INCO Concerted Action - Universiteit Gent
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1. Status of realization of deliverables
1.1. Deliverable 3: Participation to 2nd Regional Workshop (Puerto Varas, Chile, November 16-20, 2003)
At the occasion of the 2nd Regional Workshop, the RUG staff (Gilbert Van Stappen) reported about the realizations of the respective action plans and regional partnerships, coordinated by RUG, as conceived at the INCO workshop in Ghent, February 2002, in an oral presentation, entitled “Present studies on biodiversity of Artemia populations in Northerm Asia” (authors: G. Van Stappen, B. Marden, L. Litvinenko, I. Mirabdullayev, I. Zholdasova, Xin Naihong, P. Bossier, P. Sorgeloos).
1.2. Deliverable 5: Training of visiting scientists
During a training stay in summer 2003 by Naser Agh (responsible scientist UU, INCO partner 11) preparation was made for a paper entitled “Biogeography of Artemia in Iran” (authors: Agh, N., Van Stappen, G., Razavi Rouhani, S.M., Abatzopoulos, T. and Sorgeloos, P.), that has meanwhile been submitted to the Journal of Biogeography. In this study the authors report about the distribution of Artemia populations in Iran, their mode of reproduction, and provide ecological information about their biotopes. The effect of recent climatological fluctuations on the respective populations is equally discussed. Additionally, rDNA RFLP analyses were performed (see individual partner report CLO-DVZ, INCO partner 2) on a number of samples from Lake Urmia and adjacent sites.
2. Research on Artemia Biodiversity: action plans and partnerships; other tasks
2.1. Central Asian regional partnership and dissemination of INCO activities towards ‘associated’ INCO members
2.1.1. 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
2.1.2. Study of Artemia colonization of the Aral Sea (Uzbekistan)
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., USA.
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 the present research programme is to establish a team of biologists, limnologists, chemists, and natural resource experts from western 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.
2.2. Chinese regional partnership
2.2.1. 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. (2003).
2.2.2. Characterization of Artemia from Tibet-Qinghai plateau
Following the research work described in Year 1 Annual report RUG, the results on Artemia biodiversity on the Tibet-Qinghai plateau were orally presented at the Symposium on Aquatic Biodiversity: Past, Present, Future, Antwerp, Belgium, 11-13 August 2003, and published in the Symposium’s Proceedings:
Van Stappen, G., Sui, L., Xin, N., Sorgeloos, P. 2003. Characterisation of high-altitude Artemia populations from the Qinghai-Tibet Plateau, PR China. Hydrobiologia, 500: 179-192.
Abstract:
The brine shrimp Artemia was discovered in a number of saline lakes on the Qinghai-Tibet Plateau, widely diverging in chemical composition. Several lakes were athalassohaline, with relatively high amounts of trace elements. Common environmental factors are their high altitutde (exceeding 4500 m) and the low average annual temperatures. A number of Artemia populations in this area were analysed to assess their preference for low temperatures and an athalassohaline medium. Furthermore, their characteristics were compared with Artemia tibetiana, the species recently described for one lake in this area. All samples contained a variable mixture of parthenogenetic and bisexual individuals. A cross-breeding test of the sample from Jingyu Lake showed cross-fertility both with A. tibetiana and A. sinica. All populations showed similarities to A. tibetiana: a large cyst diameter and naupliar length, high HUFA content and a high tolerance to low temperatures, as compared to the control A. franciscana samples. These can thus be considered as recurrent characteristics of the populations from the high-altitude low-temperature environment on the Qinghai-Tibet Plateau, although further research is needed to identify their exact species status.
2.3. Characterization study of Egyptian Artemia
An ongoing study program (PhD student) in cooperation with INCO partner 3 (AUTH) focused of the characterization of Artemia strains from the Eastern Mediterranean, and more specifically on Egyptian strains (see Year 1 Annual Report RUG; see also Annual report of AUTH, INCO partner 3). The results were published in the PhD thesis by El Bermawi Nagy “Determination and identification of biological characteristics of Artemia populations from the Egyptian Nile delta for application in aquaculture”, defended at Ghent University, May 2003.
4. Other Artemia-related publications
Camargo, W.N., Ely, J.S., Sorgeloos, P. 2003. Morphometric characterization of thalassohaline Artemia franciscana populations from the Colombian Caribbean. Journal of Biogeography 30: 697-702
Dhont, J., Van Stappen, G. 2003. Biology, Tank Production and Nutritional Value of Artemia. 65-121. In: Live feeds in marine aquaculture. J.G. Støttrup, L.A. McEvoy (Eds). Blackwell Science Ltd. 318 pp.
Van Stappen, G..2003. Production, Harvest and Processing of Artemia from Natural Lakes. 122-144. In: Live feeds in marine aquaculture. J.G. Støttrup, L.A. McEvoy (Eds). Blackwell Science Ltd. 318 pp.
5. Creation of global Artemia database
RUG has proposed a template for the Artemia database. This database has been accepted by other partners. For each known Artemia site, the template provides the following data:
• Biological information on local Artemia strain
• Information on site: topography, ecology
• Available cyst samples (in resp. cyst banks of Consortium members)
• Literature references
2.6. Organisation of cyst bank
The existing cyst bank (online on INCO website) was further updated. INCO partners 6 (UBA) and 9 (RU) have transferred the data of their respective cyst banks to RUG, and the newest data have been integrated in the global cyst bank.
2.7. Updating of ISA publications
Newest ISA publications are added to existing literature database on INCO website.
2. Plans for coming year
• Genetical (rDNA RFLP) analysis of Artemia populations from Qinghai-Tibet Plateau
• Genetical (rDNA RFLP) analysis of Artemia populatons from Bohai Bay, China, to assess degree of contamination with introduced A. franciscana.
• Preparation of review article: Natural sources of brine shrimp Artemia in Western Siberia, Russia
• Further updating of cyst bank online
• Compilation of Artemia database for all Artemia sites known to occur in respective countries of INCO Artemia Biodiversity consortium.
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
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 in press.
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 neighboring 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.
Samples (adults) from Urmia lake and neighbouring salt lakes have been investigated again during the summer of 2003, during a short stay of Naser Agh (partner 11, UU). In this case the 1500 bp rDNA fragment was analysed with the HpaII enzyme. For more details, the reader is referred to his report. In summary it was found that the rDNA RFLP pattern of A. urmiana adults is similar to the patterns of parthenogenetic Artemia collected in Urmia lake or neighbouring salt ponds.
According to Abreu-Grobois & Beardmore (1991) the phylogenetic relationship of Artemia urmiana with parthenogenetic forms is suggestive of a recent single common ancestral lineage branching to both A. urmiana and the asexual forms. Browne et al (1991), on the other hand, suggested that parthenogenetic Artemia has evolved from Artemia urmiana. These findings corroborate with the mitochondrial DNA analysis. In the future it will be interesting to use, in addition to mitochondrial markers, nuclear markers to analyse individuals from Lake Urmia and its neighbourhood.
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.
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.
An examination of the available HpaII data (which is very indicative at the species level, see article in press) points in the same direction. Some lakes in Tibet seem to contain A. tibetiana, while other lakes are inhabited with parthenogenetic Artemia. Again some cyst samples seem to contain mixtures.
Also here it is advisable to analyse individuals (cysts or nauplii) and to combine the genetic data with other type of data. A detailed analysis of Tibet samples will be persued in the third year of the project.
Artemia in Latin America
The database of RFLP patterns was used to extract data (unpublished) on samples originating from America. This information was passed on the partner 14 (ULL) who used this info as input for writing a report on the current status on the genetic diversity of Artemia in Latin America.
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. This assumption could be tested in the lab of partner 3(AUTH). The reader is referred to their report for more details. In short a RFLP method was developed to distinguish the two HSP26 alleles, in such as way that the homozygote and heterozygote genotypes could be scored. It turned out that the allele frequencies in the SFB and Vinh Chau populations are not statistically different. So none of the two alleles can be used as a marker for SFB or VC individuals.
ACTIVITIES PLANNED IN the third YEAR
- Detailed analysis of the biodiversity of strains in salt lakes of Tibet
- A. franciscana as an invasive species in China
Collaborating scientists: A. Triantafyllidis, I. Kappas, A.D. Baxevanis, A. Tzika, G. Deliopoulos
During the course of the second year of the project two scientists visited the AUTH laboratory:
i) Prof. Marcos Camara from Universidade Federal do Rio Grande do Norte (INCO partner 13) from February 19th until May 10th, and
ii) Mrs. Nguyen Thi Hong Van from Can Tho University, Vietnam (INCO partner 7) from September 26th until December 19th.
The work of Prof. Marcos Camara involved the analysis of Artemia populations using molecular 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 choice of restriction enzymes that were used for RFLP analysis was based on the results gained during the first year of this project. All molecular work was done at the level of individual scoring.
More specifically, 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 and were presented in Puerto Varas (Chile) meeting.
The work of Mrs. Nguyen Thi Hong Van also involved training on molecular techniques. During her stay in AUTH the visiting scientist applied the following techniques: i) DNA extraction (CTAB-Phenol/Chloroform and Chelex protocols), ii) PCR amplification of a 294bp segment of the p26 gene iii) agarose gel electrophoresis and iv) Restriction Fragment Length Polymorphism (RFLP) analysis of amplified DNA and e) Single-Stranded Conformational Polymorphism (SSCP) analysis. RFLP data were analysed with Genepop ver. 3.4. All molecular work was also performed at the level of individuals.
The above methods aimed to discriminate between the A. franciscana San Francisco Bay (SFB) strain and that of Vietnam (VC). Preliminary results showed that the presence of a previously detected point mutation in the p26 targeted region (HinfI RFLP analysis) cannot safely discriminate between SFB and VC strains. RFLP and SSCP analyses have proven to be useful tools for identification of all three possible genotypes at the p26 region.
The visiting scientists during the course of their stay were also able to come into contact and discuss with scientists in the AUTH lab about several other techniques such as DNA sequencing, microsatellite genotyping, genetic identification of species using molecular markers etc. They were also able to take advantage of the internet and library facilities as well as the rich pre-existing bibliography concerning genetic structure of Artemia populations.
According to the conclusions and recommendations in Scientific Annual Report (Year 1, see section 3.4.2.) regarding the possible threats to Artemia biodiversity by the uncontrolled spreading of invasive Artemia species, AUTH (in collaboration with CSIC – INCO partner 4 and La Sapienza – INCO partner 5) undertook the screening of autochthonous species reported in the southwest of Spain. The aims were to
i) confirm the species status (suspected as A. franciscana) of El Pilar and El Estanquillo populations and
ii) find out if there are any differences in the genetic makeup of diploid and tetraploid parthenogenetic Artemia populations in the Spanish region
by using PCR-RFLP analyses. These analyses were continuation of previous work done by CSIC in terms of morphometry, reproductive characteristics etc.
A total of 23 individuals (males and females) from El Pilar and El Estanquillo populations were scored. Following DNA extraction, a 535 bp region of the mitochondrial 16S gene was successfully amplified by the PCR technique. Four restriction endonucleases were subsequently employed in order to digest the PCR product. These enzymes gave specific restriction profiles for the discrimination of A. franciscana, A. persimilis and A. salina. They can also distinguish South from North America A. franciscana. The RFLP analyses of both populations produced patterns typical of South America A. franciscana, which is in accordance with the CSIC characterisation.
The Odiel parthenogenetic Artemia population is a mixture of diploid and tetraploid individuals (based on CSIC results). Twenty individuals were scored for three enzymes and polymorphism was revealed for the same mitochondrial region. Individuals ascribed as diploid produced patterns different from those produced by individuals ascribed as tetraploid. This is a strong indication that the two groups belong to different strains which reinforces the initial characterisation by CSIC.
The Rocio population is comprised of bisexual A. salina and a diploid parthenogenetic strain. Twenty individuals (A. salina males and females as well as parthenogenetic females) were similarly scored for three restriction enzymes digesting the same mitochondrial region. These enzymes can discriminate A. salina from parthenogenetic strains. The individuals ascribed as A. salina gave the same patterns to those produced by a reference population of A. salina (Sfax, Tunisia). The rest individuals produced different patterns but identical to those of parthenogenetic diploid individuals from Odiel. The apparent similarity of Odiel and Rocio populations cannot be safely deduced before a greater number of restriction enzymes are employed.
As a conclusion, the RFLP analyses on the 16S mitochondrial DNA region support the characterisation of all populations performed by CSIC. Furthermore, the collaboration of AUTH and CSIC was in the framework of multidisciplinary approach for characterizing Artemia strains (as it was set at the kick-off meeting of the project in Ghent, Belgium. The results of this common effort were presented in Puerto Varas (Chile) meeting.
Our team also participated in the third regional INCO meeting that took place in Puerto Varas (Chile) with 5 presentations:
1. “2nd year progress report for INCO partner 3” by T.J. Abatzopoulos, A.D. Baxevanis, G. Deliopoulos, I. Kappas, A. Triantafyllidis & A. Tzika.
2. “Intraspecific genetic divergence between Artemia franciscana, San Francisco Bay and inoculated populations in Vietnam” by I. Kappas, T.J. Abatzopoulos, N.V. Hoa, P. Sorgeloos & J.A. Beardmore.
3. “The use of RFLP 16S rDNA analysis for detecting invasion of Artemia franciscana in Western Mediterranean region” by F. Amat, A.D. Baxevanis, A. Triantafyllidis, A. Tzika, G. Mura & T.J. Abatzopoulos.
4. “How do mitochondrially identical Artemia clones respond to different salinities?” by G. Deliopoulos, A.D. Baxevanis & T.J. Abatzopoulos.
5. “Preliminary characterization of Brazilian Artemia franciscana populations by means of mtDNA RFLP analysis” by M.R. Camara, A. Triantafyllidis, A.D. Baxevanis & T.J. Abatzopoulos.
Three Diploma Theses have been completed and defended:
1. A. Tzika, 2003. Genetic identification of Artemia species based on mtDNA-RFLP analysis, Diploma Thesis, AUTH.
2. E. Markatzinou, 2003. Detection of invasive species in Mediterranean Basin by using discriminant analysis, Diploma Thesis, AUTH.
3. G. Athanasiadis, 2003. The use of discriminant analysis based on morphometry: discriminating parthenogenetic Artemia populations in N. Aegean coastal saltworks, Diploma Thesis, AUTH.
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 492: 191-199, 2003
2. Title: “Elevated salinities may enhance the recovery of hydrated heat-shocked 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 133 (2): 103-109
3. Title: “Salinity effects on maturation, reproductive and life span characteristics of four Egyptian Artemia populations (International Study on Artemia. LXVIII)”
Authors: Baxevanis A.D., Ν. El-Bermawi, T.J. Abatzopoulos & P. Sorgeloos
Journal: Hydrobiologia (in press)
4. Title: “Species identification of Chilean Artemia populations based on mitochondrial DNA RFLP analysis”
Authors: Gajardo G., J. Crespo, A. Triantafyllidis, A. Tzika, A.D. Baxevanis, I. Kappas & T.J. Abatzopoulos
Journal: Journal of Biogeography (in press)
5. 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 (accepted)
▪ Presentations in congresses:
1. Title: “Characterization of two new Artemia parthenogenetic populations from Thrace (Greece): morphometrical analysis”
Authors: E. Markatzinou, G. Athanasiadis, A.D. Baxevanis & T.J. Abatzopoulos
Congress: 11th Panhellenic Congress of Ichthyologists, Preveza, 10-13 April 2003
Recommendations for future work:
▪ Further determination of potential molecular markers for characterizing Artemia populations.
▪ Urge partners to collect more information on diverse Artemia habitats focusing on Central and South East Asia.
▪ 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 2004:
▪ Scheduled visits by three INCO partners.
▪ Further investigation on MEDBAS Artemia populations focusing on invasion patterns (joined effort of AUTH, CSIC and La Sapienza).
▪ Defining mixed populations in S. Africa.
▪ Focusing on Asian Artemia populations.
According to the guidelines appointed during the Beijing and Puerto Varas workshops the activities of this group during year 2003, concerning the INCO A.C. priorities and previous research commitments, were focused on the following research tasks :
Tasks
1) The presence of brine shrimp Artemia franciscana in the Western Mediterranean as exotic invasive species and its dispersal by waterbirds
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 80´s until today. This availability, together with the prospection and recovering of brine shrimp cysts from waterbirds (Dunlin Calidris alpina and Godwit Limosa limosa) faeces and pellets in two areas in the Southwest of the Iberian peninsula: Cadiz Bay (Spain) and Castro Marim salterns (Portugal), enabled a research led to establish the specific and systematic adscription of these populations. The morphometric study of adult males and females, obtained from original cysts and nauplii under standard culture conditions, and the application of a multivariate discriminant analysis provided evidence on the present distribution of autochthonous brine shrimp populations as well as of foraneous A. franciscana populations. Autochthonous populations are 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. Cooccurrence 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 foraneous A. franciscana populations as intentional or non-intentional inoculations through salterns management, aquacultural (hatchery effluents) and 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). The RFLP analyses on the 16S mitochondrial DNA region supported the characterisation performed by partner 4 (CSIC). The combination of traditional (e.g. biometrics, morphometry, cross-breeding) and molecular (RFLP analysis) techniques seems to be a useful tool for studying invasion patterns and species status in the genus Artemia.
2) Distribution and characterisation of brine shrimp populations 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, located between 45º 55´and 49º 18´ S, were visited, and Artemia presence, as living populations and/or cysts, was registered in nine of them.
Collected cysts allowed to hatch living nauplii which were grown till adulthood under standard culture conditions.The morphometrics of adult males and females studied by multivariate discriminant analysis, provided evidence that all these populations belonged to the species A. persimils. In this moment the available information from Artemia distribution in Argentina allows to venture the presence of A. franciscana in 7 localities north to 36º S, and of A. persimilis in 25 localities south to 36º S. This A. persimilis distribution in Argentina can be linked to the presence of the same species in the localities of Laguna Amarga (Torres del Paine National Park) and Laguna de los Cisnes (Isla Grande) in the Chilean Patagonia and Tierra del Fuego, respectively.
This material is presently under study to establish cysts (hydrated and decapsulated) biometrics, chorion thickness and floatability, as well as nauplii biometrics. PUFA content from these cysts is being under research, aiming to compare PUFA profiles from original or autochthonous cysts and cysts obtained in the laboratory under specific and monitored culture conditions, in order to discern the influence of genotypic versus phenotypic (environmental) components in these PUFA contents.
Reproductive output or performance in terms of ovoviviparism and oviparism with regard to other mother life span characteristics: prereproductive and reproductive phases, number of broods and brood size, number of offspring per brood and per female, recovery time between broods, etc., bound to environmental conditions and laboratory culture conditions, are under study for different populations of both brine shrimp species present in Argentina. The fitness characterisation of these populations (species) must provide preliminary information on the fate of them and their present or future biogeographic distribution if they could have competed or could compete henceforth in circumstances of cooccurence or sympatrical presence or distribution.
3) Distribution and characterisation of 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 in collaboration with INCO partner 4 (CSIC).
Laboratory brine shrimp populations obtained after hatching El Adhibet, Sijoumi and Sahline autochthonous cysts 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 another 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 another Mediterranean populations.
4) Characterisation of some brine shrimp populations from South Eastern 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 data base developed by the group INCO partner 4 (CSIC).
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.
Plans for the coming year.
Task 1. To update the cyst bank and data base of Artemia cysts samples from the Western Mediterranean region (plus Portugal) through samples provided by the cooperation supported by Spanish-Italian and Spanish-Portuguese Integrated Actions approved during 2003. Possible introduction of new data involving North African cysts samples and populations, specially from Moroco.
To develop a systematic and taxonomic approach to Mediterranean Artemia populations through male pennis morphology (SEM) (Spain-Italy) and mtDNA RFLP analyses characterization (Spain-Italy-Greece). Collaborative research with INCO partners 3 (AUTH) and 5 (LA SAPIENZA).
Task 2. To accomplish the updating of the cyst bank and data base of Artemia cysts samples from Argentinean populations sampled in the Southern Argentinean provinces of Chubut, Santa Cruz and Tierra del Fuego, supported by the Spanish-Argentinean ICI-AECI project. Foreign Affairs and Education Spanish Ministeries (Cooperación Científica con Iberoamérica).
To develop life tables under laboratory and outdoor conditions for Argentinean Artemia populations (A. persimilis and A. franciscana) in order to understand competition phenomena that could contribute to explain the evolution of wild populations in the South American Cone. To study the parental effects on the encystment rates among different geographical populations of Artemia persimilis from Argentina.
Task 3. To study the diverse factors conditioning the behaviour, diapause, hatching, nutritional value, etc., of original and laboratory obtained cyst batches under known conditions.
Task 4. To study the levels of HSP70 stress protein in adult Artemia related to environmental conditions and subsequent changes in reproduction mode: oviparism versus ovoviviparism.
To develop diverse tasks related to the visit and exchange of collaborative scientists from INCO partners 10 (SRI) and 13 (UFRN).
During 2003 the following activities started at the beginning of the year, were in progress:
1-Cooperation among different expertises aimed at characterizing new strains.
Mongolia: a new strain was studied, in cooperation with Francisco Amat (CSIC, INCO partner 4), Theodore Abatzopoulos (Aristoteles University, AUTH, INCO partner 3) and Hidetoshi Naganawa (Adjunct professor Institute of the Earth's Crust, Russian Academy of Sciences).
SEM observations revealed a pattern similar to that observed for non-Mediterranean bisexual species (Figs.1 a, b).Morphometrics and genetics are needed to ascribe the new entity to a species.
[pic] [pic]
Basal penis (a) Detail of the apical rosette of the spine (b)
Chile: preliminary SEM morphology of the 4 Chilean populations hitherto examined within the frame of a study in collaboration with Gonzalo Gajardo (University of Chile Los Lagos, ULL, INCO partner 14), revealed differences at specific level (Figs. 2, a,b,c,), some of these showing a “persimilis pattern” (Fig. 2c) whereas others are closer to the franciscana pattern (Fig.2b). Increasing of the number of individuals examines will tell us more about variability, whereas examination of the evrsible penis will hopefully clarify the taxonomic position of these entities.
[pic][pic][pic]
(a) (b) (c)
Cooperation with Dr.Laura Torrentera (university of Arizona), and with Dr.Francisco Amat (CSIC, INCO Partner 4, in progress will provide a realistic pattern as regards the possibility to tell non–mediterranean species apart,on the basis of eversible penis and morphometrics.
2-Updating of Artemia distribution
Whereas cooperation with Arabian Emirates was unsuccessful, due to several problems, cooperation with Liubov Nagorskaja (Minsk Academy of Sciences) produced very detailed information about lakes and possible Artemia find-spots of former URSS, together with an updated reference list. (Table 1).
[pic]
Moreover for some of the sites in the above list samples were provided from old collections and examined by SEM analyses.
The results produced interesting data, in that at least three different taxa seem to occur: one of these is of the Artemia salina pattern (no spine on the basal penis), the other exhibit a –non Mediterranean patter (spine on the basal penis close to that observed in the other bisexual species), the third one apparently parthenogenetic. Moreover, some sites seem to host mixed populations depending on the season sampled.
The results of this study are being prepared for publication.
However, due to the interest of widening knowledge on that huge part of Eurasia, intensive sampling should be planned with adequate funding to perform it.
3- Search for allochthonous species
Repeated sampling in Sardinia sites and in a site in Sicily gave no results as to the eventual presence of Artemia franciscana, at least as far as SEM examination of frontal knobs and penis in the individuals sampled are concerned. Confirmation by morphometrics (Francisco Amat studies) is in progress.
4-SEM studies
As long as financial support will be available from other projects SEM studies will continue, at least to complete ongoing research (250 euro/hour is presently the cost!!!!!!)
a) COMPARATIVE ECOLOGICAL REQUIREMENTS AND PHYSIOLOGICAL TOLERANCES BETWEEN SEVERAL ARGENTINIAN POPULATIONS BELONGING TO Artemia persimilis (FROM SALINAS GRANDES DE HIDALGO, SGH AND COLORADA CHICA SALINE, CCH) AND A. franciscana (FROM MAR CHIQUITA LAGOON, MCH):
We have studied the effect of diverse saline treatments (diverse end salinities and diverse saline shocks applied in different periods of the life cycle (naupliar, postmetanaupliar, adult) in A. persimilis CCH (La Pampa Province) and in A. franciscana MCH (Córdoba Province).
Diffusion of the results:
- We have presented some of our results in the V Jornadas Nacionales de Ciencias del Mar. XIII Coloquio Argentino de Oceanografía (Mar del Plata, 8-12 December, 2003):
TITLE: “Efecto de shocks salinos en distintas etapas del ciclo ontogenético de dos poblaciones argentinas del crustáceo Artemia (Branchiopoda: Anostraca).”
AUTHORS: Carolina Arbasetti, Paula Sotelano, Julieta Goenaga, Rosa Graciela Cohen.
ABSTRACT
Los crustáceos del género Artemia son habitantes frecuentes de cuerpos de agua tálaso o atálasohalinos de todos los continentes, salvo la Antártida. En Argentina están citadas dos especies: A. persimilis y A. franciscana, que se distribuyen en todo el territorio. El gran interés mundial reciente en la búsqueda y caracterización de nuevas poblaciones se debe fundamentalmente a su importancia económica en Acuicultura, que reside en el uso de Artemia como presa viva para larvas de peces y crustáceos de interés comercial. Las poblaciones de Artemia son muy plásticas ante variaciones ambientales. Toleran fluctuaciones muy marcadas de salinidad que van desde 30 a 300 g. l-1, e incluso sobreviven en forma de quistes cuando su biotopo se deseca completamente por cristalización o desecación. También son muy tolerantes a las variaciones de temperatura y contenido de oxígeno. Cuando colonizan un nuevo ambiente, se adaptan fácilmente a las condiciones climáticas locales y en pocas generaciones pueden adquirir un morfotipo propio del lugar. Con el fin de abaratar los costos de producción, algunos países con una pisci- o camaronicultura desarrolladas y carentes de poblaciones autóctonas de Artemia , recurren a la inoculación artificial. A fin de introducir la especie y el morfotipo más indicados para cada región es esencial conocer la respuesta de cada población a diversas condiciones ambientales, sus límites e intervalos de tolerancia. Esta caracterización ecológica puede proveer también valiosa información para la comprensión de la distribución biogeográfica de las especies.
En el presente trabajo, se evaluó el efecto de diversos tratamientos salinos sobre ejemplares de A. persimilis (de Laguna Colorada Chica, La Pampa: CCH) y de A. franciscana (de Laguna Mar Chiquita, Córdoba: MCH). Los animales fueron sometidos a shocks salinos desde la salinidad de eclosión o de cría hasta 4 salinidades finales: 40, 90, 150 y 220 g. l-1, en distintas etapas del ciclo ontogenético(nauplius, post-metanauplius y adulto). Se determinó el efecto de estos tratamientos sobre: supervivencia hasta la madurez sexual, longevidad y edad de madurez sexual. En ambas poblaciones, los estadios iniciales del ciclo ontogenético mostraron una tolerancia mayor a todos los tratamientos salinos que los estadios medios y adulto. CCH resultó más tolerante a los shocks salinos que MCH en las etapas metanaupliar y adulta. La salinidad de 150 g.l-1 parece ser un límite crítico para la supervivencia de ambas poblaciones. MCH alcanzó la madurez sexual antes o al mismo tiempo que CCH en todos los tratamientos. A todas las salinidades estudiadas, MCH presentó mayor supervivencia que CCH.
We are preparing a manuscript with these results.
Stay of training of Julieta Goenaga in the Laboratory of the IATS (Castellón, España, INCO partner 4) since 10 January to 15 March, 2003. This research is part of her Thesis of Graduation* to be defended in next March-April (2004).
* Thesis for achieving the grade of “Licenciada en Ciencias Biológicas in FCEN, UBA”.
During her training 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 two Argentinian Artemia populations: A. persimilis SGH and A. franciscana MCh. Going on in the same way, we have evaluated in conditions of constant salinity (90 ‰), the effect of a cold 12 h duration shock (10 °C), applied in the naupliar, in the postmetanaupliar and in adult periods. We have measured the effect of the temperature shock in each period of development by means of the survival, size and reproductive performance of both implied populations. Also, we have determined the salinity of preference for each population in conditions of constant temperature (26°C ± 1°C).
These, together with our previous results, enable us to give some explanation to the biogeographic distributional pattern of A. persimilis and A. franciscana.
Diffusion of the results:
The results of some of the experiments mentioned above, has been presented in: V Jornadas Nacionales de Ciencias del Mar. XIII Coloquio Argentino de Oceanografía (Mar del Plata, 8-12 December, 2003):
TITLE: “Efectos combinados de salinidad y temperatura sobre la supervivencia y edad de madurez sexual en dos poblaciones argentinas del crustáceo Artemia.”
(Branchiopoda: Anostraca)
AUTHORS: Julieta Goenaga*, Germán Medina López**, Francisco Amat**, Rosa Graciela Cohen*.
* Argentinian team; ** Spanish team
ABSTRACT
El camarón de la salmuera, Artemia (Crustacea: Branchiopoda: Anostraca), es un habitante típico de cuerpos de agua hipersalinos (lagunas o lagos salados) y salinas continentales o costeras del mundo, excepto la Antártida. En nuestro país se han identificado dos especies: A. franciscana, ampliamente distribuida por todo el continente americano hasta el centro de Argentina y A. persimilis, de distribución restringida al centro y sur argentinos, y a algunas localizaciones no confirmadas de Chile. Distintas poblaciones de una misma especie de Artemia se desarrollan en ambientes con grandes diferencias en salinidad y temperatura, lo que refleja una gran capacidad de adaptación a estas variables ambientales. Esta plasticidad sería consecuencia de altos niveles de variabilidad genética inter e intra poblacional, que se manifestarían fenotípicamente como variadas respuestas fisiológicas ante los cambios ambientales. Dado que la distribución de los cuerpos de agua donde habitan estos animales es discontinua y el medio natural de dispersión es por quistes (embriones criptobióticos) transportados por el viento o aves acuáticas migratorias, el aislamiento geográfico de las poblaciones podrían haber conducido a que cada una de ellas desarrollara límites e intervalos de tolerancia diferentes y propios de cada ambiente. El objetivo de este trabajo fue evaluar experimentalmente el efecto de tratamientos con diversas combinaciones de salinidad y temperatura sobre dos poblaciones argentinas de Artemia: A. persimilis de Salinas Grandes de Hidalgo (La Pampa) y A. franciscana de laguna Mar Chiquita, MCH (Córdoba). Se criaron individuos de ambas poblaciones bajo condiciones combinadas de tres temperaturas (10(, 20( y 30( C) con cuatro salinidades (30, 60, 90, 120 g. l-1). Los efectos de los tratamientos fueron medidos en función de la supervivencia (S) y la edad de madurez sexual (EMS). La S en MCH siguió la tendencia S30( > S20( > S10( (p< 0.05). A partir de 60 g. l-1 y 20 / 30 (C, SSGH y SMCH no variaron significativamente entre los 60 y 120 g. l-1, aunque siempre SMCH resultó mayor a SSGH. A 30 g. l-1, a diferencia de la SMCH, la SSGH fue muy baja o nula para cualquier temperatura (salinidad critica). En cambio MCH fue más sensible que SGH a bajas temperaturas (10(C). Para ambas poblaciones la EMS disminuyó con el aumento de la temperatura.
THESIS OF GRADUATION to be defended in March-April 2004:
TITLE: “Efectos combinados de la salinidad y la temperatura sobre la supervivencia, variables reproductivas y tamaño de adultos, en dos poblaciones argentinas del crustáceo Artemia (Branchiopoda: Anostraca).”
Autor: J. Goenaga. Director: Dr. R.G.Cohen
We have planned a conjoint paper with the Spanish team during 2004 with our biogeographical conclusions.
b) CHARACTERIZATION OF A. persimilis FROM COLORADA CHICA LAGOON.
The morphological and reproductive characterization of this population was carried out. The morphological studies were performed by means of the observation of whole specimens of A persimilis CCH and A. franciscana MCH or part of them with stereoscopic and optic microscopes and with the help of thin histological sections (Haematoxylin-Eosin coloured), in order to emphasize the structural differences between both species, in both sexes. Also the morphometry was performed. The reproductive characterization included the experimental determination of the reproductive variables in diverse saline conditions (diverse end rearing salinities, gradually or suddenly achieved) and some behavioural observations.
Diffusion of the results:
- Ph D. THESIS
TITLE: “Caracterización morfológica y reproductiva de Artemia persimilis (Crustacea Branchiopoda: Anostraca) de la laguna Colorada Chica (provincia de La Pampa).”
AUTHOR: Ximena Inés Pastorino. Director: Dr. R.G.Cohen
- 11 August, 2003.
Deposited in Hemeroteca of the Library of the Facultad de Ciencias Exactas y Naturales, UBA.
Some of the results have been already published. We are ending a manuscript about the morphologic aspects.
c) NEW ARTEMIA RECORDS FROM ARGENTINA AND CYTOGENETIC SPECIFIC IDENTIFICATION OF SOME OF THESE NEW POPULATIONS.
During 2003 we have found in collaboration with Dr. Francisco Amat, nine new Artemia localizations in Argentina. We are working in their characterization.
We have performed the cytogenetic characterization of the following new recorded Argentinean populations: Pampa de las Salinas (San Luis Province), Salinas Grandes (NW Córdoba Province), La Antigua (La Rioja Province), Salitral de la Vidriera (Buenos Aires Province).
Diffusion of the results:
-We are just ending a paper with some of these results to submit it for publication.
TITLE: “Cytogenetic analysis of four Argentine populations of Artemia (Crustacea Branchiopoda: Anostraca).”
AUTHORS: Lipko P., Papeschi A. G. & Cohen R. G.
ABSTRACT
Artemia brine shrimps inhabit hypersaline ecosystems all over the world except Antarctica. In South America two bisexual diploid species are encountered: Artemia franciscana (2n=42) distributed along America and Artemia persimilis (2n=44) restricted to Argentina and one locality in South Chile. We cited A. franciscana for the first time in our country and our results suggested a possible hybridization in Las Tunas Lagoon (Córdoba province). In the present work four new Argentinean populations of Artemia have been cytogenetically analyzed by conventional staining; AgNOR, C, and DAPI banding): Salina La Antigua (La Rioja province), Salinas Grandes (Córdoba province), Pampa de las Salinas (San Luis province) and Salitral de la Vidriera (Buenos Aires province). The former three gave similar results compatible with A. franciscana (2n=42, n=21), while Salitral de la Vidriera would correspond to A. persimilis (2n=44). While in Pampa de las Salinas and Salinas Grandes many chromocenters C+/DAPI+ were observed, in Salitral de la Vidriera only one or two small chromocenters C+/DAPI+ were detected. With the present contribution A. franciscana is cited in three new Argentinean localities.
We have planned a conjoint paper with the Spanish team during 2004 recording the new localizations.
d) CHARACTERIZATION OF SOME Artemia franciscana POPULATIONS FROM THE CÓRDOBA PROVINCE.
We have performed in collaboration with the Spanish team directed by Dr. F. Amat the characterization of the populations of Mar Chiquita Lagoon, Las Tunas Lagoon and Salinas Grandes from Córdoba, by means of Morphology, Biometry of cysts and nauplii, Morphometry of adults, Cross-breedings and Lipid profiles.
Diffusion of the results:
These results have been submitted for publication to the Journal of Biogeography (2003).
TITLE: “Further evidence and characterization of A. franciscana (Kellog, 1906) populations in Argentina.”
AUTHORS: F. Amat*, R. G. Cohen**, F. Hontoria*, J. C. Navarro*.
*Spanish team; ** Argentinian team.
ABSTRACT: This paper reports the presence of Artemia franciscana (Kellog, 1906) in Mar Chiquita salt lake and in Las Tunas lagoon, as well as at a shallow lake in Salinas Grandes (province of Córdoba). The results endorse its morphometric assimilation to other American populations of this “super-species”, together with its morphometric differentiation from the species A. persimilis (Piccinelli and Prosdocimi, 1968). The more conspicuous population from Mar Chiquita shows cross fertility with A. franciscana original from San Francisco Bay (California-USA). Both populations from Mar Chiquita and Las Tunas show cross fertility between them and reproductive isolation with A. persimilis from Hidalgo (province of La Pampa), which up today is considered to be endemic or exclusive of hypersaline ecosystems in Argentina. The reproductive characteristics displayed by the population from Las Tunas lagoon provide an approximation to the existence of an occasional hybridization between A. franciscana and A. persimilis (Papeschi et al., 2000). Artemia cysts from Mar Chiquita and Las Tunas show a fatty acid profile rich in eicosapentaenoic acid (EPA) differing markedly from the fatty acid profiles found in A. persimilis cysts.
Key words: Artemia, A. franciscana, A. persimilis, biometry, fatty acids, cross-breeding, hybridization
e) ADJUSTMENT OF THE PROTOCOLS FOR THE GENOME IN SITU HYBRIDIZATION
(GISH) TECHNIQUE
We go on working 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.
Related with this topic we have had some conversations with the Greek team in Chile Workshop in order to evaluate the possibility to send some people for training in their laboratories aiming to help in giving an important additional support or some confirmation to our hypothesis by means of molecular RFLP techniques. We have thought in this possibility also during the present year but as consequence of the economical situation of our country, young researchers are emigrating from Argentina looking for better work conditions abroad. Then it is difficult to found people who once ended the training, ensure their continuity in the Project.
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 of an important candidate for Aquaculture in the coastline of the Mekong Delta, especially in Baclieu-Soctrang area where it was 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 therefore focus on the following:
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 that related to Artemia culture through training.
2. Research activities
To have better comprehension of environmental conditions for Artemia pond culture.
I. Research on the bio-control of algae bloom due to unwanted algal species by stocking Artemia at high densities
II. To study the effect of algal quality/quatitity on the growth and reproduction of Artemia
III. To study the factors (salinity, temperature, food availability, genetics…ect.) that effect Artemia cyst production during successive generations.
IV. To study thermo-tolerance of Artemia (heat –shock protein, fatty acid profiles…ect.).
V. Socio-economic analysis of Artemia culture in the studied areas.
VI. Investigation on of soil parameters predicting N, P released from the pond bottom to prevent the problem of algal bloom in Artemia ponds.
VII. Study on the relation between soil parameters, nutrient and algae growth.
VIII. Investigation and search for solution for clay turbidity in Artemia ponds.
IX. 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 maintaining with 6 staffs in Artemia group, our tasks are transferring new culture techniques, helping the farmers who are willing 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.
4. Training and education
• Two technical staffs in our group are continuing the second year with in-service classes of Baclieu community College to get their bachelor in aquaculture.
• One staff in our group finished the first year of the Master of Science in Aquaculture Program (2002-2004) in Ghent and continuing the second year.
• Ms. Nguyen Thi Hong Van (CAF-CTU) had trained on molecular technology in Aristotle University of Thessaloniki from 26th, September 2003 to 19th December, 2003 and a collaborative research on Vietnamese Artemia strain between AUTH and CTU had been conducted during her stay. (see also point 5)
5. Collaborative research: Comparison the polymorphism on heat shock protein P26 (HSP26) between two Artemia populations, SFB and Vinh Chau strain.
I. Introduction:
Artemia is not naturally occurring in the Mekong Delta (Vietnam). Artemia fransciscana (SFB) was inoculated in Vinh Chau (VC) in 1986 in a collaborative effort between the Lab of Aquaculture and Artemia Reference Center (Belgium) and CTU (Vietnam). Since that time the Artemia population has been the subject of intensive study. The adaptation of Artemia to the local abiotic conditions has been the focus of research in the recent years. It is clear that Artemia franciscana became adapted to the local conditions through an increased thermo-tolerance. Through a ill-characterised process the Vinh Chau strain has become adapted to the local abiotic conditions, and can now be considered to be a new strain.
Thermo-tolerance in Artemia cysts from SFB and Vinh Chau (VC) is different. Vinh Chau cysts (also hydrated cysts) are more thermo-tolerant than SFB cysts. The difference in thermo-tolerance in both types of cysts corroborates with a higher concentration of hsp26 protein in VC cysts. In this study, our interesting is looking for if there is a difference in polymorphism on HSP26 between SFB and VC strain.
II. Material and method
Genomic DNA of four populations: SFB2003 (SFB strain that inoculated in VC in 2003), VC 2002 (Vinh Chau strain that inoculated from the VC cysts harvested in 2002), SFB 1258 (the original strain that used to inoculate in 1986 in VC) and VC 1993 (inoculated from the VC cysts harvested in 1993) were used in this study. The DNA target sequence was a segment of 294 bp in length, which included the partial portions of the large subunit of HSP26 genes. Oligonucleotides primers were P26F3INTRON and P26REEXONN4. The PCR products then were used for RFLPs (Restriction fractment Length Polymorphism) with HinfI and reconfirming the results by SSCPs (Single Stranded Conformation Polymorphism)
III. Results
Result of the restriction of Hinf I at locus P26 is showed in the following table:
Table 1: The genotype based on restriction of Hinf I of P26 for 4 populations: VC2002, VC1993, SFB1258, and SFB2003. Percentage are presented in parentheses
|Population |Genotype | |
| |AA |AB |BB |Total |
|VC 2002 |0 (0%) |6 (20%) |24 (80%) |30 |
|VC 1993 |0 (0%) |6 (33.3%) |12 (66.7%) |18 |
|SFB 1258 |1 (3.8%) |5 (19.2%) |20 (76.9%) |26 |
|SFB 2003 |1 (2.4%) |14 (33.3%) |27 (64.3%) |42 |
The above data showed a different between genotypes but statistical analysis (Genepope version 3.4) did not prove that differences on allele frequency as well as in genic differentiation between populations at locus P26. (table 2 and 3)
Table 2: Allele frequency of locus P26 of l studied Artemia populations
|Population |Allele |Genes |
| |A |B | |
|VC 2002 |0.100 |0.900 |60 |
|VC 1993 |0.167 |0.833 |36 |
|SFB 1258 |0.115 |0.885 |52 |
|SFB 2003 |0.190 |0.810 |84 |
Table 3: Genic differentiation for each population pair at locus P26 of studied Artemia populations (Fisher’method)
|Population pair |Chi2 |df |P- value |
|VC 2002 & VC1993 |2.058 |2 |0.35729 |
|VC 2002 & SFB1258 |0.000 |2 |1.00000 |
|VC2002 & SFB2003 |3.606 |2 |0.16484 |
|VC1993 & SFB1258 |1.232 |2 |0.54011 |
|VC1993 & SFB2003 |0.000 |2 |1.00000 |
|SFB1258 & SFB2003 |2.157 |2 |0.34016 |
No difference was found on polymorphism of locus P26, it might be that the number of samples was not enough (due to time limit) or even more target sequences should be studied in order to find out a marker that will be used to distinguishing two populations.
IV. Conclusion and recommendation
Even though no significant difference on polymorphism was found on locus P26 the study really provided a helpful opportunity for Ms. Nguyen Thi Hong Van to be trained on molecular techniques that are using in population genetics such as PCR, RFLPs, SSCPs. Studied techniques will be applied in research when her backs to Vietnam.
The short study in a foreign laboratory also gave a chance to her to broaden my knowledge, exchanging work experiences with new colleagues, friends and a promising collaboration in the future research.
6. Other activities
Prof. Dr. Patrick Sorgeloos (Gent University) had visited our College on August 2003 and discussed on future research on Artemia.
The major activity of our group during the period January – December 2003 are summarised as below:
Realised:
Continuing the cysts collecting around Tunisian Salinas and salt marches, with site characterisation and seasonal concentrations of cysts. Organisation of the cyst collections.
Realising the first cooperative research program at the Instituto de Aquicultura de Torre de la Sal (CSIC, INCO partner 4) at Castellon de la Plana Spain during two month, during this scientific training are done : morphologic and morphometric studies for 3 Tunisian artemia cysts (Eladhibet, Sijoumi et Sahline), than cross breeding experience , the results are statistically tested (multivariate analysis) and compared with other north African and European cysts. Finally lipids and fatty acid analysis are realised for the local cysts sample.
Continuing research on morphology and characterisation of Tunisian Artemia, were two master degree are finalized, the first presentation will be done earlier February 2004.
Starting a new master degree study on genetic characterisation of Tunisian Artemia, and the effect of desiccation on biometrics of Artemia
Oral communication on Artemia biochemical characterisation, at the regional workshop on marine science Tripoli Libya.
Planned:
A training program at the Department of Genetics, Development & Molecular Biology at Aristotle University of Thessaloniki (Professor T.J. Abatzopoulos; AUTH, INCO partner 3) is under discussion and we plan to realise it earlier April 2004, some genetics analysis of Tunisian cysts are fore see.
Non realised:
Unfortunately and due to VISA difficulties our participation at Chile Workshop was not possible.
Researcher: Horst Kaiser
An update on research concerning Artemia distribution in Africa
As a follow-up to a collecting trip made in South Africa at the beginning of the year, we reviewed the existing literature on Artemia for the African continent. Subsequently we updated reviews on Artemia biogeography. Africa has both sexually reproducing and parthenogenetic populations of Artemia. There are more populations recorded for the Mediterranean region, but this may be the result of more research and collecting effort. Overall, we know of 121 records of Artemia populations on the continent; species identifications and descriptions are available for only 33 populations. An estimated 36% of the populations appear to be parthenogenetic and 64% are sexually reproducing species. At this stage only a tentative species list can be presented as much of the work done on African populations needs verification using molecular techniques. Thus, applying caution in regard to the precision of the data, and considering the small number of records available, it is estimated that about 49% of the populations are A. salina, 12% can be tentatively assigned to A. franciscana and 39% of the populations described appear to be parthenogenetic Artemia. Such information is preliminary and at best can provide an estimate that must be confirmed through more research.
It is estimated that about 37% of the African continent or 11.3 million km2 are potential Artemia habitat. Water quality has been recorded for relatively few pans, and although numbers are small there appears to be a trend for most pans being dominated by Na/Mg and Sulphate. Further surveys need to be done to include more pans.
Results from a sampling trip to 33 sites in South Africa and parts of Namibia lead to several suggestions for future work on African Artemia populations. Sampling should be done repeatedly throughout the year since there are distinct dry seasons and more pans than can be accessed within one trip. Travel distances are great and collaboration with managers of salt works would be beneficial. Funding will be solicited from different sources to allow for more collections in the near future.
Cysts that have been collected this year will be analysed at Aristotle University in Greece in June/July 2004. Research papers will be prepared next year, including a review of African populations and another on molecular markers for southern African Artemia.
1. Research activities
• Continuous investigation of the Artemia resource in inland salt lakes, PR China: the cyst samples from 2 Tibet salt lakes and 2 Inner Mongolia were collected, The
• Continuous study on Artemia resources of Bohai bay; PR China: strain characterization; population composition; nutrition profile among different season and location.
• Research on HUFA profile and metabolizing 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.
• One scientist Xin Naihong attended Chile workshop and had a oral presentation “Distribution and characterization of Artemia in Bohai bay”
2. Distribution and characterization of Artemia in Bohai Bay
The Bohai bay is an important solar salt production area for China, more than 200 saltworks are over there. The saltworks in Bohai Bay provide unique environment for the production of Artemia, they are characterized by a high primary productivity providing abundant food to sustain large population of Artemia. 600 tons of Artemia cysts and several ten thousand tons of Artemia biomass are produced every year.
Before early 90’s local Artemia species in the saltworks of Bohai bay were parthenogenetic. Since 1992 inoculation of A. franciscana were firstly taken in 4 saltworks of Bohai bay (Tanggu, Hangu, Luannan and Nanpu), A. franciscana strain were dominant in those saltworks in several years after inoculation, later on due to the using of exotic Artemia strains in the aquaculture around Bohai bay, the bisexual Artemia strains were further expanded to other saltworks of Bohai bay, field observation indicated that the dominant Artemia population in salt works of Bohai bay are bisexual strains. In this study we investigated the distribution of the Artemia resources in Bohai bay, 10 Artemia samples were collected from different saltworks. The hatching ability for original and treated cysts, hatching rate, cyst diameter, length of the nauplii and fatty acid profile were analyzed.
China
Nanpu Daqinghe
Tanggu Hangu
Dagang Haixing
Zhongjie Chengkou
Lubei Zhanhua
Figure 2-1. Distribution and sampling sites of Artemia in Bohai Bay, PR China
Table 2-1. The hatching ability and hatching rate of the Artemia cysts from different saltworks.
|Name |Abbreviation |Hatching ability (%) |Hatching rate (hrs) at 25oC |
| | |Original |Treated |T0 |T10 |T90 |Ts |
|Daqinghe |DQH |23.56(3.78 |85.78(3.68 |17 |19 |28 |9 |
|Nanpu |NP |32.34(5.67 |75.64(2.24 |16 |18 |28 |10 |
|Hangu |HG |47.66(3.89 |75.64(4.37 |17 |19 |27 |8 |
|Tanggu |TG |36.87(4.12 |78.56(3.42 |15 |18 |27 |9 |
|Dagang |DG |42.98(2.46 |86.65(2.78 |15 |17 |26 |9 |
|Zhongjie |ZJ |57.67(2.79 |82.78(3.46 |14 |16 |26 |10 |
|Haixing |HX |45.78(3.49 |83.56(2.65 |16 |19 |29 |10 |
|Chengkou |CK |56.35(5.64 |82.43(4.23 |16 |18 |29 |11 |
|Lubei |LB |67.65(5.60 |86.75(2.34 |17 |19 |29 |10 |
|Zhanhua |ZH |58.60(3.56 |88.60(3.20 |17 |19 |27 |8 |
Figure 2-2. Diameter of the Artemia cysts and length of the nauplii InstarI
[pic]
Figure 2-3. HUFA content of Artemia cysts from different saltworks
3. HUFA profile and metabolizing changes of Bohai Artemia strain during enrichment and starvation
The nutritional value of the Bohai Artemia cysts (from zhanhua, Shandong, PR China, named as BH) was evaluated in the experiment. A .franciscana from Great Salt Lake (GSL) was selected as a reference species. The profile of fatty acids in newly hatched nauplii, enriched nauplii at different temperature and enriching time, enriched nauplii at different starvation time and different temperature was analyzed. After 24hr’s enrichment, the content of (n-3) HUFA, especially DHA and EPA in BH Artemia was a higher than in GSL Artemia. During the following starvation of enriched nauplii, DHA and EPA in BH Artemia were more stable than GSL Artemia. BH Artemia displayed an advanced nutritional value for aquaculture.
Table 3-1 The profile of fatty acid of BH Artemia nauplii, GSL nauplii and enrichment emulsion
|Fatty acids (mg/g dw) |BH |GSL |Enrichment emulsion |
|16:0 |27.16 |16.55 |6.94 |
|16:1n7 |36.96 |13.10 |1.70 |
|18:0 |4.94 |4.19 |1.58 |
|18:1n9+n7 |40.43 |51.33 |12.34 |
|18:2n6 |7.63 |9.14 |6.78 |
|18:3n6 |1.77 |0.75 |0.54 |
|18:3n3 |0.97 |31.16 |2.13 |
|20:4n6 (AA) |4.47 |2.32 |1.53 |
|20:5n3 (EPA) |19.83 |4.97 |85.98 |
|22:6n3 (DHA) |00 |00 |125.28 |
|DHA/EPA |0 |0 |1.46 |
Table 3-2 The profile of fatty acids of GSL enriched nauplii on 12hr’s enrichment and 24hr’s enrichment at different temperature
| Time (hrs) |12hrs |24hrs |
| | | |
|Fatty acids | | |
| |22.5℃ |25℃ |28℃ |22.5℃ |25℃ |28℃ |
|16:00 |14.45 |14.23 |11.46 |14.76 |13.48 |11.16 |
|16:1n7 |10.23 |10.68 |8.32 |9.48 |9.26 |7.77 |
|18:00 |7.33 |4.70 |5.05 |6.48 |6.37 |4.68 |
|18:1n9+n7 |44.86 |53.78 |44.53 |44.91 |50.79 |43.31 |
|18:2n6 |6.57 |8.31 |6.35 |6.47 |7.23 |6.21 |
|18:3n6 |0.40 |0.42 |0.38 |0.40 |0.50 |0.42 |
|18:3n3 |23.15 |28.24 |22.48 |21.38 |24.81 |20.31 |
|20:4n6 |2.26 |2.50 |2.71 |2.29 |3.37 |7.81 |
|20:5n3 |5.20 |7.29 |8.45 |11.72 |18.90 |15.25 |
|22:6n3 |0.87 |2.87 |4.24 |5.96 |11.55 |8.26 |
Table 3-3 The profile of fatty acids of BH enriched nauplii on 12hr’s enrichment and 24hr’s enrichment at different temperature
| Time (hrs) |12hrs |24hrs |
| | | |
|Fatty acids | | |
| |22.5℃ |25℃ |28℃ |22.5℃ |25℃ |28 |
|16:00 |14.21 |15.04 |17.07 |12.40 |16.30 |13.93 |
|16:1n7 |22.54 |24.16 |26.07 |19.99 |25.83 |20.88 |
|18:00 |4.08 |4.55 |5.34 |4.13 |6.48 |6.18 |
|18:1n9+n7 |31.42 |33.82 |38.12 |31.03 |40.52 |36.80 |
|18:2n6 |4.93 |5.57 |6.25 |5.71 |6.72 |5.97 |
|18:3n6 |0.38 |0.47 |1.49 |0.42 |0.47 |0.39 |
|18:3n3 |23.77 |21.61 |22.05 |21.07 |20.60 |19.25 |
|20:4n6 |3.66 |3.99 |4.52 |3.74 |4.73 |14.49 |
|20:5n3 |21.79 |22.68 |28.06 |27.42 |37.99 |35.49 |
|22:6n3 |7.47 |7.78 |8.37 |12.48 |19.39 |15.04 |
Table 3-4 The profile of fatty acids of enriched GSL nauplii during starvation at different temperature
| Temp (℃) |0hrs |12℃ |22℃ |
| | | | |
|Fatty acids | | | |
| | |3hrs |6hrs |12hrs |3hrs |6hrs |12hrs |
|16:0 |12.31 |11.81 |11.55 |10.91 |11.03 |11.17 |11.59 |
|16:1n7 |8.59 |8.50 |8.21 |7.44 |8.32 |7.39 |7.55 |
|18:0 |2.90 |5.72 |5.82 |3.16 |5.39 |3.26 |3.08 |
|18:1n9+n7 |45.60 |45.85 |45.38 |43.28 |43.43 |44.32 |42.75 |
|18:2n6 |7.08 |6.97 |6.64 |6.29 |6.86 |7.42 |7.18 |
|18:3n6 |25.17 |22.64 |22.50 |21.18 |22.22 |22.64 |22.50 |
|18:3n3 |5.33 |4.84 |5.01 |4.00 |4.99 |4.12 |4.05 |
|20:4n6 |2.81 |2.68 |2.69 |2.77 |2.49 |2.23 |2.12 |
|20:5n3 |18.14 |16.17 |15.89 |15.60 |15.89 |16.01 |15.92 |
|22:6n3 |9.70 |7.49 |6.95 |5.88 |7.50 |7.25 |6.03 |
Table 3-5 The profile of fatty acids of enriched BH nauplii during starvation at different temperature
| Temp (℃) |0hrs |12℃ |22℃ |
| | | | |
|Fatty acids | | | |
| | |3hrs |6hrs |12hrs |3hrs |6hrs |12hrs |
|16:0 |15.08 |15.35 |15.49 |15.76 |15.63 |14.96 |15.23 |
|16:1n7 |19.10 |19.57 |19.81 |19.91 |20.46 |20.05 |18.83 |
|18:0 |5.15 |5.47 |5.58 |5.91 |5.51 |5.41 |6.02 |
|18:1n9+n7 |33.99 |35.00 |35.73 |36.29 |36.52 |35.59 |36.09 |
|18:2n6 |8.03 |7.67 |7.94 |7.66 |7.61 |7.73 |7.19 |
|18:3n6 |3.37 |3.41 |3.76 |3.55 |3.23 |3.41 |3.09 |
|18:3n3 |2.26 |2.44 |2.38 |2.43 |1.73 |2.00 |1.75 |
|20:4n6 |5.31 |5.70 |5.41 |6.41 |4.77 |5.46 |5.83 |
|20:5n3 |34.61 |38.46 |39.16 |39.09 |37.50 |37.00 |35.48 |
|22:6n3 |17.27 |16.30 |16.19 |15.35 |15.20 |12.87 |11.36 |
Activities of Artemia and Aquatic Animals research Center, Urmia University (Partner No. 11) in the framework of INCO-DEV Project on Artemia Biodiversity are summarized below:
- Study stays as visiting scientists for 1.5 months during Aug.-Sep. 2003 at ARC, Belgium:
Naser Agh and Farzaneh Noori visited ARC in the framework of the project and performed experiments on RFLP fingerprinting technique and enrichment strategies of the Artemia urmiana nauplii. Outcome of this study visits are as follows:
- Genetic differences of Artemia populations from the Lake Urmia region:
In this experiment the RFLP fingerprinting technique, applied on a 1500 bp mitochondrial rDNA fragment, was used to study the origin and similarity of Artemia populations from the Lake Urmia region and few other parthenogenetic strains from Iran. During this work adult bisexual and parthenogenetic females from the Lake Urmia and the lagoons at peripheries of the lake, were analysed. DNA was extracted from the whole body using Wizard® Genomic Purification Kit (Promegatm, mouse tail protocol). The PCR reactions of extracted DNA were performed using one pair of primers, namely 12S-SP (5’-CTAGGATTAGATACCCTA-3’) and 16S-SP (5’CCGGTCTGAACTCAGATC-3’) in a Hybrid PCR express (Labsystem TM, Belgium). The PCR products were then purified using and Wizard! ® PCR Preps Purification system (Promegatm ), followed by a confirmation of the presence of the PCR products, using agarose electrophoresis. In order to display the polymorphism in the mitochondrial rDNA gene, each sample was digested with 4 restriction enzymes (Hpa II, Taq I, Dde I & Nde II). The digested products were separated through an agarose gel (2.5%), stained with ethidium bromide or syber gold and photographed with a Polaroid film under an Ultra Violet transilluminator. The results indicate that the bisexual and parthenogenetic Artemia individuals from Urmia Lake and other sources from Iran display identical restriction patterns, which correspond with the typical restriction pattern of parthenogenetic strains. As a result of this close genetic relationships it is suggested that the Lake Urmia could be the probable source for expansion of parthenogenetic Artemia and that these asexual forms could be derived from A. urmiana as proposed earlier by Abreu-Grobois & Beardmore (1991 and Browne et al (1991).
- Enrichment strategies of Artemia urmiana nauplii:
Nauplii of Artemia urmiana was enriched with saturated fatty acids, HUFA and Vit. C and their value in Artemia were analysed. This experiment was designed to find out a suitable enrichment method for Artemia urmiana to improve its nutritional value with respect to fatty acids and Vit. C. and to set the best enrichment strategy for this purpose. The enriched animals in another experiment were used to feed the sturgeon larvae. This is especially important as the sturgeons are listed among the endangered species and therefore proper feeding of their larvae can help in faster growth, higher survival and higher resistance against extreme conditions. Results of this experiment proved that enrichment with HUFA + Vit. C always improves performance of sturgeon larvae in compare to those received formulated feed and decapsulated cyst.
Other research and study programs performed in the framework of the project in Iran:
- Salinity competition test with Artemia populations from the Lake Urmia.
Our experiments based on competition tests at different salinities ranging from 15-80 g/l proved presence of both bisexual and parthenogenetic populations of Artemia in the Lake Urmia. The animals were compared on the basis of morphometeric and reproductive characteristics. Salinity based competition test was found to be a suitable and easy method for isolating parthenogenetic Artemia from bisexual species in a mixed cyst sample.
- Ecological assessment of the Lake Urmia
The drought condition which has caused severe increase in water salinity of the Lake Urmia continued during 2003 too. The water salinity decreased to about 255 ppt in south arm and to 280 ppt in the north arm during March – Auguest 2003. But it again increased gradually to about 290 ppt at both sides from September. Due to increase in salinity level, production of Artemia and the primary producers declined dramatically.
- Resource assessment of Artemia at Lake Urmia:
In a joint effort with the Iranian Organization for Environmental Protection, resource assessment of Artemia started from May 2003. During this project monthly sampling is programmed from different sites at both north and south arm of the lake. The sampling results indicates that there are average of 4500 cysts/m3 lake water at south and about 2500 cysts/m3 at north arm of the lake. Very little adult Artemia was observed during the sampling period and very little live biomass after October.
- Pond culture of Artemia urmiana and Artemia parthenogenetica at the vicinity of the Lake Urmia:
Due to a ban on exploitation of Artemia from Urmia lake and need of hatcheries to Artemia cyst, a project was carried out to evaluate the possibility of culturing Artemia urmiana and Artemia parthenogenetica at the vicinity of the Lake Urmia. Quarter hectare ponds were constructed for this purpose. Three ponds were used for culture of bisexual Artemia urmiana, three others for culture of local Artemia parthenogenetica and still 3 more ponds were used to culture unicellular algae. The project resulted in very good yield of cyst (25 kg/dw/h/m bisexual A. urmiana and 35 kg/dw/h/m A. parthenogenetica). Algae were produced in a continuous flow through system that resulted in a very dense production of D. tertiolecta (3.75 * 106/ml pond water). It was very interesting to observe the emergence of a dominating parthenogenetic population of Artemia in the ponds which where inoculated with nauplii hatched from Urmia Lake cysts just in previous season. This once again proves the mixed nature of the Artemia from the Lake Urmia.
- Collection of cyst samples for the Cyst Bank
As earlier years cyst samples were collected from the Lake Urmia and from the artificial ponds during this year too. The cyst samples are vacuum packed and preserved at -20°C.
Literature cited
- Abreu-Grobois, F. A. & Beardmore, J. A. (1991). Genetic characterization and intra-genetic relationships of Artemia monica and Artemia urmiana Günther. Hydrobiologia, 212: 151-168.
- Browne, R. A., Li, M., Wanigasekara, G., Simonek, S., Brownlee, D., Eiband, E. and Cowan, J. (1991). Ecological and genetic divergence of sexual and asexual (diploid and polyploidy) brine shrimp (Artemia). Advances in ecology,1, pp. 41-52.
Collaborating scientists: Jorge Castro Mejía, Aída Malpica Sánchez, Germán Castro Mejía & Ramón De Lara Andrade
The major activities related to biodiversity in Mexico during the period August-December 2003 are presented below:
Personal training
In this semester Jorge Castro Mejía is concluding the manuscript of his Master thesis about 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 the data of reproductive isolation. The title is “Morphometrical characterization of seven populations of Artemia franciscana in Mexico, in relation to its latitude, habitat, as well as its hybridization.”
In this semester Aída Malpica Sánchez is working with cysts, embryos and nauplii, to extract and to determine the protein content through electrophoretic profiles, in policrilamida gels under desnaturalized conditions for her doctoral thesis, which title is “The use of molecular and biochemical techniques in the characterization of mexican populations´of Artemia franciscana.”
During this semester, Germán Castro Mejía also finished his working in the incorporation of antibiotics (cloroanfenicol, Ciprofloxacina, Furadoine) in three stages of development of Artemia (metanaupliu, juvenile and adult) to inhibit the growth of the bacteria Aeromonas hydrophila and later on determine the necessary number of organisms that act against the bacteria. for his Master thesis which title is “Incorporation of antibiotics in Artemia franciscana, for the inhibition of the growth of Aeromonas hydrophila” and is writing the discussion of his thesis.
Cysts Bank
During this semester we updating our cysts database and sent the information to ARC
Biotope information
Also we sent to ARC and Gonzalo Gajardo, from Chile (Inco partner 14) the information about the Mexican Artemia biotopes.
Publications
During this second semester of 2003, one paper was published:
Title. “Life food in the acquaculture” Rev. Contenidos. No.48 2003 UAM
Authors: T. Castro B., R. de Lara A., G.Castro M., J. Castro M. &. A Malpica S.
Events
We presented the paper “Evaluation of crude protein in three stages of development of Artemia franciscana from different Mexican populations XXXIV National Congress of sciences and technology in foods. September, 2003 Pachuca, Hidalgo, México.
Another talk was presented in Puerto Varas, Chile in November 2003 in the Workshop of Artemia Biodiversity the title: “Morphological and biochemistry characterization of Mexican populations of Artemia franciscana as well as reproductive isolation and bioencapsulation of medications”
Consultancy
We continue advising in the Artemia culture in Las Salinas de Hidalgo, San Luis Potosí.
In the scope of INCO project Artemia biodiversity, Universidade Federal do Rio Grande do Norte (UFRN) has focussed on studying current Artemia practices and their implications for the sustainable exploitation and biodiversity of Artemia franciscana in the State of Rio Grande do Norte (RN), northeastern Brazil. Specific activities have dealt with the reproductive characterization of populations of Artemia franciscana in the coastal saltworks of RN. In addition, studies on Artemia reproductive biology, pond ecology, and aquaculture techniques have been carried out in the municipality of Grossos (RN), where a pilot farm for Artemia Research & Development has been designed and operated by UFRN in partnership with the Brazilian Shrimp Farmers Association (ABCC).
In the framework of the INCO project training courses, a study visit of Marcos R. Camara to the Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Greece (AUTH) was carried out in the period February-May 2003. His work focussed 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).
Information derived from the above mentioned activities have been discussed and presented to INCO partners during the Chile Regional Workshop (Puerto Varas, Chile) in accordance with the theme of the event (Artemia resources in Latin America).
Publications
Camara, M. R., 2003. Towards a sustainable Artemia industry in northeastern Brazil. In: Book of Abstracts, Aquaculture 2003, Salvador, Brazil. World Aquaculture Society, Baton Rouge, Louisiana, USA. p. 148.
Camara, M. R., A. Triantafyllidis, A. D. Baxevanis & T. J. Abatzopoulos, 2003. Preliminary characterization of Brazilian Artemia franciscana populations by means of mtDNA RFLP analysis. In: Book of Abstracts, Artemia Biodiversity, Chile Regional Artemia Workshop, Puerto Varas, Chile.
Oliveira, C. P. R., M. R. Camara & L. G. Reis. 2003. Utilização de náuplios de Artemia franciscana (Crustacea, Anostraca) no biomonitoramento aquático do litoral urbano da cidade de Natal (RN). In: Resumos, XIV Congresso de Iniciação Científica da UFRN, XIV Congresso de Iniciação Científica da UFRN, Natal, 2003, vol. 1, Natal: UFRN.
Reis, L. G., M. R. Camara & C. P. R. Oliveira. 2003. Monitoramento do estado reprodutivo, modo de reprodução e produtividade de cistos e biomassa de Artemia franciscana (Crustacea, Anostraca) em sistema de cultivo multifásico. In: Resumos, XIV Congresso de Iniciação Científica da UFRN, XIV Congresso de Iniciação Científica da UFRN, Natal, 2003, vol. 1, Natal: UFRN.
This report contains the major achievements of LGA for the period 2002-2003, and these mainly refer to: i) results produced as a by-product of the training activities described in previous reports;
ii) ongoing research efforts aimed at describing, at different level of resolution (from morphology to DNA), Chilean Artemia biodiversity, and iii) the organization of the Chile regional workshop.
i) Results
A manuscript accepted for publication in the Journal of Biogeography and a Master thesis in zoology are two evident accomplishments with regard to results and training. Both are based on the work Mr. Julio Crespo did as part of a 2-month training programme in Thessaloniki, Greece (partner 3). The Master thesis is due to be defended on February 24th, 2004 (University of Concepcion, VIII region, Chile) (cover included).
ii) Ongoing research
The application of new techniques to the Chilean and other Artemia samples has raised new questions and brought about chances for further north-south (Europe-DC countries) and DC-DC collaboration in relation to various aspects. Previous and new sampling campaigns provided 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 (and others) was sent, as agreed in China, to the Italian partner (Dr. Graziella Mura) for scanning microscope analysis of taxonomically relevant sexual traits (frontal knobs). After preliminary observations of few individuals, Dr. Mura has received a complete new set of animals from the main Chilean Artemia localities for population analysis.
The Pichilemu and the equivalent Argentinean location (Salitral de la Vidriera) have been identified as key latitudes for the north-south distribution of A. franciscana and A. persimilis in both countries. Consequently, the need to devote special attention to them has been on the table for discussion since the China workshop. Collaboration between the Chilean and Argentinean partners (Dr. Graciella Cohen) should be encouraged (see workshop recommendations).
All samples collected in the period have been subjected to the usual morphological, karyological, and allozyme analysis, as well as to the DNA level (RAPD’s) (results being processed). The LGA team has extended efforts to have new molecular tools available to studying the characteristic of Chilean Artemia populations and their relationship to reference samples of A. franciscana and A. persimilis, which are the two species found in Chile and the Americas. Such study benefited from a two months stay of miss Patricia Beristain (formerly trained in Ghent/Oostende in DNA analysis within the training-exchange programme of the Artemia project) in the Zoological Society of London. There she had the chance to identify, amplify and sequence a 680bp fragment of the Citochrome C subunit I (COI) from six Chilean samples (La Rinconada, Salar Llamara, Convento, Pichilemu, Laguna Amarga and Laguna de Los Cisnes), and reference samples of A. franciscana (San Francisco Bay, Macau, Brazil) and A. persimilis (Buenos Aires, Argentina). For this, multi-copies of Citochrome C subunit were obtained after PCR amplification using LCOI490 and HCO2198 primers and the 680bp fragments obtained from all samples were identified, assembled, sequenced and are being compared using the Sequencher 4.1.4 software. Preliminary findings were reported at the Chile workshp (see abstract partner 14, book of abstract Chile Regional Workshop).
Furthermore, within the field of Artemia Biodiversity Patricia Beristain, Artemia expert at ULL, spent 2 training visits to the Zoological Society of London, UK (14June-17 July, 2003; October 1-November 3, 2003), funded by the Darwin Initiative. The purpose of this visit was the training on the development and use of microsatellite markers for characterization of Artemia biodiversity. Presently data are being processed and will be published.
iii) Chile regional workshop
The Chile regional workshop took place in Puerto Varas, southern Chile, November 17-19, 2003. Two days were devoted, as in previous meetings, to presentations by each member of the consortium aimed at reporting their advances regarding project aims (see book of abstracts). The presentation were complemented by a conference given by Dr. Guillermo Chong, a professor of Geology from the Catholic University of the North (Antofagasta), on “the Saline domain of northern Chile”. The third day considered general discussions (e.g. reporting, joint publications, Artemia site database, cysts banks, and training in EU labs), recommendations and guidelines for future activities, which followed a special discussion on the status of Artemia genetic research in Latin America (included as an independent body within the general report). The book of abstract containing mini-papers (2-3 pages), distributed to all participants upon arrival, is also available as an electronic version in the project Web page.
1. Ecological study of Parthenogenetic Artemia population in Thamaraikulam and Puthalam area of South India
2. Molecular study - Genetic polymorphisms and mitochondrial RFLP of Artemia from selected South Asia regions
3. Characterization of the mixed population of Artemia from Tuticorin
4. Survey of the Artemia population in Sambar lake, Rajastan, India
1. Ecological study of Parthenogenetic Artemia population in Thamaraikulam and Puthalam area of South India
An asexual population of brine shrimp Artemia parthenogenetica from two commerical salinas at Puhalam amd Thamaraikulam of South India was sampled weekly during the 2002 - 2003 . The ecological aspects of the salt works and the population fluctuation during the period of study is given.
2. Molecular study - Genetic polymorphisms and mitochondrial RFLP of Artemia from selected South Asia regions
Artemia spp distributed in three selected regions of two South Asian countries Viz. Sri Lanka ( Puthalalm ), India (Tuticorin and Puthalam ) were compared with the Artemia of the North American and Chinese sp. for its genetic and biochemical diversity using enzyme markers, metabolism and mitrochondrial DNA. Among four strains selected from Asian strain the China strain exhibited the better stock quality and swift adaptation to changing environment. This phenomenon was assessed by metabolic enzyme expressions during hatching period of its cyst. In all experiments. Strain selected from Tuticorin exhibited quite similar phenomenon with control, which states the intermixing of native strain with North American strain. Mitrochondrial RFEL shows the maximum agreement on restriction site of BstXI between North American strain and Tuticorin. LDH polymorphisms were observed between five strains showing monomeric LDH patterns in control, Tuticorin and Srilanka. Dimprphic LDH in Puthalam and China states that there is an adaptation of carbohydrate metabolism in these strains. From the overall studies, China 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 North American strain. Further studies such as Microsatellite studies needed to establish the degree of intermixing of native with North American strain and the genetic purity of native strains of South Asia.
3. Characterization of the mixed population of Artemia from Tuticorin
Study conducted at Tuticorin region on the presence of the Artemia population reveled that in all the places in around the Tuticorin regions are having a mixed population measuring the cyst diameter of 190 to 310 um . The hatching characteristic and the culture aspects works are in progress
4.Survey of the Artemia population in Sambar lake, Rajastan, India
Survey was made in the Sambar lake during the month of September, 2003. on the already identified biotopes . During the survey no parthenogenetic Artemia population was identified. The brief study on the Sambar lake and the biota presence are studied
International Training
2. International training at the Institute de Aquicultura de Tore de la Sal (CSIC) Castellon de la Plana, Spain during June , 2003 by M. Peter Marian on the characterization of the Artemia strains from India.
Artemia workshop at Chile:
Participation of the Artemia workshop during November 2003 at Chile.
Work under progress
- the characterization and culture work on the population of Artemia from Tuticorin is going on
- the population in other parts of the India and South Asia will also be studied
- three clones of Artemia were identified from the Thamraikulam salt works; their Chromosome study will be undertaken
- the mixed population will be carefully studied for the parthenogenetic and bisexual population by separating the female and their gene pool will be conserved
Publications:
0. Characterization of Artemia parthenogeentica (KKT1 strain) from the salt works of Thamaraikulam, K.K. District, South India M. Peter Marian, J.A., Christopher and F. Amat (under preparation)
1. Life history characteristics of Artemia parthenogenetica (KKT1 strain) under laboratory conditions M. Peter Marian, C.S. Mony, (communicated)
2. Studies on the clones of Artemia parthenogenetica (KKT1 strain) rom the salt works of Tamaraikulam salt works S.M. Vijila, Rajasekar and M. Peter Marian, (In press)
Outline plans for the next year
Research:
1. Exploration and characterization of Artemia population in Northern part of India
1. Molecular characterization of the new parthenogenetic Artemia population of Thamraikulam and Puthalam
2. Characterization of the mixed population of Artemia from Northern part of India
3. Germplasm conservation of Artemia from India
Training Lab I
June, 2004
Training Lab II
September, 2004
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INCO Concerted Action
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ICA4-CT-2001-10020
Individual Partner Annual Reports
Year 2
Period 1 January-31 December 2003
INCO partner 15: IART
Partner name: Institute for Artemia Research and Training, Manonmaniam Sundaranar University, Rajakkamangalam, 629 502, Kanyakumari District, Tamil Nadu, India
tel.: 91-4652-33680; fax: 91-4652-21457; email: petermar@md5..in
Responsible scientist: Peter Marian
INCO partner 14: ULL
Partner name: Department of Basic Sciences/Laboratory of Genetics & Aquaculture, Universidad de Los Lagos, P.O. Box 933, Avenida Fuchslocher s/n, Osorno, Chile
tel. 56-64-205293; fax: 56-64-239517; email: ggajardo@ulagos.cl
Responsible scientist: Gonzalo Gajardo
INCO partner 13: UFRN
Partner name: Departamento de Oceanografia e Limnologia, Universidade Federal do Rio Grande do Norte, Campus Universitario, BR 101, 59072-970 Natal, Brazil
tel.: 55-84-2154433; fax: 55-84-6421815;email: mrcamara@ufrnet.br
Responsible scientist: Marcos R. Camara
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, Mexico
tel.: 52-5-4837151; fax: 52-5-4837469; email: cabt7515@cueyatl.uam.mx
Responsible scientist: Thalía Castro Barrera
INCO partner 11: UU
Partner name: Artemia and Aquatic Animals Research Center (AAARC), Urmia University, Shahid Beheshty Av., P.B. 57153-165, Urmia, Iran
tel: 98-441-3440295; fax: 98-441-3443442; email: n.agh@mail.urmia.ac.ir
Responsible scientist: Naser Agh
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INCO partner 10: SRI
Partner name: Salt Research Institute, Yingkou Road 831, 300450 Tanggu, Tianjin, PR China
tel.: 86-22-25301094; fax: 86-22-25897596; email: srisalt@public.tpt.
Responsible scientist: Xin Naihong
INCO partner 9: RU
Partner name: Department of Ichthyology and Fisheries Science, PO Box 94, Rhodes University, Prince Alfred Street, Grahamstown 6140, South Africa
tel.: 27-46-6038415 ; fax : 27-46-6224627 ; e-mail: h.kaiser@ru.ac.za; t.hecht@ru.ac.za
Responsible scientist: Tom Hecht
INCO partner 8: INAT
Partner name: Institut National Agronomique de Tunisie, Université de Carthage, 43 av. Charles Nicolle 1082, Tunis, Tunisia
tel. : 216-1-287110; fax: 216-1-799391;
email: romdhane.medsalah@inat.agrinet.tn
Responsible scientist: Mohamed Salah Romdhane
INCO partner 7: CTU
Partner name: Institute of Science for Aquaculture, 3rd February Street, Campus II, Can Tho University, Can Tho, Vietnam
tel.: 84-71-834307; fax: 84-71-830323; email: nvhoa@ctu.edu.vn; nvhoa.ct@bdvn.
Responsible scientist: Nguyen Van Hoa
INCO partner 6: UBA
Partner name: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Núñez, Pab. II, 4P, C1428 EHA Buenos Aires, Argentina
tel. 54-1-45763349 ; fax 54-1-45763384 ; email : cohen@bg.fcen.uba.ar
Responsible scientist: Graciela Cohen
INCO partner 5: LA SAPIENZA
Partner name: Dipartamento di Biologia Animale é dell’Uomo, Laboratorio di Zoologia Applicata, Università La Sapienza, Viale dell’ Università, 00185 Rome, Italy
tel.: 39-6-49914769; fax: 39-6-4958259; email: graziella.mura@uniroma1.it
Responsible scientist: Graziella Mura
INCO partner 4: CSIC
Partner name: Instituto de Acuicultura de Torre de la Sal (Consejo Superior de Investigaciones Científicas de España), Larval Food (Artemia), Larviculture and Ecotoxicology group, 12595 Ribera de Cabanes (Castellón), Spain
tel.: 34-964-319500; fax: 34-964-319509; email: amat@iats.csic.es
Responsible scientist: Francisco Amat
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
INCO partner 2: CLO-DVZ
Partner name: Agricultural Research Center-Ghent/Department of Sea Fisheries (CLO-DVZ), Ankerstraat 1, 8400 Oostende, Belgium
tel. 32-9-34 22 66; fax: 32-9-33 06 29; email: daan.delbare@dvz.be
Responsible scientist: Daan Delbare
INCO partner 1: RUG
Partner name: 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; email: gilbert.vanstappen@UGent.be
Responsible scientist: Patrick Sorgeloos
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