Endocrine Regulation of Lipid Synthesis in Decapod Crustaceans

The legulation of lipid synthesis in the hepatopancreas of thecrab Pachygrapsus crassipes and the crayfish Procambarus sp.was investigated. Although deatalking induces an increase inthe rate of ¹⁴C-1-acetate incorporation into lipid, injectionsof crustecdysone into intermolt animals fail to elicit a similarresponse. In addition, the increased rate of lipid synthesisinduced by destalking is unaffected by removal of the Y-organ.It would appear then that the increase in lipid synthesis characteristicof early premolt is not controlled by either crustecdysone orthe Y-organ. It is suggested that formation of acetyl-CoA byany one of a number of possible mechanisms may bethe mannerin which the increase in fatty acid synthesis is effected duringpremolt.     

Presence and development of a sensory formation in the eyestalk of the crayfish,Astacus leptodactylus (Nordmann, 1842), during its embryonic,larval and adult life

A sensory papilla is described in the eyestalk of the crayfish Astacus leptodactylus during the last embryonic stages and during larval stages by light microscopy. This region was also investigated with the scanning electron microscopy, which showed sensory hairs in the postmolt adult; they disappear during intermolt and premolt. Simultaneous cyclic changes in hair papillae are observed in the hypodermis. The possibility of a chemoreceptive function is discussed.     

Hormonal Effects on Calcium Metabolism in Crustacea

Cyclic shifts of calcium in the exoskeleton and soft tissues,as they are related to the intermolt cycle in crayfish, arereviewed. Regulatory factors, derived from the eyestalk, influencelevels of exoskeletal calcium; eyestalk extracts prepared fromanimals in premolt decrease shell calcium, while reciprocallyextracts from animals in intermolt increase it when these hormonalsources are injected into animals in the premolt stage (D₀-D₄). In addition, premolt eyestalk extract results in an increasein gastrolith calcium. In the exchange of calcium between theanimal and its environment there is evidence for differentialdepositionof recently available calcium in the exoskeleton. Further, intermoltand early premolt animals maintained in Ca⁴⁵-labelled waterfor 15 days concentrate it 4 and 3—fold in the exoskeletonand stomach, respectively. However, removal of a molt-inhibitingfactor through ablation of eyestalks results in a 20 and 40—foldincrease in incorporation inthese same sites relative to environmentalconcentrations. Treatment with mammalian parathyroid extract mobilizes bothexoskeletal and gastric calciumand leads to a rise in bloodcalcium. However, there is little or no effect on levels ofexoskeletal citric acid. Further, citric acid is higher in thecrayfish carapace during stage C, the period of mineralization,than in stage D, the period of demineralization. There are both similarities and differences between the effectsof crustacean and mammalianregulating factors with respect tothe direction and extent of mineralization. Biochemical studiesshould elucidate the mechanisms regulated by these hormones.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfish Procambarus clarkii.

The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of crayfish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Characterization of a Matrix Protein in the Gastroliths of the Crayfish Procambarus clarkii

As a first step in understanding the calcification mechanism, a matrix protein in the gastrolith of the crayfish Procambarus clarkii was purified and sequenced. The protein was insoluble in acid, but after trypsin digestion, it dissolved in 6 m urea. The trypsin-digested protein dissolved in urea solution was purified by reversed-phase HPLC and designated gastrolith matrix protein fragment. The fragment had a molecular weight of 9658 and a blocked amino terminus. It had tandemly repeated units not reported before at the central part of the sequence, with each unit being Gly-Ser-X1-X2-Phe as the most typical sequence. This peptide was found associated with chitin, a main component of the organic matrix.     

Isolation of crustecdysone (20R-hydroxyecdysone) from a crayfish (Jasus lalandei H. Milne-Edwards)

1. A small amount (2mg.) of crustecdysone, a moulting hormone of crustaceans, was isolated from 1 ton of crayfish waste. 2. The purification procedure used was developed with the aid of crustacean and insect bioassays. 3. CM-Sephadex was found to be superior to Sephadex and very effective for the chromatographic separation of crustecdysone from other non-ionic compounds. The higher efficiency of CM-Sephadex is attributed to the greater number of carboxyl groups available for hydrogen-bonding. 4. Reversed-phase chromatography, with butan-1-ol-cyclohexane mixtures as the stationary phase and water as the flowing phase, proved superior to countercurrent distribution with these solvents for the fractionation of purified extracts. 5. A second moulting hormone, deoxycrustecdysone, and the red-concentrating hormone were obtained in a partially purified form.     

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle. II. Cytochemical demonstration of Ca2+-ATPase and Mg2+-ATPase

Periodical changes in Ca2+-ATPase and Mg2+-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca2+-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt , when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg2+-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca2+-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg2+-ATPase activity. There results provide the strong evidence that Ca2+-ATPase may act strongly in the calcium transport system of crayfish molting.     

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle

Summary Periodical changes in Ca²⁺-ATPase and Mg²⁺-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca²⁺-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt, when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg²⁺-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca²⁺-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg²⁺-ATPase activity. These results provide the strong evidence that Ca²⁺-ATPase may act strongly in the calcium transport system of crayfish molting.     

Measurement of molt-inhibiting hormone titer in hemolymph of the American crayfish,Procambarus clarkii,by time-resolved fluoroimmunoassay

In order to determine the titer of molt-inhibiting hormone (Prc-MIH) in the hemolymph of the American crayfish Procambarus clarkii, a time-resolved fluoroimmunoassay (TR-FIA) was established using specific antibodies against N-terminal and C-terminal segments of Prc-MIH. The lowest limit of detection of Prc-MIH in TR-FIA was 10 amol/assay. The Prc-MIH titers in the hemolymph were 6.53 fmol/ml at the intermolt stage and 1.25 fmol/ml at the early premolt stage. This result is consistent with the long-known hypothesis that the Y-organ is inhibited by MIH during the intermolt stage, whereas the Y-organ is activated by being freed from the inhibitory regulation of MIH.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfishProcambarus clarkii

Summary The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of cryafish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

Differential growth of crayfish Procambarus alleni in relation to hydrological conditions in marl prairie wetlands of Everglades National Park, USA

Environmental conditions influence crustacean growth by affecting molt intervals and incremental increases in length and weight. In the seasonally-flooded marl prairie wetlands of eastern Everglades National Park, U.S.A., hydropattern exerts considerable influence on aquatic primary productivity, and so may influence the availability of food resources for higher trophic levels. The seasonal hydroperiod has been drastically altered by anthropogenic factors, but the impacts on the aquatic community are not well known. We studied whether differences in growth of crayfish Procambarus alleni could be detected in habitats with different hydroperiods. We first described growth patterns based on incremental increases in length and weight of crayfish on a high protein diet in the laboratory. Regression analyses indicated that growth patterns in males and females were similar. Although the intermolt period increased with age, the proportional increases in length and weight were similar through successive molts. The relationship between length and weight of crayfish was best described by a power equation for allometric growth. We then compared growth curves for crayfish subpopulations from different areas of the marl prairie. In habitats with the longest hydroperiods, crayfish weight-at-size was not significantly different from that in laboratory crayfish on the high protein diet. However, weight gain per unit increase in length in short hydroperiod sites was significantly less than in long hydroperiod sites or in the laboratory. These results indicate that crayfish productivity may be associated with hydroperiod in these stressed wetlands, and this may contribute to observed source-sink population regulation.     

Adaptation of the Common Ice Plant to High Copper and Zinc Concentrations and Their Potential Using for Phytoremediation

A facultative halophite Mesembryanthemum crystallinum L. (the common ice plant) was shown to grow successively at the high concentrations of Cu and Zn. Although 25 μM CuSO₄ or 800 μM ZnSO₄ retarded markedly plant growth, they did not interfere with the completion of plant development and the formation of viable seeds. In such plants, leaves accumulated more than 200 μg of Cu and 1700 μg of Zn per 1 g of dry weight. A damaging effect of heavy metals (HMs) was manifested in a reduced content of water in leaves and proline accumulation in them. As copper is a metal with transient valence, copper salts are more toxic than zinc salts, which was manifested in a stronger inhibition of the chlorophyll synthesis. Both HMs induced oxidative stress, as evident from increased activities of guaiacol peroxidase and lipoxygenase. Moderate Cu and Zn concentrations did not damage cell membranes in leaves, as evident from the absence of their action on electrolyte leakage either under optimum conditions or after heat treatment. A capability of a substantial HM accumulation by the common ice plant and their considerable transport to shoots (up to 50 μg of Cu and 560 μg of Zn per plant) make it possible to consider the common ice plant as a promising phytoremediator. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 848–858. Original Russian Text Copyright ? 2005 by Kholodova, Volkov, Kuznetsov.     

Direct and Indirect Shoot Organogenic Pathways in Epicotyl Cuttings of Troyer Citrange Differ in Hormone Requirements and in their Response to Light

Bud differentiation by direct organogenesis at the apical endof Troyer citrange (Citrus sinensis[L]. OsbeckxPoncirus trifoliata[L].Raf.) epicotyl cuttings inserted vertically in a semi-solidculture medium did not require hormone additions. The numberof buds regenerated was slightly, but significantly, increasedwhen the incubation was performed in the light as compared tothe dark, and by the addition of benzyladenine (BA; 2.2 to 22µM) to the medium. Bud sprouting and subsequent shootformation required the addition of BA and was increased by lightto a higher extent than bud formation. The best response wasobtained with the highest BA concentration tested (22 µM).Regeneration through the indirect organogenic pathway at thetwo edges of the epicotyl cuttings when in contact with theculture medium did not occur in the absence of benzyladenine,which was an absolute requirement for callus development. Thebest regeneration response was obtained when the explants wereincubated in the light in the presence of 4.4 µM BA andan auxin. Indole-3-acetic acid (IAA; 5.8 µM) was moreeffective in increasing shoot formation than naphthaleneaceticacid (NAA; 0.54 µM). Higher NAA concentrations inhibitedshoot formation. Incubation in the dark or increasing the BAconcentration (22 µM) increased markedly callus growth,but inhibited both bud differentiation and sprouting, almostcompletely suppressing shoot formation. The conditions duringregeneration affected the rooting of the regenerated shoots.Rooting of 86% of the shoots was achieved in a medium with 2.7µM NAA and 2.6 µM indole-3-butyric acid. All therooted explants acclimated and survived transplanting. Underthe optimal conditions tested, the proliferation rate obtainedthrough the indirect regeneration pathway ranged from 60 to86 plants per seedling. Copyright 2000 Annals of Botany Company Troyer citrange, Citrus sinensisxPoncirus trifoliata, auxins, benzyladenine, direct organogenesis, hormone requirement, indirect organogenesis, light, morphogenesis, rooting.     

EFFECTS OF VARIOUS FACTORS ON THE INCREASE OF {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM INDUCED BY GIBBERELLIN A3

The increase of -amylase activity in embryoless rice endosperminduced by the addition of gibberellin A₃ was examined undervarious conditions with an aim to establish a bioassay methodfor gibberellins. Sterilized embryoless rice endosperms were incubated in a testtube containing 0.2–1.0 ml of test solution for 4 daysat 30. -Amylase activity in the endosperm was determined bymeasuring digestion of added starch. The increase of amylaseactivity during the incubation was not affected by the additionof various vitamins, amino acids, organic acids, protease, sucrose,indoleacetic acid or kinetin. Helminthosporol, helminthosporicacid and sclerin (1–10 µg/ml) had weak promotingeffects. Under appropriate conditions, 10^–5 µg/mlof gibberellin A₃ could be detected. In double logarithmic plot,the increase in the enzyme activity was proportional to thegibberellin A₃ concentration in the range from 10^–5 to10^–2 µg/ml. (Received April 13, 1966; )     

Human growth hormone peptide 1–43: Isolation from pituitary glands

A procedure is described for isolation from human pituitary glands of a peptide with the amino acid sequence of the first 43 residues of human growth hormone, hGH (1–43). The peptide was recovered in a yield of 12 mg per 1000 pituitary glands. In a radioimmunoassay for hGH the peptide was unreactive when tested at 1 µg/ml and therefore if it does circulate, it probably has gone underected in blood. Growth-promoting activity as measured by the tibial line procedure was low but detectable; however, the log dose response was not parallel to that of hGH. Separate studies indicated the hGH (1–43) was a potent potentiator of insulin action suggesting physiologic significance. Further, because the peptide can be a cleavage product of the major form of hGH but not of its 20,000-dalton variant, importance is indicated for the multiple forms of the hormone.     

Purification and properties of alanine racemase from crayfish Procambarus clarkii

Fresh water crayfish Procambarus clarkii is known to accumulate d-alanine remarkably in muscle after seawater acclimation, accompanied by an increase in alanine racemase activity. We have purified alanine racemase from crayfish muscle to homogeneity. The enzyme is a monomeric protein with a molecular mass of 58 kDa. It is highly specific to alanine and does not racemize l-serine, l-aspartate, l-glutamate, l-valine and l-arginine. The enzyme shows the highest activity at pH 9.0 in the conversion of l- to d-alanine and at pH 8.5 in the reverse conversion. Properties such as amino acid sequence, quaternary structure, pyridoxal 5′-phosphate (PLP)-dependency, pH-dependency and kinetic parameters seem to be distinct from those of the microbial alanine racemases. Various salts including NaCl at concentrations around seawater level were potently inhibitory for the activity in both of l- to -d and d- to -l direction.     

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.