Entomophthovirus: An insect-derived iflavirus that infects a behavior-manipulating fungal pathogen of dipterans.

We report a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Berkeley Entomophthovirus, is a positive-strand RNA virus in the iflaviridae family of capsid-forming viruses, which are mostly known to infect insects. The viral RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and virus particles can be seen intracellularly in E. muscae. This virus, of which we find two closely related variants in our culture of E. muscae, is also closely related to three different viruses reported from metagenomic surveys, two of which were isolated from wild dipterans, and a third isolated from wild ticks. By analyzing sequencing data from these earlier reports, we find abundant reads aligning to E. muscae specifically in the samples from which viral reads were sequenced. These data establish a wide and perhaps obligate association with E. muscae in the wild, consistent with our laboratory data that E. muscae is the host for these closely related viruses. Because of this, we propose the name Entomophthovirus for this group of highly related virus variants. As other members of the iflaviridae have been reported to cause behavioral changes in insects, we speculate on the possibility that Entomophthovirus plays a role in the behavioral manipulation of flies infected with E. muscae.

Single-fly genome assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1 Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries.

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AIDevoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

Evolutionary analyses of intrinsically disordered regions reveal widespread signals of conservation.

Intrinsically disordered regions (IDRs) are segments of proteins without stable three-dimensional structures. As this flexibility allows them to interact with diverse binding partners, IDRs play key roles in cell signaling and gene expression. Despite the prevalence and importance of IDRs in eukaryotic proteomes and various biological processes, associating them with specific molecular functions remains a significant challenge due to their high rates of sequence evolution. However, by comparing the observed values of various IDR-associated properties against those generated under a simulated model of evolution, a recent study found most IDRs across the entire yeast proteome contain conserved features. Furthermore, it showed clusters of IDRs with common "evolutionary signatures," i.e. patterns of conserved features, were associated with specific biological functions. To determine if similar patterns of conservation are found in the IDRs of other systems, in this work we applied a series of phylogenetic models to over 7,500 orthologous IDRs identified in the Drosophila genome to dissect the forces driving their evolution. By comparing models of constrained and unconstrained continuous trait evolution using the Brownian motion and Ornstein-Uhlenbeck models, respectively, we identified signals of widespread constraint, indicating conservation of distributed features is mechanism of IDR evolution common to multiple biological systems. In contrast to the previous study in yeast, however, we observed limited evidence of IDR clusters with specific biological functions, which suggests a more complex relationship between evolutionary constraints and function in the IDRs of multicellular organisms.

Intrinsic protein disorder is insufficient to drive subnuclear clustering in embryonic transcription factors.

Modern microscopy has revealed that core nuclear functions, including transcription, replication, and heterochromatin formation, occur in spatially restricted clusters. Previous work from our lab has shown that subnuclear high-concentration clusters of transcription factors may play a role in regulating RNA synthesis in the early Drosophila embryo. A nearly ubiquitous feature of eukaryotic transcription factors is that they contain intrinsically disordered regions (IDRs) that often arise from low complexity amino acid sequences within the protein. It has been proposed that IDRs within transcription factors drive co-localization of transcriptional machinery and target genes into high-concentration clusters within nuclei. Here, we test that hypothesis directly, by conducting a broad survey of the subnuclear localization of IDRs derived from transcription factors. Using a novel algorithm to identify IDRs in the Drosophila proteome, we generated a library of IDRs from transcription factors expressed in the early Drosophila embryo. We used this library to perform a high-throughput imaging screen in Drosophila Schneider-2 (S2) cells. We found that while subnuclear clustering does not occur when the majority of IDRs are expressed alone, it is frequently seen in full-length transcription factors. These results are consistent in live Drosophila embryos, suggesting that IDRs are insufficient to drive the subnuclear clustering behavior of transcription factors. Furthermore, the clustering of transcription factors in living embryos was unaffected by the deletion of IDR sequences. Our results demonstrate that IDRs are unlikely to be the primary molecular drivers of the clustering observed during transcription, suggesting a more complex and nuanced role for these disordered protein sequences.

Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Leveraging genomic redundancy to improve inference and alignment of orthologous proteins.

Identifying protein sequences with common ancestry is a core task in bioinformatics and evolutionary biology. However, methods for inferring and aligning such sequences in annotated genomes have not kept pace with the increasing scale and complexity of the available data. Thus, in this work, we implemented several improvements to the traditional methodology that more fully leverage the redundancy of closely related genomes and the organization of their annotations. Two highlights include the application of the more flexible k-clique percolation algorithm for identifying clusters of orthologous proteins and the development of a novel technique for removing poorly supported regions of alignments with a phylogenetic hidden Markov model (phylo-HMM). In making the latter, we wrote a fully documented Python package Homomorph that implements standard HMM algorithms and created a set of tutorials to promote its use by a wide audience. We applied the resulting pipeline to a set of 33 annotated Drosophila genomes, generating 22,813 orthologous groups and 8,566 high-quality alignments.

Unified bursting strategies in ectopic and endogenous even-skipped expression patterns.

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.

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Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos.

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the use of single-nucleus RNA sequencing in Drosophila embryos prior to cellularization, here we look at gene expression in control and insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we provide a resource of candidate differentially expressed genes that might show local changes in gene expression between control and maternal dCTCF null nuclei with no detectable differential expression in bulk. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo.

Microbiome-by-ethanol interactions impact Drosophila melanogaster fitness, physiology, and behavior.

The gut microbiota can affect how animals respond to ingested toxins, such as ethanol, which is prevalent in the diets of diverse animals and often leads to negative health outcomes in humans. Ethanol is a complex dietary factor because it acts as a toxin, behavioral manipulator, and nutritional source, with both direct effects on the host as well as indirect ones through the microbiome. Here, we developed a model for chronic, non-intoxicating ethanol ingestion in the adult fruit fly, Drosophila melanogaster, and paired this with the tractability of the fly gut microbiota, which can be experimentally removed. We linked numerous physiological, behavioral, and transcriptional variables to fly fitness, including a combination of intestinal barrier integrity, stored triglyceride levels, feeding behavior, and the immunodeficiency pathway. Our results reveal a complex tradeoff between lifespan and fecundity that is microbiome-dependent and modulated by dietary ethanol and feeding behavior.

Impact of essential workers in the context of social distancing for epidemic control.

New emerging infectious diseases are identified every year, a subset of which become global pandemics like COVID-19. In the case of COVID-19, many governments have responded to the ongoing pandemic by imposing social policies that restrict contacts outside of the home, resulting in a large fraction of the workforce either working from home or not working. To ensure essential services, however, a substantial number of workers are not subject to these limitations, and maintain many of their pre-intervention contacts. To explore how contacts among such "essential" workers, and between essential workers and the rest of the population, impact disease risk and the effectiveness of pandemic control, we evaluated several mathematical models of essential worker contacts within a standard epidemiology framework. The models were designed to correspond to key characteristics of cashiers, factory employees, and healthcare workers. We find in all three models that essential workers are at substantially elevated risk of infection compared to the rest of the population, as has been documented, and that increasing the numbers of essential workers necessitates the imposition of more stringent controls on contacts among the rest of the population to manage the pandemic. Importantly, however, different archetypes of essential workers differ in both their individual probability of infection and impact on the broader pandemic dynamics, highlighting the need to understand and target intervention for the specific risks faced by different groups of essential workers. These findings, especially in light of the massive human costs of the current COVID-19 pandemic, indicate that contingency plans for future epidemics should account for the impacts of essential workers on disease spread.

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A phylogeny for the Drosophila montium species group: A model clade for comparative analyses.

The Drosophila montium species group is a clade of 94 named species, closely related to the model species D. melanogaster. The montium species group is distributed over a broad geographic range throughout Asia, Africa, and Australasia. Species of this group possess a wide range of morphologies, mating behaviors, and endosymbiont associations, making this clade useful for comparative analyses. We use genomic data from 42 available species to estimate the phylogeny and relative divergence times within the montium species group, and its relative divergence time from D. melanogaster. To assess the robustness of our phylogenetic inferences, we use 3 non-overlapping sets of 20 single-copy coding sequences and analyze all 60 genes with both Bayesian and maximum likelihood methods. Our analyses support monophyly of the group. Apart from the uncertain placement of a single species, D. baimaii, our analyses also support the monophyly of all seven subgroups proposed within the montium group. Our phylograms and relative chronograms provide a highly resolved species tree, with discordance restricted to estimates of relatively short branches deep in the tree. In contrast, age estimates for the montium crown group, relative to its divergence from D. melanogaster, depend critically on prior assumptions concerning variation in rates of molecular evolution across branches, and hence have not been reliably determined. We discuss methodological issues that limit phylogenetic resolution - even when complete genome sequences are available - as well as the utility of the current phylogeny for understanding the evolutionary and biogeographic history of this clade.

Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene.

We used live imaging to visualize the transcriptional dynamics of the Drosophila melanogaster even-skipped gene at single-cell and high-temporal resolution as its seven stripe expression pattern forms, and developed tools to characterize and visualize how transcriptional bursting varies over time and space. We find that despite being created by the independent activity of five enhancers, even-skipped stripes are sculpted by the same kinetic phenomena: a coupled increase of burst frequency and amplitude. By tracking the position and activity of individual nuclei, we show that stripe movement is driven by the exchange of bursting nuclei from the posterior to anterior stripe flanks. Our work provides a conceptual, theoretical and computational framework for dissecting pattern formation in space and time, and reveals how the coordinated transcriptional activity of individual nuclei shapes complex developmental patterns.

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