Interactions between the flavescence dorée phytoplasma and its insect vector indicate lectin-type adhesion mediated by the adhesin VmpA

AbstractThe flavescence dorée phytoplasma undergoes a propagative cycle in its insect vectors by first interacting with the insect cell surfaces, primarily in the midgut lumen and subsequently in the salivary glands. Adhesion of flavescence dorée phytoplasma to insect cells is mediated by the adhesin VmpA. We hypothesize that VmpA may have lectin-like activity, similar to several adhesins of bacteria that invade the insect gut. We first demonstrated that the luminal surface of the midgut and the basal surface of the salivary gland cells of the natural vector Scaphoideus titanus and those of the experimental vector Euscelidius variegatus were differentially glycosylated. Using ELISA, inhibition and competitive adhesion assays, and protein overlay assays in the Euva-6 insect cell line, we showed that the protein VmpA binds insect proteins in a lectin-like manner. In conclusion, the results of this study indicate that N-acetylglucosamine and mannose present on the surfaces of the midgut and salivary glands serve as recognition sites for the phytoplasma adhesin VmpA.

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Immunolabeling of grapevine flavescence dorée MLO in salivary glands of Euscelidius variegatus: a light and electron microscopy study.

Journal of Histochemistry & Cytochemistry ◽

10.1177/38.1.2294149 ◽

1990 ◽

Vol 38 (1) ◽

pp. 79-85 ◽

Cited By ~ 34

Author(s):

J Lherminier ◽

G Prensier ◽

E Boudon-Padieu ◽

A Caudwell

Keyword(s):

Light Microscopy ◽

Salivary Glands ◽

Light And Electron Microscopy ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Good Tool ◽

Tissue Samples ◽

Thin Sections ◽

Flavescence Dorée ◽

Euscelidius Variegatus

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.

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Ability of Euscelidius variegatus to Transmit Flavescence Dorée Phytoplasma with a Short Latency Period

Insects ◽

10.3390/insects11090603 ◽

2020 ◽

Vol 11 (9) ◽

pp. 603

Author(s):

Luca Picciau ◽

Bianca Orrù ◽

Mauro Mandrioli ◽

Elena Gonella ◽

Alberto Alma

Keyword(s):

Salivary Glands ◽

Infection Rate ◽

Temporal Dynamics ◽

Latency Period ◽

Flavescence Dorée ◽

Access Period ◽

Euscelidius Variegatus ◽

Inoculation Access Period ◽

Time Required

Phytoplasma transmission takes place by insect vectors through an Acquisition Access Period (AAP), Latency Period (LP) and Inoculation Access Period (IAP). Generally, phytoplasmas are believed to be transmitted more efficiently by nymphs because they need a long LP to reach the salivary glands before becoming infective. The transmission can start from adults as well, but in this case a long LP may exceed the insect’s lifespan. However, previous evidence has indicated that adults can undergo a shorter LP, even though little knowledge is available regarding the phytoplasma temporal dynamics during this period. Here, we investigate the minimum time required by the phytoplasma to colonize the vector midgut and salivary glands, and finally to be inoculated into a plant. We used the leafhopper Euscelidius variegatus to investigate the life cycle of flavescence dorée phytoplasma (FDP). Phytoplasma-free E. variegatus adults were left on broad beans (BBs) infected with FDP for an AAP of 7 days. Subsequently, they were individually transferred onto a healthy BB for seven different IAPs, each one lasting 24 h from day 8 to 14. Molecular analyses and fluorescence in situ hybridization were performed for FDP detection. FDP was found in the leafhopper midgut from IAP 1 with an infection rate reaching 50%, whereas in the salivary glands it was found from IAP 2 with an infection rate reaching 30%. FDP was also detected in BBs from IAP 4, with infection rates reaching 10%. Our results represent an important step to further deepen the knowledge of phytoplasma transmission and its epidemiology.

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Bacteriophage-Host Association in the Phytoplasma Insect Vector Euscelidius variegatus

Pathogens ◽

10.3390/pathogens10050612 ◽

2021 ◽

Vol 10 (5) ◽

pp. 612

Author(s):

Marta Vallino ◽

Marika Rossi ◽

Sara Ottati ◽

Gabriele Martino ◽

Luciana Galetto ◽

...

Keyword(s):

Crop Production ◽

Axenic Culture ◽

Pathogen Transmission ◽

Economic Losses ◽

Insect Vector ◽

Insect Vectors ◽

Electron Microscopic ◽

Flavescence Dorée ◽

Euscelidius Variegatus ◽

Complex Relationships

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.

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A Novel Bacteroidetes Symbiont Is Localized in Scaphoideus titanus, the Insect Vector of Flavescence Dorée in Vitis vinifera

Applied and Environmental Microbiology ◽

10.1128/aem.72.2.1467-1475.2006 ◽

2006 ◽

Vol 72 (2) ◽

pp. 1467-1475 ◽

Cited By ~ 67

Author(s):

Massimo Marzorati ◽

Alberto Alma ◽

Luciano Sacchi ◽

Massimo Pajoro ◽

Simona Palermo ◽

...

Keyword(s):

Salivary Glands ◽

Fat Body ◽

The Body ◽

Follicle Cells ◽

Complex Life Cycle ◽

Universal Primers ◽

Insect Vector ◽

Wine Production ◽

Major Band ◽

Scaphoideus Titanus

ABSTRACT Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

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Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane ofEuscelidius variegatusand Promotes Adhesion to Its Epithelial Cells

Applied and Environmental Microbiology ◽

10.1128/aem.02487-17 ◽

2018 ◽

Vol 84 (8) ◽

pp. e02487-17 ◽

Cited By ~ 11

Author(s):

Nathalie Arricau-Bouvery ◽

Sybille Duret ◽

Marie-Pierre Dubrana ◽

Brigitte Batailler ◽

Delphine Desqué ◽

...

Keyword(s):

Membrane Protein ◽

Salivary Glands ◽

Plant Pathogens ◽

Insect Cells ◽

Wild Plants ◽

Insect Vector ◽

Plant Host ◽

Content Type ◽

Flavescence Dorée ◽

Fluorescent Beads

ABSTRACTPhytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered theSpiroplasma citrimutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin inex vivoadhesion assays andin vivoingestion assays. VmpA specifically interacted withEuscelidiusvariegatusinsect cells in culture and promoted the retention of VmpA-coated beads to the midgut ofE. variegatus. In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCEPhytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells ofEuscelidiusvariegatus, the experimental vector of the FD phytoplasma.

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Route of a Multipartite Nanovirus across the Body of Its Aphid Vector

Journal of Virology ◽

10.1128/jvi.01998-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 6

Author(s):

Jérémy Di Mattia ◽

Marie-Stéphanie Vernerey ◽

Michel Yvon ◽

Elodie Pirolles ◽

Mathilde Villegas ◽

...

Keyword(s):

Salivary Gland ◽

Salivary Glands ◽

Plant Viruses ◽

The Body ◽

Insect Vector ◽

Vector Transmission ◽

Aphid Vector ◽

Vector Interaction ◽

Salivary Gland Cells ◽

Mode Of Transmission

ABSTRACT Vector transmission plays a primary role in the life cycle of viruses, and insects are the most common vectors. An important mode of vector transmission, reported only for plant viruses, is circulative nonpropagative transmission whereby the virus cycles within the body of its insect vector, from gut to salivary glands and saliva, without replicating. This mode of transmission has been extensively studied in the viral families Luteoviridae and Geminiviridae and is also reported for Nanoviridae. The biology of viruses within these three families is different, and whether the viruses have evolved similar molecular/cellular virus-vector interactions is unclear. In particular, nanoviruses have a multipartite genome organization, and how the distinct genome segments encapsidated individually transit through the insect body is unknown. Here, using a combination of fluorescent in situ hybridization and immunofluorescence, we monitor distinct proteins and genome segments of the nanovirus Faba bean necrotic stunt virus (FBNSV) during transcytosis through the gut and salivary gland cells of its aphid vector Acyrthosiphon pisum. FBNSV specifically transits through cells of the anterior midgut and principal salivary gland cells, a route similar to that of geminiviruses but distinct from that of luteoviruses. Our results further demonstrate that a large number of virus particles enter every single susceptible cell so that distinct genome segments always remain together. Finally, we confirm that the success of nanovirus-vector interaction depends on a nonstructural helper component, the viral protein nuclear shuttle protein (NSP), which is shown to be mandatory for viral accumulation within gut cells. IMPORTANCE An intriguing mode of vector transmission described only for plant viruses is circulative nonpropagative transmission, whereby the virus passes through the gut and salivary glands of the insect vector without replicating. Three plant virus families are transmitted this way, but details of the molecular/cellular mechanisms of the virus-vector interaction are missing. This is striking for nanoviruses that are believed to interact with aphid vectors in ways similar to those of luteoviruses or geminiviruses but for which empirical evidence is scarce. We here confirm that nanoviruses follow a within-vector route similar to that of geminiviruses but distinct from that of luteoviruses. We show that they produce a nonstructural protein mandatory for viral entry into gut cells, a unique phenomenon for this mode of transmission. Finally, noting that nanoviruses are multipartite viruses, we demonstrate that a large number of viral particles penetrate susceptible cells of the vector, allowing distinct genome segments to remain together.

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New Viral Sequences Identified in the Flavescence Dorée Phytoplasma Vector Scaphoideus titanus

Viruses ◽

10.3390/v12030287 ◽

2020 ◽

Vol 12 (3) ◽

pp. 287

Author(s):

Sara Ottati ◽

Marco Chiapello ◽

Luciana Galetto ◽

Domenico Bosco ◽

Cristina Marzachì ◽

...

Keyword(s):

Large Scale ◽

Control Measures ◽

Insect Vector ◽

Wine Production ◽

Flavescence Dorée ◽

Reverse Transcription Pcr ◽

Scaphoideus Titanus ◽

The Usa ◽

In The Wild ◽

Increasing Demand

(1) Background: The leafhopper Scaphoideus titanus is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of Vitis vinifera and, consequently, a major threat to grape and wine production in several European countries. Control measures are compulsory, but they mainly involve large-scale insecticide treatments, with detrimental impacts on the environment. One possible solution is to exploit the largely unexplored genetic diversity of viruses infecting S. titanus as highly specific and environmentally benign tools for biological control. (2) Methods: A metatranscriptomic approach was adopted to identify viruses that may infect individuals caught in the wild in both its native (United States) and invasive (Europe) areas. Reverse transcription PCR was used to confirm their presence in RNA pools and explore their prevalence. (3) Results: We described nine new RNA viruses, including members of “Picorna-Calici”, “Permutotetra”, “Bunya-Arena”, “Reo”, “Partiti-Picobirna”, “Luteo-Sobemo” and “Toti-Chryso” clades. A marked difference in the diversity and abundance of the viral species was observed between the USA population and the European ones. (4) Conclusions: This work represents the first survey to assess the viral community of a phytoplasma insect vector. The possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of S. titanus could be the answer to increasing demand for a more sustainable viticulture.

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Adaptation of an Indigenous Insect Cell Line DZNU-Bm-1 to Mitsuhashi and Maramorosch (MM) Culture Medium Supplemented with 3% Fetal Bovine Serum

Bioscience Biotechnology Research Communications ◽

10.21786/bbrc/13.2/69 ◽

2020 ◽

Vol 13 (2) ◽

pp. 842-847

Author(s):

Syed Obaid Qureshi

Keyword(s):

Cell Line ◽

Insect Cell ◽

Bovine Serum ◽

Culture Medium ◽

Fetal Bovine Serum ◽

Insect Cell Line ◽

Fetal Bovine

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Sequential production of gametes during meiosis in trypanosomes

Communications Biology ◽

10.1038/s42003-021-02058-5 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Lori Peacock ◽

Chris Kay ◽

Chloe Farren ◽

Mick Bailey ◽

Mark Carrington ◽

...

Keyword(s):

Salivary Glands ◽

Nuclear Dna ◽

Nuclear Dna Content ◽

Cell Types ◽

Tsetse Fly ◽

Binucleate Cell ◽

Insect Vector ◽

Content Ratio ◽

Different Cell Types ◽

Meiosis I

AbstractMeiosis is a core feature of eukaryotes that occurs in all major groups, including the early diverging excavates. In this group, meiosis and production of haploid gametes have been described in the pathogenic protist, Trypanosoma brucei, and mating occurs in the salivary glands of the insect vector, the tsetse fly. Here, we searched for intermediate meiotic stages among trypanosomes from tsetse salivary glands. Many different cell types were recovered, including trypanosomes in Meiosis I and gametes. Significantly, we found trypanosomes containing three nuclei with a 1:2:1 ratio of DNA contents. Some of these cells were undergoing cytokinesis, yielding a mononucleate gamete and a binucleate cell with a nuclear DNA content ratio of 1:2. This cell subsequently produced three more gametes in two further rounds of division. Expression of the cell fusion protein HAP2 (GCS1) was not confined to gametes, but also extended to meiotic intermediates. We propose a model whereby the two nuclei resulting from Meiosis I undergo asynchronous Meiosis II divisions with sequential production of haploid gametes.

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