Water Balance in the Land Crab, Gecarcinus lateralis, During the Intermolt Cycle

Gecarcinus lateralis can take moisture from a clamp substratumin amounts adequate for the needs of the entire intermolt cycle.It can also rehydrate in this way, even after severe dehydration. This crab is able to survive for many months when free waterof a wide range of salinities (0.30; = parts per thousand)is made available in a shallow dish. The crab dies within sevenweeks when the salinity of this water is 35. During proecdysisthe pericardial sacs of eyestalkless crabs become most swollenwhen the salinity of the available water is 15 or 23, and survivalduring and after ecdysis is greatest with water of 15. A crab in proecdysis shows no increase in the rate at whichwater enters following dehydration. Yet large amounts of waterare retained, particularly at the intermediate salinities. Maximalswelling of the pericardial sacs just prior to ecdysis is essentiallyequivalent in crabs with eyestalks, in eyestalkless crabs, andin eyestalkless crabs that have received an implant of centralnervous tissue. Hence, we conclude that a hormone causing theretention of water exists, but not in the eyestalks, in thebrain, or in the thoracic ganglionic mass. At ecdysis eyestalkless crabs show large increases in the dimensionsof the carapace, while crabs with eyestalks and eyestalklesscrabs that have received an implant of certain central nervoustissues show much less increase and may even show a decrease.Thus, we conclude that a hormone causing a release of waterat ecdysis is produced in the central nervous system. The advantages to the crab of a dual hormonal control of itswater balance are discussed.     

Ion Regulation in Crayfish: Freshwater Adaptations and the Problem of Molting

SYNOPSIS. Crayfish have a long evolutionary history in temperatefresh water (FW). Ion regulation is challenged by low externalconcentrations of Na, Cl, and Ca (<1 mM). In intermolt theprimary concern is Na and Cl balance; around ecdysis the emphasisswitches to Ca regulation as the cuticle is decalcified/calcified.Compared with marine crustaceans, intermolt crayfish maintaina reduced extracellular (EC) osmolality and have lower permeabilityto both ions and water. Hyperregulation involves active branchialuptake of Na and Cl and the unique ability to produce a hypotonicurine. Ion uptake involves apical electroneutral ion exchange(Na^$ for H^$; Cl^– for HCO₃–; counterions providedfrom CO₂ via carbonic anhydrase) followed by active basolateraltransport of Na via the Na pump, with Cl following passively.Reabsorption of 95% of filtered electrolytes at the antennalgland (kidney) involves similar subcellular mechanisms in amorphologically differentiated region of the distal tubule.Intermolt crayfish exhibit negative Ca balance (passive effluxunopposed by uptake) tolerable in view of the large cuticularCaCO₃ reserve. In premolt, cuticular Ca is reabsorbed. A smallamount is stored as gastroliths, the remainder is lost via branchialexcretion and in the discarded exuviae. At ecdysis, FW uptakegenerates the physical force for shedding, leaving the crayfishwith dilute hemolymph and a Ca deficiency. Levels of EC Na andCl are restored by intensive postmolt branchial uptake. Mineralizationof the soft exoskeleton involves remobilization of stored Caand branchial uptake of Ca and HCO₃. Transepithelial Ca transportinvolves Ca^2$ ATPase and Ca^2$/Na^$ exchange. The importance ofexternal electrolytes and pH in postmolt ion regulation is explored,as are some allometric considerations.     

From Sea to Tree: Saga of a Land Crab

There are advantages to be gained when an animal abandons itsancestral home, the ocean, and advances across the littoralzone to the land. But along with the advantages come the inevitableproblems. How a land crab, Gecarcinus lateralis, has solvedits problems of life on land is the subject of this paper. Itsuse of dew as a source of moisture and of its burrow as a refugefrom predators and a hostile environment; its ability to collectmoisture from a damp substrate and conduct this moisture toits branchial chambers and its gills; its capacity to retainlarge amounts of water before ecdysis and to use this waterto stretch its new soft exoskeleton and attain its proper shapeafter ecdysis are only a few of the crab's special adaptationsfor terrestrial life. Its feeding and reproductive behaviorare equally adaptive, as is its propensity for tree-climbingwhen danger threatens–or at other times. During the pastseveral decades, much interesting and important research hasbeen done on G. lateralis, as well as on other land crabs–butmuch remains to be done. Land crabs are and will undoubtedlycontinue to be promising objects of scientific research.     

Macromolecular Changes Associated with the Growth of Crustacean Tissues

Tissue mass, rate of protein synthesis, content of ribosomalRNA and rates of synthesis of ribosomal RNA have been studiedthroughout the molting cycle in the midgut gland, epithelium,and somatic muscle in the land crab, Gecarcinns lateralis. Inall tissues there is an increase in ribosomal RNA followed byan increase in the rate of synthesis of protein in the premoltperiod. Subsequently, the three tissues differed in that (a)in the midgut gland the level of ribosomal RNA and protein synthesisreturned to the intermolt rates before ecdysis whether or notthe mass of the tissue was increasing or decreasing; (b) ribosomalRNA and protein synthesis in epithelium reached a maximum ata time when epithelial cells reached a maximal size; subsequently,all three parameters decreased toward intermolt levels beforeecdysis; (c) ribosomal RNA and protein synthesis reached a maximumin the premolt period in somatic muscle while the muscle wasin fact decreasing in mass. Muscle ribosomes are very stableand appear to be conserved for weeks or months to be reusedafter ecdysis in a second burst of protein synthetic activityat the time when there is replacement and growth of new musculartissue. The relation of these events with hormonal control ofgrowth is discussed.     

Proteins of the Crustacean Exoskeleton

We describe here some of the components of the exoskeleton ofthe decapod crustacean with emphasis on the constituent proteins,including both structural and enzymatic. All four layers, butparticularly the inner three, of the exoskeletons of four brachyuranscontain high concentrations of proteins 31 kDa; the innermostmembranous layer is especially rich in such proteins. A numberof crab exoskeletal proteins resemble insect cuticle proteinsin size (M_r) and isoelectric point (pI). A further similarityis the cross reactivity of crab exoskeletal proteins with fourdifferent antibodies against cuticular proteins of two speciesof insects. One of the small M_r exoskeletal proteins in theBermuda land crab Gecarcinus lateralis has a similar distributionas a protein of similar size in the cuticle of the tobacco hornworm Manduca sexta. The partial dissolution of an old exoskeletonand formation of the two outer layers of a new exoskeleton aremajor events in readying a crustacean for the increase in sizethat occurs at each molt. Expressing both parallel and sequentialactivation of a number of genes, a single layer of epidermalcells that bounds a crustacean such as G. lateralis synthesizesspecific proteins at different stages of the intermolt cycleas the outermost epicuticle and exocuticle are formed duringproecdysis and as the endocuticle and membranous layer are formedduring metecdysis. Finally, two sets of proteinases isolatedfrom integumentary tissues of land crabs degrade the same exoskeletalproteins in vitro as are degraded in vivo during proecdysis.     

The Pericardial Sacs of Ocypode in Relation to the Conservation of Water, Molting, and Behavior

The surface area of pericardial sacs of the semiterrestrialcrab, Ocypode cordimana, is greater than that of pericardialsacs of the intertidal crabs, Ocypode macrocera and Ocypodeplatytarsis. Responses of the pericardial sacs with relationto conservation of water and molting vary with the species.In the semiterrestrial crab, Ocypode cordimana, the pericardialsacs store water during proecdysis which is used to stretchthe new cuticle after ecdysis. In contrast, the pericardialsacs of the intertidal crabs, Ocypode macrocera and Ocypodeplatytarsis, do not store water during proecdysis. But the sacsof these crabs swell for a brief period during ecdysis, apparentlyto accommodate excess fluid entering the body while the legsare being withdrawn from the old exoskeleton. Although the significanceremains unknown, deposits of uric acid are found in the pericardialsacs of Ocypode cordimana but not in the pericardial sacs ofOcypode macrocera and Ocypode platytarsis. Exposure of specimensof Ocypode cordimana to dry sand reduces the swelling of pericardialsacs, while proecdysial growth of the limbs remains unaffected.The pericardial sacs of Ocypode cordimana swell at a rapid rateif the crabs are maintained in darkness rather than in light.Implications of the present findings are discussed with relationto the habitat of the crabs.     

Radial Water and Solute Flow in Roots of Zea mays: III. EFFECT OF TEMPERATURE ON THO AND ION TRANSPORT

THO influx, ³⁶Cl^– influx and efflux and ²²Na influx throughmaize cortex sleeves were assayed under different temperaturesranging from 5 to 40 °C. The highest Q¹⁰ and H values forTHO influx were found between 12 and 32 °C. ³⁶Cl effluxand ²²Na influx were almost temperature independent. ³⁶Cl influxwas greatly increased above 20°C. These results are consistentwith previous conclusions that water flow through maize sleevesis metabolically mediated and that chloride is actively pumpedinwards.     

IONIC COMPOSITION AND ELECTRIC RESPONSE OF LAMPROTHAMNIUM SUCCINCTUM

The concentrations of K⁺, Na⁺ and Cl^– in the cytoplasmof Lamprothamnium succinctum, a brackish water Characeae, areabout 137, 47 and 86 mM respectively. The concentration of K⁺in the cytoplasm is of the same order as that in the cell sap,while the concentrations of Na⁺ and Cl^– are much lowerthan those in the cell sap (i.e., 140 mM Na⁺, 370 mM Cl^–).In the brackish water, in which the plant grows, the internodesis never excitable electrically. However, it acquires excitabilitywhen it is kept in a mannitol-pond water. The action potentialthus elicited is accompanied by a temporary cessation or slowdown of the cytoplasmic streaming. ¹This work was supported by Research Grants from the Ministryof Education of Japan     

Potassium, sodium and Chloride in the protoplasm of characeae

Using vacuolar perfusion, which enabled us to replace the cellsap with a solution containing no k⁺, Na⁺ and Cl^–, theconcentrations of these ions in the protoplasm of three speciesof fresh water Characeae were determined. They were respectively,78, 2 and 27 mM in Nitella flexilis, 101, 9 and 31 mM in Nitellapulchella, and 112, 3 and 21 mM in Chara australis. Our previouslyreported results (3) indicating that the chloroplast layer containedmuch more Na⁺ and Cl^– than the endoplasm has been questionedin the light of the present results. ¹Present address: Department of Biology, College of GeneralEducation, Osaka University. (Received September 5, 1973; )     

The ionic hemolymph composition of the terrestrial isopod Porcellio scaber Latr. during molt

We analyzed the ionic composition of the hemolymph of Porcellio scaber in four different stages of the molt cycle using capillary electrophoresis and calcium selective mini- and microelectrodes. The main ions in the hemolymph were K⁺, Ca²⁺, Na⁺, Mg⁺, and Cl⁻. The values for total calcium obtained by means of capillary electrophoresis and calcium selective minielectrodes did not differ significantly from each other. In situ measurements of the free Ca²⁺ concentration ([Ca²⁺]) by means of calcium-selective microelectrodes indicated that Ca²⁺ is not bound in the hemolymph. During molt the [Ca²⁺] is significantly larger than during intermolt. The [Ca²⁺] increased by 13%, 19% and 18% during premolt, intramolt, and postmolt, respectively. The concentration of the other cations and of Cl⁻ decreased significantly between premolt and intramolt. Thus, the rise of the [Ca²⁺] in the hemolymph is not due to a general increase in all ions, but rather to the resorption of cuticular calcium. Furthermore, the results suggest that K⁺, Na⁺, Mg⁺, and Cl⁻are extruded from the hemolymph during and/or after posterior ecdysis. Accepted: 5 August 1997     

The Ionic Relations of Acetabularia mediterranea

The concentrations of K⁺, Na⁺, and Cl^– in the cytoplasmand the vacuole of Acetabularia mediterranea have been measured,as have the vacuolar concentrations of SO₄^–– andoxalate. The electrical potential difference between externalsolution, and vacuole and cytoplasm has been measured. The resultsindicate that Cl^– and SO₄^–– are probably transportedactively into the cell, and that active transport of Na⁺ isoutwards. The results for K⁺ are equivocal. The fluxes of K⁺,Na⁺, Cl^–, and S0₄^–– into the cell and theeffluxes of Na⁺ and Cl^– have been determined. The Cl^–fluxes are extremely large. In all cases the plasmalemma isthe rate-limiting membrane for ion movement. A technique isdescribed for the preparation of large, completely viable cellfragments containing only cytoplasm, with no vacuole.     

Inhibition of Sugar and Salt Elimination by Glands with the Amino Acid Analog p-Fluorophenylalanine

The amino acid analog p-fluorophenylalanine (FPA) inhibitedsugar and K⁺ secretion by nectary glands. FPA specifically reducedthe net excretion of Na⁺ and Cl^– by the salt glands ofthe halophyte Limonium vulgare and ³⁶Cl^–excretion by theglands of the pitcher walls of the carnivorous plant Nepenthes.Net uptake and net accumulation of Na⁺ and Cl^– by Limoniumleaf tissue and ³⁶Cl^– accumulation in Nepenthes pitchertissue were much less inhibited than excretion. The resultsare discussed in relation to literature reporting similar specificeffects of FPA on transport of ions from the symplast of barleyroots into the dead xylem elements. Limonium vulgare, Nepenthes hookeriana, salt-glands, excretion, p-fluorophenylalanine     

An Examination, by Compartmental Flux Analysis, of the Development of Sodium and Chloride Absorption Capacities in Beetroot Disks

Using beetroot, Beta vulgaris L. var. Avon Early, grown in radioactivelylabelled nutrient solutions, concentrations and fluxes of Na⁺and Cl^– were estimated for cells of freshly cut storageroot disks, by compartmental analysis of radioisotope elutionmeasurements. These values were compared with results obtainedin a similar manner from beetroot grown in non-labelled nutrientsolution and loaded instead during an aging period in ²²Na-or ³⁶Cl- labelled 1 mM NaCl solution. In accord with the generallyaccepted, but never properly tested, view, it was found thatnet Na⁺ influx followed from a reduction in efflux with agingand net Cl^– uptake depended on a marked increase in influx.However, both these important changes took place at the tonoplast,and, although aging led to a reduction of plasmalemma fluxes,at no time was entry of Cl^– into the cytoplasm, or lossof Na⁺ from the cytoplasm, significantly restricted.     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Getting out of a Tight Squeeze--Enzymatic Regulation of Claw Muscle Atrophy in Molting

The claw closer muscle of the land crab, Gecarcinus lateralis,undergoes a cyclical atrophy and restoration during the intervalbetween ecdyses. During proecdysis (stage D₀), 30–60%of the muscle protein is degraded, which reduces tissue massand facilitates withdrawal of the propodus at ecdysis. Proteinis resynthesized as the muscle grows back to its previous sizeduring metecdysis. This atrophy is specific to the claws andcan be accentuated by multiple limb autotomy. Crustacean musclescontain five cytosolic proteinases that degrade myofibrillarproteins. Four of these constitute a family of enzymes requiringCa²⁺ for activity. These calcium-dependent proteinases (CDPs)hydrolyze myofibrillar proteins in vitro and in situ and showincreased activity in atrophic claw muscles, which suggeststhat CDPs play an important role in myofibrillar protein metabolism.The fifth enzyme is a multicatalytic proteinase (MCP), a multisubunitproteolytic complex that degrades a wide range of peptide andprotein substrates. The catalytic properties of the complexare altered with low concentrations of sodium dodecyl sulfateor by brief heating at 60°C. Only the heat-activated formdegrades myofibrillar proteins. Since the CDPs hydrolyze contractileproteins about 30-fold more rapidly than the heat-activatedMCP, the MCP probably has a more limited or specialized functionin molt-induced claw muscle atrophy.     

Limb Autotomy as an Investigatory Tool: Host Molt-Stage Affects the Success Rate of Infective Larvae of a Rhizocephalan Barnacle

D. M. Skinner was the first biologist to systematically investigatethe effect of multiple limb autotomy on ecdysis in decapod crustaceans.She proposed the existence of (1) limb autotomy factor anecdysis(LAF_an) which initiates precocious molting, and (2) limb autotomyfactor proecdysis (LAF^pro) which postpones proecdysis. Meantime to ecdysis did not differ significantly among groups ofsmall juvenile Callinectes sapidus with zero, two, four, andsix limbs removed. Variance was significantly less for the groupmissing six limbs; i.e., autotomy of six limbs synchronizedthe molt-cycle. These patterns were consistent with the hypothesesthat LAF^an exerts an additive effect, i.e., more anecdysialcrabs enter proecdysis as more limbs are autotomized and thata delay of proecdysis occurs above a threshold (>4) of autotomizedlimbs. Multiple limb autotomy provides investigators with easyaccess to crabs in metecdysial molt-stages which is useful instudying interactions between rhizocephalan barnacles and theirhosts. While 71% of metecdysial C. sapidus exposed to infectivelarvae of the sacculinid rhizocephalan, Loxothylacus texanus,developed the external stage of the parasite, no similarlysizedanecdysial crab was parasitized, suggesting that these crabsare not susceptible to infection during anecdysis. Size of C.sapidus at infection was inversely proportional to the numberof ecdyses between infection and emergence of the parasite,but not correlated to final host size. These data suggest thatthere is a minimum threshold for host size; smaller hosts undergomore ecdyses before attaining the threshold. Rhizocephalansare being considered as biological control agents and thereis a need to understand how they find and infect hosts. Becauselimb autotomy is such a useful research tool, D. M. Skinner'scontributions to our understanding of how limb autotomy influencesthe crustacean molt cycle will continue to pay significant dividendsin crustacean biology.     

NaCl Uptake in Roots of Zea mays Seedlings: Comparison of Root Pressure Probe and EDX Data

The extent by which salinity affects plant growth depends partlyon the ability of the plant to exclude NaCl. To study the uptakeof NaCl into excised roots of Zea mays L. cv. ‘Tanker’,two different techniques were applied. A root pressure probewas used to record steady state as well as transient valuesof root (xylem) pressure upon exposure of the root to mediacontaining NaCl and KCl as osmotic solutes. In treatments withNaCl, pressure/time responses of the root indicated a significantuptake of NaCl into the xylem. NaCl induced kinetics were completelyreversible when the NaCl solution was replaced by an isosmoticKCl solution. This indicated a passive movement of Na⁺-saltsacross the root cylinder. Root samples were taken at differenttimes of exposure to NaCl and prepared for X-ray microanalysis(EDX analysis). Radial profiles of ion concentrations (Na⁺,K⁺, Cl^–) were measured in cell vacuoles and xylem vesselsalong the root axis. Na⁺ appeared rapidly in mature xylem (earlymetaxylem) and living xylem (late metaxylem) before it was detectablein vacuoles of the root cortex. EDX results confirmed that thekinetics observed by the pressure probe technique correspondedmainly to an influx of Na⁺-salts into early metaxylem. In latemetaxylem, the uptake of Na⁺ was associated with a decline ofK⁺. The Na⁺/K⁺ exchange indicated a mechanism to reduce sodiumfrom the transpiration stream. Ion localization, ion transport, maize, root pressure, salinity, water relations, X-ray microanalysis, Zea mays     

Methionine-enkephalin induces hyperglycemia through eyestalk hormones in the estuarine crab Scylla serrata

The hypothesis is tested that methionine-enkephalin, a hormone produced in and released from eyestalk of crustaceans, produces hyperglycemia indirectly by stimulating the release of hyperglycemic hormone from the eyestalks. Injection of methionine-enkephalin leads to hyperglycemia and hyperglucosemia in the estuarine crab Scylla serrata in a dose-dependent manner. Decreases in total carbohydrate (TCHO) and glycogen levels of hepatopancreas and muscle with an increase in phosphorylase activity were also observed in intact crabs after methionine-enkephalin injection. Eyestalk ablation depressed hemolymph glucose (19%) and TCHO levels (22%), with an elevation of levels of TCHO and glycogen of hepatopancreas and muscle. Tissue phosphorylase activity decreased significantly during bilateral eyestalk ablation. Administration of methionine-enkephalin into eyestalkless crabs caused no significant alterations in these parameters when compared to eyestalk ablated crabs. These results support the hypothesis that methionine-enkephalin produces hyperglycemia in crustaceans by triggering release of hyperglycemic hormone from the eyestalks.     

Ion Imbalance in Capsicum annuum, scabrous diminutive, a Wilty Mutant of Pepper I. SODIUM FLUXES

Leaves of the wilty pepper mutant, scabrous diminutive, accumulatemore Na+ than those of the normal genotype, when both grow inmedium containing N⁺. It seems that the regulation of Na⁺ fluxin the mutant root was modified. Net uptake of Na⁺ was muchhigher and efflux of ²²Na⁺ was lower in the mutant roots thanin the normal ones. Two possible explanations for these differencesbetween mutant and normal plants are discussed, namely (a) achange in membrane permeability and (b) a change in the mechanismof Na⁺ extrusion.