Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR.

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy.

The Bugs in the Bags: The Risk Associated with the Introduction of Small Quantities of Fruit and Plants by Airline Passengers.

Among European countries, Italy is the most exposed to the risk of biological invasions, principally for its numerous entry points (ports and airports) and for climatic conditions favorable for the acclimatization of several invasive species. Here it was assessed that the greatest threats to our agro-ecosystems come mainly from the passenger baggage in which a variety of fruits and vegetables are carried. From 2016 to 2021, large quantities of plant products were found in the luggage of passengers travelling from outside the EU and seized at the BCPs (border control posts) in the Campania region. Inspections and the following laboratory analyses were conducted on the plant material to assess the presence of exotic pests. Inspections led to several non-native species being recorded, and among the intercepted organisms, some should be considered "alarming", such as Bactrocera dorsalis, Anastrepha obliqua, and Leucinodes africensis. Despite a well-organized border inspection system, travelers transporting infested material unknowingly contribute to increasing the risk of the introduction of exotic species. Given the current situation, it is necessary to impose stricter controls and greater attention, ensuring compliance with the requirements of the new phytosanitary regulations by the actors involved in the transport of plant material. Finally, it is essential to improve awareness through a phytosanitary campaign on plant health risks, especially for people wishing to transport fruits and vegetables in their luggage.

The spread of Carpophilus truncatus is on the razor's edge between an outbreak and a pest invasion.

In 2019, in southern Italy (Campania) there was an outbreak of a sap beetle infesting stored walnut fruits. A monitoring activity started to assess the spread and impact of the pest in walnut orchards and in warehouses, and an integrative characterization led to identify the beetle as Carpophilus truncatus. This species has been in Europe for a long time, rare and harmless until recently. We show also that this species is the same recently recorded in other two continents, Latin America and Australia, where it is causing massive damage on walnut and almond fruits. The sharing of a mitochondrial haplotype among populations recorded on three continents suggests that a worldwide invasion might be ongoing. A Geographic Profiling approach has determined that the more virulent population was first introduced in Italy, and the climate conditions of areas where C. truncatus is currently widespread and harmful indicate that the entire walnuts world production is in jeopardy as this species could adapt to any of the main walnut and almond production areas.

An Integrative Study on Asphondylia spp. (Diptera: Cecidomyiidae), Causing Flower Galls on Lamiaceae, with Description, Phenology, and Associated Fungi of Two New Species.

An integrative study on some species of Asphondylia was carried out. Two species of gall midges from Italy, Asphondylia rivelloi sp. nov. and Asphondylia micromeriae sp. nov. (Diptera: Cecidomyiidae), causing flower galls respectively on Clinopodium vulgare and Micromeria graeca (Lamiaceae), are described and illustrated. The characteristics of each developmental stage and induced galls are described, which allowed the discrimination of these new species in the complex of Asphondylia developing on Lamiaceae plants. Molecular data based on sequencing both nuclear (ITS2 and 28S-D2) and mitochondrial (COI) genes are also provided in support of this discrimination. Phylogeny based on nuclear markers is consistent with the new species, whereas COI phylogeny suggests introgression occurring between the two species. However, these species can also be easily identified using a morphological approach. Phenology of host plants and gall midges are described, and some peculiar characteristics allow the complete and confident discrimination and revision of the treated species. Gall-associated fungi were identified as Botryosphaeria dothidea,Alternaria spp., and Cladosporium spp.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass.

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect's nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Biological and molecular characterization of Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae), an emerging pest of stone fruits in Europe.

The red-necked longhorn beetle (RLB) Aromia bungii (Fald.) is an emerging pest of stone fruit trees, native to East Asia, accidentally introduced in Europe (Germany and Italy) and Japan. Threatening seriously the stone fruit crops in Europe, RLB was added to both the EPPO A1 and priority pest lists of quarantine species. Molecular analyses highlighted that all specimens recovered in southern Italy share the same haplotype, different from the German one, supporting that the invasive process in Europe started from at least two independent introductions. To fill the existing gap of biological knowledge about A. bungii, several laboratory tests were carried out on specimens collected in the outbreak area of Naples (Italy). Results suggest a high biotic potential of the RLB Italian population. Females showed a short pre-oviposition period while the period of oviposition lasted about three weeks, with a rate of 24.2 eggs/day. Each female laid an average of 587.5 eggs and spawned the largest amount of eggs during the first week after emergence. Fed males live up to 62 days at 20 °C while fed females about 63 days at 25 °C. These results are crucial to draw up a multi-facet IPM approach against A. bungii in the outbreak areas.

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.

First Report of Aleurocanthus spiniferus on Ailanthus altissima: Profiling of the Insect Microbiome and MicroRNAs.

We report the first occurrence of the orange spiny whitefly (Aleurocanthus spiniferus; OSW) on the tree of heaven (Ailanthus altissima) in Bari, Apulia region, Italy. After our first observation in 2016, the infestation recurred regularly during the following years and expanded to the neighboring trees. Since then, we have also found the insect on numerous patches of the tree of heaven and other plant species in the Bari province. Nevertheless, the tree of heaven was not particularly threatened by the insect, so that a possible contribution by OSW for the control of such an invasive plant cannot be hypothesized hitherto. This work was also aimed at profiling the microbiome of OSW feeding on A. altissima. For this purpose, we used the denaturing gradient gel electrophoresis (DGGE) and the deep sequencing of small RNAs (sRNAs). Both techniques unveiled the presence of "Candidatus Portiera" (primary endosymbiont), Wolbachia sp. and Rickettsia sp., endosymbionts already reported for other Aleyrodidae. Deep sequencing data were analyzed by four computational pipelines in order to understand the reliability of the detection of fungi, bacteria, and viruses: Kraken, Kaiju, Velvet, and VelvetOptimiser. Some contigs assembled by Velvet or VelvetOptimiser were associated with insects, but not necessarily in the Aleurocanthus genus or Aleyrodidae family, suggesting the non-specificity of sRNAs or possible traces of parasitoids in the sample (e.g., Eretmocerus sp.). Finally, deep sequencing data were used to describe the microtranscriptome of OSW: 56 canonical and at least four high-confidence novel microRNAs (miRNAs) were identified. The overall miRNA abundance in OSW was in agreement with previous works on Bemisia tabaci, and bantam-3p, miR-276a-3p, miR-317-3p, miR-750-3p, and mir-8-3p were the most represented miRNAs.

First Record of an Invasive Fruit Fly Belonging to Bactrocera dorsalis Complex (Diptera: Tephritidae) in Europe.

Emerging pests are increasingly threatening fruit orchard health across the Mediterranean area. Tephritidae, representing serious threats for Europe, are numerous, and the fruit flies Bactrocera zonata and those belonging to Bactrocera dorsalis complex are among the most alarming species. These species are highly polyphagous and B. zonata has already spread to some Mediterranean countries. Due to these ongoing threats, in the Campania Region (southern Italy), a survey with traps and infested fruits analysis was performed with the aim of detecting the presence of species of Bactrocera dorsalis complex. In two mixed fruit-trees fields, some adults belonging to a species of Bactrocera were captured in traps baited with the highly attractive male lure (methyl eugenol). They were distinguished from similar-looking Bactrocera spp. by morphological and molecular comparative analyses. Considering the existing morphological keys, specimens were tentatively identified as B. dorsalis but molecular characterization with COI split them into two clades. Some specimens were grouped with B. dorsalis similar to B. kandiensis and B. kandiensis and others in a clade including B. dorsalis and B. invadens (syn. B. dorsalis). ITS1 sequences instead confirmed morphological identification. The integrative approach allowed identifying all the specimens collected as belonging to the B. dorsalis complex. This finding represents the first field interception in Europe of a member of one of the most dangerous groups of fruit flies.

A New Gall Midge Species of Asphondylia (Diptera: Cecidomyiidae) Inducing Flower Galls on Clinopodium nepeta (Lamiaceae) From Europe, Its Phenology, and Associated Fungi.

A new gall midge, Asphondylia nepetae sp. n. Viggiani (Diptera: Cecidomyiidae), causing flower gall on Clinopodium nepeta (L.) Kuntze (Lamiaceae), is described from Europe. The morphological characteristics of adult, larvae, and pupa are described and illustrated. Molecular approach (by sequencing 28S-D2, ITS2, and COI) confirmed that A. nepetae is a distinct species. The development of the gall is always associated with the presence of the fungus Botryosphaeria dothidea (Moug.: Fr.) Ces. and De Not. (Botryosphaeriales: Botryosphaeriaceae). The new species can complete several generations per year, on the flowers of the same host plant and its adults emerge from late spring to autumn. Pupae overwinter inside peculiar flower galls in a state of quiescence. The impact of the pest is highly variable with a percentage of flowers infested that ranged between 3 and 57.5% in the sampled years. Insect mortality was, at least in part, due to parasitoids that attack the young stages of the midge. Among them, the dominant species was Sigmophora brevicornis (Panzer) (Chalcidoidea: Eulophidae).

Life inside a gall: closeness does not favour horizontal transmission of Rickettsia between a gall wasp and its parasitoid.

The incidence of horizontal transmission as a route for spreading symbiont infections is still being debated, but a common view is that horizontal transfers require intimate between-species relationships. Here we study a system that meets ideal requirements for horizontal transmission: the gall wasp Leptocybe invasa and its parasitoid Quadrastichus mendeli (Hymenoptera: Eulophidae). These wasps belong to the same subfamily, spend most of their lives inside the same minute gall and are both infected by Rickettsia, a maternally inherited endosymbiotic bacteria that infects several arthropods, sometimes manipulating their reproduction, like inducing thelytokous parthenogenesis in L. invasa. Despite intimate contact, close phylogenetic relationship and the parasitoid's host specificity, we show that host and parasitoid do not share the same Rickettsia. We provide indirect evidence that Rickettsia infecting Q. mendeli may be inducing thelytokous parthenogenesis, as the symbiont is densely present in the reproductive apparatus and is vertically transmitted. Phylogenetic analyses based on 16S and gltA placed this symbiont in the leech group. The confirmed and presumed parthenogenesis-inducing Rickettsia discovered so far only infect eulophid wasps, and belong to three different groups, suggesting multiple independent evolution of the parthenogenesis inducing phenotype. We also show some degree of cospeciation between Rickettsia and their eulophid hosts.

A new species of Soikiella Nowicki (Hymenoptera: Trichogrammatidae) from Italy.

A new species of Soikiella Nowicki (Hymenoptera: Trichogrammatidae) that emerged mostly from galls induced by Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) on chestnut is described from Italy. The characterization of the new taxon, Soikiella italica Viggiani sp. n., was performed through a morpho-molecular approach. A key to the species of Soikiella is given. Emerging from the same samples were a few females of an unidentified species of Trichogramma Westwood that likely emerged from eggs of Lepidoptera associated with the galls. The true host of S. italica is also indicated to be the eggs of some insect other than D. kuriphilus that uses their galls as oviposition sites.

Genetic Diversity of the Invasive Gall Wasp Leptocybe invasa (Hymenoptera: Eulophidae) and of its Rickettsia Endosymbiont, and Associated Sex-Ratio Differences.

The blue-gum chalcid Leptocybe invasa Fisher & LaSalle (Hymenoptera: Eulophidae) is a gall wasp pest of Eucalyptus species, likely native to Australia. Over the past 15 years it has invaded 39 countries on all continents where eucalypts are grown. The worldwide invasion of the blue gum chalcid was attributed to a single thelytokous morphospecies formally described in 2004. Subsequently, however, males have been recorded in several countries and the sex ratio of field populations has been found to be highly variable in different areas. In order to find an explanation for such sex ratio differences, populations of L. invasa from a broad geographical area were screened for the symbionts currently known as reproductive manipulators, and both wasps and symbionts were genetically characterized using multiple genes. Molecular analyses suggested that L. invasa is in fact a complex of two cryptic species involved in the rapid and efficient spread of the wasp, the first recovered from the Mediterranean region and South America, the latter from China. All screened specimens were infected by endosymbiotic bacteria belonging to the genus Rickettsia. Two closely related Rickettsia strains were found, each infecting one of the two putative cryptic species of L. invasa and associated with different average sex ratios. Rickettsia were found to be localized in the female reproductive tissues and transovarially transmitted, suggesting a possible role of Rickettsia as the causal agent of thelytokous parthenogenesis in L. invasa. Implications for the variation of sex ratio and for the management of L. invasa are discussed.