Oogenesis in the tropical land crab,Gecarcinus lateralis (Freminville)

Summary The ovaries of small and large adult Gecarcinus were studied histologically and histochemically at various stages in the annual cycle. At all seasons of the year, dividing cells are seen within germinal nests in the ovary. Following division, the cells within the germinal nest enlarge and appear to move out into the stroma, forming cords of young oocytes that become encapsulated by follicle cells. Glycogen, not demonstrable in cells within the germinal nests, is present in the perinuclear cytoplasm of both young and mature oocytes. Lipid is distributed peripherally in the cytoplasm of the oocytes. Deoxyribonucleoprotein is demonstrable within the nuclei of germinal nest cells and of the young oocytes; it is not detectable within the nuclei of the large oocytes. The histological observations suggest that oogenesis occurs throughout the reproductive life of Gecarcinus.Dedicated to Professor Berta Scharrer on her 60th birthday in love, respect and admiration. — This work was supported in part by U.S.P.H.S. Training Grant GM-102.I express my thanks to the late Dr. Helen W. Deane and Dr. Dorothy E. Bliss for their help and encouragement.     

Evidence for dopaminergic and opioid involvement in the regulation of locomotor activity in the land crab Gecarcinus lateralis

1. Computerized analysis of the crabs locomotor behavior revealed an initial increase in activity followed by a gradual decrease over a 12 min observation period. 2. Dopamine, in a dose-dependent manner, inhibits locomotor activity. The effect can be antagonized with the dopamine antagonist, haloperidol. This suggests that dopaminergic influences are involved with locomotor mechanisms. 3. FK 33,824, a stable opioid analog, significantly enhances the initial excitatory locomotor activity. Naloxone, a potent opiate antagonist, can block the excitatory action induced by FK 33 824. This suggests the presence of an opioid modulation mechanism in the regulation of locomotor activity. 4. Concomitant administration of the various agents results in the behavioral characteristics of the agonist appearing when the appropriate antagonist is not present. Thus, administration of dopamine + FK 33,824 + haloperidol results in enhanced locomotor activity. 5. Concomitant dopamine and FK 33,824 administration results in enhanced locomotor activity. This suggests that the opioid mechanism is closer to the last step in affecting the organism's locomotion or in initiating activity.     

Interruption of proecdysis by autotomy of partially regenerated limbs in the land crab, Gecarcinus lateralis

In the land crab, Gecarcinus lateralis, autotomy of partially regenerated limbs before a critical stage in the premolt period results in (1) a very rapid decrease in the serum ecdysone titer, (2) a delay in the growth of partial regenerates remaining on the animal, (3) a delay in the deposition of gastroliths, and (4) a delay in cytological changes in the epidermis. Serum ecdysone titers remain low while new limb regenerates form at the sites of those removed. Ecdysone titers rise when these secondary regenerates complete basal regeneration. Premolt events, which had ceased at the time of autotomy of the partial regenerates, resume their normal patterns of development when ecdysone titers reach the level present in the serum at the time of this interruption. We propose that the effect of autotomy before a critical period is to reinitiate a normal proecdysis. The same pattern of events occurs following autotomy of partial regenerates of crabs without eyestalks, suggesting that the decrease of serum ecdysones is brought about by some mechanism other than changes in the titer of the molt inhibitory hormone.     

Actin-encoding cDNAs and gene expression during the intermolt cycle of the Bermuda land crab Gecarcinus lateralis

Two actin-encoding cDNAs (act1 and act2) from Gecarcinus lateralis have been sequenced or partially sequenced and the corresponding proteins deduced. The actl cDNA has a complete ORF; the act2 cDNA lacks most of the 5′ end of the coding region. The nucleotide (nt) sequences of both clones are very similar to act sequences of many organisms, the most closely related being from another arthropod, the silkmoth Bombyx mori. The proteins Actl and Act2 are more similar to vertebrate cytoplasmic actin isoforms (β-actins) than to vertebrate muscle actins (α-actins); they are also more similar to animal actins than to those of fungi or plants. Codon usage is strongly biased toward C or G in the third position. The deduced number of amino acid (aa) residues and calculated M_r for Actl are 376 aa and 41.94 kDa, respectively. The deduced aa sequence of Actl is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to eleven act genes in the crab genome. Northern blots probed with a segment from the 3′ UTR of actl showed a single band of approx. 1.6 kb in poly(A)⁺mRNAs from epidermis, limb bud or claw muscle and in total RNAs from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in total RNA from midgut gland. Western blots of one-dimensional gels of proteins from the four layers of the exoskeleton, epidermis, limb buds and claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were observed. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detected on Western blots of two-dimensional gels.     

Some observations on the fine structure of the sinus gland of a land crab,Gecarcinus lateralis

The dilated axon endings of the sinus glands of the brachyuran crab, Gecarcinus lateralis, are filled with homogeneously dense granules, each granule being bounded by a delicate membrane. The granules are of two orders of magnitude: 0.05 to 0.1 micro and 0.15 to 0.2 micro in diameter. Each axon ending contains granules of a nearly uniform size. Endings with granules of the larger size range predominate. Non-nervous cells endogenous to the sinus gland are scattered among the nerve endings. The cell contours are irregular, and cytoplasmic processes ramify between endings. The axons are unmyelinated, having only thin limiting membranes, and they possess many neurofibrils. Granules in preterminal portions of the axons tend to lie at the periphery of the fiber, and in some cases in chains at the core of the fiber. The granules appear to be storage and release centers for neurosecretory substances or their precursors.     

Ultrastructural study on the release of neurosecretory material from the sinus gland of the land crab,Gecarcinus lateralis

Summary Sites of release of neurosecretory material were examined in a neurohemal organ of decapod crustaceans, the sinus gland of the land crab, Gecarcinus lateralis. Such discharge into the circulation seems to occur primarily at interfaces between the neurosecretory axons and the acellular stromal sheath which is interposed between parenchyma and hemolymph. The evidence obtained from electron micrographs of adult specimens indicates that more or less intact secretory granules are released into the extraaxonal space primarily by the process of exocytosis. Synaptic-type vesicles are clustered in parts of neurosecretory axons facing the stromal sheath. Such vesicles are thought to result from rearrangement of membranes temporarily fused at the release site and to a minor degree from fragmentation of neurosecretory vesicles within the axon. The presence of nonmarginal vesicles and the occasional appearance of free intraaxonal dense material are interpreted as indications of a second, probably less frequent, mode of release of neurosecretory material.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S., administered by Dr. Berta Scharrer.I am greatly indebted to Mrs. Sarah Wurzelmann for her excellent technical assistance. I thank Dr. Dorothy E. Bliss for contributing the animals used in this study, and Mr. Murray Altmann for technical advice.     

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus)

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.     

Mechanism of Coagulation in Gecarcinus lateralis

Agglutination of cells, degranulation, and loss of cellularmembranes compose the major form of coagulation in the hemolymphof Gecarcinus lateralis. It is only after agglutination of theformed elements of the hemolymph that fibrin-like strands appear.Sodium citrate, in a concentration of 10% or more to preventcoagulation, is always inadequate to prevent cell agglutination. Multiple studies by protein electrophoresis failed to revealany differences between plasma and serum, nor did they allowus to identify a soluble protein in plasma that did not appearin serum. Crab hemolymph changes in its capacity for clottingduring the molt cycle, with the most rapid clotting occurringin the premolt period. A new protein appears in the premoltperiod, but its relation to the whole clotting mechanism isunknown. In contradistinction to vertebrate systems, citrated hemolymphdoes not clot when calcium is added. There is no relationshipthat can be demonstrated between activating systems in vertebrateplasma and clotting in the crab. It would seem that, ratherthan the vertebrate coagulating system evolving from the crustaceantype of clotting system, the development of these clotting systemshas run in parallel. The crustacean cell, in addition, appearsto be more potent than vertebrate cells in clotting systems.The comparison of human lymph to crustacean hemolymph wouldindicate that, for a given amount of cells, crustacean hemolymphclots 2 to 20 times faster than human lymph. On the other hand,agglutination of cells is a fundamental initiating step in coagulationof both human blood and crustacean hemolymph.     

Characterization of limb autotomy factor-proecdysis (LAF(pro)), isolated from limb regenerates,that suspends molting in the land crab Gecarcinus lateralis

Molting and limb regeneration are tightly coupled processes, both of which are regulated by ecdysteroid hormone synthesized and secreted by the Y-organs. Regeneration of lost appendages can affect the timing and duration of the proecdysial, or premolt, stage of the molt cycle. Autotomy of all eight walking legs induces precocious molts in various decapod crustacean species. In the land crab Gecarcinus lateralis, autotomy of a partially regenerated limb bud before a critical period during proecdysis (regeneration index <17) delays molting so that a secondary limb bud (2 degrees LB) forms and the animal molts with a complete set of walking legs. It is hypothesized that 2 degrees LBs secrete a factor, termed limb autotomy factor-proecdysis (LAF(pro)), that inhibits molting by suppressing the Y-organs from secreting ecdysone. Molting was induced by autotomy of eight walking legs; autotomy of primary (1 degrees ) LBs reduced the level of ecdysteroid hormone in the hemolymph 73% by one week after limb bud autotomy (LBA). Injection of extracts from 2 degrees LBs, but not 1 degrees LBs, inhibited 1 degrees LB growth in proecdysial animals, thus having the same effect on molting as LBA. The inhibitory activity in 2 degrees LB extracts was stable after boiling in water for 15 min, but was destroyed by boiling 15 min in 0.1 N acetic acid or incubation with proteinase K. These results support the hypothesis that LAF(pro) is a peptide that resembles a molt-inhibiting hormone.     

Fine Structure of Salt and Water Uptake in the Land-Grab, Gecarcinus lateralis

The salt-absorbing tissue is found in the respiratory lamellaeof the gills in the form of a highly interdigitated epithelium.The folds of the epithelium are supplied with mitochondria inthe form of "mitochondrial pumps." Intercellular spaces betweenthe folds also satisfy the morphological requirements for Diamond'smodel system for water transport based on his theory of standingosmotic gradients. Most of the osmoregulatory tissue is localizedin the three most posterior gills, which in turn rest on thepericardial sac. It is suggested that the pericardial sac transfersground water to the gills for salt and water absorption. Thepericardial sac serves for storage of water and as a hydraulicassist during the molt.     

Transport of Oxygen by the Blood of the Land Crab, Gecarcinus lateralis

Parameters relating to transport of oxygen were measured inthe pericardial blood and venous outflow from the first walkingleg of Gecarcinus lateralis. O₂-equilibrium curves of the hemocyaninof G. lateralis were found to be sigmoid and, at 27°C andpH 7.45, to have a half-saturation pressure of about 17 mm Hgoxygen. Average partial pressures of oxygen as measured by O₂-electrodewere 32 mm Hg in pericardial blood and 9 mm Hg in the venoussamples. Analysis of the O₂-content in corresponding samplesby the Van Slyke technique revealed an average of 2.17 volumes% O₂-capacity for whole blood, 1.45 volumes % for pericardialblood, and 0.61 volumes % for venous blood. Estimates basedon the Van Slyke analyses indicated an average pO₂ of 29 and14 mm Hg in pericardial and venous samples, respectively. Thesefigures agree fairly well with those obtained by means of O₂-electrodes.Of the oxygen carried to the tissues, about 94% is carried asoxyhemocyanin and about 6% is carried in physical solution.As the blood passes through the gills, the hemocyanin, on anaverage, becomes 80–85% saturated with oxygen and returnedfrom the tissues 18–45% saturated with oxygen. These resultsindicate that the blood of G. lateralis has a higher O₂-capacitythan the blood of most other decapod crustaceans for which similarinformation is available. In addition, the blood of G. lateralistransports more oxygen to the tissues per unit volume than doother crustacean bloods.