Surveys of Drosophila suzukii (Diptera: Drosophilidae) and Its Host Fruits and Associated Parasitoids in Northeastern China

Simple Summary Drosophila suzukii has become a globally invasive pest of thin-skinned berries and stone fruits such as strawberries, blueberries, raspberries, blackberries, and cherries. D. suzukii has caused severe economic losses to the fruit industries in more than 30 countries and has been listed as an important quarantine pest in many countries around the world. To better understand the ecology of this invasive pest for its effective management, it is essential to investigate the occurrence of D. suzukii and its wild host fruits and natural enemies in its native range. Here, we report the occurrence of D. suzukii and its wild host fruits and associated parasitoids in Liaoning, Northeast China for the first time. Four species of wild berries from non-crop habitats were found infested by D. suzukii, and two species of parasitoids (Leptopilina japonica and Asobara japonica) were discovered. Over the survey period from June to October, D. suzukii adult populations increased and peaked in August, and then declined until it was no longer detectable in October. Abstract Spotted-wing drosophila, Drosophila suzukii (Matsumura), is a worldwide quarantine pest that is currently undergoing a rapid range expansion in the Americas, Europe, and parts of Africa. It feeds and breeds on soft-skinned fruits such as raspberries, blueberries, and cherries, and can cause significant economic losses to fruit production. This study investigated the occurrence of D. suzukii and its wild host fruits and parasitoids in Liaoning, Northeast China for the first time. Sentinel traps were used to monitor D. suzukii adults, and suspected fruits were collected weekly in four different locations (Wafangdian, Faku, Fengcheng, and Shenyang). The results showed that D. suzukii were distributed in the sweet soft-skinned fruit-production areas of Liaoning, and raspberry was the most infested fruit. During the field survey, four species of wild berries from non-crop habitats were found infested by D. suzukii, and two species of parasitoids (Leptopilina japonica and Asobara japonica) were collected. D. suzukii adult-population dynamics throughout the survey period (June to October) were similar in different survey locations; adult fly populations increased and peaked in August, and then declined until the fly was no longer detectable in October.


Introduction
Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has become a globally invasive pest of berries and stone fruits, causing significant economic losses to the fruit industry in its invaded regions [1][2][3][4]. The native range of D. suzukii is probably East Asia. It was first described by Matsumura in Japan in 1931 [1], and has since been reported in eastern China [5], the Korean Peninsula [6,7], Myanmar [8], Thailand [9], and other regions in southeastern Asia [10,11]. The focus on economic losses due to D. suzukii in Japan has been concentrated on cherry and blueberry [1]. However, D. suzukii has not been considered a serious fruit pest in other southeastern Asia regions, despite recent frequent reports of damage of soft-skinned fruits by D. suzukii in cherry in China [12,13].

Collections of D. suzukii and Parasitoids
Surveys were carried out during the fruiting season for 17 consecutive weeks from June to October 2016. Twelve sentinel traps were placed (six yeast-sugar-baited traps and six uninfested-fruit-baited traps) in each collection site in the commercial or research farms. Each yeast-sugar-baited trap was placed at a height of 1-1.5 m above the ground, and the uninfested-fruit-baited trap was placed under the yeast-sugar-baited trap and covered with a lid to block direct sunlight or rain. The yeast-sugar-baited trap was made from a 1000 mL transparent plastic container (Horeca select, CN) with 20 holes (0.5 cm diameter) around the side and was filled with 300 mL of bait (21.5 g yeast, 43 g sugar, and 200 mL water). The uninfested-fruit-baited trap was made from a 500 mL plastic container (Horeca select, CN) with 10 holes (0.5 cm diameter) around the side. It was filled with uninfested fruits (banana, blueberry, and raspberry) and agar hydrocolloid (0.8%). The traps were replaced once a week and taken to the laboratory of Forestry College, Shenyang Agricultural University to sort out, count, and record the numbers of larvae, pupae, and adults of flies. Larvae and pupae of Drosophila were placed in a 500 mL transparent plastic container (Horeca select, CN) with an artificial diet cultivated at 22 • C and 70% relative humidity (RH). All adults were preserved separately in 75% ethanol for identification.
Both ripe cultivated fruits next to each sentinel trap in the commercial and research fields and ripe wild berries in natural habitats were randomly collected as potential host fruits and taken to the laboratory. Next, the fruits were weighed and placed in 500 mL plastic containers (Horeca select, CN) and incubated at 22 • C and 70% (RH). Fruit juice was constantly removed to prevent the emerged larvae from drowning. The resultant D. suzukii pupae were identified from the rotten fruits under a microscope; then, the pupae were individual placed in 1.5 mL Eppendorf tubes at 22 • C and 70% (RH) and checked for emerging parasitoids every day. Furthermore, the flies and parasitoids were preserved in 75% alcohol for identification.

Morphological Identification of D. suzukii and Parasitoids
Specimens of D. suzukii were morphologically identified according to the key and descriptions by Okada [9] and Bock & Wheeler [39]. Drosophila samples were identified based on morphology of body color, body length, wings, male's tarsal comb, and ovipositor by dissecting and observing under a microscope. Parasitoid morphological identification was carried out according to the description by Guerrieri et al. and Abram et al. that provided pictures of the genus Leptopilina spp. [40] and Asobara spp. [41]. The figitids were further confirmed by Dr. Matthew Buffington (USDA-ARS, Systematic Entomology Laboratory, Washington, DC, USA). Each parasitoid specimen was examined for its body color, body length, wings, antenna, and leg morphology under a microscope.

Statistical Analysis
All analyses were conducted using the Statistical Package for Social Sciences software (SPSS20.0 version, SSPS Inc, Chicago, IL, USA), and one-way analysis of variance (LSD multiple comparisons, p < 0.05) was used to compare different treatments. The relative abundance and sex ratio of D. suzukii were calculated as follows: Relative abundance (RA) = (the amount of D. suzukii/the total amount of Drosophilidae) × 100% Female ratio = (the amount of D. suzukii females/the amount of all D. suzukii) × 100%   [40] and Asobara spp. [41]. The figi were further confirmed by Dr. Matthew Buffington (USDA-ARS, Systematic Entomol Laboratory, Washington, DC, USA). Each parasitoid specimen was examined for its b color, body length, wings, antenna, and leg morphology under a microscope.

Statistical Analysis
All analyses were conducted using the Statistical Package for Social Sciences softw (SPSS20.0 version, SSPS Inc, Chicago, IL, USA), and one-way analysis of variance ( multiple comparisons, p < 0.05) was used to compare different treatments. The rela abundance and sex ratio of D. suzukii were calculated as follows: Relative abundance (RA) = (the amount of D. suzukii/the total amount of Dr philidae) × 100% Female ratio = (the amount of D. suzukii females/the amount of all D. suzukii) × 1

Captures of Adult D. suzukii in Sentinel Traps
A total of 406 traps were deployed and successfully recovered from four diffe locations, and D. suzukii adults were found in all locations. In total, 11,229 adults o suzukii (6697 females and 4532 males) and 34,743 adults of other Drosophila species w captured.
Different adult Drosophila species were captured from survey locations, and D. m nogaster Meigen was the most abundant species. The relative abundance (RA) of D. suz adults was less than 30% at all survey locations. The RAs of D. suzukii adults captu were 5.61%, 28.89%, 26.91%, and 18.10% from WFD, FK, FC, and SY, respectively (Fig 2, Supplementary Data S1). The RAs of D. suzukii adults captured from FK and FC w significantly higher than those from WFD and SY (F = 7.720, df = 3, 37, p < 0.05).

Captures of Adult D. suzukii in Sentinel Traps
A total of 406 traps were deployed and successfully recovered from four different locations, and D. suzukii adults were found in all locations. In total, 11,229 adults of D. suzukii (6697 females and 4532 males) and 34,743 adults of other Drosophila species were captured.
Different adult Drosophila species were captured from survey locations, and D. melanogaster Meigen was the most abundant species. The relative abundance (RA) of D. suzukii adults was less than 30% at all survey locations. The RAs of D. suzukii adults captured were 5.61%, 28.89%, 26.91%, and 18.10% from WFD, FK, FC, and SY, respectively (Figure 2, Supplementary Data S1). The RAs of D. suzukii adults captured from FK and FC were significantly higher than those from WFD and SY (F = 7.720, df = 3, 37, p < 0.05). In FC, a weekly average of 57.86 D. suzukii adults was captured, which was the highest compared to other survey locations and was significantly higher than those from WFD or SY. Conversely, WFD recorded the lowest (15.56) average weekly amount of D. suzukii adults captured in each trap compared to FK and FC and the difference was significant (Figure 3, Supplementary Data S1) (F = 1.661, df = 3, 37, p < 0.05). In FC, a weekly average of 57.86 D. suzukii adults was captured, which was the highest compared to other survey locations and was significantly higher than those from WFD Insects 2022, 13, 390 6 of 13 or SY. Conversely, WFD recorded the lowest (15.56) average weekly amount of D. suzukii adults captured in each trap compared to FK and FC and the difference was significant (Figure 3, Supplementary Data S1) (F = 1.661, df = 3, 37, p < 0.05).

Figure 2.
Relative abundance (mean ± SE) of D. suzukii adults captured from different locations. Different letters above the bars indicate a significant difference between locations (p < 0.05, LSD).
In FC, a weekly average of 57.86 D. suzukii adults was captured, which was the highest compared to other survey locations and was significantly higher than those from WFD or SY. Conversely, WFD recorded the lowest (15.56) average weekly amount of D. suzukii adults captured in each trap compared to FK and FC and the difference was significant (Figure 3, Supplementary Data S1) (F = 1.661, df = 3, 37, p < 0.05).    The population dynamics of D. suzukii adults were similar among the different locations, with one capture peak in August and then a decrease to zero by October. The peak number of D. suzukii adults captured per week per trap was 259.88 in FC, significantly higher than those from other locations; and the peak amount of D. suzukii adults captured was 24.67 in SY, which was the lowest compared to other locations. The number of D. suzukii adults captured reached its peak on 07-22 (month-day), 08-02, 08-05, 08-17, at SY, WFD, FC, and FK, respectively, and decreased to zero on 08-07, 09-06, 10-01, and 10-05, at SY, WFD, FC, and FK, respectively ( Figure 5, Supplementary Data S1). tions, with one capture peak in August and then a decrease to zero by October. The peak number of D. suzukii adults captured per week per trap was 259.88 in FC, significantly higher than those from other locations; and the peak amount of D. suzukii adults captured was 24.67 in SY, which was the lowest compared to other locations. The number of D. suzukii adults captured reached its peak on 07-22 (month-day), 08-02, 08-05, 08-17, at SY, WFD, FC, and FK, respectively, and decreased to zero on 08-07, 09-06, 10-01, and 10-05, at SY, WFD, FC, and FK, respectively ( Figure 5, Supplementary Data S1).

D. suzukii Larvae Numbers in Suspected Fruits
D. suzukii larvae were found in soft-skinned fruits, such as raspberries, blueberries, and cherries. The total numbers of D. suzukii pupae recorded were 4619, 102, and 24 from raspberry, blueberry, and cherry, respectively. The number of D. suzukii pupae/g fruit in raspberry was significantly higher than in blueberry or cherry (F = 17.055, df = 2, 48, p < 0.05) (Figure 7, Supplementary Data S1).

D. suzukii Larvae Numbers in Suspected Fruits
D. suzukii larvae were found in soft-skinned fruits, such as raspberries, blueberries, and cherries. The total numbers of D. suzukii pupae recorded were 4619, 102, and 24 from raspberry, blueberry, and cherry, respectively. The number of D. suzukii pupae/g fruit in raspberry was significantly higher than in blueberry or cherry (F = 17.055, df = 2, 48, p < 0.05) (Figure 7, Supplementary Data S1).

D. suzukii Larvae Numbers in Suspected Fruits
D. suzukii larvae were found in soft-skinned fruits, such as raspberries, blueberries, and cherries. The total numbers of D. suzukii pupae recorded were 4619, 102, and 24 from raspberry, blueberry, and cherry, respectively. The number of D. suzukii pupae/g fruit in raspberry was significantly higher than in blueberry or cherry (F = 17.055, df = 2, 48, p < 0.05) (Figure 7, Supplementary Data S1). In WFD, D. suzukii larvae were found in cherries only at the 4th week, which was the peak maturity period for cherries. In FC, D. suzukii larvae were found in blueberries at the 6th and 7th week, and many blueberries were damaged due to heavy rains in that period. In FK, D. suzukii larvae were found in raspberries during the fruit development period, and D. suzukii larvae were present in each collected raspberry. The weekly mean number of D. suzukii pupae peaked at the 12th week and then decreased to zero at the 17th week In WFD, D. suzukii larvae were found in cherries only at the 4th week, which was the peak maturity period for cherries. In FC, D. suzukii larvae were found in blueberries at the 6th and 7th week, and many blueberries were damaged due to heavy rains in that period. In FK, D. suzukii larvae were found in raspberries during the fruit development period, and D. suzukii larvae were present in each collected raspberry. The weekly mean number of D. suzukii pupae peaked at the 12th week and then decreased to zero at the 17th week in FK, and the peak number of D. suzukii pupae was 2.43/g (Figure 8, Supplementary Data S1).

D. suzukii Surveys in Wild Host Species
Wild berries of 10 different plant species were collected from non-crop habitats in Liaoning, and the list of the plant species from which D. suzukii emerged is provided in Table 2.

D. suzukii Surveys in Wild Host Species
Wild berries of 10 different plant species were collected from non-crop habitats in Liaoning, and the list of the plant species from which D. suzukii emerged is provided in Table 2.

Discussion
This was the first study that surveyed the occurrence of D. suzukii and its associated host plants and parasitoids in both crop and non-crop habitats in the major berry-fruitproduction regions in Northeastern China. Our results showed that: (1) D. suzukii occurred in raspberries, blueberries, and cherries, and raspberries appeared to be the most seriously infested crop; (2) four wild berries from non-crop habitats were also infested by D. suzukii; (3) two species of parasitoids occurred in Liaoning, China; (4) FC had the highest captures of D. suzukii adults in sentinel traps compared to other three survey locations; and (5) throughout the surveyed period (from June to October 2016), the seasonal population dynamics of D. suzukii adults were similar in different survey locations.
Many studies have showed that the number of captured D. suzukii adults varied among the different geographic locations, the host plant life cycle, temperature, and rainfall [34,42,43]. Like other colder or northern regions in Europe or North America [44], there was only one peak of adult flies per year in Liaoning. Fly populations appeared in June, increased steadily over the summer to reach a peak in August, and flies were no longer detected by October as temperatures decreased. Among the four locations, the numbers of D. suzukii adults captured in FK and FC were significantly higher, and collected fruits in these two locations were also more severely damaged than those from WFD and SY. The FK and FC cites were surrounded by woods and bushes, and these non-crop habitats had likely provided source populations of D. suzukii and affected the occurrence and distribution of the flies. In FK, the number of D. suzukii larvae collected at the end of the harvest period was higher than that of other periods, probably because those unpicked ripe fruits provided breeding sites for D. suzukii adults. Furthermore, In FC, the number of D. suzukii larvae collected in blueberries during the 4th and 5th weeks was significantly higher than in other weeks. This was likely because blueberries were mechanically injured due to the heavy rains and strong winds, and juices from the overripe, damaged, or split fruits could attract D. suzukii adults.
D. suzukii has a wide range of hosts, and many fruits are oviposition hosts, adult food sources, or provide shelter for overwintering D. suzukii [26]. In addition to commonly cultivated soft-skinned fruits, D. suzukii can also infest a variety of wild or ornamental hosts [26]. D. suzukii most likely overwinters in forests with wild hosts where refuge and nutrients are more abundant than crop fields. During the spring and early summer, small overwintering populations likely build in non-crop areas to escape monitoring and insecticides [45,46]. Surveying and identifying wild hosts, followed by removal of whole plants or fruits, can reduce D. suzukii habitats and help manage D. suzukii populations [44]. The wild berries from ten sampled plant species were collected from non-crop habitats in Liaoning, four of which were infested by D. suzukii. However, infestations in the field will depend on the level of D. suzukii populations, host plants (including fruits ripeness, age, and architecture) and relative attractiveness of other hosts in surrounding vicinity [26]. Therefore, the absence of infestation in the other six fruiting species does not necessarily indicate that they are unsuitable hosts. They are still potential wild hosts for D. suzukii, and more extensive investigations are needed in the future. Furthermore, studies on host preference by D. suzukii showed that the fly prefers soft-skinned fruits. Therefore, there is a need to determine the kinds of volatile compounds involved, which will be helpful to develop a better trap than the current yeast traps for the monitoring and control of D. suzukii in the future.
Many natural enemies, including parasitoids, predators, and entomopathogens, have been evaluated under laboratory conditions for their efficiency against D. suzukii, and some of them have the potential to be used for biological control of this pest [47,48]. In particular, some host-specific parasitoids could be promising natural enemies. There are more than 50 hymenopteran parasitoid species worldwide, attacking Drosophila species in the larval or pupal developmental stages [49]. Genetic analyses suggest East Asia is the region of origin for the D. suzukii populations that invaded North America [50]. As the origin region of D. suzukii, East Asia should be the focal region for parasitoid collections [51]. To date, no locally occurring larval Drosophila parasitoids can readily develop from D. suzukii in the invaded regions. In contrast, 19 species of larval D. suzukii parasitoids were collected from D. suzukii in East Asia [48]. Among them, L. japonica, Ganaspis brasiliensis (Ihering), and A. japonica were the dominant parasitoids [52,53].
G. brasiliensis and L. japonica are the most abundant and frequently collected larval parasitoids, and predominantly or exclusively reared from D. suzukii with reported highest parasitism rates >70% [54]. At least one genetic group of G. brasiliensis was found to be the most host-specific to D. suzukii, which has only been collected from fresh fruits infested by D. suzukii and other closely related drosophilids [38,54]. Thus, the more host-specific G. brasiliensis was currently being considered for introduction into North America and Europe [38]. L. japonica was first collected from fresh cherries in Trento, Italy in 2019, and in the coming year, L. japonica was collected from more locations there, which confirmed that L. japonica is widely established in the region. They shared more than 99% sequence similarity with specimens of L. japonica collected in Asia. This means that L. japonica was probably accidentally introduced into Italy from Asia [55]. Both G. brasiliensis and L. japonica were found established in British Columbia in 2020, probably resulting from accidental introduction [56]. Although China is one of the native ranges of L. japonica, there are few studies on it. Previous studies have only confirmed the distribution of L. japonica in Yunnan, Sichuan, and Beijing in China [38]. Further research is needed on the distribution and biological characteristics of L. japonica in China. As one of the native ranges of D. suzukii, the Chinese fruit industry has not suffered serious economic losses [2], probably due to the wide occurrence of some effective native natural enemies of D. suzukii. At least 10 larval parasitoids, A. japonica, Asobara leveri (Nixon) (Hymenoptera: Braconidae), Asobara mesocauda van Achterberg and Guerrieri (Hymenoptera: Braconidae), Asobara triangulata van Achterberg & Guerrieri (Hymenoptera: Braconidae), Asobara pleuralis (Ashmead) (Hymenoptera: Braconidae), Areotetes striatiferus Li, van Achterberg and Tan (Hymenoptera: Braconidae), G. brasiliensis, L. japonica, Tanycarpa chors Be-lokobylskij (Hymenoptera: Braconidae), and Leptopilina sp. (Hymenoptera, Figitidae), and the two pupal parasitoids, Pachycrepoideus vindemiae (Rondani) (Hymenoptera: Pteromalidae) and Trichopria drosophilae Perkins (Hymenoptera: Diapriidae) were collected in Sichuan, Yunnan, Beijing, and Hubei Provinces of China [38,53]. The current survey in Liaoning further complemented previous research on the diversity and distribution of these parasitoids in China. However, we only collected L. japonica and A. japonica, and did not find G. brasiliensis. The parasitoid species found in Liaoning were much less diverse than other warmer regions in China as well as in South Korea and Japan [38,52,53]. In the future, more extensive surveys of native parasitoids in East Asia may be needed to discover different species/strains that can establish in different climatic zones in the fly's invaded regions.