Thermosensitivity of the lobster, Homarus americanus, as determined by cardiac assay

It is generally accepted that crustaceans detect, and respond to, changes in water temperature, yet few studies have directly addressed their thermosensitivity. In this investigation a cardiac assay was used as an indicator that lobsters (Homarus americanus) sensed a change in temperature. The typical cardiac response of lobsters to a 1-min application of a thermal stimulus, either warmer (n = 19) or colder (n = 17) than the holding temperature of 15 degrees C, consisted of a short bradycardia (39.5 +/- 8.0 s) followed by a prolonged tachycardia (188.2 +/- 10.7 s). Lobsters exposed to a range of rates of temperature change (0.7, 1.4, 2.6, 5.0 degrees C/min) responded in a dose-dependent manner, with fewer lobsters responding at slower rates of temperature change. The location of temperature receptors could not be determined, but lesioning of the cardioregulatory nerves eliminated the cardiac response. Although the absolute detection threshold is not known, it is conservatively estimated that lobsters can detect temperature changes of greater than 1 degree C, and probably as small as 0.15 degrees C. A comparison of winter and summer lobsters, both held at 15 degrees C for more than 4 weeks, revealed that although their responses to temperature changes were similar, winter lobsters (n = 18) had a significantly lower baseline heart rate (34.8 +/- 4.4 bpm) and a shorter duration cardiac response (174 s) than summer lobsters (n = 18; 49.9 +/- 5.0 bpm, and 320 s respectively). This suggests that some temperature-independent seasonal modulation of cardiac activity may be occurring.     

Low-salinity stress in the American lobster,Homarus americanus,after chronic sublethal exposure to cadmium: Biochemical effects

Lobsters(Homarus americanus) were exposed to cadmium (6μg l⁻¹, 30 days) in flowing seawater, then held for 7 days in aerated “clean ” seawater at either ambient (27 ‰) or low (17 ‰) salinity. Cadmium exposure alone (ambient salinity) induced a general elevation of enzyme activity (heart, antennal gland, and muscle MDH; heart LDH and GPI), despite the probability of some clearance of cadmium from body tissues during the “clean ” seawater holding period. Low-salinity alone (non-exposed lobsters) caused a decrease of enzyme activity (AAT, LDH, GPI, PK) in most tissues examined, except for tail muscle IDH, the activity of which was increased, and MDH, which was significantly elevated above ambient controls in all tissues except heart. Most low-salinity effects were observed in tail muscle, and most cadmium effects, in heart; low-salinity effects outnumbered cadmium stress by nine to four. In heart and tail muscle of cadmium-exposed lobsters held at low salinity, each of the two stresses apparently operated to nullify the other's effects. The most prominent single biochemical response to these sublethal stresses was the elevation of MDH activity. The ratio MDH: LDH gave the clearest indication of overall relative stress.     

Activation of carbonic anhydrase in branchial cavity tissues of lobsters (Homarus gammarus) by dilute seawater exposure

Activities of carbonic anhydrase (CA) and its distribution in the branchial cavity tissues were studied in European lobsters (Homarus gammarus) from ambient seawater (SW; salinity=38 ppt, 1126 mosmol/l) and acclimated to dilute seawater (DSW; salinity=20 ppt, 548 mosmol/l). Acetazolamide inhibited dose dependently the activity of CA in homogenates of epipodites, where the inhibition constant (IC₅₀=0.12 μM) did not differ significantly from that of membrane vesicles and cytosolic fraction. In DSW-acclimated lobsters, almost 70% of total CA in tissues of the branchial cavity was found in epipodites (E) and the rest was equally distributed between branchiostegites (B) and gills (G). Upon acclimation to dilute seawater, CA activity in membrane fractions of E and B was increased 6-fold and in homogenates, respectively 5- and 13-fold compared to SW-acclimated lobsters. Exposure to DSW enhanced cytosolic CA in E (8-fold) and B (7-fold) over SW-acclimated animals. Slight activation of CA in homogenates and in partially purified membranes of G was not confirmed as a statistically significant difference between SW and DSW groups. In DSW, cytosol specific activity of CA was increased compare to the SW cytosol. These results indicate the importance of E and B in CA induction when lobsters are acclimated to DSW. In subcellular fractions from DSW-acclimated lobsters, the main proportion of 75.8% (E), 61.0% (B) of total CA activity in each of these tissues remained in cytosol portion. Partially purified membranes contained 6.8% (E) and 16.2% (B) and the remainder of 15% (E) and 27% (B) was found in mitochondrial and nuclear fractions. In gills, 49.2% and 9.0 % of total gill CA activity was found respectively in cytosol and partially purified vesicles and the rest in mitochondrial and nuclear fractions.     

Behavioral influences on the physiological responses of Cancer gracilis, the graceful crab, during hyposaline exposure

The relationship between the behavioral and physiological responses to hyposaline exposure was investigated in Cancer gracilis, the graceful crab. The status of C. gracilis as an osmoconformer was confirmed. Survival decreased with salinity: the LT(50) in 50% seawater (a practical salinity of 16, or 16 per thousand) was 31.5 +/- 22.7 h and in 25% seawater (a salinity of 8) was 8.0 +/- 0.7 h. When exposed to a salinity gradient, most crabs moved towards the highest salinity. However, in the salinity range of 55% to 65% seawater, they became quiescent. This "closure response" was also evident at low salinities: the mouthparts were tightly closed and animals remained motionless for 2 to 2.5 h. During closure, crabs were able to maintain the salinity of water within the branchial chambers at a level that was about 30% higher than that of the surrounding medium. The closure response was closely linked to a short-term decrease in oxygen uptake. During closure, oxygen within the branchial chamber was rapidly depleted, with oxygen uptake returning to pretreatment levels upon the resumption of activity. In addition to the short-term decrease in oxygen uptake, there was a longer-term bradycardia, which may serve to further reduce diffusive ion loss across the gills. By exhibiting a closure response during acute hyposaline exposure and an avoidance reaction during prolonged or severe hyposaline exposure, C. gracilis is able to use behavior to exploit areas prone to frequent episodes of low salinity.     

Immunolocalization of Na+,K+-ATPase in the organs of the branchial cavity of the European lobster Homarus gammarus (Crustacea, Decapoda)

The localization of Na+,K(+)-ATPase in epithelia of the organs of the branchial cavity of Homarus gammarus exposed to seawater and dilute seawater was examined by immunofluorescence microscopy and immunogold electron microscopy with a monoclonal antibody IgG alpha 5 raised against the avian alpha-subunit of the Na-,K(+)-ATPase. In juveniles held in seawater, fluorescent staining was observed only in the epithelial cells of epipodites. In juveniles held in dilute seawater, heavier immunoreactivity was observed in the epithelial cells of epipodites, and positive immunostaining was also observed along the inner-side epithelial layer of the branchiostegites. No fluorescent staining was observed in the gill epithelia. At the ultrastructural level, the Na+,K(+)-ATPase was localized in the basolateral infolding systems of the epipodite and inner-side branchiostegite epithelia of juveniles held in dilute seawater, mostly along the basal lamina. The expression of Na+,K(+)-ATPase therefore differs within tissues of the branchial cavity and according to the external salinity. These and previous ultrastructural observations suggest that the epipodites, and to a lesser extent the inner-side epithelium of the branchiostegites, are involved in the slight hyper-regulation displayed by lobsters at low salinity. Enhanced Na+,K(+)-ATPase activity and de novo synthesis of Na+,K(+)-ATPase within the epipodite and branchiostegite epithelia may be key points enabling lobsters to adapt to low salinity environments.     

Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus)

Potassium chloride (KCl: 330 mg/ml) was assessed as an euthanasia agent in American lobsters (Homarus americanus). Two groups of 10 lobsters (408.2 to 849.9 g) were maintained at 11.9 to 12.1 degrees C ('warm') and 1.5 to 2.5 degrees C ('cold') to evaluate the possible effect of ambient temperature on response to KCl. Death was defined as time of cardiac arrest, as viewed and measured by use of ultrasound. The KCl solution was injected (100 mg of KCl/100 g of body weight) at the base of the second walking leg to flood the hemolymph sinus containing the ventral nerve cord with potassium. Disruption of this 'central nervous system' was immediate, followed by cardiac arrest within 60 to 90 seconds. Group median ( +/- SD) baseline heart rate was 42 +/- 14 'warm' and 36 +/- 5 'cold' beats per minute. Time until cardiac arrest ranged from 35 to 90 (57 +/- 18) seconds in the 'warm' group and from 40 to 132 (53 +/- 34) seconds in the 'cold' group. There was no significant difference between group medians for either parameter. Histologic lesions were limited to mild to moderate acute degeneration, characterized by cell swelling, loss of contraction bands, and occasional mild cytoplasmic vacuolation of skeletal muscle at the injection site. Injectable KCl solution was an effective, reliable method for euthanasia of H. americanus.     

Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.     

Physiological and behavioral effects of chemoreceptors located in different body parts of the swimming crab Callinectes danae

By perfusing their branchial chambers with filtered seawater, we have developed a preparation that allows us to maintain the swimming crab Callinectes danae outside water without any major effects on its cardiac activity. This in turn allowed us to selectively stimulate chemoreceptors located in different body parts, and specifically to discriminate between the receptors located in the branchial chambers and those located in the oral region (mainly in the mouthparts, antennules and antennae). We show that a taurine solution can evoke bradycardia when applied to the oral region or to a combination of the oral region and the branchial chambers. Although the precise localization of the oral region receptors involved remains to be determined, ablation experiments show that the olfactory organs (i.e., the antennules) are not involved. Finally, we show that although stimulating the pereiopods has no effect on the animals' cardiac activity it causes the animals to move, putatively to try to grasp a piece of food, a reaction not evoked by stimulating the gills or the oral regions. Our results lend support to the idea that chemoreceptors located in different parts of the body play different functional roles in decapod crustaceans.     

Physiological responses to hyper-saline waters in sailfin mollies (Poecilia latipinna)

We examined the ionoregulatory physiology and biochemistry of the teleost sailfin molly (Poecilia latipinna), an inhabitant of salt marshes along the gulf coast, during exposure to hyper-saline waters (salinity range 35-95 ppt). Mollies were able to tightly control plasma Na(+) and Cl(-) concentrations and tissue water levels up to 65 ppt, but at higher salinities plasma ion levels began to rise and muscle water content dropped. Still, even at the highest salinity (90 ppt) plasma Na(+) and Cl(-) levels were only 32% and 39%, respectively, above levels at 35 ppt. Drinking rates at 60 ppt climbed 35%, while gut Na(+)/K(+)-ATPase (NAK) activity rose 70% and branchial NAK activity jumped 200%. The relatively small rise in drinking rate, in the face of a more than doubling of the osmotic gradient, suggests that a reduction in branchial water permeability significantly limited water loss and associated salt load. At 80 ppt, a salinity where plasma ion levels just begin to rise, drinking rate rose more rapidly, but gut and gill NAK activity did not, suggesting that mollies employed other pathways (perhaps renal) of salt excretion. At higher salinities, plasma ion levels continued to rise and muscle water content fell slightly indicating the beginnings of internal osmotic disturbances. To evaluate the energetic costs of hyper-salinity on mollies we measured the rate of O(2) consumption and found it rose with salinity, in sharp contrast to virtually all species previously examined. Interestingly, despite higher metabolism, growth was unaffected by hyper-salinity.     

Physiological and behavioral effects of chemoreceptors located in different body parts of the swimming crab Callinectes danae

By perfusing their branchial chambers with filtered seawater, we have developed a preparation that allows us to maintain the swimming crab Callinectes danae outside water without any major effects on its cardiac activity. This in turn allowed us to selectively stimulate chemoreceptors located in different body parts, and specifically to discriminate between the receptors located in the branchial chambers and those located in the oral region (mainly in the mouthparts, antennules and antennae). We show that a taurine solution can evoke bradycardia when applied to the oral region or to a combination of the oral region and the branchial chambers. Although the precise localization of the oral region receptors involved remains to be determined, ablation experiments show that the olfactory organs (i.e., the antennules) are not involved. Finally, we show that although stimulating the pereiopods has no effect on the animals' cardiac activity it causes the animals to move, putatively to try to grasp a piece of food, a reaction not evoked by stimulating the gills or the oral regions. Our results lend support to the idea that chemoreceptors located in different parts of the body play different functional roles in decapod crustaceans.     

Saline groundwater as an aquaculture medium: physiological studies on the red drum, Sciaenops ocellatus

Physiological responses of the euryhaline red drum, Sciaenops ocellatus, to chloride salt addition, low salinity, and high sulfate concentration were measured. Survival was increased by addition of calcium chloride (CaCl₂) or magnesium chloride (MgCl₂) to dilute artificial seawater (0.2 ppt salinity). Although survival and routine metabolic rates were greater in MgCl₂ treatments, growth and feed efficiency were greater in CaCl₂ treatments. Marginal metabolic scope increased when CaCl₂ or MgCl₂ were added to dilute artificial seawater. There was a strong positive linear relationship (p=0.0001, r=0.91) between fish survival and salinity of artificial seawater dilutions over the salinity range 0.1 to 3.0 ppt. Monovalent ion concentrations in red drum plasma varied; whereas, divalent ion concentrations were relatively constant. Survival and growth were not affected by high sulfate concentrations (2000 mg l^-1) in 3.0 ppt artificial seawater supplemented with either sodium sulfate or magnesium sulfate. Routine metabolic rate and marginal metabolic scope of red drum exposed to high sulfate concentrations were slightly, but not significantly, lower than those of red drum in 3 ppt artificial seawater.     

Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers. Adaptiveness of enzymes from osmoconformity to hyperregulation

Haemolymph inorganic osmolyte changes and Na,K-ATPase activities in trichobranchiate and epipodite tissues were examined in the spiny lobster Palinurus elephas gradually acclimated from seawater (SW; 38 ppt, salinity; 1291 mOsmol/l) down to dilute seawater (DSW; 20 ppt, salinity; 679 mOsmol/l). During acclimation to DSW haemolymph was only transiently hypoosmotic, becoming isosmotic to the medium over a 24-h period of acclimation. Na,K-ATPase specific activities in homogenates of the trichobranchiate gills from SW- and DSW-acclimated spiny lobsters were in the range of 2-3 μmol Pi/h/mg protein and were not significantly different. It has also been confirmed for the marine stenohaline crustaceans Maja crispata and Dromia personata that gill Na,K-ATPase maintains the same level of specific activity in SW- and DSW-acclimated crabs. The saponin-treated fraction of Na,K-ATPase activity in trichobranchiate gills was 67-89% and epipodites 63-64% over the native homogenates' activity and no differences in enzyme activities upon saponin treatment between SW- and DSW-acclimated spiny lobsters were found. Recovery of 6% and enrichment factor (1.6) of Na,K-ATPase in partially purified plasma membrane fractions of epipodites was relatively low and not different in SW- and DSW-acclimated spiny lobsters. In the hemiepipodite, negative short-circuit current was in the range from -16.7 to -22.7 μA cm(-2) and conductance varied in the range of 205-290 mS cm(-2), values which were not significantly different in spiny lobsters residing in SW or DSW. Very high conductance suggests leakiness of the hemiepipodite epithelium-cuticular complex. In contrast to the group of euryhaline hyperosmoregulating Crustacea in which activation of the specific activity of Na,K-ATPase upon acclimation to dilute seawater occurs, in marine osmoconformers there is no activation of the enzyme in dilute seawater. Based on the literature data and our own results, we have reported a correlation coefficient of 0.65 between specific activity of Na,K-ATPase and the sodium gradient (mmol Na/l; haemolymph-seawater ) between 12 species of osmoconforming and osmoregulating Crustacea. During evolution, hyperosmoregulating Crustacea have achieved internal osmolyte gradients generated by Na,K-ATPase and lowering the gill surface permeability. However these adaptive characteristics are not present in marine osmoconforming Crustacea, restraining them to migrate in the brackish water habitats.     

Effect of salinity variation and pesticide exposure on an estuarine harpacticoid copepod, Microarthridion littorale (Poppe), in the southeastern US

The harpacticoid copepod Microarthridion littorale (Poppe) was tested for interaction effects between salinity change and acute pesticide exposure on the survival and genotypic composition of a South Carolina population. Previous data suggested a significant link between a combined exposure to chlorpyrifos (CHPY) and dichloro-diphenyl-trichloroethane (DDT) and mitochondrial haplotype in the cytochrome b apoenzyme for this euryhaline species when exposed at 12-ppt salinity seawater. Our tests demonstrate a significant non-linear survival response for M. littorale to short-term immersion (24 h) in 3-, 12- and 35-ppt seawater, with copepods transferred to 12-ppt seawater having the lowest survival. There was significant statistical interaction between salinity and pesticide exposure for the dependent variable “survival.” However, changes in genetic composition of survivors were not significant, and they were complicated by extremely low survival in the pesticide/3 ppt and pesticide/36 ppt treatments. As noted for many studies of harpacticoids, males faired worse than females in all treatments, with none surviving pesticide exposure at 45 μg/l CHPY and 6 μg/l DDT.     

Changes in tissue free amino acid contents, branchial Na+/K+ -ATPase activity and bimodal breathing pattern in the freshwater climbing perch, Anabas testudineus (Bloch), during seawater acclimation

This study aimed to examine effects of short- or long-term acclimation to brackish water or seawater on the climbing perch, Anabas testudineus, which is an aquatic air-breathing teleost living typically in freshwater. A. testudineus exhibits hypoosmotic and hypoinoic osmoregulation; the plasma osmolality, [Na+] and [Cl-] of fish acclimated to seawater were consistently lower than those of the external medium. However, during short-term (1 day) exposure to brackish water (15 per thousand) or seawater (30 per thousand), these three parameters increased significantly. There were also significant increases in tissue ammonia and urea contents, contents of certain free amino acids (FAAs) in the muscle, and rates of ammonia and urea excretion in the experimental fish. The accumulated FAAs might have a transient role in cell volume regulation. In addition, these results indicate that increases in protein degradation and amino acid catabolism had occurred, possibly providing energy for the osmoregulatory acclimation of the gills in fish exposed to salinity stress. Indeed, there was a significant increase in the branchial Na+/K+ -ATPase activity in fish exposed to seawater for a prolonged period (7 days), and the plasma osmolality, [Na+] and [Cl-] and the tissue FAA contents of these fish returned to control levels. More importantly, there was a significant increase in the dependence on water-breathing in fish acclimated to seawater for 7 days. This suggests for the first time that A. testudineus could alter its bimodal breathing pattern to facilitate the functioning of branchial Na+/K+ -ATPase for osmoregulatory purposes.     

The ionic basis of cardiac activity in the bivalve mollusc Perna perna

The ionic basis of cardiac activity and aspects of excitation-contraction (E-C) coupling were investigated in the isolated heart of the bivalve mollusc Perna perna, using the sucrose-gap technique. The role of the principal ions was established employing artificial seawater, in which specific ion concentrations were modified, and ion channel blockers. The mean membrane resting potential (MP) and the action potential (AP) were -33+/-0.7 mV (n=89) and 13+/-0.3 mV (n=71), respectively. The MP potential was primarily dependent on K(+) ions. Three types of cardiac APs were identified: fast, slow and spike-plateau potentials. Cardiac activity was maintained in Na(+)- or Ca(2+)-free salines but ceased when either Cd(2+) or EDTA was added to these salines. Other Ca(2+) channel blockers reduced the amplitude and increased duration of the cardiac APs. Tetrodotoxin (TTX) and procaine did not alter the AP. The data showed that the depolarizing phase of the AP was dependent on Ca(2+) influx while the plateau phase, when present, resulted from Na(+) influx that was modulated by Ca(2+). The mechanical responses were more sensitive to changes in extracellular Ca(2+) concentration than were the electrical responses.     

Enhancement of growth of tilapia Oreochromis niloticus in iso-osmotic medium

Tilapia ( Oreochromis niloticus ) kept in 15 ppt sea water (roughly iso-osmotic salinity) had higher growth rates than fish kept in 0 ppt (freshwater) or 30 ppt seawater, but circulating level of growth hormone was highest in fish exhibiting the poorest growth rate (30 ppt seawater). Serum thyroxine concentration was highest in 15 ppt seawater. Intestinal trypsin may play a role in promoting growth in iso-osmotic salinity since its activity was highest in fish cultured in 15 ppt seawater. The results indicate that changes in the digestive power, coupled with changes in thyroxine secretion, may account for the variations in growth rate in tilapia reared under different salinities.     

Infectivity of microsporidia spores stored in seawater at environmental temperatures

To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.     

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.     

The effect of seawater composition and osmolality on hemolymph levels of methyl farnesoate in the green crab Carcinus maenas

Green crabs, Carcinus maenas, exposed to dilute seawater (e.g., 5 ppt salinity, approximately 150 mOsm/kg) have hemolymph levels of methyl farnesoate (MF) that are up to 10-fold higher than animals in isosmotic seawater (27 ppt, approximately 800 mOsm/kg). In this paper, we examine aspects of osmotic and ionic stress to identify factors involved in elevating MF levels. MF levels did not rise after exposure to concentrated seawater, so only hypoosmotic stress elevates MF. MF levels rose in animals exposed to dilute seawater containing mannitol to make it isosmotic, indicating that the hypoosmotic rise in MF is due to decreased ion concentrations. Individual ions were investigated by exposing crabs either to isosmotic seawater with low concentrations of an ion or to dilute seawater with high concentrations of an ion. Ca(2+) and Mg(2+) in combination affected MF levels. Finally, we found that the increase in MF levels was accelerated when hemolymph osmolality was precociously lowered by partially replacing hemolymph with deionized water prior to transferring animals to dilute seawater. Thus, the 6-8 h delay between exposing crabs to dilute sea water and observing an increase in MF appears to reflect the time needed for specific hemolymph ions to decrease below a threshold concentration.     

Relationship of Cell Envelope Stability to Substrate Capture in a Marine Psychrophilic Bacterium

Cells of a psychrophilic marine bacterium were found to take up a variety of amino acids from seawater. Some of the amino acids that were taken up were released when the cells were exposed to a hypotonic salt solution. The proportion that was released varied according to the amino acid. A pool of the amino acid arginine that was formed during very short periods of exposure of cells to the exogenously supplied amino acid was particularly sensitive to reductions in salinity. In general, exposure to hypotonic salt solutions also resulted in reduced amino acid uptake by the cells. Complete removal of seawater salts (SE treatment) produced obvious structural alterations in the cell envelope, resulting in an even greater reduction in amino acid uptake. Under these conditions, amino acid-binding components were released by the cells. Differential centrifugation and fluorescent antibody studies indicated that arginine-binding components are located on or near the surface of intact cells. The data suggest that substrate receptors were sensitive to reductions in seawater salt concentrations and that lesions at this level affected the organism's substrate uptake and retention capabilities.