Immediate Infection

Larvae may be infected immediately upon arrival with customer's baculovirus-infected cell culture either by syringe injection or by dosing the food surface for oral ingestion. Larvae to be injected should be injected with 5 microliters of baculovirus-infected insect cell culture containing 500 - 1000 plaque-forming units (pfu). After incubation at 28°C to 32°C infected insects can usually be harvested within 48 to 54 hours. However, the first time a gene is expressed in larvae we recommend harvest of some larvae a few hours on either side of this time frame, especially on the later side, to establish a time-line for ideal expression.

Oral Infection

Larvae may also be infected orally (per os) by a very simple procedure. First remove the small cups containing larvae from within the food-containing cups and place the insects in a cool place (4°C.). Spread 350 microliters of high titer insect cell culture stock (greater than 1 X 10⁷ pfu per ml) evenly over the food surface of each cup. Allow the liquid to soak in (about 1 hour) before transferring the larvae to the food cups. Infections per os require about 20 to 26 hours longer incubation time for protein expression than do larvae that have been injected. Again, multiple collections should be made initially to determine optimum expression time-lines.

Full-sized infected 5th instar cabbage looper larvae usually weigh 300-450 mg each at harvest and can express up to 0.4% of their body weight as recombinant protein (estimates of beta-galactosidase expressed per larvae in Entopath Inc. experiments). However, not all proteins are expressed at these levels. But recombinant expression levels in insect cell cultures (High 5 or other T. ni cell lines) often predict expression levels in larvae. Unless the expressed protein adversely effects larval growth or development, we expect that about 200 larvae should equal the expression yield from one liter of insect cells in culture.