Nuove prospettive d controllo di Bactrocera oleae (Rossi) (Diptera: tephritidae) mediante l'alterazione del suo microbioma e della sua biologia con l'utilizzo di sostanze di derivazione naturale

La mosca delle olive (Bactrocera oleae) è tra i più pericolosi parassiti degli oliveti ed è stabilmente presente nel bacino del Mediterraneo. Come accade per altri insetti, la mosca delle olive (OLF) presenta importanti simbiosi che assicurano vantaggi nello sviluppo dell'ospite. Recenti studi hanno individuato il batterio "Candidatus Erwinia dacicola" come il più importante endosimbionte, fondamentale per lo sviluppo degli stadi giovanili. Il progetto del presente Dottorato di Ricerca Industriale, in collaborazione con l'Institut de Recherche sur la Biologie de l'Insecte (IRBI) di Tours (FR) e l'azienda partner I.L.Co.A. sita in Crotone, prevedeva di valutare la capacità di alcune sostanze di derivazione naturale, tra cui Metaboliti Secondari (MS) estratti da colture di Trichoderma harzianum, di alterare la fitness di questo endosimbionte primario e quindi dell'insetto ospite. Contemporaneamente sono state saggiate, sia in laboratorio che in pieno campo, le risposte dell'OLF a delle acque affumicate (Smoke Waters – SW) ottenute durante la produzione di biochar con pirolisi a 300 °C e 500 °C a partire da cellulosa, segatura, sansa, mais e erba medica. I saggi sperimentali prevedevano saggi di ingestione, topici, elettroantennografici, olfattometrici e prove di campo completate in due condizioni pedoclimatiche differenti tra loro. Sono state completate una caratterizzazione del profilo dei composti volatili (VOCs) presenti in tutte le SW utilizzate per queste prove ed analisi molecolari sugli adulti esposti durante i saggi di ingestione, per misurare l'effettiva presenza ed attività dell'endosimbionte primario di B. oleae. In parallelo è stata valutata la presenza e l'attività di "Candidatus Erwinia dacicola" anche in adulti neosfarfallati in condizioni naturali. I Metaboliti Secondari hanno mostrato la capacità di alterare la fitness della mosca in funzione della diluizione saggiata. Il microbioma d'altro canto ha mostrato di essere naturalmente variabile in funzione del periodo di sfarfallamento degli adulti. Le acque affumicate hanno mostrato nei saggi di laboratorio vari effetti in funzione della matrice vegetale di partenza, soprattutto di repellenza, confermata poi nelle prove di campo. La caratterizzazione chimica dei VOC ha evidenziato la presenza di varie interessanti molecole, alcune anche dall'efficacia già nota, che suggeriscono la possibilità di un eventuale futuro loro impiego per il controllo diretto del dittero

Aleurocanthus spiniferus, an alien invasive threat to Europe. AssociatEd bacterial community and natural enemies

Aleurocanthus spiniferus also known as orange spiny whitefly (OSW), is a pest native to tropical Asia that in the last century has spread throughout Asia, reaching Africa, Australia, and Pacific islands. In 2008 the first European OSW population was recorded in Apulia region (South East Italy) and allowed EPPO to add the species as a quarantine threat to Europe now in the A2 list. In the following years OSW spread and invaded new territories of Italy, Croatia and Montenegro. Although OSW polyphagy is already well-known, new associations with autochthonous and allochthonous plants have been reported showing its host-shifting ability. To counteract an upcoming pan-Mediterranean invasion updated bio-ethological information of the pest and the role of possible natural enemies are essential to implement a correct IPM strategy. Field samplings have been aimed at the identification of natural enemies and the evaluation of their efficacy. Furthermore, through insect small-RNA sequencing and by Denaturing Gradient Gel Electrophoresis (DGGE) technique coupled with 16S-rRNA gene sequencing, the primary symbiotic bacteria of OSW have been identified. Sampling on natural enemies highlighted the presence of predatory species belonging to the Coccinellidae family. Besides to the almost ineffective populations of Oenopia conglobata and Clithostetus arcuatus, new findings detected scattered Delphastus sp. populations along the western coast of Italy. Both adult and larvae of this ladybird species preyed OSW developmental stages. The evaluation of the role of Delphastus sp. as biocontrol agent is underway. The first study on OSW microbiota allowed to find symbiotic bacteria commonly associated with the genus Aleurocanthus: Portiera sp., Serratia sp., Wolbachia sp., Rickettsia sp. and, although sporadically, other species. Further studies will target the functional role of these symbionts to develop an effective IPM tailored for Countries at risk

Tortoise pine scale: a new pest for Southern Italian pines

The Italian stone pine (Pinus pinea L.) is a characteristic Mediterranean plant that became a symbol of the Italian landscape. Many fungi and pests are found on this plant and in the following article we investigate a recently reported species which belongs to Coccidae family. Its scientific name is Toumeyella parvicornis (Cockerell, 1897), also known as “pine tortoise scale”. In the last years, it increased its infestation area in Naples province. Tortoise scale causes several direct and indirect damages to host plants by removing their lymph and producing honeydew. In urban areas, it causes indirect damages to underlying people and things. An important characteristic of this pest concerns its capability to have multiple generations over a single year. T. parvicornis has three nymph stages, and the third stage is the first one to have a sexual differentiation; after that stage, females increase their size until they reach their maturity while males become pupae and then winged adults able to catch females. Some IPM solutions are under investigations because only few pesticides are registered to be used on pines, but they are not very effective against T. parvicornis. Our objective is to reach a perfect natural control, as in areas of origin

Seasonal variations in the Bactrocera oleae (Rossi) (Diptera: Tephritidae) microbiome

Bactrocera oleae is well-known for the wide symbiotic relationships linked with the adult and larval stages phenology. In the whole microbiome of the fly stands out the primary bacterium "Candidatus Erwinia dacicola", usually vertically transmitted and hosted in an oesophageal diverticulum, the "Bulb," and in the gut of larvae and adults. Quantitatively, the titer is not so much high in the new-emerged adults. Still, it increases quickly during the first days until it is impossible to remove the bacterium through oral antibiotic administrations after the fifth day. Due to the peculiar characteristics of "Ca. Erwinia dacicola" and its symbiotic relation with the fly, it is commonly used as an indicator to evaluate the actual symbiotic titer. The absolute quantization of "Ca. Erwinia dacicola" was obtained with Real-Time qPCRs after an extraction of both symbionts and host DNAs from the heads and the abdomens separately, on pools of 4 females. The wild adults were obtained from olives attacked by B. oleae and collected in Portici (NA) all along the fly's activity period, from June to the end of December, for two subsequent years. The results highlighted a cyclical and seasonal variability in the symbiont presence that follows and adapts to the seasonal modifications acting on the phenology of the host and modifying its physiology due to the environmental and climatic conditions. Such results underline the need to accurately define the timing and control techniques of the fly populations based on the alteration of the fly's microbiome

Characterization of Smoke Waters and their effects on Bactrocera oleae (Rossi) (Diptera: Tephritidae) microbiome

From the pyrolysis of organic feedstock, aside from Biochar and Bio-oils, it is possible to obtain volatile fractions that can be bubbled in distilled water to obtain the "smoke waters" (SWs), rich in biological compounds and usable for energy purposes or technological applications. Overall, 10 different SWs types were generated and investigated from 5 organic feedstocks treated at 300 and 500°C, with which 2 mixtures were produced. The SWs investigated were obtained from solid olive mill waste (OLI mix) and from dried and finely crushed corn (Zea mays L.) leaf and stem; (3) alfalfa (Medicago sativa L.) hay; (4) sawdust of beech (Fagus sylvatica L.) wood; and (5) cellulose from filter paper (MIX). The SWs and the two mixtures obtained from them were characterized with GC-MS. The effect of oral administrations of OLI and MIX on the Bactrocera oleae fitness was evaluated throughout bioassays carried out with groups of wild new-emerged adults of both sexes, collected from clearly attacked olives harvested in Parco Gussone in Portici (NA). The compounds were administered through a diet of water and sugar added with known amounts of the two mixtures. The alteration effects on the microbiome of the fly were evaluated at the end of each replication. Real Time qPCRs were carried out on pools of 4 females to quantify the presence of the primary endosymbiont, "Candidatus Erwinia dacicola". The analysis were completed on DNA extracted from the head, esophageal bulb's seat, and the intestine's abdomen. The results highlight how the MIX administration limits the bacterial titer and even halves it in the gut, with strong phonological effects also in the subsequent generation. The OLI mixture promotes, instead, the symbiont proliferation both in the bulb and in the gut

Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) in Some European Countries: Diffusion, Hosts, Molecular Characterization, and Natural Enemies

After the first record in 2008 in Southeast Italy, the alien invasive and quarantine pest Aleurocanthus spiniferus (orange spiny whitefly—OSW) has gradually spread throughout Europe, infesting several new host plants in addition to the known hosts. Molecular characterization of some Italian populations and a newly found Albanian population highlighted two different haplotypes invading Europe, belonging to one of the haplogroups previously recorded in China. A predator was recorded for the first time in several fields in Italy in association with OSW and other whitefly species. It was successively identified through a morpho-molecular characterization as a Nearctic member of the tribe Serangiini, the ladybird beetle, Delphastus catalinae. This predator represents a promising biocontrol agent to manage A. spiniferus outbreaks in Italy and other invaded countries

Salivary gland transcriptome of Varroa destructor reveals suitable targets for dsRNA-mediated control of the mite

Varroa destructor is a parasitic mite of honey bees and a major driver of honey bee colony losses. By feeding on the host’s body fluids, this obligate ectoparasite has a strong impact on honey bee physiology, causing the reduction of weight and longevity, and the spread of viral pathogens. Despite its importance, many aspects of Varroa lifestyle are still obscure. During feeding activity on the host, the mite injects salivary secretions only partly identified so far. Here we performed a differential expression analysis between the transcriptome of salivary glands and the rest of the body of V. destructor, identifying 12 transcripts encoding putatively secreted proteins. Using databases such as InterPro, UniProt and NCBI, we functionally annotated this secretome, which includes proteins belonging to cysteine protease, serine protease, endochitinase and phospholipase families. Selected genes were subsequently studied from a functional point of view, by analyzing their impact on mite survival following gene silencing. Mites were soaked in dsRNA solutions targeting chitin-binding, kazal serine protease inhibitor and papain cysteine protease, for a few hours and maintained on honey bee pupae under laboratory conditions. The effect on mortality was checked daily, while gene silencing was assessed at 72 hours after treatment, by qRT-PCR. The dsRNA mix targeting the three genes simultaneously resulted effective in reducing mite survival compared to controls. Further functional characterization of these salivary components will offer new knowledge on the molecular basis of Varroa-bee interactions, on which to develop innovative and eco-sustainable mite control strategies

DNA barcoding of Toumeyella parvicornis (Hemiptera: Coccidae)

The pine tortoise scale, Toumeyella parvicornis (Hemiptera: Coccidae), is a sap-sucking pest native to the Nearctic region that affects several Pinus species, whose heavy infestations may contribute to host tree declines. Recently, the scale insect was accidentally introduced to Europe where it was first discovered in Italy in 2014 and later in southern France in 2021. Since its introduction, T. parvicornis has rapidly spread across several regions in Italy and is contributing to pine tree losses, particularly in the areas surrounding Naples and Rome. The infestation caused by the pest is of great concern as it has the potential to significantly impact the local pine forests and cause direct and indirect damage to urban areas. The lack of a reliable system to trace the origins and spread of T. parvicornis infestation limits our capacity to identify the main entry routes of this alien species and to develop effective control measures. In this study, we used the mitochondrial COI and ribosomal 28S-D2 markers to assess the genetic diversity of pine tortoise scale populations and to study its invasive process in Europe. Samples were collected from Italy, France and USA. Alignment of the 28S-D2 sequences revealed a unique haplotype, while COI showed a single haplotype in Europe which differs by five nucleotide substitutions from USA population of T. parvicornis. Moreover, our dataset differs by approximately 10% identity from the only two sequences of T. parvicornis available in GenBank at the moment, suggesting a likely erroneous taxonomic annotation of these samples collected in Canada. These results highlight the importance of morphological identification and haplotype analysis in tracing the origins and spread of invasive pests. Further studies and additional samples from different countries are needed to fully reconstruct the invasive process of T. parvicornis and to implement effective control measures

Distribution of Toumeyella parvicornis (Cockerell) nine year after its introduction in Campania Region, Italy

The pine tortoise scale Toumeyella parvicornis (Cockerell) (Hemiptera: Coccidae) is considered a major threat for pine species in Italy. Strong phytosanitary measures have been adopted by the Ministerial Decree of 3 June 2021 with the aim to contain the spread of this harmful pest in the country. Native from the Nearctic region, T. parvicornis has been recorded in Campania during 2014 infesting mainly Italian stone pine (Pinus pinea) in the province of Naples. In following years, the pine tortoise scale has been reported in different Italian regions (Abruzzo, Lazio, Puglia and Tuscany) and in 2021 a first outbreak has been recorded for France. The Plant Protection Organization of Campania region suggested the application of the first containment measures in two executive decrees in 2015 that were updated in 2021. Among different pine species P. pinea resulted to be the most susceptible host with huge infestations and high mortality rate interesting mainly pines already weakened by environmental and anthropic factors. In less than a decade T. parvicornis spread over a great part of the region, from urban areas to forest plantations by means of wind dispersal of crawlers or favored by their passive transport with vehicles. The phytosanitary surveys carried out in Campania since 2015 to 28.02.2023, reported in the database SIMFito, list as monitored 328 of 550 municipalities. 28% of the 2700 surveyed sites resulted infested. To date T. parvicornis is infesting pines in 203 municipalities of the region located along the coastal areas from northern regional limit to the gulf of Policastro to the south. The species is largely present also in the internal zones of the Avellino, Benevento and Caserta provinces. The elevation of infested areas ranges from 2 m of coastal plains to 840 m a.s.l. of hilly zones. The main infestations have been observed on Italian stone pine and Maritime pines (P. pinaster), with clear prevalence of the first species (95%). Aleppo pine (P. halepensis) infestations resulted usually inconsistent. The first serious attacks on Austrian pine (Pinus nigra) have been observed in November 2022 at Avellino. The spatial analysis on the progress of the infestations by T. parvicornis showed a spread rate of 7 km/year heading north, 8 km/years eastwards and 15 km/year southwards with new territorial records located along the main regional road network