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 complete mitochondrial DNA sequence of the crustacean Artemia franciscana

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3 downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.Correspondence to: R. Garesse     

Morphometric characterization of thalassohaline Artemia franciscana populations from the Colombian Caribbean

Aim To establish possible interpopulation relationships among Colombian Artemia franciscana (Crustacea, Anostraca) populations. Location Colombian Caribbean coast (Manaure, Galerazamba, Salina Cero and Tayrona) and a similar thalassohaline reference population from San Francisco Bay (SFB‐USA). Methods Morphometric characters of male and female cultured individuals of A. franciscana were measured. The populations were grouped according to: (1) population type (populations grouped according to two broad regions of origin: North America and the Caribbean coast), and (2) specific geographical origin (populations selected according to five specific local origins: Manaure, Galerazamba, Salina Cero, Tayrona and SFB) and evaluated using forward stepwise discriminant analysis (SPSS, Ver. 10). Results Optimal discriminant variables for males grouped by the type of population were left setae and antenna length, and for females they were abdominal length and antenna length. However, for males grouped by their specific geographical origin, the optimal variables were furca length, left setae, antenna length, eye separation, abdominal width and abdominal length, and for the females, they were furca length, abdominal length, left setae and eye separation. Male and female Colombian Caribbean populations were separated from the North American populations. However, our results show that the classification based on male characters provides better group membership than females. Main conclusions Male morphometric characters separated the type of population groups more clearly than the female characters, because all Colombian populations were correctly positioned in the Caribbean coast region and the SFB population in the North American region, with no overlapping between the two types, as was the case for the female individuals. Likewise, male individuals correctly position the Salina Cero population to its neighbouring Galerazamba population and to the other Colombian populations. In contrast, female individuals from Salina Cero did not cluster with the other Colombian coast populations (Galerazamba, Tayrona and Manaure) or with the SFB population.     

Respiration of Artemia franciscana embryos after continuous anoxia over 1-year period

Encysted embryos of the brine shrimp, Artemia franciscana, exhibit extraordinary longevity when exposed to continuous anoxia. To explore the metabolic basis of this ability, the post-anoxic respiration of embryos exposed to anoxia for periods exceeding 1 year was measured. Since anoxic metabolism might result in the accumulation of metabolic end products, an O₂ debt would be expected. Contrary to that expectation, post-anoxic embryos exhibited a marked depression in respiration rate whether embryos were hydrated under anoxic conditions or were exposed to a previous aerobic incubation and then placed under anoxia. These results, and those of previous studies, suggest that extended anoxia may bring the metabolism of these embryos to a reversible standstill.     

Estimate of the effects of ultraviolet radiation on the mortality of Artemia franciscana in naupliar and adult stages

The impact of different doses of artificial ultraviolet (UV) radiation on the growth stages of a marine zooplankton was investigated using laboratory microcosms. Mortality percentages of naupliar and adult samples of Artemia franciscana were recorded in relation to different UV doses (single exposure: 75, 150, 300, 600, 1,200, 2,400, 3,900, 7,800 J m^–2) at specific observation times after exposure (24, 48, 72, 96 and 120 h). The relationship between mortality percentage and UV dose showed significant differences in relation to the zooplankton growth stage. The elevated susceptibility of the naupliar samples to UV radiation is described through a mortality model based on a logistic equation. The data analysis shows that the slope of mortality versus dose remains the same for the two growth stages while the lethal dose in the naupliar stage was 3.3 smaller than that determined for the adult stage. The slope of the UV mortality rate versus post-incubation time was found to be significantly different (P<0.05) at low UV doses for the two life stages examined, i.e. naupliar and adult. The lower value of LD₅₀ in naupliar stages compared to that for adults confirms that in the early growth stage this marine zooplankton is more susceptible to UV radiation.     

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.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

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.     

Cytogenetic study of Artemia from Urmiah, Maharloo and Incheborun Lakes

Three populations of Artemia from Urmiah (West-Azarbaijan Province), Maharloo (Fars Province) and Incheborun (Golestan Province) Lakes in Iran were cytogenetically analyzed. In order to study the reproduction mode in Artemia populations, cyst samples reared for three successive generations (F₁--F₃). The results obtained from rearing until maturation indicated that, there are two subpopulations of Artemia in Urmiah lake (bisexual and parthenogenetic). These two subpopulations have the same diploid chromosome number (2 n =42). The results obtained from rearing and cytogenetic analysis of Artemia from Maharloo and Incheborun Lakes showed that these two populations are parthenogenetic and their chromosome number is also 2 n=42. So far, no cytogenetic differences in ploidy level have been detected among the populations investigated in this study. Very small chromosomes with metacentric, submetacentric and telocentric morphology were observed in mitotic prometaphase cells, implying that chromosomes of Artemia may be monocentric.     

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from contrasting hypersaline habitats express a conserved stress protein complement

The brine shrimp Artemia is a well known animal extremophile adapted to survive in very harsh hypersaline environments. We compared the small stress proteins artemin and p26, and the chaperone hsc70 in encysted embryos (cysts) of the New World species, A. franciscana and A. persimilis. Cysts of the former, from San Francisco Bay, USA (SFB), were used essentially as a reference for these proteins, while both species were from locations in Chile where they occur in habitats at latitudinal extremes, the Atacama desert and Patagonia. These two species are phylogenetically distant, A. persimilis being closer to the Old World species, whilst A. franciscana is considered younger and undergoing evolutionary expansion. Using western blotting we found all three stress proteins in cysts from these five populations in substantial although variable amounts. The protein profiles revealed by Coomassie staining after electrophoresis (SDS-PAGE) were similar qualitatively, in spite of marked differences in the habitats from which these populations originated, and the long time since they diverged. We interpret these findings as further evidence for the adaptive importance of these three conserved proteins in coping with the variable, but severe stresses these encysted embryos endure.     

Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance

RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relationships between Artemia monica life history characteristics and salinity

Relationships between Artemia monica life history characteristics and salinity were determined using data from four published studies and three experiments presented here. Salinity explained 40 to 93 percent of the variation in ten life history characteristics. Reductions in hatching success, survival, length, weight, ovigery, and brood size were observed as salinity increased from 76 to 168 g l^–1. Inter-brood duration, and time to hatching and reproduction were protracted as salinity was elevated. Salinity effects on life history characteristics appeared to be gradual and continuous rather than exhibiting thresholds. The one exception was naupliar survival, which was constant between 76 and 133 g l^–1 followed by a decrease above 133 g l^–1.     

Artemia monica cyst production and recruitment in Mono Lake,California, USA

Annual egg production was determined for Artemia monica in Mono Lake, California, from 1983 to 1987. Annual oviparous (overwintering cyst) production was 3 and 7 million cysts m^–2 yr^–1 in 1986 and 1987, respectively, as measured by in situ sediment traps. Cyst production for the entire five year period was calculated using Artemia census data and inter-brood duration derived from mixolimnetic temperature. These estimates ranged from 2 to 5 million cysts m^–2 yr^–1. This method underestimated annual production by 30%, when compared to estimates using sediment traps. Cyst production was similar during 1983–1986 and showed a significant increase in 1987, which was due primarily to a larger reproductive population later in the year. Recruitment into the adult populations of the following spring ranged between 1.4 to 3.2%. Overall abundance of this generation reflected the patterns in annual cyst production. Compensatory effects must operate on the second generation of each year, since summer populations were similar in all years despite differences in cyst production.     

In situ hatching of Artemia monica cysts in hypersaline Mono Lake,California

Two emergence trap designs were tested in Mono Lake, California, to measure in situ hatching of Artemia monica cysts on the lake bottom. One design incorporated a removable sample bottle; the other had a catch tube which was pumped from the surface. Both traps rested on the bottom and had a narrow gap between the collecting funnel and bottom flange to allow the chemical conditions within the trap to be similar to those outside. This gap was open during April and May but, because some animals entered from outside the area enclosed by the trap, the gap was covered with 400 µm or 800 µm screen during June and July. The two trap types without screens sampled a station in oxic water 7 m deep similarly in April and May 1985. Mean daily hatching rates from April to May 1985 ranged from 720 to 25 340 shrimp m^-2 day^-1. In contrast, mean daily hatching rates during the same period at a station in anoxic water 21 m deep were from 3 to 138 shrimp m^-2 day^-1. June and July hatching rates in the shallow station were lower than in the spring, usually less than 1000 shrimp m^-2 day^-1.     

Observations on future sea level changes in the Venice lagoon

The identification of the environmental conditions inducing different ecophysiological responses in the different strains and populations of the brine shrimp Artemia should improve the understanding of their biogeographic distribution. Nauplii from two Argentinean brine shrimp populations, Artemia persimilis from Salinas Grandes de Hidalgo (province of La Pampa) and Artemia franciscana from Laguna Mar Chiquita (province of Cordoba), were grown up until adulthood at different salinities (30, 60, 90, 120 gL⁻¹) and temperatures (12, 21, 28°C). The aim was to assess the effects of these different conditions on prereproductive life span and reproductive traits. Results evidenced that at 21 and 28°C, at any salinity, A. franciscana from Laguna Mar Chiquita attained higher survival and fecundity, after a shorter prereproductive period, than A. persimilis from Salinas Grandes de Hidalgo. These data support that A. franciscana, considered a superspecies, exhibits higher phenotypic plasticity than A. persimilis, and that A. persimilis is better adapted to lower temperatures than A. franciscana. These differences in temperature and salinity tolerance could explain the present distribution of these two species in the South Cone in South America. Handling Editor: J. Melack     

Ecological and genetic data indicate recovery of the endangered coral Acropora palmata in Los Roques, Southern Caribbean

The rapid decline of Acropora cervicornis and Acropora palmata has often been linked with coral reef deterioration in the Caribbean; yet, it remains controversial whether these species are currently recovering or still declining. In this study, the status of ten populations of A. palmata in Los Roques National Park (LRNP), Venezuela is presented. Six of these populations showed signs of recovery. Ten 80 m² belt-transects were surveyed at each of the ten reef sites. Within belt-transects, each colony was measured (maximum diameter and height) and its status (healthy, diseased or injured) was recorded. Populations in recovery were defined by a dominance of small to medium-sized colonies in densities >1 colony per 10 m², together with 75% undamaged colonies, a low prevalence of diseases (<10%), and a low density of predators (0.25 snails per colony). Based on allozyme analysis of seven polymorphic loci in four populations (N = 30), a moderate to high-genetic connectivity among these populations (F _ST = 0.048) was found with a predominance of sexual over asexual reproduction (N* : N = 1; N _go : N = 0.93–1). Both ecological and molecular data support a good prognosis for the recovery of this species in Los Roques.     

A comparative study of the fatty acid profile of Artemia franciscana and A. persimilis cultured at mesocosm scale

The goal of this study was to examine the fatty acid (FA) profile of two Artemia species, A. persimilis (Argentina) and A. franciscana (Great Salt Lake,Utah; USA) in coexistence at mesocosm scale. The experiment was carried out to 1) evaluate putative differences in the fatty acid composition of both species while they share resources and 2) to investigate the causes of such differences. Although the coexistence of these species in nature has not yet been observed, it remains possible that this situation may arise in the future mainly due to the invasive ability of A. franciscana. FA analyses were performed on individuals as well as on pooled biomasses of each species, and integrated in multivariate principal components analysis (PCA). Comparison of the relative abundance of FA between the two species revealed that interspecific differences in FA composition are greater than intraspecific variability. Higher percentages of unsaturation were found in the fatty acids of A. persimilis compared to A. franciscana, demonstrating that aside from a high phenotypic effect of diet on the FA composition of the animals, a species-specific genotypic effect should not be discarded.