Brine shrimp ( Artemia salina )唱a marine animal for simple and

JOURNAL of CHINESE CLINICAL MEDICINE VOLUME 2 |
NUMBER 4 |
April 2007
23 6
REVIEW
Brine shrimp ( Artemia salina ) 唱
a marine animal for simple and rapid
biological assays
Anubha Singh Kanwar
INTRODUCTION
Nowadays,there is a tendency that calls for substituting
the use of laboratory animals in toxicological tests due
to the high cost and the animals suffering caused by
these tests.The alternative methods include proceed唱
ings that could replace the experiments carried out with
adverse effects on vertebrates.In addition, vertebrates
can be replaced by invertebrate alternatives in different
tests .
Cyst唱
based tests have been developed from a rela唱
tively new bioassay technology, which makes use of
resting ( dormant ) stages of selected aquatic inverte唱
brate species as biological starting material
[ 7]
.The
animals, reduce the number of animals used in every
main advantage of cyst唱
based bioassays over conven唱
duce pain and stress,which correspond to the 3R prin唱
tenance of live stocks.Artemia salina can be hatched
test,or refine the existing methodology in order to re唱
ciples
[ 1]
.
Artemia salina L. ( Artemiidae ) , the
brine
shrimp,is an invertebrate component of the fauna of sa唱
tional tests is that there is no need for continuous main唱
within a short period of time ( 24 hour) and used for
routine ecotoxicity testing.The availability of eggs, the
ease of hatching them into larvae, the rapid growth of
line aquatic and marine ecosystems.It plays an impor唱
nauplii and the relative ease of maintaining a popula唱
can be used in a laboratory bioassay in order to deter唱
a simple and effective test animal in bioassays and toxi唱
tant role in the energy flow of the food chain
[3 ]
and it
mine toxicity by the estimation of the medium lethal
concentration ( LC 5 0 )
[4]
,which have been reported for
a series of toxins and plant extracts
[ 5]
.
Invertebrate species are used primarily as true
sentinel organisms on the basis of abundance,sampling
tion under laboratory conditions,make the brine shrimp
cology studies.All stages in their life cycle have been
used,and the hatching rate of the eggs after the expo唱
sure to petroleum oil,pesticides,carcinogens and other
contaminants has been used as a criterion for toxici唱
ty
[5 ]
.The commonest stage used is the period of 24 ~
facility, and wide spectrum of ecologic characteristics
48 hours after hatching.
Identification of the lethal con唱
as bioindicators or bioaccumulators is therefore imple唱
( the acute LD 5 0 ) makes the test rapid and simple.
are marginal and primarily involved developmental tox唱
MATERIALS
have clearly highlighted the interest.
A comparative study of an enzymatic activity present in
with changes in populations or communities, such cor唱
nucleoside tetraphosphates is reported.All the purine
and sensitivity to chemicals
[6 ]
.The use of invertebrates
mented before that of vertebrates,but their application
icity and genotoxicity testing.
More recent investigations
In using invertebrates to link individual responses
relations will be of great value for rapid, early warning
assessment of the environmental impact of chemicals.
The use of invertebrates in such a strategy may prevent
centration for 50 % mortality after 6 hours of exposure
Previous Studies
Artemia salina and rat liver which specifically splits di唱
and pyrimidine dinucleoside tetraphosphates tested, i.
e.diadenosine,diguanosine,dixanthosine and diuridine
tetraphosphates , are substrates of enzymes with both
D epartment of Natural Products,National Institute of Pharmaceutical Edu cation & Research,S.A.S.Nagar,Punjab 160062 , India
Corres pondence to D r Anubha Singh K anwar, Scient ist, Departmen t of N atural Products , National Insti tute of Pharmaceu tical Educu tion & Research
( NIPER ), Sector 67 ,S.
A .S N agar( Mohali) Pun jab 16006 2,India
E唱
mail :an ub ha_n ip er@ yahoo.co.
in
JOURNAL of CHINESE CLINICAL MEDICINE VOLUME 2 |
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April 2007
2 37
similar maximum velocities and Km values ( around 10
in its ecosystem.
Brine shrimp feeds on microorganisms
from the two sources is also reported similarly.Particu唱
of contaminants such as phosphorus and nitrogen.Un唱
muM ) .The inhibition by nucleotides of the enzyme
larly relevant is the strong inhibition caused by nucleo唱
side tetraphosphates which have Ki values in the nano唱
molar range.The Artemia enzyme has been observed
with a slightly lower molecular weight ( 17 ,500 ) than
such as algae and bacteria and helps rid the lake water
der optimal conditions, brine shrimp can live up to
three months or more.
However,due to changes such as
temperature and food supply in the lake, the average
life cycle is closer to 1枩
枾 months.Other variables that
the liver enzyme (21,000) and found more resistant to
can affect a brine shrimp摧
s growth and survival include
Artemia salina is named diguanosine tetraphosphatase
a role in brine shrimp hatching.
Commercially available
acidic pH.Based on previous findings,the enzyme from
( EC 3畅6.
1.
17) by the Enzyme Commission.They pro唱
pose to name this enzyme as dinucleoside tetraphos唱
phataseor, dinucleoside
tetraphosphate
nucleotide唱
hydrolase for their resemblance with each other
[ 8]
.
Brine shrimp has an additional property i.
e.posi唱
tive phototaxis and are attracted toward light
[ 9]
.
Types of Artemia Salina
There are few types of them found like Artemia salina
( Mediterranean ), and Artemia franciscana ( Ameri唱
can) ,and two parthenogenetic ( diploid and tetraploid)
strains of Artemia parthenogenetica.
They are extremely
light,pH,oxygen,and salinity of the water .Light plays
eggs of Artemia are easy to be reared and thus easily
studied alive at any time of the year .
Successive stages in the development of the brine
shrimp cyst can be used as a model for studying differ唱
entiation at the level of mRNA transcription and trans唱
lation.Each stage, however, exhibits a characteristic
protein pattern.Two low 唱
molecular唱
weight proteins
prominent in the cyst disappeared almost completely in
the nauplius stage, whereas the proportion of actin in唱
creased 3 times
[1 1]
.
Adult Artemia ( ±1 cm in length)
has an elongated body with two stalked complex eyes,a
linear digestive tract,sensorial antennulae and 11 pairs
different among themselves,in the cloned mitochondrial
of functional thoracopods.
They are extremely different among themselves, much
Artemia Salina and Biological Assays
gaster and Drosophila yakuba and in the same range of
role in the energy flow of the food chain and it can be
man /
cow.The evolutionary distance between the two
icity by the estimation of the medium lethal concentra唱
DNAs ( mtDNAs) isolated from two bisexual species.
more than the differences between Drosophila melano唱
different mammalian species such as mouse /
rat or
Artemia parthenogenetica is high and there is no clear
relationship w ith any of the bisexual species.Using a
Artemia salina L.( Artemiidae ) , plays an important
used in a laboratory bioassay in order to determine tox唱
tion ( LC 5 0 ) .This method,which determines the value
of LC 5 0 of the active compounds and extracts in saline
combination of molecular ( mtDNA) and morphological
medium
temia isolates should be actually considered as belong唱
der to evaluate toxicity, gastro唱
protective action, and
markers it is possible to conclude that all of these.Ar唱
ing to different species,even the two Artemia partheno唱
genetica diploid and tetraploidica
[1 0]
.
Life Cycle of Artemia Salina
Brine shrimp ( Artemia salina) is one of the tiny aquat唱
[ 12 ]
in mg /
ml, has been used in research on
medicinal plants carried out in different countries in or唱
other biological actions,which in some cases have been
related to pharmacological studies carried out for differ唱
ent chemical compounds
[1 3]
being a screening method
mainly for products of plant origin.
In this study the effectiveness of the bioassay of
ic animals that live in hypersaline environments.Cysts
Artemia salina L.( Artemiidae ) for predicting toxicity
first stage in the life cycle of brine shrimp after they
sults of LD 5 0 values obtained in oral acute toxicity test
are dormant brine shrimp babies, and nauplii are the
have been hatched from seed.Brine shrimps are well
adapted to the high salinity and play an important role
of plant extracts is evaluated by comparing it to the re唱
in mice.
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Inhibition of Protein Synthesis
Barbieri,et al
[1 4]
,purified the haemagglutinating lectin
from the seeds of Momordica charantia by affinity chro唱
matography on Sepharose 4B and on acid唱
treated Seph唱
the animal摧
s swimming activity, while temperature in唱
creased or decreased activity as it is raised or lowered
from ambient.The locomotory activity of small groups of
brine shrimp ( Artemia salina ) is studied
[1 8]
, under
arose 6B.It has been reported the mol.wt.115,000
conditions of high hydrostatic pressure,varying temper唱
29,000 and 28,500 and 27,000.Artemia salina ribo唱
Pressure antagonism of anesthesia is reported demon唱
and consisted of four subunits,of mol.wts.
30 ,500 and
somes preincubated with the lectin lose their capacity
to perform protein synthesis.The proteins seem to act
catalytically,since they inactivate a molar excess of ri唱
atures and exposure to several gaseous anesthetics.
strable and might represent a fundamental interaction of
these parameters in biological systems.
bosomes.
Evaluation of Brine Shrimp Bioassays for Saxitoxin
A Bioassay for Mycotoxins
the determination of paralytic shellfish poisoning toxici唱
Brine shrimp is evaluated as alternative test systems for
Brine shrimp larvae is tested as a possible simple bio唱
logical screening system to identify specimens of animal
feedstuffs that should be examined further by chemical
analytical procedures for mycotoxins
[ 15 ]
.
to saxitoxin at a dose level of 5 micrograms
[1 9]
.
Effect of Constant Magnetic Field on Development
of Artemia Eggs
Biogenesis Study of the Sodium唱
and Potassium 唱
ac唱
tivated Adenosine Triphosphatase
Developing nauplii of the brine shrimp Artemia salina
is supposed to be an excellent model system for stud唱
ying the biogenesis of the sodium唱
and potassium唱
activa唱
ATPase) .The
ted adenosine triphosphatase ( Na ,K 唱
+
ty ( saxitoxin).Brine唱
shrimp larvae are shown sensitive
+
Effect of pre唱
flight treatment with constant magnetic
field on development of Artemia eggs retrieved from
Chinese Satellite “ 8885 ” .Dry winter eggs of Artemia
salina are treated with a 30 ~60 mT inhomogeneous
constant magnetic field for 113 hour, and then carried
by Chinese Satellite “ 8885 ” to its spaceflight for 8
ATPase synthesis
nauplii exhibited a burst of Na ,K 唱
days.Their early development rate and hatching ratio
are also studied the site of synthesis of the alpha and
spectively after the accomplishment of the flight.Three
+
+
between 6 and 32 hour of development.Fisher, et al.
ATPase in brine shrimp
beta subunits of the Na ,K 唱
+
nauplii
[1 6]
+
.
Brine Shrimp ( Artemia salina) Nauplii as a Tera唱
togen Test System
are observed on the 10th,31st,65th and 227th days re唱
main results are obtained:the treatment accelerated the
development of the eggs not flown; also enhanced the
resistance of the flown eggs against unfavorable space唱
flight effects (retarding early development and lowering
the hatching ratio) ,and promoted the recovering ability
A teratogen test system is based on disruption of elon唱
of the flown eggs from the affected state;and the latent
cysts.Teratogenicity of substances has been
very durable,still appearing in the development of the
gation between 24 and 48 hour after wetting of the
served
[ 17 ]
ob唱
.Brine shrimp increase in length rapidly after
beneficial effect produced by treatment is found to be
treated eggs on the 318th day after treatment ( the
hatching.Brine shrimp are suited to testing industrial
227th day after the flight)
that can be dissolved in water at 25 ℃.
reported the shortened extremities, or abnormal thorax
Anesthetic Antagonism of the Effects of High Hy唱
aboard Apollo 16 contained one unit with eggs of the
wastes,chemical formulations,drugs,and food additives
drostatic Pressure on Locomotory Activity of the
Brine Shrimp Artemia
Both compression and exposure to anesthesia reduced
Ruther W ,et al.
[ 21 ]
[ 20 ]
.
and Graul EH,et al.
[2 2]
have
or abdomen of brine shrimp.The Biostack experiment
brine shrimp ( Artemia salina ) .The eggs are fixed in
polyvinyl alcohol foils.Nuclear emulsions and plastics
are used as track detectors.The development of 260
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April 2007
and 500 eggs hit by cosmic heavy ions were investiga唱
2 39
the in唱
flight data are limited,spectacular degrees of de唱
ted.
It differed significantly from the development of the
velopment have been achieved.
by heavy ions—and of the ground controls.From these
Different Populations of Artemia and Biological As唱
may injure the encysted blastula.This damage is found
The lethal responses to cadmium of instar II nauplii
flight controls—eggs flown in the Biostack but not hit
results it is concluded that a hit of a single heavy ion
say
to influence the gastrula formation and even the hatch唱
from eight populations of Artemia belonging to the spe唱
ing process of the nauplius.Abnormalities of the ortho唱
nanplius are observed during the development of the hit
cies Artemia franciscana,Artemia salina,Artemia persi唱
milis, and Artemia parthenogenetica have been com唱
eggs.
Artemia salina摧
s dry eggs also exposed to 80 kV唱
pared.Generalized linear models are used to fit mortali唱
both to sparsely and ionizing radiation
type of population and mortality rate.The two popula唱
X唱
rays and Kr唱
Ions,demonstrate a very high resistance
[ 23]
.
Modelling of Human Acute Toxicity from Physico唱
chemical Properties and Invertebrate Acute Toxici唱
ty by PLS Regression and Neural Network
The crustacean ( Artemia salina) is also described im唱
portant to the neural network models
[ 24]
.
The model ob唱
tained is derived from the acute toxicities together with
the physicochemical properties.For the linear type,
modeling is performed using a partial least square pro唱
ty.The model indicated a relationship among species,
tions of Artemia franciscana are the most sensitive to
cadmium toxicity ( median lethal concentration,LC 5 0 ,of
93畅3 ~142 mg /
L ), while the population of Artemia
persimilis is observed the most resistant ( LC 5 0 of 284
mg /
L ) .Differences in the phenotypic variability in
each population are assessed through the slope of the
mortality curve; A persimilis exhibited the highest di唱
versity
[ 26 ]
.
jection to latent structures ( PLS ) regression method;
The Biological Effectiveness of HZE唱
particles of
ral network are utilized.A neural network using a com唱
Biostack experiments
for the non唱
linear models,both PLS regression and neu唱
Cosmic Radiation Studied in the Apollo 16 and 17
bination of back唱
propagation and cascade唱
correlation
The Biostack experiments I and II
algorithms is applied to this study.
The Brine Shrimp,Artemia salina, Has Been Used
as a Model System to Assess Microgravity Effects
The brine shrimp, Artemia salina, has been used as a
model system to assess microgravity effects on develo唱
ping organisms.Following fertilization and early devel唱
opment,the egg can be arrested in early gastrula as a
dehydrated cyst stage that is found stable to harsh envi唱
ronments over long唱
time periods.When salt water is
added,the cysts can reactivated,and embryonic devel唱
opment and egg hatching can be seen in about 24
hours .
A series of larval molts or instars,over about a 2唱
[2 7]
are flown on
board the Apollo 16 and 17 command modules in order
to obtain information on the biological damage pro唱
duced by the bombardment of heavy high唱
energy
( HZE ) particles of cosmic radiation during space唱
flight.Such data are required for estimating radiation
hazards in manned spaceflight.Eggs of Artemia salina
are accommodated in the two Biostacks by using a spe唱
cial sandwich construction of visual track detectors and
layers of biological objects,identification of each hit bi唱
ological object is achieved and the possible biological
damage correlated with the physical features of the re唱
sponsible HZE 唱
particle.A high sensitivity to HZE 唱
par唱
ticle bombardment is observed on Artemia salina eggs;
week period, resulted in the adult crustacean.Spoo唱
90% of the embryos, which are induced to develop
closed syringe system, a bioprocessing module, in
Malformations of the abdomen or the extremities of the
ner
[2 5]
have assessed these developmental events in a
ground唱
based studies, and have conducted preliminary
in唱
orbit experiments aboard the Space Shuttle Atlantis
during the flights of STS -37 and STS -43畅Although
from hit eggs, died at different developmental stages.
nauplius are frequently induced.This has provided an
improved knowledge about radiation conditions inside
of spacecrafts,necessary to secure a maximum possible
24 0
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April 2007
protection to the astronauts.
CONCLUSION
The Brine shrimp ( Artemia salina) is found to be a
simple marine organism, easy to breed and hatch in
normal laboratory conditions.It has the complete and
effective range to test the toxicity as well as for various
assays.
So after a through review of literature,it can be
suggested one of the best and rapid biological and toxi唱
cological assays for all the labs especially dealing with
screening of drugs /
compounds /
plant extracts.
of medicinal plants in traditional medicin es in Tanzania .East Afr
M ed J ,1995 ,72 :6 61 -663 .
12 . Fumaral
F , G archit orena
M . A rtemia
salin a. R ecolecció
n,
descap sulació
n y desarroll o.
R evi sta Aquamar,1996 ,3 :22 -24.
13 .Barb ieri L ,Z amboni M ,Lorenz oni E ,et al.Inh ib ition of p rotein s yn 唱
thesis i n vitro by proteins from the seeds of Momord ica charantia
( bitter pear melon ) .Biochem J ,19 80 ,1 86:443 -452 .
14 .Prior M .
Evaluat ion of brin e shrimp ( Artemia salina) larvae as a bio唱
assay for mycotoxins in animal feedstuffs.Can J Comp M ed , 19 79 ,
43:352 -3 55 .
15 .Fish er J ,Baxter -Lowe L,Hokin L .
Site of synt hesis of t he alph a and
beta subunits of the N a, K 唱
A TPase in bri ne s hrimp nauplii .J Biol
Chem ,1984,25 9:14 217.
16 .Kerster H , Schaeff er D.Brine shrimp ( A rt emia salina ) n auplii as a
teratogen test system.Ecot oxicol Environ Saf,1 98 3,7:342 -34 9.
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( Editor HOU)