Evaluation of mixed status of Asian Artemia populations through RFLP-analysis

Transcription

1 Evaluation of mixed status of Asian Artemia populations through RFLP-analysis Concerted Action Artemia biodiversity INCO-DEV project P. Bossier 1, G. Van Stappen 1, Yu Haiying², Wang Xiaomei², Xin Naihong², D. Deloof³, D. Delbare³, P. Sorgeloos 1 1 ARC ²SRI ³CLO-DVZ

2 1. Qinghai- plateau 2. Artemia urmiana 3. Bohai Bay area

3 1. Overview experimental samples Qinghai- Plateau Lake Lagkor Co Bong Co Bozi Co Co Qen Gaize Shuanghu Nima Province ARC code Harvesting/ Winter 96/97 Oct Oct 98 Oct 98 Arrival date May 99 May 99 Nov 1 ARC code Harvesting date Autumn Lake Yangnapeng Qixiang A Jingyu L. Haiyan L. co Province Xinjiang Qinghai ARC code Harvesting/ Feb 2 Feb 2 Mar 97 Arrival date Sept 97 Nov 1 Nov 1

4 Geographical location of sites Zheng Mianping (1997). An Introduction to Saline Lakes on the Qinghai- Plateau (Kluwer) 1. Lagkor Co 2. Bong Co 3. Bozi Co 4. Co Qen 5. Gaize 6. Shuanghu 7. Nima 8. Yangnapengco 9. Qixiang 1. Jingyu L. 11. Haiyan L. 2 km Xinjiang Qinghai 11

5 Methodology RFLP of 15 bp mitochondrial rdna fragment Analysis of ± 3 individual cysts per sample: DNA extraction PCR amplification Restriction digestion Data analysis Comparison with reference database An RFLP database for authentication of commercial cyst samples of the brine shrimp Artemia spp (ISA LXX) 24. Aquaculture 231 (93-112) Bossier et al.

6 RFL P Hae III+ RFLP ms ei+ RF LP H pai I+R FL P Nd eii+ RFLP Ta qi+ RFLP Ts p5 9I+RFL P Hinf I+ RFLP Dd ei 8RF LPs UPGMA dendrogram Cyst SAMPLES! using 7 restriction enzymes parthenogenetic Artemia parthenogenetic Artemia parthenogenetic Artemia parthenogenetic Artemia parthenogenetic Artemia parthenogenetic Artemia Karabogaz-Gol, Turkmenistan Karabogaz-Gol, Turkmenistan G ahai, Q inghai Province, P.R. China Karabogaz-Gol, Turkmenistan S a yte n, K a za kh sta n U z be k si tan Bolshoe Yarovoe, Russia K a za kh sta n Bolshoe Yarovoe, Russia P a vlod a r, K az a kh sta n Jingyu (W hale Lake), Xinjiang, P.R. China T anggu, Tianjin Province, P.R. China) W u Shen Qi County,Inner Mongolia, P.R.China Bameng, Inner-Mongolia Province, P.R. China (id.) Karabogaz-Gol (RHI), Turkmenistan Karabogaz-Gol, Turkmenistan A nkiembe, Madagascar Sorkaidak, Kazakhstan parthenogenetic parthenogenetic Artemia Vineta Swakopmund, Namibia Medvezhe (Bear) Lake, Kurgan, Russia Urmia Lake, Iran Urmia Lake, Iran Urmia lake, Iran Urmia Lake, Iran parthenogenetic Artemia A. tibetiana A.franciscana A ibi Lake, Xinjiang Province, P.R. China Coqen Lake,, P.R.China T ibet, P.R. China B ozi Co,, P.R. China T ibet, P.R. China Laggor Co,, P.R. China Vinh Chau, Vietnam tibetiana San Francisco Bay, USA Vinh Chau, Vietnam Vinh Chau, Vietnam Vinh Chau, Vietnam Vinh Chau, Vietnam San F rancisco Bay (location 3), USA A.franciscana San F rancisco Bay (location 5) USA G reat Salt Lake, USA Blenheim, New Zealand G SL ' ( N.A.) DOUG 1138 G SL '99 ( N.A.) >9% G reat Salt Lake, USA G reat Salt Lake,South Arm, USA Estuario de Virrila, Peru Macau, Brazil franciscana A. monica A. persimilis parthenogenetic Artemia parthenogenetic Artemia parthenogenetic Artemia Artemia sinica A. urmiana A. salina A. salina A.s alina A. salina A. salina Mono Lake, USA B onaire Duinmeer, Neth. A ntilles Porto Arraya, Venezuela Curacao Fuik, Neth.Antilles A rgentina W u Shen qi County, Inner Mongolia, P.R. China Xiechi Lake, Yuncheng, PR China Yuncheng, P.R. China T uhum lake, Mongolia Megalon Embolon, Greece Citros, Pieria, G reece Yuncheng, Shanxi Province, P.R. China Yimeng, Inner Mongolia, P.R. China Haolebaoji, Inner Mongolia, PR China, A. sinica Urmia Lake, Iran Megrine, Tunisia W adi Natron, Egypt Sfax, Tunisia Larnaca, Cyprus Larnaca, Cyprus persimilis sinica urmiana salina

7 FIRST STEP : Hpa II digestion on total of 15 samples : Reason: previous research shows best clustering (using single enzyme) into known Artemia species by use of Hpa II 7 different haplotypes found over 15 samples m similar to patterns found in parthenogenetic cluster m m A. sinica m m m similar to patterns found in tibetiana cluster, including type sample 1347

8 NEXT STEP: analysis of several individuals of each parthenogenetic Hpa II haplotype (1-4): digestion with other restriction enzymes Taq I Nde II Hinf I Identical to pattern from tibetiana cluster in database Identical to pattern from parthenogenetic cluster in database

9 NEXT STEP: analysis of individuals of each tibetiana Hpa II haplotype (6-7): digestion with other restriction enzymes (Taq I, Nde II, HinfI): e.g.: TaqI Identical to pattern from tibetiana cluster in database

10 Type sample Lagkor Co 1347 Hpa II cysts 9 % parthenogenetic pattern 1 % tibetiana pattern Hpa II nauplii Taq I nauplii 36 % parthenogenetic pattern 64 % tibetiana pattern

11 Q-T Plateau: Conclusions 1 7 Hpa II haplotypes found in samples from Q-T Plateau 1 typical for A. sinica 2 typical for Artemia from parthenogenetic cluster (ratio of occurrence: 97-3 %) but no variability for Taq I, Nde II, Hinf I: but also present in bisexual samples, when cultured! 4 banding patterns, typical for tibetiana cluster but no variability for Taq I, Nde II, Hinf I: = A. tibetiana haplotypes, present in variable proportions in different samples

12 Q-T Plateau: Conclusions 2 Where are we now???? Samples from Q-T Plateau: when using Taq I, Nde II, Hinf I: 3 haplotypes : sinica, tibetiana and parthenogenetic when using Hpa II: sinica: no problem tibetiana: split up over 4 haplotypes parthenogenetic : split up over 2 haplotypes

13 Q-T Plateau: Conclusions 3 BUT : Which pattern(s) correspond(s) to bisexual resp. parthenogenetic INDIVIDUALS??? Cfr example Jingyu, Gaize: 4 resp. 3 % males when cultured, but 1 % parthenogenetic pattern

14 1 % parthenogenetic 1 % tibetiana mixed (with occurrence of sinica) Link to ecological-topographical conditions? => data needed! 1. Lagkor Co 2. Bong Co 3. Bozi Co 4. Co Qen 5. Gaize 6. Shuanghu 7. Nima 8. Yangnapengco 9. Qixiang 1. Jingyu L. 11. Haiyan L. A 2 km Xinjiang Qinghai 11

15 Are there 2 bisexual species?? (not incl. sinica) RFLP analysis of adults and/or different methodological approach (see further)

16 2. Artemia urmiana samples 155 & 1511: all adults showing A. urmiana morphometry A. urmiana parthenog. same methodology as samples from Q-T Plateau RFLP analysis of 3 individual cysts Tru9 I, Hpa II, Nde II, Taq I, Hae III, Hinf I (Tsp59 I)

17 1511 Hpa II Nde II

18 1511 Taq I Hinf I

19 A. urmiana : Conclusions 1 (large) majority of individuals of samples 155 and 1511 shows parthenogenetic pattern for resp. restriction enzymes few individuals show new, non- parthenogenetic, non-tibetiana pattern for resp. restriction enzyme Same parthenogenetic pattern also shows up in bisexual sample (adult males analyzed!!!) from near Aktumsuk, near Aral Sea, Uzbekistan

20 A. urmiana : Conclusions 2 = limitations of approach using mitochondrial DNA analysis need for alternative approach, e.g. NUCLEAR markers

21 2. Hebei 3. Tianjin 3. Samples from Bohai Bay area Overview of samples analyzed ARC code Saltwork (T= Tanggu H= Hebei) Tanggu (T) Hangu (T) Nanpu (H) Tanggu (T) Tanggu (T) Tanggu (T) Luannan (H) Nanpu (H) Cangzhou (H) Luannan (H) Luannan (H) harvesting/ arrival year

22 Sex ratio after culture HpaII digestion on 3 individual cysts ARC code harvesting/ arrival year H% % survival H % x % survival Male/female (n = 1-15) A. parth % A. sinica % A. franc %?

23 Example: 124 Luannan (Hebei): 28 cysts analyzed marker parthenogenetic A. sinica A. franciscana x xx xx 18 % parthenogenetic 25 % sinica 39 % franciscana 18 % franciscana (x, most probably)

24 Bohai Bay : Conclusions Additional proof Bohai Bay has been invaded from early 9s by A. franciscana Occurrence of A. sinica: natural or artificial?