INCO partner 3: AUTH
Partner name: Aristotle University of Thessaloniki, Department of Genetics, Development &
Molecular Biology, Faculty of Sciences, School of Biology, 541 24 Thessaloniki, Greece
tel.: 30-231-099 83 01; fax. 30-231-099 82 56; email: abatzop@bio.auth.gr
Responsible Scientist: Theodore J. Abatzopoulos
Collaborating scientists: A. Triantafyllidis, A. Baxevanis, A. Tzika, E. Markatzinou
In the first global workshop organized in Belgium the first meeting of the participants
took place. AUTH presented its objectives for the INCO project and the techniques that
are to be used for the study of the Artemia populations focusing on Artemia biodiversity.
During the course of the first year of the project two scientists visited the AUTH lab. i)
Julio Crespo from University de Los Lagos, Chile (INCO partner 14) from May 11th until
July 17th and ii) Dr Horst Kaiser from Rhodes University, South Africa (INCO partner 9)
from May 28th until July 8th.
The work of the invitees involved the analysis of Artemia populations using molecular
(DNA) techniques. During their stay in AUTH the visiting scientists 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 a 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. Also, information on
analysis of sequences deposited in Genbank was given and on choice of restriction
enzymes to be used for RFLP analysis. RFLP data were statistically treated with different
packages (i.e. REAP, Arlequin, Phylip). All molecular work was done at the level of
individual scoring. The visiting scientists during their course of their stay were 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. They were 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 Julio Crespo to analyse
the relationships of Chilean Artemia populations. This scientist came into first contact
with the molecular techniques using two reference populations of Artemia fransiscana
and Artemia persimilis species. After successfully applying these techniques in these
populations, five Chilean Artemia populations were further screened (i.e. Laguna Cejas,
Laguna Chaxas, Los Vilos, El Convento and Pichilemu). Three additional Chilean
populations (Torres del Paine, Iquique, Llamara) were also screened by personnel of
AUTH. The results obtained, permit the identification and assignment of these
populations to Artemia franciscana or Artemia persimilis species with a simple restriction
with specific enzymes. Information was also gained on the levels of genetic variation of
these populations (see Annex I).
Dr Horst Kaiser used the same approaches to analyse relationships among
parthenogenetic and bisexual Artemia populations from Southern Africa, i.e. Coega
(South Africa), Swakopmund (Namibia), Ankiembe (Madagascar). For comparison
reasons three Greek parthenogenetic Artemia populations were used, i.e. Citros, M.
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Embolon and Kalloni. Composite genotypes that characterize populations and species
were determined (see Annex II).
The analysis of the Artemia biodiversity, evolution and phylogeography has proceeded in
AUTH with the addition of bisexual populations from A. salina (Sfax, Tunisia) and A.
urmiana (Urmia lake), as well as extra parthenogenetic populations from Kessani, Mesi,
and Polychnitos areas in Greece. In this direction, methods have been developed to even
extract DNA from cyst samples, which do not hatch. Composite genotypes are now
available which characterize these populations-species (see Annex III).
Following the multidisciplinary approach for characterizing Artemia strains (as it was set
at the kick-off meeting in Ghent, Belgium – see six month report), morphometric
parameters were measured for the following strains/populations:
 Parthenogenetic strains: Citros, Kalloni, Kesani, M. Embolon, Mesi, Polychnitos
(Greece), Coega (South Africa), Madagascar, Namibia
 Bisexual strains: A. franciscana (SFB strain - USA), A. persimilis (Argentina), A.
urmiana (Urmia Lake, Iran), Coega (South Africa)
The morphometric variables that were measured were: total length, abdominal length,
maximal width of brood pouch, width of 3rd abdominal segment, width of head, length of
1st antenna, maximal diameter of complex eye, distance between complex eyes, length of
furca and the ratio abdominal length / total length (expressed as percentage). The
morphological data were analysed by well-known statistical tests (ANOVA, discriminant
analysis) and they revealed that the bisexual strains were very well discriminated and that
the Coega bisexual population was closer to A. salina than the rest bisexual populations.
Furthermore, parthenogenetic strains from Greece were grouped together with the
parthenogenetic strain from Urmia Lake (Iran) and they were separated from the
parthenogenetic strains from Africa (Madagascar and Namibia). It should be noted that
the grouping of Artemia populations based on morphometry was more or less the same
with the grouping proposed by genetic analysis. This reinforces the idea for
multidisciplinary approach of characterising Artemia strains (see Annex III).
Two Diploma Theses have been almost completed (are to be defended in February-March
2003). The tentative 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
AUTH participated in organizing a cyst bank in the framework of this project. Cyst data
are very scattered and cyst samples exist in different laboratories. Our team was
responsible for collecting and processing data from laboratories all over the
Mediterranean Basin (MEDBAS). The following INCO partners provided data for
organizing the MEDBAS database: AUTH (INCO partner 3), CSIC (INCO partner 4), La
Sapienza (INCO partner 5), INAT (INCO partner 8), while there was additional
contribution from ARC (INCO partner 1). The results of this effort were presented by our
team in the first regional meeting (Beijing, China) (see Annex III).
Our team also participated in the second regional INCO meeting that took place in
Beijing (China) with the presentation “Artemia characterisation based mainly on mtDNA
RFLP analyses and progress in Mediterranean Basin Regional Action” (see Annex III).
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Our studies are aiming on developing markers suitable for detection of invasive species
focusing mainly on the area of Eastern Mediterranean Basin.
In the framework of this project the following publications and presentations were
produced:

Publications:
1. Title: “Effects of salinity and temperature on the reproductive and life span
characteristics of clonal Artemia (International Study on Artemia LXVI)”
Authors: Abatzopoulos, T.J., N. El-Bermawi, C. Vasdekis, A.D. Baxevanis & P.
Sorgeloos.
Journal: Hydrobiologia (in press) (see Annex IV, uncorrected proofs)
2. Title: “Elevated salinities enhance the thermotolerance of hydrated Artemia
franciscana cysts. (International Study on Artemia. LXV)”
Authors: Abatzopoulos, T.J., G.V. Triantaphyllidis, N. Roedaki, A.D. Baxevanis, A.
Triantafyllidis & P. Sorgeloos
Journal: Belgian Journal of Zoology (accepted after revision)
3. Title: “Salinity effects on survival, growth and morphometry of four Egyptian
Artemia populations. (International Study on Artemia. LXVII)”
Authors: El-Bermawi, N., A.D. Baxevanis, T.J. Abatzopoulos, G. Van Stappen & P.
Sorgeloos
Journal: Hydrobiologia (submitted)

Presentations in congresses/workshops:
1. Title: “Elevated salinity enhances the thermotolerance of hydrated Artemia cysts.
(International Study on Artemia. LXV)”
Authors: T. J. Abatzopoulos, G.V. Triantaphyllidis, N. Roedaki, A.D. Baxevanis, A.
Triantafyllidis & P. Sorgeloos
Congress: 9th International Congress on the Zοοgeography and Ecology of Greece
and Adjacent Regions (ICZEGAR), Thessaloniki, May 2002 (see Annex V)
2. Title: “The use of multidisciplinary approaches for characterizing and biomonitoring
Artemia populations: uniformity of practices”
Authors: T.J. Abatzopoulos, A.D. Baxevanis, A. Triantafyllidis
Workshop: NATO ADVANCED RESEARCH WORKSHOP (ARW) “Artemia
biodiversity in the Newly Independent States: Current global resources and their
sustainable exploitation”, Moscow, Russia, 17-19 July 2002 (see Annex VI)
Recommendations for future work:

Further determination of potential molecular markers for characterizing Artemia
populations.
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


Urge partners to collect more pieces of information on diverse Artemia habitats.
Build a data base using mainly mtDNA analyses based on the existing species or
populations. This would be extremely useful in identifying “new” Artemia strains or
tracking down invasive species.
Establishing a frame for multidisciplinary approach in Artemia characterization.
Work plan for 2003:



Scheduled visits by three INCO partners.
Further investigation on MEDBAS Artemia populations focusing on invasion
patterns.
Defining mixed populations in S. Africa
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