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Simple Summary: To reliably identify the gut contents of plant-feeding insects, the removal of contaminant plant DNA from the insect surface is necessary. Previous approaches have used bleach and alcohol washes to achieve this. We perform a controlled baseline study on a herbivorous insect, the Meadow Spittlebug (Philaenus spumarius), to identify possible contamination that may persist after bleach washes. Despite the reported success of these methods, we find that contamination is still present, leading to unreliable results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove. We conduct a further three experiments, investigating the effectiveness of more robust bleach washes, sterilised gut excision, and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination while still detecting signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut. Dietary analysis of herbivorous insects relies on successfully eliminating surface contamination. If this cannot be performed reliably, then it will not be possible to differentiate between plants that the insect is feeding on and plants the insect has been in contact with, either directly or via pollen. Methods in the literature often use bleach and alcohol washes to remove contamination. We perform a controlled metabarcoding baseline study on a herbivorous, xylem-feeding insect, the Meadow Spittlebug (Philaenus spumarius), using Oxford Nanopore Technologies (ONT) sequencing, and identify possible contamination that persists after washes. Despite the reported success of methods in the literature, we find that contamination is still present, leading to possible false-positive results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove, and conduct a further three experiments with the goal of removing pollen from the surface of Philaenus spumarius. This study investigates the effectiveness of robust bleach/Tween/alcohol washes, sterile gut excision (including combined with Distel application), and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination and still detect signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Citrus is a global fruit crop and one of the most destructive citrus diseases is the Citrus Variegated Chlorosis (CVC) caused by the bacterium Xylella fastidiosa Wells et al. The pathogen is usually transmitted by xylem sap-feeding insects. Given the tremendous impact that a X. fastidiosa CVC invasion could have in the Mediterranean and the consequent catastrophic scenarios, we offer robust data about the potential vector's bionomics in citrus groves. Throughout 2022, we sampled two citrus groves with sweet orange trees, Citrus × sinensis (L.) Osbeck, in central Greece plus a nearby natural area with pine trees, Pinus halepensis Miller, and lentisk shrubs, Pistacia lentiscus L., to study the presence, seasonal occurrence and population dynamic of potential insect vectors of X. fastidiosa. Additionally, another thirty-five citrus fields were single sampled in 2023 to gain more reliable results about the presence and relative abundance of the potential vectors of the bacterium in a wide area. Seven potential insect vectors were recorded. Philaenus spumarius L. and Neophilaenus campestris (Fallen) were the most frequently found and abundant species in all the citrus groves. Spittlebug nymphs were observed between late March and mid-May, whereas adults were present in spring, autumn and early winter. In the natural area, N. campestris observed from summer to early autumn on the pines, suggesting that pines are among the food plants for the species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa Wells, a bacterial plant pathogen, represents a threat to many crops all around the word. Unfortunately, no effective treatments are available to reduce the infection and, to date, the most promising strategy relies on controlling the meadow spittlebug Philaenus spumarius L. one of the main vectors of X. fastidiosa. Among insecticides and repellents, botanical insecticides represent valuable candidates. Their encapsulation into stable formulations, like nanoemulsions (NEs), can boosts the efficacy and stability. We investigated the toxicity, repellent, and antifeedant activity of carlina oxide, a polyacetylene isolated from the roots of Carlina acaulis L. (Asteraceae), and its NEs, against P. spumarius adults. In addition, we carried out electroantennographic (EAG) tests to evaluate the capability of the male and female antennae to perceive carlina oxide. EAG assays demonstrated that P. spumarius antennal olfactory system of both sexes is capable of perceiving carlina oxide over a wide range of doses and that females, at the lowest doses, have significantly greater olfactory sensitivity than males (0.01 and 0.1 µg, respectively (p < 0.001)). In toxicity experiments, the percentage of adult survival in topical and fumigant trials was high, while it resulted significantly lower in ingestion trials. In olfactory tests, P. spumarius showed a slight preference for green beans treated with 1 % carlina oxide. However, no significant differences were observed between treatments when the concentration of carlina oxide was increased to 3 %. On the contrary, feeding tests showed a significant repellent effect of carlina oxide against P. spumarius adults up to 24 h after the treatment and a reduction in P. spumarius presence on treated green beans ranging from 51.2 % to 94.7 %, if compared with control green beans. Overall, this study sheds light on the possible development of effective and environmentally friendly formulations of carlina oxide to manage P. spumarius. Further studies are needed to evaluate the efficacy of this natural compound in field conditions, and to investigate its potential side effects on non-target species. [Display omitted] • Carlina oxide bioactivity was assessed on Philaenus spumarius. • A stable 3 % (w/w) carlina oxide nanoemulsion (NE) was developed. • Sexually dimorphic electroantennographic responses were noted on P. spumarius. • Carlina oxide showed antifeedant/repellent activity against P. spumarius. • Ingestion toxicity of the carlina oxide NE was detected on adults. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

The bacterium Xylella fastidiosa (Xf) is an invasive insect-borne pathogen, which causes lethal diseases to important crops including olives, citrus, almonds and grapes as well as numerous forest, ornamental, and uncultivated plants. Outbreaks of Xf-related plant diseases are currently occurring in the Mediterranean region, causing substantial losses to various agricultural sectors. Several models have recently been published to identify which regions are at highest risk in Europe; however, such models did not consider the insect vectors, which constitute the key driver of short-range Xf spread. We fitted bioclimatic species distribution models to depict the macroclimatic preferences of the meadow spittlebug Philaenus spumarius L. (1978) (Hemiptera: Aphrophoridae), the major epidemiologically relevant vector currently responsible for Xf spread in the Europe. Many regions of Western Europe and Mediterranean basin are predicted by models as highly climatically suitable for this vector, including all regions where severe Xf have occurred so far. Conversely, the driest and warmest areas of the Mediterranean basin are predicted as little suitable for P. spumarius. Models forecast that agricultural-important parts of the southern Mediterranean area might experience a substantial decrease in climatic suitability for P. spumarius by the period 2040–2060. Areas predicted as highly suitable just for the bacterium but not optimal for this vector are apparently still free of severe Xf outbreaks, suggesting that climate tolerances of P. spumarius might partly explain the current spatial pattern of Xf outbreaks in Europe and should always be considered in further risk assessments. [ABSTRACT FROM AUTHOR]

The characterization of behavioural cues involved in the location of a host-plant by a phytophagous insect allows the elaboration of tools that can be exploited in the sustainable protection of cultivated plants. In this context, the identification of the blend of Volatile Organic Compounds (VOCs) released by a host plant can be profitably used for diverting pest insects away from it and towards specific targets (plants, traps). Philaenus spumarius, the main vector of Xylella fastidiosa, i.e., the pathogen bacterium responsible for the Olive Quick Decline Syndrome (OQDS), moves to olive trees when the grass cover dries up. To date, the possible role of chemical cues regulating this passage is not known. Moreover, it is still to be tested whether VOCs are involved in the resistance to the bacterium recorded in two olive varieties, namely FS-17 and Leccino. To verify these hypotheses, we characterized the behavioural response of P. spumarius towards the two resistant (Leccino, FS-17) and three common susceptible olive trees varieties (Ogliarola, Frantoio and Rotondella) in olfactometer bioassays. The response of P. spumarius towards olive varieties was sex-dependent: males were totally unresponsive whilst females were attracted by Ogliarola, Rotondella and Frantoio, and repelled by FS-17. No preference was recorded when testing females against Leccino and clean air. The characterization and the quantification of the VOCs released by the olive varieties tested supported the behavioural observations and paved the way to discuss their possible role in the elaboration of integrated protocols for the sustainable protection of olive trees from the deadly expansion of X. fastidiosa. [ABSTRACT FROM AUTHOR]

The development of an efficient indoor rearing protocol of Philaenus spumarius is a key step for research on the meadow spittlebug, as it could reduce researchers' dependency on the availability and collection of univoltine individuals in the field. Here, we implemented the rearing protocol previously described by inducing females' parapause breakage before mating. This new step enables the completion of the life cycle of P. spumarius (adult to adult) in 149 days, potentially providing three generations per year under laboratory conditions. [ABSTRACT FROM AUTHOR]

Abstract Insect declines have been reported worldwide, although the particular causes of the declines may be complex and are poorly understood. Meadow spittlebugs were one of the most abundant insects in the coastal prairie along the California coast 40 yr ago but have largely disappeared. Evidence links this decline to changing climatic conditions, which have reduced survival of eggs and neonates. We identified several refugia where meadow spittlebug populations have persisted amidst unfavorable conditions. Protection from desiccating winds was the common attribute of these refugia. Following a wet year, adult meadow spittlebugs were able to disperse from one refuge that we studied to recolonize coastal prairie habitats, although populations declined over the next two drier years. Because of their previous high abundance, loss of meadow spittlebugs is likely to affect the functioning of this widespread habitat, including energy transfer, their host plants, and their predators. In addition, meadow spittlebugs are unusual in having been the subject of extensive physiological and long‐term ecological data, so they can serve as a bellwether species, indicating the effects of climate change.

Insect declines have been reported worldwide, although the particular causes of the declines may be complex and are poorly understood. Meadow spittlebugs were one of the most abundant insects in the coastal prairie along the California coast 40 yr ago but have largely disappeared. Evidence links this decline to changing climatic conditions, which have reduced survival of eggs and neonates. We identified several refugia where meadow spittlebug populations have persisted amidst unfavorable conditions. Protection from desiccating winds was the common attribute of these refugia. Following a wet year, adult meadow spittlebugs were able to disperse from one refuge that we studied to recolonize coastal prairie habitats, although populations declined over the next two drier years. Because of their previous high abundance, loss of meadow spittlebugs is likely to affect the functioning of this widespread habitat, including energy transfer, their host plants, and their predators. In addition, meadow spittlebugs are unusual in having been the subject of extensive physiological and long‐term ecological data, so they can serve as a bellwether species, indicating the effects of climate change. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The meadow spittlebug Philaenus spumarius, vector of the bacterium Xylella fastidiosa, relies on vibrational communication to accomplish mating: the female calls to establish a duet with a male. A deeper knowledge of the species' reproductive biology and behaviour would provide useful information for developing control techniques based on principles of 'biotremology', which studies the vibrational behaviour of animals. Playback tests were conducted on single females and male–female pairs of P. spumarius from June to October 2018, and the features of the recorded calling signals were analysed using a wavelet decomposition. Dissections were performed on females to evaluate the relationship between calling activity and ovarioles development. From August onwards, females started to emit calling signals and to develop ovarioles. Female calling activity, duration of their chirps and their responsiveness to mating increased as the season progressed, and they were correlated with ovarioles' development and presence of mature eggs. Hence, the ovarian maturation represents a key factor in association with the development of the sexual behaviour of P. spumarius females. Conversely, males produced advertisement signals soon after adult eclosion in May, but these signals were not involved in the pair formation process. Mating was achieved only when males produced courtship signals in response to female calling signals and established with them vibrational duets starting from August. Here, we provide new information regarding the P. spumarius' ethology and hypothesize that potential mating disruption techniques should consider the insect physiology and be applied when both sexes are responsive to mating signals. [ABSTRACT FROM AUTHOR]

As a vector of Xylella fastidiosa (Wells, 1987) in Europe, the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) is a species of major concern. Therefore, tools and agents to control this ubiquitous insect that develops and feeds on hundreds of plant species are wanted. We conducted a field survey of P. spumarius eggs in Corsica and provide a first report of Ooctonus vulgatus Haliday, 1833 (Hymenoptera, Mymaridae) as a potential biocontrol agent of P. spumarius in Europe. To allow species identification, we summarized the main characters distinguishing O. vulgatus from other European species of Ooctonus and generated COI DNA barcodes. Parasitism rates were variable in the four localities included in the survey but could reach 69% (for an average number of eggs that hatched per locality of 109). Based on the geographic occurrences of O. vulgatus obtained from the literature, we calibrated an ecological niche model to assess its potential distribution in the Holarctic. Obviously, several questions need to be addressed to determine whether O. vulgatus could become an effective biocontrol agent of P. spumarius in Europe. So far, O. vulgatus has been reared only from P. spumarius eggs, but its exact host-range should be evaluated to ensure efficiency and avoid non-target effect. The top-down impact of the parasitoid on vector populations should also be assessed on large data sets. Finally, the feasibility of mass rearing should be tested. We hope this report serves as a starting point to initiate research on this parasitoid wasp to assess whether it could contribute to reduce the spread and impact of X. fastidiosa in Europe.

The meadow spittlebug, Philaenus spumarius L. (Hemiptera: Aphrophoridae), is the main vector of the phytopathogenic bacterium Xylella fastidiosa in Europe, where the ST53 strain induces the olive quick decline syndrome, causing severe economic damage in southern Italy. The wide range of plant species infected by X. fastidiosa, and the wide host range of P. spumarius suggest that a large number of wild and cultivated plants may become infected by the pathogen following unintentional introduction events. Therefore, it is necessary to detail the host plant preference of the vector, in order to include preferred plants in the field, in pathogen‐targeted diagnostic efforts. This would allow the identification of main sources of X. fastidiosa acquisition by P. spumarius; such plant species represent an important target for rational disease management. Here, we investigated the host plants of P. spumarius in north‐western Italy, a region where X. fastidiosa is still not present but is regarded as a primary threat. We designed a new molecular diagnostic tool targeting chloroplast DNA, to characterize the gut content of single P. spumarius adults. The newly set up, nested PCR/sequencing‐based identification protocol was proven to be useful for retrieving sequences from the last two different host plants used by P. spumarius, even if limited persistence of intact chloroplast DNA was reported in the spittlebug gut. We propose this protocol as a new tool for supporting research on xylem feeder biology that could be particularly useful for highly polyphagous species such as P. spumarius. Furthermore, the method could help monitor X. fastidiosa invasion, and contribute to the study of vector ecology and pathogen epidemiology. [ABSTRACT FROM AUTHOR]

As a vector of Xylella fastidiosa (Wells, 1987) in Europe, the meadow spittlebug Phi- laenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) is a species of major concern. Therefore, tools and agents to control this ubiquitous insect that develops and feeds on hundreds of plant species are wanted. We conducted a field survey of P. spumarius eggs in Corsica and provide a first report of Ooctonus vulgatus Haliday, 1833 (Hymenoptera, Mymaridae) as a potential biocontrol agent of P. spumarius in Europe. To allow species identification, we summarized the main characters distinguishing O. vulgatus from other European species of Ooctonus and generated COI DNA barcodes. Parasitism rates were variable in the four localities included in the survey but could reach 69% (for an average number of eggs that hatched per locality of 109). Based on the geographic occurrences of O. vulgatus obtained from the literature, we calibrated an ecological niche model to assess its potential distribution in the Holarctic. Obviously, several questions need to be addressed to determine whether O. vulgatus could become an effective biocontrol agent of P. spumarius in Europe. So far, O. vulgatus has been reared only from P. spumarius eggs, but its exact host-range should be evaluated to ensure efficiency and avoid non-target effect. The top-down impact of the parasitoid on vector populations should also be assessed on large data sets. Finally, the feasibility of mass rearing should be tested. We hope this report serves as a starting point to initiate research on this parasitoid wasp to assess whether it could contribute to reduce the spread and impact of X. fastidiosa in Europe. [ABSTRACT FROM AUTHOR]

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 meadow spittlebug, Philaenus spumarius (Hemiptera: Aphrophoridae; Linnaeus, 1758), is considered the main vector in the ongoing Xylella fastidiosa outbreak in the olive groves of southern Italy. Identifying the factors driving the spatial distribution of P. spumarius is of primary importance to determine X. fastidiosa infection risk in healthy olive groves. Here, we investigated the local and landscape factors shaping the occurrence and abundance of P. spumarius by sampling 182 olive groves over 2 years in the Abruzzo region. We found the occurrence and abundance of the vector to be positively associated with the proportion of olive groves in the landscape and negatively with the cover of vineyards. Philaenus spumarius best responded to landscape processes at small spatial scale (125–250 m) confirming the available information regarding its dispersal ability. At the local scale, soil management and pesticide application did not affect the vector probably because both interventions were not timed according to the vector biology and ecology. Our findings suggest that the management of agricultural landscape mosaics can drive the spatial distribution of P. spumarius. In particular, the landscape composition of the close surrounding of the olive groves may potentially influence the emergence probability of local X. fastidiosa epidemics through its effects on the vector spatial distribution and activity. [ABSTRACT FROM AUTHOR]

The meadow froghopper, Philaenus spumarius L., is endemic in Italy and was not considered a harmful species until 2014, when the olive quick decline syndrome (OQDS) showed up in Apulia (southern Italy). It was immediately suspected and then verified as the main vector of Xylella fastidiosa, the bacterium responsible for the disease. Currently, EU Directives consider the fight against P. spumarius compulsory in member states and recommend Integrated Pest Management (IPM), both in uncultivated and cultivated infested areas, to minimise the environmental impact of chemical pesticides. This should be based on an improved knowledge of the vector with its seasonal trends and feeding habits linked to specific herbaceous species. In this context, our field study was aimed to improve the understanding of the vector nutritional behaviour, especially at its critical nymph stage, by monitoring its presence on different herbaceous target species, using its typical feeding foams as key indicator. The study area was in Lazio region (central Italy), dedicated to olive growing and still unaffected by the X. fastidiosa plague. Over two years, during the nymph development period, field data have been acquired over the test area and then analysed by coupling statistical (ANOVA), geographical information system (GIS) and geo-referenced field sampling approaches. Results highlighted that P. spumarius exhibits significant preferences for specific herbaceous plants, especially at its early development stages, detectable by tenuous spittle. This indicates female oviposition activity, which seems also not influenced by olive tree proximity. Furthermore, the non-host plant species identified here could be suitable for creating green barriers for limiting the vector diffusion to contiguous areas where sensible plantations are growing. In the final section, applied implications arising from the present findings for P. spumarius population management are discussed. [ABSTRACT FROM AUTHOR]

Background: Colour polymorphisms are common among animal species. When combined with genetic and ecological data, these polymorphisms can be excellent systems in which to understand adaptation and the molecular changes underlying phenotypic evolution. The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae), a widespread insect species in the Holarctic region, exhibits a striking dorsal colour/pattern balanced polymorphism. Although experimental crosses have revealed the Mendelian inheritance of this trait, its genetic basis remains unknown. In this study we aimed to identify candidate genomic regions associated with the colour balanced polymorphism in this species. Results: By using restriction site-associated DNA (RAD) sequencing we were able to obtain a set of 1,837 markers across 33 individuals to test for associations with three dorsal colour phenotypes (typicus, marginellus, and trilineatus). Single and multi-association analyses identified a total of 60 SNPs associated with dorsal colour morphs. The genome size of P. spumarius was estimated by flow cytometry, revealing a 5.3 Gb genome, amongst the largest found in insects. A partial genome assembly, representing 24% of the total size, and an 81.4 Mb transcriptome, were also obtained. From the SNPs found to be associated with colour, 35% aligned to the genome and 10% to the transcriptome. Our data suggested that major loci, consisting of multi-genomic regions, may be involved in dorsal colour variation among the three dorsal colour morphs analysed. However, no homology was found between the associated loci and candidate genes known to be responsible for coloration pattern in other insect species. The associated markers showed stronger differentiation of the trilineatus colour phenotype, which has been shown previously to be more differentiated in several life-history and physiological characteristics as well. It is possible that colour variation and these traits are linked in a complex genetic architecture. Conclusions: The loci detected to have an association with colour and the genomic and transcriptomic resources developed here constitute a basis for further research on the genetic basis of colour pattern in the meadow spittlebug P. spumarius. [ABSTRACT FROM AUTHOR]

1. Colour polymorphisms are common across animals and are often the result of complex selection regimes. Philaenus spumarius ( Linnaeus) ( Hemiptera, Aphrophoridae) shows a widely studied dorsal colour polymorphism with several described phenotypes whose variation in their occurrence and frequency, as well as their maintenance across time, have been reported. Several selective influences have been suggested to play a role, but the mechanisms underlying the maintenance of this polymorphism are still poorly understood. 2. To explore the adaptive significance of the colour polymorphism of P. spumarius, an experiment was conducted in captivity under semi-natural conditions to measure survival, reproductive success, and duration of egg maturation. 3. It was found that there was higher longevity, a higher number of oviposition events, and a higher number of eggs laid by trilineatus phenotype females than by typicus and marginellus, supporting previous reports of an increase in trilineatus frequency during the season. The duration of egg maturation did not differ among phenotypes. 4. The higher longevity and fertility of the trilineatus phenotype may compensate, for example, the higher rate of attack by parasitoids and/or higher solar radiation reflectance in this phenotype, which have already been reported in previous studies, constituting a possible mechanism for the maintenance of this polymorphism. [ABSTRACT FROM AUTHOR]

Simple Summary: Vibrational communication is widespread in insects. Since most studies of intrasexual behavior on insects have targeted few individuals, the behaviors occurring within groups have been left unexplored. Including multiple individuals in behavioral studies on vibrational communication would provide more reliable information about their intrasexual behavior. In semi-field conditions, the intrasexual behavior of the meadow spittlebug, Philaenus spumarius, was studied. Using a laser vibrometer, we recorded vibrational signals exchanged among individuals of the same sex, throughout their adult stage and during the day, from the morning to the evening. The results showed that males were less active than females and the interactions among males were less frequent than among females. Intrasexual interactions were characterized by signal overlapping in both unisex groups, in addition to signal alternating only in the case of males. This study provided a better understanding of the social behavior of P. spumarius based on vibrational signals. 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. [ABSTRACT FROM AUTHOR]

• Foregut morphometry of leafhopper and spittlebug performed with μCT and SEM. • Foregut hydrodynamics reproduced through CFD modeling. • The foregut of the spittlebug may harbor twice as many X. fastidiosa cells as the leafhopper. • Sap velocity in the foregut is 10 cm/s if only 5% of the cibarium volume is exploited. • Bacterial colonization may impact sap ingestion of a leafhopper more than the spittlebug. Xylem sap sucking insects are adapted to ingest fluids under tension. Although much has been learned about such feeding strategy, this adaptation still poses several unresolved questions, including how these insects ingest against strong xylem sap tension. Xylem sap-feeding insects are vectors of the plant pathogenic xylem-limited bacterium Xylella fastidiosa. This bacterium colonizes the cuticular lining of the foregut of vectors in a persistent manner. We used micro-computed tomography and scanning electron microscopy to investigate the foregut morphometry of two X. fastidiosa vector species: Philaenus spumarius and Graphocephala atropunctata (Hemiptera: Aphrophoridae and Cicadellidae, respectively). On the basis of morphometric data, we built a hydrodynamic model of the foregut of these two insect species, focusing on the precibarium, a region previously shown to be colonized by X. fastidiosa and correlated with pathogen acquisition from and inoculation to plants. Our data show that space in the P. spumarius functional foregut could potentially harbor twice as many cells as similar space in G. atropunctata , although the opposite trend has been observed with biological samples. Average flow velocity of ingested fluid depended on the percentage of the cibarium volume exploited for suction: if the entire volume were used, velocities were in the range of meters per second. In contrast, velocities on the order of those found in the literature (about 10 cm/s) were attained if only 5% of the cibarium volume were exploited. Simulated bacterial colonization of the foregut was analyzed in relation to hydrodynamics and pressure needed for insects to ingest. Our model is designed to represent the diameter reduction of the food canal in both insect species when infected with X. fastidiosa. Results indicated that full bacterial colonization significantly increased the mean sap-sucking flow velocity. In particular, the colonization increased the maximum section-averaged velocity in the G. atropunctata more than two times and the net pressure needed to mantain the flow in the precibarium when colonized is relevant (about 0.151 MPa) if compared to a standard xylem sap tension (1 MPa). Bacterial colonization also influenced the sucking process of the G. atropunctata , by hindering the formation of a recirculation zone (or eddy), that characterizd the flow in the distal part of the precibarium when bacteria were absent. On the other hand, considering the pressure the insect must generate to feed, X. fastidiosa colonization probably influences fitness of the G. atropunctata more than that of P. spumarius. [ABSTRACT FROM AUTHOR]

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is an important vector for the xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which is associated with olive quick decline syndrome in southern Italy. The mouthparts of Hemiptera have important roles in host plant selection, feeding behavior and for vectoring pathogens that cause plant diseases. In this study, the functional morphology of the sensory structures located on the labium tip and precibarium of P. spumarius was investigated using scanning and transmission electron microscopy. The labium tip is composed of two symmetrical sensory complexes, each with five different types of sensilla: aporous sensilla trichodea type 1 and 2; uniporous sensilla chaetica type 1 and 2; and multiporous sensilla basiconica. The precibarium of P. spumarius has two kinds of sensory structures: bulbous sensilla and papillae sensilla. In particular, two groups of sensilla are located on the epipharynx: a distal group that consists of ten papillae sensilla and a proximal group composed of six papillae sensilla and two bulbous sensilla, while the hypopharynx has only two papillae sensilla. The involvement of these sensory structures in the context of feeding behavior and pathogen transmission is discussed.