A parasitic bacterium that uses an array of dastardly tricks to favour female hosts over males is holding evolutionary biologists in thrall. Jonathan Knight enters the strange world of Wolbachia.
According to the Bible, King Herod had the blood of thousands of male children on his hands. Yet his record for gender-biased genocide pales alongside that of Wolbachia, bacteria that infect a wide variety of invertebrates including insects, spiders, crustaceans and nematode worms.
The bacteria, which live inside the cells of its hosts' reproductive systems and other tissues, can only spread from one generation to the next by invading a female's eggs. Males, to Wolbachia, are useless. Accordingly, these bacteria remove males from the populations they infect with a chilling efficiency, using strategies that range from enforced sex changes to cold-blooded murder.
Wolbachia were long regarded as a biological quirk. But now it seems that they are much more prevalent than was originally thought, and evolutionary biologists are starting to view these bizarre parasites as a unique natural laboratory. Thanks to their male-hating habits, Wolbachia might help investigations of such key questions as how species are created and go extinct, and why different organisms determine the difference between the sexes in myriad ways. “It's clear that they are a major force in the biology of a lot of invertebrates,” says Scott O'Neill, who studies the bacteria at Yale University in New Haven, Connecticut.
Wolbachia disrupt their hosts' biology in many ways. In mosquitoes, for instance, infected males can only reproduce successfully with females infected with the same strain of Wolbachia1. Although the bacteria themselves cannot hitch a ride in sperm, they alter the cells by an unknown mechanism so that the sperm can only fertilize eggs that contain the same Wolbachia strain. This 'cytoplasmic incompatibility' reduces reproduction among uninfected females, and so drives the infection through the population with each generation. Wolbachia-induced cytoplasmic incompatibility has also turned up in beetles2, wasps3, moths4 and fruitflies5.
In other species, Wolbachia makes males dispensable. Some populations of parasitic wasp in the genus Trichogramma are entirely female, reproducing parthenogenetically — without fertilization. Parthenogenesis arises from time to time in animals ranging from insects to fish and lizards. But in 1990, Richard Stouthamer of the University of California, Riverside, found that antibiotics could 'cure' parthenogenesis in Trichogramma6. Later, he revealed that the culprit was a Wolbachia infection7.
Wolbachia also convert male woodlice into females, apparently by suppressing a gland that produces a masculizing hormone8. In species including ladybirds, flour beetles and several species of fruitfly, Wolbachia simply kill males during early development9,10,11. By preventing these 'dead-end' hosts from hatching, they ensure better supplies of food for their infected daughters. “It's a bacterium that can do it all,” says Stouthamer.
Out of the shadows
Wolbachia were relatively obscure until John Werren of the University of Rochester in New York looked for them in the wild — and found them everywhere. Using the polymerase chain reaction (PCR) to copy fragments of bacterial DNA from insect tissue samples, Werren discovered evidence of Wolbachia infection in about 17% of the species he surveyed in Panama and 20% of those in the United States and Canada12.
Wolbachia might in fact be even more widespread than that. Last year, Marjorie Hoy of the University of Florida in Gainesville was working with a species of spider mite that had a skewed sex ratio highly suggestive of a Wolbachia infection. She could not reproducibly detect the bacterium with standard PCR. Only 'long' PCR, which includes a second enzyme to correct errors in copying the DNA, gave consistent results.
This led Hoy to suspect that Werren's study might have underreported Wolbachia's incidence. She teamed up with her Florida colleague Jay Jeyaprakash and collected specimens from 61 insect species and two species of spider from the field or local laboratory stocks. “We got some cockroaches and house flies and termites and dragonflies and ants — whatever was available,” says Hoy. With long PCR, 76% of the species tested positive for Wolbachia13.
Some researchers want to see further evidence before accepting that Wolbachia are so remarkably widespread. “I'd like to see that work replicated,” says Francis Jiggins of the University of Cambridge. But Jiggins has his own evidence that Werren's estimate is low.
Werren only sampled one or two individuals from each species he tested. If Wolbachia only infects a small proportion of a population, he may have missed many species that do carry the microbe. Jiggins reported last month that, at least for butterflies, this is the case14. He surveyed a large number of individuals within a small number of African butterfly species, and found that typically only 15% of the individuals are infected. “We can certainly say that Werren's is a conservative estimate,” Jiggins concludes.
If Wolbachia are as prevalent as these studies suggest, then the bacteria are likely to exert a powerful influence over the evolution of their hosts. One provocative proposal is that they might drive speciation — the emergence of new species. Speciation begins with the reproductive isolation of two populations of the same species. A mountain range, for instance, could separate populations for millennia until the accumulation of mutations in each prevents them from producing viable offspring if they ever do meet.
Cytoplasmic incompatibility could provide a similar reproductive barrier. If two groups within a population of insects are infected with two different types of Wolbachia, males from either group may be unable to fertilize females from the other. Werren believes such 'bi-directional' cytoplasmic incompatibility might allow speciation to occur. The best evidence so far comes from two species of wasp, Nasonia longicornis and N. giraulti. They look different — N. giraulti has much smaller wings — and do not interbreed. But they are closely related: DNA analyses suggest that their most recent common ancestor lived around 250,000 years ago15.
In February, Werren and his graduate student Seth Bordenstein reported that the two species were infected with different strains of Wolbachia that were causing bi-directional cytoplasmic incompatibility16. Once cured with antibiotics, the wasps could produce fertile offspring. Infection with different strains of Wolbachia appeared to be one of the first reproductive barriers to have arisen between these recently divergent species.
Divide and conquer
But some experts question whether Wolbachia-induced cytoplasmic incompatibility is, by itself, enough to cause species to diverge. Scott Turelli, an evolutionary biologist at the University of California, Davis, points out that Werren's two wasp species are geographically separated: N. giraulti lives in eastern North America, whereas N. longicornis inhabits the western part of the continent. And Stouthamer notes that cytoplasmic incompatibility can break down, for instance where insects mate repeatedly17. He believes this may allow enough gene flow to prevent co-existing populations from becoming reproductively isolated. “My feeling is that cytoplasmic incompatibility can help once speciation has started, but it can't initiate speciation,” he says.
John Jaenike, an evolutionary biologist at the University of Rochester, may have stumbled upon just such a case among fungus-eating flies. One species, Drosophila recens, collected from the eastern United States, is heavily infected with Wolbachia. Its males cannot fertilize females from another species, D. subquinaria, that lives in the Pacific northwest and is Wolbachia-free. The reverse cross — D. subquinaria males and D. recens females — does produce offspring. But in unpublished observations, Jaenike has found that D. recens females are reluctant to mate with their western relatives. He has yet to study the flies' behaviour in the zone in the central United States where their ranges overlap, but it is possible that female mating preference prevents hybridization in one direction whereas Wolbachia-induced cytoplasmic incompatibility stops it in the other.
Speciation is not the only aspect of evolution in which Wolbachia may have a hand. Greg Hurst of University College London believes the bacteria may have helped to spawn the tremendous diversity in the way in which different invertebrate species determine whether to be male or female.
One method of sex determination, found in Drosophila, depends on the ratio of X chromosomes to autosomes (all the other chromosomes except the Y chromosome). Flies with two X chromosomes and two copies of each autosome will be female, whereas a 1:2 ratio produces a male. In Trichogramma wasps, fertilized eggs become female, and unfertilized eggs become male — unless a Wolbachia infection forces them to develop as females. There are many other mechanisms in different species.
To Hurst, this is an intriguing puzzle. In other respects, sex is relatively invariant — males produce sperm and females make eggs. So why should sex determination be different? “One hypothesis is that it's driven by parasites that affect sex,” says Hurst. In other words, the struggle between the hosts' attempts to produce an even sex ratio, and Wolbachia's desire for more females than males, has led hosts to evolve a variety of means to influence sex.
In March, for instance, Stouthamer's group reported the discovery of a 'parasitic' chromosome in Trichogramma which causes females to produce only males18. This chromosome appears to restore some balance between the sexes to Wolbachia-infected wild populations of the wasp. 'Masculizing' genes have also been found in infected woodlice19.
Decline and fall
For most species, a distorted sex ratio makes reproduction less efficient. But in theory, if the distortion becomes severe enough that females struggle to find mates, it could drive populations — and even species — to extinction. Jiggins and Hurst are investigating this possibility. Two years ago, they reported cases of male-killing by Wolbachia in ladybirds from Russia and butterflies from Uganda20. Both populations were more than 90% female, and nearly all individuals were infected.
Wolbachia has so skewed the gender ratio of the Ugandan butterfly, Acraea encedon, that a rare form of courtship has arisen. Usually, males compete for the attentions of choosy females. But in A. encedon, females wait in groups for a male to flutter by and then vie for a chance to mate with him. Many females never mate. “The question is whether they could ultimately go to extinction,” says Hurst. Jiggins and Hurst are now separately studying similarly afflicted insect populations for evidence that some truly are on the brink.
A knock-on effect of male-culling might be to restrict where an insect species can live. For reasons that are not understood, Wolbachia do not transfer to the next generation as efficiently at higher temperatures21. At low temperatures, it may transmit so effectively that its male-killing habit can make populations extinct. So a population infected with Wolbachia might only survive in warmer climates. Jaenike is now testing this idea in Drosophila innubila, which lives in Arizona. He wants to know if male-killing Wolbachia are restricting the flies to life at warmer, lower elevations.
Intriguingly, just as Wolbachia are integral to the biology and evolution of their hosts, it seems that a virus known as the WO phage may be integral to the biology of Wolbachia. Shinji Masui and his colleagues at the University of Tokyo last year reported finding phage DNA sequences embedded in the chromosomes of several Wolbachia strains22.
Phages can hitch a ride from their bacterial host for generations by inserting their DNA into the host's genome. Later the phage hops back out, gets packaged in its protein coat and bursts out of the cell to infect more bacteria. Sometimes, it takes a bit of bacterial DNA with it, and Masui suspects that this may allow the WO phage to shuttle genes from one Wolbachia strain to another. If so, it could explain why closely related strains of Wolbachia can cause radically different effects on their hosts. Genes involved in male killing, feminization and parthenogenesis might have travelled from one species to another many times, mixing up the Wolbachia evolutionary tree.
Preliminary evidence that genes are jumping between Wolbachia strains is now emerging from several labs that are sequencing the Wolbachia genome. O'Neill's Yale group has nearly finished one Wolbachia strain from a nematode worm, Brugia malayi, that lives in the human lymph system and blood, and another from Drosophila melanogaster. Although the sequences are still being analysed, there appear to be many mobile genetic elements such as phage DNA and transposons, 'jumping genes' that can hop in and out of the genome, inserting randomly.
Two become one
Wolbachia has a small genome — less than 1.5 million base pairs, or about a third of the size of the genome of the common gut bacterium Escherichia coli. Intracellular parasites often seem to develop slimmed-down genomes, as they lose genes that are only required by free-living bacteria23. And some biologists wonder if Wolbachia are on the evolutionary road towards becoming one with their hosts — like the mitochondria that generate energy inside our cells, which are thought to have once been parasites.
O'Neill, for one, is not convinced. Given the deleterious effects of Wolbachia on its insect hosts, he definitely views the bacteria as parasites: “The microbe has the upper hand.” But in other invertebrates, who has the upper hand is less clear. The nematode worms that cause elephantiasis and river blindness in humans seem to need Wolbachia to survive — antibiotics kill the worms, apparently by killing their Wolbachia24.
As interest in the bacteria explodes, strains of Wolbachia that are in the process of being incorporated by their hosts may be among the evolutionary treasures waiting to be discovered. The availability of the Wolbachia genome is also expected to aid research into the mechanisms by which the bacteria manipulate their hosts — which at present are not well understood. Fans of bizarre biology and afficionados of evolution should watch this space.
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Knight, J. Meet the Herod bug. Nature 412, 12–14 (2001). https://doi.org/10.1038/35083744
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