Impact of body melanisation on contrasting levels of desiccation resistance in a circumtropical and a generalist <Emphasis Type="Italic">Drosophila</Emphasis> species

We investigated the role of cuticular lipids, body melanisation and body size in conferring contrasting levels of desiccation resistance in latitudinal populations of Drosophila melanogaster and Drosophila ananassae on the Indian subcontinent. Contrary to the well known role of cuticular lipids in water proofing in diverse insect taxa, there is lack of geographical variations in the amount of cuticular lipids per fly in both the species. In D. ananassae, quite low levels of body melanisation are correlated with lower desiccation resistance. By contrast, increased levels of desiccation resistance are correlated with quite high melanisation in D. melanogaster. Thus, species specific cuticular melanisation patterns are significantly correlated with varying levels of desiccation resistance within as well as between populations and across species. Role of body melanisation in desiccation resistance is further supported by the fact that assorted dark and light flies differ significantly in cuticular water loss, hemolymph and dehydration tolerance. However, similar patterns of body size variation do not account for contrasting levels of desiccation resistance in these two Drosophila species. Climatic selection is evidenced by multiple regression analysis with seasonal amplitude of thermal and humidity changes (Tcv and RHcv) along latitude on the Indian subcontinent. Finally, the contrasting levels of species specific distribution patterns are negatively correlated with RHcv of sites of origin of populations i.e. a steeper negative slope for D. ananassae corresponds with its desiccation sensitivity as compared with D. melanogaster. Thus, evolutionary changes in body melanisation impact desiccation resistance potential as well as distribution patterns of these two Drosophila species on the Indian subcontinent.     

Sex-specific differences in desiccation resistance and the use of energy metabolites as osmolytes in Drosophila melanogaster flies acclimated to dehydration stress

In the Indian subcontinent, there are significant between-population variations in desiccation resistance in Drosophila melanogaster, but the physiological basis of adult acclimation responses to ecologically relevant humidity conditions is largely unknown. We tested the hypothesis that increased desiccation resistance in acclimated flies is associated with changes in cuticular permeability and/or content of energy metabolites that act as osmolytes. Under an ecologically relevant humidity regime (~50 % relative humidity), both sexes showed desiccation acclimation which persisted for 2–3 days. However, only females responded to acclimation at ~5 % relative humidity (RH). Acclimated flies exhibited no changes in the rate of water loss, which is consistent with a lack of plastic changes in cuticular traits (body melanization, epicuticular lipid). Therefore, changes in cuticular permeability are unlikely in drought-acclimated adult flies of D. melanogaster. In acclimated flies, we found sex differences in changes in the content of osmolytes (trehalose in females versus glycogen in males). These sex-specific changes in osmolytes are rapid and reversible and match to corresponding changes in the increased desiccation resistance levels of acclimated flies. Further, the increased content of trehalose in females and glycogen in males support the bound-water hypothesis for water retention in acclimated flies. Thus, drought acclimation in adult flies of D. melanogaster involves inducible changes in osmolytes (trehalose and glycogen), while there is little support for changes in cuticular permeability.     

Trade-off of energy metabolites as well as body color phenotypes for starvation and desiccation resistance in montane populations of Drosophila melanogaster

Storage of energy metabolites has been investigated in different sets of laboratory selected desiccation or starvation resistant lines but few studies have examined such changes in wild-caught populations of Drosophila melanogaster. In contrast to parallel selection of desiccation and starvation tolerance under laboratory selection experiments, opposite clines were observed in wild populations of D. melanogaster. If resistance to desiccation and starvation occurs in opposite directions under field conditions, we may expect a trade-off for energy metabolites but such correlated changes are largely unknown. We tested whether there is a trade-off for storage as well as actual utilization of carbohydrates (trehalose and glycogen), lipids and proteins in D. melanogaster populations collected from different altitudes (512-2500 m). For desiccation resistance, darker flies (> 50% body melanization) store more body water content and endure greater loss of water (higher dehydration tolerance) as compared to lighter flies (< 30% body melanization). Based on within population analysis, we found evidence for coadapted phenotypes i.e. darker flies store and actually utilize more carbohydrates to confer greater desiccation resistance. In contrast, higher starvation resistance in lighter flies is associated with storage and actual utilization of greater lipid amount. However, darker and lighter flies did not vary in the rate of utilization of carbohydrates under desiccation stress; and of lipids under starvation stress. Thus, we did not find support for the hypothesis that a lower rate of utilization of energy metabolites may contribute to greater stress resistance. Further, for increased desiccation resistance of darker flies, about two-third of total energy budget is provided by carbohydrates. By contrast, lighter flies derive about 66% of total energy content from lipids which sustain higher starvation tolerance. Our results support evolutionary trade-off for storage as well as utilization of energy metabolites for desiccation versus starvation resistance in D. melanogaster.     

Quantitative genetic analysis suggests causal association between cuticular hydrocarbon composition and desiccation survival in Drosophila melanogaster

Survival to low relative humidity is a complex adaptation, and many repeated instances of evolution to desiccation have been observed among Drosophila populations and species. One general mechanism for desiccation resistance is Cuticular Hydrocarbon (CHC) melting point. We performed the first Quantitative Trait Locus (QTL) map of population level genetic variation in desiccation resistance in D. melanogaster. Using a panel of Recombinant Inbred Lines (RILs) derived from a single natural population, we mapped QTL in both sexes throughout the genome. We found that in both sexes, CHCs correlated strongly with desiccation resistance. At most desiccation resistance loci there was a significant association between CHCs and desiccation resistance of the sort predicted from clinal patterns of CHC variation and biochemical properties of lipids. This association was much stronger in females than males, perhaps because of greater overall abundance of CHCs in females, or due to correlations between CHCs used for waterproofing and sexual signalling in males. CHC evolution may be a common mechanism for desiccation resistance in D. melanogaster. It will be interesting to compare patterns of CHC variation and desiccation resistance in species which adapt to desiccation, and rainforest restricted species which cannot.     

Melanic pigmentation and light preference within and between two Drosophila species

Environmental adaptation and species divergence often involve suites of co‐evolving traits. Pigmentation in insects presents a variable, adaptive, and well‐characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies'' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light‐bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana‐N14 and D. americana‐A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light‐bodied D. novamexicana‐N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.     

A Single Gene Causes an Interspecific Difference in Pigmentation in Drosophila

The genetic basis of species differences remains understudied. Studies in insects have contributed significantly to our understanding of morphological evolution. Pigmentation traits in particular have received a great deal of attention and several genes in the insect pigmentation pathway have been implicated in inter- and intraspecific differences. Nonetheless, much remains unknown about many of the genes in this pathway and their potential role in understudied taxa. Here we genetically analyze the puparium color difference between members of the virilis group of Drosophila. The puparium of Drosophila virilis is black, while those of D. americana, D. novamexicana, and D. lummei are brown. We used a series of backcross hybrid populations between D. americana and D. virilis to map the genomic interval responsible for the difference between this species pair. First, we show that the pupal case color difference is caused by a single Mendelizing factor, which we ultimately map to an ∼11-kb region on chromosome 5. The mapped interval includes only the first exon and regulatory region(s) of the dopamine N-acetyltransferase gene (Dat). This gene encodes an enzyme that is known to play a part in the insect pigmentation pathway. Second, we show that this gene is highly expressed at the onset of pupation in light brown taxa (D. americana and D. novamexicana) relative to D. virilis, but not in the dark brown D. lummei. Finally, we examine the role of Dat in adult pigmentation between D. americana (heavily melanized) and D. novamexicana (lightly melanized) and find no discernible effect of this gene in adults. Our results demonstrate that a single gene is entirely or almost entirely responsible for a morphological difference between species.     

Resistance to environmental stress in Drosophila ananassae: latitudinal variation and adaptation among populations

Geographical variation in traits related to fitness is often the result of adaptive evolution. Stress resistance traits in Drosophila often show clinal variation, suggesting that selection affects resistance traits either directly or indirectly. Multiple stress resistance traits were investigated in 45 natural populations of Drosophila ananassae collected from all over India. There was significant positive correlation between starvation resistance and lipid content. Significant negative correlations between desiccation and lipid content and between desiccation and heat resistance were also found. Flies from lower latitudes had higher starvation resistance, heat resistance and lipid content but the pattern was reversed for desiccation resistance. These results suggest that flies from different localities varied in their susceptibility to starvation because of difference in their propensity to store body lipid. Multiple regression analysis provided evidence of climatic selection driven by latitudinal variation in the seasonal amplitude of temperature and humidity changes within the Indian. Finally, our results suggest a high degree of variation in stress resistance at the population level in D. ananassae.     

The vectoring of cactophilic yeasts by Drosophila

Summary At two locations in the Sonoran Desert, yeasts were sampled from species of Drosophila, the flies' cactus hosts, and other neighboring sources of cactophilic yeasts to determine the relation between the yeasts vectored by the fly and the yeasts found in their breeding sites. D. mojavensis, D. nigrospiracula, and D. mettleri vectored yeast assemblages significantly more similar to the yeast species found on the rot from which the flies were collected than to the yeasts found on other rots from the flies host cactus or other rotting cactus at the same site. Rots with Drosophila had fewer yeast species than those without flies, suggesting that flies were associated with younger rots. Rots with flies and the Drosophila also had more yeast species with the capability to produce ethyl acetate than rots without flies. The results support the contention that cactophilic Drosophila feed on a subset of the yeasts available in an area, and may act to maintain differences among the yeast communities found on different species of cactus.     

THE INFLUENCE OF ABDOMINAL PIGMENTATION ON DESICCATION AND ULTRAVIOLET RESISTANCE IN TWO SPECIES OF DROSOPHILA

Drosophila yakuba and D. santomea are sister species that differ in their levels of abdominal pigmentation; D. yakuba shows heavily pigmented posterior abdominal segments in both sexes, whereas D. santomea lacks dark pigment anywhere on its body. Using naturally collected lines, we demonstrate the existence of altitudinal variation in abdominal pigmentation in D. yakuba but not in D. santomea. We use the variation in pigmentation within D. yakuba and two body‐color mutants in D. yakuba to elucidate selective advantage of differences in pigmentation. Our results indicate that although differences in abdominal pigmentation have no effect on desiccation resistance, lighter pigmentation confers ultraviolet radiation resistance in this pair of species.     

Variation in adult life history and stress resistance across five species of<Emphasis Type="Italic">Drosophila</Emphasis>

Dry weight at eclosion, adult lifespan, lifetime fecundity, lipid and carbohydrate content at eclosion, and starvation and desiccation resistance at eclosion were assayed on a long-term laboratory population ofDrosophila melanogaster, and one recently wild-caught population each of four other species ofDrosophila, two from themelanogaster and two from theimmigrans species group. The relationships among trait means across the five species did not conform to expectations based on correlations among these traits inferred from selection studies onD. melanogaster. In particular, the expected positive relationships between fecundity and size/lipid content, lipid content and starvation resistance, carbohydrate (glycogen) content and desiccation resistance, and the expected negative relationship between lifespan and fecundity were not observed. Most traits were strongly positively correlated between sexes across species, except for fractional lipid content and starvation resistance per microgram lipid. For most traits, there was evidence for significant sexual dimorphism but the degree of dimorphism did not vary across species except in the case of adult lifespan, starvation resistance per microgram lipid, and desiccation resistance per microgram carbohydrate. Overall,D. nasuta nasuta andD. sulfurigaster neonasuta (immigrans group) were heavier at eclosion than themelanogaster group species, and tended to have somewhat higher absolute lipid content and starvation resistance. Yet, these twoimmigrans group species were shorter-lived and had lower average daily fecundity than themelanogaster group species. The smallest species,D. malerkotliana (melanogaster group), had relatively high daily fecundity, intermediate lifespan and high fractional lipid content, especially in females.D. ananassae (melanogaster group) had the highest absolute and fractional carbohydrate content, but its desiccation resistance per microgram carbohydrate was the lowest among the five species. In terms of overall performance, the laboratory population ofD. melanogaster was clearly superior, under laboratory conditions, to the other four species if adult lifespan, lifetime fecundity, average daily fecundity, and absolute starvation and desiccation resistance are considered. This finding is contrary to several recent reports of substantially higher adult lifespan and stress resistance in recently wild-caught flies, relative to flies maintained for a long time in discretegeneration laboratory cultures. Possible explanations for these apparent anomalies are discussed in the context of the differing selection pressures likely to be experienced byDrosophila populations in laboratory versus wild environments. This paper is dedicated to the memory of our friend and former colleague Dr Hans Raj Negi, who tragically passed away at a very young age in a road accident in November 2003.     

Eco-genetics of desiccation resistance in Drosophila

Climate change globally perturbs water circulation thereby influencing ecosystems including cultivated land. Both harmful and beneficial species of insects are likely to be vulnerable to such changes in climate. As small animals with a disadvantageous surface area to body mass ratio, they face a risk of desiccation. A number of behavioural, physiological and genetic strategies are deployed to solve these problems during adaptation in various Drosophila species. Over 100 desiccation-related genes have been identified in laboratory and wild populations of the cosmopolitan fruit fly Drosophila melanogaster and its sister species in large-scale and single-gene approaches. These genes are involved in water sensing and homeostasis, and barrier formation and function via the production and composition of surface lipids and via pigmentation. Interestingly, the genetic strategy implemented in a given population appears to be unpredictable. In part, this may be due to different experimental approaches in different studies. The observed variability may also reflect a rich standing genetic variation in Drosophila allowing a quasi-random choice of response strategies through soft-sweep events, although further studies are needed to unravel any underlying principles. These findings underline that D. melanogaster is a robust species well adapted to resist climate change-related desiccation. The rich data obtained in Drosophila research provide a framework to address and understand desiccation resistance in other insects. Through the application of powerful genetic tools in the model organism D. melanogaster, the functions of desiccation-related genes revealed by correlative studies can be tested and the underlying molecular mechanisms of desiccation tolerance understood. The combination of the wealth of available data and its genetic accessibility makes Drosophila an ideal bioindicator. Accumulation of data on desiccation resistance in Drosophila may allow us to create a world map of genetic evolution in response to climate change in an insect genome. Ultimately these efforts may provide guidelines for dealing with the effects of climate-related perturbations on insect population dynamics in the future.     

Seasonal changes in recombination characteristics in a natural population of Drosophila melanogaster

Environmental seasonality is a potent evolutionary force, capable of maintaining polymorphism, promoting phenotypic plasticity and causing bet-hedging. In Drosophila, environmental seasonality has been reported to affect life-history traits, tolerance to abiotic stressors and immunity. Oscillations in frequencies of alleles underlying fitness-related traits were also documented alongside SNPs across the genome. Here, we test for seasonal changes in two recombination characteristics, crossover rate and crossover interference, in a natural D. melanogaster population from India using morphological markers of the three major chromosomes. We show that winter flies, collected after the dry season, have significantly higher desiccation tolerance than their autumn counterparts. This difference proved to hold also for hybrids with three independent marker stocks, suggesting its genetic rather than plastic nature. Significant between-season changes are documented for crossover rate (in 9 of 13 studied intervals) and crossover interference (in four of eight studied pairs of intervals); both single and double crossovers were usually more frequent in the winter cohort. The winter flies also display weaker plasticity of both recombination characteristics to desiccation. We ascribe the observed differences to indirect selection on recombination caused by directional selection on desiccation tolerance. Our findings suggest that changes in recombination characteristics can arise even after a short period of seasonal adaptation (~8–10 generations).Subject terms: Structural variation, Evolutionary biology, Evolutionary genetics     

Sexual dimorphism for water balance mechanisms in montane populations of Drosophila kikkawai

Conservation of water is critical to the ecological success of Drosophila species living in the drier montane localities of the Western Himalayas. We observed clinal variation in desiccation resistance for both sexes of Drosophila kikkawai from an altitudinal transect (512–2226 m above sea level). Since more than 90 per cent of body water is lost through cuticular transpiration, the target of selection may be cuticular lipids or cuticular melanization. We tested whether melanic females and non-melanic males of D. kikkawai have similar mechanisms of desiccation resistance. There is clinal variation in the amount of cuticular lipids per fly in males, but not in females. By contrast, for females, elevational increase in melanization is positively correlated with desiccation resistance and negatively with cuticular water loss, but there is no variation in the amount of cuticular lipids. Thus, sexual dimorphism for the mechanism of desiccation resistance in D. kikkawai matches the water proofing role of body melanization as well as cuticular lipids.     

Starvation resistance is positively correlated with body lipid proportion in five wild caught Drosophila simulans populations

Stress resistance traits in Drosophila often show clinal variation, suggesting that selection affects resistance traits either directly or indirectly. One of the most common causes of stress for animals is the shortage or suboptimal quality of food, and individuals within many species must survive periods of starvation or exposure to nutritionally imbalanced diets. This study determines the relationship between starvation resistance, body lipid content, and lifespan in five recently collected Drosophila simulans populations from four distinct geographic localities. Despite rearing under standard nutritional conditions, we observed significant differences in starvation resistance between sexes and between localities. If body lipid proportion is included as a covariate in statistical analysis the difference between the sexes remains (slopes are parallel, with males more susceptible than females to starvation across all lipid proportions) but the effect of locality disappears. This result suggests that flies from different localities differ in their susceptibility to starvation because of differences in their propensity to store body lipid. We observed a negative relationship between lifespan and starvation resistance in both males and females, suggesting a fitness cost to increasing lipid reserves. These data raise issues about the role of diet in maintaining life history trait variation within and among populations. In conclusion, we show many similarities and surprising differences in life history traits between D. simulans and Drosophila melanogaster.     

Host Species and Environmental Effects on Bacterial Communities Associated with Drosophila in the Laboratory and in the Natural Environment

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory-reared Drosophila species. We find no evidence for host species effects in lab-reared flies; instead, lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila–associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild-caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore, we were able to identify candidate pathogens in natural fly isolates.     

Attractiveness and exploitation of decaying herbage by Drosophila in temperate woodland

Summary The Drosophila fauna of a deciduous flood plain forest rich in undergrowth near the river Isar, close to Munich, Germany, was surveyed in summer 1990. Decaying herbage baits (decay artificially induced) were set out to study the exploitation of that resource by Drosophila. Sixteen plant species belonging to several families dominant in the collecting area were tested. All attracted and produced drosophilid flies. Ten Drosophila species utilized decaying plant material as breeding sites; at least eight of the ten are polyphagous. Decaying stalks and leaves of Angelica sylvestris (Apiaceae) were examined in detail. In the case of the most frequent species of Drosophila attracted to A. sylvestris, the number of adults collected did not correlate with the number of flies emerging from the substrate. This was particularly true of D. limbata and D. phalerata. When oviposition and larval development of D. limbata and D. phalerata on A. sylvestris was tested in the laboratory, the number of offspring per female was the same in both species. The difference between these two species of the quinaria group in the exploitation of A. sylvestris in the field is therefore not due to differential suitability of the substrate.     

Fitness components of Drosophila melanogaster developed on a standard laboratory diet or a typical natural food source

Drosophila melanogaster is often used as a model organism in evolutionary biology and ecophysiology to study evolutionary processes and their physiological mechanisms. Diets used to feed Drosophila cultures differ between laboratories and are often nutritious and distinct from food sources in the natural habitat. Here we rear D. melanogaster on a standard diet used in our laboratory and a field diet composed of decomposing apples collected in the field. Flies developed on these two diet compositions are tested for heat, cold, desiccation, and starvation resistance as well as developmental time, dry body mass and fat percentage. The nutritional compositions of the standard and field diets were analyzed, and discussed in relation to the phenotypic observations. Results showed marked differences in phenotype of flies from the two types of diets. Flies reared on the field diet are more starvation resistant and they are smaller, leaner, and have lower heat resistance compared to flies reared on the standard diet. Sex specific effects of diet type are observed for several of the investigated traits and the strong sexual dimorphism usually observed in desiccation resistance in D. melanogaster disappeared when rearing the flies on the field diet. Based on our results we conclude that care should be taken in extrapolating results from one type of diet to another and especially from laboratory to field diets.     

Substrate Vibrations during Courtship in Three Drosophila species

While a plethora of studies have focused on the role of visual, chemical and near-field airborne signals in courtship of Drosophila fruit flies, the existence of substrate-borne vibrational signals has been almost completely overlooked. Here we describe substrate vibrations generated during courtship in three species of the D. melanogaster group, from the allegedly mute species D. suzukii, its sister species D. biarmipes, and from D. melanogaster. In all species, we recorded several types of substrate vibrations which were generated by locomotion, abdominal vibrations and most likely through the activity of thoracic wing muscles. In D. melanogaster and D. suzukii, all substrate vibrations described in intact males were also recorded in males with amputated wings. Evidence suggests that vibrational signalling may be widespread among Drosophila species, and fruit flies may provide an ideal model to study various aspects of this widespread form of animal communication.     

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 ontogeny of color: developmental origins of divergent pigmentation in Drosophila americana and D. novamexicana

Pigmentation is a model trait for evolutionary and developmental analysis that is particularly amenable to molecular investigation in the genus Drosophila. To better understand how this phenotype evolves, we examined divergent pigmentation and gene expression over developmental time in the dark‐bodied D. americana and its light‐bodied sister species D. novamexicana. Prior genetic analysis implicated two enzyme‐encoding genes, tan and ebony, in pigmentation divergence between these species, but questions remain about the underlying molecular mechanisms. Here, we describe stages of pupal development in both species and use this staging to determine when pigmentation develops and diverges between D. americana and D. novamexicana. For the developmental stages encompassing pigment divergence, we compare mRNA expression of tan and ebony over time and between species. Finally, we use allele‐specific expression assays to determine whether interspecific differences in mRNA abundance have a cis‐regulatory basis and find evidence of cis‐regulatory divergence for both tan and ebony. cis‐regulatory divergence affecting tan had a small effect on mRNA abundance and was limited to a few developmental stages, yet previous data suggests that this divergence is likely to be biologically meaningful. Our study suggests that small and developmentally transient expression changes may contribute to phenotypic diversification more often than commonly appreciated. Recognizing the potential phenotypic impact of such changes is important for a scientific community increasingly focused on dissecting quantitative variation, but detecting these types of changes will be a major challenge to elucidating the molecular basis of complex traits.