Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Near-infrared orientation of Mozambique tilapia Oreochromis mossambicus

Light plays a pivotal role in animal orientation. Aquatic animals face the problem that penetration of light in water is restricted through high attenuation which limits the use of visual cues. In pure water, blue and green light penetrates considerably deeper than red and infrared spectral components. Submicroscopic particles and coloured dissolved organic matter, however, may cause increased scattering and absorption of short-wave components of the solar spectrum, resulting in a relative increase of red and infrared illumination. Here we investigated the potential of near-infrared (NIR) light as a cue for swimming orientation of the African cichlid fish (Cichlidae) Oreochromis mossambicus. A high-throughput semi-automated video tracking assay was used to analyse innate behavioural NIR-sensitivity. Fish revealed a strong preference to swim in the direction of NIR light of a spectral range of 850-950nm at an irradiance similar to values typical of natural surface waters. Our study demonstrates the ability of teleost fish to sense NIR and use it for phototactic swimming orientation.     

Incorporation and metabolism of cortisol in oocytes of tilapia (Oreochromis mossambicus)

The entry and metabolism of 3H-cortisol in oocytes were investigated using isolated follicles of the tilapia (Oreochromis mossambicus) in order to examine the mechanisms of incorporation of maternal hormones into oocytes. The composition of 3H-labeled steroids in the oocyte was analyzed by high-performance liquid chromatography. A significant amount of cortisol was converted to cortisone and an unidentified molecule by the follicular layer. The contents of 3H-cortisol and 3H-cortisone in the oocyte reached an equilibrium level within 12 hr, whereas the content of the unidentified metabolite continued to increase for 36 hr. The total content of the incorporated cortisol and its metabolites was proportional to cortisol in the medium over the concentration range of 5 ng/ml to 5 microg/ml. The amounts of cortisone and the unidentified molecule increased proportionally when the concentration of cortisol in the medium was lower than 500 ng/ml, whereas they reached a plateau when the concentration of cortisol exceeded 500 ng/ml. Cortisol entry was reversible, because 90% of cortisol and cortisone in the oocyte was lost within 18 hr when the medium was changed to that without 3H-cortisol. On the other hand, 50% of the unidentified molecule was preserved at the end of the incubation. In conclusion, the entry of cortisol into the oocyte was considered to be nonspecific and due probably to simple diffusion. However, a considerable amount of cortisol (50-70%) was specifically converted to cortisone and another unidentified molecule during passage through the follicular layer.     

Metallothionein induction in early larval stages of tilapia (Oreochromis mossambicus)

Amounts of whole-body metallothionein (MT) in tilapia (Oreochromis mossambicus) larvae increased to a peak (1,500 ng mg(-1) protein) 1 d after hatching (H1), decreased rapidly thereafter, and was maintained at a constant level (700 ng mg(-1)) 3 d after hatching (H3). Waterborne Cd(2+) could stimulate MT expression in newly hatched (H0) larvae in dose-dependent and time-dependent patterns. H0 larvae, which were treated with 35 microg L(-1) Cd(2+) for 24 h, showed a 1.7-fold increase in the MT amount (174.0+/-64.7) and a 6. 5-fold increase in accumulated Cd(2+) but no significant change in Ca(2+) content, compared with the H0 control (MT, 102.6+/-48.1). H3 larvae with the same treatment revealed about a 10-fold increase in accumulated Cd(2+), a 10% decrease in Ca(2+) content, but no change in MT (261.2+/-120.0), compared with the H3 control (MT, 330+/-74.0). H0 larvae could synthesize more MT to bind Cd(2+) for detoxification in 35 microg L(-1) Cd(2+), a dose that would not affect normal physiology or survival of H0 larvae. On the other hand, 35 microg L(-1) Cd(2+) caused H3 larvae to experience hypocalcemia, an abnormal physiological condition, in which H3 larvae could not synthesize sufficient MT, thus causing greater than 25% mortality. These results indicate for the first time that the inducibility of MT by waterborne Cd(2+) is development dependent, being correlated with inconsistent sensitivities to Cd(2+) during larval development.     

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.     

Toxicological impact of Zinc Nano Particles on tilapia fish (Oreochromis mossambicus)

In this study we investigated the acute toxicity of Zinc Nano Particles (ZnO NPs) and bulk ZnO on tilapia fish (Oreochromis mossambicus). Oreochromis mossambicus was exposed to the different concentration of ZnO NPs, ZnO and mixed solution of both ZnO NPs and ZnO (20 ppb, 20 ppb, 20 ppb) respectively for 96 h. A very high impact was recorded in hematological parameters which shows significant increased (p < 0.05) in count of white WBCs and platelets in all the experimental groups compared tocontrol group. The count of RBCs, Hb, hematocrit and MCHC were significantly decreased. The remarkable changes which were recorded during this study were histopathological lesions in the gills of exposed fish including, disorganization of gill lamella, cartilaginous core disruption, lifting of epithelium, loss of secondary gill lamellae, blood congestion, fusion of secondary gills lamellae, shortening of secondary gills lamellae, atrophy and curling. Disassembly were seen in plasma membrane of liver along with blood congestion, pyknosis, necrosis, hyperplasia and formation of vacuoles. Intestinal alterations which were observed include shortening of villi, necrosis, detachment and fusion of villi and extreme goblet cells formation. It is concluded from the present study that high level of ZnO NPs, ZnO and mixed solution has a strong tendency to alter hematological parameters, histological architecture, therefore, the indiscriminate use of ZnO NPs and ZnO can subsidize in reducing the population of Oreochromis mossambicus in natural water bodies.     

Molecular cloning, expression and phylogenetic analyses of parvalbumin in tilapia, Oreochromis mossambicus

The gene expression of parvalbumin (Pvalb), a high-affinity calcium-binding protein and the major fish allergen, was significantly increased in the tilapia fry treated with methyltestosterone (MT) as examined using a subtractive hybridization assay. Using the real-time quantitative PCR, we further confirmed the increased Pvalb expression in the MT-treated tilapia fry. The 568 base pairs (bp) tilapia Pvalb (tPvalb) cDNA clone was fully sequenced and found to contain a coding region of 330 bp, which encodes a 108 amino acids protein with a molecular weight of 11,370.5 and an calculated isoelectric point of 4.56. The predicted secondary structure of tPvalb is comprised of seven alpha helices. It contains two characteristic EF-hand calcium-binding motifs, one PKC and five casein kinase II consensus phosphorylation sites. The tPvalb is highly homologous to the selected fish Pvalbs at a similarity ranging from 53% to 80%. The phylogenetic tree analysis showed that the tPvalb is closest to the Scomber japonicus Pvalb. The tPvalb was found to express in the heart, muscle, gill, kidney, brain and ovary of adult fish by RT-PCR analysis. In situ hybridization also revealed that the tPvalb was highly expressed in the hypothalamus and sarcoplasmic reticulum. A tPvalb glutathione S-transferase (GST) fusion protein was generated and digested by thrombin to remove the GST moiety. Further Western analysis showed that the tPvalb protein was cross-reacted to an anti-rat Pvalb antibody. Those results suggest that Pvalb is evolutionally conserved in tilapia.     

Cellular distributions of creatine kinase in branchia of euryhaline tilapia (Oreochromis mossambicus)

Although euryhalineteleosts can adapt to environmental fluctuation of salinity, theirenergy source for responding to changes in salinity and osmolarityremains unclear. This study examines the cellular localization ofcreatine kinase (CK) expression in branchia of tilapia(Oreochromis mossambicus). Western blot analysis ofmuscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted toseawater did not change. With the use of immunocytochemistry, three CKisoforms (MM, MB, and BB) were localized in mitochondria-rich (MR)cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To ourknowledge, this work presents the first evidence of CK expression in MRcells of tilapia gills, highlighting the potential role of CK inproviding energy for ion transport.

     

Diurnal changes in the territorial behaviour of the tilapia Oreochromis mossambicus (Peters)

The numbers of black, territorial, male tilapia in large tanks undergoes a clear diurnal cyclicity, with peak numbers seen in the afternoon. It is suggested that this cyclicity is functionally related to an observed spawning cyclicity.     

Bacterial lipopolysaccharide (LPS) and interleukin 1 (IL-1) exert multiple physiological effects in the tilapia Oreochromis mossambicus (Teleostei)

To gain insight in immuno-endocrine communication in teleosts the physiological effects of interleukin 1 and bacterial lipopolysaccharide in teleosts were investigated. Tilapia (Oreochromis mossambicus) were treated with murine interleukin 1 and E. coli lipopolysaccharide in vivo, and lipopolysaccharide was administered to pituitary lobes and head kidneys in vitro. The integument of the fish appeared to be a sensitive target for the preparations tested, since proliferation of chloride cells and of epidermal mucous cells was observed as well as an increase in epidermal thickness. These effects may relate to an acute phase-like reaction caused by the treatments. Lipopolysaccharide administration furthermore resulted in an increase in plasma free fatty acids levels. Lipopolysaccharide, but not interleukin 1, stimulated the interrenal axis of the fish, as judged by the increase in cortisol production measured in superfusion of head kidneys. In addition to these in vivo effects, lipopolysaccharide also displayed several effects in vitro. Pituitary adrenocorticotropic hormone, as well as -melanocyte stimulating hormone, release was inhibited, and the head kidney responsiveness to adrenocorticotropic hormone was inhibited after pretreatment of the tissue with the E. coli product. This latter effect coincided with the release of an unidentified -melanocyte stimulating hormone immunoreactive fraction by the head kidneys which could be stimulated by lipopolysaccharide. The data strongly support the notion that the immune system is involved in adaptive regulations in teleosts, and that immuno-endocrine interactions are phylogenetically old mechanisms.Abbreviations ACTH adrenocorticotropic hormone - AUC area under the curve - FFA free fatty acids - HPLC high-performance liquid chromatography - IL-1 interleukin 1 - LPS lipopolysaccharide/endotoxin - -MSH alpha melanocyte stimulating hormone - NIL neurointermediate lobe - POMC proopiomelanocortin - RIA radioimmunoassay - RPD rostral pars distalis     

N-Methyl-D-aspartate (NMDA) receptor subunit NR1 forms the substrate for oligomeric assembly of the NMDA receptor

The time course of the assembly of the N-methyl-D-aspartate receptor was examined in a cell line expressing it under the control of the dexamethasone promoter. These studies suggested a delay between the appearance of the NR1 and NR2A subunits and their stable association as examined by co-immunoprecipitation of NR1 and NR2A. This prompted us to examine the stability and folding of the individual subunits using nonreduced polyacrylamide gels and the sulfhydryl cross-linker BMH. Both studies showed that the NR1 subunit was expressed in a monomer and dimer form, whereas both NR2 and NR3 showed substantial aggregation on both nonreduced gels and after cross-linking. Protein degradation experiments showed that NR1 was relatively stable, whereas NR2 and NR3 were more rapidly degraded. When co-expressed with NR1, NR2 was more stable. Fluorescence recovery after photobleaching experiments showed that, under conditions of reduced ATP, the diffusion rate of NR2 and NR3 in the endoplasmic reticulum was reduced, whereas that of NR1 was unaffected. Together these data show that NR1 folds stably when expressed alone, unlike NR2 and NR3, and provides the substrate for assembly of the N-methyl-D-aspartate receptor.     

Regulation of drinking rate in euryhaline tilapia larvae (Oreochromis mossambicus) during salinity challenges

Euryhaline tilapia larvae are capable of adapting to environmental salinity changes even when transferred from freshwater (FW) to seawater (SW) or vice versa. In this study, the water balance of developing tilapia larvae (Oreochromis mossambicus) adapted to FW or SW was compared, and the short-term regulation of drinking rate of the larvae during salinity adaptation was also examined. Following development, wet weight and water content of both SW- and FW-adapted larvae increased gradually, while the dry weight of both group larvae showed a slow but significant decline. On the other hand, the drinking rate of SW-adapted larvae was four- to ninefold higher than that of FW-adapted larvae from day 2 to day 5 after hatching. During acute salinity challenges, tilapia larvae reacted profoundly in drinking rate, that is, increased or decreased drinking rate within several hours while facing hypertonic or hypotonic challenges, to maintain their constancy of body fluid. This rapid regulation in water balance upon salinity challenges may be critical for the development and survival of developing larvae.     

Evaluating the toxic potential of agrochemicals on the hypothalamic-pituitary-thyroid axis in tilapia (Oreochromis mossambicus)

Agrochemicals are a major cause of concern for the aquatic environment because of their toxicity, persistence, and tendency to accumulate in the organisms. The impact of these chemicals on aquatic organisms is due to their movement from various diffuse or point sources, which poses a great threat to aquatic fauna especially fishes, which constitute one of the major sources of protein-rich food for mankind. The present study is a first of its kind, where the toxic potential of two sublethal concentration (LC_1/10th and LC_1/25th) of four different classes of agrochemicals have been tested (Insecticide- Imidacloprid-0.074 ppm, 0.02 ppm, Fungicide-Curzate- 4.9 ppm, 1.96 ppm, Herbicide- Pyrazosulphuron ethyl-50 ppm, 20 ppm and Fertilizer-Micronutrient mixture 500 ppm, 200 ppm) on candidate markers of hypothalamus pituitary-thyroid axis (TSH, T₃, T₄, TSHβr) in Oreochromis mossambicus (tilapia) by validating hormonal level and mRNA expression. The results reveal that exposure to agrochemicals resulted in a broad range of alterations with maximum damage being caused by insecticide followed by herbicide and fungicide in that order on the thyroid axis. The results of the present study highlight the need for more detailed studies on the effects of agrochemicals that accumulate in organisms and propose that there should be a check on the rampant use of agrochemicals.