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The vector‐borne bacterium Xylella fastidiosa(Wells and Raju) causes several serious diseases to plants. Recently, different subspecies of X. fastidiosa were reported in some European countries. The risk of the bacterium’s spread on the entire European territory is very high; therefore, it has been added into the priority pest list (2019/1702/EU Regulation). The main purposes of this work were to verify the presence of potential vectors in areas at a high risk of introduction in Tuscany and to ascertain the presence of X. fastidiosa in these insect vectors. Over 4,000 Auchenorrhyncha were collected and analysed from 2015 to 2019. Among the xylem sap-feeder putative vectors, most of the insects collected belonged to the family Aphrophoridae, but also many species of leafhopper were identified. Overall, in Tuscany four species were the most represented: Philaenus spumarius(L.), Cicadella viridis(L.), Synophropsis lauri (Horvath) and Neophilaenus campestris(Fallen).In 2018 an outbreak of X. fastidiosa subsp. multiplex was reported in Monte Argentario (Grosseto province, Tuscany). In 2019 X. fastidiosa subspecies multiplex ST 87 was detected in seven P. spumarius and three N. campestris collected from the infected area.

Philaenus spumarius is the main vectors of Xylella fastidiosa in Europe. For integrated pest management, the monitoring of the pest is crucial to planning an efficient and sustainable control strategy. The monitoring of spittlebug adults is carried out mainly using the sweeping net. Nevertheless, several studies have reported its ineffectiveness in sampling insects from the olive canopy. Moreover, using the sweeping net requires quite a lot of time and a skilled operator. So far, only one study has been carried out on the attractiveness of different color sticky traps for adults of P. spumarius. The results showed that the most attractive one was yellow. Nevertheless, the use of yellow sticky traps in a field has highlighted a low efficacy in monitoring spittlebugs. In order to improve the knowledge on this topic a new studies on the attractiveness of different colors are needed. Within the Life Resilience project, a field trial, aimed at testing different color sticky traps, has been carried out in three different olive groves in Tuscany in the 2020 fall. The tested colors were white, red, blue, brown, dark green, yellow (as positive control) and transparent as negative control. For each color, two 20x25 cm double-sided sticky boards were positioned in the olive grove respectively at 50 and 100 cm from the ground. Traps were controlled every fifteen days and trapped specimens of P. spumarius and N. campestris were removed, identified, sexed and counted. Moreover, during every on-site visit a monitoring of the population density was carried out by means of the sweeping net. As previously recorded, yellow colored sticky traps showed the greatest attractiveness towards P. spumarius, especially males, while only a few N. campestris were caught by the traps overall. Further studies on the response to colors are planned in order to clarify these preliminary results., IT; PPT; claudio.cantini@ibe.cnr.it

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons. The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively. The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

Before the Xylella fastidiosa outbreak in Europe and the identification of spittlebugs as the main vectors of this xylem-inhabiting bacterium, little was known about this insect group. The need of effective control tools has highlighted the lack of knowledge on several aspects of spittlebugs’ ecology, such as their response to olfactory stimuli and the potential role of different volatile compounds as semiochemicals. Recently, the antennal sensilla fine structure and the response to several volatile organic compounds were investigated in Philaenus spumarius. Although its antennal chemoreceptors are limited, the meadow spittlebug seems to be able to recognize and react to some substances. While the study of P. spumarius chemical ecology is ongoing, for Neophilaenus campestris this topic is still uninvestigated. In order to evaluate the response of P. spumarius and N. campestris adults to volatile organic compounds, several Y-tube olfactometer bioassays were carried out. Bergamot essential oil, farnesene, nonanal and undecenal were selected through a preliminary electroantennographic examination. In addition to these compounds, insect body extracts obtained from 5 males and five females of both species were tested too. The average linear speed of the insect in the olfactometer and time spent in the two arms (treatment vs. control) were considered. In response to almost all the substances, a decrease in the linear speed for both males and females of N. campestris was evidenced in comparison to pure air. Males of both species responded to farnesene remaining for a longer time in the arm containing the substance compared to the arm with pure air. Moreover, when considering the body extracts of males or females both species showed a linear speed significantly different compared to the control tests. These results may suggest that one or more volatile compounds extracted from spittlebug bodies could play a role as semiochemicals, especially in N. campestris., IT; PDF; patrizia.sacchetti@unifi.it

In Europe, 5 Lipoptena species have been recorded, including Lipoptena fortisetosa. This species, native to Asian countries, was described as a parasite of sika deer and its appearance in Europe dates back to more than 50 years ago. Lipoptena fortisetosa has been recently reported in Italy, sharing its hosts with Lipoptena cervi. A morpho-molecular approach was developed to determine the phylogenetic interrelationship of Italian and Asian CO1 haplotypes sequenced from Lipoptena fly individuals collected in Italy, and their DNA sequences were compared with conspecifics available in GenBank; morphological key-characters (terminalia) of L. fortisetosa were compared with the original description. Two haplotypes were recorded from Italy and assigned to L. cervi and L. fortisetosa, respectively. The latter was part of the monophyletic clade L. fortisetosa, along with 2 Central European and 2 Korean haplotypes (100% identical with one of the Korean haplotypes); moreover, Italian L. fortisetosa female terminalia were consistent with the original description of Asian individuals. Pending more in-depth investigations, this study provides a first answer to the hypothesis of the recent colonization of Italy by L. fortisetosa from Asia as we did not detect any obvious and stable morphological and molecular differences in specimens from the 2 geographical areas. The presence of the sika deer in Europe was retraced and the possible route traveled by the parasite from Asia and the eco-biological factors that may have enhanced its settlement are discussed.

Investigations on microbial symbioses in Tephritidae have increased over the past 30 years owing to the potential use of these relationships in developing new control strategies for economically important fruit flies. Bactrocera oleae (Rossi)-the olive fruit fly-is a monophagous species strictly associated with the olive tree, and among all the tephritids, its symbionts are the most investigated. The bacterium Candidatus Erwinia dacicola is the major persistent resident endosymbiont in wild B. oleae populations. Its relationship with B. oleae has been investigated since being identified in 2005. This endosymbiont is vertically transmitted through generations from the female to the egg. It exists at every developmental stage, although it is more abundant in larvae and ovipositing females, and is necessary for both larvae and adults. Studying B. oleae-Ca. E. dacicola, or other B. oleae-microbe interactions, will allow us to develop modern biological control systems for area-wide olive protection and set an example for similar programs in other important food crops. This review summarizes the information available on tephritid-microbe interactions and investigates relationships among fruit flies, bacteria and host plants; however, its focus is on B. oleae and its strict association with Ca. E. dacicola to promote environmentally friendly control strategies for area-wide pest management.

Lipoptena cervi (Linnaeus, 1758), Lipoptena fortisetosa Maa, 1965, Hippobosca equina Linnaeus, 1758, and Pseudolynchia canariensis (Macquart, 1840) (Diptera: Hippoboscidae) are haematophagous ectoparasites that infest different mammal and bird species and occasionally attack humans. They are known for the health implications they have as vectors of pathogens to humans and animals, and for the injuries they inflict on their host's skin. This study focused on the morphological structures evolved by parasites in terms of their biology and the different environment types that they inhabit. To this aim, we examined four hippoboscid species, as well as their hosts' fur (ungulate and horse), and feather (pigeon) through light and Scanning Electron Microscopy (SEM) observations in order to highlight the main morphological features that evolved differently in these flies and to explain the effect of hosts' fur/feather microhabitats on the morphological specializations observed in the investigated ectoparasites. The studied species showed main convergent characters in mouthparts while remarkable differences have been detected on the antennal sensillar pattern as well as on the leg acropod that displayed divergent characters evolved in relation to the host.

Stored dates require postharvest disinfestation treatment to prevent damage caused by insects feeding on these fruits. Fruit disinfestation can be achieved with different methods, at present fumigation, heating and refrigeration being the most common pest control methods, though there are drawbacks related to product quality, process efficiency and environmental impact. The aim of this study was to define a procedure for disinfesting dates using Radio Frequency (RF) heating. RF treatments were carried out on dates of the semi-dry Siwi variety, artificially infested with the larvae, pupae and adults of the dried fruit beetle, Carpophilus hemipterus. A pilot-scale radio frequency instrument with 3.5 kW nominal maximum power and a frequency of 27.12 MHz was used to perform the experiments. The internal temperature of the dates was measured during irradiation. An insect mortality assessment was carried out both at the end of RF treatment and on the following days. The effects of a 4-, 5- and 6-min RF treatment on different stages of C. hemipterus were evaluated, finding that a 6-min exposure ensured mortality of the larvae, pupae and adults of the pest. No significant alterations in moisture content or colour of the treated dates were evident. RF treatment appeared to be a viable technique for the disinfestation of dates, due to the short treatment time together with the possibility of continuous processing.

The dried fruit beetle (Carpophilus hemipterus) is considered a key pest of dates, infesting fruits both in the field and during storage. Control measures against the species rely on the use of chemicals or heat treatments based on sunlight, hot air or radio frequency (RF) applications. Previous investigations that have aimed to define control procedures for a total disinfestation using RF treatments with different exposure durations have shown the typical behavior of adults in some trials, which, under the influence of temperature increases, started to escape from infested fruits. We focused on the application of different RF voltage-time combinations to induce the emigration of C. hemipterus adults from dates in order to produce a complete disinfestation. The results showed that the application of 2500 V RF for 8&ndash, 10 min to infested dates resulted in nearly 100% of adults escaping from fruits, thereby cleaning the commodity with low or no mortality inside and outside the dates. These achievements provide a new strategy for controlling key insect pests of dates that could be applied at an industrial scale, allowing for the quick disinfestation of fruits without affecting the fruit with harmful substances, such as residues from chemical treatments, and without unwanted side effects on date quality.

Hippoboscidae flies parasitize various animal species. Knowledge about these insects remains sparse, although they are known to cause stress and damage to their hosts, and can also accidentally infest humans, causing different sanitary risks. Research conducted in Tuscany assessing the biology and distribution of Lipoptena cervi (Linnaeus, 1758) (Diptera: Hippoboscidae), the most common ectoparasite of ungulates in Italy, revealed the presence of Lipoptena fortisetosa Maa, 1965 in Italy for the first time. This study includes a morphological comparative description of L. cervi and L. fortisetosa, emphasizing the peculiar differences between the two species to facilitate their accurate identification. The most pertinent morphological differences between the two species are highlighted, such as the external features of the antennae, distribution of bristles, and different features in the external genitalia. In both species, scanning electron microscopy of mouthparts revealed strong adaptive convergence in the feeding apparatus. Modified palps and a very thin proboscis are described in relation to feeding behaviour.

BackgroundThe olive fly,Bactrocera oleae,is the most important insect pest in olive production, causing economic damage to olive crops worldwide. In addition to extensive research onB. oleaecontrol methods, scientists have devoted much effort in the last century to understanding olive fly endosymbiosis with a bacterium eventually identified asCandidatusErwinia dacicola. This bacterium plays a relevant role in olive fly fitness. It is vertically transmitted, and it benefits both larvae and adults in wild populations; however, the endosymbiont is not present in lab colonies, probably due to the antibiotics and preservatives required for the preparation of artificial diets. Endosymbiont transfer from wildB. oleaepopulations to laboratory-reared ones allows olive fly mass-rearing, thus producing more competitive flies for future Sterile Insect Technique (SIT) applications.ResultsWe tested the hypothesis thatCa. E. dacicola might be transmitted from wild, naturally symbiotic adults to laboratory-reared flies. Several trials have been performed with different contamination sources ofCa. E. dacicola, such as ripe olives and gelled water contaminated by wild flies, wax domes containing eggs laid by wild females, cages dirtied by faeces dropped by wild flies and matings between lab and wild adults. PCR-DGGE, performed with the primer set 63F-GC/518R, demonstrated that the transfer of the endosymbiont from wild flies to lab-reared ones occurred only in the case of cohabitation.ConclusionsCohabitation of symbiotic wild flies and non-symbiotic lab flies allows the transfer ofCa. E. dacicola through adults. Moreover, PCR-DGGE performed with the primer set 63F-GC/518R was shown to be a consistent method for screeningCa. E. dacicola, also showing the potential to distinguish between the two haplotypes (htA and htB). This study represents the first successful attempt at horizontal transfer ofCa. E. dacicola and the first step in acquiring a better understanding of the endosymbiont physiology and its relationship with the olive fly. Our research also represents a starting point for the development of a laboratory symbiotic olive fly colony, improving perspectives for future applications of the Sterile Insect Technique.

The behavioural response of the sweet potato whitefly, Bemisia tabaci biotype B, to the volatiles emitted by three different species of Callistemon (C. laevis, C. pallidus and C. phoeniceus) was assayed in still-air, and Y-tube olfactometers. B. tabaci adults showed a preference for C. pallidus, while C. laevis and C. phoeniceus were less attractive to the insects when compared to a blank control or to C. pallidus. Volatile organic compounds emitted by the leaves of the three Callistemon species were analysed by solid phase microextraction coupled with GC–MS. 1,8-Cineole was detected in high percentages in C. phoeniceus and C. laevis headspaces, while linalool was abundant in the headspace of C. pallidus. Because of this difference, behavioural tests in the Y-tube olfactometer were carried out also with 1,8-cineole and linalool. Adults of B. tabaci were attracted by linalool, while they were repelled by 1,8-cineole compared with pure air or linalool. These results support the role of these volatiles in the host plant selection process by a herbivorous insect and lay the foundations for the development of sustainable control strategies based on the manipulation of insect pest behaviour.

The response of 160 European pear genotypes to the attacks of Cacopsylla pyri in natural conditions of infestation was studied in the ex situ germplasm collection of the IVALSA Institute – CNR located in Follonica (Grosseto, Italy) during three years. ANOVA showed that both the year and the period of survey have a significant effect upon pear infestation assessed by seven variables. The overall average values of the studied accessions for the variables taken into account were used for grouping the genotypes into 5 putative classes of susceptibility by cluster analysis. Four variables (number of colonies, small nymphs, large nymphs and length of the colonies) selected on the basis of discriminant analysis allowed us to classify 70% of the cultivars into the correct group of susceptibility by jackknifed classification matrix. Taking into account the mean values of the variables and their upper and lower limits all the genotypes were grouped into 5 different classes of susceptibility. On the whole, the number of highly tolerant or resistant cultivars were 8.1% of the investigate genotypes; part of them were ancient cultivars of Italian origin as ‘Eletta Morettini’, ‘Fiorenza’, ‘Gentile’, ‘Moscatellina’, ‘Pera Volpina VP7’, ‘Precoce di Cassano’ and ‘Precoce di Masi’, but there were also the French ‘Notaire Lepin’ and ‘President Loubet’, the German ‘Forelle’ and the American ‘Starking Delicious’. The remaining 91.9% of the germplasm collection showed different levels of susceptibility to psylla attack. The largest part of the genotypes (56.9%) was identified as slightly susceptible, often with minor infestation (that is some isolated larvae, few colonies and reduced honeydew) only in one or two out of three years. Several cultivars (46.3%) were scored as susceptible, suffering medium or high damage, including the widespread cultivated ‘Conference’, ‘Doyenne du Comice’, ‘Highland’ ‘Cure’, ‘Etrusca’, ‘Kaiser’, and ‘William’, these last 3 being classified as highly susceptible. The influence of the environment and growth condition of trees are discussed together with screening methods applied for the evaluation of pear susceptibility to psylla attack reported in world literature.