ENTOMOLOGIA HELLENICA 18 (2009): 3-16

Wolbachia-induced reproductive parasitism and applications

A. SARIDAKI AND K. BOURTZIS*

Department of Environmental and Natural Resources Management, University of Ioannina, 2 Seferi st., 30100 Agrinio, Greece

ABSTRACT

Insects have been reported to be associated with a broad variety of microorganisms, affecting the host biology in many different ways. Among them, , an obligatory intracellular and maternally-inherited symbiont, has recently attracted a lot of attention. Beside insects, Wolbachia are found in association with a wide variety of other invertebrate , including mites, scorpions, spiders, , filarial nematodes. Several surveys have indicated that Wolbachia may be symbiont of up to 70% of all insect species, rendering Wolbachia the most ubiquitous intracellular symbiotic organism on Earth. Wolbachia-host interactions range from many forms of reproductive parasitism to mutualistic symbioses. Different Wolbachia strains have been found to induce a number of reproductive alterations such as feminization, parthenogenesis, male-killing or cytoplasmic incompatibility. Despite their common occurrence and major effects on host biology, speciation and ecological diversity, little is known on the molecular mechanisms that mediate Wolbachia-host interactions. Recent studies focus on the potential of Wolbachia-based methods for the biological control of insect pests and disease vectors of agricultural, environmental and medical importance.

KEYWORDS: insect symbiosis, Wolbachia, parthenogenesis, feminization, male-killing, cytoplasmic incompatibility, incompatible insect technique.

Introduction on Insect Symbiosis the symbiont being vertically transmitted among generations. Intracellular symbionts Several types of insect-microbe associations of insects are divided into two groups. The are present in nature, many of which are first one, the primary symbionts, covers accountable to a more or lesser extent for symbiotic microbes that usually supply the evolutionary success of insects. hosts with nutrients and are harboured by Microbes are ubiquitous both inside and the host bacteriocyte, a special cell outside the insect bodies, inside or outside differentiated for this purpose (Buchner the insect cells and interact with their host 1965, Ishikawa 1989). Secondary in a broad variety of relationships that range symbionts, also known as “guest microbes”, from mutualism, which is beneficial to the are not restricted to a particular cell type, host, to parasitism, where the symbiont has but are present in many cell types of the a negative impact on host’s biology host insect. Unlike primary symbionts, guest (Ishikawa 2003). The most intimate microbes can colonize naive hosts through association is intracellular symbiosis, with horizontal transmission among host individuals and species. These associations

*Corresponding author, e-mail: [email protected] 4 ENTOMOLOGIA HELLENICA 18 (2009): 3-16 are typically facultative from the Windsor 2000, Hilgenboecker et al. 2008, perspective of the host and can be Werren et al. 2008). deleterious to the host (parasitism), Molecular phylogenetic analysis based beneficial only to the symbiont on the 16S rRNA gene indicated that (commensalism) or beneficial to both Wolbachia belongs to α-Proteobacteria, parties (mutualism). During the last three being evolutionary related to other decades, a novel type of symbiosis has been intracellular bacterial species of the genera described for bacteria like Wolbachia, Anaplasma, Ehrlichia and Rickettsia Cardinium, Rickettsia, Spiroplasma and (Breeuwer et al. 1992, O’Neill et al. 1992, Arsenophonus, that manipulate the host Rousset et al. 1992). A significant amount reproduction system to their advantage of Wolbachia genomic information is (reproductive parasitism). Wolbachia, the available since the genome of four best-studied symbiont of this group, will be Wolbachia strains (wMel, wRi, wPip and the focus of this review. wBm) has been completed (Wu et al. 2004, Foster et al. 2005, Klasson et al. 2008, Introduction on Wolbachia Klasson et al. 2009). The available genomic information allowed the development of two Collaborative studies between Marshall multi locus sequencing typing (MLST) Hertig, an entomologist, and Samuel systems which can be used for the Wolbach, a pathologist, on the presence and genotyping of any given Wolbachia strain identification of microorganisms in (Baldo et al. 2006, Paraskevopoulos et al. , resulted in the discovery of 2006); they have also facilitated the Wolbachia in the gonads of the Culex classification of the Wolbachia strains into pipientis mosquito in 1924 (Hertig and 10 major phylogenetic clades which have Wolbach 1924); however, the complete been named ‘supergroups’ (Werren et al. description of this symbiotic association 1995, Bandi et al. 1998, Zhou et al. 1998, was published in 1936 (Hertig 1936). For Lo et al. 2002, 2007, Rowley et al. 2004, decades, Wolbachia was known only from Bordenstein and Rosengaus 2005, Ros et al. mosquitoes; the development of PCR-based 2008, Bordenstein et al. 2009). screening methods clearly indicated that Several studies have shown that Wolbachia is widespread in nature (O’Neill Wolbachia is mainly localized in the et al. 1992). It has been demonstrated that reproductive tissues of arthropods and is Wolbachia infects up to 70% of insect responsible for the induction of a number of species, a large number of other arthropods reproductive alterations including (including spiders, scorpions, mites, feminization, thelytokous parthenogenesis, springtails, terrestrial isopods) as well as male-killing and cytoplasmic filarial nematodes (Werren et al. 1995, incompatibility (CI) (Werren 1997, Bourtzis Bandi et al. 1998, Jayaprakash and Hoy and O’Neill 1998, Bourtzis and Braig 1999, 2000, Werren and Windsor 2000, Stouthamer et al. 1999, Werren et al. 2008). Hilgenboecker et al. 2008, Werren et al. The widespread distribution of Wolbachia 2008). These studies place Wolbachia as well as the manipulation of host’s among the most common intracellular reproductive system places this symbiont bacteria known, with estimates of several among the most promising targets for million infected species (Werren et al. 1995, disease/ pest control. The aim of this review Jayaprakash and Hoy 2000, Werren and is to present the Wolbachia-induced reproductive manipulations with an

SARIDAKI AND BOURTZIS.: Wolbachia-induced parasitism and applications 5 emphasis on how this symbiont could be “feminized” males may however suffer a used for the control of insect pests and fitness disadvantage compared to genetic disease vectors. females, with males preferring to mate with genetic females (Moreau et al. 2001). A Wolbachia-induced Phenotypes feminizing Wolbachia infection with complete penetrance would eliminate phenotypic males and lead to the extinction Wolbachia is maternally inherited and has of both the host population as well as the evolved a number of strategies to ensure symbiont. Such events, although difficult to transmission by manipulating the host be observed, may occur in nature. On the reproductive system (Werren et al. 2008). other hand, populations that do persist take These strategies include: a) feminization, advantage of imperfect transmission of the conversion of genetic males into feminizing Wolbachia strains (Rigaud et al. females, b) parthenogenesis, the production 1999b) or constrain the ability of Wolbachia of diploid offspring in the absence of sexual to spread by exploiting the local scarcity of reproduction, c) male killing, the killing of males (Moreau and Rigaud, 2003). Recent infected males to the benefit of infected studies suggest that Wolbachia can also female siblings and d) cytoplasmic induce feminization in insect species as incompatibility (CI), the inability of reported for the leafhopper Zyginidia infected males to successfully fertilize eggs pullula (Hemiptera: Cicadellidae) and the from either uninfected females or from butterfly Eurema hecabe (Lepidoptera: females infected with different Wolbachia Pieridae) (Negri et al. 2006, Narita et al. types. Each of these phenotypes increases 2007). the frequency of infected females in the host Parthenogenesis, the production of population and therefore they represent female offspring in the absence of sperm bacterial adaptations to increase fertilization offers an obvious advantage to transmission of the microorganisms. Such a maternally inherited microorganism. If a parasite effects on hosts are commonly 100% occurrence is assumed, referred to as “reproductive parasitism” parthenogenesis as well as feminization (Bandi et al. 2001). doubles the potential transmission of Feminization is the most obviously Wolbachia to the offspring. Interestingly, all beneficial strategy for a maternally inherited currently documented cases of Wolbachia- bacterium such as Wolbachia. The induced parthenogenesis are found only conversion of genetic male offspring into within haplodiploid species belonging to females doubles the potential Wolbachia Thysanoptera (Arakaki et al. 2001), Acari transmission to the following generation. (Weeks and Breeuwer 2001) and However, the Wolbachia-induced Hymenoptera (Stouthamer et al. 1993, feminization is the most infrequently Zchori-Fein et al. 1995). Haplodiploidy described of the Wolbachia-induced describes the development of (diploid) phenotypes, reported most commonly in females from fertilized eggs, while several species of terrestrial isopods (haploid) males develop from unfertilized (Bouchon et al. 1998, Rigaud et al. 1999a, eggs. In this particular sex determination Michel-Salzat et al. 2001). In these isopod system, parthenogenesis may occur either hosts, Wolbachia within genetic males by complete suppression of meiosis inhibits the development of the androgenic (apomixis) or by restoration of diploidy gland and the production of the androgenic upon meiosis (automixis). Wolbachia- hormone (Azzouna et al. 2004). These

6 ENTOMOLOGIA HELLENICA 18 (2009): 3-16 induced parthenogenesis has been found to occurs between populations infected with be apomictic in mites (Weeks and Breeuwer different Wolbachia strains, when an 2001) and automictic in wasps (Zchori-Fein infected male mates with a female lacking et al. 1995). the same Wolbachia strain. The second type The killing of genetic males by of incompatibility reproductively isolates Wolbachia has been described in four two populations and may contribute to different orders namely Diptera speciation (Werren 1998, Bordenstein 2003, (Hurst et al. 2000, Dyer and Jaenike 2004), Telschow et al. 2005). Coleoptera (Fialho and Stevens 2000; The types of the incompatible crosses Majerus et al. 2000), Lepidoptera (Jiggins et lead to the assumption that there are at least al. 2000) and Arachnida (Zeh et al. 2005). two distinct functions involved in CI, the Male killing may be advantageous under “modification” and the “rescue” function limited conditions, where resource (Werren 1997, Bourtzis et al. 1998, Merçot reallocation from dead males to female and Poinsot 1998). When the female lacks siblings increases the fitness of infected the “rescue” function, the “modification” females (Hurst 1991, Hurst et al. 2003). In function of the male results in embryonic all cases detected, the Wolbachia-induced lethality. Although the exact mechanism male killing meets the above criterion. remains unclear, the incompatible Another predicted benefit would be the phenotype is associated with an asynchrony resulting avoidance of the inbreeding in the development of the male and female (Werren 1987). pronuclei probably due to impaired histone Despite the fact that cytoplasmic deposition in the male pronucleus (Lassy incompatibility (CI) is the most commonly and Karr 1996, Tram and Sullivan 2002, described reproductive abnormality induced Landmann et al. 2009). by Wolbachia, the underlying mechanism In addition to the above mentioned still remains under investigation. CI has reproductive abnormalities, Wolbachia can been described in many different arthropod positively or negatively influence other orders: Diptera (Yen and Barr 1973), aspects of host fitness. In Aedes albopictus Coleoptera (Wade and Stevens 1985), Acari (Diptera: Culicidae), fitness benefits (Breuuwer and Jacobs 1996), resulting from Wolbachia infection affect (Moret et al. 2001), Lepidoptera (Brower both fecundity and longevity (Dobson et al. 1976), Hymenoptera (Reed and Werren 2002). Both single and doubly infected 1995), Homoptera (Hoshizaki and Shimada females produce more eggs and live longer 1995) and Orthoptera (Kamoda et al. 2000). than uninfected females; no effect on males As shown in Figure 1, CI can be has been observed. It should be noted that unidirectional or bidirectional, depending on similar observations were recently reported the number of Wolbachia strains involved in for Drosophila simulans (Diptera: the phenotype (Breeuwer and Werren 1990, Drosophilidae) (Weeks et al. 2007). O’Neill and Karr 1990, Bourtzis et al. 1998, Negative effects of Wolbachia in host Bourtzis et al. 2003). Unidirectional CI longevity have been well documented due describes the embryonic lethality observed to wMelpop strain (Min and Benzer 1997). when a Wolbachia-infected male mates with Flies bearing wMelpop suffer significant an uninfected female. All the other possible reduction in longevity, most likely due to crosses are fully compatible, favoring the overproliferation of the symbiont in the relative fitness of infected females and the neuronal tissue (Min and Benzer 1997, spread of Wolbachia. Bidirectional CI

SARIDAKI AND BOURTZIS.: Wolbachia-induced parasitism and applications 7

B A FIG. 1. Schematical representation of unidirectional (A) and bidirectional (B) cytoplasmic incompatibility. Insects bearing incompatible Wolbachia strains are marked with red or black.

McGraw et al. 2002, McMeniman et al. render Wolbachia as key-player in pest 2009). control management (Table 1). Wolbachia’s Although Wolbachia successfully evades potential as novel environmentally friendly the host immune system and does not induce bio-control agent has already attracted a lot the normal antibacterial response (Bourtzis of attention (Beard et al. 1993, Sinkins et al. et al. 2000), Wolbachia infection has been 1997, Bourtzis and Braig 1999, Sinkins and shown to be a key player in host immunity. O’Neill 2000, Aksoy et al. 2001). Several In at least one host-parasitoid system, the strategies have been proposed, most of presence of Wolbachia decreases fitness in which take advantage of the induction of both the host and the parasitoid (Fytrou et al. cytoplasmic incompatibility (Bourtzis, 2006). D. simulans infected with Wolbachia 2008). is less effective in killing the eggs laid by Despite the global distribution of the parasitoid Leptopilina heterotoma Wolbachia, many important agricultural (Hymenoptera: Eucoilidae). Similarly, pests (e.g. Bactrocera oleae) and disease Wolbachia infection of L. heterotoma makes vectors (Aedes aegypti, Anopheles the parasitoid more vulnerable to the host gambiae) are not naturally Wolbachia- defenses. The exact nature of these infected. However, many studies have interactions is currently unknown. On the shown that Wolbachia can be transferred other hand, recent reports suggest that and established into a naive host resulting Wolbachia infections provide virus in the expression of the expected protection in insect hosts (Hedges et al. reproductive phenotype (Boyle et al. 1993, 2008, Teixeira et al. 2008). Braig et al. 1994, Poinsot et al. 1998, Sasaki and Ishikawa 2000, McGraw et al. Practical Applications 2001, Zabalou et al. 2004a, b, Xi et al. 2005). Based on these observations, The widespread distributions as well as the Wolbachia may serve as an important tool manipulation of host’s reproductive system for the “Incompatible Insect Technique”, the use of a symbiont-associated

8 ENTOMOLOGIA HELLENICA 18 (2009): 3-16

TABLE 1. Synopsis of Wolbachia-based applications.

Application Wolbachia-induced phenotype Wolbachia strain / host

Incompatible Insect Technique wCer2 / Ceratitis capitata Cytoplasmic Incompatibility (pest population control) (Zabalou et al. 2004a, 2009) wAlbB / Aedes aegypti Drive strategies Cytoplasmic Incompatibility (Xi et al. 2005) Control of vector-borne wMelPop / Aedes aegypti Shortening of host lifespan diseases (McMeniman et al. 2009)

reproductive incompatibility as for the useful tool to complement current control control of insect pests and disease vectors strategies. Drive systems are an important (Bourtzis and Robinson 2006). component of population replacement A successful example of stable strategies that provide mechanisms for the transinfection of a Wolbachia-free autonomous spread of desired agricultural pest has been reported for genotypes/transgenes into the targeted Ceratitis capitata (Diptera: Tephritidae) population (Dobson 2003). Besides (Zabalou et al. 2004b). Wolbachia strains autonomous transposons, primary from the host Rhagoletis cerasi (Diptera: candidates for drive strategies are bacterial Tephritidae) have been used to stably infect symbionts used as expression vehicles the Mediterranean fruit fly through (Curtis and Sinkins 1998, Turelli and embryonic injection. Crosses between Hoffmann 1999). The reproductive uninfected females and Wolbachia-infected advantage afforded by CI to Wolbachia- males resulted in 100% egg mortality, while infected females promotes the spread of the crosses between fly lines bearing different maternally inherited Wolbachia infection. Wolbachia strains were 100% Thus, desired genotypes / transgenes linked bidirectionally incompatible. The major to a Wolbachia infection would be expected advantage of the Wolbachia-based to spread into a targeted population Incompatible Insect Technique over the following the seeding of the targeted Sterile Insect Technique lies on the fact that population with proper Wolbachia-infected the insects do not have to be irradiated females. Xi et al. (2005) demonstrated the before release. However, the necessity of ability of wAlbB to spread into an A. employing an effective sexing strain of the aegypti population after seeding of an insect pest, so that only infected males are uninfected population with infected released, still remains (Bourtzis and females, reaching infection fixation within Robinson 2006). Zabalou et al. (2009) seven generations in laboratory cage tests. described a Wolbachia-infected line of the Age is a critical determinant of the VIENNA 8 genetic sexing strain of the ability of most insect vectors to transmit a medfly that carried the selectable marker range of human pathogens. This is due to temperature sensitive lethal (tsl). the fact that most pathogens require a Transferred Wolbachia induced high levels period of extrinsic incubation in the insect of CI even after the temperature treatment host before pathogen transmission can required for the male-only production. occur. This developmental period for the Genetic manipulation that reduces the pathogen often comprises a significant fitness of a pest population would provide a proportion of the expected lifespan of the

SARIDAKI AND BOURTZIS.: Wolbachia-induced parasitism and applications 9 vector. As such, only a small proportion of intramural funding from University of the population that is oldest contributes to Ioannina to K.B. pathogen transmission (Cook et al. 2008). Given this, strategies that target vector age References would be expected to obtain the most significant reductions in the capacity of a Aksoy, S., I. Maudlin, C. Dale, A.S. vector population to transmit disease. The Robinson and S.L. O'Neill. 2001. identification of insect symbionts that Prospects for control of African shorten the host lifespan would offer new trypanosomiasis by tsetse vector tools for the control of vector-borne diseases manipulation. Trends Parasitol. 17: 29- (Sinkins and O’Neill 2000). McMeniman et 35. al. (2009) reported the successful transfer of Arakaki, N., T. Miyoshi and H. Noda. 2001. wMelpop, a life-shortening strain of Wolbachia-mediated parthenogenesis in Wolbachia, into the major mosquito vector the predatory thrips Franklinothrips of dengue, Aedes aegypti (Diptera: vespiformis (Thysanoptera: Insecta). Culicidae). The association halved host life Proc. R. Soc. Lond. B. Biol. Sci. 268: span under laboratory conditions and the 1011-1016. symbiont induced complete cytoplasmic Azzouna, A., P. Greve and G. Martin. 2004. incompatibility, which should facilitate its Sexual differentiation traits in functional invasion into natural field populations. males with female genital apertures (male symbol fga) in the woodlice Concluding Remarks Armadillidium vulgare Latr. (Isopoda, Crustacea). Gen. Comp. Endocrinol. During the last decades, insect symbiosis 138: 42-49. gained a lot of attention as a widespread Baldo, L., J.C. Dunning Hotopp, K.A. phenomenon affecting the host biology in Jolley, S.R. Bordenstein, S.A. Biber, many ways. Among the bacteria related to R.R. Choudhury, C. Hayashi, M.C. insects in a positive or negative aspect, Maiden, H. Tettelin and J.H. Werren. Wolbachia is doubtless the most ubiquitous. 2006. Multilocus sequence typing Causing a broad range of reproductive system for the endosymbiont Wolbachia phenotypes to the hosts, Wolbachia is a key pipientis. Appl. Environ. Microbiol. 72: player with biological, ecological and 7098-7110. evolutionary significance. Due to its unique Bandi, C., T.J.C. Anderson, C. Genchi and properties, Wolbachia offers the potential M.L. Blaxter. 1998. Phylogeny of for development of novel and environment Wolbachia in filarial nematodes. Proc. friendly biotechnological strategies for the R. Soc. Lond. B. Biol. Sci. 265: 2407- control of insect pests and disease vectors. 2413. Bandi, C., A.M. Dunn, G.D. Hurst and T. Acknowledgments Rigaud. 2001. Inherited microorganisms, sex-specific virulence and reproductive parasitism. Trends This paper is dedicated to the memory of Parasitol. 17: 88-94. Prof Michael Majerus†. This work was Beard, C.B., S.L. O'Neill, R.B. Tesh, F.F. supported by grants from the European Richards and S. Aksoy. 1993. Community’s Seventh Framework Modification of arthropod vector Programme CSA-SA_REGPROT-2007-1 under grant agreement no 203590 and

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Wolbachia – Επαγόμενος αναπαραγωγικός παρασιτισμός και εφαρμογές

Α. ΣΑΡΙΔΑΚΗ ΚΑΙ Κ. ΜΠΟΥΡΤΖΗΣ

Τμήμα Διαχείρισης Περιβάλλοντος και Φυσικών Πόρων, Πανεπιστήμιο Ιωαννίνων, Σεφέρη 2, 30 100 Αγρίνιο, Ελλάδα

ΠΕΡΙΛΗΨΗ

Οι συμβιωτικές σχέσεις εντόμων-μικροοργανισμών (και ιδιαίτερα βακτηρίων) είναι ένα ευρέως διαδεδομένο φαινόμενο με ποικίλες συνέπειες στη βιολογία του ξενιστή, θετικές ή αρνητικές. Ένα από τα πιο διαδεδομένα και καλά μελετημένα βακτήρια είναι το βακτήριο Wolbachia. Το βακτήριο Wolbachia είναι ένα υποχρεωτικά ενδοκυττάριο και μητρικά κληρονομούμενο βακτήριο που συμβιώνει με ένα μεγάλο εύρος ειδών ασπόνδυλων όπως ακάρεα, αράχνες, ισόποδα, νηματώδεις της φιλαρίασης και κυρίως έντομα. Μελέτες έχουν δείξει ότι το βακτήριο Wolbachia πιθανώς συμβιώνει με το 70% όλων των ειδών των εντόμων, καθιστώντας το ως τον πιο διαδεδομένο συμβιωτικό οργανισμό του πλανήτη. Οι αλληλεπιδράσεις του βακτηρίου με τον ξενιστή κυμαίνονται μεταξύ της αμοιβαίας συμβίωσης και της επαγωγής αναπαραγωγικών ανωμαλιών όπως θηλυκοποίηση, παρθενογένεση, θανάτωση των αρσενικών ατόμων ή κυτταροπλασματική ασυμβατότητα. Παρά την ευρεία εξάπλωση και τις επιδράσεις του βακτηρίου στη βιολογία του ξενιστή, στην ειδογένεση και στην οικολογική ποικιλότητα, λίγα είναι γνωστά για τους μοριακούς μηχανισμούς της αλληλεπίδρασης του ξενιστή με το συμβιωτικό αυτό βακτήριο. Πρόσφατες μελέτες εστιάζουν στη δυνατότητα ανάπτυξης μεθόδων βιολογικού ελέγχου εντόμων γεωργικής, περιβαλλοντικής ή ιατρικής σημασίας, οι οποίες βασίζονται στις ιδιότητες του βακτηρίου Wolbachia.