Neuroendocrinology of Osmoregulation in Decapod Crustacea

Experimental evidences for neuroendocrine control of osmoreregulationin decapod crustaceans are mounting. The eyestalk system, brainand thoracic ganglionic centers, and the pericardial organ appearto be involved in this control. Evidences based on experimentationwith eyestalk removal and the injection of extracts of neuroendocrinecenters are presented. Neuroendocrine extracts affect the movementsof salts and water in the gills, stomach, intestine and antennalglands. The pericardial organ material may affect osmoregulationby increasing rate of circulation and influencing salt movement. Three factors have been partially separated from CNS tissues.A factor of the freshwater crayfish, and other freshwater decapods,increases the influx of salt. Two factors from an estuarinecrab influence the movement of water, an acetone-soluble factorincreasing its influx and a water-soluble factor decreasinginflux and increasing efflux. The factors may be involved inthe adaptation of the animals to their osmotic environments. None of the factors have yet been identified in circulationnor in effector tissues. Future research must place specialemphasis on the identification of the CNS factors in circulationas well as in secretory cells and effector tissues to establishtheir true hormonal nature.     

Bihormonal control of oogenesis in the freshwater prawn, Macrobrachium kistnensis

The hormonal control of oogenesis has been investigated in the freshwater prawn, Macrobrachium kistnensis. Experiments using bilateral eyestalk ablation and injection of eyestalk extract supported the presence of a gonad inhibiting factor in the eyestalk of female prawns. Injections of brain and thoracic ganglion extract in normal female prawns and in those subjected to bilateral eyestalk ablation revealed the presence of a gonad stimulating factor in the central nervous tissue.     

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation

The blood–brain barrier (BBB) is a barrier that prevents free access of blood‐derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood‐derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet‐derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis‐associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low‐molecular‐mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis. Copyright © 2013 John Wiley & Sons, Ltd.     

Salt Tolerance in the Triticeae: Growth and Solute Accumulation in Leaves of Thinopyrum bessarabicum

Gorham, J., McDonnell, E., Budrewicz, E. and Wyn Jones, R. G.1985. Salt tolerance in the Triticeae: growth and solute accumulationin leaves of Thinopyrum bessarabicum.—J. exp. Bot. 36:1021–1031. The diploid wheatgrass Thinopyrum bessarabicum was found towithstand prolonged exposure to 350 mol m^–3 NaCl in hydroponicculture. During the gradual addition of salt to the externalmedium, osmotic adjustment was rapidly achieved by the accumulationof Na and Cl. Following osmotic adjustment constant leaf Naand Cl concentrations were maintained, and K was retained ata high level. Thinopyrum bessarabicum may be described as anosmoconformer, adjusting its internal osmotic pressure to 400–500mOsmol kg^–1 above that of the external medium in hydroponicculture. Both slower shoot initiation and reduced leaf lengthcontributed to the reduced growth rates at higher salinities.Leaf width was not affected. Increasing salinity resulted inincreases in leaf concentrations of phosphate, glycinebetaine,sucrose and proline, and in decreases in the concentrationsof nitrate, sulphate, magnesium, calcium, total amino acidsand organic acids. Thinopyrum bessarabicum exhibits salt tolerancecharacters which may be useful in wheat breeding. Key words: Salt stress, solute accumulation, osmotic adjustment, Thinopyrum     

Hormonal Control of the Molt Cycle in the Fiddler Crab Uca pugilator

Comparison of the circulating steroids in the blood of crabslacking neurosecretory eyestalk centers and crabs with thosecenters intact (but lacking several walking legs) has revealeddifferences in vivo that can be attributed to eyestalk factors.It is concluded that eyestalk factors (including the putativemolt-inhibiting hormone, MIH): 1) exert control in vivo overproduction of 25-deoxyecdysone by the crab Y-organ, 2) controlcyclic steroid production throughout anecdysis and parts ofproecdysis and 3) are not solely responsible for the increasedsteroid production that occurs during late proecdysis. Usingthe data presented here and previously published data, a simplefeedback model for the control of Y-organ activity is proposed.The model suggests that the Y-organs of the crabUca pugilatorare modestly active during anecdysis but become further activatedduring late proecdysis. The increased activation requires morethan eyestalk removal and may involve additional extra-eyestalkfactors.     

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.     

Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method

A calcein fluorescence quenching method was applied to measure osmotic water permeability in highly differentiated primary cultures of brain astrocytes from wild-type and aquaporin-4 (AQP-4)-deficient mice. Cells grown on coverglasses were loaded with calcein for measurement of volume changes after osmotic challenge. Hypotonic shock producing twofold cell swelling resulted in a reversible 12% increase in calcein fluorescence, which was independent of cytosolic calcein concentration at levels well below where calcein self-quenching occurs. Calcein fluorescence was quenched in <200 ms in response to addition of cytosol in vitro, indicating that the fluorescence signal arises from changes in cytosol concentration. In astrocytes from wild-type CD1 mice, calcein fluorescence increased reversibly in response to hypotonic challenge with a half-time of 0.92 ± 0.05 s at 23°C, corresponding to an osmotic water permeability (P_f) of 0.05 cm/s. P_f was reduced 7.1-fold in astrocytes from AQP-4-deficient mice. Temperature dependence studies indicated an increased Arrhenius activation energy for water transport in AQP-4-deficient astrocytes (11.3 ± 0.5 vs. 5.5 ± 0.4 kcal/mol). Our studies establish a calcein quenching method for measurement of cell membrane water permeability and indicate that AQP-4 provides the principal route for water transport in astrocytes. water transport; aquaporin-4; fluorescence quenching; brain swelling; cerebral edema     

Neurohormonal Integration of Osmotic and Ionic Regulation

There is evidence in crustaceans that neuroendocrine centers,including the eyestalk, brain, thoracic ganglionic mass, andpericardial organ, produce factors that affect osmotic and ionicregulation. Understanding of the processes responsible for osmoticadjustment in the intact animal, such as regulation of permeability,active uptake of ions, and respiratory and cardiovascular alterations,has increased substantially in the past few years. However,interaction of neuroendocrine factors with the target tissuesand systems is just beginning to be investigated. There is evidencethat content of lipids and activity of enzymes are importantin osmoregulation, and neuroendocrine effects on these metabolicprocesses are worthy of study. In addition, there are some crustaceansin which osmoregulatory ability varies developmentally. Furtherinvestigations of such animals is also necessary. Progress inour understanding of neuroendocrine influences on osmoregulationdepends upon further purification of active factors from neuroendocrinecenters and hemolymph, and upon development of appropriate assayson which to test them.     

Sea snake skin: Permeable to water but not to sodium

Summary Fasting yellow-bellied sea snakes (Pelamis) have a very low rate of exchange of Na with sea water. Influx and efflux are balanced at a value near 8 moles/100 g h. This is only a fraction of the rate of exchange found in marine fish. Na influx is due to uptake in the head region; dermal and cloacal influx are minimal. The impermeability of the skin to Na has been confirmed in isolated preparations. The outer keratin layer seems to be the primary barrier, since the shed skin alone is also impermeable. Na efflux can be increased to 140 m/100 g h by salt injections, and secretion by the sublingual salt gland can account for all of this loss. Fasting snakes are not in water balance in sea water. There is a net loss of water amounting to about 0.4% body wt/day that probably occurs mainly through the skin. The major osmotic problem ofPelamis in sea water seems to be water balance, not salt balance. Differences in salt gland size among sea snakes might be related to differences in skin permeability to water associated with dermal respiration. The importance of the skin as a permeability barrier suggests that the frequent skin shedding of sea snakes may be related to maintenance of low water permeability as well as to prevention of growth by marine fouling organisms.     

Salinity--stress and Desiccation in Intertidal Worms

SYNOPSIS. Intertidal worms (oligochaetes, polychaetes, sipunculids)inhabiting beaches and tidal fiats both on the open sea coastand in estuaries may be exposed to significant tidal as wellas seasonal variations in salinity. However, there are veryfew measurements of the actual variations in salinity encounteredby worms in nature. Behavior (irrigation of burrow, verticalmovements in burrows, migration in gradients of salinity)maybe important in determining to which of the available salinitiesin a tidal cycle the worms may be exposed. In response to rapidlowering of salinity, worms gain water and lose salts, theseprocesses combining in diluting the internal fluids. Internalilution occurs more slowly in euryhaline species than in stenohalinespecies. Worms fully adapted to salinities lower than 30 % seawater may be hyperosmotic (demonstrated for six species of Nereidae).Mechanisms involved in hyperosmotic regulation include activetransport of salts(demonstrated in Nereis diversicolor), reductionofthe permeability of the body surface to salts and perhapsto water, and perhaps production of hypo—osmotic urine.Sipunculids can tolerate considerable loss of water rom dehydrationand concomitant increases in osmotic concentration of the bodyfluids. It is suggested that worms exposed to significant tidalvariations in salinity may seldom be in osmotic equilibriumwith their external medium.     

Regulation of the Crustacean Mandibular Organ

The crustacean mandibular organ (MO) produces methyl farnesoate(MF), a juvenile hormone-related compound thought to have rolesin crustacean reproduction and development. Therefore, the controlof MF production by the MO has been of considerable interest.Current evidence indicates that the MO is negatively regulatedby peptides present in the eyestalk (MO inhibiting factor, MO-IH).Several eyestalk neuropeptides have been identified that inhibitMF synthesis by MO incubated in vitro. The amino acid sequencesof these MO-IH peptides are similar to peptides in the crustaceanhyperglycemic hormone (CHH) family of neuropeptides. In addition,there appears to be a compound in the eyestalk that lowers hemolymphlevels of MF in vivo but does not directly affect the MO invitro. The inhibition of MF synthesis by eyestalk peptides involvesthe inhibition of farnesoic acid O-methyl transferase, the lastenzyme in the MF biosynthetic pathway. The activity of thisenzyme is affected by cyclic nucleotides, suggesting that thesecompounds may be involved in the signal transduction pathwaymediating the effects of MO-IH.     

In vitro effects of both dopaminergic and enkephalinergic antagonists on the ovarian growth of Cherax quadricarinatus (Decapoda, Parastacidae), at different periods of the reproductive cycle

The in vitro effect of both spiperone (dopaminergic antagonist) and naloxone (enkephalinergic antagonist), was assayed on small pieces of ovary dissected from C. quadricarinatus females, with the eventual addition of some neuroendocrine organs, such as thoracic ganglion or eyestalk tissue. The incorporation of tritiated leucine by the ovary was measured in order to estimate the ovarian growth. During the post-reproductive period, both mentioned antagonists were able to significantly stimulate the ovary in the presence of thoracic ganglion, but did not produce any significant effect in the preparation containing ovary and eyestalk tissue, or only ovary. No significant effects of the assayed antagonists were noted during the pre-reproductive period. These results were in accordance with previous models describing the neuroendocrine control of crustacean reproduction, and represent new findings about the hormonal context in different periods of the reproductive cycle of crayfish. Besides, by means of the experimental combination of the tested antagonists with dopamine or met-enkephalin, a new model dealing with the interaction of these two neurotransmitters on the hormonal secretion of thoracic ganglion has been proposed.     

Current progress in shrimp endocrinology-a review

One problem in aquaculture is obtaining brood because many commercially important species are incapable of spontaneous maturation under artificial conditions. Commercial shrimp hatcheries commonly use eyestalk ablation to stimulate gonadal maturation in shrimps. Research has been conducted on the inhibition of reproductive maturation by hormones originating in the eyestalk glands and on other endocrine sources (e.g.,brain, thoracic ganglion, ovary, mandibular organ, androgenic gland and Y-organs) to determine their roles. Alternate techniques for acceleration of gonad maturation through the use of synthetic hormones or neurotransmitters may benefit aquaculture. Neurohormones and neuroregulators have been shown to accelerate gonadal maturation but an effective delivery technique must be developed for use in a large-scale aquaculture operation.     

Further Studies on Growth and Osmoregulation of Sugar Beet Leaves under Low Salinity Conditions

In previous work, Nunes and Dias (1980) demonstrated that lowsodium concentrations in the root medium of intact or decapitatedyoung sugar beet plants grown under controlled conditions modifiedleaf water relations and increased leaf area and dry weight.The present study confirms these findings and presents furtherresults concerning the effect of salt on the concentrationsof the main osmotic substrates and on the structural and chemicalfractions of the cell dry weight. Increases of water and turgor potentials (0.25 MPa and 0.4 MPa,respectively) and a small decrease in osmotic potential (0.16MPa) were found in the leaves of salt treated plants. In theseplants, osmotic potentials estimated from the concentrationof ions and organic solutes in the leaf sap agree with thosemeasured showing that the observed increase in sodium concentrationmay account for the small decrease in the osmotic potential.No changes were detected in the concentration of orthophosphateor malic acid but total acidity of the leaf sap from salt treatedplants was significantly lower. It was found that all the main components of cell dry matter(total protein, soluble sugars, pigments and crude cell wall)contributed to the dry weight increase in the salt treated plants.Among the polysaccharide fractions of the cell wall, pectinsincreased significantly relative to hemicellulose and cellulose. Key words: Sugar beet, Sodium chloride, Growth, Osmoregulation     

Hormonal control of renal functions in insects

Insect Malpighian tubules secrete an isosmotic, KCl-rich primary urine containing low concentrations of most other blood solutes. Neuropeptide diuretic hormones (DH), possibly related to vasopressin, stimulate tubular fluid secretion by 2- to 200-fold in response to water loading, e.g., feeding. DH acts on tubules through cyclic AMP (cAMP) to stimulate salt transport without measurable change in osmotic permeability. Changes in composition of tubular secretion after stimulation and the possible control of DH release are discussed. Most of the water, ions, and metabolites in tubular secretion are normally reabsorbed by active mechanisms in the rectum, where the urine may finally become either hyposmotic or strongly hyperosmotic to the blood. A newly discovered neuropeptide, chloride transport-stimulating hormone, controls (via cAMP) reabsorption of the principal salt by stimulating K-dependent, electrogenic transport of Cl- across the apical cell border. Passive net absorption of K+ is thereby enhanced. Diuretic and antidiuretic factors may control osmotic permeability of the rectal wall and thereby influence the osmotic concentrations of the rectal absorbate and final urine. The increased recycling of a KCl-rich fluid through the Malpighian tubule-rectal system after feeding probably serves to clear the body of unwanted substances ingested with, and produced by, metabolism of the meal.     

Recovery of Germination from Different Osmotic Conditions by Four Halophytes from Southeastern Spain

Seeds of four halophytes from southeastern Spain (the perennialsHalocnemum strobilaceum,Arthrocnemum macrostachyum and Sarcocorniafruticosa, and the annual Salicornia ramosissima) were exposedto iso-osmotic stress with NaCl, MgCl₂, MgSO₄and Na₂SO₄. Theosmotic potential that enforced dormancy in the majority ofseeds was determined. The percentage of ungerminated seeds thatrecovered when they were transferred to distilled water didnot differ significantly from the percentage germination ofseeds in distilled water controls, irrespective of the saltused to impose the stress. Therefore, the factor responsiblefor the dormancy of seeds seems to be the decrease in osmoticpotential. Such osmotic pretreatments promoted an increase inthe rate of recovery germination which, inArthrocnemum macrostachyumand Sarcocornia fruticosa, doubled their rates of germinationcompared to distilled water controls. The osmotic potentialsof the soils where the four species occur naturally were morenegative than those allowing seeds to germinate after osmoticstress. Copyright 2000 Annals of Botany Company Arthrocnemum macrostachyum, Halocnemum strobilaceum, halophytes, osmotic stimulation, recovery of seed germination, Salicornia ramosissima, Sarcocornia fruticosa.     

Water transport and ion-water interaction in the gramicidin channel

The diffuse permeability and the diffusion coefficient of water (Dw) in the gramicidin channel is determined from the osmotic water permeability of the channel and "single file" pore theory. Dw is about 7% of the self-diffusion coefficient of bulk water. The diffusion coefficient of a single water molecule alone in the channel is also determined and is about equal to the value in bulk water. This provides an estimate of the mobility of water on the channel walls in the absence of water-water interaction. Since the gramicidin channel walls should be representative of uncharged polar protein surfaces, this result provides direct evidence that the presence of a cation in the channel reduces the hydraulic water permeability by a factor ranging from 60 for Tl+ to 5 for Na+. The diffusion coefficient of a cation (Dc) in the channel is estimated and compared with Dw. For Na+ it is found that Dc approximately equal to Dw, which implies that the movement of the row of water molecules through the channel determines the local mobility of Na+. Thus, it seems that short range ion-wall interactions are not important in determining the channel conductance for Na+. In contrast, for Li+, local ion-wall interactions probably do limit the conductance.     

The osmotic behaviour of frogs eggs and young tadpoles

Summary Determinations of total osmotic concentration on eggs ofRana temporaria by the vapour pressure method and of chloride by theWigglesworth ultra micro-technique show a rapid fall during the first few hours, from 120 to 95 mM total concentration and, until the blastopore closes, a further slight fall to about 80 mM. Thereafter the total concentration rises rapidly, while the chloride concentration remains unaltered or (later) rises very gradually.The eggs swell considerably at first and then more gradually and the changes in concentration until the budding of the gills are due at least in the main to osmotic uptake of water.The permeability, as determined both by rate of swelling and in special experiments with heavy waterbecomes greatly reduced by fertilization.The initial permeability is calculated to correspond to a minute Number of 140 days, while later it is reduced to about 5 1/2 years.When the gills begin to develop permeability for water again increases and the weight rises by osmotic inflow of water. The kidneys become functional and an active uptake of salt (probably located in the gills) prevents a reduction of the osmotic concentration.The fluid in the chorionic cavity is very slightly hypertonic to the surrounding water.The degree of swelling of the egg mucus depends upon the salt concentration in the surrounding water. The swelling is enormous in distilled water and seems to be specifically inhibited by calcium.     

A trematode metacercaria encysted in the nerves of the dungeness crab, Cancer magister

Of 188 Dungeness crabs, Cancer magister, 8 contained unidentified trematode metacercariae encysted in various components of the nervous system, including the thoracic ganglion, brain, lamina ganglionaris of the eyestalk, and major nerves arising from the thoracic ganglion. A single cyst was present in the available tissue sections of 7 of the crabs and no behavioral abnormalities were exhibited. One crab with multiple cysts in major nerves near the thoracic ganglion was markedly ataxic. The cysts and included worms distorted, compacted, and destroyed nervous tissue, and occupied most of the nerves where present. Host response was minimal, but some cysts invoked massive hemocytic accumulations near the infection site. Infections probably seriously affect nerve impulse transmission and accounts for the lethargic behavior of the crab with multiple cysts in major nerves. The present report is the first record of digenetic trematode infections in the Dungeness crab and the apparent restriction of the worm to nervous tissue is unusual if not unique in the Digenea. Because of the absence of grossly recognizable lesions and the small samples excised at necropsy, both the incidence and intensity of infection in Dungeness crab populations are almost certainly higher than indicted by our data.     

Increase of water permeability and recovery of water impermeability of the frog urinary bladder

The experiments carried out on the urinary bladder of the frog Rana temporaria L. have shown that the period of recovery of water impermeability after an increase of osmotic water permeability induced by arginine-vasotocin, desmopressin, or cAMP depends on the degree of increase of the osmotic permeability but not on the nature of the substance stimulating the increase of osmotic water permeability. The removal of the hormone in the absence of autacoids fails to recover the water impermeability. After cessation of the vasotocin action the water permeability decrease is delayed if phospholipase A2 and cyclooxygenase are inhibited by qiunacrine and voltaren, respectively. An agonist of V1-receptors has no effect on dynamics of the recovery of water impermeability. This recovery has been shown to depend on PGE2 concentration in the serosal solution after the hormone removal. The obtained results indicate that decrease of water permeability depends not only on removal of vasotocin or cAMP but also on involvement of autacoids.