Biopesticides

Biopesticides David HL Bishop NERC Institute of Virology and Environmental Microbiology, Oxford, UK Two subjects have dominated the biopesticide literature over the past year. One concerns the use of engineered and natural baculovirus insecticides; the other concerns insect-specific bacterial toxins. The proceedings of a recent symposium on Bacillus thuringiensis and related bacterial toxins have been published. Soon to be reported are the successful field trials of an engineered baculovirus insecticide that encodes an insect-specific scorpion toxin.

Current Opinion in Biotechnology 1994, 5:307-311 Introduction Biopesticides are potential alternatives to chemical insecticides, particulady when insect resistance or untoward environmental effects are associated with the use of chemicals. Even so, chemical insecticides have dominated the pest control market since the 1960s and will continue to d o so for the foreseeable future. They have a number of major advantages, including the low costs of production, the breadth of target species and the ease of application. Biopesticides account for an increasing, albeit small, part of the market (<5%). In specific areas, they offer an environmentally safe and costeffective alternative to chemicals. In other areas, either they are not cost effective, or appropriate biopesticides have yet to be identified. The use of baculovirus insecticides has found favour in certain countries (e.g. Brazil, China and the former Soviet Union) for particular crops and pests, and where labour costs are low. In the UK, such insecticides have been used with considerable success to control Panolisflammea (pine beauty moth), or Neodiprion sertifer (pine sawfly) infestations in Scottish forests. Bacterial toxins are used to control pests of a number of crops, for example, spruce budworm in Canadian forests. Nevertheless, worldwide, biopesticides are used on only a few million hectares of crops and forests each year. Paradoxically, the specificity of biopesticides is both their advantage and their disadvantage. On the one hand, this specificity means that non-target species (beneficial insects, other invertebrates, vertebrates and plants) are not affected. On the other, the target specificity restricts their use where, for example, a variety of pests are infesting a particular crop. The relatively slow speed of action and higher production costs are also factors that limit the use of biopesticides. Genetic engineering is being explored in a number of laboratories to address some of the problems associated with

biopesticides. The main objective is to retain the beneficial features of a biopesticide, while redressing some of the problems (e.g. slow speed of action). This review surveys all the literature from the past few years in the field of baculovirus insecticides and provides a brief account of the recent findings concerning bacterial toxins that exhibit insecticidal properties.

Baculovirus insecticides A number of reviews on progress in the genetic engineering of baculovirus insecticides have b e e n published recently [1,2,3°,4,5°]. These are complemented by work on the ecology and use of natural baculovirus insecticides [6°,7-9]. Some of the most successful baculovirus insecticides are those that are occluded by polyhedrin protein (i.e. nuclear polyhedrosis viruses) or by granulin protein (i.e. granulosis viruses), because these viruses are relatively stable in the environment. In relation to the genetic engineering of baculoviruses, a major issue is whether it is possible to improve their insecticidal activities. The insecticidal activity of a virus can be measured by a number of criteria, such as the dose of the virus required to produce a lethal outcome (e.g. LD50), the lethal time (e.g. LT50), or the effects on feeding ability and plant damage. For recombinant viruses, the principal purpose of engineering is to limit damage to the plant by the target insect. Ideally, this must be determined in field sitautions; however, laboratory assessments can be made to select suitable candidates. Although baculoviruses have genomes of doublestranded DNA, many natural baculoviruses have proved to be difficult, or impossible, to clone by plaque assays. This has hampered the engineering of a number of viruses that are potentially important biocontrol agents. The majority of work on genetically engineered

Abbreviations AcNPV--Autographa californica nuclearpolyhedrosisvirus; BmNPV--Bomby× mori nuclear polyhedrosisvirus; BT--Bacillus thuringiensis, EGT---ecdysteroidUDP-glucosyltransferase;JHE--juvenile hormoneesterase.

© Current Biology Ltd ISSN 0958-1669

307

308

Environmental biotechnology baculoviruses has, therefore, involved the Autograpba californica nuclear polyhedrosis virus (AcNPV) and the Bombyx mori nuclear polyhedrosis virus (BmNPV) because these viruses may be plaque cloned. While foreign genes may be expressed in lieu of the AcNPV polyhedrin genes, BmNPV polyhedrin genes, or other resident genes (e.g. plO), the ability to prepare multiple gene expression vectors allows foreign genes to be expressed in addition to the natural baculovirus genes. Polyhedrin-negative viruses are infectious w h e n injected into the body cavities of larvae, but exhibit a much reduced infectivity w h e n introduced per os. Thus, the preferred baculovirus recombinant insecticides either express foreign genes and are occluded by virtue of the expression of the natural polyhedrin gene, or are recombinants that are co-encapsidated with a wild-type virus. In 1991, it was reported that an occluded engineered derivative of AcNPV (i.e. AcST-3), which contained a gene encoding an insect-specific toxin of the North African (Algerian) scorpion (Androctonus australia, Arachnidae; Scorpionidae), exhibited a significantly improved insecticidal activity for susceptible larvae (e.g. Trichoplusia nO w h e n compared with the wildtype virus [10-12]. The A. australis toxin affects sodium conductance in nerve cells, but only in insects; it has no effect on mammalian cells. In host range studies with AcST-3, no significant difference in host range was identified between the parent and recombinant virus, even though the expressed toxin can affect a variety of insect species [3°]. This is because the host range is specified by the virus and not by the introduced toxin. An AcNPV derivative with a straw itch mite toxin gene has also been reported to exhibit an improved insecticidal activity [13,14°]. The most effective construct was a virus in which a dual promoter was employed for toxin expression. When mixtures of the occluded mite and scorpion recombinants were used to infect susceptible T. ni larvae, however, it was found that the effect of the viruses in combination was no better than that obtained using either recombinant virus alone (M Hirst, personal conarnunication). In studies reported previously in 1988, a recombinant AcNPV that expressed a Buthus eupeus scorpion toxin failed to demonstrate any improvement in insecticidal activity [15]. In another approach, no improvement in efficacy was observed with a recombinant AcNPV that expressed an insect eclosion h o r m o n e gene [16]. In contrast, expression of a synthetic diuretic hormone by a polyhedrin-negative BmNPV following injection into silkworm larvae was reported to give a small, but significant, improvement in insecticidal activity [17]. Expression of Heliotbis virescens juvenile h o r m o n e esterase fJHE) by a recombinant AcNPV has b e e n shown to have some effect on the feeding ability of first instar T. ni larvae, but not for later instars [18]. Further work demonstrated that the expressed JHE protein had a relatively short half-life in infected insects. In view of this, a gene was constructed that produced a more stable JHE. This was shown to exhibit an improved insectici-

dal activity [19,20°°]. Comparisons of the mite, scorpion and improved JHE recombinants have all shown similar levels of improvement of insecticidal activity (M Hirst, personal comnmnication). Removal of the AcNPV ecdysteroid UDP-glucosyltransferase (EGT) gene has b e e n reported to increase the insecticidal activity of the derived virus w h e n compared with the parent virus [21]. EGT degrades the insect ecdysteroid moulting hormones, resulting in inhibition of ecdysis (moulting). An EGT-negative virus allows moulting to occur and, interestingly, kills the host faster than the wild-type virus, hence resulting in less feeding damage. Little benefit was derived, however, w h e n an EGT-negative virus was constructed that also expressed the H. virescensJHE [14°]. Studies to assess the effects of inclusion of Bacillus thuringiensis (BT) toxin genes into AcNPV have shown that the expression of such toxins provides no significant benefit, even though active toxin is made [21,22]. It is presumed that this is because the BT toxin acts on the insect midgut, rather than on internal cells and tissues (where the virus replicates). The results reviewed above all concern laboratory analyses that have yet to be replicated in field assays. In part, this is due to the concern that risks may be associated with the use of engineered viruses in the environment. In 1993, permission was given to scientists at the Institute of Virology and Environmental Microbiology, Oxford, UK, to field test AcST-3, the scorpion toxin virus. The results of these studies will be published in 1994. They provide the first evidence that this engineered virus is an improved insecticide in the field (,IS Cory et aL, unpublished data). Thus, there is strong reason to expect that genetically engineered virus insecticides will become a commercial reality in the future, provided that no unacceptable risks are identified.

Bacterial toxins A number of bacteria make insecticidal toxins. Some are specific for Lepidoptera, others for Diptera, Coleoptera, etc. The subject has been reviewed in a recently published book, as well as in other articles [24°°,25,26°°]. In the past three years, several new bacterial insecticidal genes h a v e been described that encode toxins with particular host specificities [27-31]. Also, polymerase chain reaction procedures have been developed that allow n e w insecticidal genes to be identified on the basis of the conserved sequences that are present in certain toxin genes [32,33]. In 1991, the three-dimensional structure of the BT toxin ($-endotoxin) was published [34]. This has enabled an understanding of the mechanism of action of the toxin, revealing those domains of the protein that are probably involved in binding to a cell receptor and those that are involved in the formation of a pore in the target cell membrane (eventually resulting in cell death and lysis).

BiopesticidesBishop This landmark in o u r understanding of h o w such bacterially encoded toxins affect their target species will no doubt lead to other major advances in the future. Furthermore, it will provide further insight into h o w these toxins work, in particular what determines their host specificity. One of the most interesting developments from the past few years has b e e n the mounting evidence for synergism b e t w e e n the activities of certain, but not all, combinations of bacterially encoded insecticidal toxins [35°-37°]. H o w this occurs is not clear, but it will be an important subject for fftture investigations. Moreover, data have b e e n obtained that support the concept that chaperonins may b e involved in the formation of the crystalline inclusions of certain toxins [38°]. It has recently b e e n shown that new forms of B. tburingiensis can be prepared by transduction or electrotransformation of DNA containing toxin genes [39°]. With the use either of plasmids with specific toxin genes and acrystalliferous hosts of the parent species, or of shuttle vectors and foreign hosts, the opportunities to investigate toxin formation, to study toxin properties, a n d to prepare new bacterial species for biocontrol p u r p o s e s are potentially limitless. This has recently b e e n demonstrated with some of the bacterial toxins specific for mosquitoes. Both the mosquitocidal activity of toxins from B. thuringiensis and B. sphaericus species, and the genetic determinants of the host ranges of their toxins, have b e e n investigated [40,41°,42°,43°°,44,45°,46"]. Mosquitoes are important vectors of a number of diseases, including those caused by viruses and other parasites. The control of these and other haematophagous arthropods is a major challenge for the pesticide industry. Some of the recent reports discuss the properties of toxins cloned into alternative bacterial hosts, including those that are sympatric with mosquito larvae [42°,43"°,44,45°]. Of particular interest are the reports in which hosts, such as filamentous cyanobacteria (Anabaena spp. [43"']), unicellular cyanobacteria (Agmenellum quadruplicarum and Anacystis nidulans [44,45°]), or Caulobacter crescentus [46"], h a v e b e e n engineered for mosquito control by the incorporation of B. thuringiensis or B. sphaericus toxin genes. The data suggest that it will be possible to d e v e l o p alternative biopesticides based on the properties o f bacteria that have different lifecyd e s and ecological niches to those of the original host from which the recombinant genes were isolated.

resistance is observed w h e n a subset of the species is selected that has a natural (genetic) ability either to resist, or not to be affected by, the insecticide. Once susceptible counterparts have b e e n eradicated through the use of chemical insecticides, these resistant species emerge as the dominant members. Will the same p h e n o m e n o n occur through the use of natural or genetically engineered bacterial or viral insecticides? Already, insect resistance has b e e n reported following both the use of BT toxins as biopesticides and the introduction of genes encoding BT toxins into crops to prevent insect damage (see [47"']). This raises questions about the future use of BT toxins in agriculture and h o w such resistance might be prevented b y appropriate m a n a g e m e n t practices, or by the development of better (e.g. multivalen0 insecticides [47°°]. The possibility that resistance may develop raises both regulatory and scientific issues, issues that it w o u l d be wise to address before such insecticides are used more widely in agriculture. We must ensure that the use of biopesticides based on BT toxins or other insecticidal toxins does not follow the course of chemical insecticides. So far, no evidence for resistance (in a genetically stable form) by insects targeted by virus insecticides has ever b e e n demonstrated, either in the laboratory or in the field. Whether this results from an inability of the insect to evolve faster than the evolving virus population, or whether it simply results from the fact that viruses have not b e e n used in the amounts and manner that will produce selection of resistant species is, at present, unknown. Viruses evolve faster than their hosts b y virtue of their genetic composition and their replication rates. In general, their survival depends on replication at the expense of the host cells. These factors m a y mitigate against resistance developing by simple changes to cell-surface proteins, or other components of a cell that a virus utilizes. On the other hand, viruses gain entry into a host through a limited number of mechanisms. Where a single type of receptor protein provides a port of entry into a cell (e.g. a surface protein on a midgut cell), there is no reason w h y the loss of such a receptor should not provide resistance to infection, provided that the receptor is not essential or irreplaceable to the insect. Clearly, the issue of whether insect pests can e v o k e resistance to virus biocontrol agents and their genetically engineered derivatives deserves further study.

References and recommended reading Conclusions Considerable progress has occurred during the past few y e a r s i n the d e v e l o p m e n t and assessment of genetically engineered viruses and bacteria for biocontrol a n d biopesticide purposes. Despite this, the issue of whether insect resistance may develop following their use in the field still needs to be considered and addressed. With a chemical insecticide, it is believed that

Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •of outstanding interest 1.

BishopDHL, Cory JS, Possee RD: The Use of Genetically Engineered Virus Insecticides to Control Insect Pests. In Release

of GeneticallyEngineered and OtherMicroorganisms.

Edited by Day M, Fry JC. Cambridge: Cambridge University Press; 1992:137-146.

309

310

Environmental biotechnology 2.

Possee RE), Cayley PJ, Cory JS, Bishop DHL: Genetically Engineered Viral Insecticides: New Insecticides with Improved Phenotypes. Pesticide Sci 1993, 39:109-115.

Possee RD, Hirst M, Jones LD, Bishop DHL, Cayley PJ: Field Tests of Genetically Engineered Baculoviruses. In Opportunities for Molecular Biology in Crop Protection, monograph 55. Edited by Beadle DJ, Bishop DHL, Copping LG, Dixon GK, Holloman DW. Famham: British Crop Protection Council; 1993:23-36. The preparation, properties and field trials of engineered AcNPV baculovirus insecticides are described. The review includes viruses engineered to express insect-specific toxins or hormones.

expresses the mite toxin. The data suggest that the use of a hybrid late and very late promoter is best. 15.

Carbonell LF, Hodge MR, Tomalski MD, Miller LK: Synthesis of a Gene Coding for an Insect-Specific Scorpion Neurotoxin and Attempts to Express it Using Baculovirus Vectors. Gene 1988, 73:409-418.

16.

Eldridge R, O'Reifiy DR, Miller LK: Efficacy of a Baculovirus Pesticide Expressing an Eclosion Hormone Gene. Biol Control 1992, 2:104-110.

17.

Maeda S: Increased Insecticidal Effect by a Recombinant Baculovirus Carrying a Synthetic Diuretic Hormone Gene. Btochem Biopby Res Comm 1989, 165:1177-1183.

18.

Hammock BD, Bonning BC, Possee RD, Hanzlik TN, Maeda S: Expression and Effects of the Juvenile Hormone Esterase in a Baculovirus Vector. Nature 1990, 344:458--461.

19.

Eldridge R, O'Reilly DR, Hammock BD, Miller LK: Insecticidal Properties of Genetically Engineered Baculoviruses Expressing an Insect Juvenile Hormone Esterase Gene. Appl Env Microbiol 1992, 58:1583-1591.

3. •

4.

Possee RD, King LA, Weitzmann MD, Mann SG, Hughes DS, Cameron IR, Hirst ML, Bishop DHL: Progress in the Genetic Modification and Field-Release of Bacuiovirus Insecticides. In The Release o f Genetically Modified Microogantsms--REGF2¢I 2. Edited by Stewart-Tull DES, Sussman M. New York and London: Plenum Press; 1992:47-58.

5. •

Bonning BC, Hammock BD: Development and Potential of Genetically Engineered Viral Insecticides. Biotechnol Genet Eng Rev 1992, 10:455-489. This comprehensive review covers the background concerning the use of baculovirus insecticides, the procedures for preparing recombinants, and the phenotypes of a variety of recombinants that have been developed to provide improved insecticides. It describes why recombinant baculoviruses may be adopted for insect pest control programmes in the future. 6. •

Cory JS: Biology and Ecology of Baculoviruses. In Opportun/ties for Molecular Biology in Crop Production, monograph 55. Edited by Beadle DJ, Bishop DHL, Copping L, Dixon GK, Holloman DW. Farnham: British Crop Protection Council; 1993:3-9. Provides a general review of the ecology of natural baculoviruses in relation to their insect hosts. 7.

8.

O'Reilly DR, Miller LK: Improvement of a Baculovirus Pesticide by Deletion of the EGT Gene. Blotechnology 1991, 9:1086--1089.

22.

Speight MR, Kelly PM, Sterling PH, Entwistle PF: Field Application of a Nuclear Polyhedrosis Virus Against the BrownTail Moth, Euproctts chrysorrhoea (L) (Lep., Lymantriidae). J Appl Entomol 1992, 113:295-306.

Merryweather AT, Weyer U, Harris MPG, Hirst M, Booth T, Possee RD: Construction of Genetically Engineered Baculovirus Insecticides Containing the Bacillus thurtngtensls Subsp. kurstakl HD-73 Delta Endotoxin. J Gen Virol 1990, 71:1535-1544.

23.

Bourner TC, Vargas-Osuna E, Williams T, Santiago-Alvarez, Cory J: A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control ofAgrotis segetum (Lepidoptera: Noctuidae) in Maize. Btocontrol Set Technol 1992, 2:315-326.

Martens JWM, Honer G, Zuidema D, van Lent, JWM, Visser B, Vlak JM: Insecticidal activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells. Appl Env Mtcrobtol 1990, 56:2764-2770.

24. ,..

Entwistle PF, Forkner AC, Green BM, Cory JS: Avian Dispersal of Nuclear Polyhedrosis Virus After Induced Epizootics in the Pine Beauty Moth Panolis flammea, (Lepidoptera: Noctuidae). Biol Control 1993, 3:61--69.

10.

Stewart LMD, Hirst M, Lopez Ferber M, Merryweather AT, Cayley PJ, Possee RD: Construction of an Improved Baculovirus Insecticide Containing an Insect-Specific Toxin Gene. Nature 1991, 352:85-88.

12.

13.

Bonning BC, Hirst M, Possee RD, Hammock BD: Further Development of a Recombinant Baculovirus Insecticide Expressing the Enzyme Juvenile Hormone Esterase from Heliothis virescens. Insect Blochem Mol Biol 1992, 22:453-458. JHE from H. virescens is modified to provide a stabilized form of the enzyme. When JHE was expressed in a recombinant AcNPV, a significant improvement in insecticidal properties was noted compared with the wild-type enzyme. 21.

9.

11.

20. °,.

Maeda S, Volrath SL, Hanzlik TN, Harper SA, Majima K, Maddox DW, Hammock BD, Fowler E: Insecticidal Effects of an Insect-Specific Neurotoxin Expressed by a Recombinant Baculovirus. Virology 1991, 184:777-780. McCutchen BF, Choudary PV, Crenshaw R, Maddox D, Kamita SG, Palekar, Volrath S, Fowler E, Hammock BD, Maeda S: Development of a Recombinant Baculovirus Expressing an Insect-Selective Neurotoxin: Potential for Pest Control. Btotechnologv 1991, 9:848--862. Tomalski MD, Miller LK: Insect Paralysis by BaculovirusMediated Expression of a Mite Neurotoxin Gene. Nature 1991, 352:82-85.

Tomalski MD, Miller LK: Expression of a Paralytic Neurotoxin Gene to Improve Insect Baculoviruses as Biopesticides. BLotechnology 1992, 10:545-549. A variety of promoters are employed to assess which provides the optimal improvement of phenotype of a recombinant AcNPV that

Entwistle PF, Cory JS, Bailey, MJ, Higgs S: Bacillus thuringienisis, an Environmental Btopesttctde: Theory and Practice. Chichester: Wiley; 1993. This book is an excellent collection of papers from a conference on B. thurlngtensts and related toxins, describing their genetics, genetic engineering and use as insecticides. 25.

H6fte H, Whiteley HR: Insecticidal Crystal Proteins of Bacillus thurtngier~ls. Microbtol Rev 1989, 53:242-255.

26. ee

Gill SS, Cowles EA, Pietrantonio P: The Mode of Action of Bacillus thurlngtenMs Endotoxins. Annu Rev Entomol 1992, ~7:615-636. This review describes current knowledge concerning the mode of action of BT toxins. 27.

Chambem JA, Jelen A, Gilbert MP, Jany CS, Johnson TB, Gawron-Burke C: Isolation and Characterization of a Novel Insecticidal Crystal Protein Gene from Baclllua Subsp. alzawal. J Bacterlol 1991, 173:3966-3976.

28.

Gleave AP, Broadwell AH, Hedges RJ, Wigley PJ: Cloning and Nucleotide Sequence of an Insecticidal Crystal Protein Gene from Bacillus thuringlenMs DSIR732 Active Against Three Species of Leafroiler (Lepidoptera: Tortricidae). NZJ Crop Horttc Set 1992, 20:27-36.

29.

Gleave AP, Hedges RJ, Broadwell AH: Identification of an Insecticidal Crystal Protein from Bacillus thurlnglensls DSIR517 with Significant Sequence Differences from Previously Described Toxins. J Gen Mtcrobtol 1992, 138:55-62.

30.

Tailor R, Tippet J, Gibb G, Pells S, Pike D, Jordan L, Ely S: Identification and Characterization of a Novel Bacillus

14. •

Biopesticides Bishop 311 thuringlensls 6-End•toxin Entomocidal to Coleopteran and Lepidopteran Larvae. Mol Microbiol 1992, 6:1211-1217. 31.

Visser B, Munsterman E, Stoker A, Dirkse WG: A Novel Bacillus thuringiensis (;erie Encoding a Spodoptera exigua° Specific Crystal Protein. J Bacte~ol 1990, 172:6783-6788.

32.

Bourque SN, Valero JR, Mercier J, Lavoie MC, Levesque RC: Multiplex Polymerase Chain Reaction for Detection and Differentiation of the Microbial Insecticide Bacillus thuringtensis. Appl Env Microbiol 1993, 59:523-527.

33.

Gleave AP, williams R, Hedges RJ: Screening by Polymerase Chain Reaction of Bacillus thuringiensis Serotypes for the Presence of cryV-Like Insecticidal Protein Genes and Characterization of a cryV Gene Cloned from B. thuringiensis Subsp. kurstaki. Appl Env Microbiol 1993, 59:1683-1687.

34.

Li J, Carroll J, Ellar DJ: Crystal Structure of Insecticidal 8End•toxin from Bacillus thuringiensis at 2.5-~ resolution. Nature 1991, 353:815-821.

35. •

A n g s u t h a n a s o m b a t C, Crickmore N, Ellar DJ: Comparison of Bacillus thuringiensls Subsp. israelensls CryIVA and CrylVB Cloned Toxins Reveals Synergism in Viw~. F ~ S Microbiol Lett 1992, 94:63-68. Recombinant B. thuringiensis hosts cured of toxin genes, but expressing recombinant cryIVA and cryIVB genes, form inclusions of the mosquitocidal toxins. The expressed proteins, after solubilization, were tested o n mosquito cell lines and selected larvae. Synergism b e t w e e n the activities of the toxins were demonstrated using certain mosquito larvae. Tabashnik BE: Evaluation of Synergism A m o n g Bacil~ lus thuringiensis Toxins. Appl Env Mtcrobtol 1992, 58:3343-3346. Synergisim is demonstrated b e t w e e n the CtyA a n d CryIVB toxins of B. thur~ngiensL~ using Aedes aeRvpti larvae. No synergism w a s demonstrated w h e n lepidopteran larvae were treated with different CryI toxins. A simple test is described to evaluate synergism a m o n g toxins. 36. •

37. •

C h a n g C, Yu YM, Dai SM, Law SK, Gill SS: High-Level cry/VD and cytA Gene Expression in Bacillus thurlngiensis Does Not Require t h e 20-Kilodalton Protein and the Coexpressed Gene Products Are Synergistic in their Toxicity to Mosquitoes. Appl Env Microbtol 1993, 59:815--821. A 20 kDa protein e n c o d e d on plasmids that harbour crtA and crylVD BT toxin g e n e s in B. thuringiensis is required for efficient production of CrytA in Escher~chia coll. These experiments demonstrated that this 20 kDa protein is not required for CrytA/CryIVB toxin synthesis in an acrystalliferous B. thurlengtenMs strain. Synergism b e t w e e n toxin synthesis is demonstrated and evidence for a chaperonin protein is obtained and related to more efficient toxin expression. 38. •

Crickmore N, Ellar DJ: Involvement of a Possible Chaperouin in the Efficient Expression of a Cloned CrylIA y-End•toxin Gene in Bacillus thuringiensis. Mol Mtcrobiol 1992, 6:1533-1537. Evidence that supports the concept o f the involvement of chapero n i n s in the formation of crystalline inclusions of BT toxins is presented. 39. •

Lecadet MM, Chaufaux J, Ribier J, Lereclus D: Construction of Novel Bacillus tburingiensis Strains with Different Insecticidal Activities by Transduction and Transformation. Appl Env Mtcrobtol 1992, 58:840--849. Both transduction a n d electrotransformation procedures are e m ployed to introduce insecticidal toxin g e n e s into certain strains o f B. thuringtensis, allowing the construction of bacteria with n e w combinations o f end•toxins.

40.

Yu YM, Ohba M, Gill SS: Characterization of Mosquitocidal Activity of Bacillus thuringiensis Subsp. fukuokaensis Crystal Proteins. Appl Env Microbiol 1991, 57:1075-1081.

41. •

Berry C, Hindley J, Ehrhardt AF, Grounds T, De Souza I, Davidson EW: Genetic Determinants of Host Ranges of Bacillus sphaericus Mosquito Larvicidal Toxins. J Bacter~ol 1993, 175:510-518. Reports the differential toxicity, the temporal patterns of mortality, and sublethal effects of the binary larvicidal toxins of B. sphaertct~. Expression o f mutant toxins in E. coli h a s s h o w n that a region of the 41.9 kDa c o m p o n e n t is responsible for specifying host range, but that the overall toxicity is a function of both the 41.9 kDa and 51.4 kDa components. 42. •

Chang C, Dai SM, Frutos R, Federici BA, Gill SS: Properties of a 72-Kilodalton Mosquitocidal Protein from Bacillus thuringiensis Subsp. morrlsoni PG-14 Expressed in B. thuringiensis Subsp. kurstaki by Using the Shuttle Vector pHT3101. Appl Env Microbiol 1992, 58:507-512. The shuttle vector pHT3101 is e m p l o y e d to express high levels of BT CryIVD inclusions in a mutant B. th~tr~ngiens~ host. 43. ••

Xudong X, Renqiu K, Yuxiang H: High Larvicidal Activity of Intact Recombinant Cyanobacterium Anabaena sp. PCC 7120 Expressing Gene 51 and Gene 42 of Bacillus sphae~ lcus sp. 2297. FEMS Microbiol Lett 1993, 107:247-250. The nitrogen-fixing filamentous cyanobacterium is employed as a host to express B. sphae~cus derived mosquitocidal toxins using a shuttle vector. Laboratory and field tests were conducted, d e m o n strating that the expressed proteins are highly toxic to larvae of Culex pipiens a n d less toxic to larvae of Anopheles MnenMs. 44.

Angsuthanasombat C, Payim S: Biosynthesis of 130-Kilodalton Mosquito Larvicide in the Cyanobacterium Agmenellum quadruplicatum PR-6. Appl Env Mtcrobiol 1989, 55:2428--2430.

45. •

Murphy RC and Stevens SE: Cloning and Expression of the cry/VD Gene of Bacillus thuringiensls Subsp. israelensis in the Cyanobacterium Agmenellum quadrupllcatum PR6 and Its Resulting Larvicidal Activity. Appl Env Mtcrobtol 1992, 58:1650-1655. The expression of the cryIVD gene from B. thur~ngtensis as a translation chimaera in a cyanobacterium species is reported, with the highly expressed toxin exhibiting a significant larvicidal effect on Culex ptptens. The expressed prc,xJuct appeared to be relatively stable, possibly d u e to the fact that it was a chimaera, involving a c o m p o n e n t of a native cyanobacterium protein. 46. •

Thanabalu T, Hindley J, Brenner S, Oei C and Berry C: Expression of the Mosquitocidal Toxins of Bacillus sphaet, icus and Bacillus thurlngiensls Subsp. israelenMs by Recombinant Caulobacter crescentus, a Vehicle for Biological Control of Aquatic Insect Larvae. Appl Env Mtcrobtol 1992, 58:905-910. Expression o f various mosquitocidal toxins are reported in a Caulobacter species.

47. McGaughey WH, Whalon ME: Managing Insect Resistance to o• Bacillus thurlngiensis Toxins. Science 1992, 258:1451-1455. The discovery that insects can adapt in the laboratory and field to BT toxins, including those expressed in transgenic plants, raises the question o f what are the appropriate m e t h o d s for using such biocontrol species in pest m a n a g e m e n t . These issues are discussed in relation to both policy and procedures for pest management.

DHL Bishop, NERC Institute of Virology a n d Environmental Microbiology, Mansfield Road, Oxford O X l 3SR, UK.