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UBC Theses and Dissertations

Population ecology of Trichoplusia ni in greenhouses and the potential of Autographa californica nucleopolyhedrovirus… Cervantes, Veronica Beatriz 2005

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P O P U L A T I O N E C O L O G Y O F TRICHOPLUSIA NI I N G R E E N H O U S E S A N D T H E P O T E N T I A L O F A UTOGRAPHA  CALIFORNICA  N U C L E O P O L Y H E D R O V I R U S F O R THEIR C O N T R O L by VERONICA BEATRIZ CERVANTES Ingeniera Agronoma, Universidad de Buenos Aires, 2000  A THESIS S U B M I T T E D IN P A R T I A L F U L L F I L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE  in T H E F A C U L T Y OF G R A D U A T E STUDIES (PLANT SCIENCE)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A November 2005  © Veronica Beatriz Cervantes, 2005  Abstract The cabbage looper  Trichoplusia ni is the major lepidopteran pest i n tomato, cucumber  and pepper greenhouses i n British Columbia. T. ni has developed resistance to Bacillus  thuringiensis (Btk) in some greenhouses and this compromises the effectiveness of this widely used bioinsecticide for the control o f loopers. The production in greenhouses continues almost year round and loopers can be present i n the crop very early i n the season. Few other biological control options are available for this important pest, so new alternatives that are compatible with integrated pest management programs are required. In this work I explored the overwintering success o f cabbage loopers and found that populations could survive winter i n greenhouses but not outside. This poses a great risk for continued resistance o f loopers to Btk. I then evaluated the potential of Autographa californica nucleopolyhedrovirus ( A c M N P V ) in controlling cabbage looper larvae that are resistant to Btk. I found that larvae resistant to Btk were twice as susceptible to A c M N P V as were those from a non-resistant colony. I also studied i f host plants could alter the susceptibility o f T. ni to A c M N P V and the speed o f k i l l o f the virus. I conducted bioassays with susceptible cabbage loopers on the three main crops cultivated in greenhouses as well as on artificial diet. Susceptibility did not vary among the hosts. Despite the fact that larval growth was highest on cucumber and lowest on pepper no biologically meaningful differences were found i n speed o f k i l l . These parameters could be further influenced by the behaviour and differential consumption o f host plants by larvae. Observations i n greenhouses showed that on tomato plants larvae tended to forage in the lower portion o f the plants while on peppers they tended toward the top o f the plants. This agrees with experimental results that showed larvae tended to go to the top o f  pepper plants, the middle o f cucumber plants and the bottom o f tomato plants. Position was not affected by whether larvae were infected with A c M N P V but the behaviour o f larvae could influence the distribution o f virus on different crops. I conclude that A c M N P V has much potential as an alternative to Btk and that greenhouse level evaluations should proceed.  Table of Contents Abstract  ii  Table o f Contents  iv  List o f Tables  vii  List o f Figures  ix  Acknowledgements  x  Statement o f Co-authorship  xi  CHAPTER 1 1.1 Introduction  1  2.1 References  5  C H A P T E R 2. The overwinter survival o f cabbage loopers as a potential factor i n their continued resistance to Bt 2.1 Introduction  7  2.2 Materials and Methods  10  2.3 Statistical Analysis  16  2.4 Results  16  2.5 Discussion  19  2.6 Conclusions  22  2.7 References  33  C H A P T E R 3: Potential o f A c M N P V as an alternative for control o f cabbage loopers that are resistant to Bt 3.1 Introduction  35  3.2 Materials and Methods  38  3.3 Statistical Analysis  41  3.4 Results  41  iv  3.5 Discussion  44  3.6 Conclusions  47  3.7 References  51  C H A P T E R 4: Does host plant affect the susceptibility o f  Trichoplusia ni to A c M N P V ? 4.1 Introduction  54  4.2 Materials and Methods  56  4.3 Statistical Analysis  60  4.4 Results  61  4.5 Discussion  62  4.6 Conclusions  66  4.7 References  75  C H A P T E R 5: The effect o f host plants on cabbage looper behaviour as a predictor o f the potential o f A c M N P V for their control. 5.1 Introduction  78  5.2 Materials, Methods and Results  80  5.3 Discussion  90  5.4 Conclusions  97  5.5 References  107  C H A P T E R 6: General conclusion and future work  112  A P P E N D I X 1:  Mass rearing o f cabbage loopers on artificial diet  113  A P P E N D I X 2:  Artificial diet recipe  114  A P P E N D I X 3:  Outside temperature records  115  A P P E N D I X 4:  Level o f Resistance to Bt'm GIP colony  118  A P P E N D I X 5:  V i r a l counting  119  v  A P P E N D I X 6:  Virus dilution procedure  120  A P P E N D I X 7:  Larval growth and pupal weights on host plants  121  A P P E N D I X 8:  Hanging leaf technique  136  List of Tables  Table 2.1.  Records o f cabbage looper found on weeds  26  Table 2.2.  Number o f males/1000m captured inside greenhouses  27  Table 2.3.  Efficacy o f organophosphate insecticides on T. ni pupae  28  Table 2.4.  Effect o f winter temperatures on adult emergence i n greenhouses  30  Table 2.5.  Effect o f winter temperatures on adult wing deformity in G H s  31  Table 2.6.  Temperature records o f two consecutive winters (Dec-Jan)  32  Table 3.1.  Cross-resistance (%) mortality, L D 5 0 S and slopes per replicate  48  Table 3.2.  Cross-resistance  49  Table 4.1.  LD50S,  Table 4.2.  L D s , CI and slopes on different hosts (pooled replicates)  68  Table 4.3.  Time to death among doses on different hosts  69  Table 4.4.  Time to death for dose 5 on different hosts ( 1 replicate GIP)  LD50S  and CI (combined replicates)  95% C I and % mortality per replicate on different hosts  50  st  Table 4.5a. Time to death for dose 5 on different hosts ( 2 Table 4.5b. Time to death multiple comparisons ( 2  nd  nd  replicate GIP)  replicate GIP)  Table 4.6a. Time to death for dose 5 on different hosts ( 2 Table 4.6b. Time to death multiple comparisons ( 2  nd  nd  replicate G L E N )  replicate G L E N )  67  70 71 72 73 74  Table 5.1  Water content o f different host plants  103  Table 5.2  Records o f visual monitoring o f T. ni inside greenhouses  104  Table 5.3  Behaviour o f healthy and virus infected T. ni on different crops  105  Table A2.1 Artificial diet recipe  114  Table A 2 . 2 Preparation o f artificial diet dry mix  114  vii  Table A4.1 Level o f resistance to B t by resistant and susceptible colonies  118  Table A7.1 T. ni larval weight (5 day-old) on different hosts  129  Table A 7 . 2 T. ni larval weight (5 day-old) on parental crops and artificial diet  130  Table A 7 . 3 T. ni larval weight o f different populations on artificial diet  131  Table A 7 . 4 Pupal weights o f different T. ni populations  135  viii  List of Figures  Fig. 2.1 .a. Pheromone trap catches o f T. ni in pepper greenhouse  24  Fig. 2.1 .b Pheromone trap catches o f T.ni in tomato greenhouse  25  Fig. 2.2.  T. ni pupal mortality and adult deformity at constant 9°C  29  Fig. 3.1  Cross-resistance: development time to pupation  50  Fig. 5.1  Fresh leaf consumption by T. ni larvae (by weight ranges)  99  Fig. 5.2  Fresh leaf consumption by T. ni larvae on different crops  100  Fig. 5.3  Leaf area consumption by T. ni larvae on different crops  101  Fig. 5.4  Leaf area consumption by T. ni larvae (by weight ranges)  102  Fig. A3.1 Records o f outside minimum temperatures  116  Fig. A 3 . 2 Records o f outside average temperatures  117  Fig. A7.1 Larval weights o f T. ni colonies (A= cucumber colony, B=tomato colony, C=pepper colony) maintained on different crops  132  Fig. A 7 . 2 Comparison o f T. ni larval weights within crop and among colonies (A,B,C,D)  133  ix  Acknowledgements I am very grateful to my supervisor, Dr. Judith Myers, for her personal, professional, and financial support, and for her extraordinary patience. It was a great pleasure to be part o f her lab. I also want to thank my committee members, D r Murray Isman and D r David Theilmann, for their guidance with my project and my directed studies. A big thank goes to the Biocontrol Network for their financial support and for training new scientists in Canada. I was very fortunate to be one of them. I also want to thank Jenny Cory for her constructive comments and revisions on my viral chapters.  I w i l l never forget the opportunity that A l i d a Janmaat and Judy gave me as soon as I arrived in Canada. Words w i l l never be enough to express my gratitude. Thanks A l i d a for being a cornerstone in my research and for staying beside me i n difficult moments. A very special thanks for Tahmineh Ziaei one o f my laboratory assistants for all the support and help she gave me. A l s o thanks to my other assistants Ross, Heather, and Audrey for working so hard and making this project possible. Thanks to Jessamyn Manson for teaching me about bioassays and viral countings.  Thanks to Linda D e l l i Santi, Jaromir Zemman, M i r k a Pennors, Ruben Houweling, E d Basilaga, Peter Voogt for all the help received for my research and for letting me in the greenhouses to carry out my project.  Finally I want to thank my family, especially my parents that even not having an academic education envisioned the importance o f studying English. I also want to thank my husband, Javier, for his patience and kindness especially during the last stages o f my thesis and to my son, Matias, for being the best baby a student m o m could ever wish. Y o u both are the light o f my life, thanks for accompanying and encouraging me throughout all this time.  Statement of Co-authorship  Papers from this manuscript have not been submitted yet although some o f the chapters w i l l be submitted in the near future. I w i l l have my supervisor as a co-author in all the papers for her contribution o f ideas and professional guidance i n the development o f my research project.  xi  Chapter 1 Introduction The cabbage looper, Trichoplusia ni (T.ni) Hiibner (Lepidoptera, Noctuidae), is a generalist insect (Sutherland and Greene 1984) and it is the major lepidopteran pest in tomato, cucumber and pepper greenhouses in British Columbia (Manson 2003 and Janmaat 2004). This pest causes damage by larval feeding, mainly on the crop foliage. In tomato greenhouses, when T. ni is present at high density, the cabbage looper can damage the fruit. In cucumber greenhouses however, the insects feed on young fruits even when the density is low, making them unmarketable and causing considerable economic loss. The greenhouse industry in British Columbia contributes about 11% o f the province's total agriculture production value, with a farm gate value o f $250 million and thus this 1  pest can be o f considerable importance.  Greenhouse growers in British Columbia control insect and disease pests mainly with the aid o f biological control agents and occasionally with pesticides that have low impact to the environment. Regrettably, there are few other biological options available for the control o f cabbage loopers inside greenhouses as alternatives to  Bacillus thuringiensis  var kurstaki (Btk). The cabbage looper can cycle monthly in greenhouses throughout the growing season (Janmaat 2004) and females can lay between 300 ( M c E w e n and Hervey 1960) to over 1,000 eggs (Sutherland 1966). Therefore, several applications o f Bt per year are needed to control the pest. The repeated use of this bioinsecticide had led to the development o f resistance in cabbage loopers in greenhouses (Janmaat and Myers, 2003).  1  British Columbia Greenhouse Growers Association's web page, www.bcgreenhouse.ca  This is aggravated by the almost year round production o f vegetables, which is extended by the use o f supplemental artificial light in some greenhouses. This long growing season can have a tremendous effect in continuing the resistance to Btk. Therefore, there is a need for the development o f a new alternative for the control o f T. ni that is compatible with the integrated pest management program that growers are currently carrying out.  Nucleopolyhedroviruses ( N P V s : family Baculoviridae) are pathogens that mainly infect lepidopteran larvae. They are double stranded D N A viruses with enveloped, rod-shaped nucleocapsids incorporated into a protein matrix called the occlusion body (OB) (Granados and Federici 1986). They have a great potential as alternative for pest control (Moscardi 1999, Granados and Federici 1986). They occur in nature (Granados and Federici 1986, Podgwaite 1985), are safe for humans and environment (Granados and Federici 1986, Entwistle 1983), can be transmitted both horizontally (Evans 1986, Andreadis 1987) and vertically (Kukan 1999, Myers et al. 2000, Fuxa et al. 2002), and can co-evolve with the pest ( M i l k s 1996, in review Cory and Myers 2003). These viruses infect the insects following the ingestion o f the viral particles (Evans and Entwistle 1987). This means that the insect has to feed on the treated crop to become infected. Therefore, some level o f damage has to be tolerated by the growers i f they are to be used. Two nucleopolyhedroviruses that infect the cabbage looper are the singly embedded nucleopolyhedrovirus o f  Trichoplusia ni ( T n S N P V ) and the multiply embedded  nucleopolyhedrovirus o f  Autographa californica ( A c M N P V ) . They vary i n their host  range, with T n S N P V being specific for cabbage looper and A c M N P V infecting many species in several families o f Lepidoptera including T. ni (in review Cory and Myers 2003). For this thesis I worked with A c M N P V , the virus with the broader host spectrum.  2  The main objective o f my thesis is to describe the population persistence, dynamics and behaviour o f T. ni i n vegetable greenhouses in British Columbia to explore the potential of A c M N P V as an alternative for the control o f cabbage loopers. To accomplish my objective I focused my research to answer the following questions:  1- What is the source o f cabbage loopers to greenhouses in British Columbia? 2-  What are the population dynamics o f T. ni inside greenhouses?  3-  H o w effective might A c M N P V be for controlling T. ni populations that are resistant to  Btk?  4- Does the crop type affect the susceptibility o f cabbage loopers to the virus? 5- H o w might the consumption behaviour of larvae in each crop affect the performance o f the viral agent? These questions are addressed throughout the thesis. In Chapter 2,1 discuss the population dynamics o f T. ni inside commercial greenhouses. The main purpose o f this chapter is to explore alternative sources to the immigration o f cabbage loopers into the greenhouses that better help to explain the early presence o f the pest and the appearance of resistance to Btk inside greenhouses.  In Chapter 3,1 explore the susceptibility o f /M-resistant and non-resistant T. ni populations to A c M N P V . I conducted laboratory bioassays to test for cross-resistance between A c M N P V and Btk.  Chapter 4 includes a series o f bioassays performed with A c M N P V on different crops. The main focus o f this chapter is to find out i f host plant can influence the performance of A c M N P V .  3  Chapter 5 addresses the differences in growth and behaviour o f cabbage loopers when foraging on different crops. This information is valuable as a complement o f bioassays and better predicts the potential o f the virus in a more realistic scenario as well as the occurrence o f horizontal transmission.  Chapter 6 discusses all the results and addresses future work.  1.2. References  Andreadis, T . G . 1987. Transmission. In Epizootiology o f insect diseases. Fuxa and Tanada [eds]. Wiley, N e w York, p 159-176. Cory, J. S., and J. H . Myers. 2003. The ecology and evolution o f insect baculoviruses. Annu. Rev. E c o l . E v o l . Sys. 34:239-272. Entwistle, P.F. 1983. Viruses for insect pest control. Span 26:21-34 (n.s). In M i l k s 1996. Evans, H . 1986. In the biology o f baculoviruses. V o l 2. Chapter 4. See Granados and Federici. Evans, H . F . and P.F. Entwistle. 1987. V i r a l diseases. In Epizootiology o f insect diseases. Fuxa and Tanada [eds]. Wiley, N e w York, p 257-322. Fuxa, J.R., A . R . Richter, A . O . A m e e n , B . D . Hammock. 2Q02. Vertical transmission of T n S N P V , T n C P V , A c M N P V , and possibly recombinant N P V in Trichoplusia J. Invertebr. Pathol. 79:44-50  ni.  Granados, R. R., and B . A . Federici. 1986. The Biology o f Baculoviruses. V o l 1 and 2. C R C , Biological properties and molecular biology. Boca Raton F L . Janmaat, A . F., and J. Myers. 2003. Rapid evolution and the cost o f resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni. Proc. R. Soc. London B:2263-2270. Janmaat, A . F . 2004. The evolution o f resistance to Bacillus thuringiensis i n greenhouse Trichoplusia ni populations. Ph.D. Thesis - Depart, o f Zoology, University o f Bristish Columbia, Vancouver. Kukan, B . 1999. Vertical transmission o f nucleopolyhedrovirus i n insects. J. Invertebr. Pathol. 74:103-111. Manson, J. 2003. The effect o f food plant on the efficacy o f a nucleopolyhedrovirus o f Trichoplusia ni. Honours Biology. Faculty o f Science - University o f British Columbia. M c E w e n , F . L . and G . E . R . Hervey. 1960. Mass-rearing o f the cabbage looper, Trichoplusia n i , with notes on the biology in the laboratory. A n n . Entomol. Soc. Amer. 53:229-234. M i l k s , M . L . 1996. The implications o f Cabbage Looper (Trichoplusia ni) - Nuclear polyhedrosis virus coevolution for biological control. Ph D . Thesis -Dept. o f Zoology- University o f British Columbia, Vancouver.  5  Moscardi, F. 1999. Assessment o f the application o f baculoviruses for control o f Lepidoptera. Annu. Rev. Entomol. 44:257-289 Myers, J . H , R . Malakar and J.S. Cory. 2000. Sublethal nucleopolyhedrovirus infection effects on female pupal weight, egg mass size, and vertical transmission in gypsy moth (Lepidoptera: Lymantriidae). Environ. Entomol. 29:1268-72. Podgwaite, G . D . 1985. Strategies for field use o f baculoviruses. Maramosch and Sherman [eds], V i r a l insecticides for biological control. Academic Press, Inc., N e w York, p 775-797. Sutherland, D . W . S. 1966. Biological Investigation o f Trichoplusia ni (Hubner) and other Lepidoptera damaging cruciferous crops in Long Island, N e w York. N . Y . Agric. E x p . Stn. Ithaca M e m . 399. Sutherland, D . W . S., and G . L . Greene. 1984. Cultivated and w i l d host plants, in G . L . Green, [ed]. Suppression and Management o f Cabbage Looper Populations. U S D A Tech B u l l # 1684- Chapter 1.  6  Chapter 2  The overwinter survival of cabbage loopers as a potential factor in their continued resistance to Btk  2.1 Introduction The cabbage looper,  Trichoplusia ni, is a migratory pest o f subtropical origin and is  currently o f major concern i n tomato, pepper and cucumber greenhouses in British Columbia where cabbage loopers are the major lepidopteran pest. Some T. ni greenhouse populations have developed resistance to  Bacillus thuringiensis var kurstaki (Btk), the  most widely used bioinsecticide inside greenhouses (Janmaat and Myers 2003) . This resistance makes their management an increasing challenge. The production o f greenhouse crops runs almost year round with the exception o f a 2-6 week clean-up period i n which the greenhouse is unheated and cleaning procedures take place including the use o f organophosphate insecticides ( D i b r o m ® (active ingredient: Naled) and D D V P - 1 0 F S ® (a.i.: Dichlorvos). The source o f T. ni in greenhouses is uncertain. Roofs o f greenhouses are vented and these could allow entrance o f moths from the outside. However, cabbage loopers can be found as early as January-February in greenhouses while they normally do not occur outside until M a y . Adult cabbage loopers can fly long distances and this accounts for much o f the spread of the pest northward into Canada in the spring and early summer, and southward late in the summer and fall (Mitchell and Chalfant 1984). A long distance movement experiment  7  carried out using black light traps on o i l platforms located several kilometers off shore in the G u l f o f M e x i c o found that large numbers o f cabbage loopers were able to travel at least 161 k m in a single flight (Debolt et al. 1984).  In spite o f this, mark-recapture  experiments showed the maximum distance for moth recaptures to be 14.5 k m (Debolt et al. 1984) Adult flight, however, is not the only means by which cabbage loopers may be able to extend their northerly range each season (Poe and Workman 1984). For example, Lingren et al. (1979) proposed that other forms o f dispersal could occur, such as the transportation of immature forms (eggs, larvae, and pupae) with plants from nurseries to agricultural production areas. Survival through the winter might also occur for larvae feeding on ornamental plants  located near heated  residences  or greenhouses.  These residual  populations could contribute significant numbers o f cabbage loopers much earlier in the spring than would dispersal from the south. The cabbage looper is recorded as feeding on over 160 species o f w i l d and cultivated plants, in 36 families (Sutherland and Greene 1984). The possibility exists for the persistence o f cabbage loopers on various plants, such as weeds, in the vicinity o f greenhouses (Lingren et al. 1979). For adjacent weeds to serve as refuges,  cabbage  loopers must be able to overwinter outside the greenhouses. The northern extent o f cabbage looper overwintering is unclear. Sirrine (1894) indicated that the cabbage looper could overwinter in the pupal stage in northern and central United States, but Sutherland (1966) reported that the insect could not overwinter near Long Island, N Y . Elsey and Rabb (1970) stated that loopers probably did not often overwinter  8  in North Carolina. Furthermore, Poe and Workman (1984) claimed that loopers are likely to overwinter i n greenhouses i n northern areas. Uncertainty exists about  the potential sources  o f cabbage  loopers to  vegetable  greenhouses i n British Columbia other than immigration from the south. The aim o f this study was to elucidate those sources. I asked the following questions: 1) can cabbage loopers survive over winter outside the greenhouses on weeds? 2) is T. ni able to persist through the unheated period inside greenhouses  and survive the  organophosphate  applications used for the clean-up? and 3) could the reintroduction o f moths occur with contaminated seedlings early in the season? The answers to these questions can yield important information that can be implemented as management practices to lessen the impact o f this pest i n commercial crops. B y reducing and delaying the introduction of moths to greenhouses, the continued need for spraying cabbage loopers with Bacillus  thuringiensis w i l l be reduced and thus also the development of the resistance to Bt.  2.1.1 Growing season and greenhouse clean-up  Crops are established in greenhouses at the beginning o f the production season with seedlings bought from nurseries. I monitored plants for cabbage looper larvae weekly through all developmental stages, starting from seedlings with 3-4 true leaves (end o f December- early January) until the end o f the production season (late November - mid December). Between the end o f the growing season and the beginning o f the next season a clean-up process takes place.  9  During this period the greenhouses remain unheated and several sanitation procedures are carried out. This unheated period varies in length among the greenhouses ranging between 2-6 weeks. Although the type and extent o f sanitation procedures also varies with each greenhouse, the clean-up generally involves removing all the plants, changing the plastic that covers the soil, replacing sawdust bags, rockwool blocks and strings, applying organophosphate  insecticides, washing all irrigation system  components,  powerwashing walls and structures, and turning off the heating system. Not all o f these procedures are necessarily carried out by all the growers and in some cases the activities overlap, which means that part o f the greenhouse is being cleaned while the new seedlings are being introduced to the areas already cleaned.  2.2 Materials and methods 2.2.1 Monitoring of cabbage looper populations with pheromone traps a. Outside greenhouses To determine i f T. ni were present outside the greenhouses before being detected inside, pheromone traps (Wing Trap II, containing cabbage looper lures supplied by Phero Tech Inc., Delta, B C . Canada) were placed close to greenhouses with 2 or 3 traps per greenhouse side to cover wind flow coming from all cardinal points. Lures were replaced every three weeks during the spring and summer and every 4 weeks in autumn and winter. Sticky trap inserts were replaced when more than 3 adults were trapped. Male captures were recorded almost every week.  10  b. Inside greenhouses Pheromone traps and visual monitoring (explained below) were conducted inside a pepper  (Capsicum anuum 'Triple 4') and two tomato (grafted Lycopersicum esculentum  'Rapshodie') greenhouses in Langley, British Columbia from June 2002 until June 2003. Only a portion (15,000 m ) o f the pepper greenhouse was monitored with one pheromone 2  2  trap/625 m . T w o tomato greenhouse units (one o f 7,000 m and the other one o f 12,000 m ) located i n the same field plot were sampled with one pheromone trap/1000 m . Catches in traps were recorded weekly and the sticky trap inserts were replaced when more than 3 adults were trapped. The traps remained before, during and after the winter clean-up took place. The clean-up in the tomato greenhouses consisted o f all the sanitation procedures previously described and the insecticide used was D I B R O M ® . In the pepper greenhouse only D D V P - 1 0 F S ® was applied and none o f the other sanitation practices except pulling out the crop were carried out.  2.2.2 Visual monitoring of cabbage loopers a. On weeds outside greenhouses In August 2002, I observed adult  T. ni feeding on fireweed (Epilobium angustifolium)  outside greenhouses. I then began to monitor the w i l d vegetation around the greenhouses for any developmental stages o f cabbage loopers. This visual monitoring was done weekly on weed patches surrounding the greenhouses and near the pheromone traps. Most predominant weeds or turf components were observed. Plant patches were carefully  11  checked and the total time involved in this visual monitoring was approximately one hour per visit.  b. On crops inside greenhouses In addition to the pheromone trapping, twenty plants were visually searched for cabbage looper stages every week. Six o f those plants were in the rows near the glass walls because I observed a concentration o f loopers in that area and this may have been associated with the vicinity o f the primary heating pipes. The rest o f the plants sampled were arbitrarily picked from other bays in the greenhouse. When the new seedlings were brought in from the nursery for planting, I conducted a more exhaustive sampling; 100 plants were completely checked and one row o f plants from each bay was monitored by looking for visible signs o f previous looper damage that could have occurred in the nurseries.  2.2.3 Overwintering survival of cabbage looper pupae inside greenhouses a. Susceptibility of cabbage looper pupae to the organophosphate insecticides DIBROM® and DDVP-10FS® To test the efficacy o f insecticide treatment I placed pupae in the greenhouses and observed their survival after insecticide application. O f the two tomato  greenhouses  selected for this pupal survival experiment, one was treated with D D V P - 1 0 F S ® as the clean-up insecticide and the other one with D I B R O M ® .  Insecticide applications were  carried out by greenhouse authorized personnel. A t the latter greenhouse two D I B R O M ® applications a week apart, allowed two repetitions o f the efficacy o f this insecticide. The  12  product was applied at a rate o f 10 ml/100 m with spray equipment on to cold heating 3  pipes. Following application, pipes were heated to 4 1 ° C for 4 hours. In the case of D D V P - 1 0 F S ® , the product was fogged at 500 ml/1000 m o f space using electric fogging 3  equipment. Pupae used for these experiments were obtained from laboratory culture. Larvae o f T. ni were reared on artificial diet until pupation following the mass rearing procedure described by Ignoffo (1963) (see Appendices 1 and 2). Pupae were kept within their silken cocoons. Pupae were placed in muslin bags approximately 8 cm wide x 5 cm in height. Five pupae (~2 day-old) were placed inside each bag and were sealed with staples to prevent the escape o f any emerging adults. These bags were placed in the commercial greenhouses the day prior to the insecticide application. The number o f bags placed in each greenhouse varied with the experiment and the availability o f pupae (see details in Tables 2.3 and 2.4). Bags were labeled with a colour tag from which they were hung on the greenhouse structure or a plant. Coloured tags reminded the greenhouse operators not to remove the bags during the clean-up. Bags were collected 48 hours later. The greenhouses remained closed for 24 hr during the insecticide treatment then they were ventilated for another  day without heat. Control pupae were kept at the U B C  experimental greenhouse at 20°C ± 2°C and were not exposed to the insecticides. After collection from the commercial greenhouse, pupae were placed in a controlled environment chamber at 2 6 ° C and the emergence o f adults was recorded daily. Pupae were left i n their cocoons to determine whether insecticide deposits could alter the survivorship o f the adults. Emerged adults were caged in the same type cages used for adult rearing (see Appendix 1) to determine egg production o f survivors and to test i f  13  reduced fertility occurred following exposure to the insecticides. A 10% sucrose solution was provided as a food source for adults inside the cages and paper towelling was used as an oviposition substrate. This paper towel was changed every other day, and two egg sheets in the peak o f the oviposition period were collected to test for egg hatchability. Subsets o f ca. 25 eggs were separated and placed inside 170 m l Styrofoam cups and kept at 26°C for 2 days. The number o f neonates hatched was recorded. For survivors o f D I B R O M ® a total o f 158 eggs in 6 subsets were tested and for D D V P - 1 0 F S ® survivors 287 eggs were checked in 10 subsets.  b. Survival of cabbage looper pupae to winter temperatures This experiment was carried out in the same greenhouses as the previous experiment and pupae were reared i n the laboratory using the same protocol. Five plastic 21 m l cups with one 2 day-old pupa each were placed in 455 ml paper cups that had a metal screen on one side and were covered with a plastic l i d . These cups were distributed inside the greenhouses following the powerwashing and were kept there for the entire period in which the greenhouse remained unheated (2 weeks in one greenhouse and 5 weeks and 1 day in another greenhouse). Cups were collected the day prior to reheating. Pupae with their respective cocoons were kept in the individual plastic cups inside the control environment chamber at 22°C. Adult emergence was recorded.  2.2.4 Overwintering survival of cabbage looper pupae outside greenhouses During the winter 2002-2003 pupae were placed outside, in the vicinity o f the tomato and pepper greenhouses, i n Langley. O n November 2  n d  2002, 25 pupae were collected from  14  I  laboratory colonies and cocoons removed. These were placed individually in 29 m l cups in the periphery o f the pepper greenhouse both in open sites as well as in some more protected ones (i.e. under tree canopy, close to walls). During the monitoring routine inside the tomato greenhouse, 32 pupae were collected on November 13 2002 and were th  kept inside their cocoons with part o f the leaf material attached. Those pupae were also placed inside individual plastic 29 ml cups and distributed in the vicinity o f the greenhouses to check for survivorship at outside temperatures. These were left outside until M a y 2003 to determine i f they would emerge at the same time that the first adults were trapped in the outside pheromone traps. After that time, non-emerged pupae (all o f them i n this case) were brought to the laboratory and were kept at 2 6 ° C to check for adult emergence. During the winter 2003-2004, 36 pupae were again kept outside the tomato greenhouses in Langley. Pupae from larvae reared in the laboratory were kept with their cocoons. They were placed outside the greenhouses in individual 29 ml cups on December 6  th  2003. Pupae were collected on January 10 2004 and were kept in the th  laboratory at 22°C until adult emergence.  2.2.5 Overwintering survival of cabbage looper pupae in the laboratory at 9°C I carried out this experiment to corroborate my previous findings o f overwinter survival inside greenhouses. A temperature o f 9°C was chosen from the mean average temperature observed inside greenhouses during the winter o f 2002-2003 and 2003-2004. A total o f 189 pupae o f T. ni with their silky cocoons were obtained from laboratory cultures and  15  were individually placed i n 28.4 m l plastic cups. From those, 27 pupae were kept as controls and the remaining 162 were labeled in subsets o f 27 as 1, 2, 3, 4, 5 or 6 weeks. A l l pupae were randomly assigned to 4 trays that were maintained in the refrigerator at 9°C. After one week, the 27 pupae designated as 1 week were removed from the trays and exposed to an acclimation period, which consisted o f keeping the pupae at 16°C for 3 days, then at 20°C for one day. The following day the temperature was raised to 22°C for one day, and finally pupae were maintained at 26°C until adult emergence.  This  procedure was repeated every week for each subset for six weeks.  2.3. Statistical Analyses Statistical analyses were done with J M P i n 4.04. (Student Version o f S A S Institute) and the tests used are indicated for each specific case in the results section. The level o f significance (a-value) i n all tests was 0.05.  2.4. Results T. ni were already established outside and inside both greenhouses in June 2002, when the pheromone trapping study started (Fig. 2.1a and 2.1b). For the pepper greenhouse, no cabbage looper males were collected outside from late October (28 ) until late A p r i l th  (29 ). This pepper greenhouse was located close to a nursery that produced vegetables th  (tomato, pepper, cucumber and crucifers) as well as ornamental seedlings and plants. Early in the season it is a common practice for the nursery to put the seedlings o f crucifer  16  crops outside to acclimate. The first male moth catches outside coincided with the presence o f the cole crop seedlings in the adjacent nursery field. The tomato greenhouse had no field cole crops i n the vicinity, and the main agricultural activities were hay, corn, and berry production. The greenhouse was surrounded by trees, which could interfere with the immigration o f cabbage looper moths from outside this plot. Even though the main peak o f moth catches for this location was i n September and October, possibly associated with a great flux o f moths coming from inside the greenhouse, no males were captured in the winter ( M i d November until M a y 29 ). The th  visual monitoring o f weeds started late i n August 2002 and from September 4 a variety th  of cabbage looper stages were found on different weed species. A l l instars, including eggs, were collected and reared on artificial diet in the laboratory until adult emergence for identification. The alfalfa looper, cabbage  looper  at  immature  Autographa californica is very similar to the  stages,  but adults  from  both  species  are easily  distinguishable. A l l collected individuals were cabbage loopers, and no A. californica were found. A summary o f all instars recorded and their host weeds is presented in Table 2.1. After November 2 7  th  2002, no loopers were observed on weeds outside. The pepper  greenhouse was surrounded by mowed grass that eliminated possible weed refuges for cabbage loopers.  The results o f pheromone trapping at the end o f the season inside the greenhouses are shown i n Table 2.2. T. ni captures were higher i n the second tomato greenhouse (Tomato 2) than i n Tomato 1 and Pepper greenhouses. During the unheated period no looper adults were caught i n the greenhouses but several days after the temperature was increased for the new growing season, adults were trapped inside the Tomato 2 and Pepper  17  greenhouses, indicating that the density o f loopers before the clean-up and the quality of the clean-up are potentially influencing the presence o f the pest early in the season. The pepper greenhouse only received a D D V P - 1 0 F S ® application and even though it had the lowest density o f loopers before the clean-up, they were still present at the beginning of the season. This suggests that the treatment was not effective. The fact that no loopers were caught i n the traps during the clean-up could indicate that they are able to survive in the pupal stage and that they emerge after heating is reestablished or that adults do not fly at cool temperatures. The insecticides used in the clean-up procedure were ineffective in reducing the survival of pupae o f the cabbage looper (Table 2.3). They did not k i l l the pupae and they also did not suppress adult fertility. The mean hatchability o f eggs laid by moths that survived D I B R O M ® and D D V P ® was 91.5% ± 2.5 and 90.1% ± 1.6 respectively. Pupal survival declined with an increase in the length o f exposure to 9°C in the laboratory (Fig. 2.2). A positive and significant linear relationship was found between the percentage mortality and time o f exposure (r = 0.97, F =186.8; df = 5,6; pO.OOOl). 2  Moreover, wing abnormality o f emerging adults also increased with the length o f exposure to cold temperatures but in this case, a negative and significant quadratic relationship was found between the percentage o f normal winged adults and the length o f exposure (r = 0.92, F = 21.6; df = 4,6; p = 0.007). In addition, while survival o f the pupae exposed for two weeks to winter temperatures inside greenhouses was similar to controls, 82% vs 90% (Table 2.4, Wilcoxon-Test % = 0.053; df=1; p = 0.82), only 42.5% 2  survived after 5 weeks (Wilcoxon-Test x  =  18.8; df =1; pO.OOOl) o f which 62% were  18  deformed adults (Table 2.5). Finally, none o f the pupae placed outside survived the winter in either 2002-2003 or 2003-2004. Temperature records outside during those winters are given i n Table 2.6 and Appendix 3 (Fig. A.3.1 and A.3.2). It is very unlikely that T. ni is able to survive outside and to migrate to the greenhouses early in the season.  2.5. Discussion Cabbage looper pupae were unable to survive winter conditions i n the vicinity o f Vancouver, British Columbia during two consecutive years, even though the winter in 2002-2003 was mild compared to the conditions registered for the winter in 2003-2004. This agrees with the results o f Sutherland (1966) and Elsey and Rabb (1970), but differs from those o f Sirrine (1894, 1899), Dustan (1932), and Walker and Anderson (1936) who found that T. ni overwinters in northern and central United States in the pupal stage. The field season o f cole crops started later in 2003 compared to 2002. This delay in planting could explain the lower number o f T. ni that were trapped adjacent to the pepper greenhouse in June 2003 compared to 2002. That no loopers were trapped outside during the winter and that no pupae survived to adulthood after exposure to outside winter temperatures, strongly suggests that cabbage loopers are not migrating to the greenhouses from outside i n British Columbia in early spring. However the efficiency o f pheromone lures could be lowered at cold temperatures and may reduce the probability o f capturing looper males (Gaston et al. 1971, Sower et al. 1973). Inside greenhouses, however, temperatures can reach 2 1 ° C i n the daytime during the unheated period (unpublished temperature records from greenhouses).  19  Moths were not apparently introduced with plants. The visual monitoring o f seedlings at the beginning o f the season showed no signs o f previous damage. The fact that Tomato 1 and Tomato 2 greenhouses received the seedlings from the same propagator, and that Tomato 1 had no loopers until late M a y while Tomato 2 had loopers present almost immediately after planting, also strongly suggests that loopers were not imported with the seedlings that year but persisted through the clean-up.  The insecticides used during the clean-up procedure are apparently ineffective for controlling the early pupal stage o f cabbage loopers. More than 93% o f deployed pupae survived the D D V P ® application and almost 85% survived the D I B R O M ® application (Table 2.3). These insecticides did not affect the fecundity and fertility o f the adults. While these insecticides may still be effective for other stages o f the loopers or other greenhouse pests (Smith et al. 1970), they are not effective against cabbage looper pupae. Henneberry and Kishaba (1967) reported that moths o f the cabbage looper held at a constant 10°C were able to live at least 30 days in the presence o f food. Therefore adults, i f not controlled with an insecticide, could be another source o f carry-over in those greenhouses with shorter unheated clean-up periods. Although winter temperatures inside greenhouses after 5 weeks were detrimental to pupal survival, in some cases they were not sufficient to eliminate the pest completely (Table 2.4).  Exposure o f pupae to 9°C in the laboratory increased the frequency o f adults with abnormal wings. Similar effects were observed in the greenhouses. For the 2 week unheated period inside greenhouses, the percentage o f wing deformity o f moths was not significantly higher than for controls and was half that o f individuals maintained at 9°C  20  constant in the refrigerator for 2 weeks. After 5 weeks, the percentage o f wing deformity of moths emerging from pupae stored inside greenhouses was 62% compared to 100% in the refrigerator experiment. Toba et al. (1973) found increased adult deformity at cold temperatures (7.3°C - 12 °C constant) and Grau and Terriere (1967) observed deformed adults after exposure o f pupae to high temperature (30°C). Interestingly, they found that deformity only occurred i n larvae reared on artificial diet while those reared on bean plants produced normal adults after pupae were exposed to 30°C. These results indicate that complete extrapolation from  laboratory experiments  to greenhouses  are  not  necessarily valid. Shorey and Farkas (1973) showed that T. ni males, that had had their wings removed, were capable o f finding mates by walking trails on foot. This shows that even deformed adults can be capable of reaching females to mate. However, the success of finding mates w i l l certainly depend on the pest density.  These findings are important for understanding the potential risk that a carry-over from one season to another has in the continued resistance to Bt inside greenhouses. The development o f resistance is strongly associated with the number o f Bt applications the population has previously received and fitness costs o f Bt resistance results in a loss o f resistance i n about 2-8 generations once the selection pressure is removed (Janmaat and Myers 2003). The more the T. ni population density can be reduced at the end o f the season the higher the chances to delay the first Bt application the following season, giving the population a chance to lose the resistance acquired during the previous season. This shows the importance that a good quality clean-up can have in reducing the risk o f continuing or developing resistance to this broadly-used biopesticide.  21  2.6. Conclusions This study demonstrates that: 1- winter conditions in British Columbia do not prevent T. ni from overwintering inside greenhouses, 2- the efficacy o f the insecticides used during the clean-up o f greenhouses in British Columbia are insufficient to k i l l the early pupal stage o f T. ni which can consequently remain inside the greenhouse to the following season, 3- the population density o f the cabbage looper and the clean-up quality may be related to the overwintering success, 4- the carry-over from one season to another is one source o f cabbage loopers early in the season and it is therefore a threat for continued resistance to  Bacillus thuringiensis. A s a consequence, growers can improve and  implement several other tools in their management plan for the control o f this pest such as: 1- use other insecticides rather than Bt at the beginning o f the season when other biological agents have not yet been released into the greenhouse or at the end o f the season when the biocontrol agents are not o f primary importance. This w i l l lead to a reduction o f the population density before the clean-up takes place, 2-intensify clean-up strategies, do not skip any step in the clean-up procedure, 3- keep the unheated period as long as possible, or keep the greenhouse heated for a longer period with no plants before the insecticide application in order to facilitate adult emergence since they are susceptible to the insecticides, 4-use light or pheromone traps at the beginning o f the season and monitor the seedlings before planting for the early detection o f the pest in order to adopt appropriate measures o f control at the right time.  Further investigations could involve studies o f the inside overwintering success o f cabbage looper populations resistant to Bt, the combined effect o f organophosphate  22  insecticides and winter temperatures, as well as more exhaustive research on the susceptibility o f different stages o f the cabbage loopers to these products.  23  Figure 2.1a. Pheromone trap catches outside (grey bars) and inside (black bars) a pepper greenhouse in the vicinity o f Langley, B . C . in 2002-2003. The months o f the year from June 2002 until June 2003 are indicated on the X-axis. The black dotted horizontal bar indicates the months i n which no cabbage looper males were collected outside. The black plain horizontal bar shows the period in which the clean-up took place inside the greenhouse. Small arrows indicate when Bt applications were made inside the greenhouse.  PEPPER GH 2002-2003  Jun  Jul  Aug  Sep  Oct  Nov  Dec  Jan  Feb  Mar  Apr  May  Jun  24  Figure 2.1b. Pheromone trap catches outside a tomato (grey bars) and inside two tomato greenhouse units (grey dashed and black bars) in the vicinity o f Langley, B . C . in 20022003. The months o f the year from June 2002 until June 2003 are indicated on the X-axis. Black dashed horizontal bar indicates the months in which no cabbage looper males were collected outside. Black plain horizontal bar shows the period in which the clean-up took place inside the G H . Small arrows indicate the Bt applications made inside G H s  TOMATO 2002-2003 24  Jun  Jul  Aug  Sep  Oct  Nov  Dec  Jan  Feb  Mar  Apr  May  25  Jun  Table 2.1. Records o f cabbage looper found on weeds during visual monitoring in the proximity o f the tomato greenhouses (unless indicated) in Langley, B . C .  T. ni Instar  Weed  1  Fireweed  Adults and eggs  04/09/2002  Adults, eggs and L 2  27/09/2002  Adults  27/09/2002  L 2 and L 5  18/09/2002  Epilobium angustifolium Red Clover  Trifolium pratense  Date  Dandelion  Taraxacum officinale W i l d lettuce  Lactuca sp.  18/09/2002 L3 to L 5 27/09/2002  Plantain  Plantago sp.  L 3 , L 5 and Pupae  L3 Volunteer tomato  Lycopersicum esculentum  L3 to L 5  1  L2= Second Instar Larva; L3=Third Instar Larva; L5-Fifth Instar Larva  2  Collected in the vicinity of the Pepper greenhouse.  12/11/2002  11/11/2002  2  27/09/2002  26  Table 2.2. Number o f males / 1000m captured in pheromone traps inside 3 greenhouses 2  in Langley, B . C . before (heated), during (unheated) and after (heated) clean-up.  Greenhouse  Before  After  During 5 days*  12 days*  Pepper  1.1  0  0.13  0.2  Tomato 1  1.6  0  0  0  Tomato 2  3.3  0  0.5  1.5  * Male captures obtained 5 and 12 days after seedlings were planted.  27  Table 2 . 3 . Efficacy o f the organophosphate insecticide applications on the T. ni pupal stage inside commercial greenhouses. Mean percentage o f adults emerged from pupae following treatment. Statistical results from Wilcoxon Test. Same letters indicate treatments that are not statistically different.  Mean % o f Insecticide  Wilcoxon test Adults alive ± (SE)  DIBROM®  DDVP®  C*  83.3 ±(3.33) a  6  j*  84.8 ± (2.89) a  25  C*  95.0 ±(5.00) a  4  93.0 ±(2.03) a  23  %2= 0.17; p = 0.69  %2= 0.14; p = 0.70  * T = treated; C = control ** N = Total number o f cups with 5 pupae inside  28  Fig. 2.2. Relationship between T.ni pupal mortality and adult deformity and the length o f exposure to a constant 9 ° C temperature i n the laboratory. Week 0 represents the control group that were not exposed to 9°C. % Mortality = -6 + 2.30 * days; r =0.97; F =186.8; df = 5,6; p O . 0 0 0 1 . 2  % Normal adults = 59.64-1.98 * days + 0.05 (days-21) ; r = 0.92; F= 21.6; df = 4,6; 2  2  p = 0.007.  r = 0.97  r = 0.92 2  • % Mortality • % Normal  week  29  Table 2.4. Mean percentage o f T. ni adults emerged from pupae after exposure to winter temperatures inside greenhouses. Statistical results from W i l c o x o n Test. Different letters indicate treatments are statistically different.  Length of  Mean % of  Exposure  Adults alive ± (SE)  2 Weeks  5 Weeks  Wilcoxon Test  c*  c*  90.0 ±(4.5) a  10  81.7±(8.3)a  12  90.0 ±(4.1) a  14  42.5 ± (6.6) b  24  %2 = 0.05; p =0.82  %2= 18.8; pO.OOOl  * T = treated; C = control ** N = Total number o f cups with 5 pupae inside  30  Table 2.5. M e a n percentage o f adults emerged with deformed wings after exposure to winter temperatures inside greenhouses. Statistical results from W i l c o x o n Test. Different letters indicate treatments are statistically different.  Mean % o f  Length of  Wilcoxon Test Exposure  2 Weeks  5 Weeks  Deformity ± (SE)  c*  c*  22 ±(5.6) a  10  27 ±(5.1) a  12  33 ±(3.8) a  14  62±(5.2)b  24  %2 = 0.39; p =0.53  X2= 10.8; p = 0.001  * T = treated; C = control ** N = Total number o f cups with 5 pupae inside  31  Table 2.6. Comparison.of temperatures recorded by Environment Canada at Vancouver Airport between Dec 1 and Jan 10 o f two consecutive winters (2002/2003 and st  th  2003/2004).  Maximum  Minimum  Mean minimum  Mean Average  Temperature  Temperature  Temperature  Temperature  Winter 2002/2003  13.5 °C  -2.9 °C  2.67 °C  5.5 °C  Winter 2003/2004  11.9 °C  -12.2 °C  0.37 °C  3.28 °C  32  2.7 References Debolt, J. W . , T. J. Henneberry, W . W . Wolf, and P. D . Lingren. 1984. Release, recovery and dispersal o f adults. Suppression and management o f cabbage looper populations.USDA Tech B u l l #1684. Chapter 3. Dustan, A . G . 1932. Vegetable insects and their control. Canad. Dept. A g r . Entomol. B u l l 32:43-44. Elsey, K . D . , and R. L . Rabb. 1970. Analyses o f the seasonal mortality o f the cabbage looper i n North Carolina. A n n . Entomol. Soc. A m . 63:1597-1604. Gaston, L . K . , H . H . Shorey, and C . A . Saario. 1971. Sex pheromones o f noctuid moths. X V I I I . Rate of Evaporation of model compound o f Trichoplusia ni sex pheromone from different substrates at various temperatures and its application to insect orientation. A n n . Entomol. Soc. A m . 64:381-384. Grau, P. A . , and L . C . Terriere. 1967. A temperature-development factor for normal wing development in the cabbage looper, Trichoplusia ni (Lepidopetera:Noctuidae). Ann. Entomol. Soc. A m . 60:549-552. Henneberry, T. J., and A . N . Kishaba. 1967. Mating and oviposition o f the cabbage looper i n the laboratory. J. Econ. Entomol. 60:692-696. Janmaat, A . F., and J. Myers. 2003. Rapid evolution and the cost o f resistance to Bacillus thuringiensis i n greenhouse populations o f cabbage loopers, Trichoplusia ni. Proc. R. Soc. London B.2263-2270. Lingren, P. D . , T. J. Henneberry, and A . N . Sparks. 1979. Current knowledge and research on movement o f the cabbage looper and realted species p.394-405 (n.s.). in G . L . Green, editor. Suppression and management o f cabbage looper populations. See page 18 - U S D A Tech Bull# 1684. Mitchell, E . T., and R. B . Chalfant. 1984. Biology, behaviour and dispersal o f adults, in G . L . Green, editor. Suppression and management o f cabbage looper populations. U S D A Tech B u l l # 1684 - Chapter 2. Poe, S. L . , and R. B . Workman. 1984. Potential for disersal by winter shipments o f vegetables, ornamental plants, and cut flowers. Suppression and management of cabbage looper populations. U S D A Tech B u l l # 1684 - Chapter 4. Shorey, H . H . , and S. R. Farkas. 1973. Sex pheromones o f Lepidoptera. 42. Terrestrial odor-trial following by pheromone-stimulated males o f Trichoplusia ni. A n n . Entomol. Soc. A m . 66:1213-1214.  33  Sirrine, F. A . 1894. Insects affecting late cabbage. N . Y . State Agric. E x p . Stn. B u l l 83:557-571. Sirrine, F. A . 1899. Spraying mixture for cauliflower and cabbage worms II. N . Y . Rept. Entomol. 6:389-413. Smith, F. F., A . K . Ota, and A . L . Boswell. 1970. Insecticides for control o f the greenhouse whitefly. J. Econ. Entomol. 63:522-527. Sower, L . L . , R. S. Kaae, and H . H . Shorey. 1973. Sex Pheromone o f Lepidoptera. X L I . Factors limiting potential distance o f sex pheromone communication i n Trichoplusia ni. A n n . Entomol. Soc. A m . 66:1121-1122. Sutherland, D . W . S. 1966. Biological Investigation o f Trichoplusia ni (Hubner) and other Lepidoptera damaging cruciferous crops in Long Island, N e w York. N . Y . Agric. E x p . Stn. Ithaca M e m . 399. Sutherland, D . W . S., and G . L . Greene. 1984. Cultivated and w i l d host plants, in G . L . Green, editor. Suppression and Management o f Cabbage Looper Populations. U S D A Tech B u l l # 1684- Chapter 1. Toba, H . H . , A . N . Kishaba, R. Pangaldan, and P. V . V a i l . 1973. Temperature and the development o f the cabbage looper. Ann. Entomol. Soc. A m . 66:965-973. Walker, H . G . , and L . D . Anderson. 1936. Control o f cabbage worms. Truck Exp. Sta. B u l l 93:1381-1384.  34  Chapter 3  Potential of AcMNPV as an alternative for control of cabbage loopers that are resistant to Btk  3.1 Introduction  Resistance to insecticides has a great impact on the control o f insect pests. It leads to economic loss, increased environmental risk as a consequence o f the use o f repeated and high doses, and uncertainty about the performance o f new products (Wood 1999). Insect resistance to a specific synthetic or biological insecticide can lead to positive or negative consequences. First, fitness costs associated with resistance might make the pest more susceptible to other causes o f mortality and pathogens ( M i l k s and Myers 2003) and this could suppress the maintenance o f resistance. Development o f cross-resistance to other insecticides with a similar mode o f action however could negatively reduce the number o f alternatives available for pest resistance management.  The use o f  Bacillus thuringiensis (Bt) has greatly increased since its commercial release  in 1938. It is the most widely used bioinsecticide in organic and integrated pest management production accounting for 90% o f the bioinsecticide market (Chattopadhyay et al. 2004). During recent years, its use has been expanded even more with the incorporation o f the Bt toxin gene into genetically modified crops. Tabashnik et al. (1990) reported the appearance o f resistance to  Bt by the diamondback moth, Plutella xylostella  35  in the field in Hawaii, and Janmaat and Myers (2003) reported the evolution o f resistance to Bt by the cabbage looper, Trichoplusia ni, in commercial greenhouses o f the Fraser Valley, B . C . Canada. Other species have been selected for resistance to Bt in the laboratory (in review Ferre & V a n Rie, 2002). Greenhouse growers i n British Columbia deal with high levels o f resistance to Bt in cabbage loopers, the major lepidopteran pest in that system. Therefore, the need is great to develop new strategies to maintain T. ni population levels below economic damage thresholds. If 5/-based products become ineffective due to resistance, organic farmers w i l l have lost this valuable resource (McGaughey et al. 1998) Greenhouses represent an ideal environment for the selection for insecticide resistance because insect populations can continuously reproduce during most o f the year. Thus they require repeated and higher applications o f insecticides. Overwintering o f cabbage loopers in greenhouses, as shown i n Chapter 2, makes the management o f resistance even more difficult. These factors appear to contribute to the rapid increase o f resistant individuals. The aim o f "resistance management" is not to stop resistance entirely, but to slow its development and extend an insecticide's useful lifespan as long as possible (Comins 1977). According to Croft (1990) there are three goals o f resistance  management:  avoiding resistance when and i f possible, delaying resistance as long as possible, and making resistant populations revert to susceptibility. Management strategies  could  involve the use o f more than one biocontrol agent in order to reduce the selection pressure o f each agent and to slow or avoid the development o f resistance. Studies  36  conducted by Janmaat and Myers (2003) showed that the resistance o f cabbage loopers to  Bacillus thuringiensis increases with an increase in the number o f Bt applications. In addition, resistant individuals reverted to susceptibility in several generations when growers stop spraying Bt. These results greatly support the idea that using an alternative bioinsecticide to break down the resistance to Bt is a viable strategy.  Although the use o f baculoviruses seems to fit within the proposed strategy, the goal of this chapter is to test for cross-resistance in  T. ni between Bacillus thuringiensis var  kurstaki and a potential alternative bioinsecticide: a nucleopolyhedrovirus o f the alfalfa looper Autographa  californica ( A c M N P V ) . I investigated i f individuals resistant to Bt are  as susceptible to virus as individuals that lack Bt resistance. Resistance to one pathogen could possibly increase the susceptibility o f T. ni to other pathogens. Demonstration o f a lack o f cross-resistance or even better, increased susceptibility o f resistant individuals to A c M N P V would support the recommendation for future development o f A c M N P V as a component o f a Bt resistance management plan.  Objectives 1) To determine i f cross-resistance between A c M N P V and Btk occurs in T. ni populations. 2) To determine i f the resistance to Btk confers a higher susceptibility o f T. ni to AcMNPV.  37  3.2 Materials and Methods Three bioassays were conducted using two T. ni colonies: both derived from descendants of insects collected from the Gipaanda (GIP) greenhouse in 2001 by A l i d a Janmaat and collaborators. In the first generation, this colony was found to be extremely resistant to Bt compared to the laboratory colony (RC). Subsequently a G I P - resistant population (GIPR) was established by continuous selection with Bt almost every generation (See selection method described in Janmaat 2004 p. 62). A GIP - susceptible population (GIPS) was established by maintaining the colony without any further selection with Bt. This colony became susceptible to Bt after several generations. B y the time o f the experiment, the level o f resistance to Bt o f G I P - R and GIP-S, measured as L C s , was found to be 5 0  35,486 I U V m l and 2,180 I U / m l respectively. Further details about the method used to. estimate the level o f resistance o f the GIP colonies are given i n Appendix 4.  Bioassays Susceptible and resistant colonies were reared following the procedure described in Appendix 1. A description o f the Bt selection protocol is given in Janmaat (2004) page 62. Egg sheets were collected and put out to hatch in 4 L plastic buckets after 2-4 days of collection. After 2 days at 2 6 ° C , neonates were transferred to 175 m l cups containing artificial diet (25 neonates/cup), prepared following the recipe shown in Appendix 2. After 5 days, m i d to late second instar larvae (L2) were starved i n individual 21 m l plastic cups for 3 h and were then randomly assigned to each o f the treatments.  1  IU= International Units  38  The multiple nucleopolyhedrovirus o f Autographa  californica ( A c M N P V ) was provided  by Dr. Martin Erlandson (Saskatoon Research Centre), and originated from samples isolated from infected T. ni larvae collected in Fraser Valley greenhouses. The viral concentration i n the stock was rechecked by viral occlusion body counting with a Neubauer hemocytometer (see Appendix 5). The final viral doses used for the three replicates were 2.7, 6.6, 13.4, 27 and 67 PIBs/larva. The viral doses were presented to larvae on cucumber (cv. Natika ez) leaf discs. After consuming the leaf disc completely individuals were kept on artificial diet for the rest o f the experiment (Forschler et al. 1992; Manson 2003; Cook et al. 2003).  Procedure Viral dilutions o f A c M N P V , prepared following the protocol explained in Appendix 6, were removed from the freezer several hours before dosing and thawed at room temperature inside a 455 m l paper cup with l i d to avoid the incidence o f light and therefore degradation o f the viral dilutions.  Cucumber leaves, cv. Natika ez, were collected from plants cultivated at the U B C Horticultural greenhouse.  Plants had approximately 6-8 leaves at the time o f the  experiment. M i d d l e leaves were used for the experiment. Leaves were collected in a tray covered with a damp paper towel on the bottom and a transparent plastic l i d to prevent desiccation.  Six trays per colony were labeled to individualize each treatment (Control, D I , D2,...,D5) and each tray was provided with a damp paper towel and 30 pieces o f damp  39  filter paper (Whatman N ° l ) , o f 1.8 c m . Using a cork borer # 2 ( 0 = 4 mm), cucumber 2  leaf discs were cut and placed on top o f the filter paper pieces, one leaf disc per piece. Once the dilutions were thawed, 2 u.1 o f viral dilution was added to each leaf disc using a micropipette. In the case o f the control treatment, 2 ul o f distilled water was added to each leaf disc. The doses were added systematically from lowest to highest to avoid contamination. The drop o f virus solution was allowed to dry at room temperature. This required between 3-4 hours. The paper towel was kept moist to avoid leaf water loss. After drying, each leaf disc with its filter paper was transferred into individual plastic 21 ml cups. One starved larva was placed inside each plastic container. Thirty larvae were used for each dose. Larvae were allowed to feed on the leaf disc for 24 h at 26°C in the growth room.  After 24 h, all larvae that finished eating the leaf disc were included in the experiment and were transferred to individual 30 m l cups filled with 2.5 m l o f artificial diet and kept at 24°C inside a growth chamber. Mortality was recorded for the first time 48 h after the day o f the infection, and after that, twice a day. Survivors were transferred to new diet every 5-7 days, until pupation. Date o f death, pupation date, date o f adult emergence and pupal weight were recorded.  The experiment was repeated 3 times.  40  3.3 Statistical Analysis The analysis o f L D o was done using G E N S T A T 5 Release 4.1 (1998) by fitting the 5  results to the Probit Analysis outlined by Finney (1971). Before obtaining the LD50 for each o f the colonies, data were corrected for natural mortality in the control treatment as necessary (see Table 3.1). A t-test was conducted for the analysis o f pupal weight by sex using J M P i n 4.04 (2002). For the analysis o f the time to pupation I performed a Kruskal-Wallis test for nonparametric data using J M P i n 4.04 (2002). Finally, time to death was analyzed with a survival analysis performed with J M P i n 4.04 (2002) where L o g Rank and W i l c o x o n tests were used to look for differences between treatments. The alpha level was set at 0.05 for all tests unless otherwise specified.  3.4 Results a. Analysis of Mortality The first step i n the analysis o f mortality consisted o f correcting for natural mortality that occurred in the control groups. A s shown in Table 3.1, the natural mortality in control groups was less than 5% for each colony in repetitions 1 and 2, and therefore, there was no need for mortality correction. However, in repetition 3 mortality exceeded 5% and Abbot's formula was used to correct those values (Abbot 1925).  After mortality corrections were applied in repetition 3, a Probit Analysis was carried out with Genstat 5 to check for differences among replicates. There were no differences  41  among replicates for both colonies. For the susceptible colony, the deviance ratio was 1.54 with a p = 0.214 while for the resistant colony the deviance ratio was 0.68 with a p= 0.505. Therefore, the results o f the three replicates for each colony were combined.  The last step was to look for significant differences between the resistant and the susceptible colonies. Results after statistical analysis showed that there are significant differences between the colonies (deviance ratio: 16.13, pO.OOOl). Table 3.2 shows the values o f LD50 and their confidence intervals expressed as the number o f polyhedra inclusion bodies (PIBs)/larva for each colony.  The confidence intervals derived from Genstat 5 and shown in Table 3.2 do not overlap for the two colonies. The individuals resistant to Bt are twice as susceptible to A c M N P V as individuals susceptible to Bt.  b. Time to Death The time to death was analyzed using a survival curve obtained with J M P i n for the dose that caused more than 80% o f mortality in both colonies (dose 5: 67 PIBs/larva). This non-parametric  statistical  analysis  was  chosen  because data  were  not normally  distributed. Time to death decreased with increased dose from 9.0 days with dose 1 to 5.3 days with dose 5 for the resistant colony (x  2  Log Rank Resistant  = 31.38, df = 4, pO.OOOl) and  from 9.1 days with dose 1 to 5.0 days with dose 5 for the susceptible colony (x Susceptible  (X  =  2  Log Rank  28.9, d f = 4, pO.OOOl). N o differences were detected by dose between colonies  LogRank Dose  p = 0.97; x  2  1 = 0.160, p = 0.69; X  Log Rank Dose 4  Log Rank Dose 2  = 1 -67, p = 0.20;  £  L  o  g  = 0.024, p = 0.88; %  Rank Dose 5  Log Rank Dose 3  = 0.016,  = 0.73, p = 0.39). The reason for  42  choosing the dose that killed more than 80% o f the individuals is based on potential commercial applications. J M P i n performs both a W i l c o x o n and a Log-Rank test to detect differences i n survivorship between treatments. N o differences i n time to death were detected between the susceptible and the resistant colonies with 5.0 ± 0.17 days and 5.3 ± 0.22 days respectively (x  c. Tim^  2  Log Rank =  0.73, p= 0.39).  to Pupation  The time from second instar to the pupal stage for untreated controls is shown i n Fig. 3.1. A  non-parametric test, Wilcoxon-Kruskal Wallis, was performed with J M P i n 4.04  because data were not normally distributed.  Individuals susceptible to Bt reached pupation at 15.8 days ± 0.2 days while resistant individuals required 20.9 days ± 0.4 days. These differences are statistically significant (X = 69.48; d f = 1; p = <0.0001) and suggest a fitness cost associated with resistance.  d. Pupal Weight A t-test was performed to look for differences in pupal weights. Pupal weights o f males and females were analyzed separately and were ln-transformed to make data normal before the t-test analysis. Despite the differences i n the time to pupation, the pupal weights o f the control groups for the resistant and susceptible colonies were the same (weight-Resistant  f aies  weight-Resistant  i  m a  era  e s  =  225.4 ± 5 . 5 mg; weight-Susceptible  = 250.1 ± 7.7 mg; weight-Susceptible  m a  i  e s  f  ema  ies  =  227.9 ± 6.4 mg;  = 254.2 ± 4.9 mg). Even  though resistant individuals had slightly smaller pupal weights, those differences were not statistically significant ( t  fem  aies  = -0.202; df = 66; p Itl = 0.8406; t  m a ! e s  = -0.467; df =  43  67; p Itl = 0.6357).  3.5 Discussion  Both, A c M N P V and Bt are ingestion, non-systemic bioinsecticides. Midgut cells and the peritrophic membrane ( P M ) may play a key role in the infection process o f both biocontrol agents. In spite o f these similarities, there are important differences in the in  vivo pathway necessary for these agents to k i l l the host. The lack o f cross-resistance can be attributed to differences in the mechanism o f infection o f Bt and A c M N P V . The experimental results show that the 16X resistance to  Bacillus thuringiensis was not  associated with an increased resistance o f 71 ni to A c M N P V but resistant individuals were slightly (2X) more susceptible to the virus. These findings are in accordance with other examples in which cross-resistance was evaluated but the other way around, for populations resistant to viruses and not to Bt. For example F u x a and Richter (1990) reported that the resistance to the  Spodoptera frugiperda multiple nucleopolyhedrovirus  ( S f M N P V ) did not affect the susceptibility o f  Spodoptera frugiperda to either Bt or the  chemical methyl parathion, and M i l k s and Myers (2003) showed that there was no crossresistance between the singly embedded nucleopolyhedrovirus o f the cabbage looper ( T n S N P V ) and  Bacillus thuringiensis in populations o f T. ni resistant to T n S N P V . One  possible explanation is that midgut proteases are essential for toxin formation in the Bt in  vivo pathway, while i n the case o f baculoviruses, proteases are responsible for the inactivation o f occlusion derived viruses (ODVs). After liberation, O D V s must rapidly pass through the peritrophic membrane to avoid degradation by proteases (Elam et al. 1990). According to Oppert et al. (2000), midgut extracts from Indian meal-moth, Plodia  44  interpunctella resistant to Bt had lower proteolytic activity than extracts from susceptible insects and these had a reduced capacity to activate Cry 1 A c protoxin. These findings may indicate that i f lower proteolytic activity confers resistance to Bt, this could reduce the availability o f enzymes capable o f degrading the O D V s and facilitate their passage through the peritrophic membrane.  Another possible explanation is that the susceptibility o f lepidopteran larvae to N P V is influenced by the rate at which the midgut cells are sloughed off (Keddie et al. 1989; Washburn et al. 1995; Washburn et al. 1998; Hoover et al. 2000). Individuals that develop more rapidly may slough off these cells before the virus spreads to other tissues thus reducing infection ( M i l k s and Myers 2002). Originally, the greater resistance o f rapidly growing larvae was thought to be related to the increasing weight o f the larva (review i n Cory and Myers 2003). However, comparisons o f oral with intra-hemocoelic inoculation demonstrated that this resistance did not occur when virus was injected but was related to infection through the midgut (Teakle et al. 1986).  I did not record the larval weight o f controls at the time o f the infection. However, the time to pupation could be used as a measure o f developmental rate. Janmaat and Myers (2003) showed that one indicator o f reduced fitness o f T. ni resistant to Bt is the slower larval growth o f resistant individuals compared to susceptible ones. Although larvae o f both colonies were the same age at the time o f infection, there were differences in the time to pupation o f larvae i n the control group (Fig. 3.1) indicating that resistant individuals developed more slowly compared to susceptible individuals. Furthermore, only the larvae that finished eating the leaf disc in 24 h were included in the experiments.  45  For the susceptible colony the final number o f larvae included was 30% higher than for the resistant colony, supporting the possibility that resistant individuals could have been smaller at the moment o f dosing. However, the fact that the smaller individuals, that had not finished eating, were not included in the experiment is likely to have compensated for differences in initial larval size. Secondly, to assess the level o f resistance to Bt, larvae o f the same age were used for the experiment. Resistant individuals, even though they may have been smaller, were sixteen times more resistant to Bt than the susceptible individuals. If cross-resistance had been present in the bioassays with A c M N P V , then a similar level o f resistance to the virus might have been expressed, but this was not the case. Ultimately, in a greenhouse that has both susceptible and resistant individuals, susceptible individuals w i l l grow more rapidly than resistant ones. For instance, this could be another advantage in the management o f resistance to Bt with A c M N P V as an alternative agent, because individuals susceptible to Bt w i l l be more likely to survive the application o f the virus and to spread the susceptible genes, and the resulting population w i l l then be killed by the next Bt application following the virus.  Another parameter o f primary importance is the time it takes the virus to k i l l the pest. Implementation o f Baculoviruses for pest control has been slow because they are slow to k i l l insects compared to synthetic insecticides (Granados and Federici 1986). In spite o f the slower developmental time exhibited by the resistant individuals, the time to death for susceptible versus resistant populations was much the same. The higher susceptibility o f the resistant colony seems to compensate for slower viral replication, associated with longer development time. Thus, susceptible and resistant individuals have the same time to death.  46  3.6 Conclusions Given that no cross-resistance occur and that resistant individuals to Bt are twice as susceptible to A c M N P V these biocontrol agents might complement each other and might be compatible for their use i n a rotation strategy in greenhouses. Further investigations should focus on experimental applications in greenhouses, with both agents alone in a rotational schedule as well as in mixtures.  47  Table 3.1. Values o f control mortality (%), L D s (PIBs/larva) and slopes for each 5 0  replicate o f susceptible and resistant colonies obtained with P R O B I T  ANALYSIS  (Genstat 5).  Colony  GIP-S  GIP-R  Replicate  LD  5 0  (95 % C I )  Slope (s.e)  Control Mortality %  1  13 (9-19)  0.68 (0.13)  2.8  2  14(10-20)  0.72 (0.14)  3.2  3  19(14-23)  0.65 (0.15)  7.2*  1  6(4-10)  0.52 (0.17)  4.6  2  7(4-10)  0.67 (0.19)  5.1  3  8(5-12)  0.86 (0.21)  9.4*  * Mortality in these repetitions had to be adjusted by Abbott's correction formula before further analysis.  48  Table 3.2. L D  5 0  values and confidence intervals obtained with Probit Analysis for each  colony, with repetitions combined.  PIBs/ larva Lower 95%  LD  5 0  Upper 95%  Slope ± s.e.  Susceptible  12  15  19  0.66 ± 0.06  Resistant  5  7  10  0.68 ± 0 . 0 7  49  F i g . 3.1. Development time to pupation from second instar o f larvae resistant (R) and susceptible (S) to Bt. Data obtained from untreated group and analyzed by Wilcoxon / Kruskall - Wallis in J M P i n 4.04. Asterisk indicates a statistical difference, x  2 =  69.4; p =  <0.0001;a = 0.05  Colony  50  3.7. References Abbott, W . S. 1925. A method for computing the effectiveness o f an insecticide. J. Econ. Entomol 18:265-267. Chattopadhyay, A . , N . B . Bhatnagar, and R. Bhatnagar. 2004. Bacterial Insecticidal Toxins. Crit. Rev. M i c r o b i o l . 30:33-54. Comins, H . N . 1977. The development o f insecticide resistance in the presence o f migration. J. Theor. B i o l . 64:177-197. Cook, S. P., R. E . Webb, J. D . Podgwaite, and R. C . Reardon. 2003. Increased mortality of gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) exposed to Gypsy moth nuclear polyhedrosis virus in combination with phenolic gycoside salicin. J. Econ. Entomol. 96:1662-1667. Cory, J. S., and J. H . Myers. 2003. The ecology and evolution o f insect baculoviruses. Ann. Rev. Ecol., E v o l . Sys. 34:239-272. Croft, B . A . 1990. Developing a philosophy and program o f pesticide resistance management. Pages 277-296 in H a l l , editor. Pesticide Resistance in Arthropods. Roush, R.T., Tabashnik, B . E . , N e w York. Elam, P., P. V . V a i l , and F. Schreiber. 1990. Infectivity of Autographa californica nuclear polyhedrosis virus extracted with digestive fluids o f Heliothis zea, Estigmene acrea, and carbonate solutions. J. Invert. Pathol. 55:278-283. Finney, D . J. 1971. Probit Analysis, 3rd edition. Cambridge University Press. Fuxa, J. R., and A . R. Richter. 1990. Response o f nuclear polyhedrosis virus-resistant Spodoptera frugiperda larvae to other pathogens and to chemical insecticides. J. Invert. Pathol. 55:272-277. G E N S T A T . 1998. Release 4.1. in Rothamsted Experimental Station, Lawes, Agricultural Trust: Harpenden, U K . Granados, R. R., and B . A . Federici. 1986. The Biology o f Baculoviruses. V o l 2. C R C , Practical applications for insect control. Boca Raton. F L . Hoover, K . , J. O. Washburn, and L . E . Volkman. 2000. Midgut-based resistance o f Heliothis virescens to baculovirus infection mediated by phytochemicals in cotton. J. Insect Physiol. 46:999-1007. Janmaat, A . F., and J. Myers. 2003. Rapid evolution and the cost o f resistance to Bacillus thuringiensis in greenhouse populations o f cabbage loopers, Trichoplusia ni. Proc. R. Soc. London B:2263-2270.  51  Janmaat, A . F. 2004. The evolution of resistance to Bacillus thuringiensis in greenhouse Trichoplusia ni populations. Ph.D. Thesis - Depart, o f Zoology, University o f Bristish Columbia, Vancouver. J M P I N , V . 2002. S A S Institute, Inc. Cary, N C , U S A . in, (Distributed by Duxbury Press). Keddie, B . A . , G . W . Aponte, and L . E . Volkman. 1989. The pathway o f infection o f Autographa californica nuclear polyhedrosis virus i n an insect host. Science 243:1728-1730. Manson, J. 2003. The effect o f food plant on the efficacy o f a nucleopolyhedrovirus o f Trichoplusia ni. Honours Biology. Faculty o f Science - University o f British Columbia. McGaughey, W . H . , F. Gould, and W . Gelernter. 1998. ifr resistance management. Nat. Biotech. 16:144-146. M i l k s , M . L . , and J. Myers. 2003. Cabbage looper resistance to a nucleopolyhedrovirus confers cross-resistance to two granuloviruses. Environ. Entomol. 32:286-289. M i l k s , M . L . , and J. H . Myers. 2002. Costs and stability o f cabbage looper resistance to a nucleopolyhedrovirus. E v o l . Ecol.:369-385. Oppert, B . , R. Hammel, J. E . Throne, and K . J. Kramer. 2000. Fitness costs o f resitance to Bacillus thuringiensis in the Indian-meal moth, Plodia interpunctella. Entomol. exp. appl. 96:281-287. Tabashnik, B . E . , N . L . Cushing, N . Finson, and M . W . Johnson. 1990. Field  development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera:Plutellidae). J. Econ. Entomol. 83:1671-1676. Teakle, R. E . , J. M . Jensen, and J. E . Giles. 1986. Age-related susceptibility of Heliothis punctiger to a commercial formulation of nuclear polyhedrosis virus. J. Invert. Pathol. 47:82-92. Washburn, J. O., B . A . Kirkpatrick, E . Haas-Stapleton, and L . E . Volkman. 1998. Evidence that the stilbene-derived optical brightener M 2 R enhances Autographa californica M nucleopolyhedrovirus infection o f Trichoplusia ni and Heliothis virescens by preventing sloughing of infected midgut epithelial cells. Biol.Control 11:58-69. Washburn, J. O., B . A . Kirkpatrick, and L . E . Volkman. 1995. Comparative pathogenesis of Autographa californica M Nuclear Polyhedrosis Virus i n larvae o f Trichoplusia ni and Heliothis virescens. Virology 209:561-568.  52  Wood, E . 1999. Ecologia y Manejo de la Resistencia de insectos a insecticidas. Ministerio de Asuntos Agrarios (Prov. Bs. As.) - Serie T e c n i c a N 2:1-25. 0  Chapter 4  Does host plant affect the susceptibility of Trichoplusia ni to AcMNPV?  4.1 Introduction  Baculoviruses are entomopathogens that have been used as microbial pesticides to control pests i n many crops. Nucleopolyhedroviruses are one o f the two genera i n the family Baculoviridae that have been successfully implemented in the biological control o f pests in forestry and agriculture (Fuxa 1987; Moscardi 1999). A number o f factors can affect the performance o f these natural pathogens for the control o f pests. Temperature, for example, plays a key role i n regulating the time to k i l l but not host mortality. Higher temperatures cause faster deaths (van Beek et al. 2000; Ignoffo 1966; Frid 2002). Other factors such as the age at which larvae become infected ( M i l k s 1996; Bianchi et al. 2001), the quantity o f virus consumed, and virus-food plant interactions (Forschler et al. 1992; Hoover et al. 1998; Farrar and Ridgway 2000; Bianchi et al. 2001) can alter the susceptibility o f the insect pest to the viruses.  Because the route o f entry o f baculoviruses is oral, it is assumed that factors affecting virulence must act i n the insect's midgut environment within a short time (Hoover et al. 1998). The effects o f host plants could result from direct antagonism (Kushner and Harvey. 1962) or synergy between leaf compounds and microbes. Plant chemistry can  54  modulate infection in the gut and nutrient content can determine host survival (review in Cory and Myers 2003). Plants can also influence virus interactions i n other ways: plant architecture affects virus persistence, and the palatability o f the plant can modify the mobility o f insect hosts and virus acquisition through levels o f consumption (review in Cory and Myers 2003). Virus-plant chemical interactions can reduce larval susceptibility to baculoviruses (Richter and Abdel-Fattah 1987; Keating et al. 1988; Forschler et al. 1992;  Santiago-Alvarez and Ortiz-Garcia 1992; Hoover et al. 1998a), or increase the  susceptibility o f larvae (Forschler et al. 1992; Hoover et al. 1998a). For example, larvae of  Spodoptera exigua were more susceptible to S e M N P V when feeding on plants (tomato  and chrysanthemum) in comparison to artificial diet (Bianchi et al. 2001). Manson (2003) found that the susceptibility o f T. ni to T n S N P V varied with the crop on which the larvae were infected. If the efficacy o f baculoviruses in a greenhouse situation is consistent with laboratory bioassays, virulence and speed o f action determined in the laboratory may be used to indicate baculovirus efficacy at the crop level (Bianchi et al. 2000).  Cabbage loopers are susceptible to A c M N P V , a virus first isolated in  Autographa  californica, the alfalfa looper. Here I analyzed the susceptibility o f cabbage loopers to A c M N P V on the three major crops cultivated in greenhouses in British Columbia (tomato, cucumber and bell pepper), as well as on artificial diet, i n order to be able to predict the potential o f A c M N P V i f used in larger scale greenhouse experiments.  55  Objectives  1- T o find the doses o f A c M N P V that could effectively control T. ni on tomato, cucumber and pepper. 2-  To investigate i f there are interactions between A c M N P V and the host plant that changes the insect's susceptibility to the virus.  3-  To find the virus doses that minimize the time to death on each crop.  4-  To test i f the susceptibility o f virus to larvae fed on artificial diet can be extrapolated to crop plants.  4.2 Materials and Methods Bioassays were conducted using  Trichoplusia ni that originated from three different  colonies ( T O M F 3 , G L E N , GIP). A l l colonies were reared following the procedure explained in Appendix 1.  These colonies were established in the laboratory after  collection from commercial greenhouses.  T O M F 3 was collected from a tomato greenhouse in Langley, B C and was kept on tomato leaves for 7 generations and then transferred to artificial diet for 3 generations prior to the experiments.  G L E N colony came from a pepper greenhouse and GIP from a tomato greenhouse in the Fraser Valley o f British Columbia. These colonies were originally established by A l i d a Janmaat (Janmaat and Myers 2003) and have been kept in the laboratory on artificial diet for several years.  56  The three colonies were tested for susceptibility to N P V at different times. In the first trial I used T O M F 3 and cucumber leaves because larvae were less susceptible to T n S N P V on cucumber (Manson 2003). This colony was lost due to very l o w fecundity and failed egg hatch before it was possible to repeat the experiment and include the other crops. Further bioassays were conducted with G L E N . This colony was very robust, however, only two repetitions could be carried out before the colony crashed due to a virus outbreak. T w o further replicates were then performed with the GIP colony.  Bioassays For most o f the bioassays I used four different food sources: tomato, cucumber, pepper and artificial diet discs on which I offered the virus to the host. A l l crops were grown from seeds inside the U B C Horticulture greenhouse. Tomato, "Maribel ez T m C 5 V F 2 F r w i " , and pepper,  Lycopersicum esculentum  Capsicum annuum L . "Triple 4 ez", seedlings  were obtained by sowing seeds on Redi-earth (35-45% peat + 55-65% vermiculite) inside individual cells o f seedling trays (72 cells per tray). Cucumber,  Cucumis sativus  "Natika ez", seeds were directly sown in four inch, disposable, plastic pots filled with potting m i x (regular peat 75%, regular perlite 25%, N - P - K - starter; supplied by West Creek Farm- Fort Langley). A l l containers were drenched with a 1% fungicidal solution of N o - D a m p ® (Oxine Benzoate 2.5%) to prevent damping-off and other early fungal diseases. A l l pots were placed i n a hot and humid bedding bench inside the greenhouse until the emergence o f the seedlings. They were then moved to a regular bench until they had approximately 2 true leaves. A t that time, plants were transplanted to five inch plastic pots filled with potting m i x . Plants were kept at 24°C ± 2°C without any supplementary lighting and a regular flooding fertirrigation with 120 ppm o f 15-5-15 C a l - M g Scotts  57  Petters Excel® fertilizer provided daily in the fall and winter and twice a day during spring and summer. Pepper plants were used after 2-3 months o f being sown. Tomato plants were used after 1.5-2 months o f being sown and cucumber plants were used after  1-1.5 months,  depending on the season. In the case o f peppers, plants had 9-13 leaves, but only new completely expanded leaves were used to dose the virus. Cucumber leaves were collected from the middle position from plants having between 5-8 leaves. Tomato leaves were collected from the bottom o f the plants, however senescent leaves were avoided. The decision to pick leaves from different parts o f the plant depending on the crop comes from the observation o f the pest's foraging behaviour on each crop in both, commercial and experimental greenhouses (see Chapter 5 for more details). Artificial diet was prepared following the recipe described in Appendix 2.  Egg sheets o f each colony were collected and allowed to hatch i n 4 L plastic buckets after 2-4 days o f collection. After 2 days at 26°C, neonates were transferred to 170 m l cups containing artificial diet (25 neonates/cup). After 5 days larvae were starved in individual 21 m l plastic cups for 3 h and then randomly assigned to treatments.  The multiply embedded nucleopolyhedrovirus o f  Autographa californica ( A c M N P V )  was provided by Dr. Martin Erlandson (Saskatoon Research Centre, A A F C ) , and originated from samples isolated from infected caterpillars collected from Fraser Valley greenhouses. The viral concentration in the stock was rechecked by viral occlusion body counting using a Neubauer hemocytometer (see Appendix 5).  58  The virus doses were offered to larvae on discs o f artificial diet and leaves cut with a cork borer #2 ( 0 = 4 mm). One control and four to five viral doses per food source were used. The doses changed with the colony. For G L E N and T O M F 3 they were 1, 2.7, 27 and 268 PIBs/larva after viral recounting, while for GIP they were 2.7, 6.6, 13.4, 27 and 67 PIBs/larva after viral recounting. The doses used for G L E N  and T O M F 3  before  recounting were suggested by Martin Erlandson according to previous experiments he had done, however a great variation within LD50 and confidence intervals were detected so doses were adjusted for the bioassays conducted with GIP colony.  After completely consuming the contaminated disc the individuals were kept on artificial diet for the rest o f the experiment (Forschler et al. 1992; Manson. 2003; Cook et al. 2003).  Infection Technique  V i r a l dilutions o f A c M N P V , prepared following the protocol explained in Appendix 6, were removed from the freezer several hours before dosing and thawed at room temperature inside a 455 m l paper cup with lid to avoid the incidence o f light and therefore degradation o f the viral dilutions.  Leaves o f the three crops were collected from plants cultivated at the U B C Horticulture greenhouse placed onto a tray covered with a damp paper towel on the bottom and a transparent plastic lid to prevent desiccation.  Six trays per crop were labeled to individualize each treatment (Tomato Control, Tomato D I , Tomato D 2 , . . . , Tomato D 5 , Pepper Control,..., Pepper D 5 ,  etc.). The same  59  procedure explained in page 38 (Chapter 3 - Cross resistance) was used to set up the bioassays. Mortality, date o f death and pupation date were recorded. The total experiment consisted o f 2 repetitions for G L E N (tomato and pepper) and GIP on all the host plants, and one repetition for T O M F 3 (cucumber) and G L E N (diet and cucumber).  Artificial diet discs - Two drops o f 1 m l o f artificial diet each were spread on a microscope slide and a cover slip was placed on top o f the drops and pressed down until the drops touched each other and the diet became a thin layer o f about 0.5 m m in thickness. The artificial diet was prepared following the procedure explained in Appendix 2. - Ten microscope slides were placed inside a 9 m m diameter Petri dish and the Petri dish was kept refrigerated to facilitate handling after solidifying. Once the diet was solid, discs were obtained using a cork borer #2. Each slide yielded approximately 23 discs. - Discs were removed with a spatula from the slide and placed on top o f each filter paper piece.  4.3 Statistical Analysis The analysis o f LD50 was done using G E N S T A T (1998) by fitting the results to the Probit Analysis outlined by Finney (1971). Time to death was analyzed with a survival analysis performed with J M P i n 4.04 where L o g Rank and W i l c o x o n tests are used to look for differences between treatments. The significance level (a value) for all statistical tests was set at 0.05.  60  4.4 Results a. Analysis of Mortality  The analysis o f mortality yielded no significant differences among the LD50S for larvae from G L E N and GIP colonies infected with A c M N P V on leaf discs from different host plants.  The confidence intervals obtained with Genstat 5 after combining replicates  overlapped (Tables 4.1 and 4.2), indicating no significant differences among food plants with corrections for natural mortality in the control groups being automatically performed by the program. Furthermore, an analysis o f deviance was performed with Genstat 5, with mortality corrections calculated using Abbott's formula for those treatments with more than 5% o f mortality in control groups. N o differences were detected for the four food sources used for viral inoculation with this method (dev ratio  = G  L  E  N  2.29, p = 0.08; dev  ratio G I P = 0.53, p = 0.66).  L D 5 0 estimates for G I P had the lowest variation and this indicates that the adjustment o f doses, which was done prior to the onset o f the experiment with this colony, yielded more consistent results. In spite o f that, no differences were detected among food disc types.  b. Time to Death  Significant differences i n speed o f k i l l were found among doses, with the time to death decreasing with an increase in the dose for all food sources and colonies (Table 4.3). Therefore the time to death was analyzed for dose 5, which caused the highest mortality (more than 90%) for all the colonies and plant types. The reason for choosing this dose is based on potential commercial application (targeting a high level o f mortality).  Data  61  were non-parametric and therefore analyzed with a survival curve performed with J M P i n 4.0.4. The analysis o f time to death between replicates for GIP colony for dose 5 on each crop yielded significant differences on tomato (x on pepper (x  2  Log-Rank  2  Log-Rank  =  15.05; df= 1; p= 0.0001) and  = 7.28; df= 1; p= 0.007). Therefore the analysis for the GIP colony  data was done with a separate survival curve for each replicate. N o differences among plant types were found for GIP replicate #1 (Table 4.4), however, there were some differences for GIP replicate #2 (Table 4.5a). Multiple comparisons were carried out with the Sidak procedure (Hardin et al. 1996) to determine on which crops time to death was statistically different (Table 4.5b).  N o differences were observed for G L E N colony between replicates for dose 5 i n either pepper (x  2  Log-Rank  = 2.65; df= 1; p= 0.1037) or tomato leaf discs (x  2  Log-Rank  = 2.23; df= 1;  p= 0.1353), so time to death data were combined for both replicates. A survival curve, and Sidak test for multiple comparisons were performed  (Table 4.6a and  4.6b  respectively).  The mean number o f days to death for dose 5, varied from 4.5 to 6.1 days among plant type, colony and replicate. Although differences in speed o f k i l l were detected among food sources, colonies varied in their response. However, the maximum variation among crops was 0.5 and 0.6 o f day for G L E N and GIP # 2 respectively.  4.5 Discussion The host plant can play an important role in mediating the susceptibility o f lepidopteran larvae to baculoviruses (Fuxa, 1982; Fuxa and Geaghan, 1983; Keating and Yendol,  62  1987; Richter et al. 1987; Duffey et al. 1995; Hoover et al. 1998a,b; A l i et al. 1998). However, according to my results, the susceptibility o f T. ni larvae to A c M N P V appeared to be unaffected by the type o f food ingested at the time o f infection. These findings concur with a study done by Forschler et al. (1992) on the susceptibility o f Helicoverpa zea to its nucleopolyhedrovirus ( H z N P V ) when larvae were fed the virus on cotton, tomato and artificial diet and remained on artificial diet pre and post infection. However, these results contradict many other studies i n which the susceptibility o f larval hosts to nucleopolyhedroviruses changed with the food plant species (Richter and Abdel-Fattah 1987;  Keating et al. 1988; Santiago-Alvarez and Ortiz-Garcia 1992; A l i et al. 1998;  Hoover et al. 1998a; Farrar and Ridgway 2000).  One possible explanation for my findings is that as T. ni is highly susceptible to A c M N P V , any variation with food plant might be hard to detect. Other lepidopteran hosts,  Spodoptera exigua, Heliothis virescens and Helicoverpa zea, have been found to  vary in their susceptibility to A c M N P V on different host plants (Hoover et al. 1998; Hoover et al. 1998a; Hoover et al. 1998b; Bianchi et al. 2000; Hoover et al. 2000). According to Washburn et al. (1995) T. ni is highly susceptible to A c M N P V while H.  virescens is very susceptible and H. zea is highly resistant. In many other studies, other hosts and N P V s were used and the  LD50S  or  LC50S  were always much higher than those  found here (Richter and Abdel-Fattah 1987; Keating et al. 1988; Forschler et al. 1992; Santiago-Alvarez and Ortiz-Garcia 1992; Hunter and Schultz 1993; A l i et al. 1998; Farrar and Ridgway 2000). Richter and Abdel-Fattah (1987) and A l i et al. (1998) found the susceptibility o f  S. frugiperda and heliothine Lepidoptera to vary among different crop  plants, however no differences were detected for highly virulent viruses similar to my  63  results with A c M N P V .  Another factor that could have reduced my ability to detect differences in susceptibility among food sources is the stage at which the larvae were infected. Most studies in the literature were conducted on larvae in their 4  th  instar. A t this stage, larvae are likely to be  less susceptible to the virus, and therefore variation in the susceptibility with host foliage is more likely to be revealed.  The size o f the leaf disc or diet disc must not be overlooked. The leaf discs I used were small (4 m m 0 ) compared to the ones most authors used (5 - 9 m m 0 ) . Richter et al. (1987) stated that it is likely that the differences in susceptibility were related to the pest being stressed by consumption o f less suitable host plants, although the possibility o f an antiviral agent in one or more o f the plant species cannot be ruled out. It could be that a threshold amount o f antagonistic or synergistic compounds must be present in the leaves to detect differences and in such a small piece o f leaf the proportion is not adequate. So the question that arises then is: would results have been different i f larvae were maintained on the different plant foliage for the whole experiment or at least for a longer period o f time before and after infection? Forschler et al. (1992) found that host plants did not affect the activity o f H z S N P V against corn earworm larvae unless the foliage was fed to larvae for the 24 h before administration o f the virus. They found significant differences on cotton when larvae were fed continously and were dosed on crops, however, no differences were detected between tomato and artificial diet in any o f their experiments.  Richter et al. (1987) conditioned larvae to the plants by rearing them  individually from the first instar o f one generation through the second instar o f the next  64  generation before testing. However, Schultz et al. (1992) stated that neither food quality before nor after infection influence mortality, and only the quality o f foliage entering the gut in concert with the virus influences infection.  In many studies larvae were starved for 18-24 h prior to bioassays to void their guts from previous food (Keating et al. 1988; Hunter and Schultz 1993; Hoover et al. 1998; Hoover et al. 1998a). I only starved the larvae for 3 h prior to infection to assure that they would be hungry enough to finish eating the whole leaf /diet disc in 24 h. Even though the voiding o f the guts could possibly help to detect differences among host plants, that situation would be very unlikely to occur outside laboratory conditions.  Other factors could possibly regulate the acquisition o f virus i n real situations and could therefore modify the susceptibility o f the host to A c M N P V in commercial crops. Those include: the amount o f leaf consumed on each crop that w i l l i n turn determine the actual amount o f virus ingested, the foraging behaviour that can help the insect escape from the virus, and the persistence o f the virus on the different crops that is also determined by plant architecture and leaf morphology.  Regarding the speed o f k i l l , even when differences were detected for some o f the repetitions, the highest difference among crops (cucumber, tomato and pepper) was never more than 0.6 o f a day, a difference that would not be relevant in an agricultural situation. Post infection food source however is very likely to regulate the speed o f k i l l . Hoover et al. (1998) stated that the faster the larvae grow on a host plant the quicker they die, due to an increase o f viral replication. This indicates that in order to realistically evaluate speed of k i l l , it would have been better to test the larvae on the same food source for the whole  65  experiment given that great differences in growth rates exist among the food sources tested (see Appendix 7). Moreover, other factors such as consumption and foraging behaviour may also play very important roles in affecting the speed o f k i l l .  4.6 Conclusions N o differences in susceptibility o f T. ni to A c M N P V associated with host plant were detected i n laboratory bioassays. In addition, no biologically meaningful differences in time to death occurred in these bioassays. However, even though bioassays are a good tool for basic research, the realistic scenario should not be overlooked.  Bioassays must be complemented with experimental applications in greenhouses to more accurately predict the performance o f A c M N P V on the different crops.  66  Table 4.1. L D  5 0  and 9 5 % confidence intervals (95% CI) in PIBs/larva, slope for the  relationship between dose and mortality and % mortality o f controls for replicates o f T. ni colonies inoculated with A c M N P V on different diet discs obtained using P R O B I T A N A L Y S I S (Genstat 5).  Plant type  Slope-s.e  % Control mortality  X Fit o f line  10(8-17)  0.91 (0.16)  2.8  78.62; p O . O O l  2  11 (7-22)  0.66 (0.12)  3.3  62.28; p O . O O l  1  18(10-23)  0.66 (0.15)  10.7  24.15; p O . O O l  2  12(9-17)  0.90 (0.16)  3.5  45.53; p<0.001  1  17(10-30)  0.58(0.10)  10.2  56.61; p O . O O l  2  19(11-32)  0.67 (0.11)  9.3  70.79; p O . O O l  1  11(7-17)  0.81 (0.16)  8.3  34.52; p O . O O l  2  14(11-19)  1.01 (0.16)  0  56.91; p O . O O l  TOM-F3  1  10(6-18)  0.57(0.11)  9.7  35.49; p O . O O l  GLEN  1  12(8-19)  0.69 (0.10)  0  73.57; p O . O O l  1  11 (8-19)  0.64 (0.14)  7.9  23.03; p O . O O l  2  13 (10-17)  0.90 (0.16)  6.3  51.88; p O . O O l  1  20 (12-32)  0.77 (0.12)  0  76.03; p O . O O l  1  11 (7-22)  0.52 (0.16)  9.6  14.55; p O . O O l  2  12(9-18)  0.82 (0.14)  8.0  44.86; p O . O O l  Colony  GLEN TOMATO GIP  GLEN PEPPER GIP  CUCUMBER  GIP GLEN DIET GIP  * LD  5 0  Rep#  LD  1  5 0  (95% CI)*  2  (95% CI) automatically adjusted for control mortality by Genstat 5.  67  Table 4.2. Mean L D , and confidence intervals in occlusion bodies o f A c M N P V per 5 0  larva and slopes for each colony obtained with PROBIT A N A L Y S I S (Genstat 5), combined data used for GIP and G L E N colonies.  Colony  Confidence Interval  Diet  Pepper  Tomato  N/A  N/A  N/A  12  20  18  11  Low95%  8  12  13  8  Up95%  19  32  27  17  dev ratio:0.84 p=0.473  0.69±0.10  0.77±0.12  0.62±0.07  0.76±0.10  LD50  12  11  12  13  Low95%  10  9  9  10  Up95%  15  14  15  17  0.78±0.10  0.68±0.10  0.92±0.11  0.81±0.11  LD  TOMF3  50  10  Low95%  6  Up95%  18  Slope  0.57±0.11  LD  GLEN  Cuke  50  Slope  GIP  Slope dev ratio: 1.19 p=0.313  68  T a b l e 4.3. Analysis o f time to death among doses by colony, repetition and crop performed using survival curves and L o g Rank Test i n J M P i n 4.0.4  GIP #1 Cuke  X2 =32.7; df = 4 ; p < 0.0001; range D1 - D5 = 9.3 - 5.0 days  Diet  X2 =17.0; df = 4 ; p = 0.002; range D1 - D5 = 8 . 3 - 4 . 7 days  Pepper  X2 =26.3; df = 4 ; p < 0.0001; range D1 - D5 = 9.2 - 4.8 days  Tomato  X2 =22.7; df = 4 ; p = 0.0001; range D1 - D5 = 8 . 7 - 4 . 5 days  GIP #2 Cuke  X2 =22.7; df = 4 ; p = 0.0001; range D1 - D5 = 8.9 - 5.0 days  Diet  X2 =15.3; df = 4 ; p = 0.0041; range D1 - D5 = 9 . 0 - 4 . 9 days  Pepper  X2 =12.9; df = 4 ; p < 0.0110; range D1 - D5 = 8.8 - 5.6 days  Tomato  X2 =15.9; df = 4 ; p = 0.0030; range D1 - D5 = 8.7 - 5.4 days  GLEN #1 Cuke  X2 =9.95; df = 3 ; p < 0.0413; range D1 - D5 = 8.5 - 5.8 days  Diet  X2 =9.77; df = 3 ; p = 0.03; range D1 - D5 = 9.5 - 6.1 days  Pepper  X2 =9.68; df = 3 ; p < 0.021; range D1 - D5 = 8.6 - 5.7 days  Tomato  X2 =32.4; df = 3 ; p < 0.0001; range D1 - D5 = 9 - 5.2 days  GLEN #2 Pepper  X2 =20.5; df = 3 ; p = 0.0001; range D1 - D5 = 9.6 - 5.3 days  Tomato  X2 =19.8; df = 3 ; p = 0.0006; range D1 - D5 = 9.5 - 5.4 days  69  Table 4.4. Mean time to death in days (± S E M ) o f Trichoplusia ni that fed A c M N P V (dose 5) on discs o f different food sources for the first replicate o f GIP colony. 2  X  Log Rank  =2.7; d f = 3; p = 0.4394. Same bold letters indicate no significant statistical  differences.  GIP #1 Cuke  5.0 ± 0.21 a  Diet  4.7 ± 0.20 a  Pepper  4.8 ± 0.22 a  Tomato  4.5 ± 0.25 a  70  Table 4.5a. Mean time to death i n days (± S E M ) o f Trichoplusia ni that fed A c M N P V (dose 5) on discs o f different food sources for the second replicate o f GIP colony. 2 X  Log Rank  = 13.97; d f = 3; p = 0.0029. Same bold letters indicate no statistical differences after multiple comparisons (see Table 4.5b.) GIP #2 Cuke  5.0 ± 0.14 a  Diet  4.9 ± 0.16 a  Pepper  5.6 ± 0.13 b  Tomato  5.4 ± 0.18 b  71  Table 4.5b. Multiple comparisons for non-parametric data o f time to death among leaf disc types for G I P colony replicate #2 using the Sidak procedure, with a y-level or comparison wise-error rate o f 0.0042 (Hardin et al. 1996).  Group  X2*  P  Significance **  Cuke vs Diet  0.06  0.4263  NS  Cuke vs Pepper  10.16  0.0014  S  Cuke vs Tomato  11.03  0.0009  S  Diet vs Tomato  13.02  0.0003  S  Diet vs Pepper  11.40  0.0007  S  Pepper vs Tomato  0.75  0.3867  NS  * Chi-squared values with their associated probabilities (p) obtained after performing a Wilcoxon/Kruskal Wallis test in J M P i n for each pair. ** Significance (S) based on the y-level value = 0.0042.  72  Table 4.6a. Mean time to death in days (± S E M ) o f Trichoplusia ni that fed A c M N P V (dose 5) on discs o f different food sources for the second replicate o f G L E N colony.  2 X  Log Rank  = 17.4; d f = 3; p = 0.0006. Same bold letters indicate no statistical differences after multiple comparisons (see Table 4.6b)  GLEN  Cuke  5.8 ± 0.09 be  Diet  6.1 ± 0.16 c  Pepper  5.5 ±0.10 ab  Tomato  5.3 ±0.10 a  73  Table 4.6b. Multiple comparisons for non-parametric data of time to death among leaf disc types for G L E N colony using the Sidak procedure, with a y-level or comparison wise-error rate o f 0.0042 (Hardin et al. 1996).  Group  %2*  P  Significance **  Cuke vs Diet  2.66  0.1028  NS  Cuke vs Pepper  7.9  0.005  NS  Cuke vs Tomato  10.4  0.0012  S  Diet vs Tomato  15.6  <0.0001  S  Diet vs Pepper  11.80  0.0006  S  Pepper vs Tomato  0.61  0.4359  NS  * Chi-squared values with their associated probabilities (p) obtained after performing a  Wilcoxon/Kruskal Wallis test in J M P i n for each pair.  ** Significance (S) based on the y-level value = 0.0042.  74  I-  4.7 References  A l i , M . I., G . W . Felton, and S. Y . Young. 1998. Influence o f interespecific and intraspecific host plant variation on the susceptibility o f heliothines to a Baculovirus. B i o l . Control 12:42-49. Bianchi, F. J. J. A . , N . N . Joosten, J. M . Vlak, and W . van der Werf. 2000. Greenhouse evaluation o f dose-time mortality relationships o f two nucleopolyhedroviruses for the control o f beet armyworm, Spodoptera exigua, on chrysanthemum. B i o l . Control 19:252-258. Bianchi, F. J. J. A . , N . N . Joosten, J. M . Vlak, and W . van der Werf. 2001. The influence of greenhouse chrysanthemum on the interaction between the beet armyworm, Spodoptera exigua, and the baculovirus S e M N P V : parameter quantification for a process-based simulation model. J. A p p l . Entomol. 125:557-562. Cook, S. P., R. E . Webb, J. D . Podgwaite, and R. C . Reardon. 2003. Increased mortality o f gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae) exposed to Gypsy moth nuclear polyhedrosis virus in combination with phenolic gycoside salicin. J. Econ. Entomol. 96:1662-1667. Cory, J. S., and J. H . Myers. 2003. The ecology and evolution o f insect baculoviruses. Annu. Rev. E c o l . E v o l . Sys. 34:239-272. Farrar, R. R., and R. L . Ridgway. 2000. Host plant effects on the activity o f selected nuclear polyhedrosis viruses against the corn earworm and beet armyworm (Lepidoptera: Noctuidae). Environ. Entomol. 29:108-115. Finney, D . J. 1971. Probit Analysis, 3rd edition. Cambridge University Press. Forschler, B . T., S. Y . Young, and G . W . Felton. 1992. Diet and the susceptibility o f Helicoverpa zea (Noctuidae: Lepidoptera) to a nuclear polyhedrosis virus. Environ. Entomol. 21:1220-1223. Fuxa, J. R. 1987. Ecological considerations for the use o f entomopathogens in I P M . Annu. Rev. Entomol. 32:225-251. G E N S T A T . 1998. Release 4.1. in Rothamsted Experimental Station, Lawes, Agricultural Trust: Harpenden, U K . Hardin, M . J., J. L . Willers, and T. L . Wagner. 1996. Nonparametric multiple comparisons o f survivorship distributions. J. Econ. Entomol. 89:715-721. Hoover, K . , S. A . Alaniz, J. L . Yee, D . M . Rocke, and B . D . Hammock. 1998a. Dietary protein and Chlorogenic A c i d Effect on Baculoviral Disease o f Noctuid  75  (Lepidoptera: Noctuidae) Larvae. Environ. Entomol. 27:1264-1272. Hoover, K . , K . T. Kishida, L . A . DiGiorgio, J. Workman, S. A . Alaniz, B . D . Hammock, and S. S. Duffey. 1998b. Inhibition o f baculoviral disease by plant-mediated peroxidase activity and free radical generation. J. Chem. E c o l . 24:1949-2001. Hoover, K . , M . J. Stout, S. A . Alaniz, B . D . Hammock, and S. S. I. Duffey. 1998a. Influence o f induced plant defenses in cotton and tomato on the efficacy o f baculoviruses on noctuid larvae. J. Chem. E c o l . 24:253-271. Hoover, K . , J. O. Washburn, and L . E . Volkman. 2000. Midgut-based resistance o f Heliothis virescens to baculovirus infection mediated by phytochemicals i n cotton. J. Insect Physiol. 46:999-1007. Hoover, K . , J. L . Yee, J. C . Schultz, D . M . Rocke, B . D . Hammock, and S. S. Duffey. 1998b. Effects o f plant identity and chemical constituents on the efficacy o f a baculovirus against Heliothis virescens. J. Chem. E c o l . 24:221-252. Hunter, M . D . , and J. C . Schultz. 1993. Induced plant defenses breached? Phytochemical induction protects an herbivore from disease. Oecologia 94:195-203. Keating, S. T., W . G . Yendol, and J. C . Schultz. 1988. Relationship between susceptibility o f gypsy moth larvae (Lepidoptera: Lymantriidae) to a baculovirus and host plant foliage constituents. Environ. Entomol. 17:952-958. Manson, J. 2003. The effect o f food plant on the efficacy o f a nucleopolyhedrovirus o f Trichoplusia ni. Honours Biology. Faculty o f Science - University o f British Columbia. M i l k s , M . L . 1996. The implications o f Cabbage Looper (Trichoplusia ni) - Nuclear polyhedrosis virus coevolution for biological control. P h D . Thesis -Dept. o f Zoology- University o f British Columbia. Moscardi, F. 1999. Assessment o f the application of baculoviruses for control o f Lepidoptera. A n n u . Rev. Entomol. 44:257-289. Richter, A . R., and M . Abdel-Fattah. 1987. Effect o f host plant on the susceptibility o f Spodoptera frugiperda (Lepidoptera: Noctuidae) to a nuclear polyhedrosis virus. Environ. Entomol. 16:1004-1006. Santiago-Alvarez, C , and R. Ortiz-Garcia. 1992. The influence o f host plant on the susceptibility o f Spodoptera littoralis (Boisd.) (Lep., Noctuidae) larvae to Spodoptera littoralis N P V (Baculoviridae, Baculovirus). J.Appl.Ent. 114:124130.  76  van Beek, N . , P. R. Hughes, and A . H . Wood. 2000. Effects o f incubation temperature on the dose-survival time relationship o f Trichoplusia ni larvae infected with A c M N P V . J. Invert. Pathol. 76:185-190. Washburn, J. O., B . A . Kirkpatrick, and L . E . Volkman. 1995. Comparative pathogenesis of Autographa californica M Nuclear Polyhedrosis Virus i n larvae o f Trichoplusia ni and Heliothis virescens. Virology 209:561-568.  77  Chapter 5 The effect of host plants on leaf consumption and movement of cabbage loopers as potential influences on infection by AcMNPV  5.1 Introduction The cabbage looper, Trichoplusia ni, is a generalist insect that feeds on over 160 species of plants in 36 families (Sutherland and Greene 1984). It is the major lepidopteran pest in cucumber, pepper and tomato greenhouses i n British Columbia, however its growth and behaviour differ with the host crop.  Despite the fact that nucleopolyhedroviruses ( N P V s ) have been shown to be effective for the control o f insect pests (Granados and Federici 1986, Moscardi 1999), extrapolation of results from laboratory experiments on interactions between viruses and hosts do not necessarily apply to the field situation. Efficacy o f N P V s for control o f lepidopteran pests not only has to do with the ability o f the virus to k i l l the pest i n the laboratory, but also with the probability that the pest w i l l ingest a lethal dose o f viral occlusion bodies capable o f causing infection and death. Thus foraging behaviour and development time are important factors to consider when attempting extrapolate laboratory experiments to greenhouse situations. For example, in a study o f  Spodoptera exigua in chrysanthemum  greenhouses, Bianchi et al. (2001) found that larval development time on chrysanthemum plants was 36% greater than that on artificial diet. Moreover, development can differ from one crop to another associated with differences in food quality and the presence o f  78  antiherbivore compounds. Sutherland (1966) found that larval development o f cabbage loopers on potted pepper plants took 11-15 days longer than those on potted cabbage plants.  Foliage consumption affects the amount o f nutrients and viral particles ingested and can regulate larval growth and development time. A characteristic o f  S. exigua on artificial  diet is that later instars are more resistant than early instars to virus. Bianchi et al. (2000) found however, that the increased resistance o f later instars o f  S. exigua to its N P V did  not occur on the chrysanthemum greenhouse crop. They suggested that the increase in foliage consumption o f later instars resulted in higher doses o f the virus and this compensated for their reduced susceptibility to the viruses. Furthermore, variation in the dose o f viral particles ingested can affect both: a) the level o f infection achieved and b) the time it takes the virus to k i l l the host (Ignoffo 1964). Ignoffo (1964) found that for T. ni as much as 4.3 days difference in time to death occurred with doses ranging from 25,000 PIBs o f T. ni singly embedded nucleopolyhedrovirus ( T n S N P V ) / u l diet to 350 PIBs T n S N P V / u l diet. The highest dose killed T. ni in 3.7 days while the lowest required 8 days.  Moreover, as N P V s can be horizontally transmitted, the behaviour o f infected larvae i f different from healthy ones, can greatly affect the spread o f N P V s and the chances for the pest to encounter viral particles. If, as is sometimes the case, infected larvae climb to the upper parts o f the plants to die (stated by Fuxa 1987 and Moscardi and Carvalho 1993, shown by Vasconcelos et al. 1996 and Goulson 1997, review in Cory and Myers 2003) new upper leaves that are not sprayed, can still potentially become contaminated by the  79  PIBs released after the liquefaction o f dead larvae (horizontal transmission). This can reduce the number o f applications o f virus required to keep the pest under control.  Several biological and behavioural characteristics o f the insect and their interactions with the plants and virus must be studied to determine the potential o f N P V s to effectively control cabbage loopers inside a greenhouse environment. The aim o f this chapter is to explore the differences in consumption and behaviour o f cabbage loopers fed on the three main crops cultivated i n greenhouses: tomato, bell pepper and cucumber. Firstly I w i l l focus on consumption rates and the water content o f leaves that could be factors in the differences i n growth rates o f larvae reared on the three crops (see Appendix 7 and Janmaat 2004). Finally, I w i l l describe the behaviour o f larvae on the different crops based on visual monitoring in greenhouses as well as movement experiments with healthy and infected caterpillars carried out in the U B C Horticulture greenhouse.  Objective To describe consumption and behavioural patterns o f cabbage loopers fed on different host crops as factors that might influence the potential o f A c M N P V for the control o f loopers in vegetable greenhouses.  5.2 Materials, Methods and Results The materials, methods and results w i l l be described separately for each experiment. The statistical analyses were performed with J M P i n 4.04 (Student Version o f S A S Institute). The level o f significance (a-value) was 0.05 in all tests.  80  5.2.1 Leaf consumption experiment The aim o f this experiment was to test for differences in leaf consumption by T. ni larvae from three different colonies. Each o f the colonies was originally collected from commercial greenhouses. The colonies from the pepper and the tomato greenhouses were maintained on artificial diet for more than 2 years and the colony coming from a cucumber greenhouse was maintained on artificial diet for five generations before the experiment.  Leaf discs o f 9.6 c m were cut using a plastic cutter o f 3.5 cm in diameter. Leaf discs 2  were assigned to plastic trays containing six holes per tray. Four trays per treatment were included in each o f the three repetitions. O n the bottom o f each hole, a filter paper disc o f the same area as the hole was provided and was dampened with 5 drops o f distilled water. The vials were maintained inside a tray provided with a damp paper towel on the bottom and a plastic l i d on top to prevent leaf desiccation. Each hole was individually identified and each leaf disc was weighed and its surface was measured with a leaf area meter (LIC O R , model Li-3000).  Larvae from each colony were 8 days old at the time o f the experiment. Larvae were reared at 26 °C ± 1°C on artificial diet in 170 m l Styrofoam cups (15 larvae/cup). A wide range o f larval sizes was collected from each cup. Larvae were individually starved for 3 hours prior to the onset o f the experiment. Leaf discs were cut and let sit for 4 hours at room temperature before recording the fresh weight and leaf area. Larvae were weighed after starvation and the weight was written on each o f the plastic containers. In order to test the same range o f weights for all the crops, groups o f three individuals per colony  81  with similar weights were put aside to assign one o f the three individuals to each crop for the experiment. Larvae were then assigned to each o f the holes containing a leaf disc and the larval weight was recorded. Larvae were kept inside a growth chamber at 26°C for seven hours and then the leaf discs were weighed, scanned for area and the final larval weight was recorded. The experiment was repeated three times, and the three repetitions were one week apart. Plants were grown at the U B C Horticulture greenhouse as previously described for other experiments. Leaves were collected from top, middle and bottom sections o f the plants for pepper, cucumber and tomato respectively to reflect the position in the plant where larvae are normally encountered feeding i n commercial crops.  I calculated the grams o f leaf consumed, the amount o f surface area consumed, and the weight gained for each individual. Larval weights were bracketed into three ranges: range 1 from 0.02 grams to 0.05, range 2 from 0.06 to 0.09 grams and range 3 from 0.1 grams to 0.15 grams and data from the three replicates were combined to have a higher sample size for each range. Larval weights within ranges and above 0.15 grams were not included i n the analysis. Larval weights within ranges and among colonies and crops were homogeneous (Range 1: 2.2, p  c o l o  Pcoiony=  „ = 0.12; Range y  x = 0.034 g, N=62, F  2: x = 0.076  g, N=100, F  0.23, Range 3: x= 0.12 g, N = 92, F , 2  8 9 c r 0  p=  2  , 9 crop^  0.22, p  crop  = 0.8; F , 9 colony =  , 7crop=  2.54, p  crop  = 0.08; F ,  2  5  9  0.69, p  crop  2  5  2  = 0.50; F | C O  o n y  9 7  = 1-48,  = 0.23,  Pcolony 0.78). =  The analysis o f grams o f leaf consumed and c m consumed were analyzed with a 2  Factorial analysis, while food conversion index data (weight gained / g o f leaf consumed) were non parametric and were analyzed with a Wilcoxon test.  82  Results The amount o f leaf ingested varied significantly within the three ranges o f larval weight (Fig 5.1). Heavier larvae ingested more mass o f fresh leaf o f all the crops (F ge ran  =  46.7,  p< 0.0001, df=18, 236). N o differences were detected among colonies (Fcoiony^.O, p =0.137, df=18, 236) and no significant interactions occurred between colony and crop (F oiony*crop 1.41, p=0.23,df=l 8, 236). However, larvae consumed significantly more =  C  cucumber than tomato and pepper ( F  c r o p  = l 11.86, p O . 0 0 0 1 , df=l 8, 236), see Fig. 5.2. O n  average, cucumber fed larvae consumed 3.5 times more fresh leaf weight after seven hours than larvae reared on tomato and pepper with the level o f consumption being similar for the last two crops.  For leaf area consumed (Fig. 5.3 and 5.4), all the colonies were similar ( F | n y 0.88, =  C0  0  p=0.41, df=18 ,236), but showed a significantly higher level o f consumption o f cucumber leaves, followed by tomato leaves and then by pepper leaves (F p=279.3, p O . O O O l , cr0  df=18, 236). O n average, larvae reared on cucumber consumed about 10 times more leaf area than larvae reared on pepper and almost 5 times more than larvae reared on tomato. Although leaf discs were the same size in diameter they did not weigh the same. In addition the size o f leaf veins varies among crops and T. ni do not feed on those although they contributed to the leaf disc weight. Cucumber leaf discs were on average lighter than tomato and pepper in that order ( x ucumber= 146 mg ± 0.003 mg, x omato= 160 mg ± 0.002 C  mg and  x  pe  t  pper= 184 mg ± 0.003 mg). These differences were statistically significant (F=  56.51, p< 0.0001, df=2, 251). Differences were also detected among larval weight categories ( F n e 62.96, p O . O O O l , df=18, 236) with heavier larvae consuming about 3.3 =  ra  g  83  times more surface than larvae in the lowest range (Fig. 5.4).  Regarding the efficiency o f food conversion (ECI) as a function o f the larval weight, no differences were detected among colonies (x = 0.61, df=2, p= 0.752). Differences were 2  detected among weight range categories (x =8.08, df=2, p=0.0175) and crops (x 17.64, 2  2=  df=2, p=0.0001). Overall, there was a trend that larvae fed on pepper had higher food conversion efficiencies than larvae fed on tomato and cucumber ( E C I p p e r 0.63 ± 0.053, =  pe  ECI  tomato  =  0.50 ± 0.052, E C I  C  ucumber=  0.44 ± 0.048). In the case of weight ranges, heavier  larvae had higher food conversion indexes compared to smaller larvae with E C I 0.61± 0.063, E C I  ran e2 g  = 0.53± 0.049 and E C I  r a n g e  i  r a  nge3  =  = 0.44± 0.051.  5.2.2 Water content experiment Differential growth rates o f larvae on different plants could possibly be related to the water content o f leaves. To determine i f the water content o f leaves at different positions on plants was governing the foraging behaviour o f larvae observed in the greenhouses, plants in commercial greenhouses were arbitrarily divided vertically into three thirds and leaves were collected from each o f the three positions (top being the first third o f the plant, middle the second third and bottom the last third) in four consecutive visits. Leaves were collected from the same commercial greenhouses (one cucumber, one tomato and one pepper greenhouse). Fifty leaves per position were brought to the laboratory each time, in a cooler with ice packs to keep the leaves fresh, and then one leaf disc per leaf was cut with a cork borer #13 (1.53 cm 0 ) . Leaf discs were weighed, and placed in individually labeled paper envelopes, and dried in an oven at 65 °C for 72 h. Dry weights were then recorded and the water content (mg o f water), mg o f water per c m , mg o f dry 2  84  weight/cm and % water were calculated.  Results The fresh weight o f leaf discs did not differ among visits for the cucumber crop (F k = CU  e  1.89, p= 0.13,df=3,513). Fresh weight did differ among collections for the pepper crop (F epper P  =  53.28, p O . 0 0 0 1 ,df=3,595) but maintained the same trend among positions in all  the replicates. For tomato leaves differences in fresh weight among collections were found  (F mato t0  =  66.42, p <0.0001,df=3,594) and in two of the replicates the fresh weight  was higher for bottom leaves and for the other two replicates weight was higher for middle leaves. In all the cases differences were not greater than 15% among replicates and therefore, the disc weights o f all the replicates were pooled together for further analysis. A summary o f all the results obtained in this experiment is shown i n Table 5.1.  The fresh weight o f leaf discs differed with the location o f the leaf on the crops. For cucumbers, discs weighed significantly more in the bottom position followed by the middle and the top. The same significant trend occurred for pepper. However for tomatoes, even though differences were detected, bottom and middle leaves had similar average weights but they were heavier than discs cut from leaves from the top (Table 5.1).  Differences in water content also occurred with leaf position. For cucumbers, differences were detected between bottom leaves and the other two positions, with no significant differences between middle and top. For pepper, water content o f bottom and middle leaf discs did not differ, but they were significantly higher than that o f leaf discs collected at  85  the top position. For tomato leaves all positions differed, with the bottom leaf discs having the highest percentage o f water, followed by the middle and the top discs. The amount o f water (in mg) per mg o f dry matter was compared for each crop and all positions. For cucumber, discs from the bottom portion had significantly lower content o f water per mg o f dry matter with no differences between middle and top positions. For pepper discs, bottom and middle position registered the same ratio while they differed from the top position ratio. For tomatoes, the water content to dry leaf weight ratio varied among all positions with a decrease in the ratio from bottom to top.  5.2.3 Behaviour of cabbage loopers on different hosts  a. Visual monitoring for larval location inside greenhouses Once a week, visual monitoring o f 20 plants was done inside a tomato and a pepper 1  greenhouse for an entire year with the aim o f determining which part o f the plants was preferred by cabbage looper larvae in each o f the crops. Plants were arbitrarily divided into three portions (top, middle and bottom) and the number o f individuals and their developmental stages were recorded. The accumulated number o f individuals per month was calculated.  Results Table 5.2 shows percentage and total number o f individuals o f all the instars recorded in a tomato and a pepper greenhouse during each month o f the year. In the case o f the tomato greenhouse, cabbage loopers were mainly found on the bottom portion o f the  1  Except after the clean-up (in January) when 100 plants were checked.  86  plants until August. However, as the population increased and Bt was sprayed, moths began laying eggs i n the upper portion o f the plants. O f all the individuals collected inside the greenhouse (N=902), 73% were found at the bottom and middle portion while only 27%, primarily eggs and first instars, were found on top o f the plants (% = 8.3, 2  p=0.016). In the case o f the pepper greenhouse (N=453) 87% o f the individuals were found on the top portion regardless o f the instar and the remaining 13% were found in the middle and bottom portions (x =336, p<0.0001). 2  b. Larval movement on plants at the UBC greenhouse Objectives: 1) To describe larval foraging behaviour on each crop. 2) To test i f healthy caterpillars behave in the same way as infected larvae in order to predict the potential for horizontal transmission on each crop.  Materials and Methods:  A colony o f cabbage loopers maintained on cucumber leaves for 3-5 generations was used for the movement experiments in all the crops. Three day-old eggs were allowed to hatch. After two days, neonates were transferred to Styrofoam cups containing artificial diet and remained there for six days. Six day-old larvae were given a viral dose o f 2 ul o f a solution containing 1.7 x 10 PIBs o f A c M N P V per m l on cucumber leaf discs that 5  were cut with a cork borer #2 (4 m m 0 ) . Controls were dosed with 2 u.1 o f distilled water on leaf discs. Larvae were allowed to feed on discs for 24 hr and were then released in numbers o f one per plant on the last leaf at the bottom o f the plant. Extra control and  87  treated larvae were maintained feeding on the crop as back ups for replacements. Assessment started one day after the release. Whenever the released larva was not found during two consecutive assessments, the larva was replaced in the position where it was found the last time. Replacements were only made during the first 3 days o f the experiment. After that time no new releases were made.  The total number o f expanded leaves o f each plant at the moment o f the release was recorded. The assessments were done twice a day (morning and evening) and the total number o f expanded leaves o f each plant, larval stage and larval position were recorded each time.  Plants for this experiment (grafted tomato "Rapshodie", sweet bell pepper and English cucumber) were donated by Houweling Nursery Limited, and were brought into the U B C greenhouse and maintained on a flooding bench provided with 120 ppm o f 15-5-15 C a l M g Scotts Petters E x c e l ® fertilizer for the rest o f the experiment. Plants with at least 6 expanded leaves were used for the experiment and were potted i n 1 gallon plastic pots. Plants were strung up with cotton threads from the G H structural wires in order to maintain plants upright and to avoid instability o f the pots caused by bench flooding. A n unfolded paper bag was placed at the bottom o f each plant to give falling larvae the opportunity to climb up again to the plant. The last expanded leaf on each plant at the moment o f the release was tagged with colored flagging tape. To record the position of larvae, new expanded leaves were indicated relative to the leaf number above the last expanded leaf at the moment o f the release, e.g., L f - 1 was the first expanded leaf above the color flagging tape, and L f - 2 indicated the 2  n d  newly expanded leaf above the  88  colored flagging tape. Plants were separated 1 m from each other to prevent larvae from moving onto another plant. Fifteen control and treated plants were used for each replicate. Plants hosting virus infected caterpillars and control larvae were arranged alternately on the bench.  Two replicates were conducted for each crop, but each crop and replicate was tested at a different time. A contingency table was used to analyze the data. Each replicate and crop was analyzed separately. The null hypothesis was that the position o f the larva on the plant would be independent o f treatment (control vs. virus).  Results  For all crops and replicates I fail to reject the null hypothesis, indicating that the larval position on the plant was independent o f the virus treatment (Table 5.3). Infected larvae behaved i n the same way as the control larvae. However, a greater proportion o f the larvae were found on the middle of the plants in cucumber crop p<0.0001; x  cucumber2  56.16, p O . O O O l ; 25.5, p O . O O O l ;  =  2  p e  33  53.6,  30.16, pO.OOOl), bottom of the plant in tomato crop (x  x tomato2  %  (x^cucumberi  pper2  =  =  tomatoi  69.7, pO.OOOl) and top o f the plants in pepper crop  =  (x pepperi 2  =  6.13, p= 0.045). In the case of the cucumber and pepper crops  there was a trend for larvae to start moving up after the release while in the tomato crop larvae mainly remained on the leaf where they had been released. However, in the pepper crop the pattern was more diverse with larvae feeding up to the very top o f the plant and most leaves had bites taken from them. Overall, approximately 20 % o f larvae were replaced in the first 3 days. Mostly all larvae became established when they reached the 4 instar. A 90 -100% mortality was achieved in the virus treatment between 5 and 9 th  89  days after infection, and when that happened the experiment was terminated.  5.3 Discussion Larval growth and feeding behaviour o f T.ni differed considerably on the three crops under study. A s tomato and pepper belong to the same family, Solanaceae, these host plants were expected to be more similar than cucumber that is i n the family Cucurbitaceae. However, this was not the case. The three plant species differ in their nutritional requirements and growth rates. Hydroponic solutions for each crop contain different amounts o f macronutrients such us nitrogen, phosphorous and potassium, as well as calcium, magnesium, sulfate, manganese, boron, and copper . In commercial greenhouses, pepper plants are grown at higher densities per square meter (3.3 -3.5 pi / m2) followed by tomato (2.2-2.5 pi / m 2 ) and cucumber (1.2-1.4 pl/m2) . The planting 2  2  2  density can be used as an indirect measurement o f the crop growth rate, the higher the density the slower the growth o f the crop. Besides, cucumber greenhouses can have 2-3 crops during the year while tomato and pepper greenhouses have only one. Interestingly, T. ni growth follows the crop growth rate. In addition, the three crops have different architectures, leaf morphologies and chemical compositions (Everett 1981, Atherton and Rudich 1986, Bosland and Votava 2000, Choudhury and Copland 2003). Cucumber plants have alternate undivided palmately lobed leaves and branched or branchless tendrils (Everett 1981). Tomato plants have compound leaves arranged alternately. Each leaf has 1 terminal leaflet and up to 8 large lateral leaflets with smaller folioles  Greenhouse Vegetable Production Guide for commercial growers.(1993-94 Edition). Province of British Columbia, Ministry of Agriculture, Fisheries and Food. 2  90  interspersed with the large leaflets. Leaflets are irregularly lobed with toothed edges and covered with glandular and non-glandular hairs (Atherton and Rudich 1986). Pepper plants are more compact and more erect than tomato plants and have shiny, glabrous simple leaves (Bosland and Votava 2000). Choudhury and Copland (2003) showed that the lamina o f cucumber leaves had 1.5 times and 5 times more hairs than the same surface o f tomato and pepper leaves respectively. However all the hairs were non glandular in the case o f cucumber, while 100% were glandular in pepper leaves and only 33% were glandular in tomato. The dominant secondary compounds present i n cucumber, pepper and tomato plants are cucurbitacins, capsaicinoids and tomatine respectively. Heterogeneity in leaf morphology, leaf size, nutritional chemistry and secondary chemistry can affect plant-herbivore interactions (Denno and M c Lure 1983, Price 1991, Jones et al. 1993, Suomela et al. 1995, Hartley and Jones 1997).  Among the experiments conducted, the larval weight gained and the development time of T. ni were higher and faster on cucumber leaves followed by tomato and pepper (see Appendix 7). These results agree with (Janmaat 2004 and Janmaat and Myers 2005). However, Sutherland (1966) found a great difference in the development time o f T. ni among crucifers and cucumber and peppers, although he was unable to detect differences between cucumber and tomato. He found that it took T. ni approximately 24 days to develop from egg to adult on cucumber and tomato and 32 days on pepper (no temperature mentioned).  Water and nitrogen are fundamental requirements in insect nutrition and are an important component o f tissues (Wigglesworth 1984). According to Bernays and Chapman (1994)  91  caterpillars prefer food with high water content, and given a choice o f food o f the same species they w i l l tend to select the foliage with the highest water content. These findings are in accordance with the results obtained from the movement experiment and the visual monitoring o f larvae i n greenhouses together with leaf water content measurements for 71 ni feeding on tomato and cucumber plants. O n those crops, larvae mainly foraged in the position in which leaves had the greater water content. This was not the case however for larvae on pepper plants. Despite the fact that differences in water content o f tomato, pepper and cucumber foliage were detected, those differences were not greater than 10% independent o f the position. Therefore, water alone was not sufficient to explain the great differences in consumption and behaviour observed in the experiments. However, it partially explained the differences i n development time given that larvae ate almost 4 times greater mass o f cucumber leaves, and consequently water, than tomato or pepper leaves after seven hours.  Given that water alone cannot fully explain the differences in behaviour and consumption rates o f larvae, plant chemistry must be responsible for this differential behaviour. The amount o f primary and secondary compounds in plants can be regulated by the addition of supplemental nutrients. Fertilization typically increases foliar nitrogen levels (Minkenburg and Ottenheim 1990, Estiarte et al. 1994, Wilkens et al. 1996) and decreases the leaf phenolic content (Estiarte et al. 1994, Orians et al. 2002). Phenolics are widely considered deterrents, antifeedants or toxins that can change the nutritional quality of plant tissues for herbivores (Lambers 1993). Consequently, differences in foliar nitrogen can have big effects on herbivore behaviour and growth (Minkenburg and Ottenheim 1990).  92  Capsaicinoids are phytochemicals present in pepper plants. They are the phenols associated with the pungency found in hot peppers and are synthesized by the placenta in the fruits, although they have also been found in the stems and leaves o f the plants when fruits are present (Estrada et al. 2002). Estiarte et al. (1994) showed that pepper plants fertilized with higher levels o f nitrogen had a lower content o f phenolics, a higher quality of leaf and Helicoverpa armigera ate more on fertilized plants. Raven and Smith (1976) stated that the allocation o f nitrates taken up by the root system is mainly to the shoots. This generates an increased concentration o f nitrogen and depletion o f carbon in growing points that retards the production o f lignin and phenols in shoots and favours it in the roots. These findings might be useful to explain why cabbage loopers mainly forage in younger leaves (top position) o f pepper plants that might have greater digestibility and palatability than older leaves.  The most remarkable secondary compounds present in cucumber are the tetracyclic triterpenoids: cucurbitacins. Cucurbitacins are responsible for the bitter taste o f leaves, fruits and roots in Cucurbitaceae. The amount o f cucurbitacins in fruits is affected by the addition o f nitrogen. Kano and Goto (2002) showed that the occurrence o f bitterness in cucumber fruits was higher in plants cultivated with twice as much nitrogenous fertilizer than control plants. Cucurbitacins are thought to be potent feeding deterrents for insects not adapted to them (Tallamy et al. 1997), but they are also well known as kairomones for the cucumber beetles Diabrotica (Chrysomelidae: Galerucinae: Luperini) in which they promote compulsive feeding (Chambliss and Jones 1966, Sharma and H a l l 1973a, Metcalf et al. 1980, Ferguson et al. 1983). Tallamy (1997) found that exogenous addition of cucurbitacins to cucumber leaf discs did not deter the cabbage looper and stated this  93  might be an adaption from previous exposure o f low doses o f cucurbitacins i n crucifers. It might be that cucurbitacins together with other compounds act as phagostimulants for the cabbage looper. It might also be that the consumption levels on cucumber are a measure o f the maximum feeding potential for T. ni, and secondary compounds in pepper and tomato plants are acting as feeding deterrents.  The foliage and fruit o f the tomato plant also contain polyphenol oxidases and peroxidases (Felton et al. 1989). These tomato foliar enzymes oxidize an array o f endogenous compounds including caffeic acid, chlorogenic acid, rutin, cumaric acid, cinnamic acid and guaiacol (Felton and Duffey 1991). The oxidized forms o f these compounds, quinones, react with amino acids that contain nucleophilic centres (-SH, N H 2 ) ; this reaction (alkylation) reduces the digestibility o f dietary protein and the bioavalability o f amino acids (Felton et al. 1989). Felton and Duffey (1991) and Felton et al. (1992) reported reduced larval growth o f some lepidopteran species when tomato foliar protein was pretreated with peroxidase and chlorogenic acid and incorporated in artificial diet. However, when the amount o f alkylatable amino acids is significantly greater than the phenolic concentration, the reduction in larval growth is negligible (Felton et al. 1992). Insects adapted to tomato feeding can also cope with the detrimental effects o f oxidative enzymes with the aid o f catalase. Catalase activity was detected in the midgut tissues and regurgitate o f several lepidopteran pests o f the tomato plant and when purified catalase was added to tomato foliage, the peroxidase activity was eliminated and the leaves were superior as larval food compared to untreated foliage (Felton and Duffey 1991). In spite o f these proposed mechanisms, I was unable to find differences in larval growth among the colony originally adapted to tomato plants compared to the ones  94  coming from pepper and cucumber. Furthermore there was a trend for the pepper colony to grow faster when exposed to the tomato crop (Appendix 7).  It is important to note that cabbage loopers seem to feed without problems inside commercial greenhouses and they can cause considerable damage to all o f the crops. A s previously stated, fertilization can greatly affect the leaf chemistry composition and therefore the ability o f the pest to forage on the crop. In commercial greenhouses plants have balanced nutrition throughout the year and macro and micronutrients are supplied together with adequate and constant amounts o f water and increased CO2. Elevated CO2 tends to increase photosynthetic rates, plant growth, and C : N ratio due to a decrease in nitrogen content and an increase in the availability o f carbon (soluble sugars, starch) that induces the accumulation o f carbon-based defenses (Cure and A y c o c k 1986, Bazzaz 1990, K i m b a l l et al. 1994, Williams et al. 1998, Chen et al. 2005). Osbrink et al. (1987) reported that leaf-chewing insect herbivores exhibited compensatory increases i n foliar consumption rate or a delay in development when reared on plants grown with elevated CO2 environments. Orians et al. (2002) demonstrated that nitrogenous fertilization applied to isolated lateral roots in tomato generates heterogeneity i n leaf morphology, phenolic chemistry and side-shoot growth. Leaflets in direct connection to the supplemented roots were larger and had lower levels o f rutin and chlorogenic acid than did leaflets lacking direct vascular connection. A n overall assay o f foliar chemical compounds (primary and secondary chemistry) is essential to understanding the complex relationship among specific compounds (Duffey and Isman 1981, Isman and Duffey 1982, Felton e t a l . 1987).  95  It is possible that the differences i n nutrient supplementation to the plants in the Horticulture greenhouse at U B C are responsible for the differences i n behaviour and pupal weights observed i n commercial greenhouses. The pupal weights o f individuals collected in commercial greenhouses were higher than the ones collected after feeding on foliage from plants grown at the U B C greenhouses (See Table A7.1). In addition the low weight o f pupae grown on plants in the old U B C greenhouse where plants were not fertilized indicates a potential impact on leaf quality among those plants (Appendix 7). The leaf weight per c m also varied. Weights o f leaves collected inside commercial 2  tomato, cucumber and pepper greenhouses were heavier compared to the ones obtained i n the Horticulture greenhouse. Pupal weights o f cabbage loopers i n tomato greenhouses may reflect the consistency in plant quality throughout the year given that they did not vary in 11 collections made year round. Nevertheless pupal weights o f cabbage loopers collected from commercial tomato greenhouses were higher than those from commercial pepper greenhouses. Lower pupal weights are associated with reduced fitness (Janmaat 2004, Janmaat and Myers 2005). This indicates that in spite o f the balanced nutrition, pepper plants are a poorer quality resource for the cabbage looper compared to tomato.  Finally, no differences were detected in the behaviour o f healthy and virus infected caterpillars. These results contradict the statements o f Evans (1986), Fuxa (1987), and Moscardi and Carvalho (1993) who reported that infected caterpillars climb to the upper parts o f the plants to die and the findings o f Vasconcelos et al. (1996) i n Mamestra  brassicae, the cabbage moth, and Wahl 1909 (n.s., cited in Vasconcelos 1996) in Lymantria monacha, the nun moth. Both species are at least gregarious at early stages of their life cycles. The fact that the cabbage looper has no gregarious behaviour and that  96  only one larva per plant was used i n my experiments may indicate that this mechanism is triggered in the presence o f other individuals as an ecological adaptation to avoid the contamination o f other individuals o f the same species. In their absence there is no reason to invest energy to climb up the plants. Given the different foraging behaviour i n the three crops under study, climbing to the top o f the plants w i l l definitely not be beneficial on pepper where healthy larvae are normally found foraging at the top o f the plants. In addition whether a behavioural response is an adaptation w i l l depend on the strength o f selection. The occurrence o f viral disease in greenhouse populations is l o w and w i l l not be a strong selective force. Furthermore, as remarked by Myers and Cory (2003), upward movement might not necessarily enhance virus fitness in all hosts.  5.4 Conclusions  Cucumber, tomato and pepper plants differ in quality as food sources for the generalist herbivore T. ni. T. ni grows faster and consumes more foliage in cucumber, followed by tomato and pepper. Moreover, larvae are mainly found foraging in the middle, bottom and top position o f cucumber, tomato and pepper plants respectively. This differential behaviour can greatly influence the performance o f A c M N P V i n commercial greenhouses. I predict better and faster killing in a cucumber crop given the amount o f food ingested and the rapid larval growth rate, a longer persistence on tomato crop where the larvae tend to feed at the bottom o f the plants, and the highest challenge on pepper crop where it w i l l be difficult to k i l l young instars given the amount o f leaf consumed and where a more frequent application might be necessary to counteract the tendency o f the  97  pest to avoid contact with the viral particles by foraging at the top o f the plants.  Fig. 5.1. Average fresh leaf consumed after 7 hours by T. ni larvae in three different ranges o f weight. Consumption calculated as an average o f three colonies fed on three crops. Larval weight range 1 includes larval weights between 0.02 and 0.05 grams o f body weight, range 2 between 0.06 and 0.09 grams o f body weight and range 3 between 0.1 and 0.15 grams o f body weight. F=46.7, p O . O O l , N = 254, df ei=18, 236, q = 2.36, mod  60  -i  50 O  E  3 (0  c o u n  40 30  0)  "S ro E  20 10  1 Larval weight range  a = 0.05  99  F i g . 5.2. Average fresh leaf consumed i n milligrams after 7 hours by T. ni larvae fed on three different crops: cucumber (C), pepper (P) and tomato (T). Consumption calculated as an average o f all the colonies for all larval weights. F = l 11.86, p<0.001, N = 254, dfmodei=18, 236, q = 2.35, a =  0.05  100  F i g . 5.3. Average leaf area consumed in c m after 7 hours by T. ni larvae fed on three 2  different crops: cucumber (C), pepper (P) and tomato (T). Consumption calculated as an average o f all the colonies for all larval weights. F=279.3, p<0.001, N = 254, d f d e i l 8 , =  mo  236, q= 2.36, a =0.05.  5 4.5 4  3.5 3  2.5 2  1.5 1  0.5 0  T host crop  101  F i g . 5.4. Average leaf area consumed in c m after 7 hours by T. ni larvae in three 2  different ranges o f weight. Consumption calculated as an average o f three colonies fed on three crops. Range 1 includes larval weights between 0.02 and 0.05 grams o f body weight, range 2 between 0.06 and 0.09 grams o f body weight and range 3 between 0.1 and 0.15 grams o f body weight. Different letters indicate significant differences after Tukey test. F=62.96, p O . O O l , N = 254, df  mod  ei=18, 236, q = 2.36, a =0.05  102  Table 5.1. Water content experiment results per crop. Statistical analyses were done with an A N O V A in J M P i n 4.04 and confidence intervals were used to determine differences among positions within crops. Values are given as mean ± S E M . Different letters indicate significant differences among positions within crop.  Fresh disc weight (mg)  Water content (%)  Water (mg) / dry matter (mg)  32.46 ± 0.47 a  82.5 ± 0 . 3 a  5.1 ± 0 . 1 a  middle  37.13 ± 0 . 4 8  b  80.8 ± 0.3 b  4.8 ± 0 . 1 a  bottom  39.84 ± 0.44 c  79.7 ± 0.4 b  4.2 ± 0 . 1 b  statistics  F= 67.7,p<0.0001, df=2, 514  F = 25.01, p<0.0001,df=2, 514  F=19.4, p< 0.0001, df= 2, 514  top  Cucumber  top  53.49 ± 0.73  a  82.9 ± 0.2 a  5.1 ± 0 . 1 a  middle  63.85 ± 0 . 8 3  a  85.9 ± 0.2 b  6.4 ± 0 . 1 b  bottom  64.04 ± 1 . 0 1 b  88.0 ± 0.3 c  7.6 ± 0 . 1 c  statistics  F=46.0, p< 0.0001, df= 2, 595  F = 126.1,p<0.0001,df=2, 595  F =181.4 p< 0.0001, df= 2, 595  Tomato  top  44.1 ± 0 . 5 4  a  81.1 ± 0.3 a  4.6 ± 0 . 1 a  middle  48.1 ± 0 . 5 4  b  83.1 ± 0.2 b  5.1 ± 0 . 1 b  bottom  52.9 ± 0 . 5 3  c  83.4 ± 0.2 b  5.1 ± 0 . 1 b  F = 25.94, p O.0001, df = 2, 596  F =15.24, p< 0.0001, df = 2, 596  Pepper  statistics  F = 64.8, pO.OOOl, df= 2, 596  Table 5.2. Percentage o f cabbage looper instars found at top, middle and bottom portion of tomato and pepper plants in commercial greenhouses for each month o f the year.  Crop Tomato  Pepper  Month  Top %  JUN  0  0  100  30  83  17  0  148  JUL  0  29  71  51  100  0  0  93  AUG  16  30  54  202  86  0  14  7  SEP  33*  47  20  172  93  7  0  30  OCT  35*  2  44  21  299  80  20  0  48  NOV  54*  3  33  13  95  100  0  0  22  DEC  0  0  0  0  0  0  0  0  JAN  0  9  91  23  100  0  0  6  FEB  0  50  50  4  100  0  0  17  MAR  0  0  100  1  0  0  0  0  APR  0  0  100  4  65  22  13  31  MAY  0  0  100  21  84  16  0  51  1  Middle % Bottom % # Individuals  Top %  Middle % Bottom % # Individuals  * : 60% o f total individuals observed on top position were eggs and first instars 1  * : 86% o f total individuals observed on top position were eggs and first instars 2  * : 65% o f total individuals observed on top position were eggs and first instar. 3  104  T a b l e 5.3. Percentage o f cabbage looper larvae treated with A c M N P V , " V i r u s " or untreated "Control", found on top, middle or bottom position o f cucumber, pepper or tomato plants.  N 15 107 49 6 105 38  % 9 63 28 5 70 25  Statistical analysis  top middle bottom top middle bottom  N 4 20  % 4 18  Statistical analysis  top middle bottom top middle bottom  86 3 15 91  78 3 14 83  top middle  N 155 87  % 60 34  Cucumber* 1 CONTROL  VIRUS  Tomato # 1 CONTROL  VIRUS  Pepper* 1 CONTROL  VIRUS  bottom  16  6  top  119  63  middle  50  27  bottom  20  10  X = 3.89, p=0.14 2  X = 0.99 p =0.61 2  Statistical analysis  X = 4 . 6 1 p=0.10 2  105  C o n t i n u e d T a b l e 5.3.  Cucumber* 2 CONTROL  VIRUS  top middle bottom top middle bottom  Tomato # 2 CONTROL  VIRUS  top middle bottom top middle bottom  N 6 65 8 3 89 16  N 2 20 111 0 12 83  %  Statistical analysis  8 82 10 3 82 15  X = 2.98, p =0.23  % 2 15 83 0 13 87  2  Statistical analysis  X = 0.68 p =0.41 * 2  * After grouping top and middle in the same category to avoid suspect X (more than 20% of data below 5)  Pepper # 2 CONTROL  top middle bottom  VIRUS  top middle bottom  N 78 42 38 65  % 50 26 24  44  50 31  23  19  Statistical analysis  X = 2.62 p =0.27 2  106  5.5 References Atherton, J. G . , and J. Rudich. 1986. Tomato Crop. Chapman and H a l l , N e w York, London. Bazzaz, F. A . 1990. The responses o f natural ecosystems to the rising global C O 2 levels. Annu. Rev. E c o l . Sys. 21:167-196. Bernays, E . A . , and R. F. Chapman. 1994. Host-Plant selection by phythophagous insects. Chapman and H a l l , N e w York, London. Bianchi, F. J. J. A . , N . N . Joosten, J. M . V l a k , and W . van der Werf. 2000. Greenhouse evaluation o f dose-time mortality relationships o f two nucleopolyhedroviruses for the control o f beet armyworm, Spodoptera exigua, on chrysanthemum. B i o l . Control 19:252-258. Bianchi, F. J. J. A . , N . N . Joosten, J. M . Vlak, and W . van der Werf. 2001. The influence of greenhouse chrysanthemum on the interaction between the beet armyworm, Spodoptera exigua, and the baculovirus S e M N P V : parameter quantification for a process-based simulation model. Journal o f Applied Entomology 125:557-562. Bosland, P. W . , and E . J. Votava. 2000. Peppers: Vegetable and spice capsicums. C A B I Publishing, N e w York, U S A . Chambliss, O. L . , and C . M . Jones. 1966. Cucurbitacins: specific insect attractants in Cucurbitaceae. Science 153:1392-1393. Chen, F., G . W u , F. Ge, M . N . Parajulee, and R. B . Shrestha. 2005. Effects o f elevated C O 2 and transgenic B t cotton on plant chemistry, performance, and feeding o f an insect herbivore, the cotton bollworm. The Netherlands Entomological Society  Entomol. exp. appl. 115:341-350. Choudhury, D . A . M . , and M . J. W . Copland. 2003. Influence o f plant structural complexity on the searching behaviour of the egg parasitoid Anagrus atomus (Linneaus) (Hymenoptera: Mymaridae). Pakistan J. o f B i o l . Sci. 6:455-460. Cory, J. S., and J. H . Myers. 2003. The ecology and evolution o f insect baculoviruses. Annu. Rev. E c o l . E v o l . Sys. 34:239-272. Cure, J. D . , and B . Aycock. 1986. Crop responses to carbon dioxide doubling: a literature survey. Agric. Forest Meteorol. 38:127-145. Denno, R. F., and M . S. E . M c Lure. 1983. Variable plants and herbivores in natural and managed systems. Academic Press, N e w York, N e w York, U S A . Duffey, S. S., and M . B . Isman. 1981. Inhibition o f insect larval growth by phenolics in  107  glandular trichomes o f tomato leaves. Experientia 37:574-576. Estiarte, M . , I. Filella, J. Serra, and J. Penuelas. 1994. Effects o f nutrient and water stress on leaf phenolic content o f peppers and susceptibility to generalist herbivore  Helicoverpa armigera (Hubner). Oecologia 99:387-391. Estrada, B . , M . A . Bernal, J. Diaz, F. Pomar, and F. Merino. 2002. Capsaicinoids in vegetative organs o f Capsicum annuum L . in relation to fruiting. J. Agric. Food. Chem. 50:1188-1191. Evans, H . F. 1986. Ecology and epizootiology o f baculoviruses. in The Biology o f Baculoviruses, V o l 2 (See Granados and Federici). Everett, T. H . 1981. The N e w Y o r k Botanical Garden Illustrated Encyclopedia o f Horticulture. V o l 3:939. Felton, G . W . , K . Donato, R. M . Broadway, and S. S. Duffey. 1992. Impact o f oxidized plant phenolics on the nutritional quality o f dietary protein to a noctuid herbivore, Spodoptera exigua. J. Insect Physiol. 38:277-285. Felton, G . W . , K . Donato, R. J. D e l Vecchio, and S. S. Duffey. 1989. Activation o f plant foliar oxidases by insect feeding reduces nutritive quality o f foliage for noctuid herbivores. J. Chem. E c o l . 15:2667-2694. Felton, G . W . , and S. S. Duffey. 1991. Protective action o f midgut catalase in Lepidopteran larvae against oxidative plant defenses. J. Chem. E c o l . 17:17151732. Felton, G . W . , S. S. Duffey, P. V . V a i l , H . K . Kaya, and J. Manning. 1987. Interaction o f nucleopolyhedrosis virus with catechols: Potential incompatibility for host-plant resistance against noctuid larvae. J. Chem. Ecol. 13:947-957. Ferguson, J. E . , E . R. Metcalf, R. L . Metcalf, and A . M . Rhodes. 1983. Influence o f cucurbitacin content i n cotyledons o f cucurbitaceae cultivars upon feeding behaviour o f Diabroticina beetles (Coleoptera: Chrysomelidae). J. Econ. Entomol. 76:47-51. Fuxa, J. R. 1987. Ecological considerations for the use o f entomopathogens in I P M . Annu. Rev. Entomol. 32:225-251. Goulson, D . 1997. Wipfelkrankheit: modification o f host behaviour during baculoviral infection. Oecologia 109:219-228. Granados, R. R., and B . A . Federici. 1986. The Biology o f Baculoviruses. V o l 1. C R C , Biological properties and molecular biology. Boca Raton F L .  108  Hartley, S. E . , and C . G . M . Jones. 1997. Plant chemistry and herbivory, or why the world is green. Pages 283-324 in J. C . [ed], editor. Plant Ecology. 2nd Edition. Blackwell Science, Cambridge, Massachusetts, U S A . Ignoffo, C . M . 1964. Effects o f temperature on mortality o f Heliothis zea larvae exposed to sublethal doses o f nuclear-polyhedrosis virus. J. Invert. Pathol. Notes:290-292. Isman, M . B . , and S. S. Duffey. 1982. Phenolic compounds in foliage o f commercial tomato cultivars as growth inhibitors to the fruitworms, Heliothis zea. J. Amer. Hortic. Soc.l07:167-170. Janmaat, A . F. 2004. The evolution of resistance to Bacillus thuringiensis in greenhouse Trichoplusia ni populations. University o f Bristish Columbia, Vancouver. Janmaat, A . F. and J. H . Myers. 2005 The cost of resistance to Bacillus thuringiensis varies with the host plant o f Trichoplusia ni. Proc. Royal Soc. London B . 2721031-1038 Jones, C . G . , R. F. Hopper, J. S. Coleman, and V . A . Krischik. 1993. Control o f systematically induced herbivore resistance by plant vascular architecture. Oecologia 93:452-456. Kano, Y . , and H . Goto. 2002. Relationship between the occurrence o f bitter fruit (Cucumis sativus 1. cv. kagafutokyuri) and the content o f amino acid, protein and H M G - C o a reductase in the leaf. Acta Hort.(ISHS) 575 http://www.actahort.org/books/575/575 94.htm:797-803. K i m b a l l , B . A . , R. L . Lamorte, R. S. Seay, P. J. Pinter, R. Rokey, D . J. Hunsaker, W . A . Dugas, M . L . Heuer, J. R. Mauney, G . R. Hendrey, K . F. L e w i n , and J. Nagy. 1994. Effects o f free-air CO2 -enrichment on energy balance and evapotranspiration o f cotton. Agric. Forest Meteorol. 70:259-278. Lambers, H . 1993. Rising CO2 and secondary plant metabolism, plant-herbivore interactions and litter decomposition. Vegetatio 104/105:263-271. Metcalf, R. L . , A . Metcalf, and A . M . Rhodes. 1980. Cucurbitacins as kairomones for diabroticite beetles. Proc. Natl. Acad. Sci. U S A 77:3769-3772. Minkenburg, O. P. J. M . , and J. J. G . W . Ottenheim. 1990. Effect o f leaf nitrogen content of tomato plants on preference and performance o f a leafmining fly. Oecologia 83:291-298. Moscardi, F. 1999. Assessment o f the application o f baculoviruses for control o f lepidoptera. A n n u . Rev. Entomol. 44:257-289.  109  Moscardi, F., and R. C . Z . Carvalho. 1993. Consumo e utilizacao de folhas de soja por Anticarsia gemmatalis. Hub. (Lepidoptera: Noctuidae) infectada, em diferentes estadios larvais, por seu virus de poliedrose nuclear. A n n . Soc. Entomol. Bras. 22:267-280. Orians, C . M . , M . Ardon, and M . A . Basma. 2002. Vascular architecture and patchy nutrien availability generate within-plant heterogeneity in plant traits important to herbivores. Amer. J. Bot. 89:270-278. Osbrink, W . L . A . , J. T. Trumble, and R. E . Wagner. 1987. Host suitability o f Phaseolus lunata for Trichoplusia ni (Lepidoptera: Noctuidae) i n controlled carbon dioxide atmospheres. Environ. Entomol. 16:639-644. Price, P. W . 1991. The plant vigor hypothesis and herbivore attack. Oikos 62:244-251. Raven, J. A . , and F. A . S. Smith. 1976. in Brown et al.1984 (n.s.). N e w Phytol.:425-431. Sharma, G . C . , and C . V . H a l l . 1973a. Relative attractance o f spotted cucumber beetles to fruits o f fifteen species o f Cucurbitaceae. Environ. Entomol. 2:154-156. Suomela, J., P. Kaitaniemi, and A . Nilson. 1995. Systematic within-tree variation in mountain birch leaf quality for a geometrid, Epirrita autumnata. E c o l . Entomol. 20:283-292. Sutherland, D . W . S. 1966. Biological Investigation o f Trichoplusia ni (Hubner) and other Lepidoptera damaging cruciferous crops in Long Island, N e w York. N . Y . Agric. E x p . Stn. Ithaca M e m . 399. Sutherland, D . W . S., and G . L . Greene. 1984. Cultivated and w i l d host plants, in G . L . Green, editor. Suppression and Management o f Cabbage Looper Populations. U S D A Tech B u l l # 1684- Chapter 1. Tallamy, D . W . , J. Stall, N . Ehresman, P. M . Gorski, and C . E . Mason. 1997. Cucurbitacins as feeding and oviposition deterrents to insects. Environ. Entomol. 26:678-683. Vasconcelos, S. D . , J. S. Cory, K . R. Wilson, S. M . Sait, and R. S. Hails. 1996. Modified behaviour i n baculovirus-infected lepidopteran larvae and its impact on the spatioal distribution o f inoculum. B i o l . Control 7:299-306. Wigglesworth, V . B . 1984. Insect physiology, 8th edition. Chapman and H a l l , London, N e w York. Wilkens, R. T., J. M . Spoerke, and N . E . Stamp. 1996. Differential responses o f growth and two soluble phenolics o f tomato to resource availability. Ecology 77:247-258.  110  Williams, R. S., D . E . Lincoln, and R. J. Norby. 1998. Leafage effects o f elevated C 0 grown white oak leaves on spring-feeding lepidopterans. Global Change Biology 4:235-246. 2  Ill  Chapter 6 Conclusions The fact that the cabbage looper, Trichoplusia ni (T. ni), can overwinter inside greenhouses and cope with the insecticides applied during the clean-up process poses a  great risk for continuing the resistance to Bacillus thuringiensis (Bt).  Autographa californica nucleopolyhedrovirus ( A c M N P V ) has a great potential to be implemented for the management o f Bt resistance in a rotation application strategy in greenhouses given that no cross-resistance was detected.  Despite the fact that susceptibility o f T. ni to the virus did not vary with the host plant, the differences in behaviour and foraging capacity shown by the pest on cucumber, tomato and pepper indicate that specific doses and application schedules might be required for each crop to fit with the different pest-crop interactions.  Future work Further research should be done to explore the differences in efficacy, speed o f k i l l , persistence and horizontal transmission of  Autographa californica nucleopolyhedrovirus  in experimental and commercial greenhouses. Another area o f interest is the different chemical composition o f leaves o f the three crops that might relate to differences in pest behaviour. Finally, studying the ability o f resistant cabbage loopers to overcome the clean-up process and understanding the adult immigration dynamics to greenhouses are essential to manage the resistance to Bt in protected crops.  112  Appendix 1 Mass rearing of cabbage loopers The cabbage looper colonies used for the experiments described i n this thesis were reared from egg hatching until adulthood through mass rearing in a controlled temperature room kept at 26°C ± 1 °C and 16:8 hr ( L : D ) on semi-synthetic artificial diet (see Annex 2) unless specified. Eggs were surface sterilized by spraying with a 0.2% sodium hypochlorite solution. Egg sheets were placed inside 4 L plastic buckets. After neonates hatched, 15 neonates were transferred to 170 m l Styrofoam cups filled with 20 m l o f artificial diet. A new container with fresh artificial diet was provided almost every week, depending on the season o f the year. Larvae were kept inside these cups until pupation. Pupae were collected, their silky cocoons removed and they were finally soaked in a 0.6% sodium hypochlorite solution for about 6-10 minutes. Pupae were then rinsed in distilled water and let to dry on a paper towel. Pupae, (70-100) for the first two years and (150-200) for the last year, were placed in cylindrical wire mesh cages (Ignoffo  1963) wrapped with paper towel as an  oviposition substrate. A 10% sucrose solution was provided inside the cage for adult feeding. Egg sheets were collected every 2-3 days after females started laying eggs. After bleaching the egg sheets, and once they dried at air temperature, egg sheets were kept inside a refrigerator at 9 ° C until used.  113  Appendix 2 Artificial Diet Recipe A.2.1  Preparation of Dry Mix A.2.2  Dry M i x  154 g  Keep refrigerated until used  Vanderzant vitamin mix  14g  Ingredient  A m o u n t (g)  Wheat o i l germ  2 ml  Wheat germ  420  Distilled water  500 m l  Cellulose  252  Streptomycin sulphate  0.06 g  Wesson salt mix  84  Agar  8.25 g  Casein  294  Distilled water  365 m l  Sucrose  154  Cholesterol  25  Ascorbic acid  28  Sodium alignate  42  Sorbic acid  10  Alfalfa meal  80  Preparation procedure 1- Bring 365 m l of water to boil i n a pot. 2- A d d 8.25 g o f Agar  3- In a separate bowl mix the rest of the ingredients listed in table A2.1 4- M i x thoroughly with a mixer and add mixture into the pot with the agar. Turn oven to minimum temperature. 5- M i x everything again with the mixer and start filling the cups. 6- Place the cups with a 30 degrees angle of inclination on the border o f a tray until diet cools and solidifies.  114  Appendix 3  Outside temperature records for December-January 2002-2003 and 20032004  11  F i g . A.3.1 Outside minimum temperatures (in °C) between December 1 and January 10 st  th  for the two consecutive winters 2002/2003 and 2003/2004 °C  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 December  January  Winter 02/03: Max.Temp: 13.5°C - Min. Temp: -2.9°C - Mean Min Temp: 2.7°C - Mean Average Temp: 5.5°C Winter 03/04: Max.Temp: 11.9°C - Min. Temp: -12.2°C - Mean Min Temp: 0.4°C - Mean Average Temp: 3.3°C  116  F i g . A.3.2 Outside average temperatures (in °C) between December 1 and January 10' st  for the two consecutive winters 2002/2003 and 2003/2004  117  Appendix 4 Level of Resistance to Bt in GIP colony  The level o f resistance to Bt o f the two GIP populations (GIP-Resistant and GIPSusceptible) was determined by a bioassay carried out before running the cross-resistance experiment. This bioassay was conducted by A l i d a Janmaat and Jessamyn Manson, as part o f another experiment. Six day old larvae o f both colonies were exposed to increasing doses o f Bt. The bioinsecticide was diluted and added to the artificial diet on which larvae were put to feed. Larvae were let to feed on that diet for 48 hr, and after that period, the larval mortality was recorded. The table A.4.1 shows the results o f the Probit analysis for both colonies. GIP-Resistant is 16.5 times more resistant to Bt than the susceptible population. Off-spring o f the same parents tested in this bioassay were used for the cross resistance experiments detailed in Chapter 3.  Table N° A.4.1  Colony  LC50(IU/ml)  Lower 95% (IU/ml) Upper 95% (IU/ml)  GIP-S  2180  1574  3018  GIP-R  35846  25840  49728  118  Appendix 5 Viral counting 1. A haemocytometer was used for viral counting. 2.  A viral solution diluted 10 times from the original stock (1/10) was used to proceed with counting.  3.  The 1/10 solution was mixed with a Vortex machine before each new counting.  4.  10 pi o f 1/10 solution was added with a micropipette on to each side o f the haemocytometer.  5. 5 squares o f 0.2 m m were counted at a time per side and an average was calculated. This procedure was repeated 10 times. A grand average was calculated for the ten time countings.  #1  #2  #3  #4  #5  #6  #7  #8  #9  #10  Sq 1  38  32  30  30  31  28  20  23  18  18  Sq2  37  27  29  31  35  36  18  21  25  27  Sq3  25  24  27  36  28  29  18  18  13  16  Sq4  25  32  30  32  25  31  25  22  26  20  Sq5  32  34  30  37  36  32  26  20  23  19  31.4  27.8  29.2  33.2  31  31.2  21.4  20.8  21  20  Average (# PIBs)  G r a n d A v e r a g e = 26.7 P I B s 6. C e l l density (per ml) was calculated as the cell density (# PIBs counted)/grid volume. 7. Grid volume was calculated with the following formula= 0.2 x 0.2 x 0.1 m m grid = 4 x 10~ ml. 6  Cell density (per ml) = 26.7 PIBs/ 4 x 10 " ml = 6.7 x 10 PIBs/ml i n a 1/10 solution. 6  6  Therefore, the density i n the stock is: 6.7 x 10 PIBs/ml x 10 = 6.7 x 1 0 PIBs/ml 6  7  119  Appendix 6 Virus dilution procedure 1. Stock 3.8 2. 1/10 3. 1/50 4. 1/100 5. 1/500 6. 1/1000 7. 1/2000 8. 1/5000 9. 1/10000 10. 1/20000 11.1/50000 12. 1/100000 13. 1/200000 14. 1/400000  x 10 200 800 2000 800 2000 1600 800 2000 1600 800 2000 2000 1000  s  ul of ul o f ul o f ul o f ul o f ul o f ul o f ul o f ul o f jxl o f ul o f ul o f ul o f  Initial dilutions  1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.  3.8 x 10 3.8 x 10 7.6 x 10 3.8 x 10 7.6 x 10 3.8 x 10 1.9 x 10 7.6 x 10 3.8 x 1 0 1.9 x 10 7.6 x 10 3.8 x 10 1.9 x 10 0.95 x 10 8  7  6  6  5  5  5  4  4  4  3  3  3  3  PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml  solution solution solution solution solution solution solution solution solution solution solution solution solution  1 2 3 4 5 6 7 8 9 10 11 12 13  in in in in in in in in in in in in in  1.8 3.2 2.0 3.2 2.0 2.4 3.2 2.0 2.4 3.2 2.0 2.0 3.0  ml ml ml ml ml ml ml ml ml ml ml ml ml  of of of of of of of of of of of of of  distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled distilled  water water water water water water water water water water water water water  Final dilutions (after correction by counting) 6.7 6.7 1.3 6.7 1.3 6.7 3.3 1.3 6.7 3.3 1.3 6.7 3.3 1.7  x x x x x x x x x x x x x x  10 10 10 10 10 10 10 10 10 10 10 10 10 10  7  6  6  5  s  4  4  4  3  3  3  2  2  2  PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml PIBs/ml  Doses 5, 7, 11 and 13 were used in susceptibility experiments for T O M F 3 and G L E N colonies. Doses 7, 8, 9,10 and 11 were used in susceptibility experiments GIP colony as well as the cross-resistance experiments.  Appendix 7 Larval growth and pupal weights on different host plants  Experiment 1 Is the larval growth the same on the three crops and does the relationship vary with the background experience of the colony? Three colonies o f cabbage loopers were tested in this experiment: R C (laboratory colony), GIP (originally collected from a Tomato G H ) and G L E N (originally collected from a Pepper G H ) . A l l o f these colonies had been maintained in the laboratory on artificial diet for several generations, however the generation previous to the onset o f the experiment they were reared on tomato plants.  Three crops: tomato  (Lycopersicum esculentum), pepper (Capsicum annuum) and  cucumber (Cucumis sativus) were used to feed the larvae through out the experiment. Plants were grown from seeds in the U B C greenhouse using the same varieties and growing techniques explained i n previous chapters. Leaves were collected from the plants without discriminating any leaf stratum.  Eggs (4 day old) were put to hatch and neonates were collected after 2 days.  Fifteen neonates were placed inside 455 m l paper cups provided with one leaf o f tomato, pepper or cucumber. Twenty-five cups per colony and per crop were prepared. Cups were maintained at 26°C ± 1 °C inside a controlled temperature room. After 3 days larvae were randomly selected and transferred to Styrofoam cups provided with a crop leaf using the  121  hanging leaf technique (See Appendix 8 for hanging leaf rearing technique). Ten cups with 3 larvae per treatment were prepared. The larval weights after 5 days were recorded and results are shown i n Table A 7 . 1 . Data were normally distributed so a factorial analysis was performed with crop and colony as factors. The existence o f interactions among those factors was explored. The average larval weight per cup was taken into account for statistical purposes to avoid pseudoreplication. Crop leaves were replaced every other day and larvae were maintained until pupation.  Results The factorial analysis produced a significant model (F=139.8, p O . 0 0 0 1 , df= 8, 76). Larval weights varied with food plant (F= 549.33, p < 0.0001, df=2) but colonies did not differ significantly (F=T.79, p=0.1735, df=2) and almost no significant interactions occurred between colonies and food plants (F= 2.49, p= 0.051, d f =4). Because colonies did not differ, larval weight data were combined for each o f the crops. The results obtained with the A N O V A and Tukey test are shown i n Table A . 7 . 1 . Overall, after 5 days of feeding on the same crop, cucumber larvae weighed 3.9 times more than tomato larvae and 15.2 times more than pepper larvae. A t day 10, larvae in the cups containing cucumber leaves entered prepupation. Only one piece o f cucumber leaf was insufficient to maintain 3 large larvae and therefore larvae in the cucumber treatment began pupating before they reached their maximum potential. None o f the individuals fed on pepper survived until pupation. O n tomato only 3 larvae pupated for G L E N colony and 2 for GIP colony. N o pupae were collected from R C colony on tomato.  The factorial analysis for pupal weights was significant (F2.9, p=0.025, d f = 5, 36)  122  Differences between male and female pupal weights o f larvae reared on cucumber leaves were detected (F = 9.47, p=0.004, df= 1, 39) and no significant differences were found among colonies (F= 0.34, p= 0.714, df= 2, 38) nor did significant interactions occur between sex and colony (F=0.57, p = 0.5687, df= 2, 38). The mean pupal weight ± S E M of females and males was 170.7 mg ± 5.3 mg and 205.4 mg ± 8.1mg respectively.  Experiment 2  Is artificial diet a better food source than cucumber, tomato and pepper leaves and does previous exposure to the host plant influence this? Larvae o f G L E N , G I P and R C were released on plants o f the three crops and maintained in an old U B C greenhouse facility irrigated manually with water only. After one generation, pupae were collected and caged and offspring were used to set up a new experiment. Pupae o f G L E N colony were collected from cucumber and pepper plants, pupae o f GIP colony were obtained only from tomato plants and pupae o f R C were collected from cucumber and tomato plants.  I tested each colony on artificial diet and also on the crop they had fed on previously. For example: G L E N was tested on cucumber ( G L E N - C - F 1 ) and pepper foliage ( G L E N - P - F 1 ) while R C was tested on cucumber ( R C - C - F 1 ) and tomato ( R C - T - F 1 ) and GIP was tested on tomato (GIP-T-F1). For the artificial diet, 80 larvae per colony were maintained individually i n 30 m l plastic cups with 2.5 m l o f artificial diet. Diet was replaced every 5 days until prepupation. In the case o f crops, 90 larvae were maintained individually in 50 ml plastic cups provided with a piece o f crop leaf until they reached the second instar.  123  After this, larvae were transferred to 170 m l Styrofoam cups provided with a leaf o f crop with the hanging leaf technique. Leaves were collected from the bottom, middle and top portion o f plants i n the case o f tomato, cucumber and pepper plants respectively according to the foraging behaviour reported by growers in a survey conducted at the beginning o f my project. Both types o f cups were kept inside a tray supplied with a damp paper towel on the bottom and a l i d on top o f the tray to prevent leaf desiccation. Leaves were replaced every other day. Larval weight was recorded at day 5. Larvae were checked every other day until pupation. Pupal weight was recorded.  Results The analysis within colony performed with t-test yielded significant differences between artificial diet and each o f the crops (Table A.7.2). Weights o f larvae fed on artificial diet were greater than on pepper and tomato except for GIP T O M F l colony for which the trend was the same but the difference was not significant. The opposite was true for larvae fed cucumber leaves that were significantly heavier than those fed artificial diet.  Differences were found among colonies on artificial diet (F= 14.83, p O . O O O l , df = 4, 343) after A N O V A and Tukey test for multiple comparisons (Table A.7.3). Larvae on artificial diet from R C parents fed tomato were significantly heavier as were larvae fed artificial diet o f G I P parents that had fed on tomato. N o significant differences were detected among the other colonies.  A factorial analysis (F= 71.9, df=7, 440, pO.OOOl) yielded significant differences in pupal weights between sexes ( F  s e x  - 51.4, p< 0.0001, df=T) with higher weights for males  124  than females, also differences among crops ( F p 1 3 9 . 3 , p< 0.0001, df= 3) and no =  cr0  interactions between sex and crop  (F *crop sex  = 2.1, p = 0.102, df = 3) meaning that the  difference i n pupal weight by sex was maintained in all the crops. The mean pupal weights (± S E M ) were 236.1 mg ± 1.8 mg and 255.8 mg ± 2.0 mg for females and males fed artificial diet, 201.6 mg ± 4.1 m g and 236.3 mg ± 4.4 m g for females and males fed tomato, 197.6 mg ± 8 . 0 mg and 224.0 mg ± 6 . 5 mg for females and males fed pepper and 175.4 mg ± 4.4 mg and 192.3 mg ± 3 . 9 mg for females and males fed cucumber.  Pupal weights and pupal success show differences i n food quality. Higher pupal weights are associated with better fitness. These results show that i n spite o f the faster growth o f larvae on cucumber leaves followed by artificial diet, tomato and pepper, pupal weights were higher on artificial diet. Surprisingly pupal weights were also higher on tomato and pepper than on cucumber. However the pupal success was 78% on artificial diet, 45% on cucumber, 24% on tomato and 17% on pepper. This indicates that even when larvae grow more slowly on artificial diet than cucumber this food source ensures the highest pupal weight and survivorship.  Experiment 3: Does experience modify the larval growth of caterpillars previously adapted to each food source? Three colonies collected from cucumber, tomato and pepper greenhouses were maintained on hanging leaves on their parental crops for six, five and six generations respectively. Plants that were grown at the U B C Horticulture greenhouse as explained in  125  other sections o f this thesis were used for this experiment. Leaves were collected from different parts o f the plant according to previously observed behaviour on each crop. In the case o f cucumber, leaves were collected from the middle portion o f the plant, for tomato from the bottom portion o f the plant and for pepper from the top o f the plants. Larvae were kept at 24°C ± 1°C and 16L:8D photoperiod inside a Conviron growth chamber. Thirty larvae were individually maintained in 170 m l Styrofoam cups with the hanging leaf technique for the crop comparison and i n 30 m l plastic cups with 2.5 m l o f artificial diet for the larvae tested on artificial diet. Leaves were replaced every other day and diet was replaced every 5 days. Larval weights were measured at day 12. Pupal success was very poor for tomato and pepper treatments. Only one repetition was done; the pepper colony was lost due to a virus outbreak. The statistical analysis was performed first with a factorial analysis with colonies and crops as the main factors and their interactions. Larval weights were In transformed before the analysis. This greatly improved the fit to the model. Secondly, an A N O V A and a Tukey test were used to look for differences among crops and among colonies.  Results  The factorial anaylis model was significant (F=64.6, df= 11, 170, pO.OOOl). Larval weights were significantly different among crops (F = 207.8, p < 0.0001, df=3) and interactions between crops and colonies were detected (F = 4.8, p = 0.0001, df=6) but no differences were detected among colonies (F = 1.8, p = 0.16, df=2). This means that not all the colonies performed the same way in all the crops. The differences among crops were significant for each o f the colonies. There was a trend for larvae to weigh more on  126  cucumber, followed by artificial diet, tomato and pepper regardless o f the colony (Fig. A.7.1 A , B , C ) . To look for differences due to parental experience in the crops, an A N O V A and Tukey tests were performed on all the colonies for each o f the crops (Fig. A.7.2 A , B , C , D.). N o significant differences were detected among colonies for larvae kept on the cucumber, pepper and tomato crop (Fig. A . 7 . 2 A , A . 7 . 2 B , A . 7 . 2 C ) . However, there was a trend for higher larval weight for larvae coming from the pepper colony on tomato and pepper crops. O n artificial diet (Fig. A.7.2D.), larvae coming from the pepper colony were significantly lighter than larvae coming from cucumber and tomato colonies.  For all the colonies, pupation started at day 14 for larvae maintained on cucumber leaves, 16 days for those on diet, 20 days for those on tomato leaves and 28 days for those on pepper. The pupation success varied with the colony however, on average a 62% o f success was recorded on cucumber leaves, 78% on artificial diet, 17% on tomato and 0% on pepper except for the colony coming from pepper for which the pupal success was 10%.  Pupal Weights Pupal weights obtained from individuals pupated in greenhouses as well as i n the laboratory is given i n Table A.7.4 along with the previous history o f each colony, the food used for maintenance and the differences among generations. Data were analyzed in J M P i n 4.0.4 with the appropriate test for each case.  Pupae collected from greenhouses  During visual monitoring for cabbage loopers in greenhouses, pupae were collected and  127  kept inside a cooler until returned to the laboratory. Pupae were sexed and weighed. A total o f 271 pupae were collected from the tomato greenhouse over 11 visits and 29 pupae from the pepper greenhouse on three different visits. Data were normally distributed so they were analyzed in J M P i n 4.0.4 with a factorial analysis with sex and date as main effect factors and their interactions.  Overall, no differences were detected on pupal weights among collections i n either greenhouse reflecting consistency in the food quality inside greenhouses year round. The pupal weight o f individuals obtained from the tomato greenhouses were heavier than the ones collected from the pepper greenhouse and the sex ratio was approximately 50F:50M in both cases. A s pupal weight is also indicative o f the food quality, these results show that the tomato crop is a better food source than pepper crop for the cabbage looper.  Pupae collected from colonies maintained in the laboratory Pupal weights were recorded from individuals coming from the laboratory colony and also from colonies collected in commercial greenhouses and maintained in the laboratory on artificial diet and/or crops. For all the crops pupal weights obtained when individuals were reared with the hanging leaf technique inside the laboratory were higher than those reared on plants. The fact that individuals were reared individually (without competition) in the cups against 5-6 larvae/plant and that no fertilization was applied to the plants maintained in the old U B C greenhouse where plants were grown might contribute significantly to those differences. However, pupal weights and survivorship rates varied considerably within crops and among colonies and generations with the hanging leaf technique. This might reflect the inability o f loopers to compensate for the lack o f some  128  nutrients or the excess o f others given that one leaf is arbitrarily assigned to each cup and the fact that the crop nutrition was not specifically adapted to their continously changing requirements.  Table A . 7 . 1 . M e a n larval weight and S E M (in mg o f larvae per cup) o f five day old T. ni larvae reared on cucumber (C), tomato (T) and pepper (P) leaves. Different letters indicate significant differences detected among crops with a Tukey test.  ANOVA  Crop  F= 505.4 p< 0.0001  C  12.49 a  0.31  T  3.17  b  0.34  df= 2.82  P  0.82  c  0.05  Mean (mg)  Std Error (mg)  Tukey  q=2.39  129  Table A.7.2. Mean larval weight and S E M of 5 day-old larvae of five different T. ni populations reared on parental crop and artificial diet. Each colony was analyzed separately. Different letters indicate significant differences between food source within each colony.  Colony  ANOVA  Crop  Mean (mg)  Std Error (mg)  RC-TOM-F1  t= 3.53 p=0.0006 df=121  D  6.9 a  0.4  T  4.8 b  0.4  t= 0.56 p=0.57 df=116  D  5.0 a  0.4  T  4.3 a  0.3  t= 15.44 p<0.0001 df=125  D  3.7 b  0.3  C  13.1 a  0.4  t= 7.48 p<0.0001 df=122  D  3.7 a  0.3  P  1.6 b  0.2  t= 22.9 p<0.0001 df=129  D  3.7 a  0.2  GIP-TOM-F1  Differences among crops  RC-C-F1  GLEN-P-F1  GLEN-C-F1  C  14.8  b  0.4  130  Table A.7.3. Mean larval weight o f 5 day-old larvae o f five different T. ni populations reared on artificial diet. Different letters indicate significant differences detected with Tukey test for multiple comparisons.  ANOVA  F= 14.83 p< 0.0001 df=4, 343  Colony  Mean (mg)  Std Error (mg)  RC-TOM-F1  6.9.a  0.4  GIP-TOM-F1  5.0 b  0.4  RC-C-F1  3.7 c  0.3  GLEN-P-F1  3.7 c  0.3  GLEN-C-F1  3.7 c  0.2  Tukey  q=2.74  13  Figure A . 7 . 1 . M e a n larval weights o f 12 day-old T.ni larvae from three different colonies and maintained for the experiment on cucumber (C), tomato (T), pepper (P) leaves or artificial diet (D). Different letters indicate significant differences after Tukey test.  CUCUMBER colony ~ 200 E,  a F=27.02 p< 0.0001 N=55 df=3, 51 q= 2.66  £ 150  1 5 100  I Si 50 c nj <u  cucumber  diet  0  E  tomato  pepper  host  TOMATO colony ~ 200 o>  F=14.07 p< 0.0001 N=68 d£=3,64 q=2.64  E, £ 150 O)  B  1  5 100 n  £ 2 50 c n <u  0  E  cucumber  diet  tomato  pepper  host  PEPPER colony ~. 200 oi  E. £ 150  F=67.1 p< 0.0001 N=58 d£= 3, 55 q=2.65  O)  s  1 0 0  H  n t Si 50  b bc  c  to  CO  E  0  cucumber  diet  tomato  pepper  host  132  Figure A.7.2. Mean larval weights o f 12 day-old T. ni larvae from three different colonies maintained for the experiment on cucumber (C), tomato (T), pepper (P) leaves or artificial diet (D).  Pepper _ 15 O)  ro 10  '53  F=3.06, p= 0.07 df= 2, 24, N=27  Colony m Cuke • Pep •  Tom  n E  133  Figure A.7.2. (Cont.) Mean larval weights o f 12 day-old T. ni larvae from three different colonies maintained for the experiment on cucumber (C), tomato (T), pepper (P) leaves or artificial diet (D). Different letters indicate significant differences after Tukey test.  Tomato 40  n  E ,  SZ  30 -  F=1.12 p< 0.3415 df= 2,25 N=27  ^ Cuke  o >  "55  5  «  20 -  n 10 c n a> E  Colony  • Pep  1-  • Tom  0-  134  T a b l e A.7.4. Mean pupal weights ± S E M o f different T.ni populations maintained under different rearing conditions. Colony  Collected  Crop of Maintenance  T(°C)  RC  Laboratory  Artificial diet  Com. GH  Tomato GH  GLEN  Generation  Pupal Weight (mg)  Sex ratio % Survival  Statistics  Female-  Male  26 +/-1  244.8 ± 5.7  262.5 ± 5.2  46F:54M  Tomato  11 collections year round  243.2 ± 2.2  270.4 ± 3.4  47F:53M  Artificial diet (Pep GH)  Tomato (Plants)  20-35  F1.F2  195.5 ±5.9  217.3 ±6.1  52F:48M  15,10  GIP  Artificial diet (Tom GH)  Tomato (HL)  24  F1  222.8 ±7.1  252.7 ± 7.9  52F:48M  15  t = -2.48, p=0.0276, N=15  GIP  Artificial diet (Tom GH)  Tomato (Plants)  20-35  F1  186.0 ±8.3  214.4 ±7.8  55F:45M  20  t = -2.81. p=0.0261, N=30  Tom  Tomato GH  Tomato (HL)  24  F1.F2  220.0 ±4.9  249.4 ± 5.4  55F:45M  58,27  Pepper  3 collections (Apr, Jun, Jul)  205.2 ± 4.8  222.7 ± 3.7  45F:55M  Com. GH  Pepper  GH  90  Fsex= 30.58, p< 0.0001 Fdate=1.65, p<0.092 Fsex*date= 0.44, p<0.92, N=271  Pepper GH  Pepper (HL)  22  181.2 ±5.2  212.9 ±5.5  50F:50M  86  Pep  Pepper GH  Pepper (HL)  26  162.4 ±5.0  185.1 ±4.0  41F:59M  60  F3.F4  204.7± 5.8  224.3 ± 4.7  54F:46M  29,25  Pep  Pepper GH  Pepper (HL)  24 F5.F6  194.3 ±4.2  205.5 ± 5.5  61F:29M  50,18  F1.F2  221.1 ±4.1  236.6 ± 5.0  65F:35M  50,29  F1  195.5 ±9.8  218.3 ±2.7  57F:43M  34  F2  165.3 ±4.3  204.5 ± 5.6  63F:37M  55  F3  234.6 ± 4.3  252.2 ± 3.9  45F:55M  86  F1  178.3 ±6.2  205.5 ± 5.7  46F:54M  48  Artificial diet (Pep GH)  Cucumber (HL)  Cucumber  Cucumber GH  Cucumber (HL)  Cucumber  Cucumber GH  Cucumber (Plants)  24  24  20-35  Fsex=6.5, p = 0.02 Fgen=1.37, p=0.26 Fgen*sex=0.28, p= 0.60, N=25  Fsex=14.95, p = 0.0002 Fgen=1.14, p=0.287 Fgen*sex=0.16, p= 0.69, N=134 Fsex=7.17, p=0.0129 Fdate=1.44, p= 0.2401 Fsex*date= 0.111, p=0.74, N=29  Pep  GLEN  Fsex= 5.44, p=0.025, N=82  Fsex= 31.91, p=0.0001 Ftemp =23.3, p=0.0001, N=131 Fsex=11.66, p = 0.0008 Fgen=3.58, p=0.015 Fgen*sex=0.36, p= 0.77, N=167 Fsex=5.8 p = 0.02 Fgen=0.84, p=0.36 Fgen*sex=0.05, p= 0.83, N=78 Fsex=13.08, p = 0.0004 Fgen=78.7, p <0.0001 Fgen*sex=2.65, p= 0.07, N=148  t = -3.26, p=0.0021,N=48  HL^Individuals reared with the hanging leaf technique, Plants= Individuals grown on potted plants in old U B C greenhouse.  Appendix 8 Hanging leaf technique Materials: - L 2 instar larvae - 170 m l Styrofoam cups and plastic lids - 60 m l plastic cups - Paper Clips #3 - Plastic Tray with transparent l i d - Damp paper towel Method: 1- Remove the bottom o f the 170 m l Styrofoam cups with the aid o f a cutter.  y  2- Open the paper clips, as it is indicating in the draw, to imitate a hanger shape, f 3- Place the damp paper towel on the bottom o f the tray. 4- Cut the crop leaves and keep them in a high humidity environment until their usage to prevent desiccation. 5- Hang the piece o f or complete leaf by the extreme numbered as " 2 " 6- Pass the hanger with the leaf by the extreme numbered as " 1" through the small hole in the l i d (The orifice comes from the manufacturer). 7- Seal the cup with the l i d , the leaf w i l l be hanging i n the centre o f the Styrofoam container. 8- Introduce one L 2 larvae from the opened bottom o f the cup.  136  1  9- Cover the bottom with a 60 m l cup. Invert the container i n order to keep the cup in its regular vertical position. 10- Put the container inside the tray.  This technique w i l l avoid the contact o f the larvae with the frass while eating. The leaf w i l l remain free o f frass. Feces w i l l be collected in the 60 m l cup at the bottom o f the container. The leaves o f the three crops can be maintained fresh for 2 days or more depending on the rearing temperature.  This system is adequate to rear larvae from second instar on. Neonates are highly susceptible to low humidity conditions, so they could be reared with another technique up to that stage. Neonates are reared, following A l i d a Janmaat's technique, individually in 60 m l plastic cups containing a piece o f leaf. Cups are covered with plastic transparent lids and placed inside the same trays used for the other technique (with the damp paper towel on the bottom). The piece o f leaf is replaced every two days and the paper towel redamp when required.  137  

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