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

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. 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, alfa-phellandrene, and alfa-terpinene in males. The olfactory system of both sexes proved to be sensitive to changes in stimulus concentration and compound structure. 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

Natural enemies of armored scales (Hemiptera: Diaspididae) and soft scales (Hemiptera: Coccoidae) in Chile: molecular and morphological identification.

Scale insects (Hemiptera: Sternorrhyncha: Coccomorpha) are key pests of agricultural crops and ornamental plants worldwide. Their populations are difficult to control, even with insecticides, due to their cryptic habits. Moreover, there is growing concern over the use of synthetic pesticides for their control, due to deleterious environmental effects and the emergence of resistant populations of target pests. In this context, biological control may be an effective and sustainable approach. Hymenoptera Chalcidoidea includes natural enemies of scale insects that have been successfully used in many biological control programs. However, the correct identification of pest scale species and their natural enemies is particularly challenging because these insects are very small and highly specialized. Integrative taxonomy, coupling DNA barcoding and morphological analysis, has been successfully used to characterize pests and natural enemy species. In this study, we performed a survey of parasitoids and predators of armored and soft scales in Chile, based on 28S and COI barcodes. Fifty-three populations of Diaspididae and 79 populations of Coccidae were sampled over the entire length of the country, from Arica (18˚S) to Frutillar (41˚S), between January 2015 and February 2016. The phylogenetic relationships obtained by Bayesian inference from multilocus haplotypes revealed 41 putative species of Chalcidoidea, five Coccinellidae and three Neuroptera. Species delimitation was confirmed using ABGD, GMYC and PTP model. In Chalcidoidea, 23 species were identified morphologically, resulting in new COI barcodes for 12 species and new 28S barcodes for 14 species. Two predator species (Rhyzobius lophantae and Coccidophilus transandinus) were identified morphologically, and two parasitoid species, Chartocerus niger and Signiphora bifasciata, were recorded for the first time in Chile

A viral chitinase enhances oral activity of TMOF

In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor (. Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors

Identification of the Red-Necked Longhorn Beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) with real-Time PCR on frass

Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae), the red-necked longhorn beetle is native to eastern Asia, where it is an important wood-borer of fruit and ornamental species of the genus Prunus. A. bungii is a quarantine pest in the European Union, following its accidental introduction and establishment in Germany and Italy, and is currently included in the list of priority pests. To confirm its infestations in outbreak areas, adult or larval specimens are needed to perform morphological or molecular analyses. The presence of A. bungii larvae inside the attacked trees makes the collection of specimens particularly difficult. Thus, we present two diagnostic protocols based on frass analysis with real-time PCR (probe and SYBR Green). The results obtained show that a non-invasive approach for detecting the presence of this harmful invasive pest can be a reliable and accurate alternative diagnostic tool in phytosanitary surveys, as well as to outline a sustainable pest management strategy

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

Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs../Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals

COEXISTENCE OF TWO EFFECTIVE PARASITOIDS OF THE WHITE PEACH SCALE PSEUDAULACASPIS PENTAGONA (HEMIPTERA: DIASPIDIDAE): THE ROLE OF THE HOST STAGE AND TEMPERATURE.

ENCARSIA BERLESEI: (Howard) and Pteroptrix orientalis (Silvestri) are endoparasitoids of Pseudaulacaspis pentagona (Targioni Tozzetti), that are sympatric in Campania (Italy). The influence of host stages on several components of E. berlesei fitness and the effect of mating status on the production of Pteroptrix orientalis progeny were evaluated at 25 degrees C. Parasitization of earlier host stages resulted in an increase in the development time and a decrease in progeny number of E. berlesei. Adult parasitoids were largest when young female stages were parasitized. Pupal mortality did not differ among host stages. Mated female P. orientalis produced a greater number of progeny and proportionally fewer sons (13.6% of the total progeny) than did virgin females (21.7%). The reproductive potential of both parasitoids was also evaluated at four regimes of constant temperature (20, 24, 26 and 30 degrees C). Encarsia berlesei attained rm values nearly double those of P. orientalis at 20, 24 and 26 degrees C, whilst at 30 degrees C the two parasitoids achieved the same rm value, since at this temperature E. berlesei suffered a high pupal mortality. Sex ratio of P. orientalis, expressed as proportion of males, varied significantly between 20 and 24 degrees C only

The spread of the non-native pine tortoise scale toumeyella parvicornis (Hemiptera: Coccidae) in europe: A major threat to pinus pinea in southern Italy

Invasive pests are considered a major threat to biodiversity, conservation and agriculture. The Italian peninsula is a major site of intensive commercial exchange and transport of plants and goods, being consequently one of the European countries most invaded by alien insects. Hemiptera Coccomorpha are the largest group of non-native species recorded in Europe. For example, in the last 70 years more than 50 scale insect species have been accidentally introduced into Italy, 50% of which are now well established. This study was conducted to investigate the biology and the damage of the non-native pine tortoise scale Toumeyella parvicornis Cockerell (Hemiptera: Coccidae) accidentally introduced a few years ago into southern Italy. T. parvicornis is multivol-tine in the invaded territories, being able to complete at least three generations per year, overwintering in the adult female stage. Oviposition periods during 2015-2017 surveys occurred from late April to end of May, from July to first half of August, and from mid-September to November. Fecundity was positively correlated to body size of gravid females and varied among the generations. Investigations on natural control by autochthonous species showed a seasonal activity of Metaphycus flavus (Hymenoptera: Encyrtidae), parasitizing mainly immature male individuals. The morpho-molecular approach confirms the hypothesis of an ongoing shift of parasitoid populations from other indigenous soft scales to the invasive one. Unfortunately, the low level of natural control was ineffective in hampering the spread of T. parvicornis, and preventing the dieback of local pine species, Pinus pinea, as observed in all invaded areas