ARTICLE IN PRESS

CBA-08728; No of Pages 6

Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

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

Two highly diverged New World Artemia species, A. franciscana and

A. persimilis, from contrasting hypersaline habitats express a
conserved stress protein complement
James S. Clegg a,, Gonzalo Gajardo b
a
b

Bodega Marine Laboratory and Section of Molecular and Cellular Biology, University of California, Davis, Bodega Bay, CA 94923, USA
Laboratory of Genetics and Aquaculture. Universidad de Los Lagos. P.O. Box 933, Osomo, Chile

a 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

a b s t r a c t
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 ve populations in substantial although variable
amounts. The protein proles 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 ndings as further evidence for the adaptive importance
of these three conserved proteins in coping with the variable, but severe stresses these encysted embryos
endure.
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 reects welldeveloped 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 environmental conditions females generally produce free swimming nauplii,
whilst production of cysts is often the method used in stressful
environments. However, there are signicant exceptions to this
generality, and control of the mode of reproduction is complex (see
Nambu et al., 2004, 2007, 2008, who also evaluate the 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
Corresponding author. Tel.: +1 707 875 2010; fax: +1 707 875 2009.
E-mail address: jsclegg@ucdavis.edu (J.S. Clegg).

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 conrmed their divergence (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 hsc70, artemin and p26 to evaluate any
qualitative and quantitative differences in cysts from these sites, two
of which are at the Southern extreme of Artemia distribution worldwide.

1095-6433/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2009.04.613

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613

ARTICLE IN PRESS
2

J.S. Clegg, G. Gajardo / Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

2. Materials and methods

2.1. Sources of Artemia cysts and habitat characteristics
San Francisco Bay: Dried encysted gastrula embryos (cysts) of A.
franciscana from the South 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
10 C. Before use, the dried frozen embryos, still under nitrogen gas,
were equilibrated at room temperature for 5 days. Hatching assays
(see Clegg, 1997) were performed in ltered aerobic seawater (SW) at
room temperature (~22 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 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 (50 29' South, area:of 6 km2; 3 m maximum depth) and Cisnes lagoons (53 17'South, area: b0.1 km2; ~1 m
depth) in Torres del Paine National Park, Patagonia. Two A. franciscana
habitats were sampled, namely, Chaxa (23 02' S, area N1 km2, N1 m
depth) and Cejas lagoons (23 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, and extremely windy conditions. Amarga has higher salinity 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 ats). 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, groundwater inow) and outputs (evaporation, drain losses) determine the
salt content of the brines Temperature is a key factor affecting Artemia populations, although differences in ionic strength and composition also must be considered. In Patagonia, Soto et al. (1994) and Saijo
et al. (1995) recorded surface water temperatures between 3 and 15
C, these being similar to air temperatures. Over a six year period
temperatures ranged between 2 C (winter, July) and 12.2 C
(summer, January).
In contrast, ambient temperatures in the salterns of the Atacama are
higher, typical of a subtropical and arid climate. Water surface temperatures vary between 16.5 (winter) and 22.5 C (summer) with signicant
day/night temperature uctuations (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 the northern locations (Chaxas and Cejas
lagoons) 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 was progressively replaced by articial seawater (35 ppt) over
a period of about two months, during which cysts were collected. Thus,
the period of cyst collection did not extend more than two generations
in culture. Animals were fed with sufcient densities of the alga Dunaliella tertiolecta. Cysts were air dried before being shipped to Bodega
Bay for analysis which was done within a month of receipt. The genetic
and reproductive aspects of both species of Chilean Artemia have been
described by Gajardo et al. (1995, 1998, 2002).

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

1, 3 and 4 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).
2.2. Cyst preparation, electrophoresis (SDS-PAGE) and western blotting
Cysts were hydrated overnight in seawater (SW) on ice, rinsed
quickly on cloth lter 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 1 of homogenizing buffer (HB): 1M NaCl in 0.05M Tris, pH 7.4,
containing a protease inhibitor cocktail (Complete Mini from Roche
Diagnostics GmbH). Known volumes of homogenates were centrifuged
(2000 g for 5 min at 2 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 2 sample buffer (Laemmli, 1970) and boiled for 5 min.
Insoluble shell fragments were then removed from pellet preparations
by centrifugation (2000 g, 3 min). Equivalent volumes of S and P 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 1:2000
for 1 h as the primary antibodies. Horseradish peroxidase-conjugated
anti-rabbit IgG (1:1250, 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 1:1,250 for 1 h. The secondary
antibody was the same as above. Chemiluminescence was detected
with Super Signal 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 developed
western membranes or color-inverted Coomassie stained gels.
2.3. Heat shock
Approximately 200 mg of hydrated cysts (SFB and Chilean sample 2)
were added to 35 ml of aerated seawater pre-heated to 45 C in plastic
tubes contained in a water bath (Lauda RM20, 0.05 C). After 30 min
incubation the cysts were ltered and processed at once for SDS-PAGE
(no recovery period).
2.4. Analysis of total protein in SFB cysts
To determine protein concentrations P and S fractions were made
to 0.2 N NaOH and incubated at 37 C for 1 h. After centrifuging at
2000 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
3.1. 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

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613

ARTICLE IN PRESS
J.S. Clegg, G. Gajardo / Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

Fig. 1. Coomassie-stained gel after SDS-PAGE of extracts of Chilean cysts (samples 14)
and three preparations of cysts from the San Francisco Bay, SFB (AC). Pellets and
supernatants were prepared in the same way for all cysts as described in the Materials
and Methods section. A volume (7.5 l) equal to the same mass of cyst equivalents
(0.75 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.

Bay (SFB) and in cysts from Chile (samples 14, 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 and P fractions from the three groups of SFB cysts
(A, B, C), in mg protein .100 mg wet wt cysts 1, were: S = 5.65 0.11
standard error of the mean (SEM) and P = 11.30 0.37 SEM.
3.2. 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,
chiey yolk proteins. Hsc70, artemin and p26 are detected specically by
antibodies in the lower panels of Fig. 2. 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
signicance of pellet hsc70 later in the paper. The level of hsc70 in
Chilean cysts is the same or higher than that in SFB cysts (A, B, C), a result
that 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 Fig. 1 where their amounts are much 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.
3.3. 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

Fig. 2. Western immunoblotting to detect hsc70, artemin and p26 in Chilean cysts
(samples 14) and 3 preparations of SFB cysts (AC). The top panel is the Ponceaustained membrane after transfer. The middle and bottom panels are membranes after
incubation with antibodies against hsc70, artemin and p26.

bands of all three proteins. Likewise, errors for the 3 bands of SFB cysts
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 supernatant trapping. On the other hand the supernatant fractions of samples 1 and 2 contain more hsc70 than the other preparations.
In Fig. 3 the ODs for artemin and p26 match the images as perceived visually on the Ponceau-stained blot. But how to reconcile

Fig. 3. The bands in the westerns of Fig. 2 were analyzed by densitometry and the unitless 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 (AC). Chile cyst samples are 14.

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613

ARTICLE IN PRESS
4

J.S. Clegg, G. Gajardo / Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

Fig. 4. Optical densities (OD) of artemin and p26 bands in Coomassie-stained gels. Four
samples of Chilean cysts (14) and three independent preparations of SFB cysts (AC)
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.

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 in A, B and C. Then we took the OD values for the four
samples of Chilean cysts as a percentage of the A, B, C mean values and
present the results in Table 1.
In all cases the amounts of artemin and p26 in samples 14 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. 14.
3.4. Heat shock
As mentioned previously, the scarcity of Chilean cysts has been a
real problem. Only sample 2 cysts were sufcient 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 hsc70
in 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

Table 1
Optical density (O.D.) of artemin and p26 bands in Chilean cyst extracts using SFB cysts
(A, B, C) as a comparison.
O.D. as a % of A, B, C mean value
Preparation
Coomassie:
Artemin
p26
Western:
Artemin
p26

Group 1

Group 2

Group 3

Group 4

63
40

60
29

61
48

76
45

59
35

63
15

83
42

88
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.

original descriptions (Clegg et al., 1995, 1999; Liang et al., 1997a,b;

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 15% of the total non-yolk protein of these embryos, and
neither has been detected in any other life cycle stage beyond the rst
day or two of larval life (Jackson and Clegg, 1996; Crack et al., 2002).
There is good evidence that p26 plays an important role 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 detected in any of the other samples (Tanguay et 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 study.
However, 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). Thus, knowing the 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 C and the average daily high water temperature during the reproductive season is about 15 C. In a similar comparison, cysts of
A. sinica collected at 1300 m in Inner Mongolia contained signicantly

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613

ARTICLE IN PRESS
J.S. Clegg, G. Gajardo / Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

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 tolerance and stress protein levels comes from the use of A. franciscana
cysts from SFB to inoculate articial ponds in the Mekong Delta of
Vietnam in which daily water temperatures usually reached 38 C,
whereas SFB water temperatures rarely exceeded 20 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 difcult. 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 (Figs. 1
and 4). Recall that the amounts of cyst extract applied per well in all
gels is the same, namely the equivalent of 0.6 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 shells.
Such 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 don't 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
signicant 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 specic
interest here: hsc70, artemin and p26. The hsc70 family is a much
studied stress protein / molecular chaperone and we show that this
protein 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.
We return to the matter of hsc70 being found in the pellet 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 anoxia and heat shock. Since it has been well documented
that the pellet fractions contain all cyst nuclei (Willsie and Clegg,
2002) the possibility exists that Chilean cyst samples 1 and 2 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 translocation 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 are of value since
they contribute to a continuing catalogue of the presence and even
level 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, J.A., Clegg, J.S., Sorgeloos, P., 2002. Artemia: Basic and
Applied Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Amat, F., Hontoria, F., Ruiz, O., Green, A., Sanchez, M.I., Figuerola, J., Hortas, F., 2005. The
American brine shrimp as an exotic invasive species in the western Mediterranean.
Biol. Invasions 7, 3747.
Beladjal, L., Mertens, J., Clegg, J.S., 2008. Biochemical and biophysical aspects of the
tolerance of anhydrobiotic crustacean embryos to very high temperatures. J. Therm.
Biol. 33, 117127.
Baxevanis, A.D., Kappas, I., Abatzopoulos, Th., 2006. Molecular phylogenetics and
asexuality in the brine shrimp Artemia. Mol. Phylogenet. Evol. 40, 724738.
Browne, R.A., Wanigasekera, G., 2000. Combined effects of salinity and temperature on
survival and reproduction of ve species of Artemia. J. Exp. Mar. Biol. Ecol. 244,
2944.
Browne, R.A., Sorgeloos, P., Trotman, C.N.A., 1991. Artemia Biology. CRC Press, Boca Raton.
Chen, T., Amons, R., Clegg, J.S., Warner, A.H., MacRae, T.H., 2003. Molecular characterization
of artemin and ferritin from Artemia franciscana. Eur. J. Biochem. 270, 137145.
Chen, T., Villeneuve, T., Garant, K., Amons, R., MacRae, T.H., 2007. Characterization of
artemin, a ferritin homologue synthesized in Artemia embryos during encystment
and diapause. FEBS J. 274, 10931101.
Clegg, J.S., 1997. Embryos of Artemia franciscana survive four years of continuous anoxia:
the case for complete metabolic rate depression. J. Exp. Biol. 200, 467475.
Clegg, J.S., Trotman, C.N.A., 2002. Physiological and biochemical aspects of Artemia
ecology. In: Abatzopoulos, Th. J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.),
Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp. 129170.
Clegg, J.S., Jackson, S.A., Warner, A.H., 1994. Extensive intracellular translocations of a
major protein accompany anoxia in embryos of Artemia franciscana. Exp. Cell Res.
212, 7783.
Clegg, J.S., Willsie, J.K., Jackson, S.A., 1999. Adaptive signicance of a small heat shock/
alpha-crystallin protein in encysted embryos of the brine shrimp, Artemia
franciscana. Am. Zool. 39, 836847.
Clegg, J.S., Hoa, N.V., Sorgeloos, P., 2001. Thermal tolerance and heat shock proteins in
encysted embryos of Artemia from widely different thermal habitats. Hydrobiologia
466, 221229.
Clegg, J.S., Jackson, S.A., Liang, P., MacRae, T.H., 1995. Nuclear-cytoplasmic translocations
of protein p26 during aerobic-anoxic transitions in embryos of Artemia franciscana.
Exp. Cell Res. 219, 17.
Clegg, J.S., Jackson, S.A., Hoa, N.V., Sorgeloos, P., 2000. Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana from San Francisco Bay
and southern Vietnam. J. Exp. Mar. Biol. Ecol. 252, 8596.
Clegg, J.S., Campagna, V., 2006. Comparisons of stress proteins and soluble carbohydrate
in encysted embryos of Artemia franciscana and two species of Parartemia. Comp.
Biochem. Physiol. B 145, 119125.
Crack, J.A., Mansour, M., Sun, Y., MacRae, T.H., 2002. Functional analysis of a small heat
shock/alpha-crystallin protein from Artemia franciscana: Oligomerization and
thermotolerance. Eur. J. Biochem. 269, 110.
Decleir, W., Moens, L., Slegers, H., Sorgeloos, P., Jaspers, E., 1987. Artemia Research and its
Applications, vol. 2. Universa Press, Wetteren, Belgium.
De Graaf, J., Amons, R., Mller, W., 1990. The primary structure of artemin from Artemia
cysts. Eur. J. Biochem. 193, 737750.
De Herdt, E., Slegers, H., Kondo, M., 1979. Identication and characterization of a 19-S
complex containing a 27,000-Mr protein in Artemia salina. Eur. J. Biochem. 96,
423430.

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613

ARTICLE IN PRESS
6

J.S. Clegg, G. Gajardo / Comparative Biochemistry and Physiology, Part A xxx (2009) xxxxxx

Demergasso, C., Casamayor, E.O., Chong, G., Galleguillos, P., Escudero, Pedrs-Ali, C.,
2004. Distribution of prokaryotic genetic diversity in athalassohaline lakes of the
Atacama Desert, Northern Chile. FEMS Microbiol. Ecol. 48, 5769.
Gajardo, G., Wilson, R., Zuiga, O., 1992. Report on the occurrence of Artemia in a saline
deposit of the Chilean Andes. Crustaceana 63, 169174.
Gajardo, G., Beardmore, J.A., 1993. Electrophoretic evidence suggests that the Artemia
found in Chile is A. franciscana Kellogg. Hydrobiologia 257, 6571.
Gajardo, G., Da Conceicao, Weber, L., Beardmore, J.A., 1995. Genetic variability and
interpopulational differentiation among Artemia strains from South America.
Hydrobiologia 302, 2129.
Gajardo, G., Colihueque, N., Parraguez, M., Sorgeloos, P., 1998. International study on
Artemia. LVIII. Morphological differentiation and reproductive isolation of Artemia
populations from South America. Int. J. Salt Lake Res. 7, 133151.
Gajardo, G., Kappas, I., Abatzopoulos, T.J., Beardmore, J.A., 2002. Evolution and
speciation. In: Abatzopoulos, Th. J., Beardmore, J.A., Clegg, J.S., Sorgeloos, P. (Eds.),
Artemia: Basic and Applied Biology. Kluwer Academic Publishers, Dordrecht, The
Netherlands, pp. 225250.
Henics, T., 2003. Extending the stressy edge: molecular chaperones irting with RNA.
Cell Biol. Intern. 27, 16.
Jackson, S.A., Clegg, J.S., 1996. The ontogney of low molecular weight stress protein p26
during early development of the brine shrimp, Artemia franciscana. Develop. Growth
Differen. 38, 153160.
Kaiser, H., Gordon, A.K., Paulet, T.G., 2006. Review of the African distribution of the brine
shrimp genus Artemia. Water S.A. 32, 597604.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680685.
Liang, P., Amons, R., MacRae, T.H., Clegg, J.S., 1997a. Purication, structure and molecular
chaperone activity in vitro of Artemia p26, a small heat shock/-crystallin protein.
Eur. J. Biochem. 243, 225232.
Liang, P., Amons, R., Clegg, J.S., MacRae, T.H., 1997b. Molecular characterization of a small
heat-shock/-crystallin protein from encysted Artemia embryos. J. Biol. Chem. 272,
1905119058.
Lorsch, J.R., 2002. RNA chaperones exist and DEAD box proteins get a life. Cell 109,
797800.
MacRae, T.H., Bagshaw, J.C., Warner, A.H., 1989. Biochemistry and Cell Biology of Artemia. CRC Press, Boca Raton.

Nambu, Z., Tanaka, S., Nambu, F., 2004. Inuence of photoperiod and temperature on
reproductive mode in the brine shrimp, Artemia franciscana. J. Exp. Zool. 301A,
542546.
Nambu, F., Tanaka, S., Nambu, Z., 2007. Inbred strains of brine shrimp derived from
Artemia franciscana lineage, RAPD analysis, life span, reproductive traits and
mode, adaptation, and tolerance to salinity changes. Zool. Sci. 24, 159171.
Nambu, Z., Tanaka, S., Nambu, F., Nakano, M., 2008. Inuence of temperature and
darkness on embryonic diapause termination in dormant Artemia cysts that have
never been desiccated. J. Exp. Zool. 309A, 1724.
Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E., 1980. The Brine Shrimp. Artemia, vol. 2.
Pilla, E.J.S., Beardmore, J.A., 1994. Genetic and morphometric differentiation in Old
World bisexual species of Artemia. Heredity 73, 4756.
Qiu, Z., Bossier, P., Wang, X., Bojikova-Fournier, S., MacRae, T.H., 2006. Diversity, structure
and expression of the gene for p26, a small heat shock protein from Artemia. Genomics
88, 230240.
Saijo, Y., Mitamura, O., Tanaka, M., 1995. A note on the chemical composition of lake
water in the Laguna Amarga, a saline lake in Patagonia, Chile. Int. J. Salt Lake Res. 4,
165167.
Soto, D., Campos, H., Steffen, W., Parra, O., Zuiga, L., 1994. The Torres del Paine lake
district (Chilean Patagonia): a case of potentially N-limited lakes and ponds. Arch.
Hydrobiol. 99, 181197.
Sun, Y., MacRae, T.H., 2005. Small heat shock proteins: molecular structure and
chaperone function. Cell. Mol. Life Sci. 62, 24602476.
Tanguay, J.A., Reyes, R.C., Clegg, J.S., 2004. Habitat diversity and adaptation to environmental stress in encysted embryos of the crustacean Artemia. J. Biosci. 29, 489501.
Van Stappen, G., 2002. Zoogeography. In: Abatzopoulos, T.H., Beardmore, J., Clegg, J.S.,
Sorgeloos, P. (Eds.), Artemia: Basic and Applied Biology. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp. 171224.
Warner, A.H., MacRae, T.H., Bagshaw, J.C., 1989. Cell and Molecular Biology of Artemia
Development. Plenum Press, New York.
Warner, A.H., Brunet, R.T., MacRae, T.H., Clegg, J.S., 2004. Artemin is an RNA-binding
protein with high thermal stability and potential RNA chaperone activity. Arch.
Biochem. Biophys. 424, 189200.
Willsie, J.K., Clegg, J.S., 2002. Small heat shock protein p26 associates with nuclear
lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells. J. Cell.
Biochem. 84, 601614.

Please cite this article as: Clegg, J.S., Gajardo, G., Two highly diverged New World Artemia species, A. franciscana and A. persimilis, from
contrasting hypersaline habitats express..., Comp. Biochem. Physiol. A (2009), doi:10.1016/j.cbpa.2009.04.613