Repeated Duplication of Argonaute2 Is Associated with Strong Selection and Testis Specialization in Drosophila.

Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. Here we reconstruct the history of Ago2 duplications across the D. obscura group and use patterns of gene expression to infer new functional specialization. We show that some duplications are old, shared by the entire species group, and that losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura We find that while the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogs have independently specialized to testis-specific expression. Using population genetic analyses, we show that most testis-specific paralogs have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogs. We speculate that the repeated evolution of testis specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. Our study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in nonmodel species.

Twenty-Five New Viruses Associated with the Drosophilidae (Diptera).

Drosophila melanogaster is an important laboratory model for studies of antiviral immunity in invertebrates, and Drosophila species provide a valuable system to study virus host range and host switching. Here, we use metagenomic RNA sequencing of about 1600 adult flies to discover 25 new RNA viruses associated with six different drosophilid hosts in the wild. We also provide a comprehensive listing of viruses previously reported from the Drosophilidae. The new viruses include Iflaviruses, Rhabdoviruses, Nodaviruses, and Reoviruses, and members of unclassified lineages distantly related to Negeviruses, Sobemoviruses, Poleroviruses, Flaviviridae, and Tombusviridae. Among these are close relatives of Drosophila X virus and Flock House virus, which we find in association with wild Drosophila immigrans. These two viruses are widely used in experimental studies but have not been previously reported to naturally infect Drosophila. Although we detect no new DNA viruses, in D. immigrans and Drosophila obscura, we identify sequences very closely related to Armadillidium vulgare iridescent virus (Invertebrate iridescent virus 31), bringing the total number of DNA viruses found in the Drosophilidae to three.

The Discovery, Distribution, and Evolution of Viruses Associated with Drosophila melanogaster.

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont--which is known to be protective against virus infections in Drosophila--we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.

Novel Drosophila viruses encode host-specific suppressors of RNAi.

The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary 'arms race' may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.

Successive increases in the resistance of Drosophila to viral infection through a transposon insertion followed by a Duplication.

To understand the molecular basis of how hosts evolve resistance to their parasites, we have investigated the genes that cause variation in the susceptibility of Drosophila melanogaster to viral infection. Using a host-specific pathogen of D. melanogaster called the sigma virus (Rhabdoviridae), we mapped a major-effect polymorphism to a region containing two paralogous genes called CHKov1 and CHKov2. In a panel of inbred fly lines, we found that a transposable element insertion in the protein coding sequence of CHKov1 is associated with increased resistance to infection. Previous research has shown that this insertion results in a truncated messenger RNA that encodes a far shorter protein than the susceptible allele. This resistant allele has rapidly increased in frequency under directional selection and is now the commonest form of the gene in natural populations. Using genetic mapping and site-specific recombination, we identified a third genotype with considerably greater resistance that is currently rare in the wild. In these flies there have been two duplications, resulting in three copies of both the truncated allele of CHKov1 and CHKov2 (one of which is also truncated). Remarkably, the truncated allele of CHKov1 has previously been found to confer resistance to organophosphate insecticides. As estimates of the age of this allele predate the use of insecticides, it is likely that this allele initially functioned as a defence against viruses and fortuitously "pre-adapted" flies to insecticides. These results demonstrate that strong selection by parasites for increased host resistance can result in major genetic changes and rapid shifts in allele frequencies; and, contrary to the prevailing view that resistance to pathogens can be a costly trait to evolve, the pleiotropic effects of these changes can have unexpected benefits.

Host phylogeny determines viral persistence and replication in novel hosts.

Pathogens switching to new hosts can result in the emergence of new infectious diseases, and determining which species are likely to be sources of such host shifts is essential to understanding disease threats to both humans and wildlife. However, the factors that determine whether a pathogen can infect a novel host are poorly understood. We have examined the ability of three host-specific RNA-viruses (Drosophila sigma viruses from the family Rhabdoviridae) to persist and replicate in 51 different species of Drosophilidae. Using a novel analytical approach we found that the host phylogeny could explain most of the variation in viral replication and persistence between different host species. This effect is partly driven by viruses reaching a higher titre in those novel hosts most closely related to the original host. However, there is also a strong effect of host phylogeny that is independent of the distance from the original host, with viral titres being similar in groups of related hosts. Most of this effect could be explained by variation in general susceptibility to all three sigma viruses, as there is a strong phylogenetic correlation in the titres of the three viruses. These results suggest that the source of new emerging diseases may often be predictable from the host phylogeny, but that the effect may be more complex than simply causing most host shifts to occur between closely related hosts.

Genetic variation affecting host-parasite interactions: different genes affect different aspects of sigma virus replication and transmission in Drosophila melanogaster.

In natural populations, genetic variation affects resistance to disease. Knowing how much variation exists, and understanding the genetic architecture of this variation, is important for medicine, for agriculture, and for understanding evolutionary processes. To investigate the extent and nature of genetic variation affecting resistance to pathogens, we are studying a tractable model system: Drosophila melanogaster and its natural pathogen the vertically transmitted sigma virus. We show that considerable genetic variation affects transmission of the virus from parent to offspring. However, maternal and paternal transmission of the virus is affected by different genes. Maternal transmission is a simple Mendelian trait: most of the genetic variation is explained by a polymorphism in ref(2)P, a gene already well known to affect resistance to sigma. In contrast, there is considerable genetic variation in paternal transmission that cannot be explained by ref(2)P and is caused by other loci on chromosome 2. Furthermore, we found no genetic correlation between paternal transmission of the virus and resistance to infection by the sigma virus following injection. This suggests that different loci affect viral replication and paternal transmission.

Synteny and chromosome evolution in the lepidoptera: evidence from mapping in Heliconius melpomene.

The extent of conservation of synteny and gene order in the Lepidoptera has been investigated previously only by comparing a small subset of linkage groups between the moth Bombyx mori and the butterfly Heliconius melpomene. Here we report the mapping of 64 additional conserved genes in H. melpomene, which contributed 47 markers to a comparative framework of 72 orthologous loci spanning all 21 H. melpomene chromosomes and 27 of the 28 B. mori chromosomes. Comparison of the maps revealed conserved synteny across all chromosomes for the 72 loci, as well as evidence for six cases of chromosome fusion in the Heliconius lineage that contributed to the derived 21-chromosome karyotype. Comparisons of gene order on these fused chromosomes revealed two instances of colinearity between H. melpomene and B. mori, but also one instance of likely chromosomal rearrangement. B. mori is the first lepidopteran species to have its genome sequenced, and the finding that there is conserved synteny and gene order among Lepidoptera indicates that the genomic tools developed in B. mori will be broadly useful in other species.