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.

1. Methods for small-scale laboratory rearing of G3 Ganaspis brasiliensis
<ol>
	<li>Prepare host diet.
	<ol>
		<li>Add 600 mL of distilled water to a 1,500 mL glass container, and heat the water on a hot plate.</li>
		<li>Add 88.6 g of commercially available dry diet (made of agar, Brewer&#39;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).</li>
		<li>Add 300 mL of distilled water into the ...

<ol>
	<li>Asplen, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+biology+of+spotted+wing+drosophila+(Drosophila+suzukii):+a+global+perspective+and+future+priorities.">Invasion biology of spotted wing drosophila (Drosophila suzukii): a global perspective and future priorities.</a> Journal of Pest Science. 88 (3), 469-494 (2015).</li><li>Tait, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+suzukii+(Diptera:+Drosophilidae):+A+decade+of+research+towards+a+sustainable+integrated+pest+management+program.">Drosophila suzukii (Diptera: Drosophilidae): A decade of research towards a sustainable integrated pest management program.</a> Journal of Economic Entomology. 114 (5), 1950-1974 (2021).</li><li>Kirschbaum, D. S., Funes, C. F., Buonocore-Biancheri, M. J., Su&#225;rez, L., Ovruski, S. M., Garcia, F. R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+ecology+of+Drosophila+suzukii+(Diptera:+Drosophilidae).">The biology and ecology of Drosophila suzukii (Diptera: Drosophilidae).</a> Drosophila suzukii management. , 41-92 (2020).</li><li>Wang, X. G., Lee, J. C., Daane, K. M., Buffington, M. L., Hoelmer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+of+Drosophila+suzukii.">Biological control of Drosophila suzukii.</a> CAB Reviews. 15, 054 (2020).</li><li>Lee, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+of+spotted-wing+drosophila+(Diptera:+Drosophilidae):+Current+and+pending+tactics.">Biological control of spotted-wing drosophila (Diptera: Drosophilidae): Current and pending tactics.</a> Journal of Integreated Pest Management. 10 (1), 13 (2019).</li><li>Wang, X. G., Lee, J. C., Daane, K. M., Hoelmer, K. A., Garcia, F. R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+of+spotted-wing+drosophila:+An+update+on+promising+agents.">Biological control of spotted-wing drosophila: An update on promising agents.</a> Drosophilia suzukii management. , 143-167 (2020).</li><li>Daane, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+exploration+of+parasitoids+of+Drosophila+suzukii+in+South+Korea+as+potential+classical+biological+agents.">First exploration of parasitoids of Drosophila suzukii in South Korea as potential classical biological agents.</a> Journal of Pest Science. 89 (3), 823-835 (2016).</li><li>Giorgini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+for+native+parasitoids+of+Drosophila+suzukii+in+China+reveals+a+diversity+of+parasitoid+species+and+narrow+host+range+of+the+dominant+parasitoid.">Exploration for native parasitoids of Drosophila suzukii in China reveals a diversity of parasitoid species and narrow host range of the dominant parasitoid.</a> Journal of Pest Science. 92 (2), 509-522 (2019).</li><li>Girod, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+parasitoid+complex+of+D.+suzukii+and+other+fruit+feeding+Drosophila+species+in+Asia.">The parasitoid complex of D. suzukii and other fruit feeding Drosophila species in Asia.</a> Scientific Reports. 8 (1), 11839 (2018).</li><li>Kacsoh, B. Z., Schlenke, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+hemocyte+load+is+associated+with+increased+resistance+against+parasitoids+in+Drosophila+suzukii,+a+relative+of+D.+melanogaster.">High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster.</a> PLoS ONE. 7 (4), 34721 (2012).</li><li>Buffington, M. L., Forshage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redescription+of+Ganaspis+brasiliensis+(Ihering,+1905),+new+combination+(Hymenoptera:+Figitidae),+a+natural+enemy+of+the+invasive+Drosophila+suzukii+(Matsumura,+1931)(Diptera:+Drosophilidae).">Redescription of Ganaspis brasiliensis (Ihering, 1905), new combination (Hymenoptera: Figitidae), a natural enemy of the invasive Drosophila suzukii (Matsumura, 1931)(Diptera: Drosophilidae).</a> Procedings of the Entomoogical Society of Washington. 118 (1), 1-13 (2016).</li><li>Biondi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+olfactory+responses+of+Asobara+japonica+(Hymenoptera:+Braconidae)towards+fruits+infested+by+the+invasive+spotted+wing+drosophila.">Innate olfactory responses of Asobara japonica (Hymenoptera: Braconidae)towards fruits infested by the invasive spotted wing drosophila.</a> Journal of Insect Behavior. 30 (5), 495-506 (2017).</li><li>Biondi, A., Wang, X. G., Daane, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+preference+of+three+Asian+larval+parasitoids+to+closely+related+Drosophila+species:+implications+for+biological+control+of+Drosophila+suzukii.">Host preference of three Asian larval parasitoids to closely related Drosophila species: implications for biological control of Drosophila suzukii.</a> Journal of Pest Science. 94 (2), 273-283 (2021).</li><li>Girod, P., Rossignaud, L., Haye, T., Turlings, T. C. J., Kenis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Asian+parasitoids+in+larvae+of+Drosophila+suzukii+feeding+on+blueberry+and+artificial+diet.">Development of Asian parasitoids in larvae of Drosophila suzukii feeding on blueberry and artificial diet.</a> Journal of Applied Entomology. 142 (5), 483-494 (2018).</li><li>Wang, X. G., Nance, A. H., Jones, J. M. L., Hoelmer, K. A., Daane, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspects+of+the+biology+and+reproductive+strategy+of+two+Asian+larval+parasitoids+evaluated+for+classical+biological+control+of+Drosophila+suzukii.">Aspects of the biology and reproductive strategy of two Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii.</a> Biological Control. 121, 58-65 (2018).</li><li>Wang, X. G., Biondi, A., Daane, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+responses+of+three+candidate+Asian+larval+parasitoids+evaluated+for+classical+biological+control+of+Drosophila+suzukii.">Functional responses of three candidate Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii.</a> Journal of Economic Entomology. 113 (1), 73-80 (2020).</li><li>Wang, X. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Asobara+japonica+as+a+potential+biological+control+agent+for+the+spotted+wing+drosophila,+Drosophila+suzukii.">Assessment of Asobara japonica as a potential biological control agent for the spotted wing drosophila, Drosophila suzukii.</a> Entomologia Generalis. 41, 1-12 (2021).</li><li>Hougardy, E., Hogg, B. N., Wang, X. G., Daane, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+thermal+performances+of+two+Asian+larval+parasitoids+of+Drosophila+suzukii.">Comparison of thermal performances of two Asian larval parasitoids of Drosophila suzukii.</a> Biological Control. 136, 104000 (2019).</li><li>Wang, X. G., Hogg, B. N., Hougardy, E., Nance, A. H., Daane, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+competitive+outcomes+among+three+solitary+larval+endoparasitoids+as+candidate+agents+for+classical+biological+control+of+Drosophila+suzukii.">Potential competitive outcomes among three solitary larval endoparasitoids as candidate agents for classical biological control of Drosophila suzukii.</a> Biological Control. 130, 18-26 (2019).</li><li>Daane, K. M., Biondi, A., Wang, X. G., Hogg, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+host+ranges+of+three+Asian+larval+parasitoids+of+Drosophila+suzukii.">Potential host ranges of three Asian larval parasitoids of Drosophila suzukii.</a> Journal of Pest Science. 94 (4), 1171-1182 (2021).</li><li>Girod, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+specificity+of+Asian+parasitoids+for+potential+classical+biological+control+of+Drosophila+suzukii.">Host specificity of Asian parasitoids for potential classical biological control of Drosophila suzukii.</a> Journal of Pest Science. 91 (4), 1241-1250 (2018).</li><li>Seehausen, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+cryptic+parasitoid+species+reveals+its+suitability+as+a+biological+control+agent.">Evidence for a cryptic parasitoid species reveals its suitability as a biological control agent.</a> Scientific Reports. 10 (1), 19096 (2020).</li><li>Nomano, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+differentiation+of+Ganaspis+brasiliensis+(Hymenoptera:+Figitidae)+from+East+and+Southeast+Asia.">Genetic differentiation of Ganaspis brasiliensis (Hymenoptera: Figitidae) from East and Southeast Asia.</a> Applied Entomology and Zoology. 52 (3), 429-437 (2017).</li><li>Kasuya, N., Mitsui, H., Ideo, S., Watada, M., Kimura, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological,+morphological+and+molecular+studies+on+Ganaspis+individuals+(Hymenoptera:+Figitidae)+attacking+Drosophila+suzukii+(Diptera:+Drosophilidae).">Ecological, morphological and molecular studies on Ganaspis individuals (Hymenoptera: Figitidae) attacking Drosophila suzukii (Diptera: Drosophilidae).</a> Applied Entomology and Zoology. 48 (1), 87-92 (2013).</li><li>Matsuura, A., Mitsui, H., Kimura, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+on+distributions+and+oviposition+sites+of+Drosophila+suzukii+(Diptera:+Drosophilidae)+and+its+parasitoids+on+wild+cherry+tree+in+Tokyo,+central+Japan.">A preliminary study on distributions and oviposition sites of Drosophila suzukii (Diptera: Drosophilidae) and its parasitoids on wild cherry tree in Tokyo, central Japan.</a> Applied Entomology and Zoology. 53 (1), 47-53 (2018).</li><li>Abram, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+records+of+Leptopilina,+Ganaspis,+and+Asobara+species+associated+with+Drosophila+suzukii+in+North+America,+including+detections+of+L.+japonica+and+G.+brasiliensis.">New records of Leptopilina, Ganaspis, and Asobara species associated with Drosophila suzukii in North America, including detections of L. japonica and G. brasiliensis.</a> Journal of Hymenoptera Research. 78, 1-17 (2020).</li><li>Puppato, S., Grassi, A., Pedrazzoli, F., De Cristofaro, A., Ioriatti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+of+Leptopilina+japonica+in+Europe.">First report of Leptopilina japonica in Europe.</a> Insects. 11 (9), 611 (2020).</li><li>Dalton, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+survival+of+Drosophila+suzukii+under+simulated+winter+conditions+of+the+Pacific+Northwest+and+seasonal+field+trapping+in+five+primary+regions+of+small+and+stone+fruit+production+in+the+United+States.">Laboratory survival of Drosophila suzukii under simulated winter conditions of the Pacific Northwest and seasonal field trapping in five primary regions of small and stone fruit production in the United States.</a> Pest Managagement Science. 67 (11), 1368-1374 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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&#39;s native ranges in East Asia7,8,9, as well as effective but newly associated parasitoids in the fly&#39;s invaded regions in the Americas and Europe4,5,6. In the fly&#39;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&#39;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 Novkovi&#263; &#38; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Active dry yeast</td>
			<td>Fleischmanns Yeast, Cincinatti, OH, USA</td>
			<td>None</td>
			<td>Used to cover fruit to reduce mold growth and enhance the frui attraction to the flies</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Merk Life Science S.r.l., Milan, Italy</td>
			<td>A1296 - 5KG</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>Bleach solution</td>
			<td>Clorox Company, Oakland, CA, USA</td>
			<td>None</td>
			<td>Used to disinfect flesh fruit</td>
		</tr>
		<tr>
			<td>Blue stopper</td>
			<td>Azer Scientific, Morgantown, PA, USA</td>
			<td>ES3837</td>
			<td>Used for sealing the tube while allowing ventilation for insects</td>
		</tr>
		<tr>
			<td>Blueberries</td>
			<td>Grocery Store, Newark, DE, USA</td>
			<td>None</td>
			<td>Provided as host fruit for the flies (various other fruit can also be used)</td>
		</tr>
		<tr>
			<td>BugDorm insect rearing cage (W24.5 x D24.5 x H63.0 cm)</td>
			<td>Mega View Science Co. Ltd., Taichung, Taiwan</td>
			<td>4E3030</td>
			<td>Used for rearing parasitoids (parasitism cage)</td>
		</tr>
		<tr>
			<td>BugDorm insect rearing cage (W32.5 x D32.5 x H32.5 cm)</td>
			<td>Mega View Science Co. Ltd., Taichung, Taiwan</td>
			<td>4E4590</td>
			<td>Used for rearing flies</td>
		</tr>
		<tr>
			<td>BugDorm insect rearing cage (W32.5 x D32.5 x H32.5 cm)</td>
			<td>Mega View Science Co. Ltd., Taichung, Taiwan</td>
			<td>4E4545</td>
			<td>Used for rearing parasitoids (eclosion cage)</td>
		</tr>
		<tr>
			<td>Chicken wire (0.64 cm, 19 gauge)</td>
			<td>Everbilt, OH, USA</td>
			<td>308231EB</td>
			<td>Used to lift up the fruit to allow maximum parasitoid oviposition</td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>Grocery Store, Trento, TN, Italy</td>
			<td>None</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>Dental cotton roll (1 x 3.8 cm)</td>
			<td>Gima S.p.A., Gessate, MI, Italy</td>
			<td>35000</td>
			<td>Used for providing water to the parasitoids within the storage container</td>
		</tr>
		<tr>
			<td>Drosophila diet</td>
			<td>Frontier Scientific, Newark, DE, USA</td>
			<td>TF1003</td>
			<td>Custom diet used to rear flies</td>
		</tr>
		<tr>
			<td>Drosophila vial narrow, Polystirene (2.5 x 9.5 cm)</td>
			<td>VWR International, LLC., Radnor, PA, US</td>
			<td>75813-160</td>
			<td>Used for providing water to the parasitoids within the cage</td>
		</tr>
		<tr>
			<td>Drosophila vial plugs, Cellulose acetate (2.5 cm)</td>
			<td>VWR International, LLC., Radnor, PA, US</td>
			<td>89168-886</td>
			<td>Used for providing water to the parasitoids within the cage</td>
		</tr>
		<tr>
			<td>Erlenmeyer flask (250 mL)</td>
			<td>Carolina Biological, Burlington, NC, USA</td>
			<td>731029</td>
			<td>Used for rearing flies and parasitoids</td>
		</tr>
		<tr>
			<td>Falcon-style centrifuge tube (50 mL)</td>
			<td>VWR International, LLC., Radnor, PA, US</td>
			<td>VWRI525-0611</td>
			<td>Modified to ship adult parasitoids</td>
		</tr>
		<tr>
			<td>Foam stopper</td>
			<td>Jaece Industries, North Tanawanda, NY, USA</td>
			<td>L800-C</td>
			<td>Used for sealing the flasks while allowing ventilation for insects</td>
		</tr>
		<tr>
			<td>Honey</td>
			<td>Grocery Store, Newark, DE, USA</td>
			<td>None</td>
			<td>Provided as food for parasitoids</td>
		</tr>
		<tr>
			<td>Identi-Plug plastic foam stopper</td>
			<td>Fisher Scientific Company, L.L.C., Pittsburg, PA, US</td>
			<td>14-127-40E</td>
			<td>Used as feeder for parasitoids and to seal the storage container</td>
		</tr>
		<tr>
			<td>Industrial paper towel</td>
			<td>Grocery Store, Newark, DE, USA</td>
			<td>None</td>
			<td>Provided as a pupation substrate for pupae and mitigated moisture</td>
		</tr>
		<tr>
			<td>Micron mesh fabric (250 mL)</td>
			<td>Industrial Netting, Maple Grove, MN, USA</td>
			<td>WN0250-72</td>
			<td>Used to make ventilation lid for insects</td>
		</tr>
		<tr>
			<td>Nutritional yeast (flakes)</td>
			<td>Grocery Store, Trento, TN, Italy</td>
			<td>None</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>Paper coaster (10.2 cm)</td>
			<td>Hoffmaster, WI, USA</td>
			<td>35NG26</td>
			<td>Porvided as pupation substrate for flies and parsitized pupae</td>
		</tr>
		<tr>
			<td>Plastic cup (&#216; 13.3 cm, 800 mL)</td>
			<td>Berry Superfos, Taastrup, Denmark</td>
			<td>Unipak 5134</td>
			<td>Modified to store adult parasitoids</td>
		</tr>
		<tr>
			<td>Plastic lid (&#216; 13.3 cm)</td>
			<td>Berry Superfos, Taastrup, Denmark</td>
			<td>PP 2830</td>
			<td>Modified to store adult parasitoids</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Merk Life Science S.r.l., Milan, Italy</td>
			<td>P1386 - 1L</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Grocery Store, Trento, TN, Italy</td>
			<td>None</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>Soup cup with lid (475 mL)</td>
			<td>StackMan, Vietnam</td>
			<td>DC1648</td>
			<td>Used for parasitized larvae to pupate</td>
		</tr>
		<tr>
			<td>Soybean flour</td>
			<td>Grocery Store, Trento, TN, Italy</td>
			<td>None</td>
			<td>Used to prepare the Standard Drosophila Medium</td>
		</tr>
		<tr>
			<td>White felt washer (0.64 cm thick, 5 mm ID x 20 mm OD)</td>
			<td>Quiklok, Lincoln, NH, US</td>
			<td>WFW/.25 x 5 x 20 mm</td>
			<td>Used as feeding ring for parasitoids</td>
		</tr>
	</tbody>
</table>

methods for rearing the parasitoid ganaspis brasiliensis, a promising biological control agent for the invasive drosophila suzukii

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.

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 &#177; 4.9 offspring per flask) with 4...

Watch this Scientific Journal Video about Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii at JoVE.com

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

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.

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

Native to East Asia, the spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), has established widely in the Americas, Europe, ...

Long term research and use of a bio-control agent depend on the availability of effective and economical rearing techniques, and this protocol has proven to be efficient for rearing this parasitoid. There is currently enormous interest in rearing this parasitoid for research or field release, and the protocol will benefit future long-term research and use of this promising bio-control agent. First users may need to understand some basic biology of the involved insects through reading the relevant references provided in the article and some practices of the methods.For making the host diet, first, prepare the liquid portion of the diet as described in the manuscript. Next, add 25 to 30 grams of mashed blueberries to an empty 250-milliliter flask. Then, pour the liquid into the flask with the liquid just covering the top of the mashed blueberries.Next, for rearing the host Drosophila suzuki, first cut a piece of absorbent paper towel and twist it at the center. Then, place the twisted middle section of the paper towel in the diet flask. For retaining moisture, use distilled water to wet the paper towel and the diet.Then, to transfer sexually mature adult flies, carefully remove the stopper of a fly colony flask and invert it into the diet flask. After a week of this exposure, hold the flies in an environmental chamber for three weeks for fly emergence. For exposing host larva to parasitoids, first remove the adult flies and the paper towel from the flask, leaving the fly eggs and larvae.To serve as a pupation substrate for the larvae, fold a new absorbent paper towel and insert it into the flask. Then aspirate six female and male pairs of the parasitoid G3 Ganaspis brasiliensis into the flask and streak a thin layer of honey on the bottom of the foam stopper. For collecting and storing the parasitoids, remove the adults after five days.Then store the flask in the environmental chamber for 35 days under the conditions mentioned earlier. With the emergence of adult wasps, aspirate them three times per week. For holding the wasps, first place a paper towel moistened but not saturated with distilled water at the bottom of a 2.5-by-9.5 square centimeter Drosophila vial.Then, transfer about 60 wasps to the vial and mark the emergence date. Store the vial in the environmental chamber. Twice a week, streak a thin layer of honey on the bottom of the foam stopper of the vials.First, arrange large scale rearing of Drosophila suzuki by placing 1, 500 to 2, 000 sexually mature adult flies at a 50:50 sex ratio in a large, knitted, mesh-covered cage. Then, place a plate with water-soaked cotton and four to six Petri dishes with standard Drosophila medium in the cage. Twice every week, replace the exposed Petri dishes with fresh ones.Place each infested Petri dish without the lid in an 800-milliliter plastic cup and cover the cup with a fine mesh. Then, incubate the cups at 23 degrees Celsius and 75%relative humidity for 12 to 15 days. After incubation, transfer newly hatched adult Drosophila suzuki to a rearing cage.Next, begin preparing the host larvae by rinsing blueberries in cold water for one minute and soak the fruits in a basin filled with a bleach solution, diluted to 5%for three minutes. Then, fill nine-centimeter Petri dishes with the washed blueberries. In the late afternoon hours, put the Petri dishes in the rearing cage and leave them overnight.The next morning, gently tap on the Petri dishes to dislodge flies from the fruits and remove the fruits from the rearing cage. Meanwhile, prepare a parasitism cage and a wasp emergence cage for the parasitoid G1 Ganaspis brasiliensis. Both the cages should have a thin string below the ceiling for suspending one or more feeders.In each cage, suspend a Drosophila vial containing water and seal with a cellulose acetate plug upside down from the ceiling every five to seven days, depending on the relative humidity. Immediately after taking out infested blueberries from the Drosophila suzuki cage, put them in the parasitism cage with 1, 500 to 2, 000 Ganaspis brasiliensis wasps for two to three days. Next, collect the fruits in an 800-milliliter plastic cup with absorbent paper at the bottom.Incubate the open cup in the emergence or eclosion cage at 21 degrees Celsius and 65%relative humidity for at least 28 days. During the second and third weeks of incubation, check the cage weekly for early host eclosion. To facilitate successive parasitoid collection, remove the adult flies.At the end of the fourth week of incubation, provide a feeder and a water source to the cage. Collect about 10 to 15%of the adult wasps and transfer them to the parasitism cage to replace old and unproductive wasps. From the remaining wasps of the eclosion cage, store up to 700 adults at a 50:50 sex ratio with an 800-milliliter plastic cup containing a two-milliliter tube filled with water and sealed with a dental cotton roll at the bottom of the cup.Store the cup with a marked emergence state in an environmental chamber for up to one month. For shipping parasitoids, place about 200 adults in a 50 milliliter conical tube. Then, put all the tubes in an insulating shipping container along with ice packs.This protocol resulted in successful small-scale rearing of G3 Ganaspis brasiliensis, a parasitoid agent, against the agricultural pest Drosophila suzuki in the laboratory. Offspring production remained unaffected by differences in parasitoid density and exposure time. Large-scale generation of G1 Ganaspis brasiliensis could also be achieved, with the production beginning five weeks after the initial exposure to the host Drosophila suzuki.From week 8 to week 22, the parasitoid production became proportional to the amount of fruit exposed to them. It is critically important to maintain a suitable rearing temperature, control the humidity, and always provide food for adult insects. The methods could be further improved by optimizing the rearing densities of host and parasitoid to maximize the reproduction of wasps and by exploring the possibility of rearing this parasitoid on alternative hosts.

methods-for-rearing-parasitoid-ganaspis-brasiliensis-promising

Research

JoVE Journal

Biology

3.9K visualizações.  Fondazione Edmund Mach. A pesquisa de longo prazo e o uso de um agente de biocontrole dependem da disponibilidade de técnicas eficazes e econômicas de criação, e este protocolo tem se mostrado eficiente para a criação desse parasitoide. Atualmente, há um enorme interesse em criar este parasitoide para pesquisa ou liberação de campo, e o protocolo beneficiará futuras pesquisas de longo prazo e uso deste promissor agente de biocontrole. Os primeiros usuários podem precisar entender alguma biologia básica d...