Chloride Cell Density Research Articles

Characterization of chloride cells coupled with immunolocalization of Na [formula omitted] K[formula omitted] ATPase in fishes of different migratory guilds of Indian Sundarbans

The Indian Sundarbans is a highly vulnerable ecosystem exposed to climate change with salinity intrusion, increasing cyclones as well as anomalies in rainfall and temperature that create an adverse impact on the physiology of resident and migratory fishes. Changes in the density and morphology of chloride cells are essential adaptive characters in response to varying salt level in the environment. Therefore, the present study attempts to compare the morphology, localization and abundance of chloride cells in the fishes representing different migratory guilds of the Matla River estuary of Sundarbans. The variations in the respective expression of the Na+K+ATPase α1 protein in chloride cells could comprehend the physiological adjustments across different migratory fish guilds. As observed in the present study, the chloride cells were concentrated at the base of the secondary lamellae in estuarine fishes whereas they occupied the interlamellar position for freshwater, air-breathing and amphidromous fishes, with the variations of such localizations being very prominent. The mean length of chloride cells was significantly higher in estuarine migratory fishes as compared to the amphidromous and freshwater species. However, the abundance of these cells was significantly higher in the estuarine migratory species and freshwater Labeo rohita compared to amphidromous ones. The combined patterns in abundance and immunolocalization of Na+K+ATPase in chloride cells illustrated a unique pattern and functional plasticity in the ion-transporting epithelia of the freshwater and estuarine fishes with important mechanisms of osmoregulatory ability. Hence, the study provides a clue to how osmoregulatory adjustments could be linked to the adaptive strategies in the ichthyofaunal communities of a mangrove estuary as vulnerable as Sundarban. Further studies may facilitate a better understanding of the fish physiology in formulating efficient management efforts to conserve aquatic resources in such vulnerable environments with a view to a certain climate change.

Effects of salinity level on the activity of chloride cell and mucus secreting cell in the gill of the female Shortfin molly, Poecilia mexicana Steindachner, 1863

Ovoviviparous poeciliid fishes have been relatively well studied in the unique reproductive strategy, but their osmoregulatory system largely remains unknown. In this study, we conducted a short-term (7 days) lab experiment to investigate the effect of different salinity levels from 0 (freshwater) to 50 ppt (mesosaline) on the number of chloride cells and mucus secreting cells of female Poecilia mexicana. Chloride cells were found at high density along the epithelial lamellae, whereas mucus secreting cells were also concentrated in the gill raker epithelium. More interestingly, the average density of chloride cells and the mucus secreting cell were significantly increased at high salinity levels (P < 0.05). While further validation by immunohistochemistry is warranted, integrative data from our study suggested that the potential function of the osmoregulatory mechanism/strategy was supported by chloride and mucus secreting cells of female P. mexicana gill.

Salinity influence on growth, osmoregulation and energy turnover in juvenile pompano Trachinotus marginatus Cuvier 1832

The effects of salinity on growth and parameters related to osmoregulation and energy turnover were evaluated in juvenile pompano Trachinotus marginatus. In a first experiment, fish were reared at different salinities (4, 8, 12, 16 and 20ppt) for 15days. No fish mortality was observed. Also, no significant difference in fish size (length and weight), hematocrit and plasma glucose concentration was observed among treatments. Although a significant increase in plasma Na+, Cl− and K+ concentrations was noted with increasing water salinities, while no significant changes in plasma osmolality were observed. The isosmotic point was calculated as 357.5mOsm/kg H2O, corresponding to the osmolality of the ambient water at salinity 13.1ppt. These findings indicate that juvenile pompanos T. marginatus acclimated to 20ppt are fully acclimated to a wide range of salinities (4–20ppt) after 15days of transfer to the new salinity condition. Also, they show that the plasma isosmotic point of juvenile T. marginatus (13ppt) is within the range of values reported for some euryhaline teleost fish from different habitats (10–14ppt). In a second experiment, fish were reared at different salinities (3, 6, 12 and 32ppt) for 28days. No significant difference in survival, feed conversion, Fulton's condition factor, body weight coefficient of variation, feed intake and intestinal trypsin activity was observed among salinity treatments. However, fish reared at 32ppt showed lower body weight, specific growth rate, oxygen consumption, and gill Na+,K+-ATPase activity than those reared at 3 and 6ppt. Also, their gills showed higher chloride cell area, epithelium thickness, chloride cell density and mucous cell density than those of fish reared at 3, 6 and 12ppt. These findings indicate that juvenile pompano T. marginatus is euryhaline in the range of salinities tested (3–32ppt) and grows better when reared at low salinities (3–6ppt) for 28days. This performance is associated with a good osmoregulatory ability resulting from structural and biochemical adaptive responses of the gill epithelium to salinity changes paralleled by increased aerobic metabolism and elevated use of liver carbohydrate store. Although it would be cost effective to maintain higher salinities in coastal aquaculture systems given the proximity to the marine environment, our findings support the idea of rearing juvenile pompano T. marginatus in low salinities (3–6ppt) to improve growth. Statement of relevancePompano rearing performance is improved in low salinities.

Cytological comparison of gill chloride cells and blood serum ion concentrations in kutum (Rutilus frisii kutum) spawners from brackish (Caspian Sea) and fresh water (Khoshkrood River) environments

Abstract The size and number of chloride cells and serum ion concentrations in kutum, Rutillus frisii kutum Nordman, from brackish (Caspian Sea) and fresh water (Khoshkrood River) environments were studied to gain a better understanding of osmoregulation in this species. Twenty mature kutum specimens were collected from the Caspian Sea (Anzali coasts, 8.49 ppt salinity and 12.4°C temperature) and 20 specimens from Khoshkrood River (0.18 ppt salinity and 18°C temperature). Gill samples were analyzed histologically and concentrations of Na+, Cl- , K+, and Mg2+ ions were determined in the blood serum. Concentrations of Na+, Cl- , K+, and Mg2+ ions and osmotic pressure in mature kutum from brackish water were significantly higher than in specimens from fresh water. The average size and number of chloride cells in the fish from seawater were considerably larger than those from fresh water. The mean size of chloride cells was 6.89 ± 1.16 μm in brackish water samples and 5.1 ± 0.81 μm in river samples. The average number of chloride cells in brackish and river water samples were 16.92 and 6.57, respectively. The density and size of chloride cells increased with increases in salinity

Ambient salinity and osmoregulation, energy metabolism and growth in juvenile yellowtail kingfish (Seriola lalandiValenciennes 1833) in a recirculating aquaculture system

The effects of salinity on plasma osmolality, branchial chloride cell density, feed consumption and conversion and growth performance of yellowtail kingfish (Seriola lalandi) were evaluated. Fish (11.6 ± 0.6 g) were kept for 29 days at 14, 18, 22, 26 (experimental) and 30 g L−1 (control) salinity in independent, pilot-scale recirculation aquaculture systems. No differences in plasma osmolality or chloride cell numbers in gills were observed, pointing to a strong osmoregulatory capacity in the juveniles. Fish at 14, 18 and 22 g L−1 (7.61 ± 0.19, 7.61 ± 0.01 and 7.61 ± 0.13% day−1, respectively) had higher growth rates than fish at 26 and 30 g L−1 (7.10 ± 0.05 and 6.97 ± 0.06% day−1 respectively). The higher growth rate at lower salinity resulted from increased feed intake; feed conversion was not different. An evaluation of the impact of salinity on growth rate of on-growing stages (till market size) seems warranted to assess whether the profitable effects of low salinity persist in later stages of this important aquaculture species.

Lead accumulation and its effects on the branchial physiology of Prochilodus lineatus

The purpose of this work was to determine the tissue accumulation of lead (Pb) and its effects on osmoregulatory processes of the freshwater fish Prochilodus lineatus. Juvenile fish were exposed to Pb (from 1.7 to 0.7mg of dissolved Pb L(-1)) for 6, 24 and 96h and Pb accumulation was analyzed in the gills, liver, kidneys, blood cells and muscle. The following parameters were also analyzed: hematologic (hemoglobin content, hematocrit and number of red blood cells), metabolic (blood glucose), endocrine (blood cortisol), osmo ionic (plasma osmolality and Na(+), K(+), Cl(-) and Ca(+2) concentrations), gill enzymes (Na(+)/K(+)-ATPase and carbonic anhydrase), chloride cell (CC) density and CC location in the gills. Pb accumulated in all the analyzed tissues, with the kidneys showing the highest concentration, followed by the gills and liver. The lowest Pb concentrations were found in blood cells and muscle. Pb promoted an increase in blood glucose after 6 and 24h exposure. Gill Na(+)/K(+)-ATPase was inhibited after 24h of exposure, but its activity was restored at 96h, probably due to the increase in CC in gill lamellae. Plasma Na(+) was reduced after 6 and 96h, while K(+) concentrations increased at all the experimental times. Fish exposed to Pb showed reduced plasma Ca(+2) at all experimental periods. Hematologic parameters remained unchanged. Overall, this study demonstrated that Pb interferes in osmoregulatory processes of P. lineatus and the proliferation of CC in the gills is a response in order to reestablish adequate ion concentrations.

Physiological effects of gasoline on the freshwater fish Prochilodus lineatus(Characiformes: Prochilodontidae)

The purpose of this work was to evaluate the effects of the water-soluble fraction of gasoline (WSFG) on the Neotropical freshwater fish Prochilodus lineatus. The WSFG was prepared by mixing gasoline in water (1:4) and animals were exposed for 6, 24 and 96h to 5% diluted WSFG or only to water. After exposure, blood was collected from the caudal vein and the gills were removed. The following parameters were analyzed: hematological (hemoglobin, hematocrit, number of red blood cells), osmo-ionic (plasma Na+, Cl- and K+ and plasma osmolarity), metabolic (total plasma proteins and glucose), endocrine (cortisol), density and distribution of chloride cells [CC] in the gills (immunohistochemistry), and branchial Na+/K+-ATPase (NKA) activity. Hemolysis was found to occur after 96h exposure to WSFG, as indicated by the decrease in the hematological parameters analyzed, followed by an increase in plasma K+. Secondary stress response was revealed by the occurrence of hyperglycemia in the three periods of exposure, despite the absence of significant increases in the plasma cortisol. The exposure to WSFG also caused an increase in the quantity of CC and in plasma Na+, after 24h, as well as in the enzymatic activity of NKA and plasma osmolarity, after 24h and 96h. These results indicate that fish exposed to the WSFG showed physiological adjusts to maintain their osmotic balance. However, the increase in the quantity of CC in the lamellae may interfere in the gas exchange impairing respiration.

Effects of the herbicide atrazine in neotropical catfish (Rhamdia quelen)

The exposure to a world-wide used herbicide atrazine (ATZ) (96h exposure to 2, 10, and 100μgL−1), was investigated on the freshwater fish Rhamdia quelen through a multi biomarker approach. Liver histopathology revealed leukocyte infiltration, hepatocyte vacuolization like steatosis and necrosis areas, leading to raised lesion index levels in all tested concentrations. The increase of free melanomacrophage numbers was observed. Gill filaments revealed considerable loss of the microridges on pavement cells at 10 and 100μgL−1 of ATZ, and a significantly increased of chloride cell (CC) number and density on apical surface area at 100μgL−1 of ATZ. CAT, GST, GPx, and GR activities were inhibited by all tested concentrations. GSH levels were reduced in individuals exposed to 100µgL−1. Osmoregulatory function was also disturbed. We observed an increase of plasma magnesium concentrations at 10µgL−1. Additionally the inhibition of branchial carbonic anhydrase activity was observed at 100µgL−1. In the kidney, carbonic anhydrase activity decreased only in the group exposed to 2µgL−1. These results suggest that ATZ, represents a potential ecotoxicological hazard and can be hepatotoxic and nephrotoxic even low concentrations. The current study was the first to show the nephrotoxic effect of ATZ in fish. Besides, in Brazil, the environmental protection agency (CONAMA) establishes that the maximum allowed level of dissolved ATZ in water is 2μgL−1, but the present results showed that this concentration may cause histopathological, biochemical and physiological changes in R. quelen.

Gill damage to juvenile orange-spotted grouper Epinephelus coioides (Hamilton, 1822) following exposure to suspended sediments

Juvenile Epinephelus coioides were exposed to three nominal concentrations (8, 32, 128 mg wet sediments L−1) of suspended sediments (SS) from Port Shelter (PS), Mirs Bay (MB) and Victoria Harbour (VH) for 10 and 30 days using semi-static system. Sediments from VH contained higher concentrations of Cu and polycyclic aromatic hydrocarbons (PAHs) than sediments from PS and MB. Gill damages including lamellar blood sinus dilation and vascular congestion were prevalent after just 10 days of exposure to SS. Fish exposed to SS at the highest concentration of 128 mg L−1 showed higher incidence of lamellar aneurism. Hyperplasia in the base of lamellae was recorded in fish that had been exposed to contaminated SS from VH. Significant increases in the density of chloride cells and mucous cells were found on the gills of fish that had been exposed to 128 mg L−1 of SS from PS. Clogging of gills by SS produced hypoxic-like responses in fish. Polluted sediments from VH produced addictive or synergistic effects between SS and chemical contaminants on fish.

Ontogenetic changes in location and morphology of chloride cells during early life stages of the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water

Ontogenetic changes in the location, size, density and morphology of chloride cells in the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water are described using Na(+) /K(+) -ATPase immunohistochemistry, light microscopy (LM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). The pattern of chloride cell distribution changed during development under both treatments, with chloride cell density decreasing significantly from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly thereafter (P < 0·05). Chloride cells were always denser in fresh- than in brackish-water larvae. In both treatments, chloride cells located on the outer operculum and tail showed a marked increase in size with age, but cells located on the abdominal epithelium of the yolk sac and the inner operculum showed a significant decrease in size (P < 0·05). Chloride cells from brackish-water adapted larvae from 1 day post-hatch onwards were always significantly larger (P < 0·05) than those from freshwater-adapted larvae. SEM revealed structural differences in chloride cell apical morphology according to environmental conditions. There appears to be clearly defined temporal staging of the appearance of adaptive mechanisms that confer an ability to cope with varying environmental conditions during early development.

How aluminium exposure promotes osmoregulatory disturbances in the neotropical freshwater fish Prochilus lineatus

The aim of this study was to understand the effects of the interaction between aluminium and low pH in a native fish species Prochilodus lineatus. Thus, juveniles of this neotropical fish species were exposed to 196 μg L −1 of dissolved aluminium in acid water (Al group), only to acid water (pH group) or to water with neutral pH (CTR group) for 6, 24 and 96 h. Al effects were evaluated with regard to hematological parameters (hemoglobin, hematocrit and red blood cell number), plasma ions and osmolarity, density and distribution of chloride cells (CC), Na +/K +–ATPase activity in the gills, metabolic (protein and glucose) and endocrine (cortisol) parameters. The fish exposed to Al had increased hematological and metabolic parameters in relation to the CTR group after all periods of exposure. In fish exposed to Al for 24 and 96 h plasma ions and osmolarity were significantly lower and the identification of the enzyme Na +/K +–ATPase by immunohistochemistry indicated a reduction in the number of CC in the gills. Enzyme activity was 50% lower in fish exposed to Al in all experimental times. Taken together these results showed that acute exposure to Al causes an ionic unbalance, probably related to the effects of Al on Na +/K +–ATPase activity, on the distribution and number of chloride cells in the gills as well as the effects associated with the stress response caused by the presence of the metal.

Fish louseArgulus funduli(Crustacea: Branchiura) ectoparasites of the euryhaline teleost host,Fundulus heteroclitus, damage the ion-transport capacity of the opercular epithelium

Killifish ( Fundulus heteroclitus (L., 1766)) collected in the wild and kept in full-strength seawater were naturally parasitized by the ectoparasite Argulus funduli Krøyer, 1863, a copepod fish louse that creates inflamed skin lesions on the opercular epithelium and host gills. We assessed the damage done by lesions by counting the density of mitochondria-rich cells by fluorescence microscopy and by measuring Cl–secretion rate electrophysiologically using control (no lesions) and affected isolated opercular epithelia, often as paired left and right membranes from a single fish. Epithelia with lesions had a significantly reduced Cl–secretion rate, and in the lesions, the density of chloride cells was near zero. Contralateral membranes without lesions from infested fish had transport rates not significantly different from membranes taken from uninfected control animals, indicating no overcompensation on the contralateral membranes. Healthy control and infested animals were transferred to hypersaline conditions (twice seawater). Infested and control animals all survived transfer and had elevated plasma Na+and plasma osmolality. Infested animals failed to significantly elevate Imto the same level as healthy animals and there was a difference in hematocrit. Happily, the hypersaline challenge also resulted in detachment and death of adult A. funduli. We conclude that Argulus lesions impair salt transport in affected membranes but do not significantly affect survival on hypersaline challenge, and that hypersaline exposure is a successful treatment for A. funduli infestation in these strongly euryhaline teleosts.

16.P14. Chloride cell density in the gills of the erythrinid fish, Hoplias malabaricus, after exposure to deionized water and hypoxia

16.P14. Chloride cell density in the gills of the erythrinid fish, Hoplias malabaricus, after exposure to deionized water and hypoxia

Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1α and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway

Redd1, a recently discovered stress-response gene, is regulated by hypoxia via hypoxia-inducible factor 1 (HIF-1) and by DNA damage via p53/p63; however, the signaling pathway by which its expression is induced by hypoxia has not been elucidated. In the present study, we demonstrated that the expression of Redd1 in response to hypoxia (1% O 2), hypoxia-mimetic agent, cobalt chloride (CoCl 2) and high cell density (HCD) requires coactivation of HIF-1α and Sp1. CoCl 2 and HCD induced the activation of HIF-1α and Sp1 in HeLa cells, and siRNAs targeting HIF-1α and Sp1 abrogated Redd1 expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and by a dominant-negative PI3K mutant reduced the expression of Redd1 and activation of HIF-1α and Sp1 by CoCl 2 and HCD. Also, suppression of Akt activation blocked the expression of Redd1 and the activation of HIF-1α and Sp1 by CoCl 2 and HCD. Furthermore, we found that the induction of Redd1 expression by CoCl 2 can be mediated by activation of Sp1 in HIF-1α-deficient cells but that a higher level of Redd1 expression is achieved when these cells are transfected with HIF-1α. These results demonstrate that hypoxic condition-and HCD-induced expression of Redd1 is mediated by coactivation of Sp1 and HIF-1α downstream of the PI3K/Akt signaling pathway.

Cortisol receptor blockade and seawater adaptation in the euryhaline teleost Fundulus heteroclitus

To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10 per thousand) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg(-1)) or high dose (200 mg.kg(-1)). Fish were transferred to 1.5 x seawater (45 per thousand) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors.

Análise morfométrica das células de cloro de Poecilia vivípara expostas a frações da folha e da casca do caule de Caryocar brasiliensis

Fractions of Caryocar brasiliensis leaf and bark, tested as molluscicidal, fought the intermediary host of schistosomiasis. The ecological niche of this mollusk is related to water sources. Besides this, a fungitoxic action of the ethyl-acetate fraction (leaf) was verified, exhibiting high activity towards Paracoccidioides brasiliensis . The gill epithelium of Poecilia vivipara (Cyprinodontiformes, Poeciliidae) was used to test the toxicity of these fractions to animal cells. Quantitative analyses demonstrated a decrease in the density of chloride cells exposed to the leaf fractions and an increase in those exposed to bark fractions. The leaf and bark fractions induced a decrease in the area of the chloride cells (CC) in the gill filament. The perimeter of the CC did not suffer significant changes in face of these fractions. The aqueous fraction of the leaf is more toxic to the guaru. Bark fractions are less debilitating, and can be employed in the control of cell populations, showing high efficacy at it

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.

Gill Cellular Changes Induced by Copper Exposure in the South American Tropical Freshwater Fish Prochilodus scrofa

The cellular changes in gill tissue induced by exposure to copper were studied in the tropical freshwater fish Prochilodus scrofa, with emphasis on chloride and pavement cells. Damage to gills included epithelial changes such as lifting, rupture, peeling of lamellar epithelium, lamellar fusion, hyperplasia, and cellular hypertrophy. Cell degeneration by necrosis and apoptosis was intense in fish exposed to 25 and 29 μg Cu L−1. Pavement cells showed microridge reduction on their surface. Chloride cells proliferated in the lamellar epithelia close to the onset of the lamellae. However, no changes in total chloride cell density in contact with the water were observed. The chloride cell apical area of fish exposed to copper increased, but only fish exposed to 25 μg Cu L−1 showed significant increase in the chloride cell fractional area. At this water copper concentration, almost 60% of the chloride cells were apoptotic. Necrotic chloride cells increased with copper in water, reaching 70% in fish exposed to 29 μg Cu L−1 (=LC50 calculated for this species). Pavement and chloride cell proliferation and hypertrophy on lamellar epithelia increased the thickness of the water–blood barrier. Our findings suggest severe impairment of ion regulation and gas transfer of fish exposed to copper.

Dynamics of pavement cell-chloride cell interactions during abrupt salinity change in Fundulus heteroclitus.

Freshwater-adapted killifish (Fundulus heteroclitus) opercular epithelia were dissected and subjected to blood-side hypertonic bathing solution in Ussing-style chambers to simulate the increase in blood osmolality during migration to sea water. Conversely, seawater-acclimated killifish opercular epithelia were subjected to hypotonic bathing solutions to simulate the initial stages of migration to fresh water. Freshwater-acclimation (hypertonic stress) induced a rapid (approximately 30 min) increase in membrane conductance (G(t)) from 3.10+/-0.56 to 7.52+/-1.15 mS x cm(-2) (P<0.01, N=27), whereas seawater-acclimation (hypotonic stress) induced a rapid decrease in G(t) from 8.22+/-1.15 to 4.41+/-1.00 mS x cm(-2) (P<0.01, N=27; means +/- S.E.M.). Control seawater-acclimated membranes had a density of apical crypts (where chloride cells are exposed to the environment; detected by scanning electron microscopy) of 1133+/-96.4 crypts x mm(-2) (N=12), whereas the hypotonically shocked specimens had a lower crypt density of 870+/-36.7 crypts x mm(-2) (P<0.01 N=10; means +/- S.E.M.). Hypertonic shock of freshwater membranes increased crypt density from 383.3+/-73.9 (N=12) to 630+/-102. 9 crypts x mm(-2) (P<0.05; N=11; means +/- S.E.M.). There was no change in density of chloride cells, as detected by fluorescence microscopy; hence, osmotic stress changes the degree of exposure, not the number of chloride cells. Cytochalasin D (5.0 micromol x l(-1)) completely blocked the conductance response to hypotonic shock and the reduction in apical crypt density measured by scanning electron microscopy, while phalloidin (33 micromol x l(-1)), colchicine (3x10(-4)mol x l(-1)) and griseofulvin (1.0 micromol x l(-1)) were ineffective. Actin imaging by phalloidin staining and confocal microscopy revealed extensive actin cords in pavement cell microridges and a ring of actin at the apex of chloride cells. We conclude that the actin cytoskeleton of chloride cells is required to maintain crypt opening and that osmotic shock causes chloride cells to adjust their apical crypt size.

Surface ultrastructure of the gills of the mullets Mugil curema, M. liza and M. platanus (Mugilidae, Pisces).

Mugil curema, M. liza, and M. platanus were collected from the southeastern and southern coast of Brazil. The second gill arches were analyzed by scanning electron microscopy and histology. The highest density of chloride and mucus-secreting cells was observed in the gill filaments of M. liza and M. platanus. Spines are scarce and were found only in the pharyngeal region of M. curema. The dorsal angle of curvature of the simple projections is most reduced in the rakers of M. liza and M. platanus. The raker borderline on the internal side of the arches of M. curema has grooves that do not occur in the other two species. On the external side of the branchial arches, the borders of the rakers of M. liza and M. platanus are smooth. The shape of the rakers is characteristic for each species: in M. curema, it resembles the letter "D"; in M. liza, it is trapezoidal, and in M. platanus, it is triangular. Thus there is a morphologic similarity between M. liza and M. platanus, and both differ from M. curema. All three species show elongated and extremely elaborated rakers that are placed next to each other and turned toward the opercular cavity. There are few taste buds and only several mucus-secreting cells along the whole pharyngeal region. These characteristics suggest that these species do not select food chemically but obtain it mechanically with the rakers and aggregate it with mucus.