Habitat use and a molecular approach to analyze the diet of Drosophila suzukii

The invasive pest species spotted wing drosophila (SWD), Drosophila suzukii, native to Southeastern Asia, has established in the Americas and Southern Europe. In 2008 it was reported for the first time in California, Italy and Spain (Hauser, 2011; Calabria et al., 2012; Cini et al., 2012). In 2009, it spread through France and since 2011, it is also known from Germany (Vogt et al., 2012). Currently D. suzukii occurs in several American (USA, Mexico and Brazil) and European countries (Asplen et al., 2015). Drosophila suzukii is an extremely polyphagous pest species. It feeds and reproduces on a large number of cultivated soft fruits as well as on a wide range of wild fruits from more than 20 plant families (Mitsui et al., 2010; Poyet et al., 2015; Briem et al., 2016). In contrast to other Drosophilidae widely spread in America and Europe, it oviposits in ripening and ripe fruits. Female individuals use their serrated ovipositor to cut the skin of the fruit and deposit their egg directly underneath (Mitsui et al., 2010). Infested fruits quickly collapse due to larval feeding and become unmarketable. Under rearing conditions at the JKI Dossenheim (23 °C and 60% RH) it completes its life cycle within 10 to 12 days. From Japan, it is known that D. suzukii can build up to 13 generations per year in field (Kanzawa, 1939). Drosophila suzukii overwinters as adult in a reproductive diapause at sheltered sites, especially in forests and hedges, as shown by monitoring traps (Briem et al., 2015). Since this invasive pest is active at mild days in winter and spring it needs food resources during that time. To investigate which nutritional resources are used, field monitoring, laboratory bioassays and molecular techniques were applied to identify wild host plants potentially sustaining D. suzukii during winter and spring. The occurrence of D. suzukii at landscape level and its remigration into orchards was monitored all year round. Additionally, an automatic trap was developed to examine the diurnal activity of D. suzukii. No-choice assays with unwounded and wounded mistletoe berries were established to investigate egg laying, feeding activity and survival of D. suzukii. For investigating the attraction of D. suzukii to trees parasitized with mistletoe, we analyzed the volatile organic compounds (VOCs) of unwounded and wounded mistletoe berries by GC-MS and identified the main components. Feeding experiments (FEX) were established to examine the digestion time and to develop a molecular approach to identify food sources during winter and spring. Additionally, bleaching experiments (BEX) were conducted in order to decontaminate the flies’ body surface which should not negatively affect the DNA of the gut content. After 24 h of starvation the individuals for FEX were individually deep-frozen at -80 °C and prepared for PCR assays whereas individuals for BEX were contaminated with homogenized mistletoe before deep-freezing. Additionally, the digestion time of ingested sugars was examined by HPLC. 181 During fall and early winter, catches in monitoring traps indicated a shift of fly activity away from orchards towards forests and hedges. Significantly higher numbers of flies were captured in the canopy of Pinus sylvestris (scots pine tree) parasitized with mistletoe, Viscum album subsp. laxum, compared to unparasitized trees. From April onwards we found females with maturing and mature eggs coinciding with ripe mistletoe berries. In no-choice assays we investigated whether unwounded or wounded mistletoe berries may serve as a host for D. suzukii. Under laboratory conditions (23 °C, 60% RH, L:D 16:8), a higher number of eggs were laid in wounded than in unwounded berries and more adults emerged from wounded than from unwounded berries as well. Further, adult D. suzukii survived more than eight days when only mistletoe berries and water were offered. Under laboratory conditions eight adult individuals emerged from 1,100 field-collected mistletoe berries in 2015 sampled at two sites. In 2016, 2,000 individuals emerged from 9,700 mistletoe berries sampled at the same sites. The odor spectrum identified from unwounded and wounded mistletoe berries using a GC-MS was comparable to other berry odors. However, unwounded and wounded berries differed significantly in the quantity of 11 of the 32 identified VOCs. Further, we succeeded in identifying ingested chloroplast-DNA in the gut content of field-caught D. suzukii. Our feeding experiments showed that females ingest more food and consequently more sugars and DNA than male individuals. However, after 12h post-feeding most of the sugars and the DNA was digested in both sexes. Results of BEX showed that the flies’ body surface was decontaminated whereas the chloroplast DNA of the gut content stays unaffected. The combination of the presented field studies and laboratory assays will provide important information to identify the nutritional resources of D. suzukii in winter and spring, and to get a better understanding of D. suzukii behavior on host plants in the course of a day. This will support the development of new and efficient control strategies.