Free amino acid pools as effectors of osmostic adjustment in different tissues of the freshwater shrimp macrobrachium olfersii (crustacea,decapoda) during long-term salinity acclimation

To examine osmotic regulation during long-term acclimation to a hyperosmotic medium, hemolymph osmolality, [Na⁺] and total protein, tissue hydration, and free amino acid (FAA) pools in abdominal muscle, gills, central nervous tissue and hemolymph were quantified in the diadromous freshwater (FW) shrimp, Macrobrachium olfersii, during direct exposure to 21‰S seawater over a 20-day period. Hemolymph osmolality and [Na⁺] reach stable maxima within 24?h while total protein is unchanged. Muscle and nerve tissues rapidly lose water while gills hydrate; all tissues attain maximum hydration (+5%) by 5 days, declining to FW values except for gills. Total FAA are highest in muscle, reach a maximum by 2 days (+64%), declining to FW values. Gill FAA increase by 110% after 24?h, diminishing to FW values. Nerve FAA increase 187% within 24?h, and remain elevated. Hemolymph FAA decrease (?75%) after 24?h, stabilizing well below the FW concentration. During acclimation, muscle glycine (+247%), gill taurine (+253%) and proline (+150%), and nerve proline (+426%), glycine (+415%) and alanine (+139%) increase, while hemolymph leucine (?70%) decreases. Total FAA pools contribute 10–20% to intracellular (22–70?mmol/kg) and 0.5–2.4% to hemolymph (3–7?mOsm/kg) osmolalities during direct acclimation from FW. These data emphasize the modest participation of FAA pools in intracellular osmotic regulation during physiological adaptation by M. olfersii to osmotic challenge, accentuating the role of anisosmotic extracellular regulation, suggesting that, during the invasion of freshwater by the Crustacea, dependence on intracellular adjustment employing FAA as osmotic effectors, has become progressively reduced.     

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.     

Adaptive shifts in osmoregulatory strategy and the invasion of freshwater by brachyuran crabs: evidence from Dilocarcinus pagei (Trichodactylidae)

To evaluate putative adaptive changes underpinning the invasion of freshwater by the Brachyura, this investigation examines anisosmotic extra and isosmotic intracellular osmoregulatory capabilities in Dilocarcinus pagei, a neotropical, hololimnetic crab, including its embryonic and juvenile phases. All ontogenetic stages show a remarkable ability to survive a high salinity medium (25 per thousand, 750 mOsm/kg H2O, 350 mm Na+, 400 mM Cl-). Adults hyper-regulate hemolymph osmolality up to isosmoticity at 744 mOsm kg/H2O (24 per thousand), [Na+] and [Cl-] becoming isoionic at 449 (22 per thousand) and 256 mM (16 per thousand), respectively. Hemolymph (420+/-39 mOsm/kg H2O) and urine (384+/-44 mOsm/kg H2O) are isosmotic in adults held in freshwater, and after 5-days exposure to 25 per thousand (787+/-9 mOsm/kg H2O and 777+/-43 mOs/kg H2O, respectively); D. pagei does not produce dilute urine. Total free amino acid (FAA) concentrations in embryos (14.9+/-1.2), juveniles (32.8+/-0.1) and adult muscle (10.9+/-2.1 mmol/kg wet weight) in freshwater are 30-fold less than in brackish/marine Crustacea, suggesting that FAA constitute a useful parameter to evaluate adaptation to freshwater. On acclimation to 25 per thousand, total FAA increase by approximately 100% in embryos and in adult muscle and nerve tissue and hemolymph, owing to large increases in proline, arginine and/or alanine. However, effective FAA contribution to intracellular osmolality increases only in embryos, from 3 to 4.5%. These findings suggest that gill-based, anisosmotic extracellular regulation has supplanted isosmotic intracellular regulatory mechanisms during the conquest of freshwater by the Brachyura, and indicate that D. pagei may be an old, well-adapted inhabitant of this biotope.     

Intra- and extracellular osmotic regulation in the hololimnetic Caridea and Anomura: a phylogenetic perspective on the conquest of fresh water by the decapod Crustacea

We investigate extra- and intracellular osmoregulatory capability in two species of hololimnetic Caridea and Anomura: Macrobrachium brasiliense, a palaemonid shrimp, and Aegla franca, an aeglid anomuran, both restricted to continental waters. We also appraise the sharing of physiological characteristics by the hololimnetic Decapoda, and their origins and role in the conquest of fresh water. Both species survive salinity exposure well. While overall hyperosmoregulatory capability is weak in A. franca and moderate in M. brasiliense, both species strongly hyporegulate hemolymph [Cl⁻] but not osmolality. Muscle total free amino acids (FAA) increase slowly but markedly in response to the rapid rise in hemolymph osmolality consequent to hyperosmotic challenge: 3.5-fold in A. franca and 1.9-fold in M. brasiliense. Glycine, taurine, arginine, alanine and proline constitute ≈85% of muscle FAA pools in fresh water; taurine, arginine, alanine each contribute ≈22% in A. franca, while glycine predominates (70%) in M. brasiliense. These FAA also show the greatest increases on salinity challenge. Muscle FAA titers correlate strongly (R = 0.82) with hemolymph osmolalities across the main decapod sub/infraorders, revealing that marine species with high hemolymph osmolalities achieve isosmoticity of the intra- and extracellular fluids partly through elevated intracellular FAA concentrations; freshwater species show low hemolymph osmolalities and exhibit reduced intracellular FAA titers, consistent with isosmoticity at a far lower external osmolality. Given the decapod phylogeny adopted here and their multiple, independent invasions of fresh water, particularly by the Caridea and Anomura, our findings suggest that homoplastic strategies underlie osmotic and ionic homeostasis in the extant freshwater Decapoda.     

Triggering of cryoprotectant synthesis in the woolly bear caterpillar (Pyrrharctia isabella Lepidoptera: Arctiidae)

Isabella tiger moths (Pyrrharctia isabella) overwinter as caterpillars (i.e., woolly bears) that can survive freezing at moderate subzero temperatures. We observed an increase in hemolymph osmolality for field-collected woolly bears during October (325 +/- 47 to 445 +/- 27 mOsmol/liter) and tested the influence of temperature and moisture levels on cryoprotectant production. Laboratory acclimation was done at 5 degrees C in moist conditions and at 25 degrees C acclimation in both dry and moist conditions. Body water contents were diminished by dehydration at 25 degrees C for 4 days (57 +/- 4%). Caterpillars collected in early October did not alter their hemolymph osmolality during cold acclimation, but caterpillars increased by 45% (to 647 +/- 90 mOsmol/liter) after 4 days at 5 degrees C following their collection in late October. Hemolymph composition was markedly changed in caterpillars experiencing dehydration at 25 degrees C (1042 +/- 200 mOsmol/liter; 507 +/- 225 mmol glycerol/liter), whereas caterpillars showed no change in their hemolymph composition when kept moist at 25 degrees C. Our experiments reveal that both dehydration and cold acclimation rapidly induce cryoprotectant synthesis in P. isabella caterpillars. J. Exp. Zool. 286:367-371, 2000.     

Respiratory and osmoregulatory responses of the hermit crab, Clibanarius vittatus (Bosc), to salinity changes

Intertidal hermit crabs were stepwise acclimated to 10, 20, and 30‰ salinity (S) and 21 ± 1 °C. Hemolymph osmolality, sodium, chloride, and magnesium were isosmotic (isoionic) to ambient sea water at 30‰ and hyperosmotic (hyperionic) at 20 and 10‰ S, while hemolymph potassium was significantly hyperionic in all acclimation salinities. Total body water did not differ significantly at any acclimation salinity. Oxygen uptake rates were higher in summer-than winter-adapted crabs. No salinity effect on oxygen consumption occurred in winter-adapted individuals. Summer-adapted, 30‰ acclimated crabs had a significantly lower oxygen consumption rate than those acclimated 10 and 20‰ S. Crabs exposed to 30 10 30‰ and 10 30 10‰ semidiurnal (12 h) and diurnal (24.8 h) fluctuating salinity regimes showed variable osmoregulatory and respiratory responses. Hemolymph osmolality followed the osmolality of the fluctuating ambient sea water in all cases, but was regulated hyperosmotically. Hemolymph sodium, chloride, and magnesium concentrations were similar to hemolymph osmolality changes. Sodium levels fluctuated the least. Hemolymph potassium was regulated hyperionically during all fluctuation patters, but corresponded to sea water potassium only under diurnal conditions. The osmoregulatory ability of Clibanarius vittatus (Bosc) resembles that reported for several euryhaline brachyuran species. The time course of normalized oxygen consumption rate changed inversely with salinity under semidiurnal and diurnal 10 30 10‰ S fluctuations. Patterns of 30 10 30‰ S cycles had no effect on oxygen consumption rate time course changes. The average hourly oxygen consumption rates during both semidiurnal fluctuations were significantly lower than respective control rates, but no statistical difference was observed under diurnal conditions.     

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.     

Effects of eyestalk ablation on carbonic anhydrase activity in the euryhaline blue crab Callinectes sapidus: neuroendocrine control of enzyme expression

Carbonic anhydrase (CA) activity in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to acute low-salinity transfer and treatment with eyestalk ablation (ESA) in an attempt to elucidate potential regulatory mechanisms of salinity-mediated CA induction. ESA alone resulted in an approximate doubling of CA activity in the posterior, ion-transporting gills of crabs acclimated to 35 ppt. Transfer of intact crabs to 28 ppt, a salinity at which the blue crab is still an osmotic and ionic conformer, had no effect on CA activity, but treatment with ESA prior to transfer resulted in a 5-fold increase. Hemolymph osmolality was unaffected by ESA. There was a 7-fold induction of CA activity in posterior gills of intact crabs transferred from 35 to 15 ppt, and this was potentiated by about 100% by ESA. Hemolymph osmolality was slightly elevated in the ESA-treated crabs. CA activity in anterior gills did not increase in response to any treatment. Hemolymph concentrations of methyl farnesoate (MF) were measured for all experimental animals. MF concentrations were undetectable in all intact crabs, regardless of salinity. Treatment with ESA resulted in elevated levels of hemolymph MF, but these levels were still relatively low and unrelated to salinity. These results suggest that CA induction is under the control of a regulatory substance located in the eyestalk. This substance appears to be a CA repressor, keeping CA expression at low levels in the gills of crabs acclimated to high salinity. Exposure to low salinity, or treatment with ESA, removes the effects of this putative repressor and allows CA induction to occur.     

Ionic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha

Dreissena polymorpha, an invasive freshwater bivalve, displays physiological characteristics that reflect its ancestry in brackish water, yet it has limited ability to withstand modest increases in salinity. We examined changes in hemolymph ion concentrations and acid-base variables in mussels transferred to and incubated in 10% artificial seawater (ASW) for 7 days and then returned to pondwater (PW) for a further 7 days. Hemolymph was sampled (10 animals per sample period) every 4 h for the first 24-h incubation and at 72 h and 168 h for both the transfer to 10% ASW and the transfer back to PW. The initial response to transfer to 10% ASW was a rapid attainment of an apparent isoosmotic steady state, with most hemolymph ion concentrations rising and attaining steady state within 12 h. Hemolymph magnesium rose more slowly, and hemolymph calcium declined despite an increase in its concentration in the bathing medium. Hemolymph pH rose significantly during the first 24 h, from 7.96 to 8.25, as a result of increases in bicarbonate; pH subsequently returned to normal through increases in PCO2. When animals were returned to PW after 7 days' incubation in ASW, the response of the major hemolymph ions was largely the reverse of that effected by the transfer to ASW. Hemolymph pH was not altered significantly until after 72 h in PW, when declines in bicarbonate lowered the pH to 7.73. Strong ion difference (SID) was related significantly to hemolymph pH. Hemolymph calcium and magnesium showed a reciprocal relationship throughout both transfer and incubation. Solubility interactions between sulfate and calcium and magnesium may be important in determining calcium availability in solution. The Na/K ratio in hemolymph was maintained within relatively narrow bounds throughout the procedure and may contribute to the mussels' ability to volume-regulate during an osmotic challenge. Overall, the responses of D. polymorpha to modest changes in salinity were largely the result of passive processes.     

Osmotic and ionic hemolymph concentrations of bathyal and abyssal amphipods of Lake Baikal (Siberia) in relation to water depth

We studied the adaptive variations of the hemolymph concentrations in relation to water depth and pressure using deep-dwelling amphipods from Lake Baikal. Hemolymph osmolality was determined in six bathyal and abyssal species immediately after capture when values come closest to the habitat concentrations. In three species, hemolymph osmolalities correlated positively with depth of capture. Prevalent ions in the hemolymph are sodium and chloride. Lactate, our indicator for capture stress, was highest after trawling (2–6 mM) and lowest after retrieval from cages (0–0.6 mM). Acclimation to different pressure was studied by exposing the specimens to different water depths over several days. Hemolymph concentrations did not change after acclimation to surface pressure in the sublittoral Acanthogammarus albus, a native also to shallow water, but decreased by 30–80 mosmol/kg H₂O in the bathyal and abyssal species Acanthogammarus grewingki, Acanthogammarus reicherti, and Parapallasea lagowskii. Similarly, hemolymph osmolality decreased in A. reicherti and P. lagowskii originating from deep water, when acclimated to reduced water depth, and, in A. reicherti hemolymph osmolality reached its original high value when returned to the depth of capture. Higher hemolymph osmolalities and NaCl concentrations, demonstrated here for the first time, may provide selective advantages to abyssal species. Accepted: 24 August 2000     

Effect of hypo- and hypersaline conditions on osmolality and Na+/K+-ATPase activity in juvenile shrimp (Litopenaeus vannamei) fed low- and high-HUFA diets

Fatty acid composition of cellular membranes can modify permeability and can modulate the activity of Na(+)/K(+)-ATPase. Although highly unsaturated fatty acids (HUFA) improve survival and osmoregulatory capacity to low salinities in penaeid shrimp, the possible mechanisms have not been established. For this purpose the influence of HUFA supplementation in diet (2.9 vs. 34% HUFA proportion to total fatty acids) on osmoregulatory responses of juvenile Litopenaeus vannamei submitted to an acute (15 h) or chronic exposure (21 days), to low (5 g L(-1)) and high salinities (50 g L(-1)) was analyzed. Shrimp fed the high-HUFA diet, had higher concentration of main HUFA (20:5n-3 and 22:6n-3) in polar lipids of gills. Osmotic pressure in hemolymph was significantly affected by salinity in acute (640, 751, 847 mOsm/kg for 5, 30 and 50 g L(-1), respectively), and chronic exposure (645, 713, 814 mOsm/kg), but variations between them were small compared to environmental salinity (206, 832, 1547 mOsm/kg), indicating that osmoregulation was achieved in a matter of hours. An increase in Na(+)/K(+)-ATPase activity was observed only after a chronic exposure to low salinity. Free amino acids (FAA), mainly alanine and arginine, were higher at 30 (control) and 50 g L(-1) in accordance to their role as organic osmolites. Neither osmotic pressure, Na(+)/K(+)-ATPase activity, nor FAA was affected by HUFA supplementation. However, higher water content in gills of shrimp exposed to low salinities was counteracted by increased HUFA content, which could be a result of changes in water permeability of gills. The osmoregulatory capacity of penaeid shrimp to low and high salinities was achieved within 15 h of acclimation and did not depend on HUFA supplementation in the diet.     

Effects of photoperiod on gluconeogenic activity and total lipid concentration in organs of crabs, Neohelice granulata, challenged by salinity changes

Abstract. This study assessed the effects of long (LD) or short (SD) days on the conversion of [¹⁴C]-glycerol to [¹⁴C]-glucose and total lipid concentration in organs of the crab Neohelice granulata challenged by a change in external salinity. In the 20‰-acclimated crabs, no difference was found in the concentration of total lipids in the muscle, hepatopancreas, gills, or hemolymph between crabs acclimated to SD or LD. In SD crabs, the total lipid levels in the anterior and posterior gills did not decrease during an osmotic challenge. Only in the posterior gills did the total lipid levels decrease during acclimation to the 34‰ medium in LD animals. The total lipid concentration in the hemolymph decreased after 1 d of osmotic stress in SD, and increased in the hepatopancreas. In LD crabs, the lipid contents decreased gradually in muscle, and in the hepatopancreas on day 3 after transfer to 34‰ medium. In 20‰-acclimated crabs, the gluconeogenesis activity in both sets of gills was higher in LD than in SD animals. The gluconeogenesis capacity decreased in both sets of gills on the first day of osmotic challenge in SD, and in the posterior gills on the third day in LD crabs. These results suggest that in organs of N. granulata , photoperiod affects the metabolic adjustments to an osmotic challenge.     

Patterns of hemolymph osmoregulation in three desert arthropods

Summary Hemolymph osmoregulation was examined in three species of desert arthropods: a tenebrionid beetle,Eleodes hispilabris, a vejovid scorpion,Paruroctonus aquilonalis, and a spirostreptid millipede,Orthoporus ornatus. During desiccation, beetles regulated hemolymph osmolality and scorpions tolerated increasing osmolality. Millipedes displayed both osmotic regulation and tolerance patterns depending on sex and on duration of desiccation. Rehydration after desiccation depressed blood osmolality of male scorpions and beetles below levels found for freshly collected specimens. This was not the case in millipedes. Seasonal osmolality changes were studied and recorded among field-collected scorpions and beetles. Patterns of regulation and tolerance of hemolymph osmolality appear to vary among different kinds of desert arthropods.     

The physiological response of larval <Emphasis Type="Italic">Chironomus riparius</Emphasis> (Meigen) to abrupt brackish water exposure

The physiological response of larval Chironomus riparius was examined following direct transfer from freshwater (FW) to brackish water (BW; 20% seawater). Endpoints of hydromineral status (hemolymph Na⁺, Cl⁻, and K⁺ levels, hemolymph pH, body water content, and whole body Na⁺/K⁺-ATPase and V-type H⁺-ATPase activity) were examined 1, 3, 5, 12 and 24 h following BW transfer. Larvae transferred from FW to FW served as a control. Hemolymph Na⁺ and Cl⁻ levels increased following BW transfer. Hemolymph pH was initially regulated, but significantly decreased after 24 h in BW. Changes in hemolymph ions were not caused by osmotic loss of water from the hemolymph, since larvae tightly regulated total body moisture content. Furthermore, salinity did not affect hemolymph K⁺. When larvae were transferred to BW, Na⁺/K⁺-ATPase (NKA) activity did not significantly alter relative to FW control animals. In contrast, V-type H⁺-ATPase (VA) activity in C. riparius significantly decreased in BW. In FW-reared C. riparius, whole body NKA and VA activities were equivalent. However, in the isolated gut with intact Malpighian tubules of FW-reared C. riparius, VA activity was significantly greater than whole body while NKA activity was equivalent. This suggested that gut and/or Malpighian tubule VA activity contributes significantly to whole body VA activity and that a decline in whole body VA activity in BW may be closely linked to alterations in the physiology of gut and Malpighian tubule tissue. Taken together, data indicate that VA is important for ion uptake in FW and that the NKA does not play a major role in regulating ion homeostasis when larvae are acutely exposed to BW.     

Temporal changes in liver carbohydrate metabolism associated with seawater transfer in Oreochromis mossambicus

The metabolic aspects of ionic and osmotic regulation in fish are not well understood. The objective of this study was to examine changes in carbohydrate metabolism during seawater (SW) acclimation in the euryhaline tilapia (Oreochromis mossambicus). Hepatic activities of three key enzymes of the intermediary metabolism, phosphofructokinase, glycogen phosphorylase and glucose 6-phosphate dehydrogenase, together with glycogen content and plasma glucose concentration were measured at 0, 0.5, 1, 2, 3, 6, 12, 24, 48 and 96 h after the direct transfer of tilapia from fresh water (FW) to 70% SW. Plasma growth hormone, prolactin₁₇₇ and prolactin₁₈₈, Na⁺ and Cl⁻ concentrations were also measured. Plasma Na⁺ and Cl⁻ levels were highest at 12 h, but returned to FW levels at 24 h after transfer, suggesting the tilapia were able to osmoregulate within 24 h after transfer. Plasma glucose levels were significantly higher in 70% SW than in FW during the course of acclimation, especially in the early stages. Hepatic enzyme activities and glycogen content did not change significantly during the acclimation period. Our results suggest the possibility that glucose is an important energy source for osmoregulation during the acclimation to hyperosmotic environments in O. mossambicus.     

Time-course changes in the expression of Na, K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia (Oreochromis mossambicus) during freshwater acclimation

Changes in expression of Na, K-ATPase (NKA) and morphometry of mitochondrion-rich (MR) cells in gills of tilapia were investigated on a 96-hr time course following transfer from seawater (SW) to fresh water (FW). A transient decline in plasma osmolality and Na+, Cl- concentrations occurred from 3 hrs onward. Gills responded to FW transfer by decreasing NKA activity as early as 3 hrs from transfer. This response was followed by a significant decrease in the NKA isoform alpha1-mRNA abundance, which was detected by real-time PCR at 6 hrs post transfer. Next, a decrease of alpha1-protein amounts were observed from 6 hrs until 24 hrs post transfer. Additionally, during the time course of FW transfer, modifications in number and size of subtypes of gill MR cells were observed although no significant difference was found in densities of all subtypes of MR cells. These modifications were found as early as 3 hrs, evident at 6 hrs (exhibition of 3 subtypes of MR cells), and mostly completed by 24 hrs post transfer. Such rapid responses (in 3 hrs) as concurrent changes in branchial NKA expression and modifications of MR cell subtypes are thought to improve the osmoregulatory capacity of tilapia in acclimation from hypertonic SW to hypotonic FW.     

Crayfish freshwater adaptation starts in eggs: ontogeny of osmoregulation in embryos of Astacus leptodactylus

Osmoregulation was studied throughout the embryonic development of Astacus leptodactylus. Egg-carrying females were held in freshwater (FW) and in three dilute seawater media (200, 400, 600 mosm kg(-1), 6.8, 13.6, 20.4 per thousand salinity). In FW, changes in peri-embryonic fluid (PEF) and (when available) embryonic hemolymph osmolality were followed from newly-laid eggs to hatching (for an embryonic eye index, EI, of 430-450 microm) and in first-stage juveniles. The PEF and/or hemolymph osmolality remained stable at about 360-380 mosm kg(-1) from early to late (EI 410 microm) embryos; it decreased prior to hatching (EI 420 microm) and in newly-hatched juveniles, down to 290 mosm kg(-1). Artificial opening and removal of the egg membranes, followed by direct exposure to FW, demonstrated that the ability to hyper-osmoregulate, and consequently to survive, in FW appears in embryos with EI > or = 410 microm, i.e., only a few hours or days before hatching. Following a transfer to the dilute seawater media, the PEF/hemolymph osmolality increased slowly over 18-20 days and became isosmotic with the external media at 13.6 and 20.4 per thousand. The embryos died at EI 380-395 microm in these media, and only at 6.8 per thousand was the development completed until successful hatch. These results demonstrate that (1) the embryos become able to osmoregulate in FW shortly before hatching, (2) the embryos are osmo-protected in the eggs during their development, (3) embryonic development and hatching are possible up to a salinity of 7 per thousand. These results are discussed in relation to freshwater adaptation of crayfish.     

Salinity-mediated carbonic anhydrase induction in the gills of the euryhaline green crab, Carcinus maenas

The euryhaline green crab, Carcinus maenas, is a relatively strong osmotic and ionic regulator, being able to maintain its hemolymph osmolality as much as 300 mOsm higher than that in the medium when the crab is acclimated to low salinity. It makes the transition from osmoconformity to osmoregulation at a critical salinity of 26 ppt, and new acclimated concentrations of hemolymph osmotic and ionic constituents are reached within 12 h after transfer to low salinity. One of the central features of this transition is an 8-fold induction of the enzyme carbonic anhydrase (CA) in the gills. This induction occurs primarily in the cytoplasmic pool of CA in the posterior, ion-transporting gills, although the membrane-associated fraction of CA also shows some induction in response to low salinity. Inhibition of branchial CA activity with acetazolamide (Az) has no effect in crabs acclimated to 32 ppt but causes a depression in hemolymph osmotic and ionic concentrations in crabs acclimated to 10 ppt. The salinity-sensitive nature of the cytoplasmic CA pool and the sensitivity of hemolymph osmotic/ionic regulation to Az confirm the enzyme's role in ion transport and regulation in this species. CA induction is a result of gene activation, as evidenced by an increase in CA mRNA at 24 h after transfer to low salinity and an increase in protein-specific CA activity immediately following at 48 h post-transfer. CA gene expression appears to be under inhibitory control by an as-yet unidentified repressor substance found in the major endocrine complex of the crab, the eyestalk.     

Physiological responses to emersion in the intertidal green crab,Carcinus maenas (L.)

The green shore crab, Carcinus maenas, undergoes on average 6?h periods of emersion during each low-tide cycle during the summer months. Under those conditions, the crab is cut off from its normal water environment and is exposed to potential stress from a suite of environmental and physiological changes: dehydration, compromised gas exchange and resultant internal hypoxia and hypercapnia, thermal stress, and ammonia toxicity. This study examined the comprehensive responses of the green crab in water and to a 6?h emersion period laboratory simulation of a tidal cycle followed by a 1?h re-immersion period, measuring indicators of dehydration, hemolymph osmolality, oxygen uptake, hemolymph acid–base status, heart and ventilatory rate, and hemolymph ammonia and ammonia excretion. Green crabs showed physiological responses of varying magnitude to 6?h of emersion. Individuals were found in the field exclusively under rocks and large clumps of seaweed where temperatures were approximately half those of exposed surfaces and relative humidity was about twice as high as ambient air. During emersion, crabs lost less than 5% of their wet weight, and hemolymph osmolality did not increase significantly. Oxygen uptake continued in air at about 50% of the control, aquatic values; and the gills continued to be ventilated by the scaphognathite, albeit at lower rates. Hemolymph lactate concentrations increased, indicating a shift to a greater reliance on anaerobic metabolism to support energetic needs. A slight acidosis developed in the hemolymph after 1?h of emersion, but it did not increase thereafter. Ammonia concentrations in the hemolymph were unchanged, as ammonia was volatilized by the gills and excreted into the air as NH₃ gas. These results show that the green crab copes with emersion by seeking refuge in microhabitats that mitigate the changes in the physical parameters of intertidal emersion. Physiologically, desiccation is avoided, cardio-respiratory processes are maintained at reduced levels, and hemolymph acid–base balance is minimally affected. Ammonia toxicity appears to be avoided by a shift to excreting NH₃ gas directly or indirectly to air.     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.