Kinetics of osmoregulation in the crab Carcinus maenas

When shore crabs Carcinus maenas are transferred from 11 to 38 S at 11°C, new constant levels of hemolymph freezing points and of concentrations of Na, K, Ca, and Mg in the hemolymph are accomplished within 24h. From a decrease in serum protein and in serum free amino acids and an increase in the relative amounts of individual essential free amino acids in the body fluids of whole crabs, a participation of proteolytic activities and a transport of amino acids from hemolymph to cell is deduced; the stationary concentration of total intracellular free amino acids increases up to a nearly two-fold value, compared with the concentration in crabs remaining in diluted sea water. Also, the low molecular neutral sugars increase in whole crabs after high salinity stress, reaching values of more than two-fold initial concentrations within a period of 10 days. This increase is fully accounted for by a 6.7-fold increase in trehalose levels. The processes of increasing the concentrations of low molecular organic material seem to be slower than those of establishing new osmotic pressures in the hemolymph. The oxygen consumption decreases by 30 to 45% to new constant values within 8 to 12 h after the salinity change from 11 to 38, reflecting similar kinetics as the establishment of new osmotic pressures in the hemolymph.     

Einfluß von 2,4-dinitrophenol auf die atmung und die Konzentration einiger metabolite bei embryonen des herings Clupea harengus

In rearing experiments with herring eggs (temperature=14.0°±0.1°C; salinity=15‰), oxygen consumption under normal conditions and after addition of 2,4-DNP (concentration=0.1 mM/l; pH=8.1) was measured over the period of embryonic development by means of the Wabburg-technique. Additionally, the concentration of low molecular sugars, polysaccharides, free amino-acids, and adenosintriphosphate (ATP) was determined. The oxygen consumption increases during embryonic development; this increase is not linear. Periods of high intensity of oxygen consumption are followed by others with only slight increase. Immediately before hatching, the respiration curve distincly declines (Fig. 1). Under the influence of 2,4-DNP (dinitrophenol), the embryos increase their respiration intensity after a short period of incubation. The maximum rise in percentage over the normal values reaches up to 400% at the beginning of gastrulation, falls to 50% even before the locking of the blastopore, and decreases slightly to about 30% until hatching. The immense decline in the percentage increase in respiration following the addition of 2,4-DNP at the end of the first day of development is caused by the rapid increase in normal respiration. After poisoning with DNP at different stages of development, the uncoupled respiration curves are normally almost equal. This holds both for the temporal position of the respiration maxima (about 12 to 24 h after poisoning), and for the absolute amounts of the increased respiration over the normal values (5 to 7 μl/h/100 embryos). Excluded from these regularly repeated findings are two stages of development: (1) the stage of epiboly after exceeding the yolk equator until shortly before locking of the blastopore (26 to 32 h after fertilization at 14°C); (2) the period at the end of the 4th day of development when the eyes become pigmented (100 to 120 h after fertilization). These two stages are characterized by the fact that, at the moment of poisoning, the normal respiration shows retarded activity. On the other hand, these two stages are well able to undergo periods of development in which long-living embryonic deformations can occur after uncoupling of respiration with 2,4-DNP. The content in low molecular sugars and polysaccharides decreases slightly in the course of embryonic development and, following the addition of 2,4-DNP, decreases considerably during the first 24 h. After 48 h, accelerated decomposition of carbohydrates continues. Under the influence of 2,4-DNP, the embryos metabolize more carbohydrates in 1 day than during the whole normal development period. The changeover of the metabolism to increased decomposition of carbohydrates can be explained as a dislocation of the energetic sources from the respiration chain to glycolytic phosphorylation. In accordance with these facts, the concentration of free amino-acids, almost equal during normal embryonic development, remains unchanged under the influence of 2,4-DNP.     

Heavy metal action on transintegumentary absorption of glycine in two annelid species

Absorption of ¹⁴C-glycine from ambient sea water across the body surface of the oligochaete Enchytraeus albidus Henle was, after 6 h preincubation, significantly reduced in the presence of 0.1 to 0.15 ppm mercury, 0.25 ppm copper, 1.5 ppm silver, and 2 ppm cadmium. Addition of heavy metal salts reduced maximum uptake rates (V _max ), without affecting transport constants (K _t ). Influx of ¹⁴C-glycine remained unchanged in the presence of up to 10 ppm aluminium, chromium, iron, lead, molybdenum, vanadium, and zinc. Effects of up to 150 ppm nickel, manganese, cobalt and selenium were negligible. Inhibition of glycine absorption by mercury and cadmium in the polychaete Nereis diversicolor Müller is almost identical with results presented for E. albidus. Transintegumentary solute absorption in soft-bodied marine invertebrates obviously represents a suitable biological function for studies on heavy metal toxicity. The close relationship between rates of heavy metal absorption, inhibition of transintegumentary glycine uptake, and acute toxicity is discussed.     

Photocatalytic degradation of trinitrotoluene and other nitroaromatic compounds

The photocatalytic degradation of 2,4,6-trinitrotoluene and ten other nitroaromatic compounds in aerated TiO₂ suspensions has been studied. The following order of reactivity was observed: nitrotoluenes > nitrobenzene > dinitrotoluenes . dinitrobenzenes > 2,4,6-trinitrotoluene > 1,3,5-trinitrobenzene, which reflects the known influence of nitro groups towards the attack of electrophilic reagents on the aromatic molecule.     

Na-K-ATPase generates an active transport potential in the gills of the hyperregulating shore crab Carcinus maenas

Perfusing and bathing isolated gills of shore crabs Carcinus maenas with artificial saline or sea (brackish) water enabled us to determine potential differences (PDs) between ambient bathing medium and perfusion solution. Establishment of diffusional PDs was avoided by employment of the same solution on the internal and external side. The PDs measured were therefore of an active nature. We compared the properties of the PDs with the well-known properties of the Na-K-ATPase: dependence on biological energy (ATP), on salinity and sodium concentration, susceptibility of PD to depletion of internal K and to the addition of 5 mM internal ouabain. Considering also the magnitude of the PDs measured, the results obtained indicate that it is the Na-K-ATPase that generates an active transport potential for Na in the gills of shore crabs. This PD represents the driving force for the active uptake of Na in crabs that hyperregulate their body fluids when they inhabit environments of reduced salinity regimes or fluctuating salinities in tidal estuaries. This process counteracts diffusional losses of Na in crabs exposed to dilute media.     

Physiological role of branchial carbonic anhydrase in the shore crabCarcinus maenas

Excretion of total CO₂ and uptake of sodium and chloride ions across the branchial epithelium of the posterior gills of the shore crabCarcinus maenas, collected from Kiel Bay (Baltic Sea) in 1989, were measured using isolated perfused gill preparations. Total CO₂ effluxes depended on the HCO ₃ ^- concentration of the internal perfusate in a saturable mode and were inhibited by internally and externally applied acetazolamide at 10^–4 M. Potential differences between hemolymph space and medium did not change significantly during experimental treatments. Neither a bicarbonate gradient (6 mM) directed from the internal perfusate to external bath solution nor symmetrically applied 10^–4 M acetazolamide significantly influenced the influxes of Na⁺ and Cl^–. Results confirmed the role of carbonic anhydrase in CO₂ excretion but called into question the assumed functioning of the enzyme in branchial ion transport processes.     

Sodium regulation in the shore crab Carcinus maenas as related to ambient salinity

The activity of Na–K-ATPase was measured in crude homogenates prepared from various organs (leg muscle, pincer muscle, heart, testes, digestive gland, hypodermis, gills 1–9) of shore crabs, Carcinus maenas L., acclimated to salinities ranging between 10 and 50 S (in steps of 10 S). In all salinities tested, Na–K-ATPase activity was highest in posterior gills 7–9 (10–12 mol P_i mg protein^-1 h^-1), followed by anterior gills 1–6 (ca. 2.5 mol P_i mg protein^-1 h^-1) and the other organs (in most cases far below 2mol P_i mg protein^-1 h^-1). In gills only, Na–K-ATPase activity was salinity-dependent, with the highest values in the lowest salinities and vice versa. In gills 7–9, Na–K-ATPase activity was increased more than threefold following a reduction in salinity from 50 to 10 S. Na–K-ATPase activity, expressed as percentage of total ATPase activity, amounted to 60–80% in gills, about 60% in hypodermis and 20–40% in the other organs. Ouabain, a specific inhibitor of Na–K-ATPase activity, reduced serum osmolalities in crabs kept at 9–10 S only when injected into the hemolymph (1 and 5 · 10^-5 M), but had no effect when dissolved in ambient water (10^-4 M). The results obtained underline that crustacean gills are the main organs for ionic regulation, and confirm the hypothesis of the central role of the Na–K-ATPase in active Na uptake as the basic mechanism of hyperregulation in dilute media. Reduction of serum osmolalities following injection of ouabain into the hemolymph confirms previous reports on localization of the sodium pump in the basolateral parts of epithelial cells.     

Inhibition of Na+/K+-ATPase and of active ion-transport functions in the gills of the shore crab Carcinus maenas induced by cadmium

Inhibition of Na⁺/K⁺-ATPase from gill plasma membranes of the shore crab Carcinus maenas by cadmium was investigated and compared with inhibitory effects by known antagonists (ouabain and Ca²⁺). For comparative considerations the Cd²⁺-inhibition of the enzyme from dog kidney was also tested. Na⁺/K⁺-ATPase from dog kidney and from crab gill differed greatly in sensitivity against ouabain. The inhibition constant K _i of the dog enzyme amounted to 9.1 × 10⁻⁷ mol l⁻¹, i.e. more than 300-fold smaller than the K _i of 2.9 × 10⁻⁴ mol l⁻¹ determined for the crab enzyme. Ca²⁺ inhibited the activity of Na⁺/K⁺-ATPase from crab gill plasma membranes with a K _i of 4.3 × 10⁻⁴ mol l⁻¹. The Na⁺/K⁺-ATPase from crab gill was inhibited by Cd²⁺ with a K _i of 9.1 × 10⁻⁵ mol l⁻¹. Cd²⁺ inhibited the Na⁺/K⁺-ATPase from dog kidney with a K _i (6.4 × 10⁻⁵ mol l⁻¹) comparable to that observed in the crab gill enzyme. Under experimental conditions Cd²⁺-inhibition of Na⁺/K⁺-ATPase was irreversible. Repeated washing, centrifugation and homogenization of the plasma membranes (four times) with Cd²⁺-free buffer did not restore any activity lost in the presence of 1 × 10⁻³ mol l⁻¹ Cd²⁺. Since ouabain-insensitive (nonspecific) ATPases in the plasma membrane fraction of crab gills were inhibited by Cd²⁺ in the same way as Na⁺/K⁺-ATPase, the heavy metal is considered as an unspecific ATPase inhibitor. Comparing these results with literature data on Cd²⁺-binding to electrophoretically separated proteins suggests that Na⁺/K⁺-ATPase is a Cd²⁺-binding enzyme. The results obtained on Na⁺/K⁺-ATPase were reflected by Cd²⁺-inhibition of the branchial ion-transport functions depending on this enzyme. The transepithelial short-circuit current of isolated gill half lamellae, a direct measure of area-specific active ion uptake, and the transepithelial potential difference of isolated, perfused whole gills, also indicative of active ion uptake, were inhibited by the heavy metal in a time- and dose-dependent mode. Remarkably these inhibitions were also irreversible. These findings are ecologically and biomedically significant: even when the actual environmental or tissue concentrations measured are low, biological microstructures such as Na⁺/K⁺-ATPase may accumulate the heavy metal by tight binding over prolonged periods until the first inhibitory effects occur. Received: 25 June 1997 / Accepted: 25 August 1997     

Average nanorough skin surface of the pilot whale (Globicephala melas, Delphinidae): considerations on the self-cleaning abilities based on nanoroughness

In aquatic environments, the biofouling process is assumed to initiate from the conditioning layer of absorbed organic carbon residues on wetted surfaces. Microfouling organisms attach to this conditioning layer, building up a biofilm on which further biofouling proceeds. In dolphins, biofouling reduces hydrodynamic efficiency and may negatively affect health if not managed. In the present study we examined the skin surface of the pilot whale (Globicephala melas). Employing cryo-scanning electron microscopic techniques combined with various sample preparations, the skin displayed an average nanorough surface characterized by a pattern of nanoridge-enclosed pores; the average pore size (approximately 0.20 µm²) was below the size of most marine biofouling organisms. Further, the implications of this type of surface to the self-cleaning abilities of the skin of pilot whales are discussed, based on reduced available space for biofouler attachment, the lack of any particular microniches as shelters for biofoulers, and the challenges of turbulent water flow and liquid-air interfaces during surfacing and jumping of the dolphin.