Baculoviruses: Members of Integrated Pest Management Strategies

Integrated Pest Management (IPM) can be defined as “an ecologically based pest control strategy that relies heavily on natural mortality factors and seeks out control tactics that disrupt these factors as little as possible” (Flint & Bosch, 1981). IPM began to be applied because the extensive use of chemical insecticides show different types of environmental damages, as development of resistant insects, the appearance of new pests, injury to bird and mammals populations and human health damage due to the release of toxic waste on environment and food contamination. The aims of IPM are to protect crops, with minimum cost and risk for humans, animals and ecosystems. The development and application of IPM requires the knowledge of how the ecosystem influences on pest insects and its natural control agents, and how to modify this environment to control particularly pest insects and avoid the related chemical agent's problems.


Introduction
Integrated Pest Management (IPM) can be defined as "an ecologically based pest control strategy that relies heavily on natural mortality factors and seeks out control tactics that disrupt these factors as little as possible" (Flint & Bosch, 1981).IPM began to be applied because the extensive use of chemical insecticides show different types of environmental damages, as development of resistant insects, the appearance of new pests, injury to bird and mammals populations and human health damage due to the release of toxic waste on environment and food contamination.The aims of IPM are to protect crops, with minimum cost and risk for humans, animals and ecosystems.The development and application of IPM requires the knowledge of how the ecosystem influences on pest insects and its natural control agents, and how to modify this environment to control particularly pest insects and avoid the related chemical agent's problems.

Integrated pest management (general overview)
IPM applies different tactics, like pest resistant plants, use of entomopathogens such as bacteria and viruses, and strategies that involves cultural, physical, mechanical, biological and chemical control.The use of these combined tactics reduces the chances of generating resistance and insect survival.

Pest resistant plants (transgenesis using plant genes)
Plants have a vast metabolic capability and produce many secondary chemicals which are toxic, anti-nutritional, or aversive to species might otherwise be potential predators (Norris & Kogan, 1980).Examples include the pyrethrins from chrysanthemums and alkaloids like nicotine from tobacco.Other compounds implicated in protection from insect attack include the terpenoids, steroids, flavonoids, phenolic, glucosinolates, cyanogenic glycosides, rotenoids, saponins and non protein amino acids (Gatehouse et al., 1991).As secondary compounds are the products of multi-enzyme pathways which involve the interaction of many gene products, such defense system are in most cases too complex to be used in plant genetic engineering (Dawson et al., 1989;Hallahan et al., 1992).However, a few plant defense mechanisms are based on the product of a single gene, and the target site of many is the insect digestive system.Most of these types of single genes are suitable for gene transfer.Being of plant origin, they have the advantage that they are likely to have a high degree of compatibility with the metabolic system of the transgenic host plant.
Crops resistant to insect attack offer an alternative strategy of pest control upon chemical pesticides.Transgenic plant technology can be a useful tool in producing resistant crops, by introducing novel resistance genes into a plant species.Several different classes of plant proteins have been shown to be insecticidal towards a range of economically important insect pests.Genes encoding insecticidal proteins have been isolated from various plant species and transferred to crops by genetic engineering.Amongst these genes are those that encode: 1. Protease inhibitors (serine and cysteine).The damage of leaves of certain solanaceous plants, either by insect feeding or mechanical wounding, induced the synthesis of protease inhibitors (Green & Ryan, 1972;Shumway et al., 1976;Walker-Simmons & Ryan, 1977;Brown et al., 1985).The first gene of a plant successfully used to be transferred to another plant was a trypsin inhibitor (cowpea trypsin inhibitor, CpTI) (Pusztai et al., 1992;Graham et al., 1995;Xu et al., 1996).As an example of a commercial deployment of a proteinase inhibitor transgene to date, could be mentioned the culture of genetically engineered cotton varieties in China.These varieties express two transgenes to improve cotton protection, Bt toxins against lepidopteran larvae and CpTI.In 2005, Bt/CpTI cotton was grown on over 0.5 million hectares (Gatehouse, 2011).2. Inhibitors of -amylase.This enzyme is able to catalyse the hydrolysis of -1-4 glycosidic bonds, transforming polysaccharides into mono and disaccharides (Grossi- de-Sá & Chrispells, 1997;Franco et al., 2002;Pelegrini et al., 2006).Interference of the carbohydrate absorption could be a way to reduce the insect pest feeding (Yamada et al., 2001).Leguminous seeds are known as rich sources of proteinaceous α-amylase inhibitors (α-AIs) (Payan, 2004).Expression of -amylase inhibitors (α-AIs) from both scarlet runner bean (Phaseolus coccineus) and common bean (Phaseolus vulgaris) has been shown to be effective in transgenic plants, showing high protection against seed weevils in pea (Shade et al., 1994;Schroeder et al., 1995), azuki bean (Sarmah et al., 2004), chickpea (Sarmah et al., 2004;Ignacimuthu & Prakash., 2006), cowpea (Solleti et al., 2008) and coffee (Barbosa et al., 2010).3. Lectins.This group of carbohydrate-binding proteins constitutes entomotoxic factors present in many plant species.During the last decade a lot of progress was made in the study of the properties of a few lectins that are expressed in response to phytophagous insects.Based on their activity towards pest insects, these proteins have a high potential for use in pest control strategies.For example, the use of plant lectins has been applied to control numerous pests: melon fruit fly larvae (Kaur et al., 2009); Aedes aegypti larvae, which has developed tolerance to many other insecticides (Coelho et al., 2009); and the cotton leafworm Spodoptera littoralis, an economically important caterpillar in agriculture and horticulture (Hamshou et al., 2010).

Entomopathogen bacteria (Bacillus thuringiensis)
Bacillus thuringiensis is a Gram positive bacteria belonging to Eubacteria.It was isolated in the early twentieth century in Japan from dead larvae of the silkworm.This bacteria, produces spores and crystalline bodies composed of one or more proteins with insecticidal activity (Schnepf et al., 1998, Sedlak et al., 2000).The crystalline toxins, named Cry δ-endotoxins, exist in a variety of forms: bipyramidal, spherical, rhomboidal, cuboidal and irregular, among others, and are active against a large number of insect groups as well as nematodes and protozoa.Today there are over 40 groups of Cry proteins (Crickmore et al., 2009).The δendotoxins are synthesized as an inactive pro-Cry-toxin and when they are ingested by the larvae feeding on plant debris or soil, the inclusions are solubilized in the alkaline conditions of the digestive tract of the larvae and are converted by the action of the insect proteases in active peptides (Feitelson et al., 1992;Schnepf et al., 1998).Active toxin is recognized by a specific receptor; it binds to microvilli of intestinal cells (Gazit et al., 1998;Gerber & Shai, 2000), and generates ion channels.The natural ion imbalance dissipates and the pH of medium diminishes causing osmotic cell lysis and larva ceases to feed (Schnepf et al., 1998).Moreover, tissue destruction allows mixing the contents of gastrointestinal tract with hemolymph.Both phenomena favor the germination of bacterial spores, resulting in the death of the larva few days after ingestion crystals (Aranda et al., 1996;Schnepf et al. 1998;Crickmore et al., 2009).
There are two main approaches to use Bt as a pest control agent: 1. Preparations based on living or dead Bt containing spores and crystals are sprayed on crops, as if it were a conventional insecticide.This strategy are currently used in the United States, Europe, Argentina and Mexico as a biological control for insect and other invertebrate pests (mites, nematodes, flatworms and protozoa) that affect crops of corn, potato, tomato, sorghum, rice, coffee, beans, sugar cane, among others (Neppl, 2000).
The application of Bt in insect control is not exempt from the emergence of resistance.
Using of a combination of toxins reduce resistance to individual toxins, maintains a populational balance and prevents the prevalence of resistant variants (Georghiou & Wirth, 1997;Ives et al., 2011;Yang et al., 2011).Research and years of use have shown that the employment of Bt products is not hazardous to non-target arthropods, birds, fish, mammals, or environment (EPA, 2008).2. The genes that encode different Cry proteins have been used to generate genetically modified (GM) plants.Transgenic plants are resistant to the attack by insect pests.The most widely GM crops commercialized so far include mainly maize, cotton and rice.The use of this modified crops has been approved in several countries, including United States, Brazil, Argentina, Canada, Australia, Spain, South Africa, among other (James, 2011).Considering that insecticidal crystal proteins can be released continuously into the soil in different forms during the growing period of Bt-plants (Zhou et al., 2011), biosafety of the use of genetically modified plants is always questioned.However, data regarding the development and commercial use of transgenic Bt varieties have shown that the currently available Bt crops have no direct detrimental effects on non target organism due to their narrow spectrum of activity.In addition, the use of these modified crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and has clear benefits on the environment and farmer health.Consequently, Bt crops can be a useful component of IPM systems to protect the crops from targeted pests (Yu et al., 2011).

Entomopathogen viruses (Baculoviruses)
The insect viruses are intracellular parasites that can only reproduce inside a susceptible insect host.They are valuable natural control agents, providing a secure control, effective and sustainable in a variety of insect pests.The virus particles are present in the environment and usually can be found on the surface of plants or in the soil.Insects become infected by consuming plant material contaminated with viral particles on the surface or by contact with the soil.Baculoviruses are the most common type of insect viruses.It has been reported that infect over 600 species of insects worldwide.Most baculovirus infect caterpillars, the immature form of moths and butterflies.Naturally, these viruses are potent regulators of the population of many caterpillar pests, but the use as a tool for biological control in agriculture is limited by biological or technical reasons.
1. Advantages.Insect viruses are very safe to handle, since they are not infectious to organisms other than their natural hosts.Moreover, most insect viruses have a high specificity, so that the risk of affecting non-target beneficial insects is very low.2. Disadvantages.Most insect viruses have a low speed of action on their insect host, during this time the plague is still eating and damaging.Insect death is also dose dependent, and very high doses are often necessary for adequate control.Usually, viruses are very effective against early larval stages; the late larval stages are less susceptible to virus infection.Virus particles exposed to sunlight or high temperatures are rapidly inactivated.In addition, some cultural practices can affect viral persistence, hiding the viral particles in the soil.

Baculovirus biology
Baculoviridae is a viral family that infects insects.Their genomic material is composed by double strand, circular, super coiled DNA, with a size ranging from 80 to 180 kpb.These viruses are classified in four genera: Alphabaculovirus (nucleopolyhedroviruses -NPVs-that specifically infect insects of lepidopteran order), Betabaculovirus (granuloviruses -GVs-that specifically infect insects of lepidopteran order), Gammabaculovirus (NPVs that specifically infect insects of hymenoptera order) and Deltabaculovirus (NPVs that specifically infect insects of diptera order) (Jehle et al. 2006).The virus genome is packaged within a rodshaped nucleocapsid which is further surrounded by a lipoprotein envelope to form the virus particle.This structure is then occluded at very late stages by a crystalline matrix (Occlusion Body or OB) largely comprising for a single occlusion protein (about 28 kDa), which serves to protect it in the environment.During the infection cycle two different phenotypes are generated: budded virus (BVs, responsible for the systemic cell to cell infection) and occluded-derived virus (ODVs, responsible for the host to host infection) (Figure 1).
The most common way of insect host primary infection is by ingestion during larval feeding of contaminated foliage (Figure 2).Following virus ingestion, the OBs are dissolved in the high pH conditions (pH 8.5 to 11) of the insect midgut, releasing the virus particles (ODVs) into the gut lumen (Granados & Lawler, 1981;Pritchett et al., 1981;Pritchett et al., 1984;Rohrmann, 2008).Released viruses bind to the columnar epithelial cells and enter the tips of the microvilli on the apical brush border of cells (Kuzio et al., 1989;Faulkner et al., 1997;Haas-Stapleton et al., 2004).Following fusion between cell and virus membranes, the nucleocapsids are released into the cytoplasm and are transported to the nucleus, where viral DNA transcription and replication occurs.Into the cytoplasm, nucleocapsids are transported from the basal membrane to the haemocoel, acquiring the host derived membrane and virus encoded proteins (Washburn et al., 2003).Secondary infection is achieved by BVs produced from the midgut cells.Thus, the viruses spread to other insect tissues including the fat body, endodermis, muscle sarcolemma and nerve ganglia (Harrap, 1970;Washburn et al., 2003).Prior to death larvae become creamy in colour, cease feeding and show limited movement.In most baculovirus infections, the host tissues break down as a result of the expression of virus-encoded chitinase and cathepsin proteins (Ohkawa et al., 1994;Hawtin et al., 1995;Slack et al., 1995).Then, OBs are released into the environment following the rupture of the insect cuticle; 10 9 OBs may be released from a single larva, and may remain viable in the environment for several years, until ingestion by another host larva resumes replication cycle (Evans & Harrap, 1982) (Figure 2).

Wild type baculoviruses
A large number of Lepidoptera are pests of economically important crops such as soybean, sunflower and cotton, among others.Alpha-and Betabaculoviruses are very useful tools for control of lepidoteran pests.These viruses have a narrow host range, are safe for other insects and organisms in the environment (Szewczyk et al, 2006), and have strong pathogenicity and virulence.Other advantages are the stability in the environment for long periods and that can be applied using simple methods.Because of the great advantages of baculoviruses as biological control tools and their safety for the ecosystem, their use in IPM is accepted and is steadily increasing.Currently, different baculovirus are used in pest control management worldwide.
In North America, the use of baculoviruses as a pest control agent started as early as 1930 with the protection of pine trees with Diprion hercyniae NPV (Bird & Buek, 1961).Subsequently, this strategy was use to protect numerous commercial crops, including alfalfa, cabbage, corn, cotton, lettuce, soybean, tobacco and tomato (Granados & Federici, 1986).Another baculovirus that is currently used to control pest infection is Helicoverpa zea Simple Nucleopolyhedrovirus (HzSNPV).This virus infects several species belonging to the genera Heliothis and Helicoverpa, which include a wide range of crop pests (Chakrabarti et al., 1999).HzSNPV provides a tool for control against the bollworm, soybean, sorghum, corn, tomatoes and beans.In the 90´s of the latest century the virus was registered under the name GemStar TM and marketed by Thermo Trilogy Company.This virus produced in United States is also marketed in Australia by Aventis Crop Science and it is now used to protect crops against Helicoverpa armigera (Mettenmeyer, 2002), which attack near 200 crops including cotton, soybeans, chickpeas, sunflower, snuff, pepper, and corn, among others.
HaSNPV has been adopted for large-scale viral pesticide production in China and has been used extensively to protect crops of cotton (Zhang et al., 1995).
In South America, probably the most successful program of using baculovirus in pest control is the program implemented in Brazil since 1980 to control the velvet bean caterpillar Anticarsia gemmatalis (pest of soybean) (Moscardi, 1989(Moscardi, , 1999)).In this program, the Anticarsia gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV) is used as a biological insecticide against Anticarsia gemmatalis larvae, and by 2005 the treated area reached 2 million hectares (Szewczyk et al., 2006).One advantage of using this virus is that it is highly virulent and only needs to be applied once, whereas chemical insecticides should be applied twice.In addition, the use of this virus is 20 to 30% less expensive than chemical insecticides.Recently, a pilot plant was constructed in Embrapa Soja, Londrina, in order to improve the laboratory process and train people in the production of virus.This laboratory will be able to inoculate 20,000 to 30,000 larvae per day (Szewczyk et al., 2006).
Moreover, the Cydia pomonella Granulovirus (CpGV) has been used as pest control in crops of apples and pears.Its use has increased in Europe and North America since 2000 and is used in about 100,000 ha in those continents.Currently there are several commercial preparations that include Cyd-X, Virosoft CP4 (North America), Carpovirusine TM (France), Madex TM y Granupon TM (Switzerland), Granusal TM (Germany) and Virin-CyAP (Russia) (Rohrman, 2008).In Argentina, Cydia pomonella is a pest of pear, apple and walnut crops.The National Institute of Agricultural Technology (INTA; Argentina) in agreement with the Natural Plant Protection company (France) developed a bio-insecticide called Carpovirus to control that pest, using a formulation based on CpGV.
For an additional example of baculovirus employed as tools of biological control and pest management, can be mentioned Spodoptera exigua Multiple Nucleopolyhedrovirus (SeMNPV).Formulations of this virus are being used to protect crops of sweet peppers in Spain against Spodoptera exigua.The company Biocolo SRL is responsible for the commercial production of this bio-insecticide with the capacity to treat 50,000 larvae per day.The introduction of this biological insecticide has helped to multiply more than 15 times the area of biologically protected crops grown, reaching 23,000 ha in 2008 (Caballero et al., 2009).

Modified baculoviruses
Despite the advantages that baculovirus have, there are also some limitations for their use as pest control agents.Some of these limitations are the high costs of in vivo production and the low persistence in very sunny conditions (Ignoffo et al, 1977).Also, the virus must be used in early insect development stages, because in the late stages the insects are more resistant to infection (Washburn et al., 2003).Another limitation is their slow speed to kill the pest (Ignoffo et al, 1992).To avoid this, recombinant baculovirus have been developed and offer attractive alternatives to broad-spectrum chemical control.These recombinant viruses can express specific toxins (Inceoglu et al, 2001), hormones (Elvira et al, 2010) or enzymes (Gramkow et al, 2010) and are much more efficient than the wild-type virus in speed to kill.
1. Expression of insect-selective toxin.The aait gene obtained from Androctonus australis scorpion is one of the most promising toxins to be expressed in baculovirus.A recombinant virus containing this gene showed to be 40% faster in killing larvae than the wild type and a reduction of host feeding by 60% (Cory et al., 1994;Inceoglu et al., 2001).The site of action of this neurotoxic polypeptide is one insect sodium channel.
Lepidopterous larvae infected with an AaIT-expressing baculovirus reveal symptoms of paralysis identical to those induced by injection of the native toxin (Elasar et al., 2001) and many of the physiological effects are very similar to those of pyrethroid insecticides which also act at the same target (Gordon et al., 1992).Other useful insect-selective neurotoxins are SFI1 (obtained from a European spider Segestria florentina) and ButaIT (derived from the South Indian red scorpion Mesobuthus tamulus (Wudayagiri et al., 2001).Some toxins could exert a cooperative effect when they are co-expressed, such as LqhIT1 and LqhIt2, obtained from Leiurus quinquestriatus scorpion (Regev et al., 2003).2. Expression of insect hormone genes.Disruption, over expression or inactivation of one or more insect hormones results in abnormal growth, feeding cessation and/or death.So, the insertion of genes that encode insect hormones were the first strategies used to generate genetically modified baculovirus.A recombinant virus of Bombyx mori MNPV (BmNPV) that encodes an active diuretic hormone (DH) showed to be 20% faster in killing larvae than wild type virus (Maeda et al., 1989).Later, by the deletion of egt gene, which prevents the larval molt, the mutant virus resulted to be 30% faster in killing larvae and in a considerable reduction in food intake than wild type virus (O´Reilly & Miller, 1991).Also, this gene may be replaced by an exogenous gene and enhance the insecticidal activity (Arif, 1997;Popham et al., 1997;Sun et al., 2004).

Baculovirus safety
Alpha-and Betabaculoviruses are not infectious for predatory or beneficial insects outside of the order Lepidoptera, or toward other non-targeted organisms (Black et al., 1997;Szewczyk et al., 2006).Baculoviruses do not replicate in mammals, birds, fish, amphibians, reptiles and other vertebrates (Barsoum et al., 1997).Moreover, different studies have concluded that the transgenes that encode toxins or other foreign proteins in recombinant baculovirus increase the speed to kill the specific target, but do not confer any selective ecological advantage compared with the wild-type baculovirus.(Cory et al., 1997;Lee et al., 2001).Another concern associated with GM organisms is the possibility of genetic recombination that results in the "jump" of the recombinant baculovirus foreign gene to another organism.If the pesticide based on a recombinant baculovirus is used long enough and in sufficiently high concentrations in the field, it is expected that genetic recombination could occur.In both the field and in laboratory conditions, this phenomenon is expected to be higher among highly homologous baculoviruses infectious to the same host (Hajos et al., 2000).However, it is important to note that the recombinant virus with a phenotype more virulent than the wild type has disadvantages, since they produces fewer progeny and are rapidily out-competed in the ecosystem (Inceoglu et al., 2001).

Future prospects
In order to improve the use of baculovirus in pest control management, various methodologies are being studied and developed.Among these we can mention: 1. Expression of fusion proteins to expand the host range.Because ButaIT toxin alone exhibits weak oral toxicity, and as an alternative to the use of recombinant baculovirus different strategies have been developed based on the generation of fusion proteins.
One of the proteins used is a lectin, which functions as a carrier (GNA, Galanthus nivalis agglutinin) and the other protein is a toxin.A fusion protein comprising GNA and SFI1 has been shown to have insecticidal effects on both lepidopteran and homopteran plant pests (Fitches et al., 2004;Down et al., 2006) while a fusion protein combining GNA with ButaIT has also been shown to have insecticidal activity against lepidopteran larvae.The injection data reported show that fusion proteins containing SFI1 and ButalT are insecticidal towards a range of insects including lepidopteran, dipteran, coleopteran and dictyopteran pests (Fitches et al., 2010).2. Antisense strategies.A new approach to the development of integrated pest control is the use of technologies based on iRNA (Gordon & Waterhouse, 2007;Price & Gatehouse, 2008).For efficient pest control methods using iRNA in the field, the major challenge is the development of easy and reliable methods for production and delivery of dsRNA.The dsRNA can be produced in vivo using a bacteria system or can be synthesized in vitro.Other way to produce dsRNA is using transgenic plants that express the desired antisense.The delivery of dsRNA can be done by spraying dsRNA on the crop plants or by feeding insects.In a recent study feeding heat-killed bacteria that produced dsRNA or in vitro synthesized dsRNA was used to silence five target genes in Colorado potato beetle, Leptinotarsa decemlineata.It was observed that the loss of function of these target genes caused larval mortality and significantly decreased insect growth (Zhu et al., 2011).In a similar study, it was used a synthetic iRNA to inhibit a mitochondrial electron transport of Plutella xylostela, causing prominent insect mortality (Gong et al., 2011).The use of different target genes could minimize the risk of resistance development.Offtarget effects and species specificity of dsRNA are two major potential issues that need to be addressed.However, the same way as was observed with Caenorhabditis elegans and other plant nematodes (Price & Gatehouse, 2008), in insects, described methods to feed dsRNA to a range of different insect species demonstrated that even highly conserved genes can be exploited to trigger speciesspecific iRNA without affecting non-target species (Whyard et al. 2009).3. Combined control methods.The use of synergism between two different pathogens is a strategy that could be useful to conduct pest control strategies.To evaluate this possibility it have been conducted studies using Spodoptera exigua larvae and assessed the effect of using a baculovirus (Spodoptera exigua NPV) and a parasitoid (Microplitis pallidipes) to control this pest (Jiang et al., 2011).The results obtained indicate that when M. pallidipes-parasitised S. exigua larvae were infected by SeMNPV, the virus did not affect the developmental period of M. pallidipes in the host, and most parasitoids completed development, possibly because parasitism by M. pallidipes reduced larval sensitivity to the virus.The results of this study also indicate that M. pallidipes is an important vector of SeMNPV and contributes to natural epizootics of the virus.Female parasitoids that had developed or oviposited in virus-infected hosts, or that emerged from cocoons contaminated with virus, were able to transmit infective doses of virus to healthy host larvae (Jiang et al., 2011).

Conclusions
Considering the amount of baculovirus species t h a t h a v e b e e n i s o l a t e d s o f a r , i t s development as bio-pesticides has not been commensurate with all its potential.Most of the viruses found in commercial phase are produced by small or medium companies or by the users themselves, as is the case of USDA Forest Service (US Department of Agriculture), CIP (International Potato Center) or EMBRAPA (Empresa Brasilera de Pesquisa Agropecuaria).
Among the factors limiting their commercialization it has been noted that they are too specific, they present a slow speed of action, they have a low persistence in the field, and are costly to produce infecting larva.Also, the approaches that involve in vitro production processes in insect cell cultures are still in development stage.
An entomopathogenic product will be considered as a viable alternative in pest control if it meets control with the same speed, ease of use, at the same cost than a chemical insecticide.This way, do not take into account their unique capabilities: the ability to replicate in their host and be dispersed in culture, the ability to synergistically act with natural enemies, and availability to be produced locally or regionally.Faced with this situation have been conducted many studies to increase the speed of action, to extend the host range and maintain their safety to non-target organisms.This involves the generation of genetically modified baculoviruses and the use of different viral formulations (Cherry & Williams, 2001).
Baculovirus survival in the environment can be affected by temperature, pH, moisture, the presence of additives, exposure to UV light, and by the action of some plant metabolites such as peroxidases, that generate free radicals (Hoover et al., 1998;Zhou et al., 2004).
Regarding the safety of genetically modified baculovirus, a recombinant HzNPV carrying the aait gene was not pathogenic for bees, birds, fish, and other vertebrates (Szewczyk et al., 2006).Natural enemies of the larvae, like parasitoids and predators, were not adversely affected by ingesting individuals infected with recombinant viruses (Li et al., 1999;Smith et al., 2000;Boughton et al., 2003).So far, neither has been shown that the transgene be transferred from baculovirus to other organism or virus (Inceoglu et al., 2001).On this basis, there is no evidence that a recombinant baculovirus is more dangerous than the corresponding wild type.
Finally, knowledge about the biology of baculoviruses suggests that bioinsecticides based on formulations containing these viruses have much lower risk to the environment than the classic chemical insecticides (Szewczyk et al., 2006).

Fig. 1 .
Fig. 1.Alphabaculovirus phenotypes.Occlusion bodies (OBs) containing occlusion derived virus (ODVs) responsibles for the primary infection in the midgut cells and Budded virus (BVs) that spread the infection to other larval tissues.

Fig. 2 .
Fig. 2. Infection cycle.NPV biological cycle.A. Larva ingests a baculovirus-contaminated leaves.B. The virions are released and bind to midgut cell microvilli.C. Into columnar cells the nucleocapsids are transported to the nucleus, where the viral DNA is released and the BVs are generated.D. Nucleocapsids can pass through the cell to the haemocele.E. Viral particles enter into susceptible tissue cells and initiate a replication cycle to produce a new BV generation.F. Later, the OBs are generated into each infected cell.G. Cell lysis occurs, larval integument breaks, and the OBs are released to be eaten by other larva and thus restart the infection.

Fig. 3 .
Fig. 3. Baculoviral bioinsecticide.A. Bioinsecticide spreading on leaves.B. Larvae feeding a contaminated leaf.C. Infected larvae.D. Dead larvae.Released virus will permit the resume of the infection cyle.
This work was supported by research funds from Agencia Nacional de Promoción Científica y Técnica (ANPCyT) and Universidad Nacional de Quilmes.PDG is member of the Research Career of CONICET (Consejo Nacional de Ciencia y Tecnología); MNB holds a postdoctoral fellowship of CONICET.