Selection for abdominal tergite pigmentation and correlated responses in the trident: a case study in Drosophila melanogaster

In Drosophila melanogaster, abdominal tergite pigmentation and the appearance of a trident‐shaped thoracic pattern exhibit similar biogeographical variation and sensitivity to temperature. These pigmentation traits may be under common selection pressure in natural populations or may be genetically correlated. To investigate the nature of this interaction, replicated populations of D. melanogaster were selected for increased or decreased melanization of the abdominal tergites for 40 generations. Selection for abdominal tergite pigmentation leads to correlated changes in trident formation. Although selection was performed only on female flies, male pigmentation also responded to selection. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 287–294.     

A Genome-Wide,Fine-Scale Map of Natural Pigmentation Variation in Drosophila melanogaster

Various approaches can be applied to uncover the genetic basis of natural phenotypic variation, each with their specific strengths and limitations. Here, we use a replicated genome-wide association approach (Pool-GWAS) to fine-scale map genomic regions contributing to natural variation in female abdominal pigmentation in Drosophila melanogaster, a trait that is highly variable in natural populations and highly heritable in the laboratory. We examined abdominal pigmentation phenotypes in approximately 8000 female European D. melanogaster, isolating 1000 individuals with extreme phenotypes. We then used whole-genome Illumina sequencing to identify single nucleotide polymorphisms (SNPs) segregating in our sample, and tested these for associations with pigmentation by contrasting allele frequencies between replicate pools of light and dark individuals. We identify two small regions near the pigmentation genes tan and bric-à-brac 1, both corresponding to known cis-regulatory regions, which contain SNPs showing significant associations with pigmentation variation. While the Pool-GWAS approach suffers some limitations, its cost advantage facilitates replication and it can be applied to any non-model system with an available reference genome.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Pleiotropic effects of regulatory variation in tan result in correlation of two pigmentation traits in Drosophila melanogaster

Traits with a common genetic basis frequently display correlated phenotypic responses to selection or environmental conditions. In Drosophila melanogaster, pigmentation of the abdomen and a trident‐shaped region on the thorax are genetically correlated. Here, we used a pooled replicated genomewide association approach (Pool‐GWAS) to identify the genetic basis of variation in thoracic trident pigmentation in two Drosophila melanogaster populations. We confirmed the previously reported large effect of ebony and the association of the cosmopolitan inversion In(3R)Payne. For the first time, we identified tan as another major locus contributing to variation in trident pigmentation. Intriguingly, the regulatory variants of tan that were most strongly associated with female abdominal pigmentation also showed a strong association with trident pigmentation. We validated this common genetic basis in transgenic assays and found qualitatively similar effects on trident and abdominal pigmentation. Further work is required to determine whether this genetic correlation is favoured by natural selection or reflects a neutral by‐product of a shared regulatory architecture.     

Catecholamine metabolism and in vitro induction of premature cuticle melanization in wild type and pigmentation mutants of Drosophila melanogaster

The major pathway leading to adult cuticle melanization in Drosophila melanogaster has been investigated by a combination of biochemical and genetic approaches. By comparing catecholamine pools in newly emerged flies and in frass (excreta) collected 1 to 4 days after eclosion from wild type with those obtained from several pigmentation mutants, the major flow of catecholamines through the pathway to an unidentified final catabolite was determined. We also demonstrate that incubation with dopamine in vitro induces premature melanization in wild type unpigmented pharate adults several hours before the developmentally programmed onset of melanization, supporting the hypothesis that the availability of catecholamines may be the limiting factor determining the onset of melanization and that the major enzymatic activities that act downstream of dopa decarboxylase in the pathway are deposited into the cuticle before pigmentation begins. In vitro melanization studies with various pigmentation mutants that are associated with critical enzymatic steps in Drosophila catecholamine metabolism are consistent with their proposed function and suggest a central role of N-β-alanyldopamine in adult cuticle pigmentation. © 1996 Wiley-Liss, Inc.     

Abdominal pigmentation in Drosophila melanogaster females from natural Indian populations

Females of Drosophila melanogaster collected from five geographically distant populations in India were analysed for the intensity of pigmentation in the 5th, 6th and 7th segments of the abdomen. In all three segments, this intensity was found to vary among individuals of any given population, and, furthermore, different populations differ with respect to this phenotypic trait. Statistical analysis revealed significant intra- and interpopulational variation. A clinical pattern was detected: females from populations closer to the equator tended to have lighter cuticle, in which case differences between the three segments could not be detected and all three segments responded both independently and jointly to latitudinal variation, as indicated by a statistically significant positive correlation between latitude and pigmentation score. This is the first report on abdominal pigmentation analysis in natural populations of D. melanogaster that provides evidence that phenotypic flexibility reflects temperature differences, as a result of which abdominal pigmentation shows geographic differentiation.     

The Complex Contributions of Genetics and Nutrition to Immunity in Drosophila melanogaster

Both malnutrition and undernutrition can lead to compromised immune defense in a diversity of animals, and “nutritional immunology” has been suggested as a means of understanding immunity and determining strategies for fighting infection. The genetic basis for the effects of diet on immunity, however, has been largely unknown. In the present study, we have conducted genome-wide association mapping in Drosophila melanogaster to identify the genetic basis for individual variation in resistance, and for variation in immunological sensitivity to diet (genotype-by-environment interaction, or GxE). D. melanogaster were reared for several generations on either high-glucose or low-glucose diets and then infected with Providencia rettgeri, a natural bacterial pathogen of D. melanogaster. Systemic pathogen load was measured at the peak of infection intensity, and several indicators of nutritional status were taken from uninfected flies reared on each diet. We find that dietary glucose level significantly alters the quality of immune defense, with elevated dietary glucose resulting in higher pathogen loads. The quality of immune defense is genetically variable within the sampled population, and we find genetic variation for immunological sensitivity to dietary glucose (genotype-by-diet interaction). Immune defense was genetically correlated with indicators of metabolic status in flies reared on the high-glucose diet, and we identified multiple genes that explain variation in immune defense, including several that have not been previously implicated in immune response but which are confirmed to alter pathogen load after RNAi knockdown. Our findings emphasize the importance of dietary composition to immune defense and reveal genes outside the conventional “immune system” that can be important in determining susceptibility to infection. Functional variation in these genes is segregating in a natural population, providing the substrate for evolutionary response to pathogen pressure in the context of nutritional environment.     

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.     

Complex patterns of cis‐regulatory polymorphisms in ebony underlie standing pigmentation variation in Drosophila melanogaster

Pigmentation traits in adult Drosophila melanogaster were used in this study to investigate how phenotypic variations in continuous ecological traits can be maintained in a natural population. First, pigmentation variation in the adult female was measured at seven different body positions in 20 strains from the Drosophila melanogaster Genetic Reference Panel (DGRP) originating from a natural population in North Carolina. Next, to assess the contributions of cis‐regulatory polymorphisms of the genes involved in the melanin biosynthesis pathway, allele‐specific expression levels of four genes were quantified by amplicon sequencing using a 454 GS Junior. Among those genes, ebony was significantly associated with pigmentation intensity of the thoracic segment. Detailed sequence analysis of the gene regulatory regions of this gene indicated that many different functional cis‐regulatory alleles are segregating in the population and that variations outside the core enhancer element could potentially play important roles in the regulation of gene expression. In addition, a slight enrichment of distantly associated SNP pairs was observed in the ~10 kb cis‐regulatory region of ebony, which suggested the presence of interacting elements scattered across the region. In contrast, sequence analysis in the core cis‐regulatory region of tan indicated that SNPs within the region are significantly associated with allele‐specific expression level of this gene. Collectively, the data suggest that the underlying genetic differences in the cis‐regulatory regions that control intraspecific pigmentation variation can be more complex than those of interspecific pigmentation trait differences, where causal genetic changes are typically confined to modular enhancer elements.     

Abdominal pigmentation and growth temperature in Indian Drosophila melanogaster: Evidence for genotype-environment interaction

Phenotypic variability for abdominal pigmentation in females of an Indian natural population ofDrosophila melanogaster was studied using isofemale lines and by rearing the larvae and pupae at 4 different temperatures ranging from 20–30°C. The dark pigmented area was found to increase in all the three segments when the growth temperature decreases. A significant positive correlation was detected for the occurrence of dark pigmentation in the 5th and 6th segments in each growth temperature but for other comparisons the correlation was not regular. Analysis of variance (ANOVA) was carried out both for individual segments over different growth temperatures and also for each temperature over the three abdominal segments and in all cases found to be statistically significant. The results are quite different from the earlier observation in FrenchDrosophila melanogaster and suggest that genes controlling pigmentation are temperature dependent; temperature could affect post-transitional events involved in pigmentation. The present findings also clearly indicate that significant genotype-environment interaction exists, responsible for the production of desired phenotype at the opportune moment during the life span of a species.     

Patterns of Nucleotide Diversity at the Regions Encompassing the Drosophila Insulin-Like Peptide (dilp) Genes: Demography vs. Positive Selection in Drosophila melanogaster

In Drosophila, the insulin-signaling pathway controls some life history traits, such as fertility and lifespan, and it is considered to be the main metabolic pathway involved in establishing adult body size. Several observations concerning variation in body size in the Drosophila genus are suggestive of its adaptive character. Genes encoding proteins in this pathway are, therefore, good candidates to have experienced adaptive changes and to reveal the footprint of positive selection. The Drosophila insulin-like peptides (DILPs) are the ligands that trigger the insulin-signaling cascade. In Drosophila melanogaster, there are several peptides that are structurally similar to the single mammalian insulin peptide. The footprint of recent adaptive changes on nucleotide variation can be unveiled through the analysis of polymorphism and divergence. With this aim, we have surveyed nucleotide sequence variation at the dilp1-7 genes in a natural population of D. melanogaster. The comparison of polymorphism in D. melanogaster and divergence from D. simulans at different functional classes of the dilp genes provided no evidence of adaptive protein evolution after the split of the D. melanogaster and D. simulans lineages. However, our survey of polymorphism at the dilp gene regions of D. melanogaster has provided some evidence for the action of positive selection at or near these genes. The regions encompassing the dilp1-4 genes and the dilp6 gene stand out as likely affected by recent adaptive events.     

Molecular population genetics and evolution of Drosophila meiosis genes

While many functional elements of the meiotic process are well characterized in model organisms, the genetic basis of most of the natural phenotypic variation observed in meiotic pathways has not been determined. To begin to address this issue, we characterized patterns of polymorphism and divergence in the protein-coding regions of 33 genes across 31 lines of Drosophila melanogaster and 6 lines of Drosophila simulans. We sequenced genes known to be involved in chromosome segregation, recombination, DNA repair, and related heterochromatin binding. As expected, we found several of the genes to be highly conserved, consistent with purifying selection. However, a subset of genes showed patterns of polymorphism and divergence typical of other types of natural selection. Moreover, several intriguing differences between the two Drosophila lineages were evident: along the D. simulans lineage we consistently found evidence of adaptive protein evolution, whereas along the D. melanogaster lineage several loci exhibited patterns consistent with the maintenance of protein variation.     

Comparative copulation anatomy of the Drosophila melanogaster species complex (Diptera: Drosophilidae)

The Drosophila melanogaster species complex consists of four species: D. melanogaster, D. simulans, D. sechellia and D. mauritiana. To identify these closely related species, researchers often examine the male genitalia, especially species‐specific shapes of the posterior process, as the most reliable and easily observable character. However, compared to genetic aspects, the evolutionary significance of the posterior process and other genital parts remains largely unexplained. By comparing genital coupling among these species, we revealed that the posterior processes, which are hidden under the female abdominal tergite VII when genital coupling is established, mesh with different parts of the intersegmental membrane between the tergite VIII and the oviscapts and that this membrane region broadens in a species‐specific manner. Furthermore, in D. simulans and D. sechellia, this membrane region is likely to incur wounds from the sharply pointed tip of the posterior process. On the basis of the use and functions of these and other genital parts, we discuss possible evolutionary forces underlying the diversification of genitalia in this group.     

Stage-Specific Effects of Candidate Heterochronic Genes on Variation in Developmental Time along an Altitudinal Cline of Drosophila melanogaster

Background

Previously, we have shown there is clinal variation for egg-to-adult developmental time along geographic gradients in Drosophila melanogaster. Further, we also have identified mutations in genes involved in metabolic and neurogenic pathways that affect development time (heterochronic genes). However, we do not know whether these loci affect variation in developmental time in natural populations.

Methodology/Principal Findings

Here, we constructed second chromosome substitution lines from natural populations of Drosophila melanogaster from an altitudinal cline, and measured egg-adult development time for each line. We found not only a large amount of genetic variation for developmental time, but also positive associations of the development time with thermal amplitude and altitude. We performed genetic complementation tests using substitution lines with the longest and shortest developmental times and heterochronic mutations. We identified segregating variation for neurogenic and metabolic genes that largely affected the duration of the larval stages but had no impact on the timing of metamorphosis.

Conclusions/Significance

Altitudinal clinal variation in developmental time for natural chromosome substitution lines provides a unique opportunity to dissect the response of heterochronic genes to environmental gradients. Ontogenetic stage-specific variation in invected, mastermind, cricklet and CG14591 may affect natural variation in development time and thermal evolution.     

Two genomic regions together cause dark abdominal pigmentation in Drosophila tenebrosa

Pigmentation is a rapidly evolving trait that is under both natural and sexual selection in many organisms. In the quinaria group of Drosophila, nearly all of the 30 species have an abdomen that is light in color with distinct markings; D. tenebrosa is the exception in that it has a completely melanic abdomen with no visible markings. In this study, we use a combination of quantitative genetic and candidate gene approaches to investigate the genetic basis of abdominal pigmentation in D. tenebrosa. We find that abdominal pigmentation is invariant across wild-caught lines of D. tenebrosa and is not sexually dimorphic. Quantitative genetic mapping utilizing crosses between D. tenebrosa and the light-colored D. suboccidentalis indicates that two genomic regions together underlie abdominal pigmentation, including the X-chromosome and an autosome (Muller Element C/E). Further support for their central importance in pigmentation is that experimental introgression of one phenotype into the other species, in either direction, results in introgression of these two genomic regions. Finally, the expression of the X-linked gene yellow in the pupae exactly foreshadows the adult melanization pattern in the abdomen of both species, suggesting that changes in the regulation of yellow are important for the phenotypic divergence of D. tenebrosa from the rest of the quinaria group. These results contribute to a body of work that demonstrates how changes in expression of highly conserved genes can cause substantial phenotypic differences even between closely related species.     

The molecular genetics of clinal variation: a case study of ebony and thoracic trident pigmentation in Drosophila melanogaster from eastern Australia

Widespread pigmentation diversity coupled with a well‐defined genetic system of melanin synthesis and patterning in Drosophila provides an excellent opportunity to study phenotypes undergoing evolutionary change. Pigmentation variation is highly correlated with different ecological variables and is thought to reflect adaptations to different environments. Several studies have linked candidate genes from Drosophila melanogaster to intra‐population variation and interspecific morphological divergence, but less clearly to variation among populations forming pigmentation clines. We characterized a new thoracic trident pigmentation cline in D. melanogaster populations from eastern Australia, and applied a candidate gene approach to explain the majority of the geographically structured phenotypic variation. More melanized populations from higher latitudes tended to express less ebony than their tropical counterparts, and an independent artificial selection experiment confirmed this association. By partitioning temperature dependent effects, we showed that the genetic differences underlying clinal patterns for trident variation at 25 °C do not explain the patterns observed at 16 °C. Changes in thoracic trident pigmentation could be a common evolutionary response to climatically mediated environmental pressures. On the Australian east coast most of the changes appear to be associated with regulatory divergence of the ebony gene but this depends on temperature.     

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.     

Gene-by-Temperature Interactions and Candidate Plasticity Genes for Morphological Traits in Drosophila melanogaster

Understanding the genetic architecture of any quantitative trait requires identifying the genes involved in its expression in different environmental conditions. This goal can be achieved by mutagenesis screens in genetically tractable model organisms such as Drosophila melanogaster. Temperature during ontogenesis is an important environmental factor affecting development and phenotypic variation in holometabolous insects. In spite of the importance of phenotypic plasticity and genotype by environment interaction (GEI) for fitness related traits, its genetic basis has remained elusive. In this context, we analyzed five different adult morphological traits (face width, head width, thorax length, wing size and wing shape) in 42 co-isogenic single P-element insertional lines of Drosophila melanogaster raised at 17°C and 25°C. Our analyses showed that all lines differed from the control for at least one trait in males or females at either temperature. However, no line showed those differences for all traits in both sexes and temperatures simultaneously. In this sense, the most pleiotropic candidate genes were CG34460, Lsd-2 and Spn. Our analyses also revealed extensive genetic variation for all the characters mostly indicated by strong GEIs. Further, our results indicate that GEIs were predominantly explained by changes in ranking order in all cases suggesting that a moderate number of genes are involved in the expression of each character at both temperatures. Most lines displayed a plastic response for at least one trait in either sex. In this regard, P-element insertions affecting plasticity of a large number of traits were associated to the candidate genes Btk29A, CG43340, Drak and jim. Further studies will help to elucidate the relevance of these genes on the morphogenesis of different body structures in natural populations of D. melanogaster.