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.

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A. glabripennis specimens from infested plants are too expensive and time consuming for routine screening. A noninvasive diagnostic tool based on frass discrimination is therefore crucial for the rapid identification of A. glabripennis at different development stages in the host. This article describes a rapid diagnostic protocol based on loop-mediated isothermal amplification (LAMP). DNA extracted from A. glabripennis frass was amplified with both visual and real-time LAMP and compared with those of nontarget species. The results show that the method is reliable and accurate and therefore could be a promising diagnostic tool in phytosanitary surveys.

Anoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach.

A molecular and morphological analysis of the species of Aclees native to Taiwan allowed for recognition of a new species, that is described here as Aclees aenigmaticus Meregalli & Boriani, sp. n. The study also revealed that the species of Aclees that has been introduced into Italy and France has identical morphology and mitochondrial cox-1 gene sequence with specimens of A. taiwanensis from Taiwan. Taiwan is thus assumed to be the country of origin of the specimens introduced into Europe. A key to the species of Aclees present in Taiwan is provided. A lectotype is designated for Aclees foveatus Voss, 1932, and some remarks on A. hirayamai Kôno, 1932 are given. Pagiophloeus visayus Heller, 1929 from the Philippines is transferred to the genus Aclees, comb. n.