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Additional file 4: Table S3. Comparative mock community composition.

Additional file 11: Table S6. Microbial transcripts detected in RNA sequencing datasets from silk, venom and/ or ovary glands.

Additional file 3: Table S2. Sequencing Primers.

Additional file 2: Table S1. PCR 16S rRNA gene V1-V2 Primer Sequences.

Additional file 5: Figure S2. QIIME 2 Microbial Alpha-Diversity Plots. Alpha-Rarefaction results for Faith-PD (a), Observed OTUs (b), and Shannon Index (c) with Depth of Coverage at 8000 rarified sequences.

Additional file 7: Figure S4. QIIME 2 Alpha Diversity Group Significance Testing. Box plots of alphadiversity tests grouped by base sample type. Overall Kruskal-Wallis p-value = 0.00028 (for all groups). (a) Observed OTUs, (b) Faith Phylogenetic-Diversity Index, (c) Shannon Index, (d) Pielou Evenness Index.

Additional file 9: Table S5. Listings of ovary gland (a) and fat tissue (b) specific microbiota.

Additional file 6: Figure S3. Taxa Plots per Tissue Type – Level 3, Class. Major microbial constituents by class (> 2%) within each whole spider and tissue sample for each species (replicates grouped via mean-ceiling). Sample types as follows: Whole Spiders, Cephalothorax, Venom Glands, Ovary, Silk Glands, and Fat Tissue.

Additional file 8: Tables S4. Listings of venom gland (a) and silk gland (b) specific microbiota.

Repetitive DNA sequences that encode polyglutamine tracts are prone to expansion and cause highly deleterious phenotypes of neurodegeneration. Despite this tendency, polyglutamine tracts (“polyQs”) are conserved features of eukaryotic genomes. PolyQs are the most frequent protein-coding homotypic repeat in insect genomes, and are found predominantly in genes encoding transcription factors conserved from Drosophila through human. Although highly conserved across species, polyQ lengths vary widely within species. In D. melanogaster, polyQs in 25 genes have more alleles and higher heterozygosity than all other poly-amino acid tracts. The heat shock protein Hsp70 is a principal suppressor of polyQ expansions and may play a key role in modulating the phenotypes of the alleles that encode them. Hsp70 also promotes tolerance of natural thermal stress in Drosophila and diverse organisms, a role which may deplete the chaperone from buffering against polyQ toxicity. Thus in stressful environments, natural selection against long polyQ alleles more prone to expansion and deleterious phenotypes may be more effective. This hypothesis can be tested by measuring the phenotypic interactions between Hsp70 and polyQ transgenes in D. melanogaster undergoing natural thermal stress, an approach which integrates comparative genomics with experimental and ecological genetics.

Organismic evolution requires that variation at distinct hierarchical levels and attributes be coherently integrated, often in the face of disparate environmental and genetic pressures. A central part of the evolutionary analysis of biological systems remains to decipher the causal connections between organism-wide (or genome-wide) attributes (e.g., mRNA abundance, protein length, codon bias, recombination rate, genomic position, mutation rate, etc) as well as their role-together with mutation, selection, and genetic drift-in shaping patterns of evolutionary variation in any of the attributes themselves. Here we combine genome-wide evolutionary analysis of protein and gene expression data to highlight fundamental relationships among genomic attributes and their associations with the evolution of both protein sequences and gene expression levels. Our results show that protein divergence is positively coupled with both gene expression polymorphism and divergence. We show moreover that although the number of protein-protein interactions in Drosophila is negatively associated with protein divergence as well as gene expression polymorphism and divergence, protein-protein interactions cannot account for the observed coupling between regulatory and structural evolution. Furthermore, we show that proteins with higher rates of amino acid substitutions tend to have larger sizes and tend to be expressed at lower mRNA abundances, whereas genes with higher levels of gene expression divergence and polymorphism tend to have shorter sizes and tend to be expressed at higher mRNA abundances. Finally, we show that protein length is negatively associated with both number of protein-protein interactions and mRNA abundance and that interacting proteins in Drosophila show similar amounts of divergence. We suggest that protein sequences and gene expression are subjected to similar evolutionary dynamics, possibly because of similarity in the fitness effect (i.e., strength of stabilizing selection) of disruptions in a gene's protein sequence or its mRNA expression. We conclude that, as more and better data accumulate, understanding the causal connections among biological traits and how they are integrated over time to constrain or promote structural and regulatory evolution may finally become possible.

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25–55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species—but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.

TTR knockdown with patisiran in patients taking a tetramer stabilizer. (a) Baseline TTR levels by stabilizer use (all cohorts). (b) TTR knockdown by patisiran (0.3Â mg/kg cohorts; error bars represent SEM). SEM: standard error of the mean; TTR: transthyretin. (DOCX 260 kb)

Mean (±SD) plasma concentration–time profiles of patisiran following 1 h intravenous infusion (semi-log scale) for: (a) First (Day 0) and (b) second (Day 21/28) doses. Q3W: every 3 weeks; Q4W: every 4 weeks; SD: standard deviation. (DOCX 196 kb)

Correlation of serum TTR knockdown with change from baseline in serum vitamin A and RBP. RBP: retinol binding protein; TTR: transthyretin. (DOCX 235 kb)

Supplemental materials. (DOCX 36 kb)

To examine how the duration of laboratory domestication may affect Drosophila stocks used in studies of thermotolerance, we measured expression of the inducible heat-shock protein Hsp70 and survival after heat shock in D. melanogaster strains recently collected from nature and maintained in laboratory culture for up to 50 or more generations. After an initial increase in both Hsp70 expression and thermotolerance immediately after transfer to laboratory medium, both traits remained fairly constant over time and variation among strains persisted through laboratory domestication. Furthermore, variation in heat tolerance and Hsp70 expression did not correlate with the length of time populations evolved in the laboratory. Therefore, while environmental variation likely contributed most to early shifts in strain tolerance and Hsp70 expression, other population parameters, for example genetic drift, inbreeding, and selection likely affected these traits little. As long as populations are maintained with large numbers of individuals, the culture of insects in the laboratory may have little effect on the tolerance of different strains to thermal stress.