Trichopria drosophilae ( Diapriidae ) and Leptopilina heterotoma ( Figitidae ) , native parasitoids of Drosophila suzukii , confirmed in Slovenia

The Spotted-wing drosophila (SWD), Drosophila suzukii (Matsumura, 1931) (Diptera, Drosophilidae) was recorded for the first time in Slovenia in autumn 2010. Shortly thereafter, it turned out to be one of the most important insect pests of soft and stone fruit in Slovenia and elsewhere. Within the expert work in the field of plant protection, more precisely within task inventarisation of beneficial organisms for biological control, the presence of indigenous D. suzukii parasitoids was investigated in 2018. Sentinel traps baited with D. suzukii larvae and pupae in banana slices enriched with artificial food medium for drosophilids were used for inventorying D. suzukii parasitoids in raspberries. The pupal parasitoid Trichopria drosophilae (Perkins, 1910) (Hymenoptera: Diapriidae) and the larval parasitoid Leptopilina heterotoma (Thompson, 1862) (Hymenoptera: Figitidae) were recorded parasitizing D. suzukii for the first time in Slovenia in August 2018 in Central Slovenia (Ljubljana).


INTRODUCTION
The spotted wing drosophila, Drosophila suzukii (Matsumura, 1931), SWD, Diptera, Drosophilidae) originally reported in Japan in 1930, almost at the same time (2008) invaded North America (California) and Europe (Italy, Spain) and emerged as an alien pest of soft fruits (Cini et.al., 2012).While other Drosophila species feed on rotten and damaged fruits, D. suzukii females possess a serrated ovipositor that allows egg deposition into undamaged fruits causing great harvest losses (Sasaki and Sato, 1995).The fruit fly D. suzukii is a highly polyphagous invasive pest with many host plants both cultivated and wild soft-skinned fruits, allowing it to spread rapidly and with high dispersal rate (Cini et. al., 2014).Damage is caused by larvae feeding within the soft tissue of the fruits.Subsequently, secondary fungal or bacterial infections may further promote fruit deterioration.Economic losses of fruit production were reported for USA (California) $390 million (Bolda et.al, 2010) and Italy (Trentino) € 3.3 million (Ros et. al., 2013).
Chemical control methods for fruit flies have low efficiency (Bruch et. al., 2011).Therefore, biological control using parasitoids might play an important role as an alternative to synthetic chemical insecticides.For effective use in biological control programmes it is important to promote the use of indigenous natural enemies from the newly invaded areas, also due to strict regulations of European legislation (Barratt et al., 2018;Van Lentern, 2012).Most studied larval parasites of Drosophila were of the genera Leptopilina and Asobara and the pupal parasites Spalangia, Pachycrepoideus and Trichopria (Fleury et. al., 2009).A generalist pupal parasitoid Pachycrepoideus vindemmiae (Rondani, 1875) (Hymenoptera Pteromalidae), a major natural enemy of D. melanogaster (Martelli), was recently found to be also associated with D. suzukii in USA (Brown et al., 2011) and Europe (Rossi Stacconi et al., 2013;Chabert et al., 2012, Knoll et al., 2017).European pupal parasitoid Trichopria drosophilae was also found to attack and develop on D. suzukii (Mazzetto et al., 2016).The fact that both parasitoids attack the invasive spotted wing drosophila was also reported in Italy (Rossi Stacconi et al., 2013), Spain (Gabarra et al., 2014) and California (Wang et al., 2018).Further, they could be adapted to different climatic conditions.The aim of this survey was to identify the presence of indigenous D. suzukii parasitoid species in Slovenia (Central Europe) via field surveys.

Insect rearing
Studies were conducted at the Agricultural Institute of Slovenia, in Ljubljana, Slovenia.Flies of D. suzukii were reared in 30×30×30 cm plastic insect rearing cages (BugDorm-1; Mega View Science, Taiwan) in a growth chamber in D: L cycles of 14:10 h at 21 °C and 77 ± 3 % relative humidity.The flies were provided with tap water and solid artificial food medium (20 g agar, 20 g sugar, 10 g wheat flour, 50 g dry baker's yeast, 500 ml tap water, 400 g grated organic apples, 500 ml organic apple juice, 50 ml apple vinegar and 4 g nipagin (methyl 4-hydroxybenzoate, Sigma-Aldrich).

Preparation of sentinel traps
Larval and pupal D. suzukii parasitoids were sampled using sentinel traps as described elsewhere (Miller et. al., 2015;Rossi Stacconi et. al., 2013) with small modification: Plastic cups (125 ml) containing fresh banana slices (60-70 g) were exposed for 1 to 3 days to D. suzukii (Diptera: Drosophilidae) flies in rearing cages.During that time the females laid eggs in banana slices.Once removed from the oviposition, infested banana slices were maintained for 5 to 7 days in the laboratory at room temperature to allow development of larvae and pupae.Afterward the larvae and the remainder of the eaten banana slices were transferred into new 500 ml plastic containers and enriched with (10 g) artificial food medium for drosophilids.In each infested plastic container one dental cotton tampon (Tosama, Domžale, Slovenia) was placed for absorbing excess liquid of contents.At the end containers were covered with mesh dimensions (0.8 × 0.8 mm) through which parasitoids could pass but not the flies.Each container was placed inside a funnel trap (green lid/green funnel/transparent bucket; catalogue number: 30201) from Pherobank, Netherlands and exposed to natural enemies in the environment.

Laboratory and field observation
Sentinel traps were set to a height of 1 to 1.5 m from the ground into raspberry plants.After 5 to 7 days of field exposure, the containers with the potentially parasitized SWD larvae and pupae were removed from the funnel trap and additionally coated with fine mesh gauze that prevents the passage of the parasitoids.They were transferred to a growth chamber and held at 22 °C and 77 ± 3 % relative humidity, 14 : 10 L : D photoperiod and observed weekly for another eight weeks for emergence of parasitoids.In central Slovenia (Ljubljana) three sentinel traps were field-exposed simultaneously for one week to natural fauna from the second half of June to October in 2018.

RESULTS AND DISCUSSION
The  A lot of parasitoids are reported to attack various Drosophilidae species, and the majority of them are larval parasitoids such as the most generalist parasitoid Leptopilina heterotoma (Fleury et al., 2009), which we also found parasitizing D. suzukii in our region.It is a solitary koinobiont parasitoid that attacks first and second stages of Drosophila larvae (Fleury et al., 2009).
The cosmopolitan pupal endoparasitoid Trichopria drosophilae attacks many Drosophilidae species, including D. suzukii, and could potentially be a good biological control agent for this important pest (Chen et al., 2018).It is idiobiont parasitoid whose host range is known to be limited to Drosophilae (Wang et al., 2016).

CONCLUSION
This paper contributes to the knowledge of the wide spread of native beneficial organisms of D. suzukii such as the paleartic larval parasitoid Leptopilina heterotoma and cosmopolitan pupal endoparasitoid Trichopria drosophilae.Results promote awareness of the importance of further field studies to investigate parasitoid adaptation to local agroecosystems and its potential for wider use in biological control.
pupal parasitoid Trichopria drosophilae (Hymenoptera: Diapriidae) and the larval parasitoid Leptopilina heterotoma (Hymenoptera: Figitidae) were recorded attacking spotted wing drosophila for the first time in Slovenia in the summer 2018 in Central Slovenia (Ljubljana).Traps baited with D. suzukii larvae or pupae exposed in the field were attacked with both species during 23 to 27 July 2018 (30 th calendar week).The peak flight of L. heterotoma in a growth chamber was recorded one month later 23 th August 2018, when more than 30 individual emerged from larvae D. suzukii.Only two individuals of Trichopria drosophilae were caught on sentinel traps.