Castniid moths (Lepidoptera: Castniidae) display a butterfly-like reproductive behavior, i. when the two sexes are in close proximity [3C5]. The Castniidae is usually a small family (113 species explained) of bright-colored day-flying moths which occurs in the Neotropics, Southeast Asia and Australia [6, 7]. Currently, it is grouped within the superfamily Cossoidea with six families: Brachodidae, Cossidae, Dudgeoneidae, Metarbelidae, Ratardidae, and Sesiidae [7C10]. The Neotropical species of castniids mimic many butterflies coexisting in the same habitat in form, colors and habits , an unprecedented case in Lepidoptera between two phylogenetically distant groups . This fact has granted the Neotropical castniids the name butterfly-moths, whereas the Australian and Asian species are commonly known as sun moths. In addition, castniid males are 13103-34-9 supplier territorial displaying perching behavior as many butterfly males [11C14]. Castniid females, as butterfly females, appear to have lost their abdominal glands, and therefore they do not release long-range pheromones to attract conspecific males. This was initially hypothesized by Sarto i Monteys and Aguilar  and later evidenced in studies carried out around the Castniid Palm Borer (Burmeister) [12, 13], recently launched to Europe from South America. However, against this evidence, Delle-Vedove et al. (2014)  claimed that sexually mature females release a pheromone to attract males in a moth-butterfly hybrid strategy implying both chemical and visual clues. To clarify this point and to shed light on its sex behavior, we present our latest findings around the chemical communication of this insect, which could also be useful for the development of a strategy to control this invasive species. Materials and methods Insects Live cocoons of were collected at the 13103-34-9 supplier beginning of summer time (late June- early July) at Bziers and Saint-Guiraud (Hrault, SE France) in 2013 and 2014 respectively, and at Santa Llogaia del Terri (Girona, NE Spain) in 2015C2016. Cocoons were found hidden in the upper a part of trunks of either Canary island date palms hort. ex lover Chabaud (French sites) or Chinese windmill palms (Hook.) H. Wendl. (Spanish site), and were placed in insectaries (50 25 32 cm3) of the ICTA-Autonomous University of Barcelona facilities at laboratory heat (22C25C). Insectaries were checked daily and hourly from 8:00 to 14:00 h to look for adult emergences, which only occur in the morning. After emergence, adults were sexed, tagged for age records, and either placed in semi-field conditions within a 1.20 1.50 2.10 m3 wire mesh cage, located in a nearby forest where sexes could restrictively fly and mate at will, or refrigerated at 18C in labelled 500 cm3 plastic material containers. In summer time 2015, a wild adult populace 13103-34-9 supplier of was found in a small palm plot (L., California fan palm (Lindl.) H.Wendl., and Date palm (L.) Mill.). male specimens were photographed while perching (S1 Fig), so that they could be singled out and identified in the next days. Wing and body size combined with wing spots and marks were sufficient to 13103-34-9 supplier help identify the males in the perching zone. The specimens were monitored undisturbed in this natural habitat for 3C4 days a week for 4 consecutive weeks. While perching, which is done mostly on palm leaves, males may also do rubbing (observe below). In three cases (two with one young male, and one with an old male) we were able to single out the palm leaf surfaces on leaves, which had been rubbed or not (control) by males (observe above), were covered with Gimap5 1 mL of hexane/g of vegetal material, and subjected to the same process as for the body extracts. For ovipositor extracts, 6 groups of virgin females, 4 of mated females and 4 of during-mating females, each of them containing two ovipositors, were obtained from females of similar age. Then, 10 L of a 0.5 ng/L of dodecyl acetate in hexane as internal standard (IS) were added, and the extracts concentrated to 1C2 L for analysis. Terminalia and male glands were analyzed individually and the two pairs of wings or legs of the same insect were considered for the extracts. Extracts were analyzed by GC-MS in splitless mode on a Finnigan Trace 2000 GC system (Thermo Fisher Scientific) coupled to a Trace MS quadrupole mass spectrometer (Thermo Fisher Sci.) working in electron impact (EI) mode. To 290 L of extract were added 10 L of a 60 ng/L answer of dodecyl acetate (12:Ac) or (Z)-9-tetradecenol (Z9-14:OH) in hexane as Is usually. Quantification 13103-34-9 supplier of the amounts of farnesals and (E,Z)-2,13-octadecadienol (E2,Z13-18:OH) was carried out through a calibration curve with solutions of these chemicals in hexane at concentrations 1, 2.5, 5, 10 and 20.