Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines.

Jing Jing Khoo,Catherine Hartley,Grant L Hughes,Alistair C Darby,Fang Shiang Lim,Alexandra Beliavskaia,Alaa M Al-Khafaji,Mulya Mustika Sari Zulkifli,Benjamin L Makepeace,Timothy J Kurtti,Nurul Aini Husin,Lesley Bell-Sakyi,Sazaly Abubakar

doi:10.3390/microorganisms8070988

Abstract

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

Highlights

Wolbachia is a genus of obligate intracellular endosymbiotic gram-negative bacteria of the family Anaplasmataceae in the order Rickettsiales
The I. scapularis cell lines ISE6 [49] and ISE18 [50] and the I. ricinus cell line IRE11 [51] were maintained at 28 ◦C or 32 ◦C in L-15C300 medium supplemented with 10% tryptose phosphate broth (TPB), 10% foetal bovine serum (FBS) and 0.1% bovine lipoprotein (MP Biomedicals, Solon, OH, USA) [52]
The I. scapularis cell line IDE8 [50] was maintained in flat-sided culture tubes (Nunc, Thermo Fisher, Loughborough, UK) at 32 ◦C in L-15B medium [53] supplemented with 10% TPB, 10% FBS, 0.1% bovine lipoprotein, 2 mM L-glutamine and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin)

Summary

Introduction

Wolbachia is best known for its ability to induce five distinct reproductive manipulations in arthropod hosts (cytoplasmic incompatibility (CI), induction of parthenogenesis, male killing, feminisation and meiotic drive), all of which favour its spread by reducing resource competition from males (a dead-end host) or imposing a fitness cost on uninfected females [6,7,8,9]. These parasitic phenotypes appear to be largely confined to the “pandemic” supergroups A and B that infect ~50% of terrestrial arthropod species [10,11]. Wolbachia form obligate and putatively beneficial relationships with their hosts, including strains from supergroups C and D in nematodes and E in springtails [12,13]

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