How to overcome osmotic stress? Marine crabs conquer freshwater. New insights from modern electrophysiology

In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na⁺ and Cl^? in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na⁺ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na⁺ and Cl^? independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na⁺-dependent short-circuit current with externally Cl^?-free saline indicate that active Na⁺ uptake proceeds in a frog-skin-like mode via apical Na⁺-channels and the basolateral Na⁺/K⁺-pump. The nature of a negative short-circuit current with external Cl^?-saline indicates that active and Na⁺-independent Cl^? uptake is driven by an apical V-type H⁺-pump and proceeds via apical Cl^?/ HCO₃ ^?-exchange and basolateral Cl^?-channels.     

Active NaCl absorption across split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) adapted to different salinities

Split lamellae of posterior gills of Eriocheir sinensis adapted to fresh water, brackish waters (9 or 18‰) or seawater (36‰) were mounted in Ussing chambers, and transepithelial short-circuit currents and conductances were measured with salines, containing approximately in vivo-like NaCl concentrations. Active sodium and chloride absorption (I_Na and I_Cl), the transcellular conductances and the leak conductance were identified with external amiloride and/or DIDS. Split gill lamellae of crabs adapted to fresh water displayed similar magnitudes of I_Na and I_Cl with 10 mmol l⁻¹ NaCl in the external medium (internally haemolymph-like NaCl saline). Augmenting external NaCl (50 mmol l⁻¹) resulted in an increase of I_Cl, whereas I_Na decreased. Split gill lamellae of crabs adapted to brackish waters (external NaCl of 125 and 225 mmol l⁻¹, respectively) showed lower currents than preparations of freshwater crabs (50 mmol l⁻¹ external NaCl). With split gill lamellae of seawater crabs no currents were detected (450 mmol l⁻¹ NaCl on both sides). The transcellular conductances showed similar changes as the currents. The leak conductance of split gill lamellae of crabs adapted to fresh or brackish waters was low (0.3–0.8 mS cm⁻²), whereas it was much higher (7 mS cm⁻²) with preparations of seawater crabs.     

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na⁺/K⁺-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na⁺/K⁺-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na⁺/K⁺-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills.In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na⁺ + K⁺-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.     

Ultrastructural changes in the gill epithelium of the green crab Carcinus maenas in relation to the external salinity

Observation of semi-thin and ultrathin sections performed in the gills of green crabs (Carcinus maenas) kept in 100% and in dilute 30% sea water respectively reveals marked differences between the six anterior and the three posterior pairs of gills. The anterior gill lamellae are almost entirely lined by a thin pavement epithelium (0.9 to 3 mum thick) which does not undergo any noticeable change when crabs are acclimated from full to dilute sea water. This supports the view it is chiefly involved in the respiratory function. In addition to the pavement epithelium, the posterior gills exhibit small areas corresponding to a thick prismatic epithelium (10 mum) the ultrastructure of which is similar to that of most of the so-called 'salt transporting epithelia'. When submitted to reduced external salinity, this epithelium undergoes structural changes consisting of elaboration of an extensive apical plasma membrane infolding system, enlargement of the subcuticular compartment and close association of mitochondria with basolateral membrane infoldings. Pilaster cells exhibit ultrastructural features of either thin (respiratory) or thick (salt transporting) epithelial differentiation according to their localization within the gill. Their peculiar organization suggests they ensure, in addition, mechanical reinforcement of the gill lamellae against blood hydrostatic pressure. The fact that salt-transporting epithelium areas do not exceed, at most, 30% of the total lamellar surface is probably related to the weak osmoregulatory capabilities of the shore crab.     

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crabEriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na⁺/K⁺-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na⁺/K⁺-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.     

Epithelial remodeling and claudin mRNA abundance in the gill and kidney of puffer fish (<Emphasis Type="Italic">Tetraodon biocellatus</Emphasis>) acclimated to altered environmental ion levels

In water of varying ion content, the gills and kidney of fishes contribute significantly to the maintenance of salt and water balance. However, little is known about the molecular architecture of the tight junction (TJ) complex and the regulation of paracellular permeability characteristics in these tissues. In the current studies, puffer fish (Tetraodon biocellatus) were acclimated to freshwater (FW), seawater (SW) or ion-poor freshwater (IPW) conditions. Following acclimation, alterations in systemic endpoints of hydromineral status were examined in conjunction with changes in gill and kidney epithelia morphology/morphometrics, as well as claudin TJ protein mRNA abundance. T. biocellatus were able to maintain endpoints of hydromineral status within relatively tight limits across the broad range of water ion content examined. Both gill and kidney tissue exhibited substantial alterations in morphology as well as claudin TJ protein mRNA abundance. These responses were particularly pronounced when comparing fish acclimated to SW versus those acclimated to IPW. TEM observations of IPW-acclimated fish gills revealed the presence of cells that exhibited the typical characteristics of gill mitochondria-rich cells (e.g. voluminous, Na⁺-K⁺-ATPase-immunoreactive, exposed to the external environment at the apical surface), but were not mitochondria-rich. To our knowledge, this type of cell has not previously been described in hyperosmoregulating fish gills. Furthermore, modifications in the morphometrics and claudin mRNA abundance of kidney tissue support the notion that spatial alterations in claudin TJ proteins along the nephron of fishes will likely play an important role in the regulation of salt and water balance in these organisms.     

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l⁻¹ NH₄Cl in the perfusion saline and concentrations of NH₄Cl in the bath that were stepwise increased from 0 to 4000 μmol · l⁻¹ allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l⁻¹ NH⁺ ₄) through the gill epithelium against ambient concentrations of up to 800 μmol · l⁻¹, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l⁻¹. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm⁻²), thus representing practically no influx barrier for ions including NH⁺ ₄. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm⁻²) which may reduce passive NH⁺ ₄ influxes at high ambient levels. Accepted: 14 October 1998     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Is there a chloride ATPase in the gills of the shore crab Carcinus maenas?

Summary In gills of the shore crab Carcinus maenas an ATPase activity was found which was stimulated by bicarbonate and inhibited by low concentration of oligomycin and thiocyanate. This ATPase was activated by small hydrated alkali cations, i.e., activation was absent in the presence of Li⁺, small in the presence of Na⁺, and highest in the presence of K⁺ (K _m=4 mM). Inhibitor studies using ouabain, NEM, and vanadate suggest that this ATPase is different from (Na⁺+K⁺)-ATPase, the H⁺-ATPase of organelles, or an E ₁ E ₂-type ATPase represented by the H⁺/K⁺-ATPase in gastric mucosa. Results obtained by differential and density gradient centrifugation indicate that this ATPase is located in crab gill mitochondria, a location ruling out its direct participation in transepithelial ion transport. Since the ATPase lacked specific Cl^--activation it is not considered to be a Cl^- pump but a mitochondrial F ₁ F ₀-ATPase. Specific activities of mitochondrial ATPase and (Na⁺+K⁺)-ATPase were of comparable magnitude. Both ATPases were greatly increased in gills of crabs acclimated to brackish water (salinity 10) compared to crabs maintained in sea water (30). These results imply that low salinity-induced modifications in branchial tissues include mechanisms for active ion uptake as well as the elements for provision of cellular energy.Abbreviations ATPase adenosine triphosphatase - HEPES N-(2-hydroxyethyl)-1-piperazine-N(2-ethanesulfonic acid) - LDH lactate dehydrogenase - NADH reduced nicotinamide adenine dinucleotide - NEM Niethylmaleimide - PEP phosphoenolpyruvate - PK pyruvate kinase - TRIS TRIS (hydroxymethyl)aminomethane - S salinity     

Monitoring of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase mRNA expression in the cinnamon clownfish, <Emphasis Type="Italic">Amphiprion melanopus</Emphasis>, exposed to an osmotic stress environment: profiles on the effects of exogenous hormone

We examined changes in the expression of Na⁺/K⁺-ATPase mRNA in the gills of the cinnamon clownfish using quantitative real-time PCR in an osmotically changing environment [seawater (35 psu; practical salinity unit, 1 psu ≈ 1‰) → brackish water (17.5 psu) and brackish water with prolactin]. The expression of Na⁺/K⁺-ATPase mRNA in gills was increased after the transfer to brackish water, and the expression was repressed by prolactin treatment. Also, activities of gill Na⁺/K⁺-ATPase and plasma cortisol levels increased after the transfer to brackish water and were repressed in brackish water with prolactin treatment. Na⁺/K⁺-ATPase-immunoreactive cells were almost consistently observed in the gill filaments, but absent from the lamella epithelia. The plasma osmolality level decreased in brackish water, but the level of this parameter increased in brackish water with prolactin treatment during salinity change. These results suggest that the Na⁺/K⁺-ATPase gene plays an important role in osmoregulation in gills, and prolactin improves the hyperosmoregulatory ability of cinnamon clownfish in a brackish water (hypoosmotic) environment.     

Organization of a phyllobranchiate gill from the green shore crab Carcinus maenas (Crustacea,Decapoda)

Summary The phyllobranchiate gills of the green shore crab Carcinus maenas have been examined histologically and ultrastructurally. Each gill lamella is bounded by a chitinous cuticle. The apical surface of the branchial epithelium contacts this cuticle, and a basal lamina segregates the epithelium from an intralamellar hemocoel. In animals acclimated to normal sea water, five epithelial cell types can be identified in the lamellae of the posterior gills: chief cells, striated cells, pillar cells, nephrocytes, and glycocytes. Chief cells are the predominant cells in the branchial epithelium. They are squamous or low cuboidal and likely play a role in respiration. Striated cells, which are probably involved in ionoregulation, are also squamous or low cuboidal. Basal folds of the striated cells contain mitochondria and interdigitate with the bodies and processes of adjacent cells. Pillar cells span the hemocoel to link the proximal and distal sides of a lamella. Nephrocytes are large, spherical cells with voluminous vacuoles. They are rimmed by foot processes or pedicels and frequently associate with the pillar cells. Glycocytes are pleomorphic cells packed with glycogen granules and multigranular rosettes. The glycocytes often mingle with the nephrocytes. Inclusion of the nephrocytes and glycocytes as members of the branchial epithelium is justified by their participation in intercellular junctions and their position internal to the epithelial basal lamina.     

Metallothionein-like proteins in the blue crab Callinectes sapidus: Effect of water salinity and ions

The effect of water salinity and ions on metallothionein-like proteins (MTLP) concentration was evaluated in the blue crab Callinectes sapidus. MTLP concentration was measured in tissues (hepatopancreas and gills) of crabs acclimated to salinity 30 ppt and abruptly subjected to a hypo-osmotic shock (salinity 2 ppt). It was also measured in isolated gills (anterior and posterior) of crabs acclimated to salinity 30 ppt. Gills were perfused with and incubated in an isosmotic saline solution (ISS) or perfused with ISS and incubated in a hypo-osmotic saline solution (HSS). The effect of each single water ion on gill MTLP concentration was also analyzed in isolated and perfused gills through experiments of ion substitution in the incubation medium. In vivo, MTLP concentration was higher in hepatopancreas than in gills, being not affected by the hypo-osmotic shock. However, MTLP concentration in posterior and anterior gills significantly increased after 2 and 24 h of hypo-osmotic shock, respectively. In vitro, it was also increased when anterior and posterior gills were perfused with ISS and incubated in HSS. In isolated and perfused posterior gills, MTLP concentration was inversely correlated with the calcium concentration in the ISS used to incubate gills. Together, these findings indicate that an increased gill MTLP concentration in low salinity is an adaptive response of the blue crab C. sapidus to the hypo-osmotic stress. This response is mediated, at least in part, by the calcium concentration in the gill bath medium. The data also suggest that the trigger for this increase is purely branchial and not systemic.     

Response to environmental salinity of Na-KATPase activity in individual gills of the euryhaline crab Cyrtograpsus angulatus

The occurrence, localization and response to environmental salinity changes of Na⁺-K⁺ATPase activity were studied in each of the individual gills 4-8 of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Na⁺-K⁺ATPase activity appeared to be differentially sensitive to environmental salinity among gills. Upon an abrupt change to low salinity, a differential response of Na⁺-K⁺ATPase activity occurred in each individual gill which could suggest a differential role of this enzyme in ion transport process in the different gills of C. angulatus. With the exception of gill 8, a short-term increase of Na⁺-K⁺ATPase specific activity was observed in posterior gills, which is similar to adaptative variations of this activity described in other euryhaline crabs. However, and conversely to that described in other hyperregulating crabs, the highest increase of activity occurred in anterior gills 4 by 1 day after the change to dilute media which could suggest also a role for these gills in ion transport processes in C. angulatus. The fact that variations of Na⁺-K⁺ATPase activity in anterior and posterior gills were concomitant with the transition to hyperregulation indicate that this enzyme could be a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab. The results suggest a differential participation of branchial Na⁺-K⁺ATPase activity in ionoregulatory mechanisms of C. angulatus. The possible existence of functional differences as well as distinct regulation mechanisms operating in individual gills is discussed.     

Uptake of cadmium through isolated perfused gills of the Chinese mitten crab, Eriocheir sinensis

Using the perfusion method, we compared cadmium accumulation and influx across the gills of the euryhaline Chinese mitten crab Eriocheir sinensis, exposed to 4.8 microM cadmium in the incubation medium (OUT). Cadmium influx was not observed across posterior gills while it ranged from 0.15 to 6.82 nmol Cd g(-1) gill w.w. h(-1) across anterior ones. For these respiratory gills, a strong increase (40 times) was observed when calcium was removed in both incubation and perfusion media while the lack of sodium in the perfusion medium resulted in a 46 times decrease. For crabs acclimated 15 days to artificial seawater, cadmium influx across anterior gills showed a 21 times decrease when compared with freshwater acclimated ones. On the other hand, after 3 h of perfusion, we detected cadmium accumulation in both types of gills, ranging from 3.8 to 68 nmol Cd g(-1) gill w.w. in anterior gills and from 2.1 to 39 nmol Cd g(-1) gill w.w. in posterior ones. Such accumulations represent between 61.3 and 100% of the total uptake of cadmium through the gills. From these results, we suggest that cadmium can penetrate more easily into the hemolymph space through the 'respiratory' type epithelium present in the anterior gills but absent in the posterior ones. This metal uptake is likely to occur at least in part through the same pathways as calcium. On the contrary, cadmium seems to be sequestered inside the posterior gills, perhaps in the cuticle of the salt-transporting type epithelium.     

Active uptake of sodium in the gills of the hyperregulating shore crabCarcinus maenas

Isolated posterior gills of shore crabs,Carcinus maenas, previously acclimated for at least 1 month to brackish water of 10 S, were connected with an artificial hemolymph circulation by means of thin polyethylene tubings. Gills were symmetrically perfused and bathed with 50 % sea water. Transepithelial potential differences (PDs) and fluxes of sodium between medium and blood were measured under control conditions and following reductions of PDs by means of 5 mM internal (blood side) ouabain, 0.5 mM internal and external (bathing medium) NaCN or by exhaustion of energy reserves along with a prolonged perfusion period of more than 9 h. In these experiments²²Na was used as tracer. Each of the three modes of reducing transepithelial potential differences resulted in a decrease in sodium influxes from 500–1000 µmoles g^–1 h^–1 to 250–400 µmoles g^–1 h^–1. The findings suggest that sodium influx, which normally greatly exceeds efflux, was diminished by its active component. The remaining non-inhibitable influx equals efflux values. Our findings thus indicate that efflux is completely passive, while influx has — beside a passive component of efflux magnitudes — an additional active portion which is much larger than the passive component. Since ouabain is a specific inhibitor of the Na-K-ATPase, our results confirm previous findings (Siebers et al., 1985) that the basolaterally located Na-K-ATPase generates the transepithelial potential difference in the gills, which is inside negative by about 6–12 mV. Inhibition of the active portion of sodium influx by internal ouabain along with reduced PDs suggests that transepithelial PDs generated by the branchial sodium pump are the driving force for active sodium uptake in hyperregulating brackish water crabs.     

Salinity-induced changes in the fine structure of the gills of the semiterrestrial estuarian crab, Uca uruguayensis (Nobili, 1901) (Decapoda, Ocypodidae)

The posterior gills of Uca uruguayensis are mostly lined with a thick tissue which presents the characteristics of a typical salt-transporting epithelium. Electron microscope analysis of gill tissue from crabs acclimated to both low (2.5 per thousand) and high (44 per thousand) salinity showed significant development of the basolateral membrane interdigitations with numerous mitochondria and conspicuous apical membrane infoldings. In high-salinity acclimated crabs, the basolateral interdigitations extended to the apical membrane. Under these conditions, apical infoldings were expanded laterally (forming wide subcuticular spaces), while the apical infoldings of low-salinity adapted animals appeared as regular leaflets. Septate desmosomes were also much more developed in low-salinity exposed animals than in those kept under high-salinity conditions. These morphological observations were analyzed for correlation with the currently-accepted ion hyporegulation model for crustaceans, which is mainly based on transcellular sodium flow. In this study, we propose an ion hyporegulation model involving apical paracellular sodium flux.     

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     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

Differential responses in gills of euryhaline tilapia, Oreochromis mossambicus, to various hyperosmotic shocks

Euryhaline tilapia (Oreochromis mossambicus) survived in brackish water (BW; 20‰) but died in seawater (SW; 35‰) within 6 h when transferred directly from fresh water (FW). The purpose of this study was to clarify responses in gills of FW tilapia to various hyperosmotic shocks induced by BW or SW. In FW-acclimated tilapia, scanning electron micrographs of gills revealed three subtypes of MR cell apical surfaces: wavy-convex (subtype I), shallow-basin (subtype II), and deep-hole (subtype III). Density of apical surfaces of mitochondrion-rich (MR) cell in gills of the BW-transfer tilapia decreased significantly within 3 h post-transfer due to disappearance of subtype I cells, but increased from 48 h post-transfer because of increasing density of subtype III cells. SW-transfer individuals, however, showed decreased density of MR cell openings after 1 h post-transfer because subtype I MR cell disappeared. On the other hand, relative branchial Na⁺/K⁺-ATPase (NKA) α1-subunit mRNA levels, protein abundance, and NKA activity of the BW-transfer group increased significantly at 6, 12, and 12 h post-transfer, respectively. In the SW-transfer group, relative mRNA and protein abundance of gill NKA α1-subunit did not change while NKA activity declined before dying in 5 h. Upon SW transfer, dramatic increases (nearly 2-fold) of plasma osmolality, [Na⁺], and [Cl⁻] were found prior to death. For the BW-transfer group, plasma osmolality was eventually controlled by 96 h post-transfer by enhancement of NKA expression and subtype III MR cell. The success or failure of NKA activation from gene to functional protein as well as the development of specific SW subtype in gills were crucial for the survival of euryhaline tilapia to various hyperosmotic shocks.     

The transepithelial potential difference in the gills of the fiddler crab,Uca tangeri: Influence of some inhibitors

Summary Euryhaline Crustacea living in dilute media, counterbalance the salt loss by active absorption of NaCl across the gill epithelium. To investigate the mechanisms involved in salt absorption, transeptithelial potential difference (PD_te) was measured in isolated, perfused gills of the fiddler crab,Uca tangeri. The influence of some specific inhibitors of epithelial ion transport on the PD_te was tested.With symmetrical conditions on both sides of the epithelium, the posterior gills ofUca tangeri showed a spontaneous PD_te of +5 to +10 mV, that is an active transport potential which was positive on the bath side as referred to the hemolymph side. This potential decreased considerably after application of KCN or 2,4-dinitrophenol (DNP) to the perfusion saline.Omission of K⁺ from the perfusion saline or addition of ouabain led to a reversible drop of the PD_te, suggesting that the absorption of Na⁺ and also of Cl^– is driven by the (Na⁺+K⁺)ATPase located in the basolateral membrane of the epithelial cells.Perfusion of the hemolymph space with saline containing diphenylamine-2-carboxylate (DPC) or the loop diuretic furosemide resulted in a decrease of the PD_te.After application of amiloride to the bath saline the PD_te increased. Half-maximum response to amiloride was reached at a concentration of about 10^–5 mol·l^–1. This suggests that one of the Na⁺ pathways across the apical membrane may consist of Na⁺ channels.Abbreviations PD _te transepithelial potential difference - DPC diphenylamine-2-carboxylate - R _ps resistance of perfusate shunt - R _te transepithelial resistance - R _in input resistance - DNP 2,4-dinitrophenol Parts of this study have been reported at the 1st Congress of Comparative Physiology and Biochemistry, Liège 1984, and at the Vth European Colloquium on Renal Physiology, Frankfurt, 1985