Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Summary

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

Abstract

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa over the last decade, becoming a devastating pest of various soft-skinned fruits in its invaded regions. Biological control, especially by means of self-perpetuating and specialized parasitoids, is expected to be a viable option for sustainable area-wide management of this highly mobile and polyphagous pest. Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae) is a larval parasitoid that is widely distributed in East Asia, and has been found to be one of the most effective parasitoids of D. suzukii.

Following rigorous pre-introduction evaluations of its efficacy and potential non-target risks, one of the more host-specific genetic groups of this species (G1 G. brasiliensis) has been approved recently for introduction and field release in the United States and Italy. Another genetic group (G3 G. brasiliensis), which was also commonly found to attack D. suzukii in East Asia, may be considered for introduction in the near future. There is currently enormous interest in rearing G. brasiliensis for research or in mass-production for field release against D. suzukii. This protocol and associated video article describe effective rearing methods for this parasitoid, both on a small scale for research and a large scale for mass-production and field release. These methods may benefit further long-term research and use of this Asian-native parasitoid as a promising biological control agent for this global invasive pest.

Introduction

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa^1,2. The fly is extremely polyphagous, being capable of utilizing various cultivated and wild fruits with soft and thin skins in its native and invaded regions^1,2,3. Current management strategies for this pest rely heavily on the frequent use of insecticides that target adult flies in crop fields when susceptible fruit are ripening. Repeated sprays are often used, possibly due to consistent spillover of reservoir fly populations from non-crop habitats and lack of effective natural enemies resident in the invaded regions^1,4. Biological control, especially by means of self-perpetuating specialized parasitoids, may help suppress fly populations at the landscape level and play a critical role for sustainable area-wide management of this highly mobile and polyphagous pest^4,5,6.

Over the past decade, researchers have focused efforts to discover co-evolved parasitoids of Drosophila suzukii in the fly's native ranges in East Asia^7,8,9, as well as effective but newly associated parasitoids in the fly's invaded regions in the Americas and Europe^4,5,6. In the fly's newly invaded regions, commonly occurring larval Drosophila parasitoids, such as Asobara c.f. tabida (Nees) (Hymenoptera: Braconidae), Leptopilina boulardi (Barbotin et al.), and L. heterotoma (Thompson) (Hymenoptera: Figitidae), are unable to develop from or have low parasitism levels on D. suzukii due to the fly's strong immune resistance¹⁰. Only some cosmopolitan and generalist pupal parasitoids such as Pachycrepoideus vindemiae (Rondani) (Hymenoptera: Pteromalidae) and Trichopria drosophilae (Perkins) (Hymenoptera: Diapriidae) in North America and Europe, and Trichopria anastrephae Lima in South America can readily develop from this fly⁴. In contrast, explorations in East Asia have discovered a number of larval parasitoids from D. suzukii^4,5,6. Among them, Asobara japonica Belokobylskij, Ganaspis brasiliensis Ihering, and Leptopilina japonica Novković & Kimura are the dominant larval parasitoids^7,8,9,11. In particular, the two figitids (L. japonica and G. brasiliensis) were the major parasitoids predominantly found in fresh fruits infested by D. suzukii and/or other closely related drosophilids in natural vegetation^7,8,9. These three Asian larval parasitoids were imported to quarantine facilities in the USA and Europe, and evaluated for their relative efficiency^{12,13,14,15,16,17}, climatic adaptability¹⁸, potential interspecific competitive interactions¹⁹, and, most importantly, host specificity^8,20,21,22.

Quarantine evaluations showed that Ganaspis brasiliensis was more host-specific to Drosophila suzukii than other tested Asian larval parasitoids, although it likely consists of different biotypes or cryptic species with varying host specificity^{8,21,22,23,24}. Nomano et al.²² grouped Ganaspis individuals from different geographical regions into five genetic groups (named as G1-G5) based on molecular analyses of the mitochondrial cytochrome oxidase I gene fragment. The G2 and G4 groups are reported only from a few south Asian tropical locations, and the G5 group was reported from Asia and other regions (e.g., Argentina, Brazil, Hawaii, and Mexico) from unknown host(s) (Buffington, personal observation). Field collections of wild fruits infested by D. suzukii in South Korea⁷, China⁸, and Japan^9,23,25 found G1 alone or a mixture of specimens representing groups G1 and G3. The two groups seem to be sympatric and co-exist on the same host plants inhabited by D. suzukii and other closely related host flies. Nonetheless, some differences have been observed between the two groups, with G1 seemingly having a higher degree of host- or host-habitat-specificity to D. suzukii than G3, although they both attack a number of closely related species in the quarantine tests^21,22. Further detailed molecular analyses may help determine the species status, especially for the G1 and G3 groups. This study refers to them as G1 G. brasiliensis and G3 G. brasiliensis. Some early studies also named the G1 G. brasiliensis as G. cf. brasiliensis^14,21,22. The G1 G. brasiliensis has recently been approved for field release against D. suzukii in the USA and Italy (several other European countries are also currently considering its introduction), while the G3 G. brasiliensis may be considered for field release in the near future. Recent surveys also found adventive populations of both L. japonica and G1 G. brasiliensis in British Columbia, Canada²⁶, and Washington State, USA (Beers et al., unpublished data), and adventive L. japonica populations in Trento province, Italy²⁷.

Given the significant interest in the development of biological control programs for Drosophila suzukii management and the substantial biological control potential of adventive and deliberate introductions of Ganaspis brasiliensis, there is a need to develop efficient rearing methods for this larval parasitoid for future long-term research and/or field release. This protocol and associated video article describe two sets of rearing methods for this parasitoid: (1) small-scale laboratory rearing in flasks using a mixture of host fruit (blueberry) and artificial diet for the culture of D. suzukii. The methods were developed using G3 material originally collected from Kunming, China⁸. (2) Mass rearing for field release in large cages using host fruit (blueberry) for the culture of D. suzukii. The genetic group used for the large-scale rearing was G1 stock originating in Tokyo, Japan^9,22. Other scales of rearing methods, such as using vials or small containers for both groups, are also briefly discussed.

Protocol

1. Methods for small-scale laboratory rearing of G3 Ganaspis brasiliensis

1. Prepare host diet.
   1. Add 600 mL of distilled water to a 1,500 mL glass container, and heat the water on a hot plate.
   2. Add 88.6 g of commercially available dry diet (made of agar, Brewer's yeast, corn meal, methyl paraben, and sucrose) or prepare diet using the formula published by Dalton et al.²⁸ (see step 2.1.2).
   3. Add 300 mL of distilled water into the dry diet and stir the diet mixture thoroughly.
   4. Add the mixture to the boiling water.
   5. Allow the liquid diet on the hot plate to boil for 10 min while periodically stirring the mixture to prevent it from burning.
   6. Allow the diet to cool at room temperature for 30 min while stirring it occasionally to distribute the release of heat evenly and prevent the diet from solidifying on the surface.
   7. Measure 6.7 mL of 95% EtOH in one container and 3.5 mL of 1 M propionic acid solution in another container.
   8. Once the diet has cooled, add the EtOH and then the propionic acid solution, stirring thoroughly after each addition.
   9. Prepare blueberries (purchased from the local market) by rinsing them in cold water, then in a sodium hypochlorite bleach solution (diluted to 5%), and cold water again.
   10. Dry the fruit with a paper towel and manually mash them until the skin on each fruit is broken and the juices and flesh of the fruit are exposed.
   11. Add 25-30 g of mashed blueberries to each 250 mL flask. Tap the sides of the flask to ensure the interior bottom of the flask is covered with an even layer of mashed blueberries.
   12. Pour the prepared diet into each flask so that it just covers the top of the mashed blueberries.
   13. Add foam stoppers to the necks of the flasks and allow the diet to solidify at room temperature (Figure 1).
   14. Once the diet has solidified, use it immediately or store at 5 °C for up to 3 weeks.
2. Rear host Drosophila suzukii.
   1. Remove the stored diet from the refrigerator and allow it to equilibrate to the ambient room temperature, or use freshly made diet.
   2. Cut a piece of absorbent paper towel (e.g., 5 cm x 20 cm) and twist it in the center. Place the twisted middle section of the paper towel in the flask (Figure 1).
   3. Wet the paper towel and surface of the diet with distilled water to retain moisture.
   4. Transfer sexually mature adult flies from the current colony fly flasks to a new diet flask by carefully removing the stopper on the old flask and quickly inverting the flask and aligning the opening of the old flask with the new flask.
   5. Gently tap on the side of the old flask to induce the flies to drop into the new flask. Ensure that there are ~25-30 mating pairs of D. suzukii in the new flask. Once there are enough flies in the new flask, quickly flip the old flask upright and replace the stoppers on both flasks.
   6. Repeat the transfers of flies until no flies are left in the old flasks. If necessary, combine or collect flies from more than one old flask into a new flask to ensure there are enough flies (20-30 pairs) per flask.
   7. Hold the new flasks after a week of exposure to adult flies at suitable conditions (21 °C, 16 L: 8 D photoperiod, 60%-80% relative humidity [RH%]) in an environmental chamber for 3 weeks for fly emergence.
3. Expose host larvae to parasitoids.
   1. Take a flask (see step 1.2.7) containing fly eggs and larvae after removing any adult flies and the twisted paper towel from the flask.
   2. Fold a piece of absorbent paper towel in half and put it in the flask as a pupation substrate for parasitized larvae.
   3. Aspirate six female and male pairs of G3 G. brasiliensis into each flask (Figure 1). Streak a thin layer of honey on the bottom of the foam stopper.
   4. Leave the parasitoids in the flask for 5 days.
   5. After a 5-day exposure, remove the parasitoids and hold the flasks under conditions described above in an environmental chamber for 35 days until the expected wasp emergence.
4. Collect and store adult parasitoids.
   1. During the second and third weeks of incubation, check the flasks weekly for early host emergence and remove the adult flies.
   2. Once adult parasitoids start to emerge, aspirate them three times per week and hold them in drosophila vials (e.g., 2.5 cm x 9.5 cm) (Figure 1).
   3. Place a small piece of paper towel moistened, but not saturated, with distilled water at the bottom of the vial.
   4. Add ~60 parasitoids to each vial and label the vial with the emergence dates. Streak a thin layer of honey on the bottom of the foam stopper, twice per week. Store the vials with adult parasitoids under the conditions described above in the environmental chamber for up to a month if not used sooner.
   5. Remoisten the paper in the vial once every 4-7 days or replace the paper towel if there are signs of mold.

2. Methods for large-scale rearing of G1 Ganaspis brasiliensis

1. Implement a large-scale rearing of host Drosophila suzukii.
   1. Rear D. suzukii within large, knitted mesh-covered cages (e.g., 50 cm x 50 cm x 100 cm) each containing 1,500-2,000 sexually mature adult flies (sex ratio 50:50) (Figure 2).
   2. Prepare the Standard Drosophila Medium (SDM) by boiling all the ingredients (6 g of bacteriological agar, 75 g of cornmeal, 17 g of nutritional yeast, 15 g of saccharose, 10 g of soybean flour, 10 mL of propionic acid) in 1 L of distilled water for 10 min while periodically stirring the mixture to prevent it from burning²⁸.
   3. Let the mixture cool down for 5 min and add 5 g of ascorbic acid.
   4. Pour the freshly cooked SDM into 9 cm Petri dishes and allow the medium to solidify at room temperature before closing the plates.
   5. Stack up the SDM Petri dishes, wrap the stack with aluminum foil, and store the dishes at 4 °C for up to 2 weeks.
   6. Within each rearing cage, place a plate with water-soaked cotton and four to six Petri dishes with SDM (Figure 2).
   7. Twice per week, replace the infested SDM Petri dishes with fresh ones.
   8. Place the infested SDM Petri dishes without lids individually into plastic cups (13.3 cm diameter or 800 mL), close each cup with a covering of fine mesh (<0.5 mm), and incubate for 12-15 days at 23 °C and 75% RH (Figure 2).
   9. Transfer the newly hatched D. suzukii adults from the plastic cups to the rearing cages.
2. Prepare host larvae.
   1. Rinse the blueberries in cold water for 1 min, and soak the fruits in a basin filled with a bleach solution (diluted to 5%) for 3 min.
   2. Drain the bleach solution and fill the basin with cold water to rinse the blueberries. Mix gently by hand for at least 30 s.
   3. Repeat step 2.2.2 with fresh water at least three times to remove bleach residues and other arthropods (e.g., mites, thrips) that may be present on the fruit.
   4. Place the fruit on a tray with several layers of absorbent paper towels and carefully tilt the tray back and forth, rolling the berries around to dry them.
   5. Prepare several 9 cm Petri dishes (either the top or the bottom halves, facing up) and fill each one with the washed blueberries (15-25 fruits per plate depending on the fruit size).
   6. During late afternoon hours, expose the Petri dishes to sexually mature adult flies within the host rearing cages (see step 2.1) and leave them overnight.
   7. The next morning, remove the Petri dishes from the host rearing cages by gently blowing or tapping on them to dislodge the flies on the fruits and use the infested fruit for the rearing of parasitoids (see step 2.4).
3. Implement a large-scale parasitoid rearing.
   1. Use two types of cages to rear the parasitoid: one for parasitism and another for wasp emergence.
   2. Ensure that the parasitism cage is cubic (e.g., 45 cm each side) with a clear plastic panel on the front for observing insect activity, two 18 cm sleeve openings in the front panel for the addition or removal of insects and the replacement of food material, and fine polyester netting (e.g., 96 x 26 mesh) on the top and sides for ventilation.
   3. Make the emergence cage smaller (e.g., 30 cm each side), with a single sleeve opening on two opposite sides and a clear plastic panel on the front for visibility (Figure 2).
   4. Ensure that both cage types have a thin string that hangs below the ceiling from which to suspend one to several feeders (Figure 2).
      NOTE: A feeder consists of a large cylindric foam stopper (9 cm diameter) covered with scattered honey droplets, and can be placed on the cage floor or hung from the cage ceiling (Figure 2).
   5. Within each cage, provide water in a straight-wall drosophila vial (2.5 cm x 9.5 cm) sealed with a cellulose acetate plug (2.5 cm diameter) every 5-7 days depending on the RH. Hang the vial upside-down from the cage ceiling (Figure 2).
4. Expose the host larvae to the parasitoids.
   1. Expose the host-infested fruit within the Petri dishes to G1 G. brasiliensis immediately after D. suzukii overnight infestation (see step 2.2.7).
   2. Leave the 10-15 Petri dishes of infested fruit in the parasitization cage containing 1,500-2,000 wasps for 2-3 days.
   3. Use plastic cups (13.3 cm diameter or 800 mL) with layers of absorbent paper on the bottom to collect the fruit containing the parasitized hosts (Figure 2).
   4. Place the open cups in the eclosion cage and incubate for at least 28 days at 21 °C and 65% RH (Figure 2).
   5. During the second and third weeks of incubation, check the cage weekly for early host eclosion and remove the adult flies to facilitate the successive collection of parasitoids.
   6. At the end of the fourth week of incubation, add a feeder and a water source to the cage.
5. Collect and store the adult parasitoids.
   1. Once parasitoid emergence starts, collect a portion (10%-15%) of the adults and transfer them back to the parasitism cage to replace old unproductive individuals.
   2. Collect and store the remaining parasitoids in plastic cups (13.3 cm diameter or 800 mL) (Figure 3A).
   3. Place a tube (2 mL) filled with water and sealed with a dental cotton roll (1 cm x 3.8 cm) at the bottom of the cup (Figure 3A).
   4. Close the cup with a modified lid fitted with a removable foam stopper (3.5 cm diameter) as a feeder substrate and a mesh-covered hole for ventilation (Figure 3B).
   5. Add up to 700 adults to each cup (sex ratio 50:50), label the cup with the emergence date, and store it in an environmental chamber (17 °C; 65% RH) until used, or for up to 1 month (Figure 3B).
6. Ship the adult parasitoids.
   1. Use conical tubes (50 mL) to ship the adult parasitoids.
   2. Pierce a ventilation hole (8 mm diameter) on the cap and cover it with a fine mesh net (Figure 3C).
   3. Add a cellulose acetate feeding ring on the inside of the cap (Figure 3C).
   4. Prepare a saturated sucrose solution using distilled water, apply a few drops on the feeding ring, and let it absorb the liquid.
   5. Place a fan-shaped piece of absorbent paper towel within the tube (Figure 3D).
   6. Add ~200 adult parasitoids to each tube, and place the tubes in an insulated shipping container together with ice packs.

Results

Figure 4 shows representative results of the small-scale laboratory rearing of G3 Ganaspis brasiliensis using two different parasitoid densities (six or 10 pairs) and two different exposure times (5 or 10 days) at the quarantine facility of the USDA-ARS Beneficial Insects Introduction Unit (Newark, Delaware). There were 14 replicates for each combination of parasitoid density and exposure time. In total, the 64 flasks produced 4,018 wasps (71.7 ± 4.9 offspring per flask) with 49.5% ...

Discussion

Long-term research and subsequent field releases of a biological control agent depend on the availability of effective and economical rearing techniques. The described methods in this study have proven to be efficient protocols for both small-scale and large-scale rearing of Ganaspis brasiliensis. The small-scale rearing protocol has been developed over several years to optimize the amount of labor and reduce specialized equipment needed to maintain the parasitoid and host colonies simultaneously. It is suitable...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Active dry yeast	Fleischmanns Yeast, Cincinatti, OH, USA	None	Used to cover fruit to reduce mold growth and enhance the frui attraction to the flies
Bacteriological agar	Merk Life Science S.r.l., Milan, Italy	A1296 - 5KG	Used to prepare the Standard Drosophila Medium
Bleach solution	Clorox Company, Oakland, CA, USA	None	Used to disinfect flesh fruit
Blue stopper	Azer Scientific, Morgantown, PA, USA	ES3837	Used for sealing the tube while allowing ventilation for insects
Blueberries	Grocery Store, Newark, DE, USA	None	Provided as host fruit for the flies (various other fruit can also be used)
BugDorm insect rearing cage (W24.5 x D24.5 x H63.0 cm)	Mega View Science Co. Ltd., Taichung, Taiwan	4E3030	Used for rearing parasitoids (parasitism cage)
BugDorm insect rearing cage (W32.5 x D32.5 x H32.5 cm)	Mega View Science Co. Ltd., Taichung, Taiwan	4E4590	Used for rearing flies
BugDorm insect rearing cage (W32.5 x D32.5 x H32.5 cm)	Mega View Science Co. Ltd., Taichung, Taiwan	4E4545	Used for rearing parasitoids (eclosion cage)
Chicken wire (0.64 cm, 19 gauge)	Everbilt, OH, USA	308231EB	Used to lift up the fruit to allow maximum parasitoid oviposition
Cornmeal	Grocery Store, Trento, TN, Italy	None	Used to prepare the Standard Drosophila Medium
Dental cotton roll (1 x 3.8 cm)	Gima S.p.A., Gessate, MI, Italy	35000	Used for providing water to the parasitoids within the storage container
Drosophila diet	Frontier Scientific, Newark, DE, USA	TF1003	Custom diet used to rear flies
Drosophila vial narrow, Polystirene (2.5 x 9.5 cm)	VWR International, LLC., Radnor, PA, US	75813-160	Used for providing water to the parasitoids within the cage
Drosophila vial plugs, Cellulose acetate (2.5 cm)	VWR International, LLC., Radnor, PA, US	89168-886	Used for providing water to the parasitoids within the cage
Erlenmeyer flask (250 mL)	Carolina Biological, Burlington, NC, USA	731029	Used for rearing flies and parasitoids
Falcon-style centrifuge tube (50 mL)	VWR International, LLC., Radnor, PA, US	VWRI525-0611	Modified to ship adult parasitoids
Foam stopper	Jaece Industries, North Tanawanda, NY, USA	L800-C	Used for sealing the flasks while allowing ventilation for insects
Honey	Grocery Store, Newark, DE, USA	None	Provided as food for parasitoids
Identi-Plug plastic foam stopper	Fisher Scientific Company, L.L.C., Pittsburg, PA, US	14-127-40E	Used as feeder for parasitoids and to seal the storage container
Industrial paper towel	Grocery Store, Newark, DE, USA	None	Provided as a pupation substrate for pupae and mitigated moisture
Micron mesh fabric (250 mL)	Industrial Netting, Maple Grove, MN, USA	WN0250-72	Used to make ventilation lid for insects
Nutritional yeast (flakes)	Grocery Store, Trento, TN, Italy	None	Used to prepare the Standard Drosophila Medium
Paper coaster (10.2 cm)	Hoffmaster, WI, USA	35NG26	Porvided as pupation substrate for flies and parsitized pupae
Plastic cup (Ø 13.3 cm, 800 mL)	Berry Superfos, Taastrup, Denmark	Unipak 5134	Modified to store adult parasitoids
Plastic lid (Ø 13.3 cm)	Berry Superfos, Taastrup, Denmark	PP 2830	Modified to store adult parasitoids
Propionic acid	Merk Life Science S.r.l., Milan, Italy	P1386 - 1L	Used to prepare the Standard Drosophila Medium
Saccharose	Grocery Store, Trento, TN, Italy	None	Used to prepare the Standard Drosophila Medium
Soup cup with lid (475 mL)	StackMan, Vietnam	DC1648	Used for parasitized larvae to pupate
Soybean flour	Grocery Store, Trento, TN, Italy	None	Used to prepare the Standard Drosophila Medium
White felt washer (0.64 cm thick, 5 mm ID x 20 mm OD)	Quiklok, Lincoln, NH, US	WFW/.25 x 5 x 20 mm	Used as feeding ring for parasitoids