Biology of Anomura II (A. Asakura et al., eds.), Crustacean Research, Special Number 6: xxx‒xxx, 2006

Expression and structure of stress chaperon hsp90 in terrestrial decapods, Coenobita (Anomura: Coenobitidae) and Chiromantes (Brachyura: Sesarmidae)

Oleg Gusev, Tracy A. Ziegler and Masayuki Saigusa

Abstract .—This study describes aspects of the biology of heat shock proteins 90kDa (hsp90) in terrestrial decapods, Coenobita pur-pureus, C. brevimanus (Anomura: Coenobitidae) and Chiromantes haematocheir (Brachyura: Sesarmidae). These species possess highly devel-oped terrestrial adaptations on both morphologi-cal and physiological levels. We have analyzed the pattern of expression for the gene coding hsp90: molecular chaperon in both embryos and the muscle tissues of adults of these species in response to heat stress. While the minimum tem-perature for initiation of the synthesis of excess of hsp90 mRNA was different between embryos and adults from the different climatic regions, the range of resistance to high temperature shock was nearly the same (6–7°C). Furthermore, the structure of the stress-factor binding domain in hsp90 coding gene showed convergent patterns by possessing a conservative amino acid sequence in decapods experiencing the same annual tempera-ture regimes in the subtropical, temperate and boreal climatic regions.

INTRODUCTION

Two separate lines of decapod crustaceans have successfully invaded the terrestrial envi-ronment (for review, see Schmitz & Harrison, 2004) the hermit crab family Coenobitidae (Anomura) (for review, see Burggren & McMahon, 1988; Greenaway, 2003) and the Grapsoidea (Brachyura) (Adamczewska & Morris, 2000; Anger & Shubart, 2005). Decapods that spend more or less of their time in the terrestrial environment have been sum-marized into five grades, T1-T5 (Burggen & McMahon, 1998), according to degree of ter-

restrial adaptation. Both Coenobita spp. and Chiromantes haematocheir belong to the T4 group, since they are found in a terrestrial envi-ronment as adults, but still depend on the ma-rine environment for planktonic larval develop-ment.

Recent studies of the concomitant effects of thermal acclimation on both heat and cold tol-erance have been made in many species: e.g., crabs (Stillman, 2003), planarians (Tsukuda & Ogoshi, 1978), earthworms (Hanumante, 1977), fl ies (Ohtsu et al., 1999), lobsters (Spees et al., 2003), fi sh (Hernandez & Buckle, 2002), crayfish (Layne et al., 1985), and copepods (Bradley, 1978; Voznesensky et al., 2004). Taken together, the results suggest that, even if they are close systematically, organisms exposed to the same climatic conditions will display different body temperatures, effective metabolic temperatures, and patterning of heat stress response and acclimation (Helmut et al., 2002).

Compared to other arthropods, terrestrial decapod crustaceans have a relatively low re-sistance to high temperature stress and water loss (Schmitz & Harrison, 2004). These two problems were overcome by the development of physiological and behavioral adaptations, including daily migrations (Imafuku, 2002; Morris, 2002), complicated breeding and lar-vae release behaviors (Saigusa & Terajima, 2000; Imafuku, 2002; Brodie, 2002), changes in respiration, in circulatory systems, and in the dynamics of molting and food digestion (McMahon & Burggren, 1988; Morris, 2002).

An important question is whether evolu-tionary adaptation at phenotypic and genotypic

2 O. Gusev et al.

levels affects acclimation and general com-pensatory abilities of terrestrial crustaceans to environmental temperature changes. Nearly all members of Coenobitidae use behavioral means to avoid overheating, but the tempera-ture threshold for such behavior was shown to be higher in diurnally active species, rather then in nocturnal and more terrestrial species (Burggren & McMahon, 1988; Greenaway, 2003). Meostatic temperature of the hemo-lymph in terrestrial hermit crabs and brachy-uran crabs is generally higher then in their lit-toral relatives, while the oxygenation level does not show a remarkable difference (Burggren & Mahon, 1988). Furthermore, the preferred average body temperature and the temperature of hemolymph, as well as total hemolymph oxygen capacity shows a tendency to be higher in terrestrial rather than in marine species (Burggren & McMahon, 1988; Schmitz & Harrison, 2004).

The question of genetypic basements for the physiological plasticity during heat acclimation in crustaceans was partially answered using temperature-stress specifi c proteins (molecular chaperons) as markers of the compensatory abilities of the organism to overcome dam-age caused by excess environmental stress on a cellular level. Heat shock proteins (hsp) are the most studied group of molecular chaperons involved in the refolding of proteins damaged by external stresses (Soti & Scermely, 2003; Robert, 2003). This group includes four major types of proteins (hsp27, hsp70, hsp90 and hsp100), according to their molecular weight and functions (Feder & Hoffman, 1999). Hsps are particularly significant as they represent a mechanism by which an organism can buffer the impact of environmental temperature on the protein pool without having to employ special-ist protein isoforms to withstand high tempera-tures (Somero, 1995). The investment in hsp’s, as the likelihood of higher temperatures from global climate change looms, may be an effec-tive strategy for intertidal and terrestrial crusta-ceans.

Crustaceans and some other inhabitants of the intertidal zone, which is characterized by

the highest temperature and water level varia-tion, were shown to possess three broad catego-ries of variation of response. First, total cellular levels of hsp’s varied with season (Hofmann & Somero, 1995; Buckley et al., 2001) and with laboratory acclimation (Tomanek & Somero, 1999; Buckley et al., 2001). Second, although the stress response displays an element of plas-ticity, some components appear to be fixed, and are a characteristic of a particular species. Different sets of congeners of rocky intertidal marine invertebrates have different stress re-sponses despite being acclimated to the same temperature (e.g., Hofmann & Somero, 1995; Tomanek & Somero, 2002). Third, the stress response varies in natural populations and close species across environmental gradients and with thermal habitat (Helmuth & Hofmann, 2001). Moreover, there is a link between the structure of certain domains in major groups of constitutively expressed and stress-induced chaperons and temperature-adaptive molecular response in the species with different latitudi-nal biogeographic patterning (Horowitz, 2001; Somero, 2005; Mahroof, et al., 2005).

In this study, we investigated the effect of temperature on the viability and expression of the molecular chaperon hsp90, showing mixed cytosolic and stress-response nature of the adults and embryos of two species of land hermit crabs Coenobita and a brachyuran crab Chiromantes haematocheir. To analyze the pos-sible infl uence of primary and secondary struc-ture of hsp90 on the induction of increasing levels of this protein in response to heat shock, we cloned a nucleotide sequence corresponding to the particular region in the second functional ATP-dependent domain of hsp90. This is one of the potential markers of environmental-conditions based on changes in the functional structure of hsp90 (Schnaider et al., 1999; Zhao et al., 2005). This domain is regarded as the binding site for the heat shock factor (hsf), a protein controlling chaperon activity of hsp90 and intensity of its synthesis under stress con-ditions. The activity of hsf closely depends on the level of damage caused by high temperature and other stresses (Westwood & Wu, 1993;

Stress protein hsp90 in terrestrial decapods 3

Rabindran et al., 1994). We also analyzed the primary genetic structure of the heat factor binding site in hsp90 coding gene in a number of decapod crustaceans from different tempera-ture regimes within various climatic regions.

MATERIALS AND METHODS

Field samplingThe main target species Coenobita purpu-

reus, C. brevimanus and Chiromantes haema-tocheir, as well as the other decapod species for comparative purposes, were collected as fol-lows from three regions in Japan with different annual temperature fluctuation in both water and air.

Subtropical species were collected at Iriomote-jima Island, Ryukyu (124°E, 24°N), with daily fluctuation of water temperature of about 24–26ºC on the surface and in shallow water and air temperature of about 28–30 ºC during the season of collection (April–June, 2004–2005). Ovigerous females of land hermit crabs Coenobita purpureus were collected on the beaches and one C. brevimanus was col-lected in the rain forests about 1–1.5 km from the shore. Adult specimens of Melicertus lati-sulcatus (Dendrobranchiata: Penaeidae) were collected in the littoral zone during low-tide. Specimens of the searmid crab Chiromantes de-haani (Brachyura: Sesarmidae) were collected in the mangroves areas during nocturnal low tides.

To represent the temperate region, Inland Sea species were collected in the Kasaoka district, Okayama Prefecture (133°E, 36°N), with daily fluctuation of water temperature of about 20–23ºC in surface and shallow water, and air temperature of about 25–27 ºC dur-ing the season of collection (April–June, 2004–2005). Ovigerous females of Chiromantes haematocheir (Brachyura: Sesarmidae) were collected in coastal habitats. Hemigrapsus san-guineus (Brachyura: Grapsidae) were collected from a rocky littoral zone during low tide. Mud shrimps Upogebia major (Thalassinidea: Upogebiidae) were obtained from intertidal mud fl ats.

Samples of boreal species, Hyas araneus (Brachyura: Majidae), Sclerocrangon boreas (Caridea: Crangonidae) and Pagurus pubescens (Paguroidea: Paguridae), were collected by deep creeping (depth ~ 80–100 meters) in the Kandalaksha Gulf, White Sea, Russia (32–34°E, 65–66°N). The water temperature at the collect-ing point depth was 4 ºC and this value remains constant throughout the year at these depths. No specifi c acclimatization procedures were further applied to the adults and the embryos.

Heat stress treatmentFor the excess temperature treatment, eight

replicate experiments were conducted. The em-bryo clusters (at the stage of development close to hatching; weight ~ 1 g;1200–1300 embryos) were dissected from the abdomen of females of Chiromantes and Coenobita and immediately placed in a 1.5 ml tube, which was preheated to the experimental temperature and fi lled with ar-tifi cial sea water (10‰). Temperature treatments of the embryos were conducted in block incuba-tors. Adult females were placed in cylindrical plastic containers (10 cm in diameter and 25cm in height) and experiments were conducted in preheated hybridization incubators (HB-80 Taitec).

Both embryos and females were treated with a range of temperatures (24ºC–40ºC) for 120 min, then returned to room temperature (RT) (26°C) for 2 hours for a recovery period. Next, a sample of muscle tissues from the female crabs was isolated and immediately frozen with liquid nitrogen. Whole embryo clusters were fi xed in the same way.

RNA and DNA analysis Total RNA was extracted from both em-

bryos and tissues of the adults. Samples were pulverized with a mortar and pestle under liquid nitrogen, and the total RNA was extracted and purified with an RNeasy Midi Kit (QIAGEN) according to the manufacturer’s protocol. To prevent possible DNA contamination in the RNA, the samples were subjected to DNase treatment using a DNA-free Kit (Ambion). Furthermore, the poly(A)+RNA was purified

4 O. Gusev et al.

Primer Sequence Direction Source

Oligonucleotides used for isolation of partial sequence hsp90 coding gene for mRNA expression analysis

S-HSP90-F 5’-GGCAGGTCACGAACGTGTGT-3’ -3’ forward

S-HSP90-R 5’-GTAACCTTGTCGGCCACCAG-3’ reverse

Oligonucleotides used for isolation of sequence of hsf-binding domain of hsp90 coding gene

S-HSF-F 5’-CTACTACATCACTGGCGAGA-3’ forward

S-HSF-R 5’-ACACCACCTCGAAGCCACG-3’ reverse

Oligonucleotides used for isolation of partial sequence of beta-actin for mRNA expression analysis S-ACT-F 5’-CACCACTGGGATGACATGGAG-3’ forward

S-ACT-R 5’-AAGGAAGGCTGGAAGAGGG-3’ reverse

from the total RNA, obtained in the previous step, using the QuickPrep Micro mRNA purifi -cation kit (Amersham-Pharmacia).

Genomic DNAs were prepared in 70% ethanol from the tissues of the specimens, us-ing the method described by Blin & Stafford (1976). The purified genomic DNA was sus-pended in TE (10 mM Tris-HCl/ 1 Mm EDTA, pH 8.0) and stored at –20 °C until used.

The mRNA expression patterns of hsp90 were examined by RT–PCR using the conser-vative region of about 500 bp in all species. Primers, length of the products, and sources for the primers are shown in Table 1. One μg of total RNA was extracted, as described above, from every experimental group of embryos and treated with two units of DNase I to remove all trace amounts of genomic DNA. These samples were subjected to reverse-transcription using a First Strand cDNA Synthesis kit (Roche Diagnostics). PCR’s were carried out in a total volume of 20 μl of solution containing 1xPCR reaction buffer, 150 μM dNTP mix, 0.5 U of Taq DNA polymerase, 2 mM MgCl

2, each

of the primers at 0.3–0.4 μM, and 100 ng of cDNA (or 1 μM of genomic DNA for isolation of the unique domain region of hsp90). The amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems) programmed for 35 cycles of 94°C (1 min), 57°C (1 min), and 67°C (4 min), followed by elongation for 10 min at 72ºC. Aliquots were

removed from each reaction during the PCR every 4 cycles starting from the 15th cycle (i.e., 15th, 19th, 23rd, 27th, 30th) and PCR products were separated on a 2% agarose gel. Levels of mRNA’s expression were estimated using GeneAnalyzer Pro Software by comparison of the intensity of the PCR bands on the different stages of PCR reactions standardized by inten-sity of actin bands. The difference in expression was further confirmed by PCR reaction using 1, 1/5 and 1/125 embryos-equivalent cDNA’s according to Chen et al. (2002).

The variable part (approximately 50 nucleo-tides) in the putative hsf-binding domain in hsp90 gene was amplifi ed from genomic DNA as a template under similar conditions, using oligonucleotide primers synthesized on the ba-sis of conservative flanking regions (Table 1) and further cloned.

Plasmid DNA was purified from clones in the subtracted library, and the inserts were sequenced using a Thermo Sequenase Cycle Sequencing kit (Amersham-Pharmacia) with M13 forward (–20) and M13 reverse prim-ers specifi c to the fl anking regions of a multi-cloning site in the pCR2.1-TOPO cloning vec-tor according to the manufacturer’s direction. They were analyzed with an automatic DNA sequencer, the DSQ-2000L (Shimadzu). For the calculation of molecular mass and primer design and analysis, we used Vector NTI Suite 9 (InforMax) computer software.

Table 1. A list of nucleotide primers used in this study for isolation of partial gene sequences.

Length of product

500 bp AY528900.1

48 bp AY528900.1

498 bp AY074923.1

Stress protein hsp90 in terrestrial decapods 5

Statistical testsThe level of variation of the calculated

values of all gene expression data were log 10 transformed prior to one-way analysis of vari-ance (ANOVA) to normalize variance, and a post-hoc analysis (Dunnett’s test) was conduct-ed to confi rm the signifi cance in the differences of the expression level. Signifi cant differences were accepted at P<0.05 (Sigma Stat Statistical Software).

RESULTS

We compared the expression of chaperon genes in response to high temperature stress in

embryos and muscle tissues of the adult crab Chiromantes haematocheir and hermit crabs Coenobita purpureus and C. brevimanus (Figs. 1 and 2). We detected strong differences in hsp90 gene expression between Coenobita and Chiromantes. The differences in the value of temperature threshold for initiation of synthesis of excess of hsp90 and level maximum resis-tance to the heat stress were apparent in both embryos (Fig. 1) and in adult animals (Fig. 2).

In the embryos of Ch. haematocheir, significant differences in hsp90 mRNA levels were detected in the group exposed to29°C, while in both species of Coenobita, the

Fig. 1. Effect of heat shock on the level of relative hsp90 mRNA in the embryos. A: typical expression pattern (RT-PCR) of the hsp90 gene in the embryos of Chiromantes haematocheir (ChH); Coenobita brevimanus (CB) and Coenobita purpureus (CP). B: calculated relative level of hsp90 mRNA in the embryos. Each bar represents mean value of mRNA level standardized by the level of actin mRNA (one-way ANOVA, P< 0.05). White bars correspond to Chiromantes haematocheir, light-grey bars to Coenobita brevimanus and dark-gray bars to Coenobita purpureus. Animals were exposed to high temperature for 2 hours and allowed to recover for 2 hours under RT. Zero values indicate lethal cases.

6 O. Gusev et al.

minimum temperature for increasing of level of hsp90 was 33–34°C (ANOVA, P=0.0017, Fig. 1). The lethal level of high temperature exposure, which caused mortality in more than 95% of embryos after a recovery period (2 hours after exposure), was detected to be 34–35˚C for the embryos of Ch. haematocheir and 38–40˚C for both species of Coenobita. The level of expression of the hsp90 gene was also different between the adult muscle tissues of both hermit crabs and Chiromantes. The minimum temperature for a signifi cant increase in the level of hsp90 mRNA in the tissues of Ch. haematocheir was found to be 30–31°C and the lethal exposure temperature was 36–38°C (ANOVA, P=0.0012, Fig. 2). The values for

C. brevimanus and C. purpureus were 34–36°C and 40–43°C respectively (ANOVA, P=0.003, Fig. 2). The range of excess temperature resis-tance (from initiation of appearance of excess hsp90 mRNA to death due to heat shock) in Ch. haematocheir was 7–8°C for the embryos and adults. For both Coenobita species, the range of excess temperature resistance was 6–7°C for embryos and 7–9°C for the adults.

We successfully cloned regions correspond-ing to an hsf-binding site in the hsp90 coding gene. We found corresponding sequences from 9 species of decapod crustaceans (see Materials and Methods).

The sequence corresponding to the heat factor binding site (amino acids from 485 to

Fig. 2. Effect of heat shock on the expression of hsp90 gene in adults. A: typical expression pattern (RT-PCR) of the hsp90 gene in the muscle tissues of adults Chiromantes haematocheir (ChH); Coenobita brevimanus (CB) and Coenobita purpureus (CP). B: calculated relative level of hsp90 mRNA in the muscle tissues of the adults. Each bar represents mean value of mRNA level standardized by the level of actin mRNA (one way ANOVA, P< 0.05). White bars correspond to Chiromantes haematocheir, light-grey bars to Coenobita brevimanus and dark-gray bars to Coenobita purpureus. Animals were exposed to high temperature for 2 hours and allowed to recover for 2 hours under RT. Zero values indicate lethal cases.

Stress protein hsp90 in terrestrial decapods 7

500 from the whole sequence of hsp90 gene of Ch. haematocheir, GenBank accession num-ber AY528900.1) consisted of 48 nucleotides, coding for 16 amino acids. No variation was observed in the number of amino acids among all species examined. At the same time, the alignment of obtained sequences revealed the presence of amino acid sequences unique for the decapods from the same climatic region (Fig. 3). The combination of Ala-Val-Thr in the middle of the domain and additional Arg was observed only in the species from the boreal White Sea. The species from Iriomote-jima Island possessed a unique combination of Ser-Ser (or Ser-Glu in case of Ch. dehaani) and additional Glu (E) instead of Val, which was found in all other species we examined. The do-main sequence of H. sanguineus and U. major revealed the presence of Thr instead of Lys (K) in these species. At the same time, all sequences possessed a combination of amino acids, which appear to be conserved, and is necessary for the posttranslational modification of the domain: Ser-Gly-x-Arg on the N-terminal end and Arg-x-x-Lys on its carboxyl end.

DISCUSSION

We compared heat tolerance of embryos and adults of three terrestrial decapods. The se-lected group experiences the most complicated

of stressful conditions, since they live in envi-ronments close to their thermal and desiccation tolerance limits on land where the fl uctuations of temperature and humidity are much higher than what they would experience in the aquatic environment (Burggren & McMahon, 1988; Greenaway, 2003). Coenobita is an exclusively subtropical and tropical hermit crabs, while Chiromantes inhabits both subtropical and temperate zones where the annual and daily temperatures show much higher fluctuation and lower mean values. Chiromantes haema-tocheir spends the coldest month in an inactive hibernation (Saigusa, 1981). At the same time, the maximum temperatures that both groups experience through their active periods are the same: 30-35 ºC (Saigusa , 2001). Numerous studies with truly marine and intertidal organ-isms show that crustaceans have high physi-ological capacity to adjust their metabolism to low temperatures, while heat resistance is limit-ed due to the often irreversible protein damage caused by heat (Spees et al., 2002b, 2003). One of the goals of this study was to connect the data of temperature exposure in nature and a predictable molecular response, the heat shock response in these species using hsp90 mRNA, as a molecular measure of reversible damage in crustaceans (Spees et al., 2002a; Cimino et al., 2002; Ravaux et al., 2003).

We observed a remarkable difference in

Fig. 3. Multiple alignment of the deduced amino acid sequence of the hsf-binding domain in the hsp90 isolated from selected species of the decapod crustaceans from 3 different climatic regions. The conservative amino acids across all species are highlighted in black; regions found only across the species from the same climatic regions are highlighted in gray.

8 O. Gusev et al.

the value of temperature shock which causes a molecular response in increasing of the level of hsp90 mRNA between subtropical hermit crabs and estuarine crabs from temperate zones (Figs. 1, 2). While the range of resistance to the high temperature in both embryos and adults of all three species was found to be 6-9˚C, the value of minimum temperature required for the initiation of the synthesis of hsp90 RNA was remarkably higher in both Coenobita species compared with Chiromantes (34–36ºC and 32–33 ºC respectively), but there were no differ-ences between the two species of hermit crabs.

The two species of Coenobita chosen by us for this experiment are different in their ecol-ogy. C. brevimanus is one of the most terrestrial hermit crabs among Coenobita and rarely can be found near the shore; this species is noc-turnal and has limited access to water or the shoreline. In contrast, C. purpureus is common near the sea and is diurnally active, thus they experience higher temperatures during the day. C. purpureus will fill its shell with sea water as a behavioral means to compensate for high temperature stress (Burggren & McMahon, 1988; Imafuku, 2002). Our results indicate that both species achieve preferable body thermal conditions using different behavioral strategies.

Furthermore, we found a clear difference between temperature values for the initiation of the expression of hsp90 gene between embryos and adults of the same species. In all cases, the embryos were less tolerant to high temperatures then adults (Fig. 2). The minimum temperature for initiation of the expression of hsp90 gene in the embryos of Coenobita was higher than in the embryos of Chiromantes: 34˚C and 29˚C respectively (Fig. 2B). To the best of our knowledge, there have been no studies specifi -cally focused on the molecular response to tem-perature stress in embryonic or larvae stages of crustaceans. However, data obtained using the embryos of brine shrimp Artemia suggest that generally, embryos of crustaceans have a lower threshold for the initiation of the expression of the main groups of chaperon proteins in re-sponse to various stresses than adults (MacRae, 2003; Tanguay et al., 2004). Since both females

and embryos are naturally being exposed to the same thermal regime, differences in the resis-tance to the heat might suggest the presence in the land terrestrial decapods of specifi c mater-nal care to prevent overheating of the embryos. A similar mechanism of maternal care was well described in amphipods (Dick et al., 1998). The presence of the shell, which forms a “buffering chamber” for keeping embryos in land hermit crabs, assumes that Coenobita and Chiromantes should have different strategies for avoiding high temperatures around the egg mass. Such behavioral patterns in these crabs and hermit crabs have previously been described by a num-ber of authors (Neil & Elwood, 1985; Brante et al., 2003). There are also a number of docu-mented examples of the existence of an interac-tion between females and embryos of crabs in order to adjust timing of development (Ikeda et al., 2006) and provide grooming and antimi-crobial treatment of egg mass (Tankersley et al., 2002; Brante et al., 2003; Ruiz-Tagle et al., 2003). Much less is known about female – egg mass interaction in the case of hermit crabs, since the abdomen is covered by the shell and direct access to the egg mass is diffi cult. Land hermit crabs keep sea or fresh water inside of the shell for both ionic and temperature bal-ance (Burggren & McMahon, 1988), but the fi ne mechanisms of maternal care about the egg mass in the aspect of thermal control are still to be elucidated.

Another objective of our study was to analyze the primary structure of hsf-binding domain in hsp90 coding gene of decapod crus-taceans (Fig. 3). The hsf-binding site acts spe-cifi cally as an acceptor of the heat stress factor (hsf), which is the first molecular response to the stress on the cellular level and mediates up-regulation of the main chaperons (Zhao et al., 2005). Analysis of hsf-binding domain in hsp90 gene of the six species of decapods examined revealed that two tetrads (Ser-Gly-x-Arg and Arg-x-x-Lys, Fig. 3) are necessary for the for-mation of the hsf-binding pocket itself and the mature chaperon dimer, and these tetrads are conserved. At the same time, the unique combi-nations of amino acids were found to be com-

Stress protein hsp90 in terrestrial decapods 9

mon for boreal species, living under isothermal 4ºC conditions (Ala-Val-Thr and additional Arg), crustaceans from littoral zone of subtropi-cal islands (Ser-Ser (Glu) and additional Glu, instead of Val) and one from temperate zone (Thr instead of Lys, Fig. 3). All three patterns might suggest possible convergent processes in the evolution of the structure and the dynamics of chaperon activity of hsp90 gene under the infl uence of the thermal regimes of the habitats of the crustaceans: one with less annual fluc-tuation in hot subtropical zone (Chiromantes dehaani, Coenobita brevimanus and Melicertus latisulcatus), one with high annual fluctua-tion in the warm temperate zone (Chiromantes haematocheir, Hemigrapsus sanguineus and Upogebia major) and one in the cold depth of the boreal sea with isothermal conditions of 4-5ºC (Hyas araneus, Sclerocrangon boreas and Pagurus pubescens).

Further studies are now needed to clarify the interaction between the primary structure of heat shock proteins and the molecular pathway leading to the differences in the stress response. For example, small colonies of Chiromantes haematocheir can be found in subtropical regions, which make this species a target for further investigations of the infl uence of envi-ronmental conditions on acclimatization and its interaction with the evolutionary patterns in the gene structure of molecular chaperons in the closely related species of decapod crustaceans.

Acknowledgments. —We want to sincerely thank Dr. K. Wada and Dr. Christopher Tudge for their valuable advice during preparation of this paper. We thank two unknown review-ers for their suggestions for improvement of the manuscript. This study was supported by a grant of Long-range Research Initiative (LRI) provided by Japan Chemical Industry Association (JCIA).

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Addresses: OG,* MS, Laboratory of Behavior, Evolution, and Environment, Graduate School of Natural Science and Technology, Okayama University, Tsushima 3–1–1, Okayama 700-8530, Japan. E-mails: (OG) ogusev@mail.ru, (MS) dns16401@cc.okayama-u.ac.jp; TAZ, Nicholas School of the Environment and Earth Sciences, Duke University Marine Laboratory, 135 Duke Marine Lab Road, Beaufort, NC 28516, U.S.A.

*Author for correspondence