Indian Journal of Geo-Marine Sciences Vol. 44(11), November 2015, pp. 1712-1715

Loss of indigenous brine Artemia parthenogenetica due to the invasion by American species Artemia franciscana at Thamaraikulam salt pan

Gayathri Valsala1, Shiburaj Sugathan1* & Hari Bharathan2 1 Division of Microbiology, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Thiruvananthapuram-695 562, India. 2 Post Graduate Department of Zoology and Research Centre, Sree Narayana College, Kollam-691 001, India. *[E-mail: [email protected]]

Received 30 September 2014; revised 06 November 2014

Artemia, a widely used aquatic live feed inhabits hyper saline environments like salt pans. Artemia parthenogenetica is the native species found in Indian salt pans and is being displaced by an exotic species Artemia fracinscana, which is imported as a commercial live feed for aquaculture. The present study provides evidence of complete invasion of Thamaraikulam salt pan by A. franciscana, which in previous studies was found to be inhabited only by native A. parthenogenetica. Artemia nauplii and cysts were collected from Thamaraikulam salterns and cultured in lab. Total RNA was isolated from the cysts produced and cDNA was synthesised. The species was identified to be A. franciscana upon analysis of cytochrome c oxidase subunit I gene and P26 gene sequences amplified from the cDNA. Presence of native parthenogenetic strain was not detected even on repeated trials.

[Keywords: Artemia, A. parthenogenetica , Bio-conservation, P26, Cytochrome c oxidase I]

Introduction the bisexual A. franciscana. Thamaraikulam salt pan The Artemia or is an anostracan (8°06'N 77°29'E) in Nagercoil, Tamil Nadu was widely used as live feed in aquaculture. selected for the study since it was reported to be free Being highly osmotolerant, their natural habitats are from bisexual species5. The Artemia population at hypersaline environments like coastal or inland Thamaraikulam was characterized by John et al. salterns1,2. Indian populations of Artemia is mainly (2004) and was reported to be parthenogenetic6. Artemia parthenogenetica and have been reported Temperature and salinity tolerance data suggest that from about 19 different areas, including Bhayander, the native strain can tolerate a wide range of Didwana, Jamnagar, Karsewar Island, Kutch, temperatures from 22 to 30°C, but the salinity Mithapur, Pattanamaruthur, Spic Nagar, Thirispuram, tolerance is highly limited to 35 ppt. According to Tuticorin, Nagercoil, Vadala, Vedaranyam, adult morphometric characters, they were found to be Veppalodai and Vivar3. Artemia franciscana is a closer to Egyptian asexual populations7. Vasudevan highly invasive commercial brine shrimp species, (2012) studied the biometrical, morphological and harvested mainly from the Great Salt Lake (GSL) and biochemical characteristics of parthenogenetic San Francisco Bay (SFB) salt works in USA and has Artemia population at Thamaraikulam8. It was resulted in the displacement of native Artemia species reported in a study carried out during 2002–2003, that throughout the world. The high demand for imported the indigenous parthenogenetic Artemia species at Artemia cysts has led to the introduction of A. Chennai and Tuticorin salt pans were eliminated by franciscana into sites previously occupied by native the introduction of exotic bisexual strain, while those Artemia species. In India, culture of Sanfracisco Bay at Vedaraniyam and Nagercoil were parthenogenetic strain of A. franciscana was introduced by Bharath in nature5,9,10. Salt chemicals Industries, Gujarat during early 80’s4. Since different species of Artemia exhibits very similar morphology, it is essential to use molecular Materials and Methods techniques to determinate the species, for Samples used in this study were originally collected conservation studies. In the present study, two marker for a comparative study on the small heat shock genes, mitochondrial gene cytochrome c oxidase I proteins from the native parthenogenetic species to (COI) and genomic small heat shock protein gene P26 GAYATHRI et al.: LOSS OF INDIGENOUS BRINE SHRIMP ARTEMIA PARTHENOGENETICA 1713

were used for species identification. COI gene is 50uL diethyl pyrocarbonate treated water and stored widely used as a DNA barcode to identify at -20 °C. species because its mutation rate is often fast enough to distinguish closely related species and also because Results its sequence is conserved among conspecifics. cDNA was prepared using SuperScript® III Reverse Adult Artemia were collected from the Transcriptase kit (Invitrogen, USA) using oligo(dT) Thamaraikulam salterns during March, July, primers. The COI gene was amplified from cDNA November 2013. Presence of males in the collected using the primers COIF 5`- ATT CTA CGA ATC sample indicated the presence of bisexual strain of ACA AGG ATA TTG G - 3` and COIR5`- TAC ACT Artemia. They were cultured in autoclaved, 0.22 µm TCA GGA TGG CCA AAA AAT CA - 3` and small vacuum filtered seawater. The cultures were grown at heat shock protein P26 gene using primers P26 AF 5`- room temperature on the bench top with moderate TAC GGA GGA TTT GGT GGT ATG - 3` and P26 aeration and were fed with unicellular brown algae AR 5`- ATT GTT GAT CTT GCT GGA GTT G - 3`. Isochrysis galbana daily. Cysts produced were EmeraldAmp® (Takara) mix was used for PCR collected, washed, dried and stored at -20° C. amplification with the following temperature profiles Total RNA was isolated from 100mg cyst samples and conditions: 1 min at 98 °C, 30 cycles of 10 s at 98 using TRIzol reagent (Life Technologies Invitrogen, °C, 30 s at 55 °C, 1 min 30 s at 72 °C and a final USA)11. Cysts were homogenized in 1 mL TRIzol extension of 7 min at 72 °C. Total reaction volumes of reagent using micropestle at room temperature and 25 µl consisted of 0.5 µl template DNA, 12.5 µl then passes through a 2.5mL syringe. Homogenized Emerald mix, 1 µl of each primer (10 µM). samples were incubated at room temperature for 5 Three sets of COI and P26 gene samples were min, centrifuged and the supernatant was extracted sequenced at the Regional Facility for DNA once with chloroform: isoamyl alcohol (24:1) and the Fingerprinting, Rajiv Gandhi Centre for RNA was precipitated using 100% ethanol (ice cold). Biotechnology (Thiruvananthapuram, Kerala). The pellet was washed with 70% ethanol, dissolved in

Table 1: DNA sequence of COI and P26 gene

COI gene sequence:

1 AAACCTTTAA ATTTTTTGGG GGCTTGAGCA GGTATAGTTG AGGAACTTCT TTAAGAATGC 61 TCATTCGAGC AGAGTTGGGT CAACCAGGTT CCCTGATTGG CGATGAACAA GTATATAATG 121 TTATTGTGAC AGCTCATGCA TTTATTATAA TTTTTTTCAT GGTTATACCA ATCTTGATTG 181 GGGGATTTGG TAACTGGCTA GTACCCATTA TATTGGGGGC CCCGGATATA GCATTTCCCC 241 GGTTAAATAA TTTAAGATTT TGAATACTTC CACCATCCTT GACTCTTCTC TTGGCCAGAT 301 CTATAGTTGA GAGAGGTGCA GGAACTGGAT GAACAGTTTA TCCCCCTCTA TCCTCAGCCA 361 TTGCCCATGC CGGACCTTCT GTAGATTTAG CTATTTTCTC GCTTCATTTA GCTGGAGTTT 421 CCTCTATCTT AGGGGCTGTA AATTTTATTA CTACTATCAT TAATATACGA CCCCAGTCAA 481 TATCTATTGA CCGTATACCT CTCTTCGTCT GAGCAGTAGG AATCACCGCC GTTCTTCTCC 541 TTTTATCACT TCCAGTCCTA GCGGGGGCTA TTACTATACT GTTAACTGAT CGTAACTTAA 601 ATACTTCTTT CTTCGACCCC GCAGGTGGTG GGGATCCCAT CCTTTATCAA CATTTATTTT 661 GATTTTTTGG CCATCCGGAA ATTTAAAA

P26 gene sequence: 1 CTCTGATCAT TTGGATTTGG TGGCTTCGGA GGTGGCATGG ACCTTGATAT TGACAGGCCC 61 TTCCGGAGAA GAATGATGAA AAGAGGTCCA GATACCAGCA GGGCTTTAAA GGAGTTAGCT 121 ACTCCTGGGT CTTTGAGGGA CACAGCTGAT GAATTTCAAG TTCAGCTAGA TGTTGGCCAC 181 TTTTTACCAA ACGAAATTAC AGTCAAGACA ACCGACGATG ATATTCTTGT CCATGGCAAA 241 CATGACGAGC GGTCTGATGA ATATGGACAC GTCCAAAGAG AATTTCGACG ACGATACAGA 301 CTCCCAGAAC ATGTCAAACC AGAATCTGTG TCATCTACTT TGTCATCAGA TGGTGTCTTA 361 ACTATCCATG CTCCGAAAAC TGCTTTAAGC TCACCAACAG AACGTATCGT ACCCATCACA 421 CCAGCGCCAG CTGTTGGAAG GATTGAAGGG GGAACTACAG GTACTACTAC AGGCAGTACA 481 GCTAGTTCAA CTCCAGCAAG ATCAACAATG AAAA 1714 INDIAN J. MAR. SCI., VOL. 44, NO. 11 NAVEMBER 2015

the scorable trials, while parthenogens displaced A. salina in 98% of the trials12,13. Studies have shown that, the presence of A. franciscana among native Artemia leads to the disappearance of native populations within a few years due to competitive exclusion14,15. A. franciscana cysts have been introduced to Brazil, Australia16, Philippines, Thailand17, India, Sri Lanka18 and Vietnam19. There are reports on the invasion of A. franciscana in Portugal20 France21, Spain, Italy and Morocco22,23. Such invasions are usually accompanied by rapid extinctions of the native Artemia populations24, 14, 15. Even though the deleterious effects of exotic A. franciscana on native Artemia biodiversity have been proved25 and suggestions on control and management put forward,14,26 no actions have been taken to limit the spread of A. franciscana. The ability of A. franciscana to outcompete native species is due to their higher resistance to parasitism by avian cestodes, which reduce the fecundity of brine and increase bird predation27,28.

Conclusion The present study indicates the wipe out of the native A. parthenogenetica by A. franciscana, from Thamaraikulam salt pan, a location previously reported to be inhabited only by the Fig. 1: 1.5% Agarose gel showing PCR amplification of native species. Species identification was based COI gene (lane 1 and 2) and P26 gene (lane 3 and 4) from Artemia cDNA with molecular weight marker (M). on the sequence analysis of two marker genes, cytochrome c oxidase subunit I and P26. By better understanding of the sources, mode and patterns of All three sets of samples gave the same sequences invasion and colonization of A. franciscana, strategies (Table 1) and were analyzed using the National can be developed for the conservation of endemic Center for Biotechnology Information BLAST search species of Artemia in hypersaline ecosystems in program (http://www.ncbi.nlm.nih.gov/). Both COI India. and P26 sequences showed 99% similarity to A. franciscana genes. This is definitive proof that the Acknowledgement Thamaraikulam salt pan, which contained only This study was financially supported by Council of indigenous species of Artemia, has now been Scientific & Industrial Research (CSIR). outcompeted by the invasive foreign species A. franciscana. References 1. Bowen, S.T., Fogarino, E.A., Hitchner, K.N, Dana, G.L., Discussion Chow, V.H.S., Buoncristiani, M.R. & Carl, J.R., Ecological Laboratory competition experiments using intra and isolation in Artemia: population differences in tolerance of anion concentrations. J. Crust. Biol., 5(1985) 106-129. interspecific variability and food level variation have 2. Bowen, S.T., Buoncristiani, M.R. & Carl, J.R., Artemia shown the out competition of parthenogenetic habitats: ion concentrations tolerated by one superspecies. populations by A. franciscana populations in 91% of Hydrobiologia, 158(1988) 201-214.

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