Drosophila melanogaster occurs in diverse climatic regions and shows opposing clinal changes in resistance to heat and resistance to cold along a 3000 km latitudinal transect on the eastern coast of Australia. We report here on variation at a polymorphic 8 bp-indel site in the heat shock hsr-omega gene that maps to the right arm of chromosome 3. The frequency of the genetic element marked by the L form of the gene was strongly and positively associated with latitude along this transect, and latitudinal differences in L frequency were robustly associated with latitudinal differences in maximum temperature for the hottest month. On a genetic background mixed for genes from each end of the cline a set of 10 lines was derived, five of which were fixed for the L marker, the absence of ln(3R)P and 12 kb of repeats at a second polymorphic site at the 3′ end of hsr-omega, and five that were fixed for the S marker, ln(3R)P and 15 kb of hsr-omega repeats. For two different measures of heat tolerance S lines outperformed L lines, and for two different measures of cold tolerance L lines outperformed S lines. These data suggest that an element on the right arm of chromosome 3, possibly ln(3R)P, confers heat resistance but carries the trade-off of also conferring susceptibility to cold. This element occurs at high frequency near the equator. The alternate element on the other hand, at high frequency at temperate latitudes, confers cold resistance at the cost of heat susceptibility.
Anderson, A. R., et al. (2005). “The latitudinal cline in the In(3R)Payne inversion polymorphism has shifted in the last 20 years in Australian Drosophila melanogaster populations.” Molecular Ecology 14(3): 851-858.
Clinal variation has been described in a number of inversions in Drosophila but these clines are often characterized by cytological techniques using small sample sizes, and associations with specific genes are rarely considered. Here we have developed a molecular assay for In(3R)Payne in Drosophila melanogaster from eastern Australia populations. It shows in repeated samples that the inversion cline is very tightly associated with latitude and is almost fixed in tropical populations while relatively rare in temperate populations. This steep cline has shifted in position in the last 20 years. The heat shock gene, hsr-omega, located centrally inside the inversion sequence, shows a different clinal pattern to In(3R)Payne. These results suggest strong ongoing selection on In(3R)Payne over the last 100 years since the colonization of Australia that is partly independent of hsr-omega.
Anderson, P. R., et al. (1987). “Observations on the Extent and Temporal Stability of Latitudinal Clines for Alcohol-Dehydrogenase Allozymes and 4 Chromosome Inversions in Drosophila-Melanogaster.” Genetica 75(2): 81-88.
Previously we have presented evidence of large-scale latitudinal clines in the frequencies of four chromosome inversions and alleles at six enzyme loci in populations of D. melanogaster in Australasia, Asia and North America. Subsequent sampling by others in Japan and western U.S.A. has failed to repeat this observation for the steepest of the clines (alcohol dehydrogenase and the four chromosome inversions). We argue that this failure reflects the few populations and small latitudinal range sampled in these later studies. From extensive sampling over a long latitudinal transect in Australasia we here document Adh and inversion clines which are virtually identical to those originally obtained in different Australian populations four years earlier. We also repeat our observation that the Adh cline is largely independent of the cline in the linked inversion In(2L)t. We therefore retain our original conclusion that these polymorphisms are subject to natural selection. However the new Australasian data do not indicate an association between Adh and maximum rainfall which had been evident in the earlier data for Australasia, Asia and North America. We therefore retract our claim that the selective agent on Adh is related to rainfall.
The laboratory has yielded many long-lived mutants of several model-organisms in the past few years. Many of the resulting claims for extended longevity have been nuanced or shown to be restricted to specific conditions, including environments and genetic backgrounds. Here, we test whether the long-lived mutant fruit fly methuselah (mth(1)) displays its apparent superiority in longevity and stress resistance in different environments, at different ages and in correlated traits. The results demonstrate that stress resistance at different times in life is not consistently higher in the mutant relative to its progenitor strain (w(1118)). Furthermore, the mth(1) genotype only leads to an increase in longevity in an environment where reproduction is not stimulated. Also, virgin and mated life span were compared and showed that mating negatively affects life span, especially in the mth(1) individuals. This reduced the life span enhancing effect of the mutation to zero. This apparent environment and mating dependent trade-off between longevity and reproduction supports the disposable soma theory of ageing. We conclude that these data can only provide limited information on natural variation. The data show the need to uncover the full complexity of variation in such traits in natural environments. (c) 2006 Elsevier Inc. All rights reserved.
Barnes, P. T., et al. (1989). “Genotype-by-Environment and Epistatic Interactions in Drosophila-Melanogaster – the Effects of Gpdh Allozymes, Genetic Background and Rearing Temperature on Larval Developmental Time and Viability.” Genetics 122(4): 859-868.
The possible role of temperature as a component of natural selection generating the latitudinal clines in Gpdh allele frequencies in natural populations of Drosophila melanogaster was examined. Effects of rearing temperature (16″, 22″ and 29″) and of Gpdh allozymes (S and F ) on larval developmental time and viability were measured. Eight genetic backgrounds from each of three populations (continents) were used to assess the generality of any effects. Analyses of variance indicated significant temperature effects and allozyme-by-genetic background interaction effects for both characters. Viability showed significant genetic background effects, as well as significant temperature- by-allozyme and temperature-by-allozyme-by-population interactions. In general, the S/S genotype was significantly lower in viability than the F/F and F/S genotypes at extreme temperatures (16″ and 29 “), with no significant differences at 22 “. However, each population had a slightly different pattern of viability associated with temperature, and only the Australian population showed a pattern that could contribute to the observed cline formation. Although the same two interactions were not significant for developmental time, examination of the means showed that the S/S genotype had a slightly faster rate of development at 16” than the F/F genotype in all populations (by an average of 0.25 day or 1.1%). The low temperature effect on developmental time is consistent with the clines observed in nature, with the S allele increasing in frequency with higher latitudes. The results for both viability and developmental time are consistent with the interpretation of Gfdh as a minor polygene affecting physiological phenotypes, as indicated by previous work with adult flight metabo- lism. Finally, it is proposed that the temperature-dependent antagonistic effects of the allozymes on viability us. developmental time and flight metabolism may be the underlying force giving rise to the – worldwide polymorphism.
Clines may either be selectively maintained or be the by-product of nonadaptive processes related to population structure and history. Drosophila melanogaster populations on the east coast of North America show a latitudinal cline in the frequencies of two common electrophoretically distinguishable alleles at the alcohol dehydrogenase locus (Adh), designated Adh-S and Adh-F. This cline may either be adaptive or an artifact of a possible recent dual founding of North American D. melanogaster populations in which frequencies of Adh alleles differed between founder populations. By means of a high resolution restriction-mapping technique, we studied the distribution of 113 haplotypes derived from 44 polymorphic DNA markers within the Adh region in 1533 individuals from 25 populations throughout the cline. We found significant clinal differentiation at the polymorphism determining the mobility-difference causing amino acid replacement between Adh-F and Adh-S alleles. Hitchhiking was limited, despite extensive linkage disequilibrium, and other sites did not vary clinally. Such a pattern of differentiation implies that selection is responsible for the cline. To investigate whether selection acts only on the Adh-F/S site, we performed a ”selective equivalence” test under the assumption that all variability within the specified allelic class is selectively neutral. This revealed selective equivalence among Adh-S-bearing haplotypes, whose frequencies showed no differentiation throughout the cline, implying high levels of frequency-homogenizing gene flow. Geographical heterogeneity among Adh-F-bearing haplotypes implied the action of selection on one or more additional variants in linkage disequilibrium with Adh-F. In a further study of a subset of the data (n = 1076 from 18 populations), we found a combined insertion/deletion polymorphism, designated del1, located in the 5′ adult intron and in linkage disequilibrium with Adh-F, to show more marked clinal variation than Adh-F/S. Although the unequivocal identification of the precise target(s) of selection requires further study, we suggest that clinal selection may be acting epistatically on the Adh-F/S and del1 polymorphisms.
To examine whether recent evolutionary history affects the expression of Hsp70, the major heat-induced-heat shock protein in Drosophila melanogaster, we measured Hsp70 expression, thermotolerance, and hsp70 gene number in replicate populations undergoing laboratory evolution at different temperatures. Despite Hsp70’s ancient and highly conserved nature, experimental evolution effectively and replicably modified its expression and phenotype (thermotolerance). Among five D, melanogaster populations founded from a common ancestral population and raised at three different temperatures tone at 18 degrees C, two each at 25 degrees C and 28 degrees C for twenty years, Hsp70 expression varies in a consistent pattern: the replicate 28 degrees C lines expressed 30-50% less Hsp70 than the other Lines at a range of inducing temperatures. This modification was refractory to acclimation, and correlated with thermotolerance: the 28 degrees C lines had significantly lower inducible tolerance of 38.5 degrees C and 39 degrees C. We verified the presence of five hsp70 genes in the genome of each line, excluding copy number variation as a candidate molecular basis of the evolved difference in expression. These findings support the ability of Hsp70 levels in D. melanogaster populations to change over microevolutionary time scales and implicate constancy of environmental temperature as a potentially important selective agent.
To determine whether and how laboratory and natural selection act on the hsp70 (70-Kd heat-shock protein) genes of Drosophila melanogaster, we examined hsp70 allele frequencies in two sets of populations. First, five populations reared at different temperatures for more than 20 years differentially fixed both a large insertion/deletion (indel) polymorphism at the 87A7 hsp70 cluster (“56H8″/”122”) and a single nucleotide polymorphism at the 87C1 hsp70 cluster. In both cases, the 18degreesC and 25degreesC populations fixed one allele and the 28degreesC populations the other, consistent with previously described evolved differences among these populations in Hsp70 expression and thermotolerance. Second, we examined 56H8 and 122 frequencies in a set of I I populations founded from flies collected along a latitudinal transect of eastern Australia. The 56118 allele frequencies are positively associated with latitude, consistent with maintenance of the 56H8/122 polymorphism by natural selection. Thermal extremes and average values are negatively correlated with latitude. These results suggest that natural selection imposed by temperature and thermal variability may affect hsp70 allele frequencies.
Based on data from 70 literature sources, the frequencies of common alleles at six allozyme loci were examined in Drosophila melanogaster populations from five geographic regions: North America (including Central America), South America, Europe-Africa, Asia, and Australasia. The analyzed loci were Adh, Odh, Gpdh, G6pd, Pgd, and Est-6, which have been previously reported by other authors to show latitudinal variation in North America, Eurasia and Australasia. We found five parallel latitudinal dines for Adh(F) and three dines for Gpdh(S) in five geographic regions as well as four dines for G6pd(F), three dines for Est-6(S), and two dines for Odh(F) and Pgd(F) in four regions (data from South America for G6pd, Odh, Est-6, and Pgd were not available). Such pattern of variation confirmed the possibility that considered allozyme polymorphisms are maintained by climatic selection. Significant differentiation of mean allele frequencies among geographic regions was in agreement with current evidence on history of D. melanogaster worldwide dispersion.
Chen, B., et al. (2007). “Abundant, diverse, and consequential P elements segregate in promoters of small heat-shock genes in Drosophila populations.” Journal of Evolutionary Biology 20(5): 2056-2066.
The present study extends evidence that Drosophila heat-shock genes are distinctively evolvable because of insertion of transposable elements by examining the genotypic diversity and phenotypic consequences of naturally occurring P element insertions in the proximal promoter regions of two small heat-shock genes. Detailed scrutiny of two populations revealed 16 distinctive P transposable elements collectively segregating in proximal promoters of two small heat-shock genes, Hsp26 and Hsp27. These elements vary in size, orientation and insertion site. Frequencies of P element-containing alleles varied from 5% to 100% in these populations. Two Hsp26 elements chosen for detailed study, RsP26 and D2Pm, reduced or abolished Hsp26 expression respectively. The RsP26 element increased or did not affect inducible tolerance of high temperature, increased fecundity, but decreased developmental rate. On the other hand, the D2Pm element decreased thermotolerance and fecundity. In lines subjected to experimental evolution, the allelic frequency of the RsP26P element varied considerably, and was at lower frequencies in lines selected for increased longevity and for accelerated development than in controls. Transposable element insertions into small Hsp genes in Drosophila populations can have dramatic fitness consequences, and therefore create variation on which selection can act.
The Drosophila melanogaster gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. in the nematode Caenorhabditis elegans, insulin/IGF signaling regulates adult longevity. We found that mutation of chico extends fruit fly median Life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of Life-span was not a result of impaired oogenesis in chico females, nor was it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of Life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved.
Collinge, J. E., et al. (2006). “Altitudinal patterns for latitudinally varying traits and polymorphic markers in Drosophila melanogaster from eastern Australia.” Journal of Evolutionary Biology 19(2): 473-482.
Altitudinal changes in traits and genetic markers can complement the studies on latitudinal patterns and provide evidence of natural selection because of climatic factors. In Drosophila melanogaster, latitudinal variation is well known but altitudinal patterns have rarely been investigated. Here, we examine five traits and five genetic markers on chromosome 3R in D. melanogaster collected at high and low altitudes from five latitudes along the eastern coast of Australia. Significant altitudinal differentiation was observed for cold tolerance, development time, ovariole number in unmated females, and the microsatellite marker DMU25686. Differences tended to match latitudinal patterns, in that trait values at high altitudes were also found at high latitudes, suggesting that factors linked to temperature are likely selective agents. Cold tolerance was closely associated with average temperature and other climatic factors, but no significant associations were detected for the other traits. Genes around DMU25686 represent good candidates for climatic adaptation.
Previous studies have found non-neutral patterns of nucleotide polymorphism in the promoter and coding regions of Est6 in D. melanogaster. Coding region polymorphism peaks around two closely linked replacement differences associated with the EST6-F/EST6-S allozyme polymorphism. The promoter contains two common, highly diverged haplotype groups, P1 and P7, that differentially affect Est6 expression. Allozyme studies have also revealed latitudinal clines in EST6-F and EST6-S frequencies that recur across continents. Here we analyse nucleotide polymorphisms across the promoter and the region of peak coding sequence polymorphism in 10 Australian populations along a 25 degrees latitudinal gradient in order to examine the basis for the allozyme clines. As with the earlier studies, we find an excess of intermediate to high frequency variants in both the P1/P7 region and around the two EST6-F/EST6-S replacements in some populations. The two EST6-F/EST6-S replacement polymorphisms show latitudinal clines whereas the P1 and P7 groups of promoter haplotypes do not. However the strongest clines are for three co-segregating silent site polymorphisms in a 4 bp stretch at the 3′ end of the sequenced region. Monte Carlo simulations show that the clines for those three sites can explain all others in the data but none of the others can explain those three. Thus the allozyme clines may not reflect selection on either the P1/P7 polymorphism or the two replacements previously associated with the EST6-F/EST-S difference.
The desaturase-2 (desat2) locus of Drosophila melanogaster has two alleles whose frequencies vary geographically: one (the “Z” allele) is found primarily in east Africa and the Caribbean, and the other (the “M” allele) occurs in other parts of the world. It has been suggested that these alleles not only cause sexual isolation between races. but that their distribution reflects differential adaptation to climate: Z alleles are supposedly adapted to tropical conditions and M alleles to temperate ones. This has thus been viewed as a case of reproductive isolation evolving as a pleiotropic byproduct of adaptation. Here we reinvestigate this presumed climatic adaptation, using transgenic lines differing in the nature of their desat2 alleles. We were unable to replicate earlier results showing that carriers of M alleles are uniformly more cold resistant and less starvation resistant than carriers of Z alleles. It is thus doubtful whether the distribution of these alleles reflects natural selection involving climate. Mating studies of transgenic lines show some evidence for sexual isolation due to desat2. However, work on other, wild-type lines, as well as observations on the nature of sexual isolation, suggest that this conclusion-and thus the relationship between this locus and mating discrimination between races of D. melanogaster-may also be doubtful.
To study the genetic differentiation and inversion clines in Indian natural populations of Drosophila melanogaster, 14 natural populations (6 from the north and 8 from the south) were screened for chromosome inversions. The chromosomal analysis revealed the presence of 23 paracentric inversions, which include 4 common cosmopolitan, 4 rare cosmopolitan, 2 recurrent endemic, and 13 unique endemic (new inversions detected for the first time) inversions. The difference in karyotype frequencies between populations from the north and south were highly significant and the level of inversion heterozygosity was higher in populations from the south. Statistically significant negative correlations were found between each of the four common cosmopolitan inversions and latitude. These findings are in accord with results from other worldwide geographic regions and show that Indian populations of D. melanogaster have undergone considerable genetic differentiation at the level of inversion polymorphism.
In an effort to characterize further the patterns of selection and adaptive evolution at the methuselah locus in Drosophila species, we extended an analysis of geographical variation to include single nucleotide polymorphisms (SNPs) in adjacent genes on either side of the mth locus, and examined the molecular variation in a neighbouring methuselah paralogue (mth2). An analysis of 13 SNPs spanning a region of nearly 19 kilobases surrounding the mth locus demonstrated that a clinal pattern associated with the most common mth haplotype does not extend to adjacent gene loci, providing compelling evidence that the clinal pattern results from selection on as yet unidentified sites associated with the functional mth locus. mth2 exhibited a significant pattern of adaptive divergence among D. melanogaster, D. simulans and D. yakuba similar to that seen at mth . However, K-a : K-s ratios indicate a difference in levels of functional constraint at the two methuselah, loci with mth2 exhibiting a five- to six-fold reduction in levels of amino acid divergence relative to mth.
The Drosophila Turandot A (TotA) gene was recently shown to encode a stress-induced humoral factor which gives increased resistance to the Lethal effects of high temperature. Here we show that TotA belongs to a family of eight Tot genes distributed at three different sites in the Drosophila genome. All Tot genes are induced under stressful conditions such as bacterial infection, heat shock, paraquat feeding or exposure to ultraviolet light, suggesting that all members of this family play a role in Drosophila stress tolerance. The induction of the Tot genes differs in important respects from the heat shock response, such as the strong but delayed response to bacterial infection seen for several of the genes.
The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3], Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6], Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9], Here we describe a novel systemic response to stress in Drosophila, The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fatbody and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.
Feder, M. E., et al. (1996). “Effect of engineering Hsp70 copy number on Hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster.” Journal of Experimental Biology 199(8): 1837-1844.
To determine how the accumulation of the major Drosophila melanogaster heat-shock protein, Hsp70, affects inducible thermotolerance in larvae and pupae, we have compared two sister strains generated by site-specific homologous recombination, One strain carried 12 extra copies of the Hsp70 gene at a single insertion site (extra-copy strain) and the other carried remnants of the transgene construct but Lacked the extra copies of Hsp70 (excision strain), Hsp70 levels in whole-body lysates of larvae and pupae were measured by ELISA with an Hsp70-specific antibody, In both extra-copy and excision strains, Hsp70 was undetectable prior to heat shock, Hsp70 concentrations were higher in the extra-copy strain than in the excision strain at most time points during and after heat shock. Pretreatment (i.e. exposure to 36 degrees C before heat shock) significantly improved thermotolerance, and this improvement was greater and more rapid in larvae and pupae of the extra-copy strain than in those of the excision strain, The experimental conditions resemble thermal regimes actually experienced by Drosophila in the field. Thus, these findings represent the best evidence to date that the amount of a heat-shock protein affects the fitness of a complex animal in the wild.
Heat shock genes are considered to be likely candidate genes for environmental stress resistance. Nucleotide variation in the coding sequence of the small heat shock genes (hsps ) hsp26 and hsp27 from Drosophila melanogaster was studied in flies originating from the Netherlands and eastern Australia. The hsp26 gene was polymorphic for an insertion/deletion of three extra amino acids and two nonsynonymous changes in all populations. The hsp27 gene exhibited two nonsynonymous changes and three synonymous mutations. The hsp26 polymorphism showed a latitudinal cline along the east coast of Australia. This pattern was not confounded by the fact that the shsps are located in the inversion In(3 L)P which also shows a latitudinal cline in eastern Australia. A similar latitudinal cline was found for the previously described variation in hsp23 , while frequencies of hsp27 alleles did not change with latitude. These findings suggest that variation at two of the shsps or closely linked loci are under selection in natural populations of D. melanogaster.
Fujikake, N., et al. (2005). “Alternative splicing regulates the transcriptional activity of Drosophila heat shock transcription factor in response to heat/cold stress.” Febs Letters 579(17): 3842-3848.
Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock proteins in response to various types of stress. Although HSF1 activity is regulated by its post-translational modifications, alterations in mRNA expression have also been suggested. We here identified three new alternatively spliced isoforms of Drosophila HSF (dHSF) mRNA, named dHSFb, dHSFc, and dHSFd. We found that the ratio of dHSFb increases upon heat exposure, while that of dHSFd increases upon cold exposure. The dHSFc and dHSFd isoforms showed greater transcriptional activity than the other isoforms. Our findings suggest that alternative splicing regulates the transcriptional activity of dHSF.
One general approach for assessing whether phenotypic variation is due to selection is to test its correlation with presumably neutral molecular variation. Neutral variation is determined by population history, the most likely alternative explanation of spatial genetic structure, whereas phenotypic variation may be influenced by the spatial pattern or selection pressure. Several methods for comparing the spatial apportionment of molecular and morphological variation have been used. Here, we present an analysis of variance framework that compares the magnitudes of latitudinal effects for molecular and morphological variation along a body size dine in Australian Drosophila populations. Explicit incorporation of the relevant. environmental gradient can result in a simple and powerful test of selection. For the Australian dine, our analysis provides strong internal evidence that the dine is due to selection.
Gene expression during cold acclimation at a moderately low temperature (15 degrees C) was studied in Drosophila melanogaster using a subtraction technique. A gene homologous to senescence marker protein-30 (SMP30), which has a Ca2+-binding function, was up-regulated at the transcription level after acclimation to 15 degrees C. This gene (henceforth referred to as Dca) was also expressed at a higher level in individuals reared at 15 degrees C from the egg stage than in those reared at 25 degrees C. Moreover, DCA mRNA increased at the senescent stage in Drosophila, although SMP30 is reported to decrease at senescent stages in mammals. in situ hybridization to polytene chromosomes revealed that the Dca gene was located at 88D on chromosome 3R. The 5′ flanking region of this gene had AP-1 (a transcription factor of SMP30) binding sites, stress response element and some other transcription factor binding sites. The function of DCA was discussed in relation to the possible regulation of cytosolic Ca2+ concentration. (C) 2000 Elsevier Science Ltd. All rights reserved.
Gene expression during recovery at 25 degreesC (rearing temperature) after cold shock (0 degreesC) was studied in Drosophila melanogaster using a subtraction technique. A novel gene (Frost, abbreviated as Fst) was considerably up-regulated during recovery after cold shock. In addition, a prolongation of cold shock was more effective for induction. In contrast to cold shock, Fst gene did not respond to heat shock. This gene is apparently the same as the unidentified gene, CG9434. Fst has high internal repeats not only in nucleotide but also in amino acid sequences. In addition, FST protein has a proline-rich region. The deduced amino acid sequence revealed a modular structure; i.e., a signal peptide in the N-terminal region followed by a long hydrophilic region. Therefore, this protein is likely to be directed into ER and secreted into extracellular space. (C) 2001 Elsevier Science B.V. All rights reserved.
To understand the role of adaptation in speciation, one must characterize the ecologically relevant phenotypic effects of naturally occurring alleles at loci potentially causing reproductive isolation. The desaturase2 gene of Drosophila melanogaster is such a locus. Two geographically differentiated ds2 alleles underlie a pheromonal difference between the Zimbabwe and Cosmopolitan races. We used a site-directed gene replacement technique to introduce an allele of ds2 from the Zimbabwe population into Cosmopolitan flies. We show that the Cosmopolitan allele confers resistance to cold as well as susceptibility to starvation when the entire genetic background is otherwise identical. We conclude that ecological adaptation likely accompanies sexual isolation between the two behavioral races of D. melanogaster.
To probe the role of natural selection in species origin, we performed a DNA polymorphism survey of the Drosophila melanogaster desaturase2 (ds2) locus. ds2 is responsible for a cuticular hydrocarbon difference between two behaviorally isolated races-Zimbabwe (Z) and Cosmopolitan (M). The ds2 allele prevalent in the Z populations is functional, while the allele from the M populations harbors a 16-bp deletion Upstream of: the gene which knocks out its expression. We find a signature of positive selection ill the ds2 promoter, but not in the control gene, sas. This signature appears to be confined to the derived M population. We also find that the selection has been recent because the gene retains a signature of a selective sweep evidenced by the departure of Fay and Wu’s H test from neutral expectation. We also find that ds2, as well as its duplicate pair ds1, has been maintained in the Drosophila genus for at least 40 Myr without any sign of adaptive change. Taken together with previous Molecular genetic evidence, our results Suggest that ds2 is one of the genes responsible for adaptive divergence of the Z and M races of D. melanogaster.
Background: A major challenge of modern biology is to understand the networks of interacting genes regulating complex traits, and the subset of these genes that affect naturally occurring quantitative genetic variation. Previously, we used P-element mutagenesis and quantitative trait locus (QTL) mapping in Drosophila to identify candidate genes affecting resistance to starvation stress, and variation in resistance to starvation stress between the Oregon-R ( Ore) and 2b strains. Here, we tested the efficacy of whole-genome transcriptional profiling for identifying genes affecting starvation stress resistance.
The ability to withstand periods of scarce food resources is an important fitness trait. Starvation resistance is a quantitative trait controlled by multiple interacting genes and exhibits considerable genetic variation in natural populations. This genetic variation could be maintained in the fact of strong selection clue to a trade-off in resource allocation between reproductive activity and individual survival. Knowledge of the genes affecting starvation tolerance and the subset of genes that affect variation in starvation resistance in natural populations would enable its to evaluate this hypothesis from a quantitative genetic perspective. We screened 933 co-isogenic P-element insertion lines to identify candidate genes affecting starvation tolerance. A total of 383 P-element insertions induced highly significant and often sex-specific mutational variance in starvation resistance. We also used deficiency complementation mapping followed by complementation to mutations to identify 12 genes contributing to variation in starvation resistance between two wild-type strains. The genes we identified are involved in oogenesis, metabolism, and feeding behaviors, indicating a possible link to reproduction and survival. However, we also found genes with cell fate specification and cell proliferation phenotypes, which implies that resource allocation during development and at the cellular level may also influence the phenotypic response to starvation.
Thirty-one Japanese populations of D melanogaster were surveyed for the frequency of polymorphic inversions. Four common cosmopolitan inversions (2Lt, 2RNS, 3LP, 3RP), four rare cosmopolitan inversions (2LA, 3LM, 3RC, 3RMo) and two recurrent endemic inversions (2LW, 3LY) were detected from the most populations. Their frequencies were positively correlated, that is, when a population carried a polymorphic inversion with high frequency, it also carried the other polymorphic inversions with high frequency. When the average frequency of inversions was compared geographically, a north-south cline, significantly higher to the south, was obtained when all 31 populations were considered, but it reversed in direction when four southern island populations were excluded. With respect to longitude, similarly, the slope of the cline is reversed when the four southern islands are excluded. Several populations were also examined seasonally or yearly for total inversion frequency. We could not detect a clear and repeatable seasonal change in the Mishima population. However, the average frequency of inversions has drastically decreased in the Katsunuma and other populations during the past 18 years, although it was rather stable in the last 8 years. When we compared the frequency order among the common cosmopolitan inversions, old populations were 2Lt > 2RNS > 3RP > 3LP but recent populations were 3RP > 2RNS > 2Lt > 3LP; there has been a relative decrease of 2Lt and increase of 3RP. This phenomenon was not restricted to the Katsunuma but was also observed in many United States populations
Four common cosmopolitan inversions (2Lt, 2RNS, 3LP, 3RP), four rare cosmopolitan inversions (2LA, 3LM, 3RC, 3RMo) and two recurrent endemic inversions (2L W, 3LY) were found to be distributed in Japanese natural populations of Drosophila melanogaster. Moreover, seventeen unique endemic inversions were rather randomly detected in many natural populations as well as in stocks that originated from small number of wild caught flies. The frequency of inversions was generally higher in Southern populations than in Northern populations, especially in the case of In(3R)P. The polymorphic inversions did not show any heterosis in the present analysis. The frequencies of inversion heterozygotes were almost equal to the expectaions with a few exceptions of a scarcity of the heterozygotes over the expectation. In the Katsunuma population, some inversions decreased remarkably within ten years, which resulted in the change of abundance order among four common cosmopolitan inversions. This kind of change might have occurred in the Shiojiri population. Both populations are very interesting in that new inversions are increasing in frequency and replacing the old ones.
Heat shock transcription factor (HSF) is a transcriptional activator of heat shock protein (hsp) genes in eukaryotes. In order to elucidate the physiological functions of HSF in Drosophila, we have isolated lethal mutations in the hsf gene. Using a conditional allele, we show that HSF has an essential role in the ability of the organism to survive extreme heat stress. In contrast to previous results obtained with yeast HSF, the Drosophila protein is dispensable for general cell growth or viability. However, it is required under normal growth conditions for oogenesis and early larval development. These two developmental functions of Drosophila HSF are genetically separable and appear not to be mediated through the induction of HSPs, implicating a novel action of HSF that may be unrelated to its characteristic function as a stress-responsive transcriptional activator.
The cellular heat stress response is well studied in Drosophila in respect to the role of heat shock proteins (Hsp). Hsps are molecular chaperones, highly expressed during and after exposure to numerous stress types. Hsps are all regulated by a common transcription factor, the heat shock factor (HSF), and it is known that HSF is controlling other, so far uncharacterised, heat-responsive genes. In this study, we investigate whether novel candidate genes for heat resistance, identified by microarray experiments, are regulated by HSF. The microarray experiments recently identified several strongly upregulated genes in response to a short, non-lethal heat treatment in Drosophila melanogaster. To test whether or not a subset of these genes are HSF-induced, we studied 11 currently unannotated genes using quantitative polymerase chain reaction on HSF mutant flies with a non-functional HSF at elevated temperatures. We found indication of HSF regulation in most of the studied genes, suggesting a role of these unknown genes in heat tolerance. Surprisingly, some of the genes seemed to be upregulated independent of HSF function. The high induction in response to heat, which mimics the expression profile of Hsps, implies a role in the cellular heat response of these genes as well.
Kelty, J. D. and R. E. Lee (2001). “Rapid cold-hardening of Drosophila melanogaster (Diptera : Drosophilidae) during ecologically based thermoperiodic cycles.” Journal of Experimental Biology 204(9): 1659-1666.
In contrast to most studies of rapid cold-hardening, in which abrupt transfers to low temperatures are used to induce an acclimatory response, the primary objectives of this study were to determine (i) whether rapid cold-hardening was induced during the cooling phase of an ecologically based thermoperiod, (ii) whether the protection afforded was lost during warming or contributed to increased cold-tolerance during subsequent cycles and (iii) whether the major thermally inducible stress protein (Hsp70) or carbohydrate cryoprotectants contributed to the protection afforded by rapid cold-hardening, During the cooling phase of a single ecologically based thermoperiod, the tolerance of Drosophila melanogaster to Ih at -7 degreesC increased from 5+/-5 % survival to 62.5+/-7.3 % (means +/- S.E.M., N=40-60), while their critical thermal minima (CTmin) decreased by 1.9 degreesC. Cold hardiness increased with the number of thermoperiods to which flies were exposed; i.e. flies exposed to six thermoperiods were more cold-tolerant than those exposed to two. Endogenous levels of Hsp70 and carbohydrate cryoprotectants were unchanged in rapidly cold-hardened adults compared with controls field at a constant 23 degreesC. In nature, rapid cold-hardening probably affords subtle benefits during short-term cooling, such as allowing D. melanogaster to remain active at lower temperatures than they otherwise could.
Knibb, W. R., et al. (1981). “Chromosome Inversion Polymorphisms in Drosophila-Melanogaster .1. Latitudinal Clines and Associations between Inversions in Australasian Populations.” Genetics 98(4): 833-847.
Nineteen Australasian populations of Drosophila melanogaster have been screened for chromosome inversion polymorphisms. All 15 of the inversion types found are paracentric and autosomal, but only four of these, one on each of the major autosome arms, are common and cosmopolitan. North-south clines occur, with the frequencies of all four of the common cosmopolitan inversions increasing toward the equator. These clines in the Southern Hemisphere mirror north-south clines in the Northern Hemisphere, where the frequencies of all four of the common cosmopolitan inversions again increase towards the equa- tor.-While few of the Australasian populations show significant disequi- librium between linked common cosmopolitan inversions, those that do in- variably have excesses of coupling gametes, which is consistent with other reports. We also find non random associations between the two major autosomes, with the northern populations in Australasia (those with high inversion frequencies) tending to be deficient in gametes with common cosmopolitan inversions on both major autosomes, while the southern populations in Australasia (low inversion frequencies) tend to have an excess of this class of gametes.- The clines and the non random associations between the two major autosomes are best interpreted in terms of selection operating to maintain the common cosmopolitan inversion polymorphisms in natural populations of D. melanogaster.
Leemans, R., et al. (2000). “Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays.” Proceedings of the National Academy of Sciences of the United States of America 97(22): 12138-12143.
Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses high-density oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on:the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that high-density oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos.
Lerman, D. N. and M. E. Feder (2001). “Laboratory selection at different temperatures modifies heat-shock transcription factor (HSF) activation in Drosophila melanogaster.” Journal of Experimental Biology 204(2): 315-323.
The magnitude and time course of activation of the heat-shock transcription factor (HSF) differ among Drosophila melanogaster lines evolving at 18 degreesC, 25 degreesC or 28 degreesC for more than 20 years. At lower heat-shock temperatures (27-35 degreesC), flies from the 18 degreesC population had higher levels of activated HSF (as detected by an electrophoretic mobility shift assay) than those reared at 25 degreesC and 28 degreesC. At higher temperatures (36 and 37 degreesC), however, the 28 degreesC flies had the highest levels of HSF. These differences persisted after one generation of acclimation at 25 degreesC, suggesting that phenotypic plasticity was limited. In addition, larvae from the 28 degreesC lines activated HSF less rapidly after a 35 degreesC heat shock than those from the 18 degreesC and 25 degreesC populations. These results are similar but not identical to previously reported differences in expression of Hsp70 (the major heat-inducible stress protein in Drosophila melanogaster) among the experimental lines. We conclude that HSF activation evolves rapidly during laboratory culture at diverse temperatures and could play an important role in the evolution of the heat-shock response.
Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was perform ed, The mutant Line methuselah (mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate-binding protein-coupled seven-transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span.
To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39 degrees heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from similar to 5 min to >20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and hsp68 contribute to natural heritable variation for hardened heat resistance.
The hsr-omega gene of Drosophila melanogaster produces RNA products both constitutively and at elevated levels in response to heat stress. A single-nucleotide difference in this gene that has been detected using denaturing gradient gel electrophoresis (DGGE) is responsible for an hsr-omega(a/b) polymorphism, and selection experiments have indicated an association between the hsr-omega(a) allele and susceptibility to heat stress. Since allele frequency estimates for population surveys using PCR and DGGE for single flies would be relatively time-consuming and expensive, we here develop a quantitative competitive-PCR method using mass-grind genomic DNA preparations for this purpose. Geographical and temporal variation of allele frequency at the hsr-omega locus in Australian populations of D. melanogaster are examined. Regular samples from a southern population through a summer season suggested stability of hsr-omega(a) frequency. Field populations sampled from a similar to 2,250 km north-south transect along eastern Australia revealed a strong positive association between the frequency of hsr-omega(a) and latitude, and marked spatial autocorrelation. Using appropriate analyses, strong associations between population differences in hsr-omega(a) frequencies and differences in temperature and rainfall measures, after controlling for latitudinal differences, support the idea that the dine in hsr-omega(a) frequency may be attributable to some form of climatic selection.
McKechnie, S. W., et al. (1998). “Both allelic variation and expression of nuclear and cytoplasmic transcripts of Hsr-omega are closely associated with thermal phenotype in Drosophila.” Proceedings of the National Academy of Sciences of the United States of America 95(5): 2423-2428.
Inducible heat shock genes are considered a major component of the molecular mechanisms that confer cellular protection against a variety of environmental stresses, in particular high temperature extremes. We have tested the association between expression of the heat shock RNA gene hsr-omega and thermoresistance by generating thermoresistant lines of Drosophila melanogaster after application of two distinct regimes of laboratory selection. One set of lines was selected for resistance to knockdown by heat stress and the other was similarly selected but before selection a mild heat exposure known to increase resistance (heat hardening) was applied. A cross between resistant and susceptible lines confirmed our earlier observation that increased thermal tolerance cosegregates with allelic variation in the hsr-omega gene. This cosegregating variation is attributed largely to two haplotype groups. Using quantitative reverse transcription-PCR, we find evidence for divergent phenotypic responses in the two selection regimes, involving both structural and regulatory changes in hsr-omega. Lines selected after hardening showed increased levels of the cytoplasmic transcript but decreased levels of the nuclear transcript. Lines selected without hardening showed decreased levels of the cytoplasmic transcript. The allelic frequency changes at hsr-omega could not by themselves account for the altered transcription patterns. Our results support the idea that the functional RNA molecules transcribed from hsr-omega are an important and polymorphic regulatory component of an insect thermoresistance phenotype.
Twenty different natural populations of Drosophila melanogaster were sampled to determine the frequencies of inversions. Based on their frequencies and geographical distributions, the inversions could be classified as follows: (1)Common cosmopolitan inversions that are present in many populations in frequencies exceeding five percent and that may exhibit frequency clines over large geographical regions; (2) Rare cosmopolitan inversions that occur throughout the species range but usually at frequencies below five percent and that may be absent in many populations; (3) Recurrent endemic inversions that are found in several adjacent populations in frequencies usually not exceeding one or two percent; and (4) Unique endemic inversions that are found only among the progeny of a single individual and that may represent one aspect of the syndrome termed “hybrid dysgenesis”. Four common cosmopolitan inversions that exhibit highly significant clines in populations in the eastern United States are In(2L)t, In(2R)NS, In(3L)P and In(3R)P
Michalak, P., et al. (2001). “Genetic evidence for adaptation-driven incipient speciation of Drosophila melanogaster along a microclimatic contrast in “Evolution Canyon,” Israel.” Proceedings of the National Academy of Sciences of the United States of America 98(23): 13195-13200.
Substantial genetic differentiation, as great as among species, exists between populations of Drosophila melanogaster inhabiting opposite slopes of a small canyon. Previous work has shown that prezygotic sexual isolation and numerous differences in stress-related phenotypes have evolved between D. melanogaster populations in “Evolution Canyon,” Israel, in which slopes 100-400 m apart differ dramatically in aridity, solar radiation, and associated vegetation. Because the canyon’s width is well within flies’ dispersal capabilities, we examined genetic changes associated with local adaptation and incipient speciation in the absence of geographical isolation. Here we report remarkable genetic differentiation of microsatellites and divergence in the regulatory region of hsp70Ba which encodes the major inducible heat shock protein of Drosophila, in the two populations. Additionally, an analysis of microsatellites suggests a limited exchange of migrants and lack of recent population bottlenecks. We hypothesize that adaptation to the contrasting microclimates overwhelms gene flow and is responsible for the genetic and phenotypic divergence between the populations.
For insects, temperature is a major environmental variable that can influence an individual’s behavioral activities and fitness. Drosophila melanogaster is a cosmopolitan species that has had great success in adapting to and colonizing diverse thermal niches. This adaptation and colonization has resulted in complex patterns of genetic variation in thermotolerance phenotypes in nature. Although extensive work has been conducted documenting patterns of genetic variation, substantially less is known about the genomic regions or genes that underlie this ecologically and evolutionarily important genetic variation. To begin to understand and identify the genes controlling thermotolerance phenotypes, we have used a mapping population of recombinant inbred (RI) lines to map quantitative trait loci (QTL) that affect variation in both heat- and cold-stress resistance. The mapping population was derived from a cross between two lines of D. melanogaster (Oregon-R and 2b) that were not selected for thermotolerance phenotypes, but exhibit significant genetic divergence for both phenotypes. Using a design in which each RI line was backcrossed to both parental lines, we mapped seven QTL affecting thermotolerance on the second and third chromosomes. Three of the QTL influence cold-stress resistance and four affect heat- stress resistance. Most of the QTL were trait or sex specific, suggesting that overlapping but generally unique genetic architectures underlie resistance to low- and high-temperature extremes. Each QTL explained between 5 and 14% of the genetic variance among lines, and degrees of dominance ranged from completely additive to partial dominance. Potential thermotolerance candidate loci contained within our QTL regions are identified and discussed.
Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance or Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression, In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We Found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF. Such its Hsps, although occurring following LTCA. are not of major importance for cold stress resistance and neither for RCH not, high-temperature knock-down resistance in D. melanogaster. (c) 2005 Elsevier Ltd. All rights reserved.
Knockdown resistance to high temperature is an ecologically important trait in small insects. A composite interval mapping was performed on the two major autosomes of Drosophila melanogaster to search for quantitative trait loci (QTL) affecting knockdown resistance to high temperature (KRHT). Two dramatically divergent lines from geographically different thermal environments were artificially selected on KRHT. These lines were crossed to produce two backcross (BC) populations. Each BC was analysed for 200 males with 18 marker loci on chromosomes 2 and 3. Three X-linked markers were used to test for X-linked QTL in an exploratory way. The largest estimate of autosome additive effects was found in the pericentromeric region of chromosome 2, accounting for 19.26% (BC to the low line) and 29.15% (BC to the high line) of the phenotypic variance in BC populations, but it could represent multiple closely linked QTL. Complete dominance was apparent for three QTL on chromosome 3, where heat-shock genes are concentrated. Exploratory analysis of chromosome X indicated a substantial contribution of this chromosome to KRHT. The results show that a large-effect QTL with dominant gene action maps on the right arm of chromosome 3. Further, the results confirm that QTL for heat resistance are not limited to chromosome 3.
Norry, F. M., et al. (2007a). “Knockdown resistance to heat stress and slow recovery from chill coma are genetically associated in a quantitative trait locus region of chromosome 2 in Drosophila melanogaster.” Molecular Ecology 16(15): 3274-3284.
In insects, two ecologically relevant traits of thermal adaptation are knockdown resistance to high temperature (KRHT) and chill-coma recovery (CCR). Chromosome 2 of Drosophila melanogaster was tested for quantitative trait loci (QTL) affecting both CCR and KRHT in backcrosses between homosequential lines that are fixed for the standard (noninverted) sequence of this autosome. These lines were obtained by artificial selection on KRHT and subsequent inbreeding from a stock that was derived from a single wild population. Heat-induced expression of the 70KD heat-shock protein (Hsp70) was also examined for variation between the lines. Composite interval mapping was performed for each trait on each reciprocal backcross, identifying one QTL region in the middle of chromosome 2 for both KRHT and CCR. The largest estimates of additive effects were found in pericentromeric regions of chromosome 2, accounting for 10-14% (CCR) and 10-17% (KRHT) of the phenotypic variance in BC populations. No QTL was found in the region of the heat-shock factor (hsf) gene. However, the two parental lines have diverged in the heat-induced Hsp70 expression. Distribution of KRHT QTL on chromosome 2 was similar between this study based on crosses between lines selected from a single wild population and previous work based on crosses between selection lines from different continents. Colocalized QTL showed a trade-off association between CCR and KRHT, which should be the result of either multiple, tightly linked trait-specific genes or a single gene with pleiotropic effects on the traits. We discuss candidate loci contained within the QTL regions.
Knockdown Resistance to High Temperature (KRHT) is an adaptive trait of thermotolerance in insects. An interval mapping was performed on chromosome X of Drosophila melanogaster to search for quantitative trait loci (QTL) affecting KRHT. A backcross population was obtained from two lines that dramatically differ for KRHT. Microsatellites were used as markers. Composite interval mapping identified a large-effect QTL in the region of band 10 where putative candidate genes map. To further test for this QTL a set of recombinant (but non-inbred) lines was obtained from backcrosses between the parental lines used for the interval mapping. Recombinant line analysis confirmed that one QTL is targeted by band 10. We identify and discuss candidate loci contained within our QTL region.
Geographic variation in Esterase-6 (Est-6) and Phosphoglucomutase (Pgm) gene frequencies in Australasian populations of Drosophila melanogaster are compared with analogous data collated from 16 previous reports for North America and Europe/Asia. A large-scale latitudinal cline is found on all three zoogeographic zones for Est-6 and overall, Est-61.00 frequency increases from about 20 per cent around 20 degrees latitude to about 80 per cent approaching 50 degrees latitude. In contrast, there is no consistent evidence for the latitudinal cline in Pgm gene frequencies in any of the three zones with Pgm1.00 frequency generally about 85 per cent and Pgm1.20 and Pgm0.70 frequencies each between 5 per cent and 10 per cent. The consistent Est-6 clines are attributed to latitudinal selection gradients but not consistent correlations are found between Est-6 gene frequencies and maximum or minimum temperature or rainfall which might be associated with these gradients. The directions of the Est-6 clines in fact run counter to expectations based on the in vitro thermostabilities of the respective allozymes
Oakeshott, J. G., et al. (1984). “Population-Genetics of the Metabolically Related Adh, Gpdh and Tpi Polymorphisms in Drosophila-Melanogaster .1. Geographic-Variation in Gpdh and Tpi Allele Frequencies in Different Continents.” Genetica 63(1): 21-29.
Among Australasian populations from above 32.5” latitude there is a significant negative relationship between GpdhF frequency and distance from the equator which is not explained by gametic disequilibrium with the linked inversion Zn(2L)t. This is consistent with the associations reported earlier for GpdhF among populations covering comparable latitudes in North America and Europe/Asia. By contrast, Tpi allele frequencies are found to be significantly associated with distance from the equator in Australasia but not North America or Europe/ Asia. The Tpi pattern in the different zones is essentially the same as that reported earlier for the Acph polymorphism, which maps only 0.2 CM away from the Tpi locus. There are now ten enzyme polymorphisms in D. melanogaster which have been screened for latitudinal associations in Australasia, North America and Europe/ Asia. Allele frequencies at six of these loci show significant relationships with distance from the equator which are consistent across all three zones. These lati- tudinal associations are more prevalent for Group II than Group I enzymes. Values of genie heterozygosity averaged over the ten polymorphic loci and eleven other monomorphic systems do not vary with latitude but differ substantially between zones. Values of Nei’s genetic distance between North American and European/ Asian populations calculated from all 2 1 systems are equivalent to subspecific differences elsewhere in the genus.
In view of the world-wide latitudinal cline of the Adh and alpha Gpdh allozyme frequencies of Drosophila melanogaster and the interactions between these loci, experiments were performed to study the phenotypic effects of these loci. Starvation resistance, oxygen consumption, body weight, protein content and triglycerides content were measured in flies with all possible combinations of Adh and alpha Gpdh alleles. Genotypic differences were found for survival time under food deprivation, body weight, protein content and triglycerides content. Oxygen consumption did not differ significantly between genotypes. A significant positive correlation was observed between triglycerides content and starvation resistance. Body weight, protein content and triglycerides content decreased significantly during starvation but no rate differences between genotypes could be found. It is argued that genotypic differences in starvation resistance and triglycerides content can play a role in the maintenance of the world-wide dine of Adh and alpha Gpdh allele frequencies.
Understanding the significance of electrophoretic variation is of interest for both ecological and evolutionary genetics. Although there has been a very active neutralist – selectionist debate about the patterns of electrophoretic variation in natural populations, it is only recently that charged amino acids have been shown to be important in enzyme adaptation. In this study we carried out a broad electrophoretic survey of amylase variation in 150 species of Drosophilids. The distribution of amylase electromorphs was found to be correlated with the geographical origin of the flies. Generally the faster migrating variants are found in warmer temperatures. There is also a correlation with the feeding habits of the species, in particular, fungus feeders consistently showed a deviating pattern of electrophoretic mobility. These correlations between ecological diversity and electrophoretic patterns indicate that at least some of the changes in charged amino acids are adaptive, and result from selection to cope with specific environments.
Cold hardening treatment – a brief exposure to low temperatures – can protect certain insects against subsequent exposure to temperatures sufficiently low to cause damage or lethality. Microarray analysis to examine the changes in transcript abundance associated with cold hardening treatment (0 degrees C for 2 h followed by 30 min recovery at 25 degrees C) was undertaken in Drosophila melanogaster in order to gain insight into this phenomenon. Transcripts associated with 36 genes were identified, a subset of which appeared to be also differentially expressed after heat shock treatment. Quantitative RT-PCR was used to independently determine transcript abundance of a subset of these sequences. Taken together, these assays suggest that stress proteins, including Hsp23, Hsp26, Hsp83 and Frost as well as membrane-associated proteins may contribute to the cold hardening response.
Rako, L., et al. (2007). “Candidate genes and thermal phenotypes: identifying ecologically important genetic variation for thermotolerance in the Australian Drosophila melanogaster cline.” Molecular Ecology 16(14): 2948-2957.
Clinal variation in traits often reflects climatic adaptation; in Drosophila melanogaster clinal variation provides an opportunity to link variation in chromosomal inversions, microsatellite loci and various candidate genes to adaptive variation in traits. We undertook association studies with crosses from a single population of D. melanogaster from eastern Australia to investigate the association between genetic markers and traits showing clinal variation. By genotyping parents and phenotyping offspring, we minimized genotyping costs but had the power to detect association between markers and quantitative traits. Consistent with prior studies, we found strong associations between the clinal chromosomal inversion In(3R)Payne and markers within it, as well as among these markers. We also found an association between In(3L)Payne and one marker located within this inversion. Of the five predicted associations between markers and traits, four were detected (increased heat, decreased cold resistance and body size with the heat shock gene hsr-omega S, increased cold resistance with the inversion In(3L)Payne), while one was not detected (heat resistance and the heat shock gene hsp68). In a set of eight exploratory tests, we detected one positive association (between hsp23a and heat resistance) but no associations of heat resistance with alleles at the hsp26, hsp83, Desat 2, alpha-Gpdh, hsp70 loci, while cold resistance was not associated with Frost and Dca loci. These results confirm interactions between hsr-omega and thermal resistance, as well as between In(3L)Payne and cold resistance, but do not provide evidence for associations between thermal responses and alleles at other clinally varying marker genes.
The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question of whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits. We addressed this question in Drosophila melanogaster, in which the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments(1). Here we demonstrate that single P-element insertions in the intergenic region between the gustatory receptor 5a (Gr5a, also known as Tre)(2-4) and trapped in endoderm 1 (Tre1)(5), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, life span, and resistance to starvation and heat stress. Mutations in this region interact epistatically with downstream components of the insulin signaling pathway. Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences that intergenic transposable elements can contribute to quantitative trait phenotypes.
We report the characterization of a novel Drosophila circadian clock-regulated output gene, takeout (to). The to amino acid sequence shows similarity to two ligand binding proteins, including juvenile hormone binding protein, to mRNA is expressed in the head and the cardia, crop, and antennae-structures related to feeding. to expression is induced by starvation, which is blocked in all arrhythmic central clock mutants, suggesting a direct molecular link between the circadian clock and the feeding/starvation response. A to mutant has aberrant locomotor activity and dies rapidly in response to starvation, indicating a link between locomotor activity, survival, and food status. We propose that to participates in a novel circadian output pathway that conveys temporal and food status information to feeding-relevant metabolisms and activities.
Examination of the phenotypic effects of specific mutations has been extensively used to identify candidate genes affecting traits of interest. However, such analyses do not reveal anything about the evolutionary forces acting at these loci, or whether standing allelic variation contributes to phenotypic variance in natural populations. The Drosophila gene methuselah (mth) has been proposed as having major effects on organismal stress response and longevity phenotype. Here, we examine patterns of polymorphism and divergence at mth in population level samples of Drosophila melanogaster. D. simulans. and D. yakuba. Mth has experienced an unusually high level of adaptive amino acid divergence concentrated in the intra- and extracellular loop domains of the receptor protein, suggesting the historical action of positive selection on those regions of the molecule that modulate signal transduction. Further analysis of single nucleotide polymorphisms (SNPs) in D. melanogaster provided evidence for contemporary and spatially variable selection at the mth locus. In ten surveyed populations, the most common mth haplotype exhibited a 40% dine in frequency that coincided with population level differences in multiple life-history traits including lifespan. This clinal pattern was not associated with any particular SNP in the coding region. indicating that selection is operating at a closely linked site that may be involved in gene expression. Together, these consistently nonneutral patterns of inter- and intraspecific variation suggest adaptive evolution of a signal transduction pathway that may modulate lifespan in nature.
We exposed adult male Drosophila melanogaster to cold, desiccation or starvation, and examined expression of several genes during exposure and recovery. Frost was expressed during recovery from cold, and was up-regulated during desiccation. Desiccation and starvation (but not cold) elicited increased expression of the senescence-related gene smp-30. Desat2 decreased during recovery from desiccation, but not in response to starvation or cold. Hsp70 expression increased after 1 h of recovery from cold exposure, but was unchanged in response to desiccation or starvation stress, and Hsp23levels did not respond to any of the stressors. We conclude that D. melanogaster‘s responses to cold and desiccation are quite different and that care must be taken to separate exposure and recovery when studying responses to environmental stress.
Sorensen, J. G. and V. Loeschcke (2001). “Larval crowding in Drosophila melanogaster induces Hsp70 expression, and leads to increased adult longevity and adult thermal stress resistance.” Journal of Insect Physiology 47(11): 1301-1307.
In this study we show for the first time that moderate high larval density, induces Hsp70 expression in Drosophila melanogaster larvae. Larval crowding led to both increased mean and maximal longevity,in adults of both sexes. Two different measures of heat-stress resistance increased in adult flies developed at high density compared to flies developed at low density. The hardening-like effect of high larval density carried over to the adult life stage. The hardening memory (the period of increased resistance after hardening) was long compared to hardening of adult flies, and possibly lasts throughout life. The increase in resistance in adults following development at high larval density seemed not to be connected to Hsp70 itself, since Hsp70 expression level in adult flies after hardening was independent of whether larvae developed at low or high densities. More likely, Hsp70 may be one of many components of the stress response resulting in hardening. (C) 2001 Elsevier Science Ltd. All rights reserved.
The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36 degrees C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early-upregulated, early downregulated, and late-upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.
Here, we report a detailed analysis of changes in gene expression in Drosophila melanogaster selected for ecologically relevant environmental stress resistance traits. We analysed females from seven replicated selection regimes and one control regime using whole genome gene expression arrays. When compared with gene expression profiles of control lines, we were able to detect consistent selection responses at the transcript level in each specific selection regime and also found a group of differentially expressed genes that were changed among all selected lines. Replicated selection lines showed similar changes in gene expression (compared with controls) and thus showed that 10 generations of artificial selection give a clear signal with respect to the resulting gene expression profile. The changes in gene expression in lines selected for increased longevity, desiccation and starvation resistance, respectively, showed high similarities. Cold resistance-selected lines showed little differentiation from controls. Different methods of heat selection (heat survival, heat knock down and constant 30 degrees C) showed little similarity verifying that different mechanisms are involved in high temperature adaptation. For most individual selection regimes, and in the comparison of all selected lines and controls, the gene expression changes were exclusively in one direction, although the different selection regimes varied in the direction of response. The responses to selection restricted to individual selection regimes can be interpreted as stress specific, whereas the response shared among all selected lines can be considered as a general stress response. Here, we identified genes belonging to both types of responses to selection for stress resistance.
Stalker, H. D. (1980). “Chromosome-Studies in Wild Populations of Drosophila-Melanogaster .2. Relationship of Inversion Frequencies to Latitude, Season, Wing-Loading and Flight Activity.” Genetics 95(1): 211-223.
In the midwestern and eastern U.S. populations of Drosophila melanogaster, the Standard gene arrangements show higher frequencies in the north than in the south. In a Missouri population, and to a lesser extent in a south Texas population, the frequencies of Standard chromosomes regularly rise during the cold season and drop during the warm season, thus paralleling the north-south frequency differences. In the Missouri population in 1976 and 1978, wild males were tested far their ability to fly to bait at different ambient temperatures. In both years, males flying in nature in the temperature range of 13″ to 15″ showed significantly higher frequencies of Standard chromosomes than did those flying in the 16″ to 28″ range. Wild males flying at 13″ to 15″ also have different tharax/wing proportions and significantly lower wingloading indices than do those flying at 16″ to 28″. Moreover, wild flies homozygous Standard in 2R and/or 3R have significantly lower wing-loading indices than flies carrying inversions in these arms. Thus, wild flies with high frequencies of Standard chromosommes are karyotypically northern, are selectively favored during the cold season, have a relatively low wing-load and are most capable of flying at critically low ambient temperatures.-In summary, in Missouri, presence or absence o€ the common cosmopolitan inversions is an important factor in low temperature adaptation, and at least part of the adaptive mechanism involves control of thorax/wing proportions and thus control of wing-loading.
The capacity to moderate internal and external stress is arguably the central function regulating senescence in whole-animal ageing. During ageing, molecular chaperones such as heat-shock proteins are thought to combat stress-related senescent dysfunction. In transgenic Drosophila melanogaster, with varying copy numbers of the gene hsp70 encoding heat-shock protein hsp70, we found that heat-induced expression of hsp70 increased lifespan at normal temperatures. Only a brief, low level of expression was required to obtain a long-term improvement in survival.
Umina, P. A., et al. (2006). “An independent non-linear latitudinal cline for the sn-glycerol-3-phosphate (alpha-Gpdh) polymorphism of Drosophila melanogaster from eastern Australia.” Genetical Research 87(1): 13-21.
Latitudinal variation of the polymorphic sn-glycerol-3-phosphate (alpha-Gpdh) locus in Drosophila melanogaster has been characterized on several continents; however, apparent clinal patterns are potentially confounded by linkage with an inversion, close associations with other genetic markers that vary clinally, and a tandern alpha-Gpdh pseudogene. Here we compare clinal patterns in alpha-Gpdh with those of other linked markers by testing field flies from eastern Australian locations collected in two separate years. The a-Gpdh variation exhibited a consistent non-linear cline reflecting an increase in the alpha-Gpdh(F) allele at extreme latitudes. This pattern was not influenced by the In(2L)t inversion wherein this locus is located, nor was it influenced by the presence of the alpha-Gpdh pseudogene, whose presence was ubiquitous and highly variable among Populations. The alpha-Gpdh pattern was also independent of a cline in allozyme frequencies at the alcohol dehydrogenase (Adh) locus, and two length polymorphisms in the Adh gene. These results suggest clinal selection at the alpha-Gpdh locus that is partially or wholly unrelated to linear climatic gradients along the eastern coast of Australia.
Geographical clines in genetic polymorphisms are widely used as evidence of climatic selection and are expected to shift with climate change. We show that the classic latitudinal cline in the alcohol dehydrogenase polymorphism of Drosophila melanogaster has shifted over 20 years in eastern coastal Austratia. Southern high-latitude populations now have the genetic constitution of more northerly populations, equivalent to a shift of 40 in latitude. A similar shift was detected for a genetically independent inversion polymorphism, whereas two other linked polymorphisms exhibiting weaker clinal patterns have remained relatively stable. These genetic changes are likely to reflect increasingly warmer and drier conditions and may serve as sensitive biomarkers for climate change.
Vandelden, W. and A. Kamping (1989). “The Association between the Polymorphisms at the Adh and Alpha-Gpdh Loci and the in(2l)T Inversion in Drosophila-Melanogaster in Relation to Temperature.” Evolution 43(4): 775-793.
Substantial allele-frequency changes were observed at the Adh and aGpdh loci in a seminatural population of Drosophila melanogaster kept in a tropical greenhouse during 1972- 1985.Further analysis of the changes at the Adh and aGpdh loci showed that linkage disequilibrium between these loci occurred for a prolonged period due to the presence of Zn(2L)t, a long inversion on the left arm of the second chromosome. We observed increases in the frequencies of Zn(2L)t and of short inversions on the left arm of the second chromosome in subpopulations kept at 29.5OC or 33°C. These inversion-frequency increases were accompanied by an increase in AdhS and a decrease in otGpdhS frequency. In populations kept at 20°C and 25″C, inversion frequencies decreased, while otGpdhS allele frequencies decreased at 25°C and increased at 20°C. At 33″C, eggto- adult survival of individuals possessing Zn(2L)t, either in the homokaryotypic or the heterokaryotypic state, was higher than that of the other karyotypes of identical allozyme constitution (i.e., AdhS aGpdhf). Thus it seems that Zn(2L)t has a selective advantage at high temperature. We argue that the observed changes in allele frequencies at the Adh and otGpdh loci are, in part, due to genic selection and are not merely the result of selection acting on the chromosome rearrangements and hitchhiking of the allozymes. The results are discussed with respect to the latitudinal clines found for Zn(2L)t, Adh, and aGpdh
Many organisms show latitudinal variation for various genetically determined traits. Such dines may involve neutral variation and originate from historical events or their maintenance may be explained by selection. For Drosophila melanogaster, latitudinal variation for allozymes, inversions, and quantitative traits has been found on several continents. We sampled D. melanogaster populations in Panama and along a transect of 40 latitudinal degrees on the west coast of South America. Negative correlations with latitude were found for Adh(S) and alpha Gpdh(F) allele frequencies and for the frequency of the cosmopolitan inversion In(2L)t in Adh(S) alpha Gpdh(F) chromosomes. A positive correlation existed between wing length and latitude. Significant correlations were found between these traits and climatic variables like temperature and rainfall. The observed dines show considerable resemblance to those found on other continents. Gametic disequilibrium between Adh(S) and alpha Gpdh(F) occurred predominantly at higher latitudes and was caused by the presence of In(2L)t. The reasons for the clinaI. distributions are discussed and it is argued that selection is the most likely explanation. However, the exact nature of the selective force and the interactions of allozymes with each other and with In(2L)t are complex and not fully understood. In tropical regions In(2L)t-containing genotypes have higher fitness than ST/ST and Adh and alpha Gpdh hitchhike with the inversion, but there is also evidence for balancing selection at the Adh locus.
Clinal variation is common for enzymes in the glycolytic pathway for Drosophila melanogaster and is generally accepted as an adaptive response to different climates. Although the enzyme phosphoglucomutase (P(;M) possesses several allozyme polymorphisms. it is unique in that it had been reported to show Ilo clinal variation. Our recent DNA sequence investigation of Pgm found extensive cryptic amino acid polymorphism segregating with the allozyme alleles. In this study, we characterize the geographic variation of Pgm amino acid polymorphisms at the nucleotide level along a latitudinal dine in the eastern United States. A survey of 15 SNPs across the Pgm gene finds significant clinal differentiation for the allozyme polymorphisms as well as for many of the cryptic amino acid polymorphisms. A test of independence shows that pervasive linkage disequilibrium across this gene region call explain many of the amino acid dines. A single Pgm haplotype defined by two amino acid polymorphisms shows the strongest correlation with latitude and the steepest change in allele frequency across the dine. We propose thar clinal selection at Pgm ma) in part explain the extensive amino acrid polymorphism at this locus and is consistent with a multilocus response to selection in the glycolytic pathway.
The nature of genetic variation for Drosophila longevity in a population of recombinant inbred lines was investigated by estimating quantitative genetic parameters and mapping quantitative trait loci (QTL) for adult life span in five environments: standard culture conditions, high and low temperature, and heat-shock and starvation stress. There was highly significant genetic variation for life span within each sex and environment. In the analysis of variance of life span pooled over sexes and environments, however, the significant genetic variation appeared in the genotype X sex and genotype X environment interaction re rms. The genetic correlation of longevity across the sexes and environments was not significantly different from zero in these lines. We estimated map positions and effects of QTL affecting life span by linkage to highly polymorphic roo transposable element markers, using a multiple-trait composite interval mapping procedure. X minimum of 17 QTL were detected; all were sex and/or environment-specific. Ten of the QTL had sexually antagonistic or antagonistic pleiotropic effects in different environments. These data provide support for the pleiotropy theory of senescence and the hypothesis that variation for longevity might be maintained by opposing selection pressures in males and females and variable environments. Further work is necessary to assess the generality of these results, using different strains, to determine heterozygous effects and to map the life span QTL to the level of generic loci.
Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer’s disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, we isolated a fly homolog of ApoD, Glial Lazarillo (GLaz). In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O-2) as well as a 29% extension of lifespan under normoxia. These files also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, we subjected the flies to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This and the accompanying paper by Sanchez et al.  (in this issue of Current Biology) are the first to manipulate the levels of an ApoD homolog in a model organism. Our data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases.
Weeks, A. R., et al. (2002). “Dissecting adaptive clinal variation: markers, inversions and size/stress associations in Drosophila melanogaster from a central field population.” Ecology Letters 5(6): 756-763.
Many organisms show latitudinal variation for quantitative traits that is assumed to be due to climatic adaptation. These clines provide an opportunity to study the genetics of the adaptive process both at the phenotypic and the underlying molecular levels. Yet researchers rarely try to link variation in quantitative traits to their underlying molecular genetic basis. We describe a novel approach for exploring the genetic basis for clinal variation in size and stress traits in Drosophila melanogaster. We look for associations between genetic markers and traits that exhibit clinal patterns on the east coast of Australia using a single, geographically central population. There are strong associations between markers found within In( 3R) Payne and variation in size, suggesting that this inversion explains much of the clinal variation in this trait. We also find that development time is associated with the Adh allozyme locus, cold resistance is negatively associated with the In(3L) Payne inversion and a genetic marker for Hsp70, a heat-shock protein, is associated with heat resistance. Finally we discuss the importance of inversions in clinal variation for quantitative traits and for identifying quantitative trait loci.
Zinke, I., et al. (1999). “Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system.” Development 126(23): 5275-5284.
We have isolated a Drosophila mutant, named pumpless, which is defective in food intake and growth at the larval stage, pumpless larvae can initially feed normally upon hatching. However, during late first instar stage, they fail to pump the food from the pharynx into the esophagus and concurrently begin moving away from the food source, Although pumpless larvae do not feed, they do not show the typical physiologic response of starving animals, such as upregulating genes involved in gluconeogenesis or lipid breakdown. The pumpless gene is expressed specifically in the fat body and encodes a protein with homology to a vertebrate enzyme involved in glycine catabolism. Feeding wild-type larvae high levels of amino acids could phenocopy the feeding and growth defects of pumpless mutants, Our data suggest the existence of an amino acid-dependent signal arising from the fat body that induces cessation of feeding in the larva, This signaling system may also mediate growth transition from larval to the pupal stage during Drosophila development.
We have identified genes regulated by starvation and sugar signals in Drosophila larvae using whole-genome microarrays. Based on expression profiles in the two nutrient conditions, they were organized into different categories that reflect distinct physiological pathways mediating sugar and fat metabolism, and cell growth. In the category of genes regulated in sugar-fed, but not in starved, animals, there is an upregulation of genes encoding key enzymes of the fat biosynthesis pathway and a downregulation of genes encoding lipases. The highest and earliest activated gene upon sugar ingestion is sugarbabe, a zinc finger protein that is induced in the gut and the fat body. Identification of potential targets using microarrays suggests that sugarbabe functions to repress genes involved in dietary fat breakdown and absorption. The current analysis provides a basis for studying the genetic mechanisms underlying nutrient signalling.