Gene expression and alternative splicing dynamics are perturbed in female head transcriptomes following heterospecific copulation.

BACKGROUND: Despite the growing interest in the female side of copulatory interactions, the roles played by differential expression and alternative splicing mechanisms of pre-RNA on tissues outside of the reproductive tract have remained largely unknown. Here we addressed these questions in the context of con- vs heterospecific matings between Drosophila mojavensis and its sister species, D. arizonae. We analyzed transcriptional responses in female heads using an integrated investigation of genome-wide patterns of gene expression, including differential expression (DE), alternative splicing (AS) and intron retention (IR). RESULTS: Our results indicated that early transcriptional responses were largely congruent between con- and heterospecific matings but are substantially perturbed over time. Conspecific matings induced functional pathways related to amino acid balance previously associated with the brain's physiology and female postmating behavior. Heterospecific matings often failed to activate regulation of some of these genes and induced expression of additional genes when compared with those of conspecifically-mated females. These mechanisms showed functional specializations with DE genes mostly linked to pathways of proteolysis and nutrient homeostasis, while AS genes were more related to photoreception and muscle assembly pathways. IR seems to play a more general role in DE regulation during the female postmating response. CONCLUSIONS: We provide evidence showing that AS genes substantially perturbed by heterospecific matings in female heads evolve at slower evolutionary rates than the genome background. However, DE genes evolve at evolutionary rates similar, or even higher, than those of male reproductive genes, which highlights their potential role in sexual selection and the evolution of reproductive barriers.

Host use and host shifts in Drosophila.

Over a thousand Drosophila species have radiated onto a wide range of feeding and breeding sites. These radiations involve adaptations for locating, accepting, and growing in hosts with highly differing characteristics. In a number of species, owing to the availability of sequenced genomes, particular steps in host specialization and genes that control them, are being identified. Many cases of specialization involve the ability to detoxify some component of the host. Examples include Drosophila sechellia and the octanoic acid in Morinda citrifolia, alpha-amanitin in mycophagous drosophilids, and the alkaloids in cactophilic species. Owing to the known ecologies of many species for which genomes exist, the Drosophila model system provides an unprecedented opportunity to simultaneously examine the genes underlying HOST LOCATION, HOST ACCEPTANCE and HOST USE, the types of selection acting upon them and any coevolutionary interactions among the genes underlying these steps.

Developmental and Transcriptomal Responses to Seasonal Dietary Shifts in the Cactophilic Drosophila mojavensis of North America.

Drosophila mojavensis normally breeds in necrotic columnar cactus, but they also feed and breed in Opuntia fruit (prickly pear) which serves as a seasonal resource. The prickly pear fruits (PPFs) are much different chemically from cacti, mainly in their free sugars and lipid content, raising the question of the effects of this seasonal shift on fitness and on gene expression. Here we reared 3 isofemale strains of D. mojavensis collected from different parts of the species' range on semi-natural medium of either cactus or PPF and measured the development time, survival, body weights, and desiccation resistance. All these parameters were affected by diet and by interaction with strain and or sex. Interestingly, however, there appear to be tradeoffs: flies developed faster in prickly pear and the emerging adults were heavier, but those having grown in cactus were more resistant to desiccation. We also evaluated the gene expression of emerging male and female adult flies using RNA-Seq. While more genes were down-regulated in PPF than up-regulated in both sexes, the sexes did differ in expression patterns. The majority of the genes that were preferentially expressed comparing PPF versus cactus underlie metabolism. Genes involved with carbohydrate and lipid metabolism, as well as with the amino acid serine, and their relationship to growth and development reflect the ways in which these dietary differences affect the flies.

Transcriptional responses of ecologically diverse Drosophila species to larval diets differing in relative sugar and protein ratios.

We utilized three ecologically diverse Drosophila species to explore the influence of ecological adaptation on transcriptomic responses to isocaloric diets differing in their relative proportions of protein to sugar. Drosophila melanogaster, a cosmopolitan species that breeds in decaying fruit, exemplifies individuals long exposed to a Western diet higher in sugar, while the natural diet of the cactophilic D. mojavensis, is much lower in carbohydrates. Drosophila arizonae, the sister species of D. mojavensis, is largely cactophilic, but also utilizes rotting fruits that are higher in sugars than cacti. We exposed third instar larvae for 24 hours to diets either (1) high in protein relative to sugar, (2) diets with equal amounts of protein and sugar, and (3) diets low in protein but high in sugar. As we predicted, based upon earlier interspecific studies of development and metabolism, the most extreme differences in gene expression under different dietary conditions were found in D. mojavensis followed by D. arizonae. No differential expression among diets was observed for D. melanogaster, a species that survives well under all three conditions, with little impact on its metabolism. We suggest that these three species together provide a model to examine individual and population differences in vulnerability to lifestyle-associated health problems such as metabolic syndrome and diabetes.

Early events in speciation: Cryptic species of Drosophila aldrichi.

Understanding the earliest events in speciation remains a major challenge in evolutionary biology. Thus identifying species whose populations are beginning to diverge can provide useful systems to study the process of speciation. Drosophila aldrichi, a cactophilic fruit fly species with a broad distribution in North America, has long been assumed to be a single species owing to its morphological uniformity. While previous reports either of genetic divergence or reproductive isolation among different D. aldrichi strains have hinted at the existence of cryptic species, the evolutionary relationships of this species across its range have not been thoroughly investigated. Here we show that D. aldrichi actually is paraphyletic with respect to its closest relative, Drosophila wheeleri, and that divergent D. aldrichi lineages show complete hybrid male sterility when crossed. Our data support the interpretation that there are at least two species of D. aldrichi, making these flies particularly attractive for studies of speciation in an ecological and geographical context.

Evolution of GSTD1 in Cactophilic Drosophila.

GSTD1 is an insect glutathione S-transferase that has received considerable attention because of its role in detoxification of xenobiotic compounds, specifically pesticides and plant allelochemicals involved in detoxification, or in the use of some substrates as a nutritional source. GSTD1 has been implicated in the adaptation to a new cactus host in Drosophila mojavensis and thus constitutes an interesting candidate to study ecological genetics of adaptation in Drosophila. We conducted population genetic and molecular evolution analyses of the GstD1 gene in the context of association with different cactus hosts (Opuntia sp. vs. Columnar) in nine Drosophila species from the repleta group. We observed strong evidence of selection in GstD1 from D. hexastigma. This species is associated with a diverse set of columnar cacti with very complex chemistries. GstD1 sequences from D. hexastigma show evidence of a recent selective sweep, and positive selection at one residue just outside of the active site of the enzyme. The substitution (Q116T) at the site under selection leads to a conformational change in the enzyme that could have important consequences for substrate binding efficiency. Our results suggest that GSTD1 from D. hexastigma may have evolved improved substrate binding in order to adapt to the diverse chemical environments that this species encounters in the wild.

Ectoparasitic mites and their Drosophila hosts.

Only two parasite interactions are known for Drosophila to date: Allantonematid nematodes associated with mycophagous Drosophilids and the ectoparasitic mite Macrocheles subbadius with the Sonoran Desert endemic Drosophila nigrospiracula. Unlike the nematode-Drosophila association, breadth of mite parasitism on Drosophila species is unknown. As M. subbadius is a generalist, parasitism of additional Drosophilids is expected. We determined the extent and distribution of mite parasitism in nature Drosophilids collected in Mexico and southern California. Thirteen additional species of Drosophilids were infested. Interestingly, 10 belong to the repleta species group of the subgenus Drosophila, despite the fact that the majority of flies collected were of the subgenus Sophophora. In all cases but 2, the associated mites were M. subbadius. Drosophila hexastigma was found to have not only M. subbadius, but another Mesostigmatid mite, Paragarmania bakeri, as well. One D. hydei was also found to have a mite from genus Lasioseius attached. In both choice and no-choice experiments, mites were more attracted to repleta group species than to Sophophoran. The extent of mite parasitism clearly is much broader than previously reported and suggests a host bias mediated either by mite preference and/or some mechanism of resistance in particular Drosophilid lineages.

Population Genetics of Overwintering Monarch Butterflies, Danaus plexippus (Linnaeus), from Central Mexico Inferred from Mitochondrial DNA and Microsatellite Markers.

Population genetic variation and demographic history in Danaus plexippus (L.), from Mexico were assessed based on analyses of mitochondrial cytochrome c oxidase subunit I (COI; 658 bp) and subunit II (COII; 503 bp) gene segments and 7 microsatellite loci. The sample of 133 individuals included both migratory monarchs, mainly from 4 overwintering sites within the Monarch Butterfly Biosphere Reserve (MBBR) in central Mexico (states of Michoacán and México), and a nonmigratory population from Irapuato, Guanajuato. Haplotype (h) and nucleotide (π) diversities were relatively low, averaging 0.466 and 0.00073, respectively, for COI, and 0.629 and 0.00245 for COII. Analysis of molecular variance of the COI data set, which included additional GenBank sequences from a nonmigratory Costa Rican population, showed significant population structure between Mexican migratory monarchs and nonmigratory monarchs from both Mexico and Costa Rica, suggesting limited gene flow between the 2 behaviorally distinct groups. Interestingly, while the COI haplotype frequencies of the nonmigratory populations differed from the migratory, they were similar to each other, despite the great physical distance between them. Microsatellite analyses, however, suggested a lack of structure between the 2 groups, possibly owing to the number of significant deviations from Hardy-Weinberg equilibrium resulting from heterzoygote deficiencies found for most of the loci. Estimates of demographic history of the combined migratory MBBR monarch population, based on the mismatch distribution and Bayesian skyline analyses of the concatenated COI and COII data set (n = 89) suggested a population expansion dating to the late Pleistocene (~35000-40000 years before present) followed by a stable effective female population size (Nef) of about 6 million over the last 10000 years.

Genome Evolution in Three Species of Cactophilic Drosophila.

We report genomes of two species of cactophilic Drosophila: Drosophila arizonae and D. navojoa These two are the closest relatives of D. mojavensis, forming the D. mojavensis cluster. D. mojavensis and D. arizonae diverged from D. navojoa ∼5.8 Mya, while the split between D. arizonae and D. mojavensis is more recent, at 1.5 Mya. Together the three genomes provide opportunities to examine genomic changes associated with speciation and host shifts in this ecologically defined group of flies. The three species are also separated by fixed inversion differences in three of their six chromosomes. While the levels of nucleotide divergence in the colinear chromosomes are significantly lower than in the inverted chromosomes, consistent with a past role of the inversions in preventing gene flow, the patterns differ among the inverted chromosomes when the locations of nucleotides inside or outside of the inversions are considered. For Muller element E, there is greater divergence external to the inversion breakpoints. For Muller A, the divergence is slightly higher inside the inversions, while for Muller B, the breakpoints and hence the difference in substitutions in relation to the inversions could not be determined. The differences among the inverted chromosomes, especially once the breakpoints are clearly established, could aid in dating the origins of the inversions.

The secret lives of Drosophila flies.

Flies of the genus Drosophila, and particularly those of the species Drosophila melanogaster, are best known as laboratory organisms. As with all model organisms, they were domesticated for empirical studies, but they also continue to exist as wild populations. Decades of research on these flies in the laboratory have produced astounding and important insights into basic biological processes, but we have only scratched the surface of what they have to offer as research organisms. An outstanding challenge now is to build on this knowledge and explore how natural history has shaped D. melanogaster in order to advance our understanding of biology more generally.

Parents Without Partners: Drosophila as a Model for Understanding the Mechanisms and Evolution of Parthenogenesis.

Of 40 Drosophila species screened to date, a majority have shown some ability to at least initiate parthenogenetic development. In one case, Drosophila mangebeirai, natural populations are entirely female, making it the only obligate parthenogenetic species of Drosophila Only a few of the species that exhibit the ability to undergo early embryonic development of unfertilized eggs successfully respond to selection for parthenogenetic production of adult flies. Laboratory strains of parthenogenetic Drosophila mercatorum have been created by artificial selection on multiple occasions, but the proportion of eggs undergoing development to adulthood has never exceeded 8%. Selection produces gains in the number of unfertilized eggs undergoing early development, but the majority arrest at the embryonic or first larval instar stages. Four components to successful parthenogenesis include (1) a female's propensity to lay unfertilized eggs, (2) the ability of the eggs to restore diploidy, (3) the ability of the parthenogenetically produced diploid embryo to complete larval development and pupation, and (4) the existence of genetic variability within and among Drosophila species in the frequency of parthenogenesis suggests the existence of multiple steps in its evolution and offers a way to explore the genetics of this unusual reproductive strategy.

The 19 genomes of Drosophila: a BAC library resource for genus-wide and genome-scale comparative evolutionary research.

The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125-168 kb), low nonrecombinant clone content (0.3-5.3%), and deep coverage (9.1-42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny.

"Cost" of virginity in wild Drosophila melanogaster females.

Laboratory studies have revealed a significant "cost of mating" to Drosophila melanogaster females in the form of reduced longevity. The effect is attributable to nonsperm components of the ejaculate. Female D. melanogaster are known to mate up to six times in nature, and given that they do not typically remate daily, it raises the question as to the extent to which the longevity of wild mated females is reduced. Here I addressed this question by comparing the longevity of wild virgin females, collected as they emerged from rotting fruit, to the longevity of randomly collected mature females at the same site. Because the randomly collected females all were inseminated and were fully pigmented at the time of collection, they already were older than the virgins when the experiment began. Contrary to expectations from laboratory studies, the older, mated females lived significantly longer than the virgins. Rather than a "cost of mating," there appears to be a "cost of virginity" to female D. melanogaster in the wild.

Sexual isolation and mating propensity among allopatric Drosophila mettleri populations.

Drosophila mettleri is found in deserts of North America breeding in soil soaked by the juices of necrotic cacti. Saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) are the usual host cacti in Mexico and Arizona, while prickly pear (Opuntia spp.) is used by an isolated population on Santa Catalina Island off the southern California Coast. Populations of D. mettleri show significant local genetic differentiation, especially when geographical isolation is coupled with host shifts. We tested for evidence of sexual isolation among allopatric populations of D. mettleri using a variety of choice and no-choice tests. Populations exhibited significant differences in mating propensity, which translated into significant deviations from random mating. While in some cases these deviations were consistent with sexual isolation, in others, negative assortative mating was observed. No relationship between degree of genetic differentiation and the appearance of sexual isolation was detected.

Drosophila biology in the genomic age.

Over the course of the past century, flies in the family Drosophilidae have been important models for understanding genetic, developmental, cellular, ecological, and evolutionary processes. Full genome sequences from a total of 12 species promise to extend this work by facilitating comparative studies of gene expression, of molecules such as proteins, of developmental mechanisms, and of ecological adaptation. Here we review basic biological and ecological information of the species whose genomes have recently been completely sequenced in the context of current research.

Sperm fate and function in reproductive isolation in Drosophila.

Central to the study of speciation is the identification of the isolating mechanisms that reduce gene flow. Of special interest are those isolating mechanisms that are the earliest to arise. Sperm fate and function play a major role in two distinct types of reproductive isolation in the genus Drosophila. First, postcopulatory-prezygotic (PCPZ) incompatibilities between the male ejaculate and a heterospecific female reproductive tract can result in a reduction in sperm fertilisation success. Although PCPZ incompatibilities have, until recently, received little attention, overwhelming diversity in reproductive morphology and biochemistry indicates that they may play an important role in speciation in this genus. The second type of isolating mechanism that affects sperm occurs in the testes of hybrid males produced by heterospecific matings. These individuals often suffer from dysfunction in spermatogenesis, resulting in the production of aberrant sperm. Hybrid male sterility and hybrid inviability are examples of postzygotic (PZ) reproductive isolation. The observation that hybrid sterility is pervasive among males of all taxonomic groups is known as Haldane's rule. Here we discuss both the evolutionary origins, and functional causes of both PCPZ incompatibilities, which affects sperm fate and function in females, and one type of PZ incompatibility, hybrid male sterility, or sperm dysfunction in hybrid males. Although these two mechanisms of isolation are quite distinct, they are similarly caused by breakdowns in epistatic interactions which occur in the encounter between two divergent genomes. Molecular, cytological, and empirical data are discussed, as is relevant evolutionary theory.

Evolutionary genetics of reproductive behavior in Drosophila: connecting the dots.

Species of the genus Drosophila exhibit enormous variation in all of their reproductive behaviors: resource use and specialization, courtship signaling, sperm utilization, and female remating. The genetic bases of this variability and its evolution are poorly understood. At the same time, Drosophila comparative genomics now has developed to a point at which approaches previously only possible with D. melanogaster can be exploited to address these questions. We have taken advantage of the known phylogenetic relationships of this group of flies not only to place these behaviors in an evolutionary framework, but to provide a roadmap for future genetic studies.

Sympatry, allopatry and sexual isolation between Drosophila mojavensis and D. arizonae.

Populations of the North American cactophilic fruitfly Drosophila mojavensis and its sibling species D. arizonae exist both in sympatry and in allopatry. Females of D. arizonae, regardless of their population of origin, are effectively completely isolated behaviorally from D. mojavensis males. On the other hand, females of D. mojavensis from the sympatric populations in Sonora, Mexico exhibit significantly stronger premating isolation from D. arizonae males than do D. mojavensis females from allopatric populations from the Baja California peninsula. Earlier studies interpreted these limited observations as support for reinforcement. Since the time of those studies, additional allopatric populations of D. mojavensis have been collected from southern California and from Santa Catalina Island, off the coast of southern California. Here, we tested the prediction that if sympatry is in fact associated with increased isolation in D. mojavensis, these additional allopatric populations also should show, relative to the sympatric ones, less isolation from D. arizonae. Our results are consistent with this prediction and suggest that isolation is in fact stronger in sympatry.

Perspective: female remating, operational sex ratio, and the arena of sexual selection in Drosophila species.

As commonly observed among closely related species within a variety of taxa, Drosophila species differ considerably in whether they exhibit sexual dimorphism in coloration or morphology. Those Drosophila species in which male external sexual characters are minimal or absent tend, instead, to have exaggerated ejaculate traits such as sperm gigantism or seminal nutrient donations. Underlying explanations for the interspecific differences in the presence of external morphological sexual dimorphism versus exaggerated ejaculate traits are addressed here by examining the opportunity for sexual selection on males to occur before versus after mating in 21 species of Drosophila. Female remating frequency, an important component of the operational sex ratio, differs widely among Drosophila species and appears to dictate whether the arena of sexual selection is prior to, as opposed to after, copulation. Infrequent female mating results in fewer mating opportunities for males and thus stronger competition for receptive females that favors the evolution of male characters that maximize mating success. On the other hand, rapid female remating results in overlapping ejaculates in the female reproductive tract, such that ejaculate traits which enhance fertilization success are favored. The strong association between female remating frequency in a given species and the presence of sexually selected external versus internal male characters indicates that the relationship be examined in other taxa as well.