Recombinant baculoviruses for insect control

Transcription

1 Pest Management Science Pest Manag Sci 57:981±987 online: 2001) DOI: /ps.393 Recombinant baculoviruses for insect control Ahmet B Inceoglu, Shizuo G Kamita, Andrew C Hinton, Qihong Huang, Tonya F Severson, Kyung-don Kang and Bruce D Hammock* Department of Entomology and Cancer Research Center, University of California, One Shields Avenue, Davis, CA 95616, USA Abstract: Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host speci city. Baculoviruses have been genetically modi ed to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are ef cacious in the laboratory, greenhouse and eld and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will nd a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control. # 2001 Society of Chemical Industry Keywords: baculovirus; biopesticide; recombinant; toxin; hormone; review; safety 1 INTRODUCTION Baculoviruses are highly bene cial viruses. They do not infect man or plants, but they do provide effective natural biological control of many insect species. 1 Along with bacteriophages, baculoviruses are among the most useful tools for the study of basic virology and biology. Baculoviruses are also versatile vectors for the expression of proteins for basic research and medical applications. As expression vectors, baculoviruses possess numerous advantages including extremely high rates of expression and expression of products with authentic biological activity. 1,2 In North America, the use of baculoviruses for pest insect control started as early as 1930 with the protection of pine trees with Diprion hercyniae Htg) nucleopolyhedrovirus NPV). 3 Subsequently, numerous commercial crops, including alfalfa, cabbage, corn, cotton, lettuce, soybean, tobacco and tomato have been protected with varying degrees of success by applying baculoviruses. 4 Perhaps the greatest success with baculoviruses has been obtained for the protection of approximately one million hectares of soybean in Brazil against the velvetbean caterpillar. 5 In the USA, several baculoviruses have been registered as biological pesticides, 6 but their commercial use has been severely limited, partly due to their slow speed of kill compared to classical synthetic chemical insecticides. 7 Currently a number of wild-type baculoviruses are used for the protection of forest, vegetable and stored product pests Table 1). The availability of effective and relatively cheap chemical insecticides has adversely impacted not only baculovirus research and application but also biological control in general. The occurrence of insecticide resistance and environmental concerns, however, have been stimulatory phenomena for biological control. Although the number of target sites in insects is considered to be numerous, few of these targets limited mostly to the nervous system) have been exploited by the past and current generations of chemical insecticides. Today, not only are the discovery and registration costs of new insecticides * Correspondence to: Bruce D Hammock, Department of Entomology, University of California, One Shields Avenue, Davis, CA 95616, USA bdhammock@ucdavis.edu Based on a paper presented at the Conference Insect Toxicology 2000, organized by John E Casida and Gary B Quistad, and held at the University of California at Berkeley, USA on July, 2000 Contract/grant sponsor: NIEHS; contract/grant number: RO1 ES02710 Contract/grant sponsor: USDA; contract/grant number: ; contract/grant number: Contract/grant sponsor: University of Ankara, Turkey Contract/grant sponsor: NIH (Received 18 January 2001; revised version received 12 June 2001; accepted 3 July 2001) # 2001 Society of Chemical Industry. Pest Manag Sci 1526±498X/2001/$

2 AB Inceoglu et al Table 1. A summary of baculoviruses currently in use for pest insect control Pathogen Trade name s) Target host s) Registration status Country NPV Mamestrin Mamestra brassicae, Helicoverpa, Plutella, Yes France Lobesia spp. Genetically engineered NPV Ð Various species of Lepidoptera In progress USA Adoxophyes orana GV Capex Adoxophyes orana Yes Germany AfMNPV AfNPV- Heliothis, Helicoverpa and Spodoptera Yes USA AcMNPV Gusano Various species of Lepidoptera Yes USA CpGV Cyd-X, Madex, Granupom Cydia pomonella Yes USA, Germany, Spain, France Switzerland, HzSNPV GemStar Helicoverpa spp. Yes USA Oryctes NPV Ð Oryctes beetles Not necessary Maldive Islands, Philippines, Samoa, Tonga, Fiji, Indonesia SeMNPV SPOD-X Spodoptera exigua Yes The Netherlands, USA, Thailand Spodoptera littoralis NPV Spodopterin Spodoptera littoralis Yes Lymantria dispar NPV Disparvirus Lymantria dispar Yes Canada, USA Anticarsia gemmatalis NPV Polygen Multigen Anticarsia gemmatalis Yes Brazil Data compiled from Copping and Menn 7 and Society of Invertebrate Pathology web page Abbreviations: NPV, nucleopolyhedrovirus; MNPV, multiple nucleocapsid NPV; SNPV, single nucleocapsid NPV; GV, granulovirus; Af-Autographa falcifera; Ac- Autographa californica; Hz- Heliothis zea, Se- Spodoptera exigua. increasing, but also there is concern over the possible occurrence of cross-resistance to the new active ingredients. Thus, the availability and diversity of tools for insect pest control is not increasing at the same pace as contemporary insect evolution. This of course limits our ability to manage pest insect populations. One approach to exploit new target sites in the insect is the employment of microbial insecticides, including, for example, bacteria, fungi, viruses and nematodes. Microbial insecticides are often similar to synthetic chemicals in terms of formulation and application methods, and have found a robust niche in certain cropping situations. Unlike chemical pesticides, microbial insecticides typically have the potential to stay in the environment and can provide prolonged pressure on insects. This potential to persist and to even recycle does, however, lead to other considerations. In particular, the use of genetically engineered crop plants expressing insecticidal toxins from Bacillus thuringiensis Berliner Bt endotoxin) has introduced a new dimension of complexity to the problem and is further confusing producers and regulatory agencies throughout the world. Resistance against this family of insecticidal proteins is more and more common, especially with the extensive use of genetically modi- ed plants expressing Bt toxins. In one case, the use of Bt toxin against the diamondback moth has been abandoned due to resistance. 8 Recent problems with public acceptance of transgenic crops such as Bt corn for human consumption are also important considerations. In the absence of resistance management systems and alternative toxins, the life expectancy of this technology will be short. These arguments support the conclusion of a recent NRC report 9 arguing that integrated insect pest management is most powerful when there is a diverse tool box available to the pest management specialist. The value of the baculovirus as a complementary or alternative method of pest control becomes more apparent within the framework above. Baculoviruses have many attractive advantages including narrow speci city, adequate pathogenicity, ease of genetic manipulation, minimal residue problems, in vivo and in vitro production capability, and compatibility with integrated pest management programs. Baculoviruses, however, are not devoid of potential problems for widespread commercial use, including relatively slow speed of kill, narrow speci city, instability in the eld, high production costs and short shelf life compared with chemical pesticides. Within the last decade many of these problems have been addressed and, to a degree, solved by laboratories in academia and industry. In this paper, we would like to emphasize strategies that have been developed to improve speed of kill and related ecological consequences. We wish to provide an overview of the status of recombinant baculoviruses for insect control in the context of pesticide chemistry. The reader is also referred to several reviews in the eld. 1,7,10,11 2 BACULOVIRUS BIOLOGY Baculoviruses Baculoviridae) possess doublestranded, circular DNA genomes of about 88±180 kilobases in size. 12 Two genera, nucleopolyhedrovirus NPV) and granulovirus GV), make up the Baculoviridae. Both NPVs and GVs are speci c to the larval stage of their insect hosts. A unique feature of baculoviruses is that they have adapted both to ef cient replication in the insect host and dormancy outside the host ie in the environment) by producing two morphotypes, budded virus BV) and occluded virus OV). In the NPV, a large number of OVs are `micro-encapsulated' in a crystalline protein structure called polyhedron plural polyhedra) which protects them from environmental damage and aids in the oral delivery of infectious virus to the susceptible host. 1 Following ingestion of the polyhedron by the host larvae, the crystalline matrix is rapidly degraded in the alkaline midgut environment and the OVs are released. The OVs are free to infect the midgut cells 982 Pest Manag Sci 57:981±987 online: 2001)

3 Recombinant baculoviruses for insect control or possibly further disseminate via the tracheal system. 13 Early in the infection cycle, within 15h post-infection hpi), the cell releases hundreds of BVs. BVs can subsequently infect other cells rapidly. By 24hpi a late time post-infection), progeny production switches to the formation of OVs which accumulate within the infected cell nucleus. In the case of NPVinfected larvae, a critical level of viral infection and accumulation of virions occurs, generally by 5 days pi, resulting in death. In the case of GVs this period is generally extended to 7±14 days. NPV-infected larvae swell slightly and may become pale due to the accumulation of OVs, and often climb toward the upper foliage of the host plant where they die. 3 GENETIC MODIFICATION OF BACULOVIRUSES The NPV polyhedron is primarily composed of a single protein, termed polyhedrin. This protein is expressed under an exceptionally strong promoter at a late stage of infection. Since the polyhedrin promoter is so active, the polyhedrin protein accounts for more than 50% of the cell mass at a late stage of infection, and polyhedra are easily visible under light microscopy. In cultured insect cells, the polyhedrin protein is not essential for virus replication because the BV is the infective morphotype. The Summers 14 and Miller 15 laboratories were the rst to take advantage of the polyhedrin promoter's unusually high activity and non-essential nature of polyhedrin protein in tissue culture. They used the polyhedrin promoter as the driver for expression of the heterologous gene product and the lack of polyhedra as a marker for successful gene insertion. Since then there have been numerous improvements in the technology, and baculovirus expression vectors are in common use. The most commonly used baculovirus expression vector is Autographa californica NPV AcMNPV), followed by the silkworm Bombyx mori L) NPV BmNPV). 2 Unlike most baculoviruses, AcMNPV has a relatively wide host range, which includes several major pest insect species. GVs may also be used for genetic modi cation, but so far GVs are more dif cult to modify due to the lack of an adequate cell line which supports ef cient replication. 3.1 Expression of insect hormone genes Insect growth is tightly regulated by a handful of major and minor hormones. Disruption, overexpression, untimely expression or inactivation of one or more of these hormones result in abnormal growth, feeding cessation and/or death. Maeda and his colleagues at the Zoecon Research Institute were the rst to overexpress an insect hormone in the host larvae using the baculovirus as a protein expression vector in order to disrupt the normal hormonal balance. 16 They expressed functionally active diuretic hormone DH) of Manduca sexta Joh in silkworm larvae using a recombinant BmNPV, named BmDH5, that expressed the DH gene. BmDH5 infection caused a 30% reduction in hemolymph volume and killed larvae about 20% faster than wild-type BmNPV infection. This improvement in speed of kill was assumed, but not proven, to be due to diuresis. Other insect hormones that have been expressed by NPVs in larvae include the eclosion hormone EH) of M sexta 17 and prothoracicotropic hormone PTTH) of B mori. 18 Functionally active EH and PTTH were produced by the recombinant baculoviruses, but expression of neither of these hormones produced any signi cant improvement in speed of kill. Another target of the insect endocrinological system is juvenile hormone JH). JH regulates various aspects of insect development, and in lepidopteran larvae regulates the onset of metamorphosis at the nal larval molt. Juvenile hormone esterase JHE) is the endogenous insect enzyme which regulates JH titers during the nal larval instar. The JHE gene has been cloned from several insects including the tobacco budworm, Heliothis virescens F, M sexta, and Tenebrio molitor Land inserted into the AcMNPV genome Hinton AC and Hammock BD, unpublished). A recombinant AcMNPV expressing JHE from H virescens shows a slight improvement in speed of kill. 19 However, it was later observed that JHE is rapidly cleared from the hemolymph of both infected and uninfected insects by a receptor-mediated uptake mechanism. 20,21 Thus, the unusually short half-life of the native form of JHE in the hemolymph is likely to be a limiting factor in the insecticidal ef cacy of JHE overexpression and is a current area of research. At present, in our laboratory the primary sequence and three-dimensional structures of JHE from both lepidopteran M sexta and B mori) and non-lepidopteran T molitor and Drosophila melanogaster Meig) insects are being compared and common structural motifs are being explored as sites of recognition for a receptor-mediated recognition process. Such potential target sites are also being subjected to mutagenesis in order to improve the in vivo half-life of the JHE and theoretically improve the insecticidal effect of JHE overexpression in the infected larvae. 3.2 Expression of insect-selective toxins Prior to the development of BmDH5, Carbonell et al 22 attempted to express an insect-selective scorpion toxin gene BeIT from Buthus eupeus using a recombinant AcMNPV. The expression of BeIT did not, however, improve speed of kill. Genes encoding Bt endotoxins have also been engineered into recombinant AcMNPVs, 23,24 but these constructs also did not show any increase in insecticidal ef cacy compared to wild-type virus. Perhaps this was to be expected because the mode of action of Bt toxins is at the level of the midgut, whereas the baculovirus replicates poorly in the midgut. Unlike BeIT, expression of AaIT, a highly potent insect-selective toxin from the scorpion Androctonus australis Hector increases speed of kill by about 40% 25±27 and decreases feeding damage to cotton by 60% due to a `fall down' Pest Manag Sci 57:981±987 online: 2001) 983

4 AB Inceoglu et al effect. 28,29 Fall down is caused by the inability of the infected larvae to control muscle coordination. Interestingly, the site of action of AaIT is the insect sodium channel and many of the physiological effects of AaIT are very similar to those of pyrethroid insecticides which also act at the insect sodium channel. 30 However, the actual site of action within the sodium channel is predicted to be different, since AcMNPV expressing AaIT is equally active against pyrethroidresistant and pyrethroid-sensitive H virescens. 31 With the signi cant improvement in insecticidal ef cacy resulting from AaIT expression, insect-selective toxins such as LqhIT2 from the venom of other scorpions, 32,33 spider, 34 sea anemone 34 and mites 35±37 have been expressed using the baculovirus. These other toxin genes should expand the effective host range and increase the insecticidal ef cacy of baculovirus biopesticides even further Fig 1). Certainly the baculovirus system represents a simple way to test for the toxicity of a peptide or protein before it is engineered into more complex systems. 3.3 Deletion of an endogenous baculovirus gene Another elegant approach to improve baculovirus ef cacy, used by O'Reilly and Miller at the University of Georgia, is the deletion of an endogenous gene which encodes ecdysteroid UDP-glucosyltransferase egt). The egt gene product regulates the endocrine system of the insect host by enzymatically modifying the molting hormone, ecdysone. 38 In the wild-type baculovirus the egt gene product prolongs the larval stage of the insect by inactivating ecdysteroids. This longer larval stage results in about a 30% increase in the yield of progeny virus compared to egt-deleted Figure 1. A general overview of the improvements in speed of kill of recombinant baculoviruses in comparison to wild-type AcMNPV. Note that the methods used for the determination of lethal times differ between laboratories. Bars represent percentage increase in speed of kill. Abbreviations: WT, wild-type; Bt, Bacillus thuringiensis endotoxin; 24 BeIT, Buthus eupeus insect toxin; 22 EH, eclosion hormone; 57 DH, diuretic hormone; 16 JHE, juvenile hormone esterase; 19 JHE KK, stabilized JHE construct; 21 egt-, ecdysteroid UDP-glucosyltransferase deletion mutant; 39 AaIT, Androctonus australis insect toxin 1; 26 Txp-1, insect toxin from Pyemotes tritici; 35 LqhIT2, Leiurus quinquestriatus insect toxin 2; 32 Double construct, LqhIT2-LqhaIT simultaneous expression (Chejanovsky N, Inceoglu B and Hammock BD, unpublished). virus. 39 Deletion of the egt gene from AcMNPV can result in a 30% improvement in speed of kill and 40% reduction in food consumption compared to the wild type virus. 39 American Cyanamid now BASF) has recently eld-tested egt gene-deleted baculovirus constructs and have found similar improvements in speed of kill and reduction in feeding damage as was found in laboratory testing. 40 Although the actual mechanism by which egt gene-deletion improves speed of kill is unknown, one recent study indicates that the egt gene product may induce a hormonal effect which increases virus-speci c protein synthesis, resulting in faster virus transmission. 41 Premature or inappropriate molting induced by the egt gene-deleted virus may also result in feeding cessation and/or premature death. With the complete genome sequencing of at least seven baculoviruses including AcMNPV, 42 Orgyia pseudotsugata MNPV, 43 Lymantria dispar NPV, 44 Spodoptera exigua MNPV, 45 Helicoverpa armigera SNPV, 46 Xestia c- nigrum GV 47 and Plutella xylostella GV, 48 other nonessential genes may also be identi ed as targets for deletion in order to generate a quicker acting or replicating virus. 4 SAFETY OF BACULOVIRUSES Evaluation of the potential risks associated with recombinant organisms should include at least two levels of enquiry: the organism which is genetically modi ed and the fate of the recombinant material. At the organismal level, several studies have unanimously concluded that baculoviruses are safe for use as pestcontrolling agents. 40,49 These reports showed that most baculoviruses are not infectious toward predatory or bene cial insects outside of the order Lepidoptera, or toward other non-targeted organisms. Based partly on these studies, non-recombinant baculoviruses have been registered as insecticides with the US Environmental Protection Agency. 6 Baculoviruses also do not replicate in mammalian cells and appear not to be able to ef ciently enter the mammalian cell nucleus. 50 Furthermore, the ecological consequences of the release of recombinant baculoviruses have been experimentally addressed in terms of the competitive characteristics of recombinant versus wild-type baculoviruses, both in the greenhouse microcosm 40,51 and in the eld. 29,52 Basically, all of these studies have concluded that genes encoding, for example, JHE or AaIT accelerate the speed of kill of the recombinant baculovirus but do not confer any selective ecological advantage in comparison to the wild-type baculovirus. In other words, compared with the wild-type AcMNPV, the recombinant AcMNPV shows reduced tness, indicating once again that the risk factors associated with recombinant baculoviruses are low. Another concern associated with recombinant organisms is the potential of genetic recombination resulting in the foreign gene eg the AaIT gene) `jumping' from the recombinant baculovirus to an- 984 Pest Manag Sci 57:981±987 online: 2001)

5 Recombinant baculoviruses for insect control other organism. Several key factors exclude or limit the occurrence of genetic recombination between donor and recipient DNAs, including physical proximity ie localization within the same compartment within a single cell), similar modes of replication and degree of homology. 40 However, if a recombinant baculovirus pesticide is used long enough and at high enough concentrations in the eld, it is expected that genetic recombination can eventually occur. In the eld, as in laboratory conditions, 53±55 such an occurrence is expected to be highest between highly homologous baculoviruses ie recombinant baculovirus donor and wild-type baculovirus recipient) that are infectious within the same host. The key question in terms of safety, however, is whether such recombination will result in an environmentally detrimental trait that will become xed in the population. We believe, as shown in the laboratory and eld experiments described above, that it will not. There is a strong negative selection pressure against any organism that obtains a gene that improves speed of kill such that the recipient virus will be quickly outcompeted by the wild-type. 5 FUTURE PROSPECTS Farmers throughout the world have traditionally been encouraged by commercial, government and consumer interests to use synthetic chemical pesticides for a `fast and effective x' to pest problems. After 30-plus years of the use and abuse of chemical pesticides, all of these interests, as well as the farmer, now understand that chemical insecticides have a limited life and that excessive and repeated use leads to resistance, pest resurgence and environmental problems. Effective pest management requires a diversity of tools and the exibility that these tools will bring. The baculovirus is a good example of an effective tool which allows diverse approaches that are in many ways more similar to chemical pesticides than to classical biocontrol agents. Most of the disadvantages of baculovirus pesticides, some of which have been addressed in this paper, have been or can be addressed by molecular biology and well-designed ecological studies. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Many developing countries that lack the economic resources to produce or purchase synthetic chemical pesticides can locally produce biocontrol agents such as the baculovirus either recombinant or nonrecombinant) at minimal costs. Furthermore, innovative steps towards making baculoviruses more pro table are being taken simultaneously throughout the world. 56 Considering that some approaches to the large-scale production of baculoviruses can be rather labor-intensive processes, the cost-bene t analysis may favor developing countries where labor costs are cheaper. Additional factors, including environmental concerns over the misuse of insecticides and crossresistance of major groups of insecticides favor wildtype and recombinant baculoviruses, especially in developing countries. One classic example of the successful use of baculoviruses is found in Brazil. 5 The Brazilian success should encourage other nations to implement their own programs, taking into consideration their regional crops, cropping systems and culture. Another continuing area of debate with regard to genetically modi ed organisms, including recombinant baculoviruses, is public acceptance and perception. The public needs to be better informed on risks and bene ts of baculovirus insecticides, especially in comparison with synthetic chemical insecticides and their risks. With this knowledge, we feel that the relative risks of recombinant baculoviruses are far lower than those posed by many chemical insecticides and transgenic crops, while offering clear bene ts in terms of environmentally safe insect pest control. ACKNOWLEDGEMENTS This research was supported in part by the NIEHS RO1 ES02710) and USDA and ). AB Inceoglu is a recipient of a fellowship from the University of Ankara, Turkey. TF Severson is a recipient of a NIH Training Grant in Biotechnology. 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