Delayed onset of midline netrin expression in Artemia franciscana coincides with commissural axon growth and provides evidence for homology of midline cells in distantly related arthropods

Although many similarities in arthropod central nervous systems (CNS) development exist, differences in midline cell formation and ventral nerve cord axonogenesis have been noted in arthropods. It is possible that changes in the expression of axon guidance molecules such as Netrin, which functions during commissural axon guidance in Drosophila and many other organisms, may parallel these differences. In this investigation, we analyze this hypothesis by examining Netrin accumulation during development of the brine shrimp Artemia franciscana, a branchiopod crustacean. An Artemia franciscana netrin (afrnet) orthologue was cloned. An antibody to the afrNet protein was generated and used to examine the pattern of afrNet accumulation during Artemia development. Despite differences between Drosophila and Artemia nerve cord development, examination of afrNet accumulation suggests that this protein functions to regulate commissure formation during Artemia CNS development. However, detection of afrNet at the midline and on commissural axons occurs at a relatively later time point in Artemia as compared with Drosophila. Detection of afrNet in a subset of midline cells that closely resemble Netrin-expressing cells at the Drosophila midline provides evidence for homology of midline cells in arthropods. Expression of Netrins in many other tissues is comparable, suggesting that Netrin proteins may play many conserved roles during arthropod development.     

Developmental and comparative aspects of brine shrimp tubulin.

Tubulin from embryos of the brine shrimp Artemia has been purified to apparent homogeneity by chromatography on phosphocellulose P11 and DEAE-cellulose, (NH4)2SO4 fractionation and assembly-disassembly of microtubules. Peptide mapping indicated that Artemia and bovine brain tubulin were very similar in spite of differences in the electrophoretic behaviour of tubulin from these two organisms. Isoelectric focusing and two-dimensional gel electrophoresis were used to resolve and identify several Artemia isotubulins . The isotubulin composition and the quantity of tubulin did not change during pre-emergence development of Artemia embryos. Formation of microtubules with tubulin purified from embryos at different stages of development did not require glycerol or microtubule-associated proteins and formation of structurally normal microtubules was actually hindered by glycerol and Mg2+. The characteristics of Artemia tubulin, in concert with the unusual life history of Artemia, suggest that this organism will be very useful for the study of tubulin gene expression and tubulin utilization during embryo development.     

Isolation of cDNA clones coding for mitochondrial 16S ribosomal RNA from the crustacean Artemia

cDNA clones coding for Artemia mitochondrial 16S ribosomal RNA (rRNA) have been isolated. The clones cover from nucleotide 650 of the RNA molecule to its 3' end. The comparison of Artemia sequence with both vertebrate and invertebrate mitochondrial 16S rRNA sequences has shown the existence of regions of high similarity between them. A model for the secondary structure of the 3' half of Artemia mitochondrial 16S rRNA is proposed. The size of the rRNA molecule has been estimated at 1.35 kb. Despite the similarity of the Artemia gene to insect rRNA in size, sequence and secondary structure, the G + C content of the Artemia gene (42%) is closer to that of mammals than to the insect genes. The number of mitochondria in Artemia has been estimated at 1500 per diploid genome in the cyst and 4000 in the nauplius. In contrast, the amount of mt 16S rRNA is constant at all stages of Artemia development.     

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 occurrence of juvenile pigmentation abnormalities in plaice (Pleuronectes platessa) larvae fed on enriched and unenriched Artemia salina nauplii

The effect of diet on the frequency of malpigmentation was investigated in laboratory-reared plaice ( Pleuronectes platessa ) fed on either enriched or unenriched Artemia salina nauplii . The juveniles fed continuously on unenriched Artemia during their larval period showed 29.0% normal pigmentation whereas those fed solely on enriched Artemia , showed 63% normal pigmentation. The possibility of a critical period for pigment development was tested by changing the diet of larvae at different ages. Results suggested that a critical period is less important than the duration of time fed on a nutritionally complete diet.     

Composition of fat acids in three Mexican populations of Artemia franciscana from epicontinental waters

In this paper is presented the percentage of fatty acids composition of three Artemia franciscana Mexican populations of epicontinentals waters; two are from natural environments (Coahuila and San Luis Potosf) and one (Texcoco) is a culture fed with Spirulina. Determination of fatty acids composition in each population, was performed by extraction of total lipid by the soxhlet method and the fatty acids methyl esters were determined by gas chromatography. The results show that Artemia of Texcoco contains the six fatty acids recommended for the culture of fish and crustaceans (16:0; 16:1; 18:1; 18:2w6; 18:3w3 and 20:5w3); Artemia from San Luis Potosi showed the poorest content in these acids and Artemia from Coahuila, although it showed a wide profile, it lacks the linolenic acid. When comparing results among the three populations with ecological data that have been published, it can be pointed out that the environment is decisive for this crustacean; Artemia from Texcoco fed with Spirulina showed the largest variety of fatty acids; the other two populations are wild, and lives in different habitats, Artemia of Coahuila is found in waters that are rich in sulfates and Artemia of San Luis Potosf lives in evaporation saltern ponds, built with stone blocks and therefore with scarce phytoplankton growth. Both Artemia populations showed deficiencies in essential fatty acids, mainly the last one.     

Critical involvement of dietary docosahexaenoic acid in the ontogeny of schooling behaviour in the yellowtail

To investigate involvement of the central nervous system in the ontogeny of behaviour, diets of differing quality were used to rear yellowtail Seriola quinqueradiata larvae and juveniles. Artemia nauplii enriched with oleic acid (OA), eicosapentaenoic acid (EPA), or two different concentration levels of docosahexaenoic acid (DHA) were fed to yellowtail larvae (L^T 7 mm; 13 days old) for 12 days, and their behavioural development was analysed together with growth, survival, activity and fatty acid composition. Yellowtail fed with DHA–enriched Artemia showed mutual attraction behaviour at 11 mm L^T, while those fed with OA– or EPA–enriched Artemia did not show this behaviour at the same size. While fish in the OA group showed poor growth, survival and activity index, fish in the EPA group were similar to the two DHA groups. In addition, most fish tested, including the OA group, showed clear optokinetic responses. Fatty acid composition of the diet was reflected in that of the fish body. Therefore, dietary DHA in the larval stage is considered to be essential for the development of schooling behaviour in the yellowtail.     

Characterization of the microtubule proteome during post-diapause development of Artemia franciscana

The microtubule proteome encompasses tubulin and a diverse group of proteins which associate with tubulin upon microtubule formation. These proteins either determine microtubule organization and function or their activity is influenced by microtubule association. To characterize the microtubule proteome in Artemia franciscana, tubulin assembly was induced with taxol in vitro after 0 and 12 h of post-diapause development. Proteins obtained by extraction of microtubules with 0.5 M NaCl were electrophoresed in two-dimensional gels and analyzed by mass spectrometry. Fifty-five proteins were identified with 10 of these occurring at both developmental stages, and multiple isoforms were observed for some proteins of the Artemia proteome. Their functions include roles in membrane transport, metabolism, chaperoning and protein synthesis, thus reflecting physiological properties of encysted Artemia such as stress resistance and the ability to rapidly initiate post-diapause development. For example, chaperones may protect tubulin during encystment and facilitate folding in metabolically active embryos. Additionally, the interaction of metabolic enzymes with microtubules funnels reaction intermediates, potentially enhancing efficiency within biochemical processes. This study represents the first systematic characterization of a crustacean microtubule proteome. Although it is difficult to be certain that all protein associations documented herein occur in vivo, the results suggest how protein-protein interactions contribute to cytoplasmic organization while implying how Artemia embryos resist stress and remain capable of development once diapause terminates.     

Independent temporal expression of two N-substituted aminoacyl-tRNA hydrolases during the development of Artemia salina.

Encysted embryos of the crustacean Artemia salina contain an enzymatic activity which hydrolyzes N-acetylphenylalanyl-tRNA to N-acetylphenylalanine and tRNA. The enzyme apparently does not hydrolyze other free or N-substituted aminoacyl-tRNAs. The levels of this enzyme do not significantly change during embryonic and early larval development. In contrast, an unspecific hydrolase active on several N-substituted aminoacyl-tRNAs is practically absent in the encysted embryos and during embryogenesis and appears abruptly during larval development. The independent temporal expression of these two hydrolases during Artemia salina differentiation makes this organism siuitable for the study of the physiological role of these enzymes.     

Early dolichyl sugar synthesis during differentiation of non-growing brine-shrimp (Artemia salina) embryos.

Incubation of cell-free extracts of Artemia salina with sugar nucleotides resulted in the formation of dolichyl derivatives. These enzymic activities were detected early in the development of the encysted Artemia embryos. Dolichyl sugars seemed to act as intermediates in the glycosylation of proteins, which may be involved in the initiation of cellular differentiation.     

Molecular chaperones, stress resistance and development in Artemia franciscana

Embryos of the brine shrimp, Artemia franciscana, either develop directly into swimming larvae or are released from females as encysted gastrulae (cysts) which enter diapause, a reversible state of dormancy. Metabolic activity in diapause cysts is very low and these embryos are remarkably resistant to physiological stresses. Encysting embryos, but not those undergoing uninterrupted development, synthesize large amounts of two proteins, namely p26 and artemin. Cloning and sequencing demonstrated p26 is a small heat shock/alpha-crystallin protein while artemin has structural similarity to ferritin. p26 exhibits molecular chaperone activity in vitro, moves reversibly into nuclei during stress and confers thermotolerance on transformed organisms, suggesting critical roles in cyst development. The function of artemin is unknown. Encysted Artemia also contain an abundance of trehalose, a disaccharide capable of protecting embryos. Artemia represent a novel experimental system where the developmental functions of small heat shock/alpha-crystallin proteins and other stress response elements can be explored.     

Diguanosine 5',5'-P1,P4-tetraphosphate and other purine nucleotides inhibit endoribonuclease VI from Artemia

The activity of the endoribonuclease VI from Artemia is sensitive to several purine nucleotides. The enzyme is non-competitively inhibited by diguanosine tetraphosphate (Ki = 75 microM), a nucleotide abundant in Artemia encysted gastrulae and located in the same particulate fraction as the gastrular ribonuclease. Diguanosine triphosphate and diadenosine tetraphosphate are less efficient inhibitors (Ki congruent to 200 microM). The ribonuclease is non-competitively inhibited by 5'-AMP (Ki = 10 microM) and 5'-GMP (Ki = 50 microM) but is insensitive to the corresponding 5'-phosphates of cytosine and uridine. Other purine mononucleotides inhibit the enzyme activity less efficiently. The modulation of the enzyme activity by these nucleotides is discussed in relation with the changes in ribonuclease activity during early development of Artemia.     

Immunodetection of thiol proteinase levels in various populations of Artemia cysts and during development

An immunodetection assay on Western blots has been used to determine the thiol proteinase content and composition in cysts from 12 populations of the brine shrimp Artemia. Our results showed no differences in the subunit composition of the thiol proteinase among cysts from eight bisexual strains and four parthenogenic strains, and confirmed an earlier finding that the proteinase is composed of two subunits of 25.9 and 31.5 kilodaltons. In contrast, we found that Artemia cysts from parthenogenic strains contain 17.1 ng/cyst of the thiol proteinase, while cysts from bisexual strains contain 8.2 ng/cyst of the thiol proteinase. Also, there was a good linear correlation (r = 0.863; p less than 0.001) between the thiol proteinase content and cyst mass. Embryo fractionation experiments showed that 82% of the thiol proteinase was in the cytosol, while 14 and 4%, respectively, were in the nuclei/yolk platelets and mitochondria/lysosome fractions. Measurements of the thiol proteinase content of developing Artemia embryos showed that the proteinase content was relatively constant during early development, suggesting that the activity of the thiol proteinase gene(s) may be constitutive and not developmentally regulated in Artemia embryos.     

Yolk platelets in artemia embryos: are they really storage sites of immature mitochondria?

We have used semi-quantitative polymerase chain reaction (PCR) technology to determine the mitochondrial DNA (mtDNA) content of yolk platelets isolated from embryos of the brine shrimp, Artemia franciscana, and ultrastructural analysis of yolk platelet formation to determine whether these organelles contain mitochondria as reported previously. Using six different isolation and purification protocols, we found one yolk platelet preparation to be devoid of mtDNA, while four yolk platelet preparations contained mtDNA ranging from 16.4 to 85 pg/10(6) yolk platelets. One preparation contained 600 pg mtDNA per 10(6) yolk platelets. Based on our PCR analyses, the mtDNA component of Artemia yolk platelets represented 0.16-4.5% of the total DNA isolated from the platelets. We calculated that Artemia yolk platelets contain, on average, approximately 1.78 molecules of mtDNA/platelet. Direct analysis of mtDNA in "free" mitochondria isolated from yolk platelet-free preparations of Artemia embryos and newly hatched larvae yielded 0.76-0.80 ng/animal. Based on these values, the mtDNA content of yolk platelets was approximately 0.2% of total mtDNA in Artemia embryos. Microscopic analysis of yolk platelet formation during oogenesis in Artemia failed to show the inclusion of mitochondria during the assemblage of yolk platelets. The "mitochondria-like" structures that appear in yolk platelets during their utilization lack the well defined inner and outer membranes characteristic of mitochondria making it unlikely that the yolk platelet inclusions are mitochondria. Our results from PCR technology and ultrastructure analysis demonstrate that mtDNA in yolk platelets of Artemia franciscana embryos is a minor component of the total mtDNA in the embryo, and they fail to support the notion that yolk platelets in Artemia are a major source of immature mitochondria for development.     

Review of the biogeography of the genus Artemia (Crustacea, Anostraca)

In this study, we report on the known Artemia habitats worldwide. Recent literature information is incorporated about the taxonomic status of the various populations studied. The genus is composed of di-, tri-, tetra- and pentaploid parthenogenetic populations and of the following bisexual species: A. franciscana franciscana , A. franciscana monica , A. franciscana sp., A. persimilis , A. salina , A. urmiana , A. sinica and A. sp. from Kazakhstan. The problems of characterizing new brine shrimp populations are discussed. In view of the great importance of Artemia as part of the live food chain for the culture of fish and shellfish larvae and the present cyst shortage from the market, the need for commercial exploitation and development of new Artemia sources is now, more than ever, necessary.     

Induction of multiple proteases during the early larval development of Artemia salina

Extracts from dormant and developing embryos of Artemia salina contain almost undetectable levels of protease activity. However, after hatching of the nauplii, there is a high increase in the activity of the extracts on different proteolytic substrates. The activities correspond to four different enzymes, A, B, C, and D, that have a sequential timing of induction during the early larval development. The enzymes show a different elution pattern after chromatography on DEAE-cellulose and a different sensitivity to protease inhibitors. Enzyme A is strongly inhibited by phenylmethylsulfonyl fluoride, while the other enzymes are mostly insensitive. Enzymes A, B, and C are sensitive at low concentrations of the soybean trypsin inhibitor. The temporal induction of these enzymes is an example of the expression, at the biochemical level, of the developmental program of Artemia salina.