Preliminary RFLP analysis of mtDNA from southern African Artemia populations Horst Kaiser, Department of Ichthyology and Fisheries Science Rhodes University, South Africa and Alexander Triantafyllidis, Athanasios Baxevanis, and Theodore J. Abatzopoulos; Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Greece

Bisexual –Several species with a wide distribution range Parthenogenetic –Different ploidy levels –Genetics is poorly described Mixed populations Artemia life histories

Potentially interesting southern African Artemia populations Namibia: Smallest recorded cysts Coega: Undescribed Madagascar

The current project: Thessaloniki, Greece Main topic: Artemia biodiversity –Sub-topic: Genetic structure of southern African populations –Technique development for Artemia

Project objectives To develop a data base of genetic markers for comparison with other populations. To find genetic markers for African Artemia populations. Getting accustomed to molecular techniques

Problems with morphometrics as a tool to differentiate species Different species may be very similar morphometrically. Results are sensitive to rearing conditions. Requires strict standardisation.

Molecular techniques Using the16s sub-unit of the mitochondrial rDNA –Maternal inheritance –Small size (540 base pairs) –Stable gene arrangement –Fairly well conserved

Individuals from five populations were used for genetic analysis Three parthenogenetic populations from Greece Namibia (Swartkopmund) Madagascar (near Toliara) South Africa (Coega; Cerebos salt works)

RFLP Restriction Fragment Length Polymorphism The molecular geneticist’s version of a jigsaw puzzle - without knowing the number of pieces.

The RFLP recipe Use only the best Artemia: gently squashed and homogenised. For best results use one shrimp at time. Prepare with a liberal dose of proteinase (keep in the oven overnight). Wash in phenol and chloroform, then spin. Add a few picograms of primers and a touch of Taq enzyme…. continued

The RFLP recipe “Cook” while varying the temperature (PCR) Add assorted enzymes to cut the PCR product into smaller units. Add the rDNA pieces to a bed of gel. Let run on gel until well separated. Serve results in a table.

PCR product from 17 different individuals 1.5% Agarose gel, ethidium bromide stain, ~ 540 base pairs (bp) Reference ladder: 100 bp Individuals

Restriction of PCR product using the Dpn II enzyme 120 bp 180 bp 240 bp 100 bp Ladder

Restriction of PCR product using the Taq I enzyme Artemia parthenogenetica (Greek population) Artemia parthenogenetica (Greek population)

Restriction of PCR product using the Hae III enzyme Six individuals from the Coega population(s) show different restriction patterns: An indication of a mixed population?

Examples of different restriction patterns for two enzymes

Haplotypes of different populations Greek (1) P*DBAAABABC7 Greek (2) PCCCAABABD7 Greek (2) PCBCAABABD2 Greek (3) PDBAAABABE9 Namibia (P)DBAAABABC8 Madagascar (P)DBAAABABC10 CoegaCBCAAAABD7 CoegaAAAACAABC1 CoegaABAACAABC9 A. franciscanaAAAAAAAAA- *P = A. parthenogenetica ; numbers are frequencies.

Artemia franciscana Bisexual component from Coega A. parthenogenetica (Greece) A. parthenogenetica (Madagascar) A. parthenogenetica (Greece) A. parthenogenetica (Namibia) Parthenogenetic component from Coega A. parthenogentica (Greece) Phylogram based on restriction site patterns

Interesting preliminary results There is a considerable genetic distance between the two populations from the Coega salt works (South Africa) Populations with different life histories appear to co-exist.

Questions and tasks for next year Do the environmental requirements differ between the populations? Which species represents the bisexual component? Survey and study bird migration routes.