Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Microbial Insecticides
Baculoviruses as Biocontrol Agents

MECHANISM OF ACTION

Baculoviruses are rod-shaped Viruses with a double-stranded DNA genome that infect A wide variety of invertebrates. Different subgroups of this family are pathogenic to insect orders such as Lepidoptera, Hymenoptera, Diptera, Neuroptera, Trichoptera, Coleoptera, and Homoptera. Some of them play an important role in controlling the populations of certain insect pests under natural conditions. Baculoviruses were used in North America starting in the 1930s, prior to the advent of chemical pesticides in the 1960s, to control forest pests, including the European pine sawfly (Neodiprion sertifer), and they continue to be used, albeit on a smaller scale, to control gypsy moth (Lymantria dispar) populations (Table 15.3). The baculovirus virion has a cylindrical nucleocapsid enclosing its DNA. Once inside The Nucleus of an infected Cell, baculovirus particles aggregate to form a compact Structure enclosed within a crystalline protein matrix. This matrix consists primarily of the protein polyhedrin. Upon the death of infected insects, millions of polyhedral particles are released into the environment. When ingested by other insects, they are exposed to an alkaline environment in the gut, which dissolves the matrix and releases infectious Viral Particles. These particles penetrate the insect's midgut Cells, pass through the Cytoplasm into the nucleus, where, after nucleocapsid uncoating, viral Replication and the assembly of new viral particles occur. Some of these particles enter the insect's hemolymph by budding from The Plasma Membrane of infected cells, and subsequently spread to other Organs. Typically, the insect dies after about 10 rounds of viral replication, i.e., approximately 5 to 6 days post-infection, with polyhedrin accounting for up to 25% of the insect's dry weight.

Class="center">Table 15.3. Some insect pests controlled using baculoviruses

One of the advantages of baculoviruses as biocontrol agents is their host Specificity. On one hand, this means that a given baculovirus can be used to control only specific insect pests. On the other hand, because baculoviruses have coevolved with their insect hosts over thousands of years, they have adapted to overcome host defense mechanisms. Consequently, resistance to these viruses develops extremely rarely—much less frequently than to B. thuringiensis. Moreover, insects resistant to baculoviruses rapidly lose this resistance once they are no longer exposed to the viruses.

Farmers and growers prefer to use a single insecticidal agent that targets a variety of insect pests rather than multiple different insecticides. This means that for baculoviruses to be more widely adopted, their host range must be expanded.

It has been shown that when an insect cell is simultaneously coinfected with two baculovirus strains, their replication sometimes yields new strains with slightly different properties than the parental viruses. This is caused by Homologous Recombination between the DNA of the different viruses. A detailed study of this phenomenon revealed that recombination occurs within a DNA region of only 79 NUCLEOTIDES located in the p143 helicase Gene. This region likely determines the host range of different baculoviruses. Given these findings, it is reasonable to assume that substituting certain nucleotides within this region will allow the engineering of baculoviruses with broader specificity.

Enhancing Biocontrol through Introduction/32.html">Genetic Engineering

Baculoviruses do not act instantaneously. Depending on the conditions, an infected insect may take anywhere from several days to several weeks to die. To accelerate this process, attempts have been made to increase baculovirus virulence by introducing foreign genes whose expression weakens or kills the infected insect (Table 15.4). One experiment proposed using a gene whose expression in the host insect cells disrupts its normal life cycle. It is known that a drop in juvenile hormone levels in larvae initiates pupation and causes them to stop feeding. This decrease occurs due to an increase in the level of a specific esterase that catalyzes The conversion of the biologically active methyl ester form of juvenile hormone into its inactive acid form. Inhibiting esterase activity leads to the in vivo accumulation of active juvenile hormone, causing larvae to remain in the active feeding stage longer and consequently reach giant sizes. It was reasonable to assume that artificially increasing juvenile hormone esterase levels would lower endogenous active juvenile hormone levels, leading to premature cessation of feeding. Furthermore, it was known that reducing the active feeding time of larvae minimizes crop damage.

Table 15.4. Effects of certain genes introduced into baculoviruses to enhance their insecticidal activity¹⁾

¹⁾ From Maeda, Curr. Opin. Biotechnol. 6: 313—319, 1995.

To test this hypothesis, experiments on cloning and expressing the juvenile hormone esterase gene had to be conducted first. The enzyme was isolated from the insect Heliothis virescens (tobacco budworm) and purified. Its Amino Acid Sequence was determined, and an oligonucleotide corresponding to one of the protein segments was synthesized and used as a Hybridization probe. The coding sequence for juvenile hormone esterase was isolated from an H. virescens cDNA library and inserted into the baculovirus genome under viral transcriptional control. After treating first-instar larvae of Trichoplusia ni (cabbage looper) with this recombinant baculovirus, the insect's juvenile hormone levels decreased, and larval growth was significantly retarded compared to control larvae treated with wild-type baculovirus.

Unfortunately, the applicability of this approach to enhance the insecticidal efficacy of baculoviruses is limited because the reduction in larval feeding due to elevated juvenile hormone esterase levels occurs only During the first larval instar. Larvae at other developmental stages are much less sensitive to baculovirus Treatment. Thus, baculoviruses engineered to express the juvenile hormone esterase gene are effective only when the majority of the pest population is in the first instar, which is nearly impossible to achieve under natural conditions.

Another approach to enhancing the insecticidal efficacy of baculoviruses is based on incorporating a gene encoding a potent insect-specific toxin into the viral genome, to be expressed during the viral infection cycle. A gene for an insect-specific neurotoxin from the North African scorpion, Androctonus australis Hector, was introduced into one of the baculovirus strains. This neurotoxin, which has no effect on mice, blocks sodium channel transport in the Neurons of target insects, leading to paralysis and death. Insects infected with the baculovirus carrying the scorpion neurotoxin gene caused 50% less damage to the leaves of control plants compared to insects infected with the wild-type baculovirus.

Cloning and expression of the cDNA of a toxin from the Israeli yellow scorpion, Leiurus quinquestriatus Hebraeus, in the Autographa californica baculovirus reduced the time required to kill 50% of the control insect larvae from 120 to 78 hours. Furthermore, 120 hours post-infection, insect larvae treated with the recombinant virus gained threefold less weight compared to larvae treated with the wild-type virus. Thus, the recombinant baculovirus not only accelerated the death of infected larvae but also significantly reduced the ability of the insects to damage plants.

Recently, a recombinant A. californica baculovirus producing the insect-specific neurotoxin of Androctonus australis was tested under field conditions. While previous laboratory experiments showed a 25–50% reduction in the time required to kill T. ni insects, the recombinant baculovirus demonstrated an even greater effect in controlled field trials (Fig. 15.7): it killed the insects faster, reduced damage to cabbage plants, and decreased the pest population in the subsequent reproductive cycle. Regardless of the efficacy demonstrated by a specific recombinant baculovirus in laboratory experiments, the primary obstacle to its large-scale application is the high cost of its formulations and the difficulty of dissemination. Baculoviruses are obligate parasites; they can replicate only within a living host or in insect cell culture. The cost of baculovirus formulations, whether recombinant or wild-type, is much higher than that of chemical insecticides. Nevertheless, biological insecticides may find wider application when considering the adverse environmental impacts of chemical insecticides—that is, when weighing their cost against the benefits they provide.

Fig. 15.7. Survival of T. ni larvae after treating cabbage leaves with wild-type baculovirus or a recombinant baculovirus expressing a scorpion neurotoxin gene. Only insect larvae were applied to control plants. The lower the survival rate, the more larvae died, indicating higher treatment efficacy. Plants were treated with baculoviruses only once at the beginning of the experiment.