INCO Concerted Action
Artemia Biodiversity:
Current global resources and
their
sustainable
exploitation
INCO
Concerted
Action
ICA4-CT-2001-10020
Artemia Biodiversity:
Current global resources and
their sustainable exploitation
Summary of Final Report
ICA4-CT-2001-10020
Period
1
January
2002-31
December
Annexes
Period 1 January 2002-31 December 2004
1
2004
MEETING REPORTS
Work Package & Deliverable 1:
Global Workshop
Ghent University, Belgium, February 5-7, 2002
“Planning of Consortium Strategy”
1. Introductory Note
The Global workshop, first concrete realization of the INCO Project, was organized at an
early stage, shortly after the official start of the Project on January 1, 2002, and was intended
to be the formal and practical ‘kick-off’ meeting of the Project.
Keywords of the Workshop:
 general topics of species description and species characterization, population
description, biodiversity
 intercalibration of methodologies
 planning of other workshops
 planning of training courses
2. Programme
February 5: Oral presentations
February 6: Discussions
a.m.
 General objectives of INCO project; planning of activities
 Objectives of stays of visiting scientists
 Objectives of regional workshops
p.m.
 Discussions of a.m: continued
 Project management: financial aspects, reporting
February 7: Discussions
a.m.
 Regional workshops: practical planning
 Conclusions and recommendations
3. Participants and oral presentations
The purpose of the presentations was mainly to acquaint the audience with the expertise of all
attendants’ Artemia expertise and to assess how complementarity and interaction of Project
activities could be conceived.
a) INCO Members
1.
RUG :
Sorgeloos Patrick, Laboratory of Aquaculture & Artemia Reference Center, Ghent
University, Belgium: “Welcome and introduction”
Van Stappen Gilbert,Laboratory of Aquaculture & Artemia Reference Center, Ghent
University, Belgium: “Artemia research at the Artemia Reference Center”
2.
CLO-DVZ :
2
Bossier Peter, Agricultural Research Center-Ghent/Department of Sea Fisheries, Ostend,
Belgium: “A database for Artemia authentication”
Wang Xiaomei, Agricultural Research Center-Ghent/Department of Sea Fisheries, Ostend,
Belgium
3.
AUTH:
Abatzopoulos Theodore, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, Thessaloniki,
Greece: “Running research projects at AUTH on Artemia and other organisms”
Baxevanis Thanos, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, Thessaloniki,
Greece
4.
CSIC:
Amat Francisco, Instituto de Acuicultura de Torre de la Sal (Consejo Superior de
Investigaciones Científicas de España), Ribera de Cabanes (Castellón), Spain: “Artemia
biodiversity: current global resources and their sustainable exploitation”
5.
La Sapienza : none
6.
UBA: none
7.
CTU:
Nguyen Van Hoa,Institute of Science for Aquaculture, Can Tho University, Can Tho,
Vietnam: “Artemia pond culture, strain characterisation and its approach in Vietnam”
8.
INAT:
Romdhane Mohammed, Institut National Agronomique de Tunisie, Université de Carthage,
Tunis, Tunisia: “Status of the Artemia biodiversity in Tunisian salt areas”
9.
RU:
Hecht Tom, Department of Ichthyology and Fisheries Science, Rhodes University,
Grahamstown, South Africa
Kaiser Horst, Department of Ichthyology and Fisheries Science, Rhodes University,
Grahamstown, South Africa: “Characterisation of southern African Artemia populations, and
the use of Artemia as a vector for probionts and hormones”
10.
SRI:
Xin Naihong, Salt Research Institute, Yingkou Road 831, 300450 Tanggu, Tianjin, PR
China: “Artemia resources and their development in China”
11.
UU:
Agh Naser, Artemia and Aquatic Animals Research Center, Urmia University, Iran: “Studies
on Artemia populations from Iran”
12.
UAM-X:
Castro Mejia Jorge, Universidad Autónoma Metropolitana-Xochimilco, Depto. El Hombre y
su Ambiente, , Mexico D.F., Mexico: “Artemia research in the Universidad Autonoma
Metropolitana-Xochimilco, Mexico”
13.
UFRN:
Camara Marcos, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio
Grande do Norte, Natal, Brazil: “Artemia research at Universidade Federal do Rio Grande
do Norte (UFRN), Natal, Brazil”
14.
ULL:
Gajardo Gonzalo, Department of Basic Sciences/Laboratory of Genetics & Aquaculture,
Universidad de Los Lagos, Osorno, Chile: “Artemia characterization, species and
speciation”
15.
IART:
Maryan Peter, Institute for Artemia Research and Training, Manonmaniam Sundaranar
University, Rajakkamangalam, India: “Research on Artemia in the Institute for Artemia
Research and Training”
b) Guest Speakers (see below: ‘Involvement of Associated INCO partners):
3
Beardmore John, University College of Swansea, School of Biological Sciences, University
of Wales, Swansea UK
Boyko Elena, Tyumen State Agricultural Academy - The Siberian Science-Research and
Project-Construction Institute of Fishery Tyumen, Russia: “Authentication of Artemia
from Russia”
Brendonck Luc, Laboratory Aquatic Ecology, Catholic University of Leuven, Belgium:
“Adaptations to life in hyper-oligotrophic ephermeral pools”
Clegg Jim, Bodega Marine Laboratory, Section of Molecular and Cellular Biology,
University of California, Davis, Bodega Bay, USA: “The diversity of Artemia habitats is
reflected in the biochemical repertoire of this remarkable organism”
Criel Godelieve, Department of Anatomy, Embryology and Histology, Ghent University,
Belgium: “Morphological tools to help distinguishing Artemia species and populations”
De Meester Luc, Laboratory Aquatic Ecology, Catholic University of Leuven, Belgium:
“Dispersal, habitat size and genetic differentiation among zooplankton populations”
MacRae Tom, Department of Biology, Dalhousie University, Halifax, N.S., Canada:
“Microtubule proteins and chaperones: molecular technologies applied to study of the
brine shrimp, Artemia franciscana”
Marden Brad, Research and Development, Utah Strategic Alliance, Utah, USA: “Recovery
of the Great Salt Lake, USA, brine shrimp (Artemia franciscana) population”
4. Conclusions and Recommendations: Planning of Consortium Strategy
At the occasion of the Global Workshop, a number of concrete working guidelines were
agreed upon, intending to contribute to the achievement of the Project’s objectives.
4.1. Involvement of ‘Associated’ INCO partners
The Project Consortium (PC) has the intention to involve, throughout the Project period, a
number of experts in the field of Artemia study who are excluded from formal partnership to
the INCO project (e.g. non-eligible countries affiliation; retired from active professional
activity). Their role is envisaged as follows:
1. participation to Project events (Workshops), pending availability of alternative cq
own financial resources;
2. access to information about Project’s activities; feedback and advice towards PC, thus
providing added value to the PC’s scientific expertise
Additionally, for regional workshops, the participation of local or regional Artemia experts is
highly recommended, and the respective Consortium members (CM), responsible for the
Project’s regional workshops (INCO partners 10, 11 and 14) have committed themselves to
invite a maximum of guest speakers (by alternative financing) to upgrade the scientific
relevance of the event.
4.2. Identification of ‘Action Plans’ and ‘Partnerships’
The Consortium Members realized that scientific progress in various fields of Artemia
biodiversity would benefit from:
1. identification of concrete points where action needs to be taken;
2. subdivision of the Consortium into smaller workable task forces.
The CM thus decided to identify a number of concrete actions ("action plans") and to create a
number of task forces or partnerships ("thematic" and "regional"), each working in an
informal way around certain concrete themes. Consortium members (and ‘associates’) can
join these respective task forces on a voluntary basis. For practical convenience, a coordinator
has been appointed for each task force. Each task force identifies its own priorities and
strategies, in line with the Project’s objectives. The task force coordinators agree to report
4
briefly about their activities at the occasion of the 6 month-reports and the various workshops;
here feedback from the entire Consortium is to be given, and the Project Coordinator ensures
that there is mutual information flow between the groups, and that the Project Objectives are
used as common denominator for all task forces.
Action plans (with abbrevation of participating CM’s; coordinator in bold):
1. Database: ARC, AUTH, CSIC, SRI, ULL, IART
2. Cyst bank: ARC, CSIC, SRI, UU, Jim Clegg
3. Study visits (visiting scientists): ARC, AUTH, CSIC
4. Publications, Reports, Website, Code of Conduct for conservation of biodiversity and
sustainable exploitation: John Beardmore, ARC, AUTH, RU
Regional partnerships:
1. Africa: RU, ARC, AUTH, CSIC, INAT
2. China: SRI, ARC, CLO-DVZ, AUTH, UU
3. Iran: UU, ARC, CLO-DVZ, AUTH, CSIC
4. Mediterranean: AUTH, CSIC, LA SAPIENZA, INAT
5. Central Asia: ARC, CLO-DVZ, UU, Brad Marden, Elena Bojko
6. Latin America: ULL, CLO-DVZ, CSIC, UBA, UMA-X, UFRN
7. South Asia: IART, ARC, CTU, UU
Thematic partnerships:
1. Adaptation/Speciation: UFRN, CLO-DVZ, CSIC, CTU, RU, SRI, UMA-X, ULL
2. Diapause: AUTH, CLO-DVZ, IART, La Sapienza, Tom McRae, Jim Clegg
3. Plasticity: CSIC, INAT, RU, UMA-X
4.3. Planning of Workshops and Training (Study visits)
The timing for the First regional Workshop (Work Package and Deliverable 2) was set at
September 2002. As for the training programme, an essential part to reach the Project’s
objectives, it was agreed among CM, conform to the recommendations of the Commission
evaluating the Project Proposal, that the training programme should be tailored according to
the needs of both the ‘trainees’ and the hosting institutes. Moreover, CM agreed that the stays
of the visiting scientists should not be restricted to trainings in se, but should focus on
concrete points of collaborative research for the benefit of both host and trainee, and should
maximally result in such concrete output like joint publications.
5
Work Package & Deliverable 2:
First Regional Workshop
Beijing, China, September 23-26, 2002
1. Introductory Note
For reasons of optimal logistics and of maximal participation by guest speakers, the
organisers (SRI, Salt Research Institute, Tanggu) decided to organize the Workshop in
Beijing (Zhongyang Hotel) instead of at their premises in Tanggu.
Keywords of the Workshop:
 Artemia resources of China (coastal and inland)
 threats for local populations by introduction of foreign species
 initial evaluation of scientists’ visits
2. Programme
September 23: a.m.-p.m. Oral presentations
September 24: a.m. Oral presentations
September 25: Excursion to Tanggu; visit to Salt Research Institute, Tanggu Saltworks and
Fish Farm
September 26: a.m.-p.m. Discussion
Participants and oral presentations:
Li Shusheng (Director Salt Research Institute): “Welcome and Introduction of Salt Research
Institute”
a) INCO Members
1.
RUG:
Sorgeloos Patrick, Laboratory of Aquaculture & Artemia Reference Center, Ghent
University, Belgium: “Introduction to INCO project”
Van Stappen Gilbert, Laboratory of Aquaculture & Artemia Reference Center, Ghent
University, Belgium:“INCO activities at Artemia Reference Center”
2.
CLO-DVZ:
Bossier Peter, Agricultural Research Center-Ghent/Department of Sea Fisheries, Ostend,
Belgium: “A rDNA-RFLP database for identifying commercial Artemia samples”
3.
AUTH:
Abatzopoulos Theodore, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, Thessaloniki,
Greece: “Artemia characterisation based on mtDNA RFLP analyses”
Baxevanis Thanos, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, Thessaloniki,
Greece
4.
CSIC:
Amat Francisco, Instituto de Acuicultura de Torre de la Sal (Consejo Superior de
Investigaciones Científicas de España), Ribera de Cabanes (Castellón), Spain: “Artemia
biodiversity: current global resources and their sustainable exploitation”
Nieves Sanz Maria, Instituto de Acuicultura de Torre de la Sal (Consejo Superior de
Investigaciones Cientificas de España), Ribera de Cabanes (Castellón), Spain
5.
La Sapienza:
6
Mura Graziella, Dipartamento di Biologia Animale é dell'Uomo, Laboratorio di Zoologia
Applicata, Università La Sapienza, Rome, Italy: “Use of morphological characters as a tool
for species separation”
Giampaolo Rossetti, Dipartamento di Biologia Animale é dell'Uomo, Laboratorio di
Zoologia, Università La Sapienza, Rome, Italy
6.
UBA:
Rosa Graciela Cohen de Sanchez, Universidad de Buenos Aires Facultad de Ciencias
Exactas y Naturales, Departamento de Ciencias Biológicas, Buenos Aires, Argentina: “An
overview of the team's activities”
7.
CTU:
Van Hong Nguyen Thi, Institute of Science for Aquaculture, Can Tho University, Vietnam:
“Artemia culture in Southeast Asia: emphasis to Vietnam”
8.
INAT:
Romdhane Mohammed, Institut National Agronomique de Tunisie, Université de Carthage,
Tunis, Tunisia
9.
RU:
Kaiser Horst, Department of Ichthyology and Fisheries Science, Rhodes University,
Grahamstown, South Africa: “Preliminary RFLP analysis of mtDNA from southern African
Artemia”
10.
SRI:
Xin Naihong, Salt Research Institute, Tanggu, Tianjin, PR China,
Sui Liying, Salt Research Institute, Tanggu, Tianjin, P.R. China: “Characterization of
Artemia from Qinghai-Tibet Plateau Salt lakes”
Yu Xiuling, Salt Research Institute, Tanggu, Tianjin, PR China: “Characterization of 4
Tibetan Artemia strains and HUFA profile during enrichment and starvation”
Jin Zhiling, Salt Research InstituteTanggu, Tianjin, P.R. China,
Chen Ziqiang, Salt Research Institute, Tanggu, Tianjin, P.R. China
11.
UU:
Agh Naser, Artemia and Aquatic Animals Research Center, Urmia University, Iran:
“Coexistence of bisexual and parthenogenetic Artemia populations in Lake Urmia”
12.
UAM-X:
Thalia Castro, Universidad Autónoma Metropolitana-Xochimilco, Depto. El Hombre Y su
Ambiente, Mexico: “Progress in Artemia study at UMA-X”
13.
UFRN:
Camara Marcos, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio
Grande do Norte, Natal, Brazil: “Ecological threats to Artemia franciscana Kellogg
(Crustacea; Anostraca) populations in the coastal saltworks of Rio Grande do Norte,
northeastern Brazil”
14.
ULL:
Gajardo Gonzalo, Department of Basic Sciences/Laboratory of Genetics & Aquaculture,
Universidad de Los Lagos, Osorno, Chile: “Artemia diversity and evolution in South
America: new results and ideas in the framework of the Artemia Biodiversity project”
15.
IART:
Maryan Peter, Institute for Artemia Research and Training, Manonmaniam Sundaranar
University, Rajakkamangalam, India: “Status of Artemia production in the coastal salt works
of India”
b) Guest Speakers & other attendants:
As this workshop focuses on Artemia from China, a number of Chinese participants from
other institutes/universities had been invited:
Chen Shao-zhong, Hangu Saltworks, Hangu, Tianjin: “The application of Artemia and
development of new Artemia product”
7
Dong Zhihua, China National Salt Industry Corporation, Beijing
Jia Qinxian, Open Laboratory of Saline Lake Resources and Environment, The Chinese
Academy of Geological Sciences, Beijing: “Improvement of insect ecdysome on the hatching
rate of Artemia eggs”
Kong Fanjing, Research & Development Center of Salt lake and Epithermal Deposits,
Chinese Academy of Geological Sciences, Beijing:“Genetic diversity of halophilic organism
in Chinese salt lakes and application prospect for plant salt tolerance genetic engineering”
Liang Peilin, China National Salt Industry Corporation, Beijing
Lin Jiahua, China National Salt Industry Corporation, Beijing
Li Yanan, Marine Science and Engineering College, Tianjin Science and Technology
University, Tanggu, Tianjin
Liu Fengqi, Life Sciences College, Nankai University, Tianjin
Sun Jingxian, Dalian Fisheries University, Dalian: “Some ecological parameters of Artemia
parthenogenetica from Gahai Lake and its use in resource exploitation”
Wang Xiaomei, Tianjin Agriculture University
Xing Kezhi, Tianjin Agriculture University
Zeng Hui, Life Sciences College, Nankai University, Tianjin: “The cloning of the Brine
Shrimp DNA fragments correlated to the Bombyx mori doublesex gene”
Zhang Fu, Marine Science and Engineering College, Tianjin Science and Technology
University, Tanggu, Tianjin: “Preliminary study on the effect of Na/Mg ratio of artificial
seawater and natural seawater on hatching ability of Artemia cysts”
Zhang Yanmei, China National Salt Industry Corporation, Beijing
Zheng Mianping, R&D Center of Saline Lakes and Epithermal Deposits, Chinese Academy
of Geological Sciences, Open Laboratory of Saline Lake Resources and Environment
Research, Ministry of Land and Resources, Beijing.
Furthermore, the delegation of partner 11 (UU) had been reinforced by a delegation of the
Iranian Fisheries Company, Dept. Public Relations and International Affairs, Tehran, Iran:
Amir Shoahassani Lashidani
Mahmoud Hafezieh
3. Conclusions and Recommendations
3.1. Artemia resources of China (coastal and inland)
Special efforts have been devoted in recent years to the inventorization of Chinese salt lakes
in general, and more specifically to Artemia sites. Half of the total lake area of China is
reported to consist of saline lakes (mostly in the inland provinces of Inner Mongolia,
Xinjiang, Qinghai and Tibet). There are more than 1000 saline lakes (salinity > 3.5 ppt) in
China, with a total area of more than 50,000 km², of which 534 have been the subject of
investigation. An overview of these salt lakes can be found in literature, as well as overviews
on the occurrence and use of Chinese Artemia. One of the key problems in identifying
Chinese Artemia sites remains the high incidence of misunderstandings or mistakes when
transcribing Chinese names into Latin characters. Therefore a new, numerical, identification
system for Chinese Artemia sites has been proposed in literature, taking into account the
province and the inland or coastal location of the site.
The Artemia biodiversity of PR China shows a complex pattern. The prevailing mode of
reproduction in the coastal habitats in China is parthenogenesis, though in recent years some
populations are mixed with, or have been outcompeted by, introduced A. franciscana, as a
consequence of the vicinity of aquaculture activities in the area and/or deliberate inoculations.
Numerous parthenogenetic populations also exist in inland lakes and especially in the big salt
lakes of Xinjiang and Qinghai provinces. In continental China an endemic species occurs, A.
8
sinica. The recent exploration of new Artemia biotopes in PR China has resulted in the
description of a new bisexual species, A. tibetiana found in Lagkor Co Lake, on the high
plateaus of Tibet. Bisexuals are also found in other lakes on the Qinghai-Tibet plateau, though
their species status has not been ascertained yet. According to recent data, they co-occur in
these habitats with parthenogenetic strains.
China can thus be considered as a scale-model for global Artemia biodiversity phenomena:
occurrence of several bisexual species into widely diverging and extreme habitats, cooccurrence of bisexual and parthenogenetic strains, introduction of allochthonous species and
local populations being outcompeted by invasive strains. Similarly, the study of Artemia in
China covers all possible scientific aspects with immediate relevancy for the local Chinese
situation: Artemia biodiversity, taxonomy and genetics, production in solar saltworks,
application of cysts and biomass in aquaculture. The Salt Research Institute (INCO partner
10) is in a privileged position to act as ‘Reference Center’ for Artemia study, thanks to its cyst
bank and to its network of contacts with saltworks, aquaculture operations, governmental
agencies elsewhere in China (allowing to obtain Artemia cyst samples even from extremely
remote areas), and with research institutes experienced in up-to-date Artemia research
techniques abroad and in China itself; the advanced status of the study of Chinese Artemia
strains and its interdisciplinary approach is illustrated by recent papers in the field of DNA
fingerprinting and authentication, and by the use of allozyme electrophoresis to assess the
inter- and intra-populational genetic variation in Chinese bisexual populations. The broad
scope and importance of Artemia research in China is reflected by the participation to the
Beijing Workshop of representatives from the salt production sector (Hangu Saltworks; China
National Salt Industry Corporation), ecologists and geologists specialized in salt lake study
(Laboratory of Saline Lake Resources and Environment; Research & Development Center of
Salt lake and Epithermal Deposits, Chinese Academy of Geological Sciences), aquaculturists
and marine biologists (Marine Science and Engineering College, Tianjin Science and
Technology University; Dalian Fisheries University), and geneticists (Life Sciences College,
Nankai University, Tianjin; Tianjin Agriculture University).
Further study of Artemia in China should focus on the following axes of research:
 Characterization of inland Artemia strains, with focus on bisexual species and/or from
extreme habitats (Qinghai-Tibet Plateau, Inner Mongolia) (cooperation with ARC, CLODVZ, AUTH, UU): patterns of coexistence, adaptation to ecological conditions (e.g. by
study of dominant heat shock proteins), HUFA metabolism and other aquaculture related
characteristics
 Artemia resources of coastal areas in China, with focus on Bohai Bay area (cooperation
with ARC, CTU, UU): contamination of local populations by invasive A. franciscana;
dispersal patterns, potential production and aquaculture applications.
 Genetic database for authentication of Artemia samples (with ARC, CLO-DVZ, AUTH)
 Further updating and upgrading of cyst bank of Chinese cyst sources.
3.2. Threats for local populations by introduction of foreign species
The first inoculation in 1977 in the Macau saltworks, Natal, northeastern Brazil, with nauplii
hatched out of San Francisco Bay cysts, was extremely successful, and brine shrimp soon
spread out over 3000 ha of nearby saltworks. As a result of the introduction of A. franciscana
in solar saltworks for improved salt production and/or for harvesting cysts and biomass for
use in the aquaculture industry, seasonal A. franciscana farming is practised in many tropical
and subtropical countries such as the Philippines, Thailand, Vietnam, Sri Lanka. However,
due to the particular climatic conditions of these countries, the Artemia populations are not
permanent and principally annual inoculations are required.
9
Permanent populations of this species, however, have established themselves in climatic
conditions allowing perennial brine shrimp occurrence, such as in Brazil, Australia, China,
Egypt, Portugal, etc.
Initially, the process of Artemia inoculation was considered as unambiguously positive: salt
lakes were considered as having little biogeographical variability and low biodiversity. Now it
is widely recognized that salt lakes are unique and well-balanced ecosystems, and that there
are differences in their fauna between continents and regions, and even locally, according to
their salinity fluctuations, water permanence, seasonality etc. Many salt lakes are of
intermediate or small size, and this very specific environment, including the food chain with
Artemia and waterfowl, is very vulnerable for deterioration by human intervention. Threats to
salt lakes are numerous, and mainly consist of desiccation by drainage or diversion of
influents, or pollution. For coastal saltworks urbanization projects (industry, harbour
infrastructure, tourism, expansion of residential areas), represent a supplementary threat,
especially in industrialized or industrializing countries. If an original Artemia gene pool
disappears, a basic potential of genetic improvement is destroyed as well.
What are the possible effects of Artemia introduction on other organisms inhabiting natural
saltwater bodies? While this practice frequently ensures social and economic benefits,
particularly in developing countries, it also bears certain risks. An obvious effect is that
competition with local (or nearby) strains or species of Artemia may occur which may lead to
the extinction of some genotypes, or at worst, of one of both competitors. Competition
experiments suggest that A. franciscana may outcompete other bisexual species and the
parthenogenetic strains. The effect of one introduction will not remain local but may have
consequences over large areas (cfr Brazil), following dispersion by wind and local waterbirds.
The resolution put forward at the 2nd International Symposium on Artemia, Antwerp,
Belgium, in September 1985, therefore is still valid: “…the 2nd International Symposium
resolves that all possible measures be taken to ensure that the genetic resources of natural
Artemia populations are conserved; such measures include the establishment of gene banks
(cysts), close monitoring of inoculation policies, and where possible the use of indigenous
Artemia for inoculating Artemia-free waters’.
The INCO members recognize the possible threat to Artemia biodiversity by the uncontrolled
spreading of invasive Artemia species. At present the geographical areas where this
phenomenon has been observed are still confined, but it may be expected that the problem
might increase at mid- and long-term, and that possibly invasion already has taken place in
areas that have not been the subject of study yet. Presently only A. franciscana (mainly the
San Francisco Bay strain) has been reported as allochthonous invasive species in certain areas
of Europe and Asia: A. franciscana has been reported in salterns in Spain (near Portugese
Algarve where the species has been deliberately introduced) and in southern France, often in
the vicinity of aquaculture operations (CSIC). In Italy, in spite of high aquaculture activity, no
report has been made of A. franciscana (La Sapienza), but the species has further been
reported for the Bohai Bay area in China (SRI), certain areas in India (IART), in Kenya (RU)
and an occasional observation for the surroundings of Lake Urmia (UU). Theoretically in
future a similar threat may be posed by the San Francisco Bay strain in areas of North and
South America where local A. franciscana strains occur, or by non-franciscana strains (e.g.
parthenogenetic) known to be processed and used in aquaculture activities worldwide (be it
more limited than A. franciscana).
Within this context, the study of invasive A. franciscana in the Mediterranean Basin can be
considered as a case study, with different angles of approach realized by the INCO partners
CSIC, AUTH, La Sapienza and INAT. A screening of available samples in the respective cyst
10
banks and of possible newly sampled sites will reveal the extent of contamination of
Mediterranean Artemia habitats, using different research tools: morphological characteristics,
molecular (DNA) markers suitable for discriminating between A. franciscana and the local A.
salina and parthenogenetic strains; active transport of Artemia cysts by waterfowl, through
analysis of cysts obtained from faeces, culture experiments of joint Mediterranean and A.
franciscana populations in order to assess competition patterns in function of environmental
parameters. These results can be complemented with the results, obtained by the recent
construction of an Artemia database of identifying RFLP patterns based on a mitochondrial
rDNA fragment that has proved to be a quick and reliable molecular tool for the
authentication of Artemia cysts samples. This technique, which allows discriminating
between populations below the species level, may have high potential, especially as it is being
worked out for working with single cysts or individuals. In cases of controversy (coexistence
of species cq by invasion of allochthonous strains, or cases where uncertainty about the
species status of the population exists) the technique might bring decisive evidence. The
results may be complemented by information originating from other disciplines in a
multidisciplinary cooperation with other institutes: e.g. the study of the behaviour of wading
birds that frequent saline water bodies in Africa, focusing on migration patterns and their role
in disperson of cysts, by e.g. capturing birds to investigate stomach contents and inspect
feathers for cysts, and by using molecular marking techniques to identify Artemia
populations, and to identify which natural populations of Artemia are more vulnerable to
invasions by introduced Artemia species in Africa.
3.3. Initial evaluation of scientists’ visits
As the number of scientists’ visits being realized was limited at the time of the 1 st Regional
Workshop, few conclusions could be drawn
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Work Package & Deliverable 3:
Second Regional Workshop
Puerto Varas, Chile, November 17-19, 2003
1. Introductory Note
The 2003 Chile workshop was held in Puerto Varas from 17 to 19 November. This city is
located by the lake Llanquihue, one of the largest Andean lakes in Chile, close to Puerto
Montt, the capital of the 10th region. The area concentrates aquaculture activities, particularly
salmon farming. The headquarters (and freshwater facilities) of many local and international
aquaculture companies are located in Puerto Montt. This city also houses multinational feed
meal companies.
Keywords of the Workshop:
 Artemia resources of Latin Americ
 Genetics and speciation
 Intermediate evaluation of training courses
Welcome word by Workshop organizers
Since the brine shrimp Artemia is normally associated with tropical and subtropical areas, one
could be intrigued to see an Artemia meeting being held in southern Chile, where temperate
rainforest and Araucarian lakes of glacial origin are (or used to be in the case of forest) the
usual landscape. But Chile is quite diverse in terms of climate, hydrobiological and geological
conditions and these are barriers that establish clear-cut zoogeographic areas. In the North the
Atacama desert, one of the driest in the world, imposes severe restrictions to life; the central
zone is marked by the influence of the four seasons, whilst the extreme South, the Chilean
Patagonia, is referred as one of the most environmentally sensitive and biologically diverse
ecosystems in the world, which provides a wide variety of unique habitats for a unique
diversity of species.
Throughout more than 4,000 kilometres of coastline, salt lakes and lagoons, saline deposits
and evaporitic basins as well as solar salt works are found from sea level to heights of about
3,800 meters in the Andes Mountains. The two striking environmental extremes, the Atacama
desert in the pre-altiplanic region and Torres del Paine in Chilean Patagonia, are the biotopes
for the two New World Artemia species, A. franciscana and A. persimilis, respectively. Hence
Chile is a perfect location for a meeting on Artemia biodiversity. Moreover, Artemia locations
in the South American continent have received increasing attention lately, which explains the
participation of 3 groups from the continent in this INCO project.
We are grateful to Patricia Beristain for her collaboration to the organization of the workshop
and Myriam Angulo for secretarial services. Additional support from Universidad de Los
Lagos (Research and Foreign Affairs units) is greatly appreciated.
We trust that the group of people gathered and the beautiful setting of Puerto Varas are the
required ingredient for a fruitful meeting as well as for informal discussions and friendship.
G. Gajardo & G. Van Stappen
2. Programme
Monday November 17
12
Hotel Cabañas del Lago, Salón Petrohue
09.00-09.30: Welcome and opening comments
Gonzalo Gajardo and Gilbert Van Stappen
Oral Presentations:
Chair: G. Cohen
Co-chair: G. Gajardo and G. Van Stappen
09.30-10.00 Gilbert Van Stappen (INCO partner 1)
Present studies on biodiversity of Artemia populations in Northern Asia
(G. Van Stappen, B. Marden, L. Litvinenko, I. Mirabdullayev, I. Zholdasova, Xin Naihong, P.
Bossier, P. Sorgeloos)
10.00-10.30 Peter Bossier (INCO partner 1 & 2)
Polymorphism in Artemia species as revealed with mitochondrial and nuclear markers
(D. Delbare, P. Bossier, S. Dooms, G. Van Stappen, P. Sorgeloos, Zhijun Qiu, Th. H.
MacRae)
11.00-11.30 Theodore Abatzopoulos (INCO partner 3)
Preliminary data on intraspecific genetic divergence between Artemia franciscana, San
Francisco Bay and inoculated populations in Vietnam
(I. Kappas, Th. J. Abatzopoulos, Nguyen Van Hoa, P. Sorgeloos, J. A. Beardmore)
11.30-12.00 Athanasios Baxevanis (INCO partner 3)
How do mitochondrially identical Artemia clones respond to different salinities?
(G. Deliopoulos, A.D. Baxevanis, T.J. Abatzopoulos)
12.00-12.30 Francisco Amat (INCO partner 4&3)
The use of RFLP 16S rDNA analysis for detecting invasion of Artemia franciscana in
Western Mediterranean region
(F. Amat, A.D. Baxevanis, A. Triantafyllidis, A Tzika, G. Mura, T.J. Abatzopoulos)
12.30-13.00 Francisco Amat (INCO partner 4)
Artemia biodiversity: current global resources and their sustainable exploitation
(F. Amat)
Oral Presentations (cont’d)
Chair: T. Castro
Co-chair: G. Gajardo and G. Van Stappen
14.00-15.00 Guillermo Chong
The saline domain of Northern Chile
(G. Chong)
15.30-16.00 Rosa Graciela Cohen (INCO partner 6)
Record of new Artemia populations and contribution to the morphological and
ecological characterization of some Argentinean populations
(R. G. Cohen1, C. Arbasetti, X. I. Pastorino, J. Goenaga, F. Amat)
16.00-16.30 Mohammed S. Romdhane (INCO partner 8)
Strain characterisation and climate effect on Artemia from Tunisia
(M.S. Romdhane)
13
16.30-17.00 Horst Kaiser (INCO partner 9)
Artemia research in southern Africa: A report on a sampling expedition through parts of
South Africa and Namibia
(H. Kaiser, T. Hecht)
17.00-17.30 Xin Naihong (INCO partner 10)
Distribution and characterization of Artemia in Bohai Bay
(Xin Naihong, Li Yanan)
17.30-18.00 Naser Agh (INCO partner 11)
Diversity of Artemia populations at Lake Urmia
(N. Agh, P. Sorgeloos, T. Abatzopoulos, G.Van Stappen, L. Sanders, D. Delbare, P. Bossier)
Tuesday November 18
Oral Presentations (cont’d)
Chair: M. Camara
Co-chair: G. Gajardo and G. Van Stappen
09.00-09.30 Thalía Castro (INCO partner 12)
Morphological and biochemical characterization of Mexican populations of Artemia
franciscana, and data on reproductive isolation and bioencapsulation of medications
(J. Castro, A. Malpica, G. Castro, T. Castro, R. De Lara)
09.30-10.30 Marcos Camara (INCO partner 13)
Preliminary characterization of Brazilian Artemia franciscana populations by means of
mtDNA RFLP analysis
(M. R. Camara, A. Triantafyllidis, A. D. Baxevanis, T. J. Abatzopoulos)
11.30-12.00 Patricia Beristain (INCO partner 14)
Preliminary comparison of DNA sequences of the cytochrome c oxidase subunit I (COI) in
Chilean Artemia populations
(P. Beristain, S. M. Funk, G. Gajardo)
12.00-12.30 Peter Marian (INCO partner 15)
Report of activities partner 15
14.00. Guided tour to aquaculture facilities (private and Universidad de los Lagos)
Wednesday November 19
General discussion
Chair: G. Gajardo & G. Van Stappen
3. Participants
Abatzopoulos Theodore, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, 541 24
Thessaloniki,
Greece,
Tel:
30-231-998301,
Fax:
30-231-998256,
e-mail:
abatzop@bio.auth.gr
Amat Francisco, Instituto de Acuicultura de Torre de la Sal (Consejo Superior de
Investigaciones Científicas de España), Ribera de Cabanes (Castellón), Spain, Tel : 34-964319500, Fax : 34-964-319509, e-mail : amat@iats.csic.es
14
Baxevanis Athanasios, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, 541 24
Thessaloniki, Greece, Tel: 30-231-998301, Fax: 30-231-998256
Beristain Patricia, Laboratory of Genetics & Aquaculture, Universidad de Los Lagos, P.O.
Box 933, Osorno, Chile, Tel: +56-64-205285, Fax: +56-64-239517, e-mail:
pberista@ulagos.cl
Bossier Peter, Laboratory of Aquaculture & Artemia Reference Center, Ghent University,
Rozier 44, B-9000 Gent, Belgium, Tel: 32-9-2643754, Fax: 32-9-2644193, e-mail:
Peter.Bossier@UGent.be
Camara Marcos, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio
Grande do Norte, Campus Universitario, BR 101, 59072-970 Natal, Brazil, Tel:: 55-842154433, Fax: 55-84-6421815, e-mail: mrcamara@ufrnet.br
Castro Thalía, Departamento El Hombre y su Ambiente, Universidad Autónoma
Metropolitana-Xochimilco, Distrito Federal, Calz. Delz. Del Hueso No. 1100. col. Villa
Quietud, Mexico, 04960, D.F. Mexico. Tel: +52-5-4837151, Fax: +62-5-4837469, e-mail:
cabt7515@cueyatl.uam.mx
Castro Jorge, Departamento El Hombre y su Ambiente, Universidad Autónoma
Metropolitana-Xochimilco, Distrito Federal, Calz. Delz. Del Hueso No. 1100. col. Villa
Quietud, Mexico, 04960, D.F. Mexico. Tel: +52-5-4837151, Fax: +62-5-4837469, e-mail:
camj7509@cueyatl.uam.mx
Chong Guillermo, Departamento de Ciencias Geológicas, Universidad Católica del Norte,
Av. Angamos 0610, Antofagasta, Chile, Tel: +56-55-355951, Fax: +56-55-355977, e-mail:
gchong@socompa.ucn.cl
Cohen de Sanchez Rosa Graciela, Departamento de Biodiversidad y Biología Experimental,
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
Universitaria, Núñez, Pab. II, 4° Piso, C 1428EHA, Buenos Aires, Argentina, Tel: +54-11-4576-3349, Fax: +54-11-4-576-3384, e-mail: cohen@bg.fcen.uba.ar
Gajardo Gonzalo, Department of Basic Sciences/Laboratory of Genetics & Aquaculture,
Universidad de Los Lagos, P.O. Box 933, Osorno, Chile, Tel: 56-64-205293, Fax: 56-64239517, e-mail: ggajardo@ulagos.cl
Hauva Mayda, Laboratory of Genetics & Aquaculture, Universidad de Los Lagos, P.O. Box
933, Osorno, Chile, Tel: 56-64-205013, Fax: 56-64-239517,
Kaiser Horst, Department of Ichthyology and Fisheries Science, Rhodes University,
Grahamstown, South Africa, Tel: 27-46-6038415, Fax: 27-46-6224827, e-mail:
h.Kaiser@ru.ac.za
Malpica Sánchez Aida, Departamento El Hombre y su Ambiente, Universidad Autónoma
Metropolitana-Xochimilco, Distrito Federal, Calz. Delz. Del Hueso No. 1100. col. Villa
Quietud, Mexico, 04960, D.F. Mexico. Tel: +52-5-4837151, Fax: +62-5-4837469, e-mail:
amalpica@cueatl.uam.mx
Marian Peter, Institute for Artemia Research and Training, Manonmaniam Sundaranar
University, Rajakkamangalam, 629 502, Kanyakumari District, Tamil Nadu, India, Tel: 914652-33680, Fax: 91-4652-21457, e-mail: petermar@md5.vsnl.net.in
Triantafyllidis Alexandros, Aristotle University of Thessaloniki, Department of Genetics,
Development & Molecular Biology, Faculty of Sciences, School of Biology, 541 24
Thessaloniki, Greece, Tel: 30-231-998301, Fax: 30-231-998256,
Van Stappen Gilbert, Laboratory of Aquaculture & Artemia Reference Center, Ghent
University,
Belgium,
Tel:
32-9-2643754,
Fax:
32-9-2644193,
e-mail:
gilbert.vanstappen@UGent.be
Xin Naihong, Salt Research Institute, Yingkou Road 831, 300450 Tanggu, Tianjin, PR
China, Tel: 86-22-25301094, Fax: 86-22-25897596, e-mail: srisalt@public.tpt.tj.cn
4. Workshop Conclusions and Recommendations
15
The Chile regional workshop took place in Puerto Varas, southern Chile, November 17-19,
2003. As in previous meetings the first two days were devoted to presentations by each
member of the consortium aimed at reporting their progress regarding the project aims. 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.
4.1. Genetic research in Latin America
The first publications on Artemia genetic resources from some Latin American countries can
be associated to the work of Beardmore (Beardmore & Abreu-Grobois, 1983) and the training
of people in the UK (in collaboration with the Artemia Reference Center of the RUG, INCO
project coordinator, Belgium) on the biology and population genetics of Artemia. The basic
characterisation work considered species identification by means of karyological and
allozyme traits. Thereafter, and coupled to the advantages of Artemia for evolutionary studies
along with the variety and peculiar characteristics of the Artemia habitats in Latin America
(see Van Stappen, 2002), interesting questions in relation to adaptation and speciation have
been raised and tackled (reviews by Abreu-Grobois, 1987; Gajardo et al. 2002).
The Artemia Biodiversity project has greatly expanded this initial effort by gathering people
from key countries in the region, making available new tools (DNA markers), and training
people in their use for initial characterisation of local Artemia resources. The availability of
new genetic markers has also allowed to set a database on global Artemia resources (PCRRFLP), either for authentification of commercial samples or quantification and comparison of
global Artemia biodiversity (Bossier et al., 2004). This database offers valuable (comparable)
information to identify new samples and populations, track down their origin and to study the
phylogenetic and phylogeographic pattern of the superspecies A. franciscana. Last but not
least, the availability of new genetic markers has allowed, in certain cases (Chile, for
example) to confront the DNA data against those obtained with more traditional genetic tools
(cytogenetic and starch electrophoresis) (Gajardo et al., in press).
4.2. Status of the genetic characterization of the New World Artemia species found in
Latin American countries.
Rapid shaping and reshaping of the genetic structure of Artemia populations in nature is
expected at different geographic scales due to the island-like nature of Artemia environments
and the occurrence of successive cycles of changes in population size. Hence the
differentiation of local populations as a by-product of restricted gene flow, selection to the
local environment and genetic drift is common. Not surprisingly, Artemia populations are
highly heterogeneous genetically and display great variability in life history traits throughout
their distribution (Gajardo et al., 2002). The identification, management and conservation of
these resources are therefore highly topical for the assessment of Artemia biodiversity.
Nonetheless, genetic characterisation has been limited so far in Latin America, and this is in
part due to the shortage of people trained in the concepts and tools of population genetics and
evolutionary biology.
Two bisexual species, namely A. franciscana and A. persimilis, are found in the New World.
The former is widely distributed over numerous localities in North, Central and South
America, whilst A. persimilis was until very recently believed to be confined to some
localities in Argentina (Triantaphyllidis et al., 1998). However, the finding of the two species
in Chile (Gajardo et al. 1998, 1999) and later on in Argentina (Papeschi et al. 2000), two
16
countries isolated by the Andes mountains and sharing quite similar latitudinal range all
along, has set a new scenario to study the evolutionary relationships between them. The
following lines are a brief account of the genetic knowledge of both species in these countries.
a) The case of Chile
Artemia populations from most of the Chilean sites listed by Van Stappen (2002) have been
characterised karyologically (Colihueque & Gajardo, 1996; Gajardo et al., 2001), by the use
of allozyme markers (Gajardo & Beardmore, 1993, Gajardo et al., 1995, 1999), and by
laboratory cross-fertility tests (Gajardo et al., 2001ab). These studies demonstrated that A.
franciscana is the most widely distributed species in Chile (and in adjacent countries, see
Gajardo et al., 1995), either in coastal or inland lagoons and salt lakes between regions I and
VI (between 20 and 32° latitude south) (Gajardo et al., 1992, Gajardo & Beardmore, 1993).
The occurrence of A. persimilis in Chile is, however, geographically disjunct (below 40°
latitude south). The Artemia persimilis from Chile differs from A. franciscana by the presence
of one extra pair of chromosomes (2n = 44) and a lower number of chromocentres (Alu I-type
of repetitive DNA) (Gajardo et al., 2001). A broadly good correspondence exists between
morphological and genetic (mostly allozymic) traits (see Gajardo & Beardmore, 2001) with
regard to species demarcation. However, there are some populations that tend to diverge from
A. franciscana and/or resemble A. persimilis. Hence, the development and use of new genetic
markers (PCR-RFLPs) in the framework of the Artemia Biodiversity project has been useful
to investigate whether the conclusions drawn from traditional comparative tools (Gajardo &
Beardmore, 2001) are congruent with the pattern of genetic divergence depicted by RFLP
analysis of mtDNA. Such study (Gajardo et al., in press) has shown that no mitDNA
haplotypes are shared between the two species. Five restriction enzymes produced speciesspecific patterns, enabling the unambiguous assignment of populations to species. Very high
(100%) bootstrap values supported the clustering of haplotypes in two groups corresponding
to the two species. The two species were clearly differentiated with average sequence
divergence between them of 12.3%. High genetic differentiation was also found among conspecific populations of A. franciscana with an FST estimate of 91%.
The mtDNA results of this study show a broadly similar pattern to those of previous allozyme
and nuclear DNA analyses, with both New World species appearing as highly divergent. The
presence of A. persimilis in southern Chile (Chilean Patagonia) was confirmed. Hence, a
species previously regarded as geographically restricted mainly in Argentina, appears
expanding its range. Populations of A. franciscana appear highly structured with a level of
inter-population genetic differentiation much higher at mtDNA than previously reported with
allozymes. These populations form two groups according to their geographical distribution.
The identification of population-specific genetic markers for A. persimilis and A. franciscana
will help to tackle further aspects related to the speciation patterns of these species.
b) The case of Argentina
Different localities have been surveyed in Argentina and characterised by means of
cytogenetic methods (Papeschi et al., 2000), a study that suggested the presence of A.
franciscana in this country. But coincidentally to what occurs in Chile, the species is
geographically segregated from A. persimilis. New populations (Pampa de las Salinas, San
Luis; Salinas Grandes, Córdoba; La Antigua, La Rioja and Salitral de la Vidriera, Buenos
Aires) are currently being analysed by different techniques such as conventional staining, C,
DAPI and Nor-Ag banding (G. Cohen, pers. com.). The results obtained so far suggest: i) the
haploid and diploid numbers as well as the chromocentre numbers in interphasic cells of
nauplii are compatible with Artemia franciscana (first three populations). These populations
exhibit between 1 to 4 nucleoli in somatic cells implying at least two pairs NOR; ii) the
population of Salitral de la Vidriera showed the number and size of chromosomes
corresponding to A. persimilis. In spite of the fact that the reference population (A. persimilis
17
from Salinas Grandes de Hidalgo, La Pampa) lacked completely heterochromatin, the
population of Salitral de la Vidriera showed one or two small-sized chromocenters, revealing
some intraspecific differences.
From what has been so far observed, a latitudinal limit between the Argentinean populations
assigned to A franciscana and to A. persimilis seems to exist. Hence 35 °- 36° S seems to be
the southern limit for the distribution of A. franciscana. Below this latitude populations
belong to A. persimilis. This is in agreement to what has been reported for Chile (see above).
However, there are exceptions and so the Las Tunas population (33° 44´S) would very likely
be a location where occasional hybridization could be occurring (as hypothetised in Papeschi
et al., 2000) between both species, according to morphological data of adults, biometry of
cysts, cytogenetic and cross-breeding data (not published). This hybridization may be due to
the permanent contribution of cysts from the closer populations of both species to Las Tunas
Lagoon by means of winds or waterfowls. Since individuals with the morphotype of A.
persimilis were not ever observed in several Las Tunas Lagoon samplings, probably this
population is made up of hybrids and introgressants in addition to the parental form, A.
franciscana. Therefore the existence of a wide zone, rather than a narrow one, is believed to
exist where the distribution of both species overlaps (a hybrid zone). As the Pichilemu
population (Chile) is located at this latitudinal range, and since the specific identification of
this population has been rather controversial, the hypothesis of hybridization could be also
valid for this location. Immediate plans consider joining efforts between the Argentinean and
Chilean INCO partners in order to address this interesting problem.
Presently, we are looking for some new techniques to confirm the presence of hybridization in
Argentina, such as the Genome In Situ Hybridization technique (GISH). This technique will
allow to assess genome affinity between A. franciscana and A. persimilis, and to obtain a
better characterization of the heteromorphic bivalents in supposedly hybrid individuals. So far
DNA from A. franciscana from Great Salt Lake, USA, A. persimilis from Salinas Grandes de
Hidalgo and DNA from individuals from Mar Chiquita Lagoon has been isolated, quantified
and labelled by nick translation with biotin 14-dUTP (Bionick Labeling system, GIBCO
BRL). Nauplii of A. franciscana and A. persimilis (12-24 h) previously squashed in acetic
acid have been subject to the in situ technique using the labeled DNA of each species on the
slides of the same species, in order to adjust the hybridization conditions. We have obtained
good results in A. franciscana slides, but in A. persimilis the hybridization signal was rather
weak. Our next steps are aimed at improve the results with A. persimilis and to perform the
cross-hybridization between both species (i.e. A. franciscana DNA hybridized on A.
persimilis slides and vice versa) (data not published).
c) Other countries: Brazil, Mexico and Colombia
In contrast to the area involved and the Artemia sites already described, the genetic
characterisation in these countries is relatively scarce. Camara et al. (2003) studied the
molecular genetic diversity of three A. franciscana populations (Macau, Galinhos and Areia
Branca/Grossos) from northeastern Brazil, by means of RFLP (Restriction Fragment Length
Polymorphism) analysis of their mtDNA. Four restriction enzymes, which had already been
found to give species-specific haplotypes for A. franciscana, differentiate its southern or
northern origin and indicate variation within populations of A. franciscana (unpublished
data), were used. Fragment patterns observed among these Brazilian Artemia populations in
part of the 16S RNA region showed a consistent homogeneity as only one single composite
haplotype (AAAA) occurred in 70 individuals scored. The RFLP data obtained in this
preliminary characterization confirmed that the feral populations of A. franciscana found in
the state of Rio Grande do Norte, northeastern Brazil, belong to the A. franciscana
superspecies. In addition, their proposed origin from San Francisco Bay (USA) cysts, was
18
clearly demonstrated. This second information corroborated a previous report by Gajardo et
al. (1995) based on allozyme evidence derived from the Macau population.
Mexican Artemia populations from the Yucatan peninsula have been analysed by starch
electrophoresis (Torrentera, 1993) and cytogenetic traits (Torrentera & Abreu-Grobois, 2002),
and these data show that Mexican populations are quite distinct from the reference A.
franciscana samples (San Francisco Bay and Great Salt Lake, USA). Hence, according to
these authors, Mexican Artemia, or at least those populations from the Yucatan Peninsula,
would conform a well-differentiated group within the A. franciscana superspecies.
On the other hand, the Mexican partner of the consortium has sent samples to the Artemia
Reference Center (RUG, INCO partner 1, Belgium) to be analysed by RFLP. The preliminary
results are discussed further.
4.3. The Latin American samples in the context of global genetic Artemia (RFLP
database) study
The dendrogram (not included) summarizes the results that have been obtained on Artemia
samples from the American continent, considering the RFLP pattern of the 1500bp
mitochondrial rDNA fragment (see Bossier et al., 2004). It is based on the restriction enzyme
(RE, HpaII) for which all samples have been checked with positive results (e.g. produce
restriction fragments that are common to all A. franciscana samples), though a number of
other RE have been tested. All analyses were made using a small amount of cysts as starting
material, except for the samples from Chili, where individual adults were used. Although
these results are quite preliminary, especially those obtained on Mexican and Chilean
samples, the HpaII restriction enzyme is very useful. Most of the A. franciscana samples
display an identical pattern and especially a 240 bp HpaII fragment seems to be typical to A.
franciscana. So far only one sample from Coahuila State in Mexico seems to make an
exception to the rule.
The dendrogram confirms the presence of two species on the continent, namely A.
franciscana and A. persimilis. The latter does not seem to be restricted to Argentina but can
be found west of the Andes in Chile below 35 ° latitude South.
All the individual restriction digests and the summarizing dendrogram confirm that the
diversity within A. franciscana is big. It seems to be possible to distinguish between A.
franciscana from San Francisco Bay and Great Salt Lake using the HaeIII (except for samples
1287). The two samples from Canada on the other hand seem to be surprisingly different from
the other samples of North America. The Mexican samples are very diverse as well. The
summarizing dendrogram seems to suggest that one group of samples is more related to
samples from North America, while another group of samples, including those originating
from Coahuila, Oxacaca, Sinaloa and Yucatan state display more similarity to samples from
Venezuela and one sample from the Netherlands Antilles. The Chilean samples cluster
together. On the basis of the current data it is difficult to speculate about their relationship to
other samples.
4.4. Evaluation and Recommendations


The 1500 bp rDNA fragment displays, as detected with a limited number of
restriction enzymes, a high diversity among samples from American continent.
An exhaustive RFLP analysis of this fragment or sequencing data on that fragment
could constitute the appropriate input for a detailed phylogenetic and
phylogeographic analysis of the superspecies A. franciscana.
19





It was shown that the PCR amplification of the fragment can be obtained starting
from total DNA extracted from a single cyst, which does not necessarily has to be
viable. This opens the possibility to perform this phylogenetic study on the basis of
cyst material kept in the collection of various members of the INCO Artemia
biodiversity project.
The data gathered (see Fig. 1) could constitute a good guide for choosing the
locations for such a study. They also seem to indicate that salt lakes in Mexico might
play a pivotal role in the phylogeography of A. franciscana.
A crucial merit of the Artemia Biodiversity project is to have trained South American
scientists on genetic techniques and basic genetic concepts.
More regional, DC-DC interaction is advisable in the future, as some expertise and/or
techniques to share are actually available, or will be available sometime soon.
In spite of the irrefutable value of molecular genetic research, other aspects of
Artemia research should not be neglected, e.g. ecology, biogeography, etc.
Understanding the evolutionary biology of Artemia requires the complementary input
of the study of the environment, distribution etc.
4.5. References
Abreu-Grobois, F.A. 1987. A review of the genetics of Artemia. In: P. Sorgeloos, D.A.
Bengtson, W. Decleir and E. Jaspers (eds.), Artemia Research and its Applications Vol. 1,
Morphology, Genetics, Strain characterization, Toxicology, Universa Press, Wetteren,
Belgium, pp. 61-99.
Beardmore, J. A., Abreu-Grobois, F. A. 1983. Taxonomy and evolution in the brine shrimp
Artemia. In G. S. Oxford and D. Rollinson (eds), Protein polymorphism: adaptive and
taxonomic significance. The systematic association Special Vol. nº 24, academic Press,
London, pp 153-164.
Bossier, P., Wang, X., Catania, F., Dooms, S., Van Stappen, G., Naessens, E., Sorgeloos, P.
2004. An RFLP databse for authentication of commercial cyst samples of the brine shrimp
Artemia spp. (International Study on Artemia LXX). Aquaculture, 231: 93-112.
Camara, M. R., Triantafyllidis, A., Baxevanis, A.D., Abatzopoulos, T.J. 2003. Preliminary
characterization of Brazilian Artemia franciscana populations by means of mtDNA RFLP
analysis. In: Abstracts, Artemia Biodiversity, Chile Regional Workshop, November, 2003,
Puerto Varas, Chile.
Gajardo, G., Beardmore, J.A. 1993. Electrophoretic evidence suggests that the Artemia found in
Chile is A. franciscana Kellogg. Hydrobiologia, 257:65-71.
Gajardo, G., Beardmore, J. A. 2001. Coadaptation: lessons from the brine shrimp Artemia,
“the aquatic Drosophila (Crustacea, Anostraca). Rev. Chil. Hist. Nat., 74: 65-72
Gajardo, G., Da Conceiçao, M., Weber, L. Beardmore, J.A. 1995. Genetic variability and
interpopulational differences in Artemia strains from South America. Hydrobiologia 302: 2129.
Gajardo, G., Wilson, R., Zuñiga, O. 1992. Report on the occurrence of Artemia in a saline
deposit of the Chilean Andes. Crustaceana (Holland), 63 (2): 169-174.
Gajardo, G., Beardmore, J. A. Sorgeloos, P. 2001a. International Study on Artemia. LXII.
Genomic relationships between Artemia franciscana and A. persimilis, inferred from
chromocentre numbers. Heredity 87 (2): 172-177.
20
Gajardo, G., Colihueque, N., Parraguez, M., Sorgeloos, P. 1998. International study on Artemia.
LVIII. Morphological differentiation and reproductive isolation of Artemia populations from
South America. International Journal of Salt Lake Research, 7 (2): 133-151.
Gajardo, G., Mercado, C., Beardmore, J. A., Sorgeloos, P. 1999. International Study on
Artemia. LX. Allozyme data suggest that a new Artemia population in southern Chile (50ª 29’
S; 73º 45’ W) is A. persimilis. Hydrobiologia, 405: 117-123.
Gajardo, G., Parraguez, P., Beardmore J.A., Sorgeloos, P. 2001b. Reproduction in the brine
shrimp Artemia: evolutionary relevance of cross-fertility tests. Journal of Zoology 253: 25-32.
Gajardo, G., Abatzopoulos, Th. J., Kappas, I., Beardmore, J. A. 2002. Evolution and
speciation. In: Artemia: Basic and Applied Biology, 225-250. Abatzopoulos T.J. et al. (eds).
Kluwer Academic Publishers, Dordrecht, the Netherlands
Gajardo, G., Crespo, J., Triantafyllidis, A., Tzika, A., Baxevanis, A., Kappas, I.,
Abatzopoulos, Th. J. (in press). Species identification of Chilean Artemia populations based
on mitochondrial DNA RFLP analysis. Journal of Biogeography.
Papeschi, A. G., Cohen, R. G., Pastorino, X. I., Amat, F. 2000. Cytogenetic proof that the
brine shrimp Artemia franciscana (Crustacea, Branchiopoda) is found in Argentina. Hereditas
133: 159-166.
Torrentera, L. 1993. Ecology and evolution of Yucatan Peninsula Artemia. Ph. D thesis.
University of Wisconsin-Madison, Madison, USA: 104 pp.
Torrentera, L., Abreu-Grobois, F. A. 2002. Cytogenetic variability and differentiation in
Artemia (Branchiopoda: Anostraca) populations from the Yucatan Peninsula, Mexico.
Hydrobiologia 486: 303-314.
Triantaphyllidis, G.V., Abatzopoulos, T.J., Sorgeloos, P. 1998. Review of the biogeography
of the genus Artemia (Crustacea, Anostraca). Journal of Biogeography 25: 213-226.
Van Stappen, G. 2002. Zoogeography: 171-224. In: Artemia: Basic and Applied Biology.
Abatzopoulos, Th. J. et al. (eds). Kluwer Academic Publishers, Dordrecht, the Netherlands
5. Action plan INCO Consortium final year: Joint activities
In order to promote the visibility of the INCO Artemia Biodiversity Consortium as a group,
and to strengthen the links between Consortium Members beyond the project period, the
Consortium partners agreed to proceed to the realization of a minimum of 2 joint publications,
and to continue with the integration of Cyst Bank and Artemia Site databases. The
Consortium considers these publications and databases to represent a direct and essential
contribution to the realization of the Project Objectives, as specified sub 1.3 of this report.
5.1. Publication 1




Objective: Compilation/Manual of Protocols and Recommendations (exact title
still to be defined) for Artemia characterisation studies
Collective authorship of 15 partners
No separate authorship for individual chapters/protocols
To be finalized towards end of Project period
21


Format: Special Issue of journal, e.g. Hydrobiologia, Public Library of Science,
Journal of Biological Research
Timing and deadlines have been discussed and agreed upon by Consortium
Partners
List of protocols/chapters
(in tentative order of appearance; tentative titles)







Preface/Introduction (to be written by non-Consortium Artemia expert(s), e.g. J.
Clegg, T. MacRae, J. Beardmore
Artemia sampling, abiotic and biotic factors, sample preservation; diapause
(responsible: partner 1)
Genetics and molecular markers:
 cytogenetics (responsible: partner 3)
 cross-breeding (responsible: partner 14)
 molecular markers (responsible: partner 3)
Biometrics and morphometrics (responsible: partner 4)
 cysts (responsible: partner 1)
 nauplii (responsible: partner 12)
 adults (responsible: partner 4)
 culture conditions (responsible: partner 4)
 Scanning Electron Microscopy (responsible: partner 5)
 statistical analysis of data (responsible: partner 12)
Phenotypic response under different environmental conditions (responsible:
partner 6)
Sustainable exploitation (responsible: partner 1)
Artemia conservation and invasion patterns (responsible: partner 14)
5.2. Publication 2




Publication in peer-reviewed journal (e.g. Trends in Ecology and Evolution)
Working title: “A. franciscana: the invasive species in the genus”
Joint authorship of all INCO partners.
Concrete planning, timing and outline to be made at 3rd Regional Workshop
(Urmia, Iran, October 2004)
5.3. Databank of Artemia Cyst Samples
 Objective: to make contents of all individual cyst collections available online
 Presently available: Cyst collections of ARC, SRI, AUTH, CSIC, Sapienza,
INAT, UU
5.4. Databank of Artemia Sites
 Objective: to design a Microsoft Access based database of known Artemia sites;
worldwide status of knowledge early 2004
 Minimum to be accomplished within project period: database for countries of
INCO members (= Greece, Spain, Italy, Tunisia, Mexico, Chile, Brazil,
Argentina, Vietnam, China, India, South Africa, Iran).
 Consortium Members discussed and agreed on procedure to design software
template, to make data available on occurrence of Artemia in respective countries,
and to introduce data into Databank.
22
Work Package & Deliverable 4:
Third Regional Workshop
Urmia, Iran, September 21-25, 2004
1. Introductory Note
This concluding workshop differed from the previous ones as it was declared open for a much
broader national and regional audience. Participation was supported for scientists from nonProject member countries, UK, USA, Russia, Iraq, Pakistan, Kazakhstan and Uzbekistan, to
report about the status of Artemia in their respective countries in order to add more scientific
inputs to the workshop and collect more information about Artemia biodiversity in the Middle
East and Central Asia. The workshop was further attended by a large number of interested
Iranian scientists and students; 37 papers were presented in the workshop, 14 by the Iranians
and 23 by other INCO member and non-member participants from other countries. In addition
to EU-INCO funding, the workshop was supported financially by the Iranian Center for
Research and International Collaborations, the Iranian Research Organization for Science and
Technology, the Iranian Organization for Protection of Environment, and Urmia University.
The workshop was attended by over 150 people from 20 countries.
Keywords of the Workshop:
 Artemia resources of Central Asia
 Sustainable exploitation
 Final evaluation of training courses and previous workshops
A separate workshop was organized for all those interested on Resource Assessment of
Artemia populations in inland salt lakes.
2. Programme
Presentations
Chair of all sessions: Prof. John Beardmore
Tuesday, September 21
Opening Ceremony and Welcome Addresses
17:30-18:00 G. Sadeghi (Chancellor, Urmia University, Iran)
N. Agh (Workshop Director, Urmia University, Iran)
18:00-18:15 M. Ebtekar (Vice-President, Islamic Republic of Iran & President, Iranian
Organization for Protection of Environment)
18:15-18:25 L. Saeedi (Managing Director, Iranian Fisheries Organization, Iran)
18:25-18:35 S. Rezvani (Chairman, Iranian Fisheries Research Organization, IFRO, Iran)
18:35-18:55 P. Sorgeloos (Project Coordinator, Ghent University, Belgium)
18:55-19:10 C.E. Nauen (Representative of the International Scientific Cooperation, European
Commission, Belgium)
19:10-19:20 M.S. Mohammad (President, University of Erbil, Iraq)
19:20-19:30 J. Beardmore (Honorary President of the workshop, University of Swansea, UK)
Wednesday, September 22
23
Methodological Aspects of Artemia Biodiversity Studies
8:30-9:00
J. Abatzopoulos (Aristotle University of Thessaloniky, AUTH, Gerece)
Title: The contribution of DNA markers in Artemia biodiversity and future
perspectives
9:00-9:25
F. Amat (Instituto de Acuicultura Torre de la Sal, CSIC, Spain)
Title: Artemia biodiversity: current global resources and their sustainable exploitation
9:25-9:50
G. Mura (Universita La Sapienza, Italy)
Title: General remarks on the usefulness and limits of morphological characters in
species separation within the genus
Artemia Biodiversity in Latin America
9:50-10:20
T. Castro Barrera* (Universidad Autonoma Metropolitana, UAMX, Mexico), M.J.
Castro (UAMX, Mexico), M.G. Castro (UAMX, Mexico), S.A. Malpica (UAMX,
Mexico), A. De Lara (UAMX, Mexico)
Title: Cross breeding and morphometric studies in seven Artemia franciscana strains
from Mexico
10:20-10:45 G. Gajardo* (University of Los Lagos, ULL, Chile), P. Beristain (ULL, Chile)
Title: Evolutionary biology and Artemia biodiversity in Chile: how much do we know
now?
Artemia Biodiversity in Africa
11:05-11:30 H. Kaiser (Rhodes University, RU, South Africa)
Title: Artemia biodiversity in Africa - current status of Artemia distribution with a
view towards further work
11:30-11:55 M.S. Romdhane* (Ecosystèmes et Ressources Aquatiques, INAT, Tunisia), H. Ben
Naceur (INAT, Tunisia), S. Hamrouni (INAT, Tunisia), A. Ben Rejeb Jenhani
(INAT, Tunisia), M. El Cafsi (INAT, Tunisia)
Title: Biological and biochemical characterisation of Artemia from Tunisian wetlands
Iran: the Artemia Populations of the Lake Urmia Ecosystem
11:55-12:25 T.J. Abatzopoulos (Aristotle University of Thessaloniky, AUTH, Greece), G.V.
Triantaphyllidis (AUTH, Greece), G. Criel (Ghent University, Belgium), A.D.
Baxevanis* (AUTH, Greece), G. Van Stappen (Ghent University, Belgium), P.
Sorgeloos (Ghent University, Belgium)
Title: Artemia urmiana Günther: reproductive and lifespan characteristics, cyst and
naupliar biometrics, HUFA profiles, chorion structure and cyst buoyancy
12:25-12:45 A. Karbasi (Directorate of Shakid Kalantari Free way on Lake Urmia, Iran)
Title: Environmental impact assessment of Urmia Lake Causeway project
12:45-13:05 R. Seddighi (Atomic Energy organization of Iran, Iran)
Title: Effect of He-Ne Laser on hatching characteristics of Artemia urmiana
13:05-13:20 M. Hafezieh (Iranian Fisheries Research Organization, IFRO, Iran)
Title: Effects of environmental changes on reproduction mode of Artemia urmiana in
Lake Urmia
14:30-14:55 F. Noori* (Urmia University, UU, Iran), N. Agh (UU, Iran)
Title: Introducing a different enrichment material for improving HUFA levels in
Artemia urmiana nauplii and juvenile
14:55-15:20 O. Tabiee* (Azad University of Arsanjan, Iran), G. Azari Takami (Tehran
University, Iran), N. Agh (Urmia University, Iran)
Title: The effect of different HUFA enrichment emulsions on the nutritional value of
Artemia urmiana
24
15:20-15:45 A. Eimanifar* (Iranian Fisheries Research Organization, IFRO, Iran), S. Rezvani
(IFRO, Iran), J. Carapetian (Urmia University, Iran)
Title: A molecular PCR-RFLP and morphological study on floating cyst stock of
Artemia urmiana from Urmia Lake
Thursday, September23
Artemia Biodiversity in Asia
8:30-9:00
S.K. Nasiri (University of Baghdad, Iraq)
Title: Studies about Artemia from Iraq
9:00-9:30
M. Peter Marian* (M.S. University, IART, India), J.A. Christopher John (IART,
India), M.M. Babu (IART, India), G. Immanuel (IART, India), T. Sivaram (IART,
India)
Title: Threat to the Indian Artemia biodiversity by aquaculture and chemical
industries: need for Artemia genome conservation in South Asia
9:30-10:00
R. Sultana* (Food & Marine Resources Research Center, Pakistan), W. Ali, (Food &
Marine Resources Research Center, Pakistan), S.G. Abbas Shah (Food & Marine
Resources Research Center, Pakistan)
Title: On the Artemia populations from Pakistan
10:00-10:30 X. Naihong* (Salt Research Institute, SRI, China), Y. Xiuling (SRI, China)
Title: Strain characterization of Artemia from 9 inland salt lakes of China
11:00-11:30 A.D. Baxevanis (Aristotle University of Thessaloniki, AUTH, Greece), G.V.
Triantaphyllidis (AUTH, Greece), A. Triantaphyllidis (AUTH, Greece), I.
Kappas* (AUTH, Greece), T.J. Abatzopoulos (AUTH, Greece)
Title: Artemia tibetiana: truth and errors on its species status
11:30-12:00 P. Bossier* (Ghent University, Belgium), G.Van Stappen (Ghent University,
Belgium), Y. Haiying (Salt Research Institute, SRI, China), W. Xiaomei (SRI,
China), X. Naihong (SRI, China), D. Deloof (Sea Fisheries Department, CLO-DVZ,
Belgium), D. Delbare (CLO-DVZ, Belgium), P. Sorgeloos (Ghent University,
Belgium)
Title: Evaluation of mixed status of Asian Artemia populations through RFLP-analysis
12:00-12:30 I. Mirabdullayev* (Institute of Zoology, Uzbekistan), N.I. Jumaniyozova (Urgench
State University, Uzbekistan)
Title: Biodiversity and distribution of Artemia in Uzbekistan
Artemia Populations from Solar Saltworks
14:00-14:30 M.R. Camara (Universidade Federal do Rio Grande do Norte, UFRN, Brazil)
Title: Is small-scale Artemia farming a sustainable alternative to brine shrimp
harvesting in Brazilian saltworks?
14:30-15:00 H. Nguyen Van* (Cantho University, CTU, Vietnam), N. Thi Hong (CTU, Vietnam)
Title: Adaptation of Artemia franciscana in Vinchu
Sustainable Exploitation of Artemia Resources in Central Asia
15:00-15:30 G. Van Stappen* (Ghent University, Belgium), H. Negarestan* (Iranian Fisheries
Research Organization, IFRO, Iran), N. Agh (Urmia University, UU, Iran), R.
Manaffar (UU, Iran), B. Agashbar (UU, Iran), P. Sorgeloos (Ghent University,
Belgium)
Title: Artemia resources in Lake Urmia, past and present
15:30-15:50 L. Vdovchenko (Siberian Research and Design Institute of Fisheries, Russia)
Title: Artemia in lakes of Western Siberia
25
15:50-16:10 E. Boyko (Siberian Research and Design Institute of Fisheries, Russia)
Title: Identification of Artemia from Russia
16:10-16:30 A. Kurtulus (INVE, Kazakhstan)
Title: Exploitation of Artemia resources from Kazakhstan
Friday, September 24
Artemia Biodiversity Project members
8:30-17:00
Artemia Biodiversity concluding discussions
Participants other than Project members
8:00-14:00
Excursions to Badin Abad rainbow trout proliferation and culture complex near
Sardasht City
Saturday, September 25
Resource Assessment Workshop
8:00-8:40
B. Marden (Parliament Fisheries LCC, USA)
Title: Resource assessment methods applied in GSL, Russia and Central Asian
countries
8:40-9:00
N. Agh (Urmia University, UU, Iran)
Title: Laboratory analysis and calculations method for resource assessment of Artemia
9:00-10:00
Open discussion about resource assessment
10:00-14:00 Excursion to Lake Urmia by ship & demonstration of sampling methods
3. Participants
Abatzopoulos Theodore, Department of Genetics, Development & Molecular Biology,
Faculty of Sciences, School of Biology, Aristotle University of Thessaloniki, 541 24
Thessaloniki,
Greece,
Tel:
30-231-998301,
Fax:
30-231-998256,
e-mail:
abatzop@bio.auth.gr
Abbas Shah Seyed Ghulam, University of Kerachi, Pakistan
Abbasi Sepideh, Shiraz University, Shiraz, Iran
Agh Naser, Artemia & Aquatic Animals Research Center, Urmia University, Urmia, Iran
Ahmadi Reza, Iranian Fisheries Research Organization (IFRO), Urmia, Iran
Akbarpour Mehrzad, Faculty of Veterinary Medicine, Islamic Azad University, Urmia, Iran
Ali Akbari Mohammad Taghi, Bonab Radiation Research Center, Bonab, Iran
Amat Francisco, Instituto de Acuicultura de Torre de la Sal (Consejo Superior de
Investigaciones Científicas de España), Ribera de Cabanes (Castellón), Spain, Tel : 34-964319500, Fax : 34-964-319509; e-mail : amat@iats.csic.es
Amin Dezaye Ahmed Anwar, Salahaddin University, Erbil, Iraq
Amini Hamid, Environment Protection Organization of Iran, Tehran, Iran
Aminzadeh Narges, Center for International Resaerch and Collaborations, Ministry of
Science, Research & Technology, Tehran, Iran
Asadpour Yosefali, Natural Resources Research Center, Urmia, Iran
Asem Alireza, Faculty of Sciences, Department of Biology, Razi University, Kermanshah,
Iran
Atashbar Behrooz, Artemia & Aquatic Animals Research Center, Urmia University, Urmia,
Iran
Ayatollahi Mohammad, Environment Protection Organization of Iran, Tehran, Iran
Azari Takami Ghobad, Faculty of Veterinary, University of Tehran, Iran
Baldoras Mariabaty, Artemia & Aquatic Animals Research Center, Urmia, Iran
Barzani Abdulradha Kh., Director General of Veterinary Organization, Kurdistan, Iraq
26
Baxevanis Athanasios, Department of Genetics, Development & Molecular Biology, Faculty
of Sciences, School of Biology, Aristotle University of Thessaloniki, 541 24 Thessaloniki,
Greece, Tel: 30-231-998301, Fax: 30-231-998256
Beardmore John, University College of Swansea, School of Biological Sciences, University
of Wales, Swansea, UK
Beardmore Patricia, Swansea, UK
Bossier Peter, Laboratory of Aquaculture & Artemia Reference Center, Ghent University,
Rozier 44, B-9000 Gent, Belgium, Tel: 32-9-2643754, Fax: 32-9-2644193; e-mail:
Peter.Bossier@UGent.be
Boyko Elena, Tyumen Academy of Agricultural Sciences, Russia
Camara Marcos, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio
Grande do Norte, Campus Universitario, BR 101, 59072-970 Natal, Brazil, Tel:: 55-842154433, Fax: 55-84-6421815, e-mail: mrcamara@ufrnet.br
Castro Thalía, Departamento El Hombre y su Ambiente, Universidad Autónoma
Metropolitana-Xochimilco, Distrito Federal, Calz. Delz. Del Hueso No. 1100. col. Villa
Quietud, Mexico, 04960, D.F. Mexico. Tel: +52-5-4837151, Fax: +62-5-4837469; e-mail:
cabt7515@cueyatl.uam.mx
Dadjoi Saleh, Environment Protection Organization of Iran, Tehran, Iran
Dehghani Abbasali, Educational Center for Fishery Sciences of Persian Golf, Bandar Abbas,
Iran
Eimanifar Amin, Iranian Fisheries Research Organization (IFRO), P.O. Box 57135-1367,
Urmia, Iran
Elmi Amir Mohammad, Environment Protection Organization of Iran, Tehran, Iran
Falahatkar Bahram, University of Tarbiat Modarres, Tehran, Iran
Falahati Afagh, Fisheries Company of Iran, Fars Province, Shiraz, Iran
Farhangi Mehrdad, Tehran University, Tehran, Iran
Feiz Bakhsh Reza, Iranian Fisheries Organization, Tehran, Iran
Gajardo Gonzalo, Department of Basic Sciences/Laboratory of Genetics & Aquaculture,
Universidad de Los Lagos, P.O. Box 933, Osorno, Chile, Tel: 56-64-205293, Fax: 56-64239517,
e-mail: ggajardo@ulagos.cl
Ghaeni Mansoreh, Shahid Beheshti University, Tehran, Iran
Ghaforian Hossein, Director of Nuclear Research Center, Tehran, Iran
Hafezieh Mahmood, Iranian Fisheries Research Organization (IFRO), P.O. Box 14155-6116,
Tehran, Iran
Haggirad Mansour, Iranian Fishery Organization, Zanjan, Iran
Haji Salim H., University of Duhuk, Duhuk, Iraq
Hami Tabari Ahmad, Ecology section, Fisheries Research Center of Golestan province
(GFRC), P.O. Box 139, Gorgan, Iran
Harirchi Gooya, Center of International Research and Collaboration, Ministry of Science,
Research & Technology, Tehran, Iran
Hong Van Ngueyn Thi, Institute for Marine Aquaculture (IMA), Agriculture College, Can
Tho University (CTU), Vietnam
Hosseini Alireza, Environment Protection Organization of Iran, Tehran, Iran
Hosseini Najde Geramy Ebrahim, Artemia and Aquatic Animals Research Center, Urmia
University, Urmia, Iran
Jafarzadeh Ahamad, Director of Bonab Radiation Center, Bonab, Iran
Javeri Hayfa, University of Baghdad, Baghdad, Iraq
Kaiser Horst, Department of Ichthyology and Fisheries Science, Rhodes University,
Grahamstown, South Africa, Tel: 27-46-6038415, Fax: 27-46-6224827; e-mail:
h.Kaiser@ru.ac.za
27
Kappas Illias, Department of Genetics, Development & Molecular BIology, School of
Biology, Faculty of Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki,
Greece
Karbasi Abdulreza, Directorate of Shahid Kalantari Free Way on Lake Urmia, Iran
Khalid Asmat M., University of Duhuk, Duhuk, Iraq
Kurtulus Ali, INVE, Aktau, Kazakhstan
Lofti Govarchin Ghale Vahid, Artemia & Aquatic Animals Research Center, Urmia, Iran
Maaulood Bahram Kh., Deputy MInister of Agriculture, Kurdistan region, Iraq
Mahini Siamak, Iranian Fishery Organization, Tabriz, Iran
Malekpour Saadollah, Iranian Fishery Organization, Kerman, Iran
Manaffar Ramin, Artemia & Animals Research Center, Urmia University, Urmia, Iran
Mansouri Seyedeh Mansoreh, Iranian Fishery Organization, Bandar Abbas, Hormozgan,
Iran
Marden Brad, INVE Aquaculture, INVE Technologies, 5859 North Cottonwood Canyon
Rd., Mountain Green, Utah, 84050, USA
Marian Peter, Institute for Artemia Research and Training, Manonmaniam Sundaranar
University, Rajakkamangalam, 629 502, Kanyakumari District, Tamil Nadu, India, Tel: 914652-33680, Fax: 91-4652-21457; e-mail: petermar@md5.vsnl.net.in
Masnadani Saeid, Iranian Fishery Organization, Bandar Abbas, Hormozgan, Iran
Masoumian Mahmood, Iranian Fisheries Research Organization, (IFRO), P.O. Box 141556116, Tehran, Iran
Mehrannazdad Reza, Iranian Fisheries Research Organization, (IFRO), Urmia, Iran
Mohaghegh M.R., Agriculture and Natural Resources Research Center of Qom, P.O. Box
195, Qom, Iran
Mohammad Sadik Mohammad, Salahaddin University, Erbil, Kurdistan, Iraq
Mohammadyari Ali, Artemia & Aquatic Animals Research Center, Urmia, Iran
Mojahed Ekbatani Laela, Natural Resources Department, University of Tehran, Tehran,
Iran
Monsef Hamid, Jihad Keshavarzi, Tehran, Iran
Moshtaghian Abdomajid, Fisheries Company of Iran, Fars Province, Shiraz, Iran
Mikeal Ali, University of Duhuk, Duhuk, Iraq
Mirabdullayev Iskandar, Institute of Zoological Sciences, Tashkent, Uzbekistan
Mirzarar Seyed Saeid, Faculty of Veterinary, Tehran University, Tehran, Iran
Mura Graziella, Universita La Sapienza, Roma, Dipartimento di Biologia Animale e
dell'Uomo, Laboratorio di Zoologia Applicata, Italy
Naihong Xin, Salt Research Institute, Yingkou Road 831, 300450 Tanggu, Tianjin, PR
China, Tel: 86-22-25301094, Fax: 86-22-25897596, e-mail: srisalt@public.tpt.tj.cn
Nasiri Sufian K., University of Baghdad, Baghdad, Iraq
Nazarnazhad Habib, Faculty of Natural Resources, Urmia University, Urmia, Iran
Negarestan Hossein, Iranian Fisheries Research Organization, (IFRO), P.O. Box 141556116, Tehran, Iran
Nohe Khan Hamid, Bonab Radiation Research Center, Bonab, Iran
Noori Farzaneh, Artemia & Aquatic Animals Resaerch Center, Urmia University, Urmia57153, Iran
Noori Uhmer M., University of Duhuk, Duhuk, Iraq
Ownagh Abdulghaffar, Faculty of Veterinary Madicine, Urmia University, Urmia, Iran
Parsa Abdorahman, Iranian Fishery Organization, Chabahar, Iran
Pazouki Jamileh, Shahid Beheshty University, Tehran, Iran
Pourafshar Kazem Ghasemi, Iranian Fishery Organization, Hormozgan, Iran
Pourhokmi Ali Mohammad, Educational Center of Kolahi Port, Minab, Iran
Pour Reza Mohammad Hossein, Iranian Fishery Organization, Tehran, Iran
Qadiri Ali, Bonab Research Center, P.O. Box 56515-196 Bonab, Azarbayjan, I.R. Iran
Rabbani Mohammad, Iranian Nuclear Energy Organization, Tehran, Iran
28
Rashed Alwand Tahir, Salahaddin University, Erbil, Iraq
Razeghi Mohammad Ebrahim, Natural Resources Research Center of West Azarbaijan,
Urmia, Iran
Rezvani Sohrab, Iranian Fisheries Research Organization, (IFRO), Tehran, Iran
Romdhane Mohamed Salah, Institut National Agronomique de Tunisie (INAT), Université
de Tunis, Tunisia
Sanz Maria, Spain
Sadighi Bonabi Rasoul, Department of Physics, Sharif University of Technology, P.O.Box
11365-9161, Tehran, I.R. Iran
Sulayman Fahry Omar, Salahaddin University, Erbil, Iraq
Sultana Razia, Food & Marine Resources Research Center, PCSIR Labs Complex Karachi,
Karachi-75280, Pakistan
Tabiee Omid, Islamic Azad University, Arsanjan, Shiraz, Fars, Iran
Tehrani Aliasghar, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Tizkar Babak, High Educational Center of Mirza Kochek Khan, Rasht, Iran
Van Hoa Nguyen, Institute for Marine Aquaculture (IMA), Agriculture College, Can Tho
University (CTU), Vietnam
Van Stappen Gilbert, Laboratory of Aquaculture & Artemia Reference Center, Ghent
University,
Belgium,
Tel:
32-9-2643754,
Fax:
32-9-2644193;
e-mail:
gilbert.vanstappen@UGent.be
Vdovchenko Marina, Tyumen Academy of Agricultural Sciences, Russia
Yarmohammadi Mahtab, International Sturgeon Research Institute, Iranian Fishery
Research, Organization, Rasht, Iran
Yahya Zadeh Mir Yosef, Natural Research Center of West Azarbaijan, Urmia, Iran
Yakhchali Mohammad, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Yeganehkari Ataollah, Institute for Scientific-Applied Higher Education of Jihad Keshvarzi,
Tehran, Iran
Zabihpour Asadoolah, Iranian Fishery Organization, Ghazvin, Iran
4. Workshop Conclusions and Recommendations
Artemia offers a somewhat different approach to biodiversity that is often evaluated as species
richness. Although the genus is not relevant in terms of species, it represents a good example
of how biodiversity can be distributed in the highly structured population genetic architecture
of species. By describing and understanding the factors causing population genetic differences
within a species, over time and space, we approach the making of new species (speciation), in
other words, the origin of biodiversity. Both the species concept (important to cataloguing
biodiversity) and the speciation process are the framework for analysis. The latter should be
taken into consideration for restoring and/or conserving natural Artemia biodiversity. This is
particularly important as Artemia gene pools are often perturbed by man as a consequence of
the species’ aquaculture importance.
A. franciscana as invasive species
a) Bohai Bay area, China
The previous reports have indicated that species authentication making use of HpaII RFLP of
the mitochondrial rDNA is possible. This knowledge was used to verify the presence of A.
franciscana in the salt works of the Bohai Bay. The occurrence of A. franciscana in the Bohai
Bay was confirmed by genetic analysis and dates back to at least 1991. The genetic data
furthermore suggest that in a lot of samples from the Bohay Bay A. sinica is occurring. The
finding is totally new. It is not clear if this is naturally or the result of human release. The cooccurrence of 3 species in cyst samples is possible.
29
b) Western Mediterranean
The tasks developed during 2003 were based on the availability in the partners’ cysts bank of
cyst samples collected in the Western Mediterranean area from the beginning of the 80´s until
present. This availability, together with the prospection and recovering of brine shrimp cysts
from waterbird faeces and pellets in the Southwest of the Iberian Peninsula, enabled further
research, which provided evidence on the present distribution of autochthonous brine shrimp
populations (A. salina and diploid parthenogenetic Artemia) as well as of allochthonous A.
franciscana populations in the Western Mediterranean basin.
All saltworks in Italy (insular and peninsular) were again surveyed in order to collect animals
and cysts for analysis and updating of cystbank and distribution. This prospection showed the
presence of introduced A. franciscana in Margherita di Savoia salterns (confirmed by
different techniques: morphometry, biometry, morphology, molecular markers), while the
prospection in Sardinia and Sicily did not show the presence of that exotic species. In
Sardinia it was possible to verify the presence of bisexual autochthonous A. salina
populations free or co-occurring with parthenogenetic diploid populations. In San Pietro
island a site was sampled, producing a mixed bisexual A. persimilis plus parthenogenetic
diploid population. This A. persimilis population could be a remnant of that found in San
Bartolomeo by Piccinelli and Prosdocimi (1968) used for the first description of this species.
In Sicily it was possible to verify the generalized presence of autochthonous Artemia salina
populations in the two important saltwork complexes still under exploitation (several
saltworks, visited and sampled at previous occasions have been abandoned since).
The prospection conducted in Portugal showed a generalized presence of A. franciscana
populations in the operational salterns in the southern region. Also in Portugal there has been
a drastic decrease of operational saltworks throughout the past decade, most of them being
abandoned or changed into rice fields or aquaculture facilities.
c) South Asia: India and Sri Lanka
Continued research of coastal Indian populations in Tuticorin province showed increased
occurrence (sex ratio study and mitochondrial RFLP analyses) of mixed parthenogeneticbisexual Artemia (in contrast to a prospection made in 1996, when only parthenogenetic
Artemia were found).
Artemia in Sri Lanka has been classified as parthenogenetic, occurring naturally in coastal
salterns, but a survey of the area (see annexes, CTU) showed an increasing presence of
bisexual Artemia, as a consequence of inoculation of San Francisco Bay A. franciscana in
certain saltworks, and its dispersion over a larger area through the local migrating avifauna.
Artemia biodiversity in Africa
The genetic characterization of South African Artemia populations was continued. Six of
them were partially characterised using molecular techniques (PCR-RFLP). Preliminary
results revealed that the majority of the sampled sites had mixed Artemia populations
(parthenogenetic and bisexual strains). It should be noted that the existing literature on
Artemia biodiversity in South Africa is very limited and this effort is expected to provide new
and valuable information. As for North Africa, the cysts sampling and environment
measurement has been continued around Tunisian salinas and salt marshes. Nevertheless, as
the season 2003/2004 was exceptionally rainy; some sebkhas reveal a drastic change from
saline to fresh water, and less cysts were collected.
Artemia biodiversity in southern South America
30
As stated above, the genus Artemia represents a good example of how biodiversity can be
distributed in the highly structured population genetic architecture of species. The case of
Chile and Argentina is an example of this. Thus, our focus has firstly been to highlight the
relevance and uniqueness of Chilean biodiversity in relation to global Artemia resources. The
initial expectation that Chile might have interesting opportunities to offer in relation to
speciation and biodiversity studies (see previous reports) has been confirmed. Results
obtained in 2004 can be summarized as follows:
 New genetic markers (RFLP and mitochondrial DNA sequences) were assessed,
and so the genetic tools available for analysis as well as the existing database on
Chilean resources have been expanded.
 A key new morphological trait has been also added (scanning microscopy of
frontal knobs), hence the Chilean database now includes information from DNA
sequences to 3D-morphology.
 Progress in molecular analysis revealed high haplotype diversity of Chilean
populations and population-specific haplotypes.
 Populations of A. franciscana are genetically heterogeneous as revealed by the
two independent groups resulting from the cluster analysis based on mitDNA
sequences. Chilean populations are even separated from those in other locations.
Cooperation, also with alternative, national funding, allowed the development of broad
research on the biodiversity status of the species of the genus Artemia in Argentina. Previous
results were published reporting on the presence of A. persimilis and A. franciscana
populations in Argentinean hypersaline ecosystems. The present information available allows
to support the actual presence of Artemia franciscana in 5 biotopes and of A. persimilis in, at
least 21 biotopes. In this study also indications were found for the presence of A. persimilis in
Peru, and in the Chilean Tierra del Fuego, exceeding the distribution of Argentinean A.
persimilis populations towards the North and South of the American South Cone,
respectively.
The available cyst samples from these diverse populations allowed developing several
research lines dealing with:
 cyst and naupliar biometrics in order to explain the different cyst buoyancy levels
found for Argentinean A. franciscana and A. persimilis populations; bigger size of A.
persimilis nauplii at more southern latitudes may accord to thermo-ecological rules;
 quantitative and qualitative identification of HUFA levels, aiming to correlate these
profiles to species specific conditioning; marine or inland origin of brines, shoreline,
sea level or high altitude biotopes; phenotypic influence of the diet versus a possible
genotypic effect, etc. Within the same field of research, a study was initiated on the
nutritional value of a diet of different unicellular algae on survival, growth,
maturation, reproduction and HUFA levels of A. persimilis and A. franciscana.
 differentiation of fitness traits between Argentinean (plus Chilean and Peruvian)
populations of A. persimilis and A. franciscana, and among populations within
species, which should allow to establish a general view of phenotypic plasticity and
response of the different populations (species) to environmental conditions: effect of
environment on life-history traits like maturation period, length of reproductive
period, brood size, total offspring production, brood numbers, interbrood interval,
offspring type and quality (oviparous/ ovoviviparous) which can help to hypothesise
on the present distribution and biogeography of both species in this region.
 correlation between morphological and genetic characteristics of Artemia populations
with respect to species discrimination and identification, based on mitochondrial
DNA RFLP analysis: identification of population-specific genetic markers for A.
persimilis and A. franciscana, helping to describe aspects of their biodiversity and
speciation patterns.
31
Furthermore, in order to determine the genome affinity between A. franciscana and A.
persimilis preliminary assays have been performed of Genomic In Situ Hybridization (GISH)
on Argentinian A. franciscana and A. persimilis populations. The statistical analysis revealed
significant differences in the percentage of heterochromatin among populations, and
suggested that the heterochromatin content measured as percentage of heterochromatin should
be a more reliable trait for intra- and interspecific comparisons than the number of
chromocentres. However, additional studies should be carried out in order to validate the use
of this parameter as a more reliable cytogenetic marker. The amount of heterochromatin could
be related to environmental (geographical or ecological) conditions rather than to taxonomic
categories.
In order to help in the specific identification of American Artemia populations, the
morphology of the structures of both sexes involved in the amplexus was studied and
compared between specimens of A. franciscana and A. persimilis by means of microscopic
and histological observations. The usefulness of the shape and size of the frontal knob as
morphological and morphometrical characters in specific taxonomy of the Artemia
populations was confirmed, and the shape of the distal article of the male antenna was added
as a reliable specific character.
The Pichilemu area 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, hence the need to devote special attention to these areas in future
collaboration.
Artemia biodiversity in continental Asia
a) Lake Urmia area, Iran
Samples from Urmia lake have been investigated making use of RFLP analysis of the 1500 bp
fragment (individual cysts were scored). In summary it looked like the RFLP patterns from
individual cysts from an Urmia lake sample are closely related to the RFLP pattern of
parthenogenetic Artemia. Yet individuals from these samples, when raised to adulthood,
display an approximately 50/50 sex ratio, and adults of both sexes have the typical
morphology of A. urmiana. So it looks like the rDNA fragments from parthenogenetic
individuals are closely related to the rDNA from A. urmiana. These data together with those
obtained on samples from the Qinghai-Tibet Plateau illustrate that a multi-marker approach
applied on sexually mature individuals will be needed to unravel the phylogenetic relationship
between these Artemia species.
A preliminary phylogeny based on mit DNA sequences, performed by INCO partner 14
(ULL) including data on haplotype variability for parthenogenetic types, showed A. urmiana
clustering together with a parthenogenetic sample, and the same occurs with A. tibetiana. This
result are open to discussion in view of the lack of purity of samples available in the ARC
cyst bank, which can be quoted as an important result of the project.
Continued genetic study of Artemia populations from Lake Urmia area is planned in the
framework of a PhD, which will focus on present Artemia populations and on historical cyst
samples using different genetic markers.
Field resource assessment at Lake Urmia has been continued through 2004: monthly sampling
is performed from 19 sampling sites. Results indicate that Artemia cysts hatch in Lake Urmia
end of March initially at the far south and towards the areas where rivers terminate to the lake.
Artemia density reaches its peak by end of July/early August. Salinity levels have dropped
from over 300 ppt in 2002 to 280 ppt in 2004 – which is still too high to obtain abundant
Artemia production, but the increasing levels of rainfall during the last two years are
32
promising. Tests were run with local sturgeon species to assess local Artemia urmiana,
enriched with different levels of highly unsaturated fatty acids and vitamins, as larval food for
these fish species.
b) Aral Sea
Cooperation with laboratories in Tashkent and Nukus, Uzbekistan was continued and through
mutual visits a critical mass of Artemia expertise at the partner labs was generated. Joint
research will focus on description and characterization of the Aral Sea Artemia population, on
the development of a population model for the Aral Artemia resource, and on the definition of
resource management recommendations.
c) PR China
A number of Artemia strains from inland salt lakes of the People’s Republic of China were
analysed in terms of cyst and naupliar biometrics, hatching characteristics and nutritional
profile. All parthenogenetic Artemia strains displayed a larger cyst and naupliar size
compared to bisexual strains. The hatching characteristics widely fluctuated from strain to
strain, as well as the nutritional profile.
Special attention was given to the Qinghai-Tibet Plateau. To evaluate the mixed status of
Artemia populations originating from this area, samples were subjected to RFLP-analysis of a
1500 bp mitochondrial rDNA fragment (individual cysts were scored). Based on the RFLP
patterns, the species composition of the populations from the resp. sites was as follows: in two
samples all individual cysts displayed the parthenogenetic pattern, in five samples uniquely
Artemia tibetiana was found, whereas the other samples proved to be a mixture of
parthenogenetic patterns with A. tibetiana and/or A. sinica patterns in various proportions.
Among the parthenogenetic patterns, one particular HpaII haplotype was dominant, while a
second haplotype was rare (equal to or below 3%). The frequency of the four A. tibetiana
haplotypes was variable from sample to sample. However, the parthenogenetic pattern(s) also
showed up in samples proving predominantly bisexual, when being cultured in the laboratory.
The presumed close genetic relationship between the local parthenogenetic and bisexual
species, and the intra-species polymorphism, illustrate the need for cautious interpretation of
the results and for a multi-faceted and well-balanced analytical approach when the issue of
possible co-existence of eastern Old World bisexual and parthenogenetic populations is
addressed.
References
Bossier, P., Wang, X., Catania, F., Dooms, S., Van Stappen, G., Naessens, E., Sorgeloos, P.
2004. An RFLP databse for authentication of commercial cyst samples of the brine shrimp
Artemia spp. (International Study on Artemia LXX). Aquaculture, 231: 93-112.
Piccinelli, M. and Prosdocimi, T (1968) Descrizione tassonomica delle due specie Artemia
salina L. e Artemia persimilis n. sp. Istituto Lombardo, Accademia di Scienze e Lettere,
Rendiconti B, 102: 170-179.
Joint activities INCO Consortium beyond project period
As described in the Annual report year 2 (2003) the Consortium had reached an agreement on
the realization of common publications, to be realized after the project lifetime.
Publication 1:
 Objective: Compilation/Manual of Protocols and Recommendations for Artemia
characterisation studies
33




Title: “Artemia biodiversity: protocols and guidelines for study and
sustainability”
Collective authorship of 15 partners
Original timing, set at Chile workshop (2003) was too optimistic. Therefore a
new timing was agreed upon at Urmia workshop. The consortium aims at a
finalization of this document within 2005.
The following draft chapters have been compiled and can be downloaded from
the INCO website:
1. Artemia sampling, sample preservation, diapause termination techniques
2. Genetics and molecular markers
1. Cytogenetic analyses and methodologies
2. Cross breeding
3. Genetic approaches to the analysis of Artemia biodiversity
3. Biometrics and morphometrics
1. Cysts and naupliar biometrics
2. Culturing conditions
3. Discriminant analysis on adult Artemia morphology
4. SEM techniques
5. Statistical analysis
4. Phenotypic response under different environmental conditions
5. Sustainable exploitation
6. Artemia conservation and invasion patterns
Publication 2:
 Publication in peer-reviewed journal
 Working title: “A. franciscana: the invasive species in the genus”
 Joint authorship of all INCO partners.
 Initiative for publication to be taken by AUTH (INCO partner 3).
34
PAPERS AND PUBLICATIONS
(not included in annual reports)
Integrated system of Shrimp-Artemia-Salt in the Mekong delta Vietnam
Nguyen Van Hoa, Nguyen Thi Hong Van and Nguyen Thi Ngoc Anh
Aquaculture Compendium – CAB International
Abstract:
Traditional solar saltworks appeared in Vinh Chau (Soc Trang) and Bac Lieu since 60’s (Do
Van Hoang, 1998). However, crude salt are mainly served for human consumption (salted
fish, fish sauces etc.) therefore its demand is not considerable. Moreover, large amount of
crude salt was produced yearly in the site and thus excess the need for local consumption.
Consequently, salt produced this season could not be sold totally and make difficulty for salt
farmers as their income was reduced. To the end of 80’s, Artemia culture technique in
saltfields for cyst production was developed by College of Aquaculture and Fisheries (CAF),
Cantho University and proved to be profitable compared to traditional salt production
(Nguyen Van Hoa, 1991; Vu Do Quynh, 1997). Therefore the technique was immediately
transferred to farmers and larger production scale was developed year by year. The paper
describes the system of shrimp, Artemia and salt production with the aim to introduce to
saltfarmers a new production system which helps to improve their living standards.
Socio-economics of Artemia culture in the Mekong delta, Vietnam
Nguyen Van Hoa, Nguyen Thi Hong Van and Nguyen Thi Ngoc Anh
Aquaculture Compendium – CAB International
Abstract:
Traditional solar saltworks exclusively performed in Vinh Chau (Soc Trang) and Bac Lieu
since 60’s (Do Van Hoang, 1998). However, low and not sustainable income made salt
production less attractive to salt farmers as a whole. To the end of 80’s Artemia production in
the area was taken place under the technical advice of College of Aquaculture and Fisheries,
Cantho University. Artemia production provides not only cysts to shrimp/fish hatcheries but
also to improve considerable salt farmers income (Vu Do Quynh, 1997). Through out the
years, nonetheless, cyst production shows its unstable but rather varies from place to place
and from season to season and especially depended on investment scale, pond management.
Moreover, cyst price usually fluctuated and to be market driven which in turn depended on
the remaining cyst stock from shrimp feed companies, and thus make more difficult for
farmers to decide if they should invest for Artemia production for the coming season or not.
Besides, paper also presents pros and cons for a sustainable production of Artemia in the area.
Culture technique of Artemia biomass in the saltfields, Vietnam
Nguyen Thi Ngoc Anh, NguyenVan Hoa and Nguyen Thi Hong Van
Aquaculture Compendium – CAB International
Abstract:
Culture technique of Artemia biomass in the saltfields has been studied successfully by Can
Tho University and applied in pilot-scale for several years, the productivity ranged from 0.7-1
metric ton wet weight/ha/month or 2-4 tons/ha/crop (4-5 months of culture). Beside live
Artemia biomass, cyst harvest also obtained 20-30 kg cyst wet weight/ha (yielding 30-50% of
the oriented - cyst production). However, biomass culture has not been developed and its use
is still limited in Vietnam, due to its nutritional values have not been understood well, and
lack of necessary technology for processing, transporting and preservation of live Artemia
biomass.
Artemia cyst production in saltpans of the Mekong delta, Vietnam
35
Nguyen Thi Hong Van, Nguyen Van Hoa and Nguyen Thi Ngoc Anh
Aquaculture Compendium – CAB International
Abstract:
The Mekong delta in South Vietnam with their long coastlines extending through: Tien
Giang, BenTre, Tra Vinh, Long An, Soc Trang, Minh Hai and Kien Giang provinces.
Traditional salt production is the major income for most of the local farmers who lives along
the coastlines despite of salt price is low and unstable from year to year. The introduction of
Artemia originated from the San Francisco Bay, USA into the MeKong delta saltfields,
especially in Soc Trang and Bac Lieu provinces have opened a new trend in diversification of
aquaculture species and also creating a better income for local farmers compare with salt
production.
In Bac Lieu and Soc Trang provinces, Artemia cyst production is preferred and normally,
Artemia are stocked in salt-street from crystallizer to evaporation areas, even reservoir areas
where the salinity can be kept more than 70ppt upwards. The culture period last 5-6 months in
the dry season and the cyst yields vary from 40 kg/ha/crop up to 150kg/ha/crop depending
very much on locality, culture technique and the experiences of Artemia culturing farmers.
36
Aristotle University of Thessaloniki
Faculty of Science – School of Biology
Department of Genetics, Development & Molecular
Biology
Thessaloniki, GR 541 24, Greece
Genetic characterization of Vietnamese Artemia
franciscana populations based on p26 gene
Mrs. Nguyen Thi Hong Van
Institute of Science for Aquaculture, 3rd February Street, Campus
II, Can Tho University, Can Tho, Vietnam (INCO partner 7)
26th September – 19th December, 2003
37
SUMMARY REPORT
I.
-
II.
Purpose of visit
To receive training on DNA molecular techniques in order to apply them on studies in
Vietnam.
Strains of A. franciscana are to be investigated for the presence of a previously detected single
base mutation in a region of the p26 gene encoding for a small heat shock protein. Genetic
analysis will be performed on the following A. franciscana samples: SFB (inoculated in Vinh
Chau in the year 2003), SFB-1258 (the source population used for inoculation more than 20
years ago), two VC populations harvested in 1993 and 2002, respectively. The particular
mutation will be evaluated for its potential use as a discriminant DNA marker between the SFB
and VC strains. DNA analyses on SFB-1258 and VC-1993 populations were accomplished by
Dr. I. Kappas.
Training content
Ethanol-preserved samples of adults from strains SFB-2003 and VC-2002 were brought from
Vietnam.
1. DNA extraction
Two different DNA extraction methods were used. The first was a method using Chelex resin and the
second involved treating samples with phenol and chloroform Artemia and a mix thereof.
1.1 Chelex protocol
Artemia individuals that had been stored in ethanol were dried on paper and kept in 500μl of distilled
water in 2ml Eppendorf tubes on a rotor for at least 24 hours to wash out ethanol. After this, they were
homogenized in 200μl of 10% Chelex solution. Ten μl of Proteinase K were added to Chelex to
remove proteins and the tubes were boiled at 100oC to deactivate Proteinase K. Extracted DNA was
stored at 4oC.
1.2 CTAB – Phenol/Chloroform protocol
This procedure consisted of the following steps:
(a) Homogenization of the whole animal in 500µl 2x CTAB buffer. The CTAB is used to break the
cell walls and proteins.
(b) Lysis of cells and membranes with proteinase treatment. Usually 10µl of Proteinase K (20mg/ml)
are added and the mix is vortexed well.
(c) Protein degradation by incubating the samples in water-bath at 55°C for at least 2 -4 hrs. Then
500µl of phenol were added and the samples were put on the rotor for 10min. It followed a spinning
step at 13000rpm for 5min. The top layer was removed and transferred in a new tube to which 250µl of
phenol and 250µl of chloroform:isoamyl-alchohol (24:1) were added and the whole procedure was
repeated. In the final step, 500µl of chloroform were added followed by rotation, spinning and removal
of top layer.
(d) DNA precipitation by addition of 500 – 800µl of ice-cold absolute ethanol. Samples were put at 20°C for 2 hrs and then spun for 15min at 13000rpm. The supernatant was carefully decanted off and
the pellet washed with 70% ethanol for at least one hour or overnight by placing the tube on a rotary
mixer. After that, samples were spun for 10min, the ethanol was carefully removed with a micropipette
and the DNA pellet was vacuum dried at 37°C for 10 – 15min.
(e) Re-suspension of DNA in 50µl of dH2O (or TE buffer). Samples were kept at 4°C for later use.
39
2. PCR amplification
The DNA target sequence was a segment of 294 bp in length which included the partial portions of the
large subunit of p26 genes. Oligonucleotides primers were P26F3INTRON and P26REXON4. Primers
were supplied by Invitrogen. For each amplification, the following mix was used to make a total
reaction volume of 20µl for each sample: 2.0µl of 10x reaction buffer, 1.0µl of MgCl 2 (50mM), 0.2µl
of dNTPs, 0.2µl of each primer, 0.4µl of Taq DNA polymerase, 2.0µl template DNA and 14µl of
dH2O.
The PCR was carried out using an automatic thermocycler (Techgene- FT Gene E2D model) with the
following program:
1) one preliminary denaturation at 94ºC for 4min
2) 32 amplification cycles each consisting of 30sec at 94ºC, annealing at 60ºC for 30sec and
primer extension at 72ºC for 30sec
3) one final extension at 72ºC for 5min
Amplification products were checked on 1.5 % agarose gels for purity and correct size and stored at
4ºC for further use.
3. Results
The restriction enzyme used for the analysis of the p26 fragment (294bp) was Hinf I
(5’CTNAG3’;3’GANTC5’) (BioLabs, Inc). Cleavage of this specific region of p26 would produce
different patterns (Table 1), according to known sequences kindly provided by Prof. T. MacRae
(Dalhousie University, Canada).
Table 1: Fragment patterns for HinfI observed in Artemia populations (VC and SFB) in part of
the p26 region
Genotype
AA
AB
BB
Fragment sizes ------- 201
------- 201
-------- 201
------- 70
------- 93
-------- 93
------- 23
------- 70
------- 23*
*The fragment of 23bp could not be visualised on the agarose gel
The reaction mix consisted of 5.0 µL of the PCR product, 1.5 µL of the 10x reaction buffer and 2.0 µL
of the specific restriction enzyme (Hinf I) and 1.5 µL of dH2O (total reaction volume was 10 µL per
sample). The reaction mixtures (samples) were then incubated at 37oC in an oven for at least 6 hours
(or even overnight) to ensure complete digestions.
40
Fig 1: Fragment patterns for HinfI observed in Artemia populations (VC and SFB) in part of the p26
region. (AA: rank 3; AB: rank 12 and 13; BB: the rest; Marker: rank 8; Uncut (294bp) P26
fragment: rank 9
3.1 Single Stranded Conformation Polymorphism (SSCP)
SSCP is one of the most widely used and practical approaches for the detection of mutations in DNA
as well as for detection and analysis of DNA variation. The advances of this method are speed and low
cost compared with other techniques. However, it is highly sensitive and based on the results,
sequencing of the screened fragment may be needed.
This method relies on the mobility of a single stranded DNA molecule in a non-denaturing gel
depending on its structure (conformation). Differences in conformation will result in differences in
mobility, evident as separate bands on the gel. Usually, the fragments used for SSCP analysis range
from 100-300 base pairs.
-
Acrylamide gel preparation: 8% acrylamide gel was prepared from 18ml of 30%
acrylamide stock (29g acrylamide + 1g bis-acrylamide dissolved in 70ml of dH2O and
kept in refrigerator) 5ml of TBE x5 buffer and 47ml of dH2O. To the mixture then they
were quickly added 210µL of 20%APS solution together with 49µL of TEMED
(ethylene diamine) and the liquid was immediately poured between the glass plates.
After that the gel was kept at room temperature for 2 hours or put at 4°C overnight for
later use. In case glycerol was used, 14ml of 50% glycerol stock were added (reducing
the dH2O accordingly).
-
Sample preparation: 1.2-1.5 µL of PCR product, 2µL of formamide and 6µL of
loading buffer were added, the samples were spun shortly and put in a PCR machine
for denaturation at 99oC for 12 min. They were then immediately removed and placed
on ice for 5 min before loading on gel.
-
Electrophoresis condition: Gels were normally run on SSCP equipment with TBE
buffer x1 (running buffer) at 80A and 200V and 4oC for 5-6 hours.
-
Staining: After electrophoresis the gel was carefully removed from the plate and
stained. This procedure consisted of 3 steps:
i. Gel washing: the gel was washed twice with a solution of dH2O (358ml),
absolute ethanol (40ml) and acetic acid (2ml) for 3 min by using a shaker.
ii. Silver staining: in this step the gel was firstly stained with 200ml 1% silver
nitrate (AgNO3) solution on a shaker for 10 min and then washed well with
dH2O.
iii. Buffer staining: the gel was stained in a buffer (consisting of 3g NaOH, 800
µL formaldehyde and 0.02g NaBrH4 dissolved in 200ml of dH2O at 50oC).
Stronger shaking was applied until the bands on the gel appeared.
41
- Gel storage: After the third step, the gel was dried with tissue paper, sealed in a plastic
bag and stored at 4°C.
-
-
DNA from four populations, VC-2002 (30 inds), SFB-2003 (42 inds), VC-1993 (18
inds) and SFB-1258 (26 inds) was isolated by both methods (CTAB and Chelex) and
used for PCR amplifications at the p26 region. The VC-1993 and SFB-1258
populations come from the DNA collection of Dr. Ilias Kappas (AUTH, Greece).
Results of the restrictions with HinfI at the p26 locus are shown below.
Table 2: Genotypes of the p26 locus for 4 populations based on restriction digests with HinfI
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
Statistical analysis (GENEPOP, version 3.4) did not yield any significant differences in allele
frequencies as well as in genic differentiation between populations at locus p26 (Tables 3 and 4,
respectively).
Population
VC-2002
VC-1993
SFB-1258
SFB-2003
Table 3: Allele frequencies at locus p26 in all Artemia populations
Allele
Genes
A
B
0.100
0.900
60
0.167
0.833
36
0.115
0.885
52
0.190
0.810
84
Table 4: Pairwise genetic differentiation tests (Fisher’s method) at locus p26 for all Artemia
populations studied
2
Comparison
χ
Df
P- value
VC-2002 vs. VC-1993
VC-2002 vs. SFB-1258
VC-2002 vs. SFB-2003
VC-1993 vs. SFB-1258
VC-1993 vs. SFB-2003
SFB-1258 vs. SFB-2003
2.058
0.000
3.606
1.232
0.000
2.157
2
2
2
2
2
2
0.35
1.00
0.16
0.54
1.00
0.34
4. Conclusions
Although no significant differences were found between populations, this study provided a helpful
opportunity for training on molecular techniques, commonly applied in population genetics (such as
PCR, RFLP, SSCP). These investigations will be continued through the joined research between
AUTH (INCO partner 3), CTU (INCO partner 7) and other INCO members.
42