ARTEMIA BIODIVERSITY,



Abstracts – INCO Members

INCO partner: 1

Present studies on biodiversity of Artemia populations in Northerm Asia

G. VAN STAPPEN, B. MARDEN, L. LITVINENKO, I. MIRABDULLAYEV, I. ZHOLDASOVA, XIN NAIHONG,

P. Bossier, P. Sorgeloos

Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent University, Rozier 44, B-9000 Gent, Belgium, tel. 32-9-264 37 54; fax 32-9-264 41 93

1. Distribution of Artemia in lakes of South-Siberia

Topography and climatological aspects

A survey of Artemia habitats in south Siberia resulted in the registration of 90 sites, distributed over the Altai region (total area of Artemia lakes 1280 km2), Kurgan region (123 km2), Omsk region (92 km2) and Novosibirsk region (92 km2). Most of these lakes are small to medium sized, generally ranging between 1 and 10 km² surface area. All sites are located in a relatively flat arid or semi-arid zone, merging into the semi-deserts of Kazakhstan in the south. The climate is typically continental with low winter temperatures (water temperature may drop below –19°С; ice layer may be 15-20 cm thick in winter, only above 250 ppt no ice formation occurs) and relatively warm summer temperatures (water temperatures up to 25-30°C and even exceeding 40°C in shallow lakes). Because of the shallowness of most lakes, generally no temperature stratification occurs.

Over a 6-year observation period (1995-2001), considerable fluctuations in salinity have been observed as a consequence of fluctuations in precipitation (e.g. doubling of salinity in Medvezhye Lake in the period 1995-2000, followed by a very rainy 2001). Some lakes show total desiccation every summer; others periodically dry out, e.g. in years with reduced rainfall and/or increased evaporation. Precipitation is limited (250-400 mm annually) and generally occurs as snowfall. Increase of salinity and/or elevated temperatures in summer may result in total mortality of brine shrimp in some lakes. There are a few low-salinity lakes where Artemia occurred in the past, but due to increased precipitation it is presently not found. Furthermore in several lakes with relatively low salinity Artemia faces competition with other zooplanktonts; lakes with Artemia as monoculture are a minority.

Hydrology and ionic composition

Depth of the lakes is generally in the order of 2-3 m at most; most lakes are thalassohaline, though carbonate and sulfate lakes are found; about 2/3 of the lakes have an average salinity below 150 ppt.

Phytoplankton

Phytoplankton species diversity is limited, and cell densities are overall low, reflected in high water transparencies. All species identified sofar are ubiquitous species of brackish water and salt lakes. In terms of abundance, green (and to a lower extent blue-green) algae prevail, generally making up 70-90 % of total phytoplankton biomass. The seasonal dynamics of phytoplankton biomass was monitored in a number of lakes. Generally phytoplankton densities peak in spring (April-May) before maximal Artemia densities are reached, and a second, smaller, peak is observed just before winter (October-November) when the Artemia population has declined.

Artemia population dynamics

The growth and reproduction period for Artemia (> 4-5°C water temperature) is limited to the period end of April-early October. According to present data, Artemia produces 3 to 4 generations within this period; in small and shallow lakes, subject to summer desiccation, only 1 or 2 generations are produced.

Parthenogenetic populations predominate; until now bisexual populations have only been found in 3 lakes. Depending on the available field samples, cyst biometrics and adult morphometrics have been determined, as well as cytogenetics for a number of populations.

Detailed Artemia population dynamics have been monitored for two consecutive years in Medvezhye lake and for one year in Nevidim and Vishnakovskoye lakes. Maximal biomass densities are observed late spring, early summer. Later on densities decrease, presumably as a consequence of food depletion. Brood sizes are generally relatively low, reflecting the low productivity of the lakes. In small shallow lakes Artemia distribution is very much dependent on weather conditions. Small commercial quantities of cysts are harvested in several lakes

2. NATO Collaborative Linkage Grant

“Artemia colonization of the Aral Sea: hope for a dying ecosystem”

ARC cooperation with 1) Laboratory of Ichthyology and Hydrobiology, Institute of Zoology, Tashkent, Uzbekistan; 2) Institute of Bioecology of the Karakalpak Branch of the Uzbek Academy of Sciences, Nukus, Uzbekistan; 3) Inve Aquaculture, Inc.

Background

The Aral Sea has been claimed to be one of the worst human-induced ecological disasters of this century. Water management alternatives, the introduction of drought tolerant crop strains, less wasteful irrigation methods and other means of mitigating the economic losses and ecological catastrophe for the Aral Basin have been proposed and water conservation measures for the entire Aral Basin have been initiated. But these efforts to increase the Aral Sea elevation, to reduce the level of salinity, and reverse the ecological demise of the region have been largely unsuccessful. The contemporary Aral Sea is essentially a hypersaline lake with near-total elimination of species of freshwater origin.

The economic consequences of the Aral crisis have had devastating financial and health impacts on local economies and communities. Any means of economic recovery for these communities would be a most welcome and much needed benefit. Prudent commercial exploitation of an emerging Artemia population could provide income and employment. It is unknown, however, whether the current hydrobiological and hydrochemical status and primary productivity of the Aral Sea is sufficient to support a stable Artemia population. Planning for immediate cyst and/or biomass harvesting at this stage is premature as this could jeopardize the future potential for a viable industry. Detailed ecological information is clearly needed in order to evaluate the feasibility and potential benefits of commercial exploitation of Artemia.

Scope and research programme

The scope of this grant is to establish a team of biologists, limnologists, chemists, and natural resource experts from NATO countries and from Uzbekistan to document the ecological characteristics of the Aral Sea, more specifically the hydrobiological and hydrochemical status of the Aral Sea as it relates to the successful colonization by an Artemia population. A monitoring programme is taking place, focusing on Aral Sea developments in hydrochemistry, phytoplankton and its ongoing gradual colonization by a parthenogenetic population.

3. Contamination of Bohai Bay (PR China) samples with A. franciscana

Standard culture experiments with Artemia samples from the Bohai Bay area in China, harvested as early as 1980 until now, have been performed in order to perform a preliminary assessment of the gradual contamination of the autochthonous A. parthenogenetica populations by introduced A. franciscana. Provisional results show that from ± 1991 onwards, all available samples are at least predominantly bisexual, with percentages of males fluctuating between 31 and 50 %. Before 1991, ‘typical’ male percentages in the order 0.1-0.3 % are observed. As especially older samples (1980’s and early 90’s) often show no hatchability anymore, these data will be complemented and refined by identification of the species status of individual cysts, according to the authentication method developed by Bossier et al.

INCO partner: 1 & 2

Polymorphism in Artemia species as revealed with mitochondrial and nuclear markers

Daan Delbare1, Peter Bossier1, Stefania Dooms 1,2, Gilbert Van Stappen2, Patrick Sorgeloos2, Zhijun Qiu3, Thomas H. MacRae3

1 Agricultural Research Center-Ghent/Department of Sea Fisheries (CLO-DVZ), Ankerstraat 1, 8400 Oostende, Belgium

2 Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent University, Rozier 44, B-9000 Gent, Belgium

3 Department of Biology, Dalhousie University, Halifax, N.S., Canada

As reported in previous meetings a database of RFLP patterns of a 1500 bp mitochondrial rDNA fragment has been constructed. This database contains now RFLP patterns (using 8 restriction enzymes)on more than 100 samples. A subset of these samples is now used as reference database for authenticating unknown samples at the species level. The 1500 bp mitochondrial rDNA fragment can be amplified from either a single cyst or a small amount of cysts. Detailed analysis of this reference database revealed that the RFLP patterns of one single restriction enzyme (HpaII) is sufficient to differentiate between the species present in the reference database. Samples grouping to certain species were characterised by the presence of unique marker fragments. The HpaII restriction patterns displayed two fragments, namely 1200 and 240 bp, that were present in all strains from the “franciscana” group and absent in all other samples, making them putative markers for that group. In addition all Artemia samples originating from North America displayed an unique 541 bp HinfI fragment. Other groups also displayed unique restriction fragments: samples from the “sinica” group originating from China showed unique 1486 bp TaqI and 464 bp Hinf I fragments. The 317 bp HinfI, the 273 DdeI and the 205 bp HaeIII fragments were characteristic for the “salina” group. Finally the “parthenogenetic” group was characterised by the presence of a typical 297 bp HpaII fragment. A single A. persimilis sample is characterised by two HpaII (656 and 611 bp) and two DdeI fragments (453 and 421 bp) that were unique for this sample. It remains to be established if these fragments are real markers for this group, through the analysis of other A. persimilis populations (collaboration with partner 14, Gonzalo Gajardo, Chili seems to confirm this). These markers and the typical HpaII restriction patterns can now be used as an easy tool to identify Artemia at the species level. As it is possible to perform the analysis on a single cyst, it becomes feasible to study the populations dynamics in cases of syntopic occurrence of bisexual and parthenogenetic species.

The use of mitochondrial markers is in a considerable amount of samples hampered by the occurrence of “double” restriction patterns. These double restriction patterns are characterised by a restriction banding pattern of which the total sum of the visualised bands sizes is bigger than the 1500bp. Sufficient evidence has been gathered that shows that the observed phenomenon is not the consequence of a partial restriction digest. The phenomenon even occurs in mitochondrial DNA amplified from a single individual. The 1500 bp rDNA fragment as well as a smaller cyt B fragment is affected by this phenomenon. Recent analysis on parthenogenetic individuals from Urmia lake and surroundings (collaboration with partner 11) has also in these samples confirmed this phenomenon. It is suspected that this is the result of either heteroplasmy or pseudogenes.

The Hsp26 gene can be amplified from cysts by RT-PCR using a primer couple developed in the lab of Tom MacRae. Polymorphism was detected by RFLP (see previous report). The PCR fragments have now been sequenced at the lab of Tom MacRae. Part of that sequence is shown below. The genetic distance between these sequences will be shown at the meeting. Of particular interest is the polymorphism that was found at one particular nucleotide (marked in yellow in the figure), differentiating Artemia franciscana sampled in North America and in Vietnam (Vinh Chau). As the latter are more thermotolerant it is now investigated whether this difference is a marker for the Vinh Chau strain, by verifying much more individuals. In addition at the lab of Tom MacRae the genomic HSP26 sequence has been established. This opens the possibility to amplify a fragment containing the putative polymorphic nucleotide starting from a single individual or cyst (a protocol for doing RT-PCR on a single cyst has not been developed so far)

INCO partner: 3 (I)

Preliminary data on intraspecific genetic divergence between Artemia franciscana, San Francisco Bay and inoculated populations in Vietnam

Ilias Kappas1, Theodore J. Abatzopoulos1, Nguyen Van Hoa2, Patrick Sorgeloos3 & John A. Beardmore4

1 Department of Genetics, Development & Molecular Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

2 Institute of Marine Aquaculture, University of Can Tho,Vietnam

3 Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, Ghent B-9000, Belgium

4 School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, Wales, UK

The main objective in this study was to investigate the microevolutionary changes that have taken place since the introduction of Artemia franciscana (SFB) into Vietnam. Temperature is a key factor, expected to have a bearing on the genetic architecture of Artemia populations in the area of Vinh Chau saltworks, Vietnam. Therefore, the hypothesis in question dealt with the response of the source population (A. franciscana, SFB) to a novel environment. In addition, the speed of this response was tested through comparisons of early and late Vietnamese Artemia samples.

The methodological approach consisted of the examination of genetic differences at three different levels. A number of reproductive characters were tested for intrapopulation differentiation at 26°C and 30°C. Also, a battery of allozyme loci, routinely employed in Artemia population genetics, were screened for variation within, and differentiation between, Artemia populations from Vietnam. Finally, the same populations were assayed by way of Restriction Fragment Length Polymorphism (RFLP) analysis of a region of the mitochondrial (mt) DNA.

Materials and Methods

The samples of A. franciscana used in this study consisted of A. franciscana, San Francisco Bay (SFB) considered as the inoculum (source) population, the Vinh Chau (VC) strain, present in Vinh Chau saltfields since inoculation (1986) and strains of Year 1 (Y1), Year 3 (Y3) and Year 4 (Y4), originated after one, three and four culture seasons in Vinh Chau, respectively.

Strain Y2 (after two culture seasons in Vinh Chau) was also used initially but it was subsequently dropped from the study due to crash.

Reproductive characters

Cyst samples of each strain were hatched (according to Sorgeloos et al., 1986) and young nauplii were transferred to cylindroconical tubes until they reached maturity. Mating pairs (in Falcon tubes) for each strain were set up in a salinity of 80ppt and two temperatures (26°C and 30°C). Five reproductive characters were daily recorded: number of cysts per female, number of nauplii per female, total number of offspring per female, number of broods per female and number of encycted broods per female. Data for each variable were analysed through a two-way ANOVA.

Allozymes

Twenty enzyme-coding loci were scored in all samples. The TFPGA-1.3 (Miller, 1997) software was used to calculate common measures of genetic variability and differentiation as well as to construct a UPGMA dendrogram of populations.

MtDNA

Following DNA extraction (Bardakci & Skibinski, 1994) samples were loaded on an automatic thermocycler for amplification of a 2963 bp long mtDNA target sequence. Eight restriction endonucleases were employed to assess variation in the amplified region. Two computer software, REAP, version 4.0 (McElroy et al., 1992) and NTSYS®, version 1.2 (Applied Biostatistics, Inc.) were used to obtain mtDNA variability indices and dendrograms.

Results and Discussion

There is ample evidence of divergence in reproductive traits between the source A. franciscana, SFB population and the founded ones in Vietnam as well as between the latter. Temperature appears to be invariably involved in the observed differentiation between samples, either as a single factor or interactively with respect to the particular strain. At the temperature of 30°C the VC strain showed significantly higher reproductive output compared to all other strains, which is indicative of its thermal adaptation.

Similar differentiation to that observed with reproductive characters was evident in the allozymic survey. The source population and the Vietnamese strains showed detectable genetic differences and patterns of genetic differentiation comparable to those found at an initial stage of divergence between geographic populations in nature. In addition, there was no evidence of genetic impoverishment through the successive generations of Vietnamese Artemia, a fact particularly important for the success of inoculation schemes.

Unlike allozymes, strong indications of reduction in mtDNA gene diversity were obtained in the Vinh Chau populations. In particular, the VC strain displayed the smallest number of haplotypes and the lowest level of haplotype diversity compared to the rest. It seems that a global composite haplotype is driven through time (year classes) to near fixation (VC). The application of new molecular markers and tools (see Abatzopoulos et al., 2002) has revealed patterns of evolution previously undetected and has boosted considerably fine-scale genetic investigations.

Overall, the “sequential” culture scheme used in Vinh Chau ponds favours the gradual accumulation of genetic adaptations. The different strains show considerable differentiation brought about by temperature. This is also supported by Clegg et al. (2000) who found a similar pattern in the thermal adaptation of cysts samples derived form the same strains.

References

Abatzopoulos Th.J, Beardmore JA, Clegg JS, & Sorgeloos P. 2002. Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht.

Bardakci F, & Skibinski DOF. 1994. Application of the RAPD technique in tilapia fish – species and subspecies identification. Heredity 73 (2): 117-123.

Clegg JS, Jackson SA, Hoa NV, & Sorgeloos P. 2000. Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana, from San Francisco Bay and southern Vietnam. Journal of Experimental Marine Biology and Ecology 252: 85-96.

McElroy D, Moran P, Bermingham E, & Kornfield I. 1992. REAP – the restriction enzyme analysis package. Journal of Heredity 83: 157-158.

Miller MP. 1997. TFPGA version 1.3, Department of Biological Sciences, Northern Arizona University, Box 5640, Flagstaff, AZ 86011-5640, USA.

Sorgeloos P, Lavens P, Léger P, Tackaert W, & Versichele D. 1986. Manual for the culture and use of brine shrimp Artemia in Aquaculture. State University of Ghent, Belgium.

INCO partner 3: (II)

How do mitochondrially identical Artemia clones respond to different salinities?

G. Deliopoulos, A.D. Baxevanis & T.J. Abatzopoulos

Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece

The brine shrimp Artemia consists of a number of sexual species and a large number of obligatory parthenogenetic strains. Polyploid parthenogens are characterized by apomixis (i.e. meiosis is totally suppressed), while diploid Artemia populations are characterized by automixis (i.e. capable of limited meiotic recombination); therefore, the former are considered monoclonal and the latter polyclonal (Barigozzi, 1974; Abatzopoulos et al. 2002, 2003).

The majority of the studies, that have estimated environmental and genetic components of variance for life span and reproductive traits of Artemia, have focused on temperature and salinity; these are the most important abiotic factors affecting the life history of hypersaline organisms (Barata et al. 1996; Browne and Wanigasekera, 2000; Abatzopoulos et al. 2003).

Μonoclonal populations produced by apomictic parthenogenetic Artemia (i.e. lineages derived from the same mother) could be used successfully for studying phenotypic traits (such as life span and reproductive characteristics), although this approach has been poorly utilized until very recently (Browne et al. 2002; Abatzopoulos et al. 2003).

In this study, we tried to document the phenotypic expressions (ten life history traits) of two genotypic lineages exposed to variable levels of salinity; these lineages came from the same parthenogenetic population (but from different females) and they were identical according to RFLP-mtDNA analysis.

Materials and Methods

Two Artemia clones were isolated from the parthenogenetic tetraploid population of Polychnitos saltworks (Lesbos island, Aegean Sea, Greece – Triantaphyllidis et al. 1993).

Culture conditions

Stock culture of Polychnitos population (1 individual / 4ml) was kept in 1L cylindroconical glass jars maintained at 24oC. The salinity of the culture medium was 60 g l-1. Animals were fed with 75% of yeast-based diet LANSY-PZ (INVE Aquaculture NV, Belgium) and 25% Dunaliella tertiolecta according to Abatzopoulos et al. (2003).

Experimental design

Twenty five females were isolated in equal of 50 ml cylindroconical tubes before they reached adulthood. After their first brood, five nauplii from each individual were sacrificed for DNA extraction (see mtDNA-RFLP analysis section). Extracted DNA was used for RFLP-mtDNA analysis; this technique revealed that there were two different clades of mtDNA in the same population (clade 1 and clade 2, respectively). Two individuals were chosen from clade 1 (A and B, respectively) for the continuation of the experiment. These two individuals produced clones which were used for recording ten life span and reproductive characteristics (total lifespan, pre-reproductive, reproductive and post-reproductive period, number of broods, days between broods, number of nauplii, number of encysted embryos, offspring per brood and offspring per reproductive day) under three different salinities (50, 100 and 150 g l-1; temperature was constant at 24±1oC). Reproductive and life span characteristics were analyzed by using non-parametric tests (Kruskal-Wallis), since the ANOVA assumptions were not fulfilled. The significance level was set to 0.05.

mtDNA-RFLP analysis

DNA was extracted using the CTAB protocol described in Hillis et al. (1996). Part of the 16S rRNA gene was amplified using the universal primers L2510 and H3080, described by Palumbi et al. (1996). PCR products were digested with 9 restriction endonucleases, electrophoretically separated in 1.5% agarose gel, stained with ethidium bromide, visualized and photographed under UV light.

Results and Discussion

Six reproductive and four life span characters from the parthenogenetic clones A and B at three different salinities are summarized in Table 1. Statistical analysis using Kruskal-Wallis test indicated that significant differences exist between clones in each salinity treatment for the majority of the characters studied (see Table 1). More specifically, at 50 g l-1 clone A and B were statistically different in eight out of ten characters studied; clone A presented statistically lower values than clone B in total life span, reproductive period, post reproductive period, number of broods, days between broods, number of nauplii, offspring per brood and offspring per reproductive day. It is worth noting that at 50 g l-1 both clones have produced no encysted embryos. At 100 g l-1, eight out of the ten scored characters were statistically different; clone A and B appeared to have similar values only in post-reproductive period and in number of encysted embryos; at the same salinity, clone A outperformed clone B. At 150 g l-1, the same characteristics were statistically different between the two clones but in this case clone B seemed to be the fittest.

In conclusion, the two investigated clones (A and B) appear to have different response to the elevation of salinity. There is no supporting evidence that clones, which appear to be identical in terms of mtDNA, should exhibit similar reproductive performance. However, mtDNA can be a useful tool when assessing verified clonal lineages (i.e. offspring produced by a single parthenogenetic apomictic female).

References

• Abatzopoulos T.J. et al., 2002. Artemia: Basic and Applied Biology, Kluwer Academic Publishers.

• Abatzopoulos T.J. et al., 2003. Hydrobiologia 492: 191-199.

• Barata C. et al., 1996. J. Exp. Mar. Biol. Ecol. 196: 329-340.

• Barigozzi C., 1974. Evol. Biol. 7: 221-252.

• Browne R.A. & G. Wanigasekera, 2000. J. Exp. Mar. Biol. Ecol. 244: 29-44.

• Browne R.A. et al., 2002. J. Exp. Mar. Biol. Ecol. 267: 107-119.

• Hillis D.M. et al., 1996. Molecular Systematics, Sinauer Associates.

• Palumbi S.R. et al., 1996. Simple Fool’s Guide to PCR, University of Hawai.

• Triantaphyllidis G.V. et al., 1993. Int. J. Salt Lake Res. 2: 59-68.

Table 1. Mean values (±S.D.) of various reproductive and life span characteristics for the two parthenogenetic clones A and B reared at 3 different salinities (temperature was 24±1oC). Significant differences were determined by Kruskal-Wallis test (p 90 %) prior to analysis.

Macau (MAC) was chosen as the site where Artemia franciscana was originally inoculated in RN in 1977 (Camara, 2001). Galinhos (GAL) and Areia Branca/Grossos (ABG) are equidistant (about 100 km) from Macau. In addition, Galinhos saltworks act as wintering grounds or passage areas for a diverse and abundant avifauna. Thus the possible role of migratory birds as contributors to gene flow within and between populations of Artemia in RN would be better estimated in Galinhos.

DNA was successfully extracted from 70 collected specimens using modified protocols for CTAB (Estoup et al., 1996) (65 extractions) and a simplified Chelex method (Tagart et al., 1992) (5 extractions) for total DNA isolation from Artemia. PCR amplification was carried out using an automatic thermocycler (Mastercycler EppendorfTM). The mtDNA target sequence was a segment of ca. 550 bp in length, which included a partial portion of the large subunit ribosomal RNA genes (16S rDNA) (Valverde et al., 1994).

Genetic diversity (variability) of experimental populations was assessed by Restriction Fragment Length Polymorphism (RFLP) analysis of PCR amplified mitochondrial DNA (mtDNA). Four restriction enzymes (BioLabs Inc.) were used. These enzymes have already been found to give species-specific haplotypes for Artemia franciscana, differentiate its southern or northern origin and indicate variation within populations of Artemia franciscana (unpublished data).

Results and discussion

Fragment patterns observed among Brazilian Artemia populations in part of the 16S RNA region showed a consistent homogeneity as only one single composite haplotype (AAAA) occurred in all individuals scored.

Unpublished data have already shown that Artemia franciscana presents the same composite haplotype (AAAA) found in the present study. Thus, in spite of the relatively conservative region of the mtDNA studied and the limited number of restriction enzymes used, the RFLP data obtained in this preliminary characterization confirmed that the feral populations of Artemia franciscana found in the state of Rio Grande do Norte, northeastern Brazil, belong to the Artemia franciscana superspecies. In addition, their proposed origin, from San Francisco Bay cysts, was clearly demonstrated. This second information corroborates a previous report by Gajardo et al. (1995) based on allozyme evidence derived from the Macau population.

References

Camara, M. R., 2001. Dispersal of Artemia franciscana Kellogg (Crustacea; Anostraca) populations in the coastal saltworks of Rio Grande do Norte, northeastern Brazil. Hydrobiologia 466: 145-148.

Estoup, A. C. R. Largiadièr, E. Perrot & D. Chourrout, 1996. Rapid one-tube DNA extraction for reliable PCR detection of fish polymorphic markers and transgenes. Molecular Marine Biology and Biotechnology 5(4): 295-298.

Gajardo, G., M. Da Conceicao, L. Weber & J. A. Beardmore, 1995. Genetic variability and interpopulational differences in Artemia strains from South America. Hydrobiologia 302: 21-29.

Taggart, J. B., R. A. Hynes, P. A. Prodöhl & A. Ferguson, 1992. A simplified protocol for routine total DNA isolation from salmonid fishes. Journal of Fish Biology 40: 963-965.

Valverde, J.R., B. Batuecas, C. Moratilla, R. Marco & R. Garesse, 1994. The complete mitochondrial DNA sequence of the crustacean Artemia franciscana. Journal of Molecular Evolution 39, 400-408.

INCO partner: 14

preliminary ANALYSIS of DNA sequences of the cytochrome c oxidase subunit I (COI) in chilean artemia populations.

Patricia Beristain 1,2, Stephan M. Funk 2 & Gonzalo Gajardo 1

1. Laboratory of Genetics & Aquaculture, Universidad de Los Lagos, Osorno, Chile.

2. Zoological Society of London, Institute of Zoology, London.

According to possibilities we have considered different levels of analysis, from morphology to DNA, to understand the origin and evolutionary pattern of Artemia species from Chile and adjacent countries in the South American continent (Gajardo & Beardmore, 2001; Gajardo et al, 1995, 1998, 1999, 2000, 2001, 2002). Focus has been placed in the two New World sibling species, A. franciscana and A. persimilis, found in Chile and Argentina, both representing interesting models to evaluate the pattern of intraspecific genetic variation and differentiation as well as genetic differences at the species level. While these kind of multi-level studies are good to documenting Artemia biodiversity in the continent, the availability of a wide range of genetic markers facilitates the characterization, exploitation, management and conservation of valuable local genetic resources.

The finding of A. persimilis in southern Chile, geographically segregated from A. franciscana, which is the dominant species in both the country and the continent, has raised interesting questions that we have investigated by using different approaches. Particularly, the genetic structure of A. franciscana has been studied throughout the species range, confronting data produced at different levels of analysis. This report considers preliminary findings of a study aimed at sequencing and comparing a 680bp fragment of the cytochrome C Oxydase subunit I (COI) in the Chilean Artemia populations.

Six Artemia samples from La Rinconada (RIN), Salar Llamara (LLA), Convento (CON), Pichilemu (PIC), Laguna Amarga (LAM) and Laguna de Los Cisnes (CIS), were considered as well as reference samples of A. franciscana (San Francisco Bay, SFB, USA; Macau, MAC, Brazil) and A. persimilis (ARC code 1321) (kindly provided as cysts by the Artemia Reference Center, ARC, Ghent). Adult female of each population (n=20) were fixed in absolute ethanol (99.9 %) until required for analysis.

DNA extraction

DNA extraction from each individual followed the protocol of QIAampTM tissue extraction kit (Qiagen), whilst amplification of the 680-bp fragment of the mitochondrial (mt) cytochrome c oxidase subunit I was carried out in a reaction volume of 25( containing 2.5 mM Mg+2, 0.2 (M of each dNTP’s, 0.2(g of Bovine Serum Albumin (BSA), 0.25 unit of BiotaqTM polymerase, 1X of 10X PCR Buffer (QIAgen) and 0.25(M of each primer: LCOI490 (5’-GGT-CAA-CCA-ATC-ATA-AAG-ATA-TTG-G-3’) and HCO2198 (5’TAA-ACT-TCA-GGG-TGA-CCA-AAA-AAT-CA-3’) (Folmer et al., 1994).

Sequencing the mtDNA cytochrome c oxidase subunit I of Artemia

The purified PCR products of each population (n=10) were subjected to sequencing reaction containing 0.67(l ABI BigDye terminator v3.1, 3.3(l Better Buffer, 2 (l of LCOI490 or HCO2198, 2(l of template adjusting the sample with double distillated water to a final volume to 10(l. The thermal regime for sequencing consisted of 1cycle of 3 min at 96(C and 30 cycles of 15 sec at 96(C, 10 sec at 50(C and 4 min at 60(C. The DNA was precipitated with ethanol and the pellet were diluted in deionized formamide and loading buffer. The diluted samples were denaturated at 95(C during 1.5 min and loaded on a polyacrylamida gel. The gel matrix was prepared using 12.6 gr urea, 3.5(l longer ranger, 3.5(l 1XTBE and 18.2(l double distillated water. To gel solution, 180(l of APS and 18(l of TEMED were added. The products were separated electrophoretically using an ABI PRISM ™377 DNA sequencer (Perkin Elmer).

Results and discussion

Fig. 1 shows gels with the identification of the 680 pb COI fragment in the Chilean and the reference Artemia samples considered, whilst Fig. 2 shows the sequence of a partial fragment in an individual from Convento, a coastal lagoone in Central Chile. The statistical analysis of the full sequence in all samples will be produced shortly, and these results will complement those done in collaboration with the ARC (Dr. Peter Bossier).

Figure 1: PCR of Artemia samples

Cloning of Artemia samples from Pichilemu

Since not all individuals from Pichilemu showed a clear sequence pattern on the 680 bp fragment, PCR products of this sample sample were inserted into the bacterial cells by using TA Cloning® kit (Invitogen) and replicated. After several cycles of ligation/transformation the transformed cells containing the inserted DNA sequence were selected (Fig. 2)

Mitochondrial DNA is a valuable marker to indicate maternal gene flow since the somewhat confounding effect of recombination is avoided (Avise, 1994). Hence, the level of variability between samples depends on their degree of genetic isolation, and this turns out to be a powerful tool when closely related (sibling) species like A. franciscana and A. persimilis are compared. As noted by Bossier (partner 1, 3) and also in aphids (Sunnucks et al., 1996), a confounding factor in the use of mt DNA is the presence of multiple copies of certain mtDNA genes, which is reflected in a double-banding pattern, or as an unexpected sequence pattern. The fact that the sample from Pichilemu exhibited a weird pattern needs further attention, but also confirms our previous findings showing this is a key location that seems to set be a natural barrier for the southward distribution of A, franciscana and A. persimilis nortward. The use of new genetic markers will contribute to further investigate this transitional zone, which was among the problems highlighted in the China workshop.

FIGURE 2: COI SEQUENCE FRAGMENT OF 500BP OF ARTEMIA FROM CONVENTO

References

• Avise, J.C. (1994) Molecular markers, natural history and evolution. New York, Chapman & Hall.

• Cox, A. and Hebert, P. 2001. Colonization, extinction and phylogeographic patterning in a freshwater crustacean. Molecular ecology 10: 371-386.

• Folmer, O., Black, M., Hoeh, W., Lutz, R. and Vrijenhoek, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294-299.

• Lavens, P. and Sorgeloos, P. 1996. Manual on the Production and Use of Live Food for aquaculture. FAO Fisheries Technical Paper 361.

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Pichilemu 1-8 SFB 1-8

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