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Page 7 5. Once they emerge as adults, allow the males and females at least 24–48 hours together before sub-culturing. This will ensure that the males and females have had a chance to copulate. 6. In order to subculture, obtain a clean, empty culture tube. 7. Obtain the stock culture of Nasonia and tap the tube on the table so the Nasonia fall to the bottom. Remove the cotton plug from the stock and invert the empty culture tube over it. Nasonia will naturally crawl up from the stock culture tube into the empty culture tube. Make sure that the lips of the tubes are lined up so that the Nasonia do not escape. 8. Allow some Nasonia to enter the empty culture tube. Make sure that females are present because they will be the Nasonia that parasitize the hosts (see Section III, Part C: Virgin Isolation and Sex Determination). Note: Alternatively, you can temporarily immobilize them by placing the culture tube in the refrigerator for fifteen to twenty minutes. This will slow the Nasonia down enough so that you can transfer them by hand to new culture tubes, etc. 9. Quickly replace the cotton plugs in both tubes. 10. Tap the new culture tube on the table so the Nasonia fall to the bottom. Remove the cotton plug and add several hosts. Replace the cotton plug. 11. Label the new culture tube appropriately with the Nasonia strain and the date. 12. Allow two or three days for the females to lay their eggs. Depending on conditions, the larvae will pupate after 9–10 days. After this time, a parasitized host can be cracked open, and the Nasonia offspring can be isolated before reaching adulthood (see Section III, Part C: Virgin Isolation and Sex Determination). 13. Within 14–15 days after the pupa is parasitized (if incubated at room temperature with 24 hours of light), adult offspring will emerge. Important! If you are working with various strains of Nasonia, make sure that you only work with one strain at a time. Thoroughly clean your work station of one strain before working with other strains. 14. Dispose of Nasonia as needed (see Section IV, Part D: Disposal). C. Nutrition 1. Adult Nasonia can live without food for three or four days, so plan accordingly. If you need to feed the emerged Nasonia, you can add a drop of 4% sugar solution (4 g sucrose/100 mL of water) or honey to the culture tube. For best results, use the Nasonia as soon as they reach adulthood so feeding is not an issue.