John H. Werren

Wolbachia: master manipulators of invertebrate biology

Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These alphaproteobacteria endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm–egg incompatibility. They can also move horizontally across species boundaries, resulting in a widespread and global distribution in diverse invertebrate hosts. Here, we review the basic biology of Wolbachia, with emphasis on recent advances in our understanding of these fascinating endosymbionts.

How many species are infected with Wolbachia? – a statistical analysis of current data

Wolbachia are intracellular bacteria found in many species of arthropods and nematodes. They manipulate the reproduction of their arthropod hosts in various ways, may play a role in host speciation and have potential applications in biological pest control. Estimates suggest that at least 20% of all insect species are infected with Wolbachia. These estimates result from several Wolbachia screenings in which numerous species were tested for infection; however, tests were mostly performed on only one to two individuals per species. The actual percent of species infected will depend on the distribution of infection frequencies among species. We present a meta-analysis that estimates percentage of infected species based on data on the distribution of infection levels among species. We used a beta-binomial model that describes the distribution of infection frequencies of Wolbachia, shedding light on the overall infection rate as well as on the infection frequency within species. Our main findings are that (1) the proportion of Wolbachia-infected species is estimated to be 66%, and that (2) within species the infection frequency follows a ‘most-or-few’ infection pattern in a sense that the Wolbachia infection frequency within one species is typically either very high (>90%) or very low (<10%).

Evolution and Phylogeny of Wolbachia: Reproductive Parasites of Arthropods

Wolbachia are cytoplasmically inherited bacteria found in reproductive tissues of many arthropod species. These bacteria are associated with reproductive alterations in their hosts, including parthenogenesis, reproductive incompatibility and feminization. A fine-scale phylogenetic analysis was done using DNA sequences from ftsZ, a rapidly evolving bacterial cell-cycle gene. ftsZ sequences were determined for 38 different Wolbachiastrains from 31 different species of insects and one isopod. The following results were found: (i) there are two major division of Wolbachia (A and B) which diverged 58-67 millions years before present based upon synonymous substitution rates; (ii) a general concordance is found between the ftsZ and 16S rDNA phylogenies, indicating that these represent bacterial strain (rather than simply gene) phylogenies; however, a possible example of recombination between A and B division bacteria may have occurred in the feminizing Wolbachia present in an isopod; (iii) extensive horizontal transmission of Wolbachia has occurred between insect taxa, including different insect orders; one strain in particular (designated Adm) shows extensive recent horizontal transmission; (iv) there is an association between the Wolbachiafound in a parasitic wasp (Nasonia) and its fly host (Protocalliphora), suggesting exchange of bacteria between these species; (v) parthenogenesis induction has evolved several times among the Wolbachia; and (vi) some insects harbour infections with more than one Wolbachia strain, even within individual insects.

Widespread Lateral Gene Transfer from Intracellular Bacteria to Multicellular Eukaryotes

Although common among bacteria, lateral gene transfer—the movement of genes between distantly related organisms—is thought to occur only rarely between bacteria and multicellular eukaryotes. However, the presence of endosymbionts, such as Wolbachia pipientis, within some eukaryotic germlines may facilitate bacterial gene transfers to eukaryotic host genomes. We therefore examined host genomes for evidence of gene transfer events from Wolbachia bacteria to their hosts. We found and confirmed transfers into the genomes of four insect and four nematode species that range from nearly the entire Wolbachia genome (>1 megabase) to short (<500 base pairs) insertions. Potential Wolbachia-to-host transfers were also detected computationally in three additional sequenced insect genomes. We also show that some of these inserted Wolbachia genes are transcribed within eukaryotic cells lacking endosymbionts. Therefore, heritable lateral gene transfer occurs into eukaryotic hosts from their prokaryote symbionts, potentially providing a mechanism for acquisition of new genes and functions.

Wolbachia infection frequencies in insects: evidence of a global equilibrium?

Wolbachia are a group of cytoplasmically inherited bacteria that cause reproduction alterations in arthropods, including parthenogenesis, reproductive incompatibility, feminization of genetic males and male killing. Previous general surveys of insects in Panama and Britain found Wolbachia to be common, occurring in 16–22% of species. Here, using similar polymerase chain reaction methods, we report that 19.3% of a sample of temperate North American insects are infected with Wolbachia, a frequency strikingly similar to frequencies found in two other studies in widely separated locales. The results may indicate a widespread equilibrium of Wolbachia infection frequencies in insects whose maintenance remains to be explained. Alternatively, Wolbachia may be increasing in global insect communities. Within each of the three geographic regions surveyed, Hymenoptera are more frequently infected with A group Wolbachia and Lepidoptera more frequently infected with B group Wolbachia.

Distribution of Wolbachia among Neotropical Arthropods

Wolbachia are a group of cytoplasmically inherited bacteria that cause reproduction alterations in arthropods, including parthenogenesis, reproductive incompatibility and feminization of genetic males. Two major subdivisions of Wolbachia (A and B) occur. Wolbachia are found in a number of well-studied insects, but their overall distribution in arthropods has not been well studied. A survey for Wolbachia in 157 Panamanian neotropical arthropod species was done using a polymerase chain reaction assay. Wolbachia were detected in 26 of 154 insect species (16.9%) and zero of three arachnids (0%). Extrapolating to the estimated total number of insect species present globally (10—30 million), an estimated 1.69—5.07 million insect species are infected with these bacteria, making Wolbachia an extremely abundant bacterial group. Wolbachia were found in each of the major insect orders examined, including Coleoptera (6/57 infected), Diptera (5/14), Hemiptera/Homoptera (1/7), Hymenoptera (6/23) Lepidoptera (7/43) and Orthoptera (1/8). Of the 26 positives, eight species were found to be singly infected with A group Wolbachia, nine singly with B group Wolbachia and nine doubly infected with both A and B group Wolbachia. Double infections occur at significantly higher frequencies than expected by chance. The abundance of Wolbachia further supports their potential importance as a mechanism for rapid speciation in insects.

Multilocus Sequence Typing System for the Endosymbiont Wolbachia pipientis

ABSTRACT The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/ .

Sex Ratio Adaptations to Local Mate Competition in a Parasitic Wasp

Females of the parasitic wasp Nasonia vitripennis adjust the sex ratio of their broods according to whether they are a first or second wasp to parasitize a host. The first wasp produces a strong daughter bias. The second wasp adjusts the prorportion of sons to the relative level of local mate competition, as predicted by a natural selection model. The results provide a quantitative test of sex ratio theory.

Male-killing Wolbachia in two species of insect

The inherited bacterium Wolbachia spreads through the manipulation of host reproduction, and has been suggested to be an important factor in arthropod evolution, from host speciation to the evolution of sex–determination systems. Past work has shown that members of this group may produce cytoplasmic incompatibility, feminize genetically male hosts, and induce host parthenogenesis. Here, we report an expansion of the range of reproductive manipulations produced by members of this clade, recording Wolbachia strains that kill male hosts during embryogenesis in two host species, the ladybird Adalia bipunctata, and the butterfly Acraea encedon. Both male–killing bacteria belong to the B group of Wolbachia. However, phylogenetic analyses were unable to resolve whether the bacteria in the two species are monophyletic, or represent independent origins of male–killing among the B–group Wolbachia. We also found significant divergence within the wsp gene of Wolbachia strains found in different A. bipunctata individuals, suggesting this host species contains two Wolbachia strains, diverged in wsp sequence but monophyletic. Our observations reinforce the notion that Wolbachia may be an important agent driving arthropod evolution, and corroborates previous suggestions that male–killing behaviour is easily evolved by invertebrate symbionts.

Wolbachia-induced incompatibility precedes other hybrid incompatibilities in Nasonia.

Wolbachia are cytoplasmically inherited bacteria that cause a number of reproductive alterations in insects, including cytoplasmic incompatibility, an incompatibility between sperm and egg that results in loss of sperm chromosomes following fertilization. Wolbachia are estimated to infect 15–20% of all insect species, and also are common in arachnids, isopods and nematodes. Therefore, Wolbachia-induced cytoplasmic incompatibility could be an important factor promoting rapid speciation in invertebrates, although this contention is controversial. Here we show that high levels of bidirectional cytoplasmic incompatibility between two closely related species of insects (the parasitic wasps Nasonia giraulti and Nasonia longicornis) preceded the evolution of other postmating reproductive barriers. The presence of Wolbachia severely reduces the frequency of hybrid offspring in interspecies crosses. However, antibiotic curing of the insects results in production of hybrids. Furthermore, F1 and F2 hybrids are completely viable and fertile, indicating the absence of F1 and F2 hybrid breakdown. Partial interspecific sexual isolation occurs, yet it is asymmetric and incomplete. Our results indicate that Wolbachia-induced reproductive isolation occurred in the early stages of speciation in this system, before the evolution of other postmating isolating mechanisms (for example, hybrid inviability and hybrid sterility).