Effects of starvation and - or -alanine administration on the free - and -alanine levels in the muscle and hepatopancreas of the crayfish, Procambarus clarkii

Changes of - and -alanine and other free amino acids were examined in muscle and hepatopancreas of the crayfish Procambarus clarkii before and after a 1-month starvation in freshwater, 50% seawater, and seawater. Total free amino acids and - and -alanine in both tissues decreased during starvation. The largest decrease was found in the animals starved in freshwater. In 50% seawater and full-strength seawater, the ratio of -alanine to total alanine increased in both tissues, although the level was reduced a little. A large dose of -alanine administered into the tail muscle of freshwater crayfish was converted to -alanine and -alanine returned to the control level within a week. A large amount of -alanine was transported from muscle to hepatopancreas. -Alanine injected into freshwater crayfish was converted to -alanine, but was not transported to the hepatopancreas. In the case of -alanine injection into the muscle of crayfish acclimated to 50% seawater, large amounts of - and -alanine were maintained in the muscle during the experimental period of 4 days. These data indicate that -alanine is metabolized actively in crayfish tissues and accumulated as an important osmolyte during osmotic stress.     

Hepatopancreas gluconeogenesis during anoxia and post-anoxia recovery in Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets

C. granulata is a semiterrestrial crab that lives in the mesolittoral and the supralittoral zones of estuaries and faces hypoxia and anoxia when exposed to atmospheric air. The carbohydrate or protein content of the diets administered to the crabs induced different metabolic adjustments during anoxia and post-anoxia recovery period. During the first hour in anoxia a marked increase in L-lactate concentration in hemolymph was induced, followed by a reduction in its levels accompanied by two peaks in hepatopancreas gluconeogenic capacity. Anoxia exposure did not induce a reduction in the hepatopancreas phosphoenolpyruvate carboxykinase activity in either dietary group. Our results suggest that in anaerobiosis this crab uses the conversion of lactate to glucose in hepatopancreas to maintain the acid-base balance and the glucose supply. In post-anoxia recovery, the fate of L-lactate is the hepatopancreas gluconeogenesis in high protein maintained crabs. On the other hand, in the crabs maintained on carbohydrate-rich diet the L-lactate levels decreased gradually in the hemolymph during the post-anoxia recovery; however, the hepatopancreas gluconeogenesis did not increase. In both dietary groups, an increase in the gluconeogenic capacity of hepatopancreas occurred at 30 h of post-anoxia recovery.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

The role of the antennal glands in ion and body volume regulation of cannulated Penaeus monodon reared in various salinity conditions

Urinary production rate and the osmotic and ionic concentrations in both urine and hemolymph were measured in cannulated intermolt Penaeus monodon which were either abruptly transferred from 45 ppt seawater to 15 ppt seawater (Experiment 1) or acclimated to 5, 25 and 45 ppt seawater (Experiment 2). In Experiment 1, urinary magnesium concentration fell dramatically from 228 to 30 mEq/l within 4 h post-transfer, but 8 h after transfer, U/H (urine/hemolymph) ratios stabilized at between 1.0 and 2.5. Sodium was higher in urine than in hemolymph during the first 24 h after transfer, while potassium was lower in urine than in hemolymph until 72 h after transfer, which suggests that sodium and potassium concentrations are regulated by the antennal gland after an abrupt change in media. In Experiment 2, the urinary production rate of P. monodon decreased as salinity increased, suggesting that the antennal glands also regulate body volume. In the acclimated shrimps of Experiment 2, the antennal glands did not appear to regulate osmolarity or the concentration of chloride, sodium, potassium, and calcium ions, but as salinity increased, U/H ratios of magnesium increased from 2.3 to 13.5, and active secretion by the antennal gland accounted for 57 approximately 93% of the total magnesium excretion through urine. These results suggest that active secretion of magnesium by the antennal gland enable this shrimp to maintain hypoionic levels of magnesium in the hemolymph.     

急性低氧/复氧胁迫对克氏原螯虾抗氧化-能量代谢的影响

为研究低氧/复氧胁迫对克氏原螯虾(Procambarus clarkii)抗氧化及能量代谢的影响,将克氏原螯虾暴露于(1.0±0.1) mg/L急性低氧胁迫和后续(6.8±0.2) mg/L复氧环境中,于低氧胁迫1h、6h及复氧1h、12h分别采集肝胰腺、鳃和血淋巴,研究低氧/复氧胁迫下克氏原螯虾抗氧化-能量代谢酶的活力变化,分析鳃和肝胰腺组织的超微结构改变。在低氧胁迫下,肝胰腺和血淋巴中SOD酶活力显著下降(P<0.05);复氧以后,肝胰腺、血淋巴及鳃组织中SOD酶活力均出现了显著上升(P<0.05)。SOD酶活力变化可能与复氧过程中超氧阴离子自由基的过量产生有关。在复氧12h后,血淋巴和鳃组织中MDA含量均出现了显著性增加(P<0.01),提示机体细胞在复氧胁迫下产生了脂质过氧化。在低氧胁迫下,肝胰腺、鳃和血淋巴中ACP、AKP酶活力显著上升(P<0.05);在复氧12h后,肝胰腺和鳃组织中ACP酶活力显著降低(P<0.01)。显示低氧/复氧胁迫影响了机体的非特异性免疫应答。在急性低氧胁迫下,肝胰腺、血淋巴与鳃组织中的LDH含量和总ATPase活力均显...     

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.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28 degrees C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.     

Metabolic responses of the white shrimp, Penaeus vannamei, to ambient ammonia

Juvenile shrimp were individually exposed during 24 h to 0.007 (control), 0.36, 1.07, and 2.14 mmol/l total ammonia-N at 28°C and 39 ppt salinity. After 22 h of ammonia-N exposure, oxygen consumption was measured for 2 h, and then hemolymph, hepatopancreas, and muscle tissues were sampled. Oxygen consumption, and levels of lactate and glycogen in the hepatopancreas increased significantly at the highest ammonia-N concentration. Concentration of oxyhemocyanin, acylglycerol, and cholesterol in hemolymph, and lactate in muscle decreased significantly in the group exposed to the highest ammonia levels. The changes observed in hemolymph and tissue metabolic fuels suggest a reduced use of carbohydrate through anaerobic metabolism and an increase in the use of lipids to satisfy the metabolic demand.     

Antioxidant defenses and oxidative stress parameters in tissues of mud crab (Scylla serrata) with reference to changing salinity

The effects of salinity (10, 17 and 35 ppt) on O₂ consumption, CO₂ release and NH₃ excretion by crabs and oxidative stress parameters and antioxidant defenses of its tissues were reported. An increase in salinity caused a decrease in O₂ consumption and CO₂ release and an increase in ammonia excretion by crabs. Lipid peroxidation, protein carbonyl, H₂O₂ levels and total antioxidant capacity of the tissues elevated significantly at 35 ppt salinity except in abdominal muscle where H₂O₂ content was low. Ascorbic acid content of tissues was higher at 17 ppt salinity than at 10 and 35 ppt salinities. With increasing salinity, a gradual decrease in SOD, an increase in catalase, no change in GPx and a decrease followed by an increase in GR activities were recorded for abdominal muscle. While for hepatopancreas, an increase followed by a decrease in SOD and catalase, decrease in GPx and GR activities were noticed with increasing salinity. In the case of gills, a decrease followed by an increase in SOD, a decrease in catalase and GPx and an increase in GR activities were noted when the salinity increased from 10 ppt to 35 ppt. These results suggest that salinity modulation of oxidative stress and antioxidant defenses in Scylla serrata is tissue specific.     

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.     

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.     

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l.During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.     

Growth, Fine Structure and Cyst Formation of a Microbial Mat Ciliate: Pseudocohnilembus pusillus (Ciliophora, Scuticociliatida)

ABSTRACT Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.     

Immune responses of Litopenaeus vannamei to thermal stress: a comparative study of shrimp in freshwater and seawater conditions

The effects of hypothermal (22–16?°C) and hyperthermal (22–28?°C) stress on the immune system responses of the shrimp Litopenaeus vannamei cultured in either freshwater or seawater were measured and compared. The following immune system indicators were measured for comparison: total hemocyte count (THC), activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase (SOD), and malondialdehyde (MDA) content. Thermal stress significantly decreased THC in both freshwater and seawater shrimp within 6–12?h (P?0.05). After hypothermal stress, all shrimp had a significantly lower THC level than their prechallenge levels (P?0.05). Under both types of thermal stress, the activity of PO, NOS, and SOD first increased and then decreased. After 48?h of thermal stress, shrimp PO and NOS activity decreased in both freshwater and seawater. After 48?h of thermal stress, the reduction in the SOD activity in the hemolymph of freshwater shrimp was greater than that in seawater shrimp. During exposure to stress, the MDA content in freshwater shrimp was significantly higher than in seawater shrimp, which demonstrated that lipids in freshwater shrimp were more susceptible to peroxidation than those in seawater shrimp, particularly at low temperatures. Large temperature fluctuations, particularly sudden cooling, should be avoided when rearing L. vannamei because of high rates of lipid peroxidation and decreased immunity. These effects are more marked in freshwater than in seawater.     

Salinity tolerance and osmoregulation of the arctic marine amphipods Onisimus litoralis (Kroyer) and Anonyx nugax (Phipps)

Summary The gammarid amphipod Onisimus litoralis, which inhabits arctic and subarctic intertidal and under-ice habitats, is a euryhaline hyperosmotic regulator. It survives 10 d exposures to salinities from 5 to 55 ppt. It hyperregulates its hemolymph osmolality during 3 h exposures to dilutions of 33 ppt seawater and remains hyperosmotic for at least 2 w. The hemolymph is isosmotic to the medium after 12 h exposures to salinities higher than 33 ppt. The gammarid amphipod Anonyx nugax, which inhabits arctic and subarctic subtidal areas, tolerates salinities from 23 to 45 ppt with little mortality. Unlike Onisimus, however, it is an osmoconformer and its hemolymph becomes isosmotic to all dilute salinities within its tolerance range after 12 h and to concentrated media after 3 h. The salinity tolerances and osmoregulatory abilities of both species are reflected in their distributions in the field.     

Tolerance of seahorse Hippocampus kuda (Bleeker) juveniles to various salinities

In line with current conservation efforts, some success in the captive breeding of the seahorse Hippocampus kuda (Teleostei: Syngnathidae) has been achieved. To evaluate the salinity tolerance of these hatchery‐bred juveniles, 9‐week‐old H. kuda were transferred without prior acclimatization from ambient full strength seawater (32–33 ppt) to salinities ranging from freshwater to 85 ppt. Survival, growth, and total body water content were determined after 4 and 18 days of exposure. Juvenile H. kuda are able to survive in dilute seawater (15 ppt) for at least 18 days without any compromise in growth (both wet and dry body weight), survival, and total body water. Fish abruptly transferred to freshwater succumbed within 4–24 h, while survival of 5 ppt‐reared fish decreased to ca. 65% in 18 days. Although 10 ppt‐reared seahorses had growth and survival comparable with the control (30 ppt seawater), total body water was significantly elevated indicating reduced adaptability. The upper limit of H. kuda salinity tolerance was 50 ppt. Fish reared at salinities ≥55 ppt succumbed within 24 h. Like several other marine teleosts, growth and survival of juvenile H. kuda tended to peak in diluted seawater salinities of 15 and 20 ppt. These results indicate the possibility of growing hatchery‐bred H. kuda in brackishwater environments.     

Tolerance of Pinus taeda and Pinus serotina to low salinity and flooding: Implications for equilibrium vegetation dynamics

Questions: 1. Do pine seedlings in estuarine environments display discrete or continuous ranges of physiological tolerance to flooding and salinity? 2. What is the tolerance of Pinus taeda and P. serotina to low salinity and varying hydrologic conditions? 3. Are the assumptions for ecological equilibrium met for modeling plant community migration in response to sea‐level rise? Location: Albemarle Peninsula, North Carolina, USA. Methods: In situ observations were made to quantify natural pine regeneration and grass cover along a salinity stress gradient (from marsh, dying or dead forest, to healthy forest). A full‐factorial greenhouse experiment was set up to investigate mortality and carbon allocation of Pinus taeda and P. serotina to low‐salinity conditions and two hydrology treatments over 6 months. Treatments consisted of freshwater and two salinity levels (4 ppt and 8 ppt) under either permanently flooded or periodically flushed hydrologic conditions. Results: Natural pine regeneration was common (5–12 seedlings per m²) in moderate to well‐drained soils where salinity concentrations were below ca. 3.5 ppt. Pine regeneration was generally absent in flooded soils, and cumulative mortality was 100% for 4 and 8 ppt salinity levels under flooded conditions in the greenhouse study. Under weekly flushing conditions, mortality was not significantly different between 0 and 4 ppt, confirming field observations. Biomass accumulation was higher for P. taeda, but for both pine species, the root to shoot ratio was suppressed under the 8 ppt drained treatment, reflecting increased below‐ground stress. Conclusions: While Pinus taeda and P. serotina are commonly found in estuarine ecosystems, these species display a range of physiological tolerance to low‐salinity conditions. Our results suggest that the rate of forest migration may lag relative to gradual sea‐level rise and concomitant alterations in hydrology and salinity. Current bioclimate or landscape simulation models assume discrete thresholds in the range of plant tolerance to stress, especially in coastal environments, and consequently, they may overestimate the rate, extent, and timing of plant community response to sea‐level rise.     

Lipids as energy source during salinity acclimation in the euryhaline crab Chasmagnathus granulata dana, 1851 (crustacea-grapsidae)

Lipids seem to be the major energy store in crustaceans. Moreover, they are extremely important in maintaining structural and physiological integrity of cellular and sub cellular membranes. During salinity adaptation, energy-demanding mechanisms for hemolymph osmotic and ionic regulation are activated. Thus, the main goal of this work was to verify the possible involvement of lipids as an energy source in the osmotic adaptation process. The estuarine crab Chasmagnathus granulata was captured and acclimated to salt water at 20 per thousand salinity and 20 +/- 2 degrees C, for 30 days. After acclimation, crabs were divided into groups of ten and transferred to fresh water (0 per thousand ), salt water at 40 per thousand salinity, or maintained in salt water at 20 per thousand salinity (control group), without feeding. Before and seven days after the salinity change, wet weight and lipid concentration in gills, muscle, hepatopancreas, and hemolymph were determined according to the colorimetric assay of sulphophosphovanilin. Results show that hepatopancreas lipids were not mobilized during osmotic stress regulation. Gill and muscle lipids were significantly lower in crabs subjected to hypo-osmotic stress than those subjected to the hyper-osmotic stress or maintained at the control salinity. Our results point to the occurrence of lipid mobilization and involvement of these compounds in the osmotic acclimation process in C. granulata, but with differences between tissues and the osmotic shock (hypo or hyper) considered.     

The concentrations of antioxidant compounds in the hepatopancreas, the gills and muscle of some freshwater crayfish species

We examined the concentrations of vitamin E (vit E), sulphydryl groups (-SH), total protein and protein electrophoretic profiles in the hepatopancreas, the gills and muscle of three freshwater crayfish species: Noble crayfish (Astacus astacus), stone crayfish (Austropotamobius torrentium) and spiny cheek crayfish (Orconectes limosus). Vit E concentration in the hepatopancreas of O. limosus was lower compared to A. astacus, while in the gills of O. limosus it was lower compared to both A. astacus and A. torrentium. The concentration of -SH groups was lower in the hepatopancreas of A. astacus compared to A. torrentium and O. limosus. In the gills of A. astacus and A. torrentium the concentration of -SH groups was higher compared to O. limosus. Protein concentration was higher in the hepatopancreas of A. torrentium compared to A. astacus and O. limosus. A lower protein concentration in muscle of A. torrentium was found compared to O. limosus and A. astacus. Electrophoretic analysis of proteins indicated species and tissue specifities between investigated crayfish species. Our results represent the first study of its kind and provide the basis for future studies that will consider our reported parameters as potential biomarkers for biomonitoring of basic environmental conditions and some anthropogenic impacts.