Hepatopancreas gluconeogenesis during hyposmotic stress in crabs Chasmagnathus granulata maintained on high-protein or carbohydrate-rich diets

The incorporation of [14C]-alanine or [14C]-lactate into glucose was measured in hepatopancreas fractions from Chasmagnathus granulata crabs adapted to a high protein or a carbohydrate-rich diet and submitted or not (control group) to hyposmotic stress. Gluconeogenic capacity and phosphoenolpyruvate carboxykinase (PEPCK) activity increased during acclimation to a dilute medium in C. granulata hepatopancreas. In intact animals, high hemolymph urea levels occurred for the high-protein regimen and for crabs fed both diets and submitted to hyposmotic stress. It could be that the amino acids released during hyposmotic stress are deaminated in the hepatopancreas, and that the carbon chains are used as substrate for gluconeogenesis. Hepatopancreas gluconeogenesis seems to be one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress in C. granulata.     

Hepatopancreas gluconeogenesis and glycogen content during fasting in crabs previously maintained on a high-protein or carbohydrate-rich diet

The present study assessed the effect of different fasting times on the in vitro gluconeogenic capacity of Chasmagnathus granulata crabs previously adapted to a high-protein (HP) or carbohydrate-rich (HC) diet using the incorporation of [U-(14)C]l-lactate or [U-(14)C]l-alanine into glucose. We also recorded haemolymphatic glucose and hepatopancreatic glycogen levels. In the HP group, on the third day of fasting there were decreases in the synthesis of glucose from (14)C-alanine and in haemolymph glucose. After 15 days of fasting, haemolymph glucose and hepatopancreatic glycogen levels were maintained by an increase in the conversion of (14)C-alanine into glucose. However, after 21 days of fasting the gluconeogenic capacity was decreased and hepatopancreas glycogen concentration was reduced. In the HC group, hepatopancreatic glycogen was the energy source during the first 6 days of fasting. Gluconeogenesis from (14)C-lactate decreased after 6 days of fasting, remaining low until 21 days of fasting. The conversion of (14)C-alanine into glucose was increased after 15 days fasting and hepatopancreatic glycogen was raised in relation to that present after a 6-day fasting. In both dietary groups the stabilization in the levels of haemolymph glucose after 21 days fasting may result from a reduction in metabolic rate during restricted feeding.     

Effects of anoxia and post-anoxia recovery on carbohydrate metabolism in the jaw muscle of the crab Chasmagnathus granulatus maintained on carbohydrate-rich or high-protein diets

The present study assesses the effects of 1-h anoxia and 3-h post-anoxia recovery period on the activity of pyruvate kinase (PK), ¹⁴CO₂ produced from ¹⁴C-glucose and ¹⁴C-lactate, ATP, and glycogen levels in the jaw muscle of Chasmagnathus granulatus fed either a carbohydrate-rich (HC) or high-protein (HP) diet.In the HC control group the jaw muscle PK activity was higher than in HP-fed crabs. In jaw muscle from control HP-fed crabs the lactate oxidation was higher than in HC-fed animals. We observed increased PK activity and ATP concentration and a reduction in the glycogen concentration, ¹⁴CO₂ production from ¹⁴C-lactate in HP-fed crab jaw muscle during anoxia. In crabs fed an HC diet the PK activity decreased in anoxia, the ¹⁴CO₂ production from ¹⁴C-glucose increased, and the ¹⁴CO₂ production from ¹⁴C-lactate did not change.During recovery, a low oxidation capacity for lactate was found in jaw muscle of both dietary groups. PK activity and ¹⁴CO₂ production from ¹⁴C-glucose were high during the recovery period only in the jaw muscle from HP-fed crabs.Recovery decreased the ATP concentration of both dietary groups as compared to anoxia and normoxia, and did not restore the glycogen concentration in the jaw muscle.     

Effect of starvation and refeeding on amino acid metabolism in muscle of crab Neohelice granulata previously fed protein- or carbohydrate-rich diets

The present study assesses the effects of starvation and refeeding on 1-[¹⁴C]-methyl aminoisobutyric acid (¹⁴C-MeAIB) uptake, ¹⁴C-total lipids, ¹⁴CO₂ production from ¹⁴C-glycine, ¹⁴C-protein synthesis from ¹⁴C-leucine and Na⁺–K⁺-ATPase activity in jaw muscle of Neohelice granulata previously maintained on a carbohydrate-rich (HC) or high-protein (HP) diet. In N. granulata the metabolic adjustments during starvation and refeeding use different pathways according to the composition of the diet previously offered to the crabs. During starvation, ¹⁴CO₂ production from ¹⁴C-glycine, and ¹⁴C-protein synthesis from ¹⁴C-leucine were reduced in HC-fed crabs. In crabs maintained on the HP or HC diet, ¹⁴C-total lipid synthesis increased after 15 days of starvation. In crabs fed HP diet, ¹⁴C-MeAIB uptake and Na⁺–K⁺-ATPase activity decreased in refeeding state. In crabs refeeding HC diet, ¹⁴C-MeAIB uptake and ¹⁴CO₂ production decreased during the refeeding. In contrast, the ¹⁴C-protein synthesis increased after 120 h of refeeding. In both dietary groups, ¹⁴C-total lipid synthesis increased during refeeding. Changes in the carbon amino acid flux between different metabolic pathways in muscle are among the strategies used by this crab to face starvation and refeeding. Protein or carbohydrate levels in the diet administered to this crab modulate the carbon flux between the different metabolic pathways.     

Carbohydrate metabolism in the central nervous system of the megalobulimus oblongus snail during anoxia exposure and post-anoxia recovery

The effects of anoxic exposure and the post-anoxia aerobic recovery period on carbohydrate metabolism in the central nervous system (CNS) of the land snail Megalobulimus oblongus, an anoxia-tolerant land gastropod, were studied. The snails were exposed to anoxia for periods of 1.5, 3, 6, 12, 18, or 24 hr. In order to study the post-anoxia recovery phase, snails exposed to a 3-hr period of anoxia were returned to aerobic conditions for 1.5, 3, 6, or 15 hr. Glycogen and glucose concentrations in the CNS, hemolymph glucose concentration, and glycogen phosphorylase (active form, GPa) activity in the CNS were analyzed. Anoxia does not significantly affect the concentration of CNS glucose but induces hyperglycemia and a reduction of CNS GPa activity. The glycogen concentration was decreased at 12 hr of anoxia; however, by 18 and 24 hr in anoxia, the glycogen content was not significantly different from basal control values. During the post-anoxia period, the reduction in GPa activity and the increased hemolymph glucose concentration induced by anoxia returned to control values. These results suggest that the CNS of M. oblongus may use hemolymph glucose to fulfill the metabolic demands during anoxia. However, the hypothesis of tissue metabolic arrest cannot be excluded.     

Effects of environmental anoxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata

We investigated the effects of anoxia (8 h) and different periods of reoxygenation (20 and 40 min) on the oxidative balance in anterior and posterior gills of the crab Chasmagnathus granulata. Enzyme activity of catalase and GST was increased in the gills of the animals submitted to anoxia, and SOD activity was decreased. These enzymes returned approximately to control levels during the anoxia recovery time. These results demonstrated enzyme activities change with variations in environmental oxygen levels. The posterior gills showed a higher antioxidant enzyme activity than anterior gills. In the gills, there were no changes in the non-enzymatic antioxidant system (TRAP) during anoxia. On the other hand, during anoxia recovery, an increase of TRAP in both gills was observed. Anoxia does not change lipid peroxidation (TBARS) in the gills. During anoxia recuperation, an increase in levels of TBARS was observed. Thus the results demonstrate that C. granulata has a similar strategy of preparation for oxidative stress as observed in other intertidal species, enabling the crabs to survive in an environment with extreme variations in physical and chemical characteristics, such as salt marshes.     

Effect of hyper or hypo-osmotic conditions on neutral amino acid uptake and oxidation in tissues of the crab Chasmagnathus granulata

We investigated the transport of ¹⁴C-methylaminoisobutyric acid (¹⁴C-MeAIB) and ¹⁴C-alanine oxidation in hepatopancreas and jaw muscle of Chasmagnathus granulata submitted to 24, 72, and 144 h of hypo- or hyperosmotic stress. While ¹⁴C-MeAIB uptake increased in jaw muscle and hepatopancreas from crabs submitted to hyperosmotic stress, it did not change in tissues from animals submitted to hypo-osmotic stress. Incubation of jaw muscle and hepatopancreas from control groups with 1 mM ouabain did not decrease ¹⁴C-MeAIB uptake. However, ouabain prevented ¹⁴C-MeAIB uptake in hepatopancreas at 24 h of hyperosmotic stress. In contrast, in jaw muscle from crabs submitted to the same conditions, ¹⁴C-MeAIB uptake was not prevented by ouabain in the incubation medium. Jaw muscle from the control group produced four times more ¹⁴CO₂ from ¹⁴C-alanine than the hepatopancreas. During hypo-osmotic stress, amino acid oxidation does not seem to be one of the pathways implicated in the decrease of the amino acid pools in hepatopancreas and jaw muscle. In contrast, during hyperosmotic stress the reduction in ¹⁴C-alanine oxidation appears to be one of the mechanisms involved in the increase of the amino acid pool in the hepatopancreas.     

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.     

Inter-organ relationships between glucose, lactate and amino acids in rats fed on high-carbohydrate or high-protein diets

1. Inter-organ relationships between glucose, lactate and amino acids were studied by determination of plasma concentrations in different blood vessels of anaesthetized rats fed on either a high-carbohydrate diet [13% (w/w) casein, 79% (w/w) starch] or a high-protein diet [50% (w/w) casein, 42% (w/w) starch]. The period of food intake was limited (09:00-17:00h), and blood was collected 4h after the start of this period (13:00h). 2. Glucose absorption was considerable only in rats fed on a high-carbohydrate diet. Portal-vein-artery differences in plasma lactate concentration were higher in rats fed on this diet, but not proportional to glucose absorption. Aspartate, glutamate and glutamine were apparently converted into alanine, but when dietary protein intake was high, a net absorption of glutamine occurred. 3. The liver removed glucose from the blood in rats fed on a high-carbohydrate diet, but glucose was released into the blood in rats fed on the high-protein diet, probably as a result of gluconeogenesis. Lactate uptake was very low when amino acid availability was high. 4. In rats on a high-protein diet, increased uptake of amino acids, except for ornithine, was associated with a rise in portal-vein plasma concentrations, and in many cases with a decrease in hepatic concentrations. 5. Hepatic concentrations of pyruvate and 2-oxo-glutarate decreased without a concomitant change in the concentrations of lactate and malate in rats fed on the high-protein diet, in spite of an increased supply of pyruvate precursors (e.g. alanine, serine, glycine), suggesting increased pyruvate transport into mitochondria. 6. High postprandial concentrations of plasma glucose and lactate resulted in high uptakes of these metabolites in peripheral tissues of rats on both diets. Glutamine was released peripherally in both cases, whereas alanine was taken up in rats fed on a high-carbohydrate diet, but released when the amino acid supply increased. 7. It is concluded that: the small intestine is the main site of lactate production, and the peripheral tissues are the main site for lactate utilization; during increased ureogenesis in fed rats, lactate is poorly utilized by the liver; the gut is the main site of alanine production in rats fed on a high-carbohydrate diet and the liver utilizes most of the alanine introduced into the portal-vein plasma in both cases.     

Stress response in Drosophila subobscura. II. Puff activity during anoxia and recovery from anoxia

When individuals of Drosophila subobscura at 0 hr prepupa are submitted to anoxia (4 hr and 24 hr, respectively), their puffing pattern is very similar to that shown by individuals at the moment of development in which treatment began. The same expression of genes (the same puffing pattern and the same protein pattern) is induced in this species by recovery from anoxia as well as by heat shock treatment at 31 degrees C.     

Physiological and biochemical changes in frog sciatic nerve during anoxia and recovery

Frog (Rana pipiens) sciatic nerve was incubated, with and without stimulation, in an oil bath. The correlation between changes in the magnitude of the compound action potential (α and β) and changes in metabolites, particularly energy reserves, during anoxia and recovery from anoxia was studied. The time to extinction of the action potential in anoxia was frequency-dependent. The action potential could not be restored, nor its extinction delayed, by washing the nerve in O₂-free Ringer's solution. Therefore, in this system extracellular K⁺ accumulation was not a significant factor in blocking impulse conduction. At the time of complete nerve block resulting from anoxia (90 min at rest), ATP, P-creatine and glucose were 30, 10 and 10 per cent, respectively, of initial levels. Glycogen did not fall below 42 per cent of control levels even after 5 h of anoxia. Changes in the levels of energy reserves during anoxia were used to calculate the metabolic rate of nerves at rest and during stimulation. In one series of experiments, the resting metabolic rate was 0·12 mequiv. of ‘high-energy phosphate’ (～P)/kg/min. Stimulation increased the metabolic rate to 0·22 mequiv. of ～P/kg/min at 30 Hz and to 0·29 mequiv. of ～P/kg/min at 100 Hz. The change in metabolic rate when the nerve passed from the resting to the stimulated state was quite abrupt, an observation suggesting that the slow transition observed with methods monitoring O₂, consumption was largely instrumental. In nerve stimulated to exhaustion in the absence of O₂, neither ATP nor P-creatine had fully recovered within 60 min after O₂, was readmitted, although the action potential reached supranormal levels 15 min after return to O₂. The ratio of lactate: pyruvate, which increased as expected during anoxia, paradoxically increased even further after O₂, was readmitted. The rate of energy utilization during recovery was 0·30 mequiv. of ～P/kg/min. Nerves stimulated at 100–200 Hz in O₂, exhibited no changes in levels of P-creatine, ATP or lactate, an observation implying that the nerve could not be made to use ～P faster than oxidation of glucose could provide it. This meant that the maximal metabolic rate was not limited by the rate of supply of chemical energy. Instead, the limitation may have arisen as a result of a limited rate at which ionic imbalance can result from stimulation or a limited pump capacity of the axonal membrane. Nerves stimulated at 200 Hz in O₂ for 20 min and then transferred to an O₂-free environment without further stimulation exhibited an increase in the rate of energy utilization (nearly two-fold) over the resting rate, a finding that suggested a metabolic (ionic?) debt as a result of activity which could not be met even though the energy supply was adequate. Therefore, restriction of energy expenditure by a limiting pumping rate seemed to be the most likely explanation. The resting metabolic rate of frog sciatic nerve was only one-quarter to one-third of the rate for rat sciatic nerve, when compared at the same temperature (25°C).     

Effects of long-term feeding of high-protein or high-fat diets on the response to exercise in the rat

The aim of this work was to find by which mechanisms an increased availability of plasma free fatty acids (FFA) reduced carbohydrate utilization during exercise. Rats were fed high-protein medium-chain triglycerides (MCT), high-protein long-chain triglycerides (LCT), carbohydrate (CHO) or high-protein low-fat (HP) diets for 5 weeks, and liver and muscle glycogen, gluconeogenesis and FFA oxidation were studied in rested and trained runner rats. In the rested state the hepatic glycogen store was decreased by fat and protein feeding, whereas soleus muscle glycogen concentration was only affected by high-protein diets. The percentage decrease in liver and muscle glycogen stores, after running, was similar in fat-fed, high-protein and CHO-fed rats. The fact that plasma glucose did not drastically change during exercise could be explained by a stimulation of hepatic gluconeogenesis: the activity of phosphoenolpyruvate carboxykinase (PEPCK) and liver phosphoenolpyruvate (PEP) concentration increased as well as cyclic adenosine monophosphate (AMPc) while liver fructose 2,6-bisphosphate decreased and plasma FFA rose. In contrast, the stimulation of gluconeogenesis in rested HP-, MCT- and LCT-fed rats appears to be independent of cyclic AMP.     

Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.     

Effect of fasting and refeeding on gluconeogenesis and glyconeogenesis in the muscle of the crab Chasmagnathus granulatus previously fed a protein- or carbohydrate-rich diet

The present study investigated the participation of the muscle gluconeogenic and glyconeogenic pathways in lactate metabolism after 15 fasting and during different periods of refeeding in Chasmagnathus granulatus previously maintained on a carbohydrate-rich (HC) or high-protein (HP) diet. In C. granulatus the metabolic adjustments during the fasting use different pathways according to the composition of the diet previously offered to the crab. During fasting, the gluconeogenic capacity is reduced in crabs maintained on the HC diet. In animals maintained on the HP diet, an increase in activity of the glyconeogenic pathway occurs after 15 days of fasting. In the animals fed HC diet, the glyconeogenesis is one of the pathways responsible for maintenance of the lactate levels in the fed and refeeding states. In crabs fed on the HP diet, the gluconeogenesis and glyconeogenesis pathways are involved in the reduction of lactate levels during the refeeding period. This study shows that protein or carbohydrates levels in the diet previously administrated to the crabs modulate the gluconeogenesis, glyconeogenesis in muscle and lactate concentration in the hemolymph in fed, fasting and refeeding states.     

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda,Grapsidae)

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30‰. The corresponding 96-h LC₅₀ of cadmium was 2.69 mg l⁻¹ at 2.5‰, and >50 mg l⁻¹ at 30‰. Cadmium only caused a significant lower CA activity than control at 2.5‰. EC₅₀ for CA inhibition was estimated to be 1.59 mg l⁻¹ at 2.5‰. Statistical differences in Na⁺ hemolymphatic levels (P<0.05) were only detected at 2.5‰, between 0 and 1.25 mg l⁻¹ of cadmium, but no statistical differences were observed for Cl⁻ levels at any assayed salinity. As CA inhibition registered at 2.5‰ was followed by only changes in Na⁺ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC₅₀) were 2.15×10⁻⁵, 1.62×10⁻⁵, 3.75×10⁻⁶ and 4.4×10⁻¹⁰ M for cadmium, copper, zinc and AZ, respectively. The comparison with IC₅₀ values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.     

Regional changes in amino acid levels of the neonate rat brain during anoxia and recovery

The aim of this study was to compare the changes in amino acids (alanine, aspartate, GABA, glutamate, glutamine, glycine, serine taurine) that are produced in different regions of the neonate brain (telencephalon, diencephalon cerebellum, brain stem) following a survivable period of anoxia and after the re-establishment of air respiration. Anoxia provoked different responses in the different regions. The changes during the anoxic period were as follows. In the brain stem there was a decrease in aspartate, in the telencephalon there was a significant increase in GABA and alanine and a decrease in aspartate, in the diencephalon, glutamate and GABA increased, and in the cerebellum, glycine and alanine levels were enhanced. The changes during recovery were even more dissimilar. Here the greatest shifts were seen in the brain stem with increases in glutamine, GABA, aspartate, glycine, serine, alanine, and taurine. In the telencephalon glutamate fell and alanine increased, in the diencephalon GABA increased, and in the cerebellum, glutamate fell while glycine and alanine increased. In none of the major brain regions did the pattern of changes in neurotransmitters correspond to that seen in anoxic tolerant species.     

Renal lesions in streptozotocin-induced diabetic rats maintained on onion and capsaicin containing diets

Onion (Allium cepa) powder and capsaicin, the pungent principle of red pepper (Capsicum annum) were added in the amounts of 3 g% and 15 mg%, respectively, to the diet of streptozotocin-induced diabetic Wistar rats for 8 weeks. The presence of renal lesions was assessed by the extent and quality of proteinuria and by the leaching of renal tubular enzymes into the urine. Renal integrity was assessed by measuring the activities in the kidney tissue of several key enzymes of carbohydrate metabolism and of polyol pathway, transaminases, and ATPases. Data on enzymuria and proteinuria, activities of kidney ATPases present in diabetic patients, suggested that dietary onion caused significant beneficial modulation of the progression of renal lesions in the diabetic rats. These findings were also corroborated by histologic examination of kidney sections. Dietary capsaicin did not have any favorable influence on renal pathology in diabetes. It is inferred that this beneficial ameliorating influence of dietary onion on diabetic nephropathy may be mediated through onion's ability to lower blood cholesterol levels and to reduce lipid peroxidation.     

Regulation of ion transport by pH and [HCO3-] in isolated gills of the crab Neohelice (Chasmagnathus) granulata

Posterior isolated gills of Neohelice (Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO3-] and pH. Elevating [HCO3-] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference (Vte), a measure of ion transport. The elevation in [HCO3-] also induced a switch from acid secretion (-43.7 +/- 22.5 microequiv.kg(-1).h(-1)) in controls to base secretion (84.7 +/- 14.4 microequiv.kg(-1).h(-1)). The HCO3(-)-induced Vte increase was inhibited by basolateral acetazolamide (200 micromol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The Vte response to HCO3(-) did not take place in Cl(-)-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in Vte, which was matched by a net increase in acid secretion of 67.8 +/- 18.4 microequiv kg(-1) h(-1). This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na+-free conditions, but it still took place in Cl(-)-free saline. Therefore, the cellular response to low pH is different from the HCO3(-)-stimulated response. We also report V-H+-ATPase- and Na+-K+-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na+/H+ exchangers and Na+-K+-ATPase and apical anion exchangers participate in the HCO3(-)-stimulated response, while CA, apical V-H+-ATPase and basolateral HCO3(-)-dependent cotransporters mediate the response to low pH.     

Relationships among salinity, egg size, embryonic development, and larval biomass in the estuarine crab Chasmagnathus granulata Dana, 1851

We studied interrelationships between initial egg size and biomass, duration of embryogenesis at different salinities, and initial larval biomass in an estuarine crab, Chasmagnathus granulata. Ovigerous females were maintained at three different salinities (15‰, 20‰ and 32‰); initial egg size (mean diameter), biomass (dry weight, carbon and nitrogen) as well as changes in egg size, embryonic development duration, and initial larval biomass were measured.

Initial egg size varied significantly among broods from different females maintained under identical environmental conditions. Eggs from females maintained at 15‰ had on average higher biomass and larger diameter. We hypothesise that this is a plastic response to salinity, which may have an adaptive value, i.e. it may increase the survivorship during postembryonic development. The degree of change in egg diameter during the embryonic development depended on salinity: eggs in a late developmental stage were at 15‰ significantly larger and had smaller increment than those incubated at higher salinities. Development duration was longer at 15‰, but this was significant only for the intermediate embryonic stages. Initial larval biomass depended on initial egg size and on biomass loss during embryogenesis. Larvae with high initial biomass originated either from those eggs that had, already from egg laying, a high initial biomass (reflecting individual variability under identical conditions), or from those developing at a high salinity (32‰), where embryonic biomass losses were generally minimum. Our results show that both individual variability in the provisioning of eggs with yolk and the salinity prevailing during the embryonic development are important factors causing variability in the initial larval biomass of C. granulata, and thus, in early larval survival and growth.