Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

The Vao virus is commonly used as a DNA vector used in combination with insect cell lines for protein expression. However, the fact that this virus efficiently infects insects with lethal consequences also makes it an effective insecticidal agent. In Brazil, for example, wild type balo virus is applied to more than 1 million hectares of soybean crops annually for control of the velvet bean caterpillar to enhance their insecticidal efficacy, Balo viruses have been genetically engineered with genes encoding insect specific toxins that are active within the HemosIL of the insect.

In this video, we demonstrate methods for oral infection of lepidopteran larvae with bacao virus in order to analyze insecticidal efficacy. Hi, I am from Blind Boning laboratory in the Department of Ophthalmology and the Iowa State University, And I'm Wendy Sparks also of the boning lab. Today we'll show you two procedures for VA virus infection of lter and larvae.

First harran will show you how to infect neonate larvae using a droplet feeding bioassay. And then I will show you how to infect mid to late instar larvae using a diet blood bioassay. So let's get started.

Droplet feeding bioassays can be used for the determination of the lethal dose of abacavir and survival. Time of balo virus infected larvae. Early instar or neonate larvae are generally used for this assay.

Before starting, make sure you have diet cups with the appropriate diet on hand. Also, prepare the appropriate virus Dilutions include green food coloring dye to a 4%concentration by volume. In order to determine the lethal viral concentration, the dilution should give a range of mortality between one to 99%Preliminary bioassays may be required to determine an appropriate range of virus concentrations after vortexing the tube to prevent settling of the polyhedra, make two concentric circles of small droplets of the virus solution in a 60 millimeter Petri dish.

For each concentration of virus used, remember to also set up a negative control mock infection. Treatment next, transfer 25 to 30 neonate larvae to the middle of the concentric circles using a sterile paintbrush. Place the lid on the dish and seal with paraform to prevent the larvae from escaping.

Ideally, use different paintbrushes for the transfer to each virus type to prevent contamination between treatments. Allow the dish to stand at room temperature for 10 to 15 minutes during which time larvae drink. The solution larvae that have fed can be identified by green coloration of the anterior gut resulting from the green food coloring dye transfer only those larvae that have fed to one ounce plastic cups, which contain diet transfer one larvae per cup.

After transferring the larvae, cover the cups with lids and incubate a 28 degrees Celsius 24 hours after infection. Eliminate any cups containing dead larvae. These early deaths likely result from handling injury.

When using this bioassay to determine lethal dose, the larvae should be checked every two to three days until the mock infected larvae and any surviving virus treated larvae have purated or have begun to borrow in order to pupate virus infected larvae will remain on the top of the food and then die after a few days. Dead larvae will sometimes mein and turn black larvae that die from infection essentially become bags of polyhedra, which will completely dissolve. If mechanically disturbed at this time, score the larval mortality in each treatment.

Lethal concentration bioassays should be replicated three or four times on separate occasions. Use 30 individuals per dose for each virus or control treatment. Use an appropriate probate analysis program such as polo or SAS to calculate lc 50 values, 95%confidence intervals and the appropriate statistics.

This droplet feeding assay can also be used to determine ST 50 survival time values. In this case, larvae mortality is scored every four to eight hours with more frequent observations. If the rate of mortality is high survival time bioassays are performed using an lc 99 dose of virus median survival times and the 95%confidence limits are calculated using the Kaplan Mayer estimator using an s plus or SAS program.

Now that Harang has shown you the droplet feeding bioassay, I'll show you the diet plug bioassay In diet plug bioassay. Mid to late Instar larvae are fed on a diet plug treated with a known dose of virus to determine the lethal dose or LD 50 on the day prior to conducting this assay. Remove the diet from the cup to starve larvae overnight.

This step will increase the speed with which the larvae consume the virus. Inoculated diet cube the next day, prepare the diet by cutting it into small cubes about two cubic millimeters. If the diet cubes are too small, they will dry out.

If the diet cubes are too large, the larvae will not be able to consume the entire diet cube overnight. Now add one to two microliters of polyhedra suspension to each diet cube. The suspension contains 4%by volume food coloring dye.

Let the suspension soak into the diet for five to 15 minutes. Remember to also prepare a mock virus treated diet cube as a negative control. Next, transfer one diet cube to each fourth instar larvae in each cup.

Then place cups in the incubator overnight at 28 degrees Celsius the next day, discard any larvae that have not completely eaten the diet cube. Then add a large piece of diet to each cup for the remaining larvae. Maintain larvae at 28 degrees Celsius, adding additional diet as necessary until the mock infected larvae and the surviving virus infected larvae have ated or begun to pupate.

At this time, record the numbers of dead and surviving insects. We'll just show you two methods for infecting insects with back virus, bleeding, feeding biopsy, and the diet plaque biopsy. When doing this procedure, it's important to remember that the cadavers vao virus killed larvae are typically fragile and can easily rupture releasing virus.

So sterilize work surfaces and dispose of contaminated gloves and disposable materials in an appropriate manner to prevent contamination. So that's it. Thanks for watching and good luck with your experiments.