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Artemia BIODIVERSITY Powered By Docstoc
					INCO partner 4: CSIC

Partner name: Instituto de Acuicultura de Torre de la Sal (Consejo Superior de Investigaciones Científicas de España), Larval Food (Artemia), Larviculture and Ecotoxicology group, 12595 Ribera de Cabanes (Castellón), Spain tel.: 34-964-319500; fax: 34-964-319509; email: Responsible scientist: Francisco Amat

According to the guideliness appointed during the Ghent and Beijing workshops the activities of this group during year 2002, concerning the INCO A.C. priorities and previous research commitments, were focused on the following research tasks :

1) The updating of a cyst bank and a data base of Artemia cysts samples developed and kept by this group. The present collection held at the IATS-CSIC contains about 290 samples: 151 from Western Europe, 28 from Asia, 16 from Africa and 95 from the Americas. The updating of the cyst bank is mainly focused now to the study of samples from countries neighbouring Spain, especially Portugal, France, Italy and North Africa countries. Cysts samples from Portugal, France, Italy and Spain were checked and verified in order to assess their present status in terms of maintenance, viability and species composition of the populations. Portugal: 21 samples collected or dated before 1991, coming from the geographical regions of Algarve (South of Portugal) and Tejo-Sado rivers estuary did not hatch at all. The biometrics of these cyst samples was performed (micrometer & Coulter Counter) giving an average cyst diameter about 237 m. Two samples from the Sado river estuary: Bonfim (1996) and Rio Frio (1993), as well as two samples from the Aveiro salterns (1991, 1993) hatched. Nauplii obtained were grown up to adulthood producing A. franciscana populations. The average diameter of these cysts was about 24 7-248 m. On June 2002 a research in cooperation with the Estación Biológica de Doñana (CSIC, Spain) allowed to update three samples from the Algarve region (Castro Marim, Olhao and Ludo) that were added to the Portuguese cysts collection. Once hatched, their nauplii produced also A. franciscana populations. It is possible to conclude preliminarily that Portugal is completely invaded by A. franciscana. Spanish-Portuguese cooperation is being supported by an Integrated Action funded for 2003, which will allow to develop a wide and updated prospection of Portuguese salterns. France: No cysts samples from France were available until 2002. The cooperation with the University of Montpellier (Dr. Th. Lenormand) provided the availability of updated cyst samples (June 2002) from Southern France: Sete-Listel (Roussillon) and Aigües Mortes (Camargue-Salins de Giraud). Once hatched and grown up, these cysts produced A. franciscana populations. Aigües Mortes laboratory population showed the presence of about


1.6 % A.parthenogenetica (diploid). Samples of preserved specimens collected in the wild : Hyères and Pesquiers (Toulon) and Fos (Marseille) (Basse Provence) showed the exclusive presence of A. franciscana populations. Italy: Cysts samples from Italy were obtained mainly through the cooperation with Dra. Graziella Mura from the University of Rome. From these cyst samples, about 11 collected or dated before 1988 did not hatch at all, while 7 dated between 1988 and 2002 hatched and their nauplii produced autochthonous Mediterranean populations: Samples from Tarquinia (Viterbo), Cagliari, Carloforte and SanAntioco (Sardinia) salterns produced A. salina populations. Carloforte also showed the presence (< 1 %) of A.parthenogenetica (diploid). Samples from Margherita di Savoia (Apulia-Foggia) salterns produced mixed parthenogenetic populations, about 60-70% tetraploid and 30-40 % diplod. Samples from Comacchio salterns ( 1985 and ARC 371) did not hatch at all. The biometrics of these cysts samples were performed (micrometer & Coulter Counter) giving an average cyst diameter about 248-249m for A. salina samples, 261-262m for A.parthenogenetica (diploid) samples, and 276-277 m for Comacchio samples, presumably A.parthenogenetica (tetraploid). No A. franciscana populations were obtained from Italian Artemia cyst samples. The obtention of pure strains (diploid and tetraploid) cyst samples from mixed populations (Carloforte and Margherita di Savoia) is in progress. Spanish-Italian cooperation is being supported by an Integrated Action funded for 2003, which will allow to develop a wide and updated prospection of Italian salterns. Spain: A few samples from the South of Spain, mainly located in the area neighbouring Cadiz-San Fernando (El Estanquillo and El Pilar salterns), where several aquaculture plants were settled, produced also A. franciscana populations cooccurring and competing with autochthonous strains (A. parthenogenetica). Updated (2001-2002) samples from Nuestra Señora del Rocio salterns (Sanlucar de Barrameda, Cadiz) and Odiel salterns (Huelva) produced only populations from autochthonous species (A.salina and A.parthenogenetica, diploid and tetraploid). These salterns are located between the Spanish Cadiz-San Fernando area and the Portuguese Algarve area where A. franciscana populations were introduced. Cooperative research in progress with the Biological Station of Doñana (Sevilla, Spain) is trying to establish the status of active transport of Artemia cysts through water fowl. Cysts obtained from faeces and pellets produced by these birds are showing the presence of autochthonous A. parthenogentica (diploid) and A. franciscana populations. The suspected exclusive presence of A. fransicana in Portugal and France is endangering the biodiversity of Spanish autochthonous populations in the South of Spain (Cádiz and Huelva provinces) at short-medium term, and of the Mediterranean Spanish autochthonous populations at medium-long term.


2) The distribution of Artemia persimilis and Artemia franciscana populations in Argentina. The present results obtained from a cooperative research project with the University of Buenos Aires (Argentina) show the presence of A. franciscana populations North of latitude 36º S (Mar Chiquita, Salinas Grandes de Ambargasta, Las Tunas) and the presence of A. persimilis populations South of latitude 37º S (La Pampa, Buenos Aires, Rio Negro provinces) The population of A. franciscana from Las Tunas lagoon (33º44´S) evidences a phenomenon of hybridisation or introgression: 50 % males show n = 22 chromosomes and 50 % show n = 21 chromosomes. Laboratory crossbreeding experiments show reproductive isolation between A. franciscana from Mar Chiquita and Las Tunas when crossed with A. persimilis from Salinas Grandes de Hidalgo (La Pampa province). In spite of the reproductive isolation showed by Mar Chiquita (A.franciscana) and Hidalgo (A.persimilis) in the interpopulational crossbreeding experiments, it is possible to draw the evidence of the fulfilment of Haldane´s rule (Cohen et al., 1999). Crosbreeding experiments involving A.franciscana males mated with A. persimils females do not produce any viable offspring at all, while those involving A.persimilis males mated with A. franciscana females produce sometimes a few number of hybrid males. Some hybrid males attain adulthood but show certain morphological abnormalities like bent antennae claspers, curved abdominal segments, misshape of the penis, nanism, etc. Several of them show perfect viability and development. Research is in progress trying to evidence their fertility or sterility when mated with A. franciscana and A. perimilis females. Laboratory populations (outdoor – 5000 L tanks) of A. franciscana and A. persimilis in competition show different reproductive output in terms of oviparism and ovoviviparism. Under suboptimal conditions of food availability A. persimilis shifts immediately to oviparism, while A. franciscana always keeps moderate levels of ovoviviparism (>13%). Under these same conditions cooccurring populations of both species show different reproductive output. A. franciscana females show always higher fecundity than A. persimilis females. These results explain a competitive development under which A. franciscana population outcompetes completely A. persimilis.

3) Presence of rare males in diploid parthenogenetic Artemia populations from the Old World. Rare males were found in 24 populations of Artemia parthenogenetica (diploid) from the Old World. These males are morphologically different from those present in bisexual species. This “atavism” is more frequent in parthenogenetic populations from Central Eurasia (Iran, Kazakhstan, Ukraina, Mongolia) and less frequent, or scarce, in peripheral Western (Iberian peninsula, Africa) and Eastern (China) populations. Although rare males seem useless in reproductive strategies for parthenogenetic females, they are not sterile in fact. They can mate and fertilize females from the bisexual strains Artemia urmiana (lake Urmia in Iran) and Artemia sinica (lake Yuncheng, Shanxi, China), producing live offspring. It is suspected that this rare male atavism could help to explain the origin of 3

parthenogenetic strains (polyphyletic origin) from hybridization between two of the Asiatic bisexual species (A. urmiana and A. sinica) or from lineages leading to present-day Asiatic Artemia , or through a monophyletic mechanism similar to that one theoretically argumented in cladocerans, which could provoke the transition from cyclic to obligate parthenoegenesis (Hebert, 1978) involving mutations that suppress meiosis during ephippial egg formation (in cladocerans) but that sporadically could fail to suppress spermatogenesis. Then, as pointed out by Hebert (1987), male reproduction in obligately parthenogenetic clones of Daphnia (D.pulex, D.pulicaria, D. middendorffiana) may serve as a vector in the displacement of sexual populations (Browne and Bowen, 1991). Research in progress is trying to unveil the status of consecutive hybrid generations produced mating rare males of parthenogentic origin with A. urmiana or A. sinica females, in terms of postmating isolation, production of abortive and viable offspring, survival rates of the viable offsprings, etc. References Browne, R.A. and Bowen, S.T. 1991. Taxonomy and populations genetics of Artemia. In: R.A. Browne, P. Sorgeloos and C.N. Trotman (eds) Artemia Biology, pp. 221-235. CRC, Boca Ratón, Florida. Cohen, R.G; Amat. F; Hontoria. F; Navarro, J.C. 1999. Preliminary characterization of some Artemia populations from La Pampa and Buenos Aires provinces. Int. J. Salt Lake Research 8: 329-340. Hebert, P.D.N. 1978. The population biology of Daphnia. Biol. Rev. 53: 387-426. Hebert, P.D.N. 1987. Genotypic characteristics of the Cladocera. Hydrobiologia 145: 183193.

4) Ecotoxicological studies. This research was developed in order to establish the effects of pesticides in Artemia and fish , and to develop a characterization of different populations and species of Artemia related to the acute response of nauplii to cadmium toxicity. The acute toxicity of the organophosphorous pesticides dichlorvos and chlorpyrifos was evaluated for the nauplii of two different species of Artemia (A. salina and A. parthenogenetica) in terms of in vivo effect on cholinesterase (ChE) activity. Results obtained evidenced that both Artemia species are resistant to these pesticides and that they are able to survive with more than 80% ChE inhibition. However, A. parthenogenetica is more resistant than A. salina. Considering these preliminary results it was studied the accumulation and transfer of chlorpyrifos in an experimental aquatic two level food chain using two species of Artemia (A. franciscana and A. parthenogenetica) and the small fish Aphanius iberus. Artemia adults contaminated by exposure to the pesticide in water were used as live prey for Aphanius. Concentrations of pesticide accumulated in fish, and biomagnification factor values showed a 4

continuous decrease during the bioaccumulation phase, probably due to the physicochemical characteristics of the organophosphorous pesticide, to the biotransformation ability of fish and to the progressive adaptation of fish metabolism to toxic exposure. Levels of stress protein HSP70 were measured in the fish as a general biochemical response, which were significantly higher in fish fed contaminated Artemia than in the control fish fed uncontaminated Artemia. The lethal response to cadmium of instar II nauplii from eight populations of Artemia belonging to the species A. franciscana, A. salina, A. persimilis and A. parthenogenetica showed that there is a relationship between species, type of population and mortality rate of their nauplii. The two populations of A. franciscana were the most sensitive to cadmium toxicity, while the population of A. persimilis from Hidalgo (Argentina) was the most resistant. These results suggest that habitat peculiarities and historical origin of the populations (biodiversity) may have a significant influence on their response to cadmium toxicity.

5) Enrichment and larviculture research Different liposome formulations, including several combinations of membrane composition, type of vesicle (multilamellar and large unilamellar vesicles), preparation method, and vehiculated nutrient, have been assayed as bioencapsulation products to enrich Artemia nauplii in nutrients for fish larvae. The stability of the liposome preparations under conditions of use as enrichment product has been tested using water soluble fluorescent markers as leakage indicators. The content of the fatty acids and lipid classes bioencapsulated in Artemia nauplii with liposomes has been analyzed by gas and thin layer chromatography, respectively, and compared with other enrichment products. The effect of the liposome enriched Artemia nauplii used as food for fish larvae has been evaluated in sea bass cultures. Liposomes with high content in polyunsaturated fatty acids leak out more than 50% of their aqueous phase in less than 2 hours, unless they are stabilized with cholesterol and formed as large unilamellar vesicles. These last vesicles hold 70% of the encapsulated material for 8 hours. Liposome enriched nauplii in this study reflect the influence of the enrichment products, however, they are far from the commercial emulsion (Super Selco) in terms of docosahexaenoic acid content, except for the nauplii enriched with liposomes made of pure krill phospholipid extract by the method of detergent solubilization. The liposome enriched nauplii show a higher amount of polar lipids in contrast to the feed enriched with emulsions. The larvae fed liposome enriched nauplii have only a slightly lower docosahexaenoic acid content than those fed emulsion enriched nauplii. The results obtained confirm the suitable potential use of liposomes as food supplement in larviculture. Several experiences were also developed aiming to rear Octopus vulgaris paralarvae with live and inert food. The influence of the fatty acid profile of Artemia used as live food seems clearly reflected in the cultured O. vulgaris paralarvae. The effect of the fatty acid composition of food was evident in the paralarvae after 10 days. The total lipid of the cultured animals increased notably, as did the levels of monounsaturated fatty acids, to the detriment of polyunsaturated fatty acids (PUFA) and, particularly, docosahexaenoic acid (DHA). Poor growth and high mortality seemed to result from a nutritional imbalance in the fatty acid profile produced by the artificial feeding, suggesting also that the microdiet was inefficiently


digested and/or assimilated. Wild Octopus juveniles tend to lose lipids as they increase in weight.


VARÓ I. , R. SERRANO, E. PITARCH, F. AMAT, F.J. LÓPEZ, J.C. NAVARRO. 2002. Bioaccumulation of chlorpyrifos through an experimental food chain. Study of protein HSP70 as biomarker of sublethal stress in fish. Arch. Environ. Contam. Toxicol., 42: 229-235 VARÓ I. , J.C. NAVARRO, F. AMAT, L. GUILHERMINO. 2002. Characterization of cholinesterases and evaluation of the inhibitory potential of chlorpyrifos and dichlorvos in Artemia salina and Artemia parthenogenetica. Chemosphere 48 : 563-569 SARABIA R., J. DEL RAMO, I. VARÓ, J. DIAZ-MAYANS, A. TORREBLANCA. 2002. Comparing the acute response to cadmium toxicity of nauplii from different populations of Artemia. Environ. Toxicol. Chem., 21(2): 437-444.

NAVARRO. J.C and VILLANUEVA, R. 2002. The fatty acid composition of Octopus vulgaris paralarvae reared with live and inert food:deviation from their natural fatty acid profile. Aquaculture 62068 : 1-19 (in press). VARO. I, NAVARRO. J.C, AMAT. F and GUILHERMINO.L. 2002. Effect of dichlorvos on cholinesterase activity of the European sea bass (Dicentrarchus labrax). Pesticide Biochemistry and Physiology (in press). Submitted SAAVEDRA, C and AMAT.F. Parental effects on encystment in crosses between two geographical strains of Artemia franciscana., submitted to HEREDITY. AMAT. F, COHEN. R.G, HONTORIA.F, NAVARRO. J.C. Further evidence and characterization of Artemia franciscana populations in Argentina, be submitted to JOURNAL OF BIOGEOGRAPHY. MONROIG. O, NAVARRO. J.C, AMAT. I, GONZALEZ. P, AMAT. F, and HONTORIA.F. Enrichment of Artemia nauplii in PUFA, phospholipids and water soluble nutrients using liposomes, submitted to AQUACULTURE INTERNATIONAL.

Plans for the coming year. Task 1. To update the cyst bank and data base of Artemia cysts samples from the Western Mediterranean region (plus Portugal) through samples provided by the cooperation supported


by Spanish-Italian and Spanish-Portuguese Integrated Actions. Introduction of new data involving North African cysts samples and populations. To develop a systematic and taxonomic approach to Mediterranean Artemia populations through male pennis morphology (SEM) (Spain-Italy) and mtDNA RFLP analyses characterization (Spain-Italy-Greece). To develop outdoor massive autochthonous Artemia populations (Mediterranean basin) “contaminated” with A. franciscana in order to study the evolution of competition phenomena and consequences mediated by environmental conditions.

Task 2. To accomplish the updating of the cyst bank and data base of Artemia cysts samples from Argentinean populations sampled in the Southern Argentinean provinces of Chubut, Santa Cruz and Tierra del Fuego, supported by the Spanish-Argentinean ICI-AECI project. Foreign Affairs and Education Spanish Ministeries (Cooperación Científica con Iberoamérica). To develop life tables under laboratory and outdoor conditions for Argentinean Artemia populations (A. persimilis and A. franciscana) in order to understand competition phenomena that could contribute to explain the evolution of wild populations in the South American Cone. To study the parental effects on the encystment rates among different geographical populations of Artemia persimilis from Argentina.

Task 3. To study the diverse factors conditioning the behaviour, diapause, hatching, nutritional value, etc., of original and laboratory obtained cyst batches under known conditions. Task 4. To study the levels of HSP70 stress protein in adult Artemia related to environmental conditions and subsequent changes in reproduction mode: oviparism versus ovoviviparism. Task 5. To develop new delivery techniques of nutrients to marine fish larvae through liposomes and bioencapsulation into live preys. To develop diverse tasks related to the visit and exchange of collaborative scientists from INCO groups.