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BACKGROUND Aphis gossypii is a worldwide agricultural pest that causes high levels of economic losses by feeding and transmitting virus diseases. It is usually controlled by chemical insecticides, but this could lead to the selection of resistant populations. Several single nucleotide polymorphisms (SNPs) have been identified associated with insecticide resistance. Monitoring activities to detect the presence of such mutations in field populations can have an important role in insect pest management but, currently, no information on Italian strains is available. RESULTS The presence of target site mutations conferring resistance to different insecticides was analysed in Italian field collected populations of A. gossypii with an allele specific approach (QSGG, Qualitative Sybr-Green Genotyping). Primers were designed to detect mutations in genes coding acetylcholinesterase (S431F), nicotinic acetylcholine receptor (R81T) and voltage-gated sodium channel (M918L and L1014F). S431F was widespread but with high variability across populations. R81T was detected for the first time in Italy but only in two populations. The L1014F mutation (kdr) was not found, while in the samples showing the M918L two different nucleotidic substitutions were detected. Mutant allele frequencies were, respectively, 0.70 (S431), 0.31 (M918) and 0.02 (R81). Further analysis on the voltage-gated sodium channel gene showed the presence of eight haplotypes and one non-synonymous mutation in the gene coding region. CONCLUSION Multiple target-site mutations were detected within Italian populations. The combinations of genotypes observed in certain locations could affect negatively the control of this pest. Preliminary insights on the genetic structure in the Italian populations of A. gossypii were acquired. (c) 2024 Society of Chemical Industry.

BACKGROUND: The massive use of synthetic insecticides strongly affects the level of insecticide resistance in populations of Myzus persicae worldwide. The selection of target site insensitivity-mutations is particularly worrying in areas where agro-industrial crops are vulnerable to the attacks of aphids that vector viruses, as in the case of Tunisia. Knowledge of the resistance mechanisms evolved locally in this aphid pest is a prerequisite to improving and retaining the sustainability of integrated pest management strategies. RESULTS: Target site mutations were surveyed in several populations of M. persicae collected from peach and potato crops between 2011 and 2017 in three Tunisian regions using real-time allele-specific PCR. The L1014F mutation (kdr locus) was found at a moderate frequency mostly in the heterozygous state and the homozygous resistant genotype was very uncommon. The M918T mutation (super-kdr locus) was present in a few heterozygous individuals, whereas the M918L mutation was detected for the first time in Tunisia and extreme North Africa. This latter mutation was shown to be widespread and well-established in Tunisia mainly as homozygous individuals, and was more abundant on peach than on potato crops. The S431F mutation (MACE) was found in a few heterozygous individuals. No individuals carrying the R81T mutation linked to neonicotinoid resistance were detected. CONCLUSION: This study points out a critical situation for the efficacy of pyrethroid insecticides to control M. persicae populations in Tunisia. It also confirms the rapid spread of the M918L mutation which has been detected in many different areas of the Mediterranean basin. © 2022 Society of Chemical Industry.

Livestock farms represent a source of attraction for other species, which find food resources on the animals themselves, in the food supplied to them, in their manure, etc. Insect farms too can suffer infestation by different organisms living on substrates or behaving as parasites and/or predators. Breeding of the black soldier fly (BSF), Hermetia illucens (L.) requires organic materials which are attractive for other arthropods (commensals, mycetophages, scavengers, etc.). During recent years, the breeding system adopted at the Di.Pro.Ve.S. of the Università Cattolica del Sacro Cuore, Piacenza (Italy), has suffered the presence of the following ‘pests’: Megaselia scalaris (Loew) (Diptera Phoridae), Muscina stabulans (Fallén) (Diptera Muscidae), Monomorium pharaonis (L.) (Hymenoptera Formicidae) and Caloglyphus berlesei (Michael) (Astigmata Acaridae). The use of fermented fruit, vegetables or of an artificial diet to induce egg laying proved to be attractive for small flies such as M. scalaris. This species also takes advantage of the aqueous sugar solution used to feed BSF adults. Infestations by M. scalaris are worrying because its larvae can compete efficiently with those of the BSF in substrate colonisation. Likewise, M. stabulans can be attracted by the substrates, even though this species has not shown the same levels of high competitiveness as the previously mentioned species. M. pharaonis was observed to prey on eggs and newborn BSF larvae. Lastly, infestations by the mite C. berlesei were detected when conditions for the larval development of the BSF were not optimal. This species could also be harmful for the workers involved in the breeding. The establishment of insect and mite populations inside BSF rearing boxes suggests that a careful analysis should be made based on the location of the breeding facility and a series of measures should of course be adopted when this kind of structure and activities are designed and realised.

Uno degli insetti infestanti gli ambienti antropici più diffusi a livello globale è Blattella germanica (L.). In questa specie la resistenza agli insetticidi è andata crescendo negli anni e si è tramutata in uno dei maggiori ostacoli all’attuazione di efficienti strategie di controllo. Attualmente le esche insetticide con formulazione gel mantengono una buona efficacia mentre i trattamenti convenzionali che prevedono l’uso di formulazioni spray, soprattutto di piretroidi, hanno dimostrato molti limiti. In letteratura risultano numerosi casi di inefficacia per diversi principi attivi nei confronti di B. germanica, rendendolo il secondo insetto urbano più resistente nella classifica mondiale. Una possibile soluzione a questo problema può venire dall’uso di sinergizzanti come il piperonil butossido (PBO), molecole senza o con trascurabile attività insetticida che, quando sono applicate in abbinamento a vari prodotti insetticidi, ne potenziano l’efficacia inibendo i sistemi metabolici di detossificazione presenti nell’insetto bersaglio. In questo studio, eseguito nell’ambito del progetto europeo “Ecosyn” (EcoSyn - Ecofriendly synergists for insecticide formulations, finanziato dal Settimo Programma Quadro dell’Unione Europea – Contratto no 605740), il PBO e alcuni suoi nuovi derivati sono stati combinati con insetticidi di già nota efficacia, quali cipermetrina e S-metoprene, per valutarne il possibile effetto sinergico. Per le prove con il piretroide è stato sviluppato un test per contatto lasciando gli insetti a contatto della superficie trattata per 30 minuti e valutando l’effetto knock-down ogni 5 minuti. La mortalità è stata valutata dopo 24 ore. In generale i vari sinergici non hanno aumentato l’effetto knock-down ma hanno causato un significativo aumento di mortalità rispetto all’insetticida usato da solo. Uno solo dei nuovi derivati ha dimostrato, quando usato da solo, una limitata tossicità nei confronti di B. germanica. L’esperimento con S-metopre

In conservation agriculture, slugs are considered significant pests and their monitoring is a key option in the integrated pest management framework. Together with molluscicide applications, predators such as ground beetles can offer a tool for slug control in the field. Through the evaluation of slug and ground beetle monitoring strategies, this work compared their presence in conventional and conservation agricultural plots. The invasive Deroceras invadens was the dominant slug species to occur in all sampling periods. Among Carabidae, Poecilus cupreus and Pterostichus melas were the most abundant species, and Bembidion spp., Brachinus spp., and Harpalus spp. were also common. Beer-baited pitfall traps, whatever their alcoholic content, caught more slugs and ground beetles than wooden boards used as shelters. Slugs were more abundant in conventional plots than in conservation plots, possibly due to the lower presence of natural enemies such as ground beetles. Despite possible impacts on Carabidae, beer-baited pitfall traps should be considered a useful tool for slug monitoring and for the planning of molluscicide applications. Soil management such as minimum- or no-tillage and the presence of cover crops are important elements influencing both slug and ground beetle presence, possibly playing a key role in the maintenance of natural enemy populations.

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae) is a serious pest of the soybean plant, Glycine max, a major world-wide agricultural crop. We assembled a de novo genome sequence of Ap. glycines Biotype 1, from a culture established shortly after this species invaded North America. 20.4% of the Ap. glycines proteome is duplicated. These in-paralogs are enriched with Gene Ontology (GO) categories mostly related to apoptosis, a possible adaptation to plant chemistry and other environmental stressors. Approximately one-third of these genes show parallel duplication in other aphids. But Ap. gossypii, its closest related species, has the lowest number of these duplicated genes. An Illumina GoldenGate assay of 2380 SNPs was used to determine the world-wide population structure of Ap. Glycines. China and South Korean aphids are the closest to those in North America. China is the likely origin of other Asian aphid populations. The most distantly related aphids to those in North America are from Australia. The diversity of Ap. glycines in North America has decreased over time since its arrival. The genetic diversity of Ap. glycines North American population sampled shortly after its first detection in 2001 up to 2012 does not appear to correlate with geography. However, aphids collected on soybean Rag experimental varieties in Minnesota (MN), Iowa (IA), and Wisconsin (WI), closer to high density Rhamnus cathartica stands, appear to have higher capacity to colonize resistant soybean plants than aphids sampled in Ohio (OH), North Dakota (ND), and South Dakota (SD). Samples from the former states have SNP alleles with high FST values and frequencies, that overlap with genes involved in iron metabolism, a crucial metabolic pathway that may be affected by the Rag-associated soybean plant response. The Ap. glycines Biotype 1 genome will provide needed information for future analyses of mechanisms of aphid virulence and pesticide resistance as well as facilitate c

During the spring of 2016, the aphid Myzus mumecola (Matsumura) (Hemiptera: Aphididae) was detected on its preferential host plant, Prunus armeniaca L. (Rosaceae), in Italy. This was the first record of the species in Europe. Several populations of the aphid have been collected in various apricot orchards in Emilia-Romagna, in the localities of Imola, Cam- piano, Cesena and Castiglione di Cervia. The species is considered to be an apricot pest in India and East Asia. Details on the morphology, biology and distribution of this aphid species and the damage it causes are given.

In this study we measured in vitro honey bee enzyme activity in presence of the synergist piperonyl butoxide (PBO) and some new benzodioxole and dihydrobenzofuran derivatives through absorbance and fluorescence spectrophotometric assays, to determine if and to what extent detoxification systems and acetylcholinesterases are affected by these compounds. Both Italian (Italy) and Carniolan (Czech Republic) honey bees were tested. In the case of Italian honey bees the esterase activity was partially but significantly inhibited by almost all the tested products. In Carniolan honey bees only the dihydrobenzofuran derivative EN16-41 significantly inhibited esterases. Other enzymatic systems, such as cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs), often involved in xenobiotics detoxification, as well as acetylcholinesterases (AchEs) seem not to be targeted by the studied synergists. These data seem to suggest that some of the investigated PBO analogues could not be detrimental to honey bees

In the last decade several insect genomes have been sequenced, but for most the chromosomal mapping of the identified scaffolds/annotated genes is not available. The lack of this information makes it difficult to analyse various genetic aspects, including the presence of genome rearrangements and the extent of synteny within and across species. We mapped five multigenic DNA families (major and minor rDNAs, histone gene cluster, esterases and carotenoid desaturases) and seven scaffolds corresponding to 9 Mb of the soybean aphid, Aphis glycines, genome and identified loci spanning the four soybean aphid chromosomes. A comparative analysis of the localization of the annotated A. glycines genes with respect to the peach potato aphid, Myzus persicae, and the fly, Drosophila melanogaster, evidenced a lower degree of synteny between the two aphid species than in the aphid-fly comparison. Only 1.4 genes per syntenic block were observed in aphids in contrast to 2.3 genes per block in flies. This higher chromosomal rearrangement rate in aphids could be explained considering that they possess holocentric chromosomes that can favour the stabilization and inheritance of chromosomal rearrangements. Lastly, our experiments did not detect the presence of chimeric assemblies in the newly available A. glycines biotype 1 genome, differently from what reported in assembled genome of other aphid species, suggesting that chromosomal mapping can be used to ascertain the quality of assembled genomes

L’elevato uso di insetticidi di sintesi per il controllo degli insetti dannosi nel corso degli anni ha contribuito all’inquinamento ambientale e ha portato all’instaurarsi di popolazioni resistenti che rendono inefficaci i trattamenti. Il problema della comparsa e diffusione di vari meccanismi di resistenza a diversi principi attivi è stato di recente aggravato dal ritiro dal commercio di numerosi prodotti a diversa modalità d’azione. Nonostante ciò, il controllo con mezzi chimici resta comunque uno dei principali strumenti delle pratiche fitosanitarie e per questo è importante trovare approcci alternativi che possano contribuire al mantenimento dell’efficacia degli insetticidi oggi disponibili. Una possibile soluzione è rappresentata dalla combinazione degli insetticidi con molecole sinergizzanti, quali il ben noto piperonil butossido (PBO). I sinergici sono composti di per sé non tossici ma in grado di aumentare l’efficacia degli insetticidi in quanto capaci di inibire i principali enzimi detossificanti (esterasi e monoossigenasi) che costituiscono i principali strumenti di difesa degli insetti. Partendo dalle attuali conoscenze sul PBO, nuovi analoghi sono stati sintetizzati e valutati in vitro per la loro capacità di inibire esterasi e monoosigenasi di diversi insetti target, al fine di chiarire le possibili interazioni tra gli enzimi e le strutture chimiche dei sinergici sviluppati. I composti che hanno mostrato una maggior capacità inibitoria sono stati selezionati e successivamente valutati in vivo attraverso biosaggi di laboratorio su popolazioni resistenti, utilizzando formulazioni commerciali di insetticidi (piretroidi e neonicotinoidi). I dati ottenuti hanno dimostrato l’effettiva capacità sinergica di alcuni analoghi, con prestazioni diverse a seconda delle specie indagate, consentendo una riduzione della dose di insetticida da applicare per raggiungere le stesse percentuali di mortalità. Il lavoro è stato sviluppato nell’ambito del progetto e

Uno degli insetti infestanti gli ambienti antropici più diffusi a livello globale è Blattella germanica (L.). Grazie alla facilità con cui colonizza case, cucine commerciali, lavanderie ecc., è uno degli insetti più rilevanti dal punto di vista igienico-sanitario, essendo ampiamente noto il suo coinvolgimento nella trasmissione di numerose specie di patogeni oltre alle potenzialità allergeniche per l’uomo. In B. germanica la resistenza agli insetticidi è andata crescendo negli anni e si è tramutata in uno dei maggiori ostacoli all’attuazione di efficienti strategie di controllo di questo infestante. Attualmente le esche insetticide con formulazione gel mantengono una buona efficacia mentre i trattamenti convenzionali che prevedono l’uso di formulazioni spray, soprattutto di piretroidi, hanno dimostrato molti limiti. In letteratura risultano numerosi casi di inefficacia per diversi principi attivi nei confronti di B. germanica, rendendolo il secondo insetto urbano più resistente nella classifica mondiale. Una possibile soluzione a questo problema può venire dall’uso di sinergizzanti come il piperonil butossido (PBO), molecole senza o con trascurabile attività insetticida che quando sono applicate in abbinamento a vari prodotti insetticidi ne potenziano l’efficacia inibendo i sistemi metabolici di detossificazione presenti nell’insetto bersaglio. In questo studio, eseguito nell’ambito del progetto europeo “Ecosyn” (EcoSyn -Ecofriendly synergists for insecticide formulations, finanziato dal Settimo Programma Quadro dell’Unione Europea – Contratto no 605740), il PBO e alcuni suoi nuovi derivati sono stati combinati con insetticidi di già nota efficacia, quali cipermetrina e S-metoprene, per valutarne il possibile effetto sinergico. Per le prove con il piretroide è stato sviluppato un test per contatto, trattando contenitori in vetro con una miscela di insetticida sciolto in una soluzione di sinergico in acetone e lasciando gli insetti a contatto della super

Negli insetti, le resistenze “target site” nei confronti degli insetticidi piretroidi sono causate da mutazioni puntiformi nella sequenza codificante il canale del sodio. Tali meccanismi di resistenza compromettono l’efficacia di questa classe di principi attivi che hanno ancora grande importanza per combattere le infestazioni di Musca domestica L. in ambiente urbano o zootecnico. Abbinando tecniche molecolari in vitro e biosaggi di efficacia in vivo, sono state prese in esame due popolazioni raccolte in contesti ambientali differenti della provincia di Piacenza, confrontandole con una popolazione sensibile di riferimento. Le PCR allele specifiche hanno evidenziato la diffusa presenza di mutazioni ai loci kdr (L1014F e L1014H, quest’ultima nota anche come kdr-his) e super-kdr (M918T). I biosaggi hanno stimato nelle popolazioni studiate fattori di resistenza molto elevati e correlabili alle frequenze delle mutazioni individuate. I dati hanno confermato la presenza della mutazione M918T solo in contemporanea alla L1014F, come già evidenziato in studi precedenti condotti in Europa, Asia e Stati Uniti. Al contrario, la mutazione L1014H (sia essa in eterozigosi o omozigosi) non è mai stata trovata in correlazione al super-kdr. L’analisi della sequenza intronica in prossimità del locus kdr ha inoltre evidenziato una notevole varietà di aplotipi in accordo con i dati di letteratura, confermando la probabile origine multipla delle mutazioni responsabili della resistenza ai piretroidi in M. domestica.

I prodotti sinergizzanti (come il piperonil-butossido - PBO) sono molecole senza o con trascurabile attività insetticida ma che quando sono applicate in abbinamento a vari prodotti fitosanitari ne potenziano l’efficacia inibendo i sistemi metabolici di detossificazione dell’insetto bersaglio. Nell’ambito del progetto europeo “Ecosyn” (EcoSyn -Ecofriendly synergists for insecticide formulations, finanziato dal Settimo Programma Quadro dell’Unione Europea – Contratto no 605740) sono stati sviluppati e sintetizzati nuovi sinergizzanti, partendo dalla struttura molecolare del PBO. Oltre alla valutazione della capacità di queste nuove molecole di inibire i meccanismi di resistenza metabolica di varie specie di interesse agrario e veterinario si è voluto verificare se il loro utilizzo potesse avere ripercussioni negative sui pronubi e in particolare su Apis mellifera L. Sono stati considerati il classico PBO, una serie di derivati del benzodiossolo, indicati rispettivamente come EN1-213, EN1-216, EN1-218, e due derivati del 2,3-diidrobenzofurano, indicati come EN16-55 e EN16-41. E’ stata indagata la capacità di questi composti di inibire l’attività di enzimi di cui è ampiamente nota, in molte specie di artropodi, la capacità di intervenire nei processi di detossificazione e sequestro di varie classi di insetticidi. In questa fase, ancora preliminare, sono state prese in considerazione le esterasi e le glutatione S-transferasi. È stata anche indagata la possibilità che questi sinergizzanti possano avere effetti sull’attività delle acetilcolinesterasi. L’attività enzimatica è stata misurata tramite analisi spettrofotometrica in assorbanza sia in presenza che in assenza dei sinergici per poter calcolare l’attività residua, dopo preincubazione dell’omogenato con acetone o con il sinergico. I risultati preliminari indicano che in vitro l’attività degli enzimi studiati è solo parzialmente inibita dai derivati del 2,3-diidrobenzofurano (EN16-41 e EN16-55) e che no

Blattella germanica (L.) is a common pest of anthropic environments. Cockroach infestations are associated with poor sanitation, particularly around food-handling facilities, causing damages and contaminations. Furthermore, this species is responsible for transmitting microorganisms which implicate medical and hygienic problems. In this study, carried out in the framework of the European project "Ecosyn" (Ecofriendly synergists for insecticide formulations, funded by the 7th Framework Program of the European Union, contract no. 605740), the synergists PBO and some of its new derivatives were combined with two insecticides to assess any possible effect. The cypermethrin was assayed using a contact test in vials, leaving the insects with the products for 30 minutes, evaluating the knock-down effect every 5 minutes. Then, cockroaches were transferred in untreated vials and mortality was assessed 24 hours later. In general, the synergists did not influence the knock-down effect but caused a significant increase in mortality compared to the insecticide used alone. The S-methoprene effect was evaluated feeding virgin females for a week with moist baits containing insecticide and synergist. After mating with untreated males, every day mortality and appearance, abortion or hatching of ootheca were assessed. Although differences among various synergists were observed, the treated cockroaches showed a delay in ootheca production, an increase in its abortion and a reduction in the number of births.

Pyrethroid insecticides have been widely used to control housefly infestations and they still remain an important tool above all in urban and household environments. However their efficacy is affected by insecticide resistance mechanisms. In particular, some mutations in the sodium channel coding sequence are responsible for target-site resistance, which results in reduced sensitivity against this class of products. Using a molecular approach based on allele-specific PCRs we point out and characterise the presence of point mutation in “kdr” and “s-kdr” loci linked to target-site resistance in two Musca domestica populations (PNT and TRV) collected in livestock farms near Piacenza (Northern Italy). The molecular approach here reported represents an alternative to the technologies till now adopted (direct sequencing, PCR-RFLP) to investigate the above reported mutations. The presence of kdr, kdr-his and s-kdr genotypes was assessed for population PNT whilst kdr only was detected in population TRV. Dose-response bioassays evidence high resistance factors, above all for population PNT, in line with the mutation frequencies observed in the assayed populations. This is the first report that confirms the presence of different genotype combinations of target-site mutations in Italian houseflies.

In the framework of the EU-funded EcoSyn project, laboratory bioassays and biochemical analysis were performed to evaluate the performance against Musca domestica L. (Diptera, Muscidae) of novel potential synergists in combination with a pyrethroid and a growth regulator (IGR) insecticide. The well known synergist piperonyl-butoxide (PBO) was included in the list to be used as a standard reference. Bioassays has been carried out against two resistant populations evaluating the effects against adults (pyrethroid) or larvae (IGR). Synergist significantly improved the efficacy of insecticides in bioassay against housefly. A few biochemical tests were performed to measure total activity of oxidases and GSTs in the same populations and to comparison with those of a full susceptible strain (S-WHO). These enzymatic activities were always higher than those of the susceptible reference strain. It was also investigated the “in vitro” inhibitory efficacy of PBO and that of new hypothetical synergists. In almost every case the novel synergists gave various levels of oxidases inhibition and in some cases performed better than PBO. Among the potential synergist assayed just one did not inhibited oxidases. But this was the only product giving a visible level of inhibition of GST activity.

Pyrethroid insecticides combine a high efficacy against insects with a low toxicity towards warm-blooded vertebrates. For this reason they are largely used to control housefly infestations. The efficacy of these products is affected by insecticide resistance mechanisms. In particular, some mutations in the sodium channel coding sequence are responsible for target-site resistance to pyrethroid insecticides. This work presents a new molecular approach based on allele-specific PCRs to point out and to characterise this resistance mechanism in two M. domestica populations (PNT and TRV) collected near Piacenza (Northern Italy). The presence of different kdr and s-kdr genotypes was assessed for population PNT whilst kdr only was detected in population TRV. Dose-response bioassays evidenced quite high resistance factors, expecially for population PNT. This is in line with the kdr and s-kdr frequencies observed in the assayed populations.