TRANSMISSION OF A NUCLEOPOLYHEDROVIRUS IN BALSAM FIR SAWFLY (Neodiprion abietis) AND ITS IMPLICATIONS FOR INTEGRATED PEST MANAGEMENT IN WESTERN NEWFOUNDLAND by
Roger William Graves, B.Sc., B.Sc.F., RPF (NB)
Bachelor of Science Biology, Dalhousie University (1992); Bachelor of
Science Forestry, University of New Brunswick (1997)
A Thesis Submitted in Partial Fulfillment of
The Requirements for the Degree of
Master of Science
In the Graduate Academic Unit of Forestry and Environmental Management
Supervisor(s): Dr. Daniel T. Quiring, FOREM, UNB Dr. Christopher J. Lucarotti, Adjunct-FOREM/CFS
Examining Board: Dr. Renee LaPointe, HRA-FOREM/Sylvar Technologies Dr. Tillmann Benfey, Biology, UNB
External Examiner: Dr. Tillmann Benfey, Biology, UNB
This thesis is accepted by the Dean of Graduate Studies
THE UNIVERSITY OF NEW BRUNSWICK
September, 2010
© Roger W. Graves, 2010 Library and Archives Bibliotheque et Canada Archives Canada
Published Heritage Direction du 1+1 Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-87692-3
Our file Notre reference ISBN: 978-0-494-87692-3
NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distrbute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. Canada ABSTRACT
Neodiprion abietis Harris (Hymenoptera: Diprionidae) is a forest pest causing periodic, extensive defoliation in the forests of western Newfoundland. The nucleopolyhedrovirus (NeabNPV; Baculoviridae) of N. abietis is environmentally safe and efficacious for suppressing outbreak populations but can be expensive to produce on the scale required for aerial spray programs. On the premise that nucleopolyhedroviruses often spread rapidly throughout a host population, I tested whether epidemic populations of N. abietis near Corner Brook, NL, Canada could be suppressed by spraying a portion of the area to be protected. The spread of NeabNPV within cohorts of N. abietis larvae increased in a positive, density dependant manner. NeabNPV-induced mortality was slightly higher when infected larvae were introduced into first- versus third-instar cohorts, suggesting that early instars are more susceptible to NeabNPV. Aerial application of NeabNPV resulted in major declines in N. abietis densities in treated areas. High levels of NeabNPV-caused mortality also occurred in areas adjacent to the treatment areas. This suggests that the application of NeabNPV to entire stands of balsam fir may not be necessary to suppress N. abietis populations.
Key words: Neodiprion abietis, balsam fir sawfly, Abies balsamea,
nucleopolyhedrovirus, NPV, disease transmission, pest management
ii PREFACE
The two main chapters presented in this thesis are independent but related manuscripts that have been prepared for publication in the Journal of Insect Pathology
(Chapter 2), and Biocontrol (Chapter 3). The authorship of all publications will be:
Roger W. Graves', Dan T. Quiring' 2, Christopher J. Lucarotti' 2
'Population Ecology Group, Faculty of Forestry and Environmental Management,
University of New Brunswick, Fredericton, New Brunswick, Canada, E3B 5P7
2Natural Resources Canada, Canadian Forest Service - Atlantic Forestry Centre, P.O.
Box 960, Corner Brook, Newfoundland and Labrador, Canada, A2H 6J3
During the development of these manuscripts, I was primarily responsible for the design and organization of experiments, the preparation and presentation of the initial research proposal, collecting and analyzing data, and writing the manuscripts. Dan Quiring and
Chris Lucarotti were involved in all aspects of this research. Many of the ideas presented in this thesis reflect the sage advice and learned guidance given by Dan and Chris and reflect the valuable comments and suggestions provided during the preparation of these manuscripts.
iii ACKNOWLEDGMENTS
I sincerely thank my supervisors, Drs. Dan Quiring and Chris Lucarotti, for the guidance, encouragement and endless patience that got me through this endeavour. Their excellent academic feedback coupled with their approachability made my graduate studies at the University of New Brunswick enjoyable. I thank Benoit Morin for technical assistance and laboratory space but, more importantly, for his patience and humour.
Thanks to my committee members Drs. Jon Sweeney and Yvan Pelletier, who provided valuable comments this manuscript and important feedback during committee meetings.
I thank my lab mates, "The Quirlings", including: Drew Carleton, Leah Flaherty,
Heidi Fry, Rob Johns, Jonathan Leggo, Gaetan Moreau, Andrew Morrison, Lauren
Penult, Kate Van Rooyen, and "Heard's nerds", who were always supportive and provided feedback on presentations.
Thanks to everyone who helped me in the field and processing samples in the lab but, I especially thank Chris Vickers for dedication to long hours of hard work, and to
Andrew Penney for his dedication and humour. During my field seasons it was my pleasure to work out of the CFS Atlantic Pasadena Field Station in conjunction with
CFS Corner Brook, where the staff was always friendly and helpful. In particular I thank
Pamela Rideout for always having a solution to logistical problems.
I gratefully acknowledge Corner Brook Pulp and Paper for use of field sites, and the Biocontrol Network, the Canadian Forest Service, Corner Brook Pulp and Paper,
Abitibi Consolidated, Forest Protection Limited, Natural Sciences and Engineering
Research Council of Canada (Discovery grant) and the Newfoundland and Labrador
iv Department of Natural Resources for funding and logistical support during my graduate studies.
This work would never have been completed without the continuous encouragement, support and understanding given by my wife Kate. Thanks for being as solid as rock when the storms hit, and for being a joy in the good times, and most of all, thanks for being a friend.
v TABLE OF CONTENTS
ABSTRACT ii PREFACE iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS vi LIST OF TABLES viii LIST OF FIGURES ix
CHAPTER 1: GENERAL INTRODUCTION 1 Objectives of Thesis Research 5 LITERATURE CITED 6
CHAPTER 2: Transmission of a nucleopolyhedrovirus within cohorts of balsam fir sawfly (Neodiprion abietis) larvae 11 Abstract 12 Introduction 13 Materials and Methods 14 Laboratory Study 14 Field Study 16 Molecular probing for presence of NeabNPV 17 Statistical analyses 18 Results 18 Laboratory Study 18 Field Study 19 Discussion 19 Acknowledgements 22 Literature cited 22
CHAPTER 3: The spread of a nucleopolyhedrovirus in populations of balsam fir sawfly (Neodiprion abietis) following aerial application 31 Abstract 32 Introduction 32 Materials and Methods 34 Amplification and purification of NeabNPV for use in field trials 35 Field sites and study design 36 BFS sampling 38 Molecular probing for NPV 39 Statistical analyses 40 Results 41 Island Pond 2002 41 Old Man's Pond 2002 43 Discussion 45 Acknowledgements 48 Literature cited 48
vi CHAPTER 4: GENERAL DISCUSSION 60 Literature cited 65 APPENDIX A 68 CURRICULUM VITAE
vii LIST OF TABLES
Table 3-1: Island Pond 2002. Summary of Pearson correlation analyses showing weekly trends in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and juvenile sawfly densities and the prevalence of NeabNPV infection in juvenile sawfly populations 52
Table 3-2: Old Man's Pond 2002. Summary of Pearson correlation analyses showing weekly trends in the relationship between distance from the two spray swaths separated by 200 m in the treatment block and the untreated (control) block and, juvenile sawfly densities and the prevalence of NeabNPV infection in juvenile sawfly populations. The 'distance from spray' values used for the treatment block were also used for the control block, assuming that spray swaths would have occurred in the same location as in the treatment 53 LIST OF FIGURES
Figure 2-1: Mean larval mortality (+/- SEM) resulting from introducing 0, 1. 5 or 10 NeabNPV-infected N. abietis larvae into groups of first instars. Each group contained 50 larvae, a density of approximately 1330 larvae/ m2 foliage 29
Figure 2-2: Mean larval mortality (+/- SEM) resulting from introducing 1 or 10 NeabNPV-infected N. abietis larvae into groups of first or third instars. Each group contained 50 larvae, a density of approximately 220 larvae/m2 foliage 30
Figure 3-1: Map of area surrounding Corner Brook, NL, showing the location of Island Pond and Old Man's Pond NeabNPV-treatment blocks in 2002. Insets show the locations of sampling transect lines (dotted lines) and aircraft spray tracts (dashed lines) at each location. Control blocks, which were located 1 to 5 km from treatment blocks, are indicated with stars 54
Figure 3-2: Wind speed, temperature and relative humidity during the periods of NeabNPV aerial spray operations on July 22, 2002, at a) Island Pond and on July 24, 2002, at b) Old Man's Pond 55
Figure 3-3: a) Average spray deposit along two transect lines extending 400 m out from the east and west sides of a 313-ha treatment block at Island Pond following aerial application of NeabNPV on July 22, 2002. Mean (+SE) juvenile balsam fir sawfly (larvae and pupae) density (gray bars) and mean (+ SE) prevalence of NeabNPV infection (black bars) in the treatment block and the untreated control block on b) July 22, c) July 29, d) August 6 and e) August 13, 2002 56
Figure 3-4: a) Seasonal trends in the mean (+ SE) densities of juvenile (larvae and pupae) balsam fir sawflies, b) Mean (+ SE) prevalence of NeabNPV infection in juvenile sawfly populations adjacent to NeabNPV treatment (black circles) and untreated control (white circles) blocks at Island Pond. Arrow indicates aerial application of NeabNPV OBs on July 22, 2002 57
Figure 3-5: a) Average spray deposit along a transect line extending 400 m across two 50-m NeabNPV spray swaths (arrows) and a 200-m untreated zone (between arrows) at Old Man's Pond following aerial application of NeabNPV on July 24, 2002. Juvenile balsam fir sawflies (larvae and pupae) densities (gray bars) and prevalence of NeabNPV infection (black bars) in the treatment block (same transect line as a) and the untreated control block on b) July 22, c) July 29. d) August 6 and e) August 13, 2002 58
Figure 3-6: a) Seasonal trends in the mean (+ SE) densities of juvenile (larvae and pupae) balsam fir sawflies. b) mean (+ SE) prevalence of NeabNPV infection in juvenile sawfly populations adjacent to NeabNPV treatment (black circles) and
ix untreated control (white circles) blocks at Old Man's Pond. Arrow indicates aerial application of NeabNPV OBs on July 22, 2002 59
x CHAPTER 1 - GENERAL INTRODUCTION
Sawflies in the family Diprionidae (Hymenoptera: Symphyta) are phytophagous, feeding mostly on conifers in the forests of the Northern Hemisphere. They derive their common name from the serrated ovipositors which the adult females use to either partially, or fully embed their eggs into the host plant's tissues (Wallace and
Cunningham 1995). Typical diprionid population outbreaks are of short duration, lasting
3-5 years before natural enemies cause population densities to decline to low levels - rarely causing mortality among their host trees (Knerer and Atwood 1973, Wallace and
Cunningham 1995).
Balsam fir sawfly (Neodiprion abietis Harris) (hereafter BFS), an eruptive defoliator that is native to North America, is a univoltine, indigenous forest pest of balsam fir (Abies balsamea Mill.) in Newfoundland (Smith 1947, Carrol 1962, Wallace and Cunningham 1995). Egg-lay occurs from early September through to October
(Carrol 1962). The eggs over-winter within the needles and the larvae emerge in late
June to mid July. BFS larvae are highly gregarious and feed primarily on older foliage
(one year old and older) (Moreau. 2004, Parsons et al. 2003). Male larvae develop through five instars, completing development in approximately 30 days, while females may undergo 5 or 6 instars, completing development in up to 35 days (Carrol 1962). In their last instar, larvae spin cocoons on the current-year foliage, pupate inside, and emerge as adults in late August or early September.
Since the early 1990s, western Newfoundland has been experiencing the worst outbreak of BFS on record (Moreau 2006). Tens of thousands of hectares (ha) of balsam fir-dominated forests have been impacted annually (Moreau 2006). Historically, the periodic outbreaks of N. abietis in western NL usually last three to four years. However,
1 the length and severity of the current outbreak is unprecedented, and has led the local forest industries, federal and provincial government agencies to invest significant effort in suppressing BFS populations and minimize their impact on the region's most important forest resource, balsam fir (Moreau 2006; Lucarotti et al. 2007). Historically, collapses of BFS populations have been associated with epizootics of the Neodiprion abietis nucleopolyhedrovirus (NeabNPV) (Carrol 1962; Moreau 2006), a
Deltabaculovirus (Jehle et al. 2006) which was seen as a viable, environmentally-safe means to manage BFS populations.
The Baculoviridae is a family of DNA viruses characterized by double-stranded, circular genomes (85-180 kD in size), that infect only arthropods, primarily insects.
Baculoviruses are distinguished by the formation of occlusion bodies (OBs), formed within the nucleus of the host cell (nucleopolyhedroviruses, or NPVs), or in the cytoplasm (Granuloviruses, or GVs). NPV OBs may contain multiple virions (infective particles of viral DNA) in either singly- or doubly-enveloped nucleocapsids, whereas the
OBs of GVs contain singly-enveloped and singly-occluded virions. The Baculoviridae nomenclature has been modified to accommodate clades, based on the 29 core baculovirus genes (Jehle et al. 2006). NPVs infecting Lepidoptera are classed as
Alphabaculoviruses (further divided into groups I and II), GVs that only infect
Lepidoptera classed as Betabaculoviruses, NPVs that infect the Diptera as
Deltabaculoviruses, and those that infect the Hymenoptera as Gammabaculoviruses.
Baculovirus epizootics have been reported for numerous outbreaking temperate insect defoliators, but most epizootics that have been studied have been primarily those in Lepidoptera (Lymantriidae, Lasiocampidae) (Cory and Myers 2003, Cory et al. 1997,
Federici 1997). Most studies focusing on baculovirus transmission have explained the
2 transmission of infections using simplified models based on epidemiological theory
(Anderson and May 1979, Anderson and May 1981, Anderson 1991). The most crucial parts of these models include the manner in which the pathogen is transmitted between hosts and the speed and efficiency with which the pathogen is transmitted between infected and susceptible uninfected hosts. Generally, in the case of baculoviruses, the unit of inoculation is considered to be the OB, and transmission of infectious OBs is assumed to be density-dependant, i.e. linear and positively influenced by densities of both the host insect and the infectious particles of the pathogen (Anderson and May
1979, 1980, 1981, Dwyer 1992, Dwyer and Elkinton 1993, 1995). However, several investigators have found that pathogen transmission in many lepidopteran hosts does not adhere to the density-dependant assumption of the Anderson and May model (D"Amico et al. 1996, Dwyer 1991, Dwyer and Elkinton 1995, Dwyer et al. 2000, Knell et al.
1998). It has also been found that increasing larval age tends to confer greater resistance to pathogens (Dwyer 1991, Goulson and Cory 1995).
NPV infections are initiated when OBs are ingested by feeding larvae. In the larval midgut, the proteinaceous OBs dissolve, releasing the virions (occlusion-derived virions or ODVs) that attach to and enter midgut epithelial cells. In many lepidopteran species virions bud out of the cells of the gut (budded virions or BV), invading the adjacent fat bodies and other tissues and create a reservoir of OBs to be released upon the death of the individual larva (Tanada and Kaya 1993, Federici 1997). The majority of OBs produced become available for secondary infections upon lysis of the corpse. In
Lepidoptera, infected corpses act as point sources for horizontal transmission (between individuals of the same generation) of the virus, so, the individual corpse is the relevant
3 ecological unit of inoculation for virus transmission (Hails et al. 2002, Cory and Myers
2003).
In contrast, NPV infections in sawflies are limited to the midgut (Federici 1997).
Horizontal transmission of sawfly NPVs occurs through the continuous sloughing of infected midgut cells. The OBs pass directly into the larval frass and, subsequently, contaminate the foliage with OBs where other conspecific larvae may consume them.
NeabNPV replicates in the midgut epithelium of N. abietis. The manner of NeabNPV replication is similar to that of the NPVs of many lepidopteran larvae, with NeabNPV
DNA occurring between four and twenty-four hours post infection (Duffy et al. 2007).
Research and development of microbial pathogens as viable alternatives to chemical pesticides has been encouraged by public pressure for environmentally-benign pest management strategies. Baculoviridae are generally considered as ideal candidates for biological control agents (see reviews by Fuxa 1987; Moscardi 1999; Vail et al.
1999; Payne 2000; Cory and Evans 2007) because as they are naturally-occurring, host- specific (infecting only a single species or a few closely- related species), are found only in arthropods, primarily insects, cause epizootics in host-insect populations, and can persist in the environment for many years (see reviews by Fuxa 1987; Payne 2000; Fuxa
2004; Cory and Evans 2007).
A number of studies (Bird and Elgee 1957; Bird and Birk 1961; Stairs 1965;
Young and Yearian 1987 a,b; Entwistle et al. 1983; Shepherd et al. 1984; Otvos et al.
1987a, b; Fuxa and Richter 1999; Moreau et al. 2005) have been conducted to assess the effects of NPVs on insect populations. Most studies have reported that, as per Anderson and May (1981), mortality by NPVs acts in a density-dependent manner to reduce insect population densities. Although most studies have focused on the effectiveness of the
4 pathogen as a biological control agent (but see Entwistle et al. 1983; Dwyer 1992;
Dwyer and Entwistle 1995; Vasconcelos et al. 1996), such studies frequently lack detailed descriptions of the patterns of virus dispersal, and usually only list possible abiotic and biotic dispersal agents of the pathogen (see reviews by Fuxa, 1987; Cory and
Evans 2007). The extent and speed of NPV dispersal is still poorly understood.
Recent studies have shown that populations of BFS can be successfully suppressed by aerial applications of NeabNPV (Moreau et al. 2005, Lucarotti et al.
2007) as NeabNPV infection most often results in the death of infected BFS larvae
(Moreau 2004). While aerial application is generally considered to be the most efficient manner to disseminate most forest and agricultural pest control products (Payne 2000)
(including NPVs) to large areas, inundative aerial sprays are often expensive to conduct.
NPVs are relatively expensive when compared to other microbial control products such as Bacillus thuringiensis because commercial NPV production must still take place in vivo (Fuxa 2004, Cory and Evans 2007).
Objectives of Thesis Research
Under the assumption that NeabNPV follows the generally accepted premise that
NPVs spread rapidly throughout a host population, the objectives of my research were:
1) to evaluate the vulnerability of individual N. abietis larvae to NeabNPV; and 2) to determine whether treating a portion of a forest stand was sufficient to initiate NeabNPV epizootics among N. abietis larvae. If the latter were true, epidemic populations of BFS might be effectively suppressed with reduced cost and effort.
The body of this thesis is presented in formats suitable for submission to referred scientific journals. The two articles herein will evaluate transmission of NeabNPV
5 between host insects in terms of the rate of spread among individual BFS larvae among
BFS populations following aerial applications of NeabNPV.
Chapter 2 presents studies on the rate of NeabNPV transmission between N. abietis larvae in single cohorts, at the branch level. I introduced NeabNPV-infected larvae into cohorts of conspecific larvae under both field and laboratory conditions. In the field experiment, I also investigated whether the age of the N. abietis larvae influenced NeabNPV transmission within the cohort. This chapter is being prepared for submission in the Journal of Insect Pathology under the title "Transmission of a nucleopolyhedrovirus within cohorts of balsam fir sawfly (Neodiprion abietis) larvae".
Chapter 3 presents studies of aerial field trials in which I examined the spread of
NeabNPV infection in a population of BFS larvae in a 200 meter-wide untreated area between two 200 meter-wide NeabNPV treatment swaths. The spread of NeabNPV infection into a natural population of BFS adjacent to a NeabNPV-treated aerial spray block was also investigated. This chapter is being prepared for submission to the journal
Biocontrol under the title "The spread of a nucleopolyhedrovirus within populations of balsam fir sawfly (Neodiprion abietis) larvae following aerial application1'
Chapter 4 is a summary and discussion chapter which integrates the results presented in chapters 2 and 3 and places them in a pest management context.
Literature Cited
Anderson R.M. and May, R.M. 1979. Population biology of infectious diseases: Part I. Nature 280: 361-367.
Anderson R. M. and May, R.M. 1980. Infectious diseases and population cycles of forest insects. Science 210: 658-661.
6 Anderson R. M. and May, R.M. 1981. The population dynamics of microparasites and their invertebrate hosts. Philosophical Transactions of the Royal Society of London B, Biological Sciences 291:451-524.
Anderson, R. M. 1991. Populations and infectious diseases: ecology or epidemiology? the eighth Tansley lecture. Journal of Animal Ecology 60: 1-50.
Bird, F. T. and Elgee, D. E. 1957. A virus disease and introduced parasites as factors controlling the European spruce sawfly, Diprion hercyniae (Htg.), in central New Brunswick. Canadian Entomologist. 89: 371-378.
Bird, F. T. and Birk, J. M. 1961. Artificially disseminated virus as a factor controlling the European spruce sawfly, Diprion hercyniae (Htg.) in the absence of introduced parasites. - Canadian Entomologist. 93: 228-238.
Carrol, W.J., 1962. Some aspects of the Neodiprion abietis complex in Newfoundland. PhD. Dissertation, State University College of Forestry, Syracuse University.
Cory. J.S., Flails, R.S., and Sait, S.M. 1997. Baculovirus Ecology, pp. 301-339 in Miller, L.K. (ed.) The Baculoviruses. Plenum Publishing Corporation, NY.
Cory, J.S. and Myers, J.H. 2003. The ecology and evolution of insect baculoviruses. Annual Review of Ecological Systems. 34: 239-272.
Cory, J. S. and Evans, H.F. 2007. Viruses, Chap. IV-I, in Field Manual of Techniques in Invertebrate Pathology, 149-174. L.A. Lacey and H.K. Kaya (eds.), Springer, 2007.
D'Amico, V., Elkinton, J. S., Dwyer, G., Burand, J. P., and Buonaccorsi, J. P. 1996. Virus transmission in gypsy moths is not a simple mass action process. Ecology 77: 201-206.
Duffy, S.P., Becker, E. M., Whittome, B.H., Lucarotti, C.J., Levin, D.B. 2007. In vivo replication kinetics and transcription patterns of the nucleopolyhedrovirus (NeabNPV) of the balsam fir sawfly, Neodiprion abietis. Journal of General Virology 88: 1945-1951.
Dwyer, G. 1991. The roles of density, stage, and patchiness in the transmission of an insect virus. Ecology 72: 559 - 574.
Dwyer, G. 1992. On the Spatial Spread of Insect Pathogens: Theory and Experiment. Ecology 73:479-494.
Dwyer, G., and Elkinton, J.S. 1993. Using simple models to predict virus epizootics in gypsy moth populations. Journal of Animal Ecology 62: 1-11.
7 Dwyer, G., Elkinton, J.S. 1995. Host dispersal and the spatial spread of insect pathogens. Ecology 76: 1262-1275.
Dwyer, G., Dushoff, J., Elkinton, J., and Levin, S. 2000. Pathogen-driven outbreaks in forest defoliators revisited: building models from experimental data. The American Naturalist 156: 105 - 120.
Entwistle, P.F., Adams, P.H.W., Evans H.F., Rivers, C.F. 1983. Epizootiology of a nuclear polyhedrosis virus (Baculoviridae) in European spruce sawfly (Gilpinia hercyniae): spread of disease from small epicentres in comparison with spread of baculovirus diseases in other hosts. Journal of Applied Ecology 20: 473-487.
Federici, B. A. 1997. Baculovirus Pathogenesis, pp.33-59 in Miller, K.L. (ed.) The Baculoviruses. Plenum Publishing Corporation, NY, pp. 447.
Funk, C. J., Braunagel, S. C., and Rohrmann, G. F. 1997. Baculovirus Structure. In: The Baculoviruses. Pp. 7-32 in Miller, K.L. (ed.) The Baculoviruses. Plenum Publishing Corporation, NY.
Fuxa, J.R. 1987. Ecological considerations for the use of entomopathogens in IPM. Annual Review of Entomology 1987. 32: 225-51.
Fuxa, J.R. and Richter, A.R. 1999. Classical biological control in an ephemeral crop habitat with Anticarsia gemmatalis nucleopolyhedrovirus. BioControl 44: 403- 419, 1999.
Fuxa, J.R. 2004. Ecology of insect nucleopolyhedroviruses (Review). Agriculture, Ecosystems and Environment. 103: 27^3.
Goulson, D. and Cory, J.S. 1995. Sublethal effects of baculovirus in the cabbage moth Mamestra Brassicae. Biocontrol 5: 361-367.
Hails, R. S., Hernandez-Crespo, P., Sait, S. M. Donnelly, C. A., Green, B. M., and Cory, Jenny S. 2002. Transmission patterns of natural and recombinant baculoviruses. Ecology 83: 906-916.
Jehle, J.A, Blissard, G.W., Bonning, B.C., Cory, J.S., Herniou, E.A., Rohrmann, G.F, Theilmann, D.A., Thiem, S.M., and Vlak, J.M. 2006. On the classification and nomenclature of baculoviruses: A proposal for revision. Archives of Virology. 151:1257-1266.
Knell, R. J., Begon, M., and Thompson, D. J. 1998. Transmission of Plodia interpunctella granulosis virus does not conform to the mass action model. Journal of Animal Ecology 67: 592-599.
Knerer, G., and Atwood C. 1973. Diprionid sawflies: polymorphism and speciation. Science 179: 1090- 1099.Moreau, G., Lucarotti, C.J., Kettela, E. G., Thurston, G.S.,
8 Holmes, S., Weaver, C., Levin, D.B., Morin, B. 2005. Aerial application of nucleopolyhedrovirus induces decline in increasing and peaking populations of Neodiprion abietis. Biological Control 33: 65-73.
Lucarotti, C.J., Morin, B. Graham, R.I., Lapointe, R. 2007. Production, application, and field performance of Abietiv™, the balsam fir sawfly Nucleopolyhedrovirus Virologica Sinica, 22:163-172.
Moreau, G. 2004. The influence of forest management on defoliator populations: a case study with Neodiprion abietis in pre-commercially thinned and natural forest stands. PhD Dissertation, Faculty of Forestry and Environmental Management, University of New Brunswick. Pp. 166.
Moreau, G., Lucarotti, C.J., Kettela, E. G., Thurston, G.S., Holmes, S., Weaver, C., Levin, D.B., and Morin, B. 2005. Aerial application of nucleopolyhedrovirus induces decline in increasing and peaking populations of Neodiprion abietis. Biological Control 33: 65-73.
Moreau, G. 2006. Past and present outbreaks of the balsam fir sawfly: an analytical review. Forest Ecology and Management 221: 215-219.
Moscardi, F., 1999. Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology 1999. 44: 257-89.
Otvos, I. S., Cunningham, J. C. and Friskie, L. M. 1987 (a). Aerial application of nuclear polyhedrosis virus against Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough) (Lepidoptera: Lymantriidae). I. Impacts in the year of application. Canadian Entomologist 119: 697-706.
Otvos, I. S., Cunningham, J. C. and Alfaro, R. I. 1987 (b). Aerial applications of nuclear polyhedrosis virus against Douglas- fir tussock moth, Orgyia psuedotugata (McDunnough) (Lepidoptera: Lymantriidae). II. Impacts 1 and 2 years after application. Canadian Entomologist. 119: 707-715.
Payne, N.J. 2000. Factors influencing aerial insecticide application to forests. Integrated Pest Management Reviews 5: 1-10.
Shepherd, R. F., Otvos, I. S., Chorney, R. J. and Cunningham, J. C. 1984. Pest management of the Douglas-fir tussock moth (Lepidoptera: Lymantriidae): prevention of an outbreak through early treatment with a nuclear polyhedrosis virus by ground and aerial applications. Canadian Entomologist 116: 1533-1542.
Smith, H.V.E. 1947. Annual Report of The Fire Patrol to The Annual Executive meeting of The Newfoundland Forest Protection Association, November 1947. pp. 46-63. Blackmore Printing Co. Ltd. Grand Falls, Newfoundland. Blackmore Printing Co. Ltd. Grand Falls, Newfoundland.
9 Stairs, G. R. 1965. Artificial initiation of virus epizootics in forest tent caterpillar populations. Canadian Entomologist. 97: 1059-1062.
Tanada, Y., and Kaya, H.K. 1993. Insect Pathology. Academic Press. San Diego, USA.
Vail, P.V., Hostetter, D.L., Hoffman, D.F. 1999. Development of the multi-nucleocapsid nucleopolyhedroviruses (MNPVs) infectious to loopers (Lepidoptera: Noctuidae: Plusiinae) as microbial control agents. Integrated Pest Management Reviews 4: 231-257.
Vasconcelos, S.D., Cory, J.S., Wilson, K.R., Sait, S.M., Hails, R.S. 1996. Modified behaviour in baculovirus-infected lepidopteran larvae and its impact on the spatial distribution of inoculum. Biological Control 7, 299-306.
Wallace, D.R., Cunningham, J.C. 1995. Diprionid Sawflies. In: Forest Insect Pests in Canada, volume 1. Edited by J.A. Armstrong and W.G.H. Ives. Pp. 193-232.
Young, S.Y. and Yearian, W.C. 1987 (a). Intra- and inter-colony transmission of a nuclear polyhedrosis virus of loblolly pine sawfly Neodiprion taedae. Journal of Entomological Science 22: 29-34.
Young, S.Y. and Yearian, W.C. 1987 (b). Secondary transmission of a nuclear polyhedrosis virus of Neodiprion taedae [Hym.:Diprionidae] between larval colonies on loblolly pine. Entomophaga 34: 341-349.
10 CHAPTER 2
Transmission of a nucleopolyhedrovirus within cohorts of balsam fir sawfly
(Neodiprion abietis) larvae
To be submitted to: The Journal of Insect Pathology
Roger Graves', Dan T. Quiring1, Christopher J. Lucarotti1'2
'Faculty of Forestry and Environmental Management, P.O. Box 44555, University of
New Brunswick, Fredericton, New Brunswick, Canada, E3B 6C2.
2Canadian Forest Service, Natural Resources Canada, P.O. Box 4000, 1350 Regent
Street, Fredericton, New Brunswick, Canada, E3B 5P7.
11 Abstract: The spread of a nucleopolyhedrovirus (NeabNPV) within cohorts of balsam fir sawfly (Neodiprion abietis Harris) larvae was studied by introducing NeabNPV- infected larvae into single cohort groups. In the laboratory, with populations equivalent to 1300 larvae/m2 of balsam fir foliage, NeabNPV-induced mortality increased from
20% in control groups to over 80% with the introduction of one, five or 10 NeabNPV- infected larvae into treatment groups. In field studies, where populations were 220 larvae/m2 of balsam fir foliage, NeabNPV-induced mortality increased from 23% in control groups to 51% with the introduction of one NeabNPV-infected, first-instar larva and to 84% with the introduction of 10 NeabNPV-infected, first-instar larvae. When one or 10 NeabNPV-infected, third instars were introduced into cohorts of third instars mortality was not statistically significant at 60% and 63%, respectively. NeabNPV- induced mortality was higher when infected larvae were introduced into first- rather than third-instar cohorts. This was likely due to the greater susceptibility of early instars to
NeabNPV and a longer period of time for the horizontal spread of the virus if N. abietis populations are treated early in development.
Key Words: Neodiprion abietis, sawfly, nucleopolyhedrovirus, virus transmission.
12 Introduction
The Diprionidae is a family in the hymenopteran suborder, Symphyta. In North
America, many diprionid species are defoliators of conifers and represent some of the most important economic pests of northern forests (Smith 1993, Wallace and
Cunningham 1995). Typically, diprionid population outbreaks last only 3-5 years, and are brought back to low, densities by natural enemies, especially nucleopolyhedroviruses
(NPVs) (Baculoviridae) (Knerer and Atwood 1973, Smith 1993, Wallace and
Cunningham 1995).
Since the early-1990s, western Newfoundland has been experiencing the worst outbreak of balsam fir sawfly (BFS) (Neodiprion abietis Harris) on record. Tens of thousands of hectares (ha) of balsam fir (Abies balsamea (L.) Mill.)-dominated forest have been impacted annually (Moreau 2006).
BFS females oviposit into slits they cut in current-year balsam fir needles in
September and October (Carrol 1962). Eggs over-winter there and larvae emerge from late-June to mid-July the following year. Early instar balsam fir sawfly larvae are gregarious (Anstey et al. 2002) and feed on balsam fir foliage that is one-year-old and older (Moreau et al. 2003. Parsons et al. 2003). Male larvae develop through five instars. over approximately 30 days, while females undergo five or six instars and complete larval development in about 35 days (Carrol 1962). Larvae spin cocoons and pupate on the needles of balsam fir trees and emerge as adults in late-August and early September.
Historically, natural declines in BFS populations in western Newfoundland have been associated with epizootics of a species-specific NPV, hereafter referred to as
NeabNPV (Smith 1947, Carroll 1962, Moreau 2006, Duffy et al. 2006). NeabNPV has
13 been demonstrated to be an effective control agent for BFS (Moreau et al. 2005) as
NeabNPV infection most often results in larval death (Moreau 2004).
Sawfly NPV infections are initiated when occlusion bodies (OBs) are ingested by feeding larvae. In the larval midgut, the proteinaceous OBs dissolve, releasing virions that attach to and enter midgut epithelial cells. In sawflies, NPV infections are limited to the midgut (Federici 1997). Horizontal transmission (between individuals of the same generation) of sawfly NPVs occurs through the continuous sloughing of infected midgut cells, which pass directly into the larval frass and, subsequently, contaminate the foliage with OBs where other conspecific larvae may consume them.
I investigated the spread of NeabNPV within single cohorts of BFS larvae by introducing NeabNPV-infected larvae into groups of larvae under both field and laboratory conditions. Additionally, because later instars tend to be more resistant to pathogens (Dwyer 1991, Goulson et al. 1995, Dwyer et al. 1997), I investigated the effect of larval instar on NeabNPV transmission in the field when NeabNPV-infected larvae were introduced into a cohort of larvae.
Materials and methods
Laboratory study
In August, 2002, BFS cocoons were collected from field populations in a precommercially-thinned balsam fir stand (49°10*N 57°26'W) near Corner Brook in western Newfoundland. This stand was outside the leading edge of the 2002 BFS outbreak, so NeabNPV prevalence was expected to be low. Cocoons were individually placed in separate 7-mL gel capsules which were then sealed in clear plastic Ziploc bags and transported by truck to laboratory facilities at the University of New Brunswick
14 (UNB), in Fredericton, NB. Gel capsules containing cocoons were placed in a climate- controlled chamber (Conviron CMP 3032) at 18°C, -60% humidity, and a light regime of 18L: 6D until adult emergence. To further reduce the prevalence of environmental
NeabNPV, unmated adult BFS females were individually transferred to muslin sleeve cages on branches of young, 1-8 m tall, balsam fir trees in the UNB woodlot (45°52'N
66°32'W). To the best of my knowledge, this woodlot has never experienced an outbreak of balsam fir sawflies. After 4 days, 28 females had laid between nine to 70 eggs each.
In the last week of May, 2003, egg-bearing branches were cut with pruning shears and transported to the laboratory. The base of each branch was placed into a separate 750 mL standard mason jar filled with 500 ml of de-ionized water. Approximately 0.5 mL of commercial bleach (5.25% NaOCl) was added to each jar to discourage the growth of microorganisms. Branches were further pruned to 25 cm by 15 cm foliar area. Jars containing branches were placed in individual Styrofoam packing trays, separated by 5 cm with a 2.5-cm strip of Tree Tanglefoot Pest Barrier (The Tanglefoot Company) around the perimeter of each tray to ensure that larvae could not crawl between jars.
Branches were monitored and misted with water daily for 13 days while awaiting egg hatch.
On June 9, 2003, when 90% of the eggs had hatched, larval densities were adjusted to 50 larvae per branch (initial density -1330 larvae/ m2 foliated branch surface) by removing or adding larvae as required. Needles with unhatched eggs were also removed at this time. Branches were randomly assigned to the following treatment groups: (1) no larvae infected with NeabNPV (control); (2) one larva infected with
NeabNPV; (3) five larvae infected with NeabNPV; and (4) 10 larvae infected with
NeabNPV. The latter three treatments were obtained by removing one, five, or 10 larvae
15 from the non-control groups and allowing them to feed for 30 min on foliage that had been sprayed with an aqueous solution ofNeabNPV (1 x 102 OBs/mL), using a 1-liter aerosol spray bottle, before they were returned to their groups. Treated larvae were then returned to their respective groups. Similarly, control groups were fed on foliage that had been sprayed with double distilled water, and then replaced in their respective groups. Each day for the next 14 days, the number of living larvae was recorded for each group of branches and fresh foliage was added as required. All dead larvae were removed daily, individually placed in 1.5-mL polypropylene microcentrifuge tubes
(Fisher Scientific) and stored at -20°C, for molecular probing. After 14 days, any surviving larvae were individually placed in separate microcentrifuge tubes and frozen for molecular probing.
Field study
In the first week of July 2003, approximately 3000 first-instar BFS larvae were collected from a field population in an immature balsam fir (tree height 5-8 m.) stand
(49°12'N 57°33'W) beyond the leading edge of the BFS outbreak, where NeabNPV prevalence was assumed to be low. Larvae were transported to a young, precommercially-thinned balsam fir stand (49° 1 l'N 57°32'W) located approximately 7 km north-west of Deer Lake, NL where there was no history of BFS populations within the last 50 years (Moreau 2006). Groups of 50 first-instar larvae (initial density approximately 220 larvae/ m2 foliated branch surface) were placed on each of five balsam fir branches (approximately 50 cm long x 45 cm wide) on each of 12 trees, and covered with sleeve-cages, as in Parsons et al. (2006). Sleeve cages were allocated to six mid-crown branches, located in two adjacent whorls, which were arranged in a staggered pattern over the SW aspect of the trees. At the first or third instar, larvae were removed
16 from their respective groups and allowed to feed for 30 min on foliage that had been sprayed with an aqueous suspension of NeabNPV as above. Densities of zero (i.e. control), one or 10 infected larvae were randomly assigned to 10 replicates each. Larvae were then allowed to develop until mid-August, when death or pupation had occurred for all members of each group. Experimental branches bearing sleeve cages were cut with pruning shears and transported to the laboratory in Pasadena, NL, where they were examined for BFS pupae, and survival to pupation was recorded. All pupae and any remaining dead larvae in sleeve cages were placed in individual 1.5-mL polypropylene microcentrifuge tubes and stored at -20°C for molecular probing (see below).
Molecular probing for the presence of NeabNPV
All retained larvae (live and dead at the time of collection) were probed for the presence of NeabNPV DNA using Renaissance (Perkin-Elmer Life Sciences) molecular labelling and detection kits as described by Moreau et al. (2005). Briefly, fluoroscein-
N6-dATP-labeled DNA probes (typically between 300-600 bases) were produced using seven NeabNPV DNA/£coRlfragments (3.5-5.5 kb) as templates. Individual insects were thawed and homogenized in ~ 1 mL of double-distilled water in the 1.5-mL microcentrifuge tubes in which they had been stored. A 3-^iL aliquot of each sample was blotted onto Biodyne A nylon membrane (Pall, Gelman Laboratory). Positive controls of purified NeabNPV DNA or NeabNPV OBs were also spotted onto each membrane.
Membranes were soaked in denaturing solution (0.5 N NaOH, 1.5 M NaCl) and incubated at 65°C for 30 min. Membranes were neutralized in 1.5 M NaCl, 0.5 M Tris. pH 7.0 for 1 min, soaked a further 5 min in lOx SSC (Saline Sodium Citrate buffer) and air-dried on filter paper. Target DNA was bound to the membranes by exposure to
17 125mJ of UV radiation. Membranes were soaked in hybridizing solution containing the labelled probe for 18h at 65°C. Excess probe and probe bound to non-specific DNA was removed with high stringency washes and results were recorded on Kodak BioMax ML film. The lower detection limit for the probing protocol was 5 x 103 OBs (Moreau et al.
2005) implying a positive detection only for specimens where NeabNPV had replicated.
Statistical Analysis
Data from the laboratory assays were analyzed for the influence of treatment and time since treatment by repeated measures ANOVA using the General Linear Model in
SPSS™ following arcsine-transformation (to meet normality assumptions). The data failed Mauchley's test for sphericity (W(77) = 0.000, p<0.0001), indicating a violation of the assumption of equal variances between treatment groups, and therefore were adjusted using the Huyn-Feldt correction (Zar 1999, Quinn and Keough 2002). Post-hoc
Tukey tests were performed to determine whether any differences existed between the different treatment levels. For the field experiment, the effects of the number of initially infected larvae (zero (control), one or 10) and the age of the larvae at exposure to
NeabNPV (first or third instar) on mortality were analyzed by two-way ANOVA on arcsine transformed data. An apparent effect of age (instar), between the NeabNPV- treated groups was tested by one-way ANOVA on arcsine transformed data.
Results
Laboratory study
All dead larvae collected through the course of the experiment tested positive for
NeabNPV and all larvae that survived to the end of the experiment tested negative for
NeabNPV. A low level of endemic NeabNPV resulted in 20% of the dead larvae in
18 control groups testing positive for NeabNPV by the end of 14 days (Fig. 2-1). This contrasts with levels of approximately 80% NeabNPV-induced mortality, at the same time, in groups where 1, 5 or 10 infected larvae had been introduced (F3.26 = 17.6, p<0.0001). The higher rate of mortality on the branches with 1, 5, or 10 infected larvae compared to control branches, resulted in a significant interaction between time and treatment (calculated Huyn-Feldt-corrected F7.8.67.2= 10.2, p=0.0001). However, the high rate of NeabNPV-induced mortality was similar for all treated branches, regardless of whether there were 1, 5 or 10 infected larvae infected (Tukey's test p>0.05).
Field study
In the field experiments, average mortality due to NeabNPV was < 23% in control groups but mortality always exceeded 50% in cohorts into which infected balsam fir sawfly larvae were introduced (Fig.2-2). Larval mortality was significantly influenced by the number (Fi.qo=8.62,p=0.004) and the instar (F2.90- 35.99, p<0.001) of infected larvae introduced into cohorts. The average mortality (+ SEM) of larvae exposed to NeabNPV as third instars (62.0±4.5%) was lower than that of larvae exposed
as first instars (72.2±4.8%) (F|,64=9.60, P=0.003). Although the number of infected larvae introduced into a cohort appeared to influence mortality more for first than for third instar cohorts (Fig. 2-2), there was no significant interaction between larval instar at the time of NeabNPV exposure and number of infected larvae introduced into a cohort
(F2.9o=2.54, P=0.085).
Discussion
The laboratory experiment was conducted at densities similar to those found during the peak stage of an outbreak (i.e. 150-1500 BFS larvae/ m2 of foliated branch surface) (Moreau et al. 2005). In this case, the number of infected larvae introduced into
19 a cohort did not significantly influence the total number of infected larvae over the course of the experiment, although it may have influenced the rate at which disease spread within the cohort (Fig. 2-1). The introduction of higher numbers of infected larvae into comparatively small experimental arenas would contribute to an increased inoculum of secondarily-produced NeabNPV to infect other members of the cohort.
Under the high cohort densities of this experiment, even one infected larva apparently produced sufficient amounts of NeabNPV OBs in the local environment to infect many, if not all, larvae within the cohort.
Larval densities in the field experiment approximated those in outbreaking BFS populations, i.e. 150-1500 BFS larvae per m2 of foliated branch surface (Moreau et al.
2005). Generally, average larval mortality attributable to NeabNPV infection increased with the number of infected larvae introduced into a cohort, but average mortality decreased slightly when infected larvae were introduced to cohorts of third- versus first- instar larvae. First-instar cohorts may have had slightly higher rates of NeabNPV induced mortality than third-instar cohorts due to increased time for multiple cycles of
NeabNPV replication and subsequent release of OBs to occur, and/or because of higher susceptibility of first instars (Dwyer 1991, Goulson et al. 1995, Cory el al. 1997. Dwyer et al. 1997). Increased levels of infection, and subsequent mortality of larvae, in cohorts into which 10 infected larvae were introduced was presumably due to a much higher level of inoculum of OBs than in cohorts where only one infected larva was introduced.
Statistically, the influence of the number of infected larvae introduced into a cohort on larval mortality was not significantly more important for first than third instars (Fig. 2-
2); in spite of their smaller size, which likely makes first instars more susceptible to
NeabNPV, requiring a lower dose of OBs to initiate infection; and they are much more
20 gregarious than larger more robust third instars, increasing the likelihood of contact between infected and susceptible individuals.
Epizootics of NeabNPV occur at peak BFS population outbreak densities (Moreau
2004, Moreau et al. 2005). It has not been clear how these epizootics are initiated and transmitted throughout BFS populations. Other studies on NPV transmission in sawflies have shown that epizootics are initiated from persistent environmental contamination with OBs (Kaupp 1982, 1983, Olofsson 1987, 1988a,b 1989) and subsequent spread from groups of sawfly larvae within a tree and from tree to tree (Young and Yearian
1987, 1989, 1990). This study demonstrates how a NeabNPV epizootic might begin in
BFS populations with the initial infection of low numbers of BFS larvae in isolated groups.
There have been relatively few studies on NPV transmission in sawflies (Bird
1961, Young and Yearian 1987, Olofsson 1987, Olofsson 1988a,b, Young and Yearian
1989, Young 1990, Young 1998) compared to NPV transmission in lepidopteran defoliators (Ali et al. 1985, Dwyer 1991, Dwyer and Elkinton 1995, Goulson et al.
1995, D* Amico et al. 1996, Vasconcelos 1996, Dwyer et al. 1997, Knell et al. 1998,
Beisner and Myers 1999, Richards et al. 1999, Dwyer et al. 2000, Hails et al. 2002,
Steineke and Jehle 2004, D"Amico et al. 2005, Zhou et al. 2005). Many of the lepidopteran NPV studies have been based on investigations of the simplified model for disease transmission among invertebrates developed by Anderson and May (1979, 1980,
1981). In the lepidopteran NPV studies, it has often been noted that horizontal transmission of NPVs is not linearly density dependant, as the mass-action assumption of the Anderson and May model suggests (D'Amico et al. 1996, D'Amico et al. 2005,
Dwyer 1991, Dwyer and Elkinton 1995, Dwyer et al. 1997, Knell et al. 1998, Dwyer et
21 al. 2000). My results suggest that NeabNPV transmission in natural populations of BFS may follow the mass action assumption of Anderson and May (1981), at least when
NeabNPV is introduced early in larval development and at relatively low host density, roughly equivalent to that at the start of an outbreak. At higher densities, comparable to those found late in BFS outbreaks, this aspect of NeabNPV transmission seems to become less important as the impact of even one NeabNPV-infected larva on mortality within a cohort becomes more pronounced. This, in turn, suggests that a relatively small amount NeabNPV inoculum may be required to initiate an epizootic among field populations of BFS of concern to forest managers.
Acknowledgements
I thank Benoit Morin for technical assistance and lab space, Corner Brook Pulp and
Paper for use of field sites, Dr. Rob Johns for comments on an earlier version of this manuscript, and the Biocontrol Network, Canadian Forest Service, Corner Brook Pulp and Paper, Abitibi Consolidated, Forest Protection Limited, Natural Sciences and
Engineering Research Council of Canada (Discovery grant) and the Newfoundland and
Labrador Department of Natural Resources for funding and logistical support.
Literature Cited
Anderson R.M. and May, R.M. 1979. Population biology of infectious diseases: Part I.
Nature 280: 361-367.
Anderson R. M. and May, R.M. 1980. Infectious diseases and population cycles of forest
insects. Science 210: 658-661.
22 Anderson R. M. and May, R.M. 1981. The population dynamics of microparasites and
their invertebrate hosts. Philosophical Transactions of the Royal Society of
London B, Biological Sciences 291:451-524.
Anderson, R. M. 1991. Populations and infectious diseases: ecology or epidemiology?
the eighth Tansley lecture. Journal of Animal Ecology 60: 1-50.
Anstey, L.J., Quiring, D.T. and D.P. Ostaff. 2002. Seasonal changes in intra-tree
distribution of immature balsam fir sawfly (Hymenoptera: Diprionidae).
Canadian Entomologist 134: 529-538.
Beisner, E.B. and Myers J.H. 1999. Population density and transmission of virus in
experimental populations of the western tent caterpillar (Lepidoptera:
Lasiocampidae). Environmental Entomology 28: 1107- 1113.
Bird F.T. 1961. Transmission of some insect viruses with particular reference to ovarian
transmission and its importance in the development of epizootics. Journal of
Insect Pathology 3: 352-380.
Carrol, W.J., 1962. Some aspects of the Neodiprion abietis complex in Newfoundland.
PhD. Dissertation, State University College of Forestry, Syracuse University, pp.
186.
Cory. J.S., Hails, R.S., and Sait, S.M. 1997. Baculovirus Ecology, pp. 301-339 in
Miller, L.K. (ed.) The Baculoviruses. Plenum Publishing Corporation, NY.
Cory, J.S. and Myers, J.H., 2003. The ecology and evolution of insect baculoviruses.
Annual Review of Ecological Systems. 34: 239-272.
D'Amico, V., Elkinton, J. S., Dwyer, G., Burand, J. P., and Buonaccorsi, J. P. 1996.
Virus transmission in gypsy moths is not a simple mass action process. Ecology
77: 201-206.
23 D'Amico, V., Elkinton, J. S., Podgwaite, J. D., Buonaccorsi, J. P., and Dwyer, G. 2005.
Pathogen clumping: an explanation for non-linear transmission of an insect virus.
Ecological Entomology 30: 383-390.
Duffy, S. P., Young, A. M., Morin, B., Lucarotti, C. J., Koop, B. F., and Levin, D. B.
2006. Sequence analysis and organization of the Neodiprion abietis
nucleopolyhedrovirus genome. Journal of Virology 80: 6952-6963.
Dwyer, G. 1991. The roles of density, stage, and patchiness in the transmission of an
insect virus. Ecology 72: 559 - 574.
Dwyer, G., and Elkinton, J.S. 1993. Using simple models to predict virus epizootics in
gypsy moth populations. Journal of Animal Ecology 62: 1-11.
Dwyer, G., and Elkinton, J.S. 1995. Host dispersal and the spatial spread of insect
pathogens. Ecology. 76: 1262-1275.
Dwyer, G., Elkinton, J. L., and Buonaccorsi, J. P. 1997. Host heterogeneity in
susceptibility and disease dynamics: tests of a mathematical model. The
American Naturalist 150:685-707.
Dwyer, G., Dushoff, J., Elkinton, J., and Levin, S. 2000. Pathogen-driven outbreaks in
forest defoliators revisited: building models from experimental data. The
American Naturalist 156(2): 105 - 120.
Entwistle, P.F., Adams, P.H.W., Evans H.F., and Rivers, C.F. 1983. Epizootology of a
nuclear polyhedrosis virus (Baculoviridae) in European spruce sawfly (Gilpinia
hercyniae): spread of disease from small epicentres in comparison with spread of
baculovirus diseases in other hosts. Journal of Applied Ecology. 20: 473-487.
Federici, B. A. 1997. Baculovirus Pathogenesis, pp.33-59 in Miller, K.L. (ed.) The
Baculoviruses. Plenum Publishing Corporation, NY. pp. 447.
24 Fenton, A., Fairbairn, R. N., and Hudson, P.J. 2002. Parasite transmission: reconciling
theory' and reality. Journal of Animal Ecology 71: 893-905.
Funk, C. J., Braunagel, S. C., and Rohrmann, G. F. 1997. Baculovirus Structure. In: The
Baculoviruses. Pp. 7-32 in Miller, K.L. (ed.) The Baculoviruses. Plenum
Publishing Corporation, NY.
Hails, R. S., Hernandez-Crespo, P., Sait, S. M. Donnelly, C. A., Green, B. M., and Cory,
Jenny S. 2002. Transmission patterns of natural and recombinant baculoviruses.
Ecology 83: 906-916.
Herniou, E. A., Olszewski, J. A., O'Reilly, D. R., and Cory, J. S. 2004. Ancient
coevolution of baculoviruses and their insect hosts. Journal of Virology 78:
3244-3251.
Kaupp, W.J. 1982. Estimation of nuclear polyhedrosis virus produced in field
populations of the European pine sawfly, Neodiprion sertifer
(Geoff.)(Hymenoptera: Diprionidae). Canadian Journal of Zoology 61:1857-
1861.
Kaupp, W.J. 1983. Persistence of Neodiprion sertifer (Hymenoptera: Diprionidae)
Nuclear Polyhedrosis Virus on Pinus contorta foliage. Canadian Entomologist
115: 869-873.
Knell, R. J., Begon, M., and Thompson, D. J. 1998. Transmission of Plodia
interpunctella granulosis virus does not conform to the mass action model.
Journal of Animal Ecology 67: 592-599.
Knerer, G., and Atwood C. 1973. Diprionid sawflies: polymorphism and speciation.
Science 179: 1090- 1099.
25 Moreau, G. 2004. The influence of forest management on defoliator populations: a case
study with Neodiprion abietis in pre-commercially thinned and natural forest
stands. PhD Dissertation, Faculty of Forestry and Environmental Management.
University of New Brunswick. Pp. 166
Moreau, G. 2006. Past and present outbreaks of the balsam fir sawfly: an analytical
review. Forest Ecology and Management 221: 215-219.
Moreau, G., Lucarotti, C.J., Kettela, E. G., Thurston, G.S., Holmes, S., Weaver, C.,
Levin, D.B., and Morin, B. 2005. Aerial application of nucleopolyhedrovirus
induces decline in increasing and peaking populations of Neodiprion abietis.
Biological Control 33: 65-73.
Olofsson, E. 1987. Environmental persistence of the nuclear polyhedrovirus of the
European spruce sawfly in relation to epizootics in Swedish Scots pine forests.
Journal of Invertebrate Pathology 52: 119-129.
Olofsson, E. 1988. Persitence and dispersal of the nuclear polyhedrovirus of the
European spruce sawfly(Geoffroy)[Hymenoptera:Diprionidae] in a virus-free
lodgepole pine plantation in Sweden. Canadian Entomologist 120:887-892.
Olofsson, E. 1988. Dispersal of the nuclear polyhedrosis virus of Neodiprion sertifer
from soil to pine foliage with dust. Entomological experiment. Applications.
46:181-186.
Olofsson, E. 1989. Transmission of the nuclear polyhedrosis virus of the European pine
sawfly from adult to offspring. Journal of Invertebrate Pathology 54: 322-330.
Quinn, G. P., and Keough, M. J. 2002. Experimental Design and Data Analysis for
Biologists. Cambridge University Press. Cambridge, UK. 537 pp.
26 Smith, H.V.E. 1947. Annual Report of The Fire Patrol to The Annual Executive meeting
of The Newfoundland Forest Protection Association, November 1947. pp. 46-63.
Blackmore Printing Co. Ltd. Grand Falls, Newfoundland. Blackmore Printing
Co. Ltd. Grand Falls, Newfoundland.
Smith, D.R. 1993. Systematics, life history, and distribution of sawflies, pp. 3-32, in
Wagner, M.R. and Raffa, K.F. (eds.) Sawfly life history adaptations to woody
plants. Academic Press, N.Y., pp 581.
Tanada, Y., and Kaya, H.K. 1993. Insect Pathology. Academic Press. San Diego, USA.
Wallace, D.R., Cunningham, J.C. 1995. Diprionid Sawflies. Pp. 193-232 In: Forest
Insect Pests in Canada, volume 1. J.A. Armstrong and W.G.H. Ives (eds.), pp.
732.
Wilson, K., Knell, R., Boots, M., Koch-Osborne, J. 2003. Group living and investment
in immune defence: an interspecific analysis. Journal of Animal Ecology 72:
133-143.
Young, S.Y. and Yearian, W.C. 1987. Intra- and inter-colony transmission of a nuclear
polyhedrosis virus of loblolly pine sawfly Neodiprion taedae linaeris. Journal of
Entomological Science 22: 29-34.
Young, S.Y. and Yearian, W.C. 1987. Secondary transmission of a nuclear polyhedrosis
virus of Neodiprion taedae [Hym.:Diprionidae] between larval colonies on
loblolly pine. Entomophaga 34: 341-349.
Young, S.Y. and Yearian, W.C. 1990. The influence of canopy location on intercolony
transmission of nuclear polyhedrosis virus in larval populations of loblolly pine
sawfly Neodiprion taedae linaeris. Journal of Entomological Science 25: 535-
537.
27 Zar, J. H. 1999. Biostatistical Analysis, 4th Ed. Prentice-Hall, Inc. Simon and Schuster,
A Viacom Company. Upper Saddle River, New Jersey, USA. 07458. pp. 663.
28 0 infected larvae introduced (control) 1 infected larva introduced 100
90
80
70
60 - TO -e 50 £ o o 5 40 5 CO to ti LL ll) 30 •] CO
20
10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Days after NeabNPV introduction Days after NeabNPV introduction
5 infected larvae introduced 10 infected larvae introduced
5 O o w U_ LL CD co
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 2 3 4 5 6 7 8 9 10 11 12 13 14 Days after NeabNPV introduction Days after NeabNPV introduction
Figure 2-1: Mean balsam fir sawfly (BFS; Neodiprion abietis) larval mortality (+ SEM) resulting from introducing 0, 1, 5 or 10 NeabNPV-infected N. abietis larvae into groups of first instars. Each group contained 50 larvae, a density of approximately 1330 larvae/ nr foliage.
29 — First Instar I I Third Instar
0 1 10 Number of infected larvae introduced
Figure 2-2: Mean balsam fir sawfly (BFS; Neodiprion abietis) larval mortality (± SEM) resulting from introducing l or 10 NeabNPV-infected N. abietis larvae into groups of first or third instars. Each group contained 50 larvae, a density of approximately 220 tarvae/nr foliage.
30 CHAPTER 3 - The spread of a nucleopolyhedrovirus in populations of balsam fir sawfly (Neodiprion abietis) following aerial application
To be submitted to: Biocontrol
Roger Graves1, Dan T. Quiring1, Christopher J. Lucarotti1,2
'Faculty of Forestry and Environmental Management, P.O. Box 44555, University of
New Brunswick, Fredericton, New Brunswick, Canada, E3B 6C2.
2Canadian Forest Service, Natural Resources Canada, P.O. Box 4000, 1350 Regent
Street, Fredericton, New Brunswick, Canada, E3B 5P7.
31 Abstract: The nucleopolyhedrovirus (NeabNPV) of balsam fir sawfly (Neodiprion abietis Harris) is environmentally safe and efficacious for suppressing outbreak populations but can be relatively expensive to produce on the large scale required for aerial spray programs. Based on the premise that nucleopolyhedroviruses often spread rapidly throughout a host population, I tested whether we could suppress an epidemic population of N. abietis near Corner Brook, NL, Canada by spraying only a portion of the area we wished to protect. Aerial application of NeabNPV resulted in high levels of
NeabNPV-caused mortality and major declines in N. abietis densities directly under the flight lines of the spray plane as well as high levels of NeabNPV-caused mortality in areas adjacent to the treatment areas. This suggests that it may not be necessary to spray entire stands of balsam fir with NeabNPV to suppress BFS populations in an area.
Efficient transmission of NeabNPV is achieved by the combined actions of biotic and abiotic factors facilitating the horizontal spread of NeabNPV.
Key Words: Neodiprion abietis, balsam fir sawfly, Abies balsamea, nucleopolyhedrovirus, NPV, disease transmission, pest management
Introduction
Research and development of microbial pathogens as viable alternatives to chemical pesticides has been encouraged by public pressure for environmentally-benign pest management strategies. Baculoviruses are generally considered ideal candidates for biological control agents (see reviews by Fuxa 1987; Moscardi 1999; Vails et al. 1999;
Payne 2000; Cory and Evans 2007) because they: 1) are naturally-occurring, 2) are host- specific (infecting only a single species or a few closely- related species), 3) are found only in arthropods, primarily insects, 4) cause epizootics in host-insect populations, and
5) can persist in the environment for many years (see reviews by Fuxa 1987; Payne
32 2000; Fuxa 2004; Cory and Evans 2007). Baculoviruses are double-stranded DNA viruses with circular genomes of between 85 and 180 kilobases. Currently they are divided into the Alphabaculoviruses (nucleopolyhedroviruses or NPV),
Betabaculoviruses (granuloviruses or GV) (both in Lepidoptera), Deltabaculoviruses
(NPV in Diptera) and Gammabaculoviruses (NPV in Symphyta) (Jehle et al. 2006).
Many studies (Bird and Elgee 1957; Bird and Birk 1961; Stairs 1965; Young
1974; Entwistle et al. 1983; Shepherd et al. 1984; Otvos et al. 1987a, b; Fuxa and
Richter 1999; Moreau et al. 2005) have been conducted to assess the effects of NPVs on insect populations. Most studies have reported that, as per Anderson and May (1981),
NPV-induced mortality acts in a density dependent manner to reduce insect densities.
Although most studies have focussed on the effectiveness of the pathogen as a biological control agent (but see Entwistle et al. 1983; Dwyer 1992; Dwyer and Entwistle 1995;
Vasconcelos et al. 1996), such studies frequently lack detailed descriptions of the patterns of virus dispersal, and usually only list possible abiotic and biotic dispersal agents of the pathogen (see Reviews by Fuxa, 1987; Cory and Evans 2007).
Balsam fir sawfly (Neodiprion abietis Harris) (BFS) is an eruptive defoliator that is native to North America (Wallace and Cunningham 1995). Although periodic outbreaks usually last three to four years, the length and severity of the current outbreak in western Newfoundland is unprecedented, and has led to extensive efforts to manage
BFS populations and minimize their impact on the region's most important forest resource, balsam fir (Abies balsamea Mill.)(Moreau 2006; Lucarotti et al. 2007).
BFS females oviposit into slits they cut in current-year balsam fir needles in
September and October (Carrol 1962). Eggs over-winter there and larvae emerge from late-June to mid-July the following year. Early instar balsam fir sawfly larvae are
33 gregarious (Anstey et al. 2002) and feed on balsam fir foliage that is one-year-old and older (Moreau et al. 2003, Parsons et al. 2003). Male larvae develop through five instars, over approximately 30 days, while females undergo five or six instars and complete larval development in about 35 days (Carrol 1962). Larvae spin cocoons and pupate on the needles of balsam fir trees and emerge as adults in late-August and early September.
Historically, collapses of BFS populations have been associated with epizootics of the Gammabaculovirus, NeabNPV (Carrol 1962; Moreau 2006) and recent studies have shown that populations of BFS can be successfully suppressed by aerial applications of NeabNPV (Moreau et al. 2005, Lucarotti et al. 2007). While aerial applications are generally considered to be the most efficient way to disseminate most forest and agricultural pest control products to large areas, they are often expensive to conduct. NPVs are relatively expensive when compared to other microbial control products such as Bacillus thuringiemis because commercial NPV production must be in vivo (Payne 2000), as detailed by Lucarotti et al. (2007).
Aerial field trials with NeabNPV were carried out to determine whether horizontal transmission of NeabNPV in BFS larvae would allow reduced spray coverage to initiate NeabNPV epizootics in epidemic populations of N. abietis. Two experimental blocks, each with its own untreated control, were established. The first experimental block examined the spread of NeabNPV from a 313 ha aerial treatment block out into adjacent populations of naturally-occurring N. abietis. The second experimental block was assessed to determine whether or not NeabNPV could disperse into populations of
BFS within a 200-m untreated zone located between two, 200-m spray swaths.
34 Materials and methods
Amplification and purification of NeabNPVfor use infield trials
NeabNPV occlusion bodies (OBs) were applied to stands of balsam fir using two
Cessna 188B AgTruck aircraft (C-FIMY and C-GWKT) as described by Lucarotti et al.
(2007). The aircraft (owned and operated by Forest Protection Limited, Lincoln, NB,
Canada) were equipped with MicronAir (Bromyard, Herts. UK) AU4000 rotary atomizers and AG-Nav (Newmarket, ON) guidance systems. NeabNPV was applied in a 20% aqueous solution of molasses at ultra-low volumes of approximately 2.5L/ha.
Aircraft speed during applications was 177 km/h with a track flow rate of approximately
20 L/min. Spray swaths for each aircraft were approximately 25 m wide and the aircraft flew adjacent, parallel spray lines, separated by 25 m, resulting in complete coverage by the aerosol spray at ground level. For experimental applications, sufficient NeabNPV
OBs were added to the aircraft hoppers to result in an application rate of 3 x 109 OBs/ha when applied to a full ha (Lucarotti et al. 2007).
Stocks of NeabNPV OBs were produced as described by Lucarotti et al. (2007).
Briefly, NeabNPV was applied at a rate of 3 x 109 OBs/ha over balsam fir stands infested with high density populations (500+ larvae/ 45-cm branch) of N. abietis larvae, most of which were third instars. Approximately one week following NeabNPV applications and for the next 5-7 days, NeabNPV-infected larvae were collected onto plastic tarpaulins placed under individual trees, by beating mid- to lower canopy- branches with plastic leaf rakes. Larvae and needles were then transferred to 20- and 40- kg heavy paper bags (same as those used for sugar) along with a few balsam fir boughs for any larvae that were still feeding. The bags were stored upright and indoors at
35 ambient temperatures of 15-20°C for 7-10 days, when all feeding activity had ceased.
Dead larvae, needles and other debris were then transferred to 20-kg brown paper bags, stapled shut and stored at room temperature (20°C). Dead larvae were separated from needles and other debris using a blower (Lucarotti et al. 2007), hand picked from the remaining debris and then placed into 50-mL conical, plastic centrifuge tubes which were stored at -20°C. NeabNPV OBs were isolated and purified from N. abietis larvae by homogenizing larvae in a 0.3% sodium dodecyl sulphate (SDS) solution using a hand-held blender (Lucarotti et al. 2007, Moreau et al. 2005). The homogenate was filtered through a 1-mm plastic mesh and the filtrate was set aside. The solids caught in the mesh were re-suspended in 0.3% SDS, homogenized, filtered and the filtrate reserved. This process was repeated until the filtrate ran clear. The reserved filtrates were pooled, filtered twice through eight layers of cheesecloth and centrifuged at 9000 xg for 15 min. The supernatant was discarded and the NeabNPV OB pellet was re- suspended in 0.3% SDS. Centrifugation and re-suspension were repeated until the supernatant was clear. The NeabNPV pellet was suspended in water and the concentration of OBs quantified by proportional counting using serial dilutions of a suspension of latex beads (Moreau et al. 2005). The mean concentration of OBs was adjusted to 4><109 OBs/mL and then stored at 4°C to inhibit the growth of contaminating bacteria.
Field Sites and Study Design
Field trials and assessments were conducted in July and August 2002 near
Corner Brook, NL, Canada (48° 57'N: 57° 57'W) in naturally regenerated and
36 precommercially thinned stands of mixed conifers, dominated by balsam fir, but also including some black spruce (Picea mariana (Mill.) B.S.P.) and white spruce (Picea glauca (Moench.) Voss) (Moreau et al. 2006b). Two experimental blocks, each with its own untreated control block, were set up (Fig. 3-1). The purpose of the first block, near
Island Pond (Moreau et al. 2005, block 02-T2: 48°53'13.0"N: 57°52'55.9"W; control
48°53'48.5"N: 57°53'17.7"W), was to examine the spread of NeabNPV from a 313-ha aerial treatment block into populations of balsam fir sawflies adjacent to the treated block. The purpose of the second 3141-ha treatment block, near Old Man's Pond
(Moreau et al. 2005, block 02-T1: 49°3'23.3"N: 57°51'45.6"W; control 49°05'37.3"N:
57°54'49.0"W) was to determine whether or not NeabNPV could disperse and infect
BFS within a 200m zone between spray lines. Temperature, wind speed and relative humidity were monitored every 60 sec, on site, at each treatment block, during aerial applications of NeabNPV using a WatchDog 700 weather station (Plainfield, IL, USA).
The Island Pond treatment block (Fig. 3-1) was sprayed on July 22, 2002 between 06:30 and 07:45 (Fig. 3-2a). The entire 313-ha block was treated with
NeabNPV. The eastern half of the Old Man's Pond treatment block, which contained our transect line (Fig. 3-1), was treated on July 24, 2002 between 19:45 and 20:45. The western half was treated on July 25, 2002 between 07:35 and 08:20 (data not shown).
NeabNPV was applied to both blocks at track flow rates of 17.5-20L/min with swath widths of 25 m. The Old Man's Pond block was sprayed in treatment zones, 200 m wide, which were separated from each other by a 200 m wide area of untreated forest
(no treatment zones). Spray deposit in both the Island Pond and Old Man's Pond treatment blocks was monitored using 10 x 10 cm Kromakote cards placed on top of 1-m
37 wooden stakes driven into the ground adjacent to branch sample sites located at 20-m intervals along transects roughly perpendicular to the spray tracks of the aircraft
(Moreau et al. 2005) (Fig. 3-1). Kromacote cards were collected between 1 and 2 hours after the spray at Island Pond, and within an hour of the spray at Old Man s Pond.
Balsam Fir Saw fly Sampling
At Island Pond, one balsam fir tree was selected along each of two transects at distances of 0 (the edge of the treatment block), 25, 50, 100, 200 and 400m outside of the treatment block (i.e., total of 6 tress per transect). The two transect lines were established on opposite sides of the treatment block running roughly parallel to the direction of the prevailing north-east winds (Fig. 3-1) in order to account for any drift that may occur from the spray. Transects at Island Pond were sampled on a weekly basis, starting with a pre-spray sample on July 22, and following with three post-spray samples between August 1 to August 13. Two similar transect lines were established, and sampled weekly in a similar manner, in the control block. At Old Man's Pond, a 400 m long transect line that ran perpendicular to the aircraft spray lines was established.
The transect line spanned one untreated zone and extended 100 m into the two treatment zones on either side (Fig. 3-1). A similar 400-m transect line was established, and sampled weekly, in the control block (Fig. 3-1). One balsam fir tree was selected for sampling every 20 m along the transect line in both the treatment and control blocks
(i.e., total of 20 trees). Transects at Old Man's Pond were sampled on a weekly basis, starting with a pre-spray sample on July 22, and followed by three post-spray samples between July 29 to August 13. Densities of BFS larvae and/or pupae (juvenile BFS, henceforth) were determined, for both Island Pond and Old Man's Pond, in treatment and control blocks beginning immediately prior to NeabNPV application and then once
38 each week until all surviving larvae had pupated. A 45-cm branch tip was cut, using pruning shears, from one mid-crown branch of each sample tree within each block pair
(i.e. treatment and control) on the same day. Sample branches were placed into 20-kg brown paper bags for transport to the laboratory at the Canadian Forest Service Field
Station at Pasadena, NL (49°01 '29.9"N: 57°35'24.1"W) for processing. The number of larvae and cocoons on each sample branch were counted and recorded. To express balsam fir sawfly densities in terms of the number of juvenile BFS per m2 of foliage, the surface area of each branch was estimated by multiplying the 45-cm branch length by the average width of each branch. To determine the prevalence of NeabNPV, a sub- sample of 10 larvae and/or pupae (either living or dead) was randomly taken from each sample branch on each sampling date. Insects were individually placed into 1.5-mL polypropylene microcentrifuge tubes and stored at -20° for molecular probing.
Molecular probing for NeabNPV
Larvae and pupae were probed for NeabNPV using NeabNPV DNA-fluoroscein-
N6-dATP-labeled DNA probes (Renaissance, Perkin-Elmer Life Sciences). Seven
NeabNPV DNA/£coRlfragments (3.5-5.5 kb) were used as templates (Moreau et al.
2005). Individual insects were thawed and homogenized in ~ 1 mL of double-distilled water in the 1.5-mL microcentrifuge tubes they had been stored in. A 3-|jL aliquot of each sample was blotted onto Biodyne A nylon membranes (Pall, Gelman Laboratory).
Positive controls of purified NeabNPV DNA or NeabNPV OBs were also spotted onto each membrane. Membranes were soaked in denaturing solution (0.5 N NaOH. 1.5 M
NaCl) and incubated at 65°C for 30 min. Membranes were neutralized in 1.5 M NaCl,
0.5 M Tris. pH 7.0 for 1 min, soaked for 5 min in lOx SSC (saline sodium citrate buffer),
39 air-dried on filter paper and target DNA was then bound to the membranes by exposure to 125mJ of UV radiation using a BioRad GS Gene Linker™. Membranes were soaked in hybridizing solution containing the labelled probe for 18 h at 65°C. Excess probe and probe bound to non-specific DNA was removed with high stringency washes and results were recorded on Kodak BioMax ML film. The lower detection limit for the probing
-j protocol was 5 x 10 OBs (Moreau et al. 2005), implying a positive detection only for specimens where NeabNPV had replicated.
Statistical analysis
Data from the two sites were analysed separately because the NeabNPV treatments at Island Pond (entire block treated with sampling mostly outside the treatment block) differed from that at Old Man's Pond (partial block treatment with sampling within the block). The independent and interacting effects of NeabNPV application (treatment) versus no treatment (control), time of sample and distance from
NeabNPV application (a covariate with values of 0 to 400m at Island Pond and 0 to
100m at Old Man's Pond) on juvenile BFS densities and percent NeabNPV infection were evaluated using analysis of covariance (General Linear Model in Minitab -Anova tables are presented in Appendix A). Pearson's correlation coefficients were calculated to evaluate the relationships between distance from spray swaths and: 1) the level of
NeabNPV infection, and, 2) the larval density in the treatment blocks.
Results
40 Island Pond 2002
During the period of the aerial application, the temperature remained constant at
15°C, relative humidity increased from about 60 to 70% and wind speed was negligible at < 1.5 km/h (Fig. 3-2a). Spray droplets were not detected on cards beyond 25m outside
the treatment block (Fig. 3-4a), on either side, presumably due to low wind velocities at the time of application. Consequently, data from transects on both sides of the block
were pooled for analysis.
NeabNPV prevalence was negatively correlated with distance from the treatment
block in samples taken after the spray (Table 3-1). The overall trend in the change in the densities of juvenile sawflies and prevalence of NeabNPV infections over time in treatment and control is shown in Fig. 3-3.
Prior to application of NeabNPV, densities of juvenile sawflies were relatively constant along the 400-m transect lines extending out from both the treatment and control blocks (Fig. 3-4b). The densities and infection in the control plots (Figs. 3-3b-e) are represented as a mean value (+SEM) because they exhibited no discernable consistent pattern along the transects. Densities of juvenile sawflies decreased with time
(F3,80=34.64, p<0.01) from about 770 individuals/m2 before the spray to fewer than 250 individuals/m2 three weeks after the spray (Fig. 3-4e). There was a greater decrease in the density of juveniles at the edge of the treatment block than control block (F|.so=2.55,
p=0.017). The greatest reduction in juvenile sawfly densities over time occurred at sampling sites closest to the treatment block, resulting in significant interactions
between treatment and distance (Fi.80=1.66, p=0.04) and between time and treatment
(F3.8o= 3.45, p=0.024). Density of BFS larvae was not significantly affected by distance
41 from the edge of the treatment block (Fi.go-0.77, p=0.579), or interactions between time
= and distance (F3.80- 0.81, p-0.66), or between time, distance and treatment (F(3.8o> 0.81, p=0.67). There was no significant correlation between BFS density and distance from the control block but by the third week of sampling after the NeabNPV application, density was positively correlated with distance from the treated block (Table 3-1).
NeabNPV was not detected in any larvae in the control block and was only detected in three larvae, located 200 m from the treatment block, on the day before the aerial application of NeabNPV. During the weeks following the spray, the percentage of infection with NeabNPV increased with time (Fig. 3-4b-d) (F <3.80)==41.16, p<0.01). The prevalence of NeabNPV in juvenile sawflies outside the treated block rose from <5% before the spray to approximately 70% after three weeks. The prevalence of NeabNPV increased much more in juvenile sawfly larvae at the edge of the treatment than control
= block (FI,8O 192.90, p<0.01), and NeabNPV prevalence decreased with distance from the treated block (FI.8O=12.93, p<0.01). This resulted in significant interactions between treatment and time since application ofNeabNPV (F3.8o=42.51, p<0.01) and between treatment and distance from the treatment block (FI.8O=5.64, p<0.01). Similarly, the increase in NeabNPV infection along the transect outward from the treatment block resulted in a significant interaction between time and distance from the treatment block
(F3.80—6 .74, p<0.01) and by the three-way interaction between treatment, time and distance from the treatment block (F3.8o=2.34, p<0.01).
Old Man's Pond 2002
During the 50 min over which the application ofNeabNPV OBs occurred, the temperature declined from 16.5°C to 13.5°C and the relative humidity rose from 65 % to
42 82% (Fig. 3-2b). Wind speed was variable, gusting between 1.5 and 11.5 km/h and blew predominantly from the north-west. The exact position of the aircraft over the transect line could not be determined, but the greatest concentration of droplets occurred at points 80 and 280 m along the transect line and tapered off either side of those points
(Fig. 3-6a).
Initially, there was no significant correlation between larval sawfly density and distance from the spray swaths in the treatment block. An initially weak negative correlation between prevalence of NeabNPV infection and distance from the spray swaths declined with the passing of time since spray (Table 3-2). The overall trend in the change in the densities of juvenile sawflies and prevalence of NeabNPV infections over time in treatment and control is shown in Fig. 3-5.
Densities of sawfly larvae varied between approximately 100 and 500/m2 just before the application of NeabNPV (Fig. 3-6b). Thereafter, densities of juvenile sawflies decreased with time (F3.i44= 12.27, p<0.01) from an average of approximately 250/m2 before the spray to fewer than 100/m2 3 weeks later (Fig. 3-6b-e). The densities and infection in the control plots (Figs. 3-6b-e) are represented as a mean value (+SEM) because they exhibited no discernable consistent pattern along the transects. Densities declined more in the treatment block than in the control block (Fu44= 12.38, p<0.01).
Juvenile sawfly densities were influenced by distance from the spray swaths (Fi.i44=
4.20, p=0.04), declining more at sample points within or adjacent to the spray swaths than in those towards the middle of the untreated zone. A decline in juvenile sawfly densities in the untreated zone in the weeks following the spray resulted in an interaction between time and distance from the spray swaths (Fu44=4.00, p=0.01), but not between
= treatment and distance (F|.l44 0.33, p=0.57). The decline in juvenile sawfly densities in
43 the treatment block in the weeks following the spray was higher than in the control block, resulting in an interaction between time and treatment (F3.h4= 3.01, p=0.03).
Juvenile sawfly density was not influenced by the interaction between time, treatment and distance from the spray swaths (F3.h4= 0.24, p=0.87).
NeabNPV was not detected in any larvae in the control block, and was only detected in two larvae within the treatment block on the day before the aerial virus application. In contrast, NeabNPV infection increased with time following the application of NeabNPV OBs (F3.m4= 43.20, p<0.01) with 100% NeabNPV infection in juvenile sawflies at most sampling points beneath the spray swaths three weeks post spray (Fig. 3-6e). Over all, NeabNPV infection was much higher in the treatment block than in the control block (F1144- 97.42, p<0.01). Prevalence of NeabNPV infection rose on average to 50-75% in juvenile sawflies in the untreated zone between the spray swaths, resulting in a significant effect of distance from the spray swaths (F 1,144— 9.58, p<0.01). This was presumably a result of aerosol spread of NeabNPV by gusting winds during the application of NeabNPV OBs (Fig. 3-2b). The increase in incidence of
NeabNPV infection in the area between spray swaths occurred more slowly, and to a lower extent than that at sample points beneath the spray swaths, resulting in interactions between time and distance from the spray swaths (F3,i44= 4.22, p<0.01), treatment and time since treatment (F3 i44=23.53, p<0.01) and treatment and distance (Fi,|44= 7.04, p=0.01). Percent infection within the treatment block was not influenced by interactions between treatment, time, and distance from the spray swaths (F3.144= 1 -48, p= 0.22).
44 Discussion
My study demonstrates that aerial application of NeabNPV not only results in major declines in BFS densities directly under the flight lines of the aerial applications
(Moreau et al. 2006; Lucarotti et al. 2007) but also in high levels of NeabNPV-caused mortality in areas adjacent to the treatment areas. The greater declines in BFS density observed closer to the areas of aerial application are probably the result of greater
NeabNPV prevalence (bigger pool of virus) close to the treatment blocks.
Following the aerial sprays, a 'wave' of virus spread outwards from the point of aerial application was observed at both of my sites, although the wave-like pattern of disease spread seen in previous studies (Entwistle et al. 1983, Dwyer and Elkinton,
1995) was more apparent at the Island Pond site than at Old Man's Pond. The dispersal of NeabNPV into adjacent areas could have been caused by abiotic (drift, post-spray movement due to environmental factors such as wind and rain) or biotic (dispersal of infected BFS larvae, secondary inoculum built up over successive rounds of replication, or dispersal by other organisms passively carrying the virus (e.g., birds, parasitoids) (see
Reviews by Fuxa, 1987, Cory and Evans 2007).
Numerous studies have shown aerial drift of pesticides to be a major concern of aerial spray programs, with identifiable volumes of active ingredients being recorded hundreds of metres from the point of application (Yates et al. 1978, Renne and Wolf
1979, Chester and Ward 1984, Oeseburg and van Leeuwen 1990, Payne 1993, Woods et al. 2001). Teshke et al. (2001) found culturable quantities of Bacillus thurigiensis could remain airborne for up to 3.3 hours after aerial spray. In my study, moderate and variable wind conditions (Fig. 3-2b) led to drift of the NeabNPV spray into the 200m
45 wide spaces between the treated areas within the Old Man's Pond treatment block (Fig.
3-6a). This may explain the rise in prevalence of NeabNPV in the untreated areas between the aerial spray swaths. In contrast, drift from the spray plane cannot explain the spread of the virus at Island Pond, where post-spray horizontal transmission is seen to have occurred up to 400 m from the point of application (a two-fold further distance than possible at Old Man's pond). The wind speed at Island Pond was very low during the aerial spray of NeabNPV (Fig.3-2a) and kromakote cards did not show any drift greater than 25 m outside of the spray block (Fig. 3-4a), comparable to a study by Ripley et al. (1993), where aerial pesticide residues were found up to 84 m from the point of application under windless conditions. The kromakote cards at Island Pond were collected over a two hour period following the spray as the potential for moisture from evening dew to accumulate on the cards was limited. This provided a short period of time for aerosols from the spray to deposit on cards post spray, which did not occur, outside of what might be expected during the spray itself. Thus the horizontal transmission of NeabNPV outward from the area sprayed at Island Pond was facilitated by either meteorological or biotic factors post spray.
Work by Anstey et al. (2002) indicates that most N. abietis larvae move within the crown of the same tree, and my own observations on daily activity suggest that daily movements are usually limited to within a few centimetres on the same branch (Graves et al. unpublished). To the best of my knowledge, there are no studies demonstrating movement of BFS larvae between balsam fir trees. Thus, the presence of larvae infected by NeabNPV up to 100m at Old Man's Pond and 400m at Island Pond from the sprayed area is probably not due to dispersal of infected larvae from the treatment area.
46 Dwyer and Elkinton (1995) found that dispersal of the NPV of gypsy moth
(Lymantria dispar) larvae into a susceptible, healthy population from point source
introductions was not explained by larval dispersal. They observed that few first-instar
larvae ballooned further than 40 m from the point of NPV introduction, but that
comparably high proportions of larvae were infected with virus at distances of up to 120 m, three times the ballooning distance. In studies on lepidopteran pests of agricultural crops Vasconcelos et al. (1996) found that the movement of both healthy and NPV-
infected larvae only ranged from 0 to 24 cm but NPV inoculum was found up to 45 cm from a point source introduction.
The shape of the waves at my sites were somewhat irregular, similar to the multiple season work on GiheNPV in populations of Gilpina hercyniae carried out by
Entwistle et al. (1983). There, they suggested that spatial irregularities in the incidence of virus was due to secondary 'waves of infection' produced by successive generations of the insect resulting in the overlapping of multiple epicentres. Modelling work done by
White et al. (1999) indicates that the irregularities in the wave form may be attributed to seasonality of the host insect, caused by multiple epicentres of disease spread forming multiple wave fronts as subsequent rounds of the infection cycle starts within a new generation of hosts in each new season.
I postulate that these scenarios are unlikely to be operative in either of my studies as my results examine the pattern of NeabNPV dispersal within a single season. N. abietis is univoltine. Therefore I did not observe a subsequent wave of NeabNPV infection arising among the successive generations of a multivoltine species. The irregularities of form in the infection wave are, instead more likely the result of secondary replication of virus within the current generation and deposition of virus-
47 laden diarrhea by infected larvae (Graves et al. unpublished) or the expression of low levels of ambient NeabNPV in the surrounding BFS populations (as observed in the controls in Figs. 3-3 and 3-5).
In conclusion, my results suggest that inundative spray of entire stands of balsam fir with NeabNPV may not be necessary to suppress BFS populations. Instead, efficient transmission of NeabNPV may be achieved by the combined actions of natural and abiotic factors that facilitate the horizontal spread of the virus across the landscape within a single season.
Acknowledgements
I thank Benoit Morin for technical assistance and lab space, Corner Brook Pulp and
Paper for use of field sites, and the Biocontrol Network, Canadian Forest Service,
Corner Brook Pulp and Paper, Abitibi Consolidated, Forest Protection Limited, Natural
Sciences and Engineering Research Council of Canada (Discovery grant) and the
Newfoundland and Labrador Department of Natural Resources for funding and logistical support.
Literature Cited
Anderson R. M. and May, R.M. 1981. The population dynamics of microparasites and their invertebrate hosts. Philosophical Transactions of the Royal Society of London B, Biological Sciences 291:451-524.
Anstey, L.J., Quiring, D.T. and D.P. Ostaff. 2002. Seasonal changes in intra-tree distribution of immature balsam fir sawfly (Hymenoptera: Diprionidae). Canadian Entomologist.
Bird, F. T. and Elgee, D. E. 1957. A virus disease and introduced parasites as factors controlling the European spruce sawfly, Diprion hercyniae (Htg.), in central New Brunswick. Canadian Entomologist. 89: 371-378.
48 Bird, F. T. and Birk, J. M. 1961. Artificially disseminated virus as a factor controlling the European spruce sawfly, Diprion hercyniae (Htg.) in the absence of introduced parasites. - Canadian Entomologist. 93: 228-238.
Carrol, W.J., 1962. Some aspects of the Neopdiprion abietis complex in Newfoundland. PhD. Dissertation, State University College of Forestry, Syracuse University.
Chester, G. and Ward, R.J. 1984. Occupational exposure and drift hazard during aerial application of paraquat to cotton. Archives of Environmental Contamination and Toxicology 13: 551-563.
Cory, J. S. and Evans, H.F. 2007. Viruses, Chap. IV-I, in Field Manual of Techniques in Invertebrate Pathology, 149-174. L.A. Lacey and H.K. Kaya (eds.), Springer, 2007.
Dwyer, G. 1992. On the Spatial Spread of Insect Pathogens: Theory and Experiment. Ecology 73 (2). 479-494.
Dwyer, G., Elkinton, J.S. 1995. Host dispersal and the spatial spread of insect pathogens. Ecology 76: 1262-1275.
Entwistle, P.F., Adams, P.H.W., Evans H.F., Rivers, C.F. 1983. Epizootology of a nuclear polyhedrosis virus (Baculoviridae) in European spruce sawfly (Gilpinia hercyniae): spread of disease from small epicentres in comparison with spread of baculovirus diseases in other hosts. Journal of Applied Ecology 20: 473-487.
Frank, R., Ripley, B.D., Lampman, W., Morrow, D., Collins, H., Gammond, G.R., McCubbin, P. 1993. Comparative spray drift studies of aerial and ground applications 1983-1985. Environmental Monitoring and Assessment 29:167-181
Fuxa, J.R. 1987. Ecological considerations for the use of entomopathogens in IPM. Annual Review of Entomology 1987. 32: 225-51.
Fuxa, J.R. and Richter, A.R. 1999. Classical biological control in an ephemeral crop habitat with Anticarsia gemmatalis nucleopolyhedrovirus. BioControl 44: 403- 419, 1999.
Fuxa, J.R. 2004. Ecology of insect nucleopolyhedroviruses (Review). Agriculture, Ecosystems and Environment. 103: 27—43.
Jehle, J.A, Blissard, G.W., Bonning, B.C., Cory, J.S., Herniou, E.A., Rohrmann, G.F, Theilmann, D.A., Thiem, S.M., and Vlak, J.M. 2006. On the classification and nomenclature of baculoviruses: A proposal for revision. Archives of Virology. 151: 1257-1266.
49 Lucarotti, C.J., Morin, B. Graham, R.I., Lapointe, R. 2007. Production, application, and field performance of Abietiv™, the balsam fir sawfly Nucleopolyhedrovirus Virologica Sinica, 22 (2):163-172
Moreau, G., Lucarotti, C.J., Kettela, E. G., Thurston, G.S., Holmes, S., Weaver, C., Levin, D.B., Morin, B. 2005. Aerial application of nucleopolyhedrovirus induces decline in increasing and peaking populations of Neodiprion abietis. Biological Control 33: 65-73.
Moreau, G. 2006. Past and present outbreaks of the balsam fir sawfly: an analytical review. Forest Ecology and Management 221:215-219.
Moscardi, F., 1999. Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology 1999. 44: 257-89.
Oeseburg, F. and van Leeuwen, D. 1990. Dispersion of aerial agricultural sprays; model and validation. Agricultural and Forest Meteorology 53: 223-255.
Otvos, I. S., Cunningham, J. C. and Friskie, L. M. 1987 (a). Aerial application of nuclear polyhedrosis virus against Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough) (Lepidoptera: Lymantriidae). I. Impacts in the year of application. Canadian Entomologist 119: 697-706.
Otvos, I. S., Cunningham, J. C. and Alfaro, R. I. 1987 (b). Aerial applications of nuclear polyhedrosis virus against Douglas- fir tussock moth, Orgyia psuedotugata (McDunnough) (Lepidoptera: Lymantriidae). II. Impacts 1 and 2 years after application. Canadian Entomologist. 119: 707-715.
Payne, N.J. 1993. Spray dispersal from aerial glyphosate applications. Crop Protection 12 (6): 463-469.
Payne, N.J., 2000 Factors influencing aerial insecticide application to forests. Integrated Pest Management Reviews 5: 1-10.
Renne, D. S. and Wolf, M. A. 1979. Experimental studies of 2,4-D herbicide drift Characteristics. Agricultural Meteorology 20: 7—24
Shepherd, R. F., Otvos, I. S., Chorney, R. J. and Cunningham, J. C. 1984. Pest management of the Douglas-fir tussock moth (Lepidoptera: Lymantriidae): prevention of an outbreak through early treatment with a nuclear polyhedrosis virus by ground and aerial applications. Canadian Entomologist 116: 1533-1542.
Stairs, G. R. 1965. Artificial initiation of virus epizootics in forest tent caterpillar populations. Canadian Entomologist. 97: 1059-1062.
Teshke, K., Chow, Y., Bartlett, K. Ross, R., van Netten, C., 2001. Spatial and temporal distribution of airborne Bacillus thuringiensis var. kurstaki during an aerial spray
50 program for gypsy moth eradication. Environmental Health Perspectives 109 (1): 47-54.
Vasconcelos, S.D., Cory, J.S., Wilson, K.R., Sait, S.M., Hails, R.S. 1996. Modified I behaviour in baculovirus-infected lepidopteran larvae and its impact on the spatial distribution of inoculum. Biological Control 7, 299-306.
Wallace, D.R., Cunningham, J.C. 1995. Diprionid Sawflies. In: Forest Insect Pests in Canada, volume 1. Edited by J.A. Armstrong and W.G.H. Ives. Pp. 193-232.
White, A., Bowers, R.G. Begon, M. 1999. The spread of infection in seasonal insect- pathogen systems. Oikos 85: 487-498.
Woods, N., Craig, I.P., Dorr, G., Young, B. 2001. Spray Drift of Pesticides Arising from Aerial Application in Cotton. Journal of Environmental Quality 30:697-701.
Yates, W.E., Akesson, N.B., Bayer, D.E. 1978. Drift of glyphosate sprays applied with aerial and ground equipment. Weed Science 26(6): 597-604
Young, E. C. 1974. The epizootiology of two pathogens of the coconut palm rhinoceros beetle. Journal of Invertebrate Pathology 24: 82-92.
51 Table 3-1: Island Pond 2002. Summary of Pearson correlation analyses showing weekly trends in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and juvenile sawfly densities and the prevalence of NeabNPV infection in juvenile sawfly populations.
No.weeks Density Infection
Treatment Post spray N Pearson's r p-value Pearson's r p-value
control 1 12 -0.469 0.12 -0.343 0.27
control 2 12 -0.254 0.43 -0.229 0.47
control 3 12 -0.434 0.16 -0.421 0.17
NeabNPV 1 12 0.156 0.63 -0.103 0.75
NeabNPV 2 12 0.466 0.13 -0.718 0.01
NeabNPV 3 12 0.722 0.01 -0.909 0.00
52 Table 3-2: Old Man's Pond 2002. Summary of Pearson correlation analyses showing weekly trends in the relationship between distance from the two spray swaths separated by 200 m in the treatment block and the untreated (control) block and, juvenile sawfly densities and the prevalence ofNeabNPV infection in juvenile sawfly populations. The "distance from spray" values used for the treatment block were also used for the control block, assuming that spray swaths would have occurred in the same location as in the treatment.
No. weeks Density Infection
Treatment Post spray N Pearson's r p-value Pearson's r p-value
control 1 20 -0.237 0.31 0.150 0.02
control 2 20 0.505 0.62 .* .*
control 3 20 0.058 0.81 -0.408 0.07
NeabNPV 1 20 -0.418 0.07 -0.402 0.01
NeabNPV 2 20 -0.402 0.50 -0.364 0.12
NeabNPV 3 20 -0.103 0.01 -0.098 0.68
*Infection values were 0 on the second sampling date, July 29, 2002
53 •id Baraohois Ponds Old Man's Pond j± *•* IF
-k rclver's 11 i v' i] 1 : •*! i Deer L Ell 4:!•'] riM'! i!. NwJh Pond VkBt sch ;.l !i(i ach
N pve
Rubber Lake e Rapids
ssell CORNERrmsxTurc BROOK Steady firoek Lake MasseTiJrive Clarks Bros •14501 jrnfc^ond South E Ea#few*4.ake
Islaniyond
3ig Ci
Island • alley UdKBb' StaoflLake Pond 5 km Corner Brook Lake jvefPond
Figure 3-1: Map of area surrounding Corner Brook, NL, showing the location of Island Pond and Old Man's Pond NeabNPV-treatment blocks in 2002. Insets show the locations of sampling transect lines (dotted lines) and aircraft spray tracts (dashed lines) at each location. Control blocks, which were located 1 to 5 km from treatment blocks, are indicated with stars.
54 SPRAY WEATHER, NEWFOUNDLAND ISLAND POND, JULY 22, 2002 (a)
30 100 •O •I „ 90 Ul Q. 24 End 80 CO Start a 21 70 S 18 60 UJ 15 50 X IK K 40 2 9 30 £ 6 20 £ 3 10 0 — 6:00 6:15 6:30 6:45 7:00 7:15 7:30 7:45 TIME OF DAY(AM)
TEMP, C RH. % WIND SPEED, kmh
SPRAY WEATHER, NEWFOUNDLAND OLD MANS POND (1/2), JULY 24, 2002 (b)
30 100 o UJ 27 90 Ul a. 24 80 CO a 21 70 z 5 18 60 15 50 ^ uf TEMP, C - RH, % —— WIND SPEED, kmh Figure 3-2: Wind speed, temperature and relative humidity during the periods of NeabNPV aerial spray operations on July 22, 2002, at a) Island Pond and on July 24, 2002, at b) Old Man's Pond. 55 1000 n (a) Treated 600 - u- £0 o 400 - Z 200 - Treated (b) Control c _o o js > cu Z X)03 z 0 1 2 No. Weeks Post Treatment Figure 3-3: a) Seasonal trends in the mean (+ SE) densities of juvenile (larvae and pupae) balsam fir sawflies, b) Mean (+ SE) prevalence of NeabNPV infection in juvenile sawfly populations adjacent to NeabNPV treatment (black circles) and untreated control (white circles) blocks at Island Pond. Arrow indicates aerial application of NeabNPV OBs on July 22, 2002. 56 15 - rs| e 12 - o (a) 9 - ts a 6 - o i- a 3 - 80 160 240 320 400 100 100 (b) 80 80 60 - 60 40 - 40 20 - 20 0 0 80 160 240 320 400 Control C/3 c 60 - - 60 40 - 40 20 20 0 0 80 160 240 320 I400 Control 100 100 (e) 80 - 80 60 - 60 40 40 20 - 20 0 Li 160 240 320 400 Control Distance (m) Figure 3-4: a) Average spray deposit ( shown as light gray shaded area) along two transect lines extending 400 m out from the east and west sides of a 313-ha treatment block at Island Pond following aerial application of NeabNPV on July 22, 2002. Percentage of initial juvenile balsam fir sawfly (larvae and pupae) density (dark gray bars) and mean prevalence of NeabNPV infection (black bars) in the treatment block and the untreated control block on b) July 22, c) July 29, d) August 6 and e) August 13, 2002. The densities and infection in the control plots are represented as a mean value (+SEM) because they exhibited no discernable consistent pattern along the transects. 57 300 (a) Treated COu- 03 150 o 100 Z 50 - (b) 100 Treated Control 0s C .2 , > Cu Z X>CS 2 0 12 3 No. Weeks Post treatment Figure 3-5: a) Seasonal trends in the mean (+ SE) densities of juvenile (larvae and pupae) balsam fir sawflies. b) mean (+ SE) prevalence of NeabNPV infection in juvenile sawfly populations adjacent to NeabNPV treatment (black circles) and untreated control (white circles) blocks at Old Man's Pond. Arrow indicates aerial application of NeabNPV OBs on July 22, 2002. 58 12 £O (a) 73C/5 9 6 a. o 3 Qu. 80 160 240 320 400 100 - (- 100 (b) 80 - 80 60 60 40 - - 40 20 - 20 0 0 80 160 240 320 400 Control 100 i 100 (c) 80 - 80 c 60 - 60 Q O-a •-i "3 40 - 40 O a 3 20 20 o r&n ox 80 160 240 320 400 Control •2 o c D «u u (Uf-4 100 - 100 (X (d) 80 - - 80 60 60 40 - 40 20 - 20 0 il 0 80 160 240 320 400 Control (e) 60 - 160 240 Control Distance fm) Figure 3-6: (a) Average spray deposit along a transect line extending 400 m across two 50-m NeabNPV spray swaths (light gray shaded areas) and a 200-m untreated zone (between shaded areas) at Old Man's Pond following aerial application of NeabNPV on July 24, 2002. Percentage of initial juvenile balsam fir sawflies (larvae and pupae) densities (gray bars) and prevalence of NeabNPV infection (black bars) in the treatment block (same transect line as A) and the untreated control block on (b) July 22, (c) July 29, (d) Aug. 6 and (d) Aug. 13, 2002. The densities and infection in the control plots are represented as a mean value (+SEM) because they exhibited no discernable consistent pattern along the transects. 59 CHAPTER 4 - GENERAL DISCUSSION It has been suggested that the host specificity and general lethality of NPVs make them ideal candidates for biological control (Fuxa 1987, Vail et al. 1999, Fuxa 2004, Szewczyk et al. 2006, Cory and Evans 2007), and, in fact, NPVs are well known to initiate epizootics among eruptive sawfly host populations. For example, NeabNPV has previously been shown to be effective (Moreau et al. 2005, Lucarotti et al. 2007) in suppressing N. abietis using aerial spraying, which is considered the most efficient means to treat large areas with pesticides in the forestry context (Payne 2000). Unfortunately, the interest in developing NPVs as a biological control agent has often suffered from the limitations imposed by in vivo production of virus (Fuxa 1987, Cory and Evans 2007), which makes them expensive to produce for large scale applications. A strategy for increasing the cost effectiveness of aerial applications of NPVs is the exploitation of the natural ability of NPVs to spread rapidly throughout a population in order to reduce the coverage required during spraying. It has generally not been clear how NPV epizootics are initiated and transmitted throughout sawfly populations. However, other studies on NPV transmission in sawflies have shown that epizootics can be initiated from persistent environmental contamination with OBs (Kaupp 1982, 1983, Olofsson 1987, 1988a,b 1989) and subsequent spread from groups of sawfly larvae within a tree and from tree to tree (Young and Yearian 1987, Young and Yearian 1989, Young and Yearian 1990). Prior to this study, little was known of horizontal transmission rates of NeabNPV. Epizootics of NeabNPV occur at peak balsam fir sawfly population outbreak densities (Moreau 2004, Moreau et al. 2005), similar to the larval densities used in the branch level experiment described in chapter 2. Generally, the average larval mortality 60 attributable to NeabNPV infection increased with the number of infected larvae introduced into a cohort in the branch level study. The average mortality when infected larvae were introduced in cohorts of third versus first instar larvae was statistically significant in my study. One might expect higher rates of NeabNPV induced mortality in first-instar cohorts, due to an increased time for multiple cycles of NeabNPV replication and subsequent release of OBs to have occurred than when NeabNPV is introduced to third instars; or because of higher susceptibility of the less robust first instar larvae to the pathogen (Dwyer 1991, Goulson et al. 1995, Cory et al. 1997). The number of infected larvae introduced into a cohort in the laboratory (chapter 2) did not significantly influence the total number of infected larvae over the course of the experiment, although it may have had very small effects on the rate at which disease spread within the cohort. Under the high cohort densities of this experiment, even one infected larva apparently produced sufficient amounts of NeabNPV OBs in the local environment to infect many, if not all, larvae within the cohort. This study demonstrates how a NeabNPV epizootic might begin in BFS populations with the initial infection of low numbers of BFS larvae in isolated groups. In a number of studies, it has been noted that horizontal transmission of NPVs in lepidopteran hosts is not linearly density dependant, as the mass-action assumption of the Anderson and May model (1981) suggests (D'Amico et al. 1996, Dwyer 1991, Dwyer and Elkinton 1995, Knell et al. 1998, Dwyer et al. 2000). In contrast, my results suggest that NeabNPV transmission in natural populations of BFS may follow the density dependency assumption of Anderson and May (1981), at least when NeabNPV is introduced early in larval development and at relatively low host density, roughly equivalent to that at the start of an outbreak. However, at higher densities, comparable to 61 those found near the end of BFS outbreaks, this aspect of NeabNPV transmission seems to become less important. Higher BFS densities will lead to crowding, and may stimulate natural initiation of latent virus, or a greater likelihood that multiple larvae will become infected from sources of ambient NeabNPV that may be present. The field studies described in Chapter 3 demonstrate that aerial application of NeabNPV not only results in major declines in N. abietis densities directly under the flight lines of the aerial applications (Moreau et al. 2006; Lucarotti et al. 2007) but high levels of NeabNPV-caused mortality also occur in areas adjacent to the treatment blocks. NeabNPV-induced disease spreads hundreds of meters from the point of aerial application into the adjacent natural N. abietis population in a single season. Ultimately, it is unknown how much of the observed spread of NeabNPV was caused by abiotic factors (drift, post-spray movement due to environmental factors such as wind and rain) or biotic factors (dispersal of infected BFS larvae, secondary inoculum built up over successive rounds of replication, or dispersal by other organisms passively carrying the virus (e.g., birds, parasitoids) (see reviews by Fuxa, 1987, Cory and Evans 2007). However, in my study, moderate and variable wind conditions most likely led to drift of the NeabNPV spray into the spaces between the treated areas within the Old Man's Pond treatment block which would explain the rise in prevalence of NeabNPV in the untreated areas between the aerial spray swaths. In contrast, drift from the spray plane cannot explain the spread of the virus at Island Pond as the wind speed was very low during the aerial spray of NeabNPV. This was confirmed by the absence of deposits on kromakote cards placed further than 25m outside of the spray block. This is similar to a study by Frank et al. (1993), where residues from aerially applied pesticides were found up to 84 m from the point of application under windless conditions. 62 To the best of my knowledge, there are no studies demonstrating movement of N. abietis larvae between balsam fir trees. Thus, the presence of larvae infected by NeabNPV up to 100m at Old Man's Pond and 400m at Island Pond from the sprayed area is probably not due to dispersal of infected larvae from the sprayed area. During aerial sprays of pesticides from fixed wing aircraft, spray droplet sizes have been recorded as ranging from < 43-550|Jm? (for the herbicide glyphosate by Payne et al. 1993, and the insecticide diflubenzuron by Miller et al. 1996). The range of droplet sizes is a function of the spray equipment; therefore there is no reason to suspect the range of droplet sizes from aerial sprays of NeabNPV to be radically different. Given the small sizes of some of the droplets, more than adequate to house NeabNPV OBs (0.5 - 1.0 |jm), coupled with the fact that Teshke et al. (2001) found that the much larger Bacillus thurigiensis (at 1 pm by 5 |Jm) could remain airborne for more than 3 hours after aerial application; then offsite drift of some spray material is probably inevitable. In addition, copious quantities of OB-laden balsam fir sawfly frass and eventually OB- laden BFS corpses will be produced by N. abietis larvae infected by NeabNPV dying as a result of the initial spray treatment. As these sources of NeabNPV OBs are exposed to wind and rain, one would expect that the surrounding environments would become contaminated with OBs. Given the small amount of inoculum required to initiate a NeabNPV infection, and my observation that it only requires a single infected larva to initiate an epizootic within a cohort, it is my opinion that the long range dispersal of NeabNPV into the general population from the initial site of the spray treatment is primarily the result of wind and rain. NeabNPV transmission in natural populations of BFS seems to follow the mass action assumption of Anderson and May (1981), at least when NeabNPV is introduced 63 early in larval development and at relatively low host density, roughly equivalent to that at the start of an N. abietis outbreak. This is presumably due to dispersal of NeabNPV among larvae in a cohort being more dependant on biotic factors such as larval movement, where contact between infected larvae and healthy, susceptible larvae becomes more frequent with increasing larval densities (i.e. a density-dependant process). However, at higher densities, comparable to those found late in BFS outbreaks, this aspect of NeabNPV transmission seems to become less important as the impact of even one NeabNPV-infected larva on mortality within a cohort becomes more pronounced. The dispersal of NeabNPV by abiotic factors such as aerial drift or wind is not influenced by larval density. In addition, the very high virulence of NeabNPV means that only very low numbers of NeabNPV occlusion bodies are required to initiate infection in N. abietis larvae, and as I have observed, only one larva in a cohort needs to become infected to provide enough secondarily-produced occlusion bodies to infect the rest of the individuals in a cohort. Previous studies have shown that a relatively low density of NeabNPV (1 to 3 X 109 OBs/ha) is required to successfully initiate a local epizootic within a treatment block (Moreau et al. 2005, Lucarotti et al. 2007). The sprays conducted in my studies were done at the specified application rates (1X109 OBS/ ha) of abietiv (the registered commercial formulation carrying NeabNPV). My results suggest that the decreases in the densities of NeabNPV OBs per unit area of forest that may expected from spreading the spray over a bigger area (or partially treating an area), would have little impact on the transmission of NeabNPV through localized populations of N. abietis. 64 In conclusion, drift of the spray or wind dispersal of NeabNBV, combined with the high virulence of NeabNPV and gregarious behaviour within N. abietis cohorts likely allow NeabNPV to spread rapidly from the point of aerial application into the local population of N. abietis. This suggests that inundative spraying of entire stands of balsam fir with NeabNPV may not be necessary to suppress BFS populations. In fact, spraying NeabNPV with low wind speeds on site should be sufficient to initiate an epizootic and maximize viral spread throughout the N. abietis population in the local area. The potential impact of decreasing the density of NeabNPV OBs per unit area of foliage should be minimized by the high virulence of NeabNPV and how rapidly NeabNPV is communicated among the individuals in a larval cohort. My results suggest that when NeabNPV is applied early in N. abietis development, it can be an effective tool within an integrated pest management program. The rapid horizontal transmission of NeabNPV enables good suppression when only spraying a portion of an area experiencing a BFS outbreak. The efficient transmission of NeabNPV through a local population may be achieved by the combined actions of natural and abiotic factors that facilitate the horizontal spread of NeabNPV through the local forest area within a single season. Literature Cited Anderson R. M. and May, R.M. 1981. The population dynamics of microparasites and their invertebrate hosts. Philosophical Transactions of the Royal Society of London B, Biological Sciences 291:451-524. Cory. J.S., Hails, R.S., and Sait, S.M. 1997. Baculovirus Ecology, pp. 301-339 in Miller, L.K. (ed.) The Baculoviruses. Plenum Publishing Corporation, NY 65 Cory, J. S. and Evans, H.F. 2007. Viruses, Chap. IV-I, in Field Manual of Techniques in Invertebrate Pathology, 149-174. L.A. Lacey and H.K. Kaya (eds.). Springer, 2007. D'Amico, V., Elkinton, J. S., Dwyer, G., Burand, J. P., and Buonaccorsi, J. P. 1996. Virus transmission in gypsy moths is not a simple mass action process. Ecology 77: 201-206. Dwyer, G. 1991. The roles of density, stage, and patchiness in the transmission of an insect virus. Ecology 72: 559 - 574. Dwyer, G., Elkinton, J.S. 1995. Host dispersal and the spatial spread of insect pathogens. Ecology 76: 1262-1275. Dwyer, G., Dushoff, J., Elkinton, J., and Levin, S. 2000. Pathogen-driven outbreaks in forest defoliators revisited: building models from experimental data. The American Naturalist 156(2): 105- 120. Frank, R., Ripley, B.D., Lampman, W., Morrow, D., Collins, H., Gammond, G.R., McCubbin, P. 1993. Comparative spray drift studies of aerial and ground applications 1983-1985. Environmental Monitoring and Assessment 29:167-181 Fuxa, J.R. 2004. Ecology of insect nucleopolyhedroviruses (Review). Agriculture, Ecosystems and Environment. 103: 27-43. Fuxa, J.R. 1987. Ecological considerations for the use of entomopathogens in IPM. Annual Review of Entomology 1987. 32: 225-51. Goulson, D. and Cory, J.S. 1995. Sublethal effects of baculovirus in the cabbage moth Mamestra Brassicae. Biocontrol 5: 361-367. Knell, R. J., Begon, M., and Thompson, D. J. 1998. Transmission of Plodia interpunctella granulosis virus does not conform to the mass action model. Journal of Animal Ecology 67: 592-599. Lucarotti, C.J., Morin, B. Graham, R.I., Lapointe, R. 2007. Production, application, and field performance of Abietiv™, the balsam fir sawfly nucleopolyhedrovirus Virologica Sinica, 22:163-172. Moreau, G. 2004. The influence of forest management on defoliator populations: a case study with Neodiprion abietis in pre-commercially thinned and natural forest stands. PhD Dissertation, Faculty of Forestry and Environmental Management. University of New Brunswick. Pp. 166. Moreau, G., Lucarotti, C.J., Kettela, E. G., Thurston, G.S., Holmes, S., Weaver, C., Levin, D.B., and Morin, B. 2005. Aerial application of nucleopolyhedrovirus 66 induces decline in increasing and peaking populations of Neodiprion abietis. Biological Control 33: 65-73. Miller, D.R., Yendol, W.E., Ducharme, K.M., Maczuga, S., Reardon, R.C., McManus, M.A. 1996. Drift of aerially applied diflubenzuron over an oak forest. Agricultural and Forest Meteorology 80: 165- 176. Payne, N.J. 1993. Spray dispersal from aerial glyphosate applications. Crop Protection 12 (6): 463-469. Payne, N.J. 2000. Factors influencing aerial insecticide application to forests. Integrated Pest Management Reviews 5: 1-10. Szewczyk, B., Hoyos-Carvajal, L., Paluszek, M., Skrzecz, I., Lobo de Souza, M. 2006. Baculoviruses - emerging biopesticides. Biotechnology Advances 24: 143- 160 Teshke, K., Chow, Y., Bartlett, K. Ross, R., van Netten, C., 2001. Spatial and temporal distribution of airborne Bacillus thuringiensis var. kurstaki during an aerial spray program for gypsy moth eradication. Environmental Health Perspectives 109 (1): 47-54. Vail, P.V., Hostetter, D.L., Hoffman, D.F. 1999. Development of the multi-nucleocapsid nucleopolyhedroviruses (MNPVs) infectious to loopers (Lepidoptera: Noctuidae: Plusiinae) as microbial control agents. Integrated Pest Management Reviews 4: 231-257. 67 APPENDIX A Table A-l: Analysis of variance table for Island Pond, using adjusted SS for tests on the weekly trend in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and juvenile sawfly densities. Source df Seq SS Ad SS Ad MS F P Treatmt 1 32976 32976 32976 2.55 0.017 Week 3 1342046 1342046 447349 34.64 0.000 Distance 5 49406 49406 9881 0.77 0.579 Treatmt* Week 3 133638 133638 44546 3.45 0.024 Treatmt* Distance 5 106910 106910 21382 1.66 0.040 Week*Distance 15 157860 157860 10524 0.81 0.656 Treatmt* Week* Distance 15 156180 156180 10412 0.81 0.665 Error 48 619899 619899 12915 Total 95 2598916 Table A-2: Analysis of variance table for infection at Island Pond using adjusted SS for tests on the weekly trend in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and the prevalence of NeabNPV infection in juvenile sawfly populations. Source df Seq SS Ad SS Ad MS F P Treatmt 1 25431.3 25431.3 25431.3 192.90 0.000 Week 3 32244.5 32244.5 10748.2 81.53 0.000 Distance 5 8523.8 8523.8 1704.8 12.93 0.000 Treatmt*Week 3 16814.8 16814.8 5604.9 42.51 0.000 Treatmt * Distance 5 3719.1 3719.1 743.8 5.64 0.000 Week*Distance 15 13331.7 13331.7 888.8 6.74 0.000 Treatmt* Week* Distance 15 5636.4 5636.4 375.8 2.85 0.003 Error 48 6328.1 6328.1 131.8 Total 95 112029.6 68 Table A-3: Analysis of variance table for Old Man's Pond, using adjusted SS for tests on the weekly trend in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and juvenile sawfly densities. Source df Seq SS Ad SS Ad MS F P T reatmt 3 597747 597747 62056 12.27 0.000 Week 1 260549 260549 62587 12.38 0.001 Distance 1 24530 24530 21233 4.20 0.042 Treatmt*Week 3 66836 66836 15209 3.01 0.032 Treatmt* Distance 3 66432 66432 20245 4.00 0.009 Week*Distance 1 1685 1685 1685 0.33 0.565 Treatmt * Week * Di stanc e 3 3643 3643 1214 0.24 0.868 Error 144 728006 728006 5056 Total 159 1749439 Table A-4: Analysis of variance table for infection at Old Man's Pond using adjusted SS for tests on the weekly trend in the relationship between distance from the treated (NeabNPV) or untreated (control) blocks and the prevalence of NeabNPV infection in juvenile sawfly populations. Source df Seq SS Ad SS Ad MS F P Treatmt 3 47011.7 30802.0 10267.3 43.20 0.000 Week 1 44722.7 23152.7 23152.7 97.42 0.000 Distance 1 1645.3 2276.7 2267.7 9.58 0.002 Treatmt* Week 3 42386.7 16777.3 5592.4 23.53 0.000 Treatmt * Di stance 3 2812.4 3005.2 1001.7 4.22 0.007 Week*Distance 1 1674.2 1674.2 1674.2 7.04 0.009 Treatmt * Week * D i stance 3 1051.9 1051.9 350.6 1.48 0.244 Error 144 34222.4 34222.4 237.7 Total 159 175527.3 69 Meeting of the Biocontrol Network, May 5-8, 2004. Windsor Ontario, (oral presentation) Roger Graves, D.T. Quiring and C. Lucarotti. Impacts of gregariousness in balsam fir sawfly (Neodiprion abietis) on susceptibility to disease. Joint annual meeting of the Acadian and Canadian Entomological Societies, October, 2004. Charlottetown, P.E.I, (oral presentation) Roger Graves, Dan Quiring and Chris Lucarotti. The effect of viral infection on gregariousness in balsam fir sawfly populations. Annual Meeting of the Acadian Entomological Society, June 19-21, 2005. Fredericton, N.B. (poster) Roger Graves, Dan Quiring and Chris Lucarotti. The ecology of disease spread in balsam fir sawfly populations. Joint meeting of IOBC-NRS / Biocontrol Network, May 8-11, 2005. Magog-Orford, Quebec, (poster) Roger Graves, Dan Quiring and Chris Lucarotti.Does gregariousness in balsam fir sawfly influence susceptibility to natural enemies? Fifth Annual Meeting of the Biocontrol Network, February 2 -4, 2006. Kananaskis Village, Alberta, (poster) Roger Graves, Dan Quiring and Chris Lucarotti. Suppression of the balsam fir sawfly (Neodiprion abietis Harris) using a naturally-occurring nucleopolyhedrovirus. Sixty- sixth Annual Meeting of the Acadian Entomological Society, June 11-13, 2006. Kentville Nova Scotia, (oral presentation) Roger Graves, Dan Quiring and Chris Lucarotti. The use of a nucleopolyhedrovirus for the suppression of its natural host, the balsam fir sawfly (Neodiprion abielis). The IXth International Colloquium on Invertebrate Pathology and Microbial Control, 39th Annual Meeting of the Society for Invertebrate Pathology. August 27-September 1, 2006, Wuhan. China, (poster)