Efficacy of entomopathogenic fungi against Philaenus spumarius, the vector of Xylella fastidosa.

BACKGROUND: Xylella fastidiosa is an important causative agent of Olive Quick Decline Syndrome in the Apulia region of Italy. The current study evaluated the bioefficacy of three entomopathogenic fungal strains: Beauveria bassiana SGB7004, Metarhizium robertsii SGB1K, and Akanthomyces lecanii SGB4711 against Philaenus spumarius the main vector of this pathogen, under laboratory conditions. Pathogenicity bioassays were performed by dipping nymphs and adults of P. spumarius in an aqueous suspension of powdered fungal culture (PFC) or conidial suspension (CS) of the three fungal strains. RESULTS: Both B. bassiana SGB7004 and M. robertsii SGB1K affected the viability of nymphs, resulting in more than 80% mortality at 48 h post treatment, while the effect of A. lecanii SGB4711 was not statistically significant. On adults, all three biocontrol strains were effective in a time- and concentration-dependent manner. The PFCs of B. bassiana SGB7004, M. robertsii SGB1K, and A. lecanii SGB4711 at the highest concentration tested (120 mg mL-1) resulted in 97%, 83% and 27% mortality at the trial endpoint (120 h), respectively. Mycelial growth was observed on 38.5%, 37.0% and 61.5% of dead insects treated with B. bassiana SGB7004 (2.3 × 108 CFU mL-1), M. robertsii SGB1K (3.8 × 106 CFU mL-1) and A. lecanii SGB4711 (5.4 × 108 CFU mL-1), respectively. None of the PFCs of the tested strains was pathogenic when injected into nymph spittle. CONCLUSIONS: Beauveria bassiana SGB7004 and M. robertsii SGB1K significantly affected the survival of P. spumarius nymphs and adults, while A. lecanii SGB4711 was not effective on nymphs and only slightly effective against adults. © 2024 Society of Chemical Industry.

Ten Challenges to Understanding and Managing the Insect-Transmitted, Xylem-Limited Bacterial Pathogen Xylella fastidiosa.

An unprecedented plant health emergency in olives has been registered over the last decade in Italy, arguably more severe than what occurred repeatedly in grapes in the United States in the last 140 years. These emergencies are epidemics caused by a stealthy pathogen, the xylem-limited, insect-transmitted bacterium Xylella fastidiosa. Although these epidemics spurred research that answered many questions about the biology and management of this pathogen, many gaps in knowledge remain. For this review, we set out to represent both the U.S. and European perspectives on the most pressing challenges that need to be addressed. These are presented in 10 sections that we hope will stimulate discussion and interdisciplinary research. We reviewed intrinsic problems that arise from the fastidious growth of X. fastidiosa, the lack of specificity for insect transmission, and the economic and social importance of perennial mature woody plant hosts. Epidemiological models and predictions of pathogen establishment and disease expansion, vital for preparedness, are based on very limited data. Most of the current knowledge has been gathered from a few pathosystems, whereas several hundred remain to be studied, probably including those that will become the center of the next epidemic. Unfortunately, aspects of a particular pathosystem are not always transferable to others. We recommend diversification of research topics of both fundamental and applied nature addressing multiple pathosystems. Increasing preparedness through knowledge acquisition is the best strategy to anticipate and manage diseases caused by this pathogen, described as "the most dangerous plant bacterium known worldwide."

Diversity, Composition, and Specificity of the Philaenus spumarius Bacteriome.

Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) was recently classified as a pest due to its ability to act as a vector of the phytopathogen Xylella fastidiosa. This insect has been reported to harbour several symbiotic bacteria that play essential roles in P. spumarius health and fitness. However, the factors driving bacterial assemblages remain largely unexplored. Here, the bacteriome associated with different organs (head, abdomen, and genitalia) of males and females of P. spumarius was characterized using culturally dependent and independent methods and compared in terms of diversity and composition. The bacteriome of P. spumarius is enriched in Proteobacteria, Bacteroidota, and Actinobacteria phyla, as well as in Candidatus Sulcia and Cutibacterium genera. The most frequent isolates were Curtobacterium, Pseudomonas, and Rhizobiaceae sp.1. Males display a more diverse bacterial community than females, but no differences in diversity were found in distinct organs. However, the organ shapes the bacteriome structure more than sex, with the Microbacteriaceae family revealing a high level of organ specificity and the Blattabacteriaceae family showing a high level of sex specificity. Several symbiotic bacterial genera were identified in P. spumarius for the first time, including Rhodococcus, Citrobacter, Halomonas, Streptomyces, and Providencia. Differences in the bacterial composition within P. spumarius organs and sexes suggest an adaptation of bacteria to particular insect tissues, potentially shaped by their significance in the life and overall fitness of P. spumarius. Although more research on the bacteria of P. spumarius interactions is needed, such knowledge could help to develop specific bacterial-based insect management strategies.

More than 80 years without new taxa: analysis of morphological variation among members of Mexican Aeneolamia Fennah (Hemiptera, Cercopidae) support a new species in the genus.

The genus Aeneolamia includes eight described species and 32 subspecies widely distributed in America. In Mexico, two species (A.contigua and A.albofasciata) and one subspecies (A.contiguacampecheana) are recognized. In a recent study of Cercopidae in Mexico, a new species of Aeneolamia was noted from Oaxaca, Mexico based on body color and the ornamentation patterns of tegmen, without a formal taxonomic description. To test the hypothesis of an extant new taxon within the genus a comprehensive analysis of intraspecific morphological variation from 46 morphological features was performed, four related to tegmen color patterns in both sexes, six to male genitalia, and 36 continuous characters measured in specimens of both sexes of Mexican Aeneolamia from several geographical localities using traditional univariate, multivariate morphometric, and geometric morphometric methods. This is the first time that this approach has been used in Cercopidae. Aeneolamiadanpecki Castro, Armendáriz & Utrera, sp. nov. from Oaxaca showed pronounced morphological differences in tegmen coloration patterns, the shape of different elements of the male genitalia, and body measurements compared to the other Mexican members of Aeneolamia; therefore, it is described as a new species.

Translocation of Loline Alkaloids in Epichloë-Infected Cereal and Pasture Grasses: What the Insects Tell Us.

Aphids are major pests of cereal and pasture grasses throughout the world, vectoring disease and reducing plant production. There are few control options other than insecticides. Epichloë endophytes that produce loline alkaloids in their hosts provide a possible mechanism of control, with both meadow fescue and tall fescue naturally infected with loline-producing endophytes showing a resistance to Rhopalosiphum padi. We screened Elymus spp. naturally infected with endophytes that produced loline alkaloids at concentrations known to affect aphids on fescue but found no effect on these insects infesting Elymus. A synthetic loline-producing endophyte association with rye also had no effect on the aphids. After hypothesizing that the lolines were being translocated in the xylem in Elymus and rye rather than the phloem, we tested the rye and meadow fescue infected with loline-producing endophytes against a xylem feeding spittlebug. The endophyte in rye inhibited the feeding of the insect and reduced its survival, whereas the endophyte-infected meadow fescue had no effect on the spittlebug but reduced the number of aphids. Lolines applied to the potting medium of endophyte-free and endophyte-infected rye, ryegrass, and tall fescue resulted in a decrease in the aphid populations on the endophyte-free pasture grasses relative to the untreated controls but had no effect on aphid numbers on the rye. We tentatively conclude that lolines, produced in both natural and synthetic association with Elymus and rye, are partitioned in the xylem rather than the phloem, where they are inaccessible to aphids.

Potential of fungi of the genus Trichoderma for biocontrol of Philaenus spumarius, the insect vector for the quarantine bacterium Xylella fastidosa.

BACKGROUND: The meadow spittlebug Philaenus spumarius L. is the vector for the bacterium Xylella fastidiosa subspecies pauca, involved in olive quick decline syndrome (OQDS) in Salento (Italy). Control of P. spumarius is key to limiting transmission of the bacterium, and an innovative approach can be based on effective natural compounds and biocontrol agents. Entomopathogenic fungi are an important source of bioactive natural molecules that play a role in the relationship between microorganisms and insects. RESULTS: Pathogenicity bioassays, performed by dipping adults of P. spumarius in either fungal culture suspension (120 mg mL-1 ) or cell-free culture supernatant of Trichoderma chlorosporum GJS 91-150, showed, respectively, 97% and 87% death within 24 h. The effect was dose-dependent. In laboratory bioassays, the powdered fungal culture of T. chlorosporum GJS 91-150 did not exhibit pathogenic activity when injected into nymph spittle. CONCLUSIONS: T. chlorosporum GJS 91-150 affected the survival of P. spumarius adults. The lethal effect was not associated with the development of mycelium on the cuticle, but seems due, at least partly, to fungal metabolites released in the culture medium. The fungus tested here has good potential for the development of effective low-environmental impact control strategies for P. spumarius and suppression of X. fastidiosa. © 2022 Society of Chemical Industry.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae).

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

Application of vibrational signals to study and manipulate an insect vector: the case of Philaenus spumarius (Hemiptera: Aphrophoridae).

BACKGROUND: Vibrational stimuli can support pest management as they provide environmentally friendly methods to manipulate insect pest behaviors. Different vibrational stimuli were used to study and influence the behavior of the meadow spittlebug, Philaenus spumarius, the European vector of Xylella fastidiosa. In playback experiments, we tested the reactions of the spittlebug toward the male calling signals (test 1) and the male-male signal (test 2). In test 3, we evaluated the use of conspecific signals and noises to repel insects/disrupt mating. RESULTS: Test 1 provided new insights regarding the role of the male calling signal in intraspecific communication, in particular that this signal likely does not underlie aggregation or aggression toward conspecifics. Test 2 demonstrated that the male-male signal is used by males to express distress when physically interacting, whilst, when played back into a host plant, it has not any repellent effect on the spittlebug. Test 3A suggested that males exploit short silence gaps to localize the signaling partner, while test 3B showed that a continuous noise with a specific frequency range successfully disrupt mating, as only one male out of 20 localized the female on the plant. CONCLUSION: Playbacks obtained from prerecorded P. spumarius' signals were successfully used to accomplish ethological studies; even so, this approach did not show a real potential to be used as a control strategy. However, noises designed to mask the spittlebug signals significantly disrupted species mating and could integrate other techniques aimed at reducing the spread of X. fastidiosa after appropriate implementation. © 2022 Society of Chemical Industry.

Proteins from eggs of the spittlebug Mahanarva spectabilis (Hemiptera: Cercopidae) reveal clues about its diapause regulation.

Embryo development in eggs of the spittlebug Mahanarva spectabilis (Distant) (Hemiptera: Cercopidae) passes through four phases (known as S1 to S4) being stopped at S2 during diapause. Studies about the molecular basis of diapause in spittlebugs are nonexistent. Here, we analyzed proteins from non-diapausing (ND), diapausing (D) and post-diapausing (PD) eggs of the spittlebug M. spectabilis. In total, we identified 87 proteins where 12 were in common among the developmental and diapause phases and 19 remained as uncharacterized. Non-diapausing eggs (S2ND and S4ND) showed more proteins involved in information storage and processing than the diapausing ones (S2D). Eggs in post-diapausing (S4PD) had a higher number of proteins associated with metabolism than S2D. The network of protein interactions and metabolic processes allowed the identification of different sets of molecular interactions for each developmental and diapause phases. Two heat shock proteins (Hsp65 and Hsp70) along with two proteins associated with intracellular signaling (MAP4K and a serine/threonine-protein phosphatase) were found only in diapausing and/or post-diapausing eggs and are interesting targets to be explored in future experiments. These results shine a light on one key biological process for spittlebug survival and represent the first search for proteins linked to diapause in this important group of insects.

Seasonal Appearance, Abundance, and Host Preference of Philaenus spumarius and Neophilaenus campestris (Hemiptera: Aphrophoridae) in Olive Groves in Greece.

Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) is a xylem inhabiting bacterium which is exclusively transmitted by xylem sap feeding insects. Among them, Philaenus spumarius Linnaeus and Neophilaenus campestris Fallén are the most abundant species in Europe. During 2018 and 2019 a survey was conducted in olive groves in Greece aimed to improve the knowledge about the biology and ecology of those spittlebugs in areas with Mediterranean climate. Moreover, the host preference of the nymphs was studied. The nymphs of P. spumarius and N. campestris were observed between early March and middle May depending on geographic location and year. The spittlebug adults were present during two periods every year, one in spring and another one in autumn and early winter. During summer months the spittlebugs were totally absent from olive groves. Our observation on host plant selection revealed that there was a strong preference of N. campestris nymphs for plants belonging to the family Poaceae. On the contrary, P. spumarius nymphs were polyphagous. The most preferable plants for the nymphs of this species belonged to the families Asteraceae and Fabaceae. The importance of these findings for control measures for these spittlebugs is discussed.

Assessment of Nymphal Ecology and Adult Xylella fastidiosa Transmission Biology of Aphrophora nr. permutata (Hemiptera: Aphrophoridae) in California Vineyards.

Although Aphrophora nr. permutata (Hemiptera: Aphrophoridae) is a reported vector of the plant pathogen Xylella fastidiosa (Wells) (Xanthomonadales: Xanthomonadaceae), its ecology and role in Pierce's disease dynamics in coastal California vineyards are poorly understood. From 2016 to 2020, we surveyed the abundance of A. nr. permutata nymphs among potential host plants along the vineyard floor, the vineyard edges, and adjacent vegetation in vineyards in Napa and Sonoma county. In 2019 and 2020, vineyards adjacent to woodland habitat hosted larger A. nr. permutata populations than those next to riparian habitat, while in 2017 and 2018, the nymphal populations were similar among riparian and woodland sites. Among 2020 plant cover taxa, nymph abundance was positively associated with Helminthotheca echioides, Vicia sativa, and Daucus carota cover and negatively associated with Taraxacum officinale cover. In 2018 and 2019, we also tracked early-season occurrence and development of A. nr. permutata nymphs among potential host plants. Analyses showed a significant effect of site, year, and plant taxa on the first detection of nymphs and a significant effect of site and year on the estimated development time between first and fifth instars. In 2019, we conducted grapevine to grapevine X. fastidiosa transmission experiments with individuals and groups of five A. nr. permutata adults. In the transmission experiment, 5% (3 of 60) individual A. nr. permutata and 7.7% (1 of 13) of groups successfully transmitted X. fastidiosa. This study provides preliminary evidence of potential host plant associations with A. nr. permutata abundance and phenology that should be explored further with field and greenhouse-based approaches.

The complete mitochondrial genome of Eoscarta assimilis (Hemipera: Cercopidae) and phylogenetic analysis of Cercopidae.

The complete mitochondrial genome (mitogenome) of Eoscarta assimilis (Uhler, 1896) was sequenced in the current paper. The total length of the mitogenome is 17,231 bp and it consists of 37 genes including 22 transfer RNA (tRNAs), 13 protein-coding (PCGs) and 2 ribosomal RNA (rRNAs). The 13 PCGs initiated with the start codon ATN, but ND4 started with TTG. All of the PCGs ended with TAA, apart from COX3 which terminated by incomplete TAG. A ML tree based on sequences of 15 complete mitogenomes (13 Cercopidae and 2 outgroup) suggests that E. assimilis is more closely related to the genus Callitettix. The phylogenetic analysis supports the monophyly of the family Cercopidae and the genus Cosmoscarta, and the paraphyly of the subfamily Callitettixinae. This mitogenome information for E. assimilis could facilitate future evolutionary studies to related insects.

Intrasexual Vibrational Behavior of Philaenus spumarius in Semi-Field Conditions.

Insects that communicate by vibrational signals live in a complex interactive network of communication. Most studies on insect intrasexual behavior, based on plant-borne vibrational signals, have targeted few individuals. Despite their importance, behaviors that occur within groups were often overlooked. The study of multiple individuals, when insects occur in high density could simulate the environment in which they live and provide more reliable information on their behavior. In semi-field conditions, we investigated the intrasexual behavior of the meadow spittlebug, Philaenus spumarius. Vibrational signals exchanged among individuals of the same sex were recorded throughout their adult stage, from late spring to early autumn, and during the day, from the morning to the evening using a laser vibrometer. Males were less active than females throughout the season and their interactions were less frequent compared to females. Intrasexual interactions were characterized by signal overlapping in both unisex groups, in addition to signal alternating only in the case of males. In conclusion, the study of signaling behavior in intrasexual groups contributed to a better understanding of P. spumarius social behavior. We discuss the hypothesis of a possible competitive behavior between males and cooperative behavior between females.

The cibarial pump of the xylem-feeding froghopper Philaenus spumarius produces negative pressures exceeding 1 MPa.

The xylem sap of vascular plants is an unlikely source of nutrition, being both nutrient poor and held under tensions (negative pressures) that can exceed 1 MPa. But some insects feed on xylem sap exclusively, extracting copious quantities using a muscular cibarial pump. However, neither the strength of the insect's suction, nor the direct energetic cost of xylem ingestion, have ever been quantified. Philaenus spumarius froghoppers were used to address these gaps in our knowledge. Micro-CT scans of its cibarium and measurements of cibarial muscle sarcomere length revealed that P. spumarius can generate a maximum tension of 1.3 ± 0.2 MPa within its cibarium. The energetic cost of xylem extraction was quantified using respirometry to measure the metabolic rate (MR) of P. spumarius while they fed on hydroponically grown legumes, while xylem sap excretion rate and cibarial pumping frequency were simultaneously recorded. Increasing the plants' xylem tensions up to 1.1 MPa by exposing their roots to polyethylene glycol did not reduce the insects' rate of xylem excretion, but significantly increased both MR and pumping frequency. We conclude that P. spumarius can gain energy feeding on xylem sap containing previously reported energy densities and at xylem tensions up to their maximum suction capacity.

Ground Cover Management in Olive Groves Reduces Populations of Philaenus spumarius (Hemiptera: Aphrophoridae), Vector of Xylella fastidiosa.

Philaenus spumarius (Linnaeus, 1758) (Hemiptera: Aphrophoridae) is considered the main vector of Xylella fastidiosa (Wells Raju et al. 1986) (Xathomonadales: Xanthomonadaceae), agent of the Olive Quick Decline Syndrome in Southern Europe. To limit the spread of the disease, it is of primary importance to identify effective control measures against the vector. Besides chemical control, cultural practices could potentially help reducing vector activity and population density. Here, we tested the effectiveness of three different ground cover management practices in controlling vector populations in olive groves in the Abruzzo region (Central Italy). We compared tillage (two tillage operations in spring followed by two cuts in summer), frequent mowing (four cuts from spring to summer) and a control (two cuts in summer) by sampling vectors both in the ground vegetation and in the tree canopy. In late spring, after the peak of the population, tillage reduced P. spumarius density by 60%, while frequent mowing only reduced the density by 20% compared to control plots. The differences tended to disappear with time. The treatments had the same effect on the vector density in both the ground vegetation and tree canopy. The vectors were more concentrated in the ground cover at the beginning of the season while in summer both the canopy and ground vegetation had the same vector density. Our findings suggest that tillage is a viable option for the containment of P. spumarius, as frequent mowing did not achieve sufficient control efficacy.

Biofoam of Spittlebug, Poophilus costalis (Walker): Preferential Sites, Temperature Regulation, Chemical Composition and Antimicrobial Activity.

The foam produced by nymphs of Poophilus costalis on eleven different host plants belonging to eight families on St. Xavier's College campus in India was studied over five months. The chemical composition and antimicrobial activity of these biofoams were investigated. The results revealed that P. costalis preferred Theporsia purpurea and Mimosa pudica for laying their eggs and producing foam, over the other tested plants. P. costalis produce their foam on either nodes or internodes on monocotyledons (30%) (p < 0.05), whereas on dicotyledons, they produce more foam on the stems (63.8%) than on the leaves (6.2%) (p < 0.01). The number of nymphs in each piece of foam from P. costalis varied from 1 to 3 (mean = 1.8 per plant). They produced their foam (5.7 to 45.2 cm) from the ground level on a plant. The length and breadth of a piece of foam ranged from 1.0 to 3.9 cm and 0.6 to 4.7 cm, respectively. The foam tended to be cooler than the environment. Qualitative profiling showed that the foam consists of carbohydrates, including maltose; trypsin; amino acids; protease. The foam was also analyzed using a spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), gas chromatography-mass spectroscopy (GC-MS), and high-performance liquid chromatography (HPLC). The antimicrobial activity of the biofoam was the greatest against Staphylococcus aureus, the growth of which was reduced by 55.9 ± 3.9%, suggesting that the foam could be used as an antimicrobial product. However, no activities were observed against Fusarium oxysporum and Candida albicans.

Two new species of Aufidus (Hemiptera, Cercopidae) from New Guinea.

Two new species of the cercopid genus Aufidus Stål, 1863, A. alacaligus sp. nov. and A. albonigrus sp. nov., are described and illustrated, Photos of the type specimens with the illustrations of the male terminalia are provided. A key to the species found in New Guinea is presented.

Seasonal Abundance and Infectivity of Philaenus spumarius (Hemiptera: Aphrophoridae), a Vector of Xylella fastidiosa in California Vineyards.

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is a vector of the plant pathogen Xylella fastidiosa; however, its role in recent outbreaks of Pierce's disease of grapevine (PD) in California is unclear. While the phenology and ecology of P. spumarius can help determine its contributions to PD epidemics, both remain poorly described in the North Coast vineyards of California. We assessed the phenology of P. spumarius in the region. Spittlemasses were first observed in February or March, while the emergence of adult spittlebugs did not occur until April or May depending on the year. Analysis of sweep and trap data from 2016 to 2018 revealed significant effects of survey month, vineyard site, and year on adult abundance in sweep and trap surveys. Spittlebug adults were present in the vineyards from April until December, with the greatest number of adults by sweep net in May or June, whereas adults on traps peaked between July and November. Analysis of natural infectivity in groups of field-collected spittlebug adults showed significant difference in transmission rates among months. Spittlebugs successfully transmitted Xylella fastidiosa (Wells) (Xanthomonadales: Xanthomonadaceae) to potted grapevines between July and December. The greatest risk of X. fastidiosa transmission by P. spumarius was in December (60%) followed by October (30%). However, the infectivity patterns of the meadow spittlebug did not align with the historical paradigm of California North Coast PD. We discuss alternative hypotheses in which P. spumarius could play a role in the epidemiology of this disease.

Dispersal of Philaenus spumarius (Hemiptera: Aphrophoridae), a Vector of Xylella fastidiosa, in Olive Grove and Meadow Agroecosystems.

The introduction of the Xylella fastidiosa Wells bacterium into Apulia (South Italy) has caused the massive dieback of olive trees, and is threatening olive production throughout the Mediterranean Region. The key vector of X. fastidiosa in Europe is the spittlebug Philaenus spumarius L. The dispersal capabilities of P. spumarius are poorly known, despite being a key parameter for the prediction of the spread of the bacterium. In this study, we have examined the dispersal of P. spumarius adults in two different agroecosystems in Italy: an olive grove in Apulia (Southern Italy) and a meadow in Piedmont (Northern Italy). Insects were marked with albumin and released during seven independent trials over 2 yr. The recapture data were pooled separately for each agroecosystem and used to estimate the dispersal kernels of P. spumarius in the olive grove and in the meadow. The diffusion coefficient estimate for P. spumarius was higher in the meadow than in the olive grove. The median distance from the release point for 1 d of dispersal was 26 m in the olive grove and 35 m in the meadow. On the basis of our model, we estimated that 50% of the spittlebug population remained within 200 m (98% within 400 m) during the 2 mo period of high abundance of the vector on olives in Apulia. The dispersal of P. spumarius is thus limited to some hundreds of meters throughout the whole year, although it can be influenced to a great extent by the structure of the agroecosystem.

Biology and Prevalence in Northern Italy of Verrallia aucta (Diptera, Pipunculidae), a Parasitoid of Philaenus spumarius (Hemiptera, Aphrophoridae), the Main Vector of Xylella fastidiosa in Europe.

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera Aphrophoridae), the main vector of Xylella fastidiosa Wells et al. in Europe, has few known natural enemies. The endoparasitoid Verrallia aucta (Fallén) (Diptera, Pipunculidae) was first noticed a long time ago but very little is known about its biology and prevalence. In this study, the presence and prevalence of V. aucta were investigated in different regions of northern Italy, both in plain-foothill and montane zones. Parasitic larvae were identified by the dissection of spittlebug adults, P. spumarius and Neophilaenus campestris (Fallén), and by a new species-specific molecular tool targeting the ITS2 and COI genomic regions, developed in this work. A small-scale rearing was set up to gain information on the life cycle of V. aucta on its main host P. spumarius. During the four-year investigation (2016-2019) the pipunculid parasitoid displayed low prevalence, reaching a maximum parasitization rate of 17.5% (calculated over the adult spittlebug season) in vineyards of the Piemonte region. Over the whole period, no significant difference in the prevalence was found between male and female spittlebugs. Collected data and rearing observations suggest that V. aucta is monovoltine and synchronous with P. spumarius, laying eggs in newly emerged adults, developing as an endoparasitoid through two larval stages during the whole summer, and overwintering as a pupa in the soil.