The hemoglobins of Artemia salina. IV. A model for genetic control of hemoglobin 1, hemoglobin 2, and hemoglobin X

Two loci account for all genetic variation resulting in difference in electrophoretic mobility in three hemoglobins (Hb1, Hb2, and HbX) in the hemolymph of the brine shrimp. Four alleles and nine alleles have been studied. In shrimps of all genotypes and in electrophoresis in media with varying degrees of molecular sieving, Hb2 is approximately equidistant from Hb1 and HbX. A shrimp heterozygous at both loci has a three-banded Hb1, a four-banded Hb2, and a three-banded HbX. We conclude that Hb2 contains n -polypeptides and n -polypeptides. Hb1 contains 2n -polypeptides. HbX contains 2n -polypeptides. During electrophoresis, the three native hemoglobins undergo reversible dissociation to n subunits. Subunits with the same charge reassemble to migrate as molecules of the same size as the native molecules. Although there is no evidence for an additional polypeptide in the three hemoglobins, we cannot exclude such a possibility. If it exists, it is under three constraints: (1) it must be present in equal amounts in each of the three hemoglobins; (2) it must have the same molecular weight as the - and -polypeptides; and (3) it must be free of genetic variation (detectable by electrophoresis).Supported by National Institutes of Health Grant HE-11445.     

Genetic variability and interpopulational differentiation of Artemia strains from South America

Seven Artemia samples from three South American countries (Chile, Brazil, Peru) were studied by starch electrophoresis with the aim of comparing levels of genetic variation and genetic similarity to representative populations of A. franciscana (San Francisco Bay, California, USA) and A. persimilis (Buenos Aires, Argentina), which are species endemic to the New World. Based on the analysis of 22 loci, parameters measuring genetic variability were, for some populations, found to be among the highest reported for Artemia so far. The percentage of polymorphic loci varied from 31.8% (Piura, Peru; Buenos Aires) to 50% (Los Vilos and Salar de Atacama, Chile), while the observed heterozygosity varied from 0.025 (Piura) to 0.165 (Los Vilos, Chile). A dendrogram based on Nei's genetic distance (D) produced four major groups. The Argentinian form, A. persimilis; the San Francisco Bay strain together with samples from Brazil (Macau and Rio Grande do Norte) and Chile (Pichilemu and Salar de Atacama); two coastal populations from Chile (Los Vilos and Iquique) and the sample from Peru (Piura). These four groups have inter-group D values that are, in some cases, far above those normally associated with conspecific populations.     

Genetic and developmental variation of hemoglobin in the deermouse,Peromyscus maniculatus

A genetic investigation of electrophoretic hemoglobin variants of the deermouse, Peromyscus maniculatus, shows three alleles, HbI ^f, HbI^r, and HbI ^o, at a duplicated site controlling the six adult phenotypes. The HbI ^fallele has not been described previously. The hemoglobin locus is not closely linked to the albino locus. Fetal hemoglobin is distinct from any of the adult components and has a slower electrophoretic mobility. The fetal phenotype changes to the adult type between the days 15 and 18 of prenatal life.     

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.     

Interdomain linkage in the polymeric hemoglobin molecule of Artemia

Artemia has evolved the longest known concatenation of hemoglobin domains, the subunit containing nine domains and the subunit having a similar size. Translation of the cDNA sequence of the subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit.The interdomain linker sequences were 14%–64% identical (62%–91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.The position of interdomain introns and the character of a variable residue early in the linker are compatible with the nine-domain polymer having evolved through gene duplication reflected in globin domain fusion incorporating an extension specifically of the N-terminus. The multiplication of an original single-domain globin gene to give the present nine is estimated from sequence differences, allowing for multiple mutations at individual sites, to have occurred in a period at least 500–700 million years ago.Correspondence to: C.N.A. Trotman 1444     

International study on Artemia. LVI. Characterization of two Artemia populations from Namibia and Madagascar: cytogenetics, biometry, hatching characteristics and fatty acid profiles

Two parthenogenetic Artemia populations from southern Africa, one from Swakopmund saltworks (Namibia) and another from Ankiembe saltworks (Madagascar) have been studied. The population from Namibia is mainly diploid (2n=42) with few tetraploid individuals (4n=84), while the one from Madagascar was found to be triploid (3n=63). No chromocenters have been observed in either of the two populations. The Namibian population has smaller cysts and nauplii compared to those of the Madagascar population. Discriminant analysis revealed significant differences in the biometry of the adults from the two populations. The two populations exhibited very good hatching characteristics. The study of fatty acid methyl esters revealed that the Namibian population belongs to the fresh water type of Artemia showing low levels of eicosapentaenoic acid, whereas the population from Madagascar displayed exceptionally high levels of eicosapentaenoic acid, belonging to the marine water type.     

Nearshore and pelagic abundances of Artemia monica in Mono Lake,California

The spatial distribution and abundance of the brine shrimp, Artemia monica, in Mono Lake, California were determined during 1982 and 1983. Peak abundances of shrimp occur in midsummer and reach densities of 15–17 individuals l^-1 in the nearshore regions and 6–8 individuals 1^-1 in the pelagic region. The brine shrimp were non-uniformly distributed both vertically and horizontally. The coefficient of variation in shrimp abundance among stations within the nearshore region was similar to that found in the pelagic region. On two of the nine dates, nearshore densities were 3 to 4 times greater than those in the pelagic zone, and on average the brine shrimp appear to be slightly over-dispersed to the nearshore region. However, including nearshore abundances in lakewide estimates will usually result in a change of less than a 10%.     

Effects of salinity and temperature on reproductive and life span characteristics of clonal Artemia. (International Study on Artemia. LXVI)

An apomictic clone of the tetraploid parthenogenetic Artemia population from M. Embolon (Thessaloniki, Greece) was assayed for 10 reproductive and life span characteristics under laboratory conditions (in various salinity and temperature regimes). Salinity was proved to have significant impact on the majority of the characters used in this study. Discriminant function analysis gave an overall prediction of 97.32% over the three salinities (50, 80 and 120 ppt). The temperature of 30°C seemed to be an extreme one affecting significantly nearly all of the studied variables. The overall prediction according to the discriminant analysis was 94.69% among the three temperatures (22, 26 and 30°C). The clone performed best at 80 ppt and 22°C. The data presented in this study may generate useful suggestions to investigate the potentiality of using a single genetic lineage in order to visualize the effects of different environmental cues on a specific clone.     

Natural vitamin E enrichment of Artemia salina fed freshwater and marine microalgae

Three species of microalga, the freshwater Euglena gracilis and the marine Dunaliella salina and Tetraselmis suecica, were compared in terms of vitamin E enrichment and survival and growth of the brine shrimp Artemia salina. The tocopherol content was investigated using HPLC for the post-larval and pre-adult stages of Artemia fed the microalgae and the results compared to the initial content of unfed newly hatched nauplii. There was a markedly higher content of tocopherols (about two-fold) in Artemia fed Euglena. Since this microalga contains other antioxidants such as -carotene, vitamin C and glutathione, bioactive molecules such as PUFA, and the immunostimulant polysaccharide -glucan, it represents a valuable alternative for enriching the diets of Artemia and increase its nutritional value as a food item.     

Segmentation in the crustacean Artemia: engrailed staining studied with an antibody raised against the Artemia protein

We have studied the process of post-embryonic segmentation in the anostracan crustacean Artemia franciscana using a specific antibody raised against the engrailed protein of this organism. Three cephalic segments are specified during embryonic development, before larval hatching, whilst trunk (thoracic) segmentation begins after the first stage free-swimming nauplius larva has emerged from the dormant cyst. Thus, cephalic and trunk segmentation seem to be at least in part independent and superimposed processes. Trunk stripes of engrailed expression are added one at a time as segments are generated from the posterior growth zone. The first detectable decision in engrailed expression is the establishment of a line of engrailed-expressing cells, interpretable as delineating the parasegmental boundary. The subsequent widening of engrailed stripes is not correlated with cell lineage events but is probably mediated by the combination of inheritance of the active state and recruitment of new cells into the engrailed-expressing stripe.     

International study on Artemia XLIX. Salinity effects on survival,maturity, growth,biometrics, reproductive and lifespan characteristics of a bisexual and a parthenogenetic population of Artemia

Two Artemia populations, a bisexual from San Francisco Bay (California, USA) and a parthenogenetic from Tanggu area (Tianjin province, People's Republic of China) are assayed for their tolerance and fitness in various salinity levels. This study was carried out under laboratory conditions where salinity effects upon special characteristics of the two Artemia populations, such as survival, growth rate, maturation, morphology, fecundity and life duration, were recorded. This evaluation revealed that the two populations examined exhibits significant differences in their response against elevated salinity levels. Furthermore, specific biometric parameters can be a useful tool for the discrimination and/or determination of their distribution in a mixed population, which, in fact, is the case in Tanggu salt works. Artemia franciscana seems to be a more effective colonizer at higher salinities. The data presented in this study may generate useful suggestions for proper management of the solar saltworks at Tanggu although further experimentation is needed.International interdisciplinary study on Artemia populations coordinated by the Laboratory of Aquaculture & Artemia Reference Center, University of Ghent, Belgium.     

Some aspects of Artemia biology affected by cestode parasitism

Artemia individuals from a wild population, bearing cestode cysticercoids (Hymenolepididae) were kept in the laboratory during four months to observe influences of the parasite on the host biology. No differences were found between parasitized and unparasitized shrimps regarding final adult size. The higher survival of parasitized animals suggests that parasitism is not pathogenic. The most dramatic effect was host castration. Parasitized individuals showed higher total lipid levels, probably linked to carotenoid pigments conveying a red color to parasitized shrimps.     

The use of Artemia biomass sampling to predict cyst yields in culture ponds

The possibility of using biomass volume (= mean biomass present in the pond.week ^–1) to predict the total amount of harvestable cysts (= kg wet weight collected. week ^–1) produced in a culture pond by an Artemia franciscana population using a mixed model regression was evaluated for two different sampling methods; horizontal transects and vertical point samples. For transects, the following equation was found: `log (0.01 + cyst yields) = –2.05 + 0.025<sup>*</sup>(biomass volume)' with F <sub>(1, 4.87)</sub> = 8.83 and p = 0.032. For the point samples, the regression was also significant with F <sub>(1, 55.2)</sub> = 13.62 and p = 0.0005 for following equation: `log (0.01 + cyst yield) = –3.613 + 0.021^*(biomass volume). As pond effect and interaction terms did not significantly explain a significant portion of the variance for either of the sampling methods (Transects: pond: F _{(3, 14.3)} = 2.48; p = 0.103; pond^*biomass volume: F _{(3, 3.61)} = 4.63; p = 0.0976; Point samples: pond: F _{(3, 44.5)} = 0.00; p = 0.999; pond^*biomass volume: F _{(3, 44.2)} = 0.11; p = 0.954 ), the variable pond (repeated measurement factor) was not included in the final calculations for the regression equations. Although a combination of factors influences the equation, the high significance levels of the regression indicate biomass volume can be safely used to predict production trends. The low investment requirements of this method make it especially attractive for on farm use, where correctly determining the point of cyst decline will help farmers to allocate resources where needed.     

Biochemical characterization of Artemia ras p21

The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze GP₄G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.     

Characterisation of high-altitude Artemia populations from the Qinghai-Tibet Plateau,PR China

The brine shrimp Artemia was discovered in a number of saline lakes on the Qinghai-Tibet Plateau, widely diverging in chemical composition. Several lakes were athalassohaline, with relatively high amounts of trace elements. Common environmental factors are their high altitude (exceeding 4500 m) and the low average annual temperatures. A number of Artemia populations in this area were analysed to assess their preference for low temperatures and an athalassohaline medium. Furthermore, their characteristics were compared with Artemia tibetiana, the species recently described for one lake in this area. All samples contained a variable mixture of parthenogenetic and bisexual individuals. A cross-breeding test of the sample from Jingyu Lake showed cross-fertility both with A. tibetiana and A. sinica. All populations showed similarities to A. tibetiana: a large cyst diameter and naupliar length, high HUFA content and a high tolerance to low temperatures, as compared to the control A. franciscana samples. These can thus be considered as recurrent characteristics of the populations from the high-altitude low-temperature environment on the Qinghai-Tibet Plateau, although further research is needed to identify their exact species status.     

Enhanced disease resistance in Artemia by application of commercial beta-glucans sources and chitin in a gnotobiotic Artemia challenge test

The anti-infectious potential of a selection of putative immunostimulants including six commercial beta-glucans (all extracted from baker's yeast Saccharomyces cerevisiae except for Laminarin) and chitin particles were verified in Artemia nauplii by challenging them under gnotobiotic conditions with the pathogen Vibrio campbellii. Under the described experimental conditions, no differential macroscopic nutritional effect (e.g. growth) was observed among the products. Significant increased survival was observed with beta-glucan (Sigma) and Zymosan and to a lesser extent with MacroGard in challenged nauplii. A poor correlation was found between survival values of the challenged Artemia and the product compositions (such as chitin, mannose and beta-glucan content) indicating that the quality of beta-glucans (e.g. the ratio of beta-1,3 and beta-1,6 glucan, the molecular weight, the dimensional structure, type and frequency of branches), eventually in combination with other unidentified compounds, is more important than the amount of product offered. This small-scale testing under gnotobiotic conditions using freshly hatched Artemia nauplii allows for a rapid and simultaneous screening of anti-infectious and/or putative immunostimulatory polymers, and should be combined with studies on cellular and humoral immune responses in order to gain more quantitative insight into their functional properties.     

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.     

Stress resistance induced by paramylon treatment in Artemia sp.

A study was undertaken to investigate the effectiveness of paramylon extracted from the non-photosynthetic WZSL mutant of Euglena gracilis in potentiating the resistance of the brine shrimp Artemia sp. to stress conditions resulting from poor growth medium quality and daily handling. The experimental design incorporated four different treatments: I) paramylon addition/no growth medium replacement; II) no paramylon addition/no growth medium replacement; III) paramylon addition/growth medium replacement; IV) noparamylon addition/growth medium replacement. As shown by the survival curves of Artemia sp., the addition of paramylon significantly reduced susceptibility of animals to the stressors. Paramylon effectively increased also the ability of offspring to withstand poor water quality and handling damages. Without paramylon almost all offspring died before adulthood, whereas addition of paramylon allowed the animals to moult to the adult stage. Moreover, reproductive success (higher number of living offspring) was enhanced considerably in animals treated with paramylon treated under both stress conditions. These results show that paramylon extracted from Euglena represents a promising biologically active compound for aquaculture purposes. It could be used as a purified product or as component of whole cells, since the Euglena mutant, because of the high intracellular amount of paramylon it can accumulate, could be added to the feed or to water in tanks and ponds without prior processing. This revised version was published online in August 2006 with corrections to the Cover Date.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Toxicity of the blue-green alga Oscillatoria agardhii to the mosquito Aedes aegypti and the shrimp Artemia salina

The cyanobacterium Oscillatoria agardhii 27, which does not produce mammalian neuro- or hepatotoxins, was highly toxic to the larval stages of the yellow fever mosquito Aedes aegypti: its 24-h LC₅₀ values against fourth-and second-instar larvae of A. aegypti were 8.7 and 6.1 g live cells/ml, respectively. The toxin was water-soluble and was partially purified but the chemical nature of the toxic compound(s) is still unknown. Aqueous solutions were also toxic to the newborn larvae of the brine shrimp, Artemia salina, used for the bioassay. The toxic activity of these solutions decreased markedly on heating to 90°C for 15 min.J. Kiviranta is with the Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki, Helsinki, Finland; A. Abdel-Hameed is usually with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt, but is presently with the Department of Applied Chemistry and Microbiology, P.O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki, Helsinki, Finland.