Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana.

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.

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.

Deterioration of the Provox silicone tracheoesophageal voice prosthesis: microbial aspects and structural changes.

Device life of tracheoesophageal voice prostheses is limited due to deterioration of the polymers. A group of 55 postlaryngectomy patients fitted with a Provox voice prosthesis have been studied prospectively during 6 months. Thirty-seven prostheses were replaced due to a dysfunctional valve mechanism. Although colonization with Candida species was highly associated with destruction of the silicone material, other upper respiratory tract commensals, e.g. Staphylococcus aureus, were also demonstrated. Electron microscopy of the contaminated devices showed colonization and disruption of the silicone material by penetrating yeast hyphae. During the study a remarkable increase of intratracheal phonatory pressures was assessed with progressive colonization of the prostheses.

Characterization of gamma-tubulin in Artemia: isoform composition and spatial distribution in polarized cells of the larval epidermis.

Microtubule arrangement is influenced by gamma-tubulin, a soluble protein of the eukaryotic cell cytosol and a component of microtubule-organizing centers. In this study, affinity purified antibodies to gamma-tubulin were prepared and their specificity demonstrated by immunostaining of Western blots and in competitive ELISAs. When employed to label mouse fibroblasts, one or two brightly stained dots appeared in each cell, a pattern characteristic of centrosomes. Antibody 9, raised to a conserved amino-terminal peptide of gamma-tubulin, was used with TU-30 (from P. Dráber) to characterize gamma-tubulin in the crustacean, Artemia franciscana. Cell-free protein extracts from Artemia contained gamma-tubulin and it purified with alpha/beta-tubulin through several preparative steps. Probing of Western blots prepared from two-dimensional gels yielded a single isoform of gamma-tubulin in Artemia with a pI of about 5.6. Immunostaining with TAT, a general antibody to alpha-tubulin, demonstrated that Artemia possess two morphological types of immune blood cells (hemocytes) with distinctive microtubule arrays. Both the compact spherical hemocytes and the flatter, spreading cells exhibited fluorescent dots, often in pairs, when labelled with antibodies to gamma-tubulin. Microtubules in polarized cells of the epidermis were also brightly stained with antibody to alpha-tubulin, revealing interphase arrangements, anastral mitotic spindles and midbodies. Antibody 9 and TU-30 gave punctate staining patterns in interphase epidermal cell layers and they occasionally labelled midbodies. Unexpectedly, gamma-tubulin was seen only rarely at both poles of mitotic spindles in epidermal cells. The complete absence of asters and the apparent lack of gamma-tubulin at all but a small number of poles indicate that formation and structure of the mitotic spindle in epidermal cells of Artemia are unusual.

Copper induces apoptosis in Aedes C6/36 cells.

The Aedes albopictus C6/36 cell clone is used as a model system to study the effects of heavy metals on insect cells. Here we report on the effects of Cu(2+) on these cells. Similar to Cd(2+) and Hg(2+), Cu(2+) induces hyperpolymerization of the microtubules; moreover, with Cu(2+) this is followed by cell aggregation and massive apoptosis. This process, which is cell density dependent, is maximal between 0.75 and 1 mM; this is just under the LC(50) as determined by a membrane integrity test. At higher Cu(2+) concentrations, cell death occurs by necrosis. Apoptosis was ascertained by fluorescence and electron microscopy and by agarose gel electrophoresis. At 0.75 mM, apoptosis started at 18-hr exposure time and the amount of apoptotic cells increased almost linearly until 42 hr; then a plateau was reached with 70-80% apoptotic cells. This is the first report on Cu(2+)-induced apoptosis in insect cells. Possible induction mechanisms are discussed in the light of existing literature on vertebrate cells.

Selected bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio proteolyticus CW8T2.

In this study Vibrio proteolyticus CW8T2 has been identified as a virulent pathogen for Artemia spp. Its infection route has been visualized with transmission electron microscopy. The pathogen affected microvilli and gut epithelial cells, disrupted epithelial cell junctions, and reached the body cavity, where it devastated cells and tissues. In vivo antagonism tests showed that preemptive colonization of the culture water with nine selected bacterial strains protected Artemia juveniles against the pathogenic effects. Two categories of the selected strains could be distinguished: (i) strains providing total protection, as no mortality occurred 2 days after the experimental infection with V. proteolyticus CW8T2, with strain LVS8 as a representative, and (ii) strains providing partial protection, as significant but not total mortality was observed, with strain LVS2 as a representative. The growth of V. proteolyticus CW8T2 in the culture medium was slowed down in the presence of strains LVS2 and LVS8, but growth suppression was distinctly higher with LVS8 than with LVS2. It was striking that the strains that gave only partial protection against the pathogen in the in vivo antagonism test showed also a restricted capability to colonize the Artemia compared to the strains providing total protection. The in vivo antagonism tests and the filtrate experiments showed that probably no extracellular bacterial compounds were involved in the protective action but that the living cells were required to protect Artemia against V. proteolyticus CW8T2.

[Modification of the cell surface of the egg during maturation and segmentation (Artemia salina)]. / Modification de la surface cellulaire de l'oeuf en maturation et en ségmentation. (le cas d'Artemia saline).

Agglutination of Artemia eggs by con. A occurs only after fertilization. A transient sensitiveness of the sub-cortical plasma is revealed by intense vacuolization.

[Formation of the fertilization membrane of the egg inArtemia salina]. / Formation de la membrane de fécondation de l'oeuf d'Artemia salina.

The unfertilized egg ofArtemia salina is not covered with any extracellular structure. No special organelles are found in the sub-cortical plasma. From the moment of fertilization, a membrane is progressively secreted by the egg. The membranogenous substance is first seen as large granules in the smooth endoplasmic reticulum, presumably transformed within Golgi elements and extruded in vesicles liberated from the Golgi apparatus. Retained by a glycocoat or by contact with the fluid of the genital tract, it spreads out into a fertilization membrane, soon surrounding a perivitelline space. The process lasts till 1 1/2 h after fertilization.

Qualitative and quantitative changes in the carotenoids during development of the brine shrimp Artemia.

In order to study the biological fate of all-trans- and cis-canthaxanthin in the brine shrimp Artemia, a quantitative method was developed for the determination of both carotenoids and their metabolic precursors in encysted embryos (cysts), nauplii, whole animals, organs, and subcellular fractions. This method is based on nonaqueous reversed-phase chromatography, two new exhaustive extraction procedures, and the determination of proteins in the extracted residue. Hydration of Artemia cysts caused a reversible conversion of part of the all-trans- to cis-canthaxanthin. During further pre-emergence embryonic development, there was little change in the levels of both isomers. After hatching of cysts, cis-canthaxanthin was progressively isomerized to the all-trans form, while the total (all-trans + cis) canthaxanthin to protein ratio tended to remain constant. Cis-canthaxanthin rapidly became undetectable in animals fed on algae and reappeared in females at an advanced stage of the reproductive cycle. All-trans-canthaxanthin remained present during the whole Artemia life cycle in addition to its metabolic precursors echinenone and beta-carotene. The carotenoid distribution in organs and subcellular fractions indicated high affinity of cis-canthaxanthin for the female reproductive system, oocytes in general, and yolk in particular. A role for cis-canthaxanthin is suggested at an early developmental stage, i.e., in cysts, before hatching.

cis-Canthaxanthins. Unusual carotenoids in the eggs and the reproductive system of female brine shrimp artemia.

The significance of carotenoid accumulation in crustacean eggs remains obscure, particularly because neither eggs nor female animals have been found to display specific pigment patterns in relation to reproduction. We report here the first example of carotenoids found exclusively in the ovaries, the eggs, and the hemolymph, but not in the carcass of a female, reproductively active crustacean, i.e. the brine shrimp Artemia. These pigments are virtually absent in males and in immature animals and disappear very rapidly in growing nauplii following hatching of encysted embryos. Within the cysts, they are preferably localized in the yolk platelets. We have identified them as mono-cis- canthaxanthins on the basis of their mass and absorption spectra and by comparison with synthetic components. Carotenoids with the unusual cis-configuration have never been isolated from animals, nor are there reports on the occurrence of carotenoid pigments at specific sites. Our findings may thus provide a clue to a precise function for carotenoids in Artemia and, possibly, related Crustacea.