A Brief Overview of Biotechnology at DuPont Ag
and a Case Study with Recombinant Baculoviruses

Bill McCutchen, Lindsey Flexner & Gary Hollingshaus

DuPont Agricultural Products
Stine-Haskell Research Center
P.O. Box 30, Elkton Road
Newark, DE 19714

(prepared from the videotaped presentation*)

Note: The slides for this presentation will be added about the end of November.

It is important to find ways to increase food production because the world increases by 80-90 million each year and there will by 1 billion more people to feed by 2005. Therefore, food production must be doubled in the next 50 years. An important aspect of increasing food production is effective insect control. At DuPont, in addition to looking for new and novel approaches to controlling insect pests, we are doing research on the effects of baculoviruses on predators and parasitoids of insect pests.

We are currently losing from the marketplace the use of some compounds for these reasons:

• resistance,
• cost of keeping chemicals in the field,
• health effects, and
• environmental effects.

• increasing cost of discovery,
• increasing cost of production,
• more stringent registration requirements,
• liability costs, and
• resistance - an ever-present problem.

The forces that have shaped research in the past have primarily been economics, market needs, and competition in the marketplace. Increasingly, environmental factors, health and safety, and the public's perception of products is driving research. To facilitate continued advances, discovery costs of new products must be reduced. We can do this by developing cheaper, more effective synthesis procedures and more selective chemistry and by finding a new paradigm for discovery.

The Mesh Between Chemistry and Genetics

The understanding we have gained in genetics over the past decade has led to a multitude of new products and potential products. This new knowledge has led to the field of molecular biology and has led to the introduction of herbicides, disease and insect resistant crops, microbial control agents, and to a host of other new products. At DuPont we are exploring 3 major areas for biotechnology use:

• crop protection,
• the improvement of food, feed, and fiber quality, and
• as a means of diagnosing crop diseases.

The Microbial Component of Insecticide Discovery
Although DuPont is also involved in work on Bt, I will focus on baculoviruses and especially on the use and development of genetically modified baculoviruses.

Slide: Baculovirus

Of insect pathogenic viruses, both DNA and RNA in origin, only the baculovirus has no close relatives in either vertebrates or plants. This makes it a good candidate for genetic manipulation. Baculoviruses are double stranded DNA viruses with genomes from 80-200 kilobases. They are now divided into two subgroups:

• Subgroup A - nuclear polyhedrosis viruses (NPV's). These have several virions per polyhedra. The polyhedra is a proteinaceous matrix that encases the infectious units of virions.
• Subgroup B - granulosis viruses (GV's). GV's have one virion per granule.

The advantages of using baculoviruses for pest control are:

• they are non-pathogenic to vertebrates and plants;
• they are well-studied systems from both pest management and molecular biology standpoints. In fact, two new manuscripts describe in detail the entire sequence for NPVs;
• they leave no undesirable residues; and
• they have application flexibility and can not only be applied as are classical insecticides, but can be combined with chemicals in the field.

There are several limitations to baculovirus use which prevents baculoviruses from being used on a wide-scale basis in row-crop agriculture today, and at this time, neither NPV's nor GV's can compete with classical insecticides. These limitation are:

• Baculoviruses have a limited host-range (this may actually not be a limitation, depending on the intended use). Although they don't effect beneficials, they aren't broad spectrum and won't remove all potential pests.
• They are somewhat unstable in the field.
• There are regulatory problems, especially with the genetically modified viruses. (We are currently working with the agency to obtain permission to release our formulation in the field.)
• There are formulation problems.
• Baculoviruses have a slow rate-of-kill.

The genetic approaches to improving the efficacy of NPV's include:

• gene deletion of baculovirus from its genome,
• the introduction of neurohormones into the NPV,
• the introduction of enzymes into the NPV, and
• the introduction of insect-selected neurotoxins into the NPV.

Slide: Prototype toxin

The above slide is of an insect-selective toxin, AaIT, isolated from the scorpion Androctonus australis.  We inserted the gene encoding for this toxin at the University of California, Davis. There are many different classes of scorpion toxin, some of which are highly specific to the insect nervous system. Many toxins have 60-80 amino acids that are cross-linked by 4 disulfide bonds. Research being done in Israel by Zlotkin and others uses blowfly larvae to characterize the symptomology of different insect toxins.

Slide: Photograph of blowfly larva injected with toxin from scorpion The blowfly larva on the right in this photograph was injected with a toxin which produces contraction paralysis.

Slide: Diagram of virus production

This diagram presents a summary of how viruses are produced: the transfer vector on the upper right which has DNA homologous to the baculovirus genome is used. A toxin gene or other gene is inserted into this transfer vector under the control of the baculovirus promoter. Then the transfer vector is cotransfected in insect cells with the parental nuclear polyhedrosis DNA. Through homologous recombination we have the introduction of the toxin gene into the genomic viral DNA.

Slide: Polyhedrin occlusion bodies

Using a procedure called the plaque assay we can select these recombinant viruses. The round nodules in this slide represent polyhedrin occlusion bodies and we can isolate these plaques and propagate them in cell for further characterization.

Slide: Heliothis virescens  larvae paralyzed by (right) recombinant baculovirus and (left) wild virus

Host-testing is the bottom line. The second instar Heliothis virescens  larva on the right was infected with a recombinant baculovirus expressing a toxin that produces contraction paralysis. The larva on the left is at the same stage of development, but was infected with a wild virus.

The expression of the toxin produces symptoms similar to some of our classical insecticides.

Slide: Time response of second instar.

This slide shows time response of the second instar. The bottom line is have we reduced the time-to-kill, and does this corelate to reduction in crop damage? The answer is yes. Research I did at Davis shows a 30% reduction in time-to-kill, and there is a dramatic reduction in plant damage.

Slide: Cotton leaves sprayed with wild virus and with recombinant virus

In work done at UC Davis with Hammock and Maeda the cotton leaf on the right was sprayed with a wild-type virus and the Heliothis virescens  was allowed to feed. The leaf on the left was sprayed with a recominbant virus expressing AaIt, and the damage by H. virescens  is greatly reduced.

We have made dramatic improvements in the efficacy of these modified baculoviruses at DuPont over that earlier work.

Two Slides: Two soybean plants sprayed with 1) wild virus and 2) recombinant virus These soybean plants were sprayed with a wild virus and Heliothis virescens  was put on the plants. With new constructs we have substantially reduced the time-to-kill compared to the prototype AaIT virus and obtained a great increase in plant protection.

Risk to Non-Target Insects

• genetic exchange and the stability of the foreign gene inserted into the DNA;
• physical stability of the recombinant virus (i.e., is the virus itself changed by the insertion of a foreign gene); and
• damage to non-target organisms, specifically those that feed upon or develop within the target insect.

At UC Davis in collaboration with Kevin Heinz we did studies on predators and parasitoids of Heliothis virescens  exposed to recombinant viruses expressing AaIT. We looked at the green lacewing larva and the insidious flower bug in regard to this last risk. The lacewing larvae, Chrysopa carnea,  were continually fed on Heliothis virescens  larvae that were displaying advanced stages of toxin symptoms.

Slide: Development and survival rates of lacewings

Surprisingly, the development and survival rates of lacewings subjected to the three treatments showed no significant difference from those reared on uninfected H. virescens .

The endoparasitoid survival rate, using the wasp Microplitis croceipes,  also showed no difference. M. croceipes  deposits a single egg into H.virescens  and the resulting larva emerges, pupates and becomes an adult in 7-9 days. The development rate, however, differed in this way: the larvae emerged from the host significantly earlier, and the recombinant treatment produced adults with significantly smaller head-width which corresponded to the size of the parasitoid. So there is a cost associated with this treatment.

Graph: The red represents M. croceipes reared on M. virescens infected with a recombinant virus expressing AaIT, the green represents parasitoids reared on hosts infected with a recombinant virus expressing a juvenile hormone esterase, yellow represents parasitoids reared on hosts infected with a wild virus, and blue represents parasitoids reared on uninfected hosts.

Summary

The baculovirus with introduced toxin

• significantly reduced the time-to-kill host insects;
• produced no detrimental effects in predators and honeybees (as shown by studies undertaken at Davis and DuPont, in which even the virion, the infectious particle, was injected directly into the honeybee);
• affected parasitoids thusly:
  1. survival rates were not significantly different;
  2. larval emergence times were significantly shorter, causing
  3. wasp size to be significantly smaller, and
  4. mechanical transmission was documented of the NPV by adult parasitoids which had developed in infected hosts.

At DuPont, much of our research focus concerns environmental safety and public perception. We continue to develop recombinant baculoviruses and are working with the Environmental Protection Agency to have them approved. The toxicity studies on the recombinant baculovirus expressing AaIT toxin have been done and we hope to be able to field test the virus this summer.

In conclusion:

There are significant opportunities for safer, more effective products, and much of the research effort focuses on environmental conservation.

To succeed, research and development efforts must become more knowledge-based.

No single approach will guarantee success.

Development and integration of key core technologies can enhance the discovery process. Example: genetic knowledge can produce the tools necessary to develop new products.

Biotechnology-based research methods can lead to attractive alternative strategies for pest control.

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