Phage WO Research Articles

Isolation of Phage WO Particles from Wolbachia-Infected Arthropods.

Nearly all arthropod-associated Wolbachia contain intact and/or genomic remnants of phage WO, temperate bacteriophages that facilitate horizontal gene transfer, genomic rearrangement of the bacterial chromosome, and symbiotic interactions between Wolbachia and their arthropod hosts. Integrated prophage WO genomes produce active, lytic particles; but the lack of a cell-free culturing system for Wolbachia render them difficult to purify and study. This chapter describes polyethylene glycol (PEG) precipitation of phage particles from Wolbachia-infected arthropods, followed by confirmation of phage WO isolation and purification using electron microscopy and PCR.

Virus-like Particles from Wolbachia-Infected Cells May Include a Gene Transfer Agent.

Wolbachia are obligate intracellular bacteria that occur in insects and filarial worms. Strains that infect insects have genomes that encode mobile genetic elements, including diverse lambda-like prophages called Phage WO. Phage WO packages an approximately 65 kb viral genome that includes a unique eukaryotic association module, or EAM, that encodes unusually large proteins thought to mediate interactions between the bacterium, its virus, and the eukaryotic host cell. The Wolbachia supergroup B strain, wStri from the planthopper Laodelphax striatellus, produces phage-like particles that can be recovered from persistently infected mosquito cells by ultracentrifugation. Illumina sequencing, assembly, and manual curation of DNA from two independent preparations converged on an identical 15,638 bp sequence that encoded packaging, assembly, and structural proteins. The absence of an EAM and regulatory genes defined for Phage WO from the wasp, Nasonia vitripennis, was consistent with the possibility that the 15,638 bp sequence represents an element related to a gene transfer agent (GTA), characterized by a signature head-tail region encoding structural proteins that package host chromosomal DNA. Future investigation of GTA function will be supported by the improved recovery of physical particles, electron microscopic examination of potential diversity among particles, and rigorous examination of DNA content by methods independent of sequence assembly.

PWCP is a widely distributed and highly conserved Wolbachia plasmid in Culex pipiens and Culex quinquefasciatus mosquitoes worldwide

Mosquitoes represent the most important pathogen vectors and are responsible for the spread of a wide variety of poorly treatable diseases. Wolbachia are obligate intracellular bacteria that are widely distributed among arthropods and collectively represents one of the most promising solutions for vector control. In particular, Wolbachia has been shown to limit the transmission of pathogens, and to dramatically affect the reproductive behavior of their host through its phage WO. While much research has focused on deciphering and exploring the biocontrol applications of these WO-related phenotypes, the extent and potential impact of the Wolbachia mobilome remain poorly appreciated. Notably, several Wolbachia plasmids, carrying WO-like genes and Insertion Sequences (IS), thus possibly interrelated to other genetic units of the endosymbiont, have been recently discovered. Here we investigated the diversity and biogeography of the first described plasmid of Wolbachia in Culex pipiens (pWCP) in several islands and continental countries around the world—including Cambodia, Guadeloupe, Martinique, Thailand, and Mexico—together with mosquito strains from colonies that evolved for 2 to 30 years in the laboratory. We used PCR and qPCR to determine the presence and copy number of pWCP in individual mosquitoes, and highly accurate Sanger sequencing to evaluate potential variations. Together with earlier observation, our results show that pWCP is omnipresent and strikingly conserved among Wolbachia populations within mosquitoes from distant geographies and environmental conditions. These data suggest a critical role for the plasmid in Wolbachia ecology and evolution, and the potential of a great tool for further genetic dissection and possible manipulation of this endosymbiont.

Phylogenomic analysis of Wolbachia genomes from the Darwin Tree of Life biodiversity genomics project.

The Darwin Tree of Life (DToL) project aims to sequence all described terrestrial and aquatic eukaryotic species found in Britain and Ireland. Reference genome sequences are generated from single individuals for each target species. In addition to the target genome, sequenced samples often contain genetic material from microbiomes, endosymbionts, parasites, and other cobionts. Wolbachia endosymbiotic bacteria are found in a diversity of terrestrial arthropods and nematodes, with supergroups A and B the most common in insects. We identified and assembled 110 complete Wolbachia genomes from 93 host species spanning 92 families by filtering data from 368 insect species generated by the DToL project. From 15 infected species, we assembled more than one Wolbachia genome, including cases where individuals carried simultaneous supergroup A and B infections. Different insect orders had distinct patterns of infection, with Lepidopteran hosts mostly infected with supergroup B, while infections in Diptera and Hymenoptera were dominated by A-type Wolbachia. Other than these large-scale order-level associations, host and Wolbachia phylogenies revealed no (or very limited) cophylogeny. This points to the occurrence of frequent host switching events, including between insect orders, in the evolutionary history of the Wolbachia pandemic. While supergroup A and B genomes had distinct GC% and GC skew, and B genomes had a larger core gene set and tended to be longer, it was the abundance of copies of bacteriophage WO who was a strong determinant of Wolbachia genome size. Mining raw genome data generated for reference genome assemblies is a robust way of identifying and analysing cobiont genomes and giving greater ecological context for their hosts.

Widespread phages of endosymbionts: Phage WO genomics and the proposed taxonomic classification of Symbioviridae.

Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachia’s mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WO’s relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the putative origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to distinguish the four variants. We identify a novel, putative lytic cassette and WO’s association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages, including non-Wolbachia phages from aquatic environments, that includes a new family and two new genera to capture the collective relatedness of these viruses.

Muramidase, nuclease, or hypothetical protein genes intervene between paired genes encoding DNA packaging terminase and portal proteins in Wolbachia phages and prophages.

Genomes of the obligate intracellular alpha proteobacterium Wolbachia pipientis often encode prophage-like regions, and in a few cases, purified particles have been recovered. Because the structure of a conserved WO phage genome has been difficult to establish, we examined paired terminase and portal genes in Wolbachia phages and prophages, relative to those encoded by the gene transfer agent RcGTA from the free-living alpha proteobacterium Rhodobacter capsulatus. Terminase and portal proteins from Wolbachia have higher similarity to orthologs encoded by RcGTA than to orthologs encoded by bacteriophage lambda. In lambdoid phages, these proteins play key roles in assembly of mature phage particles, while in less well-studied gene transfer agents, terminase and portal proteins package random fragments of bacterial DNA, which could confound elucidation of WO phage genomes. In WO phages and prophages, terminase genes followed by a short gpW gene may be separated from the downstream portal gene by open-reading frames encoding a GH_25 hydrolase/muramidase, a PD-(D/E)XK nuclease, a hypothetical protein and/or a RelE/ParE toxin-antitoxin module. These aspects of gene organization, coupled with evidence for a low, non-inducible yield of WO phages, and the small size of WO phage particles described in the literature raise the possibility that Wolbachia prophage regions participate in processes that extend beyond conventional bacteriophage lysogeny and lytic replication. These intervening genes, and their possible relation to functions associated with GTAs, may contribute to variability among WO phage genomes recovered from physical particles and impact the ability of WO phages to act as transducing agents.

High Levels of Multiple Phage WO Infections and Its Evolutionary Dynamics Associated With Wolbachia-Infected Butterflies.

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Phage WO is the only bacteriophage known to infect Wolbachia, and may provide benefit to its host or arthropods. We screened for the presence of phage WO in Wolbachia-infected butterfly species for the first time, to investigate their diversity and evolutionary dynamics. All Wolbachia-infected butterfly species, including members of the families Hesperiidae, Lycaenidae, Nymphalidae, Papilionidae, and Pieridae, were found to harbor phage WO. Interestingly, 84% of 19 butterfly species, which were infected with a single Wolbachia strain harbored high levels of multiple phage types (ranging from 3 to 17 types), another three species harbored one or two phage types. For Wolbachia strains (ST-41, ST-19, ST-125 and ST-374) shared among various butterfly species, their host insects all harbored multiple phage types, while two Wolbachia strains (ST-297 and ST-wPcau) were found to infect one butterfly species, whose insect hosts harbored a single phage type, suggesting that horizontal transfer of Wolbachia between insects increased the likelihood of exposure to phages, resulting in increased phage genetic diversity. Twelve horizontal transmission events of phage WO were found, which shared common phage WO types among different Wolbachia strains associated with butterflies. Most horizontal transfer events involved different Wolbachia supergroups (A and B). Horizontal acquisition of phage WO might also occur between eukaryotes without Wolbachia transfer. Furthermore, 22 putative recombination events were identified in 13 of 16 butterfly species which harbored multiple phage types. These results showed that horizontal transfer of Wolbachia caused it to be exposed to the phage gene pool, and that horizontal transmission of phage WO, as well as intragenic recombination were important dynamics for phage WO genome evolution, which effectively promoted the high level of phage WO diversity associated with butterflies.

Design and Testing of Effective Primers for Amplification of the orf7 Gene of Phage WO Associated with Andricus hakonensis.

Simple SummaryAndricus hakonensis is thought to contain the most complex and diverse phage types known and should be an ideal model material for studying interactions among bacteriophages, bacteria and eukaryotes. As shown in previous studies, existing primers are not effective enough to amplify all virus groups in A. hakonensis. Based on a comprehensive analysis of all virus groups reported to date, we designed a relatively conservative primer for virus detection. This primer can accurately and efficiently detect the presence of phage WO in arthropod hosts. Using gene alignment, clear evidence was provided for the existence of hitherto unreported base deletions, which are an important cause of diversity in phage WO associated with A. hakonensis.Phage WO was first characterized in Wolbachia, an obligate intracellular Rickettsiales known for its ability to regulate the reproduction of arthropod hosts. In this paper, we focus on the study of virus diversity in Andricus hakonensis and the development of highly effective primers. Based on the existing Wolbachia genome sequence, we designed primers (WO-TF and WO-TR) to amplify the full-length orf7 gene of phage WO. Surprisingly, sequencing results showed a high abundance of other phage WO groups in A. hakonensis, in addition to the four groups previously identified. The results also showed that A. hakonensis contained most of the known types of orf7 genes (I, III, IV, V and VI) and the level of diversity of harbored phage WO was very high. Therefore, we speculated that existing primers were not specific enough and that new primers for the detection of phage WO were needed. Based on the existing orf7 gene sequence, we designed specific detection primers (WO-SUF and WO-SUR). Sequencing results showed that the primers effectively amplified all known types of phage WO. In addition to amplifying most of the known sequences, we also detected some new genotypes in A. hakonensis using the new primers. Importantly, all phage WO groups could be efficiently detected. Combined with the results of previous studies, our results suggest that A. hakonensis contains the largest number of phage types (up to 36 types). This study is novel in that it provides practical molecular evidence supporting base deletions, in addition to gene mutations and genetic recombination, as an important cause of phage WO diversity.

The impact of artificial selection for Wolbachia-mediated dengue virus blocking on phage WO

Wolbachia is currently at the forefront of global efforts to control arbovirus transmission from the vector Aedes aegypti. The use of Wolbachia relies on two phenotypes—cytoplasmic incompatibility (CI), conferred by cifA and cifB genes in prophage WO, and Wolbachia-mediated pathogen blocking (WMPB). These traits allow for local, self-sustaining reductions in transmission of dengue (DENV) following release of Wolbachia-infected A. aegypti. Here, aided by previous artificial selection experiment that generated Low and High pathogen blocking lines, we examined the potential link between WMPB and phage WO. We found no evidence that Wolbachia or phage WO relative densities predict DENV blocking strength across selected lines. However, selection resulted in reduced phage WO relative density for the Low WMPB line. The Low blocking line was previously shown to have reduced fitness as a result of selection. Through subsequent genomic analyses, we demonstrate that SNP variation underpinning selection for low blocking led to elevated frequency of potential deleterious SNPs on chromosome 1. The key region on chromosome 1 contains genes relating to cell cycle regulation, oxidative stress, transcriptional pausing, among others, that may have cascading effects on Wolbachia intracellular environment. We hypothesize that reduction in phage WO may be driven by changes in the loci directly under selection for blocking, or by the accumulation of predicted deleterious alleles in linkage disequilibrium with blocking loci resulting from hitchhiking. For the Low line with fewer phage WO, we also detected reduced expression of cifA and cifB CI genes, with patterns of expression varying between somatic and reproductive tissues. In conclusion, we propose that artificial selection for WMPB trait had corresponding impacts on phage WO densities, and also the transcription of CI-causing genes. Future studies may include a more detailed analysis of the regions the A. aegypti chromosome 1’s ability to affect WMPB and other Wolbachia-associated intrinsic factors such as phage WO.

Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

A Case of Intragenic Recombination Dramatically Impacting the Phage WO Genetic Diversity in Gall Wasps.

The phage WO was characterized in Wolbachia, a strictly intracellular bacterium causing several reproductive alterations in its arthropod hosts. This study aimed to screen the presence of Wolbachia and phage WO in 15 gall wasp species from six provinces of southern China to investigate their diversity and prevalence patterns. A high incidence of Wolbachia infection was determined in the gall wasp species, with an infection rate of 86.7% (13/15). Moreover, seven species had double or multiple infections. All Wolbachia-infected gall wasp species were found to harbor phage WO. The gall wasp species infected with a single Wolbachia strain were found to harbor a single phage WO type. On the contrary, almost all species with double or multiple Wolbachia infections harbored a high level of phage WO diversity (ranging from three to 27 types). Six horizontal transfer events of phage WO in Wolbachia were found to be associated with gall wasps, which shared identical orf7 sequences among their respective accomplices. The transfer potentially took place through gall inducers and associated inquilines infected with or without Wolbachia. Furthermore, 10 putative recombination events were identified from Andricus hakonensis and Andricus sp2, which harbored multiple phage WO types, suggesting that intragenic recombination was the important evolutionary force, which effectively promoted the high level of phage WO diversity associated with gall wasps.

Environmental Temperature, but Not Male Age, Affects Wolbachia and Prophage WO Thereby Modulating Cytoplasmic Incompatibility in the Parasitoid Wasp, Habrobracon Hebetor.

Wolbachia is an endosymbiotic bacterium found in many species of arthropods and manipulates its host reproduction. Cytoplasmic incompatibility (CI) is one of the most common manipulations that is induced when an uninfected female mates with a Wolbachia-infected male. The CI factors (cifA and cifB genes) are encoded by phage WO that naturally infects Wolbachia. Here, we questioned whether an environmental factor (temperature) or host factor (male age) affected the strength of the CI phenotype in the ectoparasitoid wasp, Habrobracon hebetor. We found that temperature, but not male age, results in reduced CI penetrance. Consistent with these results, we also found that the expression of the cif CI factors decreased in temperature-exposed males but was consistent across aging male wasps. Similar to studies of other insect systems, cifA showed a higher expression level than cifB, and male hosts showed increased cif expression relative to females. Our results suggest that prophage WO is present in the Wolbachia-infected wasps and expression of cif genes contributes to the induction of CI in this insect. It seems that male aging has no effect on the intensity of CI; however, temperature affects Wolbachia and prophage WO titers as well as expression levels of cif genes, which modulate the CI level.

DNA recombination and repair in Wolbachia: RecA and related proteins.

Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.

Distribution and Evolution of the Bacteriophage WO and Its Antagonism With Wolbachia.

The symbiosis system comprising eukaryotic hosts, intracellular bacterium Wolbachia, and temperate bacteriophages WO is widely spread through nearly half the number of arthropod species. The relationships between the three components of the system are extremely intricate. Even though the bacteriophage WO can have diverse influences on the ecology and evolution of Wolbachia, little is known about the distribution and evolution of the phages. To the best of our knowledge, this study is the first to report that in infected fig wasps (Ceratosolen solmsi, Kradibia gibbosae, and Wiebesia pumilae), the genomes of all the Wolbachia strains had only one cryptic WO prophage, which contained defects in the genomic structural modules. This phenomenon was contrary to the widely accepted understanding that Wolbachia with cryptic prophages usually possesses at least one intact WO prophage consisting of gene sequences of the head, baseplate, and tail modules, through which the prophage could form intact virions. In addition to the genetic structure features, the phylogenetic relationships of WO and Wolbachia also revealed that bacteriophage WO can horizontally spread among a certain genus or a group of insect hosts, nearly free from the restriction of the affiliation of Wolbachia. Combined with the vertical transmission along with Wolbachia, the wide spread of WO phages can be explained. Furthermore, the gender preference and functional module preference for transcriptional activity of the genes in cryptic WOs implied the antagonized coevolutionary pattern between WO prophages and their Wolbachia hosts.

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.

Transgenic Testing Does Not Support a Role for Additional Candidate Genes in Wolbachia Male Killing or Cytoplasmic Incompatibility.

Endosymbiotic bacteria in the genus Wolbachia remarkably infect nearly half of all arthropod species. They spread in part because of manipulations of host sexual reproduction that enhance the maternal transmission of the bacteria, including male killing (death of infected males) and unidirectional cytoplasmic incompatibility (CI; death of offspring from infected fathers and uninfected mothers). Recent discoveries identified several genes in prophage WO of Wolbachia (wmk, cifA, and cifB) that fully or partially recapitulate male killing or CI when transgenically expressed in Drosophila melanogaster However, it is not yet fully resolved if other gene candidates contribute to these phenotypes. Here, we transgenically tested 10 additional gene candidates for their involvement in male killing and/or CI. The results show that despite sequence and protein architecture similarities or comparative associations with reproductive parasitism, transgenic expression of the candidates does not recapitulate male killing or CI. Sequence analysis across Wmk and its closest relatives reveals amino acids that may be important to its function. In addition, evidence is presented to propose new hypotheses regarding the relationship between wmk transcript length and its ability to kill a given host, as well as copy number of wmk homologs within a bacterial strain, which may be predictive of host resistance. Together, these analyses continue to build the evidence for identification of wmk, cifA, and cifB as the major genes that have thus far been shown to cause reproductive parasitism in Wolbachia, and the transgenic resources provide a basis for further functional study of phage WO genes.IMPORTANCE Wolbachia are widespread bacterial endosymbionts that manipulate the reproduction of diverse arthropods to spread through a population and can substantially shape host evolution. Recently, reports identified three prophage WO genes (wmk, cifA, and cifB) that transgenically recapitulate many aspects of reproductive manipulation in Drosophila melanogaster Here, we transgenically tested 10 additional gene candidates for CI and/or male killing in flies. The results yield no evidence for the involvement of these gene candidates in reproductive parasitism, bolstering the evidence for identification of the cif and wmk genes as the major factors involved in their phenotypes. In addition, evidence supports new hypotheses for prediction of male-killing phenotypes or lack thereof based on wmk transcript length and copy number. These experiments inform efforts to understand the full basis of reproductive parasitism for basic and applied purposes and lay the foundation for future work on the function of an interesting group of Wolbachia and phage WO genes.

Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila.

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.

The Wolbachia mobilome in Culex pipiens includes a putative plasmid

Wolbachia is a genus of obligate intracellular bacteria found in nematodes and arthropods worldwide, including insect vectors that transmit dengue, West Nile, and Zika viruses. Wolbachia’s unique ability to alter host reproductive behavior through its temperate bacteriophage WO has enabled the development of new vector control strategies. However, our understanding of Wolbachia’s mobilome beyond its bacteriophages is incomplete. Here, we reconstruct near-complete Wolbachia genomes from individual ovary metagenomes of four wild Culex pipiens mosquitoes captured in France. In addition to viral genes missing from the Wolbachia reference genome, we identify a putative plasmid (pWCP), consisting of a 9.23-kbp circular element with 14 genes. We validate its presence in additional Culex pipiens mosquitoes using PCR, long-read sequencing, and screening of existing metagenomes. The discovery of this previously unrecognized extrachromosomal element opens additional possibilities for genetic manipulation of Wolbachia.

Genome expansion of an obligate parthenogenesis-associated Wolbachia poses an exception to the symbiont reduction model

BackgroundTheory predicts that dependency within host-endosymbiont interactions results in endosymbiont genome size reduction. Unexpectedly, the largest Wolbachia genome was found in the obligate, parthenogenesis-associated wFol. In this study, we investigate possible processes underlying this genome expansion by comparing a re-annotated wFol genome to other Wolbachia genomes. In addition, we also search for candidate genes related to parthenogenesis induction (PI).ResultsWithin wFol, we found five phage WO regions representing 25.4% of the complete genome, few pseudogenized genes, and an expansion of DNA-repair genes in comparison to other Wolbachia. These signs of genome conservation were mirrored in the wFol host, the springtail F. candida, which also had an expanded DNA-repair gene family and many horizontally transferred genes. Across all Wolbachia genomes, there was a strong correlation between gene numbers of Wolbachia strains and their hosts. In order to identify genes with a potential link to PI, we assembled the genome of an additional PI strain, wLcla. Comparisons between four PI Wolbachia, including wFol and wLcla, and fourteen non-PI Wolbachia yielded a small set of potential candidate genes for further investigation.ConclusionsThe strong similarities in genome content of wFol and its host, as well as the correlation between host and Wolbachia gene numbers suggest that there may be some form of convergent evolution between endosymbiont and host genomes. If such convergent evolution would be strong enough to overcome the evolutionary forces causing genome reduction, it would enable expanded genomes within long-term obligate endosymbionts.

Evolutionary Genetics of Cytoplasmic Incompatibility Genes cifA and cifB in Prophage WO of Wolbachia.

The bacterial endosymbiont Wolbachia manipulates arthropod reproduction to facilitate its maternal spread through host populations. The most common manipulation is cytoplasmic incompatibility (CI): Wolbachia-infected males produce modified sperm that cause embryonic mortality, unless rescued by embryos harboring the same Wolbachia. The genes underlying CI, cifA and cifB, were recently identified in the eukaryotic association module of Wolbachia’s prophage WO. Here, we use transcriptomic and genomic approaches to address three important evolutionary facets of the cif genes. First, we assess whether or not cifA and cifB comprise a classic toxin–antitoxin operon in wMel and show that the two genes exhibit striking, transcriptional differences across host development. They can produce a bicistronic message despite a predicted hairpin termination element in their intergenic region. Second, cifA and cifB strongly coevolve across the diversity of phage WO. Third, we provide new domain and functional predictions across homologs within Wolbachia, and show that amino acid sequences vary substantially across the genus. Finally, we investigate conservation of cifA and cifB and find frequent degradation and loss of the genes in strains that no longer induce CI. Taken together, we demonstrate that cifA and cifB exhibit complex transcriptional regulation in wMel, provide functional annotations that broaden the potential mechanisms of CI induction, and report recurrent erosion of cifA and cifB in non-CI strains, thus expanding our understanding of the most widespread form of reproductive parasitism.