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Wolbachia pipientis had its scientific debut nearly a century ago and has recently emerged as a target for therapeutic treatment of filarial infections and an attractive tool for control of arthropod pests. Wolbachia was known as a biological entity before DNA was recognized as the molecule that carries the genetic information on which life depends, and before arthropods and nematodes were grouped in the Ecdysozoa. Today, some investigators consider Wolbachia the most abundant endosymbiont on earth, given the numbers of its hosts and its diverse mutualistic, commensal, and parasitic roles in their life histories. Recent advances in molecular technologies have revolutionized our understanding of Wolbachia and its associated reproductive phenotypes. New models have emerged for its investigation, and substantial progress has been made towards Wolbachia-based interventions in medicine and agriculture. Here I introduce Wolbachia, with a focus on aspects of its biology that are covered in greater detail in subsequent chapters., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Eventual genetic engineering of Wolbachia will require maximizing recovery of infectious bacteria, maintaining Wolbachia in a viable state for efficient manipulation, and reinfection of host cells for propagation and expansion of recombinant progeny. Challenges to manipulating Wolbachia arise from its obligate intracellular lifestyle and inability to divide outside a host cell, requiring modifications of standard bacteriological methods. The Aedes albopictus C7-10 cell line has proven to be a good recipient for the Wolbachia supergroup B strain, wStri, from the planthopper Laodelphax striatellus; the persistently infected C/wStri1 population provides a source of wStri inoculum that can be used systematically to explore conditions that increase yields of infectious material from input Wolbachia and identify conditions conducive to Wolbachia replication. After reintroduction into naive, uninfected C7-10 cells, wStri recovery, relative to the input inoculum, is influenced by diverse conditions, such as the cell cycle arrest that follows treatment of infected host cells with the insect steroid hormone, 20-hydroxyecdysone. Pretreatment of recipient cells with mitomycin C, which cross-links DNA and inhibits host cell replication, can improve recovery from low levels of input Wolbachia. This protocol describes preparation of infectious inoculum from Aedes albopictus C/wStri1 cells and amplification of Wolbachia in mitomycin C-treated, uninfected C7-10 cells, followed by a brief description of conditions used for various small-scale manipulations of Wolbachia in infected cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The springtail, Folsomia candida, is a soil arthropod commonly used to evaluate environmental toxins. Conflicting data on the toxicity of the herbicide paraquat prompted re-evaluation of its effects on F. candida survival and reproduction. Paraquat has an LC 50 of about 80 μM when tested in the absence of charcoal; charcoal, often used in test arenas to facilitate visualization of the white Collembola, has a protective effect. Survivors of paraquat treatment fail to resume molting and oviposition, suggesting an irreversible effect on the Wolbachia symbiont that restores diploidy during parthenogenetic reproduction of this species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Wolbachia is an obligate intracellular bacterium that infects many species of insects, and has been of particular interest in recent efforts to reduce disease transmission by mosquitoes. Two aspects of Wolbachia biology underlie its applications for insect control: first, the bacterium behaves as a natural gene drive agent and, second, when introduced into mosquitoes that do not harbor Wolbachia in nature, infection reduces survival of pathogens. These properties support efforts to explore the basic biology of Wolbachia in insect cell lines, which can produce sufficient infectious material for microbiological studies and microinjection into novel hosts. When introduced into naïve C7-10 Aedes albopictus mosquito cells, the yield of Wolbachia strain wStri improves, roughly in proportion to the size of the inoculum, as exponential growth of the host cell ceases. Wolbachia yields also increase when persistently infected C/wStri1 cells or naive, newly infected cells are treated with 20-hydroxyecdysone (20E), which inhibits growth in the G1 phase of the cell cycle. These observations suggest that Wolbachia infection and replication are independent of exponential growth and mitosis of host cells. To explore yields of infectious bacteria in cells arrested prior to infection, I tested host cells pre-treated with mitomycin C, an agent that crosslinks DNA and prevents cell division that is used to produce "feeder layers" with mammalian cells. Yields of wStri per plate increased by about 50-fold relative to exponentially growing cells, and the multiplicity of infection necessary for a robust infection was reduced to a single bacterium per cell. These results suggest that Wolbachia infection and replication are supported by mitotically arrested cells and provide new insights into biological processes that influence maintenance of a widespread obligate intracellular bacterium., (© 2022. The Society for In Vitro Biology.)

Genomes of the obligate intracellular alpha proteobacterium Wolbachia pipientis often encode prophage-like regions, and in a few cases, purified particles have been recovered. Because the structure of a conserved WO phage genome has been difficult to establish, we examined paired terminase and portal genes in Wolbachia phages and prophages, relative to those encoded by the gene transfer agent RcGTA from the free-living alpha proteobacterium Rhodobacter capsulatus. Terminase and portal proteins from Wolbachia have higher similarity to orthologs encoded by RcGTA than to orthologs encoded by bacteriophage lambda. In lambdoid phages, these proteins play key roles in assembly of mature phage particles, while in less well-studied gene transfer agents, terminase and portal proteins package random fragments of bacterial DNA, which could confound elucidation of WO phage genomes. In WO phages and prophages, terminase genes followed by a short gpW gene may be separated from the downstream portal gene by open-reading frames encoding a GH_25 hydrolase/muramidase, a PD-(D/E)XK nuclease, a hypothetical protein and/or a RelE/ParE toxin-antitoxin module. These aspects of gene organization, coupled with evidence for a low, non-inducible yield of WO phages, and the small size of WO phage particles described in the literature raise the possibility that Wolbachia prophage regions participate in processes that extend beyond conventional bacteriophage lysogeny and lytic replication. These intervening genes, and their possible relation to functions associated with GTAs, may contribute to variability among WO phage genomes recovered from physical particles and impact the ability of WO phages to act as transducing agents., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Wolbachia is an obligate intracellular bacterium that has undergone extensive genomic streamlining in its arthropod and nematode hosts. Because the gene encoding the bacterial DNA recombination/repair protein RecA is not essential in Escherichia coli, abundant expression of this protein in a mosquito cell line persistently infected with Wolbachia strain wStri was unexpected. However, RecA's role in the lytic cycle of bacteriophage lambda provides an explanation for retention of recA in strains known to encode lambda-like WO prophages. To examine DNA recombination/repair capacities in Wolbachia, a systematic examination of RecA and related proteins in complete or nearly complete Wolbachia genomes from supergroups A, B, C, D, E, F, J and S was undertaken. Genes encoding proteins including RecA, RecF, RecO, RecR, RecG and Holliday junction resolvases RuvA, RuvB and RuvC are uniformly absent from Wolbachia in supergroup C and have reduced representation in supergroups D and J, suggesting that recombination and repair activities are compromised in nematode-associated Wolbachia, relative to strains that infect arthropods. An exception is filarial Wolbachia strain wMhie, assigned to supergroup F, which occurs in a nematode host from a poikilothermic lizard. Genes encoding LexA and error-prone polymerases are absent from all Wolbachia genomes, suggesting that the SOS functions induced by RecA-mediated activation of LexA do not occur, despite retention of genes encoding a few proteins that respond to LexA induction in E. coli. Three independent E. coli accessions converge on a single Wolbachia UvrD helicase, which interacts with mismatch repair proteins MutS and MutL, encoded in nearly all Wolbachia genomes. With the exception of MutL, which has been mapped to a eukaryotic association module in Phage WO, proteins involved in recombination/repair are uniformly represented by single protein annotations. Putative phage-encoded MutL proteins are restricted to Wolbachia supergroups A and B and show higher amino acid identity than chromosomally encoded MutL orthologs. This analysis underscores differences between nematode and arthropod-associated Wolbachia and describes aspects of DNA metabolism that potentially impact development of procedures for transformation and genetic manipulation of Wolbachia.

Wolbachia is an obligate intracellular Gram-negative alpha-proteobacterium that has diverse effects on reproduction of arthropod hosts, including cytoplasmic incompatibility, male killing, feminization, and parthenogenesis. Some of these effects have important potential for control of insect pests, including mosquitoes that vector pathogens of humans. In mosquitoes, and in most other arthropods, elimination of Wolbachia by antibiotic treatment has no effect on host survival and reverses the Wolbachia-associated phenotype. Elimination of Wolbachia strain wFol, which enables parthenogenetic reproduction of the Collembolan, Folsomia candida, would result in population extinction. However, F. candida adults remain viable and resume reproduction when antibiotics are removed, suggesting that wFol survives antibiotic treatment in a quiescent persister state similar to that induced by chromosomally encoded toxin-antitoxin (TA) modules in free-living bacteria. Computational approaches were used to document the presence of antitoxin genes upstream of Wolbachia RelE/ParE, Fic, and AbiEii toxin genes. Moreover, this analysis revealed that Wolbachia RatA toxin is encoded by a single copy gene associated with an ssrS noncoding RNA gene. Documentation of potentially functional TA modules expands our understanding of the metabolic capabilities of Wolbachia, and provides an explanation for variable and sometimes contradictory results of antibiotic treatments. The presence of chromosomal TA modules in Wolbachia genomes suggests that wFol, and potentially other strains of Wolbachia, can enter a quiescent persister state.

Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric profiles to define optimal conditions for harvesting an infectious inoculum. Wolbachia recovered from the cell culture supernatant after gentle pipetting of infected cells represented about 1% of the total bacterial population and were more infectious than Wolbachia that remained associated with intact cells and/or membranes after low-speed centrifugation. Optimal establishment of a robust infection in naïve cells required 6 d, at a ratio of 80 to 160 bacteria per cell. Among Aedes albopictus mosquito cell lines, an aneuploid line with a 4n + 1 karyotype was more susceptible to infection than diploid lines. These findings contribute to the in vitro manipulation of Wolbachia, illustrate some of the many factors that influence infectivity, and identify areas for future investigation.

Wolbachia pipientis (Rickettsiales; Anaplasmataceae) is an obligate intracellular alpha proteobacterium that occurs in arthropods and filarial worms. Some strains of Wolbachia can be maintained as persistent infections in insect cell lines. C/wStr1 cells from the mosquito Aedes albopictus maintain a robust infection with Wolbachia strain wStr, originally isolated from the planthopper, Laodelphax striatellus. To explore possible functions of penicillin-binding proteins expressed from the wStr genome, C/wStr1 cells were exposed to ampicillin. Absolute levels of Wolbachia increased 3.5-fold in ampicillin-treated cells and fivefold in naive cells newly infected with wStr. Because cell numbers were depressed by ampicillin treatment, Wolbachia yield on a per-cell basis increased by 15-fold. The absence of a similar effect on wAlbB in Aa23 host cells suggests that the Wolbachia strain, the presence/absence of genes encoding penicillin-binding proteins, or the interaction between wAlbB and its host cells may modulate the effects of ampicillin.

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales), is a widespread, vertically transmitted endosymbiont of filarial nematodes and arthropods. In insects, Wolbachia modifies reproduction, and in mosquitoes, infection interferes with replication of arboviruses, bacteria and plasmodia. Development of Wolbachia as a tool to control pest insects will be facilitated by an understanding of molecular events that underlie genetic exchange between Wolbachia strains. Here, we used nucleotide sequence, transcriptional and proteomic analyses to evaluate expression levels and establish the mosaic nature of genes flanking the T4SS virB8-D4 operon from wStr, a supergroup B-strain from a planthopper (Hemiptera) that maintains a robust, persistent infection in an Aedes albopictus mosquito cell line. Based on protein abundance, ribA, which contains promoter elements at the 5'-end of the operon, is weakly expressed. The 3'-end of the operon encodes an intact wspB, which encodes an outer membrane protein and is co-transcribed with the vir genes. WspB and vir proteins are expressed at similar, above average abundance levels. In wStr, both ribA and wspB are mosaics of conserved sequence motifs from Wolbachia supergroup A- and B-strains, and wspB is nearly identical to its homolog from wCobU4-2, an A-strain from weevils (Coleoptera). We describe conserved repeated sequence elements that map within or near pseudogene lesions and transitions between A- and B-strain motifs. These studies contribute to ongoing efforts to explore interactions between Wolbachia and its host cell in an in vitro system.

The plant allelochemical L-mimosine (β-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid; leucenol) resembles the nonessential amino acid, tyrosine. Because the obligate intracellular alphaproteobacterium, Wolbachia pipientis, metabolizes amino acids derived from host cells, the effects of mimosine on infected and uninfected mosquito cells were investigated. The EC50 for mimosine was 6-7 μM with Aedes albopictus C7-10 and C/wStr cell lines, and was not influenced by infection status. Mosquito cells responded to concentrations of mimosine substantially lower than those used to synchronize the mammalian cell cycle; at concentrations of 30-35 μM, mimosine reversibly arrested the mosquito cell cycle at the G1/S boundary and inhibited growth of Wolbachia strain wStr. Although lower concentrations of mimosine slightly increased wStr abundance, concentrations that suppressed the cell cycle reduced Wolbachia levels.

Conditions for flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in infected mosquito cells are described. This approach will streamline investigation of Wolbachia's interactions with host cells and facilitate identification of culture conditions that select for Wolbachia-infected cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73 kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the α-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation., (© 2014 John Wiley & Sons Ltd.)

Wolbachia is an obligate intracellular alphaproteobacterium that occurs in arthropod and nematode hosts. Wolbachia presumably provides a fitness benefit to its hosts, but the basis for its retention and spread in host populations remains unclear. Wolbachia genomes retain biosynthetic pathways for some vitamins, and the possibility that these vitamins benefit host cells provides a potential means of selecting for Wolbachia-infected cell lines. To explore whether riboflavin produced by Wolbachia is available to its host cell, we established that growth of uninfected C7-10 mosquito cells decreases in riboflavin-depleted culture medium. A well-studied inhibitor of riboflavin uptake, lumiflavin, further inhibits growth of uninfected C7-10 cells with an LC50 of approximately 12 μg/ml. Growth of C/wStr1 mosquito cells, infected with Wolbachia from the planthopper, Laodelphax striatellus, was enhanced in medium containing low levels of lumiflavin, but Wolbachia levels decreased. Lumiflavin-enhanced growth thus resembled the improved growth that accompanies treatment with antibiotics that deplete Wolbachia, rather than a metabolic advantage provided by the Wolbachia infection. We used the polymerase chain reaction to validate the decrease in Wolbachia abundance and evaluated our results in the context of a proteomic analysis in which we detected nearly 800 wStr proteins. Our data indicate that Wolbachia converts riboflavin to FMN and FAD for its own metabolic needs, and does not provide a source of riboflavin for its host cell.

Cytoplasmic incompatibility (CI) is a conditional sterility induced by the bacterium Wolbachia pipientis that infects reproductive tissues in many arthropods. Although CI provides a potential tool to control insect vectors of arthropod-borne diseases, the molecular basis for CI induction is unknown. We hypothesized that a Wolbachia-encoded, CI-inducing factor would be enriched in sperm recovered from spermathecae of female mosquitoes. Using SDS-PAGE and mass spectrometry, we detected peptides from the 56 kDa hypothetical protein, encoded by wPip_0282, associated with sperm transferred to females by Wolbachia infected males. We also detected peptides from the same protein in Wolbachia infected ovaries. Homologs of wPip_0282 and the co-transcribed downstream gene, wPip_0283, occur as multiple divergent copies in genomes of CI-inducing strains of Wolbachia. The operon is located in a genomic context that includes mobile genetic elements. The absence of wPip_0282 and wPip_0283 homologs from genomes of Wolbachia in filarial nematodes, as well as other members of the Rickettsiales, suggests a role as a candidate CI effector., (© 2013 Published by Elsevier Ltd.)

Cultured cells provide an important in vitro system for examining metabolic interactions between the intracellular bacterium, Wolbachia pipientis, and its insect hosts. To test whether Wolbachia-associated changes in antioxidant activities could provide a tool to select for infected cells, we tested the effects of paraquat (PQ) on Aedes albopictus mosquito cells. Like mammalian cells, mosquito cells tolerate PQ over a wide range of concentrations, and for considerable lengths of time, depending on cell density at the time of treatment. When mosquito cells were plated at low density and allowed to grow in the presence of PQ, we measured an LC50 of approximately 1-2 μM. Unexpectedly, cells persistently infected with Wolbachia strain wStr, from the planthopper Laodelphax striatellus, grew to higher densities in the presence of 1.5 μM PQ than in its absence. This effect of PQ was similar to the improved growth of host cells that occurs in the presence of antibiotics that suppress the Wolbachia infection. A more detailed examination of growth and metabolic sensitivity indicated that wStr is about 10-fold more sensitive to PQ than the mosquito host cells. Microscopic examination confirmed that Wolbachia levels were reduced in PQ-treated cells, and DNA estimates based on the polymerase chain reaction (PCR) indicated that Wolbachia abundance decreased by approximately 100-fold over a 10-d period. Although Wolbachia genomes encode superoxide dismutase, inspection of annotated genomes indicates that they lack several genes encoding products that ameliorate oxidative damage, including catalase, which converts the PQ byproduct, hydrogen peroxide, to molecular oxygen and water. We suggest that loss of multiple genes that participate in repair of oxidative damage accounts for increased sensitivity of Wolbachia to PQ, relative to its host cells.

Wolbachia are obligate intracellular bacteria that cause cytoplasmic incompatibility in mosquitoes. In an incompatible cross, eggs of uninfected females fail to hatch when fertilized by sperm from infected males. We used polyacrylamide gel electrophoresis and tandem mass spectrometry to identify Wolbachia proteins in infected mosquito gonads. These included surface proteins with masses of 25 and 18 kDa and the DNA binding protein, HU beta. Using reverse transcriptase polymerase chain reaction, we showed that the HU gene is transcribed in Wolbachia-infected Culex pipiens and Aedes albopictus mosquitoes. We sequenced HU genes from four Wolbachia strains and compared deduced protein sequences with additional homologs from the databases. Among the Rickettsiales, Wolbachia HU has distinct N- and C-terminal basic/acidic amino acid motifs as well as a pair of conserved, cysteine residues., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The obligate intracellular bacterium, Wolbachia pipientis (Rickettsiales: Anaplasmataceae), distorts reproduction of its arthropod hosts to facilitate invasion of naïve populations. This property makes Wolbachia an attractive "gene drive" agent with potential applications in the control of insect vector populations. Genetic manipulation of Wolbachia will require in vitro systems for its propagation, genetic modification, amplification, and introduction into target insects. Here we show that Wolbachia from the planthopper, Laodelphax striatellus, establishes a robust infection in clonal C7-10 Aedes albopictus mosquito cells. Infected cells, designated C/wStr, expressed radiolabeled proteins that were enriched in cells grown in the absence of antibiotics that inhibit Wolbachia, relative to cultures grown in medium containing tetracycline and rifampicin. Using mass spectrometry, we verified that tryptic peptides from an upregulated 24 kDa band predominantly represented proteins encoded by the Wolbachia genome, including the outer surface protein, Wsp. We further showed that resistance of Wolbachia to streptomycin is associated with a K42R mutation in Wolbachia ribosomal protein S12, and that the pattern of amino acid substitutions in ribosomal protein S12 shows distinct differences in the closely related genera, Wolbachia and Rickettsia.

Polymerase chain reaction (PCR) is often used to detect microorganisms, pathogens, or both, including the reproductive parasite Wolbachia pipientis (Rickettsiales: Anaplasmataceae), in mosquitoes. Natural populations of Culex pipiens L. (Diptera: Culicidae) mosquitoes are infected with one or more strains of W. pipientis, and crosses between mosquitoes harboring different Wolbachia strains provide one of the best-known examples of cytoplasmic incompatibililty (CI). When we used PCR to monitor Wolbachia in the Buckeye strain of Culex pipiens, and in a Wolbachia-cured sister colony obtained by tetracycline treatment, we noted false negative PCR reactions with DNA samples from infected mosquitoes; these results were inconsistent with direct microscopic observation of Wolbachia-like particles in gonads dissected from mosquitoes in the same population. Assays with diluted template often improved detection of positive samples, suggesting that DNA prepared from whole mosquitoes contained an inhibitor of the PCR reaction. We reconciled discrepancies between PCR and microscopy by systematic measurement of the PCR reaction in the presence of an internal standard. Mosquito decapitation before DNA extraction restored the reliability of the PCR reaction, allowing accurate determination of Wolbachia infection status in infected and tetracycline-cured mosquito populations, consistent with microscopic examination. Using PCR primers based on the Tr1 gene, we confirmed that the Wolbachia infection in the Buckeye strain of Culex pipiens belongs to the genotype designated wPip1. Finally, to explore more widely the distribution of PCR inhibitors, we demonstrated that DNA isolated from the cricket, Acheta domesticus (L.); the beetle, Tenebrio molitor L.; the honey bee, Apis mellifera L.; and the mosquito, Anopheles punctipennis Say also contained PCR inhibitors. These results underscore the importance of measuring the presence of inhibitors in PCR templates by using a known positive standard, and provide an approach that will facilitate use of PCR to monitor environmental samples of mosquitoes that harbor endosymbionts or pathogenic organisms.

We used Wolbachia pipientis strain wAlbB from Aedes albopictus Aa23 cells to infect clonal Ae. albopictus TK-6 cells, which are resistant to 5-bromodeoxyuridine. Infected TK-6 cells were cultured in medium containing 5-bromodeoxyuridine to select against Aa23 cells that might have persisted in the inoculum. Infected TK-6 lines retained the Wolbachia infection for 5 mo, indicating that their metabolic processes support Wolbachia growth and multiplication. To investigate early events after Wolbachia infection, we labeled infected cells with (35)S[methionine/cysteine]. Patterns of labeled proteins on sodium dodecyl sulfate gels were similar in control and infected cells, with the exception of a 29-kDa protein. Tandem mass spectrometry revealed that the 29-kDa band included alpha and beta subunits of the 26S proteasome. Independent confirmation of the up-regulation of the proteasome was established by probing Western blots with a monoclonal antibody to the proteasome-associated co-factor, ubiquitin. Wolbachia's loss of metabolic pathways for the synthesis of most amino acids and retention of pathways for their uptake and metabolism suggest that proteasome activation provides a mechanism whereby controlled degradation of intracellular host proteins would increase availability of amino acids to support establishment and maintenance of the Wolbachia infection.

The Aedes albopictus Aa23 cell line, which is persistently infected with Wolbachia pipientis strain wAlbB, tends to grow as aggregated clusters of cells that are difficult to disperse for conventional quantification based on cell number. We used A. albopictus C7-10 cells to validate conversion of methylthiazole tetrazolium (MTT) to a colored formazan product with respect to incubation time, cell number over a 40-fold range, and metabolic activity as cells enter stationary phase. Using this assay, we showed that the doubling time of Aa23 cells increases from about 45 h early after plating to more than 70 h as the cells reach stationary levels. Growth of Aa23 cells proceeds at similar rates in the presence or absence of tetracycline concentrations that decrease the abundance of Wolbachia. Insofar as the MTT assay reflects mitochondrial function, our results indicate that, in Aa23 cells, abundance of intracellular Wolbachia has no measurable effect on mitochondrial activity in the presence of tetracycline.

In vitro production of the obligate intracellular bacterium, Wolbachia pipientis, is essential to its manipulation as a genetic tool to spread transgenes within vector populations. We have adapted the Wolbachia-infected Aa23 Aedes albopictus mosquito cell line to Eagle's minimal medium, supplemented with nonessential amino acids, glutamine, and 20% fetal bovine serum. When plated at low densities, Aa23E cells grew as patchy monolayers, comprised of non-contiguous clusters of cells that gave rise to solid clumps of tightly adherent cells. Multicellular clumps eventually detached from the substrate and floated freely in the medium. Removal of Wolbachia by treatment with tetracycline did not alter the cytological properties of the host cells, which had a population doubling time of 4-5 d. The presence of Wolbachia was monitored by Giemsa staining of cytological preparations, polymerase chain reaction (PCR) amplification of Wolbachia 16S ribosomal DNA, and by simultaneous PCR amplification of ribosomal protein genes from Wolbachia and mosquito host cell genomes. Wolbachia morphology was pleomorphic, and Wolbachia DNA persisted in the culture medium for several weeks after degradation of PCR-amplifiable host cell DNA.