Microbial Control of the Culture of Artemia Juveniles through Preemptive Colonization by Selected Bacterial Strains

The use of juvenile Artemia as feed in aquaculture and in the pet shop industry has been getting more attention during the last decade. In this study, the use of selected bacterial strains to improve the nutritional value of dry food for Artemia juveniles and to obtain control of the associated microbial community was examined. Nine bacterial strains were selected based on their positive effects on survival and/or growth of Artemia juveniles under monoxenic culture conditions, while other strains caused no significant effect, significantly lower rates of survival and/or growth, or even total mortality of the Artemia. The nine selected strains were used to preemptively colonize the culture water of Artemia juveniles. Xenic culture of Artemia under suboptimal conditions yielded better survival and/or growth rates when they were grown in the preemptively colonized culture medium than when grown in autoclaved seawater. The preemptive colonization of the culture water had a drastic influence on the microbial communities that developed in the culture water or that were associated with the Artemia, as determined with Biolog GN community-level physiological profiles. Chemotaxonomical characterization based on fatty acid methyl ester analysis of bacterial isolates recovered from the culture tanks was performed, and a comparison with the initially introduced strains was made. Finally, several modes of action for the beneficial effect of the bacterial strains are proposed.     

Selected Bacterial Strains Protect Artemia spp. from the Pathogenic Effects of Vibrio proteolyticus CW8T2

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

Mixture of parthenogenetic and zygogenetic brine shrimp Artemia (Branchiopoda: Anostraca) in commercial cyst lots from Great Salt Lake, UT, USA

The brine shrimp Artemia is one of the most studied animals in the world. A large part of the knowledge of this crustacean is based on cysts harvested from two main sources; the Great Salt Lake, UT (GSL), and the San Francisco Bay salterns, CA (SFB), USA. Artemia populations from these habitats are recognized to belong to a single zygogenetic species, Artemia franciscana Kellogg, 1906. However, the GSL Artemia has been in doubt for more than a century about the existence of parthenogenetic reproduction. By using morphological, reproductive, and molecular analyses, we report that commercial GSL cyst lots contained two different brine shrimp species; a parthenogenetic (60%) and a zygogenetic (A. franciscana) (40%). From this finding, at least three hypotheses can be drawn. The parthenogenetic Artemia is native of GSL, or it was introduced to GSL, or foreign parthenogenetic cysts were mixed with A. franciscana cysts and canned for commercial distribution. Researchers using brine shrimp cysts from GSL should therefore pay careful attention to the correct identity of the species under study. The potential of an easy and unnoticed introduction of parthenogenetic Artemia into America is discussed.     

Assessment of spatial distribution and cysts resources of Artemia in late autumn in Dangxiong Co salt lake

Based on the vertically interval sampling in 25 sampling sites in Dangxiong Co salt lake in 2011, a preliminary investigation on population spatial distribution and cysts resources of Artemia in the lake has been conducted. The study achieves four new progresses. First, the average density of Artemia and Artemia cysts in the lake is 4.157 × 10³ ind. m⁻² and 8.069 × 10⁴ ind. m⁻², respectively. Among Artemia, the adults account for 60.31%; Second, different from other salt lakes in horizontal distribution, the Artemia population mainly distributes in the open water, only a little in the shallow water, and there is no distribution in the longshore area in the north part and the estuary region; Third, in vertical distribution, 44.24% of individuals intensively distribute in the upper water layer within 2.0 m, especially 0–0.2 m, where the average density of Artemia and cysts are maximum (129.488 ind. L⁻¹ and 5.728 ind. L⁻¹, respectively). A decrease of distribution density is accompanied by an increase of water depth basically, the percentage of Artemia and cysts decrease to 0.68% and 4.60%, respectively; Fourth, the cysts resources of 14.96 t in the lake are assessed using contour map. 66.35% of them distributed in 0.0–2.0 m water layer and 49.06% concentrate in the 18.21% areas of the central water; Fifth, quantity of cysts suitable for development in the lake is 2.399 ± 0.320 t, with an upper limit of 0.879 t. The study can provide a reference for the sustainable development and exploitation of Artemia in Dangxiong Co salt lake.     

Whole genome survey and microsatellite motif identification of Artemia franciscana

Artemia is an industrially important genus used in aquaculture as a nutritious diet for fish and as an aquatic model organism for toxicity tests. However, despite the significance of Artemia, genomic research remains incomplete and knowledge on its genomic characteristics is insufficient. In particular, Artemia franciscana of North America has been widely used in fisheries of other continents, resulting in invasion of native species. Therefore, studies on population genetics and molecular marker development as well as morphological analyses are required to investigate its population structure and to discriminate closely related species. Here, we used the Illumina Hi-Seq platform to estimate the genomic characteristics of A. franciscana through genome survey sequencing (GSS). Further, simple sequence repeat (SSR) loci were identified for microsatellite marker development. The predicted genome size was ∼867 Mb using K-mer (a sequence of k characters in a string) analysis (K = 17), and heterozygosity and duplication rates were 0.655 and 0.809%, respectively. A total of 421467 SSRs were identified from the genome survey assembly, most of which were dinucleotide motifs with a frequency of 77.22%. The present study will be a useful basis in genomic and genetic research for A. franciscana.     

Primary production of plankton and decomposition of organic matter in saline lakes of the crimea peninsula

The lakes under study can be classified as eutrophic-hypertrophic waterbodies according to the level of primary production. High concentrations of suspended matter (up to 7326 g/m³) and total phosphorus (up to 5625 mg/m³) were registered in the lakes. The values of the primary production of plankton and the destruction of organic matter do not depend on the absolute concentration of salts in water. Unlike the destruction of organic matter, the primary production of plankton is higher in winter than in summer. Thus, the accumulation of organic matter in saline lakes occurs mainly in winter. The negative relationship between the primary production of plankton and the biomass of the filter feeder Artemia sp. has been determined.     

Urmia Lake (Northwest Iran): a brief review

Lake Urmia (or Ormiyeh) is one of the largest hypersaline lakes in the world and the habitat of a unique bisexual Artemia species (A. urmiana). Despite this, and several other values of the lake, little literature on it has been published. The present paper is an attempt to provide a brief review on various aspects of the lake. Urmia Lake, located in northwestern Iran, is an oligotrophic lake of thalassohaline origin with a total surface area between 4750 and 6100 km² and a maximum depth of 16 m at an altitude of 1250 m. The lake is divided into north and south parts separated by a causeway in which a 1500-m gap provides little exchange of water between the two parts. Due to drought and increased demands for agricultural water in the lake's basin, the salinity of the lake has risen to more than 300 g/L during recent years, and large areas of the lake bed have been desiccated. Therefore, management and conservation of this incomparable ecosystem should be considered to improve the current condition by fisheries research institutes.     

Experimental exposure of Artemia to Hepatopancreatic parvo-like Virus and Subsequent transmission to post-larvae of Penaeus monodon

The different life stages of Artemia franciscana were experimentally exposed to Hepatopancreatic parvo-like virus (HPV), in order to evaluate the possibility of Artemia acting as reservoir or carrier for HPV. All the five developmental stages of Artemia were challenged with HPV both by immersion and oral infection routes. The viral infectivity to Artemia was studied by PCR but not much difference in mortality between control and challenge groups were observed. To confirm the vector status of Artemia for HPV, the HPV exposed Artemia were fed to postlarval forms of Penaeus monodon. Post-larvae of P. monodon were fed with HPV exposed Artemia and could get infected upon feeding on them. Mortality was observed in the post-larvae, which were fed with HPV exposed Artemia, and whereas no mortality was observed in post-larvae fed with Artemia not exposed to HPV and these post-larvae were PCR negative for HPV, as well. Results of this experiment suggest that Artemia might be a possible horizontal transmission pathway for HPV. Further research however is required with histology, immunohistochemistry and transmission electron microcopy to determine whether the Artemia are actually infected with this virus or whether they are simply mechanical carriers. This will enable us to understand better whether Artemia is a carrier of this virus and if so the mechanism involved.     

The self-diffusion of water in Artemia cysts

The self-diffusion coefficient of water molecules has been measured by nuclear magnetic resonance in cysts of Artemia over a wide range of hydration. Compared to the value for bulk water, the diffusion coefficient is reduced by a factor of 7 at the highest hydration and by approximately 100 at the lowest hydration. The results are used to evaluate various models that have been proposed to account for the reduction of water self-diffusion coefficients in complex systems.     

Cellular responses to extreme water loss: The water-replacement hypothesis

The previously advanced hypothesis that desiccation resistance involves the replacement of water adjacent to intracellular surfaces with polyhydroxy compounds has been supported by experiments on cysts of the brine shrimp, Artemia, and in a model system of albumin-glycerol-water, using nuclear magnetic resonance spectroscopy, microwave dielectrics, and density measurements. We have also considered other problems that cells face when large fractions of their total water content are removed. Observations by other investigators have indicated that a variety of mammalian cells can lose roughly 50% of their water and survive; for a given cell type death occurs if its volume is reduced below a certain minimum level. Membrane damage has previously been suggested to be a major cause of dehydration damage. We have proposed some additional plausible mechanisms that might also be involved.     

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.     

Determination of oxytetracycline in the live fish feed Artemia using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic analytical method was developed for the determination of oxytetracycline in Artemia nauplii. A solid-phase extraction protocol was used to recover oxytetracycline and the internal standard tetracycline, from the Artemia samples. Oxytetracycline was analyzed using a 150 × 4.6 mm I.D. Hypersil-ODS column, a mobile phase of acetonitrile-tetrahydrofuran-0.01 M oxalic acid buffer (pH3.0) (15:3:82, v/v), and an ultraviolet detection wavelength of 365 nm. The calibration curve of oxytetracycline in Artemia was linear (r² = 0.9998) from 0.1 to 6.4 μg/g of tissue. Using a signal-to-noise ratio of 4:1 the oxytetracycline detection limit was 10 ng/g of tissue. Mean recovery of oxytetracycline amounted to 97%, while intra-assay variability was 1.5%. Quantitative data from an in-vivo feeding study indicated an excellent uptake of oxytetracycline by Artemia, as its levels reached 25.6 μg per g of nauplii.     

Use of selected bacteria and yeast to protect gnotobiotic Artemia against different pathogens

To evaluate the potential probiotic effect of two bacterial strains towards Artemia cultured in different gnotobiotic conditions, challenge tests were performed with a virulent Vibrio campbellii or with an opportunistic Vibrio proteolyticus strain. For that purpose, three feed sources (different isogenic Saccharomyces cerevisiae mutant strains) were chosen, yielding distinct Artemia culture performances. Both bacterial strains, selected from previous well-performing Artemia cultures, were able to protect against the opportunistic V. proteolyticus, while, generally, these bacteria could not protect Artemia against V. campbellii. The quality of the feed provided (in the form of the isogenic mnn9 yeast mutant) to Artemia had a stronger influence on nauplii protection against the opportunistic and the virulent Vibrio than the addition of beneficial bacteria. This feed has a higher nutritional value for Artemia, but contains also more cell wall bound β-glucans and chitin. Data suggest that the change in the cell wall composition, rather than the overall better nutritional value, of the mnn9 strain is responsible for the protection against both Vibrios.     

Planktonic Fauna of Small Reservoirs in the Komi Republic

One hundred and eight species and forms of planktonic organisms have been revealed in three small reservoirs in the Komi Republic (the northeast of European Russia). Species that were not previously known for the water bodies of the Vychegda River Basin (Limnosida frontosa) and the Republic of Komi (Lecane mira, Macrochaetus subquadratus, Filinia longiseta limnetica, and Rhynchotalona falcata) have been found. It has been established that the richness of the planktonic fauna depends on the age of the water body: the maximum number of species is found in the oldest reservoir, namely, the Kazhim Reservoir. The planktonic communities of artificial reservoirs are distinguished by a low level of similarity of faunas both with each other and in comparison with the natural lakes of the region. The fauna distribution in the old reservoirs is uneven. The relatively large fauna richness of planktonic communities in the littoral zones is due to the water-level regime in the reservoirs.     

Attraction of Aurelia and Aequorea to prey

Previously it has been assumed that medusae make random contact with their prey. Medusae were tested in a flow-through aquarium with inflow at each end and a central outflow. Aurelia aurita and Aequorea victoria were attracted to either end of the chamber if Artemia prey were present in a screened compartment. A. aurita was also shown to be attracted to water conditioned by Artemia, and to ammonium chloride added to one or other of the inflow currents.     

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded^1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes³. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation⁴. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.     

Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.     

Identification of extremely halophilic archaea associated with adult <Emphasis Type="Italic">Artemia urmiana</Emphasis>

The brine shrimp, Artemia is the dominant macrozooplankton present in many hypersaline environments. Artemia urmiana is the only macroscopic organism in Urmia Salt Lake (Iran), and the high salinity of the lake makes it a suitable environment for halophilic archaea too. Because of common environment for Artemia and extreme halophiles; this investigation is concentrated on studying the relationship between Artemia and halophilic archaea in Urmia Lake. In this study first the procedure of arhaea isolation was done. Then, isolated strains were sub-cultured and DNA was extracted and amplified by PCR using specific primers for amplifying archaeal 16S rRNA. The amplified archeal DNA fragments were purified, and sequenced. 16S rRNA sequences were compared to known sequences using the NCBI BLAST program. Sequences relating to Halorubrum, Haloarcula and Halobacterium species were identified in Urmia Salt Lake water and Artemia adults and the phylogenetic tree of different species was constructed. Only Halorubrum species were present in association with Artemia. They belong to Halobacteriaceae family of archeae which are isolated from different salt lakes in different parts of world and we could show their existence in adult Artemia, another organism living in hypersaline enviroments.     

Cyclopoid copepods as bioindicators of eutrophication in reservoirs: Do patterns hold for large spatial extents?

Some species of copepods are sensitive to water quality oscillations from natural or anthropogenic causes. Information on basic ecological attributes such as abundance can be helpful in the context of hydric resources monitoring. Our study analyzed if the abundance of 22 copepod species of the second largest basin of South America was more associated with variables oscillating by natural or anthropic causes, contrasting among oligotrophic, mesotrophic, and eutrophic reservoirs. Our aim was to identify and understand the abundance of species with potential to monitor water quality in large scale assessments. Potential bioindicators would have different abundances in eutrophic, mesotrophic and oligotrophic sites and would not oscillate according to natural characteristics of reservoirs (water temperature, air temperature, and depth). Two species were sensitive to eutrophication and were not related to natural characteristics of reservoirs, that is, they were suitable for biomonitoring the La Plata Basin. Thermocyclops minutus negatively responded to eutrophication, while Acanthocyclops robustus responded positively. Additional exploratory analyses identified that Copepod abundance was related to total phosphorus, chlorophyll-a concentration, water transparency, total suspended matter, and depth. Metacyclops mendocinus, Acanthocyclops robustus, Mesocyclops meridianus, Mesocyclops ogunnus, and Thermocyclops decipiens were abundant in eutrophic reservoirs, and Thermocyclops minutus, and Thermocyclops inversus were associated with higher water transparency, typically oligo/mesotrophic reservoirs. Overall, we found that cyclopoids are highly affected by eutrophication, and species abundance could be used to monitor reservoirs and anticipate potential impacts on water quality in large-scale biomonitoring schemes.     

Nutritional value of the cryptophyte Rhodomonas lens for Artemia sp.

Juvenile or adult Artemia sp. are often used as live prey for the rearing of early life stages of some crustacean, fish and cephalopod species. The improvements of both Artemia growth and its biochemical composition are key issues for the suitable use of Artemia biomass in these rearing processes. In this study we evaluated the growth and survival rates of Artemia fed with the cryptophyte Rhodomonas lens in comparison with different microalgal species commonly used in aquaculture: the prasinophyte Tetraselmis suecica, the prymnesiophyte Isochrysis galbana Parke, and the eustigmatophyte Nannochloropsis gaditana. Microalgae were cultured semi-continuously in nutrient saturated conditions and with a daily renewal rate of 30% of the volume of cultures, to obtain biomass of controlled and optimized composition. Considerable differences in Artemia growth were observed, as well as in the survival rate. At day 8 of rearing, Artemia fed R. lens had the highest length (4.9 ±0.6 mm, P < 0.001), followed by individuals fed T. suecica (4.2 ± 0.7 mm), I. galbana (3.6 ± 0.7 mm) and finally those fed N. gaditana (1.5 ± 0.2 mm). The survival rate of Artemia fed N. gaditana (18 ± 3%) was much lower (P < 0.001) than values found for the remaining groups (69 to 88%). The growth rate of Artemia obtained with R. lens was in general much higher than with other microalgal diets previously reported in the literature. The higher protein content of R. lens could explain the higher growth obtained with this species, but differences of Artemia growth with the different diets could not be explained solely on the basis of the gross composition of microalgae. Factors such as cell size and digestibility all seem to contribute to the results observed. Another trial was carried out to investigate differences in Artemia growth and on its biochemical composition when fed the best two diets: R. lens or T. suecica. The fatty acid (FA) and total amino acid (AA) composition of both microalgal species and the composition of Artemia were assessed as well. As found in the first experiment individuals fed R. lens (group ARHO) grew faster than those fed T. suecica (group ATET), attaining 3.6 ± 0.3 mm and 3.2 ± 0.4 mm (P < 0.001), respectively, after 5 days of rearing. The much higher AA content obtained in R. lens may be on the basis of the higher growth obtained with this species. Protein and carbohydrate levels in Artemia juveniles were very similar in both groups (64-68% of dry weight, and 8-10%, respectively). Lipid was slightly lower in ARHO (12%) than in ATET (15%, P < 0.01). Regarding the FA composition, juveniles from group ARHO contained higher levels of eicosapentaenoic acid (EPA, 6.2%) than juveniles from ATET (4.1%, P < 0.01), whereas docosahexaenoic acid (DHA) was only found in juveniles from ARHO (1.1%). Taking into account that the daily productivity of R. lens culture was higher than, or at least equal, the remaining microalgal species this cryptophyte is confirmed as an excellent diet to optimize the growth of Artemia, as well as to improve its biochemical composition.