Physiological and metabolomic consequences of reduced expression of the Drosophila brummer triglyceride Lipase.

Disruption of lipolysis has widespread effects on intermediary metabolism and organismal phenotypes. Defects in lipolysis can be modeled in Drosophila melanogaster through genetic manipulations of brummer (bmm), which encodes a triglyceride lipase orthologous to mammalian Adipose Triglyceride Lipase. RNAi-mediated knock-down of bmm in all tissues or metabolic specific tissues results in reduced locomotor activity, altered sleep patterns and reduced lifespan. Metabolomic analysis on flies in which bmm is downregulated reveals a marked reduction in medium chain fatty acids, long chain saturated fatty acids and long chain monounsaturated and polyunsaturated fatty acids, and an increase in diacylglycerol levels. Elevated carbohydrate metabolites and tricarboxylic acid intermediates indicate that impairment of fatty acid mobilization as an energy source may result in upregulation of compensatory carbohydrate catabolism. bmm downregulation also results in elevated levels of serotonin and dopamine neurotransmitters, possibly accounting for the impairment of locomotor activity and sleep patterns. Physiological phenotypes and metabolomic changes upon reduction of bmm expression show extensive sexual dimorphism. Altered metabolic states in the Drosophila model are relevant for understanding human metabolic disorders, since pathways of intermediary metabolism are conserved across phyla.

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.

An Improved Genome Assembly for Drosophila navojoa, the Basal Species in the mojavensis Cluster.

Three North American cactophilic Drosophila species, D. mojavensis, D. arizonae, and D. navojoa, are of considerable evolutionary interest owing to the shift from breeding in Opuntia cacti to columnar species. The 3 species form the "mojavensis cluster" of Drosophila. The genome of D. mojavensis was sequenced in 2007 and the genomes of D. navojoa and D. arizonae were sequenced together in 2016 using the same technology (Illumina) and assembly software (AllPaths-LG). Yet, unfortunately, the D. navojoa genome was considerably more fragmented and incomplete than its sister species, rendering it less useful for evolutionary genetic studies. The D. navojoa read dataset does not fully meet the strict insert size required by the assembler used (AllPaths-LG) and this incompatibility might explain its assembly problems. Accordingly, when we re-assembled the genome of D. navojoa with the SPAdes assembler, which does not have the strict AllPaths-LG requirements, we obtained a substantial improvement in all quality indicators such as N50 (from 84 kb to 389 kb) and BUSCO coverage (from 77% to 97%). Here we share a new, improved reference assembly for D. navojoa genome, along with a RNAseq transcriptome. Given the basal relationship of the Opuntia breeding D. navojoa to the columnar breeding D. arizonae and D. mojavensis, the improved assembly and annotation will allow researchers to address a range of questions associated with the genomics of host shifts, chromosomal rearrangements and speciation in this group.

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.

piragua encodes a zinc finger protein required for development in Drosophila.

We isolated and characterized embryonic lethal mutations in piragua (prg). The prg locus encodes a protein with an amino terminus Zinc Finger-Associated-Domain (ZAD) and nine C2H2 zinc fingers (ZF). prg mRNA and protein expression during embryogenesis is dynamic with widespread maternal contribution, and subsequent expression in epithelial precursors. About a quarter of prg mutant embryos do not develop cuticle, and from those that do a small fraction have cuticular defects. Roughly half of prg mutants die during embryogenesis. prg mutants have an extended phenocritical period encompassing embryogenesis and first instar larval stage, since the other half of prg mutants die as first or second instar larvae. During dorsal closure, time-lapse high-resolution imaging shows defects arising out of sluggishness in closure, resolving at times in failures of closure. prg is expressed in imaginal discs, and is required for imaginal development. prg was identified in imaginal tissue in a cell super competition screen, together with other genes, like flower. We find that flower mutations are also embryonic lethal with a similar phenocritical period and strong embryonic mutant phenotypes (head involution defects, primarily). The two loci interact genetically in the embryo, as they increase embryonic mortality to close to 90% with the same embryonic phenotypes (dorsal closure and head involution defects, plus lack of cuticle). Mutant prg clones generated in developing dorsal thorax and eye imaginal tissue have strong developmental defects (lack of bristles and ommatidial malformations). prg is required in several developmental morphogenetic processes.

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.

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.