Flavescence dorée (FD), caused by FD phytoplasma (FDp) and transmitted by the leafhopper Scaphoideus titanus, is a quarantine disease that seriously threatens viticulture across Europe. Research on resistance or tolerance to FDp in grapevine is limited by the perennial nature of the host, high cultivar variability and the univoltine life cycle of the insect vector. To overcome these constraints, we employed Arabidopsis thaliana as a model host to identify genes conferring resistance to FDp. RNA sequencing (RNA-seq) was first used to identify genes deregulated in A. thaliana during infection by either the woody host pathogen FDp or the herbaceous host pathogen Chrysanthemum yellows phytoplasma (CYp). A subset of these genes was then validated by monitoring their expression in the FDp-A. thaliana pathosystem at various time points during infection. Five genes were consistently deregulated upon infection, and functional analysis of the corresponding mutants revealed two lines with significantly reduced susceptibility to FDp compared with the wild type. The double mutant combining these two genes exhibited a similar phenotype, suggesting functional convergence. Electrical penetration graph (EPG) analysis indicated that this resistance is independent of vector feeding behaviour, pointing to a genetically determined defence mechanism rather than antixenosis. These findings uncover novel components of plant defence against phytoplasma infection and establish A. thaliana as a valuable system for dissecting the molecular basis of tolerance to FDp. We discussed how the genes we identified represent promising targets for developing sustainable, phytoplasma-resistant grapevine varieties.

Abstract Growing evidence shows that eukaryotic genomes contain DNA sequences of non-retroviral RNA virus origin, yet the mechanisms underlying the generation of this virus-derived complementary DNA (vDNA) remain poorly understood, particularly in insects. Here, we demonstrate that infection with diverse non-retroviral RNA viruses induces the production of reverse-transcribed vDNA across multiple lepidopteran and hemipteran species. We further show that a Drosophila melanogaster –derived cell line actively secretes reverse-transcribed vDNA associated with extracellular vesicles (EVs), widely conserved nanoscale mediators of intercellular communication. Together, these findings reveal that infection-induced vDNA formation is a widespread phenomenon in insects and suggest that EVs may facilitate the systemic dissemination of vDNA.

Symbiotic partnerships have opened new ecological niches and contributed to the remarkable diversification of insects. The leafhopper Scaphoideus titanus, a phloem-feeding insect known to be the primary vector of Flavescence dorée phytoplasma, harbours two primary endosymbionts: the bacterium 'Candidatus Karelsulcia muelleri' and a yeast-like symbiont (YLS). While most studies on insect-associated microorganisms have focused on obligate bacterial symbionts, fungal endosymbionts, although documented for almost a century, are only now gaining renewed attention for their evolutionary and ecological significance. In this study, we integrated genomic and proteomic data with phylogenetic analyses to elucidate the functional and evolutionary features of the YLS associated with S. titanus. Using a data-independent proteomic approach supported by a newly sequenced symbiont genome, we defined the proteins expressed by the YLS that may contribute to host physiology. Comparative analyses across the five currently available YLS genomes enabled a proteome-wide phylogenetic reconstruction within the genus Ophiocordyceps, refining the evolutionary placement of these symbioses. Finally, large-scale mining of NCBI transcriptomic Sequence Read Archive datasets using a novel computational workflow, combined with an extensive literature survey, identified several new candidate insect hosts and provided a comprehensive inventory of species harbouring these fungal partners.

Entomopathogenic fungi (EPF) are promising biological control agents for the integrated pest management of Scaphoideus titanus Ball, the principal vector of Flavescence dorée phytoplasma in European vineyards. This study assessed the diversity, abundance, pathogenicity, and endophytic colonization ability of native EPF isolated from vineyard, woodland, and fallow field soils in the Piedmont Region (northwestern Italy). A total of 151 EPF isolates were recovered using selective media plating and the Galleria bait method and identified by ITS sequencing, with Metarhizium anisopliae, Metarhizium robertsii, and Purpureocillium lilacinum as the most frequent species. Pathogenicity assays revealed pronounced strain-specific differences against S. titanus. M. anisopliae BP1e and M. robertsii BP1k caused the greatest mortality, reducing insect survival to 27.2% and 40.2% at 12 days post-treatment, respectively, compared with 46.3% for the reference strain Beauveria bassiana ATCC 74040. Endophytic colonization assays also showed marked variation among strains. P. lilacinum CaS1a achieved the highest and most persistent colonization of treated leaves, reaching 79%, whereas Metarhizium isolates, particularly M. anisopliae BP1r, established consistent but more localized colonization. Penicillium citrinum CaS1d showed the highest colonization of untreated leaves (38.5% at 14 days post-treatment), suggesting greater capacity to spread beyond the application site. Beauveria isolates exhibited moderate colonization. These findings highlight the potential of indigenous EPF to combine direct pathogenicity with persistent endophytic establishment, supporting their integration into sustainable grapevine pest management. Future studies should optimize fungal formulations and evaluate single and combined strain applications under field conditions.

An infection event of the spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae) has been described, for the first time, in northwest Italy. The causative agents were two entomopathogenic fungi belonging to Entomophthorales, specifically Zoophthora radicans and, more rarely, Batkoa major. The morphological description and molecular identification of fungi have been reported, in addition to recording meteorological data that may have affected the outbreak of the infection. When massive events are ongoing, entomopathogenic fungi really behave as determinant regulators of natural populations of arthropod pests and the possibility to stress their action in this direction should be deeply investigated.

Phytoplasmas are phloem-limited plant pathogenic bacteria causing diseases in many plant species. They are transmitted by Hemipteran insect species in a persistent-propagative manner. Phytoplasmas are wall-less, and their membrane proteins are involved in pathogen internalization into host cells. We focused on the immunodominant membrane protein (Imp) of Flavescence dorée phytoplasma (FDp), a grapevine quarantine pest and a major threat to European viticulture. Scaphoideus titanus is the main natural vector of FDp to grapevine, whereas Euscelidius variegatus is commonly used as laboratory vector. Previous works indicated that recombinant Imp of two FDp strains (FD-C and FD-D) selectively interact with gut proteins from vector species rather than those from non-vectors. Here, similar patterns of interacting insect gut proteins were obtained from both vector species, following pull-down with His-tagged FDp Imps. After identification of several targets, four S. titanus and five E. variegatus proteins interacting with Imp were further characterized by measuring expression in different insect tissues and in healthy vs. infected insects. Specific RNAi silencing of two of these vector genes, namely natterin and legumain, resulted in a significant reduction of phytoplasma multiplication in insects upon pathogen acquisition, compared to control insects. Natterin displays a DM9 domain and legumain possesses a signature of G protein receptor, supporting their involvement as FDp Imp receptors. Outcomes of this work are discussed with particular attention devoted to the gain of knowledge on host/pathogen interaction as well as to the potential impact on improvement phytoplasma disease management.

Philaenus spumarius is the primary vector of the quarantine plant pathogen Xylella fastidiosa in Europe and is responsible for the devastating Olive Quick Decline Syndrome outbreak in Southern Italy. Despite its importance, little is known about its natural viral community, which could offer novel and sustainable strategies for vector control. In this three-year study, we conducted the first comprehensive characterization of the viral community of P. spumarius from multiple ecologically diverse European sites, including X. fastidiosa-affected areas in Southern Italy. Deep transcriptomic sequencing of 209 field-collected individuals pooled into 11 RNA-seq libraries revealed the presence of 26 RNA viruses. Our findings revealed a rich and structured viral community in populations from Northern Italy and France, contrasting sharply with the reduced viral diversity observed in populations from Southern Italy, where most individuals were virus-free. Temporal comparisons revealed recurrent virus-host associations over the years, and laboratory rearing provided initial insights into viral persistence and transmission dynamics. Although none of the detected viruses caused overt signs of mortality or sterility, their potential sublethal effects and ecological interactions remain unexplored. This study lays the groundwork for future research on the functional roles of insect-associated viruses and emphasizes their potential for developing sustainable, environmentally friendly approaches to managing vectors and reducing the impact of X. fastidiosa on European agriculture.

The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma associated to Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Transcriptomic studies have already provided valuable insights into the mechanisms of insect-phytoplasma interactions, but proteomics can offer immediate insights into the cellular functions and metabolic adaptations of the insect and its microbiome to the presence of this plant bacterium. Here, the generation of new genomic data of the E. variegatus holobiont was instrumental in elaborating the first comprehensive proteomic profile of its response to Flavescence dorée phytoplasma (FDp). Both data-dependent acquisition and data-independent acquisition mass spectrometry were used to explore the complex molecular interactions between the insect host, its microbial community, and the phytoplasma. Results indicated a critical role of the insect mitochondria as a shared interface exploited by phytoplasmas for survival and propagation. Additionally, it appeared that the presence of FDp had a detrimental impact on the reciprocal metabolic support between the insect host and its two primary endosymbionts, predominantly resulting in a perturbation in amino acid synthesis and exchange. Proteins upregulated in response to FDp may represent promising targets for disrupting phytoplasma acquisition and transmission, either through rationally designed agrochemicals or gene silencing approaches.

BACKGROUND:The Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) is a highly polyphagous quarantine invasive species causing severe crop damages. Its management is based on broad-spectrum insecticides and sustainable alternatives are needed. Strategies based on RNA interference (RNAi) emerged in crop protection and we aimed to explore its use to control P. japonica. RESULTS:Nine genes of P. japonica were selected as lethal candidates, based on previous wide-genome screenings on other coleopterans. To avoid off-target effects, genes showing over 80% identity with pollinator homologues were excluded and P. japonica double-stranded RNAs (dsRNAs) were designed in the least conserved portions according to alignments with Apis mellifera. When incubated in P. japonica midgut juice, dsRNAs were not degraded. Injection and plant-mediated feeding were used to deliver dsRNAs to larvae and adults. Five targets were tested, and two genes were selected as the most effective in increasing mortality, namely regulatory particle non-ATPase 6 subunit (RPN) and shibire_dynamin-like protein (SHI). A significant transcript reduction up to 21 days (RPN: 3-5 fold-change silencing) after dsRNA injection indicated that effective gene silencing occurred, as also supported by sequencing of small RNA libraries. In adults, RNAi-mediated depletion of RPN transcript reduced survival, either when insects were injected or mass-fed on vine leaves dsRNA-treated. CONCLUSION:A subunit of the 26S proteasome was indicated as promising RNAi target for dsRNA-based insecticide against the Japanese beetle. The data pave the way for the possible use of RNAi approaches to control this pest, proactively waiting for the European Union approval of exogenously applied dsRNAs. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA interference (RNAi) is double stranded RNA (dsRNA)-based gene silencing mechanism. Exogenous dsRNAs application to crops has raised as a powerful tool to control agricultural pests. In particular, several sap-feeder are important plant pathogens vectors, such as Philaenus spumarius, known as main vector of Xylella fastidiosa (Xf), causal agent of olive quick decline syndrome (OQDS) in southern Italy. Here, dsATP synthase beta (dsATP), dsLaccase (dsLacc) and dsGreen Fluorescent Protein (dsGFP) as control, were provided to spittlebug adults by microinjection or to nymphs fed on dsRNA-treated plant shoots. Treated insects were collected at different time points to monitor silencing efficiency over time, describing significant reduction of transcript levels from 8 to 24 days post treatment. Downregulation of target genes ranged from 2- to 16-fold compared to the corresponding dsGFP controls, where highest silencing effects were generally noticed for ATP synthase beta. Sequencing of libraries obtained from total smallRNA (sRNA) showed the generation of dsRNA-derived sRNAs by RNAi pathway, with majority of reads mapping exclusively on the correspondent dsRNA. Also, we characterized components of a functional RNAi machinery in P. spumarius. Further research is needed to clarify such mechanism, screen effective target lethal genes to reduce vector population and improve delivery strategies.

Flavescence dorée is an economically important vector-borne disease of grapevine in Europe caused by phytoplasmas belonging to the 16SrV ribosomal group. Expression profiles of 11 genes of the Flavescence dorée phytoplasma (FDp) were analysed over time following infection of natural ( Vitis vinifera and the leafhopper vector Scaphoideus titanus ) and experimental ( Vicia faba and Euscelidius variegatus ) hosts. Infected and symptomatic grapevine plants (Chardonnay) were sampled under natural field conditions in a productive vineyard in north-western Italy. Broad bean samples were assayed after the experimental inoculation with infectious E. variegatus . Adults of both vector species were analyzed following FDp acquisition from infected broad bean plants. The selected FDp genes were grouped according to their putative functions within different categories, namely ‘Membrane proteins’ ( imp ), ‘Regulative elements’ ( spo VG, rpo D), ‘Protein metabolism, transport and secretion’ ( tld D, ysd C, fts Y), ‘Stress response’ ( comp 83, osm C), ‘ABC transporters’ ( Co ABC) and ‘Unknown’ function ( contig 12, comp 115). All analyzed genes were expressed in the four different host species suggesting their crucial role during the FDp infection cycle. Moreover, some of them ( contig 12, Co ABC, comp 83, and imp ) might be considered essential for phytoplasma survival irrespective of the host, while comp 115 seems to be required for insect infection. We showed that FDp is metabolically more active in insects than in plants, at least according to the pool of selected genes for this study, indicating that FDp behaves differently in the two hosts compared to other phytoplasma species/strains.

Halyomorpha halys (Stal), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

RNA interference (RNAi) regulates gene expression in eukaryotes, and it is an emerging tool in crop protection by exogenous applications of double-stranded RNAs (dsRNAs) to silence the expression of essential pest genes. Nevertheless, delivery of dsRNAs to sap-sucking insects is a major challenge for RNAi applications. The present work aimed at verifying whether in leafhopper species, RNAi can be triggered by plant-mediated delivery, and providing a proof of concept towards field applications. Two phytoplasma vectors species, Euscelidius variegatus and Scaphoideus titanus (Hemiptera: Cicadellidae), were used as case study. Gene silencing can be achieved efficiently in both species through microinjection of dsRNAs, despite the technique being time consuming and inapplicable on large scale. This protocol was set as gold standard for the development of a higher throughput approach. Soaking of nymphs in a solution with co-adjuvant and dsRNAs as well as insect feeding on whole plants or detached leaves immersed in a dsRNA solution were assayed as alternative delivery strategies. Nymph soaking did not induce specific gene silencing, while plant absorption proved to be suitable to deliver both a coloured solution and control dsRNAs targeting green fluorescent protein gene. Insect feeding on detached leaves immersed in dsRNA solution was selected to test silencing of two gut-specific (legumain and natterin) and one ubiquitous (ATP synthase β) genes. The expression of the three genes significantly decreased in E. variegatus insects fed on dsRNA-treated plants. Similarly, a significant reduction of ATP synthase β transcript was measured in S. titanus fed on dsRNA-treated plants.

Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase β dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E.variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase β gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

Phytoplasmas are insect-borne pathogenic bacteria that cause major economic losses to several crops worldwide. The dynamic microbial community associated with insect vectors influences several aspects of their biology, including their vector competence for pathogens. Unraveling the diversity of the microbiome of phytoplasma insect vectors is gaining increasing importance in the quest to develop novel microbe-based pest control strategies that can minimize the use of insecticides for better environmental quality. The leafhopper Scaphoideus titanus is the primary vector of the Flavescence dorée phytoplasma, a quarantine pest which is dramatically affecting the main grape-growing European countries. In this study, the RNA-Seq data, which were previously used for insect virus discovery, were further explored to assess the composition of the whole microbial community associated with insects caught in the wild in both its native (the United States) and invasive (Europe) areas. The first de novo assembly of the insect transcriptome was used to filter the host sequencing reads. The remaining ones were assembled into contigs and analyzed by blastx to provide the taxonomic identification of the microorganisms associated with S. titanus, including the non-bacterial components. By comparing the transcriptomic libraries, we could differentiate the stable and consistent associations from the more ephemeral and flexible ones. Two species appeared to be universal to the core microbiome of S. titanus: the obligate bacterial symbiont Candidatus Sulcia muelleri and an Ophiocordyceps-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus Cardinium have been identified as another dominant member of the microbiome, but only in the European specimens. Although we are yet to witness how the interplay among the microorganisms influences the vector competence of S. titanus, this unbiased in silico characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.

The leafhopper Euscelidius variegatus Kirschbaum (Hemiptera: Cicadellidae) is a natural vector of the 'Candidatus Phytoplasma asteris' and a laboratory vector of the phytoplasma associated with grapevine Flavescence doree. Previous studies indicated that RNA interference (RNAi) by dsRNA injection efficiently works in E. variegatus and that silencing of ATP synthase beta increases mortality and impairs phytoplasma multiplication. Here, a nearly complete female sterility was observed when ATP synthase beta was silenced. The sterility was associated with the absence of mature eggs in the ovaries. On the contrary, male genitalia morphology and sperm motility were similar to those observed in E. variegatus control specimens, treated with dsRNAs targeting green fluorescent protein (dsGFP). An over-expression of hexamerin (amino acid storage protein) and cathepsin L (lysosome proteinase) was observed at transcript and protein level in dsATP-injected females in comparison with dsGFP-injected insects, in both whole body and dissected ovary samples. Conversely, the expression of other genes known to be involved in oocyte development, namely vitellogenin, perilipin and digestive cystein protein, was not altered in dsATP-injected E. variegatus insects in comparison with dsGFP ones. Possible roles of ATP synthase beta, hexamerin and cathepsin L in oocyte and egg development are discussed. Insecticide treatments against vectors are the main strategies to counteract phytoplasma diseases, with negative impact on environment and public health. RNAi is a promising sustainable approach against insect vectors and ATP synthase beta could be a valid target gene, as its silencing, besides increasing mortality and reducing phytoplasma multiplication, induces female sterility.

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and β subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase β was obtained by injection of specific dsRNAs in E. variegatus. Here we present the systemic and long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase β (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication rate when ATP synthase β was silenced. The role of the insect ATP synthase β during phytoplasma infection process is discussed.

Insect vectors transmit viruses and bacteria that can cause severe diseases in plants and economic losses due to a decrease in crop production. Insect vectors, like all other organisms, are colonized by a community of various microorganisms, which can influence their physiology, ecology, evolution, and also their competence as vectors. The important ecological meaning of bacteriophages in various ecosystems and their role in microbial communities has emerged in the past decade. However, only a few phages have been described so far in insect microbiomes. The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma causing Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Here, the presence of a temperate bacteriophage in E. variegatus (named Euscelidius variegatus phage 1, EVP-1) was revealed through both insect transcriptome analyses and electron microscopic observations. The bacterial host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), recently assigned to the genus Candidatus Symbiopectobacterium. BEV harbors multiple prophages that become active in culture, suggesting that different environments can trigger different mechanisms, finely regulating the interactions among phages. Understanding the complex relationships within insect vector microbiomes may help in revealing possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture.

Hazelnut allergy, which is characterized by symptoms that range from mild to severe, is one of the most common allergies in children throughout Europe, and an accurate diagnosis of this allergy is therefore essential. However, lipophilic allergens, such as oleosins, are generally underrepresented in diagnostic tests. We therefore sought to characterize the IgE reactivity of raw and roasted hazelnut oleosins, using the sera of hazelnut‐allergic pediatric patients.