Euryhaline Crab Research Articles

Examination of role of the AMP-activated protein kinase (Ampk) signaling pathway during low salinity adaptation in the mud crab, Scylla paramamosain, with reference to glucolipid metabolism

It is known that the marine euryhaline crab, mud crab (Scylla paramamosain), is capable of adapting to low salinity conditions through an innate osmoregulatory system. However, the influence of nutritional conditions during low salinity adaptation has not been fully elucidated. In this study, transcriptome analysis was employed to examine whether low salinity affects the AMP-activated protein kinase (Ampk) signaling pathway, and thereby carbohydrate and lipid metabolism. Further analysis revealed that low salinity activated the expression of P-Ampk and promoted glycolysis and lipolysis, which enhances glycogen and lipid utilization. Nutritional deprivation confirmed that nutrients influenced the survival and osmotic pressure regulation of mud crab under to low salinity via the Ampk signaling pathway. During adaptation to low salinity, carbohydrates were the primary energy source in the initial 7 days, followed by lipids. Further investigation showed that pharmacological stimulation of mechanistic targets in the Ampk signaling pathway using metformin could promote the osmotic pressure regulation of mud crab, confirmed the pivotal role of this pathway in low salinity adaptation. Overall, the present study demonstrated that nutrition, metabolism and osmotic pressure regulation interactively contribute to salinity adaptation in mud crab. Specifically, low salinity induced remodeling of nutritional metabolism homeostasis and promoted glycolipid catabolism, thereby providing energy for osmotic pressure regulation in mud crabs.

The role of the antennal glands and gills in acid-base regulation and ammonia excretion of a marine osmoconforming brachyuran

The excretory mechanisms of stenohaline marine osmoconforming crabs are often compared to those of the more extensively characterized euryhaline osmoregulating crabs. These comparisons may have limitations, given that unlike euryhaline brachyurans the gills of stenohaline marine osmoconformers possess ion-leaky paracellular pathways and lack the capacity to undergo ultrastructural changes that can promote ion-transport processes in dilute media. Furthermore, the antennal glands of stenohaline marine osmoconformers are poorly characterized making it difficult to determine what role urinary processes play in excretion. In the presented study, ammonia excretory processes as well as related acid-base equivalent transport rates and mechanisms were investigated in the Dungeness crab, Metacarcinus magister – an economically valuable stenohaline marine osmoconforming crab. Isolated and perfused gills were found to predominantly eliminate ammonia through a microtubule network-dependent active NH4+ transport mechanism that is likely performed by cells lining the arterial pockets of the gill lamella where critical Na+/K+-ATPase detection was observed. The V-type H+-ATPase – a vital component to transbranchial ammonia excretion mechanisms of euryhaline crabs - was not found to contribute significantly to ammonia excretion; however, this may be due to the transporter's unexpected apical localization. Although unconnected to ammonia excretion rates, a membrane-bound isoform of carbonic anhydrase was localized to the apical and basolateral membranes of lamella suited for respiration. Urine was found to contain significantly less ammonia as well as carbonate species than the hemolymph, indicating that unlike those of some euryhaline crabs the antennal glands of the Dungeness crab reabsorb these molecules rather than eliminate them for excretion.

High dietary lipid level promotes low salinity adaptation in the marine euryhaline crab (Scylla paramamosain).

The physiological processes involved in adaptation to osmotic pressure in euryhaline crustaceans are highly energy demanding, but the effects of dietary lipids (fat) on low salinity adaptations have not been well evaluated. In the present study, a total of 120 mud crabs (Scylla paramamosain, BW=17.87±1.49g) were fed control and high-fat (HF) diets, at both medium salinity (23‰) and low salinity (4‰) for 6wk, and each treatment had 3 replicates with each replicate containing 10 crabs. The results indicated that a HF diet significantly mitigated the reduction in survival rate, percent weight gain and feed efficiency induced by low salinity (P<0.05). Low salinity lowered lipogenesis and activated lipolysis resulting in lipid depletion in the hepatopancreas of mud crabs (P<0.05). Thus, HF diets enhanced the process of lipolysis to supply more energy. In the gills, low salinity and the HF diet increased the levels of mitochondrial biogenesis markers, the activity of mitochondrial complexes, and the expression levels of genes related to energy metabolism (P<0.05). Consequently, the positive effects of the HF diet on energy metabolism in mud crabs at low salinity promoted osmotic pressure regulation. Specifically, significantly higher haemolymph osmotic pressure and inorganic ion content, as well as higher osmotic pressure regulatory enzyme activity in gills, and gene and protein expression levels of NaK-ATPase were observed in crabs fed the HF diet at low salinity (P<0.05). In summary, high dietary lipid levels improved energy provision to facilitate mitochondrial biogenesis, which increased ATP provision for osmotic pressure regulation of mud crabs. This study also illustrates the importance of dietary lipid nutrition supplementation for low salinity adaptations in mud crabs.

Effects of prior acclimation on the survival, feed intake and salinity preference of a freshwater-adapted euryhaline crab

Abstract In estuarine systems, salinity changes by freshwater inflows and tides affect the survival and behaviour of euryhaline species. Juvenile Chinese mitten crabs, Eriocheir sinensis, are considered to live in fresh water during their complex life history, whilst some are found in coastal wetlands. To understand how juveniles disperse in estuarine residence, we investigated their survival, feed intake and salinity preference after acclimating in three different water situations (i.e., fresh water, salt water and alternation between fresh water and salt water). We supposed that behavioural alternation and salinity preference would play an essential role in their acclimation. Results showed no significant difference in survival and feed intake amongst the three groups of juveniles () in acclimation trials. However, the feed intake of crabs in each group was declining after changing water every three days. In the salinity preference experiment, crabs from fresh water preferred 0 and 12, whereas those from salinity and alternation treatments showed no preference. The salinity preference seemed to be modified by prior acclimation as well, and juvenile crabs could therefore survive and disperse in the different salinities. Our study helps to explain the complex distribution of juveniles in estuaries and provides arguments for the feasibility of Chinese mitten crabs being reared in low salinity waters.

Comparative Transcriptome Analysis of Eriocheir sinensis from Wild Habitats in Han River, Korea.

Eriocheir sinensis is an euryhaline crab found from East Asia to Europe and North America. This species can live in freshwater and seawater due to the unique physiological characteristics of their life cycle, which allows them to adapt and inhabit different habitats in a wide range of environments. Despite the wealth of studies focusing on adaptation mechanism of E. sinensis to specific environmental factors, the adaptation mechanisms to wild habitats with coexisting environmental factors are not well understood. In this study, we conducted a transcriptome analysis to investigate gene expression differences related to habitat adaptation of E. sinensis from two wild habitats with different environmental factors in the Han River, Korea. A total of 138,261 unigenes were analyzed, of which 228 were analyzed as differentially expressed genes (DEGs) between the two wild habitats. Among 228 DEGs, 110 DEGs were annotated against databases; most DEGs were involved in energy metabolism, immunity, and osmoregulation. Moreover, DEG enrichment analysis showed that upregulated genes were related to biosynthesis, metabolism, and immunity in an habitat representing relatively high salinity whereas downregulated genes were related to ion transport and hypoxia response in habitats with relatively low salinity and dissolved oxygen. The present findings can serve as foundation for future E. sinensis culture or conservation approaches in natural conditions.

Environmental salinity and dietary lipid nutrition strategy: Effects on flesh quality of the marine euryhaline crab Scylla paramamosain

The quality of crustaceans’ flesh has direct impact on consumers’ purchase choices, with water environment and dietary nutrition being effective ways to regulate flesh quality. The aim of present study was to investigate the impacts of water salinity (low, 4 and medium, 23) and dietary lipid source (fish oil and soybean oil) on nutritional values, texture, taste and odor of flesh of mud crab. While water salinity had no significant influence on nutritional values of crab flesh, crabs fed soybean oil displayed significantly lower contents of amino acids and n-3 PUFAs in muscle. However, crabs reared at low salinity showed reduced flesh hardness, chewiness and gumminess likely related to altered myofiber structure, that impacted muscle texture. Furthermore, low salinity and dietary soybean oil weakened umami taste and aroma characteristics of crab flesh associated with decreased contents of free amino acids, flavor nucleotides, inorganic ions and odor active compounds in flesh.

Multi-omic approach provides insights into osmoregulation and osmoconformation of the crab Scylla paramamosain

Osmoregulation and osmoconformation are two mechanisms through which aquatic animals adapt to salinity fluctuations. The euryhaline crab Scylla paramamosain, being both an osmoconformer and osmoregulator, is an excellent model organism to investigate salinity adaptation mechanisms in brachyurans. In the present study, we used transcriptomic and proteomic approaches to investigate the response of S. paramamosain to salinity stress. Crabs were transferred from a salinity of 25 ppt to salinities of 5 ppt or 33 ppt for 6 h and 10 days. Data from both approaches revealed that exposure to 5 ppt resulted in upregulation of ion transport and energy metabolism associated genes. Notably, acclimation to low salinity was associated with early changes in gene expression for signal transduction and stress response. In contrast, exposure to 33 ppt resulted in upregulation of genes related to amino acid metabolism, and amino acid transport genes were upregulated only at the early stage of acclimation to this salinity. Our study reveals contrasting mechanisms underlying osmoregulation and osmoconformation within the salinity range of 5–33 ppt in the mud crab, and provides novel candidate genes for osmotic signal transduction, thereby providing insights on understanding the salinity adaptation mechanisms of brachyuran crabs.

Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition-dependent manner.

Na+ and Cl− are the most abundant dissolved ions in seawater, constituting ~ 85% of total ions. They significantly affect the osmolality of body fluids of marine invertebrates. Seawater also contains minor ions such as Mg2+, Ca2+, K+, and SO4 2‐ , but their effects on marine organisms are unclear. This study analyzed the effects of Mg2+, Ca2+, and K+ (ambient minor cations) on survival, hemolymph ionic composition, and gene expression in the gills of three euryhaline crabs: Helice tridens, Macrophthalmus japonicus, and Chiromantes dehaani. Ambient minor cations were required for survival of H. tridens and M. japonicus under isosmotic conditions with seawater. The ambient minor cations also affected the osmolality and ionic composition of hemolymph by regulating expressions of specific genes in the gills required for Na+ uptake, such as Na+/K+ ATPase, cytoplasmic carbonic anhydrase, and Na+/H+ exchanger. Administration of carbonic anhydrase and Na+/H+ exchanger inhibitors increased the survival rate even if ambient minor cations did not exist. In contrast, under hypo‐osmotic conditions, ambient minor cations had different effects on crabs, a lethal effect on M. japonicus, and an increase of the hemolymph K+ concentration in H. tridens and M. japonicus. It is thus concluded that the effects of ambient minor cations are osmolality‐dependent. In contrast, in C. dehaani, the hemolymph ionic composition and survival rate were hardly affected by ambient minor cations, probably reflecting the habitat of this species. These results strongly indicated that C. dehaani is less susceptive to ambient minor cations compared to H. tridens and M. japonicus.

Differential modulation after feeding in different salinities and response to abscisic acid (ABA) and extracellular Ca2+ of aminopeptidase N (APN) activity in the hepatopancreas of the intertidal euryhaline crab Neohelice granulata

Modulation of aminopeptidase N (APN) activity in the digestive tract by various factors would be important to adjust digestive and absorptive processes under different physiological and (or) environmental conditions. We studied the postprandial responses at different salinities and the effect of abscisic acid (ABA) and extracellular Ca2+ on APN activity in the hepatopancreas (the main site for nutrient digestion and absorption) of the model species Neohelice granulata (Dana, 1851). Enzyme activity was determined at different times (0, 24, 48, and 72 h) after feeding in crabs acclimated either to 35 psu (osmoconformation) or 10 psu (hyper-regulation). APN activity increased around 50% at 24 h after feeding at 35 psu, whereas no changes occurred at 10 psu. Enzyme activity was also assayed in the presence of ABA (1 × 10–4 mol·L–1) or extracellular Ca2+ (1 × 10–4 mol·L–1), showing increments of 60% and 56%, respectively. The results suggest a role of APN in postprandial adjustments and its modulation by different chemical messengers by direct effect on the hepatopancreas. Moreover, to our knowledge, this work is the first to show the effect of ABA on a digestive enzyme in the digestive tract of an animal.

Two sodium pumps in the hepatopancreas of the intertidal euryhaline crab Neohelice granulata: biochemical characteristics and differential modulation after feeding

No study has been done on the existence, biochemical characteristics, and modulation of K+-independent ouabain-insensitive Na+ ATPase activity (the second sodium pump) in the digestive tract of intertidal euryhaline crabs and moreover on the coexistence and modulation under distinct physiological and (or) environmental conditions of different sodium pumps. We determined the occurrence, characteristics, and responses at different times (0, 1, 24, 48, and 120 h) after feeding upon distinct salinities of Na+ ATPase activity and Na+/K+ ATPase in the hepatopancreas of Neohelice granulata (Dana, 1851), which is a model species. The stimulation by Na+ under total inhibition of Na+/K+ ATPase activity revealed the occurrence of Na+ ATPase activity that was totally inhibited by 2 mmol·L–1 furosemide, exhibits Michaelis–Menten kinetics for ATP (apparent Km = 0.52 ± 0.16 mmol·L–1), and highest activity at around pH 7.4. In crabs acclimated to 35 psu (osmoconforming conditions), Na+ ATPase activity was highly increased (about 15-fold) (532 ± 58 nmol Pi·mg protein−1·min−1) in the hepatopancreas 48 h after feeding. In 10 psu (hyper-regulating conditions), Na+ ATPase activity decreased in the hepatopancreas 24 h after feeding (7 ± 9 nmol Pi·mg protein−1·min−1) and recovered initial values after 48 h (24 ± 35 nmol Pi·mg protein−1·min−1). Unlike Na+ ATPase, Na+/K+ ATPase activity did not change after feeding at any salinity, suggesting the specific modulation of the second sodium pump and its role in postprandial adjustments in the hepatopancreas.

Insulin-like receptors and carbohydrate metabolism in gills of the euryhaline crab Neohelice granulata: Effects of osmotic stress

The present study determined the effect of osmotic stress on the insulin-like receptor binding characteristics and on glucose metabolism in the anterior (AG) and posterior (PG) gills of the crab Neohelice granulata. Bovine insulin increased the capacity of the PG cell membrane to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and the glucose uptake in the control crab group. The crabs were submitted to three periods of hyperosmotic (HR) and hyposmotic (HO) stress, for 24, 72 and 144 h, to investigate the insulin-like receptor phosphorylation capacity of gills. Acclimation to HO for 24 h or HR for 144 h of stress inhibited the effects of insulin in the PG, decreasing the capacity of insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) and decreasing the glucose uptake. Hyperosmotic stress for the same period of 144 h significantly affected 125I-insulin binding in the AG and PG. However, HO stress for 24 h significantly reduced 125I-insulin-specific uptake only in the PG. Therefore, osmotic stress induces alterations in the gill insulin-like receptors that decrease insulin binding in the PG. These findings indicate that osmotic stress induced a pattern of insulin resistance in the PG. The free-glucose concentration in the PG decreased during acclimation to 144 h of HR stress and 24 h of HO stress. This decrease in the cell free-glucose concentration was not accompanied by a significant change in hemolymph glucose levels. In AG from the control group, neither the capacity of bovine insulin to phosphorylate exogenous substrate poly (Glu:Tyr 4:1) nor the glucose uptake changed; however, genistein decreased tyrosine-kinase activity, confirming that this receptor belongs to the tyrosine-kinase family. Acclimation to HO (24 h) or HR (144 h) stress decreased tyrosine-kinase activity in the AG. This study provided new information on the mechanisms involved in the osmoregulation process in crustaceans, demonstrating for the first time in an estuarine crab that osmotic challenge inhibited insulin-like signaling and the effect of insulin on glucose uptake in the PG.

In silico analysis and effects of environmental salinity in the expression and activity of digestive α-amylase and trypsins from the euryhaline crabNeohelicegranulata

Studies on molecular characteristics and modulation of expression of α-amylase and trypsin in the hepatopancreas of intertidal euryhaline crabs are lacking. In this work, we cloned and studied by in silico approaches the characteristics of cDNA sequences for α-amylase and two trypsins isoforms, as well as the effect of environmental salinity, on gene expression and protein activities in the hepatopancreas of Neohelice granulata (Dana, 1851), which is a good invertebrate model species. The cDNA sequence of α-amylase is 1637 bp long, encoding 459 amino acid residues. Trypsin 1 and 2 are 689 and 1174 bp long, encoding 204 and 151 amino acid residues, respectively. Multiple sequence alignment of deduced protein sequences revealed the presence of conserved motifs found in other invertebrates. In crabs acclimated at 37 psu (hyporegulation), α-amylase mRNA level and total pancreatic amylase activity were higher than at 10 psu (hyperregulation) and 35 psu (osmoconformation). Trypsin 1 mRNA levels increased at 37 psu, while trypsin 2 levels decreased at 10 and 37 psu. Total trypsin activity was similar in all salinities. Our results showed a differential modulation of α-amylase and trypsin expression and total amylase activity by salinity acclimation, suggesting the occurrence of distinct mechanisms of regulation at different levels that could lead to digestive adjustments in relation to hyperregulation and (or) hyporegulation.

Carry-over effects modulated by salinity during the early ontogeny of the euryhaline crab Hemigrapsus crenulatus from the Southeastern Pacific coast: Development time and carbon and energy content of offspring

Hemigrapsus crenulatus is a key species of coastal and estuarine ecosystems in the Southeastern Pacific and New Zealand. Since the gravid females-and their embryos-develop under conditions of variable salinity, we propose that low external salinity will be met with an increase in energy expenditures in order to maintain osmoregulation; subsequently, the use of energy reserves for reproduction will be affected. In this study, we investigate in H. crenulatus whether 1) the biomass and energy content of embryos is influenced by salinity experienced during oogenesis and embryogenesis and 2) how variation in the biomass and energy content of embryos affects larval energetic condition at hatching. Here at low salinity (5PSU), egg-bearing females experienced massive and frequent egg losses, and therefore the development of their eggs during embryogenesis was not completed. In turn, at intermediate and high salinity (15 and 30PSU) embryogenesis was completed, egg development was successful, and larvae were obtained. Consistently, larvae hatched from eggs produced and incubated at high salinity (30PSU) were larger, had higher dry weight, and had increased carbon content and energy than larvae hatched from eggs produced at intermediate salinity (15PSU). From these results, it is seen that the size and biomass of early life stages of H. crenulatus can be affected by environmental salinity experienced during oogenesis and embryogenesis, and this variation can then directly affect the energetic condition of offspring at birth. Therefore, this study reveals a “cascade effect” modulated by salinity during the early ontogeny.

Biochemical and physiological effects of nickel in the euryhaline crab Neohelice granulata (Dana, 1851) acclimated to different salinities

The estuarine crab Neohelice granulata was maintained under control condition or exposed to sublethal concentrations of dissolved Ni (measured: 128 and 1010μg/L) for 96h at different salinities (2 and 30ppt). After metal exposure, whole-body oxygen consumption was measured and tissue (hemolymph, gills, hepatopancreas and muscle) samples were collected. Control crabs acclimated to 2ppt salinity showed lower hemolymph concentrations of Na+ (33%), Mg2+ (19%) and K+ (30%), as well as increased LPO levels in anterior gills (379%), posterior gills (457%) and hepatopancreas (35%) with respect to those acclimated to 30ppt salinity. In crabs acclimated to 2ppt salinity, Ni exposure increased whole-body oxygen consumption (75%), hemolymph K+ concentration (52%), hemolymph (135%) and hepatopancreas (62%) LDH activity. Also, it reduced hemolymph Cl− concentration (16%) and muscle LDH activity (33%). In crabs acclimated to 30ppt salinity, Ni exposure increased LDH activity in hemolymph (195%), hepatopancreas (126%) and muscle (53%), as well as hemolymph osmolality (10%), Cl− (26%) and Ca2+ (20%) concentration. It also reduced hepatopancreas lipid peroxidation (20%) and hemolymph Mg2+ (29%) and K+ (31%) concentration. These findings indicate that N. granulata is hyper-osmoregulating in 2ppt salinity and hypo-regulating in 30ppt salinity, showing adjustments of hemolymph ionic composition and metabolic rates, with consequent higher oxidative damage to lipids in low salinity (2ppt). Ni effects are associated with metabolic (aerobic and anaerobic) disturbances in crabs acclimated to 2ppt salinity, while osmotic and ionoregulatory disturbances were more evident in crabs acclimated to 30ppt salinity.

Glucose homeostasis in the euryhaline crab Cytograpsus angulatus: Effects of the salinity in the amylase, maltase and sucrase activities in the hepatopancreas and in the carbohydrate reserves in different tissues.

We studied the existence, biochemical characteristics and response to different environmental salinities of amylase, maltase and sucrase activity in the intertidal euryhaline crab Cyrtograpsus angulatus (Dana, 1852) along with the response to distinct salinities of glycogen and free glucose content in storage organs. Amylase, maltase and sucrase activities were kept over a broad range of pH and temperature and exhibited Michaelis-Menten kinetics. Zymography showed the existence of two amylase forms in crabs exposed to 35 (osmoconformation) and low (6-10psu; hyper-regulation) or high (40psu) (hypo-regulation) salinities. Carbohydrases activity in the hepatopancreas and glycemia were not affected in crab exposed to different environmental salinities. In 6 and 40psu, the glycogen content in anterior gills was lower than in 35psu. In 6, 10 and 40psu, glycogen concentration in hepatopancreas, muscle and posterior gills were similar to that in 35psu. Free glucose concentration in chela muscle was higher in 6 and 40psu than in 35psu. The existence and biochemical characteristics of carbohydrases activity and the adjustments in concentration of glycogen in anterior gills and free glucose in chela muscle suggests the ability to perform complete hydrolysis of glycogenic substrates and to keep glucose homeostasis in relation to acclimation to different salinity conditions.

The euryhaline crabUca tangerishowed metabolic differences to sex and environmental salinity

This study constitutes a first attempt to investigate sex differences in osmoregulatory capacity and metabolic responses in relation to hyper- and hypo-osmoregulation in the intertidal euryhaline crabUca tangeri. Adult male and female specimens from Cadiz Bay, Spain (36°23′–37′N 6°8′–15′W), were acclimated to three different environmental salinities (12, 33 and 55 psu) during 7 days, and several parameters were assessed in haemolymph (osmolality, glucose, amino acids, triglycerides and lactate) as well as in metabolic key organs (hepatopancreas, anterior and posterior gills: glycogen, free glucose, amino acids and triglycerides). Specimens from both sex exhibited high and similar hyper- and hypo-osmoregulatory capacities. However, metabolite levels were differentially affected upon acclimation to low and high salinity in several metabolic organs and haemolymph of male and females: (i) glycogen in gills, (ii) free glucose in gills and hepatopancreas, (iii) amino acids in hepatopancreas, (iv) triglycerides in haemolymph, hepatopancreas and posterior gills, and (v) lactate in haemolymph. The results suggest the occurrence of differential metabolic adjustments upon hyper- and hypo-osmoregulation related to sex in the intertidal euryhaline crabU. tangeri.

Trypsin and N-aminopeptidase (APN) activities in the hepatopancreas of an intertidal euryhaline crab: Biochemical characteristics and differential modulation by histamine and salinity

No studies are available about biochemical characteristics and modulation (i.e. by endogenous and/or environmental cues) of trypsin (a key digestive endoprotease) in hepatopancreas of intertidal euryhaline crabs neither on the possible concomitant modulation of key ectoproteases such as aminopeptidase-N (APN) involved in final steps of protein digestion. Furthermore, nothing is still known in decapods crustaceans about the role of histamine as primary chemical messenger for modulation of main components of digestive process (i.e. proteases). We determined biochemical characteristics and investigated the effect of histamine injections; of histamine in vitro and of acclimation of individuals to low and high salinity on trypsin and aminopeptidase-N (APN) activities in the hepatopancreas of the euryhaline crab Cyrtograpsus angulatus (Dana 1851). Trypsin activity was maximal at pH7.4 and at 45°C. APN activity increased from pH6.6 to 7.6–9.0 and was maintained high at 37–45°C. Both activities exhibited Michaelis-Menten kinetics (apparent Km: trypsin=0.36mM; APN=0.07mM). The injection of 10−4M histamine decreased trypsin activity (about 40%) in hepatopancreas while did not affect APN activity. Similarly, in vitro 10-4M histamine decreased trypsin activity (about 52%) in hepatopancreas but not APN activity. Trypsin activity in the hepatopancreas was not affected by acclimation of crabs to low (10psu) or high (40psu) environmental salinity while APN activity was increased (about 200%) in 10psu. The results show the differential modulation of trypsin and APN by distinct cues and point to histamine as modulator of intracellular trypsin by direct action on the hepatopancreas.

Low salinity enhances NI-mediated oxidative stress and sub-lethal toxicity to the green shore crab (Carcinus maenas)

Nickel (Ni) is a metal of environmental concern, known to cause toxicity to freshwater organisms by impairing ionoregulation and/or respiratory gas exchange, and by inducing oxidative stress. However, little is known regarding how nickel toxicity is influenced by salinity. In the current study we investigated the salinity-dependence and mechanisms of sub-lethal Ni toxicity in a euryhaline crab (Carcinus maenas). Crabs were acclimated to three experimental salinities – 20, 60 and 100% seawater (SW) – and exposed to 3mg/L Ni for 24h or 96h. Tissues were dissected for analysis of Ni accumulation, gills were taken for oxidative stress analysis (catalase activity and protein carbonyl content), haemolymph ions were analysed for ionoregulatory disturbance, and oxygen consumption was determined in exercised crabs after 96h of Ni exposure. Total Ni accumulation was strongly dependant on salinity, with crabs from 20% SW displaying the highest tissue Ni burdens after both 24 and 96-h exposures. After 96h of exposure, the highest accumulation of Ni occurred in the posterior (ionoregulatory) gills at the lowest salinity, 20% SW. Posterior gill 8 exhibited elevated protein carbonyl levels and decreased catalase activity after Ni exposure, but only in 20% SW. Similarly, decreased levels of haemolymph Mg and K and an increased level of Ca were recorded but only in crabs exposed to Ni for 96h in 20% SW. Oxygen consumption after exercise was also inhibited in crabs exposed to Ni in 20% SW. These data show for the first time the simultaneous presence of all three modes of sub-lethal Ni toxicity in exposed animals, and indicate a strong salinity dependence of sub-lethal Ni toxicity to the euryhaline crab, C. maenas, a pattern that corresponded to tissue Ni accumulation.

Digestive flexibility in a euryhaline crab from a SW Atlantic coastal lagoon: alkaline phosphatase activity sensitive to salinity in the hepatopancreas

We studied biochemical characteristics and the response to low salinity at short and long-term after feeding of alkaline phosphatase (AP) activity in hepatopancreas of the osmoregulator crabNeohelice granulatafrom Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina) (37°32′–37°45′S 57°19′–57°26′W). The hepatopancreas exhibited a levamisole-insensitive and a levamisole-sensitive AP activity with distinct characteristics. Levamisole-insensitive activity was similar within the range of pH 7.4–9.0 and exhibited a Michaelis–Menten kinetics. Levamisole-sensitive AP activity appeared to be maximal at pH 8.5 and appeared to exhibit an allosteric kinetics. In crabs acclimated to 10 psu (hyper-regulation conditions) levamisole-insensitive and levamisole-sensitive AP activity increased (about 16-fold) over time from short term (2–4 h) to long term (120 h) after feeding while no changes occurred in crabs acclimated to 35 psu (osmoconforming conditions). The changes of AP activity along with the higher values at 120 h after feeding in 10 psu compared with those in 35 psu, and the concomitant changes in proteolytic activity, suggest a role of AP in digestive and metabolic adjustments at the biochemical level upon hyper-regulatory conditions.

Gill-specific (Na(+), K(+))-ATPase activity and α-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura).

We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.