Keywords Wolbachia, reproductive parasites, biological control, genomics. Introduction. Wolbachia spp.

Transcription

1 Application of the reproductive parasite Wolbachia to the biological control of flystrike Tim Doran and Robert Moore CSIRO Livestock Industries, Private Bag 24, Geelong, VIC, Summary Wolbachia are obligate intracellular bacterial parasites that infect an extremely wide range of invertebrates. In their insect hosts, Wolbachia induce a diverse range of effects on host reproduction, many of which lead to production of non-viable offspring and skewing of sex ratios. These reproductive parasites infect a significant number of the major insect pests of livestock and crops and also insects responsible for the spread of human infectious diseases. It has been proposed that the changes in host reproduction induced by these bacteria could be exploited to control such insect pests and disease vectors. We believe that research on Wolbachia could lead to new biological control strategies for flystrike. Our understanding of the molecular biology of Wolbachia is set to escalate with the complete genome sequence, providing an opportune time to study Wolbachia. We aim to use genomics to identify Wolbachia genes that encode proteins with the potential to be developed as insecticides. Keywords Wolbachia, reproductive parasites, biological control, genomics Introduction Wolbachia are obligate intracellular bacterial parasites that infect an extremely wide range of invertebrates, including a quarter of insect species sampled (O Neill et al., 1997; Werren, 1997). In their insect hosts, Wolbachia induce a diverse range of effects on host reproduction, many of which lead to production of non-viable offspring and skewing of sex ratios. These reproductive parasites infect a significant number of the major insect pests of livestock and crops and also insects responsible for the spread of human infectious diseases. It has been proposed that the changes in host reproduction induced by these bacteria could be exploited to control such insect pests and disease vectors (Sinkins et al., 1997). The potential to apply Wolbachia to the biological control of Lucilia will be discussed in this paper. Wolbachia spp. Wolbachia are a not-so-distant relative of Rickettsia and belong to the group of α-proteobacteria. The first strain was reported in 1924 as the unnamed rickettsia in the ovaries of the mosquito Culex pipiens and was formally named as Wolbachia pipientis (Stouthamer et al., 1999). Additional Wolbachia strains have also been named (W. postica, W. trichogrammae, and W. popcorn), but none of these are officially recognised. At the present time four major monophyletic clades of Wolbachia are recognised and referred to as Wolbachia groups A, B, C and D (Bandi et al., 1998; Werren et al., 1995). The A and B groups are found in a range of insects, mites and crustaceans, whereas groups C and D are restricted to filarial nematodes. Little is known about the genetics and biochemistry of Wolbachia because of their fastidious requirements. There is no established cell-free in vitro culturing system. Subsequently, there has been limited characterisation of Wolbachia genes other than a few loci that have been cloned and used mainly for phylogenetic purposes e.g. the wsp gene (cell surface protein) is used for the classification of W. pipientis (Braig et al., 1998). This will change dramatically when genome sequencing of several Wolbachia strains is completed in the near future (Bandi et al., 1999a). In preparation for genome sequencing, methods to rapidly purify Wolbachia chromosomal DNA for library construction have been described and the genome sizes of six different Wolbachia strains have been determined (Sun et al., 2001). Sizes of 1.4 to 1.6 Mb were observed for group A strains that infect Drosophila and 0.95 and 1.1 Mb for Wolbachia infecting nematodes. As expected, the genomes studied were all much smaller than the genomes of free-living bacteria such as E. coli (4.7 Mb) and this is typical of obligate intracellular bacteria. Studying and comparing the small genomes of these bacteria will shed light for the first time on 241

2 the molecular mechanisms used by Wolbachia to manipulate its host s reproduction. Identification of important genes in this process may provide targets to be developed in strategies to control insect pests and this will be discussed later in this paper. Effects of Wolbachia on their hosts In arthropods, Wolbachia have been implicated in several host reproductive modifications, including (A) cytoplasmic incompatibility (Hoffmann et al., 1997), (B) killing of male embryos, (C) feminisation in isopods (Rigaud, 1997) and (D) parthenogenesis in wasps (Stouthamer, 1997). Wolbachia are primarily transmitted vertically from mother to offspring and have evolved these numerous host manipulations to induce a female biased sex ratio and thus enhance transmission. Also, (E) a virulent Wolbachia strain that is pathogenic to fruit flies, and greatly reduces adult life span has been described (Min and Benzer, 1997). (A) Cytoplasmic Incompatibility (CI) The best studied and most common effect of Wolbachia on host reproduction is CI which results in embryonic death. CI is observed after mating between males infected by certain strains of Wolbachia with females that are either uninfected or infected with an incompatible Wolbachia strain. CI is widespread in insects and has been reported in different insect orders (Giordano et al., 1997), with most studies being done in Drosophila. The cause of embryonic death is an incompatibility between sperm and egg, however little is known about the molecular mechanism of CI. Infected males produce sperm that do not contain Wolbachia, therefor CI is thought to be determined by Wolbachia-induced modifications of sperm in the immature spermatids of infected males. It has been proposed that CI might be mediated by a Wolbachia encoded toxin (Bandi et al., 2001). The eggs produced by infected females are compatible with unmodified sperm and with sperm produced by males infected with the same strain. As a result, the presence of infected males in a population will prevent reproduction by uninfected females. Infected females therefore make a greater contribution of offspring to the next generation and the proportion of infected hosts increases with each successive generation. (B) Killing of Male Embryos Inherited bacteria that selectively kill male embryos are diverse and are found in a wide variety of insect hosts (Skinner, 1985; Hurst, 1997; Hurst, 1991; Hurst and Jiggins, 2000). To date, male-killing Wolbachia have been found in two taxa, Adalia bipunctata (the two-spot ladybird) and Acraea encedon (an African butterfly). Interestingly, these two host species differ in their system of sex determination, indicating that Wolbachia are relatively unconstrained with respect to the range of hosts in which they can effect male-killing. Therefore, it has been suggested that male-killing Wolbachia will turn out to be common within insects (Stouthamer et al., 1999). The method by which males are killed by Wolbachia strains is unknown. Male-killing bacteria are prevalent in insect species where unhatched eggs are consumed by siblings shortly after hatching, or where there is competition between hatched siblings for a limited resource (Hurst et al., 1997). In these cases, the death of male hosts enhances the survival of sibling female hosts. (C) Feminisation In the terrestrial isopods (woodlice), Wolbachia are responsible for sex reversal. Approximately half of the woodlice species belonging to different families are infected, each species carrying a single Wolbachia lineage (Bouchon et al., 1998). It has been suggested that Wolbachia suppress the development of the androgen gland to induce feminisation of the host (Rigaud, 1997). Again, the molecular mechanisms involved are not understood. By feminizing the male host, the maternally transmitted Wolbachia ensures its vertical transmission to offspring and the relative frequency of infected females in the population is increased (Rigaud, 1997). (D) Parthenogenesis-inducing (PI) PI Wolbachia strains were thought to be restricted to the insect order Hymenoptera (wasps), but were recently discovered in other taxa, namely mites (subclass Acari) and thrips (order Thysanoptera) (Cook 242

3 and Rokas, 2000). In these haplodiploid insect hosts, sex determination is based on ploidy; fertilised eggs produce diploid females while unfertilised eggs produce haploid males. PI Wolbachia induces the production of daughters by doubling the chromosomes of unfertilised eggs, restoring diploidy, and thus producing parthenogenic females rather than males (Stouthamer, 1997). Specifically, daughters are produced when PI Wolbachia suppresses spindle formation during anaphase of the first mitotic division, thus restoring diploidy by fusion of the two mitotic nuclei (Stouthamer, 1997). This manipulation of the host s reproduction enhances the transmission of Wolbachia to future generations. Very little is known about the molecular aspects of PI Wolbachia infection. (E) Virulence A new phenotype caused by Wolbachia infection has been described in a laboratory strain of Drosophila melanogaster that was exhibiting premature death (Min and Benzer, 1997). These flies were infected with a virulent Wolbachia variant that was multiplying rapidly in adults, causing degeneration of a variety of tissues and resulting in premature death. This strain has been named popcorn for its characteristic effect on brain cells, which become distended and rupture in response to increasing numbers of bacteria. The striking effect on its host, marks popcorn as the most phenotypically divergent Wolbachia strain identified to date in insects. Interestingly, this Wolbachia variant from Drosophila does not induce CI when crossed to uninfected females. Wolbachia and control of insect pests Because of the various effects Wolbachia has on host reproduction, these reproductive parasites have the potential to provide powerful help in controlling agricultural pests or insect vectors (Curtis and Sinkins, 1998). In fact, even before Wolbachia was recognised as the causative agent of CI, experiments were already done to use CI as a method for mosquito control (Laven, 1967). The idea was to release vast quantities of males that render the females with which they mate sterile because the incompatible matings result in no offspring. Experiments both in the laboratory and the field were promising, however the large amount of work in separating the males from females made the approach inapplicable on a large scale (Stouthamer et al., 1999). No recent work has been described that uses Wolbachia in sterile-insect release. Another two applications for Wolbachia to the control of insect pests have been proposed (Stouthamer et al., 1999). The first is to use Wolbachia to enhance the reproductive potential of parasites or predators of particular pest species (e.g. parasitoid wasps that have been used as agents for biological control because the larvae develop by eating the pest insect). Wasps infected with PI Wolbachia will have a higher population growth rate because all offspring will consist of females and therefore may be better at controlling the pest than uninfected populations of wasps. The second is to use Wolbachia as a gene vector in the host by transforming the bacteria with a gene that suppresses transmission of the pest. To achieve this, Wolbachia have to be transformed in cell culture, re-introduced back into the insect vector and then the spread of this organism must be ensured in pest populations. This is yet to be achieved (see below). Another potential way to control insect populations is to exploit the molecular tools used by Wolbachia to manipulate host reproduction. Does Wolbachia induce CI through expression of a toxin carried by spermatozoa (Bandi et al., 2001)? Do some male-killing bacteria produce a male-specific toxin? If so, could these proteins be developed as insecticides? Studying the molecular biology of Wolbachia, as well as the responses of their hosts will facilitate understanding of the processes involved in reproductive manipulations and reveal new strategies for the control of insect pests. Unfortunately, there is a barrier preventing these studies, and that is the difficulty to grow and manipulate Wolbachia in cell-free in vitro cultures. It has so far proven impossible to study the genetics of these bacteria conventionally e.g. by studying the effect of genetically defined mutants of Wolbachia on bacteria-host interactions. This barrier will be somewhat removed with the completion of the Wolbachia genome sequence. Whilst we may still not be able to genetically modify Wolbachia, we will now be able to use genomic technology to identify important genes (e.g. bioinformatics and micro-arrays) and study their expression in the host (e.g. microarrays and real time PCR). This technology will help us to characterise the host modifying chemicals produced by Wolbachia and identify their targets. The next step is to exploit these discoveries to control insect pests such as Lucilia cuprina. 243

4 Wolbachia and biological control of flystrike Flystrike control programs have traditionally relied on the use of chemical pesticides. Significant problems have arisen with the development of pesticide resistant insects and subsequently, new classes of pesticides with different modes of action are then required to control resistant strains. This situation is now compounded with increasing pressure on producers to reduce pesticide residues in their products and as a result, a rapid decline in the number of commercially available pesticides is predicted. One of the objectives of FLICS is to develop recommendations for future fly and lice control and residue minimisation R&D. We believe that research on Wolbachia fits this objective and could potentially lead to new biological control strategies. Our understanding of the molecular biology of Wolbachia is set to escalate with the complete genome sequence, providing an opportune time to study Wolbachia. Our basic research aims would be as follows: Identify if Lucilia are infected with Wolbachia. This can be done using conventional PCR. So far, 25% of insect species sampled are infected with Wolbachia, however it has been suggested that more sensitive PCR procedures may detect Wolbachia in 75% of insects (Cook and Rokas, 2000). If Lucilia do play host to Wolbachia, then the effect the bacteria may have on its reproduction, and the mode of transmission will be determined. The most common effect of Wolbachia on host reproduction is CI, which results in embryonic death. If Lucilia do not harbour Wolbachia, then can they be experimentally infected? The incongruent phylogenies of both Wolbachia and the hosts it infects, indicate that horizontal transmission between species must happen in nature (Rousset et al., 1992; O Neill et al., 1992). In woodlice, blood-toblood contact between individuals is sufficient for horizontal transfer (Rigaud and Juchault, 1995), however, the manner in which horizontal transfer takes place is unknown for other species. Further, laboratory experiments have demonstrated horizontal transmission of Wolbachia in the isopod Armadillidium vulgare following injury and exposure to hemolymph, or by microinjection of Wolbachia (Boyle et al., 1993; Braig et al., 1994). Therefore, if required, it might be possible to experimentally infect Lucilia with a CI or male killing strain of Wolbachia, or perhaps the virulent popcorn strain. Use genomics to identify the Wolbachia genes involved in CI. Drosophila is the obvious model system for infection and the CI phenotype has been well studied in this host. Also, the Drosophila melanogaster genome sequence is available and will be a valuable resource for identifying the insect target molecules of CI Wolbachia. Of the genomic tools available, we plan to use micro-arrays to study Wolbachia gene expression in the host. Upon completing the sequence of this bacteria s small genome, all open reading frames can be easily arrayed onto DNA chips. The arrays can then be probed with Wolbachia mrna isolated from infected flies and all the expressed genes identified. Male Drosophila have been identified that are infected with Wolbachia variants that do not modify host sperm and hence do not induce CI (Hoffmann et al., 1996; Mercot and Poinsot, 1998; Bourtzis et al., 1998). We aim to use the arrays to compare the expression pattern of these Wolbachia variants with a wild type CI strain, so as to identify the genes that encode the CI inducing molecules (e.g. the proposed toxin). It is these proteins that have potential to be developed as insecticides. References Bandi, C., Anderson, T.J., Genchi, C. and Blaxter, M.L. (1998). Phylogeny of Wolbachia in filarial nematodes. Proc R Soc Lond B Biol Sci. 265: Bandi, C., Slatko, B. and O'Neill, S.L. (1999). Wolbachia genomes and the many faces of symbiosis. Parasitol Today. 15: Bandi, C., Dunn, A.M., Hurst, G.D.D. and Rigaud, T. (2001). Inherited microorganisms, sex-specific virulence and reproductive parasitism. Trends in parasitology 17:

5 Bouchon, D., Rigaud, T. and Juchault, P. (1998). Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization. Proc R Soc Lond B Biol Sci. 265: Bourtzis, K., Dobson, S.L., Braig, H.R. and O'Neill, S.L. (1998). Rescuing Wolbachia have been overlooked. Nature. 391: Boyle, L., O'Neill, S.L., Robertson, H.M. and Karr, T.L. (1993). Interspecific and intraspecific horizontal transfer of Wolbachia in Drosophila. Science. 260: Braig, H.R., Guzman, H., Tesh, R.B. and O'Neill, S.L. (1994). Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart. Nature 367: Braig, H.R., Zhou, W., Dobson, S.L. and O'Neill, S.L. (1998). Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol. 180: Cook, J.M. and Rokas, A. (2000). Influential passengers come of age. Trends Genet. 16: Curtis, C.F. and Sinkins, S.P. (1998). Wolbachia as a possible means of driving genes into populations. Parasitology. 116: Giordano, 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 USA 94: Hoffmann, A.A., Clancy, D. and Duncan, J. (1996). Naturally-occurring Wolbachia infection in Drosophila simulans that does not cause cytoplasmic incompatibility. Heredity 76: 1-8. Hoffmann. A.A. and Turelli, M. (1997). Cytoplasmic incompatibility in insects. In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) Hurst, L.D. (1991). The evolution of cytoplasmic incompatibility or when spite can be successful. J Theor Biol. 148: Hurst, G.D.D. (1997). Cytoplasmic sex-ratio distorters. In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) Hurst, G.D. and Jiggins, F.M. (2000). Male-killing bacteria in insects: mechanisms, incidence, and implications. Emerg Infect Dis. 6: Laven, H. (1967). Eradication of Culex pipiens fatigans through cytoplasmic incompatibility. Nature. 216: Mercot, H. and Poinsot, D. (1998)....and discovered on Mount Kilimanjaro. Nature. 391: 853. Min, K.T. and Benzer, S. (1997). Wolbachia, normally a symbiont of Drosophila, can be virulent, causing degeneration and early death. Proc Natl Acad Sci USA. 94: O Neill, S.L., Giordano, R., Colbert, A.M., Karr, T.L. and Robertson, H.M. (1992). 16S rrna phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects. Proc Natl Acad Sci USA. 89: O Neill, S.L. (1997). In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) 245

6 Rigaud, T. and Juchault, P. (1993). Conflict between feminizing sex ratio distorters and an autosomal masculinizing gene in the terrestrial isopod Armadillidium vulgare Latr. Genetics. 133: Rigaud, T. (1997). Inherited microorganisms and sex determination of arthropod hosts. In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) Rousset, F., Vautrin, D. and Solignac, M. (1992). Molecular identification of Wolbachia, the agent of cytoplasmic incompatibility in Drosophila simulans, and variability in relation with host mitochondrial types. Proc R Soc Lond B Biol Sci. 247: Skinner, S.W. (1985). Son-killer: a third extrachromosomal factor affecting the sex ratio in the parasitoid wasp, Nasonia (=Mormoniella) vitripennis. Genetics 109: Sinkins, S.P., Curtis, C.F. and O Neill, S.L. (1997). In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) Stouthamer, R. (1997). Wolbachia-induced parthenogenesis. In Influential Passengers. (Eds SL O Neill, AA Hoffmann and JH Werren) pp (Oxford University Press) Stouthamer, R., Breeuwer, J.A. and Hurst, G.D. (1999). Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu Rev Microbiol. 53: Sun, L.V., Foster, J.M., Tzertzinis, G., Ono, M., Bandi, C., Slatko, B.E. and O'Neill, S.L. (2001). Determination of Wolbachia genome size by pulsed-field gel electrophoresis. J Bacteriol. 183: Werren, J.H., Zhang, W. and Guo, L.R. (1995). Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods. Proc R Soc Lond B Biol Sci. 261: Werren, J.H. (1997). Wolbachia run amok. Proc Natl Acad Sci USA. 94: