Sodium chloride alleviates oxidative stress and physiological responses induced by extreme winter cold in genetically improved farmed tilapia (GIFT; Oreochromis niloticus).

A 6-week trial was designed to investigate the effects of dietary sodium chloride supplementation on physiological, metabolic, and molecular stress response parameters. The findings showed that (1) there were no significant differences between sodium chloride supplementation groups (0.05S, 0.1S, and 0.15S) and the control group (P > 0.05), except for the 0.2S diet, which showed better final body weight, weight gain rate, specific growth rate, and feed conversion ratio than the control group (P < 0.05). (2) The hypothermic stress experiment results showed that the survival rates in the 0.1S and 0.15S diets were significantly higher than the control group (P < 0.05). (3) Transcription results showed that these enriched pathways in the gill were mainly energy metabolism and apoptosis pathways, while the major enrichment pathways in the liver were mainly amino acid metabolism and carbohydrate metabolism. (4) The plasma parameter results showed, compared to the control group, the 0.15S diet significantly increased the plasma GLU, TG contents, and Na+ and K+ concentrations and decreased the plasma ALT activity (P < 0.05). In addition, the 0.1S diet increased the plasma ALB content and Cl- concentration (P < 0.05). The gill Na+/K+-ATPase activity decreased markedly when the fish were fed the 0.1S and 0.15S diets (P < 0.05). The antioxidant enzyme activity results showed that the 0.1S and 0.15S diets significantly increased the T-SOD activities (P < 0.05). Gene expression results showed that compared to the control group, the 0.1S and 0.15S diets up-regulated the expression of gys, hsp70, mlcp, mlc, myosin, tnt mRNA, and down-regulated the akt, gk, and erk mRNA expression. Based on the regression analysis, the optimum dietary sodium chloride levels range from 0.10 % to 0.13 % of the diet, which could facilitate energy regulation, improve the immune response, and ultimately strengthen the cold resistance of GIFT.

Transcriptome analysis of the response of four immune related organs of tilapia (Oreochromis niloticus) to the addition of resveratrol in feed.

Resveratrol (RES) has been found to have immunological enhancement effects on Oreochromis niloticus. In O. nilocticus, the liver, spleen and kidney act as immune target tissues, while intestine works for nutrition sensing organ. In the present study, we determined RES administration on these immune tissues transcriptomic response in genetically improved farmed tilapia (GIFT), and further analyzed the relationship between transcriptomic response and intestinal microbiota. As results, hepatic hemosiderin and intestinal goblet cells significantly increased with RES addition. Kyoto encyclopedia of genes and genomes (KEGG) pathways associated with herpes simplex virus 1 infection, calcium signaling pathway, cell adhesion molecules, apoptosis, and mitogen-activated protein kinase (MAPK)/peroxisome proliferators-activated receptors (PPAR) signaling pathways were enriched. In particular, the differentially enriched genes (DEGs) associated pathways were present in different sampling tissues, times, and comparisons, interestingly, the PPAR signaling pathway was enriched with increasing time of RES addition. The assembled DEGs presented verified expression in the kidney, liver, spleen, and intestine tissues, and fabp6 was highly expressed in the intestine. Serial DEGs of fatty acid-binding proteins (fabp7, fabp7a, fabp10a) decreased in the liver and kidney, and fabp6 significantly increased in the spleen. With time, the pathways of energy metabolism, glycan biosynthesis, and metabolism decreased and increased in the intestinal metagenome. Some Candidatus branches significantly increased (C. cerribacteria and C. harrisonbacteria) and while others decreased (C. glodbacteria, etc.), whereas C. verstraetearchaeota fluctuated with RES addition. slc27a6 and dbi were negatively correlated with bacteria involved in the lipid, energy, and carbohydrate metabolism pathways. The present study suggests that RES supplementation affected lipid metabolism in immune-related organs may be related to the PPAR signaling pathway.

Carotenoid coloration and coloration-linked gene expression in red tilapia (Oreochromis sp.) tissues.

BACKGROUND: Production, marketability and consumer preference of red tilapia often depends upon the intensity of coloration. Hence, new approaches to develop coloration are now geared to improve market acceptability and profit. This study evaluated the effects of carotenoid-rich diets on the phenotypic coloration, carotenoid level, weight gain and expression of coloration-linked genes in skin, fin and muscle tissues. Carotenoids were extracted from dried Daucus carota peel, Ipomoea aquatica leaves, and Moringa oleifera leaves. Eighty (80) size-14 fish were fed with carotenoid-rich treatments twice a day for 120 days. The phenotypic effect of the carotenoid extracts was measured through a color chart. Skin carotenoid level was measured through UV-vis spectrophotometer. csf1ra, Bcdo2 and StAR expression analysis was done using qRT-PCR. RESULTS: Treatments with carotenoid extracts yielded higher overall scores on phenotypic coloration and tissue carotenoid levels. Differential expression of carotenoid-linked genes such as the elevated expression in csf1ra and lower expression in Bcdo2b following supplementation of the enhanced diet supports the phenotypic redness and increased carotenoid values in red tilapia fed with D. carota peel and I. aquatica leaves. CONCLUSIONS: Overall improvement in the redness of the tilapia was achieved through the supplementation of carotenoid-rich diet derived from readily available plants. Differential expression of coloration-linked genes supports the increase in the intensity of phenotypic coloration and level of carotenoids in the tissues. The study emphasizes the importance of carotenoids in the commercial tilapia industry and highlights the potential of the plant extracts for integration and development of feeds for color enhancement in red tilapia.

Molecular cloning and characterization of the novel adropin from tilapia (Oreochromis niloticus): Involvement in the control of food intake.

Adropin has been shown to be involved in the regulation of food intake in mice. However, the mechanism of adropin in feeding regulation is still largely unknown. Using the tilapia, Oreochromis niloticus, we identified and characterized a novel form of adropin (designated adropin-b) encoding a 68-amino acid precursor. Although adropin-b shared low amino acid identities with its tilapia paralog (designated adropin-a), synteny analysis proved that tilapia adropin is orthologous to its human counterpart. The transcripts of adropin-b were ubiquitously expressed in various tissues with the highest levels in the olfactory bulb. A decrease in adropin-b mRNA levels was observed 1 h following a meal in the olfactory bulb, hypothalamus, and optic tectum, whereas fasting for 7 days induced an increase in adropin-b mRNA levels in the olfactory bulb, hypothalamus, and optic tectum of tilapia brain. However, no changes in adropin-a mRNA levels were observed in the postprandial and fasting state. Intraperitoneal injection of tilapia adropin-b was shown to increase food consumption, but adropin-a did not affect feeding. Co-treatment of the fish with adropin-b and neuropeptide Y (NPY) had no additive effects on appetite. The appetite stimulatory effects of adropin-b appeared to be mediated by upregulating the orexigenic Npy, Orexin, and Proapelin gene expression, paralleled by inhibition of the mRNA levels of anorexigenic proopiomelanocortin (Pomc) and cocaine-amphetamine-regulated transcript (Cart) in vivo and in vitro. These observations suggested that adropin-b participated in appetite control and gene regulation of central orexigenic and anorexigenic factors in a fish model.

Scale-up production of and dietary supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus.

Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.

Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Effects of dietary supplementation with apple peel powder on the growth, blood and liver parameters, and transcriptome of genetically improved farmed tilapia (GIFT, Oreochromis niloticus).

High-density aquaculture and nutritional imbalances may promote fatty liver in genetically improved farmed tilapia (GIFT, Oreochromis niloticus), thus reducing the gains achieved by breeding. In this study, apple peel powder (APP) was used as a feed additive for GIFT. A control group (fed on a diet without APP) and five groups fed on diets supplemented with APP (at 0.05%, 0.1%, 0.2%, 0.4%, or 0.8% of the diet, by weight) were established to investigate the effects of APP on GIFT growth performance and physiological parameters, and on gene expression as determined by transcriptomic analysis. Dietary supplementation with APP at 0.2% promoted GIFT growth, reduced total cholesterol and triacylglycerol levels in the serum and liver, and decreased alanine aminotransferase and aspartate aminotransferase activities in the serum. Gene expression profiles in the liver were compared among the control, 0.2% APP, and 0.8% APP groups, and differentially expressed genes among these groups were identified. Annotation analyses using tools at the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases showed that the differentially expressed genes were mainly involved in the regulation of immunity and fat metabolism. The results showed that excessive supplementation with APP in the diet significantly inhibited the expression of insulin-like growth factor 2 and liver-type fatty acid-binding protein, and stimulated the expression of fatty acid desaturase 2, heat shock protein 90 beta family member 1, and nuclear factor kappa B. This resulted in disordered lipid metabolism and increased pro-inflammatory reactions, which in turn caused liver damage. Therefore, APP has good potential as an environmentally friendly feed additive for GIFT at levels of 0.1%-0.2% in the diet, but excessive amounts can have adverse effects.

Comparison of the growth performance and long-chain PUFA biosynthetic ability of the genetically improved farmed tilapia (Oreochromis niloticus) reared in different salinities.

To compare the growth and biosynthetic ability of long-chain PUFA (LC-PUFA) of the genetically improved farmed tilapia (GIFT) (Oreochromis niloticus) in different water salinities, an 8-week feeding trial was conducted on the GIFT juveniles at 0, 12 and 24 ‰ (parts per thousand; ppt), respectively, with three isonitrogenous (32 %) and isolipidic (8 %) diets (D1-D3). Diet D1 with fish oils (rich in LC-PUFA) as lipid source was used as the control, while D2 and D3 with vegetable oil (free LC-PUFA) blends as lipid source contained different ratios of linoleic acid (LA, 18 : 2n-6) and α-linolenic acid (ALA, 18 : 3n-3) at 4·04 (D2) and 0·54 (D3), respectively. At the end of feeding trial, the growth performance of D2 and D3 groups under all salinity treatments was as good as that of D1 group, which indicates that the GIFT juveniles may convert dietary LA and ALA into LC-PUFA to meet the requirement of essential fatty acids for normal growth and physiology. When fed the same diets, GIFT at 12 ppt had a better growth performance coupled with a higher liver and muscle arachidonic acid content than those in freshwater. Furthermore, brackish water (24 ppt) significantly promoted the mRNA levels of elongase 5 of very long-chain fatty acids (elovl5) and peroxisome proliferator-activated receptor α (pparα) in liver, when compared with freshwater. These results suggest that the GIFT may display better growth performance together with a relatively higher endogenous LC-PUFA biosynthetic ability under brackish water (12 and 24 ppt), probably through improving the expression of elovl5 and pparα in liver.

Effects of exposure to Streptococcus iniae on microRNA expression in the head kidney of genetically improved farmed tilapia (Oreochromis niloticus).

BACKGROUND: Genetically improved farmed tilapia (GIFT, Oreochromis niloticus) are susceptible to infection by Streptococcus iniae when maintained in modern intensive culture systems. GIFT are commercially important fishes that are cultured widely in southern China. The role of microRNAs (miRNAs) in the regulatory response of GIFT to S. iniae infection has been underestimated and has not yet been well studied. Head kidney has an important immune function in teleost fishes. The main aim of this study was to determine the possible function of miRNAs in head kidney of S. iniae-infected GIFT. MiRNAs are small, non-coding RNAs that regulate gene expression by binding to the 3'-untranslated regions of their target mRNAs. MiRNAs are known to regulate immune-regulated signaling and inflammatory response pathways. RESULTS: High-throughput deep sequencing of two libraries (control group [CO] and infected group [IN]) of RNA extracted from GIFT head kidney tissues generated 12,089,630 (CO) and 12,624,975 (IN) clean reads. Bioinformatics analysis identified 1736 and 1729 conserved miRNAs and 164 and 165 novel miRNAs in the CO and IN libraries, respectively. Three miRNAs (miR-310-3p, miR-92, and miR-127) were found to be up-regulated and four miRNAs (miR-92d-3p, miR-375-5p, miR-146-3p, and miR-694) were found to be down-regulated in the S. iniae-infected GIFT. The expressions of these miRNAs were verified by quantitative real-time PCR. RNAhybrid and TargetScan were used to identify complementary miRNA and mRNA target sites, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to annotate and predict potential downstream regulation of biological pathways. Seven target genes, which encode immune-related proteins (complement C3, cytidine deaminase, regulator of G-protein Rgs22, mitogen-activated protein kinase Mapk1, metabotropic glutamate receptorm GluR8, calcium-sensing receptor CaSR, and microtubule-associated protein Map1S) were predicted to play crucial roles in the GIFT response to S. iniae infection. CONCLUSIONS: S. iniae outbreaks have hindered the development of the tilapia industry in China. Understanding the miRNA transcriptome of S. iniae-infected GIFT is important for exploring the immune responses regulated by miRNAs as well as for studying novel regulated networks to prevent and treat S. iniae infections in the future.

Effects of partially replacing dietary soybean meal or cottonseed meal with completely hydrolyzed feather meal (defatted rice bran as the carrier) on production, cytokines, adhesive gut bacteria, and disease resistance in hybrid tilapia (Oreochromis niloticus ♀ × Oreochromis aureus ♂).

We formulated experimental diets for hybrid tilapia to investigate the effects of replacing dietary soybean meal (SBM) or cottonseed meal (CSM) by completely hydrolyzed feather meal (defatted rice bran as the carrier; abbreviated as CHFM), with emphasis on fish growth, the composition of adhesive gut bacteria, intestinal and hepatic immune responses, and disease resistance. A series of four isonitrogenous (33% crude protein) and isolipidic (6% crude lipid) diets were formulated to replace the isonitrogenous percentages of CSM or SBM by 6% or 12% CHFM. Quadruplicate groups of healthy and uniformly sized hybrid tilapia were assigned to each experimental diet. Fish were hand fed three times a day for 8 weeks at a rearing temperature of 25-28 °C. The growth performance of hybrid tilapia fed diets with partial replacement of dietary SBM or CSM with CHFM was comparable to the group of fish fed the control diet. The CHFM-containing diets affected the intestinal autochthonous bacterial community in similar ways. All CHFM-containing diets stimulated the expression of heat shock protein 70 in the intestine but suppressed its expression in the liver. Only the CHFM6/SBM diet stimulated the expression of interleukin-1ß in intestine, and no effects were observed in all diets to the expression of interleukin-1ß in liver. Thus, regarding the immune response in the intestine and liver, CHFM is a good alternative protein source that induces less stress in the host. CHFM did not affect disease resistance to Aeromonas hydrophila infection in hybrid tilapia. These data suggest that CHFM is a good alternative to partially replace SBM and CSM in tilapia feed.

PCR-cloning of tilapia ATP7A cDNA and its mRNA levels in tissues of tilapia following copper administrations.

We are studying the toxicity of copper to tilapia and zebrafish and have found that the copper tolerance of tilapia and the sensitivity of zebrafish were due to several proteins' regulation mechanisms that were related to the effects of reactive oxygen species, mitochondrion copper transport, and stress response. To further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, a full length cDNA of ATP7A was obtained in tilapia. Using real time quantitative PCR, the differential regulations of ATP7A in tilapia and zebrafish were studied. It was found that Cu(2+) gave a higher induction of ATP7A in tilapia than zebrafish, both in vivo and in vitro. These results suggest that the copper tolerance of tilapia may be due to higher expression level of ATP7A.

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine.

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.

Identification and genomic sequence of a ghrelin receptor (GHS-R)-like receptor in the Mozambique tilapia, Oreochromis mossambicus.

The growth hormone secretagogue-receptor (GHS-R) Is an endogenous receptor for the gut hormone ghrelin (GRLN). Two lsoforms of GHS-R have been Identified In several animals: functional GHS-R1a and a splice variant of unknown function, GHS-R1b. Here we report Identification of a GHS-R-like receptor (GHSR-LR) In the Mozambique tilapia, Oreochromis mossambicus. The cDNA is 1584 bp In length and encodes a 384-amino acid GHS-R1a ortholog. The amino acid sequence of tilapia GHS-R1a is 54, 60, 80 and 89% Identical to that of rat, chicken, pufferfish, and seabream GHS-R1a, respectively. Genomic PCR revealed that the tilapia GHS-R gene Is composed of two exons separated by a single intron. In addition, a GHS-R1b ortholog, which Is generated by alternative splicing of the GHS-R gene and contains part of the intron, was Identified and predicted to be a 298-amino acid protein. Functional analyses of tilapia GHS-R1a were conducted using mammalian HEK 293 and CHO cells, but the expected increase in intracellular calcium Ions by tilapia or rat GRLN was not observed. We found that the GHS-R1a ortholog Is expressed In greater quantities than the GHS-R1b ortholog in all tissues assayed. Further studies are required to conclude that our Identified protein Is the GHS-R for tilapia, although the gene structure and amino acid sequence showed high similarities to other GHS-R genes; thus, we designated this protein GHSR-LR.

Influence of temperature on the ontogenetic expression of neural development-related genes from developing tilapia brain expressed sequence tags.

The developing central neural circuits in teleosts are genetically controlled and temperature-initiated. We compiled a list of transcripts expressed in the developing tilapia (Oreochromis mossambicus) brain using expressed sequence tags derived from the developing brain, and investigated genes with thermosensitive ontogenetic expression. Of 1084 clones, 893 were unique genes, 445 of which were known. Fourteen of the latter were neural development-related, and the ontogenetic expression of nine was temperature-influenced. Discs large homolog 5, myelin expression factor 2, plasticity-related protein-2, tsc2 gene product-related genes, and an inhibitor of differentiation protein 2 (Id2) were differentially temperature-influenced according to their developmental stages. Endothelial differentiation-related factor 1, midkine-related growth factor b, and mitogen-activated protein kinase 14b were specifically influenced by elevated temperature, and beta-catenin-like isoform 1 by lower temperature. Neural development-related genes, particularly those with thermosensitive ontogenetic expression, might be important for developing central neural circuits in teleosts.

Two types of aromatase with different encoding genes, tissue distribution and developmental expression in Nile tilapia (Oreochromis niloticus).

We isolated a novel type of aromatase cDNA from a Nile tilapia (Oreochromis niloticus) ovary cDNA library. Because this aromatase is phylogenetically related to brain aromatase (CYP19b) of goldfish, zebrafish and sea bass, we named it tilapia CYP19b (tCYP19b). tCYP19b encodes a protein that is predicted to consist of 495 residues and have 63.8% homology with the aromatase (tCYP19a) we previously isolated from the same source. In vitro transient transfection of cultured COS7 cells demonstrated that tCYP19b codes a functional protein to catalyze estrogen production from an androgen substrate. RT-PCR and Northern hybridization analysis showed that tCYP19b was expressed at a high level in the brain and at a low level in a wide variety of other tissues, whereas tCYP19a was mainly present in the ovary and its level significantly increased during the vitellogenic stage. RT-PCR also detected tCYP19b expression in brain and gonad tissues of both female and male tilapia during sex differentiation, but tCYP19a was only found in the ovary of the fry at that period. These results suggest that tCYP19a plays a key role in sex differentiation and ovarian development. We also isolated genes of two tilapia aromatases. Based on the location of the transcription initiation site, we predicted that there is one promoter for tCYP19a and three promoters for tCYP19b. Although the two aromatase isoforms have similar gene structures in the coding region, we found that the binding regions of SF-1/Ad4 BP region, WT1-KTS and SRY, which are sex-determining factors in mammals, are present in the 5' flank region of tCYP19a but not tCYP19b. A similar situation is present in promoters of zebrafish and goldfish aromatase isoforms. This data indicates that CYP19a plays a decisive role in sex differentiation of those species. The unique presence of the ERE motif in the tCYP19b promoter and the high expression of tCYP19b in the brain support that CYP19b is mainly involved in estrogen-mediated neural estrogen synthesis.

cDNA cloning and functional expression of a Ca2+-sensing receptor with truncated C-terminal tail from the Mozambique tilapia (Oreochromis mossambicus).

The complete cDNA sequence of the tilapia extracellular Ca(2+)-sensing receptor (CaR) was determined. The transcript length of tilapia CaR (tCaR) is 3.4 kbp and encodes a 940-amino acid, 7-transmembrane domain protein that is consistent in its structural features with known mammalian and piscine CaRs. The tCaR extracellular domain includes a characteristic hydrophobic segment, conserved cysteine residues that are implicated in receptor dimerization (Cys(129) and Cys(131)) and in coupling to the transmembrane domain (nine conserved cysteine residues), and conserved serine residues (Ser(147) and Ser(169-171)) that are linked to receptor binding of Ca(2+) and L-amino acid-mediated potentiation of function. mRNA expression of tCaR was strong in kidney, brain, and gill. Weaker expression was observed in pituitary, stomach, intestine, urinary bladder, and heart. This distribution is consistent with possible physiological roles in endocrine cells, excitable tissues, and ion-transporting barrier epithelia. Expression of tCaR mRNA in kidney and intestine was salinity-dependent, suggesting a role for the receptor in iono-/osmoregulation in this euryhaline teleost species. Human embryonic kidney-293 cells transiently transfected with tCaR cDNA demonstrated dose-dependent phospholipase C activation in response to elevations in the extracellular Ca(2+) concentration ([Ca(2+)](o)). Functional activation of the mitogen-activated protein kinase cascade by high [Ca(2+)](o) was also confirmed in these cells despite the naturally occurring truncation of the receptor's intracellular tail, which removes segments variably linked in mammalian CaRs to filamin-coupled activation of mitogen-activated protein kinase cascades. Sensitivity of phospholipase C activation to [Ca(2+)](o) was dependent on the ionic strength of the bathing medium, supporting a role in salinity sensing.

Genotoxic effects of cadmium chloride and azadirachtin treated singly and in combination in fish.

The genotoxic effects of cadmium chloride (CdCl(2)) and azadirachtin (Aza) were assessed singly and conjointly in a fish, Oreochromis mossambicus, with endpoints such as chromosome aberrations, abnormal red cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen, and kidney. The primary objectives were, first, to examine if CdCl(2), a common pollutant, and Aza, a natural product of the neem plant used extensively as an 'ecofriendly' agent for many purposes, had any genotoxic effect of their own on nontarget aquatic organisms of economic importance; and second, if Aza could have any ameliorating effect on CdCl(2)-induced genotoxicity in O. mossambicus tissues. As compared with distilled water-treated controls, both CdCl(2) and Aza induced genotoxicity in O. mossambicus, the former in greater quantity than that produced by Aza. However, Cd-induced toxicity in O. mossambicus appeared to be ameliorated to some extent by Aza.

EST-based identification of genes expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus.

The hypothalamus is involved in many physiological functions in teleosts. To accelerate the molecular analysis of hypothalamic functions, a list of transcripts expressed in the hypothalamus of adult tilapia, Oreochromis mossambicus, was compiled using the expressed sequence tag (EST) strategy. Of 161 clones, 130 clones were unique genes and 31 clones were found to be redundant. Of 130 unique genes, 32.3% (42/130 ESTs) were identified as known genes and 67.7% (88/130 ESTs) as unknown genes. The functional categorization of the known genes was analyzed. Bioinformatic analysis revealed that 62 of 88 unknown genes (62/130 ESTs, 47.7%) showed a significant homology to neither nucleotide nor translated peptide sequences in the public database. These genes might be particularly expressed in the tilapia hypothalamus.

Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol.

Glutamine synthetase, an enzyme generally associated with ammonia detoxication in the vertebrate brain and with hepatic nitrogen turnover in mammals, shows substantial activities in the gastrointestinal tract of teleostean fishes. Enzyme activity is highest in the central area of the stomach and reveals a distinct distribution pattern in stomach and along the intestine of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). In all three species, intestinal activity peaks in the distal region of the intestine. The brain contains the highest titre of the enzyme (46 U g(-1) in tilapia brain versus 15 U g(-1) in tilapia stomach), but because of the relative mass of the stomach, the largest glutamine synthetase pool in tilapia body appears to be localized in the stomach. Activities in white and red muscle are very modest at 0.1% of the brain. Independent of distribution, peak activities of glutamine synthetase in selected areas of tilapia stomach and intestine are significantly (two- to fourfold) increased after a 5-day treatment with an intraperitoneal cortisol deposit. Cortisol also increases glutamine synthetase activity in tilapia liver, white and red muscle, while activities in brain remain unaffected. We cloned and sequenced the predominant transcript of tilapia stomach glutamine synthetase (about 1.9 kb), encoding a 371-amino acid peptide. The open reading frame shows considerable identity with glutamine synthetase in toadfish (92% at peptide level, 87% at nucleotide level), but possesses a longer 3'-untranslated region than the toadfish. The tilapia glutamine synthetase mRNA contains a remnant of a putative mitochondrial leader sequence, but without a conserved second site for initiation of translation. We also find evidence for additional transcripts of glutamine synthetase in tilapia, suggesting multiple genes. Finally, we present evidence for similar abundance of glutamine synthetase transcripts in all regions of rockfish intestine. The physiological significance of the presence of glutamine synthetase in teleostean intestine is discussed.

Kidney-specific chloride channel, OmClC-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney.

The kidney plays an important role in osmoregulation in freshwater teleosts, which are exposed to the danger of osmotic loss of Na(+) and Cl(-). However, ion-transport mechanisms in the kidney are poorly understood, and ion transporters of the fish nephron have not been identified thus far. From Mozambique tilapia, Oreochromis mossambicus, we have cloned a chloride channel, which is a homologue of the mammalian kidney-specific chloride channel, ClC-K. The cDNA of the channel, named OmClC-K, encodes a protein whose amino acid sequence has high homology to Xenopus and mammalian ClC-K (Xenopus ClC-K, 41.8%; rat ClC-K2, 40.9%; rat ClC-K1, 40.1%). The mRNA of OmClC-K was expressed exclusively in the kidney, and the expression level of mRNA was increased more in freshwater-adapted fish than seawater-adapted fish. The immunohistochemical study using a specific antibody showed that OmClC-K-positive cells were specifically located in the distal nephron segments. Immunoelectron microscopy further showed that immunoreaction of OmClC-K was recognizable on the structure of basolateral membrane infoldings in the distal tubule cells. The localization of OmClC-K and its induction in hypoosmotic media suggest that OmClC-K is involved in Cl(-) reabsorption in the distal tubule of freshwater-adapted tilapia.