Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Purification and characterization of phenoloxidase from brine shrimp Artemia sinica

Phenoloxidase from Artemia sinica (AsPO) was purified by Superdex 200 gel-filtration and Q Sepharose fast flow ion-exchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50°C, respectively, for its PO activity. The AsPO had an apparent K(m) value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to cysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-diphenoloxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu(2+), indicating that AsPO is most probably a copper-containing metalloenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing O-diphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.     

Lipid composition of three geographical sources of brine shrimp nauplii (Artemia sp.)

• 1.1. Percentage of triacylglycerols (TG), free fatty acids (FFA) and phospholipids (PL) in the total lipids, the fatty acid composition of each of these lipid classes, and the percentage of cholesterol were determined by gas chromatography in three geographical sources (San Francisco Bay, SFB; Chinese, CH; Colombian, COL) of brine shrim (Artemia sp.) nauplii.
• 2.2. There were no significant differences among sources of brine shrimp in total lipids, TG or FFA with means for all sources of 17.8, 65.8 and 10.9%, respectively. Percentage of phospholipid was significantly higher in SFB and CH sources of brine shrimp, 25.1 and 26.5%, respectively, than in COL 18.3%.
• 3.3. Marked differences in percentages of 18:3 (n-3) (linolenic acid) and 20:5 (n-3) (eicosapentaenoic acid or EPA) were found among brine shrimp sources, and concentration of these two fatty acids were usually inversely related within sources. The CH source contained higher concentrations of EPA ( > 9.0%) than the COL and SFB sources (< 5.0%) in all three lipid classes analyzed. No 22:6 (n-3) (docosahexaenoic acid or DHA) was found in any brine shrimp source.
• 4.4. Fatty acid compositions of the TG and PL were similar and did not differ among sources of brine shrimp, while the FFA had a lower percentage of polyunsaturated fatty acids, but was similar among sources of brine shrimp.
• 5.5. Differences in n-3 fatty acid composition indicated a difference in nutritional quality among sources of brine shrimp for feeding larval fish.

     

The primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia.

cDNA as well as amino acid sequencing has revealed the complete primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia. A comparison with the published sequences of bacterial EF-Tu, mitochondrial EF-Tu and chloroplastic EF-Tu shows that distinct areas of these polypeptide chains are conserved in evolution. The evolutionary distance between prokaryotic and eukaryotic types of EF-Tu is larger than among bacterial and organellar EF- Tus . A number of regions present in both EF-Tu and EF-G from Escherichia coli are also found in EF-1 alpha from Artemia.     

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.     

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi

Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol‐grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth‐inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing‐regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing‐regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.     

Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia.

Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.     

Exiguobacterium mexicanum sp. nov. and Exiguobacterium artemiae sp. nov., isolated from the brine shrimp Artemia franciscana

Two Gram-positive strains isolated from cysts of the brine shrimp Artemia franciscana were subjected to a polyphasic taxonomic analysis. Based on 16S rRNA gene sequence comparison and composition of isoprenoid quinones, peptidoglycan and fatty acids, these organisms are members of the genus Exiguobacterium. Both strains showed 95.9% 16S rRNA gene sequence similarity to one another. The 16S rRNA gene sequences of strain 8N(T) and 9AN(T) were 97.5% and 98.9% similar to those of Exiguobacterium aurantiacum DSM 6208(T) and Exiguobacterium undae DSM 14481(T), respectively. Based on differences in chemotaxonomic and physiological characteristics, results of DNA-DNA hybridization and automated riboprinting, two novel species of the genus Exiguobacterium are proposed, Exiguobacterium mexicanum sp. nov. (type strain 8N(T)=DSM 16483(T)=CIP 108859(T)) and Exiguobacterium artemiae sp. nov. (type strain 9AN(T)=DSM 16484(T)=CIP 108858(T)).     

Antennular sensilla of the brine shrimp, Artemia salina.

1. Scanning electron microscopy was used to characterize the external morphology of setae found on the antennules of adults and nauplii of the brine shrimp, Artemia salina (L.). The permeability of the antennular setae was studied by means of Slifer's crystal violet method. 2. Each antennule of an adult brine shrimp possessed a terminal cluster of sensory setae. Within a cluster there were two morphologically distinct kinds of sensilla, here designated type 1 and type 2. Three type 1 sensilla were observed on every antennule examined. The number of type 2 sensilla per antennule was usually four or five. 3. Type 1 sensilla of adults were 43 to 80 micrometer long and simple in external morphology. They were widest at the base, decreased in diameter gradually, and terminated as a finely tapered tip. No pores were resolved by scanning electron microscopy. 4. Type 2 sensilla of adults were shorter (shaft length, 12 to 23 micrometer) and displayed a single pore at the tip (average pore diameter, 0.4 micrometer). In thin section they were seen to possess a distinctive articular specialization of the cuticle at the base of the seta. 5. Dye penetration experiments indicated that type 2 sensilla were permeable to aqueous crystal violet, whereas type 1 sensilla were not. 6. The antennular setae of nauplii resembled type 1 sensilla in general shape, in being impermeable to crystal violet, and in lacking a terminal pore and basal articular specialization. Moreover, a total of three setae was normally present on each naupliar antennule, and the same number of type 1 sensilla was found on each adult antennule examined. If the three naupliar setae represent a developmental stage in the formation of three adult sensilla, available observations suggest that the larval setae are developmentally related to type 1, rather than to type 2 adult sensilla.     

The patterning function of purines in the brine shrimp Artemia

In two Artemia wild-type sibling species originating from the Old and New Worlds, the processes underlying the construction of the naupliar body during embryonic development and the construction of the adult body during postembryonic development are disrupted by specific nutritional deficiencies and/or the administration of metabolic inhibitors. The species-specific phenotypic outcomes of these experimental disruptions on the construction of segments and the establishment of their identity, permit us to outline a model in which Hox genes would act as intermediary cogwheels fastened to a mechanism put in gear upstream by purine-mediated processes which would trigger downstream folic acid-mediated processes. The prevalent view that Hox genes can select for different developmental programmes, is challenged by this model whose relevancy is analysed in the context of our present knowledge on the master functions ascribed to Hox genes in developmental and evolutionary processes.     

A note on the brine shrimp Artemia in Tuz Lake (Turkey)

Population density, fecundity andreproduction period of Artemia sp. in TuzLake, Turkey, beside environmental factors, such as oxygen,temperature, salinity, pH and electrical conductivity werestudied during 1993 and 1994.Artemia sp. occurred at two stations at the coast.Overwintering cysts hatched in early May, when the population was composed of 90% metanauplii (93%at Y4 and 99% at Y5) and few adult females. Peak abundanceoccurred in late May, at 106to 114 ind.m^–3.     

The development of the brine shrimp Artemia salina (L.): a morphometric approach

Growth allomentry in the development of the brine shrimp Artemia salina (L.) was studied. Body width and abdomen length show a negative allometric relation to body length ( k =0.35 and 0.72 respectively) over th eight instars studied. Antennule length shows a negative allometric relation in respect to body length, with a change in the value of the growth constant at Instar Five from 0.31 to 0.75. Thoracic limb length shows a high positive relation to body length ( k =3.0) until Instar Six, when a negative allometric phase ( k =0.4) follows. A change in the sign of the growth constant from positive ( k =0.45) to negative ( k =-1.4) is noted for the length of the 2nd antenna in relation to body length.     

Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.     

Characterisation of a bis(5'-nucleosidyl) triphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia.

1. A P1,P3-bis(5'-nucleosidyl)triphosphate pyrophosphohydrolase (Np3 Nase) has been partially purified from Artemia embryos. 2. The Np3 Nase has a native Mr of 115,000 and preferentially hydrolyses substrates of the form Np3 N. Relative rates of hydrolysis are Ap3A (Vrel = 1.0), Gp3G (Vrel = 0.71), Ap4A (Vrel = 0.08), Ap5A (Vrel = 0.09), Gp4G (Vrel = 0.3) and Gp5G (Vrel = 0.33). An NMP is always one of the products. 3. The Km values for Ap3A and Gp3G are 15 and 10 microM respectively. 4. Mg2+, Mn2+ and Ca2+ ions all stimulate the activity, while Zn2+, Co2+ and Ni2+ ions are inhibitory. 5. The activity of the Np3 Nase remains constant during pre-emergence development of encysted embryos but decreases slightly after hatching.