Isolation in Natural Host Cell Lines of Wolbachia Strains wPip from the Mosquito Culex pipiens and wPap from the Sand Fly Phlebotomus papatasi.

Lesley Bell-Sakyi,Lisa Luu,Alexandra Beliavskaia,James G C Hamilton,Catherine S Hartley,Laura Jones,Lee R Haines,Alistair C Darby,Benjamin L Makepeace

doi:10.3390/insects12100871

Abstract

Simple SummaryDiverse strains of Wolbachia bacteria, carried by many arthropods, as well as some nematodes, interact in many different ways with their hosts. These include male killing, reproductive incompatibility, nutritional supplementation and suppression or enhancement of the transmission of diseases such as dengue and malaria. Consequently, Wolbachia have an important role to play in novel strategies to control human and livestock diseases and their vectors. Similarly, cell lines derived from insect hosts of Wolbachia constitute valuable research tools in this field. During the generation of novel cell lines from mosquito and sand fly vectors, we isolated two strains of Wolbachia and demonstrated their infectivity for cells from a range of other insects and ticks. These new insect cell lines and Wolbachia strains will aid in the fight against mosquitoes, sand flies and, potentially, ticks and the diseases that these arthropods transmit to humans and their domestic animals.Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Highlights

Obligate intracellular bacteria of the genus Wolbachia are endosymbionts of a wide range of invertebrate taxa, including many species of arthropod and parasitic nematodes
The culture medium comprised L-15 (Leibovitz) medium supplemented with 10% tryptose phosphate broth (TPB), 20% foetal bovine serum (FBS), 2mM L-glutamine (L-glut), 100 units/ mL penicillin, 100 μg/ mL streptomycin and 50 μg/ mL amphotericin B (L-15; all ingredients were obtained from Invitrogen, Insects 2021, 12, x Insects 2021, 12, 871 and 50 μg/ mL amphotericin B (L-15; all ingredients were obtained from Invitrogen, Thermo Fisher, Loughborough, UK or from Sigma-Aldrich, Gillingham, UK)
A PCR targeting the cox1 gene confirmed that the cytopathic effect (CPE)/LULS50 cells belonged to the C. pipiens complex (100% identity with 99% query coverage to a sequence from a C. pipiens mosquito from Greece, GenBank accession number MN850560.1); a further ecotype analysis revealed that the cell line contained a mixture of C. p. pipiens and C. p. molestus cells and possibly cells derived from hybrids of the two ecotypes (Figure 1b)

Summary

Introduction

Obligate intracellular bacteria of the genus Wolbachia are endosymbionts of a wide range of invertebrate taxa, including many species of arthropod and parasitic nematodes. While the majority of isolations have involved the inoculation of insect-derived material into an established arthropod cell line, in a few cases the Wolbachia were cultured directly from the host during primary cell culture initiation [3,21,22]. The outcome of such isolations was either long-lived Wolbachia-infected primary cultures [22] or chronically-infected insect cell lines [3,21]

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