of CRs in PBL of stressed fish. In-vitro treatment of cortisol and LPS on isolated PBL from unstressed trout increased both IL-1 ß and CR mRNA expression. However, in PBL from stressed fish, cortisol and LPS treatment increased IL-1 ß but not CR mRNA levels. Proliferative activities estimated as in-vitro incorporation of bromodeoxyuridine (BrdU) were decreased by cortisol in PBL from the unstressed and stressed fish groups; however, LPS-stimulated proliferation was observed only in the unstressed fish. Ratios of apoptotic PBL quantified as cell fragmentation using an automated cell counter were increased by cortisol in both groups; however, LPS-stimulated apoptosis was observed only in the stressed fish. Our study reveals cortisol has immune-suppressive effects in stressed fish, irrespective of CR down-regulation and desensitization. The complexity of immune-endocrine interaction is shown by the stress-induced attenuation of LPS effects.

Post-Anesthesia Evaluation Shows No Relationship Between Body Conditions and Salinity-Choice Propensities in Anguilla japonica Glass Eels.

Anguillid eels generally exhibit catadromous migration between oceanic spawning grounds and freshwater growth habitats, but some individuals remain in coastal or estuarine saline waters for growth. This migratory plasticity had been considered to be a conditional strategy based on individual energetic status during the glass eel stage. Several studies have examined whether salinity-based habitat selection is linked to individual body conditions, but while frozen specimens of European eels showed this relationship, anesthetized samples of American eels did not. Here, we report that freezing preservation under different salinity levels influences body-condition evaluation in Japanese eels. Behavioral tests of Japanese eels did not reveal significant differences in anesthetized body conditions between those choosing saltwater and those choosing freshwater. In conclusion, the body conditions of glass-eel-stage Japanese eels are unlikely to be associated with their salinity-choice propensity.

Molecular biology and functional genomics of immune-endocrine interactions in the Japanese eel, Anguilla japonica.

Immune-endocrine interactions are an important pathogen resistance mechanism in fish. We review the immune-endocrine interactions in the Japanese eel, Anguilla japonica, with special reference to high throughput gene sequencing. These data may be relevant to the significant decrease in the eel harvest in recent years and will aid in the selection of appropriate disease-resistant strains for aquaculture. More than 1000 sequences that whose expression in elvers responded to air exposure were identified through comprehensive gene expression analysis using next-generation sequencing. These included transcription factors within the MAPK pathway. Significant changes in expression after air exposure were detected by quantitative polymerase chain reaction analysis in many genes related to disease resistance. These factors include innate immune system factors and cytokines that interact with the endocrine system during the stress response. Other applications of immune-endocrine interactions in eel culture are discussed.

Different gene expression profiles between normal and thermally selected strains of rainbow trout, Oncorhynchus mykiss, as revealed by comprehensive transcriptome analysis.

A high-temperature selected (HT) strain of rainbow trout was established from the Donaldson (DS) strain by traditional selective breeding in Japan. The aim of this study is to investigate genes related to upper temperature tolerance in this strain utilizing next generation sequencer (NGS), and to establish comprehensive and comparable datasets in brain, liver, muscle, heart and gill tissues between the HT and DS strains. After assembling, clustering and filtering, 242,530 contigs were obtained. Among them, 7624 transcripts had at least 10 counts in expression analysis in all tissues and used as references. BLASTX homology search showed that 7329 transcripts matched with known genes. Compared to the DS strain, the HT strain expressed 90, 775, 349, 188 and 194 genes 2 folds or more in brain, liver, muscle, heart, and gill, respectively in the case of fish before heat-exposure treatment. Meanwhile, the HT strain expressed 292, 363, 433, 322 and 211 genes 2 folds or more in brain, liver, muscle, heart, and gill, respectively in case of fish after heat-exposure treatment. Many of heat shock protein family genes and transcription factor AP-1 related genes were highly expressed in all tissues of the HT strain compared with the DS strain. These results suggest that these genes play key roles in upper temperature tolerance. These comprehensive and comparable datasets will offer broad visions for upper temperature tolerance in fish species.

Prolactin and upstream migration of the amphidromous teleost, ayu Plecoglossus altivelis.

Changes in mRNA levels of prolactin (PRL) during the upstream migration were examined in fry of the amphidromous fish, ayu Plecoglossus altivelis. Quantification of mRNA has been done with real-time PCR and expressed as whole body or pituitary contents depending the body size of fry. PRL mRNA levels of ayu caught in seawater of the coastal area remained low during early spring. Prior to the start of the upstream migration, the fish caught in the coastal area in mid spring showed increased levels of PRL mRNA. There were further increases in PRL levels in the fish caught in the river. Analysis of proportions revealed that there were significant differences among PRL mRNA in the fish caught in different environmental salinities. Body weight showed a positive relation with PRL mRNA in ayu caught in seawater. A landlocked population of ayu, which migrates from lake to river, showed no significant change in PRL mRNA levels before and after upstream migration. Results in this study indicate the importance of up-regulation of PRL gene expression of ayu during the upstream migration from seawater to fresh water. There is a possible relationship between body size and PRL in the early developmental stage of ayu in seawater, but not in the fish in fresh water.

Effect of short-term decrease in water temperature on body temperature and involvement of testosterone in steelhead and rainbow trout, Oncorhynchus mykiss.

The Pacific salmonid species Oncorhynchus mykiss is separated into a migratory form (steelhead trout) and a non-migratory form (rainbow trout). A decrease in water temperature is likely a cue triggering downstream behavior in the migratory form, and testosterone inhibits onset of this behavior. To elucidate differences in sensitivity to water temperature decreases between the migratory and non-migratory forms and effect of testosterone on the sensitivity, we examined two experiments. In experiment 1, we compared changes in body temperature during a short-term decrease in water temperature between both live and dead steelhead and rainbow trout. In experiment 2, we investigated effects of testosterone on body temperature decrease in steelhead trout. Water temperature was decreased by 3°C in 30min. The body temperature of the steelhead decreased faster than that of the rainbow trout. In contrast, there was no significant difference in the decrease in body temperature between dead steelhead and rainbow trout specimens. The body temperature of the testosterone-treated steelhead trout decreased more slowly than that of control fish. Our results suggest that the migratory form is more sensitive to decreases in water temperature than the non-migratory form. Moreover, testosterone might play an inhibitory role in sensitivity to such decreases.

Effect of gonadotropin-releasing hormone on phagocytic leucocytes of rainbow trout.

To clarify the role of gonadotropin-releasing hormone (GnRH) in the fish immune system, in vitro effect of GnRH was examined in phagocytic leucocytes of rainbow trout (Oncorhynchus mykiss). Gene expression of GnRH-receptor was detected by RT-PCR in leucocytes from head kidney. Administration of sGnRH increased proliferation and mRNA levels of a proinflammatory cytokine, tumor necrosis factor (TNF)-α, in trout leucocytes. Superoxide production in zymosan-stimulated phagocytic leucocytes was also increased by sGnRH in a dose-related manner from 0.01 to 100 nM. There was no significant effect of sGnRH on mRNA levels of growth hormone (GH) expressed in trout phagocytic leucocytes. Immunoneutralization of GH by addition of anti-salmon GH serum into the medium could not block the stimulatory effect of sGnRH on superoxide production. These results indicate that GnRH stimulates phagocytosis in fish leucocytes through a GnRH-receptor-dependent pathway, and that the effect of GnRH is not mediated through paracrine GH in leucocytes.

Physiological, molecular, and cellular mechanisms of impaired seawater tolerance following exposure of Atlantic salmon, Salmo salar, smolts to acid and aluminum.

We examined the physiological, molecular, and cellular mechanisms of impaired ion regulation in Atlantic salmon, Salmo salar, smolts following acute acid and aluminum (Al) exposure. Smolts were exposed to: control (pH 6.5, 3.4 micrpg l(-1) Al), acid and low Al (LAl: pH 5.4, 11 microg l(-1) Al), acid and moderate Al (MAl: pH 5.3, 42 microg l(-1) Al), and acid and high Al (HAl: pH 5.4, 56 microg l(-1) Al) for two and six days. At each time-point, smolts were sampled directly from freshwater treatment tanks and after a 24h seawater challenge. Exposure to acid/MAl and acid/HAl led to accumulation of gill Al, substantial alterations in gill morphology, reduced gill Na(+)/K(+)-ATPase (NKA) activity, and impaired ion regulation in both freshwater and seawater. Exposure to acid/MAl for six days also led to a decrease in gill mRNA expression of the apical Cl(-) channel (cystic fibrosis transmembrane conductance regulator I), increased apoptosis upon seawater exposure, an increase in the surface expression of mitochondria-rich cells (MRCs) within the filament epithelium of the gill, but reduced abundance of gill NKA-positive MRCs. By contrast, smolts exposed to acid and the lowest Al concentration exhibited minor gill Al accumulation, slight morphological modifications in the gill, and impaired seawater tolerance in the absence of a detectable effect on freshwater ion regulation. These impacts were accompanied by decreased cell proliferation, a slight increase in the surface expression of MRCs within the filament epithelium, but no impact on gill apoptosis or total MRC abundance was observed. However, MRCs in the gills of smolts exposed to acid/LAl exhibited morphological alterations including decreased size, staining intensity, and shape factor. We demonstrate that the seawater tolerance of Atlantic salmon smolts is extremely sensitive to acute exposure to acid and low levels of Al, and that the mechanisms underlying this depend on the time-course and severity of Al exposure. We propose that when smolts are exposed to acid and moderate to high Al concentrations, impaired seawater tolerance results from extensive gill Al accumulation, damage to the epithelium, reduced MRC and transport protein abundance, and a synergistic stimulation of apoptosis in the gill upon seawater exposure. When smolts are exposed to acid and low levels of Al, loss of seawater tolerance appears to be independent of these mechanisms and may result instead from a shift in the phenotype of MRCs present in the gill epithelium.

Changes in prolactin mRNA levels during downstream migration of the amphidromous teleost, ayu Plecoglossus altivelis.

Changes in mRNA levels of prolactin (PRL) during seaward migration and after experimental transfer from fresh water (FW) to seawater (SW) were examined in larvae of the amphidromous fish, ayu Plecoglossus altivelis. In the field study, ayu larvae caught in the surf zone showed lower levels of PRL mRNA than those in the river, while growth hormone (GH) levels showed no significant change. Decrease in PRL gene transcription was also observed 24h after direct transfer from FW to SW, whereas there was no significant influence of water temperature. On the other hand, there was no significant change in GH mRNA levels in relation to SW transfer or environmental temperature. In a raceway with a vertical salinity gradient, PRL mRNA levels of ayu larvae showed a significant reduction during spontaneous migration from FW to SW, which mimicked the results from the field observation and the transfer experiment, and then a gradual increase during the course of development. Whole body water and sodium contents of larvae in a salinity gradient were stable during migration to SW. Results in this study indicated the importance of regulation of PRL gene expression in the downstream migration and acclimation to SW during the early development of ayu.

Effects of insulin-like growth factor-I on non-specific immune functions in rainbow trout.

In-vitro effects of insulin-like growth factor-I (IGF-I) on superoxide production in phagocytic head kidney leucocytes (HKL) and in-vivo effects on plasma lysozyme levels were examined in the rainbow trout (Oncorhynchus mykiss). In-vitro administration of salmon IGF-I (sIGF-I), human IGF-I, and human IGF-II Increased superoxide production in zymosan-stimulated HKL. A significant effect of sIGF-I on superoxide production in HKL was observed from 0.1 to 100 nM, and was equipotent to that of salmon growth hormone. IGF receptor (IGFR) type Ib was expressed in trout HKL, and also In the brain, pituitary, liver, and gills; however, the expression of another IGFR (type Ia) was not detected in HKL. In-vivo intraperitoneal injection of sIGF-I Increased plasma levels of lysozyme, whereas ceruloplasmin or immunoglobulin M did not change. These results Indicate that IGF-I stimulates non-specific immune functions In fish.

Effects of constitutive expression of somatolactin alpha on skin pigmentation in medaka.

Somatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. Its functions have been investigated for the last decade but are still under debate. We previously reported a frame-shift mutation on SLa in a medaka mutant, color interfere (ci), which shows defects in body-color regulation, lipid storage, and cortisol secretion. In this study, we examined the effects of introducing a DNA fragment which constitutively expresses wild-type SLa under regulation of the beta-actin (Actb) promoter into the ci genome. We successfully visualized the transgene expression by taking advantage of an internal ribosome entry site (IRES) and the green fluorescent protein (GFP). The transgenic medaka, Actb-SLa:GFP, exhibited a reversed body-color phenotype of ci; more orange xanthophores and less white leucophores. We also detected more black melanophores and less silver iridophores, which indicates SLa's comprehensive role in regulating all types of pigment cells in the skin. Unexpectedly, the defects in lipid/cortisol contents remained in Actb-SLa:GFP. Therefore, the causal relationship between SLa and lipid/cortisol metabolisms relapses to an open question which needs to be reassessed by other types of experiments. Both the Actb-SLa:GFP and ci fish grow and mature similar to wild type, indicating SLa contributes little to growth regulation in spite of the fact that it binds to a teleost-specific paralog of growth-hormone receptor in vitro. The present study provides definitive evidence for SLa's principal and indispensable role in body-color regulation in medaka.

The effect of in vitro exposure to tributyltin on the immune competence of Chinook salmon (Oncorhynchus tshawytscha) leukocytes.

We evaluated the direct effects of in vitro exposures to tributyltin (TBT), a widely used biocide, on the cell-mediated immune system of Chinook salmon (Oncorhynchus tshawytscha). Splenic and pronephric leukocytes isolated from juvenile Chinook salmon were exposed to TBT (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l) in cell cultures for 24 h. Effects of TBT on cell viability, induction of apoptosis, and mitogenic responses were measured by flow cytometry. Splenic and pronephric leukocytes in the presence of TBT experienced a concentration-dependent decrease in viability in cell cultures. Apoptosis was detected as one of the mechanisms of cell death after TBT exposure. In addition, pronephric lymphocytes exhibited a greater sensitivity to TBT exposure than pronephric granulocytes. The functional ability of splenic B-cells to undergo blastogenesis upon lipopolysaccharide stimulation was also significantly inhibited in the presence of 0.05, 0.07, or 0.10 mg/l of TBT in the cell cultures. Flow cytometric assay using a fluorescent conjugated monoclonal antibody against salmon surface immunoglobulin was employed for the conclusive identification of B-cells in the Chinook salmon leukocytes. Our findings suggest that adverse effects of TBT on the function or development of fish immune systems could lead to an increase in disease susceptibility and its subsequent ecological implications.

Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout.

Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems.

Growth hormone and fish immune system.

This paper reviews the immunomodulatory effects, extra-pituitary expression and paracrine action of growth hormone (GH), and a possible role of GH/insulin-like growth factor-I (IGF-I) axis in the immune system of teleost fish. In some euryhaline fish, the activation of immune functions observed during seawater acclimation appears to be associated with the osmoregulatory action of GH. Administration of GH enhances many aspects of immune functions including non-specific defences; cytotoxic, phagocytic, haemolytic and lysozyme activities. GH also activates immunoglobulin production as a specific defense and increases ceruloplasmin levels as an acute-phase protein. The GH gene is also expressed in many extra-pituitary tissues of fish, especially in lymphoid organs and cells. Several endocrine factors appear to act on immune function through modification of GH secretion from fish leucocytes. Exposure of phagocytic leucocytes of tilapia to IGF-I in vitro stimulated proliferation and superoxide production associated with phagocytosis. Exposure to GH had no significant effect on IGF-I secretion from tilapia leucocytes, despite of the fact that they secreted significant amounts of IGF-I. GH and IGF-I appear to act in a paracrine manner in the regulation of the teleostean immune system. Further studies are necessary to characterize the interactions of GH with other endocrine and paracrine factors.

A case of isolated tricuspid valve infective endocarditis caused by Abiotrophia defectiva.

A 17-year-old man with a history of dental caries was admitted to our hospital because of 1-week high fever. There was no history of previous cardiac disease. He denied drug abuse. Blood culture was positive for Abiotrophia defectiva. Echocardiography demonstrated large vegetation attached to the anterior cusp of the tricuspid valve with moderate regurgitation. Although he was treated with antibiotics for more than 3 weeks, he had chest pain due to septic pulmonary emboli on chest computed tomography. Surgical resection of the vegetation was performed. The postoperative course was uneventful and he is doing well at the time of follow-up.

Effects of acid water exposure on plasma cortisol, ion balance, and immune functions in the "cobalt" variant of rainbow trout.

This study was undertaken to examine physiological responses to acidification of environmental water in the "cobalt" variant of rainbow trout (Oncorhynchus mykiss), which exhibits malformation of the pituitary, by following changes in plasma levels of cortisol and electrolytes, blood pH, gill Na(+), K(+)-ATPase activity, and immune functions after exposure to acid water (pH 4.5). Resting levels of plasma cortisol and lysozyme were significantly lower in the cobalt variant than in the normal trout, whereas plasma ceruloplasmin was significantly higher in the cobalt variant, suggesting that some endocrine factors, lacking or deficient in the cobalt variant, are important for the regulation of its immune functions. Blood pH was slightly but significantly lower in the cobalt variant at rest. After exposure to acid water for 24 h, both the normal trout and cobalt variant showed a significant elevation in plasma cortisol, although the increased level in the cobalt variant was still lower than that in the normal trout transferred to neutral water. No differences were seen in blood pH, plasma electrolytes, and gill Na(+), K(+)-ATPase activity between the normal trout and the cobalt variant, indicating that the cobalt variant regulates ion balance when exposed to acid water, despite malformation of the pituitary. Although the normal trout showed a reduction in plasma lysozyme level after acid exposure, there was no significant change in the cobalt trout. Adverse effects of pituitary malformation on ion balance and immune functions may be compensated by extrapituitary factors in the cobalt variant when it is exposed to acid water.