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.
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.
Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, and parts of Africa1,2. The fly is extremely polyphagous, being capable of utilizing various cultivated and wild fruits with soft and thin skins in its native and invaded regions1,2,3. 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 regions1,4. 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 pest4,5,6.
Over the past decade, researchers have focused efforts to discover co-evolved parasitoids of Drosophila suzukii in the fly's native ranges in East Asia7,8,9, as well as effective but newly associated parasitoids in the fly's invaded regions in the Americas and Europe4,5,6. 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. suzukii4,5,6. Among them, Asobara japonica Belokobylskij, Ganaspis brasiliensis Ihering, and Leptopilina japonica Novković & Kimura are the dominant larval parasitoids7,8,9,11. 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 vegetation7,8,9. These three Asian larval parasitoids were imported to quarantine facilities in the USA and Europe, and evaluated for their relative efficiency12,13,14,15,16,17, climatic adaptability18, potential interspecific competitive interactions19, and, most importantly, host specificity8,20,21,22.
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 specificity8,21,22,23,24. 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 Japan9,23,25 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 tests21,22. 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. brasiliensis14,21,22. 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, Japan9,22. Other scales of rearing methods, such as using vials or small containers for both groups, are also briefly discussed.
1. Methods for small-scale laboratory rearing of G3 Ganaspis brasiliensis
2. Methods for large-scale rearing of G1 Ganaspis brasiliensis
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% ± 1.9% female offspring. At 21 °C, adult parasitoids emerged approximately 30-35 days after oviposition. The parasitoid density and exposure time did not significantly affect the total number of offspring produced per replicate (flask) (one-way ANOVA, F3,52 = 0.379, P = 0.769) and had only a marginal effect on the percentage of female offspring (one-way ANOVA, data were logit-transformed prior to the analysis as needed to stabilize the variation, F3,52 = 2.796, P = 0.049), although the production efficiency per capita female (one-way ANOVA, F3,52 = 3.576, P = 0.020) decreased at the high parasitoid density. Exposure time of more than 5 days did not appear to increase the productivity. Increased parasitoid density similarly did not appear to increase the productivity. Therefore, the combination of six pairs and 5-day exposure times seems to be most suitable for laboratory rearing.
Figure 5 shows a 6-month productivity trend of the large-scale rearing of G1 Ganaspis brasiliensis at the quarantine facility of the Edmund Mach Foundation (Trento, Italy) in 2021. The rearing was started using 150 three-day-old, mated female wasps derived from a small-scale rearing at the quarantine laboratory of CABI in Delémont, Switzerland. More wasps from the rearing were gradually added to the parasitism cage, until reaching 1,500-2,000 individuals (sex ratio 50:50) on week 5. The occupancy of the parasitism cage was thereafter maintained at the same level by adding new wasps produced in the large-scale rearing itself. During the entire period, the parasitism cage was provided with freshly infested fruit every 2-3 days. The fruit (blueberries) was offered to G1 G. brasiliensis immediately after the overnight exposure to Drosophila suzukii. Parasitoid production started 5 weeks after the initial host exposure (Figure 5A). From week 8 to week 22, the parasitoid production was proportional to the amount of fruit exposed, averaging 0.44 ± 0.03 g/parasitoid (mean ± SE; Figure 5B). A total of 53,736 parasitoids were collected, with an average female progeny of 45.9% (range: 32.4%-79.0%).
Figure 1: A flow chart for small-scale laboratory rearing procedures of Drosophila suzukii and G3 Ganaspis brasiliensis. The left side shows the host fly rearing procedures while the right side illustrates the parasitoid rearing cycle. Please click here to view a larger version of this figure.
Figure 2: A flow chart for the large-scale rearing procedures of Drosophila suzukii and G1 Ganaspis brasiliensis. The left side shows the host fly rearing procedures while the right side illustrates the parasitoid rearing cycle. Abbreviation: SDM = standard Drosophila medium. Please click here to view a larger version of this figure.
Figure 3: Containers and tubes used to store and ship G1 Ganaspis brasiliensis from the large-scale rearing. (A) A horizontal view of the storage container showing a water tube inside the container, (B) a vertical view of the container showing a ventilated lid and a foam stopper, and (C) a ventilated lid and a piece of absorbent paper for (D) the shipping tube. Please click here to view a larger version of this figure.
Figure 4: Representative example for small-scale laboratory rearing of G3 Ganaspis brasiliensis. (A) Number of offspring produced per flask, (B) number of offspring produced per female wasp, and (C) percentage of female offspring, under two different parasitoid densities and exposure times. Values are mean ± SE, and bars bearing different letters are significantly different (ANOVA, Tukey's HSD, P < 0.05). Please click here to view a larger version of this figure.
Figure 5: Productivity trend of the large-scale rearing from its onset through week 23. (A) Bars indicate the numbers of G1 Ganaspis brasiliensis offspring collected weekly from the eclosion cages to replace old individuals in the parasitism cage (dark green) and to be stored or shipped (light green). The orange line indicates the amount of host-infested fruit (kg of blueberries) provided each week to the parasitoids within the parasitism cage. (B) Weekly ratio of the weight of host-infested fruit to the number of parasitoid offspring produced 5 weeks after exposure (i.e., grams of fruit required to produce a single adult parasitoid). Please click here to view a larger version of this figure.
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 for maintaining a colony for laboratory research or bioassays. Similar methods have been used by the authors to rear this parasitoid for quarantine evaluations of this parasitoid. The large-scale rearing protocol will allow to produce large numbers of wasps for field release, as were recently conducted in Italy. These technologies can easily be transferred to other laboratories, producers, or companies for large-scale rearing in the near future, as well as serving as the basis for further improvements in methodologies.
These protocols may also be used to rear Leptopilina japonica, as both Ganaspis brasiliensis and L. japonica are similar in terms of their host habitat preference7,8, host stage preference or reproductive biology15, thermal performance18, and foraging efficiency in laboratory bioassays13,15, as well as behavioral responses towards host-associated cues12, except that L. japonica seems to have a broader host range than that of even G3 G. brasiliensis20. Both parasitoids have been reared using similar methods as described herein. For small-scale rearing, both parasitoids can also be reared in drosophila vials, typically by transferring 20 young (1-2 days old) Drosophila suzukii larvae to a drosophila vial filled with 2 cm of artificial diet, or placing two infested fruits, each containing 5-10 young D. suzukii larvae, and exposing them to two mated female wasps for 2-3 days, both producing ~10 offspring per vial13,15,22.
As discussed above, the G1 and G3 Ganaspis brasiliensis used in this protocol may differ slightly in some host-searching behaviors and host specificity21,22. Girod et al.21 report that the Japanese G1 G. brasiliensis was more strictly specific to Drosophila suzukii, and did not appear to perform well in pure artificial diet in vials compared to its performance on host fruit. Matsuura et al.25 also reported that G1 G. brasiliensis populations collected from D. suzukii-infested cherries in Japan specialize on D. suzukii. The small-scale rearing method using diet mixed with blueberries was developed initially for rearing G3 G. brasiliensis because G1 G. brasiliensis was not available at that time. Later, this method was found to not work well for the rearing of G1 G. brasiliensis (Wang et al. unpublished data).
Therefore, for small-scale rearing of G1 G. brasiliensis, it is suggested to modify the host substrate by (1) exposing the flies directly to blueberries (or other host fruit) to collect host larvae in the fruit, and (2) transferring exposed fruit to a standard Drosophila culture medium for the larvae to develop in a low-competition environment by placing the infested fruits containing the host larvae onto the diet in the flasks for exposure to the parasitoids. This will allow the parasitized larvae to feed on the diet, especially at high host densities, and the parasitized host pupae to be collected from the paper towel. Alternatively, G1 G. brasiliensis can be reared on fruit directly in plastic containers (various sizes) by exposing 5-10 female wasps to 10-20 infested blueberries for 4-5 days, which produced up to 80 offspring per container, depending on host density (Wang et al., unpublished data). For this method, it is recommended to place the infested fruit on a raised metal mesh ("hardware cloth") to allow the parasitoids to access the infested fruit from all directions, especially if too many fruits are placed in one container. Ideally, one or two layers of infested fruit should be placed in each container for this rearing method. These alternative small-scale methods should also work well for the rearing of G3 G. brasiliensis.
Regardless of the rearing methods and scales (vial, flask, container, or cage), it is critical to maintain suitable temperature, humidity, as well as control host or female wasp age, density, and exposure time for the rearing of both G1 Ganaspis brasiliensis and G3 G. brasiliensis. Drosophila suzukii larvae developed in about 1 week under normal laboratory conditions (e.g., 22 ± 2 °C) 15. Female G. brasiliensis preferred to attack younger (1-2 day old) than older (3-4 day old) host larvae, although various ages of host larvae could be attacked15. At 22 ± 2 °C, G. brasiliensis females emerged with a substantially high proportion (~50%) of their lifetime complement of mature eggs, and the mature egg load reached a peak after 2-3 days15. Adult females survived ~20 days when provided unlimited access to hosts and began oviposition within 2 days after emergence, reaching a peak of oviposition within 5-10 days and thereafter gradually reducing oviposition15. Therefore, young (<10 days old) female wasps should be used in the rearing, but female wasps could be reused for the rearing when they are in a short supply. The parasitoid could readily develop at 21-25 °C, but temperatures below 17.2 °C appeared to trigger a facultative diapause17. It is therefore recommended to use a temperature range of 21-25 °C for optimal development of both the fly and the parasitoid.
Further, exposure time of more than 5 days will not likely increase the productivity of the parasitoid. Increased parasitoid density based on the small-scale rearing for G3 G. brasiliensis appears to not increase the productivity, possibly due to mutual interference among foraging female wasps. Six male-female pairs and a 5-day exposure time seem to be an ideal combination for the laboratory small-scale rearing, although the rearing methods may be improved in the future by further optimizing the ratio of host to parasitoid. A major mortality factor for the parasitoid seems to be related to low humidity, as many parasitoids were observed to be unable to eclose with dry substrate conditions. Adding a piece of absorbent paper towel underneath the fruit not only absorbs juices as the fruit degrades, but also provides a substrate that can be dampened to increase humidity or provide a pupation substrate for the fly.
For the small-scale rearing, a flask can maintain humidity better than a vial because the former has a narrow neck. It was also found in this study that fresh blueberries coated with a dusting of active dry yeast helped prevent the formation of mold and enhanced the attraction of the fruit to the flies. Other aspects of the parasitoid rearing that remain to be explored include (1) the possibility of rearing this parasitoid on alternative hosts or host fruits and how alternative hosts or host fruits would affect the parasitoid's efficiency, (2) factors affecting the parasitoid's offspring fitness and sex ratio, (3) the ability of this parasitoid (both G1 and G3 ) to adapt to artificial diet conditions, and (4) the genetic or behavioral changes that might occur with adaptation.
The authors have nothing to disclose.
The authors thank Lukas Seehausen and Marc Kenis (CABI, Switzerland) for kindly providing G1 G. brasiliensis. Funding in Italy was provided by Provincia Autonoma di Trento, Trento, Italy, and in the US by the National Institute of Food and Agriculture, USDA Specialty Crops Research Initiative award (#2020-5118-32140), USDA Animal and Plant Health Inspection Service (Farm Bill, fund 14-8130-0463), and USDA ARS CRIS base funds (project 8010-22000-033-00D). The USDA is an equal-opportunity provider and employer and does not endorse products mentioned in this publication.
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 |