Indian Journal of Geo-Marine Sciences
Vol. 44(11), November 2015, pp. 1712-1715
Loss of indigenous brine shrimp 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 Artemia or brine shrimp is an anostracan
crustacean widely used as live feed in aquaculture.
Being highly osmotolerant, their natural habitats are
hypersaline environments like coastal or inland
salterns1,2. Indian populations of Artemia is mainly
Artemia parthenogenetica and have been reported
from about 19 different areas, including Bhayander,
Didwana, Jamnagar, Karsewar Island, Kutch,
Mithapur, Pattanamaruthur, Spic Nagar, Thirispuram,
Tuticorin,
Nagercoil,
Vadala,
Vedaranyam,
Veppalodai and Vivar3. Artemia franciscana is a
highly invasive commercial brine shrimp species,
harvested mainly from the Great Salt Lake (GSL) and
San Francisco Bay (SFB) salt works in USA and has
resulted in the displacement of native Artemia species
throughout the world. The high demand for imported
Artemia cysts has led to the introduction of A.
franciscana into sites previously occupied by native
Artemia species. In India, culture of Sanfracisco Bay
strain of A. franciscana was introduced by Bharath
Salt chemicals Industries, Gujarat during early 80’s4.
Materials and Methods
Samples used in this study were originally collected
for a comparative study on the small heat shock
proteins from the native parthenogenetic species to
the bisexual A. franciscana. Thamaraikulam salt pan
(8°06'N 77°29'E) in Nagercoil, Tamil Nadu was
selected for the study since it was reported to be free
from bisexual species5. The Artemia population at
Thamaraikulam was characterized by John et al.
(2004) and was reported to be parthenogenetic6.
Temperature and salinity tolerance data suggest that
the native strain can tolerate a wide range of
temperatures from 22 to 30°C, but the salinity
tolerance is highly limited to 35 ppt. According to
adult morphometric characters, they were found to be
closer to Egyptian asexual populations7. Vasudevan
(2012) studied the biometrical, morphological and
biochemical characteristics of parthenogenetic
Artemia population at Thamaraikulam8. It was
reported in a study carried out during 2002–2003, that
the indigenous parthenogenetic Artemia species at
Chennai and Tuticorin salt pans were eliminated by
the introduction of exotic bisexual strain, while those
at Vedaraniyam and Nagercoil were parthenogenetic
in nature5,9,10.
Since different species of Artemia exhibits very
similar morphology, it is essential to use molecular
techniques to determinate the species, for
conservation studies. In the present study, two marker
genes, mitochondrial gene cytochrome c oxidase I
(COI) and genomic small heat shock protein gene P26
GAYATHRI et al.: LOSS OF INDIGENOUS BRINE SHRIMP ARTEMIA PARTHENOGENETICA
were used for species identification. COI gene is
widely used as a DNA barcode to identify animal
species because its mutation rate is often fast enough
to distinguish closely related species and also because
its sequence is conserved among conspecifics.
Adult Artemia
were
collected
from
the
Thamaraikulam salterns during March, July,
November 2013. Presence of males in the collected
sample indicated the presence of bisexual strain of
Artemia. They were cultured in autoclaved, 0.22 µm
vacuum filtered seawater. The cultures were grown at
room temperature on the bench top with moderate
aeration and were fed with unicellular brown algae
Isochrysis galbana daily. Cysts produced were
collected, washed, dried and stored at -20° C.
Total RNA was isolated from 100mg cyst samples
using TRIzol reagent (Life Technologies Invitrogen,
USA)11. Cysts were homogenized in 1 mL TRIzol
reagent using micropestle at room temperature and
then passes through a 2.5mL syringe. Homogenized
samples were incubated at room temperature for 5
min, centrifuged and the supernatant was extracted
once with chloroform: isoamyl alcohol (24:1) and the
RNA was precipitated using 100% ethanol (ice cold).
The pellet was washed with 70% ethanol, dissolved in
50uL diethyl pyrocarbonate treated water and stored
at -20 °C.
Results
cDNA was prepared using SuperScript® III Reverse
Transcriptase kit (Invitrogen, USA) using oligo(dT)
primers. The COI gene was amplified from cDNA
using the primers COIF 5`- ATT CTA CGA ATC
ACA AGG ATA TTG G - 3` and COIR5`- TAC ACT
TCA GGA TGG CCA AAA AAT CA - 3` and small
heat shock protein P26 gene using primers P26 AF 5`TAC GGA GGA TTT GGT GGT ATG - 3` and P26
AR 5`- ATT GTT GAT CTT GCT GGA GTT G - 3`.
EmeraldAmp® (Takara) mix was used for PCR
amplification with the following temperature profiles
and conditions: 1 min at 98 °C, 30 cycles of 10 s at 98
°C, 30 s at 55 °C, 1 min 30 s at 72 °C and a final
extension of 7 min at 72 °C. Total reaction volumes of
25 µl consisted of 0.5 µl template DNA, 12.5 µl
Emerald mix, 1 µl of each primer (10 µM).
Three sets of COI and P26 gene samples were
sequenced at the Regional Facility for DNA
Fingerprinting,
Rajiv
Gandhi
Centre
for
Biotechnology (Thiruvananthapuram, Kerala).
Table 1: DNA sequence of COI and P26 gene
COI gene sequence:
AAACCTTTAA
TCATTCGAGC
TTATTGTGAC
GGGGATTTGG
GGTTAAATAA
CTATAGTTGA
TTGCCCATGC
CCTCTATCTT
TATCTATTGA
TTTTATCACT
ATACTTCTTT
GATTTTTTGG
ATTTTTTGGG
AGAGTTGGGT
AGCTCATGCA
TAACTGGCTA
TTTAAGATTT
GAGAGGTGCA
CGGACCTTCT
AGGGGCTGTA
CCGTATACCT
TCCAGTCCTA
CTTCGACCCC
CCATCCGGAA
GGCTTGAGCA
CAACCAGGTT
TTTATTATAA
GTACCCATTA
TGAATACTTC
GGAACTGGAT
GTAGATTTAG
AATTTTATTA
CTCTTCGTCT
GCGGGGGCTA
GCAGGTGGTG
ATTTAAAA
GGTATAGTTG
CCCTGATTGG
TTTTTTTCAT
TATTGGGGGC
CACCATCCTT
GAACAGTTTA
CTATTTTCTC
CTACTATCAT
GAGCAGTAGG
TTACTATACT
GGGATCCCAT
AGGAACTTCT
CGATGAACAA
GGTTATACCA
CCCGGATATA
GACTCTTCTC
TCCCCCTCTA
GCTTCATTTA
TAATATACGA
AATCACCGCC
GTTAACTGAT
CCTTTATCAA
TTAAGAATGC
GTATATAATG
ATCTTGATTG
GCATTTCCCC
TTGGCCAGAT
TCCTCAGCCA
GCTGGAGTTT
CCCCAGTCAA
GTTCTTCTCC
CGTAACTTAA
CATTTATTTT
P26 gene sequence:
1 CTCTGATCAT
61 TTCCGGAGAA
121 ACTCCTGGGT
181 TTTTTACCAA
241 CATGACGAGC
301 CTCCCAGAAC
361 ACTATCCATG
421 CCAGCGCCAG
481 GCTAGTTCAA
TTGGATTTGG
GAATGATGAA
CTTTGAGGGA
ACGAAATTAC
GGTCTGATGA
ATGTCAAACC
CTCCGAAAAC
CTGTTGGAAG
CTCCAGCAAG
TGGCTTCGGA
AAGAGGTCCA
CACAGCTGAT
AGTCAAGACA
ATATGGACAC
AGAATCTGTG
TGCTTTAAGC
GATTGAAGGG
ATCAACAATG
GGTGGCATGG
GATACCAGCA
GAATTTCAAG
ACCGACGATG
GTCCAAAGAG
TCATCTACTT
TCACCAACAG
GGAACTACAG
AAAA
ACCTTGATAT
GGGCTTTAAA
TTCAGCTAGA
ATATTCTTGT
AATTTCGACG
TGTCATCAGA
AACGTATCGT
GTACTACTAC
TGACAGGCCC
GGAGTTAGCT
TGTTGGCCAC
CCATGGCAAA
ACGATACAGA
TGGTGTCTTA
ACCCATCACA
AGGCAGTACA
1
61
121
181
241
301
361
421
481
541
601
661
1713
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 shrimps and
increase bird predation27,28.
Conclusion
Fig. 1: 1.5% Agarose gel showing PCR amplification of
COI gene (lane 1 and 2) and P26 gene (lane 3 and 4) from
Artemia cDNA with molecular weight marker (M).
All three sets of samples gave the same sequences
(Table 1) and were analyzed using the National
Center for Biotechnology Information BLAST search
program (http://www.ncbi.nlm.nih.gov/). Both COI
and P26 sequences showed 99% similarity to A.
franciscana genes. This is definitive proof that the
Thamaraikulam salt pan, which contained only
indigenous species of Artemia, has now been
outcompeted by the invasive foreign species A.
franciscana.
Discussion
Laboratory competition experiments using intra and
interspecific variability and food level variation have
shown the out competition of parthenogenetic
populations by A. franciscana populations in 91% of
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
native species. Species identification was based
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
invasion and colonization of A. franciscana, strategies
can be developed for the conservation of endemic
species of Artemia in hypersaline ecosystems in
India.
Acknowledgement
This study was financially supported by Council of
Scientific & Industrial Research (CSIR).
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