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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.
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].
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 (mosquito) |
||||
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 wCof_A wAlbB_B wPip_B wCauA_A wCauB_B |
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 wKue_A wCauA_A |
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 wCauB_B wKue_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 C7-10B |
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 mosquito |
||||
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 wMelPop-CLA_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 mosquito |
||||
Mos-55 | wMelPop-CLA_A | infected Aa23 cells | [15] | |
Sua5B | wAlbB_B wRi_A |
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 wMel_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 |
[39] | |
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 wMel_A wMelPop_A |
infected Aa23 cells infected Aag2 cells infected Aag2 cells |
[41] | 50 passages |
Lepidopteran | ||||
BCIRL-HZ-AM1-G5 Heliothis zea |
wStri_B | L. striatellus ovary | [19] | |
Sf9 Spodoptera frugiperda |
wRi_A | D. simulans eggs | [18] | |
Sf9 Spodoptera frugiperda |
wCauB_B | Ephestia kuehniella eggs | [20] | |
Tick | ||||
Ixodes scapularis | wAlbB_B, wStri_B wCfe_F |
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] | |
Mammal | ||||
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 |