Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia

Comparative Biochemistry and Physiology, Part B 145 (2006) 119 – 125 www.elsevier.com/locate/cbpb

Comparisons of stress proteins and soluble carbohydrate in encysted embryos of Artemia franciscana and two species of Parartemia James S. Clegg a,⁎, Veronica Campagna b a

Bodega Marine Laboratory and Section of Molecular and Cellular Biology, University of California, Davis, Bodega Bay, CA 94923, USA b Department of Environmental Biology, Curtin University of Technology, GPO Box U1987, Perth, W. Australia, 6845 Received 30 January 2006; received in revised form 13 April 2006; accepted 20 April 2006 Available online 18 June 2006

Abstract We compared stress proteins (p26, artemin, hsp70) and alcohol-soluble carbohydrates (ASC) in cysts of Artemia franciscana and two as yet un-named species populations of Parartemia, the brine shrimp endemic to Australia. The small stress proteins and molecular chaperones, p26 and artemin, previously thought to be restricted to Artemia, and present in very large amounts in its encysted embryos (cysts), were also detected by western blotting in Parartemia cysts, even though roughly 85–100 million years have passed since these genera diverged. We interpret this finding as further evidence for the adaptive importance of these proteins in coping with the severe stresses these encysted embryos endure. As expected, hsp70 was present in all three groups of cysts, but apparently at somewhat lower concentrations in those of Parartemia. Based on measurements of ASC we propose that the disaccharide trehalose, critical for desiccation tolerance in many animal cells, has probably also been maintained in the metabolic repertoire of Parartemia whose cysts have well developed tolerance to severe desiccation. © 2006 Elsevier Inc. All rights reserved. Keywords: Artemia; Parartemia; Stress proteins; p26; Artemin; hsp70; Soluble carbohydrate; Cysts

1. Introduction Massive amounts of two small stress proteins, artemin (De Herdt et al., 1979; De Graaf et al., 1990) and p26 (Clegg et al., 1994) are present in stress-resistant encysted embryos (cysts) of the primitive crustacean and model system, Artemia franciscana. Both proteins have been studied reasonably well since those 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). 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 first 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 this exceptionally stress-resistant embryo (see above references, and review by Clegg and Trotman, 2002). Furthermore, recent work suggests that artemin could be a

⁎ Corresponding author. Tel.: +1 707 875 2010; fax: +1 707 875 2009. E-mail address: [email protected] (J.S. Clegg). 1096-4959/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2006.04.014

molecular chaperone for RNA (Warner et al., 2004) adding to the evidence for RNA chaperones, a subject of increasing interest (reviewed by Lorsch, 2002; Henics, 2003). Because p26 and artemin are apparently such important components of the adaptive repertoire of Artemia encysted embryos we previously examined a wide variety of invertebrates for p26 and artemin, including the resting stages of other closely related crustaceans. However, neither protein was detected in any of these samples (unpublished survey results). Thus, p26 and artemin were not detected by western blotting in cysts of the fairy shrimp Branchinecta sandiegoensis (supplied by Dr. Marie Simovich and Jake Moorad) a closely related, sister species of Artemia (see Spears and Abele, 2000) and cysts of another fairy shrimp Streptocephalus proboscideus, (supplied by Dr. Luc Brendonck). Not surprising were the negative results obtained using cysts of the more distantly related notostracan, Triops sp., and ephippia of the cladoceran, Daphnia sp. A caveat in all these studies is the well-known poor crossreactivity of antibodies against small heat shock proteins (Arrigo and Müller, 2002; Sun and MacRae, 2005). Nevertheless, until the present study there was no evidence that artemin or p26 existed outside the genus Artemia.

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The genus Parartemia, endemic to Australia, is believed to have evolved directly from Artemia, or from a common ancestor, with the estimated divergence of these genera taking place approximately 85 to 100 million years ago during the geological isolation of Australia in the late Mesozoic (Williams and Geddes, 1991; Coleman et al., 1998, 2001; Remegio et al., 2001; Van Stappen, 2002). In view of the abundance and importance of p26 and artemin in Artemia, we wondered whether these proteins had been maintained in Parartemia cysts, and undertook that study. As background, we summarize next some well-known features of Artemia cysts and include what little is known about those features in the cysts of Parartemia. The genus Artemia is found in harsh, hypersaline habitats world-wide and its biology is well known (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). We primarily use the species A. franciscana collected from salterns in the San Francisco Bay of California (SFB) but also found in the Great Salt Lake, Utah, and other locations (Van Stappen, 2002; Abatzopoulos et al., 2002). Fertilized eggs either develop directly into free-swimming larvae that are released from females, or stop developing at the gastrula stage with the embryos now being surrounded by complex shells, and often referred to as “cysts.” Encysted gastrula embryos, composed of about 4000 cells (nuclei) enter diapause, a condition of developmental arrest, characterized by a level of metabolic activity so low in these embryos that its detection is a major problem (Clegg and Jackson, 1998; Clegg and Trotman, 2002). Desiccation is often a normal part of this developmental pathway and is one of the conditions that can terminate diapause, restoring metabolism and development under permissive conditions of water content, molecular oxygen and temperature (reviewed by Clegg and Trotman, 2002). These embryos are among the most resistant of all multicellular eukaryotes to environmental insults, surviving continuous anoxia for years, while fully hydrated at physiological temperatures (Clegg, 1997), temperature extremes, as well as severe desiccation, with cycles of dehydration/re-hydration, and exposure to various forms of radiation (reviewed by Clegg and Trotman, 2002; also see Tanguay et al., 2004). Those features, plus commercial availability in large amounts, make them a useful model system in which to study the biochemistry of resistance to extreme stress in cells that I have referred to as animal extremophiles (Clegg and Trotman, 2002; Abatzopoulos et al., 2002). The majority of Parartemia species are restricted to south western Australia (Williams and Geddes, 1991). The high incidence of inland salt ponds and lakes, and their antiquity and isolation in Western Australia, has resulted in a remarkable radiation of this genus (Geddes, 1981; Remegio et al., 2001) with most species restricted to this area. It is expected that more species will be identified within the family Parartemiidae. The general characteristics of Parartemia cysts, and their developmental pathways, appear similar to those of Artemia, but we know little about the details. Although morphologically similar, the cysts of Parartemia differ from those of A. franciscana from the SFB in that the former sink to the sediment where they remain until hatching takes place. In contrast, most cysts

of A. franciscana float after being released from females into the hypersaline environment. 2. Materials and methods 2.1. Sources of Artemia and Parartemia cysts Dried encysted gastrula embryos (cysts) of A. franciscana from the South San Francisco Bay salterns were purchased from San Francisco Bay Brand, Hayward, California in 1982 and have been stored dry, under nitrogen gas at about − 10 °C, since then. The viability (hatching level) of these embryos has remained close to 90% to the present time. Before use, the dried frozen embryos, still under nitrogen gas, were equilibrated at room temperature for 5 days. Hatching assays were performed as described (Clegg, 1997) in filtered aerobic seawater (SW) at room temperature (∼22 °C) and constant laboratory light. Parartemia cysts were collected in dry surface sediment from two athalassohaline lakes: Lake Yindarlgooda in September 2001, and from Lake Miranda in April 2003. The sediment was oven dried at 40 °C, sieved through 500–180 μm Endecotte® sieves and then stored in plastic vials at room temperature. Lake Miranda is a large inland salt lake approximately 900 km NE from Perth, Western Australia, with an average background salinity of 20 g/L and a pH of about 6.3. The lake contains abundant populations of Parartemia, whose cysts will be referred to here as species A since it has yet to be named. Cysts of Parartemia species B were collected from Lake Yindarlgooda, also a large temporary inland lake 700 km E from Perth, with an average background salinity of 40 g/L and pH 7.5–8.0. These lakes are approximately 500 km apart and are influenced by anthropogenic secondary salinisation as a result of mining activities, with some sites reaching salinities up to 150 g/L. Parartemia sp. A recorded maximum hatching in salinities up to 25 g L− 1 and an average pH of 8. Parartemia sp. B hatched best at an average salinity of 32 g L− 1, pH of 7. During both trials the dissolved oxygen levels remained at approximately 7 ppm and the temperature was 20–22 °C. Consecutive repeated rewetting of the sediment resulted in continued hatching of the cyst bank; however, no specific values for the percentage of cysts that hatch were obtained. Soil samples containing Parartemia cysts were shipped to the Bodega Marine Laboratory where they were subjected to brief floatation in saturated NaCl. Most of the soil particles sank, and the floating cysts were skimmed off and placed on filter paper to absorb interstitial NaCl solution. These cysts were then rinsed quickly with distilled water and blotted with filter paper. After air drying, individual cysts, along with an unavoidable small amount of debris, were collected with a sharpened wooden splint and pooled until sufficient material was collected for study. Unfortunately, no hatching assays were carried out on the Parartemia cysts used in this study, so the percentage of dead (non-hatching) cysts is not known. The latter could be involved with the lower protein content of Parartemia cysts. However, we believe that the smaller amounts of protein in these cysts on a dry weight basis, compared to cysts of A. franciscana, are

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most likely due to the presence of non-cyst material (“soil”) that contributes to the dry mass of samples, but not their protein. 2.2. Electrophoresis (SDS-PAGE) and Western blot analysis After hydrating known dry weights of the three groups of cysts overnight in SW on ice, to suppress metabolism, they were rinsed quickly with ice-cold distilled water, blotted to remove interstitial water (Clegg, 1997) and weighed. Cysts were homogenized at 100 mg wet weight (60 mg dry weight)/mL of buffer K (5 mM MgSO4, 5 mM NaH2PO4, 40 mM Hepes, 70 mM potassium gluconate, 150 mM sorbitol, pH 7.4, containing a protease inhibitor cocktail (Complete™ Mini from Roche Diagnostics GmbH). Known volumes of homogenates were added to equal volumes of 2× sample buffer (Laemmli, 1970) and boiled for 5 min. After low speed centrifugation (1600 g, 5 min) to remove insoluble shell fragments, supernatants were electrophoresed in 12% polyacrylamide gels, and proteins detected by Coomassie blue-G staining. In these SDS-PAGE studies we applied aliquots equivalent to the same dry mass of cysts (0.15 mg) per slot because it seemed possible that protein content could also be a variable. Nevertheless, we also measured the protein content of cyst extracts to enable comparison on this basis as well. Proteins from SDS-PAGE were also transferred to nitrocellulose sheets and prepared for immunodetection using polyclonal anti-p26

Fig. 2. Homogenates of the cyst samples described in the legend to Fig. 1 were also used for Western blotting (described in Materials and methods). Results are shown in duplicate for detection of pure p26 and artemin. In this case both antibodies were incubated at the same time, prior to the secondaries. The arrow at the top right points to the relative amounts of protein applied per lane for the three samples. Amounts of protein (μg) applied per lane are 10.3 (SFB), 6.0 (A) and 8.3 (B). Low speed supernatant (sup) of A. franciscana cysts was used as a positive control for both proteins.

(Clegg et al., 1994) and anti-artemin (a gift from Herman Slegers) at 1:5000 for 1 h as the primary antibodies, and horseradish peroxidase- conjugated anti-rabbit IgG (1:5000, 1 h) as secondary. For the detection of hsp70 we used a primary antibody purchased from Affinity BioReagents, Inc. (MA3-001; 1:1,250, 2 h) which detects inducible as well as constitutive isoforms. The secondary antibody in this case was horseradish peroxidase-conjugated goat anti-rat IgG (1:5000, 1 h). Chemiluminescence was detected with Super Signal® West Pico (Pierce, Rockford, Illinois) using the Epi Chemi II Darkroom (UVP Laboratory Products). Artemin and p26 purification procedures have been described previously by Warner et al. (2004), and Viner and Clegg (2001), respectively. 2.3. Analysis of total protein and alcohol-soluble carbohydrate (ASC) To determine protein concentrations whole homogenates or low speed supernatants were made to 0.2 N NaOH and incubated at 37 °C for 1 h. After centrifuging at 1600 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. For ACS measurements, aliquots of whole cyst homogenates were made to 80% ethanol and held on ice for 2 h to precipitate glycogen and other macromolecules. After centrifuging as above, supernatants were used to determine total ASC using the colorimetric assay of Dubois et al. (1956) with glucose as standard. The results are expressed as μg glucose-equivalents per mg dry weight of cysts and also as per mg cyst protein. 3. Results

Fig. 1. SDS-PAGE and Coomassie staining of proteins in homogenates of A. franciscana cysts from San Francisco Bay (SFB), and cysts of Parartemia from Lake Miranda (A) and Lake Yindarlgooda (B). The horizontal line at the lower left points to pure p26 and the arrow above it indicates artemin (confirmed by Western blotting in Fig. 2). STDS refers to molecular mass standards. Equal volumes of homogenates were applied per lane, equivalent to the same dry weight in each of the three samples. Relative amounts of protein per slot, normalized to Artemia, are indicated by the arrow at top right. Amounts of protein (μg) applied per lane are 23.1 (SFB), 13.3 (A) and 18.6 (B). Proteins in Parartemia cyst extracts that might be similar to artemin (top asterisk) and p26 (bottom asterisk) are indicated.

3.1. SDS-PAGE and Coomassie staining Fig. 1 shows the results of SDS-PAGE and Coomassie staining of proteins in whole homogenates of A. franciscana cysts from San Francisco Bay (SFB) and Parartemia cysts from two locations in Western Australia: A = Lake Miranda; B = Lake Yindarlgooda. Pure p26 was electrophoresed in the left lane and artemin (known previously from many studies) is indicated by the horizontal arrow in the SFB preparation. The three homogenate

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J.S. Clegg, V. Campagna / Comparative Biochemistry and Physiology, Part B 145 (2006) 119–125 Table 1 Contents of protein (P) and alcohol-soluble carbohydrate (ASC) in cysts (encysted embryos) of Artemia franciscana (SFB) and those of two species populations of Parartemia designated as A and B Cyst population

Fig. 3. Western blot to detect p26 in homogenates of SFB, A and B cysts, as described in the legend to Fig. 1. Supernatants of SFB cysts (S) were used as markers and positive controls. In this case, unlike Figs. 1 and 2, the same amounts of protein (11 μg) were applied per lane for each of the three samples.

preparations represent equal wet weights of cysts per lane, and the relative amounts of protein in each lane are indicated by the horizontal arrow at the top right. Coomassie staining indicates that a protein whose mass is similar to that of artemin is present in all three samples (top asterisk), whereas a distinct protein band at 26 kDa is not obvious in Parartemia A and B extracts. One can detect several differences in the profiles of proteins between the Artemia and Parartemia preparations. Interestingly, the major yolk proteins (masses at and above ∼66 kDa) appear to have similar molecular masses in Artemia and Parartemia. The results of Western immunoblotting of these same samples are shown in Fig. 2. In this case artemin and p26 are detected specifically by antibodies, with pure p26 and artemin as positive controls (center). The SFB, A and B samples are shown in duplicate (two independent samples). The amounts of artemin and p26 cannot be compared to each other quantitatively because the affinity of anti-artemin for artemin is greater than that of anti-p26 for p26 when both are processed at the same time on the same blot, which is the case for Fig. 2. We know from many previous studies, and Fig. 1 in this paper, that the amounts of these two proteins are very similar in A. franciscana cysts based on Coomassie staining. There is some indication that antip26 appears to detect proteins in the A and B samples whose molecular masses, however, are slightly higher than SFB p26. To evaluate this further we examined westerns probed only with anti-p26 (Fig. 3).

μg ASC/mg dry wt μg P/mg dry wt μg ASC/mg P

SFB

A

B

182 266 684

111 153 725

142 213 667

SFB stands for San Francisco Bay, the origin of the A. franciscana cysts. The Parartemia cysts are from Lake Miranda (A) and Lake Yindarlgooda (B), both in Western Australia. Alcohol soluble carbohydrate (ASC) is in units of μg glucose equivalents per unit cyst dry weight, and per mg cyst protein (P).

With the complexity of detecting artemin removed, these results reveal that anti-p26 detects a protein of higher molecular mass than that of SFB p26, and this is the only protein detected on such blots. Furthermore, in this case we applied equal amounts of cyst homogenate protein, suggesting that the levels of p26 in Parartemia cysts are less than those in Artemia cysts, relative to total cyst protein. That statement is qualified by potential differences in the relative affinities of anti-p26 from Artemia for p26 in Parartemia. As we will take up in the Discussion, however, the Coomassie staining also bears directly on this issue. We examined another stress protein of major importance, hsp70 (Fig. 4). In this case, equal amounts of cyst protein were applied per lane for each of the three samples. Although SFB cysts appear to contain more hsp70 compared to cysts from both Parartemia populations, that could be due to differences in the relative affinities of the antibody used. We know that there are two constitutive isoforms of hsp70 in A. franciscana (Clegg et al., 2001) but 12% gels must be run for extended periods of time to resolve them clearly, and that was not the case in the present study (Fig. 4). 3.2. Alcohol-soluble carbohydrates and trehalose It has been shown that trehalose makes up close to 80% of the total alcohol-soluble carbohydrate, or ASC, in A. franciscana cysts (Clegg and Jackson, 1992) so we used this approach to compare cysts of Artemia and Parartemia A and B (Table 1). Ideally, it would have been best to measure trehalose levels by HPLC, as we have done previously for Artemia cysts; however, sample size was a severe constraint in the case of Parartemia cysts, so the best we could do at present was to measure the total ASC. As seen in Table 1, the amount of ASC in Artemia cysts is much greater than in cysts of Parartemia when calculated on a dry weight basis. However, the three are similar when expressed on a per mg protein basis. In view of the results shown in Table 1, and until further measurements can be made, it seems reasonable to propose that the amounts of trehalose in cysts of these three populations are similar, and all are very high. 4. Discussion

Fig. 4. Western blot to detect hsp70 in homogenates of SFB, A and B cysts. The same amount of cyst homogenate protein (13 μg) was applied per lane for the three samples. Positive controls (+C) are A. franciscana cyst supernatants.

Stress proteins and molecular chaperones are subjects of intense study, and the literature is massive, particularly in terms

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of molecular and cellular biology. This paper is not an appropriate place to attempt even superficial coverage of that literature. We refer to a book on small stress proteins by Arrigo and Müller (2002) and a very recent review by Sun and MacRae (2005) since both artemin and p26 are in that protein family, and note that there is increasing focus on the ecological and evolutionary involvement of stress proteins, and the stress response in general (reviewed by Feder and Hofmann, 1999). A major goal of our work was to determine whether the small stress proteins artemin and p26 of Artemia have been retained by the two Parartemia species populations during roughly 85– 100 million years of evolution after divergence (Williams and Geddes, 1991; Remegio et al., 2001; Van Stappen, 2002). Although recognition by antibodies does not prove identity of structure, we suggest that the simplest interpretation of our results is that p26 and artemin are both present in these Parartemia cysts (Figs. 2 and 3). Also evident from Fig. 3 are differences in the molecular mass of the p26 subunit of both A and B compared to Artemia cyst p26, indicating alterations in primary structure of this protein, the occurrence of post-translational modifications or possibly both. The amounts of artemin seem to be similar in the cysts of Artemia and Parartemia, notably in species B (Figs. 1 and 2). In contrast, the Western blot results (Figs. 2 and 3) suggest that the amounts of p26 in both A and B species of Parartemia are appreciably less than in those of A. franciscana. That result does not appear to be due to a reduction in the ability of the anti-p26 (produced from the Artemia protein) to detect this protein in Parartemia cysts because the results from the Western blotting (Figs. 2 and 3) and Coomassie staining (Fig. 1) are in fair agreement. Thus, we propose that cysts of Parartemia A and B contain the p26 sub-unit, but probably in amounts less than those of A. franciscana. Another interesting result is the apparently higher molecular mass of the p26 sub-unit from Parartemia cysts compared to that from Artemia (Fig. 3), being about 8% higher according to our estimation. For those unfamiliar with Artemia p26 we note that it is a small stress protein of native molecular mass 560 kDa (± 20) made up of 28 sub-units (Viner and Clegg, 2001). Thus, all the results in Figs. 1–3 involve the sub-units, examined by SDS-PAGE, and not the native proteins. Among the several ways to account for the higher sub-unit mass of p26 in Parartemia cysts, the simplest explanation appears to be a change in primary structure (amino acid sequence). Also, we know that the major isoform of Artemia p26 contains one phosphate per subunit (Qui et al., 2004) but an increased level of phosphorylation of Parartemia p26 could contribute only in a very minor way to sub-unit molecular mass. The lack of sufficient Parartemia cysts makes purification and study of the structure and function of their artemin and p26 impossible at the present time. We examined hsp70 (Fig. 4) primarily to determine whether there was anything unusual about this family of stress proteins in Parartemia cysts. That does not seem to be the case, at least at this level of resolution. What are the differences between the stresses encountered in nature by Artemia cysts compared to those of Parartemia? Conditions experienced by Artemia cysts in the South San

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Francisco Bay are not particularly harsh, as Artemia habitats go (see Abatzopoulos et al., 2002; Tanguay et al., 2004). Nevertheless, these animals exhibit substantial phenotypic plasticity (Browne and Wanigasekera, 2000) including their exposure to extremely 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 the former's all around superior adaptive capabilities. Cysts from the two Parartemia species were collected from sediments that had undergone extended periods of desiccation caused by a two-year drought. Both lakes have beds composed of clay, resulting in the cysts being exposed to temperatures exceeding 60 °C for extended periods of time. Thus, we draw the conclusion that at least part of the reason that cysts of Parartemia tolerate high temperatures is the presence of artemin and p26, and infer that the retention of these embryonic proteins and molecular chaperones has been the result of strong natural selection. An important common denominator of the encysted embryos of both genera is their ability to survive desiccation. Although stress proteins are probably involved in that capability, there is very strong evidence that trehalose, the non-reducing disaccharide of glucose, is of paramount importance in the desiccation tolerance of a wide variety of invertebrates (Leopold, 1986; Crowe et al., 1992, 1998) although at least one very interesting exception has been described (Tunnacliffe and Lapinski, 2003). Cysts of Artemia contain very large concentrations of trehalose, commonly about 15% of the dry cyst mass, and making up close to 85% of the total alcohol-soluble carbohydrates (ASC) that are also very high (Clegg and Jackson, 1992). Interestingly, both Parartemia cyst populations studied here contain amounts of ASC comparable to Artemia when based on total cyst protein content (Table 1). Presenting the ASC results on a mg protein basis seems appropriate because the Parartemia samples contained material other than cysts which contributed to the overall dry weight, but which did not represent biological material. Based on that result, and the desiccation-tolerance of Parartemia cysts, we suggest that natural selection has also maintained the metabolic pathway required to synthesize trehalose, and the regulatory mechanisms needed to accumulate such large amounts of this sugar, and then metabolize it away at the proper developmental time (Clegg and Trotman, 2002). One of the most astonishing feats of A. franciscana encysted embryos is their ability to endure years of continuous anoxia while fully hydrated at physiological temperatures (Clegg, 1997) a feat they seem to achieve by bringing their overall metabolism to a reversible standstill (Warner and Clegg, 2001; Hand, 1998; Hand and Podrabsky, 2000; Tanguay et al., 2004). It appears that artemin and p26 are critical components of the adaptive repertoire that enables such stability in the absence of detectable energy metabolism and macromolecular turnover. Unfortunately we know nothing about the ability of Parartemia cysts to survive prolonged anoxia. However, the shallow nature of the inland salt lakes in Western Australia is such that there is little to no thermal stratification because of sufficient water circulation.

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As a result, the temperature and dissolved oxygen levels are quite uniform throughout the water column. The sediments are, of course, another matter and are likely to be severely hypoxic or anoxic just below the sediment surface. Thus, it would seem likely that Parartemia cysts do experience those conditions when sinking to the sediments. However, no study on the viability of hydrated Parartemia cysts incubated under welldescribed anoxic conditions has been conducted. Acknowledgments The technical assistance of Joshua Tanguay is appreciated, as is the gift of anti-artemin from Herman Slegers. Research support for JSC came from the Naval Research Laboratory (DARPA) and the US National Science Foundation. VC thanks Lindsay Cahill and Caroline Vivian of Bulong Nickel who provided funding and assistance, and Dr. Jacob John for supervision and laboratory space and supplies. We are grateful to Fiona Butson and Erin Lowe for collecting the Lake Miranda (A) samples, and to Drs. Luc Brendonck, Marie Simovich, and Jake Moorad for supplying the fairy shrimp cysts referred to in the text. 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, 37–47. Arrigo, A.-P., Müller, W.E.G., 2002. Small Stress Proteins. Springer-Verlag, Berlin. Browne, R.A., Wanigasekera, G., 2000. Combined effects of salinity and temperature on survival and reproduction of five species of Artemia. J. Exp. Mar. Biol. Ecol. 244, 29–44. 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, 137–145. 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, 467–475. Clegg, J.S., Jackson, S.A., 1992. Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana. FEBS Lett. 303, 45–47. Clegg, J.S., Jackson, S.A., 1998. The metabolic status of quiescent and diapause embryos of Artemia franciscana. Arch. Hydrobiol. 52, 425–439. 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. 129–170. 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, 77–83. 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, 1–7. Clegg, J.S., Willsie, J.K., Jackson, S.A., 1999. Adaptive significance of a small heat shock/alpha-crystallin protein in encysted embryos of the brine shrimp, Artemia franciscana. Am. Zool. 39, 836–847. 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, 85–96.

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, 221–229. Coleman, M., Geddes, M.C., Trotman, C.N.A., 1998. Divergence of Parartemia and Artemia haemoglobin genes. Int. J. Salt Lake Res. 7, 171–180. Coleman, M., Matthews, C.M., Trotman, C.N.A., 2001. Multimeric hemoglobin of the Australian brine shrimp Parartemia. Mol. Biol. Evol. 18, 570–576. 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, 1–10. Crowe, J.H., Hoekstra, F.A., Crowe, L.M., 1992. Anhydrobiosis. Annu. Rev. Physiol. 54, 579–599. Crowe, J.H., Clegg, J.S., Crowe, L.M., 1998. Anhydrobiosis: the water replacement hypothesis. In: Reid, D.S. (Ed.), The properties of water in foods ISOPOW, vol. 6. Thomsen Science, London, pp. 440–455. 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., Möller, W., 1990. The primary structure of artemin from Artemia cysts. Eur. J. Biochem. 193, 737–750. De Herdt, E., Slegers, H., Kondo, M., 1979. Identification and characterization of a 19-S complex containing a 27,000-Mr protein in Artemia salina. Eur. J. Biochem. 96, 423–430. Dubois, M., Giles, K.A., Hamilton, J.K., Ribers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282. Geddes, M.C., 1981. The brine shrimps Artemia and Parartemia: comparative physiology and distribution in Australia. Hydrobiologia 81, 169–179. Hand, S.C., 1998. Quiescence in Artemia franciscana embryos: reversible arrest of metabolism and gene expression at low oxygen levels. J. Exp. Biol. 201, 1233–1242. Hand, S.C., Podrabsky, J.E., 2000. Bioenergetics of diapause and quiescence in aquatic animals. Thermochim. Acta 349, 31–42. Henics, T., 2003. Extending the “stressy” edge: molecular chaperones flirting with RNA. Cell Biol. Int. 27, 1–6. Jackson, S.A., Clegg, J.S., 1996. The ontogeny of low molecular weight stress protein p26 during early development of the brine shrimp, Artemia franciscana. Dev. Growth Differ. 38, 153–160. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Leopold, A.C., 1986. Membranes, Membranes and Dry Organisms. Cornell University Press, Ithaca. Liang, P., Amons, R., MacRae, T.H., Clegg, J.S., 1997a. Purification, structure and molecular chaperone activity in vitro of Artemia p26, a small heat shock/α-crystallin protein. Eur. J. Biochem. 243, 225–232. Liang, P., Amons, R., Clegg, J.S., MacRae, T.H., 1997b. Molecular characterization of a small heat-shock/á-crystallin protein from encysted Artemia embryos. J. Biol. Chem. 272, 19051–19058. Lorsch, J.R., 2002. RNA chaperones exist and DEAD box proteins get a life. Cell 109, 797–800. MacRae, T.H., Bagshaw, J.C., Warner, A.H., 1989. Biochemistry and Cell Biology of Artemia. CRC Press, Boca Raton. Persoone, G., Sorgeloos, P., Roels, O., Jaspers, E., 1980. The Brine Shrimp, Artemia, vol. 2. Universa Press, Wetteren, Belgium. Qui, Z., Viner, R., MacRae, T.H., Willsie, J.K., Clegg, J.S., 2004. A small heat shock protein from Artemia franciscana is phosphorylated at serine 50. Biochim. Biophys. Acta 1700, 75–83. Remegio, E.A., Hebert, P.D.N., Savage, A., 2001. Phylogenetic relationships and remarkable radiation in Parartemia (Crustacea: Anostraca), the endemic brine shrimp of Australia: evidence from mitochondrial DNA sequences. Biol. J. Linn. Soc. 74, 59–71. Spears, T., Abele, L.G., 2000. The phylogenetic relationships of crustaceans with foliacecous limbs: an 18S rDNA study of Branchiopoda, Cephalcardia and Phyllocardia. J. Crustac. Biol. 20, 1–24. Sun, Y., MacRae, T.H., 2005. Small heat shock proteins: molecular structure and chaperone function. Cell. Mol. Life Sci. 62, 2460–2476.

J.S. Clegg, V. Campagna / Comparative Biochemistry and Physiology, Part B 145 (2006) 119–125 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, 489–501. Tunnacliffe, A., Lapinski, J., 2003. Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis. Phil. Trans. Roy. Soc. Lond. 358, 1755–1771. 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. 171–224. Viner, R.I., Clegg, J.S., 2001. Influence of trehalose on the molecular chaperone activity of p26, a small heat shock protein. Cell Stress Chaperones 6, 126–135. Warner, A.H., Clegg, J.S., 2001. Diguanosine nucleotide metabolism and the survival of Artemia embryos during years of continuous anoxia. Eur. J. Biochem. 268, 1568–1576.

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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, 189–200. Williams, W.D., Geddes, M.C., 1991. Anostracans of Australian salt lakes, with particular reference to a comparison of Parartemia and Artemia. In: Browne, R.A., Sorgeloos, P., Trotman, C.N.A. (Eds.), Artemia Biology. CRC Press, Boca Raton, Florida, pp. 351–368. 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, 601–614.