Francisco Amat

Lipid conversions during enrichment of Artemia

Abstract Artemia nauplii were enriched for 24 h with radiolabelled fatty acid ethyl esters and then starved for a subsequent period of 24 h. Analyses of the distribution of radioactivity in lipids from samples taken at the end of the enrichment period and after the subsequent starvation showed that the ethyl esters were readily converted into other lipid classes, mainly triacylglycerols, during assimilation by the nauplii. The proportions of radioactivity recovered in free fatty acids and phospholipids increased during the starvation period indicating the mobilisation of fatty acids from triacylglycerols for use in catabolism and in the formation of biomembrane lipids. The distribution pattern of radioactivity from [U– 14 C ]22:6n−3 in the fatty acids of the nauplii demonstrates that Artemia are capable of retroconverting 22:6n−3 to 20:5n−3.

The American brine shrimp as an exotic invasive species in the western Mediterranean

The hypersaline environments and salterns present in the western Mediterranean region (including Italy, southern France, the Iberian Peninsula and Morocco) contain autochthonous forms of the brine shrimp Artemia, with parthenogenetic diploid and tetraploid strains coexisting with the bisexual species A. salina. Introduced populations of the American brine shrimp A. franciscana have also been recorded in these Mediterranean environments since the 1980s. Based on brine shrimp cyst samples collected in these countries from 1980 until 2002, we were able to establish the present distribution of autochthonous brine shrimps and of A. franciscana, which is shown to be an expanding invasive species. The results obtained show that A. franciscana is now the dominant Artemia species in Portuguese salterns, along the French Mediterranean coast and in Cadiz bay (Spain). Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was observed in Morocco (Mar Chica) and France (Aigues Mortes), whereas A. franciscana was not found in Italian cyst samples. The results suggest these exotic A. franciscana populations originate as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and suggest that the native species can be rapidly replaced by the exotic species.

Two novel Anemia enrichment diets containing polar lipid

Abstract Two novel Anemia enrichment diets were tested against control diets of Super Selco (SS) (Artemia Systems, INVE, Ghent) and bakers yeast. The first experimental diet consisted of tuna orbital oil emulsified with 12% herring roe polar lipid (P-T). The second test diet comprised liposomes incorporating the phosphatidylcholine (PC) fraction of herring roe. Enrichment efficiency was measured in terms of lipid class and fatty acid composition of enriched nauplii, with particular attention being paid to PC content, docosahexaenoic acid (DHA) levels and DHA:eicosapentaenoic acid (EPA) ratios. The P-T emulsion yielded Artemia nauplii with 14% DHA (% total fatty acids) and a DHA:EPA ratio of 1.8, significantly higher than in nauplii fed the other three diets. Polar lipid levels in both P-T and SS enriched nauplii were similar (32.4 mg g−1 and 34.7 mg g−1 dry body weight (DBW) of nauplii, respectively). Lower %DHA and DHA:EPA ratios were obtained in liposome-fed nauplii (2% and 0.3, respectively) However, they yielded significantly higher levels of naupliar polar lipid (40.1 mg g−1 DBW) than those fed the two oil emulsion treatments. The significance of these findings is discussed.

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 C↔T 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.

Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax)

In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass (Dicentrarchus labrax) were investigated. The characterisation of ChE and the “normal” range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (μM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the “normal” range of fingerling head and muscle ChE were 58.05±2.11 and 118.03±8.67 U/mg protein, respectively. Corresponding values for juveniles were 43.32±4.42 and 19.44±2.44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects.

Phylogeography and local endemism of the native Mediterranean brine shrimp Artemia salina (Branchiopoda: Anostraca)

There has been a recent appreciation of the ecological impacts of zooplanktonic species invasions. The North American brine shrimp Artemia franciscana is one such alien invader in hyper‐saline water ecosystems at a global scale. It has been shown to outcompete native Artemia species, leading to their local extinction. We used partial sequences of the mitochondrial Cytochrome c Oxidase Subunit 1 (COI or cox1) gene to investigate the genetic diversity and phylogeography of A. salina, an extreme halophilic sexual brine shrimp, over its known distribution range (Mediterranean Basin and South Africa) and to assess the extent of local endemism, the degree of population structure and the potential impact of traditional human saltpan management on this species. We also examined the phylogenetic relationships in the genus Artemia using COI sequences. Our results show extensive regional endemism and indicate an early Pleistocene expansion of A. salina in the Mediterranean Basin. Subsequent population isolation in a mosaic of Pleistocene refugia is suggested, with two or three refugia located in the Iberian Peninsula. Two instances of long‐distance colonization were also observed. Surprisingly, given its strong phylogeographical structure, A. salina showed a signature of correlation between geographical and genetic distance. Owing to strong ‘priority effects’, extensive population differentiation is retained, despite dispersal via migrant birds and human management of saltpans. The foreseeable expansion of A. franciscana is likely to be followed by substantial loss of genetic diversity in Mediterranean A. salina. Large genetic divergences between Mediterranean and South African A. salina suggest that the latter deserves species status.

Characterisation of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos to Artemia salina and Artemia parthenogenetica

In this study, the acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos to two different species of Artemia (A. salina and A. parthenogenetica) was evaluated. In addition, the in vivo effect of these two pesticides on cholinesterase (ChE) activity of both A. salina and A. parthenogenetica was also determined. The characterisation of the ChE, using different substrates and specific inhibitors, and the normal range of activity in non-exposed individuals were previously investigated for both species. The results obtained indicate that the ChE of A. salina is different from that of A. parthenogenetica and that both enzymes cannot be classified neither as acetylcholinesterase nor as butyrylcholinesterase since they show intermediary characteristics between the two vertebrate forms. The range of normal ChE activity was 2.65+/-0.15 U/mg protein for A. salina, and 3.69+/-0.17 U/mg protein for A. parthenogenetica. Significant in vivo effects of both pesticides on Artemia ChE activity were found, at concentrations between 5.38 and 9.30 mg/l for dichlorvos and between 1.85 and 3.19 mg/l for chlorpyrifos. Both Artemia species are resistant to these pesticides and they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina, with about a 95% reduction in its ChE activity respect to the control for nauplii exposed to the median lethal concentrations (LC50), without lethal effects after 24 h of exposure.

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.

Biogeography of the genusArtemia (Crustacea, Branchiopoda, Anostraca) in Spain

This is an updated study on the biogeographic distribution of the populations of the genusArtemia (Branchiopoda, Anostraca) in Spain, with special focus on populations inhabiting salt lagoons and inland salterns. The populations recorded (40) belong to the bisexual speciesA. tunisiana and to the asexual groupA. parthenogenetica (diploid and tetraploid strains). They usually appear in three different hypersaline ecosystems: solar salterns and lagoons filled with sea water, solar salterns and lagoons containing brines of diluted mineral salts, and lagoons filled with athalassic (endorheic) brines. The brines in salinas are chemically characterized by high Cl− concentrations, especially in inland salinas, whose brine sources are geologically associated with evaporitic formations developed in the Triassic and Lower Liassic of eastern Spain. Brines in athalassic lagoons, geologically associated with Mesozoic and Cenozoic periods, show higher levels of sulfate (up to 40–50%) as well as Mg2+. From a geographical point of view, bisexual and diploid asexual populations are mainly found in coastal and inland salinas or lagoons below 40° N, while asexual tetraploid populations are found in inland salinas and athalassic lagoons above that latitude. These populations have been biologically characterized by the morphology of their adults through multivariate analysis.

Competition between sexual and parthenogenetic Artemia: temperature and strain effects

Previously reported competition experiments among Artemia (brine shrimp) species have not incorporated abiotic variability into their experimental design. Recent field and laboratory investigations point to temperature as a critical factor in determining the biogeographic distribution and competitive ability of sexual and parthenogenetic Artemia. One sexual diploid strain A. tunisiana Bowen (S) and two A. parthenogenetica Barigozzi strains [one diploid (PD) and one tetraploid (PT)] were raised as single populations and as competitive cultures at 15 °, 24 ° and 30 °C. Cultures were scored for competitive outcome (coexistence, exclusion) and were periodically monitored for population age-structure density and female parturition mode (encysted vs. non-encysted offspring). Temperature affected competitive outcome as follows: at 15 °C, S > PD > PT; at 24 °C, PD > S > PT; at 30 °C, PD > PT > S. When physiological responses and demographic traits were examined, factors that contribute to competitive abilities could be identified. At 30 °C, S die before reproductive maturity, an obvious factor contributing to their elimination at warm temperatures. For PT more than 73% of the offspring were encysted. While this would be advantageous for reestablishing the population following adverse field conditions and/or colonization of new sites, it is disadvantageous in continuous sympatric cultures where competitor strains produce primarily non-encysted embryos.