The role of the skin of early post-hatch turbot (Scophthalmus maximus L.) in osmoregulation
To date, the structural significance of the skin of fish larvae in osmoregulation has received little attention and the evidence for salt secretion by cutaneous chloride cells is based largely on morphological observations. Thus, in the present study, a combination of microscopical and electrophysiological techniques were utilised to determine the role of the skin of early post-hatch turbot larvae in osmoregulation. A number of specialised structural features were revealed in the skin of the turbot larva with electron microscopy which would appear to provide some protection against the high osmotic and ionic gradients tending to dehydrate and salt load the body tissue and fluids. In the heterogenous epidermis, consisting of both transporting and non-transporting cells, only the shallow junctions between chloride cells and accessory cells were believed to permit ion influx and/or water loss via the paracellular pathway; the extensive junctions between adjacent pavement cells and pavement cells and neighbouring chloride cells effectively occluding the passage of ions and water through the extracellular space. Chloride cells were revealed in the skin and prebranchial epithelium of the turbot larva from hatching, but accessory cells, and thus "leaky" junctions, were only observed in association with the closely juxtaposed chloride cells in the prebranchial epithelium which, although densely packed, represented just a small area of the otherwise "tight" skin. Water and ion permeation through the external plasma membrane of the superficial pavement cells might further be impeded by the extracellular glycocalyx coat observed in TEM. In addition, the large numbers of mucous cells, which were a characteristic feature of the skin of the turbot larva, may produce a protective mucus coating of low permeability. The apparent "tightness" of the skin was reflected by the measurements of diffusional water permeability (Pdiff) from early stage larvae which suggested that the larvae of turbot were relative impermeable to water compared with the gills of adults. Nevertheless, the rates of water turnover were still sufficiently high that a net osmotic loss of water must be replaced by water uptake through drinking. The observation that the Pdiff of early stage turbot larvae increased with development substantiates earlier supposition that the drinking rates of larvae are a direct function of the permeability of the larva to water. A study of the chronology of chloride cell development utilising specific fluorochromes and electron microscopy revealed that the prebranchial chloride cells, which closely resembled the chloride cells described in the branchial epithelium of juveniles, were both numerous and well equipped to participate in active salt extrusion in turbot larvae even at hatching. In view of the early hypertrophy and proliferation of the prebranchial cells, their rapid increase in Na+,K+-ATPase binding sites, and the subsequent degeneration of the cutaneous chloride cells observed with larval development, it was concluded that the prebranchial chloride cells are the primary site for active ion excretion shortly after yolksac absorption. The potential importance of the cutaneous chloride cells in salt extrusion was also considered, but in view of the apparent lack of accessory cell associations and the small number of apical pits observed in SEM and TEM sections, questions were raised as to the significance of these cells in ion excretion. Measurements of the trans epithelial electrical potential (TEP) from early stage turbot using intracellular micro electrode techniques confirmed that the larvae of turbot maintain ionic gradients by the active extrusion of ions that enter into the body cavity down electrical or chemical gradients. The TEP was found to be largely the result of a Na+ diffusion potential with an additive electrogenic potential due to CI- transport, which was somehow functionally connected to Na+,K+ -ATPase. Furthermore, the concentration of Na+ in the external bathing medium was found to have a direct regulatory influence on the rate of CI- secretion, suggesting that the active secretion of Cl across the skin must be coupled to Na+. These conclusions are consistent with the current theories proposed for salt extrusion by the chloride cells in the adult teleost.