Multiple modes of canalization: Links between genetic, environmental canalizations and developmental stability, and their trait-specificity.

The robustness of biological systems against mutational and environmental perturbations is termed canalization. Because reducing phenotypic variability under environmental and genetic perturbations can be adaptive and facilitated by natural selection, it has been suggested that once canalization mechanisms have evolved to buffer the effects of environmental perturbations, they may act to buffer any and all sources of variation. Although whether canalization mechanisms are general or specific to the types of perturbation or phenotypic traits that they buffer is often addressed, the links between different canalization mechanisms remain unclear. In this review, three major sources of phenotypic variation, associated canalization concepts and indicators of the degree of canalization are first outlined. Then, the molecular bases of canalization mechanisms based on recent empirical studies are overviewed. Finally, the links between the underlying processes of different canalization mechanisms are explored.

Apoptosis inhibition mitigates aging effects in Drosophila melanogaster.

Aging is a natural biological process that results in progressive loss of cell, tissue, and organ function. One of the causing factors of the aging process is the decrease in muscle mass, which has not been fully verified in Drosophila. Apoptotic cell death may result in aberrant cell loss and can eventually diminish tissue function and muscle atrophy. If so, inhibition of apoptosis may prolong longevity and reduce motor function and muscle mass decline with age in Drosophila flies. Here, we used Drosophila melanogaster as study material, and induced the overexpression of Drosophila inhibitor of apoptosis protein 1 gene to inhibit apoptosis, and investigated the effect of apoptosis inhibition on the longevity and age-related declines in flight and climbing ability and muscle mass. As a result, the inhibition of apoptosis tended to mitigate the aging effects and prolonged longevity and reduced climbing ability decline with age. The current study suggests that apoptosis inhibition could mitigate the aging effects in D. melanogaster. Although such effects have already been known in mammals, the current results suggest that the apoptosis may play a similar role in insects as well.

Genome-wide association analysis of host genotype and plastic wing morphological variation of an endoparasitoid wasp Asobara japonica (Hymenoptera: Braconidae).

Accumulating evidence suggests that genotype of host insects influences the development of koinobiont endoparasitoids. Although there are many potential genetic variations that lead to the internal body environmental variations of host insects, association between the host genotype and the parasitoid development has not been examined in a genome-wide manner. In the present study, we used highly inbred whole genome sequenced strains of Drosophila melanogaster to associate single nucleotide polymorphisms (SNPs) of host flies with morphological traits of Asobara japonica, a larval-pupal parasitoid wasp that infected those hosts. We quantified the outline shape of the forewings of A. japonica with two major principal components (PC1 and PC2) calculated from Fourier coefficients obtained from elliptic Fourier analysis. We also quantified wing size and estimated wasp survival. We then examined the association between the PC scores, wing size and 1,798,561 SNPs and  the association between the estimated wasp survival and 1,790,544 SNPs. As a result, we obtained 22, 24 and 14 SNPs for PC1, PC2 and wing size and four SNPs for the estimated survival with P values smaller than 10-5. Based on the location of the SNPs, 12, 17, 11 and five protein coding genes were identified as potential candidates for PC1, PC2, wing size and the estimated survival, respectively. Based on the function of the candidate genes, it is suggested that the host genetic variation associated with the cell growth and morphogenesis may influence the wasp's morphogenetic variation.

Little effect of HSP90 inhibition on the quantitative wing traits variation in Drosophila melanogaster.

Drosophila wings have been a model system to study the effect of HSP90 on quantitative trait variation. The effect of HSP90 inhibition on environmental buffering of wing morphology varies among studies while the genetic buffering effect of it was examined in only one study and was not detected. Variable results so far might show that the genetic background influences the environmental and genetic buffering effect of HSP90. In the previous studies, the number of the genetic backgrounds used is limited. To examine the effect of HSP90 inhibition with a larger number of genetic backgrounds than the previous studies, 20 wild-type strains of Drosophila melanogaster were used in this study. Here I investigated the effect of HSP90 inhibition on the environmental buffering of wing shape and size by assessing within-individual and among-individual variations, and as a result, I found little or very weak effects on environmental and genetic buffering. The current results suggest that the role of HSP90 as a global regulator of environmental and genetic buffering is limited at least in quantitative traits.

Novel genetic capacitors and potentiators for the natural genetic variation of sensory bristles and their trait specificity in Drosophila melanogaster.

Cryptic genetic variation (CGV) is defined as the genetic variation that has little effect on phenotypic variation under a normal condition, but contributes to heritable variation under environmental or genetic perturbations. Genetic buffering systems that suppress the expression of CGV and store it in a population are called genetic capacitors, and the opposite systems are called genetic potentiators. One of the best-known candidates for a genetic capacitor and potentiator is the molecular chaperone protein, HSP90, and one of its characteristics is that it affects the genetic variation in various morphological traits. However, it remains unclear whether the wide-ranging effects of HSP90 on a broad range of traits are a general feature of genetic capacitors and potentiators. In the current study, I searched for novel genetic capacitors and potentiators for quantitative bristle traits of Drosophila melanogaster and then investigated the trait specificity of their genetic buffering effect. Three bristle traits of D. melanogaster were used as the target traits, and the genomic regions with genetic buffering effects were screened using the 61 genomic deficiencies examined previously for genetic buffering effects in wing shape. As a result, four and six deficiencies with significant effects on increasing and decreasing the broad-sense heritability of the bristle traits were identified, respectively. Of the 18 deficiencies with significant effects detected in the current study and/or by the previous study, 14 showed trait-specific effects, and four affected the genetic buffering of both bristle traits and wing shape. This suggests that most genetic capacitors and potentiators exert trait-specific effects, but that general capacitors and potentiators with effects on multiple traits also exist.

Lack of response to artificial selection on developmental stability of partial wing shape components in Drosophila melanogaster.

Developmental stability, the ability of organisms to buffer their developmental processes against developmental noise is often evaluated with fluctuating asymmetry (FA). Natural genetic variation in FA has been investigated using Drosophila wings as a model system and the recent estimation of the heritability of wing shape FA was as large as 20%. Because natural genetic variation in wing shape FA was found to localize in a partial component of the wings, heritable variation in specific parts of the wings might be responsible for FA estimation based on the whole wing shape. In this study, we quantified the shape of three partial components of the wings, and estimated the heritability of the wing shape FA based on artificial selections. As a result, FA values for the partial wing shape components did not respond to artificial selections and the heritability scores estimated were very small. These results indicate that natural additive genetic variation in FA of partial wing components was very small compared with that in a complex wing trait.

Multiple capacitors for natural genetic variation in Drosophila melanogaster.

Cryptic genetic variation (CGV) or a standing genetic variation that is not ordinarily expressed as a phenotype is released when the robustness of organisms is impaired under environmental or genetic perturbations. Evolutionary capacitors modulate the amount of genetic variation exposed to natural selection and hidden cryptically; they have a fundamental effect on the evolvability of traits on evolutionary timescales. In this study, I have demonstrated the effects of multiple genomic regions of Drosophila melanogaster on CGV in wing shape. I examined the effects of 61 genomic deficiencies on quantitative and qualitative natural genetic variation in the wing shape of D. melanogaster. I have identified 10 genomic deficiencies that do not encompass a known candidate evolutionary capacitor, Hsp90, exposing natural CGV differently depending on the location of the deficiencies in the genome. Furthermore, five genomic deficiencies uncovered qualitative CGV in wing morphology. These findings suggest that CGV in wing shape of wild-type D. melanogaster is regulated by multiple capacitors with divergent functions. Future analysis of genes encompassed by these genomic regions would help elucidate novel capacitor genes and better understand the general features of capacitors regarding natural genetic variation.

Genome-wide deficiency screen for the genomic regions responsible for heat resistance in Drosophila melanogaster.

BACKGROUND: Temperature adaptation is one of the most important determinants of distribution and population size of organisms in nature. Recently, quantitative trait loci (QTL) mapping and gene expression profiling approaches have been used for detecting candidate genes for heat resistance. However, the resolution of QTL mapping is not high enough to examine the individual effects of various genes in each QTL. Heat stress-responsive genes, characterized by gene expression profiling studies, are not necessarily responsible for heat resistance. Some of these genes may be regulated in association with the heat stress response of other genes. RESULTS: To evaluate which heat-responsive genes are potential candidates for heat resistance with higher resolution than previous QTL mapping studies, we performed genome-wide deficiency screen for QTL for heat resistance. We screened 439 isogenic deficiency strains from the DrosDel project, covering 65.6% of the Drosophila melanogaster genome in order to map QTL for thermal resistance. As a result, we found 19 QTL for heat resistance, including 3 novel QTL outside the QTL found in previous studies. CONCLUSION: The QTL found in this study encompassed 19 heat-responsive genes found in the previous gene expression profiling studies, suggesting that they were strong candidates for heat resistance. This result provides new insights into the genetic architecture of heat resistance. It also emphasizes the advantages of genome-wide deficiency screen using isogenic deficiency libraries.

Genome-wide deficiency mapping of the regions responsible for temporal canalization of the developmental processes of Drosophila melanogaster.

Developmental processes of organisms are programmed to proceed in a finely regulated manner and finish within a certain period of time depending on the ambient environmental conditions. Therefore, variation in the developmental period under controlled genetic and environmental conditions indicates innate instability of the developmental process. In this study, we aimed to determine whether a molecular machinery exists that regulates the canalization of the developmental period and, if so, to test whether the same mechanism also stabilizes a morphological trait. To search for regions that influence the instability of the developmental period, we conducted genome-wide deficiency mapping with 441 isogenic deficiency strains covering 65.5% of the Drosophila melanogaster genome. We found that 11 independent deficiencies significantly increased the instability of the developmental period and 5 of these also significantly increased the fluctuating asymmetry of wing shape although there was no significant correlation between the instabilities of developmental period and wing shape in general. These results suggest that canalization processes of the developmental period and morphological traits are at least partially independent. Our findings emphasize the potential importance of temporal variation in development as an indicator of developmental stability and canalization and provide a novel perspective for understanding the regulation of phenotypic variability.

Effects of small Hsp genes on developmental stability and microenvironmental canalization.

BACKGROUND: Progression of development has to be insulated from the damaging impacts of environmental and genetic perturbations to produce highly predictable phenotypes. Molecular chaperones, such as the heat shock proteins (HSPs), are known to buffer various environmental stresses, and are deeply involved in protein homeostasis. These characteristics of HSPs imply that they might affect developmental buffering and canalization. RESULTS: We examined the role of nine Hsp genes using the GAL4/UAS-RNAi system on phenotypic variation of various morphological traits in Drosophila melanogaster. The stability of bristle number, wing size and wing shape was characterized through fluctuating asymmetry (FA) and the coefficient of variation (CV), or among-individual variation. Progeny of the GAL4/Hsp-RNAi crosses tended to have reduced trait means for both wing size and wing shape. Transcriptional knockdown of Hsp67Bc and Hsp22 significantly increased FA of bristle number, while knockdown of Hsp67Ba significantly increased FA and among-individual variation of wing shape but only in males. Suppression of Hsp67Bb expression significantly increased among-individual variation of bristle number. The knockdown of gene expression was confirmed for Hsp67Ba, Hsp67Bc, Hsp22, and Hsp67Bb. Correlation between FA and CV or among-individual variation of each trait is weak and not significant except for the case of male wing shape. CONCLUSION: Four small Hsp genes (Hsp22, Hsp67Ba, Hsp67Bb and Hsp67Bc) showed involvement in the processes of morphogenesis and developmental stability. Due to possible different functions in terms of developmental buffering of these small Hsps, phenotypic stability of an organism is probably maintained by multiple mechanisms triggered by different environmental and genetic stresses on different traits. This novel finding may lead to a better understanding of non-Hsp90 molecular mechanisms controlling variability in morphological traits.

Reduced X-linked rare polymorphism in males in comparison to females of Drosophila melanogaster.

Natural selection is assumed to act more strongly on X-linked loci than on autosomal loci because the fitness effect of a recessive mutation on the X chromosome is fully expressed in hemizygous males. Therefore, selection is expected to fix or remove recessive mutations on the X chromosome more efficiently than those on autosomes. However, the assumption that hemizygosity of the X chromosome selectively accelerates changes in allele frequency has not been confirmed directly. To examine this assumption, we investigated current natural selection on X-linked chemoreceptor genes in a natural population of Drosophila melanogaster by comparing nucleotide diversity, linkage disequilibrium (LD), and departure from the neutrality in 4 chemoreceptor genes on 100 X chromosomes each from female and male flies. The general pattern of nucleotide diversity and LD for the genes investigated was similar in females and males. In contrast, males harbored significantly fewer rare polymorphisms defined as singletons and doubletons. When all the gene sequences were concatenated, Tajima's D showed a significant departure from the neutrality in both females and males, whereas Fu and Li's F* value revealed departure only in males. These results suggest that some rare polymorphisms on the X chromosome from females are recessively deleterious and are removed by stronger purifying selection when transferred to hemizygous males.

HSP90 as a global genetic modifier for male genital morphology in Drosophila melanogaster.

The molecular chaperone protein HSP90 has been proposed to modulate genotype-phenotype relationship in a broad range of organisms. We explore the proposed genetic modifier effect of HSP90 through a genomewide analysis. Here, we show that HSP90 functions as a genetic modifier of genital morphology in Drosophila melanogaster. We identified a large number of single-nucleotide polymorphisms (SNPs) with an HSP90-dependent effect by using genome wide association analysis. We classified the SNPs into the ones under capacitance effect (smaller allelic effect under HSP90 inhibition) or the ones under potentiation effect (larger allelic effect under HSP90 inhibition). Although the majority of SNPs are under capacitance, there are a large number of SNPs under potentiation. This observation provides support for a model in which Hsp90 is not described exclusively as a "genetic capacitor," but is described more broadly as a "genetic modifier." Because the majority of the candidate genes estimated from SNPs with an HSP90-dependent effect in the current study has never been reported to interact with HSP90 directly, the global genetic modifier effect of HSP90 may be exhibited through epistatic interactions in gene regulatory networks.

Correction: Genome-Wide Association Study on Male Genital Shape and Size in Drosophila melanogaster.

[This corrects the article DOI: 10.1371/journal.pone.0132846.].

Genome-Wide Association Study on Male Genital Shape and Size in Drosophila melanogaster.

Male genital morphology of animals with internal fertilization and promiscuous mating systems have been one of the most diverse and rapidly evolving morphological traits. The male genital morphology in general is known to have low phenotypic and genetic variations, but the genetic basis of the male genital variation remains unclear. Drosophila melanogaster and its closely related species are morphologically very similar, but the shapes of the posterior lobe, a cuticular projection on the male genital arch are distinct from each other, representing a model system for studying the genetic basis of male genital morphology. In this study, we used highly inbred whole genome sequenced strains of D. melanogaster to perform genome wide association analysis on posterior lobe morphology. We quantified the outline shape of posterior lobes with Fourier coefficients obtained from elliptic Fourier analysis and performed principal component analysis, and posterior lobe size. The first and second principal components (PC1 and PC2) explained approximately 88% of the total variation of the posterior lobe shape. We then examined the association between the principal component scores and posterior lobe size and 1902142 single nucleotide polymorphisms (SNPs). As a result, we obtained 15, 14 and 15 SNPs for PC1, PC2 and posterior lobe size with P-values smaller than 10(-5). Based on the location of the SNPs, 13, 13 and six protein coding genes were identified as potential candidates for PC1, PC2 and posterior lobe size, respectively. In addition to the previous findings showing that the intraspecific posterior shape variation are regulated by multiple QTL with strong effects, the present study suggests that the intraspecific variation may be under polygenic regulation with a number of loci with small effects. Further studies are required for investigating whether these candidate genes are responsible for the intraspecific posterior lobe shape variation.

Deficiency screening for genomic regions with effects on environmental sensitivity of the sensory bristles of Drosophila melanogaster.

Environmental canalization is defined as a reduction in the effect of external environmental perturbations on a phenotype, while phenotypic plasticity is defined as the production of different phenotypes in alternative environments. These terms describe different aspects of the same phenomenon, that is, the sensitivity of the phenotype to the environment. Genetic regulation of the environmental sensitivity has been a central topic in the field of evolutionary biology. In this study, we performed deficiency screening to detect genomic regions with effects on the environmental sensitivity of Drosophila melanogaster sensory bristles. We used a collection of isogenic deficiency strains established by the DrosDel Project for screening. We screened 423 genomic deficiencies that encompassed approximately 63.6% of the entire D. melanogaster genome. We identified 29 genomic deficiencies showing significant effects on environmental sensitivity, suggesting that multiple genomic regions may influence phenotypic variation. We also found significant correlations among the effects of deficiencies on environmental sensitivity for different bristle traits, suggesting that the same genetic mechanism can regulate environmental sensitivity of multiple traits. Current high-resolution mapping will facilitate the examination of individual candidate genes using mutations or RNAi approaches in future studies.

Deficiency mapping of the genomic regions associated with effects on developmental stability in Drosophila melanogaster.

Developmental stability is the tendency of morphological traits to resist the effects of developmental noise, and is commonly evaluated by examining fluctuating asymmetry (FA)-random deviations from perfect bilateral symmetry. Molecular mechanisms that control FA have been a long-standing topic of debate in the field of evolutionary biology and quantitative genetics. In this study, we mapped genomic regions associated with effects on the mean and FA of morphological traits, and characterized the trait specificity of those regions. A collection of isogenic deficiency strains established by the DrosDel project was used for deficiency mapping of genome regions associated with effects on FA. We screened 435 genome deficiencies or approximately 64.9% of the entire genome of Drosophila melanogaster to map the region that demonstrated a significant effect on FA of morphological traits. We found that 406 deficiencies significantly affected the mean of morphological traits, and 92 deficiencies increased FA. These results suggest that several genomic regions have the potential to affect developmental stability. They also suggest the possibility of the existence of trait-specific and trait-nonspecific mechanisms for stabilizing developmental processes. The new findings in this study could provide insight into the understanding of the genetic architecture underlying developmental stability.

Environmental stress-dependent effects of deletions encompassing Hsp70Ba on canalization and quantitative trait asymmetry in Drosophila melanogaster.

Hsp70 genes may influence the expression of wing abnormalities in Drosophila melanogaster but their effects on variability in quantitative characters and developmental instability are unclear. In this study, we focused on one of the six Hsp70 genes, Hsp70Ba, and investigated its effects on within- and among-individual variability in orbital bristle number, sternopleural bristle number, wing size and wing shape under different environmental conditions. To do this, we studied a newly constructed deletion, Df(3R)ED5579, which encompasses Hsp70Ba and nine non-Hsp genes, in the heterozygous condition and another, Hsp70Ba(304), which deletes only Hsp70Ba, in the homozygous condition. We found no significant effect of both deletions on within-individual variation quantified by fluctuating asymmetry (FA) of morphological traits. On the other hand, the Hsp70Ba(304)/Hsp70Ba(304) genotype significantly increased among-individual variation quantified by coefficient of variation (CV) of bristle number and wing size in female, while the Df(3R)ED5579 heterozygote showed no significant effect. The expression level of Hsp70Ba in the deletion heterozygote was 6 to 20 times higher than in control homozygotes, suggesting that the overexpression of Hsp70Ba did not influence developmental stability or canalization significantly. These findings suggest that the absence of expression of Hsp70Ba increases CV of some morphological traits and that HSP70Ba may buffer against environmental perturbations on some quantitative traits.

Tolerance of Drosophila flies to ibotenic acid poisons in mushrooms.

The mushroom genus Amanita has a spectrum of chemical compounds affecting survival and performance of animals. Ibotenic acid is one of such compounds found in some Amanita mushrooms. We studied the effects of ibotenic acid and its derivative, muscimol, on egg-to-pupa survival, pupation time, and pupal size in five Drosophila species (Diptera: Drosophilidae), Drosophila bizonata, Drosophila angularis, Drosophila brachynephros, Drosophila immigrans, and Drosophila melanogaster. The first three species are mycophagous and use a wide range of mushrooms for breeding, whereas D. immigrans and D. melanogaster are frugivorous. We reared fly larvae on artificial medium with 500, 250, 125, and 62.5 microg/ml of ibotenic acid and/or musimol. The three mycophagous species were not susceptible to ibotenic acid, whereas the two frugivorous species were affected. In experiments with D. melanogaster, muscimol was less toxic than ibotenic acid.

Spatial aggregation and association in different resource-patch distributions: experimental analysis with Drosophila.

1. Laboratory experiments using homogeneous resources were conducted to examine intra- and interspecific spatial egg distribution of D. simulans, D. auraria and D. immigrans in three different resource-patch distribution patterns: patchy, even and clustered. 2. Individuals of each species were introduced separately or simultaneously into the cage, into which artificial substrates were placed and allowed to oviposit for 24 h. Spatial analyses were performed with indices of intraspecific aggregation (J), interspecific association (C) and L-function based on Ripley's K-function. 3. Eggs were always spatially aggregated irrespective of species and the resource-patch distribution patterns. Spatial egg aggregation was influenced significantly by the resource-patch distribution pattern and tended to be weaker in the clustered resource-patch distribution than in the patchy or even resource-patch distribution. 4. Spatial extent of egg aggregation was always beyond the single resource patch scale, indicating aggregation of ovipositing females. 5. Interspecific association of egg distribution was absent or very weak. Thus, these results present experimental evidence of independent egg aggregation among drosophilids.