Cellular responses in the skin of the gilthead sea bream Sparus aurata L. and the sea bass Dicentrarchus labrax (L.) exposed to high ammonia.

Adult gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax were exposed for 24 and 48 h, respectively, to two concentrations of ammonia each (mean values of 3·34 and 13·10 mg l(-1) TA-N in S. aurata; 2·99 and 11·90 mg l(-1) TA-N in D. labrax). Light microscopy and computerized morphometry were used to evaluate ammonia-induced alterations in skin structure during exposure and following recovery in normal water. In S. aurata, ammonia exposure induced a concentration-dependent increase in the number (hyperplasia) of neutral mucous cells (mc), with peak values at 24 h recovery after exposure. An increase in the dispersion of melanosomes in skin melanocytes was also observed in the dermis and occasionally in the epidermis of S. aurata, with peak values at 24 h of ammonia exposure. Exposure of D. labrax to ammonia had, likewise, concentration-dependent effects on mucous secretion. Of the two types of mc in this species, there was an increase in the number of the neutral mc and a reduction in the much more numerous acid mc, with peak values at 24 and 48 h, respectively, of ammonia exposure. The more intense mucous secretion in D. labrax compared to S. aurata could be related to the lower tolerance to ammonia in D. labrax, as reported elsewhere. Finally, the increase in melanosome dispersion was less evident in D. labrax, due to highly variable control values. These morphological alterations to the skin could be useful indicators of non-specific stress in cultured fishes.

Fine structure of the skin cells of a stenohaline freshwater fish Cyprinus carpio exposed to diluted seawater.

Seawater diluted to half (1.750% salinity) is lethal for adult carps after 3 h and 15 min. At lower salinities (0.350%-0.875%), the fish survived for longer periods, but only 0.175% salinity was innocuous. In carps, adapted to 0.175% salinity, the secretory activity of pavement cells was very high and their external ridges flattened or even disappeared. Mucus secretion was conspicuous, characterized by holocriny of old cells and apparition of young ones in large numbers. The intracellular mucus droplets often coalesced. Pavement cells and mucus cells were disconnected from their neighboring tissue fabric and were sloughing off. Mitotic figures of filament cells were frequent, suggesting high turnover. Club cells appeared near the epidermal surface. The number of pinocytotic vesicles of the basal cell layer markedly decreased, indicating a possible decrease in dermis-epidermis molecular transfers. Leucocytes, mainly lymphocytes penetrated into the epidermis, where also rodlet cells appeared. The low salinity tolerance of the carp might be related among other possible factors to the absence of chloride cells in the skin.

Ultrasound-facilitated transport of silver chloride (AgCl) particles in fish skin.

Electron-dense nano-particles in aqueous suspension were administered by immersion into the epidermis of fish using ultrasound in the therapeutic range. Enhanced permeability of the tissues to the particles was achieved by acoustic cavitation, which induced a controlled level of necrosis in the outer cell layers, and by non-cavitational exposures, which widened intercellular spaces of non-necrosed tissue in deeper regions of the epidermis. Both particle concentration and penetration depth were quantified using transmission electron microscopy. While cavitation-induced perforation was necessary for particles to penetrate into the tissues, non-cavitational exposures during immersions increased the particle flux towards the skin surface, as well as the diffusion rate of the particles within the epidermis and their depth of penetration. The technique described above may potentially be applied for non-stressful, mass-administration of substances into aquatic animals, as well as the relatively new field of ultrasound-facilitated delivery in moist epithelial tissues in humans.

Ultrasound-induced intercellular space widening in fish epidermis.

Transmission electron microscopy was employed to determine the effects of therapeutic ultrasound (US) (I(sata) < or =2.2 W cm(-2), 3 MHz), sonicated at different angles and durations, on the external epithelia of fish skin. Sonication at 1.7 W cm(-2) (90 s), where the ultrasonic beam was perpendicular to the skin surface, produced minor intercellular space widening (ICSW), as well as the disruption of desmosomes connecting between the cells. Increasing the intensity to 2.2 W cm(-2) increased ICSW, the extent of which was positively correlated to the duration of exposure (30 to 90 s). Perpendicular sonication produced ICSW, almost exclusively between cells of the two outermost cell layers, parallel to the skin surface. Sonicating at 45 degrees (2.2 W cm(-2), 90 s) produced ICSW in deeper cell layers in the tissues, in which the spaces were at seemingly random orientations. Mucous cells and macrophages were also found to be damaged, as were apoptotic epidermal cells. The suggested mechanism for ICSW is the formation of transverse (shear) waves at the interface between the aquatic medium and the skin surface. The waves, which are damped out within a few cell layers, give rise to shear stresses that, in turn, cause strains that act to separate between cells and damage some of the relatively weaker cells.

Ultrasound-induced cavitation damage to external epithelia of fish skin.

Transmission electron microscopy was used to show the effects of therapeutic ultrasound (< or = 1.0 W/cm2, 1 MHz) on the external epithelia of fish skin. Exposures of up to 90 s produced damage to 5 to 6 of the outermost layers. Negligible temperature elevations and lack of damage observed when using degassed water indicated that the effects were due to cavitation. The minimal intensity was determined for inducing cellular damage, where the extent and depth of damage to the tissues was correlated to the exposure duration. The results may be interpreted as a damage front, advancing slowly from the outer cells inward, presumably in association with the slow replacement of the perforated cell contents with the surrounding water. This study illustrates that a controlled level of microdamage may be induced to the outer layers of the tissues.

Rodlet cells in the epidermis of fish exposed to stressors.

Rodlet cells (RC) were detected in the epidermis of carp (Cyprinus carpio) and trout (Oncorhynchus mykiss) exposed to stressors (e.g. acid water, heavy metals, thermal elevation, polluted water of the Rhine river, water loaded with organic manure, and distilled water), as well as after wounding. These cells were not found in the epidermis of fish kept under control conditions, thus suggesting that the appearance of RC in the epidermis is stressor-related. Immature RC, resembling mucous cells, were found in deeper epidermal zones while mature RC were found close to and at the skin surface. The latter contained several rodlets embraced within a capsule-like structure composed of filaments. At the skin surface, RC opened the filamentous capsule at the cell apex and a cytoplasmic tuft, containing the rodlets, protruded outwards into the surrounding water. Structures which appeared to be free rodlets were also observed in the water, near the ridges of the pavement cells. Cytochemical and immunocytochemical study revealed that the rodlets contain alkaline phosphatase at their periphery and peroxidase activity at their cores. These enzymes have been previously reported in mucous cells of stressed fish. All together, the location of RC, their structure, cytochemistry and direction of migration suggest that these cells form part of the non-specific defence mechanism of the skin, and possibly other epithelial tissues.

Cortisol induces stress-related changes in the skin of rainbow trout (Oncorhynchus mykiss).

The ultrastructure of the skin of rainbow trout, Oncorhynchus mykiss, was studied over a 7-day period after a single meal containing cortisol. The fish experienced increased plasma cortisol levels for 1 day. Pavement cells contained significantly more vesicles of high electron density, which were also numerous in deeper filament cells, and displayed peroxidase activity. Mitotic cells were common after 4 and 7 days. Increased apoptosis, taken to indicate accelerated ageing, was detected in both pavement and mucous cells. Newly differentiated mucous cells were found close to skin surface, and many mucous cells contained mucosomes of high electron density. The basal lamina became highly folded. The low numbers of leukocytes present in the skin did not change noticeably, but substantially more lymphocytes were apoptotic. The melanosomes in the pigment initially dispersed and subsequently reaggregated in the cell bodies of these cells. The reaggregation was accompanied by apoptosis of melanocyte extensions. The results demonstrate the ability of the hormone to regulate several of the effects observed in the skin of fish challenged by stressors. Other phenomena generally observed in stressed fish, such as pavement cell necrosis and massive leucocyte infiltration, were not found after cortisol treatment. The latter observation indicates that regulatory factors in addition to cortisol must be operative during stress.

Effects of water-borne cadmium on the skin of the common carp (Cyprinus carpio).

The skin of carp, Cyprinus carpio, was studied at the ultrastructural level after exposure of the fish to low and high concentrations of cadmium in the water (22 and 560 micrograms/L, respectively) for different periods. The effects of the low concentration of cadmium were similar to those of the high concentration, although they appeared later. The basal lamina and the skin surface became highly undulating. Chloride cells appeared between the pavement cells. Necrotic pavement cells were seen from the first day on, while apoptotic pavement cells appeared after several days. Filament cells contained many electron-transparent and electron-dense secretory vesicles. Mitotic cells were commonly seen, mainly in cells adjacent to club cells or close to the epidermal surface. Mucous cells differentiated close to the skin surface. They became elongated and synthesized highly electron-dense mucosomes. The epidermis became infiltrated by many leucocytes. As the experiment progressed, many leucocytes degenerated, and their remnants were found within macrophages and club cells. Fibroblasts displayed intense synthesis and, in fish from the low cadmium concentration, deposited a dense network of collagen fibers in the dermis. Melanosomes were located in the extensions of melanocytes. In these cells aggregation of melanosomes and apoptotic processes were common. Several of these changes were observed earlier under the impact of stressors other than cadmium. Some changes, such as the appearance of tumorlike bodies at the skin surface, the appearance of Merkel cells throughout the epidermis, and the coupling of leucocytes, may be specific for cadmium.