Molecular cloning of the cDNA encoding the β subunit of thyrotropin and regulation of its gene expression by thyroid hormones in the goldfish, Carassius auratus

A cDNA encoding the subunit of thyrotropin (TSH) was isolated from a goldfish (Carassius auratus) pituitary gland cDNA library. By comparing the sequence with other teleost TSHs, a signal peptide of 19 amino acids and a mature hormone of 131 amino acids were predicted for goldfish TSH subunits. The resulting putative mature hormone of 131 amino acids had well-conserved cysteine positions and a putative N-linked glycosylation site; homology was 51–67% with TSHs from other teleosts, 38–43% with tetrapod TSHs, but only 27 and 29% with goldfish GTH-I and -II, respectively. We also examined the effects of thyroid hormones (TH) and thiourea (TU, an inhibitor of TH production) treatments on TSH and GTH subunit gene expressions in the goldfish pituitary gland. After thyroxine (T₄) treatment, circulating T₄ concentration increased and TSH mRNA level decreased. Supressing the amount of circulating T₄ and triiodothyronine (T₃) by TU treatment increased the TSH mRNA level. Moreover, T₄ replacement therapy (simultaneous treatment of both TU and T₄) caused a high level of circulating T₄ and a low level of circulating T₃, and a decrease in the TSH mRNA level. Thus, changing levels of circulating TH exert a negative feedback on the level of TSH subunit mRNA in goldfish in vivo. On the other hand, GTH subunit mRNA levels were not affected by changes in the levels of circulating TH.     

The effect of allostatic load on hypothalamic–pituitary–interrenal (HPI) axis before and after secondary vaccination in Atlantic salmon postsmolts (Salmo salar L.)

The experiment consisted of three experimental groups: (1) “vaccine and stress”, (2) “stress and vaccine” and (3) control. All groups have previously been vaccinated 6 months prior to the start of the experiment. At the start of the experiment, the “vaccine and stress” group was vaccinated with Pentium Forte Plus for the second time (25.02.2008) and then given a daily stressor (confinement stressor 267 kg m-3 in 15 min) for a period of 4 weeks. The “stress and vaccine” group was given a similar daily stressor for 4 weeks and then vaccinated for the second time. The control group was neither stressed nor vaccinated a second time. The results indicates that fish in the “stress and vaccine” group may have entered an allostatic overload type 2 due to oversensitivity to ACTH, a reduced efficient negative feedback system with elevated baseline levels of plasma cortisol and reduced immune response with pronounced effects on the well-being of the animal. The “vaccine and stress” group may likewise have entered an allostatic overload type 1 response, with oversensitivity to ACTH and transient reduced efficient negative feedback system. This study shows that if plasma cortisol becomes elevated prior to vaccination, it could perhaps instigate an allostatic overload type 2 with dire consequences on animal welfare. To reduce the risk of compromising the animal welfare during commercial vaccination of salmon, one propose to grade the fish minimum a week prior to vaccination or grade simultaneously with vaccination. This could reduce the overall allostatic load during handling and vaccination and secure a healthy fish with intact immune response and improved animal welfare.     

Modulation of glycoprotein hormone α- and gonadotropin IIβ-subunit mRNA levels in the pituitary gland of mature male African catfish, Clarias gariepinus

The cDNAs encoding the glycoprotein hormone -subunit (GP) and the gonadotropin II-subunit (GTH II) were cloned from the pituitary gland of the African catfish, Clarias gariepinus. Using RNase protection analysis, we studied the steady-state mRNA levels of GP as well as GTH II in the pituitary gland of adult male catfish. Castration of adult male catfish resulted in a significant decrease of GTH II mRNA levels, whereas there was no change in the GP mRNA levels. Treatment of intact males with a single dose of 11-ketotestosterone (11-KT) resulted in dose-dependent increases in mRNA levels of both GP and GTH II. We conclude that 11-KT, a prominent, non-aromatizable teleost androgen, has a stimulatory effect on the pituitary mRNA levels of GP and GTH II of adult male fish.     

Ion levels in the gastrointestinal tract content of freshwater and marine–estuarine teleosts

This study investigated the relationship between ion levels (Na?, Cl?, K?, Ca2?, and Mg2?) in the fluid phase and total chyme of the contents of the gastrointestinal tract segments of freshwater and marine-estuarine teleosts collected in different salinities (0-34 ppt) in estuarine and freshwater portions of the S?o Gon?alo channel, southern Brazil. In addition, the relative contribution of feeding and osmoregulation to the ionic content of each portion of the gastrointestinal tract of fishes collected in different ambient salinities was analyzed. There was no relationship between salinity and ion levels in the fluid phase and total chyme of the segments of the gastrointestinal tract when considering all species together. However, there was a significant positive relationship between salinity and ion levels in the fluid phase and total chyme of two fish species (Micropogonias furnieri and Genidens barbus) collected in three or more different salinities. In most species, ion levels in the fluid phase and total chyme changed throughout the gastrointestinal tract, suggesting absorption, but the ionoregulatory mechanisms of the gastrointestinal tract seem to vary according to species.     

Role of thyroid hormones in tilapia larvae (Oreochromis mossambicus): II. Changes in the hormones and 5′-monodeiodinase activity during development

Thyroid hormone profiles and 5-monodeiodinase activity were determined in tilapia at different stages of early development. The results showed that both T₄ and T₃ were present in significant amounts in fertilized eggs. There was a steady decrease in both T₄ and T₃ levels during embryonic development. The levels continued to decline after hatching until around 7 days later when most of the yolk had been absorbed. The T₄ level started to rise then, suggesting that the larval thyroid had begun to produce T₄ at this time, which coincided with the period of faster growth of the larvae. The T₃ level remained fairly constant until around 20 days after which it rose significantly. In vitro determination of 5-monodeiodinase activity (5-D activity) in the whole-body homogenates of larvae showed that the enzymatic conversion of T₄ to T₃ was not detectable in eggs and 3-day-old larvae but detected in 5-day-old and older larvae. There was a gradual increase in the V_max as development proceeded indicating increasing 5-D activity during larval development. The K_m values did not differ significantly in the different stages of development. These results are discussed in relation to the growth and development of the larvae.     

Effects of salinity, dietary level of protein and 17α-methyltestosterone on growth hormone (GH) and prolactin (tPRL177 and tPRL188) levels in the tilapia, Oreochromis mossambicus

In the present study, we examined the long-term effects of environmental salinity, diet (35% and 25% crude protein) and 17-methyltestosterone (MT) on corresponding levels of pituitary and serum growth hormone (GH) and prolactins (tPRL₁₇₇ and tPRL₁₈₈) in the tilapia (Oreochromis mossambicus). We observed no discernible patterns in serum GH that would suggest an effect of salinity, diet or MT. However, serum GH levels in all treatments declined at 1 and 3h after first feeding. Serum tPRL₁₇₇ and tPRL₁₈₈ were significantly higher in freshwater (FW) than in seawater (SW) and levels were significantly affected by dietary protein. tPRL₁₇₇ levels were higher in all groups fed a 35% protein diet, but tPRL₁₈₈ levels were higher only in the groups fed the MT-treated 35% protein diet; only serum tPRL₁₈₈ levels were affected by MT. Moreover, serum tPRL₁₇₇ and tPRL₁₈₈ increased throughout the sampling time-course. Subsequent work using fasted tilapia suggests that first feeding is likely to initiate the post-prandial suppression of serum GH levels. In contrast with the picture observed in blood, pituitary glands of SW animals showed higher levels of GH than FW fish. Pituitary GH was elevated by MT in both FW and SW. We also observed that pituitary tPRL₁₇₇ and tPRL₁₈₈ levels were higher in FW fish than in SW fish; tPRL₁₇₇ and tPRL₁₈₈ levels were elevated by MT only in FW animals. To assess the somatomedin activity of plasma from FW- and SW-reared tilapia, we measured [35S]-sulfate incorporation into ceratobranchial cartilage explants in vitro. Plasma from SW-adapted tilapia showed greater activity in this assay than plasma from FW-reared tilapia, suggesting that the GH-dependent IGF bioactivity of plasma is higher in SW-reared tilapia. Collectively, these studies suggest that the growth-promoting actions of SW rearing and of MT administration in tilapia may be linked to elevations in GH and/or prolactin (tPRL₁₇₇ and tPRL₁₈₈)levels.     

Effects of light and temperature conditions on the expression of GnRH and GtH genes and levels of plasma steroids in <Emphasis Type="Italic">Odontesthes bonariensis</Emphasis> females

In this study we examined the endocrine mediation between environmental factors (temperature and photoperiod) and the brain–pituitary–gonadal axis in females of pejerrey Odontesthes bonariensis. Changes in the expression of brain gonadotropin-releasing hormones (GnRHs) and gonadotropin (GtH) subunit [follicle stimulating-β (FSH-β), luteinizing hormone-β (LH-β), glycoprotein hormone-α (GPH-α)] genes, plasma gonadal steroids [estradiol (E₂) and testosterone (T)], gonadal histology, and gonadosomatic index (GSI) in adult females exposed to combinations of short-day (8 h) or long-day (16 h) photoperiods and low (12°C) or high (20°C) temperatures after winter conditions (8 h light, 12°C) were analyzed. Pejerrey females kept under the short photoperiod had low GSIs, and their ovaries contained only previtellogenic oocytes regardless of the experimental temperature. In contrast, females exposed to the long photoperiod had high GSIs and ovaries with vitellogenic oocytes at both temperatures. These fish also showed a significantly higher expression of sGnRH, pjGnRH, cGnRH-II (the three different GnRH variants found to date in the pejerrey brain), FSH-β, LH-β and GPH-α genes and plasma E₂ levels than those at the shorter photoperiod. No significant changes were observed in plasma T levels. Based on these results, we concluded that the increase in day length but not that of temperature triggers the maturation of pejerrey females after the winter period of gonadal rest and that this occurs by an integrated stimulation of the various components of the brain–pituitary–gonad axis.     

Hypothermal effects on expression of regucalcin,a calcium-binding protein,in the livers of seawater- and fresh water–acclimated milkfish,Chanos chanos

Fish Physiology and Biochemistry - Regucalcin (RGN) is a calcium-binding protein mainly expressed in the liver. It functions in regulating activities of several calcium-dependent enzymes related to...     

Oxygen consumption in trilobite larvae of the mangrove horseshoe crab (Carcinoscorpius rotundicauda; Latreille, 1802): effect of temperature,salinity, pH,and light–dark cycle

A set of feeding trials was carried out for different microalgal species and processed diets for the culture of the New Zealand pipi, Paphies australis. Five microalgal species (Isochrysis galbana clone T-ISO, Pavlova lutheri, Tetraselmis suecica, Chaetoceros muelleri, and Thalassiora pseudonana clone 3H) and three formulated diets (baker's yeast, wheat flour, and corn flour) were fed to spat, juvenile, and adult pipi for 21 days. Unfed pipi were used in the control group. The spat and juvenile pipi showed a major shell increase with I. galbana while the greatest wet weight increase was obtained with P. lutheri. The shells of adult pipi grew better with corn flour, and the P. lutheri-fed group obtained the greatest wet weight. Results of proximate analysis in adult pipi revealed that proteins and lipids were accumulated in the tissue for all fed groups, while carbohydrate levels were depleted in all treatments including the control group. It is suggested that the gonads have developed during the experiment.     

Nucleotide sequence and mRNA expression analysis of <Emphasis Type="Italic">β</Emphasis>-actin gene in the orange-spotted grouper <Emphasis Type="Italic">Epinephelus coioides</Emphasis>

The full-length cDNA, encoding the orange-spotted grouper β-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate β-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the β-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of β-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper β-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using β-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17α–methyltestosterone (MT) had little effect on the mRNA expression of β-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting β-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.     

Cloning,expression, and induction by 17-β estradiol (E<Subscript>2</Subscript>) of a vitellogenin gene in the white cloud mountain minnow <Emphasis Type="Italic">Tanichthys albonubes</Emphasis>

Vitellogenins (Vtgs), the precursors for the yolk proteins, are very important for the embryonic development of teleosts, and have also been studied extensively as biomarkers for environmental estrogenic mimics. The cDNA for a Vtg was isolated from the liver of the female white cloud mountain minnow (Tanichthys albonubes) by 3′- and 5′-RACE methods. It is 4,171 bp in full length, and encodes a putative protein of 1,326 amino acids. This putative Vtg, designated as wcmmVtg, contains complete portions of LVI and PV, but lacks the C-terminal half of LVII and thus belongs to type I vitellogenin. In addition to the liver of the female fish, wcmmVtg was also shown to be expressed in the ovary. During ovarian development, the mRNA expression of wcmmVtg in both the liver and ovary was continuously increased from the previtellogenic to late vitellogenic stages, but then decreased significantly at post-spawning stage. In the male fish, expression of wcmmVtg mRNA was induced in the liver by treatment with E₂ (10 and 100 ng/l) for 14 days. These results suggest that the Vtg originated from the ovary of the white cloud mountain minnow may also contribute to the accumulation of yolk proteins during oocyte growth, and that the male white cloud mountain minnow is sensitive to the estrogenic treatment in terms of Vtg mRNA expression, which could also be applied to monitor the environmental estrogenic mimics.     

Effect of nitrite exposure on oxygen-carrying capacity and gene expression of <Emphasis Type="Italic">NF-κB</Emphasis>/<Emphasis Type="Italic">HIF-1α</Emphasis> pathway in gill of bighead carp (<Emphasis Type="Italic">Aristichthys nobilis</Emphasis>)

Nitrite (NO₂^?) contamination of water can severely impact the health of aquatic life and is a major concern for commercial aquaculture. In order to study the effect of nitrite on Aristichthys nobilis, we investigated the oxygen-carrying capacity, NF-κB/HIF-1α pathway, and the gill tissue structure under nitrite stress. In the current study, bighead carp (initial weight 180.05?±?0.092 g) were exposed to nitrite (48.634 mg/L) for 96 h and then for 96 h recovery test. After nitrite exposure for 6 h, hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) mRNA expression increased significantly in the gill of bighead carp (P?<?0.05). After nitrite exposure for 12 h, hemoglobin (Hb) and methemoglobin reductase (MHBR) content in blood decreased significantly (P?<?0.05); TLR4 mRNA expression increased significantly (P?<?0.05). After nitrite exposure for 24 h, methemoglobin (MetHb) content increased significantly (P?<?0.05). After recovery test, all the indicators except TLR4 mRNA expression level recovered to initial level. In conclusion, nitrite exposure can affect hemoglobin dynamics, as oxidization of nitrite by hemoglobin results in the reduction of Hb to MetHb leading to hypoxia and nitrite exposure can also result into gill tissue damage. In the face of nitrite exposure, NF-κB and HIF-1α mRNA expression level increased immediately to protect the body from oxidative damage and eased hypoxic condition caused by nitrite. It was also observed that nitrite damage is recoverable in Aristichthys nobilis, but it may be need more than 96 h.