First report of Rhabditis (Rhabditella) axei with the invasive palm borer Paysandisia archon.

Rhabditis (Rhabditella) axei is a free-living, pseudoparasitic, necromenic, and parasitic nematode, depending on the host. This species feeds mainly on bacteria present in decaying organic matter, soil, and other substrates; however, in its parasitic form, it can colonize some species of snails. Moreover, the presence of R. axei has also been detected in birds and mammals, including humans. In 2021-2023, during monitoring of the palm borer Paysandisia archon in Central Italy, R. axei emerged from dead larvae of this alien invasive moth and was extracted from palm fibres of Trachycarpus fortunei in three independent sites. The nematode was identified by morphological and morphometric analyses. Molecular analyses using SSU and LSU gene fragments were used to confirm the identification and to perform Bayesian reconstruction of the phylogeny. Each sampling site showed a unique haplotype. Concerning the pathogenicity of this nematode against insects, the test performed on Galleria mellonella larvae did not show any entomopathogenic effect. This is the first time that R. axei was found associated with P. archon, and this recurrent association was discussed.

Terahertz imaging for non-invasive classification of healthy and cimiciato-infected hazelnuts.

The development of new non-invasive approaches able to recognize defective food is currently a lively field of research. In particular, a simple and non-destructive method able to recognize defective hazelnuts, such as cimiciato-infected ones, in real-time is still missing. This study has been designed to detect the presence of such damaged hazelnuts. To this aim, a measurement setup based on terahertz (THz) radiation has been developed. Images of a sample of 150 hazelnuts have been acquired in the low THz range by a compact and portable active imaging system equipped with a 0.14 THz source and identified as Healthy Hazelnuts (HH) or Cimiciato Hazelnut (CH) after visual inspection. All images have been analyzed to find the average transmission of the THz radiation within the sample area. The differences in the distribution of the two populations have been used to set up a classification scheme aimed at the discrimination between healthy and injured samples. The performance of the classification scheme has been assessed through the use of the confusion matrix on 50 samples. The False Positive Rate (FPR) and True Negative Rate (TNR) are 0% and 100%, respectively. On the other hand, the True Positive Rate (TPR) and False Negative Rate (FNR) are 75% and 25%, respectively. These results are relevant from the perspective of the development of a simple, automatic, real-time method for the discrimination of cimiciato-infected hazelnuts in the processing industry.

Biomolecular Monitoring Tool Based on Lab-on-Chip for Virus Detection.

Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.

Effectiveness of field-exposed attract-and-kill devices against the adults of Popillia japonica (Coleoptera: Scarabaeidae): a study on duration, form and storage.

BACKGROUND: The Japanese beetle Popillia japonica Newman is an insect pest native to Japan that has spread into North America, the Azores and, recently, into continental Europe. Here, we present a study assessing the effectiveness of a long-lasting insecticide-treated net (LLIN), assembled in semiochemical-baited attract-and-kill devices (A&Ks) as a low environmental impact means to control P. japonica in the field. We compared the attractiveness of three different forms of A&Ks that were left outdoors throughout the summer, and the residence time of P. japonica landing on them. Moreover, we performed a preliminary study testing the effectiveness of new LLINs after storage. Collected data also allowed us to investigate the beetles' diel flight patterns in relation to meteorological conditions. RESULTS: Killing effectiveness of the field-exposed A&Ks declined steadily over the flight season (from 100% to 37.5%) associated with a decrease in residues of α-cypermethrin, the active ingredient in the LLINs. The different A&K forms (pyramidal, octahedral and ellipsoidal) attracted similar numbers of beetles. Individual beetles' residence time ranged from 75 to 95 s and differed slightly between A&K forms. Effectiveness of LLINs decreased by ≈30% after 1 year storage. Based on numbers landing on A&Ks, the beetles' flight activity peaked about 14:30 h and was inversely correlated with relative humidity. CONCLUSION: This study indicates that semiochemical-baited A&Ks are effective for controlling P. japonica in the field. Because of active ingredient decay, the LLINs should be replaced after 30-40 days of field exposure to ensure that the A&Ks remain fully functional. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Popillia japonica - Italian outbreak management.

Popillia japonica, a priority pest for the EU, was first detected in Northern Italy in 2014. Since its discovery, the outbreak extended over an area of more than 16,000 square kilometers in Northern Italy and Southern Switzerland. In this review, we summarize the state-of-the-art of research conducted in Italy on both the spreading capacity and control measures of P. japonica. Chemical, physical, and biological control measures deployed since its detection are presented, by highlighting their strengths and weaknesses. An in-depth study of the ecosystems invaded by P. japonica disclosed the presence and pathogenicity of natural strains of entomopathogenic fungi and nematodes, some of which have shown to be particularly aggressive towards the larvae of this pest under laboratory conditions. The Plant Health authorities of the Lombardy and Piedmont regions, with the support of several research institutions, played a crucial role in the initial eradication attempt and subsequently in containing the spread of P. japonica. Control measures were performed in the infested area to suppress adult populations of P. japonica by installing several traps (e.g., for mass trapping, for auto-dissemination of the fungus Metarhizium anisopliae, and "attract & kill"). For larval control, the infested fields were treated with commercial strains of the entomopathogenic fungus M. anisopliae and nematode Heterorhabditis bacteriophora. Future studies will aim at integrating phenological and spread models developed with the most effective control measures, within an ecologically sustainable approach.

Impact of Super-High Density Olive Orchard Management System on Soil Free-Living and Plant-Parasitic Nematodes in Central and South Italy.

The soil nematode community plays an important role in ecosystem services. The objective of this study was to assess the effect of Super-high density (SHD) olive orchards on the nematode community in five sites with different soils, climates, and cultivars. At each site, the SHD management system was compared to the adjacent olive orchard traditional (TRAD) system, in which the same soil management and phytosanitary measures were applied. Soil management was assessed by total organic carbon content (TOC), while the soil nematode community was evaluated using the nematode taxa abundances and soil nematode indicators. TOC was significantly decreased in the SHD olive orchard system compared to TRAD in the sites characterized by conventional tillage and mineral fertilization. The two-way ANOSIM analysis on nematode abundance showed no difference between the two olive management methods, instead showing only a significant difference per site mainly due to variabilities in plant-parasitic nematode assemblage. However, a negative impact of SHD management was evident in environments stressed by summer droughts and conventional tillage: the ratio of obligate plant-parasites to bacterivores and fungivores (Pp/(B+F)) was significantly higher in SHD than in the TRAD olive orchard system, and the prey-to-predator θ mass ratio showed the lowest values in the sites under organic fertilization or green manure. The canonical correspondence analysis showed that the free-living nematodes were only slightly affected by SHD olive orchards; instead, the presence of plant-parasitic nematodes families such as Telotylenchidae, Paratylenchidae, Meloidogynidae, and Criconematidae was favored, in comparison to Longidoridae, Heteroderidae, and Pratylenchidae.

New insights from the virome of Halyomorpha halys (Stål, 1855).

Halyomorpha halys (Stål, 1855) (Hemiptera: Pentatomidae), the brown marmorated stink bug, is an invasive pentatomid native to East-Asia, and introduced worldwide in recent times. It is a polyphagous pest with approximately 300 host plants, which, due to its plasticity, reproductive and feeding behavior, long-distance flight, and walking as well as human-mediated dispersal ability, is able to cause significant economic and ecological damage. In several cases pest control mediated by insecticide treatments leads to unsatisfactory efficacy, mostly due to insect recovery ability. Thus, the most promising method for the long-term management of this pest has been focused with growing emphasis on classical biological control strategies. In this framework, viruses have untill now been poorly investigated in H. halys with only a single virus described from the US territory. For this reason we investigated the virome associated with a small and well described population of H. halys from Piedmont (Italy) describing for the first time 7 new viral sequences belonging to different taxonomical groups. Further studies will be necessary to assess the biological and ecological effects the viruses have on their host. Due to the agricultural importance of this insect, the biological characterization of these viruses would give important information on the possibility to exploit viral entities as biological control agents. Finally, the presence of a such relevant number of viruses from a small population suggests a wide association between the brown marmorated stink bug and viral entities. Further studies to determine the possible exploitation of viral sequences to trace different populations are ongoing.

Olfactory responses of Trissolcus mitsukurii to plants attacked by target and non-target stink bugs suggest low risk for biological control.

In crop systems, successful management of invasive insect herbivores can be achieved through the introduction of exotic biocontrol agents, parasitoids or predators, having a coevolutionary history with the pest. To avert threats to local biodiversity, recent legislations require a risk assessment for the organism to be released. Evaluation of its ability to exploit, for host location, odours associated with target and non-target species is crucial for a better definition of its ecological host range. Using Y-tube olfactometer bioassays in a quarantine laboratory, we investigated the ability of the Asian egg parasitoid Trissolcus mitsukurii (Hymenoptera: Scelionidae) to exploit odours associated with the global invader Halyomorpha halys (Hemiptera: Pentatomidae) and with non-target stink bugs native to Southern Europe. We demonstrated that T. mitsukurii is attracted by plants exposed to feeding and egg deposition of the coevolved H. halys and the native Nezara viridula, while it is not attracted by physogastric (gravid) females or eggs alone. Remarkably, T. mitsukurii is repelled by plants bearing eggs of the beneficial Arma custos. Our results contribute to a more thorough and nuanced assessment of the potential non-target risks in the case of mass-release of parasitoids as part of a biological control programme for invasive stink bugs.

Implementing Mass Rearing of Trissolcus japonicus (Hymenoptera: Scelionidae) on Cold-Stored Host Eggs.

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), a pest of Asian origin, has been causing severe damage to Italian agriculture. The application of classical biological control by the release of Trissolcus japonicus (Ashmead) (Hymenoptera: Scelionidae), an exotic egg parasitoid, appears to be one promising solution. In Italy, releases of T. japonicus in the field were authorized in 2020. In this study, some parameters that could influence the rearing of T. japonicus in insectaries were investigated. A significantly higher production of progeny was observed on host eggs stored at 6 °C (86.5%) compared to -24 °C (48.8%) for up to two months prior to exposure to parasitism. There were no significant differences in progeny production from single females in a vial provided with only one egg mass (83.2%) or 10 females inside a cage with 6 egg masses (83.9%). The exposure of parasitoids to refrigerated (6 °C) egg masses of H. halys for 72 h led to a significantly higher production of progeny (62.1%) compared to shorter exposures for 48 (44.0%) or 24 h (37.1%). A decline in production of progeny by the same female was detected between the first (62.1%) and the second parasitization (41.3%). Adult parasitoids stored at 16 °C for up to 90 days had an 87.1% survival rate, but a significant decrease in progeny production was detected. These parameters could be adjusted when rearing T. japonicus for specific aims such as the production of individuals for field release or colony maintenance.

The complete mitochondrial genome of Trissolcus japonicus (Hymenoptera: Scelionidae), the candidate for the biological control of Halyomorpha halys (Hemiptera: Pentatomidae).

The samurai wasp Trissolcus japonicus (Ashmead, 1904) is a parasitoid hymenopteran that came into the limelight as the natural enemy of Halyomorpha halys. Here, we present the complete sequence of the mitochondrial genome of the CREATJ laboratory strain, naturally recovered in Italy in 2018. The molecule conforms to the typical model of animal mitochondrial genomes. Gene order is identical to that of its congeneric Trissolcus basalis. Phylogenetic analysis confirms its placement within monophyletic Scelionidae and Telenominae as the sister group of T. basalis.

Assessing the Distribution of Exotic Egg Parasitoids of Halyomorpha halys in Europe with a Large-Scale Monitoring Program.

The brown marmorated stink bug Halyomorpha halys is an invasive agricultural pest with a worldwide distribution. Classical biological control has been identified as the most promising method to reduce the populations of H. halys. Adventive populations of two candidates for releases, Trissolcus japonicus and Trissolcus mitsukurii, have recently been detected in Europe. To assess their distribution and abundance, a large-scale survey was performed. From May to September 2019, a wide area covering northern Italy and parts of Switzerland was surveyed, highlighting the expanding distribution of both Tr. japonicus and Tr. mitsukurii. Within four years after their first detection in Europe, both species have rapidly spread into all types of habitats where H. halys is present, showing a wide distribution and continuous expansion. Both exotic Trissolcus showed high levels of parasitism rate towards H. halys, while parasitization of non-target species was a rare event. The generalist Anastatus bifasciatus was the predominant native parasitoid of H. halys, while the emergence of native scelionids from H. halys eggs was rarely observed. The presence of the hyperparasitoid Acroclisoides sinicus was also recorded. This study provided fundamental data that supported the development of the first inoculative release program of Tr. japonicus in Europe.

Reproductive and Developmental Biology of Acroclisoides sinicus, a Hyperparasitoid of Scelionid Parasitoids.

Acroclisoides sinicus (Hymenoptera: Pteromalidae) was described in 1988 from China, but recent findings in Europe and North America within the framework of Halyomorpha halys (Hemiptera: Pentatomidae) biological control indicate a Holarctic distribution. The few records and fragmented information on A. sinicus are derived from generic observations of other species belonging to the same genus, and its biological and ethological traits are still completely unexplored. It was suspected to be a facultative or obligate hyperparasitoid of many egg parasitoid species (e.g., Scelionidae and Eupelmidae), especially those parasitizing Pentatomidae eggs. Laboratory colonies of A. sinicus were established from specimens collected in the field in Europe and the USA, which allowed us to investigate for the first time the life traits of this somewhat enigmatic species. Our studies confirmed the obligate hyperparasitoid hypothesis for species of Scelionidae but not of Eupelmidae. Laboratory studies revealed that A. sinicus is extremely selective in its host recognition as only the pupal stage of its host species is exploited for parasitization. Taking into consideration its hyperparasitoid habit, the adventive A. sinicus populations in Europe and North America may potentially be severe threats to pentatomid natural control as new components in the trophic chain of pentatomids and their parasitoid guilds.

Rearing Trissolcus japonicus and Trissolcus mitsukurii for Biological Control of Halyomorpha halys.

Halyomorpha halys is a severe agricultural pest of Asian origin that has invaded many countries throughout the world. Pesticides are currently the favored control methods, but as a consequence of their frequent use, often disrupt Integrated Pest Management. Biological control with egg parasitoids is seen as the most promising control method over the long-term. Knowledge of the reproductive biology under laboratory conditions of the most effective candidates (Trissolcus japonicus and Trissolcus mitsukurii) for optimizing production for field releases is strongly needed. Rearing of these egg parasitoids was tested by offering three different host supply regimes using new emerged females and aged, host-deprived females in different combinations. Results showed a mean progeny per female ranging from 80 to 85 specimens for T. japonicus and from 63 to 83 for T. mitsukurii. Sex ratios were strongly female biased in all combinations and emergence rates exceeded 94% overall. Cumulative curves showed that longer parasitization periods beyond 10-14 days (under the adopted rearing regimes) will not lead to a significantly increase in progeny production. However, ageing females accumulate eggs in their ovaries that can be quickly laid if a sufficient number of host eggs are supplied, thus optimizing host resources. Our data showed that offering H. halys egg masses to host-deprived female Trissolcus once a week for three weeks allowed its eggs to accumulate in the ovary, providing the greatest number of offspring within a three week span.

First report of Bursaphelenchus fungivorus (Nematoda: Aphelenchida) in Italy and an overview of nematodes associated with Crocus sativus L.

Saffron is one of the most precious spices in the world. It is derived from the dried stigmas of the Crocus sativus L. flowers. This plant is triploid sterile and propagated by corms. As a subterranean organ, the corm can come into contact with different nematodes. In this contribution Bursaphelenchus fungivorus was reported for the first time in Italy. It was found associated with C. sativus corms and characterized based on morphological and morphometrical characteristics for this species. The identification was confirmed using molecular analyses. Moreover, a review of nematodes associated with C. sativus worldwide is also provided.Saffron is one of the most precious spices in the world. It is derived from the dried stigmas of the Crocus sativus L. flowers. This plant is triploid sterile and propagated by corms. As a subterranean organ, the corm can come into contact with different nematodes. In this contribution Bursaphelenchus fungivorus was reported for the first time in Italy. It was found associated with C. sativus corms and characterized based on morphological and morphometrical characteristics for this species. The identification was confirmed using molecular analyses. Moreover, a review of nematodes associated with C. sativus worldwide is also provided.

Assessment of color response and activity rhythms of the invasive black planthopper Ricania speculum (Walker, 1851) using sticky traps.

To be effective, management strategies of invasive alien species cannot ignore their spatiotemporal behavior particularly those exerting serious damages to human activities. The black planthopper Ricania speculum is an Asian insect that has been reported as an alien invasive species in Italy, where it threatens local plant diversity, including important crops. In our work, we analyzed the activity rhythms of this species through circular statistics and the efficiency of chromotropic traps to capture adult individuals. Captures were carried out in central Italy, where the black planthopper is showing a remarkable range expansion, after its first discovery in 2009. We observed that the species was mainly crepuscular, with a high intersexual activity overlap. Activity rhythms changed between July-August and September-October, with changing heliophany, but peaked at sunset and were the lowest in the second half of the night and early morning. The insects were mostly caught by green traps, particularly in September, which is the period of egg-laying inside the leaves; conversely, orange ones were avoided, and yellow ones captured proportionally to their local availability. Strategies for controlling this species should consider concentrating trapping effort during the activity peak, using green sticky traps to enhance the capture success of each trap, with the lowest impact over non-target species.

Assessing immunocompetence in red palm weevil adult and immature stages in response to bacterial challenge and entomopathogenic nematode infection.

Parasites and pathogens can follow different patterns of infection depending on the host developmental stage or sex. In fact, immune function is energetically costly for hosts and trade-offs exist between immune defenses and life history traits as growth, development and reproduction and organisms should thus optimize immune defense through their life cycle according to their developmental stage. Identifying the most susceptible target and the most virulent pathogen is particularly important in the case of insect pests, in order to develop effective control strategies targeting the most vulnerable individuals with the most effective control agent. Here, we carried out laboratory tests to identify the most susceptible target of infection by infecting different stages of the red palm weevil Rhynchophorus ferrugineus (larvae, pupae, male, and female adults) with both a generic pathogen, antibiotic-resistant Gram-negative bacteria Escherichia coli XL1-Blue, and two specific strains of entomopathogenic nematodes (EPNs), Steinernema carpocapsae ItS-CAO1 and Heterorhabditis bacteriophora ItH-LU1. By evaluating bacterial clearance, host mortality and parasite progeny release, we demonstrate that larvae are more resistant than adults to bacterial challenge and they release less EPNs progeny after infection despite a higher mortality compared to adults. Considering the two EPN strains, S. carpocapsae was more virulent than H. bacteriophora both in terms of host mortality and more abundant progeny released by hosts after death. The outcomes attained with unspecific and specific pathogens provide useful information for a more efficient and sustainable management of this invasive pest.

Developmental stages and gut microenvironments influence gut microbiota dynamics in the invasive beetle Popillia japonica Newman (Coleoptera: Scarabaeidae).

Popillia japonica Newman (Coleoptera: Scarabaeidae) is a highly polyphagous invasive beetle originating from Japan. This insect is highly resilient and able to rapidly adapt to new vegetation. Insect-associated microorganisms can play important roles in insect physiology, helping their hosts to adapt to changing conditions and potentially contributing to an insect's invasive potential. Such symbiotic bacteria can be part of a core microbiota that is stably transmitted throughout the host's life cycle or selectively recruited from the environment at each developmental stage. The aim of this study was to investigate the origin, stability and turnover of the bacterial communities associated with an invasive population of P. japonica from Italy. Our results demonstrate that soil microbes represent an important source of gut bacteria for P. japonica larvae, but as the insect develops, its gut microbiota richness and diversity decreased substantially, paralleled by changes in community composition. Notably, only 16.75% of the soil bacteria present in larvae are maintained until the adult stage. We further identified the micro-environments of different gut sections as an important factor shaping microbiota composition in this species, likely due to differences in pH, oxygen availability and redox potential. In addition, P. japonica also harboured a stable bacterial community across all developmental stages, consisting of taxa well known for the degradation of plant material, namely the families Ruminococcacae, Christensenellaceae and Lachnospiraceae. Interestingly, the family Christensenallaceae had so far been observed exclusively in humans. However, the Christensenellaceae operational taxonomic units found in P. japonica belong to different taxonomic clades within this family.

Long-lasting insecticide-treated nets: A new integrated pest management approach for Popillia japonica (Coleoptera: Scarabaeidae).

The Japanese beetle Popillia japonica Newman is a US and EU quarantine insect pest that has recently invaded northern Italy. Its ability to rapidly spread in new areas makes this insect a threat to agriculture. In the last decades, several trials on biological control of the Japanese beetle by entomopathogenic nematodes and fungi have been carried out with variable efficacy. However, the necessity of an integrated pest management approach to improve control has arisen. Long-lasting insecticide-treated nets (LLINs) have been used to control other agricultural pests with an attract-and-kill strategy. Here, we present results from laboratory evaluation of 2 LLINs, Storanet® (BASF™) and ZeroFly® (Vestergaard™), against P. japonica adults. Both were effective in killing the beetles; however, some differences emerged if different exposure times were compared: ZeroFly® always gave 100% mortality in tests from 5-s to 30-min exposure; Storanet® showed 100% mortality only with 30-min exposure and going down to 89%-99% mortality for 5-s to 15-min exposure. A description of the paralysis process occurring at 5-s exposure is given. Possible field application of LLINs within programs of integrated pest management is discussed. Integr Environ Assess Manag 2019;15:259-265. © 2018 SETAC.

How ecosystems change following invasion by Robinia pseudoacacia: Insights from soil chemical properties and soil microbial, nematode, microarthropod and plant communities.

Biological invasions are a global threat to biodiversity. Since the spread of invasive alien plants may have many impacts, an integrated approach, assessing effects across various ecosystem components, is needed for a correct understanding of the invasion process and its consequences. The nitrogen-fixing tree Robinia pseudoacacia (black locust) is a major invasive species worldwide and is used in forestry production. While its effects on plant communities and soils are well known, there have been few studies on soil fauna and microbes. We investigated the impacts of the tree on several ecosystem components, using a multi-trophic approach to combine evidence of soil chemical properties and soil microbial, nematode, microarthropod and plant communities. We sampled soil and vegetation in managed forests, comparing those dominated by black locust with native deciduous oak stands. We found qualitative and quantitative changes in all components analysed, such as the well-known soil nitrification and acidification in stands invaded by black locust. Bacterial richness was the only component favoured by the invasion. On the contrary, abundance and richness of microarthropods, richness of nematodes, and richness and diversity of plant communities decreased significantly in invaded stands. The invasion process caused a compositional shift in all studied biotic communities and in relationships between the different ecosystem components. We obtained clear insights into the effects of invasion of managed native forests by black locust. Our data confirms that the alien species transforms several ecosystem components, modifying the plant-soil community and affecting biodiversity at different levels. Correct management of this aggressive invader in temperate forests is urgently required.

The sexuales of Cinara palaestinensis Hille Ris Lambers, the Aleppo pine aphid (Hemiptera: Aphididae Lachninae).

The male and the oviparous female of Cinara palaestinensis Hille Ris Lambers, the Aleppo pine aphid, are recorded for the first time, in populations on Pinus halepensis in Italy. Description and illustrations are provided together with additional notes on taxonomy, ecology and distribution of the species.