Heat Shock Responses of Closely Related Species of Tropical and

AMER. ZOOL., 39:877-888 (1999)

Heat Shock Responses of Closely Related Species of Tropical andDesert Fish1

LAWRENCE E. HIGHTOWER,*2 CAROL E. NORRIS,* PHILIP J. DiIoRio,t AND

EILEEN FiELDiNGf

*Department of Molecular and Cell Biology and ^Department of Ecology and Evolutionary Biology,University of Connecticut, Storrs, Connecticut 06269-3044

SYNOPSIS. Over the past twelve years, we have studied heat shock proteins in twotropical species, a half dozen desert species and a number of hemiclones of vivip-arous fishes in the genus Poeciliopsis. Heat shock protein (Hsp) isoform patternswere determined using high resolution two-dimensional polyacrylamide gels. Twofamilies of Hsps were studied in detail, the nucleocytoplasmic 70 kilodalton Hsp70family and the 30 kUodalton Hsp30 family related to a-crystallin. The temperaturedependence of Hsp accumulation was investigated using both intact fish and cul-tured cells. When the threshold temperatures were mapped onto thermal prefer-ence profiles, it was apparent that the Hsp70 threshold (33°C) was closely linkedto the most frequently selected temperatures and the Hsp30 threshold (37°C) wasclosely linked to high temperatures that fish rarely selected, indicating that fishdeploy these two molecular chaperones differently. One tropical species P. gradlisis a genetic reservoir for most of the Hsp70 isoforms of the desert species. Acquiredresistance to 41°C was strongly correlated with Hsp70 abundance for gracilis thatcontained Hsp70 isoform 3 whereas fish lacking this isoform showed similar levelsof acquired thermotolerance which did not correlate with Hsp70 abundance, sug-gesting multiple, compensating mechanisms of acquired resistance. Isoform 3 wasdegraded in cultured cells from a desert species during several hours of recoveryat normal temperature following heat shock whereas two other Hsp70 isoformswere stable. The implications of this property of isoform 3 are discussed.

INTRODUCTION the west coast of Mexico colonizing desertSmall, livebearing fishes in the genus s t r e a m s a n d headwater sites in the foothills

Poeciliopsis occupy thermally distinct hab- o f t h e S i e r r a M a d r e Occidental (Rosen anditats that range from Columbia through B a i l ey. 1963).tropical Central America to the Sonora Des- M o s t o f t h e animals used in our studiesert of northwestern Mexico and southern c a m e f r o m a genetically pedigreed aquan-Arizona. Some species live in tropical riv- u m f a c i l i ty a t t h e University of Connecticuters in a relatively stable environment in (Schultz and Schultz, 1988). Our plan waswhich temperatures vary over a narrow to characterize the heat shock response of arange and change slowly. Other species oc- representative poikilothermic vertebrate incupy harsh and unstable niches, small considerable detail and at the same time tostreams that cross subtropical desert where s u r v e y diversity in the heat shock proteinstemperatures range from 4°C to 40°C sea- <HsPs) b o t h w i t h i n and between closely re-sonally and can change rapidly, as much as l a t e d s P e c i e s - O u r diversity studies were22°C in 3 hr. Based mainly on biogeograph- concentrated on inbred strains representingical considerations, it is thought that ances- a h a l f d o z e n d e s e r t s P e c i e s a n d numeroustral tropical species migrated north along monacha-lucida hybrid species (White et

al, 1994). We later received fish from twonatural populations collected in tropical

1 From the Symposium Organismal, Ecological and southern Mexico. Subsequently these trop-Evolutionary Significance of Heat Shock Proteins and i c a l species were maintained in the aquari-the Heat Shock Response presented at the Annual , .... , , . . . . . ,Meeting of the Society for Comparative and Integra- U m f a C l l l t V a S OUtbred populations, whichlive Biology, 6-10 January 1999, at Denver, Colorado. allowed US to extend our survey of diversity

2 E-mail: [email protected] and to carry out several comparative studies

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878 L. E. HlGHTOWER

of desert and tropical species (Norris et al,1995).

The opportunity to study the thermal bi-ology of tropical and desert Poeciliopsisspecies was appealing to us, but the prob-lem was that the Poeciliopsis heat shock re-sponse was totally uncharacterized. This re-sponse was already known to be highlyconserved evolutionarily with a broad phy-logenetic distribution, so we were certainthat Hsps could be induced in Poeciliopsisand that at least some Hsps would likely beencoded in small multigene families withvery similar nucleotide sequences (High-tower et al., 1997). We chose to survey var-iation using high resolution two-dimension-al sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) to sepa-rate unfolded polypeptides obtained by de-tergent solubilization of cells and tissues. Amajor advantage of this proteomic approachis that almost all of the Hsps of an organismcan be displayed on a single gel. Even poly-peptides encoded by very similar genes canbe separated, and only expressed membersof these gene families will be scored. Poly-peptides can be positively identified by im-munoblotting and quantified using laserscanning densitometry. Disadvantages in-clude the limitation that quantitative activ-ity stains usually cannot be carried out onthe denatured polypeptides in the gels. It isalso necessary to demonstrate that the Hspisoforms represent primary gene productsand are not posttranslationally modifiedproteins. The Hsp70 major isoforms de-scribed herein appear to be derived fromdifferent genes, based on the following cri-teria: Southern blots showed that Poeciliop-sis Hsp70 isoforms are encoded in a smallmultigene gamily, radioisotopic pulse-chasestudies ruled out proteolytic processing, andphosphate radiolabeling showed that noneof the inducible isoforms is phosphorylated(Norris, 1997). Furthermore, from studiesof Mendelian inheritance of Hsp70 iso-forms, the existence of three linkage groupswas inferred from the five classes of ob-served isoform complements: linkage groupI carries the gene encoding isoform 1;group II carries the gene encoding isoform3 and group III carries the genes encodingisoforms 0 and 1 (dilorio, 1994).

Once the Hsp patterns for each speciesare in hand, this information can be used toinform molecular genetic strategies, i.e., thecloning and sequencing of specific heatshock cDNAs encoding proteins with inter-esting phylogenetic distributions or patternsof expression. This strategy was carried fur-thest for the small Hsp/a-crystallin familyin which we identified two heat inducibleclusters of proteins in the 27-30 kDa rangethat have different kinetics of induction.cDNAs encoding Hsp30 and Hsp27 werecloned and the complete coding regionswere sequenced. Further analysis of the se-quences revealed that both Hsp30 andHsp27 have signature a-crystallin domains.This published study was the first to estab-lish the presence of both of these lineagesin a single taxon (Norris et al., 1997).

RESULTS AND DISCUSSION

Poeciliopsis and temperature

When we began our investigation of var-iation in the heat shock response, the onlyinformation available was that variation incritical thermal minima, survival of acuteheat shock, and survival of acute coldshock exist in desert Poeciliopsis speciesand hemiclones (Bulger and Schultz, 1979,1982). Resistance to cold stress but notacute heat stress is clinal with the northern-most species, P. occidentalism, most resistantand the southernmost species studied, P.monacha, least resistant. The acute heatingprotocol used in these earlier studies in-volved transferring fish from 25°C to 40°Cdirectly and holding them for 30 minutesbefore returning them to 25°C. Survivalwas scored 24 hr after heating. Such acutetemperature changes do not allow fish timeto acquire thermotolerance by inducible de-fenses like the heat shock response, prior toexposure to lethal temperature. Therefore,the component of thermotolerance that de-termines the degree of damage is intrinsicthermotolerance, the ability of an organ-ism's macromolecules and cellular/tissuecomponents to resist thermal denaturation.Whether or not an organism's constitutivelyexpressed molecular chaperones such asHsc70, Hsp90 and Grp78 contribute to in-trinsic thermotolerance is a matter of spec-


POECILIOPSIS HEAT SHOCK RESPONSE 879

ulation (Leung et al, 1996). It is likelyhowever that the heat shock response wasinduced in the acutely heated fish during therecovery period where it could function asa repair system. Thus, this acute heatingprotocol probably assayed the combined ef-fects of variation in intrinsic thermotoler-ance and variation in the protein repair ca-pacity of the heat shock response. Missingwas the contribution of acquired thermoto-lerance.

The crucial first steps in assessing thiscontribution were to show that these fishpossess the ability to increase their ther-motolerance above intinsic levels under theappropriate heating regimen (figure 10 inWhite et al, 1994) and that Hsps accumu-late in fish tissues under these conditions(figure 3 in Norris et al, 1995). Our labo-ratory test of thermotolerance involvedheating fish at a constant rate (0.15°C permin) up to a lethal temperature of 41°C.This regimen was chosen to mimic the ther-mal exposure of desert species that becometrapped in shallow pools during the dry sea-son where they cannot escape relativelyrapid increases to near-lethal temperatures.This rate of temperature increase allows in-duction of the heat shock response and ac-quisition of thermotolerance before an or-ganism reaches lethal temperature, giving amore realistic measure of an organism'sthermal resistance than do acute heatingprotocols. By this test, thermal resistance inoutbred P. gracilis, which survived up to60 min at 41°C, was higher than that ineven the most resistant desert species ofPoeciliopsis, which survived approximately15 min at 41°C (dilorio, 1994). The maineffect of preconditioning heat treatments onsurvival was to extend the time that fishcould survive exposure to a lethal temper-ature. The effect on the maximum toleratedtemperature was small, an approximately1°C increase.

A protocol was also developed to deter-mine the temperatures at which accumula-tion of Hsp70 and Hsp30 can first be de-tected in fish. Induction of both of theseHsps is a threshold response in Poeciliop-sis, as will be described. Seventy-two fishfrom five species and three hemicloneswere injected intraperitoneally with 35S-me-

thionine to radioactively label newly syn-thesized proteins. Injected fish were held at25°C for time intervals prior to stress sothat incorporation times totalled 3 hr for allfish. Fish at 25°C were heated at a constantrate (0.15°C/min) to predetermined temper-atures ranging in one degree increments be-tween 30°C and 40°C and held at that tem-perature for 1 hr. At the end of the heatingperiod, fish were sacrificed, gill tissue re-moved, solubilized in lysis buffer and theproteins separated by two-dimensionalSDS-PAGE. The gels were processed byfluorography and the resulting X-ray filmswere examined visually. The threshold in-duction temperature was defined as the low-est temperature at which newly synthesized(radioactively labeled) Hsps could be de-tected. Synthesis of Hsp70 was first detect-ed at 32°C in P. occidentalis (northernmostspecies), 33°C in P. lucida, P. monacha andthree monacha-lucida hemiclones, and34°C in P. viriosa (southernmost species)and P. prolifica. Induction of Hsp30 oc-curred at 37°C in P. monacha, P. lucida, P.prolifica, and P. viriosa, but at 38°C in P.occidentalis and the three hemiclones.

The thermal profiles of Hsp70 and Hsp30induction and accumulation were developedfurther using a hepatocellular carcinomacell line, PLHC-1, derived from a liver tu-mor induced in P. lucida using a chemicalcarcinogen (Schultz and Schultz, 1985). Wehad determined previously that PLHC-1cells and primary liver cell cultures accu-mulated qualitatively the same complementof Hsp70 and Hsp30 as livers in heatshocked fish. These observations encour-aged us to carry out parallel studies in bothintact fish and cell cultures, an approachwhich became a major addition to our over-all experimental strategy. A further test ofthis strategy was to determine the thresholdinduction temperature of Hsps in PLHC-1cells for comparison to those measured infish. PLHC-1 cells were maintained at 30°Cin cell culture incubators, which was withinthe normal range (27 ± 5°C) to which fishwere exposed in the greenhouse aquaria ofour fish colony. Cell cultures were brieflylabeled with 35S-methionine after 1 hr in-cubation at various temperatures between30°C and 40.5°C. Newly synthesized (ra-


880 L. E. HlGHTOWER

32 38 4034 36

Temperature, °CFIG. 1. Quantification of the synthesis of Hsp70 (cir-cles) and Hsp30 (squares) in PLHC-1 cells as a func-tion of temperature of incubation. Duplicate cultureswere pulse-labeled with 35S-methionine after one hourat various temperatures, the cultures were solubilizedand proteins separated by one-dimensional SDS-PAGE. Fluorograms were quantified using laser den-sitometry. The optical densities in the Hsp70 andHsp30 bands were expressed as a per cent of the totaloptical density in the lane.

dioactive proteins) were separated by one-dimensional SDS-PAGE and visualized byfluorography. The resulting films werequantified by laser densitometry and the op-tical densities in the Hsp70 and Hsp30bands from duplicate cultures were ex-pressed as a percentage of the total opticaldensity in each gel lane (Fig. 1). As in thefish, Hsp70 was induced at 33°C and Hsp30was induced above 37°C in cultured cells.

The temperature dependence of Hsp ac-cumulation has several interesting features.The threshold induction temperature forHsp70 was well below the temperature atwhich Hsp30 was induced and well withinthe temperature range that P. lucida fre-quently experiences in its desert environ-ment. When the temperature increasedabove 37°C, Hsp30 was induced and therate of accumulation of Hsp70 increaseddramatically as well. We have consideredtwo different explanations for this profile.It is possible that the induction signals thatinduce Hsp70 are the same at the two tem-peratures. For example, there is consider-able evidence that the heat shock transcrip-tion factor is activated by a process that be-gins with the accumulation of damaged ordenatured proteins (Hightower, 1991). The

accumulation of denatured proteins abovethe level present in unstressed cells may be-gin at 33°C and the number of proteins un-dergoing denaturation may increase sharplyat 37°C. Alternatively, the induction signalmay be different at the two temperatures,the signal at the lower temperature beinggenerated by a conformational change in aspecific thermal sensor molecule for ex-ample and at the higher temperature bymore general protein denaturation (Leung etal, 1996).

P. dilorio combined the threshold induc-tion temperatures with data from a study byE. Fielding (1992) on thermal preferencebehavior of P. monacha (Fig. 2A) and P.lucida (Fig. 2B) to show when Poeciliopsisuses Hsp70 and Hsp30. For the thermalpreference studies, a temperature gradientof 21°C ± 1°C to 39°C ± 1°C was estab-lished in a specially designed tank with sub-mersible heaters at one end and a refriger-ation unit through which water was circu-lated at the other end. The Plexiglass tankwas partitioned into compartments all ofwhich were connected by openings in thepartitions. All compartments were aeratedand fish were provided with live brineshrimp nauplii to excess all along the gra-dient. Measurements were made on broodsof young (1-20 days old) fish 5-8 days af-ter introduction into the tank. The numberof fish present in each chamber, tempera-tures of all occupied chambers and temper-atures in the warmest and coolest chamberswere recorded at two-hr intervals from0600 hr to 2200 hr. The single asterisk inFigure 2 marks the threshold induction tem-perature of Hsp70 and the double asteriskmarks the induction temperature of Hsp30.Fish frequently crossed into regions of thetank hot enough to induce Hsp70 in theirtissues and this temperature did not pose asignificant thermal barrier. However, P.monacha was never observed in regions ofthe tank at 37°C or greater and P. lucidawas rarely observed in this thermal region.Therefore, these fish did not willingly ex-pose themselves to temperatures that in-duced Hsp30. We conclude that the induc-tion temperature for Hsp30 is closer to themaximum temperature (approximately40°C) that these fish are likely to experience


POECILIOPSIS HEAT SHOCK RESPONSE 881

21 23 25 27 29 31 33 35 37 39

A.

220

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88

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B.

220 "

198

176

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11088

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21 23 25 27 29 31 33 35 37 39

Temperature ° C

FIG. 2. Distributions of P. monacha (panel A; n =489 observations on 50 fish) and P. lucida (panel B;439 observations on 50 fish) in a temperature gradienttank expressed as the frequencies with which fish wereobserved at a particular temperature. ""Threshold in-duction temperature of Hsp70. **Threshold inductiontemperature of Hsp30. For additional information onthe thermal preference studies, see Fielding (1992).

in the wild whereas the Hsp70 inductiontemperature is closer to their optimalgrowth temperature (~31°C). These obser-vations suggest that Hsp70 may be usedroutinely by Poeciliopsis to control rela-tively minor thermal damage occurring be-tween 33 and about 36°C. Hsp70 induction,frequently used as a biomarker for acquiredthermotolerance or cytoprotection, may in-dicate here that the protective mechanismswere activated at temperatures well belowthe most damaging in order to be in placewhen fish encountered temperatures thatwould correspond to the hottest part of the

day in their natural environment. At 37°C,the accumulation of Hsp70 increased sub-stantially and was further augmented by themolecular chaperone Hsp30 which is ap-parently used primarily to survive near-le-thal temperatures.

Tropical gracilis is a genetic reservoir ofHsp70 diversity

In our survey of Hsp70 family diversityamong desert Poeciliopsis species, wefound four different patterns of heat-induc-ible Hsp70 isoforms in six species (Whiteet al., 1994). All of these species had thesame single isoform of Hsc70 and the sameGrp78 isoform in a phosphorylated and anunphosphorylated form. In subsequentanalyses, we obtained the Hsp70 family iso-form patterns from two tropical species, P.gracilis and P. fasciata, and a confamilialspecies, Gambusia affinis (Norris et al.,1995). Again, these species had the sameconstitutive isoforms as the desert Poeci-liopsis species. Evolutionary constraints onconstitutive Hsp70 homologs appear to bedifferent in different subcellular compart-ments: Endoplasmic reticulum-based Grp78proteins from diverse species are moreclosely related to one another than to nu-cleocytoplasmic Hsc70 from the same spe-cies. Heat-inducible Hsp70 is most similarevolutionarily to Hsc70 and it has been pro-posed that these two proteins compose anorthologous group, as do each of the orga-nellar homologs (Boorstein et al., 1994).The high level of conservation betweenHsp70 and Hsc70 implies conservation offunction as well. Both proteins function inthe nucleocytoplasmic compartment of cellswhere they transiently bind newly synthe-sized proteins as part of protein folding andintracellular transport pathways. Both pro-teins have conserved ATPase and peptide-binding domains with similar specificitiesfor peptides. Both localize to the same cel-lular regions after stress (Hightower andLeung, 1997). From results of domainswitching experiments with yeast Hsp70isoforms, Craig and coworkers have sug-gested that the regions of these proteins thatinteract with accessory proteins may bewhat distinguish these isoforms, not differ-ences in the ATPase or peptide-binding ac-


882 L. E. HlGHTOWER

tivities (James et al., 1997). If we acceptbiochemical diversity as an initial, mini-mum estimate of genetic diversity, then theamount of variation that we have observedamong closely related species and within P.gracilis suggests that even highly con-served proteins like Hsp70 may have con-siderable evolutionary potential, whereasthe constitutive family members, Hsc70 andGrp78, appear to be very highly constrainedevolutionarily.

The number of inducible Hsp70 isoformsfound in a single population of tropical P.gracilis was surprising. All but isoform 2among the abundant isoforms and most ofeven the minor isoforms found in the desertspecies of Poeciliopsis and in G. affiniswere found in this one tropical species. Inaddition, gracilis contained an abundantisoform (isoform 0) not found in any otherspecies. Isoform 2 was the only abundantisoform in tropical P. fasciata and desert P.latidens. A minor isoform with the same piwas found in P. gracilis, so the gene en-coding isoform 2 is likely to be present inthe gracilis genome but the expression lev-els are comparatively low. This was true ofseveral other isoforms as well for which ex-pression levels were higher in desert speciesthan in the tropical species. Isoform 3 isprobably the most common Hsp70 isoformin Poeciliopsis, being present in six of eightspecies analyzed, and it was the only abun-dant Hsp70 isoform in 21 individuals of Gaffinis collected at two different sites in Ne-vada.

Does Hsp70 abundance correlate withsurvival?

The availability of an outbred populationof P. gracilis allowed a correlation analysisof variation in acquired thermotoleranceand Hsp70 abundance. A detailed experi-mental procedure has been published (Nor-ris et al., 1995), so a brief description ofthe experiment will be given here. Lots often females from the second and third lab-oratory generations of stocks maintained byrandom breeding were used. Fish were ac-climated for one week at 30°C. Thermalstress was applied to fish in plastic contain-ers containing 250 ml of tank water per fishthat was aerated and and allowed to cool to

25°C. The tank was heated at a constant rateof 0.15°C per min up to 41°C, and fish wereheld at this temperature until they becamecompletely immobilized. The quantity"minutes at 41°C" for each fish was takenas a measure of thermal resistance that in-cluded acquired thermotolerance. Fish giv-en an acute heating, too rapid to acquirethermotolerance, survived about 25 min at41°C which is a relatively high level of in-trinsic thermotolerance. Gill tissue was re-moved immediately and solubilized in gelsample buffer. The Hsp70 isoforms wereseparated by two-dimensional SDS-PAGE,proteins were visualized by silver staining,and the resulting gels were quantified usinga laser scanning densitometer. The amountsof Hsp70 and Hsc70 were measured as totaloptical density above the local backgroundoptical density for each protein spot. TotalHsp70 was calculated by adding togetherthe optical density values for all inducibleisoforms. The amounts of Hsp70 and Hsc70in each sample were standardized againstthe amount of a nonheat inducible spotthought to be mitochondrial Hsp70. The as-sociation between Hsp70 and minutes ofsurvival at 41°C was assessed using a non-parametric statistical analysis, Spearman'srank correlation. There was a positive andnonlinear correlation between abundance ofHsp70 and survival for the entire data setthat included five different Hsp70 isoformpatterns (Figure 8 in Norris et al., 1995).

The Kruskal-Wallis nonparametric one-way ANOVA was used to test for possibledifferences in survival and Hsp70 amountsexpressed by fish possessing different iso-form patterns (Table 2 in Norris et al.,1995). Using this analysis, no differences insurvival and in total accumulation of Hsp70(P = 0.23) were found among fish with dif-ferent Hsp70 isoform complements. Fishexpressing only one isoform accumulatedas much total Hsp70 as those possessingthree isoforms, implying the existence of amechanism that applies to all of the majorisoforms in regulating hsp70 abundance.There was also no difference in minutes ofsurvival at 41°C as a function of isoformcomplement (P = 0.87) by the Kruskal-Wallis test.

The limitation of the model tested pre-


POECIUOPSIS HEAT SHOCK RESPONSE 883

viously is that compensatory mechanismsof acquired thermotolerance, some depen-dent on Hsp70 isoforms and some not,could result in about the same levels of ac-quired thermotolerance, and all of thesemechanisms may be present in an outbredpopulation, effectively masking a correla-tion with isoform patterns. Therefore we re-visited this data set specifically to test thecorrelations between acquired thermotoler-ance and Hsp70 abundance for fish contain-ing isoform 3, the most frequently encoun-tered Hsp70 isoform, and for those not con-taining this isoform. This grouping had thefurther advantage of retaining reasonablylarge N values for both subsets. The grac-ilis population that we analyzed had fivedifferent inducible Hsp70 isoform patterns.Using only the most abundant isoforms todefine these patterns, they are: a) isoform 3only, b) isoforms 1 and 3, c) isoforms 0, 1,3, d) isoform 1 only, and e) isoforms 0 and1. Subset A contained data from fish withisoform patterns containing isoform 3 (a, band c). Subset B contained data from fishwith isoform patterns (d and e) not contain-ing isoform 3. The same nonparametric sta-tistical analysis used originally on the fulldata was applied to the subsets, i.e., Spear-man's rank order correlation coefficient, rs,was calculated for the variables X = totalamount of inducible Hsp70 accumulated ingill tissue from each fish at the time ofdeath and Y = minutes at 41°C. For thedata set A containing fish with a contribu-tion from isoform 3, the null hypothesis thatthere is no association between the rankpairs was rejected at the P = 0.01 level ofsignificance (N = 21, rs = 0.58, Z = 2.59).The correlation plot is shown in Figure 3A.We conclude that there is a positive asso-ciation between survival and amount ofHsp70 for fish carrying isoform 3. This cor-relation appears to be roughly linear up toabout 5 arbitrary units of total Hsp70, anamount which may be necessary for thesefish to acquire maximum thermotolerance.Fish with more than twice this amount ofHsp70 are not more thermotolerant, andthey appear to have reached an upper limitto thermal resistance. A remarkably similarrelationship between survival of heat stressand level of expression of Hsp70 was re-

ported for clones of Rat-1 cells carrying thehuman Hsp70 gene (Favatier et al., 1997;Li and Nussenzweig, 1996). A positive cor-relation between Hsp70 levels and survivalwas also found for isofemale lines derivedfrom a single population of Drosophila me-lanogaster (Krebs and Feder, 19976).

Data set B contains isoform complementsfound only in the tropics and with no iso-form 3. The null hypothesis cannot be re-jected for data set B (N = 26, rs = 0.28, Z= 1.4 and P = 0.16), indicating little or noassociation between survival and totalamount of Hsp70 for fish that do not carryisoform 3. The correlation plot for this dataset is shown in Figure 3B. These correlationplots are consistent with the interpretationthat isoform complements containing iso-form 3 contribute to acquired thermotoler-ance, whereas those containing only iso-form 1 and both 0 and 1 do not. Can thisoutcome be reconciled with the previous re-sults of the Kruskal-Wallis analysis? Oursuggestion is that fish lacking isoform 3 areas thermoresistant as those that do and thatthey produce as much inducible Hsp70 onaverage in this particular experimental test,as supported by the Kruskal-Wallis analy-sis; however, their mechanism of acquiringthermotolerance does not include a signifi-cant contribution by Hsp70. We suggestthat another Hsp, such as Hsp30, or perhapsan Hsp-independent thermotolerance mech-anism compensates for the lack of theHsp70 isoform contribution to survival at41°C in the fish carrying the 0 and 0, 1isoform patterns, and that there is redun-dancy built into the process of acquiringthermotolerance. This possibility is sup-ported by studies in mammalian systemsthat show a strong contribution by bothHsp70 and Hsp27 to acquired thermotoler-ance (Weber, 1992) and by studies in a va-riety of organisms showing Hsp-indepen-dent mechanisms of acquired thermotoler-ance (Hall, 1983; Boon-Niermeijer et al.,1986; Easton et al, 1987; Laszlo, 1988).The results of this analysis raise the possi-bility that the Hsp70 isoforms may be qual-itatively different in their ability to contrib-ute to thermotolerance. Does the mecha-nism of thermotolerance that includes acontribution by Hsp70 isoform 3 provide an


884 L . E . HlGHTOWER

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05COBcE

10

Hsp70 arbitrary unitsFIG. 3. Panel A: Relationship between Hsp70 abundance and minutes of survival at 41°C for P. graciliscontaining inducible Hsp70 isoform 3. The amounts of the major inducible isoforms 0, 1 and 3 in the Hsp70patterns for each fish were summed as a measure of total Hsp70 accumulation. There was a positive and nonlinearcorrelation between abundance of Hsp70 in fish having Hsp70 patterns a, b and c, shown as insets, and survival(Spearman's rs = 0.58, Z = 2.59, and P = 0.01 for N = 21 fish). Panel B: Relationship between Hsp70 abundanceand minutes of survival at 41°C for P. gracilis not containing inducible Hsp70 isoform 3. The amounts of majorisoforms 0 and 1 in these patterns were summed as above. There was little or no correlation between survivaland abundance of Hsp70 in fish having Hsp70 patterns d and e, shown as insets (Spearman's r, = 0.28, Z =1.4, and P = 0.16 for N = 26 fish). Insets in Panels A and B: Hsp70 polymorphism in P gracilis. Portions ofsilver stained two-dimensional gels labeled a-e show the five different isoform patterns obtained from fish heatstressed at 41°C. Spots marked on the fiuorograms: isoform a = putative mitochondria! Hsp70; b = gill specificmarker protein; c = Hsc70; d = Grp78. Numbers identify major inducible Hsp70 isoforms, as described pre-viously (White et al., 1994). The basic end of the isoelectric focusing gel is on the left.



O

to

30-

20-

100 5 10

Hsp70 arbitrary units

FIG. 3. Continued.

advantage in desert thermal environmentsand does this explain the prevalence of iso-form 3 in desert species? More work isneeded to answer these questions, but ouranalysis indicates that they are well worthpursuing.

Hsp70 isoforms differ in protein stabilityEvidence that the isoforms of Hsp70 in

a desert species differ in at least one mo-

lecular property besides pi has already beenobtained. Cells derived from P. lucida em-bryos (PLE-1 cells) labeled after 4 to atheat shock temperature still synthesizedHsp70 isoforms 3, 5 and 7 (Fig. 4, PanelA, center gel). However, if cells were heatshocked for 5 hr and then given a 10 hrincubation at control temperature (reversal)before labeling, no residual Hsp synthesiswas observed, indicating shut-off of Hsp70


886 L. E. HiGHTOWER

Isoform #

Control 0.6 2.2 1.4HS 31.2 26.8 14.8Recovery 5.1 27.0 15.9



synthesis during the reversal period (Fig.4A, bottom panel). In the absence of syn-thesis of additional Hsp70, it was possibleto assess the stability of individual isoformsusing silver staining. When accumulation ofHsp70 under the same conditions was mea-sured by silver staining, significant accu-mulation of isoforms 3, 5 and 7 was ob-served at the end of a five hour heat shock(Fig. 4B, center panel). After a 10 hr re-covery the amounts of isoforms 5 and 7were essentially unchanged; however, verylittle of isoform 3 remained (Fig. 4B, bot-tom panel). These changes are quantified inFig. 4C.

As ancestral fish adapted to warm, ther-mally stable tropical environments movednorthward and colonized desert streams, asour scenario goes, they would have encoun-tered rapidly fluctuating temperatures, andmuch cooler temperatures than those expe-rienced in the tropics. In the desert environ-ment, frequent exposure to temperaturesthat would induce Hsp70 synthesis wouldbe followed by rapid cooling. Since induc-ible Hsp70 is thought to be deleteriouswhen expressed under non-stress condi-tions(Feder et al., 1992; Krebs and Feder,1997a), adaptation to the desert environ-ment may have involved selection for abun-dant isoforms that turn over rapidly.

ACKNOWLEDGMENTS

First and foremost, we wish to thank R.Jack and Mary E. Schultz for all thingsPoeciliopsis, specifically including years ofwork in establishing and maintaining a ge-netically pedigreed fish colony, initial stud-ies of acquired thermotolerance (RJS) andinduction, characterization and maintenanceof the hepatocellular carcinoma from whichthe PLHC-1 cell line was derived (MES).Thanks go to Craig Stockwell for providingGambusia qffinis, and to Klaus Kallman,

Don Morizot and Richard Borowsky forproviding wild-caught P. gracilis and P.fasciata.

This work was supported by a Marine/Freshwater Biomedical Sciences Centergrant (ES03848) from the NIEHS and bythe University of Connecticut ResearchFoundation. C.E.N. was a Howard HughesMedical Institute Predoctoral Fellow andP.J.D. was supported by an NSF GraduateResearch Traineeship (BIR-9256616).

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Boorstein, W. R., T. Ziegelhoffer, and E. A. Craig.1994. Molecular evolution of the hsp70 multigenefamily. J. Mol. Evol. 38:1-17.

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Heat Shock Responses of Closely Related Species of Tropical and