Assessment of the biological effect of metal ions and their complexes using Allium cepa and Artemia salina assays: a possible environmental implementation of biological inorganic chemistry.

The pollution of aquatic ecosystems due to the elevated concentration of a variety of contaminants, such as metal ions, poses a threat to humankind, as these ecosystems are in high relevance with human activities and survivability. The exposure in heavy metal ions is responsible for many severe chronic and pathogenic diseases and some types of cancer as well. Metal ions of the groups 11 (Cu, Ag, Au), 12 (Zn, Cd, Hg), 14 (Sn, Pb) and 15 (Sb, Bi) highly interfere with proteins leading to DNA damage and oxidative stress. While, the detection of these contaminants is mainly based on physicochemical analysis, the chemical determination, however, is deemed ineffective in some cases because of their complex nature. The development of biological models for the evaluation of the presence of metal ions is an attractive solution, which provides more insights regarding their effects. The present work critically reviews the reports published regarding the toxicity assessment of heavy metal ions through Allium cepa and Artemia salina assays. The in vivo toxicity of the agents is not only dose depended, but it is also strongly affected by their ligand type. However, there is no comprehensive study which compares the biological effect of chemical agents against Allium cepa and Artemia salina. Reports that include metal ions and complexes interaction with either Allium cepa or Artemia salina bio-indicators are included in the review.

Evaluation of Indonesian mangrove Xylocarpus granatum leaves ethyl acetate extract as potential anticancer drug.

Local Xylocarpus granatum leaves were extracted by ethyl acetate solvent and characterized by TLC fingerprinting and 2D 1H NMR spectroscopy to contain phenolic compounds as well as several organic and amino acids as metabolic byproducts, such as succinic acid and acetic acid. Traces of flavonoids and other non-categorized phenolic compounds exhibited intermediate antioxidant activity (antioxidant IC50 84.93 ppm) as well as anticancer activity against HeLa, T47D, and HT-29 cell lines; which the latter being most effective against HT-29 with Fraction 5 contained the strongest activity (anticancer IC50 23.12 ppm). Extracts also behaved as a natural growth factor and nonlethal towards brine shrimps as well as human adipose-derived stem cell hADSC due to antioxidative properties. A stability test was performed to examine how storage conditions factored in bioactivity and phytochemical structure. Extracts were compared with several studies about X. granatum leaves extracts to evaluate how ethnogeography and ecosystem factored on biologically active compounds. Further research on anticancer or antioxidant mechanism on cancer cells is needed to determine whether the extract is suitable as a candidate for an anticancer drug.

Selenium nanoparticle-enriched biomass of Yarrowia lipolytica enhances growth and survival of Artemia salina.

Controlling disease outbreaks is a major challenge in aquaculture farms and conventional methods are often ineffective. Nutritional supplementation and probiotic preparations help in reducing severity of such infections. The generally regarded as safe yeast (Yarrowia lipolytica) was used in the current study. A marine strain of Y. lipolytica exhibited tolerance towards sodium selenite and formed cell associated selenium nanoparticles (SeNPs). The synthesized nanoparticles were characterized by UV-vis spectroscopy, X-ray diffraction (XRD) and Field Emission Scanning Electron Microscope (FE-SEM) observations. Fourier transform infra-red (FTIR) spectroscopy indicated the role of carboxylic and amine groups in the synthesis of nanoparticles. This SeNP-enriched biomass was used as feed for the model aquaculture system, Artemia salina and compared with normal feed, baker's yeast (Saccharomyces cerevisiae). A. salina fed with SeNP-enriched biomass, showed increased survival rates (96.66%) as compared to those fed with S. cerevisiae (60.0%). The size of the larvae fed with SeNP-enriched biomass of Y. lipolytica was also found to be larger. Additionally, larval groups fed with SeNP-enriched biomass were better protected (70.0% survival) against V. harveyi infection when compared with groups fed with S. cerevisiae (24.44%). This combination of selenium in the nanoparticle form associated with the biomass of Y. lipolytica has potential application in improving health of aquaculture species in farms.

Real-time monitoring of artemin in vivo chaperone activity using luciferase as an intracellular reporter.

Artemin is an abundant thermostable protein in Artemia encysted embryos and considered as a stress protein, as its highly regulated expression is associated with stress resistance. Artemin cDNA was previously isolated and cloned from Artemia urmiana and artemin was found as an efficient molecular chaperone in vitro. Here, co-transformation of E. coli was performed with two expression vectors containing artemin and firefly luciferase for in vivo studies. The time-course of luciferase inactivation at low and elevated temperatures showed that luciferase was rapidly inactivated in control cells, but it was found that luciferase was protected significantly in artemin expressing cells. More interestingly, luciferase activity was completely regained in heat treated artemin expressing cells at room temperature. In addition, in both stress conditions, similar to residual activity of luciferase, cell viability in induced cultures over-expressing artemin was significantly higher than non-expressed artemin cells. It can be suggested that artemin confers impressive resistance in stressful conditions when introduced into E. coli cells, which is due to that it protects proteins against aggregation. Such luciferase co-expression system can be used as a real-time reporter to investigate the activity of chaperone proteins in vivo and provide a rapid and simple test for molecular chaperones.

ArHsp40, a type 1 J-domain protein, is developmentally regulated and stress inducible in post-diapause Artemia franciscana.

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.

Bioactive properties of commercialised pomegranate (Punica granatum) juice: antioxidant, antiproliferative and enzyme inhibiting activities.

Pomegranate juice and related products have long been used either in traditional medicine or as nutritional supplements claiming beneficial effects. Although there are several studies on this food plant, only a few studies have been performed with pomegranate juice or marketed products. The aim of this work is to evaluate the antioxidant effects of pomegranate juice on cellular models using hydrogen peroxide as an oxidizing agent or DPPH and superoxide radicals in cell free systems. The antiproliferative effects of the juice were measured on HeLa and PC-3 cells by the MTT assay and pharmacologically relevant enzymes (cyclooxygenases, xanthine oxidase, acetylcholinesterase and monoamine oxidase A) were selected for enzymatic inhibition assays. Pomegranate juice showed significant protective effects against hydrogen peroxide induced toxicity in the Artemia salina and HepG2 models; these effects may be attributed to radical scavenging properties of pomegranate as the juice was able to reduce DPPH and superoxide radicals. Moderate antiproliferative activities in HeLa and PC-3 cancer cells were observed. However, pomegranate juice was also able to inhibit COX-2 and MAO-A enzymes. This study reveals some mechanisms by which pomegranate juice may have interesting and beneficial effects in human health.

Iron metallodrugs: stability, redox activity and toxicity against Artemia salina.

Iron metallodrugs comprise mineral supplements, anti-hypertensive agents and, more recently, magnetic nanomaterials, with both therapeutic and diagnostic roles. As biologically-active metal compounds, concern has been raised regarding the impact of these compounds when emitted to the environment and associated ecotoxicological effects for the fauna. In this work we assessed the relative stability of several iron compounds (supplements based on glucoheptonate, dextran or glycinate, as well as 3,5,5-trimethylhexanoyl (TMH) derivatives of ferrocene) against high affinity models of biological binding, calcein and aprotransferrin, via a fluorimetric method. Also, the redox-activity of each compound was determined in a physiologically relevant medium. Toxicity toward Artemia salina at different developmental stages was measured, as well as the amount of lipid peroxidation. Our results show that polymer-coated iron metallodrugs are stable, non-redox-active and non-toxic at the concentrations studied (up to 300 µM). However, TMH derivatives of ferrocene were less stable and more redox-active than the parent compound, and TMH-ferrocene displayed toxicity and lipid peroxidation to A. salina, unlike the other compounds. Our results indicate that iron metallodrugs based on polymer coating do not present direct toxicity at low levels of emission; however other iron species (eg. metallocenes), may be deleterious for aquatic organisms. We suggest that ecotoxicity depends more on metal speciation than on the total amount of metal present in the metallodrugs. Future studies with discarded metallodrugs should consider the chemical speciation of the metal present in the composition of the drug.

Evaluation of alpha and gamma aluminum oxide nanoparticle accumulation, toxicity, and depuration in Artemia salina larvae.

In this study, Artemia salina (crustacean filter feeders) larvae were used as a test model to investigate the toxicity of aluminum oxide nanoparticles (Al2O3 NPs) on marine microorganisms. The uptake, toxicity, and elimination of α-Al2O3 (50 nm and 3.5 µm) and γ-Al2O3 (5 nm and 0.4 µm) NPs were studied. Twenty-four and ninety-six hour exposures of different concentrations of Al2O3 NPs to Artemia larvae were conducted in a seawater medium. When suspended in water, Al2O3 NPs aggregated substantially with the sizes ranging from 6.3 nm to >0.3 µm for spherical NPs and from 250 to 756 nm for rod-shaped NPs. The phase contrast microscope images showed that NPs deposited inside the guts as aggregates. Inductively coupled plasma mass spectrometry analysis showed that large particles (3.5 µm α-Al2O3) were not taken up by Artemia, whereas fine NPs (0.4 µm γ-Al2O3) and ultra-fine NPs (5 nm γ-Al2O3 and 50 nm α-Al2O3) accumulated substantially. Differences in toxicity were detected as changing with NP size and morphology. The malondialdehyde levels indicated that smaller γ-Al2O3 (5 nm) NPs were more toxic than larger γ-Al2O3 (0.4 µm) particulates in 96 h. The highest mortality was measured as 34% in 96 h for γ-Al2O3 NPs (5 nm) at 100 mg/L (LC50 > 100 mg/L). γ-Al2O3 NPs were more toxic than α-Al2O3 NPs at all conditions.

Reactive oxygen species generated by a heat shock protein (Hsp) inducing product contributes to Hsp70 production and Hsp70-mediated protective immunity in Artemia franciscana against pathogenic vibrios.

The cytoprotective role of heat shock protein (Hsp70) described in a variety of animal disease models, including vibriosis in farmed aquatic animals, suggests that new protective strategies relying upon the use of compounds that selectively turn on Hsp genes could be developed. The product Tex-OE® (hereafter referred to as Hspi), an extract from the skin of the prickly pear fruit, Opuntia ficus indica, was previously shown to trigger Hsp70 synthesis in a non-stressful situation in a variety of animals, including in a gnotobiotically (germ-free) cultured brine shrimp Artemia franciscana model system. This model system offers great potential for carrying out high-throughput, live-animal screens of compounds that have health benefit effects. By using this model system, we aimed to disclose the underlying cause behind the induction of Hsp70 by Hspi in the shrimp host, and to determine whether the product affects the shrimp in inducing resistance towards pathogenic vibrios. We provide unequivocal evidences indicating that during the pretreatment period with Hspi, there is an initial release of reactive oxygen species (hydrogen peroxide and/or superoxide anion), generated by the added product, in the rearing water and associated with the host. The reactive molecules generated are the triggering factors responsible for causing Hsp70 induction within Artemia. We have also shown that Hspi acts prophylactically at an optimum dose regimen to confer protection against pathogenic vibrios. This salutary effect was associated with upregulation of two important immune genes, prophenoloxidase and transglutaminase of the innate immune system. These findings suggest that inducers of stress protein (e.g. Hsp70) are potentially important modulator of immune responses and might be exploited to confer protection to cultured shrimp against Vibrio infection.

Predictive modeling of selenium accumulation in brine shrimp in saline environments.

Great Salt Lake, Utah, is a large, terminal, hypersaline lake consisting of a northern more saline arm and a southern arm that is less saline. The southern arm supports a seasonally abundant fauna of low diversity consisting of brine shrimp (Artemia franciscana), 7 species of brine flies, and multiple species of algae. Although fish cannot survive in the main body of the lake, the lake is highly productive, and brine shrimp and brine fly populations support large numbers of migratory waterfowl and shorebirds, as well as resident waterfowl, shorebirds, and gulls. Selenium and other trace elements, metals, and nutrients are contaminants of concern for the lake because of their concentrations in municipal and industrial outfalls and runoff from local agriculture and the large urban area of Salt Lake City. As a consequence, the State of Utah recently recommended water quality standards for Se for the southern arm of Great Salt Lake based on exposure and risk to birds. The tissue-based recommendations (as measured in bird eggs) were based on the understanding that Se toxicity is predominately expressed through dietary exposure, and that the breeding shorebirds, waterfowl, and gulls of the lake are the receptors of most concern. The bird egg-based recommended standards for Se require a model to link bird egg Se concentrations to their dietary concentrations and water column values. This study analyzes available brine shrimp tissue Se data from a variety of sources, along with waterborne and water particulate (potential brine shrimp diet) Se concentrations, in an attempt to develop a model to predict brine shrimp Se concentrations from the Se concentrations in surrounding water. The model can serve as a tool for linking the tissue-based water quality standards of a key dietary item to waterborne concentrations. The results were compared to other laboratory and field-based models to predict brine shrimp tissue Se concentrations from ambient water and their diet. No significant relationships were found between brine shrimp and their dietary Se, as measured by seston concentrations. The final linear and piecewise regression models showed significant positive relationships between waterborne and brine shrimp tissue Se concentrations but with a very weak predictive ability for waterborne concentrations<10 µg/L.

Physiological characteristics and stress resistance of great sturgeon (Huso huso) juveniles fed with vitamins C, E, and HUFA-enriched Artemia urmiana nauplii.

This study was carried out to examine the effect of Artemia urmiana nauplii enriched with HUFA, and vitamins C and E on stress tolerance, hematocrit, and biochemical parameters of great sturgeon, Huso huso juveniles. Cod liver oil (EPA 18% and DHA 12%), ascorbyl-6-palmitate and alpha-tocopherol acetate were used as lipid, and vitamin C and E sources, respectively. Beluga juveniles at the stage of first feeding (69.7 +/- 5.9 mg body weight) were randomly divided into five treatments and three tanks were assigned to each diet. All fish groups were fed non-enriched Artemia for the initial 5 days and then fed enriched Artemia for 7 days. Juveniles were fed with Artemia enriched with HUFA + 20% vitamin C (C group); HUFA + 20% vitamin E-enriched Artemia nauplii (E group); HUFA + 20% vitamin C + 20% vitamin E (C and E group); HUFA without vitamins (HUFA) and non-enriched Artemia (control). After the period of enrichment, Juveniles were fed with Daphnia sp. from the 13th to the 40th day. At day 40, the fish were transferred directly from fresh water (0.5 ppt) to brackish water (6 ppt for 4 days and 12 ppt for 2 days) and warm water (from 27 to 33 degrees C) to evaluate juvenile resistance to salinity and thermal shocks. Moreover, all treatments were separately exposed to freshwater in tanks with the same capacity as used for osmotic and thermal tests (as fresh water control). The addition of vitamins C, E, and C + E to HUFA significantly increased fish resistance to 12 ppt salinity and temperature stress tests, whereas survival was not significantly different among challenges at 6 ppt. There was no significant difference in the hematocrit index under stress conditions. Enrichment had significant influence on plasma Na(+) level in the C group on the 4th day at 6 ppt. Na(+) and Ca(2+) concentrations in C, E, and C and E groups on the 1st day at 12 ppt, and Ca(2+) level in E group on the 2nd day at 12 ppt were lower than the other groups. The glucose level in the C and C and E groups was lower than the other treatments on the 1st day at 12 ppt and the 2nd day at 33 degrees C. Regardless of Artemia enrichment, plasma ions (Na(+), K(+), Ca(2+), and Mg(2+)) and glucose concentrations in fish exposed to salinity stress tests were higher than fish in fresh water. Glucose concentration in plasma also increased after 2 days at 33 degrees C. Although most of our results were not significantly different, the use of vitamins C, E, and HUFA in Artemia enrichment can improve Juveniles tolerance under stress conditions, and regardless of enrichment, these data show that beluga juveniles are partly sensitive to high salinity and temperature.

Influence of metal ions and organic carbons on denitrification activity of the halotolerant bacterium, Paracoccus pantotrophus P16 a strain from shrimp pond

The effect of metal ions, ferric ion (Fe3+) and molybdenum ion (Mo6+) on the denitrification process of Paracoccus pantotrophus P16 grown under saline conditions was investigated. Results revealed that the dosages of added Fe3+ and Mo6+ significantly accelerated nitrate utilization and nitrite accumulation. Enzymatic studies revealed that the membrane-bound nitrate reductase and the periplasmic nitrite reductase had activities of 998 +/- 28 and 373 +/- 18 nmol (mg protein)-1 min-1, respectively after growing Paracoccus pantotrophus P16 in medium supplemented with 1.5 micron M Fe3+. If provided with 1.5 micron M Fe3+and 2.4 micron M Mo6+, the membrane-bound nitrate reductase activity increased to 6,223 +/- 502 nmol (mg protein)-1 min-1 and the periplasmic nitrite reductase was 344 +/- 20 nmol (mg protein)-1 min-1. The results indicated that an addition of Fe3+ and Mo6+ led to an overstimulation of nitrate reductase activity as compared with nitrite reductase activity. When glucose was supplied, the minimal ratio of carbon per nitrate (C/N) was 2.31 mg C/mg NO3--N with denitrification yield of 0.45 g NO3--N/g C. Addition of ethanol instead of glucose, the minimal ratio of C/N was 1.15 mg C/mg NO3--N with denitrification yield of 1.08 g NO3--N/g C.

Antimicrobial and cytotoxicity activities of the medicinal plant Primula macrophylla.

Primula macrophylla (Primulaceae) is reported as to be useful in asthma, restlessness, insomnia and fish poisoning. Antifungal and toxic activities of crude extract, fractions and a pure isolated compound exhibited statistically significant activities. Excellent antifungal activity was found in the crude extract, benzene and ethyl acetate fractions against T. longifusis and against M. canis with different MIC values. Antileishmanial activity (IC(50) = 50ug/mL) was observed as compared to standard drug Amphotericin B, and cytotoxic activity (LD(50) = 47.919microg/mL) was also found in the chloroform fraction. While pure compound 2-phenylchromone (Flavone) isolated from the chloroform fraction showed good activity (IC(50) = 25microg/mL) against Leishmania and cytotoxicity (LD(50) = 2.0116 microg/mL) in Brine Shrimp experiments. From antileishmanial and cytotoxic activity it can be concluded that 2-phenylchromone is the major compound responsible for these activities.

A new method for the study of essential fatty acid requirements in fish larvae.

This study describes a methodology with potential application in the estimation of essential fatty acid (EFA) requirements of fish larvae. Senegalese sole (Solea senegalensis) larvae were fed, from 16 days after hatching (DAH), on Artemia enriched with different oils, inducing graded dietary concentrations of DHA: (1) soyabean oil, containing no measurable amounts of DHA (NDHA); (2) fish oil, inducing a medium DHA level (MDHA, 3 g DHA/100 g fatty acids); and (3) a mixture of Easy DHA Selco and Microfeed, resulting in high DHA content (HDHA, 8 g/100 g). At 28 DAH a metabolic trial was conducted where larvae were tube fed [1-(14) C]DHA, in order to determine its absorption, retention in the gut and body tissues, as well as its oxidation. At 23 DAH the HDHA treatment induced a significantly higher larval growth, while at 32 DAH significant differences were only found between the NDHA and HDHA treatments. The absorption of tube-fed [1-(14) C]DHA was extremely high (94-95 %) and independent of feeding regime. However, in larvae fed NDHA Artemia, a significantly higher amount of label was retained in the gut compartment and a concurrently lower retention was measured in the body. A significantly higher proportion of the absorbed DHA label was oxidized in larvae fed HDHA, compared to NDHA. Based on these results, we suggest that increasing dietary supply of DHA above the larval requirement level results in its increased oxidation for energy purposes and we propose potential applications of the tube feeding methodology using radiolabelled EFA in conjunction with dose-response studies.

Identification, cloning and tissue localization of a rainbow trout (Oncorhynchus mykiss) intelectin-like protein that binds bacteria and chitin.

Intelectins are a recently identified group of animal lectins involved in innate immune surveillance. This paper describes the primary structure, expression and immunohistochemical localization of a rainbow trout plasma intelectin (RTInt). RTInt exhibited calcium-dependent binding to N-acetylglucosamine (GlcNAc) and mannose conjugated Toyopearl Amino 650 M matrices. When GlcNAc eluates from chromatography matrices were analyzed by reducing 1D PAGE and Western blots, the lectin appeared as approximately 37 kDa and approximately 72 kDa bands. Similar analysis of plasma revealed a single 72 kDa band under reducing conditions. MALDI-TOF MS demonstrated five, approximately 37 kDa isoforms (pI 5.3-6.1) separated by 2D-PAGE. A 975 bp cDNA sequence obtained by RT-PCR from liver and spleen tissue encoded a 325 amino acid secretory protein with homology to human and murine intelectins, which bind bacterial components and are induced during parasitic infections. Gene expression and immunohistochemistry detected RTInt in gill, spleen, hepatic sinusoid, renal interstitium, intestine, skin, swim bladder and within leukocytes. Direct binding assays demonstrated the ability of RTInt to bind relevant bacterial and chitinous targets. These findings suggest that RTInt plays a role in innate immune defense against bacterial and chitinous microbial organisms.

Derivation of a chronic site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA.

The purpose of this study was to develop a site-specific water quality standard for selenium in the Great Salt Lake, Utah, USA. The study examined the bioavailability and toxicity of selenium, as selenate, to biota resident to the Great Salt Lake and the potential for dietary selenium exposure to aquatic dependent birds that might consume resident biota. Because of its high salinity, the lake has limited biological diversity with bacteria, algae, diatoms, brine shrimp, and brine flies being the only organisms present in the main (hypersaline) portions of the lake. To evaluate their sensitivity to selenium, a series of acute and chronic toxicity studies were conducted on brine shrimp (Artemia franiciscana), brine fly (Ephydra cinerea), and a hypersaline alga (Dunaliella viridis). The resulting acute and chronic toxicity data indicated that resident species are more selenium tolerant than many freshwater species. Because sulfate is known to reduce selenate bioavailability, this selenium tolerance is thought to result in part from the lake's high ambient sulfate concentrations (>5,800 mg/L). The acute and chronic test results were compared to selenium concentrations expected to occur in a mining effluent discharge located at the south end of the lake. Based on these comparisons, no appreciable risks to resident aquatic biota were projected. Field and laboratory data collected on selenium bioaccumulation in brine shrimp demonstrated a linear relationship between water and tissue selenium concentrations. Applying a dietary selenium threshold of 5 mg/kg dry weight for aquatic birds to this relationship resulted in an estimate of 27 microg/L Se in water as a safe concentration for this exposure pathway and an appropriate chronic site-specific water quality standard. Consequently, protection of aquatic birds represents the driving factor in determining a site-specific water quality standard for selenium.

Development of a bioassay for phytochemicals using Daphnia pulex.

The freshwater cladoceran Daphnia pulex was explored as an alternative to Artemia salina for the biological screening of phytochemicals. This paper reports on the results of screening 27 compounds, and comparisons are made with screens using Artemia salina. The effect of miniaturisation of the assay system was investigated. Petri-dish based tests were performed for the 27 compounds using 10 daphnids at each of 5 concentrations (1 - 1000 mg l(-1)). Potassium dichromate was used as a control and the number immobile after 24 hours were counted. Results were expressed as EC(50) values. The active compounds (EC(50) < 25 mg l(-1)) comprised mainly adrenoceptor agonist alkaloids, while the cytotoxins vincristine and colchicine, and several antibiotics were relatively non-toxic towards Daphnia. The method was validated by comparative bioassay of A. belladonna fractions. Overall, this screen compared favourably with those based on Artemia. Daphnia were sensitive to a wide range of biologically active molecules including CNS-stimulants, anti-malarials, narcotics and anti-spasmodics, and the sensitivity was broader than previously seen with Artemia. The screen is perhaps limited by the lack of sensitivity to cytotoxins, but this might be overcome by altering the test criteria.

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 > or =11 micromol x 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.

The biosynthesis of omega 3 fatty acids from 18:2 omega 6 in Artemia spp.

To examine the biosynthesis of omega 3 fatty acids from 18:2 omega 6, reference Artemia (RAC III, Artemia Reference Center, Gent, Belgium) were fed rice bran supplemented with [1(-14)C]18:2 omega 6 for 2 days following 48 hours starvation since cyst hydration and 1, 2, or 3 days following 72 hours of starvation, under axenic conditions. Artemia fatty acids were analyzed by gas chromatography, AgNO3 thin layer chromatography and high performance liquid chromatography and fatty acid fractions were collected for radioisotope counts. No significant differences were observed in the omega 3 fatty acid content of Artemia cultured under axenic and xenic conditions. Radioisotope studies showed that radioactivity from [1(-14)C]18:2 omega 6 was incorporated into other fatty acids, including 18:3 omega 3, 18:4 omega 3 and 20:5 omega 3. The conversion rate was less than 5% for the two day feeding period. In the three day feeding experiment, the amount of radioactivity recovered in both 18:3 omega 3 and 20:5 omega 3 increased by 2.2- and 1.8-fold, respectively, over the three day feeding period. These results demonstrated that Artemia synthesized these omega 3 polyunsaturated fatty acids from 18:2 omega 6.