TaqMan probe assays on different biological samples for the identification of three ambrosia beetle species, Xylosandrus compactus (Eichoff), X. crassiusculus (Motschulsky) and X. germanus (Blandford) (Coleoptera Curculionidae Scolytinae).

Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.

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.

Symbiosis disruption in the olive fruit fly, Bactrocera oleae (Rossi), as a potential tool for sustainable control.

BACKGROUND: The olive fruit fly Bactrocera oleae (Rossi) (OLF) is a major agricultural pest, whose control primarily relies on the use of chemical insecticides. Therefore, development of sustainable control strategies is highly desirable. The primary endosymbiotic bacterium of OLF, 'Candidatus Erwinia dacicola', is essential for successful larval development in unripe olive fruits. Therefore, targeting this endosymbiont with antimicrobial compounds may result in OLF fitness reduction and may exert control on natural populations of OLF. RESULTS: Here, we evaluate the impact of compounds with antimicrobial activity on the OLF endosymbiont. Copper oxychloride (CO) and the fungal metabolite viridiol (Vi), produced by Trichoderma spp., were used. Laboratory bioassays were carried out to assess the effect of oral administration of these compounds on OLF fitness and molecular analyses (quantitative polymerase chain reaction) were conducted to measure the load of OLF-associated microorganisms in treated flies. CO and Vi were both able to disrupt the symbiotic association between OLF and its symbiotic bacteria, determining a significant reduction in the endosymbiont and gut microbiota load as well as a decrease in OLF fitness. CO had a direct negative effect on OLF adults. Conversely, exposure to Vi significantly undermined larval development of the treated female's progeny but did not show any toxicity in OLF adults. CONCLUSIONS: These results provide new insights into the symbiotic control of OLF and pave the way for the development of more sustainable strategies of pest control based on the use of natural compounds with antimicrobial activity. © 2020 Society of Chemical Industry.

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.

Electroantennographic Responses of Aromia bungii (Faldermann, 1835) (Coleoptera, Cerambycidae) to a Range of Volatile Compounds.

BACKGROUND: The red-necked longhorn beetle, Aromia bungii, is one of the most damaging pests of stone fruit trees. Native to the south-eastern Palearctic and Oriental regions, it invaded and is established to some extent in the Campania Region (Southern Italy). In several cerambycid species, volatile organic compounds (VOCs) have been shown to play a role in mate and host plant location. METHODS: The electroantennographic (EAG) technique was employed to explore the antennal chemoreceptivity of male and female A. bungii antennae to 90 VOCs. RESULTS: Increasing EAG amplitudes from the basal to the distal antennal segments were recorded in response to six selected plant volatiles. From the distal flagellomeres, the largest EAG responses (>0.8 mV) were elicited by 2-hexanol, octanal, sulcatone, guaiacol, sulcatol, 2,4-dimethyl-3-hexanol, 2,4-dimethyl-2-hexanone, heptanal, nonanal, (Z)-3-hexenol, and 1-heptanol in both sexes, and by linalool, (E)-2-heptenal, 1-octen-3-ol, (E)-2-octenal, 3-octanol, (E)-2-octen-1-ol, α-phellandrene, and α-terpinene in males. The olfactory system of both sexes proved to be sensitive to changes in stimulus concentration and compound structure. CONCLUSIONS: this study demonstrates the capability of A. bungii males and females to detect and discriminate among a wide range of VOCs and provides a basis for further olfactometer and field trapping experiments aimed at identifying behaviorally-active compounds useful for the implementation of semiochemical-based control strategies for this pest.

Scanning Electron Microscopy of the Antennal Sensilla and Their Secretion Analysis in Adults of Aromia bungii (Faldermann, 1835) (Coleoptera, Cerambycidae).

BACKGROUND: It has been proved that chemical signals play an important role in mating location and reproductive behavior in cerambycids; moreover, they rely on contact chemoreception for mate recognition. METHODS: Adult antennae of Aromia bungii were observed using scanning electron microscopy and adult antennal secretions were collected and analyzed with gas chromatography-mass spectrometry. RESULTS: Twelve different types of sensilla were morphologically described on the antennae of A. bungii. At least six mechanoreceptors-one gustative, one putative chemo- or thermoreceptor, and three multiporous olfactory receptors-are present on the antennae of both sexes while a receptor-type of unclear function is limited to males. Secretions associated with sensilla basiconica were observed for the first time in a cerambycid species. CONCLUSIONS: Sensilla basiconica should play a role in odor perception detecting host tree volatiles and/or pheromones. Sensilla basiconica type 1 and 2 produce a viscous material accumulating on the antennal surface. Chemical analysis of adult antennal secretions highlighted marked differences between sexes. Some of the identified compounds have been previously reported as contact pheromone components of other cerambycid species. Our observations strongly suggest sensilla basiconica as the production sites of compounds involved in mate recognition.

To divide or not to divide: An alternative behavior for teratocytes in Encarsia pergandiella (Hymenoptera: Aphelinidae).

Encarsia pergandiella (Hymenoptera: Aphelinidae) is an endoparasitoid with an unusual embryonic development compared to most of congeneric species and all other members of the superfamily Chalcidoidea. The developmental background of this wasp is based on an alecithal hydropic egg, with the embryo developing inside an extra-embryonic membrane which dissociates at hatching into special larva-assisting cells, the teratocytes. In E. pergandiella many teratocytes at hatching were multinucleated syncytial cells with no evidence of a cellular membrane separating the nuclei. These teratocytes during larval development produced smaller uninucleated teratocytes, through successive divisions obtained by progressive ingrowth of the plasmatic membrane, accompanied by appearance of degeneration symptoms, such as protrusions and blebs. As a consequence of this divisional process teratocytes showed a size reduction and an increase in number of about four times during the second day of larval development. Only on the third day of larval life teratocytes started to decrease in number, until total disappearance at larval maturation. This behaviour is in striking contrast with all other studied systems in which teratocytes do not divide and progressively decrease in number as the parasitoid larva develops.

A new embryonic pattern in parasitic wasps: divergence in early development may not be associated with lifestyle.

Comparative embryogenesis of Encarsia formosa and Encarsia pergandiella (Hymenoptera Aphelinidae), two endoparasitoids of whiteflies (Hemiptera Aleyrodidae), revealed two strongly diverging developmental patterns. Indeed, the centrolecithal anhydropic egg of E. formosa developed through a superficial cleavage, as it occurs in Nasonia vitripennis, Apis mellifera, and Drosophila melanogaster. In contrast, the alecithal hydropic egg of E. pergandiella developed through holoblastic cleavage within a specialized extra-embryonic membrane (EEM). Since this developmental pattern evolved independently in several lineages of hymenopteran endoparasitoids, departures from the superficial cleavage mode have been argued to be strongly canalized in response to a shift from ecto- to endoparasitic lifestyle. Coexistence of both developmental patterns in two congeneric species suggests that alterations of early embryonic development may not be correlated with lifestyle. In addition, embryogenesis of E. pergandiella exhibited the following developmental novelties compared to other species possessing a hydropic egg: (i) polar body derivatives early acquired a cytoskeletal boundary prior to any other cellularization event; (ii) cellularization was asynchronous, starting with an early differentiation of a single apical blastomere at the end of the third cleavage; (iii) appearance of cytoskeletal boundaries of embryo blastomeres occurred between the third and fourth cleavages; (iv) the EEM originated through asynchronous participation of three separate lineages of cleavage nuclei, one of which associated with the polar body derivatives in a syncytium. Our results confirm a scenario of high plasticity in the early developmental strategies of hymenopteran endoparasitoids.