Characterization of brine shrimp Artemia from Cam Ranh Bay in Central Vietnam

Artemia cysts collected from inoculation experiments in Cam Ranh salterns are evaluated for their potential use in aquaculture. Cyst biometrics, hatching quality, naupliar fatty acid profile and naupliar growth were measured and compared to reference Artemia strains. Cyst characteristics reveal that the parthenogenetic strain (PR China) used in inoculations, was eliminated from the environment and that the remaining brine shrimp are likely to be composed of Macau and Great Salt Lake Artemia strains, and of their cross-breds. Differences in cyst diapause deactivation characteristics between Macau and Great Salt Lake Artemia may have resulted in the disappearance of Macau Artemia during the rainy season and the persistence of Great Salt Lake Artemia during the following dry season.     

Sediment cores from Lake Urmia (Iran) suggest the inhabitation by parthenogenetic Artemia around 5,000?years ago

In Lake Urmia area, northwestern Iran, parthenogenetic Artemia and the bisexual Artemia urmiana Günther 1890 are found to occupy different ecological niches determined by salinity. Given the fluctuations of the lake over geological times, we thus hypothesized that species identification of Artemia cysts, buried in the sediments, can provide information on lake conditions in the past. Therefore, encysted embryos of Artemia were recovered from lake sediments by augering at a site near the present shoreline. Cysts and associated plant remains from two studied levels yielded radiocarbon ages in the range 5,000–6,700 YBP. For determination of the type of Artemia, the constant synonym mutation in exon-7 of the Na/K ATPase gene was verified, and the diameter of the recovered cysts was compared with that of modern cysts from the Lake Urmia region. The results show that the cysts represent a parthenogenetic type of Artemia, whose cyst diameter is somewhat different from that of present-day local parthenogenetic Artemia. The present study firstly confirms the stability of DNA in ancient Artemia cysts for molecular analysis. Moreover, it suggests variation in Lake Urmia’s conditions over time, and based on comparison with salinity preferences of contemporary Artemia populations, it more specifically suggests that Lake Urmia was a brackish lake dominated by a parthenogenetic Artemia population in the geological period sampled. It finally illustrates how, like in the study of freshwater propagule banks, paleogenetic analysis of Artemia DNA recovered from sediment cores can be used as a tool in the paleoecological study of generally highly fluctuating saline habitats.     

Variations in repetitive DNA and heterochromatin in the genus Artemia

The genus Artemia (Crustacea, Phyllopoda) is widely distributed all over the world as a result partly of natural colonization and partly of spread by birds and man. Artemia offers a very interesting model for speciation studies, since the genus comprises both bisexual sibling species and parthenogenetic populations, exhibiting different chromosome numbers (diploidy, heteroploidy and polyploidy). The finding of the clustered repetitive AluI DNA family in the heterochromatin of A. franciscana can provide a useful tool for investigating the relationship between the members of the genus Artemia at the molecular level. Sixteen strains of Artemia, comprising sibling species and parthenogenetic populations, were analysed for the presence of AluI repetitive DNA by dot-blot hybridization. The observed variation in the content of repetitive DNA together with genetical, biological and geological data, support the hypothesis that Artemia living in the New World are derived from ancestral species that evolved in the Mediterranean area.     

Solar Pond devices: free energy or bioreactors for<Emphasis Type="Italic"> Artemia</Emphasis> biomass production?

The recent exponential growth in industrial aquaculture has led to a huge increase in Artemia biomass production in order to meet increased fish production needs. The present study explores the potential use of salt gradient solar ponds (SGSPs) for production of Artemia nauplii. An SGSP is a basin of water where solar energy is trapped and collected via an artificially imposed gradient. Three zones can be identified in an SGSP: upper and lower zones, which are both convective, and a middle zone, which is intended to be non-convective. The latter acts as a transparent insulation layer and allows for storage of solar energy at the bottom, where it is available for use. The combination of salt, temperature and high transparency could make SGSPs promising bioreactors for the production of Artemia nauplii. Using particle image velocymetry (PIV) and Shadowgraph visualisation techniques, the behaviour of Artemia nauplii under critical cultivation parameters (namely, salinity, temperature and light) was monitored to determine movement velocity, and how movement of Artemia affects the salt gradient. It was observed that Artemia nauplii constantly follow light, irrespective of adverse salinity and/or temperature conditions. However, despite the substantial displacement of Artemia following the light source, the salt gradient is not disrupted. The suitability of SGSPs as bioreactors for Artemia biomass production was then tested. The results were disappointing, probably due to the lack of sufficient O₂ for Artemia survival and growth. Follow-up trials were conducted aimed at using the SGSP as a green and economically attractive energy source to induce faster hatching of cysts and improved Artemia nauplii growth. The results of these trials, and a case study of Artemia nauplii production using an SGSP, are presented. The authors constructed a Solar Pond device, which they suggest as a novel way of supplying thermal energy for Artemia biomass production in an aquaculture enterprise. Finally, the authors suggest a method of producing and collecting Artemia biomass, and of heating a fish larval tank, in an ‘ideal’ Solar Pond device, profiting from the low investment costs of using a decommissioned salt works.     

Development of paratransgenic Artemia as a platform for control of infectious diseases in shrimp mariculture

Aim: To study the accumulation and retention of recombinant proteins in Artemia gut for optimizing paratransgenic disease control in shrimp aquaculture. Methods and Results: Transgenic Escherichia coli expressing fluorescent marker proteins and the transgenic cyanobacterium Synechococcus bacillarus expressing a functional murine single chain antibody, DB3, were fed to Artemia franciscana. Stable expression and retention of several marker molecules (e.g. GFP, DS Red and DB3) up to 10 h after of feeding with E. coli were evident within the gut of Artemia. Engineered strains of S. bacillarus expressing DB3 accumulated within the gut of Artemia with detectable antibody activity for 8–10 h of feeding via ELISA, coincident with the time period of the highest density of transgenic S. bacillarus in the Artemia gut. Conclusions: Artemia fed transgenic bacteria or algae accumulated recombinant proteins for up to 10 h that retained biological activity. Co‐delivery of multiple recombinant proteins simultaneously in the gut of Artemia was also demonstrated. Significance and Impact of the Study: Expression of molecules that target infectious agents of mariculture in shrimp via commonly deployed feed organisms such as Artemia could potentially offer powerful new tools in the ongoing global effort to increase food supply.     

Anti-infectious potential of beta-mercapto-ethanol treated baker's yeast in gnotobiotic Artemia challenge test

Aim: To evaluate nutritional and anti‐infectious characteristics of the chemically treated baker’s yeast with 2‐mercapto‐ethanol (2ME) for gnotobiotically grown Artemia. Methods and Results: A selection of isogenic yeast strains was treated with 2ME and fed to gnotobiotically grown Artemia. In the first experiment the effect of the chemical treatment on the yeast nutritional value was studied. In most cases, 2ME‐treated yeast cells were better feed for Artemia than the untreated cells. In the second experiment, a small quantity of 2ME‐treated yeast cells was fed to Vibrio campbellii (VC) challenged Artemia. The 2ME‐treatment on some yeast strains (e.g. gas1, kre6 and chs3) significantly improved Artemia resistance against VC compared with the respective untreated yeast cells. Conclusion: Simple chemical treatment with 2ME could significantly improve the nutritional and anti‐infectious properties of some baker’s yeast strains for gnotobiotically grown Artemia. Significance and Impact of the Study: The gnotobiotic Artemia test system provides a unique opportunity (because of noninterference of other microbial compounds) to investigate how the yeast cell wall composition influences macro parameters (e.g. growth and survival) in an organism. In addition, gene expression studies in these gnotobiotically grown Artemia should provide further documentation on direct effects of yeast cells on the genes involved in immune functions.     

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.     

Genetic variation in sympatric populations of diploid and polyploid brine shrimp (Artemia parthenogenetica)

We examined genetic variation in sympatric diploid and polyploid brine shrimp Artemia parthenogenetica from each of three populations (China, Italy and Spain). Italian and Spanish tetraploids are closely related (I=0.964). Diploids and tetraploids within each of the two European populations are also closely related (mean I=0.905). Most alleles found in diploids also exist in sympatric polyploids. In contrast, the asexual Artemia (2N, 4N and 5N) in our study share few alleles with their close sexual relative, A. tunisiana (mean I=0.002). These results, as well as the work of other authors, strongly suggest that at least the tetraploid Artemia in our study have an autopolyploid origin.Clonal diversity of polyploid Artemia can be very high at least in some population. Both diploids and polyploids had low clonal diversities in the populations dominated by polyploids and high clonal diversities in the population dominated by diploids.The most common genotypes of sympatric diploid and polyploid Artemia frequently differed. Some alleles occurred only in diploids, while others were restricted to polyploids. These results suggest that polyploidy in Artemia has led to genetic divergence from diploid progenitors, and that ploidy-level variation must also be considered in developing an understanding of spatial and temporal allozyme polymorphism in asexual populations.     

Larval rearing of chub, Leuciscus cephalus (L.), using decapsulated Artemia as direct food

Little is known about the larviculture of the chub, Leuciscus cephalus (L.), an endangered cyprinid species endemic to European flowing waters. The use of decapsulated Artemia cysts as food for chub larviculture was investigated. After 3‐day feeding with the rotifer Brachionus calyciflorus, the larvae were fed on different diets: (i) dried decapsulated Artemia cysts, (ii) Artemia nauplii, (iii) rotifers for seven more days and then Daphnia collected from a pond, and (iv) an artificial diet. After a 24‐day rearing period, the highest survival rate was obtained with the larvae receiving decapsulated Artemia cysts. Feeding of the larvae with an artificial diet resulted in a significantly lower survival rate compared with the other groups. At the end of the experiment, the larvae fed on Artemia nauplii yielded a significantly higher mean length compared with the other groups. Feeding an artificial diet resulted in a significantly lower average weight and mean length gain compared with the other groups.     

May parthenogenesis in Artemia be attributed to Wolbachia?

Rickettsia-like maternally inherited bacteria are commonly associated with the induction of severe modifications of the reproductive behaviour of their hosts, such as parthenogenesis. Among them, the endosymbiont Wolbachia is the best known because it is widespread in many arthropods, including insects, terrestrial isopods and the exclusively aquatic ostracods. The genus Artemia (Crustacea, Anostraca) that consists of both bisexual species and parthenogenetic strains was—to our knowledge—for the first time tested for infection with Wolbachia. We screened 38 Artemia populations (parthenogenetic and bisexual) using a PCR assay. Our results clearly show that Wolbachia is not involved in the induction of parthenogenesis in Artemia.     

Diet-induced variations in fatty acid content and composition of two on-grown stages of Artemia Salina

Three species of microalgae, the freshwater Euglena gracilis and themarine Dunaliella salina and Tetraselmis suecica, were fed tothe brine shrimp Artemia salina in order to compare their suitabilityin terms of fatty acid enrichment, and their effect on the biometric parametersof the zooplankter. The fatty acid content and composition were analyzed for the post-larval and pre-adult stages of Artemia fed the algae and theresults compared to the initial content of unfed 24-hour post-hatch nauplii.Differences in the total fatty acid content occurred between the three stages,the fatty acid profile being determined by the composition of the diet. A decreasing trend for almost all the individual fatty acids occurred throughdevelopment from post-larva to pre-adult with each of the three algal diets.Biometrical differences between Artemia fed the marine algae and that fed Euglena were not consistent in the post-larval stage, but became considerable in the pre-adult stage. Artemia fed with Euglena achieved twice the weight of animals fed the marine algae and showed thehighest length. The implications for the use of on-grown Artemia as afeed in larviculture of marine and freshwater fish and crustaceans are considered.     

Development of transgenic zooplankton <Emphasis Type="Italic">Artemia</Emphasis> as a bioreactor to produce exogenous protein

Although the crustacean Artemia has been commonly used as an experimental organism and served as a live bait feed for aquaculture, gene transfer system on Artemia sp. to generate stable lines is not well developed. In this study, we optimized a condition for cyst-eletroporation and generated stable lines of transgenic A. sinica. Two expression plasmids directed by the hybrid promoters of cytomegalovirus (CMV) and medaka β-actin (Mβ) were co-electroporated on decapsulated cysts: pCMV-Mβ-GFP contained GFP reporter gene and pCMV-Mβ-ypGH contained yellowfin porgy GH (ypGH) cDNA. We examined the GFP shown in the Artemia larvae and found that the expression rate was 13.3% (3,219 out of 24,054 examined). We then chose 200 G0 founders which strongly expressed GFP to generate transgenic lines. Homozygotic strains derived from F4 generation of each transgenic line, A3 and A8, were obtained. We proved that transgenic lines A3 and A8 also harbored pCMV-Mβ-ypGH and produced recombinant ypGH with a concentration of 0.089 and 0.032 μg per 50 homozygotic nauplii, respectively. Ten live Artemia nauplii were fed daily to zebrafish larvae during 25 to 35 days of post-fertilization. The average body length gain rates of zebrafish larvae fed transgenic Artemia were 16–20% greater than those of control group, indicating the exogenous ypGH produced by transgenic Artemia is functional. Therefore, we concluded that the transgenesis on Artemia is developed, and transgenic Artemia might be highly potentially useful as a new bioreactor material for application in aquaculture and biological researches.     

Effects of Altered Light and Feeding Regimes on the Zooplankton Feeding Capability of Hydra viridis

The effects of light regime, feeding regime and tentacle number on the zooplankton feeding capability of Hydra viridis were tested in the laboratory. Feeding was measured by exposing Hydra to a known volume of Artemia salina nauplii and recording the number captured and ingested. In all cases there was a correlation between the number of Artemia captured and the number ingested. H. viridis with 7 tentacles captured and ingested more Artemia than Hydra with 6 tentacles. However, changes in light and/or feeding regimes did not alter the number of tentacles/Hydra. Varying light and feeding regimes altered the number of Zoochlorellae/cell and Hydra growth rate. There was no effect on the number of Artemia captured or ingested and no effect on the percent ingestion of captured Artemia. These data suggest that, under these conditions, zooplankton feeding by H. viridis is independent of nutritional history.     

Effect of diets containing adenosine,guanosine, inosine or xanthosine on the nucleotide content of Artemia. Influence of mycophenolic acid

Artemia uses the stored diguanosine tetraphosphate as a source of adenine and guanine nucleotides during development from the encysted gastrula to the free swimming larva. Further development of the larvae depends on a dietary source of purine rings. We have investigated the growth of Artemia in axenic cultures supplemented with 0·6 mg ml^?1 of adenosine, guanosine, inosine or xanthosine. The total protein and soluble nucleotide content of Artemia grown in the presence of adenosine, guanosine or inosine was very similar, around (2 A₂₆₀ units and 500 mg protein) and (4 A₂₆₀ units and 1000 mg protein) after 4 and 6 days of postlarval development, respectively. The nucleotide pattern of those extracts subjected to HPLC were almost identical, the major peaks corresponding to ATP, ADP and AMP. Other nucleotides, not well characterized, were also present in those extracts. Mycophenolic acid (10 μg ml^?1) inhibited the growth of Artemia (as measured by their protein and soluble nucleotide content) in the presence of adenosine and inosine as the purine source, and had no appreciable effect in the presence of guanosine. A quantitative analysis of the chromatographic peaks obtained from Artemia grown in the presence of any of the three nucleosides ± mycophenolic acid showed that the effect of the antibiotic on each one of the chromatographic peaks was very similar, suggesting that Artemia, and probably other organisms as well, tend to maintain a balance between all nucleotides and to adjust the overall level to the limiting step(s) in their rates of synthesis/interconversion. Xanthosine was not able to support the development of Artemia.     

不同营养模式下内蒙古沙漠小型盐湖卤虫种群特征与环境负载力

通过对桑根达莱淖尔卤虫的养殖实验与卵囊解剖,研究了内蒙古沙漠小型盐湖投饵、施肥与自然状态3种营养模式下卤虫的生境、种群动态、生殖特征,分析了环境对卤虫资源的负载力。结果表明:1在起始种群相同的情况下,不同营养模式对种群结构与密度有显著影响;2不同营养模式对个体生长速度影响存在差异,投饵对加快个体生长速度效果最明显,但在性成熟速度方面不同营养模式没有出现统计学显著差异;3不同营养模式对卤虫的怀卵量、卵生/卵胎生比例有显著影响;与空白组相比,投饵组平均怀卵量提高了35.52%—72.71%,施肥组提高了11.34%—26.15%;4卤虫资源的环境负载力为0.3—0.4 kg/m3,加以补充肥料,可提高到0.45 kg/m3,在投喂饲料的情况下可以达到0.5 kg/m3;5卤虫蛋白可开发量按环境负载力的1/3估计,对照组、施肥组和投饵组的相应年开发量分别为2.61—2.98 kg/m3、4.5—5.4 kg/m3和7.51—8.67 kg/m3,滞育卵产量分别为0.73、1.10 g/m3和1.17 g/m3。     

Aquaculture related invasion of the exotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> and displacement of the autochthonous <Emphasis Type="Italic">Artemia</Emphasis> populations from the hypersaline habitats of India

Autochthonous parthenogentic Artemia populations have been reported from Indian hypersaline habitats since 1950s. Exotic Artemia franciscana was imported and introduced into India as live food for aquaculture since the early eighties. To assess the present status of the Artemia populations and the possibility of invasion by the introduced A. franciscana in Indian Salinas, an extensive study was conducted using conventional and molecular approaches. Morphological and biometric observations, crossbreeding experiments and molecular and phylogenetic analysis using Internally Transcribed Spacer-1 sequence revealed the extensive presence of alien, sexual A. franciscana populations in various hypersaline areas. Individual culture experiments and crossbreeding studies further confirmed the absence of autochthonous parthenogentic Artemia populations. Lack of regional endemism in populations of distant origins was evident, indicating that the invaded populations have naturalized and are in the process of evolution. This forms the first report of invasion by A. franciscana in hypersaline habitats of Indian subcontinent and further studies are required to assess the biological implications of this invasion.     

An improved technique for sampling lotic invertebrates

The electrophoretic characterization of an Artemia population from a saline lake of the Chilean Andes with unusual ecological features (Salar de Atacama, 23° 30′ S; 68° 10′ W) was carried out with the aim of comparing its genetic variation with, and genetic similarity to, representative populations of A. persimilis (Buenos Aires, Argentina) and A. franciscana (San Francisco Bay, USA). Based on the analysis of 22 loci it is concluded that the Chilean population and that from San Francisco are conspecific (D = 0.144), while the Chilean strain and A. persimilis are congeneric species (D = 1.171), differing in more than 40% of the loci. Parameters measuring genetic variation (e.g., percentage of loci polymorphic, mean number of alleles per locus, mean heterozygosity per locus) demonstrate that values for the Chilean Artemia (50.0, 1.68, 0.126, respectively) are within the range for bisexual Artemia in general and A. franciscana in particular.     

Administration of the probiotic Lactobacillus rhamnosus IMC 501 as a strategy for the control of Vibrio bacteria in the brine shrimp Artemia

The present study aimed to address the capability of the probiotic bacterium Lactobacillus rhamnosus IMC 501^® to survive in seawater and the ability of Artemia metanauplii to incorporate it, as well as to analyse the potential effect of the probiotic as a control agent for potentially pathogenic Vibrionaceae bacteria in Artemia. The results demonstrate the ability of L. rhamnosus IMC 501^® to survive in seawater for up to 30 h. They also advocate their capability to be efficiently incorporated into Artemia metanauplii at concentrations of 10⁴ CFU per Artemia after 30 min of suspension in probiotic solution, thereby promoting a 1-log reduction in Vibrionaceae levels after 3 h. These low levels of Vibrio bacteria were maintained for about 30 min after transfer into clear seawater, a sufficient time for Artemia to be ingested by aquatic organisms. These results contribute to broaden the knowledge on the suitability of probiotics as sustainable alternatives for the prevention/reduction of diseases in aquaculture facilities.     

A model for growth of Artemia franciscana cultures based on food ration-dependent gross growth efficiencies

Laboratory cultures of Artemia franciscana grown under batch regimes at constant temperatures (28 °C) and salinity (35 g l^–1), three initial food concentrations (0.1, 0.4 and 1 M cells ml^–1), various daily food rations (0.1–9M Dunaliella tertiolecta cells Artemia ^–1), and different population densities (1–16 ind ml^–1) were used to develop a model of population growth. Growth rates and gross growth efficiencies (K ₁) were largely independent of population densities and initial food concentrations but determined by age and daily amount of food ingested. While maximum growth rates were found with the highest rations, K _{1 max} peaked at rations of 0.5 million cells d^–1 and decreased at feeding levels above this. A contour plot showing the trend relating K ₁ to Artemia size and ingestion rate in combination and was used to model growth in analogous controlled feeding conditions. Computer simulations using this model paralleled published results of final 15-day average individual sizes of Artemia. Optimal results for near constant food utilization are predicted for high initial population densities (100 Artemia nauplii ml ^–1) and daily culls of enough animals to equilibrate food demand with food availability. This strategy could permit a range of Artemia sizes harvested, maximize final individual sizes and retain high total yields (> 1.2 kg dry wt 1^–1). Effects of different culture strategies are discussed.