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Wolbachia is an intracellular bacterium that occurs in arthropods and in filarial worms. First described nearly a century ago in the reproductive tissues of Culex pipiens mosquitoes, Wolbachia is now known to occur in roughly 50% of insect species, and has been considered the most abundant intracellular bacterium on earth. In insect hosts, Wolbachia modifies reproduction in ways that facilitate spread of the microbe within the host population, but otherwise is relatively benign. In this “gene drive” capacity, Wolbachia provides a tool for manipulating mosquito populations. In mosquitoes, Wolbachia causes cytoplasmic incompatibility, in which the fusion of egg and sperm nuclei is disrupted, and eggs fail to hatch, depending on the presence/absence of Wolbachia in the parent insects. Recent findings demonstrate that Wolbachia from infected insects can be transferred into mosquito species that do not host a natural infection. When transinfected into Aedes aegypti, an important vector of dengue and Zika viruses, Wolbachia causes cytoplasmic incompatibility and, in addition, decreases the mosquito’s ability to transmit viruses to humans. 

alpha-proteobacteria reproductive parasite symbiont mosquito insect cell lines genetic manipulation cell culture
Subjects: Microbiology
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    1. Introduction

    Wolbachia is an obligate intracellular microbe first described in reproductive tissues of Culex pipiens mosquitoes nearly a century ago [1][2]. Like Escherichia coli , Wolbachia is a Gram-negative bacterium in the phylum Proteobacteria: the purple bacteria and their relatives. Proteobacteria include nine monophyletic classes representing tremendous biodiversity. Among these, the genera Ehrlichia and Anaplasma , which can cause disease in humans, are classified with Wolbachia as members of the alpha-proteobacteria, in the order Rickettsiales, family Anaplasmataceae. Wolbachia is uniquely associated with invertebrates, does not infect vertebrate hosts, and replicates only within a eukaryotic host cell. In contrast, E. coli and many familiar Gram-negative pathogens of humans classified as gamma-proteobacteria can be cultured in liquid medium and plated on solid media as free-living microbes.

    Knowledge of well-studied free-living bacteria provides an important framework for investigating the genetics and physiology of Wolbachia , now known to infect a high proportion of insect species, in addition to other arthropods and filarial worms, all members of the Ecdysozoa. Because of its widespread distribution among insects [3][4], Wolbachia provides a model system for exploring biological interactions between an intracellular microbe, the invertebrate host cells in which it resides, and the diversity of reproductive phenotypes with which it is associated [5][6]. In species that harbor Wolbachia, the bacterium is transmitted vertically, from mother to offspring, which retain the infection. In most arthropods, Wolbachia alters reproduction in diverse ways that favor its invasion of naive populations, and is sometimes considered a reproductive parasite. In contrast, Wolbachia is an essential symbiont in filarial worms [7][8][9]. In mosquitoes, Wolbachia causes a reproductive distortion called cytoplasmic incompatibility (CI), which has important applications in vector control [10].

    2. Wolbachia in Insect Cell Lines

    Wolbachia ’s obligate intracellular lifestyle complicates the biochemical and genetic analyses that could advance pest control and anti-filarial applications. Even with hosts amenable to laboratory rearing, maintenance of colonies, dissection of infected tissues, and embryonic microinjection are labor-intensive and time-consuming. Moreover, many existing laboratory colonies are highly inbred, complicating cage studies that address fitness. The utility of Wolbachia in control applications would be enhanced if the microbe could be experimentally manipulated by genetic engineering to express selectable markers, which in turn will be advanced by improving manipulation of Wolbachia in cell lines and expanding the diversity of Wolbachia strains that can be investigated in culture. A modest advance would be adaptation of a filarial strain of Wolbachia to a cell line; at present, Wolbachia -infected insect cell lines are used as a surrogate to identify new drugs that target Wolbachia for treatment of filarial diseases [11][12][13].

    The author’s research focuses on systematic exploration of Wolbachia propagation in cultured cells as a substitute for the differentiated host tissues, such as ovaries and testes, in which Wolbachia is most abundant. Cell lines used to propagate Wolbachia are listed in Table 1 , wherein supergroup designations are noted after the strain name; for example, w Pip_B indicates that w Pip is classified in supergroup B. With the exception of a single member of supergroup F from the cat flea [14], only members of supergroups A and B, sometimes called the “pandemic” supergroups, have been maintained in insect cell lines. The reader should note that, in some cases, an infected cell line may have been sub-cultured only a limited number of times and/or has a very long doubling time, and that the same cell line may have been infected with the same strain of Wolbachia by different investigators, and given a different name. An important incentive for employing cell lines was the possibility that preadaption to cultured cells might improve the likelihood that Wolbachia would establish in novel hosts infected by embryonic microinjection, and towards this end, a few lines have been maintained for several years [15]. In other cases, which are not reviewed in detail here, infected cell lines have been used to test effects of Wolbachia on viral replication in efforts that generally validate the anti-pathogen responses seen in transinfected mosquitoes. Finally, as with Wolbachia itself, a uniform descriptive label for infected cell lines remains to be developed.

    Table 1. Cell lines in which Wolbachia strains have been propagated.

    Cell Line Designation Wolbachia Strain_Supergroup Source of Wolbachia Reference Comments
    Dipteran cell lines        
    Aedes albopictus
    Aa23 wAlbB_B Aedes albopictus embryos [16] First infected cell line; established from naturally infected Ae. albopictus; one of two Wolbachia strains
    Aa23(T) wMel_A infected RML-12 cells [17] 12 passages
    Aa23(T) wRi_A
    D. simulans eggs
    D. simulans eggs
    infected Aa23 cells
    Cx. pipiens eggs
    Cadra cautella eggs
    Cadra cautella eggs
    [18] Demonstration of shell vial technique; details focus on wRi
    Aa23(T) wMelPop w1118 embryos [15] Generated wMelPop-CLA
    NIAS-AeAl-2 wStri_B
    L. striatellus ovary
    Ephestia kuehniella eggs
    Cadra cautella eggs
    [19] Infected from small inoculum; one ovary, or 80–100 eggs; Infected AeAl-2 cells form aggregates; occasional addition of uninfected cells to infected cultures
    NIAS-AeAl-2 wCau_A
    Ephestia kuehniella eggs
    Ephestia kuehniella eggs
    Ephestia kuehniella eggs
    [20] Two stages: infection and maintenance
    RML-12 wMelPop-CLA_A infected Aa23 cells [15] wMelPop transferred to cells; serial passage; reintroduction into original host by microinjection; some loss of virulence; “genetic adaptation” to improve transfer to new hosts
    RML-12 wMel_A O’Neill et al.; cited in [17] personal communication [17] Maintained for 3 years
    C6/36 wRi_A D. simulans eggs [18]  
    C6/36 wMel_A infected RML-12 cells [17] Stable; higher density than RML-12 cells
    C6/36 wAlbB_B infected Aa23 cells [21]  
    C6/36 wAlbB_B infected Aa23 cells [22]  
    C6/36 wMelPop-CLA_A RML-12-CLA [23] C6/36.wMelPop-CLA
    C6/36 wAlbB_B infected Aa23 cells [24] Virus screen
    C7-10 wStri_B NIAS-AeAl-2 [25] Called C/wStri1 line
    C7-10 wAlbB_B infected Aa23 cells [26] Infected line: C7-10B
    C7-10 wRi_A D. simulans eggs [26] Infected line: C7-10R
    C7-10R more stable, uniform than
    TK-6 (C7-10) wAlb_B infected Aa23 cells [27] Stable 5 months
    Mtx-5011-256 wStri_B C/wStri1 cells [28] Lower MOI than C7-10; aneuploidy a factor?
    Aedes aegypti
    Aag2 wAlbB_B infected Aa23 cells [29] Line called Aag2.wAlbB
    Aag2 wAlbB_B infected Aa23 cells [30] Line called w-Aag2
    Aag2 wMel_A D. melanogaster embryos [31][32] Line called Aag-2wMel
    Aag2 wMel_A
    Infected RML-12 cells
    Infected RML-12 cells
    [33] [15]
    Aa-20 wMelPop-CLA_A Not stated [34] Mos 20; CVCL_Z353; [35]
    Anopheles gambiae
    Mos-55 wMelPop-CLA_A infected Aa23 cells [15]  
    Sua5B wAlbB_B
    infected Aa23 cells
    D. simulans eggs
    [36] Best was 1/103 cells infected
    Drosophila melanogaster        
    S2 wRi_A D. simulans eggs [18]  
    S2 strain from Dm2008Wb1cells infected, D. melanogaster [37] (from abstract; Russian)
    Dm2008Wb1 primary cell culture infected, D. melanogaster [37] (from abstract; Russian)
    JW-18 wMel-Pop_A infected, D. melanogaster [13] Albendazole sulfone inhibits
    1182-48 wMelPop_A infected JW-18 cells [38] Acentriolar haploid line
    S2R+ wMelPop_A infected JW-18 cells [38] Tetraploid male cells; higher Wolbachia titers
    Lutzomyia longipalpis (sandfly)        
    LL5 wMelPop-CLA_A
    infected RML-12 cells
    infected RML-12 cells
    [39] Immune activation
    unstable; no effect on Leishmania
    Lulo wMelPop-CLA_A
    wMel_A (unstable)
    infected RML-12 cells
    infected RML-12 cells
    Culicoides sonorensis
    (Biting midge)
    W3 wAlbB_B infected Aa23 cells [40] Line W3
    W8 wAlbB_B infected Aa23 cells [40] Higher density than W3
    Hematobia irritans
    (Horn fly)
    HIE-18 wAlbB_B
    infected Aa23 cells
    infected Aag2 cells
    infected Aag2 cells
    [41] 50 passages
    Heliothis zea
    wStri_B L. striatellus ovary [19]  
    Spodoptera frugiperda
    wRi_A D. simulans eggs [18]  
    Spodoptera frugiperda
    wCauB_B Ephestia kuehniella eggs [20]  
    Ixodes scapularis wAlbB_B, wStri_B
    infected mosquito cells
    cat fleas
    [14] wStri_B, 29 passages
    wCfe_F, 2 passages
    Ixodes ricinus wAlbB_B, wStri_B infected mosquito cells [14]  
    Riphicephalus microplus wAlbB_B, wStri_B infected mosquito cells [14]  
    L929 (mouse) wStri_B L. striatellus ovary [19] Cells maintained at 28 °C
    Filarial screening        
    Aa23 wAlbB_B   [11] Anti-filarial screen
    C6/36 wAlbB_B infected Aa23 cells [12] Macrofilaricides
    JW-18 wMelPop_A D. melanogaster w1118 [13] Anti-filarial screen
    Insect cell lines in which Wolbachia has been maintained. Columns from left to right show: (1) cell lines, arranged in groups according to species from which the cell line was derived; (2) Wolbachia strain_supergroup; (3) source of the material introduced into the cell line; (4) reference; (5) brief comments.

    3. Why Cultured Cells?

    If the streamlined Wolbachia genome can be genetically engineered in the future, propagation of the altered genome will require efficient reintroduction into a host cell to allow replication and expansion of transformant populations. Use of cell lines offers a practical means of producing the large quantities of Wolbachia that will be needed to develop transformation protocols that are sufficiently robust for use in basic research and pest control applications. Although isolated examples of successful transformation of intracellular microorganisms such as Coxiella burnetti, the pathogen that causes Q fever, have been achieved, these remain labor intensive and have low frequencies of success [42]. Nevertheless, over the past two decades, remarkable progress towards cell-free culture of Coxiella has been achieved, despite its streamlined 2 Mb genome [43][44]. These successes underscore the importance of detailed attention to culture conditions and metabolic activities of obligate intracellular microbes. Wolbachia lacks pathogenicity to humans, and its genome is more extensively streamlined, relative to that of Coxiella. Nevertheless, the long evolutionary history of Wolbachia’s interaction with invertebrate hosts and its adaptations for germline transmission contribute to the value of Wolbachia as a model system for understanding the biology of obligate intracellular bacteria in invertebrate cells and manipulating their biology for control of insect pests.


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    Subjects: Microbiology
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