INCO Concerted Action

ICA4-CT-2001-10020

Second 6-monthly report

Period 1 January-30 June 2003

1

Artemia Biodiversity:Current global resources and their sustainable exploitation

2

1. Overview report

1. Introduction

During the first six months of the 2nd project year, the themes and activities, launched in the 1 st project year, were continued and consolidated. In this process, biodiversity activities and joint studies of the respective partners are being concretised along a number of specific lines of action, described below.

2. Database of Artemia sites

A lay-out has been proposed for the creation of a central Artemia database, containing both biological information on the population and topographical and ecological data on the site. Most INCO partners are involved in a constant update of the list of Artemia populations from their respective country/region and in collection of field information from these sites.

3. Cyst bank

Samples available in INCO member cyst banks were further integrated into the central cyst bank. Additionally, most INCO partners have put efforts in a renewed inventorization and more consistent classification of their respective cyst banks. In view of possible recent evolutions (impoverishment ?) of Artemia biodiversity, special attention is given to old (‘historical’) samples.

4. Study of regional biodiversity topics

4.1. Africa

Artemia biodiversity in southern AfricaFollowing the description of an interesting Artemia population from southern Africa last year, a review was compiled and new sites were identified for potential further sample collection. This was combined with the study of bird migration routes, to get a better understanding of the factors influencing Artemia distribution. A sampling program for the region was designed and cyst or biomass samples were collected from various saltpans in South Africa and Namibia.

4.2. China

Artemia biodiversity on Tibet Plateau and Bohai Bay area Samples from Artemia resources in inland salt lakes, esp. in Tibet and Inner Mongolia were further collected. Strain characterization (population composition; nutritional profile over different seasons and locations, enrichment) of Artemia populations in the Bohai Bay area was continued. First laboratory culture tests with recent and older samples were run to assess the degree contamination of Bohai Bay Artemia sites with allochthonous strains and the dispersion pattern of the latter strains in this area. These data will be complemented with future genetic characterization tests of cysts and nauplii of the respective samples.

Detailed examination of the RFLP patterns from samples of Tibet has shown a considerable variability in this area and sometimes in one lake. Future analyses will focus on individual cysts or nauplii.

4.3. Iran

Artemia biodiversity in Lake Urmia areaSalinity tolerance experiments were carried out in order to confirm earlier findings on coexistence of parthenogenetic and bisexual Artemia populatons in the Lake Urmia area.

3

Monitoring of the physico-chemical parameters of Lake Urmia and their relationship to Artemia resources has been continued. In cooperation with the Iranian Organization for Environmental Protection a detailed study on the population dynamics and resource assessment of the Artemia population at Lake Urmia has been launched. This is combined with a morphometric study of samples to identify possible subpopulations.

The RFLP patterns of adult Artemia originating from Urmia lake and neighbouring ponds and lakes have been analyzed and compared with the reference RFLP database (see further), and results were related to the possible distribution of A. urmiana and parthenogenetic populations in this area. Future study on sufficient historical samples and recently collected material will allow understanding of spatial and temporal phylogeny of Artemia in this area.

In the applied field, the use of enriched Artemia nauplii on the production of stress resistant sturgeon fingerlings has been tested. Similar experiments have been conducted to assess the effect of Artemia enrichment on growth, survival and stress resistance of rainbow trout larvae.

Cyst sampling from the Lake Urmia area and elsewhere in Iran has been continued.

4.4. Mediterranean Threats to Artemia biodiversity by introduction of allochthonous strains

Artemia biodiversity studies in the Mediterranean are focusing on the characterization of local populations and on the study of the dispersion of allochthonous Artemia (A. franciscana) in the (mainly western part of this) area.

The updating of cyst bank and database on Mediterranean Artemia cyst samples showed the availability of cyst samples from the 80s until today. Together with the isolation of cysts from water bird faeces and pellets this enables to study the potential threat of invasion by A. franciscana. Presence of this exotic species has been evidenced in salterns in Portugal, Spain, France and Morocco, (but not in Italy), where it probably has been introduced intentionally or unintentionally, as the result of aquaculture activities in the area. Intensive sampling on the Italian islands has been continued, to further assess the range of dispersal of A. franciscana.

A number of autochthonous and invasive populations were also characterised genetically (PCR-RFLP with mitochondrial genome), morphometrically and reproductively. SEM analysis was performed on specific morphological traits (frontal knob, basal and eversible penis pattern) to allow intra- and inter-bisexual species comparison.

Salinity effects on reproductive and life span characteristics of Egyptian populations were studied; moreover, a number of Tunisian populations were studied biometrically, morphometrically, nutritionally and through cross-breeding tests, in comparison with other Artemia populations from the Mediterranean. Cyst sampling was performed in Tunisian salterns.

4.5. Central Asia

A survey was made of Artemia sites in S-Siberia, between Ural in the west and the Altai area in the east, and data have been recorded on hydrology of Artemia lakes, abiotic and biotic parameters, and assessments have been made on population dynamics of these Artemia resources. A start was made with the study of the emerging and developing Artemia population in the Aral Lake (Uzbekistan, Kazakhstan), which due to progressive salinisation is gradually colonized by local and/or regional Artemia populations.

4

4.6. Latin America

Distribution of A.franciscana/persimilis in southern South America and their possible hybridization

A list of the Artemia sites in the region has been compiled together with basic information on them. The application of new techniques to the regional Artemia sites has raised new questions and brought about chances for further regional collaboration in relation to various aspects. In this regard, new sampling campaigns in the period allowed to obtain animals from key, problematic, and/or particularly interesting locations in Chile and Argentina, that were identified as key latitudes for the distribution of A. franciscana and A. persimilis in both countries. All samples collected are being subjected to the usual morphological, karyological, and allozyme analysis, as well as to the DNA level (RAPD’s). The availability of molecular markers for A. persimilis and A. franciscana suggests a future study on the probable hybridisation of both species, including laboratory crosses.

The protocols for the Genome in situ Hybridization (GISH) technique are being adjusted in order to allow determination of genome affinity between A. franciscana and A. persimilis, and to obtain a better characterization of the heteromorphic bivalents in supposedly hybrid individuals. The technique however needs further refinement, to improve the results with A. persimilis and to perform the cross hybridization between both species.

Comparative salinity/temperature tolerance tests were run with an Argentinian A. franciscana and persimilis populations.

Study of Artemia strains from MexicoThis work includes the biometrical and morphometrical study of Mexican strains, and the use of molecular and biochemical techniques in the characterization of Mexican populations. In the applied field, the incorporation of antibiotics in A. franciscana for the inhibition of growth of Aeromonas hydrophila was studied, and the contents of spore elements in various populations.

4.7. South Asia

In order to assess the possible contamination of local Artemia populations with allochthonous strains, a selection of Artemia populations from Sri Lanka and India were compared with A. franciscana and A. sinica for genetic and biochemical diversity, using enzyme markers, metabolism and mitrochondrial DNA. Future microsatellite studies need to consolidate the results obtained sofar. Morphometric analysis of cultured individuals showed the presence of exotic A. franciscana in a variety of Indian Artemia sites.

5. Sustained exploitation and preservation of biodiversity in coastal saltworks

In coastal saltworks of NE Brazil, current Artemia practices and their implications for the sustainable exploitation and biodiversity of A. franciscana populations have been studied. Specific activities deal with the reproductive characterization of local populations of A. franciscana, the genetic variability of these populations, and with the running of a pilot farm for Artemia R&D.

At the occasion of sampling in Chilean saltworks, the idea of continuous monitoring and conservation of local Artemia was promoted among local authorities and saltwork managers.

Consultancy was provided for sustainable production of Artemia in Mexican saltworks.

In Vietnam research was continued to obtain more insight into the environmental conditions relevant for Artemia pond culture: algal bloom control; selected algal species as suitable food for Artemia; soil and nutrient interaction. Extension programs among local saltwork farmers were continued. To study

5

the thermotolerance of Vietnamese strains (in comparison to the originally inoculated A. franciscana) cDNA has been sequenced; the results of this genetical study might result in the identification of a genetic marker for thermotolerance studies.

6. Training for visiting scientists and publications

See individual member reports.

6

7

Consortium member reports

1. Artemia Database

A lay-out has been proposed for the creation of the Artemia database. This database contains information about The Artemia strain(s) occurring at each site: species, reproduction (parthenogenetic or bisexual),

ploidy level (if parthenogenetic), biometrical data and availability of samples in respective cyst banks of INCO partners;

Topographical, chemical, physical and hydrological data of the Artemia site: geographical coordinates, size, depth, altitude, ionic composition, temperature regime, salinity and pH

Relevant literature references

Using information provided by INCO partner 11 (UU), the database has been implemented for Artemia sites from Iran.

2. Artemia from S-Siberia

In cooperation with Artemia experts from SibrybNIIproekt, Tyumen State Agricultural Academy, Tyumen, Siberia, a survey has been made in the period 1995-2003 of Artemia sites in S-Siberia, between Ural in the west and the Altai area in the east. The data have now been compiled into an education CDRom, containing information on: 90 Artemia sites in the area Hydrochemical description of Artemia salt lakes Description of biocoenosis of salt lakes: phytoplankton composition, benthic and planktonic

fauna; annual species dynamics, salinity tolerance of species Biological characteristics of Artemia populations, including biometrics, parthenogenetic or

bisexual reproduction, diapause characteristics Population dynamics of Artemia populations in a selection of lakes, based on field sampling in the

period 2000-2003. Long-term dynamics of Artemia stocks: estimation of standing crop and maximable harvestable

amounts

This information is to be published in a book ‘Natural resources of the brine shrimp Artemia in lakes of Western Siberia‘.

A start was made with the study of the emerging and developing Artemia population in the Aral Lake (Uzbekistan, Kazakhstan), which due to progressive salinisation is gradually colonized by local and/or regional Artemia populations.

3. Artemia from Bohai Bay, China

To assess the degree of contamination of the Bohai Bay environment with allochthonous Artemia strains, first culture tests were run with recent and older samples from this area. These data will be complemented with future genetic characterization tests of cysts and nauplii of the respective samples.

4. Publications & theses

8

INCO partner 1: RUGPartner name: Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent University, Rozier 44, B-9000 Gent, Belgiumtel. 32-9-264 37 54; fax 32-9-264 41 93; email: gilbert.vanstappen@rug.ac.beResponsible scientist: Patrick Sorgeloos

An RFLP database for authentication of commercial cyst samples of the brine shrimp Artemia spp. (International Study on Artemia LXX). Bossier et al. Aquaculture, submitted. (co-authorship with INCO partner 2, CLO-DVZ)

Characterisation of high-altitude populations from the Qinghai-Tibet Plateau, PR China. 2003. Hydrobiologia 500: 179-192 (co-authorship with INCO partner 10, SRI)

Determination and identification of biological characteristics of Artemia populations from the Egyptian Nile delta for application in aquaculture. 2004. PhD Thesis by Nagy El-Bermawi, Ghent University, Belgium. 177 pp. Containing chapters on:

Biometry, hatching characteristics and fatty acid profile of parthenogenetic Egyptian populations;

Cross-breeding tests with bisexual Egyptian Artemia; Salinity effects on maturation, reproductive and life span characteristics, survival,

growth and morphometry of Egyptian Artemia populations; Effects of salinity and temperature on reproductive and life span characteristics of

clonal Artemia; Cadmium chronic stress effects on survival, maturation, growth and reproductive

performance of Egyptian populations (several chapters published in co-authorship with INCO partner 3, AUTH).

9

Partner 2 is involved in the following actions plans and partnership agreed upon at the Gent workshop:

Regional partnership: China, Iran, Central Asia and Latin America. Thematic partnership: Adaptation and speciation.

Scientific activities:

1. An Artemia authenticating database

A paper on the authenticating database containing 8 RFLP patterns of a mitochondrial rDNA fragment has been written and submitted. At the moment the paper is being revised.

2. Artemia strains from Iran

In the summer of 2002, Naser Agh (partner 11, UU) could stay 2 weeks at the laboratory of partner 2. Adult Artemia (30) were brought along, originating from Urmia lake and neighbouring ponds and lakes. The RFLP patterns generated on these animals have now been compared with the reference database. In the majority of cases the RFLP patterns are those typically displayed by parthenogenetic Artemia (for instance with the restriction enzymes HpaI, TaqI, HinfI and HaeIII). However also untypical patterns are showing up. For instance with the restriction enzyme HpaI, one animal displayed a pattern that was new to the database. Surprisingly the NdeII patterns on all samples (also those that show a typical pattern for parthenogenetic strains with the restriction enzyme HpaI) were similar to the one displayed by ARC1230, the A. urmiana type strain. In conclusion it can be said that the neighbourhood of Urmia lake is inhabited by A. urmiana as well as parthenogenetic Artemia. The situation is however complex and could not be resolved by the limited sampling and analysis performed until now. It is advisable that in the future ARC (historical) samples and recently collected samples are analysed in sufficient number using different sources of genetic material (e.g. cyst and nauplii). Only with such an approach it will be possible to start understanding spatial and temporal phylogeny of Artemia in that area.

3. Artemia strains from Tibet

Detailed examination of the RFLP patterns from samples of Tibet have indicated that the variability in this area and sometimes in one lake is considerable.On the basis of RFLP patterns generated on a batch of cysts, double restriction enzymes patterns have been detected in the sample ARC1347.

10

INCO partner 2: CLO-DVZPartner name: Agricultural Research Center-Ghent/Department of Sea Fisheries (CLO-DVZ), Ankerstraat 1, 8400 Oostende, Belgiumtel. 32-9-34 22 66; fax: 32-9-33 06 29; email: pbossier@yucom.beResponsible scientist: Daan Delbare

This picture shows clearly that some samples from Tibet (ARC1526) display a pattern that is different from other patterns (e.g. ARC1462, 1525). It also illustrates that sample ARC1347 is probably a mixture of both patterns.

Also here it is advisable to analyse individuals (cysts or nauplii) and to combine the genetic data with other type of data.

4. HSP26 gene from Artemia

In collaboration with the lab of Tom MacRae (INCO associated partner), the cDNA sequence of 5 cDNA’s have been sequenced. The data suggest a single nucleotide difference between ARC 1258 and the Vietnam strains. This difference could become a genetic marker for these strains, facilitating ecological studies on the increase in thermotolerance in the Vietnam strains.

Activities planned in the second half of year 2

Detailed analysis of the biodiversity of strains in salt lakes of Tibet A. franciscana as an invasive species in China Analysis is the diversity in the area of lake Urmia Participation at the Chili workshop.

11

RFLP NdeII

20.00

40.00

60.00

100.00

150.00

300.00

400.00

500.00

600.00

800.00

1000

1500

2000

3000

4.00E

3

6.00E

3

A. tibetiana

unidentified Artemia

unidentified Artemia

unidentified Artemia

unidentified Artemia

unidentified Artemia

unidentified Artemia

unidentified Artemia

unidentified Artemia

Lagkor Co, Tibet, China

Beng Co, Tibet, P.R. China

Haiyan Lake, Qinghai, China

Jingyu, Xinjiang, China

Bozi Co, Tibet, China

Bolshoe Yarovoe, Altai area, Russia

Bolshoe Yarovoe, Altai area, Russia

Coqen Lake, Tibet, China

Urmia Lake area, Iran

1347

1462

1525

1524

1461

1552

1553

1526

1226

Collaborating scientists: A. Triantafyllidis, A. Baxevanis, I. Kappas, A. Tzika During the course of the first six-month period of the second year of the project, Prof. Marcos Camara from Universidade Federal do Rio Grande do Norte (INCO partner 13) visited the AUTH lab from February 19th until May 10th.The work of the visiting scientist involved the analysis of Artemia populations using molecular (DNA) techniques. During his stay in AUTH the visiting scientist gained experience in the following techniques: i) DNA extraction (both with classical phenol chloroform procedures and with quick procedures such as Chelex extraction), ii) PCR amplification of 16S rRNA mitochondrial DNA gene region iii) agarose gel preparation and electrophoresis of DNA samples and iv) Restriction Fragment Length Polymorphism (RFLP) analysis of this segment using various restriction enzymes. The choise of restriction enzymes that were used for RFLP analysis was based on the results gained during the first year of this project. The visiting scientist got familiar with different packages (i.e. REAP, Arlequin, Phylip) for treating statistically RFLP data. All molecular work was done at the level of individual scoring. The visiting scientist during his course of his stay was also able to come into contact and discuss with the scientists in the lab about numerous other techniques such as sequencing, microsatellite genotyping, genetic identification of species using molecular markers etc. He was also able to take advantage of internet and library facilities as well the rich pre-existing bibliography concerning mitochondrial DNA analysis of the genetic structure of populations and Artemia existing literature.

More analytically, the above approaches were used by the invitee Prof. Marcos Camara to analyse the relationships of Brazilian Artemia populations. This scientist came into first contact with the molecular techniques using a parthenogenetic Artemia population from M. Embolon (Thessaloniki, Greece). These techniques were successfully applied in three Artemia franciscana populations from Brazil (i.e. Galinhos, Macau and Grossos). The results obtained, verified the identification and assignment of these populations to Artemia franciscana species.

Three Diploma Theses in the framework of this project have been defended. The experimental work of the first two has been completed during the first year of the project while the third one started in the first year and completed in the second year. The titles are:

1. “Artemia in Mediterranean Basin: the use of discriminant analysis based on morphometry” by G. Athanasiadis.

2. “Invasive Artemia species in Mediterranean Basin” by E. Markatzinou.3. “Genetic identification of Artemia species by using RFLP on mtDNA” by A. Tzika

In the framework of this project and according to the final conclusions in Beijing meeting, AUTH (INCO partner 3) and CSIC (INCO partner 4) examined the possibility of A. franciscana invasion in the Mediterranean Basin. Four Artemia populations from Spain were genetically characterised by using PCR (Polymerase Chain Reaction) – RFLP (Restriction Fragment Length Polymorphism) analyses on a region of the mitochondrial genome. The CSIC laboratory has already investigated these samples in terms of morphometry, reproductive characteristics etc. The main goals of this cooperation were:

i) to confirm the species status (assigned as A. franciscana) of El Pilar and El Estanquillo populations and

ii) to find out if there are any differences in the genetic makeup of diploid and tetraploid parthenogenetic Artemia populations in the Spanish region.

12

INCO partner 3: AUTHPartner name: Aristotle University of Thessaloniki, Department of Genetics, Development & Molecular Biology, Faculty of Sciences, School of Biology, 540 06 Thessaloniki, Greecetel.: 30-31-099 83 01; fax. 30-31-099 82 56; email: abatzop@bio.auth.grResponsible Scientist: Theodore J. Abatzopoulos

In the framework of this project the following publications and presentations were produced:

Publications:

1. Title: “Salinity effects on maturation, reproductive and life span characteristics of four Egyptian Artemia populations. (International Study on Artemia. LXVIII)”Authors: A. D. Baxevanis, N. El-Bermawi, T. J. Abatzopoulos & P. SorgeloosJournal: Hydrobiologia (submitted)

2. Title: “Elevated salinities may enhance the recovery of hydrated heat-shocked Artemia franciscana cysts (International Study on Artemia. LXV)Authors: T. J. Abatzopoulos, G. V. Triantaphyllidis, N. Roedaki, A. D. Baxevanis, A. Triantafyllidis & P. SorgeloosJournal: Belgian Journal of Zoology (in press)

Presentations in Congresses/Workshops:

1. Title: “Morphometric analysis of two new parthenogenetic Artemia strains from Thrace (N. Greece)”Authors: E. Markatzinou, A. Athanasiadis, A. D. Baxevanis & T.J. AbatzopoulosCongress: 11th Congress of the Panhelenic Society of Ichthyologists, Preveza, Greece, April 2003

13

1) Updating of cyst bank and database on Artemia.

The updating of cyst bank and database on Artemia cyst samples performed by this group showed the availability of cyst samples collected in the Western Mediterranean area from the beginning of the 80s until today. This availability, together with the prospection and recovering of brine shrimp cysts from waterbirds faeces and pellets in two areas in the Southwest of the Iberian peninsula, enabled a research led to establish the present distribution of autochthonous brine shrimp populations facing the threatening development of A. franciscana populations behaving as an exotic invasive species.

The results obtained show the exclusive presence of A. franciscana populations in Portuguese salterns and in some old salterns in Cadiz bay (Spain) and in the French Mediterranean shore. Co-occurrence of autochthonous (parthenogenetic) and American brine shrimp populations was found in Morocco (Mar Chica) and France (Aigues Mortes), while A. franciscana was not found in Italian cyst samples. It is also expected to explain the origin of these foreign A. franciscana populations as intentional or non-intentional inoculations through aquacultural (hatchery effluents) or pet market activities, and to assess their ability to be dispersed by shorebirds within and between salterns in the Southwestern Iberian peninsula. (Results submitted to the EEI 2003, 1rst National Congress on Exotic Invasive Species, Leon, Spain , 4 - 7 June, 2003).

Autochthonous and invasive species present in the Southwest of Spain were also genetically characterised (PCR-RFLP) through a collaborative research with INCO partner 3 (AUTH).

2) Brine shrimp in Argentina

In concertation with INCO partner 6 (UBA) a scientist was invited to develop an experiment designed to compare some bio-ecological parameters of the Argentinean brine shrimp species: A .persimilis (Salinas Grandes de Hidalgo) and A. franciscana (Mar Chiquita). The experience consisted in the exposure of nauplii of both populations to three different temperatures (12º, 21º and 28ºC) and four different salinities (30, 60, 90, 120 g. L -1) during 12-60 days, up to attaining reproductive adulthood. The most important results were:

A. persimilis always showed high mortality at low salinities (30 g. L -1). A. franciscana always showed low survival at the low temperature (12ºC), but survived better

than A. persimilis at 21º and 28ºC A. franciscana always showed higher fecundity rates than A. persimilis. A. persimilis showed higher growth rates than A. franciscana at all temperatures. A. persimilis showed sexual maturity earlier than A. franciscana at 12ºC. For the other

temperatures the results were similar. In both species the increase of temperature is associated to an increase of survival and growth

rates, and to a decrease of pre-reproductive period and maturity fulfilment.

This cooperation with Argentina (IATS – Universidad de Buenos Aires), supported by the Spanish Government (AECI-ICI Programmes), allowed to develop a third prospecting of Artemia populations mission in the Argentinean provinces of Chubut and Santa Cruz. More than a dozen hypersaline ecosystems were visited, and Artemia presence, as living populations and/or cysts, was registered in nine of them. This material is presently under study.

14

INCO partner 4: CSICPartner 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), Spaintel.: 34-964-319500; fax: 34-964-319509; email: amat@iats.csic.esResponsible scientist: Francisco Amat

3) Brine shrimp in Tunisia

In concertation with INCO partner 8 (INAT) a scientist was invited to develop the characterisation of three autochthonous Artemia populations from Tunisia.: El Adhibet, Sijoumi and Sahline. They showed their taxonomical adscription to the Mediterranean bisexual Artemia salina.

Their characterisation was supported by: Biometrics of cysts and nauplii. Fulfilment of adult populations under standard culture conditions and morphometric

characterisation of adult specimens through multivariate analysis. Comparison of these characterisations with other strains from the Mediterranean area: El

Rocío (Spain) and Souzama (Morocco). Management of brine shrimp morphometrics data base in the Spanish group. Cross-breeding experiments among bisexual brine shrimp Mediterranean strains. Lipid and fatty acid analysis of cysts and nauplii. Relationships with other Mediterranean

populations.

4) Brine shrimp in India

In concertation with INCO partner 15 (IART) a scientist was invited to apply the morphometric characterisation of Artemia populations under standard culture conditions, according to the method applied and the database developed by the group INCO partner 4.Six samples were afforded from India: PUTHALAM cysts showed the exclusive presence of an autochthonous diploid parthenogenetic strain. THAMARIKULAM cysts did not hatch (suspected parthenogenetic). VEMBAR, VEPPELODAI, ARASARADI and THERASPURAN cysts, presumably a mixture of autochthonous diploid parthenogenetic strain and introduced American brine shrimp, showed the exclusive presence of A. franciscana. This is a new example of complete outcompeting exclusion of autochthonous strains by the American brine shrimp, behaving as an exotic invasive species.

15

Regional Partnership Mediterranean:

Within the “regional”partnership for the study of the Mediterranean area, and in accordance with the resolutions of the second workshop held in Beijing (September 2002), the objectives of the research team in La Sapienza were the following: 1-cooperation among different expertises to identify species, 2-expand present knowledge on Artemia world distribution, 3-search for allochtonous species, 4-morphological studies.

1) Cooperation among different expertises to identify species

Cooperation with the laboratory of Francisco Amat (CSIC; partner 4) to extend SEM morphological analyses to larger numbers of Artemia populations from Mediterranean and from non-Mediterranean countries. Starting collaboration also with Laura Torrentera (Arizona State University, Dept. of Plant Biology PO Box 871601, USA) to compare experiences and observations on eversible penis morphology, in order to assess the validity of this character in species separation. Cooperation was started also with Gonzalo Gajardo (ULL, partner 14) regarding populations from Latin America to try to solve some problems of species identification. Starting cooperation with Dr. Naganawa and with Theodore Abatzopoulos (AUTH, partner 3) to study Artemia populations from Mongolia (morphology and molecular genetics)

2) Updating of Artemia distribution

As to Artemia distribution in Europe, through cooperation and building a web of personal connections with Arab Emirates, Belarus and Mongolia was established, (Mura et al., articles in preparation) in order to fill the existing gaps in those parts of Eurasia. Moreover, information is being collected also as to France, where limited information was available until present days.

3) Search for allochthonous species

New intensive sampling was started in some sites in Sardinia and Sicily, in order to update the situation.

4) Morphological studies

Morphological traits such as frontal knob, basal and eversible penis pattern are being used to perform intra- and inter-bisexual species comparisons. Morphological studies have just started because of a long stop due to SEM repair. Excessive management costs are limiting the extent of studies, due to absence of specific funding

16

INCO partner 5: LA SAPIENZAPartner name: Dipartamento di Biologia Animale é dell’Uomo, Laboratorio di Zoologia Applicata, Università La Sapienza, Viale dell’ Università, 00185 Rome, Italytel.: 39-6-49914769; fax: 39-6-4958259; email: graziella.mura@uniroma1.itResponsible scientist: Graziella Mura

During the last six months our team has been working on the following topics:

- Adjustment of the protocols for the Genome in situ Hybridization (GISH) technique : in order to determine the genome affinity between A. franciscana and A. persimilis, and to obtain a better characterization of the heteromorphic bivalents in supposedly hybrid individuals we have started to adjust this technique. DNA from A. franciscana from GSL, A. persimilis from Salinas Grandes de Hidalgo and DNA from individuals from Mar Chiquita Lagoon was isolated and quantified. DNA was later labeled by nick translation with biotin 14-dUTP (Bionick Labeling system, GIBCO BRL). Nauplii of A. franciscana and A. persimilis of 12-24 h were obtained from newly hatched cysts and fixed following standard procedures. Nauplii were then squashed in acetic acid, and the cover slip was then removed by the dry-ice method and air-dried. We have applied the in situ technique using the labeled DNA of each species on the slides of the same species, in order to adjust the hybridization conditions. We have obtained good results in A. franciscana slides, but in A. persimilis the hybridization signal was rather weak. Our next steps are aimed at improving the results with A. persimilis and to perform the cross hybridization between both species (i.e. A. franciscana DNA hybridized on A. persimilis slides and vice versa).

- Collecting Campaign of new Artemia populations (not yet recorded) from near and along the coastal of South Chubut and North Santa Cruz Provinces during 20-28th May, together with Dr. Francisco Amat and our assistants. The new locations are: Lagoon near Rada Tilly (Chubut Province) 45° 55' 42,5" S, 67° 34' 21,0” W Primera Lagoon from Caleta Olivia (Santa Cruz Province) 46° 27' 01,5" S, 67° 31' 48,3" W Segunda Lagoon from Caleta Olivia (Santa Cruz Province) 46 °27' 02,0" S, 67° 32' 18,8" W Saline in Estancia La Pava (Santa Cruz Province) 47° 32' 43,6" S, 66° 38' 06,9" W Lagoon in Estancia El Caburé (Santa Cruz Province) 47° 34' 57,0" S, 66° 31' 44,0" W Salitral Bajo Pichinini in Estancia Cerro Pancho (Santa Cruz Province) 47° 45' 44,1" S, 66°

14' 0,9" W Salitral in National Route 3, km 2035 (Santa Cruz Province) 47° 28' 50,4" S, 67° 16' 18,0" W Salitral in Velódromo from San Julián (Santa Cruz Province) 49° 18' 10,4" S, 67°44' 39,1" W Laguna Seca, near San Julián (Santa Cruz Province) 49° 17' 20,3" S, 67°46' 47.0" W

We are working together, both the Argentinean and Spanish teams, in the identification of each population.

- Training: Within the framework of the INCO project, Julieta Goenaga, an advanced student of our Faculty making her Msc*, has gone for training to CSIC (Castellón, Spain, partner 4) from January 15 to March 15. In agreement with Dr. F. Amat we planned her activities in the Institute. Here, she has worked with Artemia samples from two Argentinean locations belonging to A. persimilis (Salinas Grandes de Hidalgo, La Pampa Province) and A. franciscana (Mar Chiquita Lagoon, Córdoba Province). She has performed some experiments on the combined effect of salinity and temperature, in order to establish the range of tolerance and the optimum combined condition for both parameters in those populations. Going on in the same way, we are now evaluating in conditions of constant salinity (90 ‰), the effect of a cold 12 h duration shock (10 °C), applied in the naupliar, in the postmetanaupliar 2-3 or in adult periods. We evaluate the effect of the temperature shock in each period of development by means of the survival, size and reproductive performance of both

17

INCO partner 6: UBAPartner name: Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Nunez, Pab. II, 4P, C1428 EHA Buenos Aires, Argentinatel. 54-1-45763349 ; fax 54-1-45763384 ; email : cohen@bg.fcen.uba.arResponsible scientist: Graciela Cohen

considered populations. Also, we determined the salinity of preference for each population in conditions of constant temperature (26°C ± 1°C).

The results of these experiments will be presented at two Meetings: COLACMAR (Latin-American Congress of Marine Sciences, December, 2003) and VIII Jornadas de Ciencias Naturales del Litoral y I Jornadas de Ciencias Naturales del NOA (September, 2003).

* Graduation Thesis for achieving the title of “Licenciada en Ciencias Biológicas in FCEN, UBA”.

- Publications: One paper was submitted to the Journal of Biogeography for publication:Title: “Further evidence and characterization of A. franciscana (Kellogg, 1906) populations in Argentina.”Authors: Francisco Amat, Rosa Graciela Cohen, Francisco Hontoria, Juan Carlos Navarro.

18

1. Introduction:

Artemia is a live food that has been widely used in aquaculture, especially for shrimp and fish larvae. Presently, Artemia has become one important candidate for aquaculture in the coastline of the Mekong Delta, especially in Baclieu-Soctrang area where it was firstly inoculated 20 years ago. The major problem on production of this species is that the cyst yields was decreasing by year due to the lack of knowledge in culture techniques and bioprocesses that used to happen in the culture ponds of Artemia farmers. Our research activities are aiming to fill this gap.

Objectives: Continuing research for getting better comprehension of the environmental conditions for

Artemia pond culture: algal bloom control; selected algal species as suitable food for Artemia; soil and nutrient interaction.

Improvement of knowledge and pond production by Artemia farmers through extension activities.

To establish a good research team on Artemia and environment related to Artemia culture through training.

2. Research activities

To have better comprehension of environmental conditions for Artemia pond culture. Research on the bio-control of algae bloom due to unwanted algal species by stocking

Artemia at high densities To study the effect of algal quality/quantitity on the growth and reproduction of Artemia To study the factors (salinity, temperature, food availability, genetics…etc.) that affect

Artemia cyst production during successive generations. To study thermo-tolerance of Artemia (heat –shock protein, fatty acid profiles…etc.). Socio-economic analysis of Artemia culture in the studied areas. Investigation on of soil parameters predicting N, P released from the pond bottom to

prevent the problem of algal bloom in Artemia ponds. Study on the relation between soil parameters, nutrient and algae growth. Investigation and search for solution for clay turbidity in Artemia ponds. Quantification of fertilizers (phosphorus and nitrogen) to be applied for optimal algae

biomass as a food supply for Artemia.

3. Extension programme for Artemia pond culture:

Two demonstrative stations (one in Vinh Chau-Soc Trang province and the other in Vinh Hau-Bac Lieu province) are maintained with 6 staffs in Artemia group; their tasks are to transfer new culture techniques, helping the farmers who want to work with Artemia and also try to improve the cyst yields of the whole region via this activity. More than 100ha of culture areas are under our guideline in this year.

19

INCO partner 7: CTUPartner name: Institute of Science for Aquaculture, 3rd February Street, Campus II, Can Tho University, Can Tho, Vietnamtel.: 84-71-834307; fax: 84-71-830323; email: nvhoa@ctu.edu.vn; nvhoa.ct@bdvn.vnd.netResponsible scientist: Nguyen Van Hoa

Research and investigation activities :The INAT group had continuous sampling cyst across Salinas and Sebeka’s, in particularly those of El Hadhibett (southern Tunisia) and Sahline (central Tunisia).Laboratory works are oriented to the morphobiometric characterization, and some biochemical analysis, linked to enrichment test.

Exchange and training activities :During this period we have started with the first scientific stay organized between INAT Tunisia and CSIC Spain (partner 4). A young scientist from INAT was trained for two months at CSIC with a common program and experiment plan, focusing on biometrics, cross breeding, HUFA analysis, and hatching mechanisms.

OthersUpdating local cyst bankParallel studies on Dunaliella salina from the salinas, investigated for Artemia..

20

INCO partner 8: INATPartner name: Institut National Agronomique de Tunisie, Université de Carthage, 43 av. Charles Nicolle 1082, Tunis, Tunisiatel. : 216-1-287110; fax: 216-1-799391;email: romdhane.medsalah@inat.agrinet.tnResponsible scientist: Mohamed Salah Romdhane

Researcher: Horst Kaiser

During the first year of the INCO project we described for the first time an interesting Artemia population from southern Africa. The work was done in collaboration with Aristotle University of Thessaloniki (INCO Partner 3).The population was from a salt works near Port Elizabeth in the eastern cape of South Africa. It was sampled for the first time last year, and initial results showed that the population comprised a bisexual species and a parthenogenetic component. As there were few samples available from the southern African region, a review was compiled and potentially new sites were identified for further collections. We also looked at bird migration routes to get a better understanding of the factors influencing Artemia distribution. Results from this aspect of the study will be presented in future reports.In accordance with the overall INCO project objective of describing Artemia biodiversity a sampling program for the region was designed during the first half of the year. The southern African region comprises many small salt pans and salt works. The review presented last year showed that few populations had been collected from these sites and very few cyst samples have been banked. For many sites no details have been recorded as yet. Often sampling was not done reliably and much information is still lacking (see our review presented last year). Thus, there was a need to visit new sites as well as some previously visited locations. We included salt pans in Namibia and South Africa. Distances between pans are large. Within Namibia alone, distances of more than 300 km needed to be covered to reach some of the salt pans. In South Africa, 23 pans were visited (Map 1). Cysts could not be collected from all sites. Some pans are listed as salt pans but were found to be unsuitable for Artemia due to low salinity at the time of sampling. In some cases, only adult Artemia could be sampled and they were preserved for further analysis. Many salt pans were visited for the first time as they were not in any available records. All sites were visited between 16 June and 18 July 2003. Even if no Artemia were sampled, their location and specific characteristics are now known and they will be re-visited, hopefully in early spring (i.e., September / October 2003).

21

INCO partner 9: RUPartner name: Department of Ichthyology and Fisheries Science, PO Box 94, Rhodes University, Prince Alfred Street, Grahamstown 6140, South Africatel.: 27-46-6038415 ; fax : 27-46-6224627 ; e-mail: h.kaiser@ru.ac.za; t.hecht@ru.ac.zaResponsible scientist: Tom Hecht

Map 1: A map of South Africa with sites from which Artemia or cysts were sampled (circles) or

that were visited but no samples could be collected (triangles).

A) Sites from which cysts and/or adult Artemia (S=salinity in ppt, and T=Temperature (°C) are given in brackets) were collected. All measurements were done between 7:00 a.m. and 12 a.m.1. Sundays River: previously sampled; 2. Coega: previously sampled; 3. Swartkops River 1: cysts and nauplii collected (100 ppt; 14°C); 4. Swartkops River 2: cysts and nauplii collected (75 ppt; 5°C); 5. Missionvale: cysts and nauplii collected (110 ppt; 4°C); 6. Florisbad Salt Pan: cysts and nauplii collected (4 ppt?; 6°C); 7.Renekesfontein (Hopetown): cysts and nauplii collected (80 ppt, T=12°C); 8. Brandtvlei: nauplii collected (160 ppt; 12°C); 9.Yzerfontein: nauplii collected (140 ppt; 13°C); 10. Steelworks: cysts collected; 11. Klipfontein: cysts collected; 12. Velddrif 1: cysts collected (135 ppt; 14°C); 13.Velddrif 2: cysts collected (200 ppt; 15°C).

B) Sites sampled but pans were either dry or no Artemia were found (D=dry, N=no Artemia or cysts found, U=unsuitable habitat, low salinity at the time of sampling)A) Delareyville: (D); B: Barbaspan salt pan and Leukop salt pans: (U); C) Solar saltworks around Florisbad: (N); D) Schweizer Reneke: (D); E) Kimberly: (U); F) Dealesville: (N); G) Petrusburg: (D); H) Hopetown (N); I) Upington: (N); J) Elandsbaai (N); K) Brandtvlei (N)

C) Namibian sites sampledi) Walvis Bay: nauplii collected; ii) Swakopmund: cysts and nauplii collected; iii) Henties Bay: nauplii collected.

22

2

1

35 46789

10

11

12

13

A

B

CD EF

GH

I

J K0 200

km

1. Research activities

Continuous investigation of the Artemia resources in inland salt lakes, PR China: the cyst samples from 2 Tibet salt lakes and 2 from Inner Mongolia were collected.

Continuous study on Artemia resources of Bohai bay; PR China: strain characterization; population composition; nutrition profile among different seasons and locations.

Research on HUFA profile and metabolization changes of Bohai Artemia strain during enrichment and starvation and application in larviculture of Japanese flounder.

Integration and harmonization of cyst bank in Salt Research Institute. Collection of the publications on the research of Artemia published in Chinese journal

and compiling of the publications according to the form provided by INCO Artemia Biodiversity project.

2. Exchanges of research experience within INCO Artemia biodiversity partners

Several plans on exchange of study visit were made within INCO Artemia biodiversity partners: 1 researcher from SRI will have a visit study in Aristotle University of Thessaloniki,

Greece (Partner 3) for 2 months from September, 2003, onwards. 1 researcher from Nankai University was supposed to have a visit study in Laboratory of

Aquaculture and Artemia Reference Center, Ghent University, Belgium (Partner 1) since May, 2003 (This visit was delayed by SARS)

23

INCO partner 10: SRIPartner name: Salt Research Institute, Yingkou Road 831, 300450 Tanggu, Tianjin, PR Chinatel.: 86-22-25301094; fax: 86-22-25301094; email: srisalt@public.trp.tj.cnResponsible scientist: Xin Naihong

1. Planning for the study stays as visiting scientists in the form of PhD research projects for 2 months during Aug.-Sep. 2003 at ARC-Oostende, Belgium.During this visit some Artemia samples from Iran and especially from Urmia Lake will be studied and compared on the basis RFLP technique.

2. Further studies on biology of Artemia populations from Urmia Lake:Following the earlier studies on coexistence of parthenogenetic and bisexual populations of Artemia at Lake Urmia, some more experiments were carried out in order to confirm the earlier findings. It was once again confirmed that one can very easily separate and isolate the parthenogenetic populations of Artemia from the bisexuals by culturing them at low salinities (from 30-50 ppt.). During this experiment again and again it was confirmed that the two populations coexist at Urmia Lake.

3. Monitoring the Lake UrmiaUrmia Lake had been facing a deep ecological crisis since 1999 due to insufficient rain and snowfall and this had caused severe increase in water salinity (up to 300 ppt). Fortunately there was a reasonably good rain and snowfall during last winter and the Urmia Lake recovered at least a part of its lost water (up to about 1 meter in depth) but still the water salinity has not dropped below 220 ppt. at South arm and 265 ppt at North arm. During the last 3 months we have been constantly monitoring the physio-chemical parameters of the lake water and its relationship to Artemia resources. This study will continue until the end of the year.

4. Studying population dynamics and resource assessment of Artemia at Urmia LakeThis project which is supported by the Iranian Organization for Environmental Protection as well includes a detailed study on the population dynamics and resource assessment of the Artemia population at the Lake Urmia. We have also incorporated another study to survey on probable existence of more Artemia populations on the basis of morphometric differences between samples collected from many different sites and depths. This will also be followed by verifying the genetic differences between the isolated populations with cooperation of ARC and AUTH during the coming months. So far we have been busy with monthly sampling programs and assessing the stock. On the basis of data collected so far and improving water conditions of the Lake, we predict a considerably good production yield, but yet can not predict the possibility of any commercial exploitation during this year.

5. Use of enriched Artemia for feeding sturgeon fishes on producing highly resistant sturgeon fingerlings.This project which is being done as a PhD study program at ARC and Urmia University (AAARC), evaluates the use of Vit. C + HUFA + saturated fatty acids enriched Artemia nauplii on production of stress resistant sturgeon fingerlings. The results of feeding with above enriched nauplii are compared with the results obtained by feeding the sturgeon larvae with un-enriched nauplii, decapsulated cysts and formulated feed. It was found that sturgeon fingerlings fed live Artemia (both enriched and non-enriched) had significantly better growth and survival rates and were highly resistant to stresses than those fed decapsulated cysts and formulated feed.

6. Use of enriched Artemia on increasing resistance of rainbow trout larvae to ammonia and nitrite. In an experiment local fish oil was used in combination with Vit. C to enrich Artemia nauplii and give it as starter feed to the rainbow trout larvae. Other treatments were un-enriched Artemia nauplii and

24

INCO partner 11: UUPartner name: Artemia and Aquatic Animals Research Center (AAARC), Urmia University, 165 Shahid Beheshty Av., 57153, Urmia, Irantel: 98-441-440295; fax: 98-441-440295; email: agh1960@hotmail.comResponsible scientist: Naser Agh

commercial formulated feed. The growth and survival rates of larvae were determined on days 6, 9, 12, 15 and 18. Moreover resistance of the larvae were checked against increasing nitrite and ammonia levels on days 9, 12, 15 and 18. The results indicate that the larvae receiving live food and especially enriched nauplii are much more resistant then those fed with formulated diet.

7. Collection of cyst samples for the Cyst Bank:Cyst sample collection continued especially from the Lake Urmia during last six months and some more samples are added to the cyst bank.

25

Scientific Staff:: Jorge Castro MejíaAida Malpica SánchezGermán Castro MejíaRamón de Lara Andrade

1. Research and Training

During this semester Jorge Castro Mejía has been analyzing data on the biometry of cysts, nauplii and adults, females and males from seven Mexican populations of Artemia (Yavaros, Ohuira, Juchitan, Real de las Salinas, Cuatro Ciénegas, San Luis Potosí and Texcoco), as well as data on reproductive isolation. He is writing the manuscript of his Master’s thesis scheduled to conclude in December, 2003. The title is “Morphometrical Characterization of Seven Populations of Artemia franciscana in Mexico in relation to Latitude, Habitat, and Hybridization.”

During this semester Aída Malpica Sánchez has been working on her doctoral dissertation. The title is “The Use of Molecular and Biochemical Techniques in the Characterization of Mexican Populations of Artemia franciscana.” During these six months she has identified proteins in three stages of development of Artemia (cyst, decapsulate cyst and nauplius) of nine populations. She has evaluated the content of raw protein (total nitrogen) in the sample using the MicroKjeldahl method. Then the nitrogen protein was determined by employing the Bradford method, and the profile of the proteins was analyzed by electrophoresis, under denaturalized conditions (SDS-PAGE), using the standard of well-known molecular weight at an interval of 8 to 86 kDa.

During this semester, another member of the group, Germán Castro Mejía, has been working on the incorporation of three antibiotics (Chloroamphenicol, Cyprofloxacine, and Furadoine) in three stages of development of Artemia (metanauplii, juveniles and adults) to inhibit the growth of the Aeromonas hydrophila bacteria and later on determine the number of organisms needed to act against the bacteria. This information will be in his Master’s thesis, titled “Incorporation of Antibiotics in Artemia franciscana, for the Inhibition of Growth of Aeromonas hydrophila” This thesis will be concluded in December, 2003.

During this semester we concluded advising three students in biology and they presented the following papers to get their bachelor’s degree:

“Content of Magnesium, Calcium, Sodium, Potassium and Phosphorus in Artemia franciscana, Adult Stage, of Five Mexican Populations and their Importance in Aquaculture” Student: Omar Soto Murrieta.

“Taxonomic Characterization of Three Populations (Juchitan, Oaxaca, Las Salinas, San Luis Potosí and Texcoco, State of Mexico) of Artemia franciscana in Mexico” Student: Juana Zaragoza Ríos

“Taxonomic Characterization of Three Populations (Real de Salinas, Campeche, Yavaros, Sonora, and Cuatro Ciénegas, Coahuila) of Artemia franciscana in Mexico.” Student: Araceli Castro Araujo

2. Publications

26

INCO partner 12: UMA-X Partner name: Universidad Autónoma Metropolitana-Xochimilco, Depto. El Hombre y su Ambiente, Calz. Del Hueso No. 1100. Col. Villa Quietud. México, 04960, D.F, Mexicotel.: 52-5-4837151; fax: 52-5-4837469; email: cabt7515@cueyatl.uam.mxResponsible scientist: Thalía Castro Barrera

During the first semester of this year, two papers have already been accepted for publication.

Title: “Composition of the Content of Fatty Acids in Three Mexican Populations of Artemia franciscana (Kellog, 1906), from Inland Waters”, Rev. Biología Tropical. Authors: Malpica,S.A.,T. Castro B., H. Sandoval T., J. Castro M., R. de Lara A. & G. Castro M.

Title: “The Relationship between some Microelements in Nauplii of Six Mexican Populations of Artemia franciscana with the Requirements of Fish and Crustaceans” Rev. Hidrobiológica. Authors: Castro, B. T., J. Castro M., J. Castro & B. Miramontes .

3. Consultancy

During this semester we have continued to advise on Artemia culture in Las Salinas de Hidalgo, San Luis Potosí. This saline is located inside the country and the water is extracted from underground waters. This Artemia is reproduced mainly by oviparity and the soil in this saline is particularly rich in minerals. Besides advising on the culture, we have carried out chemical and physical studies of the water and soil as well as the nutrient quality of Artemia, (contents of fatty acids, amino acids and minerals).

27

UFRN has been studying current Artemia practices and their implications for the sustainable exploitation and biodiversity of Artemia franciscana populations in the coastal saltworks of Rio Grande do Norte (RN), northeastern Brazil. Specific activities deal with the reproductive characterization of local populations of Artemia franciscana. In addition, UFRN, in partnership with the Brazilian Shrimp Farmers Association (ABCC), Brazilian Mariculture Linkage Program (BMLP), and BioArtemia Ltd., runs a pilot farm for Artemia Research & Development (reproductive biology, pond ecology, and aquaculture techniques) in Grossos (RN) since May 2001.

Information derived from the above-mentioned activities was presented at the recent meeting of the World Aquaculture Society (WAS) in Salvador (Brazil), in the following lecture: “Towards a sustainable Artemia industry in Rio Grande do Norte, northeastern Brazil”. Marcos R. Camara. In: World Aquaculture 2003, BMLP – Community Development Session, Annual Meeting of the World Aquaculture Society, Salvador, 2003.

In the framework of the INCO project, a training visit of Marcos R. Camara to AUTH took place from February to May 2003. His work focused on the analysis of Artemia populations from RN using molecular (DNA) techniques. Genetic diversity (variability) of such populations was assessed by Restriction Fragment Length Polymorphism (RFLP) analysis of PCR amplified mitochondrial DNA (mtDNA).

28

INCO partner 13: UFRNPartner name: Departamento de Oceanografia e Limnologia, Universidade Federal do Rio Grande do Norte, Campus Universitario, BR 101, 59072-970 Natal, Braziltel.: 55-84-2154433; fax: 55-84-6421815;email: mrcamara@ufrnet.brResponsible scientist: Marcos R. Camara

The group already completed the exchange/training programme with the almost simultaneous presence of two trainees in European laboratories of the Consortium last year. Results produced from both training activities were communicated to Chilean scientific societies (annual report 2002). During this period two draft manuscripts have been improved. One, based on the work of one trainee in Greece, has been distributed to the authors for final polishing and is now almost ready to be submitted. These results represents the core of a Master thesis Mr. Julio Crespo should be defending before October this year (University of Concepcion, VIII region, Chile).

Another manuscript, based on the work done in Ghent by Patricia Beristain and complemented with data produced in Chile during this period, is in an advanced stage of preparation.

According to what was agreed on in the China regional workshop, the Chilean co-ordinator of the Latin American group collected a list of the Artemia sites in the region together with basic information on them (following the protocol agreed for the Mediterranean sites). Such information is actually available in the project Web page. The application of new techniques to the Chilean and other Artemia sites has raised new questions and brought about chances for further regional collaboration in relation to various aspects. In this regard, new sampling campaigns in the period allowed to obtain animals from key, problematic, and/or particularly interesting locations in Chile, such as Pichilemu (region VII), Torres del Paine and Laguna Cisnes (Chilean Patagonia, region XII). The Pichilemu sample was sent to the Italian partner (Dr. Graziella Mura) for scanning microscope analysis of taxonomically relevant sexual traits (frontal knobs), and the same is expected to occur with the Argentinian equivalent location (Dr. Graciella Cohen), as these locations were identified as key latitudes for the distribution of A. franciscana and A. persimilis in both countries. Hence they need special attention. All samples collected are being subjected to the usual morphological, karyological, and allozyme analysis, as well as to the DNA level (RAPD’s).

The availability of molecular markers for A. persimilis and A. franciscana suggested a study on the probable hybridisation of both species, which is likely to occur in Torres del Paine and other locations (laguna Cisnes) in the Chilean Patagonia. Such a study would also consider laboratory crosses. The Belgian and Chilean laboratories have exchanged ideas for tackling this problem through a probable PhD thesis by Patricia Beristain. In the meantime she has continued her training in molecular markers which considers now microsatellites.

The 2003 Chile regional workshop was announced (see Web page), and after selecting a suitable and convenient place, local efforts have been put on matters related with the organization of the meeting. Early this year (January) the sampling campaign in Pichilemu, which is a saltwork, allowed us to promote among the local authorities and salt workers the idea of continuous monitoring and conservation of the local Artemia.

29

INCO partner 14: ULLPartner name: Department of Basic Sciences/Laboratory of Genetics & Aquaculture, Universidad de Los Lagos, P.O. Box 933, Avenida Fuchslocher s/n, Osorno, Chiletel. 56-64-205293; fax: 56-64-239517; email: ggajardo@ulagos.clResponsible scientist: Gonzalo Gajardo

1. Genetic polymorphisms and mitochondrial RFLP of Artemia from selected South Asian regions

Artemia spp distributed in three selected regions of two South Asian countries, viz. Sri Lanka (Puthalalam), India (Tuticorin and Puthalam), were compared with the Artemia of North American and Chinese sp. for its genetic and biochemical diversity, using enzyme markers, metabolism and mitrochondrial DNA. Among the four strains selected from Asia, the Chinese strain exhibited the better stock quality and swift adaptation to changing environment. This phenomenon was assessed by metabolic enzyme expressions during the hatching period of the cysts. In all experiments the strain selected from Tuticorin exhibited quite similar phenomena as the control, which indicates the intermixing of the native strain with the North American strain. Mitrochondrial RFEL shows the maximum agreement on restriction site of BstXI between the North American strain and Tuticorin. LDH polymorphisms were observed between five strains showing monomeric LDH patterns in control, Tuticorin and Sri Lanka. Dimorphic LDH in Puthalam and China indicates that there is a adaptation of carbohydrate metabolism in these strains. From the overall studies, the Chinese strain seems to be better stock and Tuticorin and Sri Lanka strains were under threat for its genetic purity since the intermixing phenomenon was observed with the North American strain. Further studies such as microsatellite studies need to establish the degree of intermixing of native with North American strains and the genetic purity of native strains of South Asia.

2. International training at the Instituto de Acuicultura de Torre de la Sal (CSIC), Castellon de la Plana, Spain, during June, 2003, by M. Peter Marian on the characterization of the Artemia strains from Vembar, Veppalodi, Therasapuram, Arasaradi of Tuticorin district region and Thamarikulam and Puthalam of Kanykumari district. See report by INCO partner 4 (CSIC).

30

INCO partner 15: IARTPartner name: Institute for Artemia Research and Training, Manonmaniam Sundaranar University, Rajakkamangalam, 629 502, Kanyakumari District, Tamil Nadu, Indiatel.: 91-4652-33680; fax: 91-4652-21457; email: petermar@md5.vsnl.net.inResponsible scientist: Peter Marian