Review of treatment methods to remove Wolbachia bacteria from

Y.-Y. Li, K. D. Floate, P. G. Fields & B.- P. Pang

Symbiosis

ISSN 0334-5114 Volume 62 Number 1

Symbiosis (2014) 62:1-15 DOI 10.1007/s13199-014-0267-1

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1 23 Author's personal copy

Symbiosis (2014) 62:1–15 DOI 10.1007/s13199-014-0267-1

Review of treatment methods to remove Wolbachia bacteria from arthropods

Y. - Y. L i & K. D. Floate & P.G. Fields & B.-P.Pang

Received: 17 October 2013 /Accepted: 15 January 2014 /Published online: 30 January 2014 # Springer Science+Business Media Dordrecht 2014

Abstract Wo l b a c h i a are intracellular bacteria that infect nu- 1 Introduction merous and diverse species including economic pests of crops and disease vectors affecting livestock and Wo l b a c h i a bacteria (α-Proteobacteria) are arguably the most humans. Research on these symbionts has identified profound successful symbionts of arthropods worldwide. They report- effects of Wo l b a c h i a on their hosts with possible application in edly infect 17 to 76 % of diverse taxa that include members of pest control. Such research often requires methods to cure Class Insecta (), Class Arachnida (mites, scorpions, infections. To facilitate future research on these bacteria, we spiders), Class Malacostraca (amphipods, isopods) and Class reviewed the Wo l b a c h i a literature to summarize results of 110 Maxillopoda (barnacles) (Baldo et al. 2007; Bouchon et al. studies spanning 62 taxa that report on treatment methods and 1998; Cordaux et al. 2012; Duron et al. 2008; Floate et al. outcomes. Application of tetracycline in diet is the most 2006; Hilgenboecker et al. 2008; Jeyaprakash and Hoy 2000; common method and is typically successful. Rifampicin is Rowley et al. 2004; Werren et al. 1995;ZugandHammerstein secondarily used, and may be successful when tetracycline is 2012). In some orders, the prevalence of infections not. Elevated temperatures can be used to eliminate infections, among species may approach 100 %; e.g., lice (Anoplura, but is not often used. Rearing hosts under crowded conditions Mallophaga) (Kyei-Poku et al. 2005). Infections also occur or starvation has been shown to reduce Wo l b a c h i a titre which in filarial nematodes (Nematoda: Class Secernentea), includ- affects maternal transmission. Application of treatment ing the causative agents for river blindness and elephantiasis methods has a number of considerations with possible impli- (Taylor et al. 2005). Given the importance of their hosts as cations for the interpretation of data. This review is intended to economic pests of crops, or as vectors of diseases affecting alert the reader to treatment options and potential non-target humans and , a tremendous amount of resources has effects. been directed towards research on Wo l b a c h i a – host interac- tions (Fig. 1). Keywords Wo l b a c h i a . Elimination . Antibiotics . The success of these bacteria reflects their ability to ma- Temperature nipulate the reproductive biology of their host to enhance the spread of infections across generations. Wo l b a c h i a are mainly transmitted from infected mothers to their offspring via egg cytoplasm (Hoffmann and Turelli 1988). Thus, manipulations Y.

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Fig. 1 Reported in 2-year increments, three metrics document the grow- ‘Floate’, but provide data through 2012. c ‘Google’ values (n=12 559) ing research emphasis on Wolbachia. a ‘Floate’ reports values (n=704) derive from Google Scholar (http://scholar.google.ca/) and report the from Floate et al. (2006). These identify original research articles located number of ‘results’ obtained using ‘Wo l b a c h i a’ as the search term for using database searches of CAB Abstracts, Biological Abstracts, and ‘articles (excluding patents and legal documents)’. Google Scholar values PubMed with ‘Wolbachia’ as the search term and subsequently vetted include original research articles, but also books, abstracts, theses and by KDF for content. Data for 2005 (n=100) is excluded. b ‘Scopus’ other items obtained from sources in addition to scientific journals. Prior values (n=1 667) derive from the Scopus database (http://www.scopus. to 1981, total values for Floate, Scopus and Google are 40, 13 and 146, com/) and identify the number of ‘articles’ that include ‘Wolbachia’ in the respectively abstract, title or keywords. They generally correspond with those for functional females. A fourth type is male-killing, in which experimental research on Wo l b a c h i a to relatively few taxa. In Wo l b a c h i a cause the death of male embryos. These manipula- a survey of 510 papers published prior to 2006 that report on tions and other effects of Wo l b a c h i a on their hosts have been Wo l b a c h i a – arthropod associations, 58 % were restricted to the subject of numerous reviews (e.g., Floate et al. 2006; taxa in four insect families: Drosophilidae (25.5), Culicidae Werren 1997; Werren et al. 2008;White2011;Engelstädter (19.4), Trichogrammatidae (8.2), Pteromalidae (5.1) (Floate and Hurst 2009;Feldhaar2011). et al. 2006). Within these families, research was limited to a The effects of Wo l b a c h i a are elegantly demonstrated by handful of genera and particularly species characterized by the treating hosts to manipulate infections and then comparing ease of laboratory culture, short generation times, high fecun- the reproductive fitness of treated versus untreated hosts. In dity, and which easily were treated with antibiotics. Several one study, antibiotic treatments were used to reduce titre levels hundred additional research articles since then have been of parthenogenesis-inducing Wolbachia in colonies of the published on Wo l b a c h i a (Fig. 1), but relatively few of them wasp Muscidifurax uniraptor (: report experimental manipulations. Pteromalidae). Whereas untreated (infected) colonies pro- Even when successful methods have been identified to duced only female progeny, colonies with decreasing levels remove Wolbachia from its host, the outcome is far from of Wolbachia produced increasingly higher proportions of certain. It has been our experience that when treatments do male progeny (Zchori-Fein et al. 2000). In a second study, work, they require continuous application to the host during antibiotic treatments were used to remove infections of CI- egg-to-adult development for two or more generations (Floate inducing Wo l b a c h i a from colonies of the confused flour bee- and Coghlin 2010; Kyei-Poku et al. 2003; Yamada et al. tle, Tribolium confusum (Coleoptera: Tenebrionidae). 2007). Thus, we were intrigued by reports that infections Subsequent crosses of uninfected females to infected males could be easily removed from T. confusum. Stevens (1989) produced no offspring, whereas all other crosses produced removed Wo l b a c h i a with 100 % success by rearing immature normal numbers of offspring (Wade and Stevens 1985). hosts for 22 days at 37 °C. Wade and Stevens (1985)removed These and similar studies (e.g., Breeuwer and Werren 1990; infections in 20 days by rearing adults on flour treated with Min and Benzer 1997; Otsuka and Takaoka 1997;Wadeand 1.25 mg tetracycline/g flour. Despite considerable effort in our Stevens 1985) emphasize the importance of being able to own lab, however, we have been unable to repeat these results experimentally cure infections to document the consequences (Y. Li, unpubl. research, see Table 1). Otsuka and Takaoka of Wo l b a c h i a infection to host reproduction and biology. (1997) cured infections of Wo l b a c h i a in the mosquito Aedes Despite its importance in clarifying Wo l b a c h i a – host rela- albopictus (Diptera: Culicidae) by exposing first-instar larvae tionships, challenges in curing infections have restricted to tetracycline (5.0 mg/ml water) for 24 h. Using this same Author's personal copy

Methods to remove Wolbachia 3

Ta b l e 1 Studies reporting on the use of antibiotics to treat infections of Wolbachia in arthropods

Taxa (common namea) Antibiotic added to diet Duration (outcome) Reference

Class Arachnida Acarina (chiggers, mites, ticks) Tetranychidae (spider mites) Bryobia praetiosa Tetracycline (1.5 mg/ml) 4 days (curedb) (Weeks and Breeuwer 2001) Panonychus mori Tetracycline (1 mg/ml) 1 generation (cured) (Hong et al. 2002) Rifampicin (0.5 mg/ml) 1 generation (cured) (Gotoh et al. 2005) Tetranychus kanzawai Tetracycline (1.0 mg/ml); rifampicin 1 generation (cured) (Gomi et al. 1997) (0.5 mg/ml) Tetranychus piercei Tetracycline (1 mg/ml) 4 generations (cured) (Zhu et al. 2012) Tetranychus urticae Tetracycline (1 mg/ml) 3 generations (cured) (Breeuwer 1997) Tenuipalpidae (false spider mites) Brevipalpus phoenicis Tetracycline (2 mg/ml) 3 days (cured) (Weeks et al. 2001) Class Entognatha Collembola (snow flies, springtails) Isotomidae Folsomia candida Ampicillin (2.27×103 mg/g) ca. 28 days (not cured) (Giordano et al. 2010) Oxytetracycline (2.27×102 mg/g) Tylosin (1.82×102 mg/g) Rifamipicin (10 mg/g) 31–34 days (reduced titrec) (Timmermans and Ellers 2009) Rifampicin (27 mg/g) 2–3 generations (cured) (Pike and Kingcombe 2009) Tetracycline (27 mg/g) 2–3 generations (not cured) Class Insecta Coleoptera (beetles) Chrysomelidae (leaf beetles) Diabrotica virgifera Tetracycline (3 mg/g) for adults 2 generations (cure) (Giordano et al. 1997) virgifera 3 generations (cure) (Barr et al. 2010) Curculionidae (weevils, snout beetles) Hypera postica Tetracycline (suspension of 100 mg in 1 generation (cured) (Klostermeyer 1978) 500 ml H20) Penicillin G (suspension of 400,000 units 1 generation (not cured) in 500 ml H20) Lissorhoptrus Tetracycline (2.5 mg/ml) 1 generation (cured) (Chen et al. 2012) oryzophilus Gentamicin (2.5 mg/ml) 1 generation (not cured) Sitophilus oryzae Tetracycline (1 mg/g) for adults 2 generations (cured) (Heddi et al. 1999) Tenebrionidae (darkling beetles) Tribolium confusum Tetracycline (1.25 mg/g) for larvae 20 days (cured) (Wade and Stevens 1985) Tetracycline (20, 30 mg/g) 3 generations (cured) Li et al. 2013 (unpublished) Tetracycline (10 mg/g) 4 generations (cured) Rifampicin (1, 2,3 mg/g) 1 generation (no cure) Diptera (gnats, mosquitoes, true flies) Culicidae (mosquitoes) Aedes albopictus Tetracycline (5.0 mg/ml) 1 generations (cured) (Otsuka and Takaoka 1997) Tetracycline (1.0 mg/ml) Adult exposure for 1 generation (cure) (Dobson and Rattanadechakul 2001) Tetracycline (1.0–5.0 mg/ml) Larval exposure for 1 or 6 (2.5 mg/ml) (Dobson and days (no cure) Rattanadechakul 2001) Aedes fluviatilis Tetracycline (0.1 mg/ml) 3 generations (10 to 14 days during each (Baton et al. 2013) generation) (cure) Aedes polynesiensis Tetracycline (0.017 mg/ml) 18–26 h (cure) (Wright and Wang 1980) Tetracycline (1 mg/ml) 3 generations (cure) (Brelsfoard and Dobson 2011) Author's personal copy

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Ta b l e 1 (continued)

Taxa (common namea) Antibiotic added to diet Duration (outcome) Reference

Tetracycline (0.25 mg/ml) 3 generations (99 % cure) (Plichart and Legrand 2005) Tetracycline (0.2 mg/ml) for female Parental generation (cure) (Crain 2013;Deanand Aedes riversi Tetracycline (0.2 mg/ml) for female Dobson 2004) Aedes pseudo scutellaris Tetracycline (1 mg/ml) for adult 4 generations (cure) (Dutton and Sinkins 2005) Armigeres subalbatus Tetracycline (0.25 mg/ml) for larvae 1 generation (cure) (Jamnongluk et al. 2000) Culex pipiens Tetracycline (0.05, 0.025 mg/ml) for larvae 1 generation (cure) (Chen et al. 2013; Yen and Barr 1973) Tetracycline (0.1–5.0 mg/ml) for larvae 1 generation (no cure) (Yen and Barr 1973) Streptomycin (0.1–5.0 mg/ml) for larvae Penicillin (0.25 mg/ml) for larvae Chloromycetin (0.1–1.0 mg/ml) for larvae Tetracycline (0.025, 0.05 mg/ml) 1 generation (cure) (Awahmukalah and Brooks 1983) Tetracycline (50 mg/ml) for larvae 24 h (cure) (Suenaga 1993) Tetracycline (0.05 mg/ml) 2 generations (cure) (Rasgon and Scott 2003) Tetracycline (0.1 mg/ml) for newly 4 generations (cure) hatched larvae Tetracycline (0.2 mg/ml) for newly 2 generations (cure) hatched larvae Drosophilidae (pomace flies, small fruit flies, vinegar flies) Tetracycline (0.3 mg/ml) for larvae 1 generation (cure) (Harcombe and Hoffmann melanogaster 2004; Hoffmann 1988) Tetracycline (0.25 mg/ml) for adults for 2rd generation (cure) (Holden et al. 1993) 3weeks Tetracycline (1 mg/ml) for second generation for 5 days Tetracycline (0.25 mg/ml) 1 or 2 generations (cure) (Min and Benzer 1997) Tetracycline (0.015 mg/ml) 2 months (cure) (Koukou et al. 2006) Tetracycline (0.25, 0.8, 1 mg/ml) 3 generations (cure) (Gazla and Carracedo 2011) Tetracycline (0.2 mg/ml) 2 generations (cure) (Silva et al. 2012) Tetracycline (0.25 mg/ml) 2 generations (cure) (Fry et al. 2004) Tetracycline (0.3 mg/ml) 2 generations (cure) (Caragata et al. 2013; Reynolds et al. 2003; Strunov et al. 2013) Drosophila Tetracycline (25 mg/ml) 3 generations (cure) (Rogina and Helfand 2013; melanogaster Wang et al. 2009) Drosophila Tetracycline (0.25 mg/ml) 1 generation (cure) (Albertson et al. 2013) melanogaster, D. simulans Drosophila simulans Tetracycline (0.3 mg/ml) 1 generation (cure) (Hoffmann and Turelli 1988; Weeks et al. 2007) Tetracycline (0.3 mg/ml) 1 generation (cure) (Binnington and Hoffmann 1989) Tetracycline (0.25 mg/ml) 2 generations (cure) (Brennan et al. 2012) Tetracycline (0.25, 0.8, 1 mg/ml) 3 generations (cure) (Gazla and Carracedo 2011) Tetracycline (0.164 mg/ml) cure (Clancy and Hoffmann 1998) Tetracycline (0.3 mg/ml) 2 generations (cure) (Poinsot and Merçot 1997) Glossinidae (tsetse flies) Tsetse fly Tetracycline (0.02 mg/ml) 1 generation (cure) (Doudoumis et al. 2012) Tachinidae (tachinid flies) Exorista sorbillans Oxytetracycline (0.02 mg/ml) 6 generations (cure) (Guruprasad et al. 2011) Tetracycline (0.015 mg/ml) 7 generations (cure) (Puttaraju and Prakash 2005) Author's personal copy

Methods to remove Wolbachia 5

Ta b l e 1 (continued)

Taxa (common namea) Antibiotic added to diet Duration (outcome) Reference

Homoptera (scale insects, treehoppers, whiteflies) Aleyrodidae (whiteflies) Bemisia tabaci Ampicillin, rifampicin, tetracycline 48 h (cure) (Ahmed et al. 2010) (0.05 mg/ml) Tetracyline (0.05 mg/ml) 48 h (reduced titre) (Zhong and Li 2013) Delphacidae (delphacid planthoppers) Laodelphax striatellus Rifampicin (1 mg/ml) 2 generations (cure) (Noda et al. 2001) Sogatella furcifera Tetracycline (1 mg/ml) 2 generations (cure) Tetracycline (0.5 mg/ml) Complete cure (Nakamura et al. 2012) Hymenoptera (ants, bees, wasps) Aphelinidae Aphytis diaspidis Rifampicin (50 mg/ml) for females 1 generation (cure) (Zchori-Fein et al. 1995) A. lingnanensis Encarsia formosa Tetracycline (1, 5 mg/ml) for adults 24 h (density reduced) (Stouthamer and Mak 2002) Encarsia hispidae Tetracycline (50 mg/ml) for adults 24 h (some adults cured, no maternal (Giorgini et al. 2009) transmissiond) Rifampicin (50 mg/ml) for adults 48 h (no maternal transmission) (Zchori-Fein et al. 2004; Giorgini et al. 2009) Encarsia inaron Rifampicin (50 mg/ml) for adults 48 h in 3 successive generations (cure) (White et al. 2009) Ampicillin or doxycycline (1 mg/ml) 48 h (reduced maternal transmission) Encarsia pergandiella Rifampicin (50 mg/ml) for adults 48 h (cure) (Hunter et al. 2003) Encarsia meritoria Tetracycline (20 mg/ml) for females 2 generations (partly cure) (Giorgini 2001) Encarsia protransvena Tetracycline (10 mg/ml) for females 2 generations (no cure) Braconidae Asobara tabida Rifampicin (0.13 to 2 mg/g) Complete cure (Dedeine et al. 2001) Tetracycline (0.13 to 2 mg/g) Ciprofloxacin (1 mg/g to 2 mg/g) No cure Gentamicin (1 mg/g to 2 mg/g) Rifampicin (2 mg/g) Complete cure (Kremer et al. 2012) Asobara spp. Rifampicin (2 mg/g) (Dedeine et al. 2005) Cotesia sesamiae Rifampicin (0.5, 1, 1.5, 2, 2.5 mg/ml) for 3 generations (cure) (Mochiah et al. 2002) host Encyrtidae (encyrtid wasps) Apoanagyrus Tetracycline (50 mg/g) 2 generations (cure) (Pijls et al. 1996) diversicornis Rifampicin (10 mg/g) Sulphadiazine (10 mg/g or 50 mg/g) No cure Eulophidae (eulophid wasps) Melittobia australica Tetracycline (0.14 mg/ml) injected in their Complete cure (Abe et al. 2003) host (figitid wasps) Leptopilina clavipes Tetracycline (5 mg/ml) at 20 °C No cure (Schidlo et al. 2002) Rifampicin (5 mg/ml) at 20 °C Tetracycline (5 mg/ml) at 25 °C Complete cure Rifampicin (5 mg/ml) at 25 °C Tetracycline (2 mg/ml) at 20 °C No cure Rifampicin (2 mg/ml) at 20 °C Tetracycline (2 mg/ml) at 25 °C Complete cure Rifampicin (2 mg/ml) at 25 °C Leptopilina heterotoma Rifampicin (0.01 to 0.1 mg/g) fed to host 1 generation (cure) (Mouton et al. 2003) larvae parasitized by wasp Rifampicin (2 mg/ml) 1 generation (cure) (Vavre et al. 2000) Leptopilina victoriae Not indicated 3 generations (cure) (Gueguen et al. 2012) Pteromalidae (pteromalid wasps) Author's personal copy

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Ta b l e 1 (continued)

Taxa (common namea) Antibiotic added to diet Duration (outcome) Reference

Muscidifurax uniraptor Rifampicin (0.1 to 100 mg/ml) Declining infection approaching 100 % (Zchori-Fein et al. 2000) at 100 mg/ml Nasonia giraulti Tetracycline (1 mg/ml) for female adults Feeding some time then allowing to lay (Breeuwer and Werren 1990) eggs for 48 h, repeat three more generations (cure) Urolepis rufipes Tetracycline (0.05 mg/ml) 4 generations (cure) (Kyei-Poku et al. 2003) Scelionidae (scelionid wasps) Te l e n o m u s n a w a i Tetracycline (50 mg/ml) Partly cure (Arakaki et al. 2000) Trichogrammatidae (trichogrammatid wasps) Trichogramma Rifampicin (5 mg/ml) 1 generation (Partial cure) (Almeida et al. 2010) atopovirilia 3 generations (cure) Trichogramma Tetracycline (0.02 mg/ml to 0.2 mg/ml) 2–3 generations (cure) (Grenier et al. 2002) cordubensis, T. evanescens, T. oleae, T. pretiosum Trichogramma chilonis, Tetracycline hydrochloride (10 and 1 or 2 generations (partly cured) (Stouthamer et al. 1990) T. deion, T. platneri, 100 mg/ml), sulfamethoxazole and T. pretiosum rifampicin (100 mg/ml) Gentamycin, penicillin G, erythromycin No cure (100 mg/ml) Lepidoptera (butterflies, moths) Crambidae Ostrinia furnacalis Tetracycline (0.6 mg/g) for larvae 1 generation (cure) (Kageyama et al. 2002; Sugimoto et al. 2010) Ostrinia scapulalis Tetracycline (0.006,0.012, 0.06, 0.12, 0.3, 1 generation (cure) (Kageyama et al. 2003b) 0.6 mg/g) for larvae Tetracycline(0.00006, 0.0006,0.0012) 1 generation (no cure) Tetracycline (0.6 mg/g) from larva to pupa (Kageyama et al. 2003a) Pieridae (orange-tips, Whites, Sulphurs) Eurema hecabe Tetracycline (1 mg/ml) for female adult Cure (Hiroki et al. 2002) Pyralidae (grass moths, snout moths) Ephestia cautella Tetracycline (0.5 mg/g) for larvae 1 generation (cure) (Kellen et al. 1981) Ephestia kuehniella Tetracycline (0.4 mg/g) 2 generations (cure) (Sasaki et al. 2002) Noctuidae (cutworms, dagger moths, noctuid moths, owlet moths, underwings) Spodoptera exempta Tetracycline (0.3 mg/g) for larvae Complete cure (Graham et al. 2012) Psocoptera (psocid) Liposcelididae Liposcelis tricolor Rifampicin (10 mg/g) 4 weeks (cure) (Dong et al. 2006) Rifampicin (3 mg/g, 0.3 mg/g) 4 weeks (not complete cure) Thysanoptera (thrips) Aeolothripidae (banded thrips, broad-winged thrips) Franklinothrips Tetracycline (50 mg/ml) for new emerged Cured (Arakaki et al. 2001) vespiformis adults Tetracycline (50 mg/ml) for second-instar Cured larvae Cephalosporin C, erythromycin, penicillin- Not cured G, streptomycin sulphate (50 mg/ml) a Preference for common names is given to those reported in the Integrated Taxonomic Information System (http://www.itis.gov/) b ‘Cure’ = authors report the elimination of infection from some or all treated individuals c ‘Reduced titre’ = authors report reduction in the level of infection in treated individuals d ‘No maternal transmission’ = treated female individuals remain infected, but do not transmit infection to their progeny e Study on the symbiotic bacteria Cardinium Author's personal copy

Methods to remove Wolbachia 7 treatment, Dobson and Rattanadechakul (2001)reportedthat flies, whiteflies, planthoppers, wasps, butterflies, moths, thrips Ae. albopictus larvae suffered high mortality with most survi- and nematodes (Table 1). Efficacy is affected by dose. vors retaining their infections. Contrasting outcomes such as Kageyama et al. (2003b)testednineconcentrationsoftetra- these caution against expectations of repeatable results. cycline ranging from 0.6×10−4 to 0.6 mg/g in diet fed to Given the need to cure infections to broaden the scope of larvae of the moth, Ostrinia scapulalis (Lepidoptera: Wo l b a c h i a research, and given the challenges in doing so, we Crambidae). Concentrations below 1.2×10−3 mg/g appeared provide here a review of treatment methods and outcomes. We to have no effect on Wolbachia, whereas infections were first compiled a list of scientific papers using various databases removed with concentrations of 0.6×10−2 to 0.6 mg/g. and online search engines; e.g., CAB Abstracts, Biological Applied over one or more generations, effective concentra- Abstracts, PubMed, Google Scholar, Scopus. We then identi- tions of tetracycline range from about 0.25 to 5 mg per g or ml fied those papers reporting efforts to cure Wo l b a c h i a -infected of diet when treating mites, beetles, flies, homopterans, lepi- arthropods and summarized their treatment outcomes dopterans and at least some species of wasps (Table 1). Other (Tables 1 and 2). Although comprehensive, this review is not taxa are more resistant. Tetracycline applied in diet at a con- exhaustive and we expect that relevant papers have been centration of 27 mg/g for two generations failed to cure overlooked. We also expect that our review is biased in favour infections in the springtail, Folsomia candida (Collembola: of successful outcomes, based on the assumption that failed Isotomidae) (Pike and Kingcombe 2009). attempts often go unreported. Nevertheless, we believe that Rifampicin is the second most commonly used antibiotic, this summary will be a useful tool to facilitate future research and has been used to cure infections of Wo l b a c h i a in mites, in Wo l b a c h i a – arthropod systems. springtails, beetles, whiteflies, planthoppers and wasps (Table 1). Effective concentrations of rifampicin can be the same, but are more often less than those for tetracycline. The 2 Antibiotic treatments same concentration (1 mg/ml) of tetracycline and rifampicin wasusedtoremoveWolbachia from the planthoppers, Our survey identified 96 papers reporting on the use of anti- Laodelphax striatellus and Sogatella furcifera (Homoptera: biotics to cure infections of Wo l b a c h i a in 35 species of arthro- Delphacidae) (Noda et al. 2001). For the mite, Panonychus pods (Table 1). Host taxa included mites (Acarina: 6 species in mori (Acarina: Tetranychidae), Wo l b a c h i a was eliminated in 1 2 families), springtails (Collembola: 1 species), beetles generation with application of 0.5 mg/ml rifampicin (Gotoh (Coleoptera: 3 species in 2 families), flies (Diptera: 10 species et al. 2005) versus application of 1.0 mg/ml of tetracycline in 4 families), whiteflies and plant hoppers (Homoptera: 3 (Hong et al. 2002). These same treatments were effective in species in 2 families), wasps (Hymenoptera: 13 species in 8 removing infections from the mite, Tetranychus kanzawai families), butterflies and moths (Lepidoptera: 5 species in 4 (Acarina: Tetranychidae) (Gomi et al. 1997). Rifampicin also families) and thrips (Thysanoptera: 1 species). Mosquitos in has been used to reduce or eliminate infections in F.candida the genus Aedes (Diptera: Culicidae) and vinegar flies in the (Pike and Kingcombe 2009; Timmermans and Ellers 2009), genus Drosophila (Diptera: Drosophilidae) were the focus of for which treatments of other antibiotics have proven ineffec- most of these studies with 13 and 17 papers published on these tive (Giordano et al. 2010). groups, respectively. Given that an estimated 3.3×106 species Antibiotics other than tetracycline and rifampicin have of arthropods may be infected with Wo l b a c h i a (Hilgenboecker been rarely tested and seem to be generally infective et al. 2008), these results emphasize that current knowledge on (Table 1). Use of ciprofloxacin and gentamicin were ineffec- how to cure infections of these bacteria derives from a small tive in removing Wo l b a c h i a from the wasp, Asobara tabida fraction of the total host taxa. (Hymenoptera: Braconidae) (Dedeine et al. 2001). Infections in F.candidawere not cured with use of tylosin, ampicillin or Classes of antibiotics Our survey identified at least 18 antibi- oxytetracycline (Giordano et al. 2010). However, oxytetracy- otics that have been assessed for efficacy in curing Wo l b a c h i a cline was used to remove Wo l b a c h i a from the uzi fly, Exorista in one or more host species (Table 1). Modes of action for sorbillans (Diptera: Tachinidae) (Guruprasad et al. 2011). these antibiotics include: i) inhibition of cell wall synthesis Antibiotics with the same mode of action may or may not (ampicillin, cephalosporin, penicillin), ii) inhibition of protein be similarly effective. Erythromycin, gentamicin, oxytetracy- synthesis (chloramphenicol, doxycycline, erythromycin, gen- cline and tetracycline each inhibit protein synthesis. When tamicin, kanamycin, minocycline, oxytetracycline, spectino- applied to E. sorbillans, the latter two compounds cured mycin, streptomycin, tetracycline, tylosin), iii) interference infections of Wo l b a c h i a at concentrations of 0.02 with nucleic acid synthesis (ciprofloxacin, rifampicin) and (Guruprasad et al. 2011) and 0.015 (Puttaraju and Prakash iv) antimetabolites (sulphonamides). 2005), respectively. Conversely, at concentrations of 2 mg/ml, By a large margin, tetracycline is the most commonly used tetracycline, but not gentamicin, was effective in removing antibiotic. It has been used to cure infections in mites, beetles, infections of Wo l b a c h i a from A. tabida (Dedeine et al. 2001). Author's personal copy

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Ta b l e 2 Summary of studies reporting on use of temperatures to cure infections of Wolbachia in arthropods

Taxa (common namea) Treatment Outcome Reference

Class Arachnida Acarina (chiggers, mites, ticks) Phytoseiidae Phytoseiulus persimilis 20 °C 48 h (cureb) (Enigl et al. 2005) 25 °C 16 h (cure) Tetranychidae (spider mites) Tetranychus piercei 34±1 °C 6 generations (mostly cure) (Zhu et al. 2012) Tetranychus urticae 32±0.5 °C 4 generations (cure) (Van Opijnen and Breeuwer 1999) Class Insecta Coleoptera (beetles) Tenebrionidae (darkling beetles) Tribolium confusum 37 °C raised larvae 22 days (cure) (Stevens 1989) Diptera (gnats, mosquitoes, true flies) Culicidae (mosquitoes) Aedes albopictus 25 °C and 37 °C raise from Density : 37 °C<25 °C (Wiwatanaratanabutr and larval to adult Kittayapong 2009) 25 °C and 37 °C Density : 37 °C《 25 °C (Wiwatanaratanabutr and Kittayapong 2006) Aedes polynesiensis 32–33 °C 5–7 days (cure) (Wright and Wang 1980) Tachinidae (tachinid flies) Exorista sorbillans 33±1 °C 6 generations (cure) (Guruprasad et al. 2011) Homoptera (scale insects, treehoppers, whiteflies) Delphacidae (delphacid planthoppers) Sogatella furcifera 30 or 35 °C during several No cure (Nakamura et al. 2012) nymphal stages Hymenoptera (ants, bees, wasps) Encyrtidae (encyrtid wasps) Apoanagyrus diversicornis 33 °C 2 generations (partial curec) (Pijls et al. 1996) 37 °C lethal to host Pteromalidae (pteromalid wasps) Urolepis rufipes 34±0.5 °C 6 generations (cure) (Kyei-Poku et al. 2003) Nasonia vitripennis 18 °C and 30 °C Reduce densities by 74 % compared to 25 °C (Bordenstein and Bordenstein 2011) Scelionidae (scelionid wasps) Te l e n o m u s n a w a i 35 °C for the last 3 days Partial cure (Arakaki et al. 2000) of pupae Psocoptera (psocids, bark lice, book lice) Liposcelididae Liposcelis tricolor 33 °C 6 generations (cure) (Jia et al. 2009) Thysanoptera (thrips) Aeolothripidae (banded thrips, broad-winged thrips) Franklinothrips vespiformis 35 °C for pupae 24 h (no cure) (Arakaki et al. 2001) a Preference for common names is given to those reported in the Integrated Taxonomic Information System (http://www.itis.gov/) b ‘Cure’ = authors report the elimination of infection from some or all treated individuals c ‘No maternal transmission’ = treated female individuals remain infected, but do not transmit infection to their progeny

Similarly, at concentrations of 50 mg/ml, tetracycline, but not Methods of application Antibiotics are normally applied in erythromycin, eliminated infections of Wolbachia from the the diet of the infected arthropod host. They have been added predatory thrips, Franklinothrips vespiformis (Thysanoptera: to honey or sugar water to treat adult wasps (Kyei-Poku et al. Aeolothripidae) (Arakaki et al. 2001). 2003; Pijls et al. 1996; White et al. 2009), to artificial diet to Author's personal copy

Methods to remove Wolbachia 9 treat fly larvae (Hoffmann 1988), or to dry diet to treat flour was achieved with a rearing temperature of 28 °C beetles (Wade and Stevens 1985). Antibiotics can also be (Hoffmann et al. 1986). added to water to treat mosquito larvae (Hong et al. 2002; Elevated temperatures also may increase the efficacy of Rasgon and Scott 2003; Yen and Barr 1973), or to treat antibiotic treatments. At 20 °C, treatments of tetracycline and phytophagous mites feeding on leaves floating on the treated rifampicin proved ineffective in removing Wo l b a c h i a from the water. The alfalfa weevil, Hypera postica (Coleoptera: wasp Leptopilina clavipes (Hymenoptera: Figitidae), whereas Curculionidae), was cured by feeding adults foliage on which curewasachievedwiththesesametreatmentsat25°C had been sprayed a water suspension of crushed antibiotic (Schidlo et al. 2002). Conversely, elevated heat used alone tablets (Klostermeyer 1978). Parasitoid wasps have also been or in combination with tetracycline failed to cure Wo l b a c h i a treated by providing females with hosts into which antibi- infections in larvae of Ae. albopictus (Dobson and oticshavebeeninjecteddirectly(Abeetal.2003; Richardson Rattanadechakul 2001). High mortality precluded use of et al. 1987), or which have been reared on diet laced with 35 °C, whereas larvae successfully reared at 32 °C retained antibiotics (Dedeine et al. 2001; Mochiah et al. 2002; their infections. Mouton et al. 2003).

4 Other methods to obtain Wo l b ac h i a-free hosts 3 Temperature treatment However, treatments need not only be antibiotics or tempera- Our survey identified a further 14 papers that used extreme tures. Rearing hosts under crowded conditions has been temperatures to treat infections of Wo l b a c h i a.Mostofthese shown to reduce Wolbachia titre in the mosquito studies examined applications of high temperatures, which A. albopictus (Wiwatanaratanabutr and Kittayapong 2009), can be effective when the lethal threshold for the symbionts and cause the loss of Wolbachia strains from D. simulans is less than that for the host. Rearing the two-spotted spider infected with multiple strains (Sinkins et al. 1995). It may also mite Tetranychus urticae (Acarina: Tetranychidae) at 32 °C for be possible to obtain Wo l b a c h i a-infected and uninfected hosts four generations achieved a cure success of 71 % that attained from field collections. This has been done for a number of 100 % after six generations (Van Opijnen and Breeuwer species including T. confusum (Wade and Stevens 1985), and 1999). Complete cure after six generations appears to be a several species of parasitic wasps in the genus Trichogramma common theme among such studies, being the time need to (Stouthamer et al. 1990). cure Wolbachia infections in the parasitic wasp Urolepis rufipes (Hymenoptera: Pteromalidae) (at 34 °C - Kyei-Poku et al. 2003), the uzi fly E. sorbillans (at 33 °C - Guruprasad 5 Other considerations et al. 2011), and the mite Te t r a n y c h u s p i e rc e i (Acarina: Tetranychidae) (34 °C - Zhu et al. 2012). Relative to individ- Detecting infections Assessing the efficacy of a given treat- uals reared at 25 °C, titres of Wolbachia were reduced by ment necessarily relies upon the ability to detect infections. rearing the mosquito Ae. albopictus at 37 °C Early research detected infections with use of microscopy (Wiwatanaratanabutr and Kittayapong 2009). (Byers and Wilkes 1970; Wright and Wang 1980; Yen and Less intuitive is the use of lower temperatures to cure Barr 1973) or crossing experiments (e.g., see Table 2 in infections. Of the few such examples, removal of Wo l b a c h i a Dobson and Rattanadechakul 2001). More recent studies typ- from the mite Phytoseiulus persimilis (Mesostigmata: ically now use molecular methods. DNA is extracted from the Phytoseiidae) was achieved by exposing the host to 20 °C host and amplified using polymerase chain reaction (PCR) for 48 h and to 25 °C for 16 h (Enigl et al. 2005). In a second and primers specific for Wo l b a c h i a genes (e.g., Braig et al. such example, Wolbachia titres were reduced in the wasp 1998). Detection of an amplified product (amplicon) provides Nasonia vitripennis (Hymenoptera: Pteromalidae) when evidence of infection. The amplicon can then be sequenced to reared at 18 versus 25 °C (Bordenstein and Bordenstein 2011). confirm its Wo l b a c h i a origin and identify the Wo l b a c h i a strain. Effective temperature varies with host species. Partial re- However, use of standard PCR methods may not detect low moval of Wolbachia was achieved by rearing the wasp titre infections such that ‘false’ negatives may be relatively Apoanagyrus diversicornis (Hymenoptera: Encyrtidae) at common within a given host taxon (Schneider et al. 2013; 33 °C, whereas a rearing temperature of 37 °C was lethal to Arthofer et al. 2009). thehost(Pijlsetal.1996). Conversely, a rearing temperature Greater confidence can be given to determinations of in- of 33 °C was ineffective in removing Wolbachia from fection status when molecular methods are supported by ex- T. confusum, whereas success was achieved with a rearing perimental studies. For example, if cytoplasmic incompatibil- temperature of 37 °C (Stevens 1989). Removal of Wo l b a c h i a ity is induced when ‘cured’ females mate with infected males, from the fly Drosophila simulans (Diptera: Drosophilidae) this provides independent evidence that the treatment has been Author's personal copy

10 Y.-Y. Li et al. successful. However, this assumes that the Wo l b a c h i a is a CI- tetracycline for 24 h and then screened with PCR assays. inducing strain. In other cases, infections may have no evident Infections were not detected in 55 % of exposed females and effect on host biology or reproduction such that comparison of none of the exposed females transmitted infections to their infected versus cured populations may not be informative progeny. In this same host, exposure to rifampicin for 48 h (Gomi et al. 1997). blocked transmission of Cardinium from infected females to their progeny (Giorgini et al. 2009; Zchori-Fein et al. 2004). A Other symbionts Although the current review is limited to duration of 48 h was also effective in eliminating infections of discussion of Wo l b a c h i a bacteria, readers should be aware that Arsenophonus and Wo l b a c h i a from adult whiteflies, Bemisi there are numerous other symbionts that occur in arthropods tabaci, exposed to tetracycline, amplicillin or rifampicin and affect their host’s reproduction. These include species in (Ahmed et al. 2010). Conversely, treatments may be required the genera Rickettsia (α-Proteobacteria) (Rickettsiaceae), for several generations. Exposure to elevated heat was re- Arsenophonus (γ-Proteobacteria) (Enterobacteriaceae), quired for six generations to remove infections of Wo l b a c h i a Cardinium (Bacteroidaceae) and Flavobacterium from the uzi fly, Exorista sorbillans (Guruprasad et al. 2011), (Flavobacteriaceae), and Spiroplasma (Spiroplasmataceae) and from the pteromalid wasp, Urolepis rufipes (Kyei-Poku (Duron et al. 2008). Only a few studies have reported on et al. 2003). methods to cure hosts of these non-Wolbachia symbionts. Variation in treatment time can be attributed to a number of Treatment with rifampicin (50 mg/ml for 48 h) was shown factors. One such factor is treatment type. Infections of to cure infections of Cardinium from adult females of the Wo l b a c h i a in U. rufipes were removed in four generations wasp, Encarsia pergandiella (Hymenoptera: Aphelinidae) with use of an antibiotic, but required six generations using (Zchori-Fein et al. 2001). Treatment with tetracycline an elevated temperature (Kyei-Poku et al. 2003). A second (50 mg/ml for 24 h) removed infections of Cardinium from factor is the life stage of the host. For the mosquito Aedes adults of E. hispida (Giorgini et al. 2009). albopictus, higher cure rates and greater host survival was achieved when antibiotic treatments were applied to adult Measuring treatment success Treatments are generally con- versus larval stages (Dobson and Rattanadechakul 2001). A sidered successful if they remove infections from exposed third factor is variation in resistance among symbionts, which individuals. However, other definitions may apply. can co-occur in the same host (Duron et al. 2008). For co- Treatments can succeed in curing a portion of exposed indi- infections of Wolbachia and Cardinium, Morimoto et al. viduals, but fail to remove infections from the population (2006) concluded that infections of the latter could be elimi- (Ahmed et al. 2010). In such cases, maternal lineages can be nated with use of penicillin-G or chloramphenicol without established from cured individuals to obtain uninfected popu- eliminating infections of the former. lations. An interesting example of this is provided by White A further factor is whether the objective is to cure an et al. (2009), who used low doses of antibiotics to uncouple individual or to cure a population. Treatments can impair co-occurring infections of Wo l b a c h i a and Cardinium in the maternal transmission of symbionts. Thus, an infected female wasp Encarsia inaron (Hymenoptera: Aphelinidae). Adult may produce both infected and uninfected individuals. females thus treated laid eggs containing either or both sym- Uninfected individuals can be isolated and used to quickly bionts, which allowed for propagation of lineages infected generate uninfected lineages (e.g. Stouthamer et al. 1990; with Wo l b a c h i a or infected with Cardinium. White et al. 2009). However, such lineages can result in In other cases, treatments may fail to remove infections genetic bottlenecks, such that differences between infected from exposed individuals, but succeed in preventing transmis- populations and uninfected lineages may partially reflect var- sion of Wo l b a c h i a to their offspring (Giorgini et al. 2009). iation in maternal host genotype. Conversely, treatments can Treatments also may succeed in reducing infection titre, but be applied to entire populations to maintain genetic diversity fail to eliminate infections. Such an outcome can still be until no further infections are detected. However, Wo l b a c h i a considered a success if the objective is to demonstrate the often confer reproductive advantages to infected female hosts. effect of titre on host-Wolbachia interactions (Zchori-Fein Their greater fecundity will tend to suppress the numbers of et al. 2000). Conversely, success may be transitory. uninfected females in the population and prolong the time Reductions in titres may be reversed in hosts one of more needed to obtain a population-level cure. generations post-treatment such that the effect of the treatment may be transitory (Stouthamer et al. 1990). Non-target effects Treatments can cause non-target effects that may confound the interpretation of data. In addition to Time to cure Treatments may need to be applied for days or Wolbachia, hosts may harbour other types of maternally- months to remove infections of symbiotic bacteria. Adult inherited symbionts, nutritional gut symbionts, or pathogens. females of the whitefly parasitoid Encarsia hispida infected Thus, a treatment to eliminate Wo l b a c h i a may also remove with Cardinium (Giorginietal.2009) were exposed to pathogenic bacteria from the host making it difficult to Author's personal copy

Methods to remove Wolbachia 11 determine if gains in host fitness are due to the absence of the Use of next-generation sequencing (NGS) is expected to symbiont, the pathogen, or both. Given their modes of action rapidly expand knowledge on the complexity of arthropod (e.g., inhibition of protein synthesis), it is not surprising that holobionts and allow for a more critical assessment of treat- hosts also may suffer direct lethal or sublethal effects of antibi- ment methods and outcomes. Whereas convention PCR otic exposure. At lower concentrations, tetracycline can cure methods are used to identify the presence of a suspected Wolbachia in the , Encarsia formosa symbiont (e.g., Wo l b a c h i a), NGS allows for both the detection (Hymenoptera: Aphelinidae), but exposure at 50 mg/ml can kill and relative abundance of all bacteria (suspected and unsus- females within 3 days (Stouthamer and Mak 2002). Antibiotic pected) in the arthropod. It is a relatively new technology that treatments also may reduce the function and density of is prohibitively expensive for most labs. However, the rapid mitochondria in the host for one or more generations uptake of this method is anticipated because of declining costs post-treatment (Ballard and Melvin 2007). Thus, it is com- and general acceptance. For example, in a survey of ants mon to provide hosts a recovery period of one or more comparing traditional methods with NGS, the latter approach generations post-treatment prior to their use in experiments. identified an additional 445 microbial operational taxonomic This allows time for the host population to reacquire gut units (OTUs) not detected with traditional techniques (Kautz bacteria and recover from direct adverse effects of the et al. 2013). treatment. It does not, however, allow hosts to reacquire With specific reference to the topic of this review, NGS will non-Wolbachia, maternally-inherited symbionts that may provide a powerful tool to assess the effect of treatments on have been eliminated by the treatment. both the target symbiont, but also on all other bacteria in the There is also the possibility that the cure may be worse than host. Results may show that effects attributed to Wo l b a c h i a the infection. Removal of Wo l b a c h i a from the collembolan may, in some cases, be due to other symbionts or possibly Folsoma candida caused female sterility (Pike and interactions among symbionts. NGS also will allow a critical Kingcombe 2009). Similarly, female Asobara tabida Nees assessment on the post-treatment period required for non- (Hymenoptera: Braconidae) cured of Wolbachia infections target symbionts to recovery from treatment effects. There were unable to develop eggs (Dedeine et al. 2001). Thus, in remains a need to expand research on a broader diversity of each case, it was impossible to maintain self-sustaining colo- arthropod taxa. nies of cured individuals. Acknowledgements We thank Agriculture and Agri-Food Canada and the Chinese Ministry of Education for providing Y. Li the opportunity to work at the Lethbridge Research Centre (LRC). We also thank Paul C. 6 Summary and future directions Coghlin (LRC) for his guidance and continuous encouragement through this project. The intimate relationship between symbionts and host is for- malized with the terms ‘holobiont’ and ‘hologenome’.The former refers to a plant or and all of its associated References microorganisms, whereas the latter refers to the collective genome of this assemblage (Zilber-Rosenberg and Abe J, Kamimura Y, Kondo N, Shimada M (2003) Extremely female- Rosenberg 2008). 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Cell Microbiol 15(9):1527–1544. doi:10.1111/cmi.12136 most common method and is typically successful. Rifampicin Almeida Rd, Lenteren Jv, Stouthamer R (2010) Does Wo l b a c h i a infection is secondarily used, and may be successful when tetracycline affect Trichogramma atopovirilia behaviour? Braz J Biol 70:435– is not. Elevated temperatures can be used to eliminate infec- 442. doi:10.1590/S1519-69842010005000016 – tions, but is not often used. The duration of treatment required Arakaki N, Miyoshi T, Noda H (2001) Wo l b a c h i a mediated partheno- genesis in the predatory thrips Franklinothrips vespiformis to eliminate Wo l b a c h i a from the host may range from days to (Thysanoptera: Insecta). Proc R Soc Lond Ser B Biol Sci generations. 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