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 dry diet and stir the diet mixture thoroughly.</li>
		<li>Add the mixture to the boiling water.</li>
		<li>Allow the liquid diet on the hot plate to boil for 10 min while periodically stirring the mixture to prevent it from burning.</li>
		<li>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.</li>
		<li>Measure 6.7 mL of 95% EtOH in one container and 3.5 mL of 1 M propionic acid solution in another container.</li>
		<li>Once the diet has cooled, add the EtOH and then the propionic acid solution, stirring thoroughly after each addition.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Pour the prepared diet into each flask so that it just covers the top of the mashed blueberries.</li>
		<li>Add foam stoppers to the necks of the flasks and allow the diet to solidify at room temperature (Figure 1).</li>
		<li>Once the diet has solidified, use it immediately or store at 5 &#176;C for up to 3 weeks.</li>
	</ol>
	</li>
	<li>Rear host Drosophila suzukii.
	<ol>
		<li>Remove the stored diet from the refrigerator and allow it to equilibrate to the ambient room temperature, or use freshly made diet.</li>
		<li>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).</li>
		<li>Wet the paper towel and surface of the diet with distilled water to retain moisture.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Hold the new flasks after a week of exposure to adult flies at suitable conditions (21 &#176;C, 16 L: 8 D photoperiod, 60%-80% relative humidity [RH%]) in an environmental chamber for 3 weeks for fly emergence.</li>
	</ol>
	</li>
	<li>Expose host larvae to parasitoids.
	<ol>
		<li>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.</li>
		<li>Fold a piece of absorbent paper towel in half and put it in the flask as a pupation substrate for parasitized larvae.</li>
		<li>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.</li>
		<li>Leave the parasitoids in the flask for 5 days.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Collect and store adult parasitoids.
	<ol>
		<li>During the second and third weeks of incubation, check the flasks weekly for early host emergence and remove the adult flies.</li>
		<li>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).</li>
		<li>Place a small piece of paper towel moistened, but not saturated, with distilled water at the bottom of the vial.</li>
		<li>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.</li>
		<li>Remoisten the paper in the vial once every 4-7 days or replace the paper towel if there are signs of mold.</li>
	</ol>
	</li>
</ol>
2. Methods for large-scale rearing of G1 Ganaspis brasiliensis
<ol>
	<li>Implement a large-scale rearing of host Drosophila suzukii.
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>Let the mixture cool down for 5 min and add 5 g of ascorbic acid.</li>
		<li>Pour the freshly cooked SDM into 9 cm Petri dishes and allow the medium to solidify at room temperature before closing the plates.</li>
		<li>Stack up the SDM Petri dishes, wrap the stack with aluminum foil, and store the dishes at 4 &#176;C for up to 2 weeks.</li>
		<li>Within each rearing cage, place a plate with water-soaked cotton and four to six Petri dishes with SDM (Figure 2).</li>
		<li>Twice per week, replace the infested SDM Petri dishes with fresh ones.</li>
		<li>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 (&#60;0.5 mm), and incubate for 12-15 days at 23 &#176;C and 75% RH (Figure 2).</li>
		<li>Transfer the newly hatched D. suzukii adults from the plastic cups to the rearing cages.</li>
	</ol>
	</li>
	<li>Prepare host larvae.
	<ol>
		<li>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.</li>
		<li>Drain the bleach solution and fill the basin with cold water to rinse the blueberries. Mix gently by hand for at least 30 s.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Implement a large-scale parasitoid rearing.
	<ol>
		<li>Use two types of cages to rear the parasitoid: one for parasitism and another for wasp emergence.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Expose the host larvae to the parasitoids.
	<ol>
		<li>Expose the host-infested fruit within the Petri dishes to G1 G. brasiliensis immediately after D. suzukii overnight infestation (see step 2.2.7).</li>
		<li>Leave the 10-15 Petri dishes of infested fruit in the parasitization cage containing 1,500-2,000 wasps for 2-3 days.</li>
		<li>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).</li>
		<li>Place the open cups in the eclosion cage and incubate for at least 28 days at 21 &#176;C and 65% RH (Figure 2).</li>
		<li>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.</li>
		<li>At the end of the fourth week of incubation, add a feeder and a water source to the cage.</li>
	</ol>
	</li>
	<li>Collect and store the adult parasitoids.
	<ol>
		<li>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.</li>
		<li>Collect and store the remaining parasitoids in plastic cups (13.3 cm diameter or 800 mL) (Figure 3A).</li>
		<li>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).</li>
		<li>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).</li>
		<li>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 &#176;C; 65% RH) until used, or for up to 1 month (Figure 3B).</li>
	</ol>
	</li>
	<li>Ship the adult parasitoids.
	<ol>
		<li>Use conical tubes (50 mL) to ship the adult parasitoids.</li>
		<li>Pierce a ventilation hole (8 mm diameter) on the cap and cover it with a fine mesh net (Figure 3C).</li>
		<li>Add a cellulose acetate feeding ring on the inside of the cap (Figure 3C).</li>
		<li>Prepare a saturated sucrose solution using distilled water, apply a few drops on the feeding ring, and let it absorb the liquid.</li>
		<li>Place a fan-shaped piece of absorbent paper towel within the tube (Figure 3D).</li>
		<li>Add ~200 adult parasitoids to each tube, and place the tubes in an insulated shipping container together with ice packs.</li>
	</ol>
	</li>
</ol>

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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, ...

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4.0K Views.  Fondazione Edmund Mach. 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.

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

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...