Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

The evolution of constitutively active humoral immune defenses in Drosophila populations under high parasite pressure.

Both constitutive and inducible immune mechanisms are employed by hosts for defense against infection. Constitutive immunity allows for a faster response, but it comes with an associated cost that is always present. This trade-off between speed and fitness costs leads to the theoretical prediction that constitutive immunity will be favored where parasite exposure is frequent. We selected populations of Drosophila melanogaster under high parasite pressure from the parasitoid wasp Leptopilina boulardi. With RNA sequencing, we found the evolution of resistance in these populations was associated with them developing constitutively active humoral immunity, mediated by the larval fat body. Furthermore, these evolved populations were also able to induce gene expression in response to infection to a greater level, which indicates an overall more activated humoral immune response to parasitization. The anti-parasitoid immune response also relies on the JAK/STAT signaling pathway being activated in muscles following infection, and this induced response was only seen in populations that had evolved under high parasite pressure. We found that the cytokine Upd3, which induces this JAK/STAT response, is being expressed by immature lamellocytes. Furthermore, these immune cells became constitutively present when populations evolved resistance, potentially explaining why they gained the ability to activate JAK/STAT signaling. Thus, under intense parasitism, populations evolved resistance by increasing both constitutive and induced immune defenses, and there is likely an interplay between these two forms of immunity.

Activation of immune defences against parasitoid wasps does not underlie the cost of infection.

Parasites reduce the fitness of their hosts, and different causes of this damage have fundamentally different consequences for the evolution of immune defences. Damage to the host may result from the parasite directly harming its host, often due to the production of virulence factors that manipulate host physiology. Alternatively, the host may be harmed by the activation of its own immune defences, as these can be energetically demanding or cause self-harm. A well-studied model of the cost of infection is Drosophila melanogaster and its common natural enemy, parasitoid wasps. Infected Drosophila larvae rely on humoral and cellular immune mechanisms to form a capsule around the parasitoid egg and kill it. Infection results in a developmental delay and reduced adult body size. To disentangle the effects of virulence factors and immune defences on these costs, we artificially activated anti-parasitoid immune defences in the absence of virulence factors. Despite immune activation triggering extensive differentiation and proliferation of immune cells together with hyperglycaemia, it did not result in a developmental delay or reduced body size. We conclude that the costs of infection do not result from these aspects of the immune response and may instead result from the parasite directly damaging the host.

Natural selection has driven the recurrent loss of an immunity gene that protects Drosophila against a major natural parasite.

Polymorphisms in immunity genes can have large effects on susceptibility to infection. To understand the origins of this variation, we have investigated the genetic basis of resistance to the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster. We found that increased expression of the gene lectin-24A after infection by parasitic wasps was associated with a faster cellular immune response and greatly increased rates of killing the parasite. lectin-24A encodes a protein that is strongly up-regulated in the fat body after infection and localizes to the surface of the parasite egg. In certain susceptible lines, a deletion upstream of the lectin-24A has largely abolished expression. Other mutations predicted to abolish the function of this gene have arisen recurrently in this gene, with multiple loss-of-expression alleles and premature stop codons segregating in natural populations. The frequency of these alleles varies greatly geographically, and in some southern African populations, natural selection has driven them near to fixation. We conclude that natural selection has favored the repeated loss of an important component of the immune system, suggesting that in some populations, a pleiotropic cost to lectin-24A expression outweighs the benefits of resistance.

Wolbachia protects Drosophila melanogaster against two naturally occurring and virulent viral pathogens.

Wolbachia is a common endosymbiont that can protect insects against viral pathogens. However, whether the antiviral effects of Wolbachia have a significant effect on fitness remains unclear. We have investigated the interaction between Drosophila melanogaster, Wolbachia and two viruses that we recently isolated from wild flies, La Jolla virus (LJV; Iflaviridae) and Newfield virus (NFV; Permutotetraviridae). Flies infected with these viruses have increased mortality rates, and NFV partially sterilizes females. These effects on fitness were reduced in Wolbachia-infected flies, and this was associated with reduced viral titres. However, Wolbachia alone also reduces survival, and under our experimental conditions these costs of the symbiont can outweigh the benefits of antiviral protection. In contrast, protection against the sterilizing effect of NFV leads to a net benefit of Wolbachia infection after exposure to the virus. These results support the hypothesis that Wolbachia is an important defense against the natural pathogens of D. melanogaster. Furthermore, by reducing the cost of Wolbachia infection, the antiviral effects of Wolbachia may aid its invasion into populations and help explain why it is so common in nature.

Wolbachia endosymbiotic bacteria alter the gut microbiome in the fly Drosophila nigrosparsa.

Wolbachia are known to cause reproductive manipulations and in some arthropod species, Wolbachia were reported to cause changes in gut microbiome. However, the effects of Wolbachia bacteria on the microbiomes of their hosts, including Drosophila flies, have not been fully accessed. Here, we checked the bacterial microbiome in guts of Wolbachia-uninfected and of Wolbachia-infected Drosophila nigrosparsa, both separated into a bleach-only (embryos bleached) and a gnotobiotic (embryos bleached and inoculated with bacteria) treatment. We observed a clear separation between the Wolbachia-infected and the Wolbachia-uninfected samples, and the infected samples had higher variation in alpha diversity than the uninfected ones. There were reductions in the abundances of Proteobacteria (Pseudomonadota), especially Acetobacter, in the infected samples of both treatments. These findings highlight that Wolbachia change the gut microbiome in D. nigrosparsa as well as that the interactions between Wolbachia and bacteria like Acetobacter need to be investigated.

Developing Wolbachia-based disease interventions for an extreme environment.

Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced egg hatch under a range of environmental conditions, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates.

Trans-regulatory changes underpin the evolution of the Drosophila immune response.

When an animal is infected, the expression of a large suite of genes is changed, resulting in an immune response that can defend the host. Despite much evidence that the sequence of proteins in the immune system can evolve rapidly, the evolution of gene expression is comparatively poorly understood. We therefore investigated the transcriptional response to parasitoid wasp infection in Drosophila simulans and D. sechellia. Although these species are closely related, there has been a large scale divergence in the expression of immune-responsive genes in their two main immune tissues, the fat body and hemocytes. Many genes, including those encoding molecules that directly kill pathogens, have cis regulatory changes, frequently resulting in large differences in their expression in the two species. However, these changes in cis regulation overwhelmingly affected gene expression in immune-challenged and uninfected animals alike. Divergence in the response to infection was controlled in trans. We argue that altering trans-regulatory factors, such as signalling pathways or immune modulators, may allow natural selection to alter the expression of large numbers of immune-responsive genes in a coordinated fashion.

A single introduction of wild rabbits triggered the biological invasion of Australia.

Biological invasions are a major cause of environmental and economic disruption. While ecological factors are key determinants of their success, the role of genetics has been more challenging to demonstrate. The colonization of Australia by the European rabbit is one of the most iconic and devastating biological invasions in recorded history. Here, we show that despite numerous introductions over a 70-y period, this invasion was triggered by a single release of a few animals that spread thousands of kilometers across the continent. We found genetic support for historical accounts that these were English rabbits imported in 1859 by a settler named Thomas Austin and traced the origin of the invasive population back to his birthplace in England. We also find evidence of additional introductions that established local populations but have not spread geographically. Combining genomic and historical data we show that, contrary to the earlier introductions, which consisted mostly of domestic animals, the invasive rabbits had wild ancestry. In New Zealand and Tasmania, rabbits also became a pest several decades after being introduced. We argue that the common denominator of these invasions was the arrival of a new genotype that was better adapted to the natural environment. These findings demonstrate how the genetic composition of invasive individuals can determine the success of an introduction and provide a mechanism by which multiple introductions can be required for a biological invasion.

A novel transposable element-mediated mechanism causes antiviral resistance in Drosophila through truncating the Veneno protein.

Hosts are continually selected to evolve new defenses against an ever-changing array of pathogens. To understand this process, we examined the genetic basis of resistance to the Drosophila A virus in Drosophila melanogaster. In a natural population, we identified a polymorphic transposable element (TE) insertion that was associated with an ∼19,000-fold reduction in viral titers, allowing flies to largely escape the harmful effects of infection by this virulent pathogen. The insertion occurs in the protein-coding sequence of the gene Veneno, which encodes a Tudor domain protein. By mutating Veneno with CRISPR-Cas9 in flies and expressing it in cultured cells, we show that the ancestral allele of the gene has no effect on viral replication. Instead, the TE insertion is a gain-of-function mutation that creates a gene encoding a novel resistance factor. Viral titers remained reduced when we deleted the TE sequence from the transcript, indicating that resistance results from the TE truncating the Veneno protein. This is a novel mechanism of virus resistance and a new way by which TEs can contribute to adaptation.

Wolbachia reduces virus infection in a natural population of Drosophila.

Wolbachia is a maternally transmitted bacterial symbiont that is estimated to infect approximately half of arthropod species. In the laboratory it can increase the resistance of insects to viral infection, but its effect on viruses in nature is unknown. Here we report that in a natural population of Drosophila melanogaster, individuals that are infected with Wolbachia are less likely to be infected by viruses. By characterising the virome by metagenomic sequencing and then testing individual flies for infection, we found the protective effect of Wolbachia was virus-specific, with the prevalence of infection being up to 15% greater in Wolbachia-free flies. The antiviral effects of Wolbachia may contribute to its extraordinary ecological success, and in nature the symbiont may be an important component of the antiviral defences of insects.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Life and Death of Selfish Genes: Comparative Genomics Reveals the Dynamic Evolution of Cytoplasmic Incompatibility.

Cytoplasmic incompatibility is a selfish reproductive manipulation induced by the endosymbiont Wolbachia in arthropods. In males Wolbachia modifies sperm, leading to embryonic mortality in crosses with Wolbachia-free females. In females, Wolbachia rescues the cross and allows development to proceed normally. This provides a reproductive advantage to infected females, allowing the maternally transmitted symbiont to spread rapidly through host populations. We identified homologs of the genes underlying this phenotype, cifA and cifB, in 52 of 71 new and published Wolbachia genome sequences. They are strongly associated with cytoplasmic incompatibility. There are up to seven copies of the genes in each genome, and phylogenetic analysis shows that Wolbachia frequently acquires new copies due to pervasive horizontal transfer between strains. In many cases, the genes have subsequently acquired loss-of-function mutations to become pseudogenes. As predicted by theory, this tends to occur first in cifB, whose sole function is to modify sperm, and then in cifA, which is required to rescue the cross in females. Although cif genes recombine, recombination is largely restricted to closely related homologs. This is predicted under a model of coevolution between sperm modification and embryonic rescue, where recombination between distantly related pairs of genes would create a self-incompatible strain. Together, these patterns of gene gain, loss, and recombination support evolutionary models of cytoplasmic incompatibility.

Constitutive activation of cellular immunity underlies the evolution of resistance to infection in Drosophila.

Organisms rely on inducible and constitutive immune defences to combat infection. Constitutive immunity enables a rapid response to infection but may carry a cost for uninfected individuals, leading to the prediction that it will be favoured when infection rates are high. When we exposed populations of Drosophila melanogaster to intense parasitism by the parasitoid wasp Leptopilina boulardi, they evolved resistance by developing a more reactive cellular immune response. Using single-cell RNA sequencing, we found that immune-inducible genes had become constitutively upregulated. This was the result of resistant larvae differentiating precursors of specialized immune cells called lamellocytes that were previously only produced after infection. Therefore, populations evolved resistance by genetically hard-wiring the first steps of an induced immune response to become constitutive.

------Widespread conservation and lineage-specific diversification of genome-wide DNA methylation patterns across arthropods.

Cytosine methylation is an ancient epigenetic modification yet its function and extent within genomes is highly variable across eukaryotes. In mammals, methylation controls transposable elements and regulates the promoters of genes. In insects, DNA methylation is generally restricted to a small subset of transcribed genes, with both intergenic regions and transposable elements (TEs) depleted of methylation. The evolutionary origin and the function of these methylation patterns are poorly understood. Here we characterise the evolution of DNA methylation across the arthropod phylum. While the common ancestor of the arthropods had low levels of TE methylation and did not methylate promoters, both of these functions have evolved independently in centipedes and mealybugs. In contrast, methylation of the exons of a subset of transcribed genes is ancestral and widely conserved across the phylum, but has been lost in specific lineages. A similar set of genes is methylated in all species that retained exon-enriched methylation. We show that these genes have characteristic patterns of expression correlating to broad transcription initiation sites and well-positioned nucleosomes, providing new insights into potential mechanisms driving methylation patterns over hundreds of millions of years.

Wolbachia affect behavior and possibly reproductive compatibility but not thermoresistance, fecundity, and morphology in a novel transinfected host, Drosophila nigrosparsa.

Wolbachia, intracellular endosymbionts, are estimated to infect about half of all arthropod species. These bacteria manipulate their hosts in various ways for their maximum benefits. The rising global temperature may accelerate species migration, and thus, horizontal transfer of Wolbachia may occur across species previously not in contact. We transinfected and then cured the alpine fly Drosophila nigrosparsa with Wolbachia strain wMel to study its effects on this species. We found low Wolbachia titer, possibly cytoplasmic incompatibility, and an increase in locomotion of both infected larvae and adults compared with cured ones. However, no change in fecundity, no impact on heat and cold tolerance, and no change in wing morphology were observed. Although Wolbachia increased locomotor activities in this species, we conclude that D. nigrosparsa may not benefit from the infection. Still, D. nigrosparsa can serve as a host for Wolbachia because vertical transmission is possible but may not be as high as in the native host of wMel, Drosophila melanogaster.

Age, tissue, genotype and virus infection regulate Wolbachia levels in Drosophila.

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host-symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus-specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell-autonomous, these effects are likely to affect the virus-blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host-symbiont-virus-dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.

Virus evolution in Wolbachia-infected Drosophila.

Wolbachia, a common vertically transmitted symbiont, can protect insects against viral infection and prevent mosquitoes from transmitting viral pathogens. For this reason, Wolbachia-infected mosquitoes are being released to prevent the transmission of dengue and other arboviruses. An important question for the long-term success of these programmes is whether viruses can evolve to escape the antiviral effects of Wolbachia. We have found that Wolbachia altered the outcome of competition between strains of the DCV virus in Drosophila. However, Wolbachia still effectively blocked the virus genotypes that were favoured in the presence of the symbiont. We conclude that Wolbachia did cause an evolutionary response in viruses, but this has little or no impact on the effectiveness of virus blocking.

Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids.

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster's immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution.