Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

While the arthropod specific baccala virus is used as a standard research tool for eukaryotic protein expression systems, it's also being developed as an environmentally safe insecticidal agent, which can kill herbivores insects such as lepidoptera in nature. Lepidopteran larvae are infected by baccala viruses when eating infected foliage. The larvae or caterpillar ingest the baccala virus in a form known as a polyhedron, which is a complex of virus particles packaged in a large crystalline protein structure that protects the virus from desiccation.

Once the larva is infected, butted virus disseminates the infection within the insect. The virus hijacks the nucleic acid and protein synthesis machinery of the host to make more virus and polyhedra. Eventually the larvae becomes so thoroughly infected with virus that they dissolve into a messy ooze of polyhedra that drips onto plant surfaces eaten by the insect, and then the cycle begins anew in order to determine the insecticidal efficacy of a particular Baca.

Avir strain micro applicator techniques and bioassays can be combined. Leid dorin larvae can be infected by injection of butted virus directly into the HemosIL or body cavity and mortality rates can be assessed. In addition, larvae can be infected with polyhedra using oral micro applicator assisted injection and the timing of infection of the gut and other tissues can be assayed.

Hi, I am Wendy Sparks in the laboratory of Brian e boning in the Department of Entomology at Iowa State University And I'm also from boning snap. Today we will show you two procedures for infecting lab duct DI with the backlog virus. Both methods use micro applicator.

Firstly, I will show you use a micro applicator to deliver body virus into the he into the heni through prole. Then when show you use the micro applicator to deliver poly header into the mid guard through mouse. So let's get started.

When developed for insecticidal purposes, balo viruses usually contain the polyhedron gene to facilitate oral infection of pest larvae. However, on occasion, there are reasons to test a polyhedron negative baccala virus for potential insecticidal effects or to bypass the gut of an insect. In this case, injection of butted virus using a micro applicator can be performed to infect larvae before starting the assay.

The titer of the working stock of butted virus should be determined using plaque assay or endpoint dilution assays. For physiological experiments, injections of five times 10 to the fourth PFU per fifth instar H VNS would be typical. To begin this bioassay load each butted virus dilution into a plastic one cc to hercule syringe with a 28.5 or 32 gauge sharp needle tip.

Usually one to three microliters of butted virus solution. With a titer of 10 to the seventh, minus 10 to the eighth per milliliter can be used for each larvae such that loading 300 to 500 microliters into the syringe is sufficient for most purposes. Next, drive air bubbles out of the syringe by holding the syringe upright to bring air to the needle tip and dispense.

Then fix the syringe onto the syringe support tube. Now set the volume to 1.0 microliter. For each droplet at the microprocessor control unit of the micro applicator, calibrate the control unit first to ensure that the correct volume is dispensed, the speed with which the droplet is dispensed can be set on a scale of zero to nine.

A range of zero to one is commonly used. Press the foot switch to ensure that the solution is dispensed appropriately. Remember that if too much solution is dispensed or if application is too slow, the volume and speed settings can be adjusted until the right volume of solution is applied at the correct rate.

I'm now ready to begin the injection for H VNS infection. Early fifth, instar larvae are typically injected the larger size of the later instars facilitates the injection process. Insert the needle tip through the plana of one of the pro legs and into the body cavity.

Note, when the needle tip inserts into the prole, change the larval body position To avoid damaging the gut and body wall, now press the foot switch four times to deliver four microliters of the virus solution into the hemo seal. After the injections are complete, return all the larvae to the 28 degrees Celsius incubator. Check frequently for dead larvae and to ensure that adequate diet is available for surviving larvae.

Even though late instars do not die from injury to the cuticle resulting from injection, the larvae should still be checked 24 hours after injection in case of damage to the gut, which could result from bacterial infection. This early mortality should be excluded from data analysis. If using the micro applicator assisted bioassay with butted virus for lethal concentration bioassays, the bioassay 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 bioassay can also be used to determine ST 50 survival time values following inoculation of a fixed dose of virus.

In this case, larvae mortality is scored every four to eight hours with more frequent observations. If the rate of mortality is high median survival times in the 95%confidence limits are calculated using the Kaplan Meyer estimator using an s plus or SAS program. Now that Harran has shown you the micro applicator assisted bioassay using butted virus.

I'll show you the assay. Using polyhedra Oral inoculation of polyhedra directly into the insect gut is used when the exact timing of infection is critical. For example, when monitoring how the virus disseminates within the insect to begin this assay suspend polyhedra in a neutrally buoyant three to two solution of glycerin and water from 10 to 300.

Polyhedra could be used for fourth instar h ance, for example. Then load this solution into a plastic or glass one CC tuberculin syringe with a 32 gauge blunt tip needle. Set the syringe on the support tube of the micro applicator looking down a microscope.

Hold the fourth or fifth in star larva, insert the needle through the mouth and into the anterior region of the midgut, then deliver the polyhedra. This technique requires some practice. First, it can be tricky to hold the larvae with gloves without squashing it.

Second, it takes some practice to navigate the needle past the mouth parts. We recommend that you practice inoculating about 30 larvae with PBS. If you do happen to puncture the gut, it will be obvious because the larvae melany and turned black quite quickly.

Again, because micro applicator assisted oral inoculation provides the exact time of infection, this method is most useful for experiments in which the infection time is important to determine how the virus spreads within the insect. For example, viruses expressing beta galactoses dase can be orally inoculated into larvae, which are then dissected at various time points to determine which tissues become infected when We'll just show you two micro applicator assisted vessels for infection. Insect love with vir, The first of which was direct inoculation via the pro Legg of butted virus, and this allows you to bypass the gut physiology and assess lethality and lethal time of the virus that you're interested in.

The second technique was direct inoculation into the midgut using polyhedra. This allows you to deliver a very precise dose directly into the midgut, as well as control for very precise timing of the event. Then you can study the mortality and other phenotypes of the virus as well.

When doing this procedure, it's important to remember that the cadavers baula 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 your watching. Good luck with your experiment.