Anatomy of an agricultural antagonist: Feeding complex structure and function of three xylem sap-feeding insects illuminated with synchrotron-based 3D imaging.

Many insects feed on xylem or phloem sap of vascular plants. Although physical damage to the plant is minimal, the process of insect feeding can transmit lethal viruses and bacterial pathogens. Disparities between insect-mediated pathogen transmission efficiency have been identified among xylem sap-feeding insects; however, the mechanistic drivers of these trends are unclear. Identifying and understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases. Here, we applied synchrotron-based X-ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap-feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue-green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy-winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae). The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex. The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions. Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities. These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

Feeding behaviour and mortality of Philaenus spumarius exposed to insecticides and their impact on Xylella fastidiosa transmission.

BACKGROUND: Insecticides are essential, though controversial tools in modern pest management. Insecticides can slow the spread of key vector-borne plant pathogens, but often lead to inconsistent results given that insecticide use is generally focused on acute toxicity under no-choice conditions. Here, we analysed the lethal (survival) and sublethal (feeding behaviour) effects of six commercial products (acetamiprid, deltamethrin, spinosad, sulfoxaflor, pyrethrin and kaolin) on Philaenus spumarius, vector of the bacterium Xylella fastidiosa. Furthermore, we assessed the impact of insecticides displaying different degrees of acute toxicity against spittlebugs (highest to lowest: acetamiprid, pyrethrin and kaolin) on the transmission of X. fastidiosa by P. spumarius under both free-choice and no-choice conditions. RESULTS: Deltamethrin, acetamiprid and to a limited extent pyrethrin significantly altered the feeding behaviour of P. spumarius. Deltamethrin and acetamiprid were highly toxic against P. spumarius, but the mortality induced by exposure to pyrethrin was limited overall. By contrast, spinosad, sulfoxaflor and kaolin did not significantly impact P. spumarius feeding behaviour or survival. Under no-choice conditions, both pyrethrin and acetamiprid reduced the X. fastidiosa inoculation rate compared with kaolin and the control. On the other hand, pyrethrin reduced transmission, but acetamiprid failed to significantly affect bacterial inoculation under free-choice conditions. CONCLUSION: Pyrethrin was the only compound able to reduce X. fastidiosa transmission under both free-choice and no-choice conditions. Xylella fastidiosa management strategy based exclusively on the evaluation of insecticide acute toxicity under no-choice conditions would most likely fail to prevent, or slow, bacterial spread. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Leafhopper feeding behaviour on three grapevine cultivars with different susceptibilities to Flavescence dorée.

Scaphoideus titanus (Ball) is a grapevine-feeder leafhopper, and the most important vector of Flavescence dorée of grapevine (FD), a disease associated with phytoplasmas belonging to ribosomal subgroups 16Sr-V-C and -D. FD is a major constraint to viticulture in several European countries and, so far, its control has relied on roguing of infected plants and insecticide applications against the vector. Detailed knowledge on different levels of the multifaceted phytoplasma-plant-vector relationship is required to envisage and explore more sustainable ways to control the disease spread. In the present work, S. titanus feeding behaviour was described on three grapevine cultivars: Barbera (susceptible to FD), Brachetto, and Moscato (tolerant to FD) using the Electrical Penetration Graph (EPG) technique. Interestingly, no differences were highlighted in the non-phloem feeding phases, thus suggesting that the tested cultivars have no major differences in the biochemical composition or structure of the leaf cuticle, epidermis or mesophyll, that can affect the first feeding activities. On the contrary, the results showed significant differences in leafhopper feeding behaviour in terms of the duration of the phloem feeding phase, longer on Barbera and shorter on Brachetto and Moscato, and of the frequency of interruption-salivation events inside the phloem, higher on Brachetto and Moscato. These findings indicate a possible preference for the Barbera cultivar, a better host for the leafhopper. Scaphoideus titanus feeding behaviour on Barbera correlates with an enhanced FDp transmission efficiency, thus explaining, at least in part, the higher susceptibility of this cultivar to FD. The mechanisms for the possible non-preference for Brachetto and Moscato are discussed, and an antixenosis is hypothesized. We propose that breeding for resistance against FD should take into account both plant traits associated with the response to the phytoplasmas and to the vector.

Design of ideal vibrational signals for stinkbug male attraction through vibrotaxis experiments.

BACKGROUND: Many groups of insects utilize substrate-borne vibrations for intraspecific communication. This characteristic makes them a suitable model for exploring the use of vibrations as a tool for pest control as an alternative to the use of chemicals. Detailed knowledge of species communication is a prerequisite to select the best signals to use. This study explored the use of substrate-borne vibrations for pest control of the brown marmorated stink bug (BMSB), Halyomorpha halys Stål (Heteroptera: Pentatomidae). For this purpose, we first identified the spectral and temporal characteristics that best elicit male responsiveness. Bioassays were conducted with artificial signals that mimicked the natural female calling signal. Second, we used the acquired knowledge to synthesize new signals endowed with different degrees of attractiveness in single- and two-choice bioassays using a wooden custom-made T stand. RESULTS: The results from this study showed that males were attracted to female signals along a high range of amplitudes, especially starting from a threshold of 100 µm s-1 , a high pulse repetition time (1 s) and frequency peak corresponding to the first harmonic (76 Hz). This resulted in an "optimal" signal for use to attract males, while the choice test in the T arena showed that this signal elicits searching behavior and attracts BMSB males towards a stimulation point. CONCLUSION: We confirm the use of vibrational signals as a strong tool for behavioral manipulation of male BMSB and suggest its possible use in the development of field traps and further management of this pest. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Behavioral effects induced by organic insecticides can be exploited for a sustainable control of the Orange Spiny Whitefly Aleurocanthus spiniferus.

The Orange Spiny Whitefly (OSW) Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) represents a new serious threat to Citrus spp., grapevine and ornamental plants in the whole Mediterranean area. Such threat urgently calls for the development of a sustainable control strategy, including insecticides compatible with biological control, and applicable also in organic citrus farming that represent an essential part of Mediterranean agricultural economy. Therefore, we evaluated the toxicity and the effects on host searching, oviposition, and probing and feeding behavior exerted on OSW by organic insecticides supposed to have limited side effects on environment and ecosystem services, i.e. sweet orange essential oil (EO), extract of Clitoria ternatea (CT), mineral oil, pyrethrin and azadirachtin. Despite none of the compounds caused a significant mortality of any of the OSW instars, we observed interesting effects on whitefly behavior: (i) EO and pyrethrin showed a relevant repellent effect, with impairment of both adults landing and oviposition on treated plants; (ii) CT and pyrethrin strongly affected probing behavior. Here, in the light of our findings, we discuss possible OSW sustainable control strategies and further research perspectives.

Transmission of Xylella fastidiosa Subspecies Pauca Sequence Type 53 by Different Insect Species.

Diseases associated with Xylella fastidiosa have been described mostly in North and South America. However, during the last five years, widespread X. fastidiosa infections have been reported in a constrained area of the Apulia region (southern Italy), in olives trees suffering a severe disease, denoted as Olive Quick Decline Syndrome (OQDS). Because many xylem sap-feeding insects can function as vectors for the transmission of this exotic pathogen in EU, several research programs are ongoing to assess the role of candidate vectors in the spread of the infections. Initial investigations identified Philaenus spumarius (L.) as the predominant vector species in the olive orchards affected by the OQDS. Additional experiments have been carried out during 2016 and 2017 to assess the role of other species. More specifically, adults of the spittlebugs Philaenus italosignus Drosopolous and Remane, Neophilaenus campestris (Fallen) and of the planthopper Latilica tunetana (Matsumura) (Issidae) have been tested in transmission experiments to assess their ability to acquire the bacterium from infected olives and to infect different susceptible hosts (olives, almond, myrtle -leaf milkwort, periwinkle). Acquisition rates determined by testing individual insects in quantitative PCR assays, ranging from 5.6% in N. campestris to 22.2% in P. italosignus, whereas no acquisition was recorded for L. tunetana. Successful transmissions were detected in the recipient plants exposed to P. italosignus and N. campestris, whereas no trasmissions occurred with L. tunetana. The known vector Philaenus spumarius has been included in all the experiments for validation. The systematic surveys conducted in 2016 and 2017 provided further evidence on the population dynamics and seasonal abundance of the spittlebug populations in the olive groves.

Distribution and Relative Abundance of Insect Vectors of Xylella fastidiosa in Olive Groves of the Iberian Peninsula.

The phytosanitary emergency caused by the spread of Xylella fastidiosa in the Mediterranean has raised demands for a better understanding of the ecology of its presumed and candidate insect vectors. Here, we present the results of a two-year survey carried out in olive groves across southern, eastern and Central Spain and northeastern Portugal. Several sampling methods were tested and compared to select the most appropriate to estimate population levels of potential vectors of X. fastidiosa. The spittlebugs Philaenus spumarius and Neophilaenus campestris (Hemiptera: Aphrophoridae) were the main species associated with olive groves. Both species were widely present on herbaceous ground vegetation within the olive groves; P. spumarius mainly associated with Asteraceae and N. campestris with Poaceae. Due to the patchy distribution of spittlebugs within the olive groves, sweep nets were the most effective and least time-consuming sampling method for the estimation of population size both in the ground cover and tree canopies. Trends in population density showed that spittlebugs can be abundant on ground vegetation but very rare on olive canopies. Spittlebugs disperse in late spring to non-cultivated hosts that act as natural reservoirs. In late fall, adults return to the olive groves for oviposition. However, olive trees may act as transient hosts for spittlebugs and high population densities of these insect vectors should be avoided in areas where X. fastidiosa is present.

EPG combined with micro-CT and video recording reveals new insights on the feeding behavior of Philaenus spumarius.

The meadow spittlebug Philaenus spumarius plays a key role in the transmission of the bacterium Xylella fastidiosa to olive in Apulia (South Italy). Currently, available data on P. spumarius feeding behavior is limited, and a real-time observation of the different steps involved in stylet insertion, exploratory probes, and ingestion, has never been carried out. Therefore, we performed an EPG-assisted characterization of P. spumarius female feeding behavior on olive, in order to detect and analyze the main EPG waveforms describing their amplitude, frequency, voltage level, and electrical origin of the traces during stylet penetration in plant tissues. Thereafter, each of the main waveforms was correlated with specific biological activities, through video recording and analysis of excretion by adults and excretion/secretion by nymphs. Furthermore, the specific stylet tips position within the plant tissues during each of the waveforms observed was assessed by microcomputer tomography (micro-CT). Additional EPG-recordings were carried out with males of P. spumarius on olive, in order to assess possible sex-related differences. P. spumarius feeding behavior can be described by five main distinct waveforms: C (pathway), Xc (xylem contact/pre-ingestion), Xi (xylem sap ingestion), R (resting), N (interruption within xylem phase). Compared to males, females require shorter time to begin the first probe, and their Xi phase is significantly longer. Furthermore, considering the single waveform events, males on olive exhibit longer np and R compared to females.

Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy.

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa-infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

Infectivity and transmission of Xylellua fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy.

Discovery of Xylella fastidiosa from olive trees with "Olive quick decline syndrome" in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.