Salinity effects on cytometrical parameters of the kidney in the euryhaline teleost Oreochromis mossambicus Peters

Study of the effects of environmental salinity on the glomerular areas and the nuclear areas of the renal tubules in tilapia showed significant differences ( P < 0.001 or 0.05) between seawater- and freshwater-adapted fish. Upon seawater adaptation, there were decreases in the glomerular areas and the nuclear areas in the main segments of the nephron: glomerular, −24.1%; 1st proximal, −17.1%; 2nd proximal, −21.5%; distal, −9.1%; collecting, −26.5%. These cytometrical changes are discussed in relation to the different osmoregulatory functions of euryhaline teleosts adapted to sea water and to fresh water.     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Response to acute changes in salinity of two different muscle type creatine kinase isoforms, from euryhaline teleost (Oreochromis mossambicus) gills

Two CKM isoforms (CKM1 and CKM2) from the gills of tilapia (Oreochromis mossambicus) were obtained after transfer from freshwater (FW) to seawater (SW, 25 ppt). Based on the 5' and 3' RACE, the identity of CKM1 and CKM2 was determined to be 59% in the 5'-untranslated region (5'-UTR) and 41.9% in the 3'-UTR. Using Northern blot hybridization with the CKM1 and CKM2 3'-UTR probes, CKM1 and CKM2 were found to be expressed in muscle, heart and gill. The levels of these two different CK isoforms (CKM1 and CKM2) were shown to be different in FW than after acute SW transfer, showing that CKM isoforms respond to changes in salinity.     

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.

     

Predictive Blood Chemistry Parameters for Pansteatitis-Affected Mozambique Tilapia (Oreochromis mossambicus)

One of the largest river systems in South Africa, the Olifants River, has experienced significant changes in water quality due to anthropogenic activities. Since 2005, there have been various “outbreaks” of the inflammatory disease pansteatitis in several vertebrate species. Large-scale pansteatitis-related mortality events have decimated the crocodile population at Lake Loskop and decreased the population at Kruger National Park. Most pansteatitis-related diagnoses within the region are conducted post-mortem by either gross pathology or histology. The application of a non-lethal approach to assess the prevalence and pervasiveness of pansteatitis in the Olifants River region would be of great importance for the development of a management plan for this disease. In this study, several plasma-based biomarkers accurately classified pansteatitis in Mozambique tilapia (Oreochromis mossambicus) collected from Lake Loskop using a commercially available benchtop blood chemistry analyzer combined with data interpretation via artificial neural network analysis. According to the model, four blood chemistry parameters (calcium, sodium, total protein and albumin), in combination with total length, diagnose pansteatitis to a predictive accuracy of 92 percent. In addition, several morphometric traits (total length, age, weight) were also associated with pansteatitis. On-going research will focus on further evaluating the use of blood chemistry to classify pansteatitis across different species, trophic levels, and within different sites along the Olifants River.     

Acute osmotic stress affects Tilapia (Oreochromis mossambicus) innate immune responses

Most of the tilapia studies are focused on its osmoregulatory mechanism. Meanwhile, less information is available about its innate immune response on fish faced with hyperosmolality. In the present study, in vivo analyses were carried out to investigate the innate immune response of Oreochromis mossambicus, transferred from freshwater to 25 ppt seawater (SW). In vivo, lysozyme activities of plasma and head kidney (HK) were increased at 1h and at 24h after transfer to SW but decreased at 8h after SW transfer. Surprisingly, the alternative complement pathway in plasma increased 8h after SW transfer. The phagocytic capacity of spleen and HK immune cells increased modestly at 1h and at 4h, after SW transfer, but the respiratory burst activity of immune cells in both HK and spleen shows an increase in superoxide release at 8h after SW transfer. Our results reveal that the transfer of fish from conditions of hypoosmolarity to hyperosmolality significantly enhances plasma lysozyme, ACP activity, and both phagocytic and respiratory burst activity. Taken together, the results indicate that exposure of tilapia to hyperosmotic conditions has immunostimulatory effects on its cellular immune reactions (phagocytosis and respiratory burst activity) and humoral reactions (lysozyme activity and complement activity).     

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.     

Continuous Exposure to Light Suppresses the Testicular Activity in Mozambique Tilapia Oreochromis mossambicus (Cichlidae)

Journal of Ichthyology - Although photoperiod plays a critical role in seasonally breeding fish, effect of altered photoperiod on reproduction of continuously breeding fish is not well understood....     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

The interrelationships between stress and osmoregulation in a euryhaline fish, Oreochromis mossambicus

Three groups of Oreochromis mossambicus, progeny of a single couple, acclimated to freshwater, seawater and iso‐osmotic water, respectively, were confined for 4 h following the same experimental procedures. Confinement stress evoked a ‘simple’ stress response that produced a significant rise in cortisol and glucose and slight osmotic disturbances for each tested salinity. It was speculated that the mechanisms securing osmotic homeostasis in this extremely euryhaline species favour it from a wide osmotic stress‐evoked disturbance.     

Expression of glucocorticoid receptor in the intestine of a euryhaline teleost, the Mozambique tilapia (Oreochromis mossambicus): effect of seawater exposure and cortisol treatment

Cortisol plays an important role in controlling intestinal water and ion transport in teleosts possibly through glucocorticoid receptor (GR) and/or mineralocorticoid receptor. To better understand the role of GR in the teleost intestine, in a euryhaline tilapia, Oreochromis mossambicus, we examined (1) the intestinal localizations of GR; (2) the effects of environmental salinity challenge and cortisol treatment on GR mRNA expression. The mRNA abundance of GR in the posterior intestinal region of tilapia was found to be higher than that in the anterior and middle intestine. In the posterior intestine, GR appears to be localized in the mucosal layer. GR mRNA levels in the posterior intestine were elevated after exposure of freshwater fish to seawater for 7 days following an increase in plasma cortisol. Similarly, cortisol implantation in freshwater tilapia for 7 days elevated the intestinal GR mRNA. These results indicate that seawater acclimation is accompanied by upregulation of GR mRNA abundance in intestinal tissue, possibly as a consequence of the elevation of cortisol levels. In contrast, a single intraperitoneal injection of cortisol into freshwater tilapia decreased intestinal GR mRNA. This downregulation of the GR mRNA by cortisol suggests a dual mode of autoregulation of GR expression by cortisol.     

Biochemical characterization of isolated branchial mitochondria-rich cells of Oreochromis mossambicus acclimated to fresh water or hyperhaline sea water

Mitochondria-rich cells have been separated from other epithelial cells of tilapia (Oreochromis mossambicus) gills by density gradient centrifugation on Percoll. During centrifugation two main bands of cells formed. The viability of the cells in both bands was high (>90%). In one band, 45–47% of the total cell number was mitochondria-rich cells. The other band contained at least 80% pavement cells, representing the majority of other gill epithelial cell types. A comparison of the activities of four enzymes involved in major metabolic and ion regulatory functions was made between these two different fractions of cells. Furthermore, the separation of gill epithelial cells and determination of enzymatic activity was carried out in tilapia after the fish were acclimated to fresh water or hyperhaline sea water (60 mg·ml^-1 S) to gain an indication of the relative contribution of mitochondria-rich cells and pavement cells to both NaCl excretion and absorption. Regardless of acclimation salinity, the activities of Na⁺/K⁺-ATPase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were significantly higher in mitochondria-rich cells than in pavement cells. However, tilapia acclimated to hyperhaline sea water possessed significantly lower carbonic anhydrase activity in mitochondria-rich cells than in pavement cells. In contrast, no significant difference of carbonic anhydrase activity was observed between the two cell fractions in tilapia acclimated to fresh water.Abbreviations ATPase adenosine triphosphatase - CA carbonic anhydrase - DASPMI dimethylaminostyrylmethylpyridinium iodine - FW fresh-water - GIDH glutamate dehydrogenase - G6PDH glucose-6-phosphate dehydrogenase - HSW hyperhaline sea water (60 mg·ml^-1) - MR cells, mitochondria-rich cells - S salinity     

Catecholamines and the abundance of blood cells in a fresh water tropical teleost (Colisa fasciatus) in relation to cold-shock.

Intraperitoneal injections of epinephrine (0.05 microng/g) elicited characteristic changes in the abundance of circulating leucocytes at selected time intervals (viz., 3, 15 and 27 min. and, later, at intervals of 48 minutes up to 363 min. and at 12 and 24 h) post-injection in Colisa. Leucocytosis was evident as 15, 27 and 75 min, and tendency towards leucocytosis was observed at 123 and 267 min.; at 3, 171, 219, 315 and 363 min. and at 12 and 24 h, the total leucocyte counts for the experimentals and controls were not significantly different. No such corresponding significant changes were observed in the abundance of circulating erythrocytes or thrombocytes. Leucocyte sequence elicited by epinephrine was also apparent in Colisa which had been exposed to a temperature of 2 degrees C for one minute. Pretreatment with phenoxybenzamine (3.0 mg/l aquarium water), an alpha-adrenergic antagonist, abolished the cold-shock leucocytic phases, except 27 min. leucocytosis which emerged unaffected. Exogenous norepinephrine (2 microng/g) and isoproterenol (0.1 microng/g) failed to elicit any significant change in the number of circulating leucocytes, erythrocytes, or thrombocytes. Clearly adrenergic mechanisms are involved in the cold-shock leucocyte stress sequence. The results suggest alpha-activating role of epinephrine during the leucocyte stress syndrome in Colisa and, apparently, epinephrine is leucocytic.     

Immunolocalization of Vacuolar-Type H(+)-ATPase in the Yolk-Sac Membrane of Tilapia (Oreochromis mossambicus) Larvae

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H(+)-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na(+) in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na(+) uptake in exchange for H(+) secretion through V-ATPase in FW-adapted tilapia during early life stages.     

The effect of prolactin on kidney structure of the euryhaline teleost Gasterosteus aculeatus during adaptation to fresh water

Summary The effect of injections of ovine prolactin on kidney structure was examined in the first 10 days following transfer of seawater sticklebacks to fresh water.In hormone injected animals as well as in controls the glomeruli increase slightly in size after transfer. The podocytes intensify the secretion of mucopolysaccharides, which is indicative of increased turnover of the components of the glomerular basal lamina. The nuclei of the podocytes become enlarged, while those of the juxtaglomerular cells decrease in size. These changes are related to the well known rise of the glomerular filtration rate following transfer to fresh water. Structural indications that prolactin is involved in the control of glomerular filtration were not found.The epithelial cells of the three nephronic segments and of the ureter become considerably better developed after transfer to fresh water. Cell height, nuclear and mitochondrial volume, and surface of the membranes of the basal labyrinth increase in all tubular epithelia, although not to the same extent. Increases are moderate in the first proximal segment, but increasingly higher for the second proximal segment, collecting duct and the ureter. Especially the growth of membrane surface of the basal labyrinth, site of ion transport mechanisms, is impressive. In controls, values characteristic for freshwater fishes are reached in 6 to 9 days for all parameters for cellular development. Prolactin injections greatly stimulate growth rates in all tubular epithelia: freshwater values are reached within 3 days. No further increase was found, however.     

New aspects of the intrafilamental vascular system in gills of a euryhaline teleost,Tilapia mossambica

Summary The non-respiratory vascular system of T. mossambica gill filaments was studied in serial longitudinal and cross sections. Comparatively few scattered vascular communications occur between the afferent filament artery and the central venous sinus (AVA_aff). The efferent filament artery, however, is connected by regularly arranged anastomoses (AVA_eff), directly, and sometimes indirectly via nutritive vessels, to the central sinus. These AVA_eff are about as numerous as lamellae counted on one side of each filament, although they diminish slightly in number towards the filament base. The relation AVA_eff to AVA_aff was 17.6:1 in the distal and 17.8:1 in the basal filamental region, while in the tip region of 7 filaments 126 AVA_eff but only 1 AVA_aff were encountered. No direct connection between the lamellar lacunae and the central sinus was detected. According to these results, non-respiratory intrafilamental blood shunting appears unlikely. AVA_eff are assumed to be the main route for blood entering the central venous sinus which would consequently flow into the branchial veins.The authors wish to express their sincere thanks to Miss Angelika Krauß for her valuable technical assistance and to Miss Erna Finger for making the photographs. Thanks are also due to Mr. W. Zeltmann for drawing Figs. 2, 5, and 8 and to Mr. K. Herzog for Fig. 7.     

Lysosomal stability in Oreochromis mossambicus (Peters) on exposure to surfactants

The three commonly used surfactants viz. anionic sodium dodecyl sulfate (SDS), cationic cetyl tri methyl ammonium bromide (CTAB) and non-ionic triton X-100 were toxic even at sub lethal levels (1 ppm for 30 days) to 0. mossambicus. Lysosomal stability index (LSI) was lowest in triton-exposed animals in vitro. In vivo, CTAB was the most toxic. SDS, the anionic surfactant was the least toxic. The possible role of surfactant structure, critical micellar concentration (CMC) and metabolism in influencing the toxicity is discussed and mechanism of action via membrane lipid peroxidation is suggested.