Isolation of Salt Glands from the Larval Brine Shrimp Artemia salina

Use of isolated cell preparations have been a powerful approachto understanding cellular and biochemical processes of a widevariety of tissues, including transporting epithelia. A methodfor isolating the extra-renal organ or salt gland from a larvalbrachiopod crustacean, the brine shrimp, has been developed.This preparation is unique as it is the first isolated epithelialcell preparation from a crustacean and is derived from an earlystage of development. As both the removal and purification protocolsdiffer markedly from previous cell isolation methods, detailsof their development have been described. This preparation hasallowed measurement of the Na$K AT Pase specific activity inthesalt gland and intestine for the first time. Use of the isolatedsalt glands will facilitate cellular and subcellular studiesof both the structure and function of this ion transportingepithelium.     

Distinctive Solvation Patterns Make Renal Osmolytes Diverse

The kidney uses mixtures of five osmolytes to counter the stress induced by high urea and NaCl concentrations. The individual roles of most of the osmolytes are unclear, and three of the five have not yet been thermodynamically characterized. Here, we report partial molar volumes and activity coefficients of glycerophosphocholine (GPC), taurine, and myo-inositol. We derive their solvation behavior from the experimental data using Kirkwood-Buff theory. We also provide their solubility data, including solubility data for scyllo-inositol. It turns out that renal osmolytes fall into three distinct classes with respect to their solvation. Trimethyl-amines (GPC and glycine-betaine) are characterized by strong hard-sphere-like self-exclusion; urea, taurine, and myo-inositol have a tendency toward self-association; sorbitol and most other nonrenal osmolytes have a relatively constant, intermediate solvation that has components of both exclusion and association. The data presented here show that renal osmolytes are quite diverse with respect to their solvation patterns, and they can be further differentiated based on observations from experiments examining their effect on macromolecules. It is expected, based on the available surface groups, that each renal osmolyte has distinct effects on various classes of biomolecules. This likely allows the kidney to use specific combinations of osmolytes independently to fine-tune the chemical activities of several types of molecules.     

Phylogenetic Analysis of Brine Shrimp (Artemia) in China Using DNA Barcoding

DNA barcoding is a powerful approach for characterizing species of organisms,especially those with almost identical morphological features, thereby helping to to establish phylogenetic relationships and reveal evolutionary histories. In this study, we chose a 648-bp segment of the mitochondrial gene, cytochrome c oxidase subunit 1 (COI), as a standard barcode region to establish phylogenetic relationships among brine shrimp (Artemia) species from major habitats around the world and further focused on the biodiversity of Artemia species in China, especially in the Tibetan Plateau. Samples from five major salt lakes of the Tibetan Plateau located at altitudes over 4,000 m showed clear differences from other Artemia populations in China. We also observed two consistent amino acid changes, 153A/V and 183L/F, in the COI gene between the high and low altitude species in China.Moreover, indels in the COI sequence were identified in cyst and adult samples unique to the Co Qen population from the Tibetan Plateau, demonstrating the need for additional investigations of the mitochondrial genome among Tibetan Artemia populations.     

卤虫(AC)_n和(AG)_n微卫星文库构建

卤虫Artemia作为重要的基础实验材料和经济动物资源,其微卫星位点具有重要的研究和应用价值。本研究使用磁珠富集法,成功构建了卤虫的(AC)_n和(AG)_n微卫星富集文库。通过对文库进行PCR方法鉴定和测序确认,所构建的(AC)_n和(AG)_n文库阳性克隆率分别为55.8%和34.5%。依据得到的微卫星序列,设计并合成了63对微卫星引物,随机以2个地理单元(青海、西藏)共18个卤虫基因组DNA为扩增模板,筛选出6对具有多态性的卤虫微卫星引物。本研究筛选出的微卫星多态位点可用于卤虫种群的遗传多样性评估、遗传图谱的构建和数量性状定位等后续工作。     

The Renal Lesion in Angiokeratoma Corporis Diffusum (Fabry's Disease)

Electron microscopic details of the glomerular and tubular lesions in a 26-year-old man with angiokeratoma corporis diffusum are presented. Though unable to concentrate urine above a specific gravity of 1.012, this patient showed preservation of the ability to acidify and alkalinize the urine following oral loads of ammonium chloride (150 mEq./day) and sodium bicarbonate (158 mEq./day) for several days. This observation is in contrast to previously reported findings and suggests that the regularly observed hyposthenuria in this disease does not depend on defects in ion transfer in the distal tubule system.     

Formation of Diapause Cyst Shell in Brine Shrimp, Artemia parthenogenetica, and Its Resistance Role in Environmental Stresses

Artemia has attracted much attention for its ability to produce encysted embryos wrapped in a protective shell when subject to extremely harsh environmental conditions. However, what the cyst shell is synthesized from and how the formative process is performed remains, as yet, largely unknown. Over 20 oviparous specifically expressed genes were identified through screening the subtracted cDNA library enriched between oviparous and ovoviviparous Artemia ovisacs. Among them, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. Lacking SGEG protein (by RNA interference) caused the cyst shell to become translucent and the chorion layer of the shell to become less compact and pultaceous and to show a marked decrease of iron composition within the shell. The RNA interference induced defective diapause cysts with a totally compromised resistibility to UV irradiation, extremely large temperature differences, osmotic pressure, dryness, and organic solvent stresses. In contrast, the natural cyst would provide adequate protection from all such factors. SGEG contains a 345-bp open reading frame, and its consequentially translated peptide consists of a 33-amino acid residue putative signal peptide and an 81-amino acid residue mature peptide. The results of Northern blotting and in situ hybridization indicate that the gene is specifically expressed in the cells of shell glands during the period of diapause cyst formation of oviparous Artemia. This investigation adds strong insight into the mechanism of cyst shell formation of Artemia and may be applicable to other areas of research in extremophile biology.Salt lakes on plateaus, are widely known as “seas of death,” because they represent one of the most hostile environments on the earth in terms of extreme salinity, high pH, anoxia, large temperature differences, and intermittent dry conditions. Hardly any animal can survive such extremes. However, one notable exception lies in the shape of a small crustacean, Artemia.Artemia, also called the brine shrimp, is an ancient species that first appeared ∼400 million years ago (1). To cope with harsh and complex habitats such as salt lakes, Artemia are able, when the circumstances become adverse, to release their offspring into a dormant, encysted state, rather than simply releasing swimming nauplius, to ensure survival. Such adverse conditions include environments where the Artemia may experience high salinity, low oxygen levels, short days, or conditions of extreme temperature variation (2, 3). These dormant cysts will keep diapause until the state is terminated by activation (triggered by factors such as desiccation, dehydration, cold or chemical treatment), at which point they resume development when appropriate and stable environmental conditions have arisen (4–7).The diapause cysts, with their greatly reduced metabolic activity, contain embryos existing as late gastrulae and are composed of ∼4000 cells that are arrested at the G₂/M phase with a complete turning off of RNA and protein synthesis (8, 9). Previous studies indicate that the resistance and resumption ability of Artemia cysts have several causes. In addition to the arrested cell cycle, it has been noted that large amounts of two molecular chaperone proteins, namely p26 and artemin, are synthesized (10–12), and a high concentration of trehalose is also accumulated (13–15). Moreover, a complicated enzyme system is also involved in the diapause and resumption mechanism, including AMP-activated protein kinase (16) and p90 ribosomal S6 kinase regulatory pathway (17–19).In addition to falling into diapause, Artemia themselves secrete a rigid noncellular shell to cope with the extreme environmental stresses before they release the diapause cysts. The complex noncellular cyst shell consists of two main regions; the outer region, secreted by the shell gland, is of hypochlorite-soluble chorion, whereas the hypochlorite-resistant inner region is formed by blastoderm cells and comprises the embryonic cuticle (5, 20, 21). The shell glands, which are composed of clusters of secretory cells, are situated at the ovisac and open into the uterus. There are many dark brown secretory granules, which probably contain chorion material and pigments such as hematin formed in the cells of the shell glands at the point where the oocytes emerge in the ovaries during the reproductive period. These are secreted out at the second day after the oocytes enter the uterus. Therefore the shell glands vary from dark brown to white, even to colorless, as reproductive cycles differ (22, 23).Microphotographs shot by Sugumar and Munuswamy (24) reveal that both the chorion and the embryonic cuticle have an exquisite structure (21). Chorion consists of two distinct layers. First, a compact outer covering is over the cyst with many radially aligned aeropyles penetrating through. This is known as the cortical layer. Second, in a cavernous region below the cortical layer is the alveolar layer, which may act as a float for the newly laid cysts. A thin supra cortical layer, probably consisting of cuticulin, covers the outer surface of the cortical layer. The embryonic cuticle, which is impermeable to nonvolatile solutes, is otherwise composed of a broad multilamellar region as a fibrous layer sandwiched between the outer and inner cuticular membranes and constructed as a tripartite structure. This forms an area of relative independence from the external environment and serves to maintain the homeostasis of inorganic ions (2). The molecular formulation of the cyst shell is complex, and details remain unclear, although it is known that the cyst shell does contain chitin, lipoprotein, hematin, and some metal elements (25–27).Besides preventing mechanical damage (28), the cyst shell also plays an important role in protecting the embryo within from other lethal environmental stresses. Previous experimental data have confirmed the protective capabilities of the cyst shell. Tanguay et al. (29) indicated that the hatching rate of intact cysts is significantly higher than the decapsulated ones after ultraviolet irradiation treatment. Hematin, the hemopigment of the cyst shell, is also demonstrated to have a light-screening function (27). Clegg (30) indicated that the cyst shell plays a critical role in desiccation tolerance, because the rate of dehydration of decapsulated cysts is much higher than intact ones in the dehydration study, and rapid water loss significantly reduces the hatching level of dehydrated cysts. Liu et al. (31) also found that intact cysts have better thermotolerance than decapsulated ones in both dry and water heating studies.In our experiments, through the in vivo gene knockdown by RNA interference, a shell gland-specifically expressed gene (SGEG) has been found to be involved in the cyst shell formation. The formed cyst shell has been demonstrated to play an important role in resistance to UV irradiation, large temperature differences, osmotic pressure, dryness, and organic solution stresses.     

A first AFLP-Based Genetic Linkage Map for Brine Shrimp Artemia franciscana and Its Application in Mapping the Sex Locus

We report on the construction of sex-specific linkage maps, the identification of sex-linked markers and the genome size estimation for the brine shrimp Artemia franciscana. Overall, from the analysis of 433 AFLP markers segregating in a 112 full-sib family we identified 21 male and 22 female linkage groups (2n = 42), covering 1,041 and 1,313 cM respectively. Fifteen putatively homologous linkage groups, including the sex linkage groups, were identified between the female and male linkage map. Eight sex-linked AFLP marker alleles were inherited from the female parent, supporting the hypothesis of a WZ–ZZ sex-determining system. The haploid Artemia genome size was estimated to 0.93 Gb by flow cytometry. The produced Artemia linkage maps provide the basis for further fine mapping and exploring of the sex-determining region and are a possible marker resource for mapping genomic loci underlying phenotypic differences among Artemia species.     

Quorum Sensing-Disrupting Brominated Furanones Protect the Gnotobiotic Brine Shrimp Artemia franciscana from Pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus Isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Adaptive Significance of a Small Heat Shock/{alpha}-Crystallin Protein (p26) in Encysted Embryos of the Brine Shrimp, Artemia franciscana

Encysted embryos of the primitive crustacean, Artemia franciscana,are among the most resistant of all animal life history stagesto extremes of environmental stress. This resistance has likelybeen acquired during evolutionary adaptation to their harshecological setting. In the laboratory, for example, some ofthese embryos survive more than 6 years of continuous anoxiawhile fully hydrated and at room temperature, a phenomenal capabilitythat can easily be related to their frequent encounters withanoxic conditions in nature. A small heat shock protein belongingto the -crystallin family of proteins, referred to as p26, appearsto play a central role in the stress resistance of these embryos.Our evidence shows that this protein is an important componentin their adaptive repertoire, and vital for the survival ofArtemia in nature.     

Distinctive signatures of recursion

Although recursion has been hypothesized to be a necessary capacity for the evolution of language, the multiplicity of definitions being used has undermined the broader interpretation of empirical results. I propose that only a definition focused on representational abilities allows the prediction of specific behavioural traits that enable us to distinguish recursion from non-recursive iteration and from hierarchical embedding: only subjects able to represent recursion, i.e. to represent different hierarchical dependencies (related by parenthood) with the same set of rules, are able to generalize and produce new levels of embedding beyond those specified a priori (in the algorithm or in the input). The ability to use such representations may be advantageous in several domains: action sequencing, problem-solving, spatial navigation, social navigation and for the emergence of conventionalized communication systems. The ability to represent contiguous hierarchical levels with the same rules may lead subjects to expect unknown levels and constituents to behave similarly, and this prior knowledge may bias learning positively. Finally, a new paradigm to test for recursion is presented. Preliminary results suggest that the ability to represent recursion in the spatial domain recruits both visual and verbal resources. Implications regarding language evolution are discussed.     

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 μg/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400 μg of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.     

Volatile Components of Roasted Shrimp

The components of the strong and favorable aroma obtained from roasted shrimp were investigated. The aroma concentrate of roasted shrimp was isolated by combining the dichloromethane extract and carbon dioxide distillation methods. The concentrate was fractionated into acidic, basic and neutral fractions. Each fraction was analyzed by combined gas chromatography-mass spectrometry. Seventy-seven compounds were identified, among which isovaleric acid, alkyl pyrazines, isovaleramide, ketones and some sulfur-containing compounds were the main and characteristic constituents of the roasted shrimp aroma.

These constituents were compared with those of the volatiles of boiled shrimp, which was prepared by using simultaneous distillation and extraction methods in a modified Likens and Nickerson’s apparatus.     

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic <Emphasis Type="Italic">Vibrio</Emphasis> Species

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8?×?10⁶ CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.     

Propagation of Geotrichum candidum in Acid Brine

Geotrichum candidum completely neutralized the acid brine and reduced its biochemical oxygen demand (BOD) by 88%. Yield of dry mycelium was 62 g per 100 g of BOD utilized.