WO2006008652A1 - Insect population control - Google Patents

Abstract

The present invention relates to methods of controlling pest populations through induction of cytoplasmic incompatibility. The invention provides a method for inducing cytoplasmic incompatibility in a pest species comprising transfer of endosymbiotic bacteria in particular wolbachia from one host species which is infected by the endosybiotic bacteria to a pest species wherein the pest is not infected with that endosymbitic bacteria.

Description

Insect Population Control

Field of the Invention The present invention relates to methods of controlling pest populations through induction of cytoplasmic incompatibility.

Background to the Invention

Insects, fungi, nematodes, protozoa, bacteria and viruses are responsible for widespread damage to crops and animals world- wide with enormous concomitant economic consequences, m addition, many human and veterinary health issues are associated with the spread of disease by insects and other pests.

To try to reduce pest-inflicted damage, resources have been devoted to the development and deployment of pesticides, which control pest populations by killing target pests. Although pesticides are in many cases effective, they are known to be toxic to life forms other than the target organism, which has important environmental consequences. Accordingly, there has been a focus on development of alternative, more biological, pest control methods that do not involve pesticides.

However, to date, the generation of these alternative control strategies has not been successful. A biotechnological strategy to control insect populations is the Sterile Insect Technique SIT (Krafsur, E.S. Journal of Agricultural Entomology 15, 303-317 (1998)), which involves the generation and mass release of sterile insects into the environment. A few members of the insect order of the Diptera have been targeted by SIT. Such methods failed in attempts to control tsetse flies carrying human and cattle trypanosomes.

The Mediterranean fruit-fly (Medfly) Ceratitis capitata is a major agricultural pest for more than 250 fruit varieties of economic importance. It is geographically widespread in tropical and temperate regions. The Medfly has been introduced relatively recently into the New World, and appears to be spreading rapidly, threatening fruit producing areas in North America (Carey, J. R., Science 253: 1369 (1991)). Since the mid 1970's, the sterile insect technique has been used for Medfly eradication and control. This method relies on the decrease in or collapse of fly populations following releases of large numbers of sterile insects over infested areas, and offers an environmentally attractive alternative to massive spraying with insecticides (Rnipling, E. F., Science 130: 902 (1959)).

However, when sterile male insects were used to control Medfly populations, it was found that although it slows Medfly population growth and may lead to its temporary collapse, it does not lead to destruction of female insects, which are responsible for crop damage. In addition, since the sterile males are rendered less fit by the irradiation treatment such that their life span and reproduction fitness is decreased, the method requires repeated releases of sterile males into the environment.

Accordingly, to date, the majority of the Medfly pest management programmes in Europe, the US and globally are still based on massive insecticide spraying.

There therefore remains a need for a control technique for insects, such as Medfly, and other pests which can selectively destroy target pests but which is environmentally more acceptable than the chemical methods involving mass spraying of toxic pesticides. It is therefore advantageous to develop methods for biological control i.e. the purposeful introduction of parasites, predators and pathogens to reduce or suppress pest populations.

A number of invertebrates are infected with endosymbiotic bacteria. Symbionts include, at one extreme, intra-cellular species permanently associated with an insect cell and of which no free-living bacterial forms have ever been found and, at the other extreme, free-living forms which are readily cultured in bacterial media in vitro.

Symbionts can be highly specific for each insect species. The functions of symbionts vary both in symbiont species and in different insects. Generally, the relationship between a specific symbiont and host is an interdependent interaction that has evolved into a stable association over time such that, in some cases, the symbionts synthesise vital insect nutrients not found in the insects single food source. One of the other functions of endosymbionts is that they appear to confer a reproductive advantage on the infected insect over the non-infected one.

Wolbachia are symbiotic, inherited bacteria of arthropods that manipulate host reproduction by using several strategies. The most widespread and, perhaps, the most prominent feature is cytoplasmic incompatibility (CI) (Stouthamer et al. Annual Review of Microbiology 53, 71-102 (1999), Werren, J.H. Biology of Wolbachia. Annual Review of Entomology 42, 587-609 (1997). CI results in embryonic mortality in crosses between insects with different Wolbachia infection status (Bourtzis et al. Nature 391, 852-853 (1998); Bourtzis, K., Braig, H. & Karr, T.L. in Insect symbiosis. (eds. K. Bourtzis & T.A. Miller) Chapter 14 (CRC Press, Florida, USA; 2003)).

Although the mechanism of CI has not yet been elucidated on the molecular level, several lines of evidence suggest that Wolbachia somehow modifies the paternal chromosomes during spermatogenesis (mature sperm does not contain the bacteria). This influences their behaviour during the first mitotic divisions and results in loss of mitotic synchrony (O'Neill, S.L. & Karr, T.L. Nature 348, 178-180 (1990); Tram, U. & Sullivan, W. Science 296, 1124-1126 (2002)).

In the past, and before the etiological connection between Wolbachia and CI was revealed (Yen, J.H. & Barr, A.R. Nature 232, 657-658 (1971)), attempts have been made to exploit CI as a method to suppress natural populations of arthropod pests in a way analogous to SIT. These early attempts involved the mass production and release of incompatible male insects to control wild populations of disease vectors such as the mosquito, Culex pipiens (Laven, H. Nature 261, 383-384 (1967)) and of agricultural pests such as the European cherry fruit fly, Rhagoletis cerasi (Boiler et al. Entomologia Experimental Et Applicata 20, 231 -2Al (1976)) and, at a smaller scale, the almond moth Cadra (Ephestia) cautella (Ηrower, J.H. Journal of Economic Entomology 73, 415-418 (1980)). However, to date, not all pest species have established Wolbachia associations and hence the full potential use in pest control has not yet been established. In particular, extensive screenings of both laboratory and natural populations of Medfly have shown that this insect pest is not infected with Wolbachia (Bourtzis et al. Insect Molecular Biology 3, 131-142 (1994)). In addition, the criteria influencing the transfer of any Wolbachia strain into any new uninfected host species such that the host species accepts, supports and transmits the infections has not been understood..

Summary of the Invention

The present invention relates to methods for the biological control of invertebrates through the introduction of cytoplasmic incompatibility (CI) by infection with an endosymbiotic bacteria (endosymbionts) such as Wolbachia.

hi particular, the present study describes the successful transfer of endosymbionts from one host species which is naturally infected by the endosymbiont to a new host species, i.e. a pest species, which is not a natural host such that cytoplasmic incompatibility is conferred.

Suitable endosymbiotic bacteria include bacteria that induce CI in a new host species. Such bacteria include those of the Bacteroidetes group (Hunter et al. Proc R Soc Lond B Biol Sci. 270(1529): 2185-90 2003). In a particularly preferred embodiment, the endosymbiotic bacteria is Wolbachia.

Accordingly, in a first general aspect of the invention, there is provided a method for inducing cytoplasmic incompatibility in a pest species comprising transfer of endosymbiotic bacteria from one host species which is infected by the endosymbiotic bacteria to a pest species wherein the pest species is not infected with that endosymbiotic bacteria.

Suitably the pest species is an insect of economic and medical importance. Preferably the pest species is selected from Medfly (Ceratitis capitata), Aedes aegypti, Anopheline mosquitoes such as Anopheles gambiae, Bactrocera oleae (olive fly), other tephritids, whiteflies and aphids, for example.

Suitably the pest species is uninfected with an endosymbiotic bacteria, hi another embodiment, the pest species is infected with a different strain of the endosymbiotic bacteria, hi a further embodiment, the pest species is not a natural host for the endosymbiotic bacteria. By this is meant that the particular strain of endosymbiotic bacteria is not normally found in the pest species in the wild or is better adapted for infection of a different insect species.

As described herein, different bacterial strains have host range limitations. For example, of the three strains infecting R. cerasi described herein, only two were transferable to different species (Medfly and Drosophila (Riegler et al. Applied and Environmental Microbiology 70, 273-279 (2004)) and one never transferred. Suitably, the strain for infection of the pest species is chosen from a natural endosymbiont of a closely related species.

hi another more focused aspect there is provided a method of inducing cytoplasmic incompatibility in Medfly comprising the steps of: a) obtaining embryonic cytoplasm from a line of a related fly species which is infected with an endosymbiotic bacteria; b) injecting said cytoplasm into Medfly embryos

Suitably, step b) represents the generation GO post infection and the method further comprises crossing GO females with uninfected males to establish an infected cell line.

hi a preferred embodiment, the endosymbiotic bacteria is Wolbachia. More preferably, the Wolbachia are two strains, wCer2 and wCer4, selected from Wolbachia infections of the cherry fruit fly Rhagoletis cerasi.

hi one embodiment, the infected fly lines are Rhagoletis cerasi. Furthermore, the present study shows that Wolbachia-indacQd CI can be used as a means for control of pest populations such as natural Medfly populations.

Accordingly, in another aspect, there is provided a method for controlling a pest population comprising introducing Wolbachia into said pest population.

Suitably the method comprises infecting target pest embryos with Wolbachia using embryonic cytoplasmic injections from a Wolbachia-infected embryo. In a preferred embodiment, the method further comprises crossing an infected individual with a non- infected individual. In an alternative embodiment, the method comprises crossing an individual infected with one strain of Wolbachia with an individual infected with another strain.

In a preferred embodiment the pest species is selected from Medfly, Aedes aegypti, Anopheles gambiae, other Anopheline mosquitoes, Bactrocera oleae (olive fly), other tephritids, whiteflies and aphids although other insect species of economic, agricultural and medical importance are also envisaged. Suitably the present invention can be used for the control of sucking, chewing and biting insects such as rice hopper, aphids, thrips, whiteflies, termites, turf insects and soil insects which attack rice, cereals, maize, potatoes, vegetables, sugar beet, soft fruit, citrus fruit, olives, cotton, hops, vines, tobacco and turf.

Biological control of insect populations through induction of CI allows elimination of pest populations in the absence of indiscriminate environmental effects seen by the wide scale application of a toxic pesticide.

In addition, biological control as described herein provides advantages over the previously used biotechnological approach of radiation of male insects and releasing those sterilised males into the environment. In particular, irradiated males generated in such an SIT approach (as described above), are generally less fit for reproduction due to the radiation causing them damage, for example, to their oesophagus such that feeding is impaired. Thus, irradiated males have a shorter life span and a lower fitness for reproduction. In contrast, insects infected with Wolbachia or other endosymbiotic bacteria have no loss in reproductive fitness.

CI can be either unidirectional or bidirectional. Unidirectional CI is typically expressed when an infected male is crossed with an uninfected female. The reciprocal cross is fully compatible, as are crosses between infected individuals. Bidirectional CI usually occurs in crosses between infected individuals harbouring different strains of

Wolbachia. Such crosses result in embryonic mortality. This is in contrast to the situation where a cross between male and female insects infected with the same strain results in the production of viable offspring.

Accordingly, the use of Wolbachia infection in insect population can be effective in a suppression scheme either where the target population is uninfected or where the target population is infected with a different strain, hi the former situation, infected males may be introduced into an uninfected target population to induce CI and thus embryonic mortality, hi the latter, if the target population is identified to be infected with one strain, males infected with an alternative strain may be introduced and CI induced.

Bidirectional CI also permits multiple releases of individuals comprising alternative strains of Wolbachia. For example, the accidental release of an infected female into the target population may result in an ineffective CI strategy as the infected females may spread into the target population such that the infected males crossing with females infected with the same strain will produce viable young. However, in the event of such accidental release, males infected with an alternative strain of Wolbachia may be generated and released in order to generate CI and embryonic mortality.

Alternative strains may be derivable from alternative host populations or generated in the laboratory.

Accordingly, in a another aspect there is provided a method to establish different lines of the novel host or pest species carrying different incompatible Wolbachia infections (bi-directional CI) as an internal control of the new CI suppression strategy. This is particularly useful in the case that one 'infection' escapes through the release of an infected female. This problem was described in the early uses of the natural Wolbachia associations, see Curtis et al. 1982.

In one embodiment, Wolbachia-mώiced CI is applicable to islands populations or isolated areas (see, for example, Neuenschwander et al., Proc. CEC/IOBC Intl Symposium Athens 1982, 366-370 (1983); and Riegler & Stauffer Molecular Ecology 11, 2425-2434 (2002))

The successful transfer of Wolbachia from a naturally infected host species to a non- naturally infected pest species supports the use of Wolbachia as a vector for the expression of genes of interest in pest species. Thus vectors may be used to drive expression of a new genotype in an insect population.

Examples of methods where bacterial symbionts are used as vectors are described, for example, in WO 94/02591 and by Beard, CB. et al. in Genetic transformation and phylogeny of bacterial symbionts from tsetse. Insect Molecular Biology 1, 123-131 (1993); Sinkins, S.P., Curtis, CF. & O'Neill, S.L. in Influential passengers. Inherited microorganisms and arthropod reproduction, (eds. S.L. O'Neill, A.A.Hoffmann & J.H. Werren) Chapter 6 (Oxford University Press, Oxford, United Kingdom; 1997); Bourtzis, K. & Braig, H. in Rickettsiae and rickettsial diseases at the turn of the third millennium, (eds. D. Raoult & P. Brouqui) 199-221 (Elsevier, Amsterdam; 1999) and Sinkins, S.P. & O'Neill, S.L. in Insect transgenesis. Methods and Applications, (eds. A.M. Handler & A.C. James) Chapter 15 (CRC Press, Boca Raton, Florida; 2000).

Accordingly, in another aspect, there is provided a method of expressing gene products in a pest population comprising introducing into said population a Wolbachia bacteria comprising a gene of interest.

hi a further aspect there is provided a use of Wolbachia as a vector for gene expression in Medfly. Detailed Description of the Invention

By "endosymbiotic bacteria" or "endosymbiont" is meant bacteria which has established a symbiotic relationship within a eukaryotic cell. In particular, "endosymbiont" in the context of the present invention are bacteria which have established a symbiotic relationship with cells of a pest population.

Wolbachia are Rickettsia-Vkβ, matrilineally inherited, obligate intracellular bacteria that infect many species of invertebrates (Werren et al. Proc R Soc Lond B Biol Sci.

1995 JuI 22;261(1360):55-63; Stouthamer et al. Annual Review of Microbiology 53,

71-102 (1999)). Phylogenetic classification of Wolbachia is within the order

Rickettsiales, family Anaplasmataceae, also containing the bacteria Ehrlichia,

Anaplasma and Neorickettsia (Dumler et al. hit J Syst Evol Microbiol. 2001 Nov;51(Pt 6):2145-65). Subgrouping of Wolbachia is ongoing, based on recently emerging genomic information. Currently, it can be grouped into six major clades, A - F, with

Wolbachia of W. bancrofti, B. malayi and Litomosoides sigmodontis in clade D, and those of Onchocerca and Dirofilaria in clade C (Lo et al. MoI Biol Evol. 2002

Mar;19(3):341-6). These are Wolbachia found in filarial parasites of importance to human health and are distinct from the clades of Wolbachia that infect arthropods

(Bandi et al. Vet Parasitol. 2001 JuI 12;98(l-3):215-38; Casiraghi et al. Parasitology.

2001 Jan;122 Pt 1:93-103.).

As used herein, a "population of target pests" or a "pest population" refers to a group of invertebrates, and particularly insects, which invade the host plant or vertebrate to feed or replicate whether delimited along species or geographical lines, or both, which it is desired to be controlled. For example, a pest population may refer to a given species of pest which infests a particular crop or vertebrate in a given geographical area. Alternatively, it may refer to all pests infesting any crop or vertebrate in a geographical area, or a given species without reference to any geographical limitation, or a population of pests which is responsible for a human or veterinary health problem, such as the spread of malaria. Target pests are the individual members of the population of pests.

"Control" as used herein refers to the limitation, prevention or reduction of population growth, i.e., by at least about 10% per generation, preferably at least about 50%, 80%, or even up to and including 100% of the pest population. Preferably, this is achieved by reducing the number of viable young individuals for example by reducing the number of eggs that hatch. Advantageously, the population of pests is eliminated.

"Population suppression" is used to describe the reduction of numbers in a population. In particular, population suppression is measured by a reduction in the number of hatched eggs.

Brief Description of the Figures

Figure 1 shows presence of Wolbachia in transinfected C. capitata embryos, ovaries and testes.

Embryos (top row): transinfected Medfly embryo undergoing synchronous mitotic divisions showing Wolbachia localization at the mitotic spindles (left); the posterior part of a transinfected Medfly embryo, where pole cells (the precursors of gonads) are being formed, showing incorporation of Wolbachia in the pole plasm (middle); uniform distribution of Wolbachia bacteria in a post-gastrulation transinfected embryo (right). Ovaries and testes (bottom row): distribution of Wolbachia during oogenesis, when oocytes start to form. The bacteria are mostly concentrated in nurse cells, presumably infecting the oocyte at later stages of oogenesis (left); large numbers of Wolbachia are present in adult testes of transinfected Medfly (middle); a sperm cyst is shown with the distal end toward the left and nuclei to the right (left). After elongation is complete, most cytoplasmic components, including Wolbachia, are stripped away from the sperm during individualization and sequestered in the waste bag (shown as a "green ball" in the left). Bacteria are visualized green-yellow and nuclei red. Scale bars 40ocm; except to the top right image which is lOOocm. Immunostaining and confocal microscopy analysis was performed as previously described (Dobson, S. L. et al. Insect Biochemistry and Molecular Biology 29, 153-160 (1999); Clark et al. Mechanisms of Development 111, 3-15 (2002); Clark et al. Mechanisms of Development 120, 185-198 (2003)).

Figure 2 shows suppression of Medfly populations using Wolbachia-indaced CL Population suppression is expressed as percentage of eggs that hatched. The numbers of the adults used as well as the number of eggs scored per cage are shown below the diagram.

Figure 3 shows Wolbachia-indaced cytoplasmic incompatibility in two transinfected lines of the Medfly Cer otitis capitata. A) Test crosses between each transinfected line and the parental naturally uninfected Benakeion strain as well as between the two transinfected lines. Crosses between 100 females (2-3 days old) and equal numbers of males (one day old) were performed in cages. B) Test crosses between tetracycline treated individuals of the line WolMed S 10.3. Crosses between 30 females and equal number of males were performed in cages, three generations (*) and five generations (**) after the tetracycline treatment. Wolbachia-indaced cytoplasmic incompatibility is expressed as percentage of unhatched eggs ± S.E.. Egg laying plates were removed daily for a period of 6 days. Hatching rates were scored 72 hours after egg collection.

Examples

Methods

Insects

Cer otitis capitato. Benakeio is an uninfected laboratory strain. A71 is an uninfected white eye mutant laboratory strain. Both strains are kept in mass in population cages at 24⁰C on standard Medfly diet.

Rhagoletis cerasi: Two natural populations of cherry fruit flies were collected from Austria and from Sicily (Italy). The fϊrsgwas doubly infected with wCerl and wCer2 and the second singly infected with wCerl¹⁸. The Sicilian population had previously been found to carry additional Wolbachia strains (M Riegler and C Stauffer, personal communication)

Embryonic cytoplasm transfer Wolbachia was transferred from naturally infected cherry fruit fly populations into the Benakeio laboratory strain of Ceratitis capitata. Microinjections were carried out using a microcapillary needle (Boehringer Femtotips). Medfly embryos were collected for 60 min, dechorionated and slightly desiccated. The cytoplasm was taken from the posterior region of donor mature oocytes and injected at the posterior pole of the recipient pre-blastoderm embryos ^{16> π}.

Establishment of the infected Ceratitis capitata lines

Females deriving from injected embryos represent the generation GO post-injection. GO females were each crossed with two Benakeio males. The progeny of transinfected GO females were kept in mass culture establishing a line.

DNA extraction, PCR and sequencing

Total DNA was extracted from single individuals following the STE boiling method.

Wolbachia was detected by PCR using the 16S rDNA Wolbachia specific primers, 99F and 994R¹⁹. At least 60 individuals from the WolMed 88.6 line and 40individuals from the WolMed S 10.3 line were screened for infections every generation. Distinction

1 51 between different Wolbachia variants was done with strain specific wsp primers . For sequencing, PCR products were amplified by the wsp primers 8 IF and 69 IR²⁹ and were sent for direct sequencing. At least 3 individuals from each established line were sequenced.

Cytoplasmic incompatibility assays and statistical analysis

CI levels were measured in two different ways, in single pair crosses and in cage populations. All the crosses were performed at 24°C. hi the first case two days old virgin females were individually crossed with one day old virgin males. The egg laying plates were removed every day and all eggs were scored for a period of 6-8 days. In the second case 100 two days old virgin females were crossed with 100 one day old virgin males in a population cage. A random sample of 500 eggs was taken every day. Hatching rates were scored 72 hours after egg collection. Embryonic mortality EM was determined as the percentage of unhatched eggs. The standard error (SD) for EM, was determined according to Sokal & Rolhf³⁰. Embryonic mortality between infected males and uninfected females was complete, which let us assume that CI is complete.

Suppression of C. capitata populations

The population suppression experiments were performed in six cages which contained equal numbers of two days old virgin uninfected females and one day old virgin uninfected males (1:1). We were then releasing one day old virgin transinfected males at different ratios 1:1:0, 1:1:1, 1:1:10, 1:1:20, 1:1:30, 1:1:50. The experiment was performed in population cages at 24°C. The first five cages were containing about 300 flies and the last one 520 flies. Egg laying plates were removed every day for a period of 6-8 days. In the first two cages a random sample of 500 eggs were kept daily, while in the rest of them all the eggs were collected. Hatching rates were scored 72 hours after egg collection. Survival was determined as the percentage of hatched eggs.

Presence of Wolbachia in transinfected C. capitata embryos, ovaries and testes Embryos. Embryos were collected and dechorionated in 50% commercial bleach for 5 min. After a quick rinse with washing buffer (0.7% NaCl, 0.3% Triton X-100) they were transferred to 1 : 1 heptane-methanol solution and shaken vigorously for a couple of minutes. They were then briefly washed three times with methanol three times with

TBST (50 niM Tris-HCl, 15OmM NaCl, 0.1% Tween, 0.05% NaN3, pH 7.5), 15 min each, were then blocked in 1% BSA in TBST and incubated with the WSP (Wolbachia

Surface Protein) antibody with a 1:500 dilution overnight at 4°C. After 3 washes with

TBST the eggs were incubated for one hour at room temperature with 1 :500 dilution of

Alexa Fluor 488 goat anti-rabbit IgG labeled antibody (Molecular Probes) and 2mg/ml

Rnase A (Sigma) in TBST. After several washes in TBST eggs were stained with 5 ocg/ml Propidium Iodide (PI) (Molecular Probes) for 20 minutes, rinsed and mounted with ProLong Antifade kit (Molecular Probes). Ovaries and testes. Ovaries from two-three days old females and testes from one day old males were removed in TBST and further dissected on glass slides. Tissue samples were flattened under a cover glass and frozen in liquid nitrogen. Cover glasses were removed using a razor blade and the slides were placed in ice-cold ethanol for 3 min and fixed in 4% paraformaldeyde for 12 min. Slides were rehydrated in TBST, blocked and incubated with antibodies and PI as previously described. ^π

Image analysis. Optical sections were taken by using a confocal laser-scanning microscope (Leica TCS-NT) and they were projected onto single images. Images were further processed using Photoshop 6.0 (Adobe) ¹².

Example 1- Determination of Wolbachia infection and CI expression in Medfly

To determine whether the Medfly can support Wolbachia infections and express CI, conventional embryonic cytoplasmic injections ^{" 7} were used for transfer of natural bacterial symbionts from a related species, Rhagoletis cerasi (Diptera, Tephritidae) , to an uninfected laboratory strain of medfly Ceratitis capitata (Diptera, Tephritidae), the Benakeion strain.

Wolbachia infected lines of R. cerasi from Sicily (Italy) and Austria were used as donors of Wolbachia-iafected embryonic cytoplasm. Eighty-eight GO isofemale lines were produced and were monitored at each generation for the presence of Wolbachia using a specific PCR assay ¹⁹. After three generations of monitoring, two out of initially eleven transinfected isofemale lines remained positive for the presence of Wolbachia, namely WolMed 88.6 and WolMed S10.3, each one being infected with a different bacterial strain as confirmed by PCR-RFLP and wsp gene sequences analysis. The line WolMed 88.6 was found to be infected with the wCer2 strain which originated from the R. cerasi Austrian population (accession number AF418557) while the line WolMed S 10.3 was found to be infected with wCer4. This Wolbachia strain was previously undetected in its original host, but originated from an island population (Sicily), which has characteristic infection types¹⁸ including new Wolbachia strains. The wCer4 strain was found to be 100% identical with the wlrr-Al strain based on partial wsp gene sequence (AF217714). After 21 generations (about 19 months) post injection, both transinfected lines are stably infected with infection rates of 100%.

Example 2 - Induction of CI by transinfected lines

Confocal microscopy analysis was performed in embryos, ovaries and testes of both transinfected medfly lines using an anti-WSP (Wolbachia Surface Protein) antiseruni20 (Fig. 1). Both lines present high infection levels and distribution patterns as in characterised Drosophila-Wolbachia associations ^21'23.

To determine whether the two transinfected lines were capable of inducing CI, test crosses were performed between each transinfected line and the parental naturally uninfected Benakeion strain. Crossing experiments in cages with 100 uninfected females and equal numbers of infected males from each transinfected line (seventh generation post injection) resulted in 100% egg mortality; the reciprocal crosses resulted in less than 32% egg mortality under identical experimental conditions (Table IA).

Similar results were obtained in cage experiments between uninfected females from the medfly strain A71 (a white eye mutant C. capitata line) and infected males from each transinfected line (sixth generation post injection, data not shown).

Crosses between individuals of the tetracycline cured WolMed S 10.3 line, three and five generations after curing, resulted in lower rates of embryonic mortality (Table IB).

It should be noted that control crosses between infected males and females resulted in high embryonic mortalities, about 65%, in both transinfected lines (Table IA). One explanation for these high embryonic mortalities could be a leaky and inefficient transmission of both wCer2 and wCer4 in medfly. However, at least 60 individuals from the WolMed 88.6 line and 40 individuals from the WolMed S 10.3 line were tested by PCR in every generation for 21 generations (over 19 months), and all individuals have been found positive for Wolbachia. Additionally, the infection status of all individuals in the single pair crosses presented below was confirmed by PCR.

Five females of generation 18 of the line WolMed 88.6 were mated to uninfected males, and the transmission of Wolbachia was assessed in 10 individuals of their offspring. All offspring carried the wCer2 strain.

Experiment 3. Persistence of Wolbachia induced CI

Persistence of Wolbachia-indaced CI was also tested by single-pair crosses. Twenty- six single pair crosses between WolMed 88.6 (wCer2) transinfected males (fifth generation post injection) and uninfected females resulted in 100% egg mortality (no hatched eggs out of 2164). Similarly, seventeen single pair crosses between WolMed SlO.3 (wCer4) transinfected males (fifth generation post injection) and uninfected females resulted again in 100% egg mortality (no hatched eggs out of 1325). The same experiments were repeated again in the tenth generation post injection providing the same results, 100% egg mortality (data not shown).

Such complete CI expression has only been observed in very few Wolbachia-infected species such as Culex pipiens9. This is the first report, to our knowledge, that a newly transinfected host species presents such stability of the infection and, at the same time, expresses 100% CL

The two transinfected rnedfry lines WolMed 88.6 (wCer2) and WolMed S10.3 (wCer4), each infected with a different Wolbachia strain, were 100% bidirectionally incompatible in appropriate genetic cross experiments performed in cages (Table 1).

To determine whether cytoplasmic incompatibility expressed by the Wolbachia infected medfly lines could be used as a potential tool for population suppression, we set up cage populations containing different ratios of uninfected females: uninfected males: transinfected males (1:1:0, 1:1:1, 1:1:10, 1:1:20, 1:1:30, 1:1:50). These experiments were performed using transinfected WolMed 88.6 (wCer2) males from generation 8-11 post injection. The laboratory cage medfly populations were suppressed by these single "releases" of incompatible males at a ratio-dependent manner. Under these conditions population suppression reached levels higher than 99% in releases of incompatible males at a ratio 1:1:50 (Fig. 2). Similar results were obtained in cage experiments using as target population the medfly white eye mutant strain A71 (data not shown). These data clearly demonstrate that Wolbachia-induced cytoplasmic incompatibility could be used as a tool for the population control of this major agricultural pest.

Our present study clearly shows that Wolbachia endosymbionts can be experimentally transferred over genus barriers into a novel host, forming associations which express complete CL The population cage experiments show that Wolbachia-induced CI (unidirectional and, importantly, bidirectional) can be used as a means for control of natural medfly populations. For effective Wolbachia-based population suppression, an efficient genetic sexing system producing males only is necessary. Such systems are available in medfly (Robinson, A.S. Geneticα 116, 5-133 (2002)).

OTHER EMBODIMENTS

All publications mentioned in the above specification, and references cited in said publications, are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims. REFERENCES

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Claims

1. A method for inducing cytoplasmic incompatibility in a pest species comprising transfer of endosymbiotic bacteria from one host species which is infected by the endosymbiotic bacteria to a pest species wherein the pest species is not infected with that endosymbiotic bacteria.

2. A method of inducing cytoplasmic incompatibility in medfly comprising the steps of: a) obtaining embryonic cytoplasm from an infected line of a related species; b) injecting said cytoplasm into medfly embryos

3. A method wherein step b) represents the generation GO post infection and the method further comprises crossing GO females with uninfected males to establish an infected isofemale line.

4. A method as claimed in any of claims 1 to 3 wherein the endosymbiotic bacteria is Wolbachia.

5. A method as claimed in claim 4 wherein the Wolbachia is a strain selected from wCer2 and wCer4.

6. A method for controlling a pest population comprising introducing Wolbachia into said pest population.

7. A method as claimed in claim 6 comprising infecting target pest embryos with Wolbachia using embryonic cytoplasmic injections from a Wolbachia-mfected embryo.

8. A method as claimed in claim 7 further comprising crossing an infected individual with a non-infected individual.

9. A method as claimed in claim 7 wherein the target pest embryos are infected with one Wolbachia strain and the embryonic cytoplasmic injections are from an embryo infected with an alternative Wolbachia strain.

10. A method as claimed in any of claims 1 to 8 wherein the pest species is selected from Medfly, Aedes aegypti, Anopheles gambiae, other Anopheline mosquitoes,

Bactrocera oleae (olive fly), other tephritids, whiteflies and aphids.