Nonoject 2

Egg solutions were prepared in 1.5 ml microcentrifuge tubes as described above. 15 μl of eggs were transferred into 50 mm diameter plastic plates with cornmeal food. Plates were incubated for 72 hr at 25°C. Borosilicate glass 3.5’ capillaries (Drummond Scientific Co. 3-000-203-G/X) were pulled to form thin needles in a needle puller (Narishige PC-10). The needle was backfilled with paraffin oil (Sigma-Aldrich #M5904) with a syringe and attached to a nanoinjector (Drummond Scientific Co. Nonoject II). Late 2nd instar and early 3rd instar larvae were carefully removed with forceps from cornmeal food plates and placed on filter paper, in groups of 20. Larvae were carefully injected with 4.6 nl of oil. After injection, _ddH₂0 was added with a brush to remove the larvae and a total of 40 larvae were transferred into a cornmeal food vial and incubated at 25°C. After 48 hr larvae were removed with a 15% w/v sugar solution and transferred into _ddH₂O droplets on top of a plastic plate. Larvae were observed under a dissecting microscope and scored for the presence of melanization in the oil droplet.

Wasp extracts were prepared by homogenizing 20 L.boulardi G486 males in 200μl of paraffin oil (Sigma-Aldrich M5904) with a pestle in a 1.5ml microcentrifuge tube. Extracts were centrifuged for 2m 30s at 500g. The supernatant was transferred into a new 1.5ml microcentrifuge tube and the centrifugation step was repeated. The resulting supernatant was used to backfill a glass needle prepared from borosilicate glass 3.5” capillaries (Drummond Scientific Co. 3-000-203-G/X) pulled in a needle puller (Narishige PC-10). The filled needle was attached to a nanoinjector (Drummond Scientific Co. Nonoject II). Second instar larvae on cornmeal food plates were obtained following the same protocol as for encapsulation assay (see above). To obtain parasitized larvae, 4 female wasps were added to food plates containing larvae for 3 hours before injection. Larvae from non-parasitized and parasitized plates were carefully moved onto filter paper in groups of 10 and injected with 4.6nl of oil containing wasp extract. After injection, ddH₂O was added to the filter paper and larvae were moved with forceps into cornmeal food vials. Vials were incubated for 48h at 25°C. 3^rd instar larvae were removed from food with 15% w/v sugar solution and dissected in PBS droplets to score for the presence of melanized oil droplets and to check that larvae exposed to the parasitoid contained a wasp egg or larva.