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The non-virulent Wolbachia strain wMel and the life-shortening strain wMelPop-CLA, both originally from Drosophila melanogaster, have been stably introduced into the mosquito vector of dengue fever, Aedes aegypti. Each of these Wolbachia strains interferes with viral pathogenicity and/or dissemination in both their natural Drosophila host and in their new mosquito host, and it has been suggested that this virus interference may be due to host immune priming by Wolbachia. In order to identify aspects of the mosquito immune response that might underpin virus interference, we used whole-genome microarrays to analyse the transcriptional response of A. aegypti to the wMel and wMelPop-CLA Wolbachia strains. While wMel affected the transcription of far fewer host genes than wMelPop-CLA, both strains activated the expression of some immune genes including anti-microbial peptides, Toll pathway genes and genes involved in melanization. Because the induction of these immune genes might be associated with the very recent introduction of Wolbachia into the mosquito, we also examined the same Wolbachia strains in their original host D. melanogaster. First we demonstrated that when dengue viruses were injected into D. melanogaster, virus accumulation was significantly reduced in the presence of Wolbachia, just as in A. aegypti. Second, when we carried out transcriptional analyses of the same immune genes up-regulated in the new heterologous mosquito host in response to Wolbachia we found no over-expression of these genes in D. melanogaster, infected with either wMel or wMelPop. These results reinforce the idea that the fundamental mechanism involved in viral interference in Drosophila and Aedes is not dependent on the up-regulation of the immune effectors examined, although it cannot be excluded that immune priming in the heterologous mosquito host might enhance the virus interference trait.

Dengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites. We used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival. The saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.

Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect. Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction. This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.

Dengue virus (DENV) is primarily vectored by the mosquito Aedes aegypti, and is estimated to cause 390 million human infections annually. A novel method for DENV control involves stable transinfection of Ae. aegypti with the common insect endosymbiont Wolbachia, which mediates an antiviral effect. However, the mechanism by which Wolbachia reduces the susceptibility of Ae. aegypti to DENV is not fully understood. In this study we assessed the potential of resident microbiota, which can play important roles in insect physiology and immune responses, to affect Wolbachia-mediated DENV blocking. The microbiome of Ae. aegypti stably infected with Wolbachia strain wMel was compared to that of Ae. aegypti without Wolbachia, using 16s rDNA profiling. Our results indicate that although Wolbachia affected the relative abundance of several genera, the microbiome of both the Wolbachia-infected and uninfected mosquitoes was dominated by Elizabethkingia and unclassified Enterobacteriaceae. To assess the potential of the resident microbiota to affect the Wolbachia-mediated antiviral effect, we used antibiotic treatment before infection with DENV by blood-meal. In spite of a significant shift in the microbiome composition in response to the antibiotics, we detected no effect of antibiotic treatment on DENV infection rates, or on the DENV load of infected mosquitoes. Our findings indicate that stable infection with Wolbachia strain wMel produces few effects on the microbiome of laboratory-reared Ae. aegypti. Moreover, our findings suggest that the microbiome can be significantly altered without affecting the fundamental DENV blocking phenotype in these mosquitoes. Since Ae. aegypti are likely to encounter diverse microbiota in the field, this is a particularly important result in the context of using Wolbachia as a method for DENV control.

Cytosine methylation is one of several reversible epigenetic modifications of DNA that allow a greater flexibility in the relationship between genotype and phenotype. Methylation in the simplest models dampens gene expression by modifying regions of DNA critical for transcription factor binding. The capacity to methylate DNA is variable in the insects due to diverse histories of gene loss and duplication of DNA methylases. Mosquitoes like Drosophila melanogaster possess only a single methylase, DNMT2. Here we characterise the methylome of the mosquito Aedes aegypti and examine its relationship to transcription and test the effects of infection with a virulent strain of the endosymbiont Wolbachia on the stability of methylation patterns. We see that methylation in the A. aegypti genome is associated with reduced transcription and is most common in the promoters of genes relating to regulation of transcription and metabolism. Similar gene classes are also methylated in aphids and honeybees, suggesting either conservation or convergence of methylation patterns. In addition to this evidence of evolutionary stability, we also show that infection with the virulent wMelPop Wolbachia strain induces additional methylation and demethylation events in the genome. While most of these changes seem random with respect to gene function and have no detected effect on transcription, there does appear to be enrichment of genes associated with membrane function. Given that Wolbachia lives within a membrane-bound vacuole of host origin and retains a large number of genes for transporting host amino acids, inorganic ions and ATP despite a severely reduced genome, these changes might represent an evolved strategy for manipulating the host environments for its own gain. Testing for a direct link between these methylation changes and expression, however, will require study across a broader range of developmental stages and tissues with methods that detect splice variants.

Competition between viruses and Wolbachia for host lipids is a proposed mechanism of Wolbachia -mediated virus blocking in insects. Yet, the metabolomic interaction between virus and symbiont within the mosquito has not been clearly defined. We compare the lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of the Wolbachia w Mel strain and dengue virus serotype 3 (DENV3). We found metabolic signatures of infection-induced intracellular events but little evidence to support direct competition between Wolbachia and virus for host lipids. Lipid profiles of dual-infected mosquitoes resemble those of DENV3 mono-infected mosquitoes, suggesting virus-driven modulation dominates over that of Wolbachia . Interestingly, knockdown of key metabolic enzymes suggests cardiolipins are host factors for DENV3 and Wolbachia replication. These findings define the Wolbachia -DENV3 metabolic interaction as indirectly antagonistic, rather than directly competitive, and reveal new research avenues with respect to mosquito × virus interactions at the molecular level. Koh, Islam, Ye et al. describe lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of Wolbachia ( w Mel) and dengue virus serotype 3 (DENV3), finding that virus modulation dominates the dual-infection lipid profile and that cardiolipins support DENV3 and Wolbachia replication. This study suggests that direct competition for lipids do not underlie Wolbachia -mediated virus blocking.

Drosophila harbor substantial genetic variation for antibacterial defense, and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life span, and reproduction. To understand the way in which insects invest in fighting bacterial infection, we selected for survival following systemic infection with the opportunistic pathogen Pseudomonas aeruginosa in wild-caught Drosophila melanogaster over 10 generations. We then examined genome-wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen. This powerful combination of techniques allowed us to specifically identify the genetic basis of the evolved immune response. In response to selection, population-level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly, however, as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection-associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and the Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. This work reveals novel pathways by which Drosophila can survive infection with a virulent pathogen that may be rare in wild populations, however, due to their cost.

Wolbachia is an intracellular bacterium that has been stably transinfected into the mosquito vector of dengue, Aedes aegypti. This inherited infection causes a range of metabolic and phenotypic alterations in the mosquito, which might be related to neuronal abnormalities. In order to determine if these alterations were caused by the manipulation of neuroamines by this bacterium, we studied the expression of genes involved in the dopamine biosynthetic pathway and also measured the amount of dopamine in infected and uninfected mosquitoes of different ages. Wolbachia-infected mosquitoes exhibit greater expression of some genes related to the melanization pathway, but not for those directly linked to dopamine production. Although dopamine levels were higher in Wolbachia-positive mosquitoes this was not consistent across all insect ages nor was it related to the previously described Wolbachia induced \"bendy\" and \"shaky\" phenotypes.

The nature and extent of mutational pleiotropy remain largely unknown, despite the central role that pleiotropy plays in many areas of biology, including human disease, agricultural production, and evolution. Here, we investigate the variation in 11,604 gene expression traits among 41 mutation accumulation (MA) lines of Drosophila serrata. We first confirmed that these expression phenotypes were heritable, detecting genetic variation in 96% of them in an outbred, natural population of D. serrata. Among the MA lines, 3385 (29%) of expression traits were variable, with a mean mutational heritability of 0.0005. In most traits, variation was generated by mutations of relatively small phenotypic effect; putative mutations with effects of greater than one phenotypic standard deviation were observed for only 8% of traits. With most (71%) traits unaffected by any mutation, our data provide no support for universal pleiotropy. We further characterized mutational pleiotropy in the 3385 variable traits, using sets of 5, randomly assigned, traits. Covariance among traits chosen at random with respect to their biological function is expected only if pleiotropy is extensive. Taking an analytical approach in which the variance unique to each trait in the random 5-trait sets was partitioned from variance shared among traits, we detected significant (at 5% false discovery rate) mutational covariance in 21% of sets. This frequency of statistically supported covariance implied that at least some mutations must pleiotropically affect a substantial number of traits (>70; 0.6% of all measured traits).

Dengue fever is the most common arboviral disease worldwide. It is caused by dengue viruses (DENV) and the mosquito Aedes aegypti is its primary vector. One of the most powerful determinants of a mosquito's ability to transmit DENV is the length of the extrinsic incubation period (EIP), the time it takes for a virus to be transmitted by a mosquito after consuming an infected blood meal. Here, we repeatedly measured DENV load in the saliva of individual mosquitoes over their lifetime and used this in combination with a breeding design to determine the extent to which EIP might respond to the evolutionary forces of drift and selection. We demonstrated that genetic variation among mosquitoes contributes significantly to transmission potential and length of EIP. We reveal that shorter EIP is genetically correlated with reduced mosquito lifespan, highlighting negative life-history consequences for virus-infected mosquitoes. This work highlights the capacity for local genetic variation in mosquito populations to evolve and to dramatically affect the nature of human outbreaks. It also provides the impetus for isolating mosquito genes that determine EIP. More broadly, our dual experimental approach offers new opportunities for studying the evolutionary potential of transmission traits in other vector/pathogen systems.