Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Yuefang Huang,John Martin,Joseph R Fauver,Jonah Kupritz,Young-Jun Choi,Wandy L Beatty,Peter U Fischer,Kurt C Curtis,Kerstin Fischer,Makedonka Mitreva

doi:10.1371/journal.pone.0250051

Yuefang Huang, John Martin + Show 8 more

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https://doi.org/10.1371/journal.pone.0250051

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Abstract

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Highlights

It is estimated that 66% of all insect species and the majority of filarial parasites that infect humans are infected/colonized with Wolbachia, obligate intracellular bacteria belonging to the order Rickettsiales [1] Wolbachia cause phenotypes such as cytoplasmic incompatibility (CI) and feminization in arthropods, or support growth and reproduction in filarial nematodes [2, 3]
The present study identified for the first time a particle-forming phage WO in North American crickets and provided the whole genome sequence of phage WOSoc
A. socius crickets screened by PCR contained Wolbachia

Summary

Introduction

It is estimated that 66% of all insect species and the majority of filarial parasites that infect humans are infected/colonized with Wolbachia, obligate intracellular bacteria belonging to the order Rickettsiales [1] Wolbachia cause phenotypes such as cytoplasmic incompatibility (CI) and feminization in arthropods, or support growth and reproduction in filarial nematodes [2, 3]. Wolbachia are abundant in male and female germlines and are enriched along the reproductive tract, and present in somatic structures (e.g., the brain and gastrointestinal tract) of select host species. The persistence of the phage despite its documented lytic activity has led to the hypothesis that phage WO provides benefit to its Wolbachia or arthropod host [10]. Phage WO provides Wolbachia with accessory genes for cytoplasmic compatibility and male killing [12, 13]

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