- Email: [email protected]
Further Wolbachia endosymbiont diversity: a tree hiding in the forest?
NEWS & COMMENT and sequences: pattern filtering, Mol. Biol. Evol. 15, 1224–1231 6 Kumar, S. and Hedges, S.B. (1998) A molecular timescale for vertebrate evolution, Nature 392, 917–920 7 Wray, G.A., Levinton, J.S. and Shapiro, L.H. (1996) Molecular evidence for deep Precambrian divergences, Science 274, 568–573 8 Ayala, F.J., Rzhetsky, A. and Ayala, F.J. (1998) Origin of the metazoan
phlya: molecular clocks confirm palaeontological estimates, Proc. Natl. Acad. Sci. U. S. A. 95, 606–611 9 Feng, D-F., Cho, G. and Doolittle, R.F. (1997) Determining divergence times with a protein clock: update and reevaluation, Proc. Natl. Acad. Sci. U. S. A. 94, 13028–13033 10 Gu, X. (1998) Early metazoan divergence was about 830 million years ago, J. Mol. Evol. 47, 369–371 11 Brasier, M. (1998) From deep time to late
Further Wolbachia endosymbiont diversity: a tree hiding in the forest? olbachia, endocellular a-proteobacteria, are probably one of the most widespread symbionts in arthropods: they are present in about 15% of insects1, in many isopod crustaceans2 and in several mites3. They manipulate the reproduction of their hosts to their own benefit. Because they generally have no other obvious detrimental effect to their host, they are considered to be ‘reproductive parasites’4. They mediate cytoplasmic incompatibility (CI) in all their host taxa, parthenogenesis in their haplo-diploid hosts and feminization in isopods. All these effects have the same goal: favouring the spread of the infection in host populations, either by providing more infected hosts of the female sex, which can transmit the infection vertically, or by decreasing the fitness of the nontransmitting (uninfected) lineages. Advances in Wolbachia knowledge have been made through a series of recent papers, all confirming that these symbionts are widespread in insects5–7, but two recent findings emphasize a significant extension in Wolbachia diversity.
W
Further manipulation of the host… A new study by G.D.D. Hurst et al.8 reveals that the sex ratio biases in two insect species, the ladybird Adalia bipunctata and the butterfly Acraea encedon, are associated with Wolbachia infection. In these species, the infected mothers produce all-female progenies that are half the size of those of the uninfected mothers. The bacteria kill all the male embryos during the early stages of embryonic development. This ability to recognize the sex of the embryo might suggest that the bacteria can detect dosage compensation in the host, but the two insects in which male-killing Wolbachia are found are of different heterogametic sex determination (males are XY in ladybirds
212
but ZZ in butterflies). Therefore the mechanism by which Wolbachia determine the host sex probably differs between the two hosts. Killing males early in development in order to spread the infection is only efficient if the host meets certain ecological conditions – conditions that enhance the survivorship or fecundity of the surviving infected females. These conditions are sibling cannibalism (the surviving females feed on dead eggs), competition between siblings for food (the death of males reduces competition) or disadvantageous inbreeding (avoided by the death of brothers)9. Given that high levels of sibling cannibalism have been found in the two insect species studied by Hurst et al., there is no doubt that Wolbachia mutants profited by killing males in these species. It is impossible to tell from molecular phylogenies constructed from two variable genes whether male-killing behaviour appeared once or twice in the Wolbachia clade. However, male-killing is not linked with a particular Wolbachia lineage: male-killing symbionts are closely related to those inducing CI, parthenogenesis or feminization (B group of the Wolbachia clade). Therefore, Wolbachia seems to be able to adapt to the particular ecological conditions they encounter in their hosts.
…and enlarged host range Another recent study10 reveals that the ability of Wolbachia to adapt to different environments is even wider than previously supposed. Wolbachia were thought to be limited to terrestrial arthropods – some other invertebrate groups and several marine crustaceans have been screened without success2,11. But, by finding these symbionts in Nematoda (filarial worms), Bandi et al.10 highlight a considerable increase of the Wolbachia host range. The worms in which Wolbachia
0169-5347/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
arrivals, Nature 395, 547–548 12 Fortey, R.A., Briggs, D.E.G. and Wills, M.A. (1996) The Cambrian evolutionary ‘explosion’: decoupling cladogenesis from morphological disparity, Biol. J. Linn. Soc. 57, 13–33 13 Canfield, D.E. and Teske, A. (1996) Late Proterozoic rise in atmospheric oxygen concentration inferred from phylogenetic and sulphur-isotope studies, Nature 382, 127–132
were found are parasites of vertebrates, some of them inducing severe diseases in humans, such as elephantiasis (caused by Wuchereria bancrofti and Brugia malayi). The Wolbachia infection cannot be considered a mere curiosity because eight species from the Onchocercidae family are infected, sometimes in several locations throughout the world (e.g. the dog parasite Dirofilaria immits). Again, molecular tools were necessary to understand Wolbachia diversity in these hosts. The Wolbachia found in nematodes formed two new groups (C and D), significantly separated from the A and B groups previously known in arthropods. As in the case of male-killing Wolbachia, the phylogenies did not clearly distinguish between monophyly or paraphyly of Wolbachia in the nematodes (i.e. whether or not Wolbachia are derived from a single common ancestor). So, the question of how nematodes acquired Wolbachia remains unsolved. Considering that nematodes are arthropod-borne parasites and that the separation time between arthropods and nematodes is more ancient than the separation of the different Wolbachia groups, the more parsimonious explanation is that an ancient horizontal transfer of the symbiont occurred between the two host phyla. Regardless of the origin of the infection, the symbiont phylogeny matches well with that of the filarial hosts, indicating a co-radiation. This suggests that the symbiotic association is more stable than in arthropods, where horizontal transfers occurred frequently1,12. Both the high prevalence of Wolbachia in these worms and the apparent stability of the symbiotic association suggest that Wolbachia plays a key role in the biology of filarial nematodes, a role yet to be discovered. Bandi et al.10 suggest that the pathogenic effect of tetracycline on nematode development (as evidenced several years ago13) is a byproduct of antibiotics on Wolbachia. This means that Wolbachia and their nematode hosts could be mutualists. This hypothesis has been tested, also very recently, by Hoerauf et al.14 Using filarial diseases in rodents as a biological
PII: S0169-5347(99)01599-2
TREE vol. 14, no. 6 June 1999
NEWS & COMMENT model, they found that tetracycline treatment eliminates the Wolbachia symbionts in the worms and results in filarial growth retardation and infertility. Parallel to this, an antibiotic therapy did not affect filarial worms naturally uninfected with Wolbachia. This strengthens the feeling that Wolbachia are necessary to their nematode hosts, and confirms that they could be helpful tools for controlling diseases caused by these worms.
A single master key or numerous keys? The findings discussed here illustrate how a clade of intracellular microorganisms can have a wide adaptive range. Wolbachia can adapt to very different ‘environments’: those living in arthropods are destroyed by temperatures above 30°C, whereas those living in filarial worms experience higher temperatures (because filarial worms are parasites of vertebrates). Above all, the evidence suggests that Wolbachia can exploit the peculiar life-history traits of the hosts they encounter. Male-killing mutants have been selected to exploit cannibalism in some of their hosts, whereas parthenogenesis-inducing mutants have been selected to exploit haplo-diploidy, and feminizing mutants were probably selected to exploit hormonal sex differentiation in crustaceans. As suggested for nematodes, Wolbachia might also evolve towards a more ‘classic’ symbiosis, becoming mutualistic to their hosts. Extending this viewpoint, CI is probably selected in hosts in which Wolbachia were unable to ‘find’ a more efficient way of increasing their fitness. Given the wide range of potential manipulations for vertically transmitted microbes4, Wolbachia endosymbionts are likely to be associated with new alterations of host reproduction in the future. However, a question remains as to which of the Wolbachia phenotypes is ancestral. Cytoplasmic incompatibility has been proposed as a good candidate12 because it is found in all arthropod taxa and requires a fundamental mechanism. Unfortunately, recent molecular phylogenies have generally failed to identify the ancestral Wolbachia because DNA sequence data, in most cases, tend to be equivocal. Consequently, the relationship between the different Wolbachia groups is always ambiguous and this is further hampered by a lack of knowledge on the precise mechanisms of Wolbachia effects. More molecular markers, a better knowledge of how Wolbachia alters host reproduction and perhaps the detection of new Wolbachia mutants are needed to answer this question. It is only since 1992, with the development of molecular tools, TREE vol. 14, no. 6 June 1999
that Wolbachia diversity has become so apparent – the variability observed at present is perhaps only a tree hiding in a forest. Another question concerns how Wolbachia adapts to the different ‘ecological niches’. Is it because they (1) use a general and simple way to manipulate host reproduction or (2) possess an innate plasticity and rapid adaptive ability. The first hypothesis depends on the fact that the most widespread phenotypes (parthenogenesis and CI) involve disruptions of mitosis during host embryonic development, a mechanism that might be used for other phenotypes. This might explain male-killing, because the death of males occurs in early stages of embryonic development. However, in this case, the phenomenon should be more complex because the symbiont can detect the sex of the embryo and specifically kill only one type. Furthermore, no mitotic disturbance seems to be involved in feminization, and different detailed analyses of CI phenotypes have revealed that mitosis is disturbed in several ways. Consequently, Wolbachia mechanisms of action probably differ according to the hosts. The second hypothesis (plasticity) relates to the fact that, by consuming and also supplying nutrients or products to host cells, all intracytoplasmic symbionts have intimate relationships with their hosts15. Wolbachia are probably no exception, a suggestion strengthened by the evidence of their mutualistic tendency in filarial worms. Here, the relationship might differ according to different host physiology, even for closely related symbionts, or because a symbiotic product might have different implications in different hosts. There might even be selection for overproduction of a particular product if it results in the symbiont being favoured. Such a phenomenon is known in Buchnera symbionts of pea aphids, where stress proteins of the GroE family are over-produced (although the release of these proteins in the host cell is not well understood)16. If endosymbiont physiology is not too limited, its numerous products could provide a variety of substrates with which selection can play. Acknowledgements I thank Greg Hurst, Claudio Bandi and Rebecca Terry for discussions and comments on this article.
Thierry Rigaud Génétique et Biologie des Populations de Crustacés, UMR CNRS 6556, Université de Poitiers, 40 avenue du Recteur, Pineau, F-86022 Poitiers Cedex, France ([email protected])
References 1 Werren, J.H., Zhang, W. and Guo, L. (1995) Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods, Proc. R. Soc. London Ser. B 261, 55–71 2 Bouchon, D., Rigaud, T. and Juchault, P. (1998) Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization, Proc. R. Soc. London Ser. B 265, 1081–1090 3 Breuwer, J.A.J. and Jacobs, G. (1996) Wolbachia: intracellular manipulators of mite reproduction, Exp. Appl. Acarol. 20, 421–434 4 Werren, J.H. and O’Neill, S.L. (1997) The evolution of heritable symbionts, in Influential Passengers: Inherited Microorganisms and Arthropod Reproduction (O’Neill, S.L., Hoffmann, A.A. and Werren, J.H., eds), pp. 1–41, Oxford University Press 5 Giordanno, R., Jackson, J.J. and Robertson, H.M. (1997) The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets, Proc. Natl. Acad. Sci. U. S. A. 94, 11439–11444 6 Hariri, A.R., Werren, J.H. and Wilkinson, G.S. (1998) Distribution and reproductive effects of Wolbachia in stalk-eyed flies (Diptera: Diopsidae), Heredity 81, 254–260 7 West, S.A., Cook, J.M., Warren, J.H. and Godfray, H.C.J. (1998) Wolbachia in two insect host–parasitoid communities, Mol. Ecol. 7, 1457–1465 8 Hurst, G.D.D. et al. (1999) Male-killing Wolbachia in two species of insects, Proc. R. Soc. London Ser. B 266, 735–740 9 Hurst, G.D.D., Hurst, L.D. and Majerus, M.E.N. (1997) Cytoplasmic sex ratio distorters, in Influential Passengers: Inherited Microorganisms and Arthropod Reproduction (O’Neill, S.L., Hoffmann, A.A. and Werren, J.H., eds), pp. 125–154, Oxford University Press 10 Bandi, C., Anderson, T.J.C., Genchi, C. and Blaxter, M.L. (1998) Phylogeny of Wolbachia in filarial nematodes, Proc. R. Soc. London Ser. B 265, 2407–2413 11 Schilthuizen, M. and Gittenberger, E. (1998) Screening mollusks for Wolbachia infection, J. Invertebr. Pathol. 71, 268–270 12 Rousset, F. et al. (1992) Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods, Proc. R. Soc. London Ser. B 250, 91–98 13 Bosshardt, S.C., McCall, J.W., Coleman, S.V., Jones, K.L., Petit, T.A. and Klei, T.R. (1993) Prophylactic activity of tetracycline against Brugia paphangi infection in Jirds (Meriones unguiculatus), J. Parasitol. 79, 775–777 14 Hoerauf, A. et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility, J. Clin. Invest. 103, 11–17 15 Schwemmler, W. and Gassmer, G., eds (1989) Insect Endocytobiosis: Morphology, Physiology, Genetics, Evolution, CRC Press 16 Hara, E. et al. (1990) The predominant protein in an aphid endosymbiont is homologous to an E. coli heat shock protein, Symbiosis 8, 271–283
213
phlya: molecular clocks confirm palaeontological estimates, Proc. Natl. Acad. Sci. U. S. A. 95, 606–611 9 Feng, D-F., Cho, G. and Doolittle, R.F. (1997) Determining divergence times with a protein clock: update and reevaluation, Proc. Natl. Acad. Sci. U. S. A. 94, 13028–13033 10 Gu, X. (1998) Early metazoan divergence was about 830 million years ago, J. Mol. Evol. 47, 369–371 11 Brasier, M. (1998) From deep time to late
Further Wolbachia endosymbiont diversity: a tree hiding in the forest? olbachia, endocellular a-proteobacteria, are probably one of the most widespread symbionts in arthropods: they are present in about 15% of insects1, in many isopod crustaceans2 and in several mites3. They manipulate the reproduction of their hosts to their own benefit. Because they generally have no other obvious detrimental effect to their host, they are considered to be ‘reproductive parasites’4. They mediate cytoplasmic incompatibility (CI) in all their host taxa, parthenogenesis in their haplo-diploid hosts and feminization in isopods. All these effects have the same goal: favouring the spread of the infection in host populations, either by providing more infected hosts of the female sex, which can transmit the infection vertically, or by decreasing the fitness of the nontransmitting (uninfected) lineages. Advances in Wolbachia knowledge have been made through a series of recent papers, all confirming that these symbionts are widespread in insects5–7, but two recent findings emphasize a significant extension in Wolbachia diversity.
W
Further manipulation of the host… A new study by G.D.D. Hurst et al.8 reveals that the sex ratio biases in two insect species, the ladybird Adalia bipunctata and the butterfly Acraea encedon, are associated with Wolbachia infection. In these species, the infected mothers produce all-female progenies that are half the size of those of the uninfected mothers. The bacteria kill all the male embryos during the early stages of embryonic development. This ability to recognize the sex of the embryo might suggest that the bacteria can detect dosage compensation in the host, but the two insects in which male-killing Wolbachia are found are of different heterogametic sex determination (males are XY in ladybirds
212
but ZZ in butterflies). Therefore the mechanism by which Wolbachia determine the host sex probably differs between the two hosts. Killing males early in development in order to spread the infection is only efficient if the host meets certain ecological conditions – conditions that enhance the survivorship or fecundity of the surviving infected females. These conditions are sibling cannibalism (the surviving females feed on dead eggs), competition between siblings for food (the death of males reduces competition) or disadvantageous inbreeding (avoided by the death of brothers)9. Given that high levels of sibling cannibalism have been found in the two insect species studied by Hurst et al., there is no doubt that Wolbachia mutants profited by killing males in these species. It is impossible to tell from molecular phylogenies constructed from two variable genes whether male-killing behaviour appeared once or twice in the Wolbachia clade. However, male-killing is not linked with a particular Wolbachia lineage: male-killing symbionts are closely related to those inducing CI, parthenogenesis or feminization (B group of the Wolbachia clade). Therefore, Wolbachia seems to be able to adapt to the particular ecological conditions they encounter in their hosts.
…and enlarged host range Another recent study10 reveals that the ability of Wolbachia to adapt to different environments is even wider than previously supposed. Wolbachia were thought to be limited to terrestrial arthropods – some other invertebrate groups and several marine crustaceans have been screened without success2,11. But, by finding these symbionts in Nematoda (filarial worms), Bandi et al.10 highlight a considerable increase of the Wolbachia host range. The worms in which Wolbachia
0169-5347/99/$ – see front matter © 1999 Elsevier Science. All rights reserved.
arrivals, Nature 395, 547–548 12 Fortey, R.A., Briggs, D.E.G. and Wills, M.A. (1996) The Cambrian evolutionary ‘explosion’: decoupling cladogenesis from morphological disparity, Biol. J. Linn. Soc. 57, 13–33 13 Canfield, D.E. and Teske, A. (1996) Late Proterozoic rise in atmospheric oxygen concentration inferred from phylogenetic and sulphur-isotope studies, Nature 382, 127–132
were found are parasites of vertebrates, some of them inducing severe diseases in humans, such as elephantiasis (caused by Wuchereria bancrofti and Brugia malayi). The Wolbachia infection cannot be considered a mere curiosity because eight species from the Onchocercidae family are infected, sometimes in several locations throughout the world (e.g. the dog parasite Dirofilaria immits). Again, molecular tools were necessary to understand Wolbachia diversity in these hosts. The Wolbachia found in nematodes formed two new groups (C and D), significantly separated from the A and B groups previously known in arthropods. As in the case of male-killing Wolbachia, the phylogenies did not clearly distinguish between monophyly or paraphyly of Wolbachia in the nematodes (i.e. whether or not Wolbachia are derived from a single common ancestor). So, the question of how nematodes acquired Wolbachia remains unsolved. Considering that nematodes are arthropod-borne parasites and that the separation time between arthropods and nematodes is more ancient than the separation of the different Wolbachia groups, the more parsimonious explanation is that an ancient horizontal transfer of the symbiont occurred between the two host phyla. Regardless of the origin of the infection, the symbiont phylogeny matches well with that of the filarial hosts, indicating a co-radiation. This suggests that the symbiotic association is more stable than in arthropods, where horizontal transfers occurred frequently1,12. Both the high prevalence of Wolbachia in these worms and the apparent stability of the symbiotic association suggest that Wolbachia plays a key role in the biology of filarial nematodes, a role yet to be discovered. Bandi et al.10 suggest that the pathogenic effect of tetracycline on nematode development (as evidenced several years ago13) is a byproduct of antibiotics on Wolbachia. This means that Wolbachia and their nematode hosts could be mutualists. This hypothesis has been tested, also very recently, by Hoerauf et al.14 Using filarial diseases in rodents as a biological
PII: S0169-5347(99)01599-2
TREE vol. 14, no. 6 June 1999
NEWS & COMMENT model, they found that tetracycline treatment eliminates the Wolbachia symbionts in the worms and results in filarial growth retardation and infertility. Parallel to this, an antibiotic therapy did not affect filarial worms naturally uninfected with Wolbachia. This strengthens the feeling that Wolbachia are necessary to their nematode hosts, and confirms that they could be helpful tools for controlling diseases caused by these worms.
A single master key or numerous keys? The findings discussed here illustrate how a clade of intracellular microorganisms can have a wide adaptive range. Wolbachia can adapt to very different ‘environments’: those living in arthropods are destroyed by temperatures above 30°C, whereas those living in filarial worms experience higher temperatures (because filarial worms are parasites of vertebrates). Above all, the evidence suggests that Wolbachia can exploit the peculiar life-history traits of the hosts they encounter. Male-killing mutants have been selected to exploit cannibalism in some of their hosts, whereas parthenogenesis-inducing mutants have been selected to exploit haplo-diploidy, and feminizing mutants were probably selected to exploit hormonal sex differentiation in crustaceans. As suggested for nematodes, Wolbachia might also evolve towards a more ‘classic’ symbiosis, becoming mutualistic to their hosts. Extending this viewpoint, CI is probably selected in hosts in which Wolbachia were unable to ‘find’ a more efficient way of increasing their fitness. Given the wide range of potential manipulations for vertically transmitted microbes4, Wolbachia endosymbionts are likely to be associated with new alterations of host reproduction in the future. However, a question remains as to which of the Wolbachia phenotypes is ancestral. Cytoplasmic incompatibility has been proposed as a good candidate12 because it is found in all arthropod taxa and requires a fundamental mechanism. Unfortunately, recent molecular phylogenies have generally failed to identify the ancestral Wolbachia because DNA sequence data, in most cases, tend to be equivocal. Consequently, the relationship between the different Wolbachia groups is always ambiguous and this is further hampered by a lack of knowledge on the precise mechanisms of Wolbachia effects. More molecular markers, a better knowledge of how Wolbachia alters host reproduction and perhaps the detection of new Wolbachia mutants are needed to answer this question. It is only since 1992, with the development of molecular tools, TREE vol. 14, no. 6 June 1999
that Wolbachia diversity has become so apparent – the variability observed at present is perhaps only a tree hiding in a forest. Another question concerns how Wolbachia adapts to the different ‘ecological niches’. Is it because they (1) use a general and simple way to manipulate host reproduction or (2) possess an innate plasticity and rapid adaptive ability. The first hypothesis depends on the fact that the most widespread phenotypes (parthenogenesis and CI) involve disruptions of mitosis during host embryonic development, a mechanism that might be used for other phenotypes. This might explain male-killing, because the death of males occurs in early stages of embryonic development. However, in this case, the phenomenon should be more complex because the symbiont can detect the sex of the embryo and specifically kill only one type. Furthermore, no mitotic disturbance seems to be involved in feminization, and different detailed analyses of CI phenotypes have revealed that mitosis is disturbed in several ways. Consequently, Wolbachia mechanisms of action probably differ according to the hosts. The second hypothesis (plasticity) relates to the fact that, by consuming and also supplying nutrients or products to host cells, all intracytoplasmic symbionts have intimate relationships with their hosts15. Wolbachia are probably no exception, a suggestion strengthened by the evidence of their mutualistic tendency in filarial worms. Here, the relationship might differ according to different host physiology, even for closely related symbionts, or because a symbiotic product might have different implications in different hosts. There might even be selection for overproduction of a particular product if it results in the symbiont being favoured. Such a phenomenon is known in Buchnera symbionts of pea aphids, where stress proteins of the GroE family are over-produced (although the release of these proteins in the host cell is not well understood)16. If endosymbiont physiology is not too limited, its numerous products could provide a variety of substrates with which selection can play. Acknowledgements I thank Greg Hurst, Claudio Bandi and Rebecca Terry for discussions and comments on this article.
Thierry Rigaud Génétique et Biologie des Populations de Crustacés, UMR CNRS 6556, Université de Poitiers, 40 avenue du Recteur, Pineau, F-86022 Poitiers Cedex, France ([email protected])
References 1 Werren, J.H., Zhang, W. and Guo, L. (1995) Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods, Proc. R. Soc. London Ser. B 261, 55–71 2 Bouchon, D., Rigaud, T. and Juchault, P. (1998) Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization, Proc. R. Soc. London Ser. B 265, 1081–1090 3 Breuwer, J.A.J. and Jacobs, G. (1996) Wolbachia: intracellular manipulators of mite reproduction, Exp. Appl. Acarol. 20, 421–434 4 Werren, J.H. and O’Neill, S.L. (1997) The evolution of heritable symbionts, in Influential Passengers: Inherited Microorganisms and Arthropod Reproduction (O’Neill, S.L., Hoffmann, A.A. and Werren, J.H., eds), pp. 1–41, Oxford University Press 5 Giordanno, R., Jackson, J.J. and Robertson, H.M. (1997) The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets, Proc. Natl. Acad. Sci. U. S. A. 94, 11439–11444 6 Hariri, A.R., Werren, J.H. and Wilkinson, G.S. (1998) Distribution and reproductive effects of Wolbachia in stalk-eyed flies (Diptera: Diopsidae), Heredity 81, 254–260 7 West, S.A., Cook, J.M., Warren, J.H. and Godfray, H.C.J. (1998) Wolbachia in two insect host–parasitoid communities, Mol. Ecol. 7, 1457–1465 8 Hurst, G.D.D. et al. (1999) Male-killing Wolbachia in two species of insects, Proc. R. Soc. London Ser. B 266, 735–740 9 Hurst, G.D.D., Hurst, L.D. and Majerus, M.E.N. (1997) Cytoplasmic sex ratio distorters, in Influential Passengers: Inherited Microorganisms and Arthropod Reproduction (O’Neill, S.L., Hoffmann, A.A. and Werren, J.H., eds), pp. 125–154, Oxford University Press 10 Bandi, C., Anderson, T.J.C., Genchi, C. and Blaxter, M.L. (1998) Phylogeny of Wolbachia in filarial nematodes, Proc. R. Soc. London Ser. B 265, 2407–2413 11 Schilthuizen, M. and Gittenberger, E. (1998) Screening mollusks for Wolbachia infection, J. Invertebr. Pathol. 71, 268–270 12 Rousset, F. et al. (1992) Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods, Proc. R. Soc. London Ser. B 250, 91–98 13 Bosshardt, S.C., McCall, J.W., Coleman, S.V., Jones, K.L., Petit, T.A. and Klei, T.R. (1993) Prophylactic activity of tetracycline against Brugia paphangi infection in Jirds (Meriones unguiculatus), J. Parasitol. 79, 775–777 14 Hoerauf, A. et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility, J. Clin. Invest. 103, 11–17 15 Schwemmler, W. and Gassmer, G., eds (1989) Insect Endocytobiosis: Morphology, Physiology, Genetics, Evolution, CRC Press 16 Hara, E. et al. (1990) The predominant protein in an aphid endosymbiont is homologous to an E. coli heat shock protein, Symbiosis 8, 271–283
213