Diversifying selection and host adaptation in two endosymbiont genomes

Background: The endosymbiont Wolbachia pipientis infects a broad range of arthropod and filarial nematode hosts. These diverse associations form an attractive model for understanding host: symbiont coevolution. Wolbachia's ubiquity and ability to dramatically alter host reproductive biology also form the foundation of research strategies aimed at controlling insect pests and vector-borne disease. The Wolbachia strains that infect nematodes are phylogenetically distinct, strictly vertically transmitted, and required by their hosts for growth and reproduction. Insects in contrast form more fluid associations with Wolbachia. In these taxa, host populations are most often polymorphic for infection, horizontal transmission occurs between distantly related hosts, and direct fitness effects on hosts are mild. Despite extensive interest in the Wolbachia system for many years, relatively little is known about the molecular mechanisms that mediate its varied interactions with different hosts. We have compared the genomes of the Wolbachia that infect Drosophila melanogaster, wMel and the nematode Brugia malayi, wBm to that of an outgroup Anaplasma marginale to identify genes that have experienced diversifying selection in the Wolbachia lineages. The goal of the study was to identify likely molecular mechanisms of the symbiosis and to understand the nature of the diverse association across different hosts. Results: The prevalence of selection was far greater in wMel than wBm. Genes contributing to DNA metabolism, cofactor biosynthesis, and secretion were positively selected in both lineages. In wMel there was a greater emphasis on DNA repair, cell division, protein stability, and cell envelope synthesis. Conclusion: Secretion pathways and outer surface protein encoding genes are highly affected by selection in keeping with host: parasite theory. If evidence of selection on various cofactor molecules reflects possible provisioning, then both insect as well as nematode Wolbachia may be providing substances to hosts. Selection on cell envelope synthesis, DNA replication and repair machinery, heat shock, and two component switching suggest strategies insect Wolbachia may employ to cope with diverse host and intra-host environments

Solving the Wolbachia Paradox: Modeling the Tripartite Interaction between Host, Wolbachia, and a Natural Enemy

Wolbachia is one of the most common symbionts of arthropods. Its establishment requires lateral transfer to and successful transmission within novel host species. However, Wolbachia performs poorly when introduced into new host species, and models predict that Wolbachia should seldom be able to establish from low initial frequencies. Recently, various symbionts, including Wolbachia, have been shown to protect their hosts from natural enemies. Hence, Wolbachia invasion may be facilitated by the dynamic interaction between it, its host, and a natural enemy. We model such an interaction whereby Wolbachia induces either complete resistance, partial resistance, or tolerance to a host-specific pathogen and also induces the common manipulation phenotype of cytoplasmic incompatibility (CI). We show that the presence of the pathogen greatly facilitates Wolbachia invasion from rare and widens the parameter space in which "imperfect" Wolbachia strains can invade. Furthermore, positive frequency-dependent selection through CI can drive Wolbachia to very high frequencies, potentially excluding the pathogen. These results may explain a poorly understood aspect of Wolbachia biology: it is widespread, despite performing poorly after transfer to new host species. They also support the intriguing possibility that Wolbachia strains that encode both CI and natural-enemy resistance could potentially rid insects, including human disease vectors, of important pathogens

Wolbachia-Mediated Antibacterial Protection and Immune Gene Regulation in Drosophila

The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections

Antiviral protection and the importance of Wolbachia density and: tissue tropism in Drosophila simulans

Wolbachia, a maternally transmitted endosymbiont of insects, is increasingly being seen as an effective biological control agent that can interfere with transmission of pathogens, including dengue virus. However, the mechanism of antiviral protection is not well understood. The density and distribution of Wolbachia in host tissues have been implicated as contributing factors by previous studies with both mosquitoes and flies. Drosophila flies infected with five diverse strains of Wolbachia were screened for the ability to mediate antiviral protection. The three protective Wolbachia strains were more closely related and occurred at a higher density within whole flies than the two nonprotective Wolbachia strains. In this study, to further investigate the relationship between whole-fly Wolbachia density and the ability to mediate antiviral protection, tetracycline was used to decrease the abundance of the high-density, protective Wolbachia strain wAu prior to viral challenge. Antiviral protection was lost when the density of the protective Wolbachia strain was decreased to an abundance similar to that of nonprotective Wolbachia strains. We determined the Wolbachia density and distribution in tissues of the same five fly-Wolbachia combinations as used previously. The Wolbachia density within the head, gut, and Malpighian tubules correlated with the ability to mediate antiviral protection. These findings may facilitate the development of Wolbachia biological control strategies and help to predict host-Wolbachia pairings that may interfere with virus-induced pathology

Evidence and Consequence of a Highly Adapted Clonal Haplotype within the Australian Ascochyta rabiei Population

The Australian Ascochyta rabiei (Pass.) Labr. (syn. Phoma rabiei) population has low genotypic diversity with only one mating type detected to date, potentially precluding substantial evolution through recombination. However, a large diversity in aggressiveness exists. In an effort to better understand the risk from selective adaptation to currently used resistance sources and chemical control strategies, the population was examined in detail. For this, a total of 598 isolates were quasi-hierarchically sampled between 2013 and 2015 across all major Australian chickpea growing regions and commonly grown host genotypes. Although a large number of haplotypes were identified (66) through short sequence repeat (SSR) genotyping, overall low gene diversity (Hexp = 0.066) and genotypic diversity (D = 0.57) was detected. Almost 70% of the isolates assessed were of a single dominant haplotype (ARH01). Disease screening on a differential host set, including three commonly deployed resistance sources, revealed distinct aggressiveness among the isolates, with 17% of all isolates identified as highly aggressive. Almost 75% of these were of the ARH01 haplotype. A similar pattern was observed at the host level, with 46% of all isolates collected from the commonly grown host genotype Genesis090 (classified as “resistant” during the term of collection) identified as highly aggressive. Of these, 63% belonged to the ARH01 haplotype. In conclusion, the ARH01 haplotype represents a significant risk to the Australian chickpea industry, being not only widely adapted to the diverse agro-geographical environments of the Australian chickpea growing regions, but also containing a disproportionately large number of aggressive isolates, indicating fitness to survive and replicate on the best resistance sources in the Australian germplasm.The research was funded by the Grains Research and Development Cooperation within project UM00052

Evidence for Metabolic Provisioning by a Common Invertebrate Endosymbiont, Wolbachia pipientis, during Periods of Nutritional Stress

Wolbachia are ubiquitous inherited endosymbionts of invertebrates that invade host populations by modifying host reproductive systems. However, some strains lack the ability to impose reproductive modification and yet are still capable of successfully invading host populations. To explain this paradox, theory predicts that such strains should provide a fitness benefit, but to date none has been detected. Recently completed genome sequences of different Wolbachia strains show that these bacteria may have the genetic machinery to influence iron utilization of hosts. Here we show that Wolbachia infection can confer a positive fecundity benefit for Drosophila melanogaster reared on iron-restricted or -overloaded diets. Furthermore, iron levels measured from field-collected flies indicated that nutritional conditions in the field were overall comparable to those of flies reared in the laboratory on restricted diets. These data suggest that Wolbachia may play a previously unrecognized role as nutritional mutualists in insects

Aligning ecological compensation policies with the Post‐2020 Global Biodiversity Framework to achieve real net gain in biodiversity

Increasingly, government and corporate policies on ecological compensation (e.g., offsetting) are requiring “net gain” outcomes for biodiversity. This presents an opportunity to align development with the United Nations Conven-tion on Biological Diversity Post-2020 Global Biodiversity Framework's (GBF) proposed ambition for overall biodiversity recovery. In this perspective, we describe three conditions that should be accounted for in net gain policy to align outcomes with biodiversity recovery goals: namely, a requirement for residual losses from development to be compensated for by (1) absolute gains,which are (2) scaled to the achievement of explicit biodiversity targets, where(3) gains are demonstrably feasible. We show that few current policies meet these conditions, which risks undermining efforts to achieve the proposed Post-2020 GBF milestones and goals, as well as other jurisdictional policy imperatives to halt and reverse biodiversity decline. To guide future decision-making, we provide a supporting decision tree outlining net gain compensation feasibility

Draft genome sequence of the male-killing Wolbachia strain wBol1 reveals recent horizontal gene transfers from diverse sources.

Background The endosymbiont Wolbachia pipientis causes diverse and sometimes dramatic phenotypes in its invertebrate hosts. Four Wolbachia strains sequenced to date indicate that the constitution of the genome is dynamic, but these strains are quite divergent and do not allow resolution of genome diversification over shorter time periods. We have sequenced the genome of the strain wBol1-b, found in the butterfly Hypolimnas bolina, which kills the male offspring of infected hosts during embyronic development and is closely related to the non-male-killing strain wPip from Culex pipiens. Results The genomes of wBol1-b and wPip are similar in genomic organisation, sequence and gene content, but show substantial differences at some rapidly evolving regions of the genome, primarily associated with prophage and repetitive elements. We identified 44 genes in wBol1-b that do not have homologs in any previously sequenced strains, indicating that Wolbachia's non-core genome diversifies rapidly. These wBol1-b specific genes include a number that have been recently horizontally transferred from phylogenetically distant bacterial taxa. We further report a second possible case of horizontal gene transfer from a eukaryote into Wolbachia. Conclusions Our analyses support the developing view that many endosymbiotic genomes are highly dynamic, and are exposed and receptive to exogenous genetic material from a wide range of sources. These data also suggest either that this bacterial species is particularly permissive for eukaryote-to-prokaryote gene transfers, or that these transfers may be more common than previously believed. The wBol1-b-specific genes we have identified provide candidates for further investigations of the genomic bases of phenotypic differences between closely-related Wolbachia strains.Peer reviewe

The Wolbachia endosymbiont as an anti-filarial nematode target

Human disease caused by parasitic filarial nematodes is a major cause of global morbidity. The parasites are transmitted by arthropod intermediate hosts and are responsible for lymphatic filariasis (elephantiasis) or onchocerciasis (river blindness). Within these filarial parasites are intracellular alpha-proteobacteria, Wolbachia, that were first observed almost 30 years ago. The obligate endosymbiont has been recognized as a target for anti-filarial nematode chemotherapy as evidenced by the loss of worm fertility and viability upon antibiotic treatment in an extensive series of human trials. While current treatments with doxycycline and rifampicin are not practical for widespread use due to the length of required treatments and contraindications, anti-Wolbachia targeting nevertheless appears a promising alternative for filariasis control in situations where current programmatic strategies fail or are unable to be delivered and it provides a superior efficacy for individual therapy. The mechanisms that underlie the symbiotic relationship between Wolbachia and its nematode hosts remain elusive. Comparative genomics, bioinfomatic and experimental analyses have identified a number of potential interactions, which may be drug targets. One candidate is de novo heme biosynthesis, due to its absence in the genome sequence of the host nematode, Brugia malayi, but presence in Wolbachia and its potential roles in worm biology. We describe this and several additional candidate targets, as well as our approaches for understanding the nature of the host-symbiont relationship

Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the α-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel–D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections