BACKGROUND
how does genotypic variation affect phenotypic variation?
and how might genes modulate the relationship between genotype and phenotype?
these are central questions in evolutionary biology.
in recent years, it has become clear that some genes play a special role in this relationship.
these genes encode chaperones, proteins that assist other proteins in folding, and that can help refold misfolded proteins  <cit> .
protein misfolding can result from mutations in protein coding regions  <cit> .
it can also result from environmental changes, such as heat stress, which can lead to protein denaturation  <cit> .
because proteins are involved in forming and maintaining every phenotypic trait, misfolded proteins often have detrimental effects on phenotypes  <cit> .
proteins that can mitigate these effects can render organisms more robust against genetic or environmental perturbations.
thus, chaperones are one of several ways in which phenotypes can become robust to genetic and environmental change  <cit> .
robustness to genetic and environmental change are often associated with one another  <cit> , although exceptions may exist  <cit> .
on evolutionary time scales, robustness to genetic change has an important consequence on the genetic constitution of a population: it allows mutations to accumulate that are not phenotypically visible, precisely because phenotypes are robust to such mutations.
the resulting genetic variation is often also called cryptic variation  <cit> .
such variation need not stay cryptic forever, however.
it can become phenotypically visible in the presence of yet other mutations or after environmental change  <cit> .
the resulting phenotypic change can be detrimental, but also beneficial, leading to new evolutionary adaptations  <cit> .
the earliest hints that cryptic variation exists, and that it can be transformed into new phenotypes came from experiments by waddington which induced new phenotypes through environmental change, and which demonstrated that the induced phenotypic changes can have a genetic basis  <cit> .
in the recent past, many further studies have demonstrated the existence of cryptic variation, and its potential phenotypic consequences.
these studies focused on a broad range of phenotypes, from molecular phenotypes to macroscopic traits of multicellular organisms  <cit> .
because chaperones help confer robustness, their activity is also a prominent cause of cryptic variation  <cit> .
an especially important chaperone is the heat shock protein hsp <dig>  which occurs in many organisms that range from microbes to humans  <cit> .
what makes hsp <dig> unique is that its "client" proteins, proteins whose integrity it helps maintain, are extremely diverse, and that they are involved in cell communication and signaling processes  <cit> .
such processes are especially important while a complex organism with many macroscopic traits develops from a single cell.
in other words, hsp <dig> affects development and thus macroscopic phenotypes.
for example, impairing hsp <dig> function through engineered mutations or pharmacological treatment-both in the laboratory-can increase phenotypic diversity in organisms as different as drosophila, arabidopsis, and zebrafish  <cit> .
this phenotypic diversity is a reflection of genotypic diversity, that is, of variation that was cryptic before hsp <dig> function was impaired  <cit> .
with some exceptions  <cit> , most experiments that studied hsp90's effects on fitness and variation buffering focus on laboratory strains  <cit> .
in addition, they use strong gain-of-function or loss-of-function mutations with dramatic, often lethal phenotypic effects  <cit> .
such mutations can usually not be maintained as homozygotes, and would rarely if ever be tolerated in a wild population  <cit> .
even disruption of hsp <dig> expression through rna interference may have large effects on phenotypes  <cit> .
other ways of manipulating hsp <dig> activity include the use of pharmaceuticals known to interact with hsp <dig>  such as geldanamycin  <cit> , which wild populations may also rarely encounter.
in contrast to such strong, artificial manipulations in the laboratory, one might expect to encounter mutations with milder effects in the wild, such as regulatory mutations that affect gene expression.
in the fruit fly drosophila, a specific class of such regulatory mutations are indeed well known to affect the expression of heat shock genes  <cit> .
these mutations are insertions of a transposable element, the p-element, into the regulatory region of heat shock genes.
such mutations have been characterized for a variety of heat shock genes, including hsp <dig>  hsp <dig>  hsp67ba, hsp <dig>  hsp <dig>  hsp <dig>  hsp <dig>  and hsrω  <cit> .
they affect gene expression, thermotolerance  <cit> , development  <cit>  and longevity  <cit> .
the likely reason why p elements often insert into these genes' regulatory regions is their high expression level, which requires a de-condensed and nucleosome-free chromatin conformation near the gene  <cit> .
such an "open" chromatin conformation makes a gene susceptible to insertion by transposable elements  <cit> .
in the fruit fly drosophila, where the role of hsp <dig> in variation buffering was first discovered  <cit> , hsp <dig> is encoded by the single copy essential gene hsp <dig>  this gene is expressed at higher levels during normal development than some other heat shock proteins, including hsp <dig>  hsp <dig>  hsp <dig>  hsp <dig>  and hsp <dig>  <cit> .
hsp <dig> is expressed constitutively, and at higher levels than required for its function under normal conditions  <cit> .
taken together, these observations make hsp <dig> a good candidate for transposable element insertions in wild populations as well.
the work we report below is the first to isolate such naturally occurring variants of hsp <dig> and to characterize their effect on gene expression and fitness.
because hsp <dig> has been associated with variation buffering, the question whether such mutants alter variation buffering is intriguing.
we show that they indeed do.
observations made with laboratory populations often do not apply to wild populations  <cit> .
this holds especially when a population's evolutionary and genomic background matter, because wild populations have a history of inbreeding and selection that is different from laboratory populations  <cit> .
for example, laboratory genetic mapping revealed a strong association between regulatory polymorphisms at the hairy locus and variation in sternopleural bristles.
however, the same association does not exist in wild-caught flies  <cit> .
a comparative study on  <dig> strains of d. melanogaster found that even central life history traits such as longevity can be highly strain-specific  <cit> .
more generally, artificially selected phenotypes can only partly predict fitness components estimated in the field  <cit> .
thus, some observations derived from laboratory studies have dubious validity until confirmed also in the wild.
in this work, we first screened more than  <dig>  flies from  <dig> world-wide drosophila populations, and identified p-element insertions near the hsp <dig> gene in three of these populations.
we verified that these insertions indeed reduce hsp <dig> gene expression.
we then showed that flies with these mutations have lower competitive fitness, as well as lower fecundity and longevity.
these effects occur in all three genetic backgrounds.
we next used inbreeding experiments, which can increase homozygosity of deleterious alleles, to reveal cryptic deleterious mutations.
these experiments showed that flies with reduced hsp <dig> expression from all three populations can buffer deleterious variation much less well.
even mild environmental stress reduced the buffering so dramatically that populations went extinct.
experiments like these would have been impossible with strong laboratory mutations.
the mild mutations we use allowed us to determine that wild-derived flies with even modest expression changes in the gene encoding hsp <dig> show altered fitness and variation buffering that can be very strong on short evolutionary time scales.
RESULTS
hsp <dig> regulatory mutations exist in three out of  <dig> natural populations
in natural populations of d. melanogaster, the promoters of heat-shock genes are especially susceptible to the insertion of transposable elements.
more than  <dig> percent of the transposable elements occurring in heat-shock promoters are p-element insertions.
the promoter of hsp <dig> is no exception  <cit> .
for example, rates of transposable element insertions in single-copy heat shock promoters are estimated to be  <dig>  times higher than the average rate of transposable element insertions in promoter regions of non-heat shock genes  <cit> .
they are up to two times higher than the rate at which other insertion/deletions occur in non-coding regions of the fly genome  <cit> .
therefore, transposon insertion is a prominent candidate source of mutations for hsp <dig>  and makes such insertions an ideal object to study natural variation in hsp <dig>  to discover naturally occurring mutations in the hsp <dig> promoter, we screened  <dig> strains derived from natural populations of d. melanogaster collected around the world .
we aimed at discovering insertion/deletion mutations through electrophoretically detected length polymorphisms in pcr products amplified from the hsp <dig> gene and its flanking regions.
the pcr method we employed for hsp <dig> gene-specific small fragment amplification  is proven to be highly sensitive in detecting any insertion/deletion mutation.
after having screened about  <dig> flies from all  <dig> populations, we did not find any insertion polymorphism in the hsp <dig> coding region.
however, we did find insertion polymorphisms in the promoter region.
they occurred in three separate populations, one from okayama, japan, a second population from tokyo, japan, and a third population from the ivory coast, africa .
sequencing of the mutated gene region showed that the mutations were caused by insertion of non-autonomous p elements, i.e., the elements contain an internal deletion, and therefore cannot encode the functional transposase needed to mobilize the elements in genome.
the p element insertions are identical in the three populations with respect to their insertion position, orientation and length as well as their flanking sequence .
the insertion is  <dig> bp long and occurred  <dig> bp upstream of the transcription starting site of hsp <dig>  the inserted p-element disrupts the characterized regulatory sequences of hsp <dig>  <cit> .
specifically, it integrated into an array of three heat-shock consensus elements that regulate hsp <dig> expression  <cit> .
its integration moved one of these elements  further away from the transcription start site, while at the same time creating an 8-bp  direct repeat of the same element .
for further analysis, we established isogenic lines homozygous for the insertion mutation with individuals from each of the three populations where they occurred .
we named their genotypes hsp83p/p.
we also established isogenic lines homozygous for the wild-type hsp <dig> gene from the three populations, and named them hsp83+/+.
we note that the two japanese populations and one african population had been collected in different years and maintained in different laboratories or stock centers , thus making cross-contamination unlikely.
although the genotypes at the insertion site are identical, and may be derived from the same insertion event, the lines have different genetic backgrounds that result from the different geographic origin of their source populations.
p-element insertion mutations reduce hsp <dig> gene expression at both normal and elevated temperatures
because the p-element integration disrupted this regulatory region, one would expect that this mutation affects hsp <dig> gene expression.
to validate this expectation, we performed real-time quantitative pcr to measure hsp <dig> mrna expression levels in third-instar larvae.
we found that at normal temperatures  hsp <dig> mrna levels in mutants were reduced to  <dig>  percent of the wild-type in the okayama lines, to  <dig>  percent of the wild-type in the tokyo lines, and to  <dig>  percent of the wild-type in the ivory coast lines .
this reduction also extended to high temperatures.
specifically, flies cultured at 28°c for one generation decreased hsp <dig> expression to  <dig>  percent of the wild-type in the okayama lines, to  <dig>  percent of the wild-type in the tokyo lines, and to  <dig>  percent of the wild-type in the ivory coast lines.
cultivation at the higher temperature  generally decreased hsp <dig> expression in both mutants and wild-type flies from the three populations .
these analyses were based on chromosomal alleles of hsp <dig>  it is possible that the observed changes in hsp <dig> expression are not caused by these alleles, but by linked alleles at other loci that are in strong linkage disequilibrium with the hsp <dig> alleles we study.
to exclude this possibility, we isolated the wild-type and mutant hsp <dig> promoter from the okayama population and cloned them upstream of a luciferase gene.
we then used the two resulting constructs to drive luciferase gene expression in transient transfection assays of two different drosophila cell lines   <cit>  .
in both cell lines, and both under normal temperatures and after heat shock at 37°c, the mutant promoter drove expression of the luciferase gene to a low level compared with the wild-type promoter.
specifically, the mutant promoter lowered luciferase gene expression between  <dig>  percent  and  <dig>  percent .
taken together, these observations show that the p-element insertion mutants we study cause a reduced expression of the hsp <dig> gene in three different genetic backgrounds.
this reduction is caused by reduced activity of the mutant hsp <dig> promoter, and not by indirect effects of alleles at loci linked to hsp <dig>
hsp <dig> mutant alleles have low frequencies in natural populations
to estimate the frequencies of the two hsp <dig> alleles  in the three source populations , we genotyped from  <dig> to over  <dig> flies from these populations .
we found no mutant homozygotes in two of the populations, and only  <dig>  percent of mutant homozygotes  in flies from the okayama population.
among the flies we examined  <dig>  percent,  <dig>  percent and  <dig>  percent were heterozygous for the p-element insertion in the okayama, tokyo and ivory coast populations, respectively.
expressed in terms of mutant allele frequencies in the sample of flies we examined, this means that the mutant hsp <dig> allele had a low frequency of  <dig>  percent in okayama flies,  <dig>  percent in tokyo flies, and  <dig>  percent in ivory coast flies.
only the genotype frequencies in the okayama population significantly departed from hardy-weinberg equilibrium .
we also attempted to identify nucleotide polymorphisms, but found only one polymorphism in a total of  <dig> bp sequenced nucleotides, both upstream and downstream of the insertion site, in  <dig> flies of the okayama population.
these observations are consistent with previous work indicating that hsp <dig> coding and regulatory regions show very little sequence polymorphisms  <cit> .
the hsp <dig> gene is located close to the breakpoint of the cosmopolitan inversion invp  <cit> .
we wished to determine if any phenotypic effects of the hsp <dig> mutation may have been indirect, caused by the inversion and its association  with the hsp <dig> mutation.
to this end, we scored the inversion invp polymorphisms in the two natural populations okayama and ivory coast.
we found no evidence of linkage disequilibrium with the hsp <dig> alleles , and only rare inversion arrangements in flies with either hsp <dig> genotype .
thus, the phenotypic effects of hsp <dig> mutations cannot be attributed to effects of the cosmopolitan inversion invp.
hsp <dig> transposon insertion mutants show lower fitness in competition assays
the low population frequency of heterozygotes for the hsp <dig> mutant allele, and in particular the near absence of mutant homozygotes in flies derived from wild populations, suggest that carriers of the mutant allele have lower fitness.
to validate this observation we let wild-type and homozygous mutant flies compete with each other in a co-culture competition assay at 25°c.
we performed two variants of this assay.
in the first, we seeded populations with  <dig> virgin wild-type flies, and another  <dig> homozygous virgin mutant flies.
thus, in this assay, the initial frequency of the mutant allele was  <dig> percent.
we propagated individuals from these populations for five generations .
in every single assay population, the mutant  allele frequency decreased over the course of the experiment .
specifically, the hsp83p allele arrived at frequencies of  <dig>   <dig> and  <dig>  percent after  <dig> generations in flies from the okayama, tokyo and the ivory coast populations, respectively.
the three populations showed significant differences in the mutant allele frequency in co-culture , indicating that genetic background also has an influence on competition outcome.
the second variant of the assay that we performed involved only individuals from the okayama population.
it started with different proportions of mutant individuals.
specifically, it involved two starting populations, each consisting of  <dig> flies, where  <dig> and  <dig> flies, respectively, were initially homozygous for the hsp83p allele.
in the first population hsp83p allele frequencies decreased by  <dig>  percent.
in the second population they fluctuated around the initial  <dig> percent.
both populations had a low hsp83p allele frequency of  <dig>  percent at the end of this experiment.
in sum, our observations suggest that in populations where mutant flies constitute a substantial fraction of the population, they are competitively inferior to wild-type flies, and their population frequency declines over several generations.
the number of individuals we used, as well as the consistent genotype frequency changes we observe in our replicate competition assays make genetic drift an unlikely sole cause of the changes we see.
mutant flies are less fecund and live less long, but are no less thermotolerant than wild-type flies
we next asked which component of fitness may be responsible for the reduced fitness of hsp <dig> mutants.
we investigated three fitness components.
the first is fecundity.
the second is longevity.
the third is resistance to environmental high-temperature stress, which we studied because hsp <dig> is known to be involved in thermotolerance.
we first estimated female fecundity by counting all eclosing adults produced by females in isofemale lines obtained from the okayama population .
on average, wild-type females had significantly higher fecundity than mutant females .
they produced three times more offspring than mutants, as shown in figure 4a.
the figure also indicates that different isofemale lines show significant differences in fecundity.
a statistical test confirms this, and also demonstrates the non-negligible effect of genetic background on fecundity .
we next determined the longevity of mutant and wild-type genotypes.
to this end, we cultured hsp83p/p and hsp83+/+ lines in fresh medium at 25°c for the whole life of individuals in these lines , and counted the fraction of individuals that survived a given number of days after eclosion .
the mutant females had an average lifespan of  <dig>  days,  <dig>  percent shorter than the average life span of wild-type females at  <dig>  days, a difference that was highly significant .
the mutant males showed a stronger reduction in average lifespan, with  <dig>  days compared to  <dig>  days in the wild-type.
this difference of  <dig>  percent is significant .
levene's test of error variances shows a significant difference in the variance of life span between wild-type and mutant lines in females , but not in males .
this indicates that the hsp <dig> mutation influences not only life span, but also its variability, at least for females.
in sum, flies, especially males, are ageing faster when hsp <dig> is mutated.
lastly, we measured the basal thermal tolerance and inducible thermal tolerance of both the mutant  and wild-type  isofemale lines.
we did not find any significant difference in basal or induced thermotolerance between the genotypes for either males or females.
.
inbreeding shows that wild-type hsp <dig> alleles buffer phenotypic expression of cryptic genetic variation better than mutant alleles
inbreeding causes increased homozygosity at multiple genomic loci, and will thus expose phenotypic effects of recessive alleles to natural selection  <cit> .
such effects are usually deleterious.
they are not apparent in outbreeding populations, where recessive alleles mostly exist in heterozygotes.
in other words, outbreeding populations contain cryptic genetic variation-variation without phenotypic effects-at loci harboring recessive alleles.
in inbreeding populations, this variation becomes phenotypically visible through its deleterious effects.
one of the best-studied aspects of hsp <dig> function in laboratory populations is the ability of hsp <dig> to buffer the phenotypic effects of genetic variation  <cit> .
with these observations in mind, we asked whether the natural hsp <dig> mutants we study here are impaired in their ability to buffer cryptic variation that can be revealed by inbreeding.
such an observation would provide evidence that hsp90's buffering ability is not just restricted to laboratory populations.
to answer this question, we established  <dig> parallel inbred fly populations.
of these populations,  <dig> were homogenous for hsp <dig> wild-type , and  <dig> for hsp <dig> mutant  genotypes, respectively.
more precisely, for each of the okayama, tokyo, ivory coast source populations, we established seven inbred populations with each of the two genotypes and propagated them at 25°c .
in each generation, we sampled  <dig> males and  <dig> females from each inbred population to create subsequent generations, and continued this process for  <dig> generations.
as a measure of inbreeding depression-the deleterious effects of inbreeding on fitness-we estimated female fecundity by mating  <dig> virgin females and  <dig> males from each generation, and counted all offspring they produced within  <dig> days .
we now discuss results from each of the three lines in turn.
first, in the inbred line from the okayama population, wild-type females did not become significantly less fecund until generation  <dig> .
in contrast, in the mutant line even one generation of inbreeding already caused a significant decrease in fecundity , and by generation four fecundity had been reduced by  <dig>  percent in the mutant.
second, in the inbred line from the tokyo population , females did not become significantly less fecund in the wild-type after four generations , indicating that the wild-type is well-buffered against short-term inbreeding.
in contrast, females in the mutant population had become significantly less fecund after four generations .
the ivory coast lines, finally, showed much lower initial fecundity than the other two inbred lines.
wild-type females showed no significant decrease in fecundity after four generations of inbreeding , whereas mutants became significantly less fecund .
in the four generations of inbreeding, the three populations showed significantly different fecundity within both the wild-type  and mutant genotypes .
this indicates that effects of genetic background are important for our analysis and cannot be neglected.
in sum, our analyses of inbred lines show that individuals with wild-type hsp <dig> alleles buffer cryptic deleterious variation more effectively than mutant individuals.
hsp <dig> thus helps buffer cryptic deleterious variation in flies derived from wild populations.
hsp <dig> is even more important for cryptic variation buffering under mild heat stress
the preceding experiments showed that mutant hsp <dig> alleles buffer cryptic deleterious variation to a lesser extent than wild-type alleles.
we next asked whether high temperatures further exacerbate this difference.
the rationale for this question is that hsp <dig> is involved in the heat stress response.
when exposed to heat stress, free hsp <dig> will be diverted from its interactions with signaling proteins to help renature excess misfolded and aggregated proteins  <cit> .
as a result, less hsp <dig> protein may be available for signaling and buffering.
to answer this question, we performed mild inbreeding at  <dig> °c with the same methods for crossing of flies within inbred lines as used in the preceding experiments.
the starting isofemale lines were also the same, except that we began to rear flies at  <dig> °c when they eclosed .
in these experiments, wild-type flies from the okayama and tokyo populations retained high fecundity over three generations of inbreeding, while wild-type flies from the ivory coast populations significantly  declined in fecundity after the first generation.
all three populations persisted over three generations of inbreeding.
in stark contrast to this persistence, lines with mutant hsp <dig> alleles from the three populations went extinct after the first generation, because their fecundity had been reduced to almost zero .
this observation underscores that hsp <dig> is important to buffer deleterious variation not only at normal temperatures, but even more so under thermal stress.
outbreeding of a previously inbred population reduces homozygosity of deleterious recessive alleles.
if our assertion that wild-type hsp <dig> buffers deleterious variation is correct, then outbreeding should restore the fecundity lost during inbreeding, and especially in mutant lines where this loss is most severe .
to ask whether this is the case, we used flies from the ivory coast population, where the fecundity loss had been especially severe at 28°c .
we crossed flies derived from the different inbred isofemale lines with each other and examined their fecundity.
in this way, we established populations that were outbred and contained either only hsp <dig> mutant alleles, only wild-type alleles , or a 1: <dig> mix of mutant and wild-type alleles .
the outbred populations show significant increases in fertility relative to their inbreeding controls .
specifically, outbreeding of populations that contained only mutant alleles  increased fecundity from zero  to  <dig> individuals per female .
this change in fecundity was highly significant , indicating that outbreeding rescued the fecundity loss during inbreeding at mild heat stress.
wild-type alleles of hsp <dig> helped increase fecundity even further.
specifically, the fecundity of outbred populations with both allele types  was  <dig> percent higher than for populations with only the mutant alleles .
outbreeding in populations with only wild-type alleles  increased fecundity by an additional  <dig> percent, to a level that was significantly higher than in the mixed cross  .
in sum, wild-type hsp <dig> alleles are much more effective in restoring the fecundity that is lost after inbreeding.
discussion
we discovered naturally occurring mutants of the hsp <dig> gene with p element insertions in the gene's proximal promoter region.
these mutants occur in three out of  <dig> populations that we examined, and at modest allele frequencies, not exceeding  <dig>  percent.
the mutations down-regulate hsp <dig> gene expression by  <dig> percent to  <dig> percent, depending on the population.
they reduce competitive fitness, female fecundity, and longevity.
we also found that hsp <dig>  strongly influences the expression of cryptic deleterious genetic variation in inbred populations.
that is, flies that carry the mutant hsp <dig> allele are much more poorly buffered against such variation than flies with the wild-type allele.
we found that even mild thermal stress can completely break down the impaired buffering associated with mutant hsp <dig>  specifically, inbred fly populations with mutant hsp <dig> alleles go extinct at modestly elevated temperatures of 28°c.
to our knowledge, this work is the first to show that naturally occurring regulatory mutations of the hsp <dig> gene-a key modulator of the stress response and cellular signaling-can affect reproductive success, reduce longevity, and reduce variation buffering in fruit flies derived from wild populations.
a previous study on naturally occurring hsp <dig> variants had identified a nonsynonymous deletion mutation in the hsp <dig> coding region, and focused on the effects of this mutation on morphological traits, not on fitness components  <cit> .
in addition, this study perturbed variation buffering only through thermal stress exposure, not through inbreeding, which is especially useful to reveal expression of recessive cryptic genetic variation.
our limited sequence polymorphism analysis found only one synonymous mutation in the hsp <dig> coding region in the flies we studied, thus making it unlikely that the same deletion mutation stands behind our observations.
our naturally occurring variants of hsp <dig> have fairly mild effects compared to some of the drastic perturbations that earlier laboratory experiments used  <cit> .
for example, the reduced expression of hsp <dig> in our mutants did not significantly affect thermotolerance in outbred flies .
working with these mutants has several benefits.
first, it avoids pharmacological manipulation or structural modification of hsp <dig>  both of which may have unknown side effects, for example, by changing the protein's molecular interaction partners  <cit> .
second, it avoids the use of mutations with drastic expression effects, such as engineered homozygous loss-of-function mutants  <cit> .
such mutants can only be maintained as heterozygotes in the laboratory  <cit> .
a continuous inbreeding experiment like the one we performed to reveal deleterious cryptic variation would be difficult with such drastic mutations, partly because homozygote offspring would be lethal.
in contrast, the mild variants we found enabled us to ask whether more hsp <dig> means better buffering  <cit> .
finally, they allowed us to study flies derived from natural populations, and to examine whether laboratory findings on hsp <dig> apply to such populations.
a potential disadvantage of working with natural populations and the mutants that they contain is that each mutant occurs in a different genetic background, and that this background can influence observations.
for example, we found that the genetic background of each geographic population influenced the competitive ability and fecundity of the flies we studied.
however, our key observations, for example that hsp <dig> can buffer deleterious variation in inbreeding lines, and that hsp <dig> mutants show reduced fitness, were consistent across all three genetic backgrounds.
p element insertions are widespread in the drosophila genome, and they are especially abundant in heat-shock genes  <cit> .
in japanese natural populations, for example, at least  <dig> distinct p element insertions exist in the heat shock gene hsp <dig>  and at least  <dig> distinct insertions exist in the heat-shock gene hsp <dig> at high population frequency  <cit> .
these observations stand in stark contrast to the low frequency of insertions into the hsp <dig> gene.
they suggest that the low frequency of p-element insertions we observe in hsp <dig> are not a consequence of a recent insertion, but that it results from natural selection against mutant hsp <dig>  our observation that these insertions are possibly associated with reduced competitive fitness, reduced fecundity, and shorter life span are fully consistent with this suggestion.
the question then arises why we observe any individuals that carry p-element insertions in our study populations.
one possible answer is that the insertion may be neutral or even beneficial when heterozygous or when at low population frequencies.
our observation that mutant allele frequencies did not decline in a competition assays seeded with only  <dig> percent of hsp83p alleles are consistent with this possibility.
moreover, the fitness effect of hsp83p may depend on the environment to which a population is adapted, as has been observed for p element insertions in other heat shock genes  <cit> .
in addition, some p-element insertions can help maintain tradeoffs between stress resistance and developmental homeostasis of flies living in a changing environment  <cit> .
unfortunately, our data does not allow us to answer which of these possibilities is responsible for the maintenance of p element insertions at modest frequencies in our study populations.
our observation that mutations in hsp <dig> affect fecundity is consistent with other reports that hsp <dig> is involved in reproductive functions.
hsp <dig> plays a critical role in the process of both oogenesis  <cit>  and spermatogenesis  <cit> .
for example, hsp <dig> rna is a component of the posterior polar plasm  <cit> , and hsp <dig> protein is required for localization of maternal mrna to the posterior pole, which is essential for development of germ cells in the drosophila embryo  <cit> .
thus, hsp <dig> is involved in the molecular pathways responsible for oogenesis and spermatogenesis in d. melanogaster.
inbreeding increases the fraction of homozygous loci in a genome.
because many alleles are recessive and deleterious in the homozygous state  <cit> , inbreeding will cause previously cryptic  deleterious variation to be expressed  <cit> .
such deleterious variation manifests itself as a reduction in one or more fitness components, such as fecundity.
in our inbreeding experiments, we found that wild-type hsp <dig> flies were better buffered against the deleterious effects of inbreeding than mutant flies.
specifically, wild-type hsp <dig> flies from all three geographic populations showed no decline in fecundity after three generations of inbreeding, and only one population showed a small decline  after four generations.
in stark contrast, mutant lines from all three geographic populations showed a significant decline in fecundity of up to  <dig> percent after between two and four generations.
genetic polymorphisms are widespread in many populations  <cit> , but the incidence of cryptic variation is usually unknown for wild populations  <cit> .
inbreeding can reveal such variation.
our experiments demonstrate that cryptic variation must be abundant in the populations we studied, because inbreeding reduced fecundity substantially.
moreover, the experiments show that expression of hsp <dig> at wild type levels can buffer the damage caused by inbreeding.
the observation that a chaperone can buffer deleterious variation is not unprecedented.
over-expression of groel, a molecular chaperone of escherichia coli, can help overcome the accumulation of deleterious mutations that occur in e. coli strains with high mutation rate  <cit> .
in other words, a chaperone can buffer these organisms against deleterious mutations  <cit> .
although the sources of deleterious variation-inbreeding and mutation accumulation-and the chaperones-hsp <dig> and groel-differ in these two organisms, chaperones have the same qualitative effect in both cases.
however, the e. coli strain in which these previous experiments were conducted is a laboratory strain.
observations made with this strain need not necessarily transfer to populations in the wild.
three main observations lead us to think that the buffering capacity of hsp <dig> is relevant for wild fly populations.
first, the flies in which we identified the effects of the hsp <dig> mutation all stem from wild populations.
second, although the buffering capacity of hsp <dig> varies with genetic background, significant buffering did occur in all three populations.
third, the regulatory mutation in hsp <dig>  is mild in its effect on expression, but strong in its effects on fitness, i.e., on fecundity, longevity, and most importantly, buffering capacity.
mutations that affect these aspects of fitness are likely to be selected against in nature  <cit> .
more specifically, because drosophila has very large effective population size in the wild  <cit> , fitness differences much smaller than we detected would be visible to natural selection  <cit> , and would lead to the eventual elimination of mutant alleles with low fitness.
CONCLUSIONS
in sum, our study shows that natural variants in the hsp <dig> gene can affect the fitness of flies derived from natural populations, regardless of their genetic background and geographic origin.
it also shows that hsp <dig> is involved in the buffering of deleterious variation in these flies, and that this role becomes even more important under heat stress.
and it demonstrates that hsp <dig> plays an important role in the life of flies in the wild.

 how does genotypic variation affect phenotypic variation? and how might genes modulate the relationship between genotype and phenotype? these are central questions in evolutionary biology. in recent years, it has become clear that some genes play a special role in this relationship. these genes encode chaperones, proteins that assist other proteins in folding, and that can help refold misfolded proteins. protein misfolding can result from mutations in protein coding regions. it can also result from environmental changes, such as heat stress, which can lead to protein denaturation. because proteins are involved in forming and maintaining every phenotypic trait, misfolded proteins often have detrimental effects on phenotypes. proteins that can mitigate these effects can render organisms more robust against genetic or environmental perturbations. thus, chaperones are one of several ways in which phenotypes can become robust to genetic and environmental change. robustness to genetic and environmental change are often associated with one another, although exceptions may exist. on evolutionary time scales, robustness to genetic change has an important consequence on the genetic constitution of a population: it allows mutations to accumulate that are not phenotypically visible, precisely because phenotypes are robust to such mutations. the resulting genetic variation is often also called cryptic variation. such variation need not stay cryptic forever, however. it can become phenotypically visible in the presence of yet other mutations or after environmental change. the resulting phenotypic change can be detrimental, but also beneficial, leading to new evolutionary adaptations. the earliest hints that cryptic variation exists, and that it can be transformed into new phenotypes came from experiments by waddington which induced new phenotypes through environmental change, and which demonstrated that the induced phenotypic changes can have a genetic basis. in the recent past, many further studies have demonstrated the existence of cryptic variation, and its potential phenotypic consequences. these studies focused on a broad range of phenotypes, from molecular phenotypes to macroscopic traits of multicellular organisms. because chaperones help confer robustness, their activity is also a prominent cause of cryptic variation. an especially important chaperone is the heat shock protein hsp which occurs in many organisms that range from microbes to humans. what makes hsp unique is that its "client" proteins, proteins whose integrity it helps maintain, are extremely diverse, and that they are involved in cell communication and signaling processes. such processes are especially important while a complex organism with many macroscopic traits develops from a single cell. in other words, hsp affects development and thus macroscopic phenotypes. for example, impairing hsp function through engineered mutations or pharmacological treatment-both in the laboratory-can increase phenotypic diversity in organisms as different as drosophila, arabidopsis, and zebrafish. this phenotypic diversity is a reflection of genotypic diversity, that is, of variation that was cryptic before hsp function was impaired. with some exceptions, most experiments that studied hsp90's effects on fitness and variation buffering focus on laboratory strains. in addition, they use strong gain-of-function or loss-of-function mutations with dramatic, often lethal phenotypic effects. such mutations can usually not be maintained as homozygotes, and would rarely if ever be tolerated in a wild population. even disruption of hsp expression through rna interference may have large effects on phenotypes. other ways of manipulating hsp activity include the use of pharmaceuticals known to interact with hsp such as geldanamycin, which wild populations may also rarely encounter. in contrast to such strong, artificial manipulations in the laboratory, one might expect to encounter mutations with milder effects in the wild, such as regulatory mutations that affect gene expression. in the fruit fly drosophila, a specific class of such regulatory mutations are indeed well known to affect the expression of heat shock genes. these mutations are insertions of a transposable element, the p-element, into the regulatory region of heat shock genes. such mutations have been characterized for a variety of heat shock genes, including hsp hsp hsp67ba, hsp hsp hsp hsp and hsrω. they affect gene expression, thermotolerance, development and longevity. the likely reason why p elements often insert into these genes' regulatory regions is their high expression level, which requires a de-condensed and nucleosome-free chromatin conformation near the gene. such an "open" chromatin conformation makes a gene susceptible to insertion by transposable elements. in the fruit fly drosophila, where the role of hsp in variation buffering was first discovered, hsp is encoded by the single copy essential gene hsp this gene is expressed at higher levels during normal development than some other heat shock proteins, including hsp hsp hsp hsp and hsp. hsp is expressed constitutively, and at higher levels than required for its function under normal conditions. taken together, these observations make hsp a good candidate for transposable element insertions in wild populations as well. the work we report below is the first to isolate such naturally occurring variants of hsp and to characterize their effect on gene expression and fitness. because hsp has been associated with variation buffering, the question whether such mutants alter variation buffering is intriguing. we show that they indeed do. observations made with laboratory populations often do not apply to wild populations. this holds especially when a population's evolutionary and genomic background matter, because wild populations have a history of inbreeding and selection that is different from laboratory populations. for example, laboratory genetic mapping revealed a strong association between regulatory polymorphisms at the hairy locus and variation in sternopleural bristles. however, the same association does not exist in wild-caught flies. a comparative study on strains of d. melanogaster found that even central life history traits such as longevity can be highly strain-specific. more generally, artificially selected phenotypes can only partly predict fitness components estimated in the field. thus, some observations derived from laboratory studies have dubious validity until confirmed also in the wild. in this work, we first screened more than flies from world-wide drosophila populations, and identified p-element insertions near the hsp gene in three of these populations. we verified that these insertions indeed reduce hsp gene expression. we then showed that flies with these mutations have lower competitive fitness, as well as lower fecundity and longevity. these effects occur in all three genetic backgrounds. we next used inbreeding experiments, which can increase homozygosity of deleterious alleles, to reveal cryptic deleterious mutations. these experiments showed that flies with reduced hsp expression from all three populations can buffer deleterious variation much less well. even mild environmental stress reduced the buffering so dramatically that populations went extinct. experiments like these would have been impossible with strong laboratory mutations. the mild mutations we use allowed us to determine that wild-derived flies with even modest expression changes in the gene encoding hsp show altered fitness and variation buffering that can be very strong on short evolutionary time scales.  hsp regulatory mutations exist in three out of natural populations in natural populations of d. melanogaster, the promoters of heat-shock genes are especially susceptible to the insertion of transposable elements. more than percent of the transposable elements occurring in heat-shock promoters are p-element insertions. the promoter of hsp is no exception. for example, rates of transposable element insertions in single-copy heat shock promoters are estimated to be times higher than the average rate of transposable element insertions in promoter regions of non-heat shock genes. they are up to two times higher than the rate at which other insertion/deletions occur in non-coding regions of the fly genome. therefore, transposon insertion is a prominent candidate source of mutations for hsp and makes such insertions an ideal object to study natural variation in hsp to discover naturally occurring mutations in the hsp promoter, we screened strains derived from natural populations of d. melanogaster collected around the world. we aimed at discovering insertion/deletion mutations through electrophoretically detected length polymorphisms in pcr products amplified from the hsp gene and its flanking regions. the pcr method we employed for hsp gene-specific small fragment amplification is proven to be highly sensitive in detecting any insertion/deletion mutation. after having screened about flies from all populations, we did not find any insertion polymorphism in the hsp coding region. however, we did find insertion polymorphisms in the promoter region. they occurred in three separate populations, one from okayama, japan, a second population from tokyo, japan, and a third population from the ivory coast, africa. sequencing of the mutated gene region showed that the mutations were caused by insertion of non-autonomous p elements, i.e., the elements contain an internal deletion, and therefore cannot encode the functional transposase needed to mobilize the elements in genome. the p element insertions are identical in the three populations with respect to their insertion position, orientation and length as well as their flanking sequence. the insertion is bp long and occurred bp upstream of the transcription starting site of hsp the inserted p-element disrupts the characterized regulatory sequences of hsp. specifically, it integrated into an array of three heat-shock consensus elements that regulate hsp expression. its integration moved one of these elements further away from the transcription start site, while at the same time creating an 8-bp direct repeat of the same element. for further analysis, we established isogenic lines homozygous for the insertion mutation with individuals from each of the three populations where they occurred. we named their genotypes hsp83p/p. we also established isogenic lines homozygous for the wild-type hsp gene from the three populations, and named them hsp83+/+. we note that the two japanese populations and one african population had been collected in different years and maintained in different laboratories or stock centers, thus making cross-contamination unlikely. although the genotypes at the insertion site are identical, and may be derived from the same insertion event, the lines have different genetic backgrounds that result from the different geographic origin of their source populations. p-element insertion mutations reduce hsp gene expression at both normal and elevated temperatures because the p-element integration disrupted this regulatory region, one would expect that this mutation affects hsp gene expression. to validate this expectation, we performed real-time quantitative pcr to measure hsp mrna expression levels in third-instar larvae. we found that at normal temperatures hsp mrna levels in mutants were reduced to percent of the wild-type in the okayama lines, to percent of the wild-type in the tokyo lines, and to percent of the wild-type in the ivory coast lines. this reduction also extended to high temperatures. specifically, flies cultured at 28°c for one generation decreased hsp expression to percent of the wild-type in the okayama lines, to percent of the wild-type in the tokyo lines, and to percent of the wild-type in the ivory coast lines. cultivation at the higher temperature generally decreased hsp expression in both mutants and wild-type flies from the three populations. these analyses were based on chromosomal alleles of hsp it is possible that the observed changes in hsp expression are not caused by these alleles, but by linked alleles at other loci that are in strong linkage disequilibrium with the hsp alleles we study. to exclude this possibility, we isolated the wild-type and mutant hsp promoter from the okayama population and cloned them upstream of a luciferase gene. we then used the two resulting constructs to drive luciferase gene expression in transient transfection assays of two different drosophila cell lines. in both cell lines, and both under normal temperatures and after heat shock at 37°c, the mutant promoter drove expression of the luciferase gene to a low level compared with the wild-type promoter. specifically, the mutant promoter lowered luciferase gene expression between percent and percent. taken together, these observations show that the p-element insertion mutants we study cause a reduced expression of the hsp gene in three different genetic backgrounds. this reduction is caused by reduced activity of the mutant hsp promoter, and not by indirect effects of alleles at loci linked to hsp hsp mutant alleles have low frequencies in natural populations to estimate the frequencies of the two hsp alleles in the three source populations, we genotyped from to over flies from these populations. we found no mutant homozygotes in two of the populations, and only percent of mutant homozygotes in flies from the okayama population. among the flies we examined percent, percent and percent were heterozygous for the p-element insertion in the okayama, tokyo and ivory coast populations, respectively. expressed in terms of mutant allele frequencies in the sample of flies we examined, this means that the mutant hsp allele had a low frequency of percent in okayama flies, percent in tokyo flies, and percent in ivory coast flies. only the genotype frequencies in the okayama population significantly departed from hardy-weinberg equilibrium. we also attempted to identify nucleotide polymorphisms, but found only one polymorphism in a total of bp sequenced nucleotides, both upstream and downstream of the insertion site, in flies of the okayama population. these observations are consistent with previous work indicating that hsp coding and regulatory regions show very little sequence polymorphisms. the hsp gene is located close to the breakpoint of the cosmopolitan inversion invp. we wished to determine if any phenotypic effects of the hsp mutation may have been indirect, caused by the inversion and its association with the hsp mutation. to this end, we scored the inversion invp polymorphisms in the two natural populations okayama and ivory coast. we found no evidence of linkage disequilibrium with the hsp alleles, and only rare inversion arrangements in flies with either hsp genotype. thus, the phenotypic effects of hsp mutations cannot be attributed to effects of the cosmopolitan inversion invp. hsp transposon insertion mutants show lower fitness in competition assays the low population frequency of heterozygotes for the hsp mutant allele, and in particular the near absence of mutant homozygotes in flies derived from wild populations, suggest that carriers of the mutant allele have lower fitness. to validate this observation we let wild-type and homozygous mutant flies compete with each other in a co-culture competition assay at 25°c. we performed two variants of this assay. in the first, we seeded populations with virgin wild-type flies, and another homozygous virgin mutant flies. thus, in this assay, the initial frequency of the mutant allele was percent. we propagated individuals from these populations for five generations. in every single assay population, the mutant allele frequency decreased over the course of the experiment. specifically, the hsp83p allele arrived at frequencies of and percent after generations in flies from the okayama, tokyo and the ivory coast populations, respectively. the three populations showed significant differences in the mutant allele frequency in co-culture, indicating that genetic background also has an influence on competition outcome. the second variant of the assay that we performed involved only individuals from the okayama population. it started with different proportions of mutant individuals. specifically, it involved two starting populations, each consisting of flies, where and flies, respectively, were initially homozygous for the hsp83p allele. in the first population hsp83p allele frequencies decreased by percent. in the second population they fluctuated around the initial percent. both populations had a low hsp83p allele frequency of percent at the end of this experiment. in sum, our observations suggest that in populations where mutant flies constitute a substantial fraction of the population, they are competitively inferior to wild-type flies, and their population frequency declines over several generations. the number of individuals we used, as well as the consistent genotype frequency changes we observe in our replicate competition assays make genetic drift an unlikely sole cause of the changes we see. mutant flies are less fecund and live less long, but are no less thermotolerant than wild-type flies we next asked which component of fitness may be responsible for the reduced fitness of hsp mutants. we investigated three fitness components. the first is fecundity. the second is longevity. the third is resistance to environmental high-temperature stress, which we studied because hsp is known to be involved in thermotolerance. we first estimated female fecundity by counting all eclosing adults produced by females in isofemale lines obtained from the okayama population. on average, wild-type females had significantly higher fecundity than mutant females. they produced three times more offspring than mutants, as shown in figure 4a. the figure also indicates that different isofemale lines show significant differences in fecundity. a statistical test confirms this, and also demonstrates the non-negligible effect of genetic background on fecundity. we next determined the longevity of mutant and wild-type genotypes. to this end, we cultured hsp83p/p and hsp83+/+ lines in fresh medium at 25°c for the whole life of individuals in these lines, and counted the fraction of individuals that survived a given number of days after eclosion. the mutant females had an average lifespan of days, percent shorter than the average life span of wild-type females at days, a difference that was highly significant. the mutant males showed a stronger reduction in average lifespan, with days compared to days in the wild-type. this difference of percent is significant. levene's test of error variances shows a significant difference in the variance of life span between wild-type and mutant lines in females, but not in males. this indicates that the hsp mutation influences not only life span, but also its variability, at least for females. in sum, flies, especially males, are ageing faster when hsp is mutated. lastly, we measured the basal thermal tolerance and inducible thermal tolerance of both the mutant and wild-type isofemale lines. we did not find any significant difference in basal or induced thermotolerance between the genotypes for either males or females.. inbreeding shows that wild-type hsp alleles buffer phenotypic expression of cryptic genetic variation better than mutant alleles inbreeding causes increased homozygosity at multiple genomic loci, and will thus expose phenotypic effects of recessive alleles to natural selection. such effects are usually deleterious. they are not apparent in outbreeding populations, where recessive alleles mostly exist in heterozygotes. in other words, outbreeding populations contain cryptic genetic variation-variation without phenotypic effects-at loci harboring recessive alleles. in inbreeding populations, this variation becomes phenotypically visible through its deleterious effects. one of the best-studied aspects of hsp function in laboratory populations is the ability of hsp to buffer the phenotypic effects of genetic variation. with these observations in mind, we asked whether the natural hsp mutants we study here are impaired in their ability to buffer cryptic variation that can be revealed by inbreeding. such an observation would provide evidence that hsp90's buffering ability is not just restricted to laboratory populations. to answer this question, we established parallel inbred fly populations. of these populations, were homogenous for hsp wild-type, and for hsp mutant genotypes, respectively. more precisely, for each of the okayama, tokyo, ivory coast source populations, we established seven inbred populations with each of the two genotypes and propagated them at 25°c. in each generation, we sampled males and females from each inbred population to create subsequent generations, and continued this process for generations. as a measure of inbreeding depression-the deleterious effects of inbreeding on fitness-we estimated female fecundity by mating virgin females and males from each generation, and counted all offspring they produced within days. we now discuss results from each of the three lines in turn. first, in the inbred line from the okayama population, wild-type females did not become significantly less fecund until generation. in contrast, in the mutant line even one generation of inbreeding already caused a significant decrease in fecundity, and by generation four fecundity had been reduced by percent in the mutant. second, in the inbred line from the tokyo population, females did not become significantly less fecund in the wild-type after four generations, indicating that the wild-type is well-buffered against short-term inbreeding. in contrast, females in the mutant population had become significantly less fecund after four generations. the ivory coast lines, finally, showed much lower initial fecundity than the other two inbred lines. wild-type females showed no significant decrease in fecundity after four generations of inbreeding, whereas mutants became significantly less fecund. in the four generations of inbreeding, the three populations showed significantly different fecundity within both the wild-type and mutant genotypes. this indicates that effects of genetic background are important for our analysis and cannot be neglected. in sum, our analyses of inbred lines show that individuals with wild-type hsp alleles buffer cryptic deleterious variation more effectively than mutant individuals. hsp thus helps buffer cryptic deleterious variation in flies derived from wild populations. hsp is even more important for cryptic variation buffering under mild heat stress the preceding experiments showed that mutant hsp alleles buffer cryptic deleterious variation to a lesser extent than wild-type alleles. we next asked whether high temperatures further exacerbate this difference. the rationale for this question is that hsp is involved in the heat stress response. when exposed to heat stress, free hsp will be diverted from its interactions with signaling proteins to help renature excess misfolded and aggregated proteins. as a result, less hsp protein may be available for signaling and buffering. to answer this question, we performed mild inbreeding at °c with the same methods for crossing of flies within inbred lines as used in the preceding experiments. the starting isofemale lines were also the same, except that we began to rear flies at °c when they eclosed. in these experiments, wild-type flies from the okayama and tokyo populations retained high fecundity over three generations of inbreeding, while wild-type flies from the ivory coast populations significantly declined in fecundity after the first generation. all three populations persisted over three generations of inbreeding. in stark contrast to this persistence, lines with mutant hsp alleles from the three populations went extinct after the first generation, because their fecundity had been reduced to almost zero. this observation underscores that hsp is important to buffer deleterious variation not only at normal temperatures, but even more so under thermal stress. outbreeding of a previously inbred population reduces homozygosity of deleterious recessive alleles. if our assertion that wild-type hsp buffers deleterious variation is correct, then outbreeding should restore the fecundity lost during inbreeding, and especially in mutant lines where this loss is most severe. to ask whether this is the case, we used flies from the ivory coast population, where the fecundity loss had been especially severe at 28°c. we crossed flies derived from the different inbred isofemale lines with each other and examined their fecundity. in this way, we established populations that were outbred and contained either only hsp mutant alleles, only wild-type alleles, or a 1: mix of mutant and wild-type alleles. the outbred populations show significant increases in fertility relative to their inbreeding controls. specifically, outbreeding of populations that contained only mutant alleles increased fecundity from zero to individuals per female. this change in fecundity was highly significant, indicating that outbreeding rescued the fecundity loss during inbreeding at mild heat stress. wild-type alleles of hsp helped increase fecundity even further. specifically, the fecundity of outbred populations with both allele types was percent higher than for populations with only the mutant alleles. outbreeding in populations with only wild-type alleles increased fecundity by an additional percent, to a level that was significantly higher than in the mixed cross. in sum, wild-type hsp alleles are much more effective in restoring the fecundity that is lost after inbreeding. discussion we discovered naturally occurring mutants of the hsp gene with p element insertions in the gene's proximal promoter region. these mutants occur in three out of populations that we examined, and at modest allele frequencies, not exceeding percent. the mutations down-regulate hsp gene expression by percent to percent, depending on the population. they reduce competitive fitness, female fecundity, and longevity. we also found that hsp strongly influences the expression of cryptic deleterious genetic variation in inbred populations. that is, flies that carry the mutant hsp allele are much more poorly buffered against such variation than flies with the wild-type allele. we found that even mild thermal stress can completely break down the impaired buffering associated with mutant hsp specifically, inbred fly populations with mutant hsp alleles go extinct at modestly elevated temperatures of 28°c. to our knowledge, this work is the first to show that naturally occurring regulatory mutations of the hsp gene-a key modulator of the stress response and cellular signaling-can affect reproductive success, reduce longevity, and reduce variation buffering in fruit flies derived from wild populations. a previous study on naturally occurring hsp variants had identified a nonsynonymous deletion mutation in the hsp coding region, and focused on the effects of this mutation on morphological traits, not on fitness components. in addition, this study perturbed variation buffering only through thermal stress exposure, not through inbreeding, which is especially useful to reveal expression of recessive cryptic genetic variation. our limited sequence polymorphism analysis found only one synonymous mutation in the hsp coding region in the flies we studied, thus making it unlikely that the same deletion mutation stands behind our observations. our naturally occurring variants of hsp have fairly mild effects compared to some of the drastic perturbations that earlier laboratory experiments used. for example, the reduced expression of hsp in our mutants did not significantly affect thermotolerance in outbred flies. working with these mutants has several benefits. first, it avoids pharmacological manipulation or structural modification of hsp both of which may have unknown side effects, for example, by changing the protein's molecular interaction partners. second, it avoids the use of mutations with drastic expression effects, such as engineered homozygous loss-of-function mutants. such mutants can only be maintained as heterozygotes in the laboratory. a continuous inbreeding experiment like the one we performed to reveal deleterious cryptic variation would be difficult with such drastic mutations, partly because homozygote offspring would be lethal. in contrast, the mild variants we found enabled us to ask whether more hsp means better buffering. finally, they allowed us to study flies derived from natural populations, and to examine whether laboratory findings on hsp apply to such populations. a potential disadvantage of working with natural populations and the mutants that they contain is that each mutant occurs in a different genetic background, and that this background can influence observations. for example, we found that the genetic background of each geographic population influenced the competitive ability and fecundity of the flies we studied. however, our key observations, for example that hsp can buffer deleterious variation in inbreeding lines, and that hsp mutants show reduced fitness, were consistent across all three genetic backgrounds. p element insertions are widespread in the drosophila genome, and they are especially abundant in heat-shock genes. in japanese natural populations, for example, at least distinct p element insertions exist in the heat shock gene hsp and at least distinct insertions exist in the heat-shock gene hsp at high population frequency. these observations stand in stark contrast to the low frequency of insertions into the hsp gene. they suggest that the low frequency of p-element insertions we observe in hsp are not a consequence of a recent insertion, but that it results from natural selection against mutant hsp our observation that these insertions are possibly associated with reduced competitive fitness, reduced fecundity, and shorter life span are fully consistent with this suggestion. the question then arises why we observe any individuals that carry p-element insertions in our study populations. one possible answer is that the insertion may be neutral or even beneficial when heterozygous or when at low population frequencies. our observation that mutant allele frequencies did not decline in a competition assays seeded with only percent of hsp83p alleles are consistent with this possibility. moreover, the fitness effect of hsp83p may depend on the environment to which a population is adapted, as has been observed for p element insertions in other heat shock genes. in addition, some p-element insertions can help maintain tradeoffs between stress resistance and developmental homeostasis of flies living in a changing environment. unfortunately, our data does not allow us to answer which of these possibilities is responsible for the maintenance of p element insertions at modest frequencies in our study populations. our observation that mutations in hsp affect fecundity is consistent with other reports that hsp is involved in reproductive functions. hsp plays a critical role in the process of both oogenesis and spermatogenesis. for example, hsp rna is a component of the posterior polar plasm, and hsp protein is required for localization of maternal mrna to the posterior pole, which is essential for development of germ cells in the drosophila embryo. thus, hsp is involved in the molecular pathways responsible for oogenesis and spermatogenesis in d. melanogaster. inbreeding increases the fraction of homozygous loci in a genome. because many alleles are recessive and deleterious in the homozygous state, inbreeding will cause previously cryptic deleterious variation to be expressed. such deleterious variation manifests itself as a reduction in one or more fitness components, such as fecundity. in our inbreeding experiments, we found that wild-type hsp flies were better buffered against the deleterious effects of inbreeding than mutant flies. specifically, wild-type hsp flies from all three geographic populations showed no decline in fecundity after three generations of inbreeding, and only one population showed a small decline after four generations. in stark contrast, mutant lines from all three geographic populations showed a significant decline in fecundity of up to percent after between two and four generations. genetic polymorphisms are widespread in many populations, but the incidence of cryptic variation is usually unknown for wild populations. inbreeding can reveal such variation. our experiments demonstrate that cryptic variation must be abundant in the populations we studied, because inbreeding reduced fecundity substantially. moreover, the experiments show that expression of hsp at wild type levels can buffer the damage caused by inbreeding. the observation that a chaperone can buffer deleterious variation is not unprecedented. over-expression of groel, a molecular chaperone of escherichia coli, can help overcome the accumulation of deleterious mutations that occur in e. coli strains with high mutation rate. in other words, a chaperone can buffer these organisms against deleterious mutations. although the sources of deleterious variation-inbreeding and mutation accumulation-and the chaperones-hsp and groel-differ in these two organisms, chaperones have the same qualitative effect in both cases. however, the e. coli strain in which these previous experiments were conducted is a laboratory strain. observations made with this strain need not necessarily transfer to populations in the wild. three main observations lead us to think that the buffering capacity of hsp is relevant for wild fly populations. first, the flies in which we identified the effects of the hsp mutation all stem from wild populations. second, although the buffering capacity of hsp varies with genetic background, significant buffering did occur in all three populations. third, the regulatory mutation in hsp is mild in its effect on expression, but strong in its effects on fitness, i.e., on fecundity, longevity, and most importantly, buffering capacity. mutations that affect these aspects of fitness are likely to be selected against in nature. more specifically, because drosophila has very large effective population size in the wild, fitness differences much smaller than we detected would be visible to natural selection, and would lead to the eventual elimination of mutant alleles with low fitness.  in sum, our study shows that natural variants in the hsp gene can affect the fitness of flies derived from natural populations, regardless of their genetic background and geographic origin. it also shows that hsp is involved in the buffering of deleterious variation in these flies, and that this role becomes even more important under heat stress. and it demonstrates that hsp plays an important role in the life of flies in the wild. 
in the laboratory, the drosophila melanogaster heat shock protein hsp can buffer the phenotypic effects of genetic variation. mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. we here study naturally occurring mutations in hsp and their effects on fitness and phenotypic buffering in flies derived from wild populations. our work shows that hsp a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress. in competition experiments, mutant flies performed much more poorly than wild-type flies. specifically, in hsp mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. the insertions we observed occur at low population frequencies, and reduce hsp gene expression. laboratory experiments either manipulate hsp activity pharmacologically, or they induce mutations with strong effects in the gene hsp the single-copy fly gene encoding hsp it is unknown whether observations from such laboratory experiments are relevant in the wild.
 in the laboratory, the drosophila melanogaster heat shock protein hsp can buffer the phenotypic effects of genetic variation. laboratory experiments either manipulate hsp activity pharmacologically, or they induce mutations with strong effects in the gene hsp the single-copy fly gene encoding hsp it is unknown whether observations from such laboratory experiments are relevant in the wild.  we here study naturally occurring mutations in hsp and their effects on fitness and phenotypic buffering in flies derived from wild populations. we examined more than flies from drosophila populations distributed world-wide for insertions or deletions of mobile dna in or near the hsp gene. the insertions we observed occur at low population frequencies, and reduce hsp gene expression. in competition experiments, mutant flies performed much more poorly than wild-type flies. mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. specifically, in hsp mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. at even slightly elevated temperatures, inbred mutant hsp populations went extinct, whereas inbred wild-type populations persisted.  our work shows that hsp a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress. 