ABSTRACT: Whole genome sequencing to study phylogenetics, population structure, genotype-phenotype associations, sex chromosome evolution and host-parasite interactions in Danaus butterflies and their relatives.
Project description:We characterized sperm from the seminal vesicles of male monarch butterflies (Danaus plexippus), in triplicate, identifying 548 high confidence proteins. As with all but the most basal lepidopteran species male monarch butterflies are sperm heteromorphic, producing fertilization competent and anucleate fertilization incompetent sperm morphs. Comparing this data to the sperm proteomes of the Carolina sphinx moth (Manduca sexta) and the fruit fly (Drosophila melanogaster) demonstrated high levels of functional coherence across proteomes, and conservation at the level of protein abundance and post-translational modification within Lepidoptera. Comparative genomic analyses revealed a significant reduction in orthology among Monarch sperm genes relative to the remainder of the genome in non-Lepidopteran insects. A substantial number of sperm proteins were found to be specific to Lepidoptera, lacking detectable homology outside this taxa. These findings are consistent with a burst of genetic novelty in the sperm proteome concurrent with the origin of heteromorphic spermatogenesis early in Lepidoptera evolution.
Project description:In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo a magnificent long-range migration. In contrast to spring and summer butterflies, fall migrants are juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants also use a time-compensated sun compass to help them navigate in the south/southwesterly direction en route for Mexico. Central issues in this area are defining the relationship between juvenile hormone status and oriented flight, critical features that differentiate summer monarchs from fall migrants, and identifying molecular correlates of behavioral state. Here we show that increasing juvenile hormone activity to induce summer-like reproductive development in fall migrants does not alter directional flight behavior or its time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby separating molecularly fall migrants from summer butterflies. Intriguing genes that are differentially regulated include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase gene. In addition, several differentially regulated genes (37.5% of total) are not annotated, suggesting unique functions associated with oriented flight behavior. We also identified 23 juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive (juvenile-hormone deficient) migrants. The results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in genomic function help define the migratory state.
Project description:Monarch butterflies (Danaus plexippus) rely on milkweeds as larval host plants. Host plant seeking and verification by female butterflies may be mediated by gustatory (GRs) and olfactory receptors (ORs). Here we employed RNA-Seq, bioinformatics and RT-qPCR techniques to identify sex- and tissue-specific gene expression. We focused on chemosensation related genes and pathways, including putative ORs, GRs, ionotropic receptors (IRs), odorant-binding proteins, chemosensory proteins, and steroid hormone mediated signaling in specific chemosensory tissues (i.e., antennae, legs and proboscis). Twelve butterflies evenly split between males and females were caught and used for RNA extraction. Tissue-specific sequencing libraries were prepared and sequenced using Illumina NovaSeq 6000 or HiSeq 3000, generating 2 billion 150-bp, paired-end reads. Many more genes and gene sets were differentially expressed between tissue types than between sexes. A total of 148 chemosensation-related genes exhibited sex- and/or tissue-biased expression. RT-qPCR of a small set of genes confirmed their differential expression between tissue types or between males and females. These findings laid a solid foundation for further investigations into the biological roles of these identified genes underlying chemosensation-mediated behaviors such as foraging and reproduction.
Project description:In the fall, Eastern North American monarch butterflies (Danaus plexippus) undergo a magnificent long-range migration. In contrast to spring and summer butterflies, fall migrants are juvenile hormone deficient, which leads to reproductive arrest and increased longevity. Migrants also use a time-compensated sun compass to help them navigate in the south/southwesterly direction en route for Mexico. Central issues in this area are defining the relationship between juvenile hormone status and oriented flight, critical features that differentiate summer monarchs from fall migrants, and identifying molecular correlates of behavioral state. Here we show that increasing juvenile hormone activity to induce summer-like reproductive development in fall migrants does not alter directional flight behavior or its time-compensated orientation, as monitored in a flight simulator. Reproductive summer butterflies, in contrast, uniformly fail to exhibit directional, oriented flight. To define molecular correlates of behavioral state, we used microarray analysis of 9417 unique cDNA sequences. Gene expression profiles reveal a suite of 40 genes whose differential expression in brain correlates with oriented flight behavior in individual migrants, independent of juvenile hormone activity, thereby separating molecularly fall migrants from summer butterflies. Intriguing genes that are differentially regulated include the clock gene vrille and the locomotion-relevant tyramine beta hydroxylase gene. In addition, several differentially regulated genes (37.5% of total) are not annotated, suggesting unique functions associated with oriented flight behavior. We also identified 23 juvenile hormone-dependent genes in brain, which separate reproductive from non-reproductive monarchs; genes involved in longevity, fatty acid metabolism, and innate immunity are upregulated in non-reproductive (juvenile-hormone deficient) migrants. The results link key behavioral traits with gene expression profiles in brain that differentiate migratory from summer butterflies and thus show that seasonal changes in genomic function help define the migratory state. A total of 40 monarch butterflies were used for the microarray analysis. Of the 40, 10 (5 male/5 female) were summer butterflies (Designated as S) and 30 were fall butterflies. The fall butterflies were further divided into three groups: 10 (5 male/5 female) were untreated (F); 10 (5 male/5 female) were treated with methoprene (M), which is a juvenile hormone analog and induces the development of reproductive organs in migrant butterflies; and 10 (5 male/5 female) were treated with vehicle only (V). We collected total brain RNA from each of the 40 butterflies. The brain RNAs were amplified and then used to probe a custom Nimblegen array that was designed to analyze the 9,417 unique cDNA sequences established in our published EST library (http://titan.biotec.uiuc.edu/cgi-bin/ESTWebsite/estima_start?seqSet=butterfly). Our main interest is to find genes involved in migration. This includes genes regulating oriented flight behavior of the butterfly and genes that regulate reproductive status. To identify these genes, we approached the microarray data in two ways. First, we identified the potential genes involved in oriented flight behavior using the following strategy. We compared the summer group to each of the three fall groups (untreated, methoprene-treated, and vehicle-treated) for males and for females, and looked for gene regulation patterns common among the three comparisons for each sex. Because the comparisons were done separately for males and females, and our behavioral data did not show significant sex differences in flight orientation, we focused on the common differentially regulated genes that were shared between males and females. Accordingly, we identified 40 cDNAs that were differentially regulated between summer butterflies and fall migrants, irrespective of sex. Second, we looked for the juvenile hormone-response genes. Again, we performed sex-specific statistical analyses, and compared the summer and the fall groups, and the methoprene-treated and vehicle-treated migrants. We then screened for shared genes between the two groups for each sex. We next examined cDNAs that were differentially regulated in both males and females, to determine juvenile hormone-regulated genes involved in more global processes (e.g., longevity and fatty acid metabolism) that would not be expected to be sex-specific. We identified 23 putative juvenile hormone-response genes that were common between males and females.