Bacteriophages in the phytoplasma insect vector Euscelidius variegatus

Plant pathogen transmission by insect vectors represent a global economic issue due to their negative impact on crop production. Insect vectors, like all other organisms, are associated with a dynamic and interactive microbial community, called microbiome, which includes not only living members (microbiota), but also elements considered to be non-living organisms (viruses, plasmids, prions, viroids, and free DNA) and the whole spectrum of molecules produced by those microbes. Insects are colonized by a diverse microbial community, such as bacteria, protozoa, fungi and viruses, which can influence their physiology, ecology and evolution as well as their competence as vectors. Insect-virus associations, in particular, are not limited to insects that act as vectors of viral pathogens. Viruses, in fact, may also be pathogenic to their insect host, cause a covert infection or infect members of the insect microbiome, as in the case of bacteriophages, i.e. viruses that infect bacteria. The environmental impact of bacteriophages in various ecosystems has been attracting considerable interest for the past ten years, but only recently their role in the dynamics of microbial communities has been investigated. However, only a few phages have been described so far in insect microbiomes (e.g. the phage APSE-1 of the pea aphid endosymbiont Hamiltonella defensa, or phage WO of Wolbachia). In this work, metatranscriptomics was integrated with classical microbiological and microscopy techniques to identify a DNA tailed-phage and its bacterial host within the microbiome of the phytoplasma insect vector Euscelidius variegatus Kirschbaum (Hemiptera, Cicadellidae). Phytoplasmas are plant pathogenic bacteria transmitted by insects, which can cause severe losses in agriculture. E. variegatus is used as a laboratory vector of the phytoplasma causing Flavescence dorée, a quarantine pest that threatens viticulture in Europe, because of the difficulties in rearing its monovoltine natural vector Scaphoideus titanus. During the electron microscope (TEM) observation of a partial viral purification obtained from E. variegatus, we serendipitously observed some bacteriophage-like particles with head-and-tail morphology typical of the viral family of Siphoviridae. In order to identify the observed viral particles, we re-analyzed two RNA-seq libraries that were originally constructed without any prior phage enrichment step to explore E. variegatus transcriptome. Stringent selection of only phage hallmark genes resulted in the identification of three transcripts coding for three putative major capsid proteins. PCR amplifications allowed us to reliably associate one of these sequences to the observed phage, which was thereafter named Euscelidius variegatus phage 1 (EVP-1). EVP-1 host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), which was recently renamed as Candidatus Symbiopectobacterium sp. This bacterial isolate proved to harbour multiple prophages that become active on axenic media; in fact, in addition to siphovirus-like particles, TEM observations of the colony also revealed the presence of phages with podovirus- and myovirus-like morphology, suggesting that different environments, namely the insect microbiome and axenic cultures, can trigger different mechanisms that finely regulate the within-host interactions among phages. Unraveling the microbiome of insect vectors and understanding the complex relationships within its components may help to unveil possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture