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Use of baculoviruses as biological insecticides

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Use of baculoviruses as biological insecticides
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  Use of aculoviruses as iological Insecticides Jenny S. Cory and David H. L. Bishop bstract Naturat y occurring baculoviruses can ~ =used o controI a wide range of insect ~'sts. Most baculoviruses are ~ed, aS biopesticides, that is, they are sprayed onto high-density pestpopula~0ns inamanner akin to the use 0f~ynthetic clieiriical pesticides. Howeve~, Other strategies that use the biological features 0f the:v~Ses ~e at~ol possible~::and Shouid increase as we e~pand our knowledge of baculovirus e~oio~ii'J~n~ order to fi0n systems todetailed study of pest behavior and the development of appropriate aoplicafion strategies. lrtdex Enti ies: Biopesticides; pest control; inseet viruses; bioassay; production; app igati0n. 1 Introduction Baculoviruses have been isolated from a wide range of invertebrates. Their development as pest control agents spans over 40 years, although records of their discovery and use date from considerably earlier 1). The baculoviruses from Lepidoptera (butterflies and moths) and Hymenoptera (sawflies only), and the now unclassi- fied nonoccluded viruses from Coleoptera (beetles) present the best options for pest control, and thus most research on baculovirus insecticides has concentrated on these orders. Baculovirus insec- ticides have been used in a wide range of situa- tions from forests and fields to food stores and greenhouses. Baculoviruses have several advantages over conventional insecticides that make them highly acceptable control agents. Probably the most important is their specificity. They have narrow host ranges (sometimes limited to one or two spe- cies), and do not infect beneficial insects, making them very suitable for use in integrated control programs. Most also possess the capacity to per- sist in the environment, which can be utilized in the development of more ecologically based long- term control programs. Baculoviruses are particu- larly suitable for use in developing countries, since they can be produced locally and their pro- duction in vivo (although labor-intensive) requires little in the way of capital expenditure. More recently, interest in baculoviruses has expanded in relation to their potential for genetic modification, primarily to increase their effi- cacy. This area is still in its infancy, and the development of genetically modified baculo- viruses will not be considered here (however, see Cory [21, Possee et al. [31, and Cory et al. [3a1), although most factors that are relevant to their use in the field will be the same as for naturally occurring baculoviruses. The use of baculoviruses as biological insecti- cides is a broad subject area. The rationale taken in this article has been to outline the pathway required for the development of a control program utilizing baculoviruses, from isolation to field tri- als, and also to describe aspects of the theoretical background see Fig. 1). The further development of baculoviruses for use in integrated control pro- grams and their commercialization are beyond the scope of this article. Author to whom all correspondence and reprint requests should be addressed. Centre for Ecology and Hydrology, Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford 0X1 3SR, UK, e-mail: JSC@mail.nerc-oxford.ac.uk Molecular Biotechnology 9 Humana Press Inc. All rights of any nature whatsoever reserved 1073-608511997/7:3/303-313/510.75  I Preliminary [ [Laboratory[ arger scale laboratory testing I development field esting Phase I Phase II Phase IV ] Integrated ield ] trials l [ ~176 1 Phase V Phase V[ [-~l evelopment of [ optimal virus I 1 l production I Development f ] systems I ] methodof Isolation and / \ | apphcatton biochemical[ /F-if[ \ / I characterization /t...J \ / , I I Formulation I\ I ~ / /I devel~ [ ~\ / [~ Appllcatioo A I strategy / \ [ Prelimlnary I / \ {I ~ [ field esting i / \ Laboratory I \ ' ' / \ I efficacy esting ] \ / \ Formulation Pest iology testing and [ behaviour / Phase III [ Field studies and I small scale rials Interaction with ther control gents Safety esting I Registration I Fig. 1. Schematic pathway for the development of baculoviruses as control agents. Numbered boxes refer to those categories dealt with in the text. 2 Materials The majority of baculoviruses have been iso- lated from Lepidoptera: over 500 to date 4). Whether this reflects a genuine host bias or is merely a product of the fact that there are large numbers of lepidopterists (compared to other insect specialists) and that baculovirus infected larvae are usually very obvious, remains to be seen. However, it does indicate that for Lepi- doptera at least, there is a high probability of iso- lating a baculovirus from a given species. Many pest-control programs rely on the isola- tion of local strains of baculoviruses. This can have some advantages: it will circumvent any problems that may occur if an exotic virus is imported for wide-scale release, and may result in the selection of a virus better adapted to a par- ticular host or ecosystem. Isolation of wild-type baculoviruses from insect pests can vary in difficulty. In families, such as the Lymantriid moths (for example, the gypsy moth, Lymantria dispar, and the Douglas fir tus- sock moth, Orgyia pseudosugata), virus epizoot- ics are common and isolates are not difficult to collect, particularly in high-density populations. The apparent rarity of epizootics in some other species, however, does not preclude the success- ful use of a baculovirus for their control. The current means of characterizing a newly isolated baculovirus is by restriction endonuclease digestion of its genomic DNA. The resulting DNA profile gives a good indication of the uniqueness (or otherwise) of the isolate. It is also highly likely that any wild-type isolate will be a mixture of sev- eral genotypically distinct varieties, for example, the lepidopteran Helicoverpa spp. nuclear poly- hedrosis viruses (NPVs) (5) and the stem borer Chilo spp. granulosis viruses (GVs) 6). The rel- evance of this heterogeneity to successful pest control has yet to be ascertained. 3 Methods Every pest:crop system will present its own unique problems, but the general approach and  areas for study will be similar for most pest con- trol programs. The most important of these areas are discussed in the following sections. 3 1 Screening and Efficacy The first step in the initiation of any pest- control program using baculoviruses is the screening of available isolates to assess their efficacy as potential control agents. This can only be accomplished by insect bioassay. Pro- duction and registration of baculoviruses also require the establishment of a standard bioassay system. Bioassay of baculoviruses basically describes the relationship between the virus and the insect in terms of overall mortality or rate of kill. The standard dose-response curve of insects to baculoviruses is sigmoidal. When setting up a bioassay, a range finding assay should be carried out first, using a large range of doses, to assess the shape of the response and to pinpoint the lin- ear region of the curve. A second assay is then set up with the doses arranged symmetrically around the lethal concentration (LC)50 or lethal dose (LD)5o point. The spacing of the doses and the number of test insects can vary, but in general, if the number of doses (and subjects) is low, the doses should be widely spaced and vice versa. Several parameters can be measured using the bioassay: The LCso and LDs0 are the most com- monly used in dose-mortality studies. The LCs0 is the concentration of virus that the test insects feed on or drink that results in 50 being killed. An LDs0 is the dose at which there is 50 mor- tality of the test insects. An LCs0 tells you less about the response than the LDs0, since in the former, the amount of virus ingested is depen- dent on the behavior of the insect and so is inher- ently more likely to vary between tests, whereas the LDs0 is independent of differences in indi- vidual behavior patterns, making this technique more suitable for comparative experiments. The most common method of analysis for dose-mor- tality data is probit analysis, which is used to determine the LDs0 and LCs0 values, although other models are also used 7,8). Analysis of the time-mortality response (time to death for 50 of the test insects) can be mea- sured either as the lethal time (LT)50, where test insects are continually exposed to virus, or as the survival time (ST)50, where the inoculum is only received at the beginning of the assay. Probit analysis is not valid for analyzing time-mortality assays, and other transforms, such as the logit, should be used for these data. Many factors will affect the outcome of bioas- says, including the instar used, temperature, the choice of insect diet, and larval weight. In order to increase the precision of the assay, it is impor- tant to minimize heterogeneity within the system, in particular, by using test insects within a very narrow weight range. The assay must be standard- ized, particularly if batches of virus are being tested against a standard, as in a potency assay. Several insect bioassay techniques have been developed, and there are numerous variants of these to account for the different feeding require- ments of particular insect species. It is not pos- sible to make valid comparisons between different bioassay techniques, since each will generate different values. The two most commonly used bioassays are described here. 3 1 I Diet Plug Assay Many Lepidoptera, and in particular, noctuid species, will feed on an artificial or semiartificial diet, which makes insect bioassays much easier to handle than when foliage is used. Details of a range of diets and the insects that have success- fully been reared on them can be found in Singh and Moore 9). 1. Place a small plug of diet (of a size suitable for the chosen instar to eat within 24 h) in the base of a 96-well microtiter plate (a larger plate will be needed for the larger instars). 2. Apply 1 IxL of the virus suspension to the diet plug with a micropipet. 3. Place larvae individually in wells and cover. It is usually best to seal the microtiter plate with damp tissues to prevent dehydration of larvae or diet, and to reduce the likelihood of escape. 4. Place the microtiter plate at constant tempera- ture for 24 h. Transfer larvae to small individual pots that contain enough diet to maintain them through to pupation. Only transfer those insects  that have completely consumed their diet plug and have therefore taken up a measured dose of virus. Seal and perforate the lids. 5. Check after 24 h to remove handling deaths, and then monitor and record daily until death or pupation. 6. With well-infected larvae, diagnosis can often be made visually. However, with early deaths or small larvae, it is often difficult to do this. The easiest method of confirming diagnosis of NPVs is to make a thin smear of the midgut region, stain with Giemsa, and observe at xl000 under oil immersion. Granulosis viruses will need further confirmation using alternative techniques, such as ELISA, dot blotting, or by the transmission electron microscope. 3.1.2. Droplet Feeding Assay This particular system was developed by Hughes and Wood 10) and has since undergone numer- ous improvements 8). The technique was devel- oped for neonates, however, it has also been successfully used for later instars lOa). Its main advantage is that the dose is ingested over a very short time, and infection can thus be regarded as synchronous. This is of particular importance in the measurement of STs0 responses. 1. Preparation and selection of the larvae: Larvae are chosen from a narrow window of hatch time, 3-5-h old. They are then selected for vigor by allowing them to climb a ramp of blot- ting paper. 2. Larvae are placed on a plastic surface that has been ringed with a 2:1 mix of mineral oil and vaseline to prevent them from escaping. The virus suspension is then placed in small drops near the larvae. The suspension should contain a small amount of blue food coloring. 3. Larvae that have fed on the virus solution should be obvious because of the blue dye in their gut. These larvae are transferred carefully to individual pots containing diet. 4. Monitor as appropriate. For LTs0 or STs0 analy- sis, this may well need to be every few hours. The bioassay carried out in this manner can be used to estimate the LCs0. The amount of fluid ingested and thereby the dose) needs to be esti- mated for evaluation of an LDs0. This can be done using 32p 10), by fluorescence spectroscopy 11), or gravimetrically. 3.2. Virus Production At present, natural viruses for pest control are produced in vivo. The use of an in vitro system for virus production is some time away, because the yield of virus/unit volume is still too low to be considered economically feasible and the virus often loses infectivity for the insect host after passage through cell culture. With larger-scale production programs such as for the corn earworm Helicoverpa zea NPV 12) and gypsy moth, L. dispar NPV 13,14), the factors affecting the optimization and cost effectiveness of in vivo virus production have been studied in more detail. The most important aspects of the produc- tion process are: choice of host, dose/instar rela- tionship, rearing conditions, and virus processing, all of which are outlined in Subheadings 3.2.1.- 3.2.4. For a more detailed discussion of these and other areas, see Shapiro 14) and Carter 15). 3.2. 1. Choice of Host It is usually preferable to use the natural virus host, which, in order to reduce the likelihood of contamination, should be reared in the laboratory rather than collected from the field. It is also a considerable advantage to use a host species that has been adapted to an artificial diet. Alternative hosts must be sought if the natural host is unsuit- able for laboratory culture, for example, where the dietary requirements are too stringent, where a long obligatory diapause is required, or if it pro- duces irritant, urticacious hairs. In these circum- stances, it is important to confirm the identity of the progeny virus, since alternative hosts might select other strains from the original mixed) inoculum or switch to a homologous virus if this is present in a latent state within the alternative host culture. 3.2.2. Instar Dose Relationship The aim of the production process is to use the minimum quantity of inoculum to produce the maximum yield and quality of virus. This will require several range-finding experiments in order to optimize the process. For instance, Kelly and  Entwistle 16) compared the use of third, fourth, and fifth instar cabbage moth, Mamestra bras- sicae larvae for NPV production. Third instars clearly gave the larger yield, which did not vary within the dose range tested (6.25 x 107-6.25 x 108 polyhedral inclusion bodies [PIBs]/mL). Other factors can also affect the viral product. For example, activity of virus appears to vary depend- ing on when it is harvested from the larvae. NPV collected from H. zea larvae was found to be more active and more abundant when it was recovered from dead larvae rather than from live ones 12). Similarly, in L. dispar, the activity of the NPV obtained from different instars varied; it was found to increase up to the fourth instar and then decrease in the fifth 17). 3.2.3. Rearing Conditions In general, the most important environmental consideration is temperature. Other factors, such as humidity, appear to have little affect on virus production, except perhaps in terms of creating favorable conditions for the growth of undesirable contaminants. The optimum temperature range appears to be between 20 ~ and 26~ Shapiro 14) investigated gypsy moth, L. dispar, NPV produc- tion at temperatures between 23 ~ and 32~ and found that at the highest temperatures, both yield and activity of the NPV were lower. Similarly, Kelly and Entwistle 16) looked at the effect of temperature on the rearing of both M. brassicae and the pine beauty moth, Panolisflammea NPVs in M. brassicae larvae over the range of 20 ~ to 30~ The results again tended to point to a reduction in yield with an increase in temperature. The other key factors for virus production are size of container and the number of larvae reared/ container. Harvesting the virus-infected larvae is the most time-consuming, and thus expensive, stage of the virus production process, so it pays to use larger containers, although this must be set against the cannibalistic nature of many species. 3.2.4. Processing The processing of virus-killed larvae and the level of purification used greatly affect the final yield. To liberate the virus from the larval bodies, the insects are blended with diluent and then fil- tered through muslin. Both the ratio of body weight to diluent and time spent blending have a significant affect on the final virus yield. Shapiro 14) showed with L. dispar NPV that using 1 g larval weight with 10 mL distilled water resulted in 95 PIB recovery after filtration. At lower dilutions, a greater proportion of the virus was lost. Similarly, with blending time, 5 s in a blender resulted in 75 PIB recovery, whereas 15 s resulted in 95 . A comparison of semipurification and full purification has been made for the production of M. brassicae and the brown tail moth, Euproctis chrysorrhoea, NPVs 16,18). Semipurification involved blending and coarse filtration, followed by low-speed contrifugation to remove debris and a higher-speed spin to pellet the virus. Both the low- and high-speed spins were repeated to ensure maximum virus recovery. For full purification, these steps were followed by centrifugation at 30,000g through a discontinuous 50-60 sucrose gradient. With M. brassicae NPV, the fully puri- fied product resulted in a loss of 25-30 of the virus and E. chrysorrhoea NPV with a 35 loss of PIBs as compared to the semipure preparation. The authors calculated that the ratio of PIBs produced to inoculum used was >1000:1 for E. chrysorrhoea NPV, 2200:1 for M. brassicae NPV, 2150:1 for O. pseudosugata NPV, and 4000:1 for L. dispar NPV. 3.3. Formulation The formulation of baculoviruses, although important, has not really received much attention. Formulation of baculovirus insecticides basically falls into two areas: that relating to storage stabil- ity and factors important to field application. Stor- age stability is very much dependent on the method of processing, but it has been little tested and there are few guidelines on it. The main aims are to produce a stable preparation in which the viability of the baculovirus is preserved or even enhanced. Most baculoviruses are processed for use as sprays, and research into the production of solid preparations is much rarer. For small-scale trials, storage stability is not usually a problem
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