Natural and Engineered Pest Management Agents - American

Chapter 23

Nuclear Polyhedrosis Viruses for Insect Control Downloaded by NORTH CAROLINA STATE UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 20, 1993 | doi: 10.1021/bk-1994-0551.ch023

John J. Hamm Insect Biology and Population Management Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 748, Tifton, GA 31793-0748

Nuclear polyhedrosis viruses have been extensively researched and are used more often for microbial control of insect pests than any of the other viruses. Their generally high degree of host specificity and ability to produce epizootics are among their most favorable attributes. The broader host ranges of a few NPVs offer hope for using a single virus to control several key pests and expands the potential market for these viruses. The rather rapid inactivation of NPVs by sunlight and the longer time required to kill pests, compared to chemical pesticides, are unfavorable attributes. In the future, the addition of feeding stimulants, UV protectants, and natural or chemical enhancers to increase infectivity of viruses may make viruses more competitive with chemicals for insect control.

There have been some very good reviews of baculoviruses, and nuclear polyhedrosis viruses in particular, in recent years (1,11,40). The process of registration of baculoviruses as pesticides was reviewed by Betz (5) and some practical factors influencing the utilization of baculoviruses as pesticides was discussed by Bohmfalk (6). Since this conference deals with pest management agents, I will concentrate on the characteristics of nuclear polyhedrosis viruses (NPVs) as pest management agents, and how these characteristics affect the strategies for their use. Characteristics of Nuclear Polyhedrosis Viruses Ingestion and Infection. The primary route of virus infection is via the alimentary tract during larval feeding. The polyhedral occlusion bodies dissolve in the alkaline midgut, releasing the virions, or enveloped nucleoThis chapter not subject to U.S. copyright Published 1994 American Chemical Society In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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capsids. Once released into the midgut, the virions are subject to degradation by the gut juices. There is a dynamic balance between the destruction of virions in the midgut and passage of virions through the peritrophic membrane and entry into the midgut epithelial cells. The envelope of the virion is believed to fuse with the cell membrane of the gut microvilli. Once inside the midgut epithelial cell, nucleocapsids migrate to the nuclear membrane where they apparently enter the nucleus through nuclear pores. The virus replicates in the nucleus. In the case of sawflies (Hymenoptera), the polyhedral occlusion bodies are produced in the midgut epithelium as that is the only tissue infected (10). In Lepidoptera and other orders, nucleocapsids or virions are not occluded in the nuclei of midgut cells but leave the nuclei and pass to the basal membrane. Nucleocapsids acquire a host-mediated envelope with a peplomere structure at one end as they bud through the basal membrane into the hemocoel. These virions infect various other tissues, including fat body, epidermis, and tracheal epithelium, where the majority of virions are occluded in the polyhedra. When larvae die, the cadavers tend to liquify or "melt," releasing the occlusion bodies into the environment. This results in a tremendous increase in the pathogen population. Obviously, from the standpoint of insect control, two of the most critical points in this cycle are the times when the occluded virus is exposed to the environment and when the virions are exposed to the contents of the midgut. Host Range. Adams et al. (1) list 523 species of insects from 52 families and 8 orders as hosts of NPVs. The majority, 455 species, are in the order Lepidoptera and 107 species are in the family Noctuidae, which contains many agricultural pests of economic importance. The order Hymenoptera has 31 species listed as hosts of NPVs, 19 of which are in the family Diprionidae and are pests of forest and shade trees. The order Diptera contains 27 species listed as hosts of NPVs, 20 of which are in the family Culicidae which are important pest mosquitoes. Thus, a great number of economically important insects are potential targets for commercially produced NPVs if the NPVs can compete with other control agents. Specificity. The specificity and safety of baculoviruses were reviewed by Doller (11) and Groner (23). All tests indicate that the NPVs do not infect man and other vertebrates or, for the most part, other orders of insects. Most NPVs are pathogenic to only a few closely related species of insects. Some NPVs have been demonstrated to infect only a single species of insect. A few broader spectrum NPVs, such as the Autographa californica NPV, the Anagrapha falcifera NPV, and the Mamestra brassicae NPV (26,72), infect many species of Lepidoptera in the family Noctuidae and several species in other families of Lepidoptera. There are a few reports of NPVs from Lepidoptera infecting insects in other orders. A low level of replication of the Autographa californica NPV was reported in a mosquito cell line, order Diptera (36). Spodoptera littoralis NPV was reported to cause a lethal

In Natural and Engineered Pest Management Agents; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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disease in two species of locusts, order Orthoptera (4) and also to infect a species of termites, order Isoptera (17). But in general it appears that NPVs can be used without endangering nontarget species, and especially beneficial species such as parasitoids and predators that help keep the pest populations down. NPVs have few deleterious effects on other biological control agents. NPVs do not directly affect predators and adult parasitoids as chemical insecticides often do, and NPVs do not infect parasitoid larvae in virusinfected hosts. However, the early mortality of host larvae, due to virus, may result in death of parasitoid larvae if the parasitoids do not have time to complete development. Parasitoids and predators can play an important role in the dissemination of viruses (25,31). Female parasitoids which sting virusinfected larvae can transmit the virus when they sting subsequent larvae. This can be particularly important in moving virus from tree to tree in the forest or from row to row in agricultural crops (25). Because the polyhedral occlusion bodies, which dissolve in the basic midgut of host larvae, are resistant to the more acid digestive tracts of predacious insects, birds, and small mammals, they remain infectious when passed out in the feces of most predators. This helps to keep the virus circulating through the environment and provides for both short and long range dissemination of virus by birds (25). Epizootic Potential. One of the main advantages of viruses as microbial control agents for insects is their epizootic potential. Fuxa and Tanada (19) define epizootic as an unusually large number of cases of disease in a host population. Epizootics are sporadic and limited in duration being character ized by a sudden change in prevalence and incidence of the disease. Two important factors are the ability of the pathogen to increase and its ability to spread though the host population (15). While only one, or a few, viral occlusion bodies may infect an insect larva, the infected larva may release billions of occlusion bodies into the environment when it dies (one large corn earworm larvae can produce approximately 6 Χ 10 occlusion bodies). When these occlusion bodies contaminate feeding sites of susceptible larvae, the disease can spread rapidly through the host population (13). 9

Environmental Stability Importance of Occlusion Body. Another important factor is the stability of the pathogen in the environment. The occlusion body provides a significant degree of protection to the virus. NPVs stored as intact occlusion bodies may retain activity for several years when stored under cool dark conditions, whereas free virions lose their infectivity within weeks or months, even when stored at 4°C (28). An envelope, containing both protein and polysac charides, surrounds the occlusion body and adds to its stability. When the occlusion body envelope is ruptured, cracks may form in the occlusion body,


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resulting in degradation of the virions (1). Thus, when producing virus it is important to allow the maximum number of occlusion bodies to complete development and be enveloped in order to maximize stability and infectivity of the viral preparation (32,33). Infected larvae may be e collected before they die in order to minimize loss of recoverable virus due to melting of the larvae and to minimize the level of bacterial contamination. However, the activity of the virus may be significantly greater when larvae are collected after they die. UV Inactivation. Most NPVs lose 50% or more of their original activity in one or two days when sprayed on plants in the field. This inactivation is associated with exposure to sunlight and more specifically, the UV portion of sunlight (28). This has led to a search for materials that could be added to viral formulations to protect them from UV inactivation. Persistence in Soil. NPVs can persist for years in the soil where the virus is protected from UV irradiation. Thus the soil acts as a reservoir of virus and provides inoculum to initiate new infections each year. Comparison of NPVs in Lepidoptera and Hymenoptera Lepidoptera. There are some important differences between NPVs of Lepidoptera and Hymenoptera. In Lepidoptera, the virus generally replicates in the midgut without producing occlusion bodies and then passes into the hemocoel where it infects a variety of tissues and produces the occlusion bodies. There is very little release of virus into the environment from the midgut, and most lateral transmission occurs after the infected larvae die and release the occlusion bodies as the cadaver melts or liquifies. Probably the most successful use of an NPV to control an agricultural crop pest is the use of NPV to control velvetbean caterpillar in Brazil (30). In this case, the government was involved in the production of limited supplies of the virus, which was distributed to extension service personnel and farmer cooperatives. The supply of virus was increased either by treating naturally occurring larvae or by releasing large numbers of larvae into soybean fields treated with the virus and collecting the dead larvae. The dead larvae were collected 7 to 10 days after treatment and frozen for further use or processing. In the early years, the virus was used by the farmers as a crude preparation. More recently, the government has been processing the virus into a kaolin-based wettable powder formulation for distribution to farmers. They still recommend that farmers collect viruskilled larvae to apply the virus to larger areas, or store frozen for use in the subsequent season. The combination of laboratory production with other less costly methods, taking advantage of low labor costs in the country, has allowed application of formulated velvetbean caterpillar NPV at a cost of US $2.00 per ha compared to US $5.00 per ha for chemical insecticides. Also, only one application of the virus is required compared to several


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applications of chemical insecticide. The estimated area treated with velvetbean caterpillar NPV in Brazil increased form 2,000 ha in the 1982-83 season to 1,000,000 ha in the 1989-90 season. Hymenoptera. In Hymenoptera the development of NPVs is restricted to the midgut. The occlusion bodies are produced in the infected midgut nuclei and are released into the lumen of the midgut as the infected cells rupture. Thus, great numbers of occlusion bodies are released into the environment in the fecal material of infected larvae. Some infected larvae survive to the adult stage. Because the adult midgut is also infected, infected adult sawflies can aid in dispersal of the virus (20). The most virulent entomopathogenic viruses described to date are NPVs infecting several species of sawflies (9,31). Currently, sawflies are relatively minor forest pests. There are no artificial diets for sawflies and it is rather expensive to rear larvae on foliage in the laboratory. Therefore, viruses are produced by spraying heavily infested plantations and harvesting the virus-killed larvae. This is relatively inexpensive, especially because only 50 virus-killed larvae are required to treat a hectare. Strategies for Using Nuclear Polyhedrosis Viruses. Introduction. Harper (24) described the goals of applied epizootiology related to different strategies for using pathogens for biological control. Permanent reduction of the general level of pest population density is the goal of classical biological control through introduction of natural enemies which persist in the new environment. While there are examples of NPVs being introduced into a new area where they have persisted in the pest population, in most cases they do not keep the pest population below the economic threshold. The European spruce sawfly, Gilpinia hercyniae, is an example of an introduced pest being brought under control by the introduction of a NPV (14). Apparently the NPV was accidentally introduced into Canada with the importation of parasites of European spruce sawfly (8). Augmentation. More commonly, NPVs are used to initiate short-term epizootics in which the pathogen may cycle and cause mortality in one to several generations (18,31). This is accomplished by augmenting the number of pathogens over those already present in the habitat. This increases disease prevalence above natural levels and can result in temporary lowering of pest population density. This differs from the temporary suppression of pest populations through application of rapidly acting entomopathogens which do not recycle, such as Bacillus thuringiensb. Bacillus thuringiensis is used in a "microbial insecticide" strategy where multiple applications of the pathogen or its toxic product are applied to compensate for the lack of epizootic potential, i.e. ability to replicate and/or survive in the field. Multiple applications of virus are often made to compensate for the time between application of the virus and recycling of the virus. However, once


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there is significant release of new virus additional applications may not be required.


Integration with Other Methods of Control IPM. Integrated pest management, or IPM, has been defined as "a system that, in the context of the associated environment and the population dynamics of the pest species, utilizes all suitable techniques and methods in as ecologically compatible a manner as possible and maintains pest population levels below those causing economic injury" (7). In an editorial in a recent Society for Invertebrate Pathology Newsletter, Mark Goettel asked the question "Whatever Happened to the Τ in IPM?" He points out that the development of Bacillus thuringiensis as a microbial control agent was expected to strengthen Integrated Pest Management but in most cases it was used as a more selective insecticide in a chemical insecticide approach to control, with the same resulting problems of resistance development. Yearian and Young (41) point out that in short term comparisons most viruses do not compare favorably with chemical insecticides, from either an economic or effectiveness standpoint, in terms of crop protection or productivity. However, insect viruses are ideally suited for IPM systems. Their host specificity limits their direct effects to target pests or closely related species. This minimizes the chances of pest population resurgence or release of secondary pests as a result of reduction in parasitoids and predators, as often occurs with the use of broad-spectrum insecticides. In IPM systems, where the objective is maintenance of pest populations below the economic threshold, not total elimination of the pest, the efficacy of many NPVs may be quite adequate. The biggest impediment to incorpora tion of insect viruses into IPM systems is the lack of an adequate supply of formulated virus (1,16,41). Population Suppression. The Heliothis NPV, Elcar, was originally registered for use only on cotton (27). From a population management standpoint, the time to get control of cotton bollworm and tobacco budworm is during the first two generations, before they are serious pests on cotton. The populations of bollworm and budworm are lowest in the first two generations when the moths emerge from over-wintering pupae in the soil and oviposit on wild flowers and weeds, before crop plants are attractive. On these wild host plants the larvae are exposed to parasitoids and predators, and thus the population does not increase significantly between the over-wintering generation and the first generation produced in the spring. The rationale for area-wide management of bollworm and budworm, with emphasis on reducing survivors in the first two generations, was presented by Knipling and Stadelbacher (29). This led to preliminary tests for area-wide treatment of wild host plants with NPV to reduce the populations of bollworm and budworm in the Delta area of Mississippi (2,3).


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A single aerial application of the Heliothis NPV (Elcar) to wild geranium, at a rate of 6 Χ 10 occlusion bodies per ha resulted in an 88% reduction in tobacco budworm and a 100% reduction in bollworm adult emergence in June (2). A single application of 3 Χ 10 occlusion bodies per ha resulted in reductions of 65% in budworms and 57% in bollworms. We need a better understanding of potential interactions between NPVs and host plant resistance. In general, smaller, less mature larvae are more susceptible to infection by NPVs than are larger, more mature larvae. Some host plants exhibit resistance through antibiosis, resulting in a reduced growth rate of insects feeding on them compared to more susceptible plants. This reduced growth rate of the insect can result in increased susceptibility of the insect to virus infection and longer exposure to parasitoids and predators. Some varieties of plants may alter the feeding sites of the insect pest or allow better coverage of virus sprays, resulting in more effective control of insects. The presence of salt glands on some plants such as cotton may increase the pH of dew on the leaves and contribute to inactivation of NPVs (13). To my knowledge, no effort has been made to screen varieties of cotton for their effects on the stability of insect viruses. In programs involving the release of sterile or substerile insects for suppression of pest populations, the released insects could be contaminated with NPV and allowed to disseminate the virus to the natural population. This would reduce the natural population, and thus the number of released insects required to obtain the desired ratio of sterile or substerile insects to wild insects. Area-wide suppression of pest populations is the goal of many control programs. This involves treating large areas that could not be treated with broad-spectrum chemical insecticides. Even a pesticide as broad-spectrum as Bacillus thuringiensis might not be as desirable as a more host-specific NPV for application to large areas if there is a single key pest to be targeted. If NPVs were used as one of a series of pest control agents it might slow the development of resistance to the various control agents. For example, the Heliothis NPV (Elcar) could be used against the first genera tion of bollworm and tobacco budworm on wild host plants, and possibly on corn, where bollworm populations expand rapidly. In subsequent genera tions, when these pests attack cotton, Bacillus thuringiensis could be used. If the pest populations reach too high a threshold, chemical insecticides could be used. This should reduce the pressure for the pest populations to develop resistance to any one of the control measures (chemical, Bacillus thuringiensis, or virus) compared to using the same control agent against all generations on all hosts. Perhaps, rather than considering NPVs as direct competitors with Bacillus thuringiensis, we should consider NPVs as tools for sustaining the usefulness of Bacillus thuringiensis and some of the chemical insecticides. 11


11


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Recent Advances Fluorescent Brighteners. Shapiro (/. Econ Entomol, in press) demonstrated the UV protectant properties of a series of optical or fluorescent brighteners. These are chemical agents added to detergents to make your clothes "whiter than white and brighter than bright." More importantly, five of the optical brighteners, including Tinopal LPW, enhanced the infectivity of the gypsy moth NPV for gypsy moth larvae even when the virus was not exposed to UV irradiation (35). Tinopal LPW (also know as Calcofluor white M2R and Fluorescent Brightener 28) is a stilbene and is believed to be a chitin synthetase inhibitor. Tinopal LPW at 0.1% enhanced the activity of gypsy moth NPV by 118-fold and at 1% it enhanced the activity by 184- to 1,670fold. Tinopal LPW, at 0.1%, enhanced infectivity of the fall armyworm NPV for fall armyworm larvae by 164- to 303,000-fold (Hamm, J. J.; Shapiro, M., /. Econ Entomol in press). Thus the optical brightener Tinopal LPW has been shown to greatly enhance the infectivity of NPVs in two important lepidopteran pests: the gypsy moth, a forest pest in the family Lymantriidae, and the fall armyworm, an agricultural pest in the family Noctuidae. Due to the unique level of enhancement of viral infectivity for lepidopteran larvae produced by this fluorescent brightener, a patent for the use of fluorescent brighteners in biological control was awarded 23 June 1992 (34). Factors Associated with Granulosis Viruses that Enhance Infectivity of NPVs. Tanada (37) reported that the Hawaiian strain of the armyworm, Pseudaletia unipuncta, granulosis virus (GV) enhanced infectivity of the armyworm NPV when they were fed simultaneously. Since then, a viral lipoprotein in the GV occlusion body was identified as the synergistic factor (SF). Preliminary tests indicated that the SF enhanced infectivity of GVs and NPVs of the armyworm, the cabbage looper (Trichoplusia ni), and the beet armyworm (Spodoptera exigua) in their respective hosts (38). Uchima et al. (39) demonstrated that the SF binds to midgut membranes and may serve as attachment sites for the enveloped virions. A different viral enhancing factor (VEF) was found associated with the GV of cabbage looper (21). This VEF acts primarily by disrupting the structural integrity of the peritrophic membrane, allowing more easy passage of virions to the midgut, and apparently also aiding in attachment of virions to the midgut epithelial cells. In addition to enhancing infectivity of cabbage looper NPV for cabbage looper, it also enhances infectivity of the Autographa californica NPV and the Anticarsia gemmatalis NPV for cabbage looper. The VEF also decreased the time to mortality (20). The potency ratios, comparing virus alone and virus with 40 ng of VEF, were 16 for TnSNPV and 10 for AgMNPV.



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The VEF is 10 times more stable than the virions to UV inactivation and is heat stable; this, plus the ability to enhance infectivity in later instars make it an attractive additive for field applications of virus (20). Granados and Carsaro (21) suggested that the genome for the VEF might be incorporated into the genome of other baculoviruses to enhance the infectivity of the baculoviruses or even into transgenic plants to increase the infectivity of naturally occurring baculoviruses. The incorporation of these enhancers, either a fluorescent brightener or one of GV products, into NPV formulations could make the NPVs more competitive with other control agents. The enhancers decrease, slightly, the time to mortality and decrease the amount of virus necessary to produce mortality. They may effectively increase the host range of some of the broad spectrum NPVs by making them more effective against pests which they can infect at high doses but against which they are only marginally effective without an enhancer. Summary In summary I would like to paraphrase from Jim Harper's message from the president in a recent newsletter of the Southeastern Biological Control Working Group: Sustainable agriculture is today's buzzword and represents a concept that is crucial for the long-term conservation of resources that we will need to feed and clothe ourselves for decades and centuries to come. Integrated pest management is one of the most technologically advanced of the many components of sustainable agriculture. Central to IPM are biological control techniques that naturally promote sustainability because they are either self-perpetuating (and thus sustainable) or are generally much less disruptive to non-target organisms in the environment than are synthetic chemical pesticides. This specificity promotes maintenance of diversity in the environment being manipulated, resulting in greater stability and ultimately greater sustainability. Nuclear polyhedrosis viruses, with their specificity and epizootic potential, fit well into the IPM approach to sustainable agriculture. But these viruses must be available as commercial products that are economically feasible for relatively small markets. NPVs should not be used as chemical insecticides have been, as the only means of control for all generations of pests on all crops. This approach leads to development of resistance to the control agent and loss of sustainability. However, if NPVs are made available for use in conjunction with other control agents (other biocontrol agents, host plant resistance, Bacillus thuringiensis, and chemical insecticides) in ways that deter development of resistance to any of these agents, then NPVs can play an important role in pest management for many years to come. A major problem is how to make profitable the registration and production of viruses for relatively small markets. If the cost of registration could be reduced significantly, that could help solve the problem.


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34. Shapiro, M.; Dougherty, Ε. M.; Hamm, J. J. 1992, U.S.Patent No. 5, 124,149. 35. Shapiro, M.; Robertson, J. L. J. Econ. Entomol. 1992, 85, 1120-1124. 36. Sherman, Κ. E.; Mcintosh, A. H. Inf. Immunity 1979, 26, 227-234. 37. Tanada, Y. J. Insect Pathol 1959,1,215-231. 38. Tanada, Y. J. Invertebr. Pathol 1985, 45, 125-138. 39. Uchima, K.; Harvey, J. P.; Omi, Ε. M.; Tanada, Y. Insect Biochem. 1988, 18, 645-650. 40. Vaughn, J. L.; Dougherty, Ε. M.; In Viral Insecticides for Biological Control, Maramorosch, K.; Sherman, Κ. E., Eds.; Academic Press, Inc.: Orlando, FL., 1985, pp 569-633. 41. Yearian, W. C.; Young, S. Y. In Microbial and ViralPesticides;Kurstak, E., Ed.; Marcel Dekker, Inc.: New York, NY., 1982, pp 387-423. RECEIVED April 30, 1993


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