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.     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

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 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.     

Characterization of polymorphic microsatellite markers in the brine shrimp Artemia (Branchiopoda, Anostraca)

The brine shrimp Artemia is a complex genus containing sexual species and parthenogenetic lineages. Artemia franciscana is native to America and its cysts (diapausing eggs) are used worldwide as a food source in aquaculture. As a consequence, this anostracan has become an invasive species in many hypersaline aquatic ecosystems of other continents. Parthenogenetic Artemia lineages occur only in the Old World. Ten and five microsatellite markers were developed to characterize two populations for A. franciscana and two populations for diploid parthenogenetic Artemia, respectively. For A. franciscana the number of alleles ranged from 11 to 58 per locus, while for parthenogens the number of alleles ranged from three to 10. The levels of heterozygosity in A. franciscana and in parthenogens ranged from 0.115 to 0.976 and from 0.000 to 0.971, respectively. These microsatellite loci showed a high population assignment power, which will be useful for future studies of population genetics and invasive processes in Artemia.     

Recognition of beta beta'-substituted and alpha beta,alpha'beta'-disubstituted phosphonate analogues of bis(5'-adenosyl) tetraphosphate by the bis(5'-nucleosidyl)-tetraphosphate pyrophosphohydrolases from Artemia embryos and Escherichia coli

A total of 13 phosphonate analogues of bis(5'-adenosyl) tetraphosphate (AppppA) have been tested as substrates and inhibitors of the asymmetrically cleaving bis(5'-nucleosidyl) tetraphosphatase (NppppNase) from Artemia and the symmetrically cleaving NppppNase from Escherichia coli. With the Artemia enzyme, the substrate efficiency of beta beta'-substituted compounds decreased with decreasing substituent electronegativity (O greater than CF2 greater than CHF greater than CCl2 greater than CHCl greater than CH2) such that AppCF2ppA and AppCH2ppA were hydrolyzed at 70% and 2.5% of the rate of AppppA, respectively. These compounds were competitive inhibitors of this enzyme with Ki values that generally also decreased with electronegativity from 12 microM for AppCF2ppA to 0.4 microM for AppCH2ppA (Km for AppppA = 33 microM). AppCH = CHppA and AppCH2CH2ppA were neither effective substrates nor inhibitors of the Artemia enzyme. Alpha beta,alpha'beta'-Disubstituted analogues were generally less effective inhibitors with Ki values ranging from 23 microM (ApCH2ppCH2pA) to greater than 1.5 mM (ApCH2CH2ppCH2CH2pA). However, they displayed a low and unexpected rate of symmetrical cleavage by the Artemia enzyme: e.g., ApCHFppCHFpA yielded ApCHFp at 3% of the rate of AppppA breakdown. Both sets of analogues were also competitive inhibitors of the E. coli NppppNase with Ki values ranging from 7 microM (AppCH2ppA) to 250 microM (ApCH2CH2ppCH2CH2pA) (Km for AppppA = 28 microM). The only alpha beta,alpha'beta'-disubstituted analogue to be hydrolyzed by the E. coli enzyme was ApCF2ppCF2pA at 0.2% of the rate of AppppA; however, several of the beta beta'-substituted compounds showed a limited degree of asymmetrical cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of brine shrimp Artemia from Cam Ranh Bay in Central Vietnam

Artemia cysts collected from inoculation experiments in Cam Ranh salterns are evaluated for their potential use in aquaculture. Cyst biometrics, hatching quality, naupliar fatty acid profile and naupliar growth were measured and compared to reference Artemia strains. Cyst characteristics reveal that the parthenogenetic strain (PR China) used in inoculations, was eliminated from the environment and that the remaining brine shrimp are likely to be composed of Macau and Great Salt Lake Artemia strains, and of their cross-breds. Differences in cyst diapause deactivation characteristics between Macau and Great Salt Lake Artemia may have resulted in the disappearance of Macau Artemia during the rainy season and the persistence of Great Salt Lake Artemia during the following dry season.     

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.     

mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina.

Cell-free protein-synthesizing extracts prepared from the brine shrimp, Artemia salina, translate methylated mRNAs. Reovirus unmethylated mRNA is inactive as a template when methylation is prevented by the inhibitor, S-adenosylhomocysteine. A salina mRNAs from both undeveloped and developed embryos contain 5'-terminal 7-methylguanosine in an inverted 5'-5' linkage through three phosphate groups to the rest of the polynucleotide chain. Removal of the 7-methylguanosine by beta elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ. Extracts of undeveloped and developed embryos methylate reovirus unmethylated mRNA at the 5' ends to form 5'-terminal structures of the type, m7G(5')ppp(5')G and m7G(5')ppp(5')Gm.     

Hydrolysis of bis(5''-nucleosidyl) polyphosphates by Escherichia coli 5''-nucleotidase.

Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'-P1,P2-diphosphate,diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied.     

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.     

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.     

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.     

Molecular phylogenetics and asexuality in the brine shrimp Artemia

Explaining cases of long-term persistence of parthenogenesis has proven an arduous task for evolutionary biologists. Interpreting sexual-asexual interactions though has recently advanced owing to methodological design, increased taxon sampling and choice of model organisms. We inferred the phylogeny of Artemia, a halophilic branchiopod genus of sexual and parthenogenetic forms with cosmopolitan distribution, marked geographic patterns and ecological partitioning. Joint analysis of newly derived ITS1 sequences and 16S RFLP markers from global isolates indicates significant interspecific divergence as well as pronounced diversity for parthenogens, matching that of sexual ancestors. Maximum parsimony, maximum likelihood, and Bayesian methods were largely congruent in reconstructing the phylogeny of the genus. Given the current sampling, at least four independent origins of parthenogenesis are deduced. Molecular clock calibrations based on biogeographic landmarks indicate that the lineage leading to A. persimilis diverged from the common ancestor of all Artemia species between 80 and 90 MYA at the time of separation of Africa from South America, whereas parthenogenesis first appeared at least 3 MYA. Common mitochondrial DNA haplotypes delineate A. urmiana and A. tibetiana as possible maternal parents of several clonal lineages. A novel topological placement of A. franciscana as a sister clade to all Asian Artemia and parthenogenetic forms is proposed and also supported by ITS1 length and other existing data.     

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.