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Development of a DNA probe for detection of nuclear polyhedrosis virus in the forest tent caterpillar… Kukan, Barbara 1992

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DEVELOPMENT OF A DNA PROBE FOR DETECTION OFNUCLEAR POLYHEDROSIS VIRUS IN THE FOREST TENTCATERPILLAR Malacosoma disstria Hbn.Barbara KukanB. Sc. (Biochemistry) University of TorontoM. Ed. (Education) Queen’s UniversityA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PLANT SCIENCEWe accept this thesis as conformingto the required standardOREUNLVERSITYOF BRITISH COLUMBIAAugust 1992© Barbara KukanIn presenting this thesis in partial fulfilment of the requirements for an advanced degree atthe University of British Columbia, I agree that the Library shall make it freely availablefor reference and study. I further agree that permission for extensive copying of thisthesis for scholarly purposes may be granted by the head of my department or by hisor her representatives. It is understood that copying or publication of this thesis forfinancial gain shall not be allowed without my written permission.Department of Plant ScienceThe University of British Columbia1956 Main MallVancouver, CanadaDate:AbstractMany species of forest Lepidoptera show eight to twelve year population cycles. Viraldisease could explain characteristics of population fluctuations in forest tent caterpillars(FTC) Malacosoma disstria Hbn. Until now, virus detection relied on microscopic examination for polyhedra of virus in smears of caterpillars or other potentially contaminatedmaterial on stained microscope slides. The main objective of this thesis was to developan efficient detection system for nuclear polyhedrosis virus (NPV) of FTC in the form ofa NPV DNA probe. Pure DNA was isolated from virions of the western tent caterpillar,digested with BamHI, and a 2,000 bp fragment which hybridized to the polyhedrin geneof Autographa californica NPV cloned into pGEM then purified for use as a probe.Samples of FTC from a peak density population in the vicinity of Prince George B.C.were tested for presence of virus using both detection systems, the DNA hybridization to aNPV DNA probe, and microscopic examination for polyhedra. Results showed variationbetween the two techniques but no consistent trend toward one technique showing moreor fewer infected individuals. Large sample sizes are necessary to overcome this variation.Because it is much easier to process large sample sizes with the DNA probe its benefitsare evident.Distribution of virus in the Prince George population of tent caterpillars was examined. Samples of different life stages of FTC adults, larvae, pupae, egg masses as wellas FTC parasitoids were tested with the DNA probe. Some individuals of all stages andparasitoids gave positive responses to the DNA probe with adults and parasitoids showing a particularly high frequency of infection. Egg mass and parasitoid contaminationcould play a role in transmission of the virus.I’Transmission of virus was tested by feeding caterpillars virus and testing adults whichsurvived and emerged for the presence of NPV. A high proportion of adults gave positiveresults.A higher proportion of smaller caterpillars were positive for NPV in six of eightcomparisons of large and small caterpillars from the same populations. This suggeststhat the virus might reduce growth of the infected caterpillar or that smaller caterpillarsdie before they reach large size which reduces the proportion of infected individuals amonglarge caterpillars. A comparison of large egg masses (many eggs) and small egg masses(few eggs) showed that fewer small egg masses were contaminated with NPV. The causeof this variation is unclear, but this relationship could be crucial to the interpretation ofchanges in egg mass size in field populations and requires further study.111Table of ContentsAbstract iiTable of Contents ivList of Tables viiList of Figures ixAcknowledgements x1 Introduction 11.1 Cycling Populations 11.2 Nuclear Polyhedrosis Virus 21.3 Life History of Forest Tent Caterpillars 41.4 Objectives 52 Materials and Methods 82.1 Probe Preparation 82.1.1 Preparation and Labelling of a Synthetic Oligonucleotide Probe 82.1.2 Isolation of NPV 102.1.3 IsolationofNPVDNA 112.1.4 Production of a Cloned NPV DNA Probe 112.2 Study Sites and Sample Preparation 122.2.1 Sample Preparation for Testing with the NPV DNA Probe . . . . 14iv4 Results4.1 Occurrence of NPV in Pupae, Adults,Forest Tent Caterpillars4.1.1 Pupae4.1.2 Adults4.1.3 Egg Masses4.2 Viral Transmission4.2.1 Virus Feeding Experiments.4.2.2 Fly ParasitoidsEgg Masses and a Parasitoid of151618191924242425262626285 Results 305.1 Impact of the Virus on the Prince George Population of Forestpillars5.1.1 Caterpillar Size 305.1.2 Association between the Occurrence of NPV and Egg Mass Size inForest Tent Caterpillars 316 Discussion6.1 Development of the NPV DNA Probe2.2.2 Preparing Labelled Probe2.3 Dot Blot Hybridization Assay2.4 Controls3 Results3.1 Preparation of an NPV DNA Probe3.2 Comparison of Viral Detection using the DNA Probe with MicroscopicExamination for PIBs 21Tent Cater-303333V6.2 Occurrence of NPV in Different Host Life Stages 356.3 Viral Transmission 386.3.1 Virus Feeding Experiments 386.3.2 Fly Parasitoids 396.4 Caterpillar Size 396.5 Association between Occurrence of NPV and Egg Mass Size in Forest TentCaterpillars 396.6 Conclusions 40Bibliography 41Appendices 46A Mean Weights of Caterpillars 46viList of Tables3.1 Comparison of percentage of caterpillars with NPV based on microscopicexamination and DNA probe detection from four areas near Prince GeorgeB.C. in 1990 and 1991. (N)=sample size and x2 is Yates corrected. . . . 234.1 Percentage of pupae with virus based on the DNA probe in 1991 244.2 Percentage of adults with virus based on the DNA probe in 1991 254.3 Percentage of egg masses from sites near Prince George B.C. positive forNPV based on the DNA probe compared to corresponding caterpillar datafrom Table 3.1 264.4 Percentage of adults developing from caterpillars fed virus in 1991 positivefor NPV based on the DNA probe 274.5 Percentage of caterpillars positive for NPV which were fed virus in 1990.Detection done using microscopic examination 274.6 Percentage of caterpillars reared from egg masses collected in early spring(1991) that were positive for NPV using DNA probe 274.7 Percentage of pupae of fly parasitoids emerging from laboratory—rearedcaterpillars collected from sites near Prince George in 1990 that were positive for NPV as indicated by the DNA probe 294.8 Percentage of pupae of fly parasitoids collected in the field from sites nearPrince George in 1991 that were positive for NPV as indicated by the DNAprobe 29vu4.9 Percentage of pupae of fly parasitoids emerging from laboratory—rearedcaterpillars collected from sites near Prince George that were positive forNPV in 1991 as indicated by the DNA probe 295.1 Percentage of caterpillars positive for NPV based on caterpillar size. Tables A.1 and A.2 (see appendix) give the weights of caterpillars in the fourareas and some indication of what is considered a small weight comparedto medium and large for both years 325.2 Mean number of eggs/mass, s, (N), for forest tent caterpillar egg massescollected from sites near Prince George, B.C 325.3 Combined 1990-91 egg masses probed positive for NPV compared in termsof egg mass size. The small egg masses came from various sites; RangeRoad=17, Truck Scale=20, Airport=O and Westlake=12. The mediumegg masses came from; Range Road=25, Truck Scale=25, Airport=3 andWestlake=8. The large egg masses were one each from Range Road, TruckScale and Airport 32A.1 Mean weight (± standard deviation) of caterpillars collected in the vicinityof Prince George B.C. 1990 46A.2 Mean weight (± standard deviation) of caterpillars collected in the vicinityof Prince George B.C. 1991 47yinList of Figures1.1 The top photograph of the four life stages of FTC; egg mass, caterpillar,pupa, adult was taken in the laboratory 1991. The bottom photograph ofthe two caterpillars on the leaf was taken in Prince George 1991 72.1 Nucleotide sequence of four NPV polyhedrin genes showing sequence selected for the oligonucleotide probe. (From Rohrmann (1986)) 92.2 Examples of stained microscope slides (napthalene black on left and triplestaining on right) and a dot blot autoradiogram. The top row of the dotblot contained the positive and negative controls. (From left to right;WTC PIBs, pure MpNPV DNA 120 ng, 12 ng, 1.2 ng, FTC PIBs, T. ni,and P. saucia.) The other rows contained larvae samples from Truck Scale1991 183.1 Restriction enzyme digestion of MpNPV DNA and detection of the polyhedrin gene by Southern blot analysis.The bands selected for cloning areindicated by the bars 20ixAcknowledgementsI would like to thank Dr. D. Theilmann and Dr. M. Isman for reading my thesis andoffering their suggestions and criticisms. Also, I would like to thank Dr. D. Theilmannfor providing the AcNPV polyhedrin gene, M. Milks for giving me Trichoplusia ni larvae,Dr. M. Isman for larvae of Peridroma saucia, Dr. J. Roland for fly parasitoid samples,and Dr. W. Kaupp for Malacosoma disstria PIBs. Jackie McPhee and Doug Sheperdprovided technical assistance for which I thank them. I owe special thanks to Dr. J.Kronstad who took me into his lab and encouraged and guided me and to Dr. J. Myers,my advisor and friend. She was always there to offer help and advice. Financial supportwas provided by the Science Council of British Columbia and NSERC for which I amgrateful.I would like to thank Debbie Cool and Brian Thomson for the final push I needed toget my thesis done and Richard Lockhart for his statistical advice. Many thanks are owedto my friends Debbie Cool, Brian Thomson, Kathy Heinrich, Brian Aispach, MalgozataDubiel, Alistair Lachlan, Greg Schlitt, Kerstin Baxter, Mimi Mekler and Bev Springwho gave me much needed emotional support through a very difficult time. Finally mydeepest thanks to my husband Alan Mekier whose love was my inspiration. This is foryou Alan.xChapter 1Introduction1.1 Cycling PopulationsFluctuating populations of forest Lepidoptera have been widely studied because theycompete with man for valuable timber resources. The cyclic dynamics of forest Lepidoptera have stimulated a number of explanatory hypotheses. Myers (1988) reviews sixgeneral hypotheses to explain the population cycles of forest Lepidoptera: genetic variation, qualitative variation, climatic release, food quality deterioration, drought stress,and disease susceptibility. Genetically controlled variation in behavior and physiologyof animals could provide the basis for self-regulation of populations [Chitty 1967], butgenetically driven self-regulation of populations has not been demonstrated [Myers 1988].Wellington (1960) attributed population fluctuations of western tent caterpillars to qualitative variation in physiology and behavior in a population but mechanisms to explainchanges in quality of insects have not been identified [Myers 1988]. Wellington et al (1975)modelled cyclic population dynamics of tent caterpillars by incorporating increased dispersal of vigorous individuals and annual variation in weather conditions, but it is notclear how variation in weather could cause cyclic population dynamics and dispersal ofhigh quality individuals should stabilize populations [Myers 1988]. Variation in foodquality as related to population cycles of insects requires more detailed experimentalanalysis [Karban and Myers 1989], but insect population declines in the absence of defoliation suggest that deterioration in food quality is not prerequisite for cyclic decline of1Chapter 1. Introduction 2populations [Myers 1988].Disease susceptibility has often been rejected as a hypothesis to explain cycling because the proportion of the population dying from disease can be small [Myers 1988].However, individuals dying of viral disease disintegrate rapidly and are not readily observable in the field. Viral disease is often reported in declining populations of forestLepidoptera [Tanada and Fuxa 1987] and could explain characteristics of populationfluctuations in western tent caterpillars (WTC) Malacosoma californicum pluviale (Dyar)and forest tent caterpillars (FTC) Malacosoma disstria Hbn. such as death of late in-stars in the first year of the decline followed by death of early instars the next year andpossibly reduced fecundity following population decline through sublethal effects [Myers1988]. Therefore the disease susceptibility hypothesis could explain cyclic populationdynamics and deserves closer analysis.Various pathogens and parasites affect forest tent caterpillar populations. Theseinclude a fungus Entomophthora crustosa, a fly parasitoid Sarcophaga aidrichi and anuclear polyhedrosis virus (MdNPV) [Carruthers and Soper 1987], [Watanabe 1987],[Tanada and Fuxa 1987] . NPV has been observed in caterpillars during peak populationyears and its effect on the fecundity and the survival of tent caterpillars could be animportant influence on the ten year population cycles [Myers 1990] and [Stairs 1965a].1.2 Nuclear Polyhedrosis VirusNuclear polyhedrosis viruses are a subgroup of the family of occluded DNA viruses calledBaculoviridae that can cause fatal disease in insects. In the environment, NPVs are commonly found on plant surfaces and in the soil, as polyhedral-shaped occlusion bodies(PIBs) that surround and protect the infectious virions. Because of their stability, limited host range, and pathogenicity to Lepidopterans and sawflies that are forest andChapter 1. Introduction 3agricultural pests, these viruses have been recognized as potential insect control agents.The baculoviruses are characterized by a large rod-shaped, enveloped nucleocapsid,and a double—stranded supercoiled DNA genome of 88 to 160 kilobase pairs [Rohrmann1986]. The matrix of the occlusion body is composed primarily of a single type of proteintermed polyhedrin. This protein has a mol. wt. of about 29,000 and is soluble in alkalinesolutions. When ingested by a susceptible lepidopteran, polyhedra dissolve in the alkalinemidgut, releasing the infectious virions. During early infection, non—occluded virions arebudded through the plasma membrane of the midgut cells into the hemolymph and spreadthroughout the body. The infection eventually reaches a final stage at which furtherocclusion bodies containing virions are produced. At this stage the host disintegratesand the polyhedra spread into the environment.The regulatory mechanism controlling the synthesis of polyhedrin is of considerableinterest because polyhedrin is produced in larger amounts than perhaps any other eukaryotic viral gene product [Rohrmann 1986]. Because of this high level of expression,the polyhedrin gene region has been used for the construction of recombinant virusescapable of expressing foreign genes at high levels. Lepidopteran NPV polyhedrins areclosely related to one another and demonstrate 85—90% amino acid homology [Rohrmann1986].One of the dominant reasons for this study of the NPV of forest tent caterpillars isto understand further the role of the virus in the cycling of the caterpillar population.The first step towards achieving this goal is to have an efficient, reliable detection systemfor the virus. This would lead to an understanding of where the pathogen occurs, andits infection rate. The main objective of this thesis is to develop an efficient detectionmethod for NPV in field populations of FTC.NPV is common in dense populations of forest Lepidoptera [Tanada and Fuxa 19871,but it is not known how NPV is maintained through the low— density population phase.Chapter 1. Introduction 4There must be some “reservoir” of virus because it reappears when the host populationis high. Possibilities are (1) that it persists through environmental contamination bypolyhedra that remain infective for caterpillars even several years following populationdecline; (2) that it is carried in some inactive form by the insects; or (3) that it persistsat a low frequency in sparse caterpillar populations killing only a few individuals eachgeneration [Evans and Entwistle 1987]. Following ingestion of polyhedra by a caterpillar,NPV replicates to produce large numbers of virions in the host. By multiplying in thehost the pathogen incurs benefits and costs. The benefit is that fitness is increased aslarge numbers of virus polyhedra are produced and released into the environment. A costis the decrease in mobility and the death of the host which terminates virus replication.The virus would benefit from having a persistent “reservoir” stage which would insureits availability for infection even at low densities of the host. Interesting questions inthis area of research are: how is the virus maintained in the host population?; where isthe persistent reservoir of NPV which is available for infection of tent caterpillars?; andis the disease involved in population cycling? In an attempt to address some of thesequestions, the present study was aimed at the development of a DNA probe which wouldmake it possible to monitor the persistence and distribution of NPV in cyclic populationsof forest tent caterpillars.1.3 Life History of Forest Tent CaterpillarsForest tent caterpillar (FTC), Malacosoma disstria, extend from British Columbia toP.E.I and from James Bay to Louisiana [Stehr and Cook 1968]. In the Prince Georgearea of British Columbia populations cycle with a periodicity of 6—13 years [Turnquist1987]. Authenticated records [MacLeod and Tyrrell 1979] of outbreaks in North Americago as far back as 1797. Outbreaks usually follow the pattern of 2—3 years of increasingChapter 1. Introduction Spopulation, 1—2 years of peak population and 1 year of decline. Population peaks, asindicated by defoliation of aspen forests, occurred in the Prince George region in 1953—1954, 1962—1964, 1973—1975, and 1989—1991 [Turnquist 1987]. During the time of thepresent study, 1990—1991, the population of caterpillars was in the outbreak phase.Egg hatch in this area occurs in May and corresponds roughly with bud burst ofthe host tree aspen, Populus tremuloides. There are five larval instars which feed overapproximately six weeks. By late June or early July the larvae form cocoons in rolledleaves and pupate. After approximately ten days to two weeks adults emerge and mate.Female moths lay a single egg mass as a band on small twigs of trees. M. disstriaoverwinters as eggs which hatch in the spring [Stehr and Cook 1968].1.4 ObjectivesThe first objective of this study was to develop a reliable assay and an effective method totest for the presence of virus. Until now virus detection relied on microscopic examinationfor PIBs of insect smears on stained slides. This work is very tedious and involves a lotof uncertainty in identification. Criteria for a good diagnostic tool would include fasterand more accurate identification of virus in a large number of samples. A DNA probe forNPV could provide an assay that is highly sensitive and capable of detecting viral DNAat very low levels [Ward et al 1987].The second objective was to compare the DNA probe assay to the previously usedtechnique of microscopic examination for PIBs by testing samples of Malacosoma disstriafor NPV. These larvae were collected from a peak density population in the Prince Georgevicinity of British Columbia.A final objective was to look at the distribution of virus in the Prince George population of FTC. This was divided into two parts. The first part included testing the probeChapter 1. Introduction 6on adults, larvae, pupae, and egg masses of FTC, and one of its parasitoids. The secondpart was to interpret some of the DNA probe results with specific reference to the statusof the population of FTC in Prince George.Chapter 1. Introduction 7Figure H: The top photograph of the four life stages of FTC; egg mass, caterpillar, pupa,adult was taken in the laboratory 1991. The bottom photograph of the two caterpillarson the leaf was taken in Prince George 1991.Chapter 2Materials and Methods2.1 Probe Preparation2.1.1 Preparation and Labelling of a Synthetic Oligonucleotide ProbeThe first probe made for the detection of NPV was an oligonucleotide probe whichconsisted of a known nucleotide sequence. Since the DNA of western tent caterpillarNPV (MpNPV) and forest tent caterpillar NPV (MdNPV) have not been sequenced,nucleotide sequences of four polyhedrin genes from Autographa californica (AcMNPV),Orgyia pseudotsugata (OpMNPV, OpSNPV), and Bombyx mon (BmNPV) were compared [Rohrmann 1986]. A conserved sequence in the 350—360 base region was selected(see Figure 2.1).The oligonucleotide probe was made by Tom Atkinson of the Biochemistry Department of UBC by small scale automated sythesis. It consisted of 21 bases with variationat 5 points in the sequence as follows:5’ ACGA(C\T)CAAGA(A\G)(G\A)T(G\A\C)ATGGA(C\T)G 3’Using T4 polynucleotide kinase the probe was end—labelled with radioactive -yATP according to conditions recommended by the supplier, Amersham Corp., Arlington HeightsIll. The reaction mixture was heated for 10 mm. at 68°C to inactivate the T4 polynucleotide kinase. Samples to be tested (2 to 100tl), were micropipetted on to a nylonmembrane (Zetabind) and air dried for 30 mm. at room temperature. The membranewas moistened by placing it on filter paper soaked in a solution of 0.5M NaOH and 1.5M8Chapter 2. Materials and MethodsRCHKPVJ(PVOp?IPVOpSNPVPbCVTnLVACtTGGACCC GCflCTGGAT6.T..6.TG I. .CTCT CTCckiTTCCTIG.I.C.A. . .C..6. .T. .C..6. .T. .A.A. 6. GCT. CC. C. Ga?...9Figure 2.1: Nucleotide sequence of four NPV polyhedrin genes showing sequence selectedfor the oligonucleotide probe. (From Rohrmann (1986)).NaC1 at room temperature for 30 mm. The filter was then air dried for at least one hr.before being placed in a plastic sealing bag. The prehybridization solution, consisting of5x SSC (lx SSC contains 0.15M sodium chloride and 0.015M sodium citrate pH 7.0),lOx Denhardts (2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 0.05MNaPO4 aiid 50% formamide, was prewarmed and added to the bag at 100tl per cm2 ofmembrane area. The bag was sealed and incubated for at least 2 hr. at 42°C. The prehybridization solution was decanted and prewarmed hybridization solution consisting of 5 xSSC, 1 x Denhardts, 0.02M NaPO4 and 50% formamide was added to the bag at lOOplper cm2 of membrane area. Denatured salmon sperm DNA (100kg per ml hybridizationsolution) and denatured labelled probe (5t1 of stock 10.2 pmoles per pI specific activityof 106 cpm per 0.1 pmole, for a 50 cm2 membrane) were added and the filter was incubated at 42°C overnight. The membrane was washed once in a solution containing 2xSSC and 0.1%(w/v) SDS (sodium dodecyl sulfate) for 15 mm. at room temperature, onceAI4WPV CTA.AGCCCAA GAACCAC?TC GCCGAACATG A6ATCCAAGA GGCEACCCTC GACCCCCTAC ACAACTACCT ACTGGCTCAC GATCCTTTCC TGGGACCCGG 200NPV.C C.A A? A.AA..G.. .AAGCA.ATGG ..T.TT A. 6..T..C..A C..Tt .A C..Op)4NPV .C C.? CTA C. .AGAG. .T.. A. .GCA. . .6 C C A. 6. .C. .6 C. .C. . .1 ... .COpSNPV CA6 Afl. .6. .C. .AGCT CAC. .6 A. .6 A C. .C. .A Vt 1..PbIV IC. .A.AT.. A.. .C.TCC C?CCCCC.A. CCCAAATT.. AC.CCTTT.A .. .ATIGCC. . .C.A.. .A. 6. .CA.C C.... CI. .1TnGV 16.6. .A C.A. .6 ATTCGCC.A. CACAATTC.. CC.A.T.AAC .. .AI.GCCA . .C.G. . .A. C. • .A. . . .A C.. .A GA. .6..?..*L?4I(PV CAAGAACCAA AA.ACTCACTC TCTTCA.AGCA AATCCCTj.AT CTIAAACCCC ACACCATCM GCIIC?CCTF GCATCCAAAG CAAA.AGACTF CTACACGGM 300I&(?V ...A T..C L.A 66.T..C C C T.AA....C AAC....GC 161CC.?...OpNJ4PV . . .A. .C. .6..?. .A 1. .C. .C . .C. .6 CA... .6 AAC. . . .GC. .C. .6 1.TAC.T...QpSNPV. ..A 1 .6. L.A 1. .C A T AlA. .A. .A AAC. . . .66 1616PbCV A TGTT .GGA.T. .6 A.. .TC?A.ACCA . .CC AT C. .CTCC MC... .GC A.. TCTTC.T...Tb(V I 1611 ..CA.L.C I C..?.. .CGG A.CC.6..A. .T..C 616. AAC....GC 1.?GC.C...ACACAGCTTC CcCAITCrTAtAccAcc.AACA AGT6ATGGAT ?I6TCAACAT CCGTCCCACT AGArCCAACC 400l A.’ C C .C.C....C. CAAA A C.C AC. .C A C .C C C.CC T. .1 . .1 I A.C C . .A.T C . .6. . .ACAC A..CACC. C C. .A.A.. .A.C AGA.TC.AC. AA.A. .A.IC CA.. .1..... ..AGAG CACC. C...?. .6.. .A.T AGC.GC.AC. 1. .6... .16 CAC AAMIPV GT?CTTACAA ATTCCTGCCC CAACACGCTC TGCCTTCCCA CCCCCACTAT CTACCTCATC ACGTGATTAC CATCCTCCAC CCTICATGGG TGG.GCAGCAA 00jPV .6. .C C. .1 IA.G. .6 .CAA C . .6. .6. .C .A. .A. .C.. A. .TA.G A. .C.AC 16..)p(V .6. .C 1... .6 CA.G. . C . .6. .6. .C I 6. .G.AC IC..OpSkPV .C....I... .. .rr... .1 6. .. .1. .C. .C. .*. .A. . .C. C..?. .A CAGC.ACPbl,V ....C 6..TACTATC. ..T I .A6A.GCTC C ..T..C I.. .C. CGC.CA6..T . ...AC.ACA .T..TCC...ToCV .G..C C...AC.ATC ...T....C. ..GC.GCGA C ..C6....C C. CCAACA...T ..C.AC.AC. .C..ACC.C.in 0.lx SSC and 0.1%(w/v) SDS for 15 mm. at room temperature, and twice in 0.lxChapter 2. Materials and Methods 10SSC and 0.1%(w/v) SDS at 60°C for 30 mm. Autoradiography with Kodak XHR filmand Kodak lightning plus intensifying screens was used for detection. The membraneand film cassette was stored overnight in a -70° C freezer and the film processed the nextday.2.1.2 Isolation of NPVWestern tent caterpillars which were infected with MpNPV through feeding leaves contaminated with viral polyhedra were frozen after death. To obtain virus, thawed caterpillars were homogenized in distilled water using a mortar and pestle, filtered throughcheesecloth, centrifuged at 1,000 rpm (JEC Centra 4B) for 5 mm. and the pellet discarded. The supernatant was centrifuged at 3,000 rpm (IEC Centra 4B) for 15 mm. andthe pellet washed 3 times with distilled water. The pellet was resuspended in distilledwater and PIBs were isolated by centrifugation on a 45-65% w/w 5-step discontinuoussucrose gradient at 15,000 rpm for 1 hr. in a Beckman L8-70M ultracentrifuge using theSW28 rotor. The PIBs were collected from a band in the 56% sucrose region with asyringe and needle. The sucrose was removed by washing and centrifugation at 3,000rpm three times with distilled water. PIBs were resuspended in water and virions werereleased from the polyhedra by adding two times the volume of 0.1M Na2CO3 pH 10and incubating at 37°C for one hr. with constant agitation. Undissolved polyhedra wereremoved by centrifugation at 5,000 rpm for 5 mm. The supernatant was collected andcentrifuged at 20,000 rpm for 30 mm. in an ultracentrifuge (Beckman L8-70 M) using anSW41 rotor. The resulting pellet contained the virions.Chapter 2. Materials and Methods 112.1.3 Isolation of NPV DNAPelleted virions were disrupted by overnight incubation at 37°C in 0.1M Tris-HC1 (pH8.0), 10 mM EDTA (ethylenediamine tetraacetic acid), 0.1% SDS and 200g/ml proteinase K. Particulate matter was removed by centrifugation at 12, 000g in an Eppendorfmicrofuge for 5 mm. The supernatant was collected and the DNA was purified by sequential extractions in TE saturated phenol (TE is 10mM Tris-HC1, pH 8.0, 1mM EDTA), TEsaturated phenol/chloroform (1:1) and chloroform. The final aqueous phase, containingthe DNA, was concentrated by ethanol precipitation and was resuspended in TE bufferto a final concentration of 0.5tg/m1. DNA was analyzed on a 0.7% agarose gel in 0.5xTBE (1 x TBE is 0.89M Tris base, 0.89M Boric acid and 2mM EDTA) using lpl of theDNA solution.2.1.4 Production of a Cloned NPV DNA ProbeThe pure DNA was digested with the restriction enzymes BamHI from BRL, EcoRI fromPharmacia, and PstI from BRL and run on a 0.7% agarose gel in lx TAE buffer (0.04MTris-acetate, 2mM EDTA) overnight at 33 volts. The DNA in the gel was transferred toa nylon membrane (Zetabind) [Southern 19751. The nylon membrane was then washedin 2 x SSC, baked at 80°C in a vacuum oven for 2 hr. and washed for 1 hr. in 0.1 x SSC,0.5% SDS at 65°C. The membrane was then dried at room temperature. The membranewas incubated with the Autographa californica virus (AcNPV) polyhedrin gene (labelledwith 32P), obtained from D.Theilmann (Agriculture Canada), to find the fragments ofDNA from MpNPV containing the homologous polyhedrin region. Seven BamHI or PstI)fragments of approximately 2,000—8,000 bp homologous to the AcNPV polyhedrin genewere cloned into the bacterial plasmid (pGEM). DNA fragments were purified usingthe Geneclean kit from BlO 101 Inc. [Struhi 1985]. The fragments were ligated intoChapter 2. Materials and Methods 12the plasmid vector pGEM-3zf(+) using 5ng pGEM, 17.5ng insert, and T4 DNA ligaseand ligase buffer from Bethesda Research Labs, Rockville Md. The ligation mixtureswere incubated for 4 hr. at 16°C. Two ttl (or ing) of each of the ligation mixtures wascombined with 20 t1 aliquots of DH5—c competent cells (made competent by calciumchloride treatment) and incubated on ice for 30 mm. The cells were transformed byheat shock in a 42°C bath for 60 sec. and 100tl of Luria broth (LB) was added. Thetransformation mixtures were plated onto LB agar plates containing ampicillin and X—gal. Plates were incubated at 37°C overnight. Desired recombinants were screened bycx—complementation and plasmid isolation [Sambrook et al 1989].White colonies were picked off the plates and placed in a sterile tube containing LBbroth, glucose, and ampicillin. These tubes were incubated at 37°C overnight. Plasmidswere purified using a modified alkaline lysis procedure [Zhou et al 1990]. Plasmids containing pGEM and the potential probe fragment were digested with either BamHI orPstI and run on a 0.7% agarose gel in 0.5x TBE. Of the seven fragments cloned onlyplasmids containing the 2,000 bp BamHI fragment or the 8,000 bp PstI fragment, andthe pGEM were inoculated into LB broth to amplify the DNA by a large scale plasmidpreparation technique [Maniatis et al 1982]. The plasmids were digested with BamHIor PstI and the inserts were separated by electrophoresis on a 1 x TAE gel, followed bypurification as described earlier.2.2 Study Sites and Sample PreparationForest tent caterpillars, egg masses, and some parasitoids were collected from four sitesin the vicinity of Prince George:(1) north end of Range Road (north of Opsika) in Prince George,(2) 3 km north of Prince George on Hwy 97 at the Truck Scale turnoff,Chapter 2. Materials and Methods 13(3) 6 km south of Prince George on Hwy 97 towards the airport on the west side onSintich Road,(4) 10 km west of Prince George at the Westlake turnoff (Blackwater Road) on theeast side Frenkel Road and on the west side Muralt Road.The Muralt site consisted of two areas, one hilltop densely populated with FTC, thathad been severely defoliated, and an adjacent sparsely populated valley. Collections weremade at this site in 1991 only. Eggs collected from the sparse site in spring 1991 did nothatch which suggests that the low density in this location was associated with high eggmortality.The caterpillars were arbitrarily chosen while walking through each site. In 1990 thecaterpillars were collected on June 22—24 and placed in groups in one litre plastic containers, while in 1991 they were placed in individual containers following collection on June15—16. In both years the caterpillars were brought back to the laboratory and separatedaccording to general size and samples of each size weighed (see Appendix A). Caterpillarswere either frozen for future analysis by DNA hybridization or fed Alder leaves (Alnusrubra) until they pupated and emerged as adults or died. Egg shells remain on treesfollowing hatching and these were collected, when found, for counting and analysis ofviral contamination.In experiments in which caterpillars were fed virus, smaller caterpillars were placedin individual cups and given 1 cm diameter leaf discs smeared with virus. Caterpillarswere third instars in 1990 and fourth and fifth instars in 1991. They were left for 24 hr.and those that did not consume the leaf disc were discarded. The viral concentration for1990 was 11 of 1.1 x i0 PIBs/mi MdNPV obtained from Dr. W. Kaupp and for 1991,2pl of 0.6 x iO PIBs/mi MpNPV obtained from WTC which had died from MpNPV.Dead caterpillars were smeared on slides and triple stained or stained with napthalene black [Kalmakoff and Longworth 1980]. These slides were examined under a lightChapter 2. Materials and Methods 14microscope to check for the presence of polyhedral inclusion bodies (PIBs). Some pupaefrom the laboratory reared individuals were frozen for analysis. Emerged adults werealso frozen for later testing by DNA hybridization. Adults and larvae of Trichoplusiani and larvae of Peridroma saucia were obtained from laboratory cultures. Samples ofsarcophagid pupae, the fly parasitoid, were collected in the field in June or saved afteremerging from FTC larvae or pupae in the laboratory. Adults and pupae of WTC werefrom late instar larvae collected from low density populations in the vicinity of Vancouver. Cyzenis albicans, a tachinid fly parasitoid of winter moth Operophthera brumata,were collected in the vicinity of Vancouver.2.2.1 Sample Preparation for Testing with the NPV DNA ProbeCaterpillars, Pupae and MothsInsects were cut into small pieces using sterilized scissors and mashed with a cleantoothpick in a tube with 100-500[tl of sterile distilled water (depending on the size ofthe insect), vortexed and left covered overnight at room temperature. The next daythe sample was mashed again with a clean toothpick and centrifuged at 1,000 rpm for5 mm. in the microfuge. The pellet was saved and the supernatant removed and spunat 14,000 rpm for 5 mm. The supernatant was removed and frozen for storage; thepellet resuspended in 50tl of sterile distilled water and this sample was used for dot blotanalysis.Egg Mass Counts and PreparationEgg masses were carefully scraped with a sterile scalpel to separate spumaline, ahard coating over the eggs, from the eggs. After the eggs were counted, each mass wasbroken into small pieces, put in 0.5 ml of sterile distilled water in an Eppendorf tubeand sonicated for 5 mm. in a Bronson 2000 sonicator. The supernatant was removedChapter 2. Materials and Methods 15from the settled debris and 0.5 ml water was again added and sonication repeated. Thesupernatant was centrifuged at 14,000 rpm in the microfuge, the supernatant was removedand the pellet was resuspended in 20tl sterile distilled water. The sample was useddirectly for dot blotting.Parasitoid PreparationParasitoid pupae from 1991 were cleaned on the outside by dipping in 10% bleachto denature any external viral contamination. A sterile needle and syringe was used totransfer the contents of the pupal case to a tube with lOOpl sterile distilled water. Pupaefrom 1990 had dried and flies did not eclose. These were surfaced cleaned by dipping in10% bleach then cut into small pieces with sterilized scissors and put in a tube with lO0t1sterile distilled water. These tubes were vortexed and left overnight at room temperature.The next day, samples were spun at 1,000 rpm for 5 mm. in the microfuge. The pellet wassaved and the supernatant removed and spun at 14,000 rpm for 5 mm. The supernatantwas removed and frozen for storage; the pellet was resuspended in 5Opl of sterile distilledwater and used for dot blot analysis.2.2.2 Preparing Labelled ProbeA final probe concentration of 10 ng probe/mi buffer was used. The diluted DNA wasboiled for 5 mm. in a boiling water bath and immediately cooled on ice for 5 mm.centrifuged in an Eppendorf tube for 5 sec. then mixed with an equivalent volume ofDNA—labelling reagent from the Amersham ECL gene detection kit. The solution wasmixed thoroughly and gluteraldehyde from the kit added in an amount equal to theDNA—labelling reagent. The Amersham ECL or enhanced chemiluminescence systeminvolved direct labelling probe DNA with the enzyme horseradish peroxidase. This wasachieved by completely denaturing the probe so that it was single stranded and negativelyChapter 2. Materials and Methods 16charged. Peroxidase complexed to a positively charged polymer was added and it formeda loose attachment to the DNA by charge attraction. Addition of gluteraldehyde causedthe formation of chemical crosslinks so the probe was covalently labelled with enzyme.The solution was vortexed for 1 sec., centrifuged 5 sec. and either incubated 10 mm. at37°C or held on ice for 10 to 15 mm. depending on the number of nylon membranes beingprocessed at one time.2.3 Dot Blot Hybridization AssayA piece of nylon membrane (Zetabind) was first soaked in lOx SSC for 5—10 mm. andthen air dried. Two to ten tl of the test sample were spotted directly on the membraneusing a homemade template and the membrane was air dried at room temperature for 30mm. The membrane was placed face up on blotting paper saturated with denaturationsolution (0.5M sodium hydroxide + 1.5M sodium chloride), incubated at 65°C for 30 mm.then neutralized by placing on blotting paper saturated in 0.5M Tris-HC1 + 1.5M NaClpH 7.5 for 5 mm. at room temperature. The membrane was washed by placing it on filterpaper saturated with lOx SSC for 5 mm. and then air dried by placing it face up on filterpaper for a few minutes then dried at 80°C in a vacuum oven for 60 mm. The membranewas washed for 1 hr. in 0.1 x SSC 0.5% SDS at 65°C (this minimized the background).The membrane was either dried and stored in sealed bags or prehybridization followedimmediately. Blocked hybidization buffer was prepared by adding 29.2 g of NaC1 and 25g blocking agent to 500 ml hybridization buffer from the Amersham kit. This solutionwas heated in a water bath at 56°C to dissolve solids. Blotted membranes were placed inplastic hybridization bags and blocked hybridization buffer added to cover the membrane(0.25 ml blocked hybridization buffer /cm2 membrane). Each bag was sealed and placedat 42°C for at least 15 mm. (with agitation).Chapter 2. Materials and Methods 17Sealed prehybridization bags were cut open at one corner and some buffer was removedand mixed with the labelled probe. This mixture was added back to the buffer in the bagwith care to avoid putting the probe directly on the membrane. The bag was resealedand incubated at 42°C overnight or for a minimum of 8 hr.The blot was removed from the hybridization buffer bag and placed in a clean container with an excess of primary wash (> 2 ml wash /cm2 membrane) containing 360gurea, 4g SDS, and 25 ml 20 x SSC per litre of solution. This was incubated with agitationat 42°C for 20 mm. Wash buffer was discarded and the wash procedure repeated. Thewash buffer was again discarded and the blot placed in a fresh container.An excess of secondary wash buffer (2x SSC) was added to the blot (> 2 ml /cm2membrane) and incubated with agitation at room temperature for 5 mm. The wash bufferwas discarded and replaced with an equivalent volume of fresh secondary wash buffer andincubated at room temperature for 5—30 mm. depending on how many membranes wereprocessed at one time.Equal volumes of detection solution 1 and detection solution 2 from the Amersham kitwere mixed (0.125 ml / cm2 membrane). Excess buffer was drained from the washed blotsand one blot put in a clean container DNA face up. Detection buffer was added directlyto the blot and then incubation proceeded for 1 mm. at room temperature. The twodetection reagents were mixed immediately prior to use. Detection reagent 1 decayedto hydrogen peroxide, the substrate for peroxidase. Reduction of hydrogen peroxideby the enzyme was coupled to the light—producing reaction by detection reagent 2. Thiscontained luminol which on oxidation produced blue light. The light output was increasedand prolonged by the presence of an enhancer. Excess detection buffer was drained andthe blot wrapped in plastic wrap. All air bubbles were eliminated. The blot was placedDNA—side up in a film cassette and exposed to film for 60 mm. The film was thenprocessed with an automated Kodak x—ray developing machine.Chapter 2. Materials and Methods 18LJFigure 2.2: Examples of stained microscope slides (napthalene black on left and triplestaining on right) and a dot blot autoradiogram. The top row of the dot blot containedthe positive and negative controls. (From left to right; WTC PIBs, pure MpNPV DNA120 ng, 12 ng, 1.2 ng, FTC PIBs, T. ni, and P. saucia.) The other rows contained larvaesamples from Truck Scale 1991.2.4 ControlsFor each membrane probed the following controls were used; positive controls were purified PIBs from WTC, pure MpNPV DNA at 120 ng, 12 ng, 1.2 ng, and purified PIBsfrom FTC. Negative controls were lab reared Trichoplusia ni and Peridroma saucia larvae prepared in the same manner as FTC samples. The ECL gene detection system wasable to detect as little as 1.2 ng of pure control DNA.(<UChapter 3Results3.1 Preparation of an NPV DNA ProbeThe most useful DNA probe for our purposes would be one that could be used for theNPVs of either WTC or FTC. As stated earlier, the first probe made for detectionof NPV was a synthetic degenerate oligonucleotide probe which consisted of a knownnucleotide sequences. The prepared degenerate synthetic oligonucleotide probe did notgive a sufficient signal for reliable detection. The control samples were not detectablewith this probe and only one tent caterpillar sample out of 25 gave a detectable result.This technique was abandoned in favor of cloning a sequence of NPV DNA to useas a probe. Cloning had several advantages. It provided a large quantity of probe, along probe, and a probe with complete homology to NPV from WTC. Chemiluminescence detection could be substituted for the radioactive detection as required for theoligonucleotide. To prepare a probe for tent caterpillar NPV, polyhedra were isolatedfrom infected western tent caterpillars (WTC) because sufficient material was available.This virus infects forest tent caterpillars and the European tent caterpillar Malacosomaneustria (J.H. Myers, personal communication) [Stairs 1989]. To make the probe, it wasnecessary to extract NPV as PIBs from dead larvae, disrupt the PIBs and isolate theDNA. When the DNA was electrophoresed on a 0.7% agarose gel in 0.5 x TBE, the gelshowed a single band of large molecular weight DNA.The polyhedrin gene is known to be highly conserved in virus from at least the three19Chapter 3. Results 20Figure 3.1: Restriction enzyme digestion of MpNPV DNA and detection of the poiyhedrin gene by Southern blot analysis.(I) Ethidium bromide stained agarose gel (0.7%) containing MpNPV DNA digestedby restriction enzymes (lane A EcoRI, lane B PstI, lane C BamHI, lane D BamHI andpBR325 with AcMNPV EcoRI—I fragment, and lane E a marker representing the DNAfragments generated by HindIII digestion of A genomic DNA).(H)Autoradiogram of a. Southern blot of the gel from (I) probed with an isolatedfragment of AcMNPV encoding the polyhedrin gene. The probe was prepared from aAcMNPV EcoRI—I DNA fragment cloned into the plasmid pBR325 restricted with BamHIand labelled with 32P. The hybridization was detected by exposing the film overnight at—70°C. The EcoRI, PstI, and BamHI lanes correspond to the same lanes in the ethidiumbromide stained gel in (I). The bands selected for cloning are indicated by the bars.IA BC 0 EIIA BC 0PstJBamHIChapter 3. Results 21species of insects sequenced to date [Rohrmann 1986]; [Smith et al 1983]. Isolationand cloning of a fragment in the polyhedrin region of NPV produces a probe from aspecific part of the viral genome, and one that probably would hybridize to both MdNPVand MpNPV since Lepidopteran NPV polyhedrins demonstrate about 90% homology asstated earlier in the introduction.The DNA was digested with restriction enzymes and the Southern blot probed witha clone of the AcNPV polyhedrin gene region to identify fragments from the MpNPVpolyhedrin region (see figure 3.1). A 2,000 bp (approximate) BamHI fragment and a8,000 bp (approximate) PstI fragment homologous to the AcNPV polyhedrin were cloned.The 2,000 bp BamHI fragment was selected as the most suitable probe because of its’smaller size.3.2 Comparison of Viral Detection using the DNA Probe with MicroscopicExamination for PIBsTo determine if the occurrence of NPV shown with the NPV DNA probe was similarto that observed in microscopic examination for PIBs, the percentage of caterpillarscollected from Prince George that tested positive with the probe was compared to thepercentage of field collected caterpillars that died of virus in the laboratory and examinedfor PIBs. Insects dying from parasitization were excluded from microscopic examination.Caterpillars from the four sites in the Prince George area in 1990 and 1991 were screenedusing both techniques.Data from the two detection methods examined in 1990 (Table 3.1), show statistically significant heterogeneity between the two techniques for caterpillars for two locations. At Range Road, the percentage of caterpillars positive for NPV was higherwith the microscopic examination technique (0.01 < p < 0.025), while caterpillars fromChapter 3. Results 22Truck Scale showed a higher frequency of NPV infection using the DNA probe technique (0.025 < p < 0.050). In 1991, percentages of caterpillars infected at RangeRoad and Truck Scale were not significantly different for the two techniques while percentages of infection at Airport were significantly higher with microscopic examination(0.01 <p < 0.025) and at Westlake with the probe method (0.01 <p < 0.025). Somesample sizes in the 1991 microscopic examination method are small so one must be cautious about interpretation. However, no pattern is apparent.A statistical comparison of the four sites in 1990 using the microscopic examinationmethod indicates that the sites are significantly different (x2 = 29.6, 3df, p < 0.001) andwith the probe method also significantly different (x2 = 18.3, 3df, p < 0.001). In 1991,a comparison of the four sites using the microscopic examination method approachessignificant difference (x2 = 7.6, 3df, 0.1 <p < 0.05) and is highly significantly differentbased on results using the probe method (x2 = 20.4, 3df, p < 0.001).A comparison of the same area over two years for the same detection technique forthe four areas indicates no significant difference in Range Road, Airport, and Westlake,while there is a significant difference in the Truck Scale samples, using the probe technique(x2 = 19.1, ldf, p < 0.001), with positive individuals being less common in 1991.In summary, there is a great deal of heteogeneity in the occurrence of virus withinand among the FTC populations. Large sample sizes are necessary to overcome thisvariation. Because it is much easier to process large numbers of samples with the DNAprobe technique, its benefits are evident.Chapter 3. Results 23Area 1990 1990 x2 1991 1991 x2Microscope Probe df=1 Microscope Probe df=1% NPV (N) % NPV (N) % NPV (N) % NPV (N)Range Road 66 (59) 39 (44) 6.60 55 (11) 44 (80) 0.12Truck Scale 47 (43) 71(38) 4.03 55 ( 9) 26 (76) 2.10Airport 15 (33) 24 (25) 0.26 38 (13) 7 (55) 6.40Westlake 16 (31) 22 (27) 0.06 17 (53) 35 (191) 5.20Table 3.1: Comparison of percentage of caterpillars with NPV based on microscopicexamination and DNA probe detection from four areas near Prince George B.C. in 1990and 1991. (N)=sample size and x2 is Yates corrected.Chapter 4Results4.1 Occurrence of NPV in Pupae, Adults, Egg Masses and a Parasitoid ofForest Tent Caterpillars4.1.1 PupaeBecause polyhedra are rarely observed in pupae, only larvae could be tested for virus bymicroscopic examination. In contrast, the viral specific DNA probe can detect virus inother life stages of forest tent caterpillars. In 1991, sufficient pupae were obtained fromrearing caterpillars from two sites to test for the presence of NPV with the DNA probe.Area Formed Malformed% NPV (N) % NPV (N)Range Road 32 (25) 14 (22)Total 23 (47)Airport male 17 (12) 30 (10)Airport female 38 (8)Total 27 (30)Overall total 25 (77)Table 4.1: Percentage of pupae with virus based on the DNA probe in 1991.While only 7% of the caterpillars from the Airport site were positive for NPV with theDNA probe, approximately 27% of the pupae were positive. Samples from Range Roadshow the opposite trend with 44% of caterpillars being positive for NPV and only 23% ofpupae being positive (see Tables 4.1 and 3.1). One may have expected lower percentages24Chapter 4. Results 25of NPV in pupae since many infected caterpillars would not survive to pupate. Table 4.1reveals that incomplete pupation does not appear to be associated with the virus sincepercentages of viral infection were not higher in malformed pupae.4.1.2 AdultsArea %NPV (N)Truck Scale 100 (5)Airport 100 (2)Westlake Frenkel 100 (10)Westlake Muralt sparse 93 (15)Total 97 (32)Table 4.2: Percentage of adults with virus based on the DNA probe in 1991.A very high percentage of adult moths were positive for NPV (see Table 4.2). Todetermine if these positive results were due to some aspect of the DNA of adult Lepidoptera, adults of other species were tested with the probe. Cabbage looper, T.ni, from alaboratory population had 63% (N=32) positive for NPV while winter moth, O.brumata,from a field population had 0% positive (N=17). Of western tent caterpillar adults tested39% were positive (N=13) for field collected caterpillars reared to adults and 26% positive(N=19) for moths from caterpillars that had been exposed to NPV but lived to pupateand emerge as adults.Overall the results show that there is variation in the detection of virus using theDNA probe on adult insect samples. However, percentages of virus infection are highestin adult forest tent caterpillars from an outbreak area (Prince George).Chapter 4. Results 264.1.3 Egg MassesThe percentages of egg masses, (see Table 4.3), positive for NPV are similar to that ofcaterpillars tested with the probe from the same site. The Truck Scale 1990 results areparticularly interesting as the egg masses were collected in the winter of 1991 (hatched1990), and therefore had been outside for many months. A x2 test comparing the % NPVin egg masses to the % NPV in caterpillars showed a significant difference in Truck Scale1990 (x2 = 6.7, df=1, 0.005 <p < 0.01) only. A x2 test comparing egg masses from allfour sites showed no significant difference between sites (x2 = 6.6, df=3, 0.05 <p < 0.10).Area Egg Mass Caterpillar%NPV (N) %NPV (N)Range Road 1991 51 (43) 44 (80)Truck Scale 1991 19 (16) 26 (76)Truck Scale 1990 37 (30) 71 (38)Westlake 1991 20 (20) 35 (191)Table 4.3: Percentage of egg masses from sites near Prince George B.C. positive for NPVbased on the DNA probe compared to corresponding caterpillar data from Table 3.1.4.2 Viral Transmission4.2.1 Virus Feeding ExperimentsThird instar forest tent caterpillars (1990) and fourth and fifth instars (1991) were fedNPV and monitored through their larval and pupal development. Some individualssurvived to the adult stage (Table 4.4) while others died of viral infection (Table 4.5). Ahigh percentage of adults that emerged from larvae fed virus gave positive responses tothe DNA probe. Caterpillars fed virus had a percentage infection rate similar to thoseof larvae collected in the field for each area in 1990. Too low a dose of virus may haveChapter 4. Results 27been provided or perhaps the virus used was no longer active. There seemed to be morecaterpillars surviving to pupation in the Airport and Westlake areas which had lowerpercentages of NPV infection (Table 4.5). This suggests that infection with NPV priorto collection may have been more important than the ingestion of virus in the laboratory.Area %NPV (N)Muralt dense 83 (6)Muralt sparse 100 (4)Westlake 100 (5)Total 93 (15)Table 4.4: Percentage of adults developing from caterpillars fed virus in 1991 positive forNPV based on the DNA probe.Area %NPV (N) pupatedRange Road 57 (30) 4Truck Scale 37 (27) 6Airport 14 (36) 21Westlake 15 (33) 19Table 4.5: Percentage of caterpillars positive for NPV which were fed virus in 1990.Detection done using microscopic examination.Area Control Exposed to Virus% NPV (N) % NPV (N)Airport 57 (7) 33 (15)Westlake 52 (25) 25 (20)Combined controls 53 (32)Combined exposed to virus 29 (35)Table 4.6: Percentage of caterpillars reared from egg masses collected in early spring(1991) that were positive for NPV using DNA probe.Chapter 4. Results 28To test for vertical transmission of virus to the next generation, late instar foresttent caterpillars were fed a virus dose in one year and overwintering eggs obtained.Caterpillars which emerged from these eggs were tested for the presence of virus duringthe third instar. These results appear in Table 4.6 and show higher percentages of virusinfection for control insects, that is, those not fed virus, than for caterpillars fed virus.Percentages are higher than in those collected from the Airport site that year and higherfor controls at Westlake. There was no significant difference between control and virusexposed in the x2 analysis for Airport or Westlake.4.2.2 Fly ParasitoidsTo determine if fly parasitoids could be transmitting NPV, sarcophagid pupae from foresttent caterpillars were tested using the DNA probe (Tables 4.7, 4.8 and 4.9). A high percentage of fly pupae were positive for virus in both 1990 and 1991. Since the parasitoidsdevelop inside the caterpillar, it is not surprising that they would be contaminated bythe virus. However, the percentage of parasitoids positive for the virus in 1990 is greaterthan that of caterpillars collected and tested that year. One parasitoid female visits morethan one caterpillar during oviposition so she could pick up virus from one caterpillar andspread it to another when ovipositing thereby infecting the host and its own offspring.Infection percentages in parasitoids therefore could be higher than in caterpillars.To see if similar results occurred in another population of FTC parasitoids, sampleswere obtained from Ontario in 1991. Seventy—nine percent of these (N=14) were positivefor NPV with the DNA probe. Adults of Cyzenis albicans, a tachinid fly parasitoid ofwinter moth, had 0% positive for NPV (N=11) which is consistent with the lack of NPVin the adult winter moth hosts (see above).Chapter 4. Results 29Area %NPV (N)Range Road 100 (20)Truck Scale 100 (39)Airport 97 (35)Westlake 100 (28)Total 99 (122)Table 4.7: Percentage of pupae of fly parasitoids emerging from laboratory—reared caterpillars collected from sites near Prince George in 1990 that were positive for NPV asindicated by the DNA probe.Area % NPV (N)Range Road 0 (3)Truck Scale 50 (2)Westlake 100 (2)Total 43 (7)Table 4.8: Percentage of pupae of fly parasitoids collected in the field from sites nearPrince George in 1991 that were positive for NPV as indicated by the DNA probe.Area % NPV (N)Range Road 100 (3)Truck Scale 50 (4)Airport 100 (4)Westlake 100 (3)Total 86 (14)Table 4.9: Percentage of pupae of fly parasitoids emerging from laboratory—reared caterpillars collected from sites near Prince George that were positive for NPV in 1991 asindicated by the DNA probe.Chapter 5Results5.1 Impact of the Virus on the Prince George Population of Forest TentCaterpillarsThe previous sections of this thesis have dealt with the distribution of virus in the FTCat Prince George B.C. The impact of virus on the host population will now be considered.Viral infection could affect a population in different ways. Virus could kill caterpillarsin early instars resulting in fewer later instars showing infection, or virus might slowdevelopment causing smaller caterpillars to have an apparently higher infection rate thanlarge ones. The DNA probe was used to test for the presence of infection in caterpillarsof different sizes. It was also used to test different—sized egg masses for the presence ofinfection.5.1.1 Caterpillar SizeThe relationship between caterpillar size and percentage of infection with NPV in 1990and 1991 are shown in Table 5.1. In two cases there is a significant statistical differencein the % NPV between small and large caterpillars; Range Road 1991 (p < 0.001) andTruck Scale 1991 (0.025 <p < 0.050) and in both of these a higher proportion of smallercaterpillars was positive for NPV. In six of eight comparisons the trend follows thisdirection.30Chapter 5. Results 315.1.2 Association between the Occurrence of NPV and Egg Mass Size inForest Tent CaterpillarsEgg masses from the Airport population appear to have more eggs per mass than thosefrom other sites, but the sample size is very small (Table 5.2). Similar percentages ofegg masses and caterpillars were infected with NPV (Table 4.3) for the different populations. However, a comparison of percentages of infection of egg masses of differentsizes (Table 5.3) shows that significantly fewer small egg masses are infected with NPV(x2 = 4.2, df=1, 0.025 <p < 0.05).Chapter 5. Results 32Area 1990 1990 x2 1991 1991 x2Small Large df=1 Small Large df=1% NPV (N) % NPV (N) % NPV (N) % NPV (N)Range Road 38 (16) 0 (10) 3.0 68 (37) 14 (35) 18.9Truck Scale 67 (12) 82 (11) 0.1 37 (38) 14 (37) 4.2Airport 30 (10) 21 (14) 0.0 15 (20) 3 (35) 1.2Westlake 30 (10) 13 (15) 0.3 29 (89) 38 (96) 1.1Table 5.1: Percentage of caterpillars positive for NPV based on caterpillar size. TablesA.1 and A.2 (see appendix) give the weights of caterpillars in the four areas and someindication of what is considered a small weight compared to medium and large for bothyears.Area Mean s Standard deviation (N)Range Road 1991 109 45.7 (43)Airport 1991 172 23.6 (4)Truck Scale 1991 100 44.4 (18)Truck Scale 1990 118 40.3 (30)Westlake 1991 96 30.9 (19)Table 5.2: Mean number of eggs/mass, s, (N), for forest tent caterpillar egg massescollected from sites near Prince George, B.C.Egg mass size % NPV (N)Small 0-100 27 (49)Medium 101-200 48 (61)Large >201 67 (3)Overall 39 (113)Table 5.3: Combined 1990-91 egg masses probed positive for NPV compared in terms ofegg mass size. The small egg masses came from various sites; Range Road=17, TruckScale=20, Airport=0 and Westlake=12. The medium egg masses came from; RangeRoad=25, Truck Scale=25, Airport=3 and Westlake=8. The large egg masses were oneeach from Range Road, Truck Scale and Airport.Chapter 6Discussion6.1 Development of the NPV DNA ProbeThe purpose of this study was to develop a DNA probe specific to NPV that could be usedto detect early viral infections in tent caterpillars and to trace the distribution of the virusamong host life stages (eggs, pupae, adults) as well as in other agents of potential spreadsuch as parasitoids. Historically NPV infection has been studied by collecting caterpillarsin the field and rearing them in the laboratory to determine if virus developed, or toprepare smears of caterpillars to look for polyhedra. While these techniques have beenwidely used in investigations of NPV in Lepidoptera and sawfiies [Evans and Entwistle1987] they are time consuming, subject to possible error through contamination in thelaboratory, and misinterpretation of early infections in which polyhedra may not yet beapparent. Detecting NPV in field samples of soil or leaves using microscopic examinationcan be extremely difficult when concentrations of polyhedra are low.It is necessary to calibrate results from the DNA probe to those from microscopicexamination for PIBs to determine consistency. Clearly there is variation between thetwo techniques from the samples of forest tent caterpillars collected over four sites intwo years. While the percentages infected varied significantly between detecting viruswith a DNA probe and microscopic examination for PIBs, there were no consistent trendstowards one technique showing more or fewer infected individuals. Several factors differedbetween caterpillars tested by the probe and those reared in the laboratory. The biggest33Chapter 6. Discussion 34difference was that caterpillars tested using the DNA probe were not segregated intothose that were parasitized and those that were not. Laboratory rearing of caterpillarsshowed that parasitization by sarcophagid flies was high in 1991 and that most of thoseflies were positive for virus using the DNA probe. Since parasitized insects were excludedfrom the total and we assume that all parasitized caterpillars were positive for NPV andwould have died of virus infection if they had not been killed by the parasitoid, this wouldhave increased the percentage infected by NPV in the laboratory—reared group used inmicroscopic examination.Another bias affecting the results was that smaller—sized caterpillars were selected forlaboratory rearing. A higher percentage of smaller caterpillars tended to be positive forNPV although this wasn’t the case for all sites. Significantly more small caterpillars werepositive for NPV in 1991 for Range Road and Truck Scale. In the two situations in whichmicroscopic examination for PIBs indicated more viral infection than in probed material(Range Road 1990 and Airport 1991), more smaller caterpillars tended to be positive forNPV with the probe than were large caterpillars. The opposite was the case for the twosites in which microscopic examination for PIBs indicated less viral infection than did theprobed samples (Truck Scale 1990 and Westlake 1991). At these sites large caterpillarsshowed more virus. Therefore part of the heterogeneity between the results using theprobe and those with microscopic examination may have been associated with caterpillarsize. Note also the small sample sizes used for microscopic examination in 1991 thatwere partially caused by the high parasitization that year. Because of the heterogeneityin distribution of virus in field populations, large sample sizes are needed to accuratelyestimate patterns of viral infection. DNA hybridization is a more appropriate techniquefor large sample sizes since microscopic examination of slides for PIBs is very labourintensive.In a laboratory study of the gypsy moth Lymantria dispar, larvae were inoculated withChapter 6. Discussion 35gypsy moth NPV (LdNPV). The occurrence of virus as detected by a DNA probe madefrom LdNPV and infection indicated by mortality observed in reared larvae were closelycorrelated [Keating et al 1989]. In the gypsy moth study virus was first detected by DNAhybridization to the probe four days after inoculation. The probe used was prepared frompurified viral DNA and radiolabelled. This probe differs from mine which was specificfor the polyhedrin gene of NPV. The authors state that their study demonstrates thefeasibility of using a DNA probe to determine virus incidence in a larval population.Because their study was a laboratory study, conditions were much more standardizedthan in studies of field populations so their results showed less heterogeneity than theforest tent caterpillars of Prince George.6.2 Occurrence of NPV in Different Host Life StagesOne of the main advantages of using the NPV DNA probe is that other types of samples,in addition to larvae, can be tested for the presence of virus. Virus was detected in pupae,adults, and egg masses of FTC.Previous studies offer mixed results on viral detection of these stages. Stairs (1965b)found virus present in both pupae and adults of the wax moth Galleria mellonella usingthe technique of sectioning insects after embedding in paraffin. Evans (1983), using thedetection technique of microscopic examination for PIBs, found some virus in pupaebut none in adults of the cabbage moth Mamestra brassicae. Whitlock (1977) detectedno death from virus in either pupae or adults of laboratory reared boliworms, Heliothisarmigera, in his mortality studies. Murray et al (1991) found NPV DNA in some pupae,none in adults, and no virus on the surface of eggs of laboratory reared gypsy moth,Lymantria dispar. They used a technique whereby DNA from mascerated abdomens orinsect hemolymph was purified and screened with a DNA probe. NPV was not detectedChapter 6. Discussion 36in any host surviving to the adult stage. Virus was fed to fourth and fifth instars as wellas larvae 4—9 days prior to pupation. These results contradict my findings with the FTCadults. However, they fed their larvae at later instar stages. It has been shown that aslarvae mature the concentration of NPV necessary to kill them also increases [Shapiroet al 1986]. Also they were using a laboratory population of insects compared to a fieldpopulation in my study. Finally, in their study DNA was purified from samples and thenhybridized to the DNA probe instead of using insect homogenates as I did.The FTC pupae tested in the present study showed the same (Range Road) or slightlyhigher (Airport) percentages of virus infection in pupae than in larvae from the same area.The important finding is that virus is present in pupae.The percentage of NPV infection in adults is very high in my FTC samples. Onewould expect the percentage of infection in adults to be reduced since many infectedcaterpillars would have died before pupation. In the laboratory FTC spend more timedeveloping to pupation, and then emerging as adults. It is possible, though unlikely, thatthey became contaminated with virus while in the laboratory. Another possibility is thatthe insects that do emerge as adults may be more resistant and/or the virus has changedin virulence. In a study by Lobinger and Skatulla (1991), passage of a virus through asusceptible Chinese race of gypsy moth led to a change in the pathogenic qualities of thatvirus strain and an enlargement of the host range. Others have suggested that resistanceof Lepidoptera might change over a population cycle [Myers 1990]. If resistance doeschange over a development cycle or over a population cycle, virus could be present butnot lethal.To determine if there was some characteristic of adults that resulted in the highfrequency of positive responses, other species of adult Lepidoptera were tested. Theseshowed some positive responses even in laboratory populations unexposed to NPV formany generations, although the percentages were not as high as for FTC. High percentageChapter 6. Discussion 37of occurrence of virus in adults in an outbreak population may be significant. Selectionpressure for more resistant strains could make the adult stage less suseptible to viraldeath but capable of maintaining and spreading the virus. This possibility requiresfurther study.In most cases the percentage of infection of egg masses with NPV corresponds to thatof the presence in caterpillars for the different areas. Egg masses are exposed for manymonths to all kinds of weather. This presence of virus argues strongly for environmentalpersistance of the virus [Jaques 1985]. Egg mass contamination could also play a role intransmission of the virus. In a study on Lymantria dispar [Murray and Elkinton 1989], theauthors concluded that in NPV—contaminated sites environmental contamination of eggmasses is an important means of transmission of NPV from one generation to the next.In another study on L. dispar [Woods et al 1989], larvae from surface—disinfected eggmasses acquired NPV when they moved over bark treated with NPV and mortality wasrelated to the degree of contamination of the bark. Bark surfaces may play an importantrole in transmitting NPV. Larvae which die from virus contaminate their environmentand virus could be spread around, by various means such as other insect species, to newlylaid egg masses. The presence of NPV on egg masses suggests the possibility that viruscould be passed on to emerging larvae when they eat their way out of the egg mass.Overall it seems that NPV persists in different life stages of the host. Many studieshave been done on the persistence of baculoviruses and these are reviewed in Jaques(1985). Detection of NPV on egg masses is consistent with previous reports.Chapter 6. Discussion 386.3 Viral Transmission6.3.1 Virus Feeding ExperimentsTransmission may be defined as the process by which a pathogen is passed from a source ofinfection to a new host [Andreadis 1987]. Transmission is horizontal when the pathogenis transferred from individual to individual but not directly from parent to offspring.Transmission is vertical when there is direct transfer of the pathogen from a parentorganism to his or her progeny or from one generation to the next. In most host insects,vertical transmission occurs entirely through the female. Infections may arise in two waysdepending on whether the passage of the pathogen occurs within the ovary (transovarian)or on the surface of the egg (transovum). Transovum transmission appears to be theprincipal method of vertical transfer of most baculoviruses [Andreadis 1987].In this thesis horizontal transmission was tested in an experiment where caterpillarswere fed virus and adults which emerged tested for NPV. A high proportion of adults thatemerged from larvae fed virus responded positively to the NPV probe, but caterpillarsfed virus had a percentage infection rate similar to those found in the field for each areawhich was far less than that found in adults. This could indicate that adults are lesssusceptible to death by NPV.Caterpillars which emerged the following year from eggs of parents fed virus as caterpillars the previous year were found to contain virus. These results suggest that viruscan be transmitted through developmental stages and into the next generation indicatingsome vertical transmission. Both transmission experiments were preliminary and pointto the need for further research using DNA hybridization detection of NPV.Chapter 6. Discussion 396.3.2 Fly ParasitoidsA high proportion of fly parasitoids gave a positive response to the NPV DNA probe.While it is to be expected that parasitoids of infected larvae could incorporate the virus,the frequency of parasitoids responding positively was greater than that of the caterpillars. It is possible that the high percentage of viral contamination in the parasitoidsresults from the host—to—host movement of their mothers transmitting virus during oviposition. The effect of such a process would be to make it more likely that a parasitizedcaterpillar would also be infected by the virus. At present there is no evidence to supportsuch a hypothesis but it could be tested using the probe. Stairs (1966) postulated thatthe fly parasitoid was a transmission agent for NPV in FTC but had no experimentaldata to support his claim.6.4 Caterpillar SizeFor the data collected in most sites small caterpillars had a higher percentage of NPVthan larger ones. Infection by virus could retard the growth of the larvae. This wouldbenefit the virus by having more time to replicate in early instars of the host. One impactof the virus on the host could be reduced growth of the caterpillar.6.5 Association between Occurrence of NPV and Egg Mass Size in ForestTent CaterpillarsThe percentage of infection in egg masses shows generally the same trends as the detectionof NPV in caterpillars using the DNA probe for the four areas studied. When percentageof infection of egg masses is examined in terms of egg mass size it appears that small eggmasses have a lower percentage of infection than other sizes. The smaller egg masses arelikely laid by smaller females, suggesting that either smaller females deposit less virusChapter 6. Discussion 40on egg masses or that fewer small females carry the virus. Smaller females may be moreresistant or less susceptible to viral infection and therefore would be less likely to contractthe disease and pass it on to the eggs. A tempting conclusion might be that the costof this resistance is reduced fecundity [Myers 1990]. However, there is another possibleexplanation. It is also possible that virus is easier to find on larger egg masses. Largeregg masses with greater surface area would carry more virus. This could increase thelikelihood of obtaining a positive response to the probe. Again, further investigation isrequired.6.6 ConclusionsEvery laboratory technique has some drawbacks and the DNA probe is no exception.Care must be taken to have the proper stringency of washes to remove all unboundprobe from the membrane. If this is not done false positives could result. This can bemonitored by the use of negative controls. Some of the dot blots came out as rings ofvarious intensity. This was probably due to diffusion of the DNA away from the centre ofthe dot. Samples which gave weak signals in detection should be tested for the presenceof viral DNA by extraction and purification of the DNA and repeating the dot blot.Despite these drawbacks, the DNA probe detection technique provides a tool forexamining virus infection in a field population of host insects. Many samples can beprocessed to compensate for the heterogeneity of the population. Use of this techniquewill allow research on NPV of tent caterpillars to be done more efficiently and over abroader scope.BibliographyAndreadis, T.G. (1987) Transmission. In Epizootiology of Insect Diseases (Ed. byJ. Fuxa and Y. Tanada), John Wiley, New York. 159—176.Carruthers, R.I., and Soper, R.S. (1987) Fungal diseases. In Epizootiology of InsectDiseases (Ed. by J. Fuxa and Y. Tanada), John Wiley, New York. 357—415.Chitty, D. (1967) The natural selection of self-regulatory behaviour in animal populations. Proc. Ecol. Soc. Aust. 2:51—78.Evans, H.F. and Entwistle, P.F. (1987) Viral Diseases. In Epizootiology of InsectDiseases (Ed. by J. Fuxa and Y. Tanada), John Wiley, New York. 257—322.Evans, H.F. (1983) The influence of larval maturation on responses of Mamestrabrassicae L. (Lepidoptera:Noctuidae) to nuclear polyhedrosis infection. Arch. Virol.75:163-170.Jaques, R.P. (1985) Stability of insect viruses in the environment. In Viral Insecticides for Biological Control. (Ed. by K. Maramorosch, and K.E. Sherman)Academic Press, Orlando. 285—360.Kalmakoff, J. and Longworth, J. (eds.) (1980) Microbial control of insect pests,Bulletin 228, New Zealand Dept. of Scientific and Industrial Research, Wellington.21—42.Karban, R. and Myers, J.H. (1989) Induced plant responses to herbivory. Annu.Rev. Ecol. Syst. 20:331—348.41Bibliography 42Keating, S.T., Burand, J.P. and Elkinton, J.S. (1989) DNA hybridization assay fordetection of gypsy moth NPV in infected gypsy moth Lymantria dispar L. larvae.Appl.Environ. Microbiol. 55,No. 11:2749—2754.Lobinger, G. and Skatulla, U. (1991) On the susceptibility of different geographicalraces of the gypsy moth Lymantria dispar L.; Lep., Lymantriidae to a nuclear polyhedrosis virus of the rusty tussock moth Orgyis antiqua Hbn.; Lep., Lymantriidae.J. Appi. Ent. 111:327—334.Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Habor Laboratory Press. 88—94.MacLeod, D.M. and Tyrrell, D. (1979) Entomophthora crustosa N. sp. as apathogen of the forest tent caterpillar, Malacosoma disstria. Can. Ent. 111:1137—1144.Murray, K.D. and Elkinton, J.S. (1989) Environmental contamination of egg massesas a major component of transgenerational transmission of gypsy moth nuclearpolyhedrosis virus (LdMNPV). J. Invert. Pathol., 53:324—334.Murray, K.D., Shields, K.S., Burand, J.P. and Elkinton, J.S. (1991) The effectsof gypsy moth metamorphosis on the development of nuclear polyhedrosis virusinfection. J. Invert. Pathol. 57:352—361.Myers, J.H. (1988) Can a general hypothesis explain population cycles in forestlepidoptera?, Adv. Ecol. Res., 18:179—242.Myers, J. (1990) Population cycles of western tent caterpillars: experimental introductions and synchrony of fluctuations, Ecology 71:986—995Rohrmann, G.F. (1986) Polyhedrin Structure, J. Gen. Virol. 67:1499—1513.Bibliography 43Shapiro, M., Robertson, J.L., and Bell, R.A. (1986) Quantitative and qualitativedifferences in gypsy moth (Lepidoptera: Lymantriidae) nucleopolyhedrosis virusproduced in different—aged larvae. J. Econ. Entomol. 79:1174—1177.Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Habor Laboratory Press 1.82—1.84.Smith, G., Viak, J. and Summers, M. (1983) Physical analysis of Autographa californica nuclear polyhedrosis virus transcripts for polyhedrin and 10,000-molecular-weight protein, J. Virol. 45:215—225.Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis., J. Mol. Biol. 98:503—517.Stairs, G.R. (1965a) Artificial initiation of virus epizootics in forest tent caterpillarpopulations. Can. Ent. 97:1059—1062.Stairs, G.R. (1965b) The effect of metamorphosis on nuclear polyhedrosis virusinfection in certain Lepidoptera. Can. J. MicrobioL 11:509—512.Stairs, G.R. (1966) Transmission of virus in tent caterpillar populations. Can. Ent.98:1100—1104.Stairs, G.R. (1989) Effects of a nuclear polyhedrosis virus isolate from Malacosomadisstria on Lymantria dispar larval growth pattern. J. Invert. Pathol. 53:247—250.Stehr, F.W. and Cook, E.F. (1968) A revision of the genus Malacosoma Hubner inNorth America (Lepitdoptera: Lasiocampidae): Systematics, biology, immaturesand parasites. U.S. Nat. Mus. Bull. 276 321 pp.Bibliography 44Struhi, K. (1985) A rapid method for creating recombinant DNA molecules.Bio Techniques, 3:452—453.Tanada, Y. and Fuxa, J.R. (1987) The pathogen population. In Epizootiology ofInsect Diseases (Ed. by J. Fuxa and Y. Tanada), John Wiley, New York. 113—158.Turnquist, R. (1987) Maps of major forest insect infestations Prince George forestregion 1944—1986. Forest Insect and Disease Survey, Report 87—11, CanadianForest Service. 1—36.Ward, V.K., Fleming, S.B., and Kalmakoff, J. (1987) Comparison of a DNA-DNAdot-blot hybridisation assay with light microscopy and radioimmunoassay for thedetection of a nuclear polyhedrosis virus., J. Virol. Meth., 15:65—73.Watanabe, H. (1987) The host population. In Epizootiology of Insect Diseases (Ed.by J. Fuxa and Y. Tanada), John Wiley, New York. 71—112.Wellington, W.G.(1960) Qualitative changes in natural populations during changesin abundance, Can. J. Zool. 38:290—314.Wellington, W.G, Cameron, P.J., Thompson, W.A., Vertinsky, I.B., Landsberg,A.S. (1975) A stochastic model for assessing the effects of external and internalheterogeneity on an insect population. Res. Popul. Ecol. 17:1—28.Whitlock, V.H. (1977) Effects of larval maturation on mortality induced by nuclearpolyhedrosis and granulosis virus infections of Heliothis armigera. J. Invert. Pathol.30:80—86.Woods, S.A., Elkinton, J.S., and Podgwaite, J.D. (1989) Aquisition of nuclearpolyhedrosis virus from tree stems by newly emerged gypsy moth (Lepidoptera:Lymantriidae) larvae. Environ. Entomol. 18:298—301.Bibliography 45Zhou, C., Yang, Y., and Jong, A.Y. (1990) Mini—prep in ten minutes. Bio Techniques, 8 No.2:172—173.Appendix AMean Weights of CaterpillarsThe following two tables are mean weights of caterpillars collected in the Prince Georgearea 1990 and 1991. The caterpillars were arbitrarily selected in the field and visuallydivided into size groups. They were weighed on a Mettler balance to estimate the actualweight differences between groups.Area mean weight in g s (N)SMALLRange Road 0.1273 0.0417 (5)Truck Scale 0.1005 0.0279 (5)Airport 0.1482 0.0383 (5)MEDIUMRange Road 0.2983 0.0712 (5)Truck Scale 0.3449 0.0790 (5)Airport 0.3725 0.0540 (5)Westlake 0.4408 0.1511 (5)LARGETruck Scale 0.5068 0.1321 (5)Airport 0.5025 0.0752 (5)Westlake 0.6956 0.1463 (5)Table A.1: Mean weight ( ± standard deviation) of caterpillars collected in the vicinityof Prince George B.C. 1990.46Appendix A. Mean Weights of Caterpillars 47Area and size mean weight in g s (N)SMALLRange Road 0.1091 0.0517 (10)Truck Scale 0.1312 0.0587 (10)Airport 0.3127 0.1215 (10)Westlake Frenkel 0.1416 0.0537 (10)Westlake Muralt dense 0.1852 0.0814 (10)Westlake Muralt sparse 0.0566 0.0822 (5)LARGERange Road 0.6426 0.1781 (10)Truck Scale 0.7474 0.1609 (10)Airport 0.8389 0.2011 (10)Westlake Frenkel 0.7806 0.1774 (10)Westlake Muralt dense 0.7567 0.1245 (10)Westlake Muralt sparse 0.6568 0.2205 (10)Table A.2: Mean weight (± standard deviation) of caterpillars collected in the vicinityof Prince George B.C. 1991.

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