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.     

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana

Summary It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an Alul DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the Alul sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.Offprint requests to: N. Landsberger     

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adiacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH₂F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.     

The gnotobiotic brine shrimp (Artemia franciscana) model system reveals that the phenolic compound pyrogallol protects against infection through its prooxidant activity

The phenolic compound pyrogallol is the functional unit of many polyphenols and currently there has been a growing interest in using this compound in human and animal health owing to its health-promoting effects. The biological actions of pyrogallol moiety (and polyphenols) in inducing health benefitting effects have been studied; however, the mechanisms of action remain unclear yet. Here, we aimed at unravelling the underlying mechanism of action behind the protective effects of pyrogallol against bacterial infection by using the gnotobiotically-cultured brine shrimp Artemia franciscana and pathogenic bacteria Vibrio harveyi as host-pathogen model system. The gnotobiotic test system represents an exceptional system for carrying out such studies because it eliminates any possible interference of microbial communities (naturally present in the experimental system) in mechanistic studies and furthermore facilitates the interpretation of the results in terms of a cause effect relationship. We provided clear evidences suggesting that pyrogallol pretreament, at an optimum concentration, induced protective effects in the brine shrimp against V. harveyi infection. By pretreating brine shrimp with pyrogallol in the presence or absence of an antioxidant enzyme mixture (catalase and superoxide dismutase), we showed that the Vibrio-protective effect of the compound was caused by its prooxidant action (e.g. generation of hydrogen peroxide, H₂O₂). We showed further that generation of prooxidant is linked to the induction of heat shock protein Hsp70, which is involved in eliciting the prophenoloxidase and transglutaminase immune responses. The ability of pyrogallol to induce protective immunity makes it a potential natural protective agent that might be a potential preventive modality for different host-pathogen systems.     

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.     

The development of the brine shrimp Artemia salina (L.): a morphometric approach

Growth allomentry in the development of the brine shrimp Artemia salina (L.) was studied. Body width and abdomen length show a negative allometric relation to body length ( k =0.35 and 0.72 respectively) over th eight instars studied. Antennule length shows a negative allometric relation in respect to body length, with a change in the value of the growth constant at Instar Five from 0.31 to 0.75. Thoracic limb length shows a high positive relation to body length ( k =3.0) until Instar Six, when a negative allometric phase ( k =0.4) follows. A change in the sign of the growth constant from positive ( k =0.45) to negative ( k =-1.4) is noted for the length of the 2nd antenna in relation to body length.     

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature,long-term anoxia,desiccation and other wide variety of stressful conditions.How the embryos survive these stresses is a very interesting and unsolved subj...     

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     

Effect of Temperature and Salinity on the Biochemical Composition of Artemia franciscana KELLOGG 1906, from Araya (Venezuela) and San José (México)

The effect of different temperature and salinity combinations on the biochemical composition of Artemia franciscana from Venezuela and Mexico, is analyzed. Temperatures were 22 ± 0.5 °C, 26 ± 0.5 °C and 30 ± 0.5°C; salinities were 30‰, 60‰, and 120‰. Chaetoceros sp. was used as food. According to Tukey's Multiple Range Analysis for the A. franciscana population from Araya and San José, there were differences in the biochemical parameters and survival percentages among treatments and between populations. A positive correlation is observed among proximate composition values and survival, total length and growth rates. The observed variations reflect a genetic component resulting from the life history of the populations, and a non-genetic component produced by the experimental conditions.     

Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii

Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.     

Effects of toxic Alexandrium species on the survival and feeding rates of brine shrimp, Artemia salina

Zooplankton is an important link between phytoplankton and higher consumers in the marine food chain. To investigate the harmful effects of the toxic dinoflagellate Alexandrium species on zooplankton, 4 strains of Alexandrium spp., isolated from the Chinese coast, were used to test the species' effects on the survival and feeding rates of the brine shrimp, Artemia salina. The experiment was designed to assess the response of A. salina in each stage of its life cycle: nauplii, metanauplii, and adult. Each experiment was conducted in a 500 ml treatment that was added. The toxic treatments consisted of single strains of A. minutum, A. catanella, and A. tamarense (Nanhai and Donghai strain), while non-toxic species (dinoflagellate Prorocentrum donghaiense and diatom Chaetoceros minutissimus) were used as control treatments. An additional phytoplankton treatment consisted of, a mixture of A. tamarense (Nanhai strain) and P. donghaiense. Alexandrium spp. species were found to have lethal effects on the brine shrimp at a density of 2000 cells/ml. All the brine shrimps died within 24-168 hours of inoculation with the 4 treatments each containing single toxic Alexandrium species. During the feeding experiment, toxic Alexandrium spp. caused a reduction in the feeding rates in all the three stages of the life cycle of A. salina, whereas this response was not obvious in the treatment involving the nontoxic species P. donghaiense. The body surface of the brine shrimp that were fed on Alexandrium species was consistently covered by a sticky floc. Mortality of A. salina was observed to increase with the occurrence of the floc. The toxicity of the paralytic shellfish poisons (PSP) produced by the Alexandrium species was not significantly correlated with the survival or the feeding rate of the brine shrimp. When A. tamarense was mixed with P. donghaiense, the lethal effect of A. tamarense decreased, as shown by an increase in the survival and the feeding rates of the brine shrimp. A. salina metanauplii were found at the life stage most sensitive to the toxic algae and hunger. In summary, toxic Alexandrium spp. were found to have lethal effects on A. salina and to restrain the brine shrimp's feeding rate. Nontoxic Prorocentrum mitigated the toxicity of Alexandrium to a certain extent. The results also imply that the sticky material on the surface of the body of the brine shrimp may have been an important lethal factor rather than the PSP toxins.     

A review of the use of the brine shrimp,Artemia spp,for teaching practical biology in schools and colleges

Brine shrimps are salt water Crustacea that are cheaply, easily, and rapidly reared in schools. In several studies they have proved to be attractive to pupils and valuable for teaching ecology and animal behaviour. Using simple and inexpensive apparatus such as plastic bottles, pipettes, sieves, and magnifiers pupils may investigate their feeding, growth, and development, observe reproductive behaviour and, by means of planned investigations, learn important lessons in animal ecology. Brine shrimps have a demonstrated usefulness for teaching and learning at every level of education — from primary, through secondary science, to undergraduate biology project work. In school, brine shrimps present fewer ethical problems than those posed by the keeping of many other laboratory animals, yet at the same time give opportunity for ethical discussion. The extensive utilitarian use of brine shrimps in research and fisheries may provide a technical and commercial link to classroom science.     

Stress tolerance during diapause and quiescence of the brine shrimp,Artemia

Oviparously developing embryos of the brine shrimp, Artemia, arrest at gastrulation and are released from females as cysts before entering diapause, a state of dormancy and stress tolerance. Diapause is terminated by an external signal, and growth resumes if conditions are permissible. However, if circumstances are unfavorable, cysts enter quiescence, a dormant stage that continues as long as adverse conditions persist. Artemia embryos in diapause and quiescence are remarkably resistant to environmental and physiological stressors, withstanding desiccation, cold, heat, oxidation, ultraviolet radiation, and years of anoxia at ambient temperature when fully hydrated. Cysts have adapted to stress in several ways; they are surrounded by a rigid cell wall impermeable to most chemical compounds and which functions as a shield against ultraviolet radiation. Artemia cysts contain large amounts of trehalose, a non-reducing sugar thought to preserve membranes and proteins during desiccation by replacing water molecules and/or contributing to vitrification. Late embryogenesis abundant proteins similar to those in seeds and other anhydrobiotic organisms are found in cysts, and they safeguard cell organelles and proteins during desiccation. Artemia cysts contain abundant amounts of p26, a small heat shock protein, and artemin, a ferritin homologue, both ATP-independent molecular chaperones important in stress tolerance. The evidence provided in this review supports the conclusion that it is the interplay of these protective elements that make Artemia one of the most stress tolerant of all metazoan organisms.     

Effect of water-borne copper on respiratory and cardiac function during the early ontogeny of the brine shrimp,Artemia franciscana Kellogg 1908 (Branchiopoda: Anostraca)

There was no direct effect of copper on the ontogeny or function of the heart of the brine shrimp Artemia franciscana in sea water (salinity= 36 mg·ml^-1, 25°C). There was, however, an indirect effect as an increase in copper concentration resulted in a reduced growth rate. There was no difference between the critical O₂ tensions of newly hatched (stage 0/1) nauplii of control and treated (<0.32 and 10.11 mol·l^-1 copper, respectively) individuals. However by developmental stages 4–6, when both the heart and thoracic gills are in the process of differentiating, respiratory performance had improved (i.e. critical O₂ tension decreased from 6.27±0.45 to 4.69±0.24 kPa) in control but not in copper-treated individuals. It is suggested that respiratory impairment of stages 4–6 individuals is unlikely to be related to differences in cardiac performance or cellular respiration. Instead it may be related to metal-related damage to newly differentiating gill tissue and/or by copper in some way compromising the normal ontogenic shift in haemoglobin O₂ affinity. Copper-related respiratory impairment develops at a critical point in brine shrimp organogenesis when a good supply of O₂ is essential for normal development and if compromised may reduce the ability of this species to survive copper exposure.Abbreviations BL body length - BW body weight - HR heart rate - HM heavy metals - SW sea water - P _c critical oxygen tension     

Effects of toxic Alexandrium species on the survival and feeding rates of brine shrimp, Artemia salina

Zooplankton is an important link between phytoplankton and higher consumers in the marine food chain. To investigate the harmful effects of the toxic dinoflagellate Alexandrium species on zooplankton, 4 strains of Alexandrium spp., isolated from the Chinese coast, were used to test the species' effects on the survival and feeding rates of the brine shrimp, Artemia salina. The experiment was designed to assess the response of A. salina in each stage of its life cycle: nauplii, metanauplii, and adult. Each experiment was conducted in a 500 ml treatment that was added. The toxic treatments consisted of single strains of A. minutum, A. catanella, and A. tamarense (Nanhai and Donghai strain), while non-toxic species (dinoflagellate Prorocentrum donghaiense and diatom Chaetoceros minutissimus) were used as control treatments. An additional phytoplankton treatment consisted of, a mixture of A. tamarense (Nanhai strain) and P. donghaiense. Alexandrium spp. species were found to have lethal effects on the brine shrimp at a density of 2000 cells/ml. All the brine shrimps died within 24-168 hours of inoculation with the 4 treatments each containing single toxic Alexandrium species. During the feeding experiment, toxic Alexandrium spp. caused a reduction in the feeding rates in all the three stages of the life cycle of A. salina, whereas this response was not obvious in the treatment involving the nontoxic species P. donghaiense. The body surface of the brine shrimp that were fed on Alexandrium species was consistently covered by a sticky floc. Mortality of A. salina was observed to increase with the occurrence of the floc. The toxicity of the paralytic shellfish poisons (PSP) produced by the Alexandrium species was not significantly correlated with the survival or the feeding rate of the brine shrimp. When A. tamarense was mixed with P. donghaiense, the lethal effect of A. tamarense decreased, as shown by an increase in the survival and the feeding rates of the brine shrimp. A. salina metanauplii were found at the life stage most sensitive to the toxic algae and hunger. In summary, toxic Alexandrium spp. were found to have lethal effects on A. salina and to restrain the brine shrimp's feeding rate. Nontoxic Prorocentrum mitigated the toxicity of Alexandrium to a certain extent. The results also imply that the sticky material on the surface of the body of the brine shrimp may have been an important lethal factor rather than the PSP toxins.