Bio logy of Anomura II (A. Asakura，e d.) ， Research，Specia lNu mber6: 109-119，2006

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

Oleg Gusev，Tracy A. Ziegler andMasayuki Saigusa

A bst ract .- Thisstudy describes aspects restrial adaptation. Both Coenobita spp.and of the biologyof heat shock proteins 90kDa Chiromantes haematocheir b巴long to the T4

(hsp90) in terrestrial decapods，Coenobita pur- group，since they are fo und in aterrestrial envi ・ pureus，C. bre vimanus (Anomura: Coenobitidae) ronment as adult s，but still depend on the ma- and Chiromantesha ematocheir (Brachyura: rine environment for planktonic larval develop- Sesarmidae). These species possess highlydevel- m en t. oped terrestrial adaptations on both morphologi- Recent studies of the concomitant effects of cal and phys iological levels. We have analyzed therrnal acclimation onboth heat and cold tol - the pattern of express ion for th ege ne coding erance have been made in m anyspecies: e.g . hsp90:mol ec ular chaperon in both embryos and ， (Sti llm an 2003) planarians (Ts ulωda the muscle tissues of aduIt sof these species in ， ， re sponse to heat stress. While the minimum tem- &Ogoshi ，19 78) ，earthworms (Hanumante， perature for initiation of the synthesis of excess 1977) ，flies (Ohtsue tal. ，1999) ，lobsters (Spees of hsp90 mRNAwas different between embryos et al. ，2003) ，fish (Hemandez & Buckle，2002) ， and aduIt sfrom the different climatic region s， crayfish (Layne et al. ，1985) ，and copepods the range of re sistance to high temperature shock (Bradley，1 978; Voznesens)くy etal .，2004) . was nearl ythe sa me (6 ーやり. Furthermore，the Taken to gether，the res ult ss uggest that ，even str ucture of the stre ss -factor binding domain in if they are close systematica l1 y，or ganisms hsp90 codin ggene showed convergent pattern sb y exposed to the same climatic conditions will possessing aconservative amino acid se quence in display different bodytemperatures ，effective decapods experiencing the sa me annual tempera- metabolic temperatur es ，and patt emingof heat ture regimes in the subtropical，te mperate and stress re sponse and ac climation (He1mut et a l.， boreal climatic regions. 2002). Compared to other ，terrestria1 decapod have arelati ve ly10 w re- INTRODUCTION sis tance to hi ghtemper at ure stress and water Two separateIi nesof decapod crustaceans 10ss (Schmitz & Harrison ，2004). These two have successfully in vaded the terrestrial envi- pr oblemswere overcome by the deve10pment ronment (for review ，see Schmitz & Harrison， of physio10 gica1 and behavioral adaptations ， 2004) the hermit family Coenobitidae including dai 1ymi gration s(I mafuku，2002; (Anomura) (for review ，see Burggren & Morris，2002) ，comp li cated breedingand 1ar- McMahon，198 8; Greenaway，2003) and vae release behaviors (Saigusa & Terajima， the Grapsoidea (Brachyura) (Adamczewska 2000;Im afuku，2002; Brodie，2002) ，changes & Morris，2000; Anger & Shubart，2005). in respiration ，in circu1atory systems，and in Decapods th at spend more or less of their time the dynamicsof molting and food di gest ion in the terrestrial environment have been sum- (McMahon & Burggren，1988 ;Morris ，2002) . marized into five grades，T 卜T 5 (Burggen & An important question is whether evo1u- McMahon，199 8) ，according to degree of ter- tionary adaptation at phenotypic and genotypic 110 O. Gusev efal levels affects acclimation and general com- the highest temperature and water level varia- pensatory abilities of terrestrial crustaceans to tion ，were shown to possess three broad catego- environmental temperature changes.Nearly ries of variation of response. First ，total cellular all members of Coenobitidae use behavioral levels of hsp's varied with season (Hofmann & means to avoid overheating，but the tempera- Somero，1995; Buckley eta l.， 2001) and with ture threshold for such behavior was shown laboratory acclimation (Tomanek & Somero， to be higher in diumally active species，rather 1999; Buckley etal .，2001). Second，although then in nocturnal and more terrestrial species the stress response displays an element of plas- (Burggren & McMahon，1988; Greenaway， ticity，some components appear to be fixed， 2003). Meostatic temperature of the hemo- and are acharacteristic of aparticular species. Iymph in terrestrial hermit crabs and brachy- Different sets of congeners of rocky intertidal uran crabs is generally higher then in their lit- marine invertebrates have different stress re- toral relatives ，while the oxygenation level does sponses despite being acclimated to the same not show aremarkable difference (Burggren temperature (e.g .，Hofmann & Somero，1995; & Mahon，1988). Furthermore，the preferred Tomanek & Somero，2002). Third，the stress average body temperature and the temperature response varies in natural populations and close of hemolymph，as well as total hemolymph species across environmental gradients and oxygen capacity shows atendency to be higher with thermal habitat (Helmuth & Hofmann， in terrestrial rather than in marine species 2001). Moreover，there is alink between the (Burggren & McMahon，1988; Schmitz & structure of certain domains in major groups Harrison，2004). of constitutively expressed and stress-induced The question of genetypic basements for the chaperons and temperature-adaptive molecular physiological plasticity during heat acclimation response in the species with different latitudi- in crustaceans was partially answered using nal biogeographic patteming (Horowitz，2001; temperature-stress speci fi. cp 附 eins (molecular Somero，2005 ;Mahroof ，et al .，2005). chaperons) as markers of the compensatory In this study，we investigated the effect of abilities of the organism to overcome dam- temperature on the viability and expression age caused by excess environmental stress on of the molecular chaperon hsp90，showing acellular leve l. Heat shock proteins (hsp) are mixed cytosolic and stress-response nature of the most studied group of molecular chaperons the adults and embryos of two species of land involved in the refolding of proteins damaged hermit crabs Coenobita and abrachyuran crab by external stresses (Soti & Scermely，2003; Chiromantes haematocheir. To analyze the pos- Robert，2003). This group includes four m 勾or sible influence of primary and secondary struc- types of proteins (hsp27，hsp70 ，hsp90 and ture of hsp90 on the induction of increasing hsp 100) ，according to their molecular weight levels of this protein in response to heat shock， and functions (Feder & Hoffman，1999). Hsps we cloned anucleotide sequence coπesponding are particularly significant as they represent a to the particular region in the second functional mechanism by which an organism can buffer ATP-dependent domain of hsp90.This is one the impact of environmental temperature on the of the potential markers of environmental- protein pool without having to employ special- conditions based on changes in the functional ist protein isoforms to withstand high tempera- structure of hsp90 (Schnaider etal .，1999; Zhao tures (Somero，1995). The investment in hsp' et al. ，2005). This domain is regarded as the s，as the likelihood ofhigher temperatures from binding site for the heat shock factor (hs f) ，a global climate change looms，may be an e汀ec- protein controlling chaperon activity of hsp90 tive strategy for intertidal and terrestrial crusta- and intensity of its synthesis under stress con- ceans ditions .The activity of hsf closely depends on Crustaceans and some other inhabitants of the level of damage caused by high temperature the intertidal zone，which is characterized by and other stresses (Westwood & Wu，1993; Stress protein hsp90 in terrestrial decapods lll

Rabindran et a l.， 1994).We also analyzed the Samples of boreal species，H yas araneus primary genetic structure of the heat factor (8rachyura: Maj idae) ，Sclerocrangon boreas binding site in hsp90 coding gene in anumber (Caridea:Crangonidae) and Pagurus pubescens of decapod crustaceans from different tempera- (Paguroidea: Paguridae)，were collected by ture regimes within various climatic regions. deep creeping (depth - 80-100 meters) in the Kandalaksha Gulf，White Sea，Russia (32-340E ， 65-660N).The water temperature at the collect- MATERIALS ANDル1 E THODS in gpoint depth was 4oC and thi svalue remains Field sampling constant throughout the year at these depths. No The main target species Coenobita purpu- specific acclimatization procedures were further reus，C. brevimanus and Chiromantes haema- applied to the adults and the embryos. tochei r， as well as the other decapod species for comparative purposes，were collected as fol- Heat stress treatment lows 什omthree regions in Japan with different For the excess temperature treatment，eight annual temperature fluctuation in both water rep1icate experiments were conducted. The em・ and air . bryo clusters (at the stage of development close Subtropical species were collected at to hatching; weight - 1g; 1200- 1300 embryos) lriomoteサima Island，Ryukyu (124 oE ，24 0N)， were dissected from the abdomen of females of with daily fluctuation of water temperature of Chiromantes and Coenobita and immediately about 24-260C on the surface and in shallow placed in a1.5 ml tube，which was preheated to water and air temperature of about 28-30 oC the experimenta1 temperature and filled with ar- during the season of collection (April-June， tificial sea water (10%0). Temperature treatments 2004-2005). Ovigerous females of land hermit ofthe embryos were conducted in block incuba- crabs Coenobita purpureus were collected on tors .Adult females were placed in cylindrical the beaches and those of C. brevimanus were plastic containers (10 cm in diameter and 25cm collected in the rain forests about 1- 1. 5km in height) and experiments were conducted from the shore. Adult specimens of Melicertus in preheated hybridization incubators (HB・80 latisulcatus (Dendrobranchiata: Penaeidae) Taitec). were collected in the littoral zone during Both embryos and females were treated with low-tide. Specimens of the sesarmid crab arange of temperatures (240C -40oC) for 120 Chiromantes dehαα ni (8rachyura: Sesarmidae) min，then retumed to room temperature (RT，26 were collected in the mangroves areas during OC) for 2hours for arecovery period. Next，a noctumal low tides. sample of muscle tissues from the female crabs To represent the temperate region，Inland was isolated and immediately frozen with liquid Sea species were collected in the Kasaoka nitrogen .Whole embryo clusters were fixed in district ，Okayama Prefecture (1330E ，36 0N)， the same way. with daily fluctuation of water temperature of about 20-230C in surface and shallow water， RNAand DNAanalysis and air temperature of about 25-270C during Total RNAwas extracted from both em- the season of collection (April-June，2004 bryos and tissues of the adults. Samples were -2005).Ovigerous females of Chiromantes pulverized with amortar and pestle under liquid haematocheir (Brachyura: Sesarmidae) were nitrogen，and the total RNAwas extracted and collected in coastal habitats .Hemigrapsus san- purified with an RNeasy Midi Kit (QIAGEN) guineus (Brachyura: Grapsidae) were collected according to the manufacturer 's protocol. To from arocky 1ittoral zone during low tide . prevent possib1e DNAcontamination in the Mud shrimps Upogebia major (Thalassinidea: RNA，the samples were subjected to DNase Upogebiidae) were obtained from intertidal treatment using aDNA-free Kit (Ambion) mud flats. Furthermore，the poly(AtRNA was purified 112 O. Gusev ela/

Table 卜A li stofnucleotide primersused in this study for isolation ofpartial gene sequences. DIrection Length of Primer Sequence pro-duct Source Oligonuc/eotides used for isolation ofpartial sequence hsp90 codingge nefor mRN Aexpr ess ion ana/ysis

S-HSP90-F 5' ・GGCAGGTCACGAACGTGTGT-3'-3' forward 500 bp AY528900.1 S-HSP90-R デーGTAACCTTGTCGGCCACCAG-3' reverse Oligonuc! eolidesus edf or isolation of sequence of hsfbindingdom αin of hsp90 coding gene S-HSF-F 5'-CTACTACATCACTGGCGAGA-3' forward 48 bp AY528900.1 S-HSF-R デー ACACCACCTCGAAGCCACG-3' reverse Oligonuc! eotidesusedfor iso/ation ofparli α/sequen ce of betα-actinfor mRN Ae xpression analysis S-ACT-F デー CACCACTGGGATGACATGGAG-3' forward 498 bp AY074923.1 S-ACT-R 5'・AAGGAAGGCTGGAAGAGGG-3' reverse

from the total RNA，obtained in the previous removed from each reaction during the PCR step ，using the QuickPrep Micro mRNApurifi- every 4cycles sta 口ing 合omthe 15th cycle (i.e .， cation kit (Amersham-Pharmacia). 15th，19th ，23rd ，2 7th ，30th) and PCR products Genomic DNAs were prepared in 70% were separated on a2% agarose ge l. Levels ethanol from the tissues of the specimens，us- of mRNA's expression were estimated using ing the method described by Blin & Stafford GeneAnalyzer Pro Software by comparison of (1976). The purified genomic DNA was sus- the intensity of the PCR bands on the different pended in TE (10 mMTris-HCI /1 Mm EDTA ， stages of PCR reactions standardized by inten- pH 8.0) and stored at -20 oC until used. sity of actin bands. The difference in expression The mRNAexpression patterns of hsp90 was further confirmed by PCR reaction using were examined by RT- PCR using the conser- 1，1/ 5and 1/1 25 embryos-equivalent cDNA's vative region of about 500 bp in all species. according to Chen etal. (2002). Primers，length of the products，and sources for The variable paロ(approximately 50 nucleo- the primers are shown in Table 1. One μg of tides) in the putative hsf-binding domain in total RNA was extracted ，as described above， hsp90 gene was amplified 合omgenomic DNA from every experimental group of embryos and as atemplate under similar conditions，using treated with two units of DNase 1to remove all oligonucleotide primers synthesized on the ba- trace amounts of genomic DNA. These samples sis of conservative flanking regions (Table 1) were subjected to reverse-transcription using and further cloned. a First Strand cDNA Synthesis kit (Roche Plasmid DNA was purified from clones Diagnostics). PCR's were carried out in atota1 in the subtracted library，and the inserts were volume of 20μ1 of solution containing 1xPCR sequenced using aThermo Sequenase C ycle reaction buffer，150μM dNTP mix，0.5 U of Sequencing kit (Amersham-Pharmacia) with

Taq DNA polymerase，2 mMMgCI 2，each M 13 forward (- 20) and M 13 reverse prim- of the primers at 0.3 - 0.4 μM，and 100 ng of ers specific to the f1 anking regions of amulti- cDNA (or 1μMof genomic DNAfor isolation cloningsite in the pCR2.トTOPOcloning vec- of the unique domain region of hsp90).The tor according to the manufacturer's direction. ampli fication was performed in aGeneAmp They were analyzed with an automatic DNA PCR System 9700 (Applied Biosystems) sequencer，the DSQ-2000L (Shimadzu). For programmed for 35 cycles of 94 0 C (1 min)， the calculation of molecular mass and primer 57 0 C (1 min)，and 67 0 C (4 min)，followed by design and analysis，we used Vector NTI Suite elongation for 10 min at 720 C. Aliquots were 9(InforMax) computer software. Stress protein hsp90 in terrestrial decapods 113

A CP CB 一一一 一b: * ChH 24 26 28 30 32 34 36 38

2EE 80 B E 。包 60 (/) 五二 』。

iii 由iii 40

担ω問〉

20 。 24 26 28 30 32 34 36 38 40 temperature of heat str ess( OC )

Fig. 1. Etfect of heatshock on the level of re lative hsp90 mRNA in the embryos. A: typical expression pattern (RT-PCR) of the hsp90 genein theembryos of C hiromantes haematocheir (ChH);Coenobita brevimanus (CB) and Coenobita purpureus (CP). B: calcu lated relative levelof hsp90 mRNA in the embryos. Eachbar repre sent smean value of mRNA level standardi ze dby th el evel of actin mRNA (one-way ANOYA， p く 0.05). White bars correspond to Chiromantes haematocheir ，li ght -grey bars to Coenobita brevimanus and dark-gray barsto Coenobifa purpureus. Animalswere expose dto hi ghtemper ature for 2hour sa nd all owedt o recoverfor 2hour su nder RT. Zero values indicate lethal cases.

Stati stical tes ts in embryos and muscletissues of the adult The level of variation of the calcul ated crab Chiromantes haematocheir and hermit values of all gene expression data were log crabs Coenobita purpureusand C. brevimanus 10transformed prior to one-way analysis of (Figs. 1，2) .We d etecte dstro ngd ifferences in variance (ANOVA) to normalize variance， hsp90 gene expression between Coenobita and and apost -hoc analysis (Dunnett's test) was Chiromantes. T hed ifferences in the value of conducted to confirm the significance in the temperature threshold for initiation of synthesis differences of the expression leve l. Significant of excess of hsp90 and level maximum resis- differences were accepted at p< 0.05 (Sigm a tance to the heat stress wereapparent in both Stat Statistical Software). embryos (Fig. 1)and ina dul tan imals (Fig. 2) . ln the embryos of Ch. haematocheir， R ESULTS significant differences in hsp90 mRNA Wecompared the expression of chaperon levels were detected in the group exposed to genes in response to hightempe rat ure stress 290 C ，whi lein bothspec ies of Coenobita，the 114 O. Gusev et al

A αH 一一切崩磁場 I B cP c |一一一樹鵬首 … 議暢E 24 26 28 30 32 34 36 38

80 B 区 E 8 g. 60 .s::。 ω 〉 t40 〉 ぷ2 2 @ 20 。 24 26 28 30 32 34 36 38 40 temperature of heat stress ('C)

Fig. 2. Effect of heat shock on the expression of hsp90 gene in adults. A: typical expression pattem (RT- PCR) of the hsp90 gene in the muscle tissues of ad ult sChiromanles haemalocheir (ChH); Coenobila 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 く 0.05). White barscorrespond to Chiromantes haematocheir，li ght-grey bars to Coenobita brevimanus and dark-gray bars to Coenobita purpureus. were exposed to hi ghtemperature for 2hours and allowed to recover for 2hours under RT. Zero valu es indicate leth alcases

minimum temperature for increasing of level C. brevimanus and 仁 purpureus were 34-360 C of hsp90 was 33-340 C (ANOVA，P = 0 .0017， and 40-43 0 C respectively (ANOVA，p = 0.003 ， Fig. 1). The lethal level of high temperature Fig. 2). The range of excess temperature resis- exposure，which caused mortalit yin more than tance (from initiation of appearance of excess 95% of embryos after arecovery period (2 hsp90 mRNA to death due to heat shock) in hours after exposure)，was detected to be 34-35 Ch. haematocheir was 7-8 0 C for the embryos 。C for the embryos of C h. haematocheir and 38 and adults. For both Coenobita species，the -40'C for both species of Coenobita. The level range of excess temperature resistance was 6 of expression of the hsp90 genewas also dif- -rc for embryos and 7-90 C for the adults. ferent between the adult muscle tissues of both Wesuccessfu ll yc1 0ned regions correspond- hermit crabs and Chiromantes. The minimum ing to an hsf-binding site in the hsp90 coding temperature for asignificant increase in the gene.We found corresponding sequences from level of hsp90 mRNA in the tissues of Ch 9species of decapod crustaceans (see Materials haematocheir was found to be 30-31 oC and and Methods). the lethal exposure temperature was 36- 380 C The sequence corresponding to the heat (ANOVA，p =0 .0012，Fig. 2). The values for factor binding site (amino acids from 485 to Stress protein hsp90 in terrestrial decapods 115

C hiromanteshaematochei r Hemigrapsus sangu加eus Upogebiamajor

Coenobita brevimanus Iriomote island (Japan] C hiromantesdehaan i subtropical(24 N ) Melic ertus latisulcatus ・

Pagurus pubescens H yasaraneus ] 附州it Sclerocrangon boreas boreal… (64-66.N ) Fig. 3. Multiple alignment of the deduced amino acid sequence of the hsf・binding domain in the hsp90 isol ate dfrom selected species of the decapod crustaceans 什om 3different climatic regions. The conservative amino acids across all species are hi ghlighted in black; regions found only across the species from the sa me climatic regions are highli ghted in gray

500 from the whole sequence of hsp90 gene of of stress 白I conditions，since they live in envi ・ Ch. haematochei r， GenBank accession num- ronments close to their thermaland desiccation ber AY528900.1) consisted of 48 nucleotides， tolerance limits on land where the f1 uctuations coding for 16 amino acids. No variation was of temperature and humidity are much higher observed in the number of amino acids among than what they wouldexperience in the aquatic all species examined. At the same time，the environment (Burggren & McMahon，1988 ; alignment of obtained sequences revealed the Greenaway，2003). Coenobita is an exclusively presence of amino acid sequences unique for subtropical and tropical hermit crabs，while the decapods from the same climatic region Chilη mantes inhabits both subtropical and (Fig. 3). The combination of Ala-Val-Thr in the temperate zones where the annual and daily middle of the domain and additional Arg was temperatures show much higher fluctuation observed only in the species from the boreal and lower mean values.Chiromantes haema- White Sea. The species from Iriomote-jima to cheir spends the coldest month in an inactive Island possessed aunique combination of Ser- hibemation (Saigusa，1981) .At the same time， Ser (or Ser-Glu in case of C h. dehαα ni) and the maximum temperatures that both groups additional Glu (E) instead of Val ，which was experience through their active periods are the found in all other species we examined. The do- same: 30-350 C (Saigusa，2001). Numerous main sequence of H. sanguineus and U. major studies with truly marine and intertidal organ- revealed the presence ofThr instead of Lys (K) isms show that crustaceans have high physi- in these species. At the same time，all sequences ological capacity to adjust their metabolism to possessed acombination of amino acids，which low temperatures，while heat resistance is limit- appear to be conserved，and is necessary for the ed due to the often irreversibleprotein damage posttranslational modification of the domain : caused by heat (Spees eta l.， 2002b，2003). One Ser-Gly-x-Arg on the N-terminal end and Arg- of the goals of this study was to connect the x-x-Lys on its carboxyl end. data of temperature exposure in nature and a predictable molecular response，the heat shock response in these species using hsp90 mRNA， SCUSSION Dl as amolecular measure of reversible damage in We compared heat tolerance of embryos crustaceans (Spees et a l.， 2002a; Cimino et al. ， and adults of three terrestrial decapods. The se ・ 2002; Ravaux eta l.， 2003). lected group experiences the most complicated We observed aremarkable difference in 116 o. Gusev etal the value of temperature shock which causes a and embryos are naturally being exposed to the molecular response in increasingof the level same thermal regime，differences in the resis- of hsp90 mRNA between subtropical hermit tance to the heat might suggest the presence in crabs and estuarine crabs from temperate zones the land terrestrial decapods of specific mater- (Figs. 1，2). While the range of resistance to the nal care to prevent overheating of the embryos. high temperature in both embryos and adults A similar mechanism of matemal care was well of all three species was found to be 6-9 0 C ，the described in amphipods (Dick et al. ，1998) .The value of minimum temperature required for the presence of the shell ，which forms a “bufferin g initiation of the synthesis of hsp90 RNA was chamber" for keeping embryos in land hermit remarkably higher in both Coenobita species crabs，assumes that Coenobita and Chiromantes compared with Chiromantes (34-360 C and 32 should have different strategies for avoiding -33 oC respectively)，but there were no differ- high temperatures around the egg mass.Such ences between the two species of hermit crabs. behavioral patterns in these crabs and hermit The two species of Coenobita chosen by us crabs have previously been described by anum ・ for this experiment are different in their ecol - ber of authors (Neil & Elwood，1 985; Brante ogy. C brevimanus isone of the most terrestrial eta l.， 2003). There are also anumber of docu- hermit crabs among Coenobita and rarely can mented examples of the existence of an interac- be found near the shore; this species is noc- tion between females and embryos of crabs in turnal and has limited access to water or the order to adjust timing of development (l keda shoreline. ln contrast，C purpureus is common eta l.， 2006) and provide grooming and antimi- near the sea and is diurnally active，thus they crobial treatment of egg mass (Ta此 ersley et experience higher temperatures during the day. al .，2002; Brante et a l.， 2003; Ruiz-Tagle et al .， C purpureus will fill its shell with sea water 2003). Much less is known about female - egg as abehavioral means to compensate for high mass interaction in the case of hermit crabs， temperature stress (Burggren & McMahon， since the abdomen is covered by the shell and 1988; Imafuku，2002). Our results indicate that direct access to the egg mass is difficul t. Land both species achieve preferable body thermal hermit crabs keep sea or fresh water inside of conditions using different behavioral strategies . the shell for both ionic and temperature bal- Furthermore，we found aclear difference ance (Burggren & McMahon，1988) ，but the between temperature values for the initiation of fine mechanisms of matemal care about the egg the expression of hsp90 gene between embryos mass in the aspect of thermal control are still to and adults of the same species. In all cases，the be elu cidated. embryos were less tolerant to high temperatures Another objective of our study was to then adults (Fig. 2). The minimum temperature analyze the primary structure of hsf-binding for initiation of the expression of hsp90 gene domain in hsp90 coding gene of decapod crus- in the embryos of Coenobita was higher than taceans (Fig. 3). The hsf・binding site acts spe- in the embryos of Chiromantes: 340 C and 29 cifically as an acceptor of the heat stress factor ・C respectively (Fig. 2B). To the best of our (hs f)， which is the first molecular response to knowledge，there hav巴 been no studies specifi- the stress on the cellular level and mediates up- cally focused on the molecular response to tem- regulation of the main chaperons (Zhao eta l.， perature stress in embryoni cor larvae stages of 2005). Analysis ofhsιbinding domain in hsp90 crustaceans. However，data obtained using the gene of the six species of decapods examined embryos of brine shrimp Artemia suggest that revealed that two tetrads (Ser-Gly-x-Arg and generally，embryos of crustaceans have alower Arg-x-x-Lys，Fig .3) are necessary for the for- threshold for the initiation of the expression mation of the hsιbinding pocket itself and the of the main groups of chaperon proteins in re- mature chaperon dimer，and these tetrads are sponse to various stresses than adults (MacRae， conserved. At the same time，the unique combi- 2003; Tanguay et a l.， 2004).Since both females nations of amino acids were found to be com- Stress protein hsp90 in te 汀 estrial decapods 117 mon for boreal species，living under isothermal Physiological and Biochemical Zoology，73: 706 4 0 C conditions (Ala-Val-Thr and additional -725. Anger Schubart D. 2005. Experimental Arg) crustaceans from littoral zone of subtropi- ，K .，& ，C. ， ， evidence of food-independent larval develop- cal islands (Ser司 Ser (Glu) and additional Glu， ment in endemic Jamaican 什eshwater-breeding instead of Val) and one from temperate zone crabs. Physiological and Biochemical Zoology， (Thr instead of Lys，Fig. 3). All three pattems 78: 246-258. might suggest possible convergent processes in Blin ，N. ，& Sta 仔' o rd ，D .w. ，1976 .A general method for isolation ofhigh molecular weight DNAfrom the evolution of the structure and the dynamics eukaryotes. Nucleic AcidsResearch ，3 :2303 - of chaperon activity of hsp90 gene under the 2308. inftuence of the thermal regimes of the habitats Bradley，B .P. ，1978. Increase in range of tempera- of the crustaceans: one with less annual fluc- ture tolerance by acclimation in the copepod Eurytemora affinis. Biological Bulletin 154 :177 tuation in hot subtropical zone (Chiromantes ， - 187. dehaani，Coenobita brevimanus and Melicertus Brante，A. ，Fernandez ，M .，Eckerle ，し， Mark，F .， latisul catus)，one with high annual fluctua- Portner，H. 0. ，& Amtz，w. ，2003. Reproductive tion in the warm temperate zone (Chiromantes investment in the crab ，Canc erse/o sus ，along haematocheir ，Hemigrapsus sanguineusand alatitudinal cline :egg production，embr yo los ses and embryo ventilation. Marine Ecology Upogebia major) and one in the cold depth of Progress Series ，521: 221 -232. the boreal sea with isothermal conditions of 4-- Brodie，R .J .，2002 .Timing of the water-to-Iand 50 C (Hyas araneus，Scl erocrangon boreas and transition and metamorphosis in the land crab Pagurus pubescens). Coenobita compressus H .Milne Edwards: ev 卜 Further studies are now needed to clarify dence that settlement and metamorphosis are de-coupled. 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