Genetic innovations in animal-microbe symbioses.

Animal hosts have initiated myriad symbiotic associations with microorganisms and often have maintained these symbioses for millions of years, spanning drastic changes in ecological conditions and lifestyles. The establishment and persistence of these relationships require genetic innovations on the parts of both symbionts and hosts. The nature of symbiont innovations depends on their genetic population structure, categorized here as open, closed or mixed. These categories reflect modes of inter-host transmission that result in distinct genomic features, or genomic syndromes, in symbionts. Although less studied, hosts also innovate in order to preserve and control symbiotic partnerships. New capabilities to sequence host-associated microbial communities and to experimentally manipulate both hosts and symbionts are providing unprecedented insights into how genetic innovations arise under different symbiont population structures and how these innovations function to support symbiotic relationships.

Evolutionary Ecology of Wolbachia Releases for Disease Control.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

Wbm0076, a candidate effector protein of the Wolbachia endosymbiont of Brugia malayi, disrupts eukaryotic actin dynamics.

Brugia malayi, a parasitic roundworm of humans, is colonized by the obligate intracellular bacterium, Wolbachia pipientis. The symbiosis between this nematode and bacterium is essential for nematode reproduction and long-term survival in a human host. Therefore, identifying molecular mechanisms required by Wolbachia to persist in and colonize B. malayi tissues will provide new essential information regarding the basic biology of this endosymbiosis. Wolbachia utilize a Type IV secretion system to translocate so-called "effector" proteins into the cytosol of B. malayi cells to promote colonization of the eukaryotic host. However, the characterization of these Wolbachia secreted proteins has remained elusive due to the genetic intractability of both organisms. Strikingly, expression of the candidate Wolbachia Type IV-secreted effector protein, Wbm0076, in the surrogate eukaryotic cell model, Saccharomyces cerevisiae, resulted in the disruption of the yeast actin cytoskeleton and inhibition of endocytosis. Genetic analyses show that Wbm0076 is a member of the family of Wiskott-Aldrich syndrome proteins (WAS [p]), a well-conserved eukaryotic protein family required for the organization of actin skeletal structures. Thus, Wbm0076 likely plays a central role in the active cell-to-cell movement of Wolbachia throughout B. malayi tissues during nematode development. As most Wolbachia isolates sequenced to date encode at least partial orthologs of wBm0076, we find it likely that the ability of Wolbachia to directly manipulate host actin dynamics is an essential requirement of all Wolbachia endosymbioses, independent of host cell species.

Functional analysis of Wolbachia Cid effectors unravels cooperative interactions to target host chromatin during replication.

Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, we show that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. Our study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity.

De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.

Creating reference animal genomes is typically a large, expensive process. Here I sequenced the genome of the black carpenter ant for only $1000 as a sole researcher in just one week. Along with the nuclear genome, I assembled the mitochondrial genome and two commensal bacteria species living within the ant. Nanopore technology also enabled epigenetic measurements from the same ant and replicated other studies showing very low DNA methylation. The reference genome compared favorably to other ant species in continuity and protein prediction accuracy. This method will allow other low-resource labs to create high quality genome assemblies with a low cost.

Differential viral RNA methylation contributes to pathogen blocking in Wolbachia-colonized arthropods.

Arthropod endosymbiont Wolbachia pipientis is part of a global biocontrol strategy to reduce the replication of mosquito-borne RNA viruses such as alphaviruses. We previously demonstrated the importance of a host cytosine methyltransferase, DNMT2, in Drosophila and viral RNA as a cellular target during pathogen-blocking. Here we report a role for DNMT2 in Wolbachia-induced alphavirus inhibition in Aedes species. Expression of DNMT2 in mosquito tissues, including the salivary glands, is elevated upon virus infection. Notably, this is suppressed in Wolbachia-colonized animals, coincident with reduced virus replication and decreased infectivity of progeny virus. Ectopic expression of DNMT2 in cultured Aedes cells is proviral, increasing progeny virus infectivity, and this effect of DNMT2 on virus replication and infectivity is dependent on its methyltransferase activity. Finally, examining the effects of Wolbachia on modifications of viral RNA by LC-MS show a decrease in the amount of 5-methylcytosine modification consistent with the down-regulation of DNMT2 in Wolbachia colonized mosquito cells and animals. Collectively, our findings support the conclusion that disruption of 5-methylcytosine modification of viral RNA is a vital mechanism operative in pathogen blocking. These data also emphasize the essential role of epitranscriptomic modifications in regulating fundamental alphavirus replication and transmission processes.

A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Wolbachia invasion dynamics of a random mosquito population model with imperfect maternal transmission and incomplete CI.

In this work, we formulate a random Wolbachia invasion model incorporating the effects of imperfect maternal transmission and incomplete cytoplasmic incompatibility (CI). Under constant environments, we obtain the following results: Firstly, the complete invasion equilibrium of Wolbachia does not exist, and thus the population replacement is not achievable in the case of imperfect maternal transmission; Secondly, imperfect maternal transmission or incomplete CI may obliterate bistability and backward bifurcation, which leads to the failure of Wolbachia invasion, no matter how many infected mosquitoes would be released; Thirdly, the threshold number of the infected mosquitoes to be released would increase with the decrease of the maternal transmission rate or the intensity of CI effect. In random environments, we investigate in detail the Wolbachia invasion dynamics of the random mosquito population model and establish the initial release threshold of infected mosquitoes for successful invasion of Wolbachia into the wild mosquito population. In particular, the existence and stability of invariant probability measures for the establishment and extinction of Wolbachia are determined.

A single mutation weakens symbiont-induced reproductive manipulation through reductions in deubiquitylation efficiency.

Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI's prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI-causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster.

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.

Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility.

The genus Wolbachia is an archetype of maternally inherited intracellular bacteria that infect the germline of numerous invertebrate species worldwide. They can selfishly alter arthropod sex ratios and reproductive strategies to increase the proportion of the infected matriline in the population. The most common reproductive manipulation is cytoplasmic incompatibility, which results in embryonic lethality in crosses between infected males and uninfected females. Females infected with the same Wolbachia strain rescue this lethality. Despite more than 40 years of research and relevance to symbiont-induced speciation, as well as control of arbovirus vectors and agricultural pests, the bacterial genes underlying cytoplasmic incompatibility remain unknown. Here we use comparative and transgenic approaches to demonstrate that two differentially transcribed, co-diverging genes in the eukaryotic association module of prophage WO from Wolbachia strain wMel recapitulate and enhance cytoplasmic incompatibility. Dual expression in transgenic, uninfected males of Drosophila melanogaster crossed to uninfected females causes embryonic lethality. Each gene additively augments embryonic lethality in crosses between infected males and uninfected females. Lethality associates with embryonic defects that parallel those of wild-type cytoplasmic incompatibility and is notably rescued by wMel-infected embryos in all cases. The discovery of cytoplasmic incompatibility factor genes cifA and cifB pioneers genetic studies of prophage WO-induced reproductive manipulations and informs the continuing use of Wolbachia to control dengue and Zika virus transmission to humans.

Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti.

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

Transcriptional response of Wolbachia-transinfected Aedes aegypti mosquito cells to dengue virus at early stages of infection.

Mosquito-borne flaviviruses are responsible for viral infections and represent a considerable public health burden. Aedes aegypti is the principal vector of dengue virus (DENV), therefore understanding the intrinsic virus-host interactions is vital, particularly in the presence of the endosymbiont Wolbachia, which blocks virus replication in mosquitoes. Here, we examined the transcriptional response of Wolbachia-transinfected Ae. aegypti Aag2 cells to DENV infection. We identified differentially expressed immune genes that play a key role in the activation of anti-viral defence such as the Toll and immune deficiency pathways. Further, genes encoding cytosine and N6-adenosine methyltransferases and SUMOylation, involved in post-transcriptional modifications, an antioxidant enzyme, and heat-shock response were up-regulated at the early stages of DENV infection and are reported here for the first time. Additionally, several long non-coding RNAs were among the differentially regulated genes. Our results provide insight into Wolbachia-transinfected Ae. aegypti's initial virus recognition and transcriptional response to DENV infection.

Sex determination systems as the interface between male-killing bacteria and their hosts.

Arthropods host a range of sex-ratio-distorting selfish elements, including diverse maternally inherited endosymbionts that solely kill infected males. Male-killing heritable microbes are common, reach high frequency, but until recently have been poorly understood in terms of the host-microbe interaction. Additionally, while male killing should generate strong selection for host resistance, evidence of this has been scant. The interface of the microbe with host sex determination is integral to the understanding of how death is sex limited and how hosts can evolve evasion of male killing. We first review current knowledge of the mechanisms diverse endosymbionts use to induce male-specific death. We then examine recent evidence that these agents do produce intense selection for host nuclear suppressor elements. We argue, from our understanding of male-killing mechanisms, that suppression will commonly involve evolution of the host sex determination pathways and that the host's response to male-killing microbes thus represents an unrecognized driver of the diversity of arthropod sex determination. Further work is required to identify the genes and mechanisms responsible for male-killing suppression, which will both determine the components of sex determination (or other) systems associated with suppressor evolution, and allow insight into the mechanism of male killing itself.

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential.

Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA's complex functional repertoire and new essential regions in CifB.

Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due, in part, to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be crucial in rescue are correspondingly crucial in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all crucial for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI.

Wolbachia and Sirtuin-4 interaction is associated with alterations in host glucose metabolism and bacterial titer.

Wolbachia is an intracellular bacterial symbiont of arthropods notorious for inducing many reproductive manipulations that foster its dissemination. Wolbachia affects many aspects of host biology, including metabolism, longevity and physiology, being described as a nutrient provisioning or metabolic parasite, depending on the host-microbe association. Sirtuins (SIRTs) are a family of NAD+-dependent post-translational regulatory enzymes known to affect many of the same processes altered by Wolbachia, including aging and metabolism, among others. Despite a clear overlap in control of host-derived pathways and physiology, no work has demonstrated a link between these two regulators. We used genetically tractable Drosophila melanogaster to explore the role of sirtuins in shaping signaling pathways in the context of a host-symbiont model. By using transcriptional profiling and metabolic assays in the context of genetic knockouts/over-expressions, we examined the effect of several Wolbachia strains on host sirtuin expression across distinct tissues and timepoints. We also quantified the downstream effects of the sirtuin x Wolbachia interaction on host glucose metabolism, and in turn, how it impacted Wolbachia titer. Our results indicate that the presence of Wolbachia is associated with (1) reduced sirt-4 expression in a strain-specific manner, and (2) alterations in host glutamate dehydrogenase expression and ATP levels, key components of glucose metabolism. We detected high glucose levels in Wolbachia-infected flies, which further increased when sirt-4 was over-expressed. However, under sirt-4 knockout, flies displayed a hypoglycemic state not rescued to normal levels in the presence of Wolbachia. Finally, whole body sirt-4 over-expression resulted in reduced Wolbachia ovarian titer. Our results expand knowledge of Wolbachia-host associations in the context of a yet unexplored class of host post-translational regulatory enzymes with implications for conserved host signaling pathways and bacterial titer, factors known to impact host biology and the symbiont's ability to spread through populations.

Infection Dynamics of Cotransmitted Reproductive Symbionts Are Mediated by Sex, Tissue, and Development.

One of the most prevalent intracellular infections on earth is with Wolbachia, a bacterium in the Rickettsiales that infects a range of insects, crustaceans, chelicerates, and nematodes. Wolbachia is maternally transmitted to offspring and has profound effects on the reproduction and physiology of its hosts, which can result in reproductive isolation, altered vectorial capacity, mitochondrial sweeps, and even host speciation. Some populations stably harbor multiple Wolbachia strains, which can further contribute to reproductive isolation and altered host physiology. However, almost nothing is known about the requirements for multiple intracellular microbes to be stably maintained across generations while they likely compete for space and resources. Here, we use a coinfection of two Wolbachia strains ("wHa" and "wNo") in Drosophila simulans to define the infection and transmission dynamics of an evolutionarily stable double infection. We find that a combination of sex, tissue, and host development contributes to the infection dynamics of the two microbes and that these infections exhibit a degree of niche partitioning across host tissues. wHa is present at a significantly higher titer than wNo in most tissues and developmental stages, but wNo is uniquely dominant in ovaries. Unexpectedly, the ratio of wHa to wNo in embryos does not reflect those observed in the ovaries, indicative of strain-specific transmission dynamics. Understanding how Wolbachia strains interact to establish and maintain stable infections has important implications for the development and effective implementation of Wolbachia-based vector biocontrol strategies, as well as more broadly defining how cooperation and conflict shape intracellular communities. IMPORTANCEWolbachia is a maternally transmitted intracellular bacterium that manipulates the reproduction and physiology of arthropods, resulting in drastic effects on the fitness, evolution, and even speciation of its hosts. Some hosts naturally harbor multiple strains of Wolbachia that are stably transmitted across generations, but almost nothing is known about the factors that limit or promote these coinfections, which can have profound effects on the host's biology and evolution and are under consideration as an insect-management tool. Here, we define the infection dynamics of a known stably transmitted double infection in Drosophila simulans with an eye toward understanding the patterns of infection that might facilitate compatibility between the two microbes. We find that a combination of sex, tissue, and development all contributes to infection dynamics of the coinfection.

Abundance and Localization of Symbiotic Bacterial Communities in the Fly Parasitoid Spalangia cameroni.

Multicellular eukaryotes often host multiple microbial symbionts that may cooperate or compete for host resources, such as space and nutrients. Here, we studied the abundances and localization of four bacterial symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, in the parasitic wasp Spalangia cameroni. Using quantitative PCR (qPCR), we measured the symbionts' titers in wasps that harbor different combinations of these symbionts. We found that the titer of each symbiont decreased as the number of symbiont species in the community increased. Symbionts' titers were higher in females than in males. Rickettsia was the most abundant symbiont in all the communities, followed by Sodalis and Wolbachia. The titers of these three symbionts were positively correlated in some of the colonies. Fluorescence in situ hybridization was in line with the qPCR results: Rickettsia, Wolbachia, and Sodalis were observed in high densities in multiple organs, including brain, muscles, gut, Malpighian tubules, fat body, ovaries, and testes, while Arsenophonus was localized to fewer organs and in lower densities. Sodalis and Arsenophonus were observed in ovarian follicle cells but not within oocytes or laid eggs. This study highlights the connection between symbionts' abundance and localization. We discuss the possible connections between our findings to symbiont transmission success. IMPORTANCE Many insects carry intracellular bacterial symbionts (bacteria that reside within the cells of the insect). When multiple symbiont species cohabit in a host, they may compete or cooperate for space, nutrients, and transmission, and the nature of such interactions would be reflected in the abundance of each symbiont species. Given the widespread occurrence of coinfections with maternally transmitted symbionts in insects, it is important to learn more about how they interact, where they are localized, and how these two aspects affect their co-occurrence within individual insects. Here, we studied the abundance and the localization of four symbionts, Rickettsia, Wolbachia, Sodalis, and Arsenophonus, that cohabit the parasitic wasp Spalangia cameroni. We found that symbionts' titers differed between symbiotic communities. These results were corroborated by microscopy, which shows differential localization patterns. We discuss the findings in the contexts of community ecology, possible symbiont-symbiont interactions, and host control mechanisms that may shape the symbiotic community structure.

Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking.

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined alpha-Mann-2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue- and time-dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti.