Evidence of Nonrandom Patterns of Functional Chromosome Organization in Danaus plexippus.

Our understanding on the interplay between gene functionality and gene arrangement at different chromosome scales relies on a few Diptera and the honeybee, species with quality reference genome assemblies, accurate gene annotations, and abundant transcriptome data. Using recently generated 'omic resources in the monarch butterfly Danaus plexippus, a species with many more and smaller chromosomes relative to Drosophila species and the honeybee, we examined the organization of genes preferentially expressed at broadly defined developmental stages (larva, pupa, adult males, and adult females) at both fine and whole-chromosome scales. We found that developmental stage-regulated genes do not form more clusters, but do form larger clusters, than expected by chance, a pattern consistent across the gene categories examined. Notably, out of the 30 chromosomes in the monarch genome, 12 of them, plus the fraction of the chromosome Z that corresponds to the ancestral Z in other Lepidoptera, were found enriched for developmental stage-regulated genes. These two levels of nonrandom gene organization are not independent as enriched chromosomes for developmental stage-regulated genes tend to harbor disproportionately large clusters of these genes. Further, although paralogous genes were overrepresented in gene clusters, their presence is not enough to explain two-thirds of the documented cases of whole-chromosome enrichment. The composition of the largest clusters often included paralogs from more than one multigene family as well as unrelated single-copy genes. Our results reveal intriguing patterns at the whole-chromosome scale in D. plexippus while shedding light on the interplay between gene expression and chromosome organization beyond Diptera and Hymenoptera.

Multiscale analysis of the randomization limits of the chromosomal gene organization between Lepidoptera and Diptera.

How chromosome gene organization and gene content evolve among distantly related and structurally malleable genomes remains unresolved. This is particularly the case when considering different insect orders. We have compared the highly contiguous genome assemblies of the lepidopteran Danaus plexippus and the dipteran Drosophila melanogaster, which shared a common ancestor around 290 Ma. The gene content of 23 out of 30 D. plexippus chromosomes was significantly associated with one or two of the six chromosomal elements of the Drosophila genome, denoting common ancestry. Despite the phylogenetic distance, 9.6% of the 1-to-1 orthologues still reside within the same ancestral genome neighbourhood. Furthermore, the comparison D. plexippus-Bombyx mori indicated that the rates of chromosome repatterning are lower in Lepidoptera than in Diptera, although still within the same order of magnitude. Concordantly, 14 developmental gene clusters showed a higher tendency to retain full or partial clustering in D. plexippus, further supporting that the physical association between the SuperHox and NK clusters existed in the ancestral bilaterian. Our results illuminate the scope and limits of the evolution of the gene organization and content of the ancestral chromosomes to the Lepidoptera and Diptera while helping reconstruct portions of the genome in their most recent common ancestor.

A de novo transcriptional atlas in Danaus plexippus reveals variability in dosage compensation across tissues.

A detailed knowledge of gene function in the monarch butterfly is still lacking. Here we generate a genome assembly from a Mexican nonmigratory population and used RNA-seq data from 14 biological samples for gene annotation and to construct an atlas portraying the breadth of gene expression during most of the monarch life cycle. Two thirds of the genes show expression changes, with long noncoding RNAs being particularly finely regulated during adulthood, and male-biased expression being four times more common than female-biased. The two portions of the monarch heterochromosome Z, one ancestral to the Lepidoptera and the other resulting from a chromosomal fusion, display distinct association with sex-biased expression, reflecting sample-dependent incompleteness or absence of dosage compensation in the ancestral but not the novel portion of the Z. This study presents extended genomic and transcriptomic resources that will facilitate a better understanding of the monarch's adaptation to a changing environment.

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.

Trichoderma atroviride from Predator to Prey: Role of the Mitogen-Activated Protein Kinase Tmk3 in Fungal Chemical Defense against Fungivory by Drosophila melanogaster Larvae.

The response to injury represents an important strategy for animals and plants to survive mechanical damage and predation. Plants respond to injury by activating a defense response that includes the production of an important variety of compounds that help them withstand predator attack and recover from mechanical injury (MI). Similarly, the filamentous fungus Trichoderma atroviride responds to MI by strongly modifying its transcriptional profile and producing asexual reproduction structures (conidia). Here, we analyzed whether the response to MI in T. atroviride is related to a possible predator defense mechanism from a metabolic perspective. We found that the production of specific groups of secondary metabolites increases in response to MI but is reduced after fungivory by Drosophila melanogaster larvae. We further show that fungivory results in repression of the expression of genes putatively involved in the regulation of secondary metabolite production in T. atroviride Activation of secondary metabolite production appears to depend on the mitogen-activated protein kinase (MAPK) Tmk3. Interestingly, D. melanogaster larvae preferred to feed on a tmk3 gene replacement mutant rather than on the wild-type strain. Consumption of the mutant strain, however, resulted in increased larval mortality.IMPORTANCE Fungi, like other organisms, have natural predators, including fungivorous nematodes and arthropods that use them as an important food source. Thus, they require mechanisms to detect and respond to injury. Trichoderma atroviride responds to mycelial injury by rapidly regenerating its hyphae and developing asexual reproduction structures. Whether this injury response is associated with attack by fungivorous insects is unknown. Therefore, determining the possible conservation of a defense mechanism to predation in T. atroviride and plants and elucidating the mechanisms involved in the establishment of this response is of major interest. Here, we describe the chemical response of T. atroviride to mechanical injury and fungivory and the role of a MAPK pathway in the regulation of this response.

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.

Re-annotation of eight Drosophila genomes.

The sequenced genomes of the Drosophila phylogeny are a central resource for comparative work supporting the understanding of the Drosophila melanogaster non-mammalian model system. These have also facilitated evolutionary studies on the selected and random differences that distinguish the thousands of extant species of Drosophila. However, full utility has been hampered by uneven genome annotation. We have generated a large expression profile dataset for nine species of Drosophila and trained a transcriptome assembly approach on D. melanogaster that best matched the extensively curated annotation. We then applied this to the other species to add more than 10000 transcript models per species. We also developed new orthologs to facilitate cross-species comparisons. We validated the new annotation of the distantly related Drosophila grimshawi with an extensive collection of newly sequenced cDNAs. This re-annotation will facilitate understanding both the core commonalities and the species differences in this important group of model organisms, and suggests a strategy for annotating the many forthcoming genomes covering the tree of life.

Exploring Effects of Sex and Diet on Drosophila melanogaster Head Gene Expression.

Gene expression depends on sex and environment. We stringently explored the contributions of these effects in Drosophila melanogaster by rearing three distinct wildtype genotypes on isocaloric diets either high in protein or sugar followed by expression profiling of heads from the sexes. By using different genotypes as replicates we developed robust sex- and diet-biased expression responses.

Lipidomic profiles of Drosophila melanogaster and cactophilic fly species: models of human metabolic diseases.

The metabolic syndrome (MetS) is associated with serious diseases and represents an important threat for global public health. The common fruit fly (Drosophila melanogaster) has served as a model organism to study physiological processes of the MetS, because central metabolic pathways are conserved among species, and because the flies are easy to cultivate in a laboratory. In nature, D. melanogaster is a fruit generalist, feeding on diets rich in simple carbohydrates. Other Drosophilids, however, have specialized on distinct resources. Drosophila mojavensis, for example, is endemic to the Sonoran Desert, where it feeds on necrotic cacti which are low in carbohydrates. Its close relative Drosophila arizonae is cactophilic as well, but is also found breeding in fruits containing simple sugars. Previous studies have shown that high-sugar diets negatively affect the larval development of D. mojavensis and increase their triglyceride content, compared to D. melanogaster. More general metabolic profiles, in response to these different diets, however, have yet to be produced for any of the species. In addition, because D. arizonae appears somewhat intermediate between D. melanogaster and D. mojavensis in its development times and survival under the above mentioned diets, its general metabolic profiles are also of interest. Thus, in the present study we ask to what extent the general metabolism of these three different Drosophila species is affected by diets of distinct protein-sugar ratios. To obtain an un-biased view on possibly novel phenomena, we combined untargeted metabolomics with Random Forest data mining.

A Distinctive and Host-Restricted Gut Microbiota in Populations of a Cactophilic Drosophila Species.

Almost all animals possess gut microbial communities, but the nature of these communities varies immensely. For example, in social bees and mammals, the composition is relatively constant within species and is dominated by specialist bacteria that do not live elsewhere; in laboratory studies and field surveys of Drosophila melanogaster, however, gut communities consist of bacteria that are ingested with food and that vary widely among individuals and localities. We addressed whether an ecological specialist in its natural habitat has a microbiota dominated by gut specialists or by environmental bacteria. Drosophila nigrospiracula is a species that is endemic to the Sonoran Desert and is restricted to decaying tissues of two giant columnar cacti, Pachycereus pringlei (cardón cactus) and Carnegiea gigantea (saguaro cactus). We found that the D. nigrospiracula microbiota differs strikingly from that of the cactus tissue on which the flies feed. The most abundant bacteria in the flies are rare or completely absent in the cactus tissue and are consistently abundant in flies from different cacti and localities. Several of these fly-associated bacterial groups, such as the bacterial order Orbales and the genera Serpens and Dysgonomonas, have been identified in prior surveys of insects from the orders Hymenoptera, Coleoptera, Lepidoptera, and Diptera, including several Drosophila species. Although the functions of these bacterial groups are mostly unexplored, Orbales species studied in bees are known to break down plant polysaccharides and use the resulting sugars. Thus, these bacterial groups appear to be specialized to the insect gut environment, where they may colonize through direct host-to-host transmission in natural settings.IMPORTANCE Flies in the genus Drosophila have become laboratory models for microbiota research, yet the bacteria commonly used in these experiments are rarely found in wild-caught flies and instead represent bacteria also present in the food. This study shows that an ecologically specialized Drosophila species possesses a distinctive microbiome, composed of bacterial types absent from the flies' natural food but widespread in other wild-caught insects. This study highlights the importance of fieldwork-informed microbiota research.

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.

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.

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.

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.

Enhanced fertility and chill tolerance after cold-induced reproductive arrest in females of temperate species of the Drosophila buzzatii complex.

Long-term exposure to low temperatures during adult maturation might decrease fertility after cold recovery as a consequence of carry-over effects on reproductive tissues. This pattern should be more pronounced in tropical than in temperate species as protective mechanisms against chilling injuries are expected to be more effective in the latter. We initially determined the lower thermal thresholds to induce ovarian maturation in four closely related Drosophila species, two inhabiting temperate regions and the other two tropical areas of South America. As expected, only temperate species regularly experience cold-inducing conditions for reproductive arrest during winter in their natural environment. Subsequently, we exposed reproductively arrested and mature females to cold-inducing conditions for reproductive arrest over a long period. Following cold exposure, tropical species exhibited a dramatic fertility decline, irrespective of reproductive status. In contrast, not only were temperate females fecund and fertile but also fertility was superior in females that underwent cold-induced reproductive arrest, suggesting that it might act as a protecting mechanism ensuring fertility after cold recovery. Based on these findings, we decided to evaluate the extent to which reproductive status affects cold tolerance and energy metabolism at low temperature. We found a lower metabolic rate and a higher cold tolerance in reproductively arrested females, although only temperate species attained high levels of chill tolerance. These findings highlight the role of cold-induced reproductive arrest as part of an integrated mechanism of cold adaptation that could potentially contribute to the spread of temperate species into higher latitudes or altitudes.

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.

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.

Postmating Reproductive isolation between strains of Drosophila willistoni.

Speciation can occur through the presence of reproductive isolation barriers that impede mating, restrict cross-fertilization, or render inviable/sterile hybrid progeny. The D. willistoni subgroup is ideally suited for studies of speciation, with examples of both allopatry and sympatry, a range of isolation barriers, and the availability of one species complete genome sequence to facilitate genetic studies of divergence. D. w. willistoni has the largest geographic distribution among members of the Drosophila willistoni subgroup, spanning from Argentina to the southern United States, including the Caribbean islands. A subspecies of D. w. willistoni, D. w. quechua, is geographically separated by the Andes mountain range and has evolved unidirectional sterility, in that only male offspring of D. w. quechua females × D. w. willistoni males are sterile. Whether D. w. willistoni flies residing east of the Andes belong to one or more D. willistoni subspecies remains unresolved. Here we perform fecundity assays and show that F1 hybrid males produced from crosses between different strains found in Central America, North America, and northern Caribbean islands are reproductively isolated from South American and southern Caribbean island strains as a result of unidirectional hybrid male sterility. Our results show the existence of a reproductive isolation barrier between the northern and southern strains and suggest a subdivision of the previously identified D. willistoni willistoni species into 2 new subspecies.