CHARACTERISATION OF INSECTICIDE RESISTANCE MECHANISMS IN ITALIAN POPULATIONS OF THE GREEN PEACH APHID MYZUS PERSICAE (SULZER)

L’afide del pesco Myzus persicae rappresenta uno degli insetti più dannosi in agricoltura. Estremamente polifago e cosmopolita, viene combattuto principalmente con trattamenti insetticidi. Nel corso degli ultimi anni neonicotinoidi e piretroidi hanno rappresentato i componenti principali delle strategie di difesa contro questa specie, ma recenti programmi di monitoraggio condotti in Sud Europa hanno rivelato la presenza di popolazioni resistenti, mettendo in dubbio l’efficacia a lungo termine di queste classi di prodotti. Il presente lavoro prende in esame la diffusione dei principali meccanismi di resistenza agli insetticidi nelle popolazioni di M. persicae presenti sul territorio italiano. La prima parte si concentra sulle resistenze target-site e considera la distribuzione delle principali mutazioni che sono state associate alla resistenza a neonicotinoidi e piretroidi. La seconda parte riguarda le resistenze metaboliche e analizza le principali classi di enzimi associate ad attività di sequestro o detossificazione delle molecole di insetticida. Infine, il progetto si focalizza sulla caratterizzazione delle possibili interazioni tra tali enzimi detossificanti e molecole sinergizzanti quali il ben noto piperonil butossido (PBO). I risultati ottenuti consentiranno di migliorare le strategie di difesa per evitare trattamenti inefficaci e mantenere il più a lungo possibile l’efficacia dei prodotti oggi disponibili per il controllo di M. persicae.The green peach aphid Myzus persicae is a globally significant crop pest, controlled mainly by chemical treatments. In recent years neonicotinoids and pyrethroids have been the main components of pest management strategies used by growers. However, recent monitoring programmes in Southern Europe have shown the widespread presence of resistant populations, posing a serious threat to the long-term efficacy of these insecticide classes. The present work aims to characterise the main biochemical and molecular mechanisms responsible for insecticide resistance in Italian populations of M. Persicae. The first part is focused on target-site resistance and consider the frequency and distribution of the main target-site mutations associated with neonicotinoid and pyrethroid resistance. The second part is related to metabolic resistance and analyses the involvement of detoxifying enzymes able to sequester or metabolise the insecticide molecules. Furthermore, the project aims to characterise possible interactions between those enzymes and synergistic compounds like the well-known piperonyl butoxide (PBO). Results obtained by this investigation will help to improve insecticide resistance management strategies, in order to avoid ineffective applications and maintain the long-term sustainability of chemical control against M. persicae

Neonicotinoid and pyrethroid target-site resistance in Italian populations of the green peach aphid (Myzus persicae).

Myzus persicae is a key pest of many crops, controlled mainly by chemical treatments. Till now neonicotinoids have been an important component of control strategies, but monitoring programmes in Southern Europe have recently shown the widespread presence of resistant populations, posing a serious threat to their long-term efficacy. This work describes the current Italian status of the main target-site mutations associated to neonicotinoid and pyrethroid insecticide resistance. R81T in the nicotinic receptor is very common in aphids from Italian specialised peach-growing areas, but it is also present in samples collected from herbaceous crops, underlying that resistant aphids, moving to secondary hosts, can affect any neonicotinoid-based management strategies. L1014F (kdr) and M918T (s-kdr) in the voltage sodium channel are present in almost all the populations collected, confirming the compromised effectiveness of this insecticide class for the control of this pest. The recently described mutation (M918L) in the s-kdr locus is present with two alternative polymorphisms (a/t or a/c) and it has been found in various genotype combinations with other target-site mutations. Our data describe a worrying situation that must be monitored, in order to avoid ineffective insecticide applications and to provide correct information to support coordinated control strategies regarding resistance management

Neonicotinoids and pyrethroids target-site resistance in Italian populations of the green peach aphid (Myzus persicae)

The green peach-potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) is one of the key pests of many agricultural and horticultural crops, causing significant damage both by direct feeding and by the transmission of many plant viruses. This polyphagous aphid is distributed worldwide and it is responsible for major economic losses. In Italy the most damaged cultivations are peach orchards and open field cultures such as sugar beet, potato and tabacco. Its control is mainly achieved by chemical treatments and in the recent years neonicotinoids and pyrethroids have been key components of the pest management strategies used by growers. Recent resistance monitoring programmes in Southern Europe have shown the widespread distribution of resistance populations of the green peach aphid, posing a serious threat to the long-term efficacy of these insecticide classes. The present work described the current Italian status of the main target-site mutations associated to neonicoitinoid and pyrethroid insecticide resistance. M. persicae populations were collected from spring to autumn 2012 and 2013 in different areas of Italy, mainly from peach orchards, primary host of this aphid, but also from a few secondary herbaceous hosts. Genomic DNA was extracted from a single specimen by a “salting-out” protocol and the presence of single point mutations was assessed with allele-specific polymerase chain reaction amplification (PASA-PCR) or pyrosequencing. The presence of the R81T mutation in the loop D region of the nicotinic acetylcholine receptor B1 subunit, linked with neonicotinoids resistance, was found in more than half of the analysed populations, with a great precentage of the homozygous resistant genotype. These data describe a critical situation especially in Emilia-Romagna, the most specialised peach-growing area of Italy. Furthermore, this mutation was also found for the first time in samples collected from herbaceous crops (eggplant and pepper), underlying that resistant aphids, spreading on secondary hosts, can affect any neonicotinoid-based management strategies. The presence of L1014F (knockdown resistance: kdr) and M918T (super-kdr: s-kdr) in the voltage sodium channel gene, that are correlated with pyrethroid resistance, was found in almost all the populations collected, confirm the compromised effectiveness of this class of insecticide for the control of this pest. In addition, one further single nucleotide polymorphism in the s-kdr locus was identified. This new non-silent mutation (M918L) was recently described only in heterozygous form and in the absence of the classic kdr or s-kdr. Its involvement in pyrethroid resistance was also shown. In our samples this mutation was found with two alternative nucleic substitutions (a/t, as described in literature, or a/c), in both heterozygous or homozygous form and also in the presence of the classic kdr and s-kdr. Our data describe a quite worrying situation that must be considered and monitored, due to the spread of the resistant populations and also to the changes in the distribution of the main target-site mutations in the last few years. The development of rapid and accurate molecolar methods to detect target-site resistance is important to determine the resistance status of this pest, in order to avoid the ineffective insecticide applications and provide correct information to support coordinated control strategies regarding resistance management

Myzus mumecola, nuovo \uabafide bianco\ubb dell\u2019albicocco

Myzus mumecola \ue8 una nuova specie di afide di origine giapponese attualmente presente in India, Cina, Taiwan e Siberia, per la prima volta segnalata in Europa. La specie \ue8 stata segnalata nel corso delle scorse annate in diversi albicoccheti della Romagna. Nel 2016, in alcune aree, sul 60-70% degli impianti \ue8 stato effettuato uno specifico trattamento fi tosanitario. Myzus mumecola \ue8 di colore verde chiaro-giallo paglierino con striature longitudinali e trasversali leggermente pi\uf9 scure. La maggioranza degli esemplari catturati nel corso del 2016 era in forma attera. Sulle giovani fogliensono presenti arrotolamenti dei lembi perpendicolari alla nervatura centrale formando spirali pi\uf9 o meno strette. Non si segnalano deformazioni sui frutti

Tradizione e innovazione nell’analisi delle impurità e degli infestanti degli alimenti: dal filth test al dna barcoding alla metagenomica

La sicurezza alimentare assume sempre più importanza sia per l’industria agro-alimentare sia per il consumatore. Igiene e qualità devono essere garantiti lungo tutta la filiera, dalla produzione al consumo. Nonostante le tecnologie di controllo attualmente disponibili, l’inquinamento da parte di infestanti, vertebrati o invertebrati, è tutt’altro che risolto. Oltre a violare i sopracitati requisiti igienici, rappresenta un limite merceologico, un danno commerciale per il produttore e un fastidio, a volte non limitato alla semplice vista ma legato alla salute, per il consumatore. L’esame degli alimenti può essere condotto con svariate tecniche, dalle più classiche diagnosi macroscopiche, ai metodi di microanalisi quali il filth-test, fino ad arrivare alle più moderne analisi del materiale genetico. Il filth-test è a tutt’oggi un prezioso strumento per la determinazione delle impurità solide di una matrice alimentare, indicatore diretto della sanità di un prodotto e indiretto dello standard igienico adottato nelle fasi di lavorazione e conservazione. Grazie a questa tecnica analitica è possibile osservare al microscopio frammenti di varia natura derivanti dall’agente contaminante. Sono di estrema importanza l’abilità dell’analista e, nel caso specifico degli artropodi infestanti, le sue conoscenze entomologiche. Una errata identificazione del contaminante può condurre ad una errata valutazione del problema e di conseguenza alla sua mancata risoluzione. Quando si incorre in simili difficoltà identificative può essere di grande aiuto un approccio molecolare. Il “DNA barcoding” è una metodica molecolare che consente l’identificazione di specie biologiche attraverso l’analisi di specifiche sequenze di DNA. La tecnica si è finora rivelata un valido supporto per il riconoscimento di macro-contaminanti animali rinvenuti nelle derrate alimentari, non identificabili con le classiche analisi morfologiche in quanto deteriorati dalla lavorazione e dallo stato di conservazione dell’alimento. Negli ultimi tempi, la metodica ha trovato ulteriore sviluppo nel “metabarcoding”, che con approccio high-throughput mira a caratterizzare tutti gli organismi presenti in matrici complesse quali acqua o suolo. Recenti lavori effettuati sugli sfarinati hanno dimostrato l’effettiva sensibilità e specificità del metodo, che rivela quindi un grosso potenziale per l’analisi della qualità degli alimenti.The quality assessment of foodstuffs is an important tool for agri-food industries: contaminated products can not be sold in the market because of sanitary problems and potential health risks for the consumers. Today, different techniques are available for the detection and identification of food contaminants. Macro-contaminations can be directly detected, whilst micro-contaminations can be discovered with filth test analyses; also, a molecular approach is possible and it is called ‘DNA barcoding’. Filth-test is a well-known method which allows the detection of arthropod fragments, hairs or other kind of solid impurities. After their finding, the final evaluation represents the most critical part, because great expertise is required to reveal their nature. This is important not only to assess the foodstuff quality, but also to infer information about the source or place of contamination in the production chain. DNA barcoding represents an alternative and useful approach for the identification of biological samples showing particular features (size, conservation or lack of diagnostic parts missed during foodstuff processing) that do not allow the use of traditional identification methods. It is based on the analysis of short and standardized fragments of genomic DNA, thanks to the development of the sequencing technologies and the availability of free on-line databases of previously characterized DNA sequences. In the last years, further advances have been made with the “metabarcoding”, a novel approach which combines the DNA barcode-base identification system together with the high-throughput ability of the next-generation sequencing (NGS) techniques. This method can be used to reveal the whole content of complex substrates like water, soil or foodstuff like flour and its derivatives, and it will be further investigated in the future for foodstuff analysis

An overview of the main pathways of metabolic resistance in insects

Insecticides have played and still fulfil a very important role in sustainable production of food, animal feed and also as protection against disease vectors. They act to suppress insect populations and, as a consequence of their use, insecticide resistance has evolved. An overview of insecticide resistance mechanisms in insects is given, focusing on the metabolic systems involved in xenobiotic metabolism in the class Insecta. Several enzyme families (e.g., esterases, mixed function oxidases, glutathione S-transferases) are involved in insecticide detoxification, sequestration and excretion and have differing relative importance within the various taxonomic groups. A brief discussion of their impact on control strategies is given

An overview of the main pathways of metabolic resistance in insects

Insecticides have played and still fulfil a very important role in sustainable production of food, animal feed and also as protection against disease vectors. They act to suppress insect populations and, as a consequence of their use, insecticide resistance has evolved. An overview of insecticide resistance mechanisms in insects is given, focusing on the metabolic systems involved in xenobiotic metabolism in the class Insecta. Several enzyme families (e.g., esterases, mixed function oxidases, glutathione S-transferases) are involved in insecticide detoxification, sequestration and excretion and have differing relative importance within the various taxonomic groups. A brief discussion of their impact on control strategies is given

Combinations of ATR, Chk1 and Wee1 inhibitors with Olaparib are active in Olaparib resistant Brca1 proficient and deficient murine ovarian cells

Simple Summary Poly(ADP-ribose) polymerases inhibitors (PARPis), including olaparib, have been recently approved for ovarian carcinoma treatment and PARPi resistance has already been observed in the clinics. With the aim of dissecting the molecular mechanisms of PARPi resistance, we generated olaparib resistant cells lines, both in a homologous recombination (HR)-deficient and -proficient background by continuous in vitro drug treatment. In the HR proficient background, olaparib resistance was caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in olaparib resistant HR-deficient cells, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. We found that combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR status. These new olaparib resistant models will be instrumental to screen new therapeutic options for PARPi-resistant ovarian tumors. Abstract Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors

Combinations of ATR, Chk1 and Wee1 Inhibitors with Olaparib Are Active in Olaparib Resistant Brca1 Proficient and Deficient Murine Ovarian Cells

Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors