The genome sequence of a drosophilid fruit fly, Drosophila limbata von Roser 1840.

We present a genome assembly from an individual male Drosophila limbata (drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 233.5 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.09 kilobases in length.

The genome sequence of a drosophilid fruit fly, Drosophila histrio (Meigen, 1830).

We present a genome assembly from an individual female Drosophila histrio (the drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 189.2 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 16.02 kilobases in length.

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.

The genome sequence of the drosophilid fruit fly, Drosophila phalerata (Meigen, 1830).

We present a genome assembly from an individual male Drosophila phalerata (drosophilid fruit fly, Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 223.9 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.14 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,973 protein coding genes.

Naturally occurring viruses of Drosophila reduce offspring number and lifespan.

Drosophila remains a pre-eminent insect model system for host-virus interaction, but the host range and fitness consequences of the drosophilid virome are poorly understood. Metagenomic studies have reported approximately 200 viruses associated with Drosophilidae, but few isolates are available to characterize the Drosophila immune response, and most characterization has relied on injection and systemic infection. Here, we use a more natural infection route to characterize the fitness effects of infection and to study a wider range of viruses. We exposed laboratory Drosophila melanogaster to 23 naturally occurring viruses from wild-collected drosophilids. We recorded transmission rates along with two components of female fitness: survival and the lifetime number of adult offspring produced. Nine different viruses transmitted during contact with laboratory D. melanogaster, although for the majority, rates of transmission were less than 20%. Five virus infections led to a significant decrease in lifespan (D. melanogaster Nora virus, D. immigrans Nora virus, Muthill virus, galbut virus and Prestney Burn virus), and three led to a reduction in the total number of offspring. Our findings demonstrate the utility of the Drosophila model for community-level studies of host-virus interactions, and suggest that viral infection could be a substantial fitness burden on wild flies.

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.

Viruses in Laboratory Drosophila and Their Impact on Host Gene Expression.

Drosophila melanogaster has one of the best characterized antiviral immune responses among invertebrates. However, relatively few easily transmitted natural virus isolates are available, and so many Drosophila experiments have been performed using artificial infection routes and artificial host-virus combinations. These may not reflect natural infections, especially for subtle phenotypes such as gene expression. Here, to explore the laboratory virus community and to better understand how natural virus infections induce changes in gene expression, we have analysed seven publicly available D. melanogaster transcriptomic sequencing datasets that were originally sequenced for projects unrelated to virus infection. We have found ten known viruses-including five that have not been experimentally isolated-but no previously unknown viruses. Our analysis of host gene expression revealed that numerous genes were differentially expressed in flies that were naturally infected with a virus. For example, flies infected with nora virus showed patterns of gene expression consistent with intestinal vacuolization and possible host repair via the upd3 JAK/STAT pathway. We also found marked sex differences in virus-induced differential gene expression. Our results show that natural virus infection in laboratory Drosophila does indeed induce detectable changes in gene expression, suggesting that this may form an important background condition for experimental studies in the laboratory.

Experimental estimates of germline mutation rate in eukaryotes: a phylogenetic meta-analysis.

Mutation is the ultimate source of all genetic variation, and over the last 10 years the ready availability of whole-genome sequencing has permitted direct estimation of mutation rate for many non-model species across the tree of life. In this meta-analysis, we make a comprehensive search of the literature for mutation rate estimates in eukaryotes, identifying 140 mutation accumulation (MA) and parent-offspring (PO) sequencing studies covering 134 species. Based on these data, we revisit differences in the single-nucleotide mutation (SNM) rate between different phylogenetic lineages and update the known relationships between mutation rate and generation time, genome size, and nucleotide diversity-while accounting for phylogenetic nonindependence. We do not find a significant difference between MA and PO in estimated mutation rates, but we confirm that mammal and plant lineages have higher mutation rates than arthropods and that unicellular eukaryotes have the lowest mutation rates. We find that mutation rates are higher in species with longer generation times and larger genome sizes, even when accounting for phylogenetic relationships. Moreover, although nucleotide diversity is positively correlated with mutation rate, the gradient of the relationship is significantly less than one (on a logarithmic scale), consistent with higher mutation rates in populations with smaller effective size. For the 29 species for which data are available, we find that indel mutation rates are positively correlated with nucleotide mutation rates and that short deletions are generally more common than short insertions. Nevertheless, despite recent progress, no estimates of either SNM or indel mutation rates are available for the majority of deeply branching eukaryotic lineages-or even for most animal phyla. Even among charismatic megafauna, experimental mutation rate estimates remain unknown for amphibia and scarce for reptiles and fish.

Variation in mutation, recombination, and transposition rates in Drosophila melanogaster and Drosophila simulans.

The rates of mutation, recombination, and transposition are core parameters in models of evolution. They impact genetic diversity, responses to ongoing selection, and levels of genetic load. However, even for key evolutionary model species such as Drosophila melanogaster and Drosophila simulans, few estimates of these parameters are available, and we have little idea of how rates vary between individuals, sexes, or populations. Knowledge of this variation is fundamental for parameterizing models of genome evolution. Here, we provide direct estimates of mutation, recombination, and transposition rates and their variation in a West African and a European population of D. melanogaster and a European population of D. simulans Across 89 flies, we observe 58 single-nucleotide mutations, 286 crossovers, and 89 transposable element (TE) insertions. Compared to the European D. melanogaster, we find the West African population has a lower mutation rate (1.67 × 10-9 site-1 gen-1 vs. 4.86 × 10-9 site-1 gen-1) and a lower transposition rate (8.99 × 10-5 copy-1 gen-1 vs. 23.36 × 10-5 copy-1 gen-1), but a higher recombination rate (3.44 cM/Mb vs. 2.06 cM/Mb). The European D. simulans population has a similar mutation rate to European D. melanogaster, but a significantly higher recombination rate and a lower, but not significantly different, transposition rate. Overall, we find paternal-derived mutations are more frequent than maternal ones in both species. Our study quantifies the variation in rates of mutation, recombination, and transposition among different populations and sexes, and our direct estimates of these parameters in D. melanogaster and D. simulans will benefit future studies in population and evolutionary genetics.

The genome sequence of a drosophilid fruit fly, Hirtodrosophila cameraria (Haliday, 1833).

We present a genome assembly from an individual female Hirtodrosophila cameraria (a drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 214.5 megabases in span. Most of the assembly is scaffolded into 4 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 15.94 kilobases in length.

The genome sequence of a drosophilid fruit fly, Drosophila funebris (Fabricius, 1789).

We present a genome assembly from an individual male Drosophila funebris (drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 181.1 megabases in span. Most of the assembly is scaffolded into 7 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.15 kilobases in length.

The genome sequence of a drosophilid fruit fly, Chymomyza fuscimana (Drosophilidae) (Zetterstedt, 1838).

We present a genome assembly from an individual male Chymomyza fuscimana (drosophilid fruit fly; Arthropoda; Insecta; Diptera; Drosophilidae). The genome sequence is 338.0 megabases in span. Most of the assembly is scaffolded into 5 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 16.47 kilobases in length.

Population-Genomic Analysis Identifies a Low Rate of Global Adaptive Fixation in the Proteins of the Cyclical Parthenogen Daphnia magna.

Daphnia are well-established ecological and evolutionary models, and the interaction between D. magna and its microparasites is widely considered a paragon of the host-parasite coevolutionary process. Like other well-studied arthropods such as Drosophila melanogaster and Anopheles gambiae, D. magna is a small, widespread, and abundant species that is therefore expected to display a large long-term population size and high rates of adaptive protein evolution. However, unlike these other species, D. magna is cyclically asexual and lives in a highly structured environment (ponds and lakes) with moderate levels of dispersal, both of which are predicted to impact upon long-term effective population size and adaptive protein evolution. To investigate patterns of adaptive protein fixation, we produced the complete coding genomes of 36 D. magna clones sampled from across the European range (Western Palaearctic), along with draft sequences for the close relatives D. similis and D. lumholtzi, used as outgroups. We analyzed genome-wide patterns of adaptive fixation, with a particular focus on genes that have an a priori expectation of high rates, such as those likely to mediate immune responses, RNA interference against viruses and transposable elements, and those with a strongly male-biased expression pattern. We find that, as expected, D. magna displays high levels of diversity and that this is highly structured among populations. However, compared with Drosophila, we find that D. magna proteins appear to have a high proportion of weakly deleterious variants and do not show evidence of pervasive adaptive fixation across its entire range. This is true of the genome as a whole, and also of putative 'arms race' genes that often show elevated levels of adaptive substitution in other species. In addition to the likely impact of extensive, and previously documented, local adaptation, we speculate that these findings may reflect reduced efficacy of selection associated with cyclical asexual reproduction.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Virus Prevalence and Genetic Diversity Across a Wild Bumblebee Community.

Viruses are key population regulators, but we have limited knowledge of the diversity and ecology of viruses. This is even the case in wild host populations that provide ecosystem services, where small fitness effects may have major ecological impacts in aggregate. One such group of hosts are the bumblebees, which have a major role in the pollination of food crops and have suffered population declines and range contractions in recent decades. In this study, we investigate the diversity of four recently discovered bumblebee viruses (Mayfield virus 1, Mayfield virus 2, River Liunaeg virus, and Loch Morlich virus), and two previously known viruses that infect both wild bumblebees and managed honeybees (Acute bee paralysis virus and Slow bee paralysis virus) from isolates in Scotland. We investigate the ecological and environmental factors that determine viral presence and absence. We show that the recently discovered bumblebee viruses were more genetically diverse than the viruses shared with honeybees. Coinfection is potentially important in shaping prevalence: we found a strong positive association between River Liunaeg virus and Loch Morlich virus presence after controlling for host species, location and other relevant ecological variables. We tested for a relationship between environmental variables (temperature, UV radiation, wind speed, and prevalence), but as we had few sampling sites, and thus low power for site-level analyses, we could not conclude anything regarding these variables. We also describe the relationship between the bumblebee communities at our sampling sites. This study represents a first step in the description of predictors of bumblebee infection in the wild.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Correction: Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

[This corrects the article DOI: 10.1371/journal.pgen.1007533.].

A new lineage of segmented RNA viruses infecting animals.

Metagenomic sequencing has revolutionised our knowledge of virus diversity, with new virus sequences being reported faster than ever before. However, virus discovery from metagenomic sequencing usually depends on detectable homology: without a sufficiently close relative, so-called 'dark' virus sequences remain unrecognisable. An alternative approach is to use virus-identification methods that do not depend on detecting homology, such as virus recognition by host antiviral immunity. For example, virus-derived small RNAs have previously been used to propose 'dark' virus sequences associated with the Drosophilidae (Diptera). Here, we combine published Drosophila data with a comprehensive search of transcriptomic sequences and selected meta-transcriptomic datasets to identify a completely new lineage of segmented positive-sense single-stranded RNA viruses that we provisionally refer to as the Quenyaviruses. Each of the five segments contains a single open reading frame, with most encoding proteins showing no detectable similarity to characterised viruses, and one sharing a small number of residues with the RNA-dependent RNA polymerases of single- and double-stranded RNA viruses. Using these sequences, we identify close relatives in approximately 20 arthropods, including insects, crustaceans, spiders, and a myriapod. Using a more conserved sequence from the putative polymerase, we further identify relatives in meta-transcriptomic datasets from gut, gill, and lung tissues of vertebrates, reflecting infections of vertebrates or of their associated parasites. Our data illustrate the utility of small RNAs to detect viruses with limited sequence conservation, and provide robust evidence for a new deeply divergent and phylogenetically distinct RNA virus lineage.

Widespread gene duplication and adaptive evolution in the RNA interference pathways of the Drosophila obscura group.

BACKGROUND: RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss-most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. RESULTS: Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. CONCLUSIONS: These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible.

A Magnesium Transport Protein Related to Mammalian SLC41 and Bacterial MgtE Contributes to Circadian Timekeeping in a Unicellular Green Alga.

Circadian clocks in eukaryotes involve both transcriptional-translational feedback loops, post-translational regulation, and metabolic, non-transcriptional oscillations. We recently identified the involvement of circadian oscillations in the intracellular concentrations of magnesium ions (Mg2+i) that were conserved in three eukaryotic kingdoms. Mg2+i in turn contributes to transcriptional clock properties of period and amplitude, and can function as a zeitgeber to define phase. However, the mechanism-or mechanisms-responsible for the generation of Mg2+i oscillations, and whether these are functionally conserved across taxonomic groups, remain elusive. We employed the cellular clock model Ostreococcustauri to provide a first study of an MgtE domain-containing protein in the green lineage. OtMgtE shares homology with the mammalian SLC41A1 magnesium/sodium antiporter, which has previously been implicated in maintaining clock period. Using genetic overexpression, we found that OtMgtE contributes to both timekeeping and daily changes in Mg2+i. However, pharmacological experiments and protein sequence analyses indicated that critical differences exist between OtMgtE and either the ancestral MgtE channel or the mammalian SLC41 antiporters. We concluded that even though MgtE domain-containing proteins are only distantly related, these proteins retain a shared role in contributing to cellular timekeeping and the regulation of Mg2+i.