WO2013026994A1

WO2013026994A1 - Mosquitoes with enhanced pathogen resistance

Info

Publication number: WO2013026994A1
Application number: PCT/GB2011/051600
Authority: WO
Inventors: Steven Paul SINKINS
Original assignee: Isis Innovation Limited
Priority date: 2011-08-24
Filing date: 2011-08-24
Publication date: 2013-02-28

Abstract

Provided are mosquitoes of the species Aedes albopictus that comprise a Wolbachia bacterium of the strain w Mel, wherein the mosquitoes have enhanced resistance to a pathogen. The pathogen may be a viral pathogen, for example dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; or yellow fever virus. The pathogen may be a nematode pathogen, for example, Dirofilaria immitis; or Wuchereria bancrofti. The bacterium may induce cytoplasmic incompatibility, in particular bidirectional cytoplasmic incompatibility. The invention also provides the use of such mosquitoes, and methods of making and using the mosquitoes.

Description

Mosquitoes with enhanced pathogen resistance

The present invention relates to mosquitoes with enhanced resistance to pathogens, and to the uses of mosquitoes as biological agents for the reduction of pathogen transmission, and to biological agents for such use. The invention also relates to methods of producing mosquitoes, and to methods of modifying populations of mosquitoes.

Introduction Dengue virus causes dengue fever in infected humans. It is estimated that between 50 million and 100 million people are infected with the dengue virus yearly, and the virus is endemic in more than 1 10 countries worldwide.

The Asian tiger mosquito Aedes albopictus, a native of south-east Asia that in recent decades has invaded Africa, the Americas and southern Europe, is an important vector of dengue virus in rural and semi-urban areas across the tropics. It is probably involved in maintaining sylvatic cycles of transmission and acting as a bridge vector from these to urban epidemic cycles; it also transmits other Flaviviruses such as yellow fever and West Nile, and the Alphavirus chikungunya. Like the primary urban dengue vector Aedes aegypti, Ae. albopictus is a day biting species and therefore not amenable to control or prevention using insecticide-treated bednets. When coupled with the absence of a vaccine for dengue, and the fact that the disease has been expanding in range, novel control methods are much needed. All known wild populations of Ae. albopictus are naturally infected with two strains of the maternally inherited bacterium Wolbachia pipientis, called ι ι/AlbA and ι ι/AlbB; Ae. aegypti is naturally uninfected with the bacterium. Recent work has shown that when an over- replicating strain of Wolbachia from Drosophila melanogaster called i i MelPop was transferred into Ae. aegypti, the transmission of dengue was blocked, and inhibition of chikungunya virus dissemination was also observed. In addition, when the ι ι/AlbB strain from Ae. albopictus was transferred into Ae. aegypti, reduced dengue susceptibility was observed.

In light of the above, it can be seen that there remains a need for the development of new agents that can be used to reduce transmission of dengue virus. It is an aim of certain aspects and embodiments of the present invention to provide mosquitoes having enhanced resistance to a pathogen. It is an aim of certain aspects and embodiments of the present invention to provide new methods of producing such mosquitoes. It is an aim of certain aspects and embodiments of the present invention to provide new agents that can be used to inhibit the transmission of pathogens. It is an aim of certain aspects and embodiments of the present invention to provide new methods of modifying populations of Aedes albopictus mosquitoes.

In a first aspect the invention provides a mosquito of the species Aedes albopictus comprising a Wolbachia bacterium of the strain i i Mel, wherein the mosquito has enhanced resistance to a pathogen. The pathogen may be a viral pathogen, such as dengue virus.

Mosquitoes in accordance with the present invention are unable to transmit dengue virus, thus making them suitable agents for use to prevent dengue virus transmission. The mosquitoes of the invention may be crossed with native mosquito populations to reduce the total pool of virus-carrying mosquitoes in a locality. To the best of the inventor's knowledge, these are the first such examples of Aedes albopictus, which is normally an important vector of dengue virus, that are unable to propagate the virus. The present invention is based, at least in part, on the inventor's surprising finding that infection of Aedes albopictus mosquitoes with the i i Mel strain of Wolbachia gives rise to mosquitoes that have a markedly enhanced resistance to pathogens, such as pathogenic viruses. The present finding is surprising since previously reported studies had shown that Aedes aegypti exhibits a major upregulation of genes associated with the immune response when infected with the wMelPop strain of Wolbachia. Infection with Wolbachia primes the mosquito's immune system, and this is likely to increase resistance to pathogens such as viruses. Ae. albopictus, because it is naturally infected with different strains of Wolbachia, is adapted to the presence of Wolbachia and thus does not exhibit this greatly enhanced (but costly) immune response. Accordingly, these previous studies (in which the Wolbachia strain wMelPop was used instead of i i Mel) would lead the skilled artisan to the incorrect conclusion that Aedes albopictus is unlikely to generate a response that effectively resists pathogens such as viruses when infected with Wolbachia bacteria generically. It seems that this property is exhibited directly by the specific i i/Mel strain of Wolbachia. It is worth noting that the i i/Mel strain of Wolbachia does not naturally occur in mosquitoes such as Aedes albopictus. Instead, Aedes albopictus is normally host to Wolbachia strains designated wAlbA and wAIbB, while the i i/Mel strain is naturally found in Drosophila melanogaster. Since Wolbachia bacteria, such as wMel, are obligate intracellular bacteria they would not be expected to transfer between species without artificial intervention (since no breeding or other natural propagation can occur between Aedes albopictus and Drosophila melanogaster).

Not only does infection of Aedes albopictus mosquitoes with wMel lead to enhanced resistance to pathogens on the part of the mosquitoes, but the bacteria also induce cytoplasmic incompatibility. This cytoplasmic incompatibility is unidirectional in the case of mating between a mosquito comprising wMel and a mosquito in which no form of Wolbachia is present, and is bidirectional in cases in which a mosquito comprising wMel mates with a mosquito comprising another Wolbachia strain. Since most naturally populations of Aedes albopictus are host to both of the wAlbA and/or wAIbB strains (naive populations of Aedes albopictus that lack any Wolbachia primarily being artificially produced) this means that in practice mosquitoes in accordance with the various aspects of the present invention will normally exhibit bidirectional cytoplasmic incompatibility when they mate with members of other populations of mosquitoes. In certain embodiments it may be preferred that mosquitoes of the invention comprising wMel also comprise wAlbA and/or wAIbB.

As described in more details elsewhere in the specification, the ability of wMel to induce bidirectional cytoplasmic incompatibility between mosquitoes of the invention and other mosquito populations is highly beneficial, since this provides a method by which wMel can extensively permeate throughout a local mosquito population. Since such mosquitoes (mosquitoes in accordance with the invention) are resistant to pathogens, and thus provide biological agents capable of inhibiting transmission of such pathogens, this provides great benefits to humans or other animals. When pathogen transmission is inhibited, incidences of infection, and so illness, among human populations are also reduced. The finding that mosquitoes comprising wMel exhibit bidirectional cytoplasmic incompatibility with mosquitoes comprising other Wolbachia strains is surprising, since there is nothing among previously published reports that would lead the skilled person to believe that this would be the case. In particular a previous report showed that the i i MelPop strain when transferred from Drosophila melanogaster into Ae. albopictus, did not produce bidirectional cytoplasmic incompatibility with mosquitoes comprising the naturally occurring ι ι AlbA and i i/AIbB strains. In a second aspect of the invention there is provided a mosquito in accordance with the present invention for use as a biological agent for the reduction of pathogen transmission. By the same token, in a third aspect the invention provides a biological agent for the reduction of pathogen transmission comprising a mosquito according to the present invention.

The mosquitoes of the invention, which comprise wMel bacteria, may be used as agents that can mate with, and thus modify biological properties of, other mosquito populations within an area.

The invention also provides, in a fourth aspect, a method of modifying a population of Aedes albopictus mosquitoes, the method comprising introducing an Aedes albopictus mosquito according to the first aspect of the invention into the population, wherein the introduction of the Aedes albopictus comprising the wMel strain of Wolbachia modifies one or more biological properties of the Aedes albopictus population.

For the present purposes "introducing" may comprise release, or any other suitable mechanism, of mosquitoes according to the present invention in an area where they are able to mate with the Aedes albopictus population to be modified. One of the biological properties of the Aedes albopictus population that may be modified by the introduction of mosquitoes according to the invention is the tendency of mosquitoes of the population to transmit a pathogen (which may optionally be achieved by enhancing resistance of the population of mosquitoes to the pathogen). Specifically, embodiments of the methods of the invention may be used to inhibit transmission of a pathogen by the Aedes albopictus population. Pathogens, the transmission of which is to be inhibited, or resistance to which is to be enhanced, include viral or nematode pathogens and may be selected from those considered elsewhere in the specification.

It is notable, the mosquitoes of the invention comprising wMel do not appear to suffer from reduced lifespan, and this represents a preferred embodiment of the invention. This provides an advantage in that the spread dynamics of Wolbachia are not adversely affected. Previous attempts to produce mosquitoes suitable for use as biological agents to reduce pathogen transmission have tended to yield short-lived insects. A mosquito comprising Wolbachia may readily be produced by infection of a suitable mosquito species using a desired Wolbachia strain. Examples of suitable techniques that may be used to infect mosquitoes with Wolbachia are described elsewhere in the specification, including the Experimental Results section.

In a fifth aspect the invention also provides a method of producing an Aedes albopictus mosquito comprising i i Mel, the method comprising treating a native population of Aedes albopictus to remove native Wolbachia bacteria; and infecting the population of treated mosquitoes with the i i Mel strain of Wolbachia. The native Wolbachia bacteria may suitably be selected from ι ι/AlbA and/or i i/AlbB.

In the context of the present disclosure a "native population" of mosquitoes (such as Aedes albopictus) may be a population present in an area where it is desired to influence biological activity of mosquitoes, for example by inhibiting transmission of pathogens by the mosquitoes. Use of a "native population" in this manner may be beneficial in that the mosquitoes may be expected to be well adapted to the environment found in the area where they are to be used. In the same context, "native bacteria" may be the bacteria, such as Wolbachia or other bacterial parasites, naturally occurring within the mosquito population.

In a suitable embodiment of the methods of invention, the treatment to remove native bacteria (such as Wolbachia) comprises antibiotic treatment of the native mosquitoes (such as Aedes albopictus). Suitably antibiotic treatment comprises injection of antibiotics, such as rifampicin, into mosquito embryos. More details of these methods are provided in the Materials and Methods section of the Experimental Results described below.

In order to further clarify the invention, and for the avoidance of doubt, certain terms used in the context of this disclosure will now be further defined. Mosquitoes

As referred to elsewhere, the present invention, in many of its' aspects and embodiments, relates to mosquitoes, methods of producing mosquitoes and also uses of such mosquitoes.

A number of the aspects of the invention specify that the mosquitoes in question are of the species Aedes albopictus, and this species represents a preferred form of mosquito in accordance with most aspects and embodiments of the invention. As described elsewhere this species is of major importance due to its role as a vector of pathogens harmful to humans and other animals, and the finding that resistance of this species to pathogens can be enhanced by the wMel strain of Wolbachia is both surprising and highly beneficial.

However, the inventor does not believe that the advantages noted are limited solely to this species of mosquito, and believe that, to a great extent, it is the wMel strain of Wolbachia that has most importance in mediating enhanced pathogen resistance, rather than the mosquito species infected by i i Mel. Accordingly, the invention provides aspects and embodiments that are not limited to this species. Certain aspects and embodiments of the invention are applicable to mosquitoes more generically, and a sixth aspect of the invention provides a mosquito comprising a Wolbachia bacterium of the strain i i Mel, wherein the mosquito has enhanced resistance to a pathogen. In a seventh aspect the invention provides a mosquito according to the sixth aspect of the invention for use as a biological agent for the reduction of pathogen transmission.

In an eighth aspect the invention provides a biological agent for the reduction of pathogen transmission comprising a mosquito in accordance with the sixth aspect of the invention.

A ninth aspect of the invention provides a method of producing a mosquito comprising a bacterium of interest, the method comprising treating a native population of mosquitoes to remove native bacteria; and crossing the population of treated mosquitoes with a line carrying the bacterium of interest.

In a tenth aspect the invention provides a method of modifying a population of mosquitoes, the method comprising introducing a mosquito comprising a bacterium of interest into the population, wherein the introduction of the mosquito comprising the bacterium of interest modifies one or more biological properties of the population of mosquitoes.

In the context of the preceding aspects, the "bacterium of interest" may be one (such as i i/Mel) that confers a desirable property to the mosquitoes, such as enhanced resistance to a pathogen. An eleventh aspect of the invention provides a mosquito comprising a bacterium of interest, wherein the bacterium of interest confers upon the mosquito enhanced resistance to a pathogen The resistance to pathogen(s) of a mosquito in accordance with this aspect of the invention, or other aspects of the invention, may be assessed with reference to resistance to the same pathogen(s) exhibited by a mosquito that lacks i i Mel (or another bacterium of interest). Within such generic aspects or embodiments particular embodiments may make use of species of mosquito such as Aedes polynesiensis. Suitable pathogens in the context of these preceding aspects of the invention may be the same as those described elsewhere in the specification, and include viral pathogens such as dengue virus and Chikungunya virus.

Except for where the context requires otherwise, any of the aspects of the invention mentioned above may make use of details of the various embodiments described in connection with aspects of the invention utilising Aedes albopictus and/or i i/Mel.

For the avoidance of doubt, a reference to "a mosquito comprising a bacterium" or "a mosquito infected with a bacterium" in the context of the present invention should, except for where the context requires otherwise, be taken as encompassing such a mosquito at any stage of its life cycle, and also as encompassing any isolated part or parts of such a mosquito. For example, a reference to a mosquito should, generally, be taken as encompassing a reference to an adult mosquito, but also to an egg, embryo, larval or juvenile form of such a mosquito. Any such reference should also be taken as encompassing isolated tissues, organs or cells of such a mosquito, so long as the parts in question contain the bacterium (which may be expected to be located in the cytoplasm). In particular, such references should also be taken as encompassing the reproductive material of such mosquitoes, since it is these tissues that mediate cytoplasmic incompatibility. While sperm of mosquito comprising a wMel bacterium will not in themselves comprise the bacterium, they will be characteristically modified as a result of the bacterium's presence in the mosquito. Except for where the context requires otherwise references to "a mosquito comprising a bacterium" or references to "a mosquito infected with a bacterium" in the various aspects and embodiments of the invention should also be taken as encompassing sperm comprising such modifications characteristic of wMel infection. i i Mel wMel is a strain of Wolbachia found in Drosophila melanogaster. The genome of wMel has been sequenced, and is described more fully in an article entitled "Phylogenomics of the Reproductive Parasite Wolbachia pipientis i i Mel: A Streamlined Genome Overrun by Mobile Genetic Elements" by Wu, et al. published in PLoS Biology (March 2004, Volume 2, Issue 3). The content of this document, in particular those aspects allowing the identification, characterisation and replication of the i i Mel strain of Wolbachia, are herein incorporated by reference. The authors of this earlier publication submitted the complete sequence for i i/Mel, and this was given the GenBank accession ID no. AE017196 (current version "AE017196.1 Gl:42410857").

As mentioned elsewhere in the specification, without wishing to be bound by any hypothesis the inventor believes that it is wMel, rather than the mosquito species that comprises wMel, that plays the major role in enhancing resistance to pathogens. Pathogens

The invention considers pathogens in a number of contexts. For example, mosquitoes in accordance with the present invention exhibit enhanced resistance to pathogens, and may be used as biological agents for the reduction of pathogen transmission. Except for where the context requires otherwise, the following considerations will be applicable to any reference to a pathogen, or pathogens, in relation to any aspect or embodiment of the invention.

In a suitable embodiment the pathogen may be a viral pathogen. Suitable examples of such viral pathogens include, but are not limited to, those selected from the group consisting of: dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; and yellow fever virus.

Alternatively or additionally, the mosquitoes of the present invention may have enhanced resistance to a nematode pathogen. Suitable examples of such a nematode pathogen include Dirofilaria immitis (which gives rise to damaging infections in hosts such as dogs or humans), and Wuchereria bancrofti (which can cause elephantitis in infected individuals). In specific embodiments an Aedes albopictus mosquito in accordance with the invention may have enhanced resistance to Dirofilaria immitis, while an Aedes polynesiensis mosquito in accordance with the invention may have enhanced resistance to Wuchereria bancrofti.

References to enhance resistance to a single pathogen or class of pathogens should generally be taken as encompassing the same activity in respect of one or more pathogens, or indeed in respect of a range of pathogens. For example, a mosquito in accordance with the present invention may have enhanced resistance to two or more viral pathogens, and additionally, or alternatively, may have enhanced resistance to two or more nematode pathogens. Similarly, a mosquito of the invention may be able to reduce transmission of two or more viral pathogens, and additionally, or alternatively, may be able to reduce transmission of two or more nematode pathogens. Cytoplasmic incompatibility

Wolbachia pipientis is a maternally inherited intracellular bacterial symbiont of invertebrates that is common in insects. It can manipulate insect reproduction by inducing sterility when individuals of different Wolbachia carrier status mate with each other, known as cytoplasmic incompatibility or CI. The basic form of CI is observed when females that do not carry Wolbachia when they mate with males that do, leading to early embryonic death of the offspring. Although it is not itself transmitted through sperm, Wolbachia modifies these sperm of carrier males so that they are unable to complete the process of fertilization. However, there is a 'rescue' effect when Wolbachia is present in the female and thus in the egg, allowing the offspring of lVo/£>ac/7/^'a-carrying females to develop normally. Thus in a mixed population of individuals with and without Wolbachia, females that do carry the bacterium will produce a greater mean number of offspring than those which do not, because they can mate successfully with all males in that population. Once a threshold population frequency of Wolbachia has been exceeded, which depends on the maternal transmission efficiency, level of CI, and any fitness costs, the reproductive advantage of harbouring Wolbachia will increase with each generation, and therefore the frequency of the bacterium will rise with increasing rapidity. Wolbachia is able to spread spatially using this mechanism of unidirectional CI. A second form known as bidirectional CI is only seen in crosses between individuals that both carry Wolbachia, but of different strains. In this case both directions of the cross are incompatible, and produce no hatching eggs. If populations carrying such mutually incompatible strains came into contact in nature, an unstable equilibrium would result, and whichever formed the local majority would be expected to reach 100% frequency while the other strain would be eliminated, because its females would more frequently encounter and mate with males with which they are compatible (see fig. 2). Bidirectional CI therefore provides a method to stably introduce Wolbachia into target populations, and once fixed it would be stable to further immigration of individuals carrying the strain with which they are incompatible. Although it would locally go to 100% frequency the introduced strain would not be expected to spread spatially, in contrast to unidirectional CI. Thus bidirectional CI gives spatial control over the process of population replacement with an introduced strain, which in some circumstances would be a very useful feature. If bidirectionally incompatible Wolbachia strains are combined to form strain superinfections, superinfected males will be incompatible with females that lack one or more of the Wolbachia strains in the male, due to the absence of the rescue factor in the egg for the strain it lacks. Because the reciprocal cross is fully compatible, these superinfections are expected to spread through populations that lack one of the strains, exactly in the same way as Wolbachia spreads through a population that does not carry the bacterium at all. The i i Mel strain occurs naturally in the fruit fly Drosophila melanogaster and its genome sequence has been published (Wu et al. 2003). The i i/MelPop strain is also naturally found in Drosophila melanogaster, and i i/Mel and i i MelPop are phylogenetically quite closely related (Sun et at. 2003). They were thought to produce the same crossing type, since lines carrying these two Wolbachia strains were compatible with each other when crossed in Drosophila (McGraw et at. 2002). The i i MelPop strain did not produce CI when introduced into Ae. albopictus and crossed with a wildtype line superinfected with the naturally occurring Wolbachia in this species, ι ι/AlbA and ι ι AlbB (Suh et at. 2009). Therefore, our finding that i i Mel-transinfected Ae. albopictus produce complete bidirectional CI when crossed with a wildtype i i/AlbA-i i AlbB-infected line was unexpected. This is an important result because it allows the introduction of Wolbachia into target populations, in a spatially controlled fashion. It also allows the future creation of a line containing with all three strains that should be able to spread itself spatially through natural populations of Ae. albopictus due to unidirectional CI. The invention will now be further described with reference to the following Experimental Results section, and the accompanying Figures and Table in which:

Figure 1 illustrates Uju.i i Mel crossing type. Experiments to characterize the crossing type of the Uju. i i/Mel line at G₆ were performed using UjuT and Ascoli strains. Error bars represent the standard error of the mean of hatch rates between females; fifteen adult males and fifteen females were used for each mass cross. A second round of crossing experiments was performed two generations later (G₈) using Uju.i i/Mel x Uju.wMel, Ascoli x Uju. i i Mel, Uju. i i/Mel x Ascoli and Uju.i i Mel x UjuT according to the same procedure as the previous experiment. No statistically significant difference was found between G₆ and G₈ of the same cross using unpaired Mests. Number of eggs counted for each generation is shown under the X-axis. Figure 2 illustrates transmission capacity (A) and titer of dengue virus in mosquito saliva (B). Three Ae. albopictus strains (Uju.wAlbA/wAIbB, UjuT, and Uju.wMel) were orally infected with dengue 2 virus using glass feeders covered with a chicken skin membrane containing the infectious blood-meal at a titer of 10⁷ FFU/mL. After blood-feeding, mosquitoes were transferred in cardboard containers and maintained in BSL3 insectaries at 28°C. After 14 days, surviving mosquitoes were tested for the presence of viral particles in saliva collected using the forced salivation technique. The number of fluorescent foci in the saliva of each mosquito was estimated on C6/36 Aedes albopictus cell culture. The transmission capacity representing the percentage of mosquitoes with infectious saliva among tested mosquitoes was also calculated. Number of fed and assayed females: Uju.wAlbA/wAIbB = 44; UjuT = 36; Uju.wMel = 44.

Figure 3 illustrates immune gene expression in Wolbachia infected and uninfected mosquitoes.

Fig. 3A: RNA was extracted from adult females of Uju.wMel, UjuT and Ascoli at 11 days post-eclosion. The expression of four Ae. albopictus orthologs for Ae. aegypti immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, PGRPS1; cecropin D, CECD; CLIP-domain serine protease, CLIPB37; and a thioester-containing protein, TEP20. Expression was normalized to the UjuT adult females. Error bars show the SEM of three biological replicates, each containing four adult females (total of 12 mosquitoes per condition). ^* = P < 0.05 compared to control using Wilcoxon tests.

Fig. 3B: Adult females were transiently infected with Wolbachia using intrathoracic injections with either i i MelPop or ι ι AlbB, or controls of either heat killed E. coli, or the buffer alone, approximately three days post-eclosion. RNA was extracted five days after injection. The expression of four Ae. aegypti immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, PGRPS1; cecropin D, CECD; CLIP-domain serine protease, CLIPB37; and a thioester-containing protein, TEP20. Orthologs for these genes in Ae. albopictus were also analyzed by qRT-PCR. Expression was normalized to non- injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing five adult females (total of 15 mosquitoes per condition). ^* = P < 0.05 compared to control using Wilcoxon tests.

Figure 5 (from Sinkins and Gould 2005) Unidirectional CI between insects carrying Wolbachia (red) and not carrying Wolbachia (green). By providing a frequency-dependent reproductive advantage to carrier females, which unlike their counterparts without Wolbachia can mate successfully with any male in this mixed population, Wolbachia can rapidly increase in frequency. Figure 6 Bidirectional CI can only occur between insects carrying different Wolbachia strains; these strains cannot stably co-exist within a population, and whichever strain is in the majority will replace the other.

Figure 7. When mutually incompatible strains are combined in a superinfection within individual insects, CI is produced when superinfected males are crossed with female insects carrying only one of the two strains. The result is unidirectional CI. The same pattern is seen when males carrying three mutually incompatible strains mate with females carrying only two of these strains.

Experimental results

Introduction

All known wild populations of Ae. albopictus are naturally infected with two strains of the maternally inherited bacterium Wolbachia pipientis, called ι ι/AlbA and ι ι/AlbB; Ae. aegypti \s naturally uninfected with the bacterium. Recent work has shown that when an over- replicating strain of Wolbachia from Drosophila melanogaster called i i MelPop was transferred into Ae. aegypti, the transmission of dengue was blocked, and inhibition of chikungunya virus dissemination was also observed. In addition, when the ι ι/AlbB strain from Ae. albopictus was transferred into Ae. aegypti, reduced dengue susceptibility was observed. Both Wolbachia strains also induced cytoplasmic incompatibility (CI) in Ae. aegypti, whereby uninfected females mated with infected males produce embryos which die shortly after fertilization. This mechanism is used by Wolbachia to spread through insect populations because, in contrast, infected females can mate successfully with either infected or uninfected males, giving them a frequency-dependent reproductive advantage. Thus the combination of viral inhibition with a built-in self-spreading mechanism provides attractive prospects for the control of dengue transmission by Ae. aegypti.

In addition to life shortening, the i i/MelPop strain also causes chronic immune upregulation in Ae. aegypti. The Toll pathway, some components of which are upregulated in Ae. aegypti in the presence of i i MelPop, has previously been shown to play a role in the control of dengue dissemination in Ae. aegypti. A general role of immune upregulation in pathogen inhibition is also supported by the knockdown of the major immune gene TEP1 which partly rescued the inhibitory effect of the presence of i i/MelPop on Plasmodium berghei development in transiently infected Anopheles gambiae. The fact that the i i/AIbB transinfection caused dengue inhibition in Ae. aegypti, while the original host of this Wolbachia strain Ae. albopictus is a fairly efficient dengue vector, suggested that there is a significant contribution of host background to the dengue inhibition phenotype, and this could be mediated by the increased immune response to Wolbachia found in a novel insect host. Therefore, it is unknown whether any Wolbachia strain could produce strong dengue inhibition in a naturally Wolbachia- infected mosquito such as Ae. albopictus, which would be expected to have acquired a degree of immune tolerance to Wolbachia over time.

The i i/MelPop strain over-replicates and can approximately halve the lifespan of both its D. melanogaster and Ae. aegypti hosts. However, a i i/MelPop transinfection into Ae. albopictus also produced a greatly reduced egg hatch from intra-strain matings, and this appeared to preclude its application to disease control in Ae. albopictus. The i i Mel strain, which is phylogenetically close to the i i MelPop variant, does not produce the life- shortening phenotype of the latter in its native Drosophila melanogaster host. However, i i/Mel can significantly delay the accumulation of RNA viruses such as Drosophila C virus in D. melanogaster. Therefore i i/Mel was selected for experimental transfer into Ae. albopictus, in order to examine whether this strain is capable of producing dengue inhibition and CI in this new host background.

Materials and Methods Mosquito Strains

The Wolbachia uninfected Ae. albopictus strain UjuT was generated by tetracycline treatment by Dr Yasushi Otsuka, Oita Medical University, Japan; the Ascoli strain of Ae. albopictus was colonized from San Benedetto del Tronto, Italy in 2006 by G. Favia and colleagues; and the Ae. aegypti Rockefeller strain originated in the Caribbean in the 1930's. All colonies and lines were kept at 27°C and 70% relative humidity, and a 12 hour light/dark cycle.

The i i/AlbA and ι ι/AlbB strains of Wolbachia were introgressed into the UjuT background for four generations by removing all male pupae from one colony of the Ascoli strain and providing an approximately equal number of UjuT males. The resulting line was thus approximately 94% UjuT nuclear background, and contained both ι ι AlbA and ι ι/AlbB. This line was only used for dengue infection, in order to partly control for any effects of host background.

Embryo Microinjection and line establishment

The i i Mel strain of Wolbachia was transferred from Drosophila melanogaster i i/¹¹¹⁸ embryos into Ae. albopictus (UjuT) by the transfer of cytoplasm. Adult Drosophila were encouraged to oviposit using apple-juice agar plates and yeast paste. Eggs were collected approximately 30mins post-oviposition. Ae. albopictus were encouraged to lay eggs by placing around fifteen females, blood-fed seven days earlier, into a small (3cm diameter, 10cm height) plastic vial with moist filter paper on the bottom. Eggs were collected approximately 30mins post-oviposition. Ae. albopictus eggs were allowed to desiccate for 15-30mins. Both donor and recipient eggs were aligned on a nitrocellulose membrane and transferred to a glass slide using double sided tape. The eggs were then covered with Voltalef oil ready for injection. Cytoplasm was aspirated from the posterior of the donor eggs using a FemtoJet microinjector (Eppendorf) and injected into the posterior of recipient eggs. After a short incubation time, eggs were transferred onto wet filter paper, stored at 100% humidity at 27⁵C for five days, and then hatched in deoxygenated water. G₀ larvae were reared using standard conditions. Females were separated 1 - 2 days after blood feeding into small plastic vials with moist filter paper on the bottom. Once females had laid eggs they were PCR assayed for the presence of Wolbachia using universal primers 81 F and 691 R (3) (Table 1 ). Following initial optimization trials, the experiment from which the line was established involved microinjection of around 100 embryos. During the period of establishment of the transinfected line, only eggs from PCR-positive females were hatched. After G6, batches were also selected for high egg hatch. Eggs from individual lVo/£>ac/7/^'a-positive females were counted, hatched (with deoxygenated water in the small plastic vials), and then 2^nd instar larvae were counted. Approximately three quarters of the broods with the highest hatch rates were pooled to form the next generation. qRT-PCR and qPCR

Gene transcription levels were tested using quantitative reverse transcription PCR (qRT- PCR). RNA was extracted from adult mosquitoes using Trizol® reagent. cDNA was generated from 1 μg of this RNA using Superscript® Vilo™ (Invitrogen). cDNA was diluted to one in 20 dilutions. The dsDNA dye SYBR® Green (Invitrogen) was used for amplicon detection in a DNA Engine thermocycler (MJ Research) with Chromo4 real-time PCR detection system (Bio-Rad). The following cycling conditions were used: 95 °C for 15 minutes, then 45 cycles of 95 °C for 10s, 59 °C for 10s, 72 °C for 20s, with fluorescence acquisition at the end of each cycle and a melting curve analysis. Quantitative PCR (qPCR) was used to determine Wolbachia copy number. DNA was extracted from adult mosquitoes using the Livak method. DNA was diluted to 100 ng/μί using a Nanodrop spectrophotometer. Thermocycler conditions and reaction chemistry followed the same protocol as for qRT-PCR. Primer pairs used for qPCR and qRT-PCR are listed in table 1 . Ae. albopictus primers were designed using sequence data generated using either degenerate primers based on Ae. aegypti sequence data (from Vectorbase) or from Ae. albopictus EST data from NCBI.

CI Crosses Crossing experiments designed to characterize the crossing type of i i/Mel were performed using UjuT, Ascoli, and Uju.i i Mel lines. All individuals were sexed as pupae. Adults were blood fed at 6 days old, and the females separated into plastic vials for individual laying. Eggs were dried and allowed to mature at 27°C and -70% RH for 5 days, counted and hatched in deoxygenated water containing algae and yeast. Larvae were fed with dried liver powder. Second instar larvae from each female were counted to give hatch rates. Females with no egg hatch were dissected to check for successful mating; egg hatch rates from females who were unmated were disregarded. Adults were given a constant supply of water and sucrose. Dengue infection

One-week-old Uju.i i Mel, UjuT and Uju.i i/AlbA/i i AIbB females were deprived of sucrose solution 24h prior exposure to the infectious blood-meal containing 10⁷ FFU (Foci Fluorescent Units) /ml_ of virus. The dengue serotype 2 virus strain used was isolated in 1974 from a human sera from Bangkok (Thailand). The artificial blood-meal provided in glass feeders covered with a chicken skin membrane and maintained at 37 ^<C, consisted of a virus suspension (1 /3 vol/vol), washed rabbit erythrocytes (2/3 vol/vol), and 5 mM ATP as a phagostimulant. Engorged mosquitoes were transferred in cardboard containers, provided with sucrose solution and maintained in BSL-3 insectaries at 28 °C for 14 days. Saliva was collected using the forced salivation technique, which consists of inserting a capillary tube containing fetal calf serum in the proboscis of females whose legs and wings had been removed. After 45 min, saliva was collected and titrated by focus fluorescent assay on C6/36 Aedes albopictus cell culture. The transmission capacity was estimated as the percentage of mosquitoes with infectious saliva among tested mosquitoes.

Wolbachia purification and intrathoracic inoculation

Wolbachia were maintained in the Ae. albopictus cell line Aa23. Cells were grown in 75- cm² culture flasks to around 50% confluence in Schneider's media containing 10% fetal bovine serum (FBS), 140 units of penicillin per ml_ and 140μg of streptomycin per ml_. Every 3-5 days cells were passaged. Wolbachia was extracted from cells and purified three days after the previous passage. The Wolbachia pellet was re-suspended in Schneider's media with 10% FBS (without antibiotics) to an optical density of OD = 0.06 at 400nm wavelength. For E. coli, controls an OD of 0.01 at 400nm was used. 69 nl_ of Wolbachia suspension (or 69 nl_ Schneider's/ E. coli suspension for the controls) was microinjected into the thorax of around three day old Ae. aegypti Rockefeller / Ae. albopictus UjuT strains using a Nanoject microinjector (Drummond). The mosquitoes were supplied with water and sucrose and left for five days prior to qRT-PCR experiments.

Creation of Wolbachia-cured lines One hour old embryos of Ae. albopictus were microinjected with100μg/mL of rifampicin. Eggs were then hatched, reared under standard conditions and adults provided with rifampicin at 30 μg mL in 10% sucrose before bloodfeedng and collection of eggs. This method provides a rapid means to create lVo/£>ac 7/^'a-cured lines, with only one generation of treatment required, in contrast to existing methods of curing reported in the literature. Males from the cured line are then mated with females carrying the i i/Mel strain, the female offspring of this cross again mated with the lVo/£>ac/7/^'a-uninfected males, and so on for several generations, in order to produce a line that carries i i Mel but has the genetic background and characteristics of the particular Ae. albopictus population to be targeted. Results

Generation of the Uju.wMel line and crossing experiments

A stable infection of i i Mel in a previously tetracycline cured Ae. albopictus strain (UjuT) was generated. Cytoplasm from D. melanogaster i i/¹¹¹⁸ was injected into UjuT, resulting in four Go females one of which was positive and produced sufficient progeny to establish an isofemale line. This line was backcrossed with UjuT males each generation in order to minimize bottlenecking and was selected for maximal maternal inheritance; the proportions that were confirmed positive for Wolbachia using PCR were 70% (n=10), 81 % (n=16), 89% (n=9) and 100% (n=10) for d to G₄ respectively. The infection has remained at 100% since G₄ (n=152, currently at G₉). The Wolbachia strain present was confirmed to be i i Mel by sequencing the wsp ( Wolbachia surface protein) gene, which showed 100% identity with published i i Mel wsp sequence.

The transinfected strain initially showed reduced hatch rates compared to wild-type females, 46.1 ±7.8% hatch for Uju.i i Mel in G₆ compared to 65.1 ±10.7% for UjuT. Following two successive generations of selection for high hatch rates, the Uju.i i/Mel x Uju.i i Mel hatch rate had risen to 56.4±9.4% by G₈ (figure 1 ). A similar effect has previously been observed in newly transinfected Drosophila and Ae. aegypti. The effects of i i/Mel on fecundity of Ae. albopictus will be characterized in detail at a later stage; however, preliminary data suggest that i i/Mel has no major negative effect on fecundity (average number of eggs laid per female = 60.3±7.0 (11 females) and 61 .2±5.0 (13 females) for Uju. i i Mel and UjuT respectively, p=0.9024, unpaired Hest).

Crossing experiments were performed to examine whether the i i/Mel strain was able to produce CI in this novel background, by crossing the Uju.i i Mel line in both directions with the uninfected UjuT and wild-type Ascoli strain (infected with Wolbachia strains ι ι/AlbA and i i/AIbB) (figure 1 ). As expected, UjuT males were compatible with all females. Males of the Uju. i i Mel line produced strong CI when mated to UjuT females (0.26% hatch). The Uju. i i Mel and Ascoli lines showed complete bidirectional incompatibility, with 0% hatch when females of either strain were mated with males of the other. Dengue infection

The transinfected Uju.wMel strain was challenged with dengue 2 virus provided in an artificial blood-meal and after fourteen days showed a complete inhibition of dengue transmission capacity, with no infectious viral particles detected in the saliva of any tested mosquito. In contrast, the superinfected Uju.wAlbA/wAIbB strain (generated by the introgression of Wolbachia from Ascoli into UjuT, in order to minimize any effect of host genetic background on DENV), and the uninfected UjuT strain were both able to transmit dengue 2 virus at day 14 post-infection (infectious rate = 27.3% and 8.3% for the Uju.wAlbA/wAIbB and the UjuT strains respectively; figure 2). Mosquito saliva contained numbers of viral particles that are in the range expected for Ae. albopictus infected with dengue virus (figure 2); average number of viral particles per saliva = 67±139 and 29±29 for the Uju.wAlbA/wAIbB and the UjuT strains respectively.

Immune gene expression in Uju.wMel

Given that the i i MelPop strain transinfection in Ae. aegypti has been shown to produce upregulation of a number of immune genes, and this could be responsible for or contribute to the viral inhibition phenotypes, the effects of the i i/Mel transinfection on transcription levels was investigated for four Aedes albopictus immune genes, selected to represent a range of immune gene categories including important antimicrobial effectors (a cecropin, a PGRP and a TEP), and also on the basis that their orthologs were previously shown to be upregulated in the presence of i i/MelPop. The transcription of immune genes was measured by qRT-PCR using G₅ Uju.wMel, and UjuT and Ascoli females 11 days post eclosion (Figure 3a). There was no significant difference in immune gene transcription between the cured UjuT strain and the i i AlbA/i i AIbB superinfected Ascoli strain. However, significant immune upregulation was observed in Uju.wMel when compared to these other two strains.

In order to compare the contribution of Wolbachia strain type with host species background, transient somatic infections of i i MelPop and ι ι/AlbB were also created in Ae. aegypti and Ae. albopictus using intrathoracic inoculation as described in and. Adult females were injected with suspensions of Wolbachia purified from Ae. albopictus cell lines (Aa23) approximately three days after eclosion. The transcription of immune genes was measured by qRT-PCR at five days post injection (figure 3b). Strong immune upregulation was observed in Ae. aegypti with both wMelPop and ι ι AlbB strains when compared to non-injected, buffer-injected and heat killed E. co//-injected controls. However, no significant immune upregulation was observed in Ae. albopictus injected with either Wolbachia strain. Concentration of Wolbachia in Uju.vjMel

The concentration of i i Mel in Uju.wMel was compared to the combined concentration of both i i/AlbA and ι ι/AlbB in the Ascoli strain using adults 11 days post eclosion. The ratio between Wolbachia WSP DNA and host S17 DNA was used to estimate the concentration of Wolbachia. The concentration of i i/Mel was found to be approximately seven times that of the total concentration of Wolbachia in the superinfected Ascoli strain (figure 4). Novel method for removing Wolbachia infections in Ae. albopictus

The microinjection of 144 Ae. albopictus embryos of the Ascoli strain with rifampicin at 100 μg/ml produced 11 sdult females which laid eggs, and none of these females or their offspring carried Wolbachia based on PCR assays. This methodology provides a marked improvement on previously reported methods of Wolbachia curing based on larval and adult treatment of insects with antibiotics (which have not been successful in curing Wolbachia from Ascoli Ae. albopictus despite several generations of treatment). Crossing the lVo/£>ac/7/^'a-cured males with i i/Mel-carrying females for six or more generations allows the introduction of the i i Mel Wolbachia into the Ascoli (Italy) genetic background, or any other genetic background of a target wild Ae. albopictus population, in order to improve the local competitiveness of the insects to be released.

Discussion Our results show that i i/Mel infection can block DENV transmission in the increasingly widespread vector species Ae. albopictus. RNA-viral inhibition by i i Mel has been previously demonstrated in Drosophila, and we have shown that this viral interference is also produced when it is transferred into Ae. albopictus. The inhibition appears to be limited to specific strains of Wolbachia, since Ae. albopictus is naturally infected with two strains of Wolbachia, ι ι AlbA and ι ι/AlbB, which themselves seem to have no inhibitory effect on the virus. This is the first time Wolbachia mediated DENV inhibition has been demonstrated in Ae. albopictus, and this result has significant implications for dengue control. Uju. i i Mel produces complete bidirectional CI with the wild-type Ascoli line (containing a i i/AlbA and ι ι AlbB superinfection, as do all known wild populations of this species). The i i/Mel crossing type unexpectedly differs from the crossing type of a i i/MelPop transinfection into Ae. albopictus, given the phylogenetic similarity between i i Mel and i i MelPop. Bidirectional CI provides a method to stably introduce Wolbachia into populations, since bidirectionally incompatible crossing types cannot stably co-exist; whichever strain is at a local majority would be at a reproductive advantage, because its females would more frequently encounter and mate with males with which they are compatible. Assuming the complete bidirectional CI will also be produced under field conditions, once the i i Mel infection reaches a population majority, it would then be expected to go to local fixation and be stable to further immigration of ι ι/AlbA / i i AlbB- infected (wildtype) individuals with which they are incompatible. Large scale female releases would not be essential for this strategy to be successful: since Aedes pupae are readily separated by sex, heavily male-biased releases could be made which would suppress the female population size at the same time. Since most populations are seasonal, appropriately timed releases at the start of the rainy season could achieve local replacement with i i Mel. Future experiments will also be conducted to examine whether the creation of a stable i i/Mel / i i AlbA / ι ι/AlbB triple infection is possible. Based on previous work we expect the crossing type of this triple infection to produce unidirectional incompatibility with the parental lines, and thus have the potential to spread i i Mel more efficiently through field populations. In any event at the initial field trial stage it would be preferable to use the bidirectionally incompatible line, in order to have better control over the geographical extent to which population replacement occurs.

There were seven times more Wolbachia in Uju.i i Mel compared to the superinfected (ι ι AlbA and ι ι/AlbB) Ascoli strain. This result is quite surprising given the relatively high concentrations found in the natural superinfection. High levels of Wolbachia are expected to be found in recently transinfected species and may decrease over time as co-adaptation of strain and host occur. It is possible that the increased density of Wolbachia had a negative effect on egg hatch rates from intra-strain matings in the early generations. The immune gene upregulation caused by i i Mel in the stably infected Uju.i i/Mel line (Figure 3a) was statistically significant but nevertheless on a much lower scale than that observed in a stably i i/MelPop-infected line of Ae. aegypti or transiently i i MelPop- transinfected Anopheles gambiae. Immune upregulation was observed when Ae. aegypti was transiently infected with ι ι/AlbB (Figure 3b), and there was no significant difference between the immune upregulation caused by i i/MelPop and ι ι AlbB in Ae. aegypti, demonstrating that this effect is not limited to the over-replicating i i/MelPop strain. However, no significant immune upregulation was observed in Ae. albopictus when transiently infected with ι ι/AlbB, one of its natural Wolbachia, or with i i MelPop. Taken together, along with our previous findings, these data suggest that whether or not the host species is naturally infected with Wolbachia is the most important factor in the level of immune response, rather than the Wolbachia strain used.

The fact that Ae. aegypti stably transinfected with ι ι/AlbB showed reduced transmission of dengue suggested a host component to the viral inhibition in that transinfection, possibly immune-related. However, the relatively modest immune upregulation observed in the transinfected Ae. albopictus Uju.wMel line, together with the fact that no dengue virus at all was observed in their saliva following challenge, suggests that priming of the host immune system may not be the most important factor in this case of viral inhibition. The Wolbachia strain used seems to be the critical consideration here. Mechanisms for direct viral inhibition by Wolbachia that could be operating include the production of reactive oxygen species by the bacterium or resource competition, for example for cholesterol (5).

Future research using later generations of the line will more thoroughly determine whether i i/Mel has any fitness or fecundity effects on Ae. albopictus similar to those demonstrated by the i i/AlbA / ι ι AlbB superinfection; however, our results so far have not shown any major effect on fecundity, unlike the significant fecundity reduction previously been observed in the i i/Pip infection of Ae. albopictus. Furthermore, the hatch rate is much higher in our i i/Mel transinfected line than that previously observed for a i i MelPop strain transinfection in Ae. albopictus, which averaged in the 10-20% range. This study has yielded an Ae. albopictus line that may provide the basis of a viable new option for dengue control in this species. It also demonstrates that both of the two main vectors of dengue globally are amenable to a lVo/£>ac/7/^'a-based strategy - improving the prospects for elimination of the disease.

Claims

1 . A mosquito of the species Aedes albopictus comprising a Wolbachia bacterium of the strain i i Mel, wherein the mosquito has enhanced resistance to a pathogen.

2. A mosquito according to claim 1 , wherein the pathogen is a viral pathogen.

3. A mosquito according to claim 2, wherein the viral pathogen is selected from the group consisting of: dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; and yellow fever virus.

4. A mosquito according to claim 1 , wherein the pathogen is a nematode pathogen.

5. A mosquito according to claim 4, wherein the nematode pathogen is selected from the group consisting of: Dirofilaria immitis; and Wuchereria bancrofti.

6. A mosquito according to any preceding claim, wherein the bacterium induces cytoplasmic incompatibility.

7. A mosquito according to claim 6, wherein the bacterium induces bidirectional cytoplasmic incompatibility.

8. A mosquito according to any preceding claim for use as a biological agent for the reduction of pathogen transmission.

9. A mosquito according to claim 8, wherein the pathogen, transmission of which is to be reduced is selected from the group consisting of: a viral pathogen (such as dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; or yellow fever virus); and a nematode pathogen (such as Dirofilaria immitis; or Wuchereria bancrofti).

10. A biological agent for the reduction of pathogen transmission comprising a mosquito according to any one of claims 1 to 9.

1 1 . A method of producing an Aedes albopictus mosquito comprising i i Mel, the method comprising treating a native population of Aedes albopictus to remove native Wolbachia bacteria; and crossing the population of treated mosquitoes with a line carrying the i i Mel strain of Wolbachia.

12. A method according to claim 1 1 , wherein the native Wolbachia bacteria is selected from the group consisting of: ι ι/AlbA; and i i AlbB.

13. A method according to claim 1 1 or claim 12, wherein the treatment to remove native Wolbachia bacteria comprises antibiotic treatment of Aedes albopictus.

14. A method according to claim 13, wherein the antibiotic treatment comprises injection of antibiotics into Aedes albopictus embryos.

15. A method according to claim 14, wherein the antibiotic injected comprises rifampicin.

16. A method of modifying a population of Aedes albopictus mosquitoes, the method comprising introducing an Aedes albopictus mosquito according to any of claims 1 to 4 into the population, wherein the introduction of the Aedes albopictus comprising the i i Mel strain of Wolbachia modifies one or more biological properties of the Aedes albopictus population.

17. A method according to claim 16, wherein the method inhibits transmission of a pathogen by the Aedes albopictus population.

18. A method according to claim 17, the wherein the pathogen, transmission of which is to be inhibited is selected from the group consisting of: a viral pathogen (such as dengue virus; Chikungunya virus; West Nile virus; St. Louis encephalitis virus; Rift Valley fever virus; or yellow fever virus); and a nematode pathogen (such as Dirofilaria immitis; or Wuchereria bancrofti).