Ultrastructural diversity in the shell of Artemia resting eggs.

The shell of Artemia resting egg, which is a delicate multilayered envelope surrounding the inside diapause embryo, plays an important role in the survival strategy of Artemia. To date, the ultrastructure of resting eggshell has been studied for only handful populations, and knowledge about the diversity of shell structure is still limited. In this paper, resting eggs from 13 Artemia populations were studied by transmission electron microscopy. Results show that the basic configuration of resting eggshell is quite conservative, but variations are not uncommon in the fine ultrastructure of each main layer of the shell (e.g., the shape and distribution of the radially oriented pores in the cortical layer; the size, number and arrangement of chambers in the alveolar layer; and the development state of outer cuticular membrane [OCM]). The ultrastructural variation of eggshell seems not to be linked with species and reproductive mode of Artemia. Resting eggs from very high habitats (4300+ m above sea level [a.s.l.]) on Qinghai-Tibet Plateau and certain tropical salterns have a hypoplastic OCM, which may be related to the adaptation to habitat conditions such as low oxygen concentration. RESEARCH HIGHLIGHTS: Comparative study on resting eggs from 13 Artemia populations reveals high diversity in the fine structure of eggshell. Resting eggs from very high (4300+ m a.s.l.) habitats commonly have a hypoplastic OCM.

Developmental toxicity of Fe3O4 nanoparticles on cysts and three larval stages of Artemia salina.

Using Artemia salina cysts (capsulated and decapsulated) and larvae (instar I, II and III) as experimental models, the potential effects of Fe3O4 nanoparticles (Fe3O4-NPs) on marine ecosystems were investigated. Hatchability, mortality and a number of ethological, morphological and biochemical parameters were selected as end-points to define the toxic responses. Data showed that the hatching rates of capsulated and decapsulated cysts were significantly decreased (p < 0.01) following exposure to 600 mg/L for 24 and 36 h. The LC50 values for instar II and III were 482 and 561 mg/L (could not be measured for instar I), and the EC50 values for swimming inhibition of instar I, II and III were 474, 365 and 421 mg/L, respectively. Effects on hatchability, mortality and swimming were accounted for Fe3O4-NPs rather than iron ion released from the NPs. Instar II larvae showed the greatest sensitivity to Fe3O4-NPs, and followed by instar III, instar I, decapsulated cysts and capsulated cysts. Body lengths of instar I, II and III larvae were decreased in dose-dependent manners. Fe3O4-NPs attached onto the gills and body surface, resulting in irreversible damages. Reactive oxygen species, malondialdehyde content, total antioxidant capacity and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities were substantially increased following exposure, indicating that toxic effects were related to oxidative stress. Mitochondrial malformation, cristae rupturing and membranous structure disruption were clearly observed after Fe3O4-NPs exposure. Fe3O4-NPs were ingested and well distributed in the gut, yolk and primary body cavity. Uptake kinetics data showed that the maximum Fe3O4-NPs content (16.4 mg/g) was reached at 30 h. The combined results so far indicate that Fe3O4-NPs have the potential to affect aquatic organisms when released into the marine ecosystems.

Siderophore coated magnetic iron nanoparticles: Rational designing of water soluble nanobiosensor for visualizing Al3+ in live organism.

This article aims to establish the judicious use of iron-binding chemistry of microbial chelators in order to functionalize the surface of iron nanoparticles to develop non-toxic nanobiosensor. Anchoring a simple siderophore 2,3-dihydroxybenzoylglycine (H3L), which bears catechol and carboxyl functionalities in tandem, on to the surface of Fe3O4 nanoparticles has developed a unique nanobiosensor HL-FeNPs which showed highly selective and sensitive detection of Al3+ in 100% water at physiological pH. The biosensor HL-FeNPs, with 20nM limit of detection, behaves reversibly and instantly. In-vivo bio-imaging in live brine shrimp Artemia confirmed that HL-FeNPs could be used as fluorescent biomarker for Al3+ in live whole organisms. Magnetic nature of the nanosensor enabled HL-FeNPs to remove excess Al3+ by using external magnet. To our knowledge, the possibility of microbial chelator in the practical development of Al3+ selective nanobiosensor is unprecedented.

Reporting a new siderophore based Ca(2+) selective chemosensor that works as a staining agent in the live organism Artemia.

A Ca(2+)-specific chemosensor involving acyclic non-ether and non-carboxylato-type metal chelating ligands is rare. The tetradentate OONO artificial receptor, HL, possessing a sulfur-containing intermediate siderophore aeruginic acid, tethered to a rhodamine 6G based signalling unit in a single molecule has been synthesized. The fluoroionophore required excitation in the visible wavelength (510 nm) and showed highly selective and sensitive detection of Ca(2+) ions in 100% water solution in HEPES buffer at physiological pH (7.4). The probe HL, with LOD as low as 70 nM, behaves reversibly and showed nearly 17-fold enhanced selectivity for Ca(2+) over other cell abundant alkali and alkaline metal ions such as Na(+), K(+), Li(+), and Mg(2+) without any intervention. Job's plot, (1)H NMR titration and ESI-MS data provided corroborative evidence in support of 1 : 1 association between HL and Ca(2+). From a wide range of transition and heavy metal ions series, HL also binds Cu(2+). However, the use of l-cysteine removes the interference from Cu(2+) and results in highly selective detection specificity of HL for Ca(2+). As a reversible "off-on-off" fluorescent chemosensor, it is possible to detect Ca(2+) at as low as 5 µM in the midgut region of the gastrointestinal tract of the live animal Artemia, a brine shrimp.

SEM study of diversity in the cyst surface topography of nine parthenogenetic Artemia (Crustacea: Anostraca) populations from China.

The cysts of nine Chinese populations of parthenogenetic Artemia were studied by scanning electron microscope. In the 270 cysts examined, 15 different morphological patterns were recognized with most of them not recorded in previous studies and the "tubercled shell surface" being the most common pattern. Results also displayed high intrapopulation variability, with the maximum of 11 patterns (in 30 cysts) recorded from the Barkol population. No positive correlation between the diversity of cyst shell patterns and ploidy compositions was found. Principal components analysis suggests higher similarity among coastal populations than among inland populations, which may be attributed to the identity of physicochemical conditions among coastal salterns and dissimilarity among inland saline lakes.

A distinct sequence in the adenine nucleotide translocase from Artemia franciscana embryos is associated with insensitivity to bongkrekate and atypical effects of adenine nucleotides on Ca2+ uptake and sequestration.

Mitochondria isolated from embryos of the crustacean Artemia franciscana lack the Ca(2+)-induced permeability transition pore. Although the composition of the pore described in mammalian mitochondria is unknown, the impacts of several effectors of the adenine nucleotide translocase (ANT) on pore opening are firmly established. Notably, ADP, ATP and bongkrekate delay, whereas carboxyatractyloside hastens, Ca(2+)-induced pore opening. Here, we report that adenine nucleotides decreased, whereas carboxyatractyloside increased, Ca(2+) uptake capacity in mitochondria isolated from Artemia embryos. Bongkrekate had no effect on either Ca(2+) uptake or ADP-ATP exchange rate. Transmission electron microscopy imaging of Ca(2+)-loaded Artemia mitochondria showed needle-like formations of electron-dense material in the absence of adenine nucleotides, and dot-like formations in the presence of adenine nucleotides or Mg(2+). Energy-filtered transmission electron microscopy showed the material to be rich in calcium and phosphorus. Sequencing of the Artemia mRNA coding for ANT revealed that it transcribes a protein with a stretch of amino acids in the 198-225 region with 48-56% similarity to those from other species, including the deletion of three amino acids in positions 211, 212 and 219. Mitochondria isolated from the liver of Xenopus laevis, in which the ANT shows similarity to that in Artemia except for the 198-225 amino acid region, demonstrated a Ca(2+)-induced bongkrekate-sensitive permeability transition pore, allowing the suggestion that this region of ANT may contain the binding site for bongkrekate.

Segmental mismatch in crustacean appendages: the naupliar antennal exopod of Artemia (Crustacea, Branchiopoda, Anostraca).

Based on traditional techniques and confocal laser scanning microscopy for external morphology, and immunohistochemistry for the muscular system, we describe here the segmental features of the antennal exopod of Artemia nauplii. Two kinds of serial elements are present, i.e. setae (with cuticular folds at their base) and ringlets (serially arranged sclerites separated by joint-like cuticular folds not extending to form complete rings around the appendage). The two series are usually not in register. The cuticular folds of the setae and of the ringlets are also sites of intermediate insertions of the three exopod muscles: as the two tegumentary structures are discordant in periodicity, this is also mirrored in the pattern of muscle insertions on the two sides of the appendage. Similar cases of segmental mismatch are known for the trunk of several arthropods, but segmental mismatch along the appendages has received very little attention. The occurrence of segmental mismatch in the naupliar appendages of both extant and fossil crustaceans is reviewed and it is suggested here to be a primitive feature of the exopods of both second antennae and mandibles. Problems in the interpretation of morphological evidence are discussed, also in relation to development and evolution of segmentation of naupliar appendages.

Structural changes of Artemia parthenogenetica (Bowen and Sterling, 1978) (Branchiopoda-Anostraca) exposed to lead acetate.

The cosmopolitan genus Artemia occurs both in saline or hypersaline waters and Artemia species are sensitive indicator organisms for environmental contamination. Scientists propose that the brine shrimp (Artemia) is a very suitable candidate for the development of identifying chemicals with adverse effects in aquatic ecosystems. In the present study, we investigated 24h the short-term toxicity of lead acetate on Artemia parthenogenetica by using electron microscopy techniques. The ultrastructural changes were studied control group and experimental group. Analysing cellular structure, structure of organelles and vacuolization were observed. The number of cells based on the toxic effects of lead acetate was increased compared with the control group.

Comparative observations on the cyst shells of seven Artemia strains from China.

The quiescent Artemia cysts of seven geographical origins in China were examined with scanning and transmission electron microscopes. SEM observations on cysts of these Artemia strains showed that the surface topography of cyst shells could be categorized into 6 types: complete smooth surface; smooth surface with sparsely distributed glabrate humps; surface with densely arranged wart-like humps that are composed of packed minute tubercles; rugged surface, with densely arranged tubercles not piling up to form larger humps; shallow-pocked surface; and surface with numerous and densely spaced pore-like fossulae. Some of the patterns were strain specific [e.g., cysts from Ga Hai (GH) are characterized by having a surface with wart-like ornaments that are composed of packed minute tubercles, rugged surface is only found in Chengkou (CK) cysts], and apparent intrastrain variation of cyst surface topography was found in Xizang (XZ), Jingyu Hu, and Xie Chi (SIN) strains. TEM studies on the ultrastructure of cyst shells revealed an apparent divergence in the structure of outer cuticular membrane (OCM) among Artemia strains. In CK, Aqqikkol Hu (AQK), SIN, and GH strains, it is a normal, asymmetrical, and multi-layered structure similar to those described in previous works. In XZ, JYH, and Lagkor Co (LGC) strains, however, the OCM is not obviously multi-layered and the borderlines between OCM and adjacent layers seem indistinct. The present results suggest that the diversity of the surface topography of Artemia cysts may be an available tool for identifying certain Artemia strains as well as for tracking the origins of some Artemia cysts, and the hypoplastic OCM may be a characteristic of the species A. tibetiana.

Ultrastructure of cyst shell and underlying membranes of three strains of the brine shrimp Artemia (Branchiopoda: Anostraca) from South India.

The cyst of Artemia has shell and membranous coverings over the embryo. The membranous coverings have special adaptive features to allow the physical changes accompanying repeated hydration and dehydration cycles that might occur and adversely influence postembryonic development. Whole and slices of cryptobiotic cysts were processed for electron microscopy to study the internal details and to compare the morphological architecture of three Artemia strains of South India. Surface topography of scanning electron microscopic (SEM) studies revealed distinct button shaped structures on the cyst of Puthalam strain. Transmission electron microscopic (TEM) studies of the cysts displayed the conventional pattern of anostracan crustaceans with outer cortex and alveolar layer, cuticular membranes, and the cytoplasmic inclusions namely nucleus, yolk droplets, lipoid bodies, and mitochondria. The prominent wavy outer cortex layer of Puthalam cysts corroborates the results of SEM studies.

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana.

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.

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.

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.

Influence of phosphorylation on isoform composition and function of a microtubule-associated protein from developing Artemia.

A novel 49 kDa protein, which exhibits nucleotide-dependent cross-linking of microtubules in vitro and localizes to ordered microtubule arrays by immunofluorescent staining, has been purified to apparent homogeneity from the brine shrimp, Artemia. Electrophoretic analysis involving isoelectric focusing and two-dimensional gels, supplemented by staining of Western blots with affinity-purified antibody, revealed that the 49 kDa protein consists of five isoforms with pI values of 6.0-6.2. The amount of 49 kDa protein increased slightly, but its isoform composition did not change significantly, during development of Artemia gastrula to third-instar larvae. Treatment with alkaline phosphatase caused the 49 kDa protein to undergo a mobility shift on gel electrophoresis, and, by use of an antibody to phosphoserine, at least two isoforms of the protein were shown to be phosphorylated. The serine phosphate, presumably added by a post-translational mechanism, did not influence binding of the 49 kDa protein to microtubules. Under conditions in which microtubules were cross-linked, the 49 kDa protein failed to interact with actin filaments. Our results demonstrate that the 49 kDa protein, like other structural microtubule-associated proteins such as tau and MAP2, is composed of several isoforms, some of which are phosphorylated. This protein has the potential to regulate the spatial distribution of microtubules within cells but does not link microfilaments to one another or to microtubules.

Development of the brine shrimp Artemia is accelerated during spaceflight.

Developmentally arrested brine shrimp cysts have been reactivated during orbital spaceflight on two different Space Shuttle missions (STS-50 and STS-54), and their subsequent development has been compared with that of simultaneously reactivated ground controls. Flight and control brine shrimp do not significantly differ with respect to hatching rates or larval morphology at the scanning and transmission EM levels. A small percentage of the flight larvae had defective nauplier eye development, but the observation was not statistically significant. However, in three different experiments on two different flights, involving a total of 232 larvae that developed in space, a highly significant difference in degree of flight to control development was found. By as early as 2.25 days after reactivation of development, spaceflight brine shrimp were accelerated, by a full instar, over ground control brine shrimp. Although developing more rapidly, flight shrimp grew as long as control shrimp at each developmental instar or stage.

Embryogenesis, hatching and larval development of Artemia during orbital spaceflight.

Developmental biology studies, using gastrula-arrested cysts of the brine shrimp Artemia franciscana, were conducted during two flights of the space shuttle Atlantis (missions STS-37 and STS-43) in 1991. Dehydrated cysts were activated, on orbit, by addition of salt water to the cysts, and then development was terminated by the addition of fixative. Development took place in 5 ml syringes, connected by tubing to activation syringes, containing salt water, and termination syringes, containing fixative. Comparison of space results with simultaneous ground control experiments showed that equivalent percentages of naupliar larvae hatched in the syringes (40%). Thus, reactivation of development, completion of embryogenesis, emergence and hatching took place, during spaceflight, without recognizable alteration in numbers of larvae produced. Post-hatching larval development was studied in experiments where development was terminated, by introduction of fixative, 2 days, 4 days, and 8 days after reinitiation of development. During spaceflight, successive larval instars or stages, interrupted by molts, occurred, generating brine shrimp at appropriate larval instars. Naupliar larvae possessed the single naupliar eye, and development of the lateral pair of adult eyes also took place in space. Transmission electron microscopy revealed extensive differentiation, including skeletal muscle and gut endoderm, as well as the eye tissues. These studies demonstrate the potential value of Artemia for developmental biology studies during spaceflight, and show that extensive degrees of development can take place in this microgravity environment.

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.

Scanning electron microscope observations of brine shrimp larvae from space shuttle experiments.

Brine shrimp are encysted as gastrula stage embryos, and may remain dehydrated and encysted for years without compromising their viability. This aspect of brine shrimp biology is desirable for studying development of animals during space shuttle flight, as cysts placed aboard a spacecraft may be rehydrated at the convenience of an astronaut, guaranteeing that subsequent brine shrimp development occurs only on orbit and not on the pad during launch delays. Brine shrimp cysts placed in 5 ml syringes were rehydrated with salt water and hatched during a 9 day space shuttle mission. Subsequent larvae developed to the 8th larval stage in the sealed syringes. We studied the morphogenesis of the brine shrimp larvae and found the larvae from the space shuttle experiments similar in rate of growth and extent of development, to larvae grown in sealed syringes on the ground. Extensive differentiation and development of embryos and larvae can occur in a microgravity environment.

High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin.

The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.