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Two highly diverged New World Artemia species A franciscana and A persimilis from contrasting hypersaline habitats express aconserved stress protein complement

James S Clegg a Gonzalo Gajardo b

a Bodega Marine Laboratory and Section of Molecular and Cellular Biology University of California Davis Bodega Bay CA 94923 USAb Laboratory of Genetics and Aquaculture Universidad de Los Lagos PO Box 933 Osomo Chile

a b s t r a c ta r t i c l e i n f o

Article history

Received 3 March 2009

Received in revised form 8 April 2009

Accepted 8 April 2009

Available online xxxx

Keywords

Artemia franciscana

Artemia persimilis

p26

artemin

hsc70

Stress proteins

The brine shrimp Artemia is a well known animal extremophile adapted to survive in very harsh hypersaline

environments We compared the small stress proteins artemin and p26 and the chaperone hsc70 in encysted

embryos (cysts) of the New World species A franciscana and A persimilis Cysts of the former from San

Francisco Bay USA (SFB) were used essentially as a reference for these proteins while both species were

from locations in Chile where they occur in habitats at latitudinal extremes the Atacama desert and

Patagonia These two species are phylogenetically distant A persimilis being closer to the Old World species

whilst A franciscana is considered younger and undergoing evolutionary expansion Using western blotting

we found all three stress proteins in cysts from these 1047297ve populations in substantial although variable

amounts The protein pro1047297les revealed by Coomassie staining after electrophoresis (SDS-PAGE) were similar

qualitatively in spite of marked differences in the habitats from which these populations originated and the

long time since they diverged We interpret these 1047297ndings as further evidence for the adaptive importance

of these three conserved proteins in coping with the variable but severe stresses these encysted embryos

endure

copy 2009 Elsevier Inc All rights reserved

1 Introduction

Artemia is a cosmopolitan microcrustacean living in hypersaline

environments found on all continents except Antarctica (Van Stappen

2002) In addition to severe hypersalinity these environments impose

a variety of other stresses The life history of Artemia re1047298ects well-

developed adaptive solutions to cope with these conditions (see

Abatzopoulos et al 2002) A key feature is the production of encysted

gastrula embryos (cysts) capable of remarkable resistance to various

stresses including severe desiccation anoxia and exposure to UV

radiation (Clegg and Trotman 2002) Under favorable environ-

mental conditions females generally produce free swimming nauplii

whilst production of cysts is often the method used in stressful

environments However there are signi1047297cant exceptions to this

generality and control of the mode of reproduction is complex (see

Nambu et al 2004 2007 2008 who also evaluatethe literature on the

subject)

Seven sexual species have been described thus far and there are

numerous parthenogenetic populations Five species are found in

Eurasia A salina (Mediterranean area) A urmiana (Iran) A tibetiana

(Tibet) A sinica (China) A spp (Pilla and Beardmore 1994 also see

Gajardo et al 2002) The New World species are A franciscana and

A persimilis the former now being widely distributed in North

Central and South America whilst A persimilis is restricted to certain

locations in Chile and Argentina Both species evolved at different

times from a common ancestor that lived in the Mediterranean area

A persimilis being older and sharing traits with Old World species

like A salina whilst A franciscana is younger and thought to be in

evolutionary expansion due to its great colonizing ability (Gajardo

et al 2002) Studies using coding and non-coding molecular markers

have con1047297rmed theirdivergence (Gajardo et al 2002 Baxevanis et al

2006) In addition Qiu et al (2006) showed that A persimilis was the

most divergent species of all sexual and parthenogenetic types based

on nucleotide sequence similarity in cDNAs of the small stress protein

p26

The occurrence of both species in Chile in highly contrasting

environmental settings that are geographically far apart such as the

Atacama Desert in the north ( A franciscana) and Patagonia in the

south ( A persimilis) (Gajardo et al 1992 Gajardo and Beardmore

1993) offers the opportunity to evaluate their adaptive capability at

the protein level Here we examined three major stress proteins

present in Artemia cysts ndash hsc70 artemin and p26 ndash to evaluate any

qualitative and quantitative differences in cysts from these sites two

of which are at the Southern extreme of Artemia distribution world-

wide

Comparative Biochemistry and Physiology Part A xxx (2009) xxxndashxxx

Corresponding author Tel +1 707 875 2010 fax +1 707 875 2009

E-mail address jscleggucdavisedu (JS Clegg)

CBA-08728 No of Pages 6

1095-6433$ ndash see front matter copy 2009 Elsevier Inc All rights reserved

doi101016jcbpa200904613

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology Part A

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e c b p a

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2 Materials and methods

21 Sources of Artemia cysts and habitat characteristics

San Francisco Bay Dried encysted gastrula embryos (cysts) of A

franciscana from theSouth San Francisco Bay salterns (referred to here

as SFB) were purchased from San Francisco Bay Brand Hayward

California in 1984 Cysts were stored dry under nitrogen gas at about

minus

10 deg C Before use the dried frozen embryos still under nitrogen gaswere equilibrated at room temperature for 5 days Hatching assays

(see Clegg 1997) were performed in 1047297ltered aerobic seawater (SW) at

room temperature (~22 deg C) and constant laboratory light and found

to be 88 quite impressive for these 25 year-old cysts We are not

aware of published water temperatures in SFB but apparently they

rarely exceed 20 deg C (personal communication from Robert Rofen of

Novalec Inc Hayward CA USA Clegg et al 2000)

Chile Artemia were collected from four sites in Chile two being

A persimilis habitats Amarga (50deg 29 South areaof 6 km2 3 m maxi-

mum depth) and Cisnes lagoons (53deg 17South area b01 km2 ~1 m

depth) in Torres del Paine National Park Patagonia Two A franciscana

habitats were sampled namely Chaxa (23deg 02 S area N1 km2 N1 m

depth) and Cejas lagoons (23deg 17 S area N1 km2 N5 m depth) in the

Atacama desert northern Chile Therefore each species is located at

latitudinal extremes with respect to the other and the two locations

experience contrasting climatic conditions On one hand sites in

Patagonia ( A persimilis) are at sea level and subject to subpolar cold

dry andextremely windy conditions Amarga hashighersalinity than

Cisnes which is a shallow and highly eutrophic lagoon These are the

southernmost Artemia sites known world wide In contrast A

franciscana habitats in Chile are athalassohaline inland lakes located

at approximately 2300 m of altitude in isolated evaporitic basins

known as salares (salt 1047298ats) The latter are found in the pre-Cordilleran

Depression (Andes Mountains) of the Atacama Desert one of the

driest places on Earth with b13 mm annual rainfall The climatic

and hydrological inputs (precipitation surface runoff groundwa-

ter in1047298ow) and outputs (evaporation drain losses) determine the

salt content of the brines Temperature is a key factor affecting Arte-

mia populations although differences in ionic strength and composi-tionalso must be considered In Patagonia Soto et al (1994) and Saijo

et al (1995) recorded surface water temperatures between 3 and 15 deg

C these being similar to air temperatures Over a six year period

temperatures ranged between 2 deg C (winter July) and 122 deg C

(summer January)

In contrast ambient temperatures in the salterns of the Atacama are

higher typical of a subtropical and arid climate Water surface tempera-

tures vary between165 (winter) and225 deg C (summer) with signi1047297cant

daynight temperature 1047298uctuations (Demergasso et al 2004)

Due to logistic limitations and remoteness of these sites normally

a one day visit is considered so collecting trips are short In any case

no cysts have been found In Amarga lagoon this is likely to be

connected with extreme winds that perhaps partially sink or suspend

cysts in the water column In thenorthernlocations (Chaxas and Cejaslagoons) cysts are normally not available

Cysts were collected only from the Cisnes lagoon in Patagonia To

obtain cysts from animals at the other three localities juveniles and

adults were collected and transported in plastic bags in a cooler to the

laboratory at Universidad de Los Lagos About 150 individuals were

immediately placed in 20 L aquaria containing water from each site

and that wasprogressively replacedby arti1047297cialseawater (35 ppt)over

a periodof about twomonths duringwhich cystswerecollected Thus

theperiod of cyst collection did not extend more than twogenerations

in culture Animals were fed with suf 1047297cient densities of the alga Du-

naliella tertiolecta Cysts were airdried beforebeingshipped to Bodega

Bayfor analysiswhichwas done withina month of receipt Thegenetic

and reproductive aspects of both species of Chilean Artemia have been

described by Gajardo et al (19951998 2002)

Because of their scarcity (less than 25 mg dry mass each of samples

1 3 and4 were available) no hatching assays were done However it is

likely that they have a high hatch level in view of their very recent

production (see Abatzopoulos et al 2002) The hatching level of

sample 2 cysts ( A persimilis) was found to be extremely low (3 nauplii

hatched from a total of 241 cysts during 10 days of incubation under

conditions conducive to hatching (Clegg 1997)

22 Cyst preparation electrophoresis (SDS-PAGE) and western blotting

Cysts were hydrated overnight in seawater (SW) on ice rinsed

quickly on cloth 1047297lter supports with ice-cold distilled water and the

supports blotted on paper towels for 2 min to remove interstitial water

(Clegg 1997) then weighed Cysts were homogenized at 100 mg wet

mass mL minus1 of homogenizingbuffer(HB) 1M NaCl in 005M Tris pH 74

containing a protease inhibitor cocktail (Completetrade Mini from Roche

Diagnostics GmbH) Known volumes of homogenates were centrifuged

(2000 g for 5 min at 2 deg C) to obtain supernatant (S) and pellet (P)

fractions The latter contain nuclei yolk platelets and shell fragments

Microfuge tubes were drained and the sides wiped to remove most

supernatant Pellets were then resuspended to their pre-centrifugation

volume with HB Known volumes of S and P were added to equal

volumes of 2times sample buffer (Laemmli 1970) and boiled for 5 min

Insoluble shell fragments were then removed from pellet preparations

bycentrifugation(2000g 3 min)Equivalent volumes ofS andP fractions

were electrophoresed in 12 polyacrylamide gels and proteins detected

by Coomassie blue-G staining or analyzed by western immunoblotting

Proteins from SDS-PAGE were transferred to nitrocellulose sheets

and prepared for immunodetection using polyclonal anti-p26 (Clegg

et al 1994) and anti-artemin (a gift from Herman Slegers) at 12000

for 1 h as the primary antibodies Horseradish peroxidase-conjugated

anti-rabbit IgG (11250 1 h) was secondary (Sigma) For detection of

hsc70 we used a primary antibody purchased from Assay Designs Ann

Arbor MI USA (Stressgen SPA-757 at 11250 for 1 h The secondary

antibody was the same as above Chemiluminescence was detected

with Super Signalreg West Pico (Pierce Rockford Illinois) using the Epi

Chemi II Darkroom (UVP Laboratory Products) The latter was also

used to determine the optical densities of bands on developedwestern membranes or color-inverted Coomassie stained gels

23 Heat shock

Approximately 200 mg of hydrated cysts (SFB and Chilean sample2)

were added to 35 ml of aerated seawater pre-heated to 45 deg C in plastic

tubes contained in a water bath (Lauda RM20 plusmn005 deg C) After 30 min

incubation the cysts were 1047297ltered and processed at once for SDS-PAGE

(no recovery period)

24 Analysis of total protein in SFB cysts

To determine protein concentrations P and S fractions were made

to 02 N NaOH and incubated at 37 deg C for 1 h After centrifuging at2000 g for 5 min aliquots of the supernatants were taken for analysis

using the Pierce BCA protein assay kit with bovine serum albumin as

standard All samples were processed at the same time to minimize

variation in protein solubilization There were not enough Chilean

cysts to measure protein however between values for the SFB cysts

and the results of Coomassie staining of gels it is possible to get a

rough idea of the relative protein content of Chilean cysts

3 Results

31 SDS-PAGE and Coomassie staining

Fig 1 shows the results of SDS-PAGE and Coomassie staining of

proteins in P and S fractions of A franciscana cysts from San Francisco

2 JS Clegg G Gajardo Comparative Biochemistry and Physiology Part A xxx (2009) xxxndash xxx

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Bay (SFB) and in cysts from Chile (samples 1ndash4 described in Materials

and Methods) We studied three independent preparations of SFB

cysts to estimate the error and variation involved in our methodology

Although the results are qualitatively similar for all preparations

differences exist such as the dark band below p26 (lower arrow

Fig 1) in SFB cyst supernatants that appears to be lacking or very

greatly reduced in the Chilean preparations Also it appears that SFB

cysts contain more total protein than the Chile cysts based on

Coomassie staining (Fig 1) That is perhaps indicated most clearly by

the relative amounts of histones (detected at and below 20 kD

molecular mass) in the four groups of Chilean cysts compared to SFB

Protein contents of S andP fractions from thethreegroups of SFB cysts

(A B C) in mg protein 100 mg wet wt cystsminus1 were S= 565plusmn 011

standard error of the mean (SEM) and P=1130plusmn037 SEM

32 Immunoblotting

The results of western immunoblotting of these samples are shown

in Fig 2 where the Ponceau-stained membrane indicates the degree of

protein transfer and in conjunction with Fig 1 shows that transfer is

complete except for a few proteins above about 80 kD molecular mass

chie1047298y yolk proteinsHsc70 artemin andp26 aredetected speci1047297cally by

antibodies in thelower panels of Fig2 Interestingly hsc70 is detected in

the pellets from Chile groups 1 and 2 above the background level (A B

and C) that is due to the fact that the pellets were not washed so all of

them contained a small amount of trapped hsc70 We will return to the

signi1047297cance of pellet hsc70 later in the paper The level of hsc70 in

Chilean cysts isthe sameor higherthanthat inSFBcysts (AB C) a resultthat differs greatly from those for artemin and p26

Results on those two proteins in the S fractions (Fig 2) seem to be

at odds with Fig1 where their amounts aremuch more similar to each

other That effect is due in part to the fact that the anti-p26 is not as

strong as the anti-artemin but also that the amount of p26 on the blot

is actually lower in some cases especially for group 2 as we will

document shortly Unlike hsc70 neither artemin nor p26 are present

in any of the pellet fractions over the background level due to trapped

supernatant

33 Optical densities of bands on western blots

Some of the quantitative issues just presented are dealt with

further in Fig 3 where the optical densities (OD) are given above the

bands of all three proteins Likewise errorsfor the 3 bands of SFBcysts

have been calculated (n=3 A B C in supernatant fractions) In terms

of hsc70 in the pellets and taking A B C as background then Chilean

samples 1 2 and 3 appear to contain hsc70 above that due to super-

natant trapping On the other hand the supernatant fractions of sam-

ples 1 and 2 contain more hsc70 than the other preparations

In Fig 3 the ODs for artemin and p26 match the images as per-

ceived visually on the Ponceau-stained blot But how to reconcile

Fig1 Coomassie-stained gel after SDS-PAGE of extracts of Chilean cysts (samples 1ndash4)

and three preparations of cysts from the San Francisco Bay SFB (AndashC) Pellets and

supernatants were prepared in the same way for all cysts as described in the Materials

and Methods section A volume (75 microl) equal to the same mass of cyst equivalents

(075 mg wet wt) was applied per lane Pre-stained protein molecular mass standards

(pss) are shown in the left lane The upper arrow at the right points to artemin and the

lower to p26

Fig 2 Western immunoblotting to detect hsc70 artemin and p26 in Chilean cysts

(samples 1ndash4) and 3 preparations of SFB cysts (AndashC) The top panel is the Ponceau-

stained membrane after transfer The middle and bottom panels are membranes after

incubation with antibodies against hsc70 artemin and p26

Fig 3 The bands in the westerns of Fig 2 were analyzed by densitometry and the unit-

less optical densities (OD) are given above (hsc70 and artemin) or below (p26) the

bands The pellet and supernatant fractions are numbered the same as in Fig 2 Plus or

minus standard errors are given for mean band OD values for the preparations of SFB

cysts (Andash

C) Chile cyst samples are 1ndash

4

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these data with the Coomassie results (Fig 1) To examine that

further we measured densities of the artemin and p26 bands in all

preparations on Coomassie-stained gels Fig 4 shows the outcome for

the artemin and p26 regions Once again we calculated errors for

artemin and p26 inA B and CThen wetook the ODvaluesforthefour

samples of Chilean cysts as a percentageof the A B C meanvalues and

present the results in Table 1

In all cases the amounts of artemin and p26 in samples 1ndash4 are less

than those in the SFB cysts In some cases the amount is much lower

(sample 2 p26) while in others comparatively high (artemin group

4) On balance artemin in the cysts from Chile is over half that in SFB

cysts while p26 is less than 50 These numbers seem to be in general

accord with the visual results of Figs 1ndash4

34 Heat shock

As mentioned previously the scarcity of Chilean cysts has been a

real problem Only sample 2 cysts were suf 1047297cient in amount to

examine the behavior of hsc70 in them compared to SFB cysts under

heat shock conditions Fig 5 shows that hsc70 in pellets from both

kinds of cysts increased as a result of heat shock even in the absence

of a recovery period As expected from Figs 2 and 3 the level of hsc70in pellets from the non-heat shocked SFB cysts was much lower than

that in group 2 cysts from Chile

4 Discussion

This paper demonstrated the presence of three proteins (p26

artemin Hsc70) that are part of the stress-resistant repertoire of

encysted Artemia embryos in four samples of Chilean cysts from

different habitats The two small stress proteins artemin (De Herdt

et al 1979 De Graaf et al 1990) and p26 (Clegg et al 1994) are

present in extremely large amounts in cysts of Artemia franciscana

from salterns in the San Francisco Bay (SFB) and the Great Salt Lake

Utah Both proteins have been studied reasonably well since their

original descriptions (Clegg et al 1995 1999 Liang et al 1997ab

Willsie and Clegg 2002 Chen et al 2003 Crack et al 2002 Tanguay

et al 2004 Warner et al 2004 Sun and MacRae 2005) Each protein

makes up 10 ndash15 of the total non-yolk protein of these embryos and

neither has been detected in any other life cycle stage beyond the 1047297rst

day or two of larval life ( Jackson and Clegg 1996 Crack et al 2002)

There is good evidence that p26playsan importantrole as a molecular

chaperone of proteins in these exceptionally stress-resistant embryos

(see above references and review by Clegg and Trotman 2002)Furthermore indirect evidence suggests that artemin might also be a

molecular chaperone for proteins (Chen et al 2007) as well as RNA

(Warner et al 2004) adding to the evidence for RNA chaperones

(reviewed by Lorsch 2002 Henics 2003)

Because p26 and artemin are such important components of the

adaptive repertoire of Artemia cysts we previously examined a wide

variety of invertebrates for these proteins including the resting stages

of other closely related crustaceans Both proteins were detected by

western blotting in the related genus Parartemia (Clegg and

Campagna 2006) albeit in different amounts However neither

protein was detectedin any of theothersamples (Tanguayet al 2004

Clegg and Campagna 2006 unpublished survey results)

It now seems likely that all species of Artemia contain these

proteins in their cysts an expectation supported in the present studyHowever this genus is found in a wide variety of habitats world-wide

that differ substantially in environmental details (see books by

Persoone et al 1980 Decleir et al 1987 MacRae et al 1989 Warner

et al 1989 Browne et al 1991 Abatzopoulos et al 2002 and the

review by Kaiser et al 2006) Thusknowingthe extent to which these

proteins vary in amount in cysts from different locations was one

motive for the present study Previous work has shown that the levels

of hsc70 artemin and p26 and the upper thermal tolerance of cysts of

A tibetiana are markedly lower than those of A franciscana from SFB

(Clegg et al 2001) A tibetiana lives on the high plateau of Tibet

at about 4500 m where the average daily air temperature is only 1 or

2 deg C and the average daily high water temperature during the repro-

ductive season is about 15 deg C In a similar comparison cysts of

A sinica collected at 1300 m in Inner Mongolia contained signi1047297cantly

Fig 4 Optical densities (OD) of artemin and p26 bands in Coomassie-stained gels Four

samples of Chilean cysts (1ndash4) and three independent preparations of SFB cysts (AndashC)

were examined Means and standard errors of the means (SEM) are given for the SFB

preparations Thus we estimate the percent error to be about 6 for artemin OD bands

and 3 for p26

Table 1

Optical density (OD) of artemin and p26 bands in Chilean cyst extracts using SFB cysts

(A B C) as a comparison

OD as a of A B C mean value

Preparation Group 1 Group 2 Group 3 Group 4

Coomassie

Artemin 63 60 61 76

p26 40 29 48 45

Western

Artemin 59 63 83 88

p26 35 15 42 50

The four groups refer to different Chilean cyst populations Preparations used for

western blotting refer to low speed supernatants (see Materials and methods section)

Fig 5 Comparison of hsc70 in SFB and Chile sample 2 cysts before (C) and after heat

shock (HS) Supernatant (S) and pellet (P) fractions (see Materials and Methods) were

analyzed by Coomassie staining (A) and western blotting (B) Asterisks over the bands

indicate pellets (nuclei) from heat shocked cysts

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lower levels of artemin and p26 but higher levels of hsc70 compared

to SFB cysts (Clegg et al 2001) In addition the upper thermal

tolerance of Mongolian and Tibetian cysts was related to these levels

so one can interpret these differences in terms of the selective

pressures of the thermal habitat of these animals although other

possibilities exist including UV radiation

Another example relating habitat temperature to thermal toler-

ance and stress protein levels comes from the use of A franciscana

cysts from SFB to inoculate arti1047297

cial ponds in the Mekong Delta of Vietnam in which daily water temperatures usually reached 38 deg C

whereas SFB water temperatures rarely exceeded 20 deg C The

Vietnamese-grown cysts showed higher thermal tolerance as well as

substantially higher levels of artemin and p26 (Clegg et al 2000 and

2001) We should note that A franciscana (SFB) exhibits a high degree

of phenotypic plasticity (Browne and Wanigasekera 2000) in the face

of severe environmental conditions (Clegg et al 2000 2001 Tanguay

et al 2004) Indeed North American A franciscana are considered by

some (Amat et al 2005) to be an invasive species in the western

Mediterranean presumably by out-competing native brine shrimp

populations due to its superior adaptive capabilities

In the present study we saw no such dramatic differences in stress

protein levels that could be related to the thermal habitats of Chilean

cysts although some uncertainty about p26 and artemin levels makes

interpretation dif 1047297cult Nevertheless there does seem to be a

relationship between thermal habitat and the levels of p26 and

artemin both being lower in samples 1 and 2 from the cooler and

higher habitat compared to samples 3 and 4 (Fig 3 and especially

Table 1) However those samples are also different species (1 and 2

persimilis 3 and 4 franciscana) so that thermal habitat may or may

not be involved with such differences

There were not enough Chilean cysts to measure their total protein

content but the results of Coomassie straining show clearly that they

contain less protein on a wet weight basis than do cysts of SFB (Figs1

and 4) Recall that the amounts of cyst extract applied per well in all

gels is the same namely the equivalent of 06 mg wet weight of cysts

The explanation we think to be most likely concerns the presence in

the Chilean cyst samples of non-cyst material that could not be

removed such as small bits of debris some of it stuck to the shellsSuch material would contribute to the wet mass of the sample but

unlikely to the protein content Another possibility is that some of the

cysts had lost integrity and as a result some of their protein content

However intact Artemia cysts that dont hatch remain impermeable to

non-volatile solutes and certainly to proteins (Clegg and Trotman

2002 Beladjal et al 2008) Furthermore there is no obvious sign of

signi1047297cant proteolytic activity on any of the gels of Chilean cyst

preparations Although no hatching levels are available for samples 1

3 and 4 the fact that they were produced in culture shortly before use

suggests high viability Therefore the integrity of cysts does not

appear to be a major factor in our results

Of more relevance are the amounts of the proteins of speci1047297c

interest here hsc70 artemin and p26 The hsc70 family is a much

studied stress protein molecular chaperone and we show that thisprotein is present at the same or greater levels in Chilean cysts

compared to those from SFB (Figs 2 3 and 5) Indeed that difference

could be even greater when considering the possibility of lower total

protein content of Chilean cysts

Wereturn to thematter of hsc70 being found in thepellet fractions

of samples 1 and 2 above the background level but not in fractions

from other Chilean and the SFB cysts (Figs 2 and 3) It is well-known

that hsc70 translocates to nuclei of various cell types under stressful

conditions such as heat shock and that includes SFB cysts (see Clegg

et al 2000 Willsie and Clegg 2002) in which such translocations

occur under anoxiaand heat shock Since it hasbeen well documented

that the pellet fractions contain all cyst nuclei (Willsie and Clegg

2002) thepossibilityexists that Chilean cyst samples 1 and2 had been

stressed at some point prior to drying perhaps as a result of severe

hypoxia or anoxia Further support for the possibility of hsc70 trans-

location was obtained from heat shock studies on sample 2 cysts

compared to those from SFB (Fig 5) Even without a recovery period

after heat shock an increase in pellet hsc70 took place in both kinds of

cysts presumably during or immediately after heat shock and prior to

analysis

Although some uncertainty exists about the absolute level of the

stress proteins studied here we believe these results areof value since

they contribute to a continuing catalogue of the presence and evenlevel of expression of these highly adaptive proteins in cysts from

different Artemia species and habitats Chile is the southern limit of

distribution for A franciscana world-wide and is also the location of

quite distinctive A persimilis habitats (sample 2) those being the

southernmost locations recorded for all Artemia thus far We hope to

carry out additional studies on Chilean Artemia in the future

Acknowledgments

The gift of anti-artemin from Professor Herman Slegers is greatly

appreciated A Fondecyt grant from Conicyt-Chile (project 1061190)

made possible Artemia collection in the Atacama Desert and in Torres

del Paine National Park

References

Abatzopoulos Th J Beardmore JA Clegg JS Sorgeloos P 2002 Artemia Basic andApplied Biology Kluwer Academic Publishers Dordrecht The Netherlands

Amat F Hontoria F Ruiz O Green A Sanchez MI Figuerola J Hortas F 2005 TheAmerican brine shrimp as an exotic invasive species in the western MediterraneanBiol Invasions 7 37ndash47

Beladjal L Mertens J Clegg JS 2008 Biochemical and biophysical aspects of thetolerance of anhydrobioticcrustacean embryos to very high temperatures J ThermBiol 33 117ndash127

Baxevanis AD Kappas I Abatzopoulos Th 2006 Molecular phylogenetics andasexuality in the brine shrimp Artemia Mol Phylogenet Evol 40 724ndash738

Browne RA Wanigasekera G 2000 Combined effects of salinity and temperature onsurvival and reproduction of 1047297ve species of Artemia J Exp Mar Biol Ecol 24429ndash44

Browne RASorgeloosPTrotman CNA1991 Artemia BiologyCRC PressBoca RatonChen TAmons RCleggJS WarnerAHMacRae TH2003 Molecularcharacterization

of artemin and ferritin from Artemia franciscana Eur J Biochem 270 137ndash145Chen T Villeneuve T Garant K Amons R MacRae TH 2007 Characterization of

artemin a ferritin homologue synthesized in Artemia embryos during encystmentand diapause FEBS J 274 1093ndash1101

CleggJS 1997 Embryos of Artemia franciscana survive fouryears of continuous anoxiathe case for complete metabolic rate depression J Exp Biol 200 467 ndash475

Clegg JS Trotman CNA 2002 Physiological and biochemical aspects of Artemiaecology In Abatzopoulos Th J Beardmore JA Clegg JS Sorgeloos P (Eds)

Artemia Basic and Applied Biology Kluwer Academic Publishers Dordrecht TheNetherlands pp 129ndash170

Clegg JS Jackson SA Warner AH 1994 Extensive intracellular translocations of amajor protein accompany anoxia in embryos of Artemia franciscana Exp Cell Res212 77ndash83

Clegg JS Willsie JK Jackson SA 1999 Adaptive signi1047297cance of a small heat shockalpha-crystallin protein in encysted embryos of the brine shrimp Artemia

franciscana Am Zool 39 836ndash847Clegg JS Hoa NV Sorgeloos P 2001 Thermal tolerance and heat shock proteins in

encysted embryos of Artemia from widely different thermal habitats Hydrobiologia466 221ndash229

CleggJS Jackson SA LiangP MacRae TH1995 Nuclear-cytoplasmic translocationsof protein p26 during aerobic-anoxic transitions in embryos of Artemia franciscanaExp Cell Res 219 1ndash7

Clegg JS Jackson SA Hoa NV Sorgeloos P 2000 Thermal resistance develop-mental rate and heat shock proteins in Artemia franciscana from San Francisco Bayand southern Vietnam J Exp Mar Biol Ecol 252 85ndash96

CleggJS Campagna V 2006 Comparisons of stressproteinsand soluble carbohydratein encysted embryos of Artemia franciscana and two species of Parartemia CompBiochem Physiol B 145 119ndash125

Crack JA Mansour M Sun Y MacRae TH 2002 Functional analysis of a small heatshockalpha-crystallin protein from Artemia franciscana Oligomerization andthermotolerance Eur J Biochem 269 1ndash10

Decleir WMoens L Slegers H Sorgeloos PJaspersE 1987 Artemia Research and itsApplications vol 2 Universa Press Wetteren Belgium

De Graaf J Amons R Moumlller W 1990 The primary structure of artemin from Artemiacysts Eur J Biochem 193 737ndash750

De Herdt E Slegers H Kondo M 1979 Identi1047297cation and characterization of a 19-Scomplex containing a 27000-Mr protein in Artemia salina Eur J Biochem 96

423ndash

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5 JS Clegg G Gajardo Comparative Biochemistry and Physiology Part A xxx (2009) xxxndash xxx

ARTICLE IN PRESS

Please cite this article as Clegg JS Gajardo G Two highly diverged New World Artemia species A franciscana and A persimilis fromcontrasting hypersaline habitats express Comp Biochem Physiol A (2009) doi101016jcbpa200904613

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httpslidepdfcomreaderfullnew-world-artemia 66

Demergasso C Casamayor EO Chong G Galleguillos P Escudero Pedroacutes-Alioacute C2004 Distribution of prokaryotic genetic diversity in athalassohaline lakes of theAtacama Desert Northern Chile FEMS Microbiol Ecol 48 57ndash69

Gajardo G Wilson R Zuntildeiga O 1992 Report on the occurrence of Artemia in a salinedeposit of the Chilean Andes Crustaceana 63 169ndash174

Gajardo G Beardmore JA 1993 Electrophoretic evidence suggests that the Artemiafound in Chile is A franciscana Kellogg Hydrobiologia 257 65ndash71

Gajardo G Da Conceicao Weber L Beardmore JA 1995 Genetic variability andinterpopulational differentiation among Artemia strains from South AmericaHydrobiologia 302 21ndash29

Gajardo G Colihueque N Parraguez M Sorgeloos P 1998 International study on

Artemia LVIII Morphological differentiation and reproductive isolation of Artemiapopulations from South America Int J Salt Lake Res 7 133ndash151Gajardo G Kappas I Abatzopoulos TJ Beardmore JA 2002 Evolution and

speciation In Abatzopoulos Th J Beardmore JA Clegg JS Sorgeloos P (Eds) Artemia Basic and Applied Biology Kluwer Academic Publishers Dordrecht TheNetherlands pp 225ndash250

Henics T 2003 Extending the ldquostressyrdquo edge molecular chaperones 1047298irting with RNACell Biol Intern 27 1ndash6

Jackson SA Clegg JS 1996 The ontogney of low molecular weight stress protein p26during earlydevelopmentof the brineshrimp Artemia franciscana Develop GrowthDifferen 38 153ndash160

Kaiser HGordonAK Paulet TG2006Review of theAfrican distributionof thebrineshrimp genus Artemia Water SA 32 597ndash604

Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680ndash685

LiangP Amons R MacRae TH CleggJS 1997a Puri1047297cation structure and molecularchaperone activity in vitro of Artemia p26 a small heat shockα-crystallin proteinEur J Biochem 243 225ndash232

LiangP Amons R CleggJS MacRae TH1997b Molecularcharacterization of a small

heat-shockα-crystallin protein from encysted Artemia embryos J Biol Chem 27219051ndash19058

Lorsch JR 2002 RNA chaperones exist and DEAD box proteins get a life Cell 109797ndash800

MacRae TH Bagshaw JC Warner AH 1989 Biochemistry and Cell Biology of Arte-mia CRC Press Boca Raton

Nambu Z Tanaka S Nambu F 2004 In1047298uence of photoperiod and temperature onreproductive mode in the brine shrimp Artemia franciscana J Exp Zool 301A542ndash546

Nambu F Tanaka S Nambu Z 2007 Inbred strains of brine shrimp derived from Artemia franciscana lineage RAPD analysis life span reproductive traits andmode adaptation and tolerance to salinity changes Zool Sci 24 159ndash171

Nambu Z Tanaka S Nambu F Nakano M 2008 In1047298uence of temperature anddarkness on embryonic diapause termination in dormant Artemia cysts that havenever been desiccated J Exp Zool 309A 17ndash24

Persoone G Sorgeloos P Roels O Jaspers E 1980 The Brine Shrimp Artemia vol 2Pilla EJS Beardmore JA 1994 Genetic and morphometric differentiation in Old

World bisexual species of Artemia Heredity 73 47ndash

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Soto D Campos H Steffen W Parra O Zuntildeiga L 1994 The Torres del Paine lakedistrict (Chilean Patagonia) a case of potentially N-limited lakes and ponds ArchHydrobiol 99 181ndash197

Sun Y MacRae TH 2005 Small heat shock proteins molecular structure andchaperone function Cell Mol Life Sci 62 2460ndash2476

Tanguay JA Reyes RC Clegg JS 2004 Habitat diversity and adaptation to environ-mental stress in encysted embryos of the crustacean Artemia J Biosci 29 489ndash501

Van Stappen G 2002 Zoogeography In Abatzopoulos TH Beardmore J Clegg JSSorgeloos P (Eds) Artemia Basic and Applied Biology Kluwer AcademicPublishers Dordrecht The Netherlands pp 171ndash224

Warner AH MacRae TH Bagshaw JC 1989 Cell and Molecular Biology of ArtemiaDevelopment Plenum Press New York

Warner AH Brunet RT MacRae TH Clegg JS 2004 Artemin is an RNA-bindingprotein with high thermal stability and potential RNA chaperone activity ArchBiochem Biophys 424 189ndash200

Willsie JK Clegg JS 2002 Small heat shock protein p26 associates with nuclearlamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells J CellBiochem 84 601ndash614

6 JS Clegg G Gajardo Comparative Biochemistry and Physiology Part A xxx (2009) xxxndash xxx

ARTICLE IN PRESS

Please cite this article as Clegg JS Gajardo G Two highly diverged New World Artemia species A franciscana and A persimilis fromcontrasting hypersaline habitats express Comp Biochem Physiol A (2009) doi101016jcbpa200904613