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Insect flight is a complex trait involved in different behaviors, from the search for sexual partners, food, or breeding sites. Many studies have postulated the adaptive advantages of certain morphological traits in relation to increased flight capacity, such as low values of wing loading or high values of wing:thorax ratio and wing-aspect ratio. However, few studies have evaluated the relationship between variables related to flight and morphological traits in Drosophila. This work aimed to study morphological traits in males and females of two pairs of sibling species: Drosophila buzzatii Patterson and Wheeler-Drosophila koeferae Fontdevila and Wasserman, and Drosophila melanogaster Meigen-Drosophila simulans Sturtevant, and to analyze its relationship with flight. We detected the highest proportion of flight time in D. koepferae and D. simulans compared to D. buzzatii and D. melanogaster, respectively. Our results also revealed sexual dimorphism, with males exhibiting a higher proportion of flight time than females. Surprisingly, we did not find a general pattern to explain the relationship between morphology and the proportion of flight time because associations varied depending upon the analyses (considering all groups together or each sex-species combination separately). Moreover, these associations explained a low percentage of variation, suggesting that other nonmorphological components related to flight, such as physiological variables, should be taken into account. This work allowed us to show the variability and complexity of an aspect of flight, suggesting that the adaptive role of the morphological traits studied might have been overestimated., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

The excessive and indiscriminate use of synthetic insecticides has led to environmental pollution, wildlife destruction, and adverse effects on human health, while simultaneously giving rise to resistance in insect pest populations. This adaptive trait is expressed through various mechanisms, such as changes in the cuticle, heightened activities of detoxifying enzymes, and alterations in the sites of action that reduce their affinity for insecticides. In this context, we associate variation in toxicological response with genomic variation, to identify genetic polymorphisms underlying the different steps of the insect (genotype)-response (phenotype)-insecticide (environment) interaction. Under this framework, our objective was to investigate the genetic factors involved in the toxicological response of D. melanogaster lines when exposed to citronellal and eucalyptol vapors (monoterpenes of plant origin). We quantified KT50 in adult males, representing the time necessary for half of the exposed individuals to be turned upside down (unable to walk or fly). Since the genomes of all lines used are completely sequenced, we perform a Genome Wide Association Study to analyze the genetic underpinnings of the toxicological response. Our investigation enabled the identification of 656 genetic polymorphisms and 316 candidate genes responsible for the overall phenotypic variation. Among these, 162 candidate genes (77.1%) exhibited specificity to citronellal, 45 (21.4%) were specific to eucalyptol, and 3 candidate genes (1.5%) namely CG34345, robo2, and Ac13E, were implicated in the variation for both monoterpenes. These suggest a widespread adaptability in the response to insecticides, encompassing genes influenced by monoterpenes and those orchestrating resistance to the toxicity of these compounds., Competing Interests: Declaration of competing interest The authors declare they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dehydration is a stress factor for organisms inhabiting natural habitats where water is scarce. Thus, it may be expected that species facing arid environments will develop mechanisms that maximize resistance to desiccation. Insects are excellent models for studying the effects of dehydration as well as the mechanisms and processes that prevent water loss since the effect of desiccation is greater due to the higher area/volume ratio than larger animals. Even though physiological and behavioral mechanisms to cope with desiccation are being understood, the genetic basis underlying the mechanisms related to variation in desiccation resistance and the context-dependent effect remain unsolved. Here we analyze the genetic bases of desiccation resistance in Drosophila melanogaster and identify candidate genes that underlie trait variation. Our quantitative genetic analysis of desiccation resistance revealed sexual dimorphism and extensive genetic variation. The phenotype-genotype association analyses (GWAS) identified 71 candidate genes responsible for total phenotypic variation in desiccation resistance. Half of these candidate genes were sex-specific suggesting that the genetic architecture underlying this adaptive trait differs between males and females. Moreover, the public availability of desiccation data analyzed on the same lines but in a different lab allows us to investigate the reliability and repeatability of results obtained in independent screens. Our survey indicates a pervasive micro-environment lab-dependent effect since we did not detect overlap in the sets of genes affecting desiccation resistance identified between labs., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

In phytophagous insects, oviposition behaviour is an important component of habitat selection and, given the multiplicity of genetic and environmental factors affecting its expression, is defined as a complex character resulting from the sum of interdependent traits. Here, we study two components of egg-laying behaviour: oviposition acceptance (OA) and oviposition preference (OP) in Drosophila melanogaster using three natural fruits as resources (grape, tomato and orange) by means of no-choice and two-choice experiments, respectively. This experimental design allowed us to show that the results obtained in two-choice assays (OP) cannot be accounted for by those resulting from no-choice assays (OA). Since the genomes of all lines used are completely sequenced, we perform a genome-wide association study to identify and characterize the genetic underpinnings of these oviposition behaviour traits. The analyses revealed different candidate genes affecting natural genetic variation of both OA and OP traits. Moreover, our results suggest behavioural and genetic decoupling between OA and OP and that egg-laying behaviour is plastic and context-dependent. Such independence in the genetic architectures of OA and OP variation may influence different aspects of oviposition behaviour, including plasticity, canalization, host shift and maintenance of genetic variability, which contributes to the adoption of adaptive strategies during habitat selection., (© 2022 European Society for Evolutionary Biology.)

It has been demonstrated that phenotypic plasticity and genotype by environment interaction are important for coping with new and heterogeneous environments during invasions. Zaprionus indianus Gupta (Diptera: Drosophilidae) is an Afrotropical invasive fly species introduced to the South American continent in 1999. This species is generalist and polyphagous, since it develops and feeds in several different fruit species. These characteristics of Z. indianus suggest that phenotypic plasticity and genotype by environment interaction may be important in this species invasion process. In this sense, our aim was to investigate the role of genetic variation for phenotypic plasticity (genotype by environment interaction) in Z. indianus invasion of the South American continent. Specifically, we quantified quantitative genetic variation and genotype by environment interactions of morphological and life history traits in different developmental environments, that is, host fruits. This was done in different populations in the invasive range of Z. indianus in Argentina. Results showed that Z. indianus populations have considerable amounts of quantitative genetic variation. Also, genotype by environment interactions was detected for the different traits analyzed in response to the different developmental environments. Interestingly, the amounts and patterns of these parameters differed between populations. We interpreted these results as the existence of differences in evolutionary potential between populations that have an important role in the short- and long-term success of the Z. indianus invasion process., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

Zaprionus indianus is a fly species native to the Afrotropical biogeographic region that invaded the South American continent 20 years ago. Its southernmost record is 34°S in areas with temperate climates with cold winters. To better understand its invasion biology, we investigated physiological responses to winter-like abiotic conditions that may be relevant in Z. indianus geographic expansion. We characterized Z. indianus females reproductive traits (ovarian maturation and fertility) and survival in response to cold treatments with summer-like and winter-like photoperiods. We also compared these traits between native (Yokadouma, Africa) and invasive (Yuto, South America) range wild-derived flies. We showed that Z. indianus females have the ability to arrest ovarian maturation and maintain fertility following recovery from cold stress. The critical temperature for ovarian maturation of this species was estimated at c. 13 °C, an intermediate value between those of tropical and temperate drosophilid species. Wild-derived females from Yuto responded to winter-like photoperiod by slowing down ovarian maturation at low but permissive temperatures of 14 °C and 16 °C and also delayed the start of oviposition after cold treatment. Yuto flies also survived better and recovered 20% faster from chill coma than flies from Yokadouma. These results are consistent with a scenario of local adaptations or phenotypic plasticity in the invaded range, and suggest that photoperiod could act as modulator of ovarian arrest. Conversely, the fact that native range flies showed higher fertility after cold recovery than females from invaded range is not indicative of local adaptation. All in all, our findings report a set of physiological responses that would enable Z. indianus expansion to temperate and cold areas, but also results that are compatible with a limitation to the invasion process., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Studying the processes affecting variation for preadult viability is essential to understand the evolutionary trajectories followed by natural populations. This task requires focusing on the complex nature of the phenotype-genotype relationship by taking into account usually neglected aspects of the phenotype and recognizing the modularity between different ontogenetic stages. Here, we describe phenotypic variability for viability during the larval and pupal stages in lines derived from three natural populations of Drosophila melanogaster, as well as the variability for phenotypic plasticity and canalization at two different rearing temperatures. The results indicate that the three populations present significant phenotypic differences for preadult viability. Furthermore, distinct aspects of the phenotype (means, plasticity, canalization, plasticity of canalization) are affected by different genetic bases underlying changes in viability in a stage- and environment-specific manner. These findings explain the generalized maintenance of genetic variability for this fitness trait., (© 2019 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2019 European Society For Evolutionary Biology.)

Changes in the environmental conditions experienced by naturally occurring populations are frequently accompanied by changes in adaptive traits allowing the organism to cope with environmental unpredictability. Phenotypic plasticity is a major aspect of adaptation and it has been involved in population dynamics of interacting species. In this study, phenotypic plasticity (i.e., environmental sensitivity) of morphological adaptive traits were analyzed in the cactophilic species Drosophila buzzatii and Drosophila koepferae (Diptera: Drosophilidae) considering the effect of crowding conditions (low and high density), type of competition (intraspecific and interspecific competition) and cacti hosts (Opuntia and Columnar cacti). All traits (wing length, wing width, thorax length, wing loading and wing aspect) showed significant variation for each environmental factor considered in both Drosophila species. The phenotypic plasticity pattern observed for each trait was different within and between these cactophilic Drosophila species depending on the environmental factor analyzed suggesting that body size-related traits respond almost independently to environmental heterogeneity. The effects of ecological factors analyzed in this study are discussed in order to elucidate the causal factors investigated (type of competition, crowding conditions and alternative host) affecting the election of the breeding site and/or the range of distribution of these cactophilic species., (© 2016 Institute of Zoology, Chinese Academy of Sciences.)

Body size is a complex character associated to several fitness related traits that vary within and between species as a consequence of environmental and genetic factors. Latitudinal and altitudinal clines for different morphological traits have been described in several species of Drosophila and previous work identified genomic regions associated with such variation in D. melanogaster. However, the genetic factors that orchestrate morphological variation have been barely studied. Here, our main objective was to investigate genetic variation for different morphological traits associated to the second chromosome in natural populations of D. melanogaster along latitudinal and altitudinal gradients in Argentina. Our results revealed weak clinal signals and a strong population effect on morphological variation. Moreover, most pairwise comparisons between populations were significant. Our study also showed important within-population genetic variation, which must be associated to the second chromosome, as the lines are otherwise genetically identical. Next, we examined the contribution of different candidate genes to natural variation for these traits. We performed quantitative complementation tests using a battery of lines bearing mutated alleles at candidate genes located in the second chromosome and six second chromosome substitution lines derived from natural populations which exhibited divergent phenotypes. Results of complementation tests revealed that natural variation at all candidate genes studied, invected, Fasciclin 3, toucan, Reticulon-like1, jing and CG14478, affects the studied characters, suggesting that they are Quantitative Trait Genes for morphological traits. Finally, the phenotypic patterns observed suggest that different alleles of each gene might contribute to natural variation for morphological traits. However, non-additive effects cannot be ruled out, as wild-derived strains differ at myriads of second chromosome loci that may interact epistatically with mutant alleles.

Drosophila buzzatii (Patterson & Wheeler), a typical cactophilic species of the repleta group, is registered for the first time emerging from Melon (Cucumis melo) in western Argentina. The analysis of inversion polymorphism and genetic diversity of mitochondrial cytochrome oxidase subunit I gene (mtCOI) provided additional evidence that corroborated the presence of a high proportion of D. buzzatii among the flies emerged from melon. This finding set the scenario for a broader range of possible hosts and host-related distribution and dispersion for this widespread species.

Many biotic and abiotic variables influence the dispersal and distribution of organisms. Temperature has a major role in determining these patterns because it changes daily, seasonally and spatially, and these fluctuations have a significant impact on an organism's behaviour and fitness. Most ecologically relevant phenotypes that are adaptive are also complex and thus they are influenced by many underlying loci that interact with the environment. In this study, we quantified the degree of thermal phenotypic plasticity within and among populations by measuring chill-coma recovery times of lines reared from egg to adult at two different environmental temperatures. We used sixty genotypes from six natural populations of Drosophila melanogaster sampled along a latitudinal gradient in South America. We found significant variation in thermal plasticity both within and among populations. All populations exhibit a cold acclimation response, with flies reared at lower temperatures having increased resistance to cold. We tested a series of environmental parameters against the variation in population mean thermal plasticity and discovered the mean thermal plasticity was significantly correlated with altitude of origin of the population. Pairing our data with previous experiments on viability fitness assays in the same populations in fixed and variable environments suggests an adaptive role of this thermal plasticity in variable laboratory environments. Altogether, these data demonstrate abundant variation in adaptive thermal plasticity within and among populations., (© 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.)

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.

In the holometabolous insect Drosophila melanogaster, genetic, physiological and anatomical aspects of olfaction are well known in the adult stage, while larval stages olfactory behavior has received some attention it has been less studied than its adult counterpart. Most of these studies focus on olfactory receptor (Or) genes that produce peripheral odor recognition. In this paper, through a loss-of-function screen using P-element inserted lines and also by means of expression analyses of larval olfaction candidate genes, we extended the uncovering of the genetic underpinnings of D. melanogaster larval olfactory behavior by demonstrating that larval olfactory behavior is, in addition to Or genes, orchestrated by numerous genes with diverse functions. Also, our results point out that the genetic architecture of olfactory behavior in D. melanogaster presents a dynamic and changing organization across environments and ontogeny.

Spatial or temporal differences in environmental variables, such as temperature, are ubiquitous in nature and impose stress on organisms. This is especially true for organisms that are isothermal with the environment, such as insects. Understanding the means by which insects respond to temperature and how they will react to novel changes in environmental temperature is important for understanding the adaptive capacity of populations and to predict future trajectories of evolutionary change. The organismal response to heat has been identified as an important environmental variable for insects that can dramatically influence life history characters and geographic range. In the current study we surveyed the amount of variation in heat tolerance among Drosophila melanogaster populations collected at diverse sites along a latitudinal gradient in Argentina (24°-38°S). This is the first study to quantify heat tolerance in South American populations and our work demonstrates that most of the populations surveyed have abundant within-population phenotypic variation, while still exhibiting significant variation among populations. The one exception was the most heat tolerant population that comes from a climate exhibiting the warmest annual mean temperature. All together our results suggest there is abundant genetic variation for heat-tolerance phenotypes within and among natural populations of Drosophila and this variation has likely been shaped by environmental temperature.

Background: The Drosophila wing represents a particularly appropriate model to investigate the developmental control of phenotypic variation. Previous studies which aimed to identify candidate genes for wing morphology demonstrated that the genetic basis of wing shape variation in D. melanogaster is composed of numerous genetic factors causing small, additive effects. In this study, we analyzed wing shape in males and females from 191 lines of D. melanogaster, homozygous for a single P-element insertion, using geometric morphometrics techniques. The analysis allowed us to identify known and novel candidate genes that may contribute to the expression of wing shape in each sex separately and to compare them to candidate genes affecting wing size which have been identified previously using the same lines., Results: Our results indicate that more than 63% of induced mutations affected wing shape in one or both sexes, although only 33% showed significant differences in both males and females. The joint analysis of wing size and shape revealed that only 19% of the P-element insertions caused coincident effects on both components of wing form in one or both sexes. Further morphometrical analyses revealed that the intersection between veins showed the smallest displacements in the proximal region of the wing. Finally, we observed that mutations causing general deformations were more common than expected in both sexes whereas the opposite occurred with those generating local changes. For most of the 94 candidate genes identified, this seems to be the first record relating them with wing shape variation., Conclusions: Our results support the idea that the genetic architecture of wing shape is complex with many different genes contributing to the trait in a sexually dimorphic manner. This polygenic basis, which is relatively independent from that of wing size, is composed of genes generally involved in development and/or metabolic functions, especially related to the regulation of different cellular processes such as motility, adhesion, communication and signal transduction. This study suggests that understanding the genetic basis of wing shape requires merging the regulation of vein patterning by signalling pathways with processes that occur during wing development at the cellular level.

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.

The inversion polymorphisms of the cactophilic Drosophila buzzatti Patterson and Wheeler (Diptera: Drosophilidae) were studied in new areas of its distribution in Argentina. A total of thirty-eight natural populations, including 29 from previous studies, were analyzed using multiple regression analyses. The results showed that about 23% of total variation was accounted for by a multiple regression model in which only altitude contributed significantly to population variation, despite the fact that latitude and longitude were also included in the model. Also, inversion frequencies exhibited significant associations with mean annual temperature, precipitation, and atmospheric pressure. In addition, expected heterozygosity exhibited a negative association with temperature and precipitation and a positive association with atmospheric pressure. The close similarity of the patterns detected in this larger dataset to previous reports is an indication of the stability of the clines. Also, the concurrence of the clines detected in Argentina with those reported for colonizing populations of Australia suggests the involvement of natural selection as the main mechanism shaping inversion frequencies in D. buzzatii.

Even though substantial progress has been made to elucidate the physiological and environmental factors underpinning differences in body size, little is known about its genetic architecture. Furthermore, all animal species bear a specific relationship between the size of each organ and overall body size, so different body size traits should be investigated as well as their sexual dimorphism that may have an important impact on the evolution of body size. We have surveyed 191 co-isogenic lines of Drosophila melanogaster, each one of them homozygous for a single P-element insertion, and assessed the effects of mutations on different body size traits compared to the P-element-free co-isogenic control. Nearly 60% of the lines showed significant differences with respect to the control for these traits in one or both sexes and almost 35% showed trait- and sex-specific effects. Candidate gene mutations frequently increased body size in males and decreased it in females. Among the 92 genes identified, most are involved in development and/or metabolic processes and their molecular functions principally include protein-binding and nucleic acid-binding activities. Although several genes showed pleiotropic effects in relation to body size, few of them were involved in the expression of all traits in one or both sexes. These genes seem to be important for different aspects related to the general functioning of the organism. In general, our results indicate that the genetic architecture of body size traits involves a large fraction of the genome and is largely sex and trait specific.

Background: Understanding the genetic architecture of ecologically relevant adaptive traits requires the contribution of developmental and evolutionary biology. The time to reach the age of reproduction is a complex life history trait commonly known as developmental time. In particular, in holometabolous insects that occupy ephemeral habitats, like fruit flies, the impact of developmental time on fitness is further exaggerated. The present work is one of the first systematic studies of the genetic basis of developmental time, in which we also evaluate the impact of environmental variation on the expression of the trait., Results: We analyzed 179 co-isogenic single P[GT1]-element insertion lines of Drosophila melanogaster to identify novel genes affecting developmental time in flies reared at 25 degrees C. Sixty percent of the lines showed a heterochronic phenotype, suggesting that a large number of genes affect this trait. Mutant lines for the genes Merlin and Karl showed the most extreme phenotypes exhibiting a developmental time reduction and increase, respectively, of over 2 days and 4 days relative to the control (a co-isogenic P-element insertion free line). In addition, a subset of 42 lines selected at random from the initial set of 179 lines was screened at 17 degrees C. Interestingly, the gene-by-environment interaction accounted for 52% of total phenotypic variance. Plastic reaction norms were found for a large number of developmental time candidate genes., Conclusion: We identified components of several integrated time-dependent pathways affecting egg-to-adult developmental time in Drosophila. At the same time, we also show that many heterochronic phenotypes may arise from changes in genes involved in several developmental mechanisms that do not explicitly control the timing of specific events. We also demonstrate that many developmental time genes have pleiotropic effects on several adult traits and that the action of most of them is sensitive to temperature during development. Taken together, our results stress the need to take into account the effect of environmental variation and the dynamics of gene interactions on the genetic architecture of this complex life-history trait.

Odour-guided behaviour is a quantitative trait determined by many genes that are sensitive to gene-environment interactions. Different natural populations are likely to experience different selection pressures on the genetic underpinnings of chemosensory behaviour. However, few studies have reported comparisons of the quantitative genetic basis of olfactory behaviour in geographically distinct populations. We generated isofemale lines of Drosophila melanogaster from six populations in Argentina and measured larval and adult responses to benzaldehyde. There was significant variation within populations for both larval and adult olfactory behaviour and a significant genotype x sex interaction (GSI) for adult olfactory behaviour. However, there is substantial variation in the contribution of GSI to the total phenotypic variance among populations. Estimates of evolvability are orders of magnitude higher for larvae than for adults. Our results suggest that the potential for evolutionary adaptation to the chemosensory environment is greater at the larval feeding stage than at the adult reproductive stage.

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F1 hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F1 hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.

Background: The rapid evolution of genital morphology is a fascinating feature that accompanies many speciation events. However, the underlying patterns and explanatory processes remain to be settled. In this work we investigate the patterns of intraspecific variation and interspecific divergence in male genitalic morphology (size and shape) in the cactophilic sibling species Drosophila buzzatii and D. koepferae. Genital morphology in interspecific hybrids was examined and compared to the corresponding parental lines., Results: Despite of being siblings, D. buzzatii and D. koepferae showed contrasting patterns of genital morphological variation. Though genitalic size and shape variation have a significant genetic component in both species, shape varied across host cacti only in D. buzzatii. Such plastic expression of genital shape is the first evidence of the effect of rearing substrate on genitalic morphology in Drosophila. Hybrid genital morphology was not intermediate between parental species and the morphological resemblance to parental strains was cross-dependent., Conclusion: Our results suggest the evolution of different developmental networks after interspecific divergence and the existence of a complex genetic architecture, involving genetic factors with major effects affecting genital morphology.

Understanding the genetic architecture of complex traits requires identification of the underlying genes and characterization of gene-by-gene and genotype-by-environment interactions. Behaviors that mediate interactions between organisms and their environment are complex traits expected to be especially sensitive to environmental conditions. Previous studies on the olfactory avoidance response of Drosophila melanogaster showed that the genetic architecture of this model behavior depends on epistatic networks of pleiotropic genes. We performed a screen of 1339 co-isogenic p[GT1]-element insertion lines to identify novel genes that contribute to odor-guided behavior and identified 55 candidate genes with known p[GT1]-element insertion sites. Characterization of the expression profiles of 10 p[GT1]-element insertion lines showed that the effects of the transposon insertions are often dependent on developmental stage and that hypomorphic mutations in developmental genes can elicit profound adult behavioral deficits. We assessed epistasis among these genes by constructing all possible double heterozygotes and measuring avoidance responses under two stimulus conditions. We observed enhancer and suppressor effects among subsets of these P-element-tagged genes, and surprisingly, epistatic interactions shifted with changes in the concentration of the olfactory stimulus. Our results show that the manifestation of epistatic networks dynamically changes with alterations in the environment.

Drosophila buzzatii and D. koepferae are two sibling species that breed on the necrotic tissues of several cactus species and show a certain degree of niche overlap. Also, they show differences in several life history traits, such as body size and developmental time, which probably evolved as a consequence of adaptation to different host plants. In this work we investigate the ecological and genetic factors affecting wing morphology variation both within and between species. Three wing traits were scored, distal and proximal wing length and width in isofemale lines reared in two of the most important host cacti: Opuntia sulphurea and Trichocereus terschekii. Our results revealed that differences between species and sexes in wing size and shape were significant, whereas the cactus factor was only significant for wing size. Intraspecific analyses showed that differences among isofemale lines were highly significant for both size and shape in both species, suggesting that an important fraction of variation in wing morphology has a genetic basis. Moreover, the line by cactus interaction, which can be interpreted as a genotype by environment interaction, also accounted for a significant proportion of variation. In summary, our study shows that wing size is phenotypically plastic and that populations of D. buzzatii and D. koepferae harbour substantial amounts of genetic variation for wing size and shape. Interspecific differences in wing size and shape are interpreted in terms of spatial predictability of the different host plants in nature.

The genetic and ecological basis of viability and developmental time differences between Drosophila buzzatii and D. koepferae were analysed using the isofemale line technique. Several isofemale lines were sampled from pairs of allopatric/sympatric populations of each species. Flies were reared in media prepared with decaying tissues of two of the main natural cactus hosts of each species. This experimental design enabled us to evaluate the relative contribution of phenotypic plasticity, genetic variation and genotype by environment interaction (G x E) to total phenotypic variation for two fitness traits, viability and developmental time. Our results revealed significant G x E in both traits, suggesting that the maintenance of genetic variation can be explained, at least in part, by diversifying selection in different patches of a heterogeneous environment in both species. However, the relative importance of the factors involved in the G x E varied between traits and populations within species. For viability, the G x E can be mainly attributed to changes in the rank order of lines across cacti. However, the pattern was different for developmental time. In D. buzzatii the G x E can be mainly accounted for by changes in among line variance across cacti, whereas changes in the rank order of lines across cacti was the main component in D. koepferae. These dissimilar patterns of variation between traits and species suggest that the evolutionary forces shaping genetic variation for developmental time and viability vary between populations within species and between species.

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 face of strong selection due 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 us 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.

We combined transcriptional profiling and quantitative genetic analysis to elucidate the genetic architecture of olfactory behavior in Drosophila melanogaster. We applied whole-genome expression analysis to five coisogenic smell-impaired (smi) mutant lines and their control. We used analysis of variance to partition variation in transcript abundance between males and females and between smi genotypes and to determine the genotype-by-sex interaction. A total of 666 genes showed sexual dimorphism in transcript abundance, and 530 genes were coregulated in response to one or more smi mutations, showing considerable epistasis at the level of the transcriptome in response to single mutations. Quantitative complementation tests of mutations at these coregulated genes with the smi mutations showed that in most cases (67%) epistatic interactions for olfactory behavior mirrored epistasis at the level of transcription, thus identifying new candidate genes regulating olfactory behavior.

Most animals depend on olfaction for survival and procreation. Odor-guided behavior is a quantitative trait, with phenotypic variation due to multiple segregating quantitative trait loci (QTL). Despite its profound biological importance, the genetic basis of naturally occurring variation in olfactory behavior remains unexplored. Here, we mapped a single Drosophila QTL affecting variation in avoidance response to benzaldehyde, using a population of recombinant inbred lines. Deficiency complementation mapping resolved this region into one female- and one male-specific QTL. Subsequent quantitative complementation tests to all available mutations of positional candidate genes showed that the female-specific QTL failed to complement a P-element insertional mutation, l(3)04276. The P-element insertion was in the intron of a novel gene, Vanaso, which contains a putative guanylate binding protein domain, is highly polymorphic, and is expressed in the third antennal segment, the major olfactory organ of Drosophila. No expression was detected in the fly brain, suggesting that Vanaso plays a role in peripheral chemosensory processes rather than in central integration of olfactory information. QTL mapping followed by quantitative complementation tests to deficiencies and mutations is an effective strategy for gene discovery that allows characterization of effects of recessive lethal genes on adult phenotypes and here enabled identification of a candidate gene that contributes to sex-specific quantitative variation in olfactory behavior.

We tested for the occurrence of oviposition acceptance for different media prepared with cactus tissues of three alternative cactus hosts: Opuntia sulphurea, O. quimilo and Trichocereus terschekii for 4 consecutive days in lines of two Drosophila buzzatii populations and one population of D. koepferae. Our results showed that the former laid significantly more eggs on both Opuntia cacti than on T. terschekii, whereas D. koepferae preferred T. terschekii. In addition, fecundity schedules differed between species: D. buzzatii laid similar numbers of eggs along the four-day sampling period, whereas D. koepferae showed an oviposition peak on the second day of egg collection on T. terschekii. We suggest that the between-species disparities observed in oviposition acceptance and fecundity schedule may be related to the temporal and spatial predictability of Opuntia versus T. terschekii (cardón) as part of the different adaptive strategies that have evolved after the split of D. koepferae and D. buzzatii from their recent common ancestor. Therefore, the willingness to accept hosts would be an important factor in the habitat selection and in the maintenance of species diversity.

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.

In this study we present the results of an analysis of differential longevity associated with Drosophila buzzatii second chromosome inversion karyotypes based on the assessment of more than 1000 individuals collected in a natural population. Comparisons of inversion frequencies between emerged and bait-collected flies showed not only that inversion arrangements were associated with differential longevity, but also that selection was sex specific. Because each individual fly was scored for thorax length and karyotype, we were able to show that longevity selection favoring larger flies coupled with the average effect of inversions on thorax length can account for the change of inversion frequencies due to longevity in females. The observed genotypic-by-sex interaction could be an important mechanism involved in the maintenance of the polymorphism. Arrangement 2Jz 3 , which was shown to impaired fecundity in two independent previous studies, exhibited a positive effect on longevity. This pattern of negative pleiotropy may be another plausible mechanism accounting for the maintenance of the polymorphism., (© 1999 The Society for the Study of Evolution.)

The inversion polymorphism of the cactophilic fly Drosophila buzzatii was studied in two natural populations. We assessed the temporal changes and microspatial population structure. We observed a significant increase in the frequency of arrangement 2J at the expense of 2ST in both populations. These gene arrangements appear to affect the life-history of flies differently. Environmental heterogeneity explains the karyotype coexistence in nature. The analysis of population structure showed that differentiation of inversion frequencies among individual breeding sites, the rotting clacodes of Opuntia vulgaris, was highly significant. The karyotypic frequencies did not depart significantly from Hardy-Weinberg expectations, neither in individual rots nor in the total population. These results suggest that the observed population structure can be easily accounted by random genetic drift.

Previous works in a colonized and an original population of Drosophila buzzatii have shown a consistent relationship between the inversion polymorphism and thorax length, a measure of body size. However, the populations studied in those reports share a close genealogical relationship as suggested by several lines of evidence. In the present paper, we revisit this issue by analysing the correlation between second chromosome arrangements and thorax length in two Argentinian natural populations (Termas de Río Hondo and Arroyo Escobar) from different biogeographic areas with different host plants. Our findings are: (1) inversion frequencies were significantly different between populations; (2) the mean thorax length of flies collected in both populations was not significantly different; and (3) we obtain confirming evidence that flies carrying 2st, the ancestral gene order, have on average a smaller body size than those carrying the derived arrangements (2j and 2jz3). These results suggest that the biometrical effect of inversions on body size previously described are due to genetic differences between arrangements and not to the close historical relationship between the populations studied in previous reports.

Previous work has shown fitness differences among chromosomal arrangements by means of selection component analysis in two Drosophila buzzatii natural populations, one of which is native to Argentina and the other a colonized population from Carboneras, Spain. Founder effects or niche shifts were proposed to explain the differences observed in the pattern of pleiotropic effects of inversions on fitness components. In this paper, we address the possible role of niche shifts by determining whether differential attraction to, oviposition on, or utilization of the rotting cladodes of two different Opuntia species (O. quimilo and O. ficus-indica) occurred among individuals carrying different second chromosome karyotypes in a natural Argentinian population. Through the analysis of more than 2500 individuals comprising five different life cycle stages associated with the necroses of these two cactus species, we found that the distributions of inversion frequencies in samples of adult flies, third instar larvae and emerging adults collected on both Opuntia species were not significantly different. Likewise, no evidence of differential oviposition was observed. These findings suggest that niche shifts cannot, solely, account for the changes observed in the Carboneras population. In addition, the selection component analysis did not reveal any significant relationship between chromosomal arrangements and the fitness components tested. These results suggest either that fitness differences might be too small to be detected or that the assumptions of the model concerning the mode of selection may not be tenable in the studied population.

Size-related phenotypic variation among second-chromosome karyotypes in Drosophila buzzatii was examined in an Argentinian natural population. For all measured traits (thorax and wing length; wing, head and face width), this inversion polymorphism exhibited a significant and (additive) linear contribution to the phenotypic variance in newly emerged wild flies. The results suggest that only overall body size, and not body shape, is affected, as no karyotypic variation was found for any trait when the effects of differences in within-karyotype size were removed with Burnaby's method. Likewise, in an experiment of longevity selection in the wild, variation in chromosomal frequencies was verified in the direction predicted on the basis of: (i) previous studies on longevity selection for body size in the wild and (ii) the pattern of chromosomal effects we observed on size. The direction of such selection is consistent with a pattern of antagonistic selection detected in previous studies on the inversion polymorphism.

D. buzzatii is a cactophilic species associated with several cactaceae in Argentina. This particular ecological niche implies that this species is faced with a non-uniform environment constituted by discrete and ephemeral breeding sites, which are colonized by a finite number of inseminated females. The genetic consequences of this population structure upon the second chromosome polymorphism were investigated by means of F-statistics in a natural endemic population of Argentina. The present study suggests that differentiation of inversion frequencies in third instar larvae among breeding sites has taken place mainly at random and selection is not operating to determine the structure of this population. The average number of parents breeding on a single pad seems to be similar to the number colonizing Opuntia ficus indica rotting cladodes in Carboneras, a derived population from Spain. There is no significant excess of heterokaryotypes within pads or in the population as a whole. The results obtained in the present study suggest that the potential role of selective versus stochastic factors relative to the among pad heterogeneity in the population here studied is different from that of the Spanish population previously reported. Potential mechanisms responsible for these differences are discussed.

The correlation between body size and longevity was tested in an Argentinian natural population of Drosophila buzzatii. Mean thorax length of flies newly emerging from rotting cladodes of Opuntia vulgaris was significantly smaller than that of two samples of flies caught at baits. The present results which might be interpreted as directional selection for longevity favoring larger flies are in agreement with previous results achieved in a Spanish natural population of D. buzzatii. Flies emerging from different substrates showed significant differences in thorax length, suggesting that an important fraction of phenotypic variance can be attributed to environmental variability. However, laboratory and field work in different populations of D. buzzatii showed a significant genetic component for thorax length variation.

We demonstrate a genetic correlation between rearrangements of the second chromosome of D. buzzatii and thorax length, as a measure of body size. The results indicate that 2j and 2jz3 arrangements are correlated with large size, whereas 2st arrangement is correlated with small size. Some inversions (2st and 2jz3) show dominant effects and others (2j/jz3) exhibit overdominance. These results show that at least 25 per cent of body size variation may be accounted for by the studied karyotypes. The possible integration of the genotypic, phenotypic and fitness levels, and also the possible implications to life-history evolution theories, are discussed. These results suggest that, under moderate to high heritability values, some kinds of chromosomal endocyclic and/or balancing selection may be valuable mechanisms for maintenance of body size variation.