Invasion of Wolbachia into Anopheles and Other Insect Germlines in an Ex vivo Organ Culture System Grant L. Hughes1., Andrew D. Pike2., Ping Xue2, Jason L. Rasgon 1 * 1 The Department of Entomology, Center for Infectious Disease Dynamics and Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America Abstract The common bacterial endosymbiont Wolbachia manipulates its host’s reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria. Citation: Hughes GL, Pike AD, Xue P, Rasgon JL (2012) Invasion of Wolbachia into Anopheles and Other Insect Germlines in an Ex vivo Organ Culture System. PLoS ONE 7(4): e36277. doi:10.1371/journal.pone.0036277 Editor: Luciano A. Moreira, Centro de Pesquisas Rene´ Rachou, Brazil Received November 28, 2011; Accepted April 1, 2012; Published April 30, 2012 Copyright: 2012 Hughes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by United States National Institutes of Health grant R21AI070178 to JLR. GLH was supported by the Johns Hopkins School of Public Health (JHSPH) Jane Welsh Russell Scholarship and a Johns Hopkins Malaria Research Institute (JHMRI) postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jlr54@psu.edu . These authors contributed equally to this work. Introduction Wolbachia are intracellular a-proteobacteria that infect approx- imately two-thirds of insect species along with numerous other arthropods [1,2]. Generally classified as reproductive parasites, Wolbachia are able to manipulate their hosts reproduction in various ways [3]. These reproductive manipulations exploit the maternal transmission route of the bacteria to the progeny of the host, facilitating the spread of Wolbachia into the host population. In addition to maternal transmission, the incongruence between the phylogenies of Wolbachia and their hosts suggests horizontal transmission has occurred repeatedly over evolutionary time, which has extended the host range of the bacteria [4,5,6]. The ability of Wolbachia to invade new species has been reproduced in the laboratory, with artificial transfers into novel insect species. Despite transinfection of Wolbachia into many new insect species, it is uncertain why some species are capable of infection yet others are seemingly not. For either horizontal transmission or transinfection to occur, Wolbachia must establish in the female germline to allow the bacteria to be transmitted to the next generation. When artificially transferred into a new host, Wolbachia are usually microinjected into the preblastoderm embryo, a scenario that is unlikely to occur in nature. However, in experiments that more closely mimic potential natural routes of horizontal transfer, Wolbachia has been shifted between insects by adult injection, hemolymph transfer and co-rearing [7,8,9]. When injected into uninfected Drosophila, Wolbachia migrate across numerous tissues to establish in the stem cell niche of the ovary [7]. Hemolymph transfer between infected and uninfected pillbugs (Armadillidium vulgare, Porcellionides pruinosus and Chaetophiliscia elongate) resulted in stable intraspecific transfer of Wolbachia and manipulation of host reproductive [8]. The capacity for Wolbachia to spread though host populations and interfere with pathogen development in insects makes them attractive agents for the control of vector-borne diseases. Currently, Wolbachia is being investigated to control malaria, a devastating disease transmitted by Anopheles mosquitoes. Wolbachia is capable of infecting Anopheles cells in vitro and Anopheles somatic tissues in vivo, and influences the expression of numerous host genes [10,11,12]. After injection into adult Anopheles mosquitoes, Wolbachia influences Plasmodium levels, with wAlbB and wMelPop reducing P. falciparum intensity [10]. Interestingly, somatic infection of An. gambiae with the wAlbB strains enhances oocyst density of P. berghei, the model murine malaria species [13]. However, all Anopheles mosquitoes lack Wolbachia infection in the wild and despite numerous attempts seem impervious to stable transinfection in the lab [14,15]. To increase the probability of infecting Anopheles mosquitoes, a variety of combinations of PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36277