Entwistle, P.F.

NERC Open Research Archive

SECTIONXRELEASEOF CAPTIVE-BREDSPECIESINTO THE WILDP.F.ENTWISTLEIVEM,OxfordRELEASEOF CAPTIVEBREDSPECIESINTO THE WILDPROLOGUEIVEM has an interest in three major aspects of the subject matter to beaddressed within this contract, viz:-It has been active in the fields of the ecology of insect baculovi ruses (seeFootnote) and the release into the environment of 'captive bred' viruses asbiopesticides for in the region of fifeteen years.For about six years IVEM has been in the forefront of developing insectbaculoviruses as fast acting biopesticides, through genetic engineering, in acautious but vigorous programme.At the same time the complex questions posed by the putativeenvironmental release of genetically engineered baculovirus pesticides have beenand are being addressed in a risk assessment context.Examples of the Release of Captive Bred Insect Viruses in the UK. 1A. the Nuclear Polyhedrosis Virus of Mamestra brassicae (MbNPV),Cabbage Moth.NPV's are largely cultured in vivo using insectary-bred insects. Laboratoryproduction of MbNPV has been in progess in IVEM for at least 15 years. Recentlythis virus has been cloned and the clones compared using restrictionendonuclease (REN) digests. There were no REN differences between 4 clonesinspected thus. However, a preliminary set of bioassays suggested one clone tobe —x10 more infective that the 'wild' type of origin. These bioassays are currentlybeing repeated. The infectivity of the 'wild type' has been carefully tested in 65species of Lepidoptera, divided between 14 familes. Fifty one percent weresusceptible but often only at high dosage levels (between >103-106 virusinclusion bodies).After many (the number has not been systematically recorded) MbNPVpassages in the laboratory this virus has been released into the field from which ithas never previously been recorded in the UK. The releases have been in twomain areas:-(i) Wytham, near Oxford in 1979. Infected larvae were placed oncabbage plants for the purpose of studying the ecology of the NPV in anagroecosystem. MbNPV would certainly persist in the soil and might even now bedetectable. No attempt has been made to see if caterpillars of other species ofLepidoptera in the area of this release subsequently showed MbNPV infections.(ii) Sutherland, Scotland. 1984-88. MbNVP is highly cross-infective tolarvae of Pine Beauty Moth, Panolis flammea. P. flammea has its own naturallyoccurring NPV. However, P. flarnmea is difficult to culture in the laboratorywhilst M. brassicae is easy and so has been used for mass productions of theseNPV's. PfNPV produced in M. brassicae has often turned out to be contaminatedwith MbNPV (reasons unknown) - these NPV's have very close homology. As itwas easier to produce pure MbNPV than PfNPV it was decided to compare thetwo as control agents of P. flammea in aerial spray trials. Consequently, MbNPVwas sprayed at several sites and, in one year, quite extensively (1986, on —300 ha).There is little doubt that since this period MbNPV has spread in P. flamtneapopulations. It has been detected in infected larvae —2Km from a sprayed site andalso as a latent infection (in eggs) 20-30 km from other sprayed sites. Currentlystudies are in progess to measure the value of the gradient of primary dispersal, aparameter we think likely to have a constant and unique value for any onehost/virus 'system' and to be at least one descriptor of spread capacity.Nine of 18 hosts species which are part of the 114 detected as part of thegeneral Lodgepole pine forset ecosystem in Sutherland were susceptible toMbNPV. However, continuous weekly sampling of Lepidoptera on vegetation inand near an MbNPV-sprayed forest in 1986 did not reveal any infected larvae. Itmust be said, though, that the population density of Lepidoptera feeding onplants other than pines is usually very low, so minimising the chances of infectionspreading, or of a single worker encountering an infection.In addition to the MbNPV isolate (which was of continental origin) a pre-commerical preparation of MbNPV was obtained from a French company(Calliope) and in 1986 sprayed on —30 ha giving quite good control of P. flammea.Again, this isolate was not subsequently detected in possible heterologous hostspecies sampled in the forest concerned.1B. The NPV of Panolis flammea (PfNPV).This virus has been maintained in the laboratory at IVEM since at least 1980being sometimes passaged through M. brassicae and less frequently through itshomologous host. From this stock it has been mass produced for field spray trialsduring the years 1982-87 and to this extent it represents a captive bred speciesliberated into the wild. Despite the fact that cloning reveals PfNPV to begenetically very variable (at least to the extent of a lot of REN differences, whichmay or may not be reflected in biological differences) and though lab testsshowed 36% of the pine forest ecosystem Lepidoptera to be susceptible, again ithas not been detected in field survey of other species nor by vigilant 0)entomologists from 1981-89. Currently the LD50 and host range of four clones isbeing studied.Pines Sawfly, Neodiprion sertifer NPV (NsNPV)It is for the general editor of this report to decide if, on the basis of thefollowing account, this example fits the needs of the review.NsNPV was first detected in Sweden and was then used in Canada forcontrol in —1955 where N. sertifer was deemed to be an exotic species.Subsequently (-1965) NsNPVwas broughtto the UK and sprayed in control trials inScotland. It is, however, uncertain that this or a similar virus type was not alreadyhere. Restriction endonuclease comparisons of a British and a North Americanisolate in 1982 revealed genomic differences. The method of NsNPV production(it has been a marketed pesticide since 1983, trade marked 'Virox') is that quitesmall but high population density areas on young pine trees are sprayed with avirus suspension in June/July and that later on the infected larvae are harvested.A subsection of this material is then used agains in the following year to producemore virus in the same way. Hence there is apparently a continous genetic linefrom year to year - but the virus is hardly captive bred in any normal sense. In anyevent no other hosts are known in the UK whilst in Canada cross-infectivity to twoother diprionid sawflies is not easily achieved.The granulosis virus (GV) of Codling Moth, Cydia pomonella (CpGV).This virus belongs to the same family as the NPV's - the Baculoviridae. It hasbeen isoalted from the 'wild' on only a very few occasions and never in the UK.The original isolate, employed extensively in field control trials on apples, pearsand walnuts throughout the world, came from Mexico. Despite its low innatefrequency CpGV is an extremely promising biopesticide. Such an apparentlycontradictory situation is not unique in virus control of pests and probably is anexample of how a virus adapted to a K-strategist host can be 'manipulated' as acontrol agent. Left alone, however, it is likely to revert to its original lowincidence status. In the UK it is known to be cross-infective to at least two othermoths, Pine Shoot moth (Rhyacionia sp.) and Pea moth (Cydia nigricana), thoughit has never been detected in these in the field. CpGV has been sprayed inorchard trials in the south of the UK in several recent years. As far as I know therehave been no attempts to look at its prevalence in the wild Lepidoptera inorchards though, as part of a safely study, antibodies were sought (withoutsuccess) in small mammals trapped in sprayed areas.Brown-tail Moth Euproctis chrysorrhoea, NPV (EcNPV).In recent years (1985-88) there has been a series of control trials, all on verysmall areas of hedgrow/bramble, in the south of England on NPV control of E.chrysorrhoea. The virus used came from a single isolate from the Isle of Grain,Kent, discovered in 1977/78 and subsequently replicated in the lab sometimes inbred and sometimes in field-collected host larvae. Hence virus used in trials hasdirect continuity with the original isolate: no new field infections have beenfound since 1977118. About 40 other species of Lepidoptera have been tested,but none is susceptible: this situation is generally regarded as surprising and noexplanation has yet been found.Releases of Genetically Engineered Autographa claifornica NPV (AcNPV).AcNPV is quite famous on two counts:It has a very wide host range, stretching across 13 families ofLepidoptera. (However, this is to be regarded with at least some caution since invery few instances has progeny virus been checked for identity; some hosts couldsimply be expressing a latent infection.)It is the central subject of baculovirus genetic investigation andengineering world wide. Laboratories everywhere seem to employ AcNPV of thesame origin and frequently exchange clones. It isa truly captive bred 'species'.It is not known if AcNPV occurs naturally in the UK - it has never beensought. The source of the original isolate is California but it is probablywidespread in North America, at least, and the epizootics which build up inpopulations Trichoplusia ni on brassica crops on the eastern seaboard in the fallare caused by an NPV with common identity. An NPV isolated from Greater Waxmoth (GaReda mellonella) in the USSRis also said to be AcNPV.A programme of genetic investigation and engineering of AcNPV has beenunderway in IVEM for —5 years. Some of this work concerns the development ofAcNPV as an expression vector for gene products of medical and veterinaryimportance (e.g. vaccines of hepatitis B, blue tongue of sheep, and etc.) and thereis, of course, no intention that such 'variants' should ever be released. Theirescape has not so far been considered, but would probably be deemed veryunlikely. However, there is a programme to enhance the lethality, etc of NPV's toinsects as improved biopesticides and in this connection the following steps havebeen taken in the UK with AcNPV:(i) Insert a junk gene (a non-coding oligonucleotide sequence) into thegenome at a non-disruptive site. This would then act as an unique tag permittingthis population of AcNPV to be traced following release using appropriategenetic probes.Do the same but attempt to destabilise the virus environmentally bydeleting the polyhedrin gene which codes for the protective virus inclusion bodyprotein but which is inessential to replication per se. This is a step towards theconcept of an engineered improved biopesticide with limited environmentalpersistence and hence enhanced 'safety'.Do similar things to (i) and (ii) but insert other marker sequences, suchas the well known 13-galactosidase gene which permits a colorimetric method ofvirus detection.Introduce genes of putative insecticidal value. The delta-endotoxingene from Bacillus thuringiensis is a now very well publicised example but othersin which there is interest are genes coding for insect growth regulators andarachnoid venins.The host range of the engineered AcNPV at stages (i) and (iii) has beenextensively investigated in the lab at IVEM. It appears to be curiously limited inview of the wide host spectrum of the 'wild' type from which it has derived andoften where a species has proved susceptible the LD50 has been very high. This'loss of activity' could be a consequence either of repeated passage in vitro or thechance selection of a clone of innately limited activity.During the past three years very restricted field releases have been madenear Oxford. These releases have taken the form of placing lab-infected larvae ofSpodoptera exigua, a frequent migrant to the UK but not resident here, ontobeet plants in a field containment facility. The function of the facility is toprevent ingress/egress of any but the most minute insects. The infected larvae arefurther confined to a central portion of the facility which itself is circumscribedwith trap crop/healthy S. exigua larvae to assessmovement of AcNPV within thecontained area. After each experimental run the facility, especially its soil, hasbeen carefully sterilised with formalin and this sterility checked through sensitivebioassays.Under these stringent conditions the following releases have been made:AcNPV with a non-coding marker sequence - 1987ditto, but with the polyhedrin gene deleted - 198813-galactosidase, polyhedrin gene deleted AcNPVThe general results have been as follows. There was no movement of AcNPVfrom the central area of the facility. Polyhedrin-minus AcNPV persisted for only afraction of the time of the intact virus whether on leaf surface or in soil. Noevidence of AcNPV spread, as active infections, was detected outside the facility.GENERAL COMMENTSHost Ranges. Many NPV's have quite wide host ranges - within theLepidoptera, at least. Other, perhaps rare instances, appear to be host-specific(e.g. EcNPV). The captive bred baculoviruses so far released into the wild in theUK have not, apparently, had any adverse effects on non-target or heterologous,hosts. It cannot at this stage of our understanding be said that this is because thereleased viruses are out-competed by their wild counterparts in any particularhost species because generally the detection of a virus depends upon an insectpopulation having been sufficiently high for long enough for disease to becomereadily manifest. Insufficient funds/attention have been invested to follow suchpossible phenomena.Genetic Recombination. Probably most lepidopteran species (and membersof other insect taxa also) have naturally associated viruses. Because of this itseems quite possible that coinfection of an individual insect with two or moreviruses is possible. In this situtation there could be genetic recombination, forwhich there is already some evidence. In the context of assessing the risks ofenvironmental releases of genetically engineered baculoviruses this is of greatinterest and of concern because it asks the question "can engineered genes be'caught' by other baculoviruses?". The much wider and more speculativequestion "can they also be caught by microorganisms in other major taxa" has yetto be given appropriate attention : it is known DNA can persist in soils where itcan be acquried by bacteria in the so-called process of 'transformation'.Risk Assessment. The risk assessment preoccupation of the last two decadeshas turned baculovirus ecology from an essentially academic study into a keytopic. Much interest is now expressed in tracing environmental pathways,particularly of NPV's and there is now little doubt that some major questions, notalready on the book, will soon be addressed. Salient amongst these are:-What is the potential impact of either captive bred wild type orgenetically engineered viruses on major food chain insects, insects significant inother areas of ecological 'balance', insects of aesthetic value?What is the likelihood of engineered genes being lost to otherbaculoviruses, and even to other microorganisms, and achieving an enhancedcapacity to (a) affect wider segments of the biological environment and (b) intheir new host, flow spatially through the environment.Many aspects of questions such as these, and also ones concerning thecomparative capacity of engineeered viruses to survive environmentally will, ofcourse have to be addressed long before their release can be contemplated. A lotcan be acheived through the use of siMulated ecostsytemcomponents, microcosms, in severe containment conditions. IVEM is alreadydeveloping appropriate microcosms in the context of EEC-funded Risk Assessmentprogrammes.Footnote. The Baculoviridae (BV's). This family of viruses is restricted to theArthropoda, apprearing to be especially prevalent in insects. The genome isdouble-stranded DNA. The virion measures about 250 x 50 nm consisting of acore of DNA surrounded by the nucleocapsid protein and enveloped in alipoprotein envelope. Three subgroups are recognised:A. Nuclear polyhedrosis viruses (NPV's). Here groups of virions areembedded in proteinaceous inclusion bodies of a size (1-5pm dia.) visible underthe compound microscope. NPV's are especially well known in Lepidoptera (over900 recorded) but also in diprionid sawflies, caddis flies and some Diptera.B. Granulosis Viruses (GV's). Each virion has its own ovoid inclusionbody (0.3 - 0.5 itm long). GV's are known only from Lepidoptera (over 100discovered).C. Non-occluded, of which the BV of Oryctes rhinoceros (CoconutRhinoceros Beetle) is best known and characterised. Tentatively, about thirtyviruses have been assigned to this subgroup. The host spectrum of the subgroupis probably wider than for subgroup 'A'.

Release of cative-bred species into the wild

http://nora.nerc.ac.uk/508466/1/O147362CR-10.pdf

Release of cative-bred species into the wild

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