Strong Positive Selection in Aedes aegypti and the Rapid Evolution of Insecticide Resistance.

Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti's biology and global population structure and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti, many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species and add to a growing body of evidence suggesting that Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

Detection of alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio Iso-Seq.

Alternative splicing is a common feature in eukaryotes that not only increases the transcript diversity, but also has functional consequences. In insects, alternative splicing has been found associated with resistance to pesticides and Bt toxins. Up to date, the alternative splicing in western corn rootworm (Diabrotica virgifera virgifera LeConte) has not been studied. To investigate its alternative splicing pattern and relation to Bt resistance, we carried out single-molecule real-time (SMRT) transcript sequencing and Iso-seq analysis on resistant, eCry3.1Ab-selected and susceptible, unselected, western corn rootworm neonate midguts which fed on seedling maize with and without eCry3.1Ab for 12 and 24 h. We present transcriptome-wide alternative splicing patterns of western corn rootworm midgut in response to feeding on eCry3.1Ab-expressing corn using a comprehensive approach that combines both RNA-seq and SMRT transcript sequencing techniques. The results showed genes in western corn rootworm are highly alternatively spliced, which happens on 67.73% of multi-exon genes. One of the alternative splicing events we identified was a novel peritrophic matrix protein with two alternative splicing isoforms. Analysis of differential exon usage between resistant and susceptible colonies showed that in eCry3.1Ab-resistant western corn rootworm, expression of one isoform was significantly higher than in the susceptible colony, while no significant differences between colonies were observed with the other isoform. Our results provide the first survey of alternative splicing in western corn rootworm and suggest that the observed alternatively spliced isoforms of peritrophic matrix protein may be associated with eCry3.1Ab resistance in western corn rootworm.

Genetic Signatures of Drug Response Variability in Drosophila melanogaster.

Knowledge of the genetic basis underlying variation in response to environmental exposures or treatments is important in many research areas. For example, knowing the set of causal genetic variants for drug responses could revolutionize personalized medicine. We used Drosophila melanogaster to investigate the genetic signature underlying behavioral variability in response to methylphenidate (MPH), a drug used in the treatment of attention-deficit/hyperactivity disorder. We exposed a wild-type D. melanogaster population to MPH and a control treatment, and observed an increase in locomotor activity in MPH-exposed individuals. Whole-genome transcriptomic analyses revealed that the behavioral response to MPH was associated with abundant gene expression alterations. To confirm these patterns in a different genetic background and to further advance knowledge on the genetic signature of drug response variability, we used a system of inbred lines, the Drosophila Genetic Reference Panel (DGRP). Based on the DGRP, we showed that the behavioral response to MPH was strongly genotype-dependent. Using an integrative genomic approach, we incorporated known gene interactions into the genomic analyses of the DGRP, and identified putative candidate genes for variability in drug response. We successfully validated 71% of the investigated candidate genes by gene expression knockdown. Furthermore, we showed that MPH has cross-generational behavioral and transcriptomic effects. Our findings establish a foundation for understanding the genetic mechanisms driving genotype-specific responses to medical treatment, and highlight the opportunities that integrative genomic approaches have in optimizing medical treatment of complex diseases.

Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing.

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

Transgenerational inheritance of diet-induced genome rearrangements in Drosophila.

Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila genome. This discovery, along with the link between rDNA, aging, and disease, high-lights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, we have discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produces similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produce offspring with fewer rDNA copies demonstrating that these effects also occur in the germline, and are transgenerationally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet.

Genomic analysis of the interaction between pesticide exposure and nutrition in honey bees (Apis mellifera).

Populations of pollinators are in decline worldwide. These declines are best documented in honey bees and are due to a combination of stressors. In particular, pesticides have been linked to decreased longevity and performance in honey bees; however, the molecular and physiological pathways mediating sensitivity and resistance to pesticides are not well characterized. We explored the impact of coumaphos and fluvalinate, the two most abundant and frequently detected pesticides in the hive, on genome-wide gene expression patterns of honey bee workers. We found significant changes in 1118 transcripts, including genes involved in detoxification, behavioral maturation, immunity, and nutrition. Since behavioral maturation is regulated by juvenile hormone III (JH), we examined effects of these miticides on hormone titers; while JH titers were unaffected, titers of methyl farnesoate (MF), the precursor to JH, were decreased. We further explored the association between nutrition- and pesticide-regulated gene expression patterns and demonstrated that bees fed a pollen-based diet exhibit reduced sensitivity to a third pesticide, chlorpyrifos. Finally, we demonstrated that expression levels of several of the putative pesticide detoxification genes identified in our study and previous studies are also upregulated in response to pollen feeding, suggesting that these pesticides and components in pollen modulate similar molecular response pathways. Our results demonstrate that pesticide exposure can substantially impact expression of genes involved in several core physiological pathways in honey bee workers. Additionally, there is substantial overlap in responses to pesticides and pollen-containing diets at the transcriptional level, and subsequent analyses demonstrated that pollen-based diets reduce workers' pesticide sensitivity. Thus, providing honey bees and other pollinators with high quality nutrition may improve resistance to pesticides.

Genome-wide patterns of genetic variation within and among alternative selective regimes.

Environmental heterogeneity has been hypothesized to influence levels of genetic variation but the effect of heterogeneity depends on (i) the form of heterogeneity, (ii) whether ecologically relevant or neutral loci are being considered, and (iii) the genetic basis of ecological adaptation. We surveyed genome-wide SNP diversity in replicate experimental Drosophila melanogaster populations with equal census sizes that evolved for 42 generations under one of four selection regimes: (i) salt-enriched environment (Salt), (ii) cadmium-enriched environment (Cad), (iii) temporally (Temp) or (iv) spatially (Spatial) variable environments. There was significant differentiation between all pairs of treatments but the greatest differentiation occurred between the two homogenous treatments (Cad and Salt). For sites likely under differential ecological selection (and those closely linked to them), the pattern of within-population diversity π followed the expectation from classic antagonistic selection theory: Spatial > Temp >S alt ≈ Cad. However, neutral diversity unlinked to selected sites followed a different pattern: Spatial>Salt ≈ Cad > Temp. As implicated by the latter result, measures of FST among replicate populations within treatments are consistent with differences in effective population sizes among selective regimes despite equal census sizes. Though there are clear changes in the rank order of treatments when contrasting selected and neutral sites with respect to π, the rank ordering of treatments with respect to FST appears reasonably consistent between site categories. These results demonstrate that alternative selective regimes affect within- and among-population diversity differently for different site types.

A network of conserved damage survival pathways revealed by a genomic RNAi screen.

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS-induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into "pathway nodes" qualitatively improved the interactome organization, revealing a highly organized "MMS survival network." We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.

A genomic response to the yeast transcription factor GAL4 in Drosophila.

The yeast transcription factor GAL4 is widely used in Drosophila genetics to misexpress genes that are under control of the yeast upstream activator sequence (UAS). Here we show that high levels of GAL4 change the expression of many Drosophila genes in a UAS-independent manner, including genes that encode components of important signaling pathways. We find that at least part of the genomic response to GAL4 appears to be caused by effects of GAL4 on stress and immune response pathways. Finally, using the transcription factor Senseless as an example, we demonstrate how an interaction between GAL4 and a GAL4-driven protein can impede the use of the GAL4/UAS system in experiments aimed at determining the transcriptional response to a misexpressed gene.

Transcriptome response to heavy metal stress in Drosophila reveals a new zinc transporter that confers resistance to zinc.

All organisms are confronted with external variations in trace element abundance. To elucidate the mechanisms that maintain metal homeostasis and protect against heavy metal stress, we have determined the transcriptome responses in Drosophila to sublethal doses of cadmium, zinc, copper, as well as to copper depletion. Furthermore, we analyzed the transcriptome of a metal-responsive transcription factor (MTF-1) null mutant. The gene family encoding metallothioneins, and the ABC transporter CG10505 that encodes a homolog of 'yeast cadmium factor' were induced by all three metals. Zinc and cadmium responses have similar features: genes upregulated by both metals include those for glutathione S-transferases GstD2 and GstD5, and for zinc transporter-like proteins designated ZnT35C and ZnT63C. Several of the metal-induced genes that emerged in our study are regulated by the transcription factor MTF-1. mRNA studies in MTF-1 overexpressing or null mutant flies and in silico search for metal response elements (binding sites for MTF-1) confirmed novel MTF-1 regulated genes such as ferritins, the ABC transporter CG10505 and the zinc transporter ZnT35C. The latter was analyzed in most detail; biochemical and genetic approaches, including targeted mutation, indicate that ZnT35C is involved in cellular and organismal zinc efflux and plays a major role in zinc detoxification.

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis.

BACKGROUND: The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. RESULTS: Here we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. CONCLUSION: This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.