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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 1: Figure S1. Study Design Flowchart. Overview of methods utilized to isolate, evaluate, and compare the microbiomes of our host spider species and their tissue samples.

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 10: Figure S4. Phylosymbiosis between Widow Phylogeny and Microbiota Dendrogram Relationships. Congruency between widow related spider phylogeny and beta-diversity dendrograms was measured via Robinson-Foulds Cluster (R-F Cluster) metric, where a value of 0 = complete congruence and 2 = incomplete incongruence. Matching Cluster (MC) Metric also accounts for incongruency between closely related branches of rooted trees and a value of 0 also indicates complete congruence between compared trees. Pink asterisks are utilized to denote phylosymbiotic relationships between the widow phylogeny and microbial dendrogram.

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)

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)

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)

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.

Low to moderate levels of stress induce a class of molecular chaperones called heat shock proteins (Hsps), which protect cells, tissues and whole organisms from more severe stress. In higher Eukaryotes, Hsp70 is one of the principle heat-induced chaperones. This response is general, and how much Hsp70 an animal produces correlates with the level of stress to which it is exposed. Nonetheless, definitively linking high Hsp70 expression as an adaptation to stress tolerance is problematic, because organisms and cells respond to stress in many ways. By molecular manipulation of Hsp70 in one animal group, Drosophila , differences in hsp70 copy number are shown to directly influence heat-induced expression of Hsp70 and tolerance of heat. However, too high an expression level of Hsp70 can harm individuals during periods of rapid growth. This strong physiological relationship between Hsp70 concentration and thermotolerance, along with Hsp70's remarkable degree of interspecific coding sequence conservation, suggest that hsp70 regulatory elements may evolve as an adaptation in diverse species to their thermal environments. To examine this possibility, correlative studies within species and research on phylogenetic covariation between these traits is reviewed with a focus on Drosophila species. However, the techniques and results discussed should broadly apply to other animal groups where evolutionary approaches can be used to test whether genetic variation in both thermotolerance and Hsp expression within and among species select locally on either hsp70 sequence and/or expression.

Thermotolerance involves more than life or death. Investigating the complexity of this trait will aid identification of its genetic contributors. We examined variation in thermally stressed walking behavior and performance in natural Drosophila melanogaster strains and strains mutant for the heat shock protein Hsp70, to determine which aspects of locomotion are affected by heat shock and genotype. We developed software for the large-scale capture, analysis, and visualization of locomotion, and determined: (1) Heat shock and thermal pretreatment significantly and differentially impact fly locomotor behavior and performance. (2) Stressed locomotion traits vary extensively among natural strains. (3) Interactions among treatments, strains, and traits are substantial and often counterintuitive. (4) Hsp70 overexpressing flies are faster and more basally thermoprotected in performance than Hsp70 null flies, but null flies are more unidirectional. (5) Natural variation in most stressed locomotion traits exceeds that caused by Hsp70 mutation, reveals uncoupling between thermoprotection of behavior and performance, and suggests significant genetic variation for trait-specific modifiers of thermotolerance.

Hsc/Hsp70-interacting protein (HIP) is a rapidly evolving Hsp70 cofactor. Analyses of multiple Drosophila species indicate that the HIP gene is duplicated only in D. melanogaster. The HIP region, in fact, contains seven distinctly evolving duplicated genes. The regional duplication occurred in two steps, fixed rapidly, and illustrates multiple modes of duplicate gene evolution. HIP and its duplicate HIP-R are adaptively evolving in a manner unique to the region: they exhibit elevated divergence from other drosophilids and low polymorphism within D. melanogaster. HIP and HIP-R are virtually identical, share polymorphisms, and are subject to gene conversion. In contrast, two other duplicate genes in the region, CG33221 and GP-CG32779, are pseudogenes, and the chimeric gene Crg1 is subject to balancing selection. HIP and HIP-R are evolving rapidly and adaptively; however, positive selection is not sufficient to explain the molecular evolution of the region as a whole.

We report multiple cases in which disruption of hsp70 regulatory regions by transposable element (TE) insertions underlies natural variation in expression of the stress-inducible molecular chaperone Hsp70 in Drosophila melanogaster. Three D. melanogaster populations from different continents are polymorphic for jockey or P element insertions in the promoter of the hsp70Ba gene. All three TE insertions are within the same 87-bp region of hsp70Ba promoter, and we suggest that the distinctive promoter architecture of hsp genes may make them vulnerable to TE insertions. Each of the TE insertions reduces Hsp70 levels, and RNase protection assays demonstrate that such insertions can reduce transcription of the hsp70Ba gene. In addition, the TEs alter two measures of organismal fitness, inducible thermotolerance and female reproductive success. Thus, transposition can create quantitative genetic variation in gene expression within populations, on which natural selection can act.

To determine whether and how laboratory and natural selection act on the hsp70 (70-Kd heat-shock protein) genes of Drosophila melanogaster, we examined hsp70 allele frequencies in two sets of populations. First, five populations reared at different temperatures for more than 20 years differentially fixed both a large insertion/deletion (indel) polymorphism at the 87A7 hsp70 cluster ("56H8"/"122") and a single nucleotide polymorphism at the 87C1 hsp70 cluster. In both cases, the 18 degrees C and 25 degrees C populations fixed one allele and the 28 degrees C populations the other, consistent with previously described evolved differences among these populations in Hsp70 expression and thermotolerance. Second, we examined 56H8 and 122 frequencies in a set of 11 populations founded from flies collected along a latitudinal transect of eastern Australia. The 56H8 allele frequencies are positively associated with latitude, consistent with maintenance of the 56H8/122 polymorphism by natural selection. Thermal extremes and average values are negatively correlated with latitude. These results suggest that natural selection imposed by temperature and thermal variability may affect hsp70 allele frequencies.

SUMMARY Drosophila melanogaster collected in sub-equatorial Africa in the 1970s are remarkably tolerant of sustained laboratory culture above 30°C and of acute exposure to much warmer temperatures. Inducible thermotolerance of high temperatures, which in Drosophila melanogaster is due in part to the inducible molecular chaperone Hsp70, is only modest in this strain. Expression of Hsp70 protein and hsp70 mRNA is likewise reduced and has slower kinetics in this strain (T) than in a standard wild-type strain (Oregon R). These strains also differed in constitutive and heat-inducible levels of other molecular chaperones. The lower Hsp70 expression in the T strain apparently has no basis in the activation of the heat-shock transcription factor HSF, which is similar in T and Oregon R flies. Rather, the reduced expression may stem from insertion of two transposable elements, H.M.S. Beagle in the intergenic region of the 87A7 hsp70 gene cluster and Jockey in the hsp70Ba gene promoter. We hypothesize that the reduced Hsp70 expression in a Drosophila melanogaster strain living chronically at intermediate temperatures may represent an evolved suppression of the deleterious phenotypes of Hsp70.

To determine how the modern copy number (5) of hsp70 genes in Drosophila melanogaster evolved, we localized the duplication events that created the genes in the phylogeny of the melanogaster group, examined D. melanogaster genomic sequence to investigate the mechanisms of duplication, and analyzed the hsp70 gene sequences of Drosophila orena and Drosophila mauritiana. The initial two-to-four hsp70 duplication occurred 10--15 MYA, according to fixed in situ hybridization to polytene chromosomes, before the origin and divergence of the melanogaster and five other species subgroups of the melanogaster group. Analysis of more than 30 kb of flanking sequence surrounding the hsp70 gene clusters suggested that this duplication was likely a retrotransposition. For the melanogaster subgroup, Southern hybridization and an hsp70 restriction map confirmed the conserved number (4) and arrangement of hsp70 genes in the seven species other than D. melanogaster. Drosophila melanogaster is unique; tandem duplication and gene conversion at the derived cluster yielded a fifth hsp70 gene. The four D. orena hsp70 genes are highly similar and concertedly evolving. In contrast, the D. mauritiana hsp70 genes are divergent, and many alleles are nonfunctional. The proliferation, concerted evolution, and maintenance of functionality in the D. melanogaster hsp70 genes is consistent with the action of natural selection in this species.

Relatively little is known about the importance of amino acid interactions in protein and phenotypic evolution. Here we examine whether mutations that are pathogenic in Drosophila melanogaster become fixed via epistasis in other Dipteran genomes. Overall divergence at pathogenic amino acid sites is reduced. However, ∼10% of the substitutions at these sites carry the exact same pathogenic amino acid found in D. melanogaster mutants. Hence compensatory mutation(s) must have evolved. Surprisingly, the fraction 10% is not affected by phylogenetic distance. These results support a selection-driven process that allows compensated amino acid substitutions to become rapidly fixed in taxa with large populations.

Background: The recent completion of the Drosophila melanogaster genomic sequence to high quality and the availability of a greatly expanded set of Drosophila cDNA sequences, aligning to 78% of the predicted euchromatic genes, afforded FlyBase the opportunity to significantly improve genomic annotations. We made the annotation process more rigorous by inspecting each gene visually, utilizing a comprehensive set of curation rules, requiring traceable evidence for each gene model, and comparing each predicted peptide to SWISS-PROT and TrEMBL sequences. Results: Although the number of predicted protein-coding genes in Drosophila remains essentially unchanged, the revised annotation significantly improves gene models, resulting in structural changes to 85% of the transcripts and 45% of the predicted proteins. We annotated transposable elements and non-protein-coding RNAs as new features, and extended the annotation of untranslated (UTR) sequences and alternative transcripts to include more than 70% and 20% of genes, respectively. Finally, cDNA sequence provided evidence for dicistronic transcripts, neighboring genes with overlapping UTRs on the same DNA sequence strand, alternatively spliced genes that encode distinct, non-overlapping peptides, and numerous nested genes. Conclusions: Identification of so many unusual gene models not only suggests that some mechanisms for gene regulation are more prevalent than previously believed, but also underscores the complex challenges of eukaryotic gene prediction. At present, experimental data and human curation remain essential to generate high-quality genome annotations.

To examine whether recent evolutionary history affects the expression of Hsp7O, the major heat-induced- heat shock protein in Drosophila melan-ogaster, we measured Hsp7O expression, thermotolerance, and hsp7O gene number in replicate populations undergoing laboratory evolution at different temperatures. Despite Hsp7O's ancient and highly conserved nature, experimental evolution effectively and replicably modified its expression and phenotype (thermotolerance). Among five D. melanogaster populations founded from a common ancestral population and raised at three different temperatures (one at 18'C, two each at 250C and 280C) for twenty years, Hsp7O expression varies in a consistent pattern: the replicate 280C lines expressed 30-50% less Hsp7O than the other lines at a range of inducing temperatures. This modification was refractory to acclimation, and correlated with thermotolerance: the 280C lines had significantly lower inducible tolerance of 38.50C and 390C. We verified the presence of five hsp70 genes in the genome of each line, excluding copy number variation as a candidate molecular basis of the evolved difference in expression. These findings support the ability of Hsp7O levels in D. inelanogaster populations to change over microevolutionary time scales and implicate constancy of environmental temperature as a potentially important se- lective agent.