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76- 18,042 SHEPPARD, Roger Floyd, 1945- EXPERIMENTAL STUDIES OF A GRANULOSIS VIRUS IN POPULATIONS OF THE CODLING MOTH, LASPEYRESIA POMONELLA (L.) (LEPIDOPTERA: OLETHREUtlDAE). The Ohio State University, Ph.D., 1976 Entomology

Xerox University Microfilms, Ann Arbor, Michigan 48106

(£_) Copyright by

Roger Floyd Sheppard

19 76 EXPERIMENTAL STUDIES OF A GRANULOSIS VIRUS IN POPULATIONS

OF THE CODLING MOTH, LASPEYRESIA POMONELLA (L.)

(LEPIDOPTERA: OLETHREUTIDAE)

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Roger Floyd Sheppard, B. S., M. S.

* * * * * *

The Ohio State University

19 76

Approved By Reading Committee:

Franklin R. Hall W. Fred Hink ______David J. Horn Adviser Department of Entomology ACKNOWLEDGMENTS

I thank my advisor, Dr. Gordon R. Stairs, for his continual guidance and encouragement during the course of this study. His suggestions and critical review of this manuscript were also invaluable. I am grateful to him for his willingness to put my problems ahead of his own interests. Loving appreciation is extended to my wife,

Sandy, for her patience and understanding during my graduate studies.

I would also like to thank Drs. Franklin R. Hall, W.

Fred Hink, and David J. Horn for serving on my reading committee and for their interest in my career. Special thanks are extended to Dr. Robert P. Holdsworth for trans­ portation to and from the orchard, providing trees for research use, and his stimulating discussions on orchard management and other areas. Many other people contributed directly to the preparation of this manuscript including

Dalene Hoppe who typed portions of the final draft, Sheila

Milligan who provided technical assistance for the scanning electron microscope, Magaret Ellis who operated the trans­ mission electron microscope, and Dwight Lynn who helped x^ith the photographs of laboratory equipment.

ii VITA

July 25, 19 45...... Born - Barberton, Ohio

19G7...... B. S., Ohio University, Athens, Ohio

1968-1971...... Biology teacher, Bellevue Senior High School, Bellevue, Ohio

19 71-19 75...... Teaching Associate, Department of Entomology, The Ohio State University, Columbus, Ohio

19 73...... M. S., The Ohio State University, Columbus, Ohio

PUBLICATIONS

Sheppard, Roger F. 19 75. The bagworm, Thyridopteryx ephemeraeformis (Haworth): A model system "for studying the principles of insect population dynamics. Bulletin of the Entomological Society of America. 21: 15 3-156.

Sheppard, Roger F. and Gordon R. Stairs. 19 76. Factors affecting the survival of larval and pupal stages of the bagworm, Thyridopteryx ephemeraeformis (Haworth). Canadian Entomologist. Tn Press.

FIELDS OF STUDY

Major Field: Entomology

Studies in Insect Ecology. Professor Gordon R. Stairs and Associate Professor David J. Horn

Studies in Insect Pathology. Professors Gordon R. Stairs and John D. Briggs and Associate Professor W. Fred Hink

Studies in Economic Entomology. Associate Professor David J. Horn and Professor Robert P. Holdsworth

i* »n * TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS...... ii

VITA...... iii

LIST OF TABLES...... vi

LIST OF FIGURES...... ix

INTRODUCTION...... 1

REVIEW OF LITERATURE...... 3

I. History of Applied Control of _L. pomonella 3

II, Possible Alternatives for Control...... 4 A. Release of Parasitoids...... 4 B. Dissemination of Pathogens...... 5 C. Autocidal Control...... 7 D. Pheromone Traps...... 7

III, A Granulosis Virus of L. pomonella...... 8

METHODS AND MATERIALS...... 16

I, Codling Moth Rearing Program...... 16

II. Virus Production...... 19

III. Orchard Population Studies...... 21 A. Study Si te ...... 21 B. Study of Population Increases...... 2 3 C. Virus Dissemination...... 25

IV. Susceptibility of Larvae to Virus...... 3 7

RESULTS...... 43

I. Population Increases in the Absence of Insecticides...... 43

iv Page

II. Effects of Virus Dissemination...... 45 A. 19 74 Studies...... 45 B. 1975 Studies...... 62

III. Analysis of Spray Deposits...... 72 A. Spray Coverage...... 72 B. Retention of Virus Activity on Apples in the Orchard...... 72 C. Microscopic Examination...... 78

IV. Susceptibility of Larvae to Virus...... 78 A. First Instar...... 78 B. Fifth Instar...... 90

DISCUSSION...... 109

SUMMARY...... 122

CONCLUSIONS...... 125

BIBLIOGRAPHY...... 12 8

, APPENDIX Laboratory and Orchard Records...... 136

V LIST OF TABLES

Table Page

1. Fate of larvae removed from apples harvested in 19 74 and subsequently reared in the laboratory...... 46

2. Fate of larvae removed from bands in 19 74 and subsequently reared in the laboratory.... 48

3. Correlation of vacated entries in control apples harvested in 19 74 to larvae collected from the bands...... SO

4. Comparison of the increase of second generation larvae and pupae in bands from treated and control trees in 19 7 4 ...... 51

5. Comparison of the increase of successful entries in treated and control trees in 1974...... 54

6 . Fruit drop from trees from August 2 7 to September 4, 19 74...... 5 7

7. Comparison of the increase of infestations in treated and control trees in 1974...... 5 8

8. Estimated mortality of larvae caused by virus in trees sprayed with 108 and 109 capsules... 60

9. Successful entries at harvest in 19 75...... 6 4

10. Effects of virus dosage, spray additives, and number of applications on successful entries in 19 75 (block 2)...... 66

11. Effect of virus dosage on feeding sites in 1975 (block 2)...... 68

12. Fate of larvae removed from apples harvested in 19 75 and subsequently reared in the laboratory...... 70

vi Table Page

13. Measurements and analyses of spray deposits on glass slides in trees during treatment with virus...... 73

14. Results of bioassay of spray deposits of virus on apples sampled at one, three, seven, and twelve days after application...... 75

15. Humber of larvae dying after 2 4 hours in experiment of bioassay of spray deposits 77

16. The LD50 values and 9 5% confidence intervals for first instar larvae treated with virus on artificial food and apples 8 3

17. Mortality of first instar larvae following ingestion of virus on artificial food...... 85

18. Mortality of first instar larvae following ingestion of virus on apples...... 86

19. The LT5Q values and 9 5% confidence intervals for first instar larvae treated with virus on artificial food...... 89

20. Mortality of fifth instar larvae (12-days old) following ingestion of virus on artificial food 9 2

21. The LT5Q and LD50 values with 95% confidence intervals for fifth instar larvae (12-days old) treated with virus on artificial food... 9 5

22. Mortality of different ages of fifth instar larvae following ingestion of virus with artificial food 9 8

23. Mortality of different ages of fifth instar larvae following per os inoculation of virus...... 99

24. The I-.T5Q values with 9 5% confidence intervals for different ages of fifth instar larvae treated with virus on artificial food 10 4

vii Table Page

25. The LT^q values with 95% confidence intervals for different ages of fifth instar larvae inoculated per os with different doses of virus ...... 10 5

26. Pupal development and adult emergence of different ages of fifth instar larvae surviving after consuming virus with artificial food...... 107

27. Pupal development and adult emergence of different ages of fifth instar larvae surviving per os inoculations of virus.... 10 8

28. Data from disseminations of virus in apple orchards by various researchers...... 110

29. Record of rearing program from January, 19 74 to December, 19 75...... 137

30. Condition of apples from trees sprayed with 10' capsules and control trees in 19 74.... 138

31. Condition of apples from trees sprayed with 108 capsules and control trees in 19 74.... 139

32. Condition of apples from trees sprayed with 109 capsules and control trees in 19 74.... 140

33. Number of larvae and pupae recovered from burlap bands on trees in 19 74...... 141

34. Number of unsuccessful and successful entries observed in 100 randomly selected apples from trees in block 2 during 19 75...... 142

viii LIST OF FIGURES

Figure Page

1. Plastic food cups and oviposition container used in the rearing program...... 17

2. Depression plate used in infection of larvae for mass propagation of virus...... 20

3. Map of sections A (rows A through F) and B of Overlook Orchard showing the experimental design used in 19 75 ...... 22

4. Map of section C of Overlook Orchard showing the experimental design used in 19 74 ...... 2 4

5. Application of virus with Solc@ Junior 410 mistblower...... 2 7

6 . Female-baited trap used to monitor flight periods of adults in 19 75...... 31

7. Apples with gelatin capsules used to contain larvae in specific areas for bioassay of activity in spray deposits...... 34

8 . Glass slide fastened in a tree with a clothes pin to monitor a spray application...... 36

9. A. Material used in per os inoculations of fifth instar larvae. B. Enlargement of a portion of A showing the glass needle mounted on the rubber cork at the end of a one ml syringe...... 41

10. Proportion of apples infested from 19 7 3 to 1975 (section B) ...... 44

11. Granulosis mortality of larvae removed from apples harvested in 19 74 and subsequently reared in the laboratory...... 47

ix Figure Page

12. Increase of second generation larvae and pupae captured in bands during 19 74...... 52

13. Increase of successful entries during 1974..... 53

14. Increase of fruit loss from trees during 1974...... 56

15. Codling moth feeding in apples harvested in 19 7 4 ...... 61

16. Comparison of adult flight periods to subsequent infestations in sections A and B during 19 75 ...... 63

17. Increase of successful entries in 1975 (block 2)...... 67

18. Codling moth feeding in apples harvested in 1975 (block 2) ...... 71

19. Relative activity of virus in the orchard environment based on a bioassay of spray deposits on apples using neonate, first instar larvae...... 76

20. Scanning electron micrograph of capsules on a glass cover slip ...... 79

21. Scanning electron micrograph of capsules (arrows) on the surface of an apple ...... 80

22. Scanning electron micrograph of capsules (arrows), skim milk (SM), and charcoal particles (C) on a glass cover slip...... 81

23. Dosage-mortality response of larvae following ingestion of virus on (A) artificial food, (B) apples with no additives, and (C) apples with additives 84

2 4. Time-mortality response of neonate, first instar larvae to various doses of virus ingested with artificial food...... 88

x Figure Page

25. Dosage-mortality response of (A) neonate, first instar and (B) fifth instar (12-days old) larvae to virus on artificial food...... 91

26. Time-mortality response of fifth instar larvae (12-days old) to different doses of virus ingested with artificial food...... 94

27. Granulosis mortality of different ages of fifth instar larvae following (A) per os inoculations of virus and (B) ingestion of virus with artificial food...... 9 7

2 8 . Time-mortality response of different ages of fifth instar larvae to different doses of virus ingested with artificial food...... 102

29. Time-mortality response of different ages of fifth instar larvae to different doses of virus administered through per os inoculations...... , 10 3

xi INTRODUCTION

The probable origin of Laspeyresia pomonella (L.) is in the area covering Europe south of the taiga zone, the Cauca­ sus, southwest Asia, and the mountains of Soviet Central

Asia (Shel1deshova, 1967). From there it has spread to nearly every place where its primary hosts, plants of the genus Malus (apples), are native or have been transported.

This includes all of Europe and the United States and por­ tions of Canada, South America, Australia, Asia, the Soviet

Union, and New Zealand. Some of the secondary hosts are apricots, quinces, peaches, walnuts, plums, and almonds

(Shel*deshova, 1967).

In Ohio, the primary host of L. pomonella is apples.

Larvae develop in fruit from May through October and eventually pupate or enter diapause under bark scales or in various sites on the ground (Wearing, 1975; Wearing and

Skilling, 1975). There are two complete generations a year and possibly a partial third generation (Cutright, 196 4).

In 196 3, a granulosis virus was isolated from codling moth larvae collected from apple and pear trees in Mexico.

Since 196 3 it has been diagnosed in laboratory cultures of

L. pomonella in the United States, Canada, Europe, and the Soviet Union (Falcon, 19 71). Laboratory tests have shown the virus to be very pathogenic for larvae, causing death

in 5 to 12 days after ingestion (Tanada, 196^0, Consider­

able interest has been shown in using the virus against I j . pomonella in orchards as an alternative to regular appli­ cations of chemical insecticides. Disseminations of virus in apple orchards in California, Ohio, Switzerland, and New

Zealand have been moderately successful in reducing populations and feeding damage (Bode, 1970; Falcon et al.,

196 8 ; Keller, 19 73; Wearing, 19751).

In the present study, relatively low concentrations of virus were disseminated in an Ohio orchard to determine the effects of low dosages on populations. Charcoal and skim milk were added to some of the spray suspensions to evaluate their ability to protect and spread the virus. The optimum time to apply virus during the growing season was also determined. In addition, the susceptibility of different larval instars was tested in the laboratory.

C. H. Wearing. 1975. Personal communication. Department of Scientific and Industrial Research Entomology Division Mt. Albert Research Center Auckland, New Zealand REVIEW OF LITERATURE

I. History of Applied Control of L. pomonella

In the nineteeth century most apple production was in small farm orchards. Chemical control was unknown so cultural and mechanical control were practiced. For example, dropped fruit were picked up and fed to hogs or boiled to destroy larvae and twisted bands of hay were wrapped around trees to catch larvae as they pupated or entered diapause

(Cutright, 1964).

Paris green and london purple were among the first chemicals used in the 1800's. Lead arsenate replaced all chemicals used for control of the codling moth at the turn of the century (Cutright, 1964). For 30 or 40 years lead arsenate was sprayed against L. pomonella until resistance appeared among different populations (Cutright, 1964). DDT was introduced in the mid-1940's and was effective against populations. However, two problems occured later with the use of DDT. Resistance to DDT developed as it did for lead arsenate and populations of red-banded leafroller, Argyro- taenia velutinana (Walker); plum curculio, Conotrachelus nenuphar (Herbst); eye-spotted bud moth, Spilonota ocellana

(Denis and Schiffermiiller) ; apple aphid, Aphis pomi De Geer; and European red mite, Panonychus ulmi (Koch) increased to damaging levels (Cutright, 196 4; Hoyt and Burts, 19 74; Lord,

19 49; Madsen and Morgan, 19 70; Oatman, 1966). Apparently, their natural predators and parasitoids were suppressed by applications of DDT (Oatman, 1966). Other chemicals such as ryania, parathion, carbaryl, and azinphosmethyl have been used with success but the possibility of resistance develop­ ing is of major concern (Hoyt and Burts, 19 74).

Modified spray programs were initiated as part of pest management systems in an attempt to alleviate some of the problems occuring with heavy insecticide use (Bastiste et al. , 1970 ; Collyer and vanGeldermalsen, 1975 ; Hamilton and

Cleveland, 195 7; Holdsworth, 19 70; Hoyt, 1969; MacLellan,

1963; Oatman, 1966; Thomas et al., 1959). Insecticides that are not toxic to beneficial arthropods and/or minimal amounts of chemicals were sprayed in the programs. Moderate to excellent success in control in the various programs was experienced. The most successful were in areas where Ij. pomonella is univoltine (Collyer and vanGeldermalsen, 19 75;

MacLellan, 196 3; MacPhee and Sanford, 1961).

II. Possible Alternatives for Control

A. Release of Parasitoids

The release of parasitoids against the codling moth in

North America have not been successful nor have native parasitoids reduced populations effectively (LeRoux, 1971), Putnam (196 3) suggested that the reason they were not

successful in Canada was because of a lack of effective

biotic controls in the native region of the codling moth

where infestations are relatively high.

The only native organisms that have been effective are

predators which includes ants (Formica fusca subsericia Say,

Solenopsis molesta (Say), Monomorium minimum (Buckley),

Aphaenogaster fulva aquia (Buckley), and Tetramorium caes-

pitum (L.)) and woodpeckers (Dendrocopos villosus villosus

(L.) and Dendrocops pubescens medianus (Swainson)) (Jaynes

and Marucci, 19 47; MacLellan, 195 8 , 1959). Ants were

observed feeding on larvae and pupae in bands in West

Virginia orchards in 193 8 , 19 39, and 19 40. Ninety percent

of larvae released on the ground in tests were killed by

ants (Jaynes and Marucci, 1947). MacLellan (1958, 1959)

observed woodpeckers feeding on overwintering larvae on

tree trunks in Nova Scotia. Woodpeckers killed an average

of 52 percent of overwintering larvae from 1950 to 1956.

B . Dissemination of Pathogens

Pathogenic fungi, nematodes, and bacteria have been isolated from and disseminated in codling moth populations.

The most complete study on fungi of larvae was conducted by Arkhipova (1965). He tested several different fungi against the codling moth and found that Beauveria glob- ulifera Pic., Metarrhizum anisopliae (Metsch.), and Beauveria bassiana (Bals.) were the most pathogenic.

The use of DDT with the fungi increased larval mortality

(Arkhipova, 1965; Gaprindashvili and Novitskaya, 1967).

It was suggested that fungi be integrated with chemical

insecticides to suppress larval populations with the reser­ vation that it is only effective when relative humidity is

high (Gaprindashvili and Novitskaya, 1967).

A nematode (Rhabditida; Steinernematidae) was isolated

from larvae in a Virginia orchard and called DD-136 (Dutky

and Hough, 1955). It was later classfied as a strain of

Neoplectana carpocapsae Weiser (Poinar, 1967). It is a

vector of the bacterium, Achromobacter nematophilus, which

kills the larvae (Dutky, 1959; Poinar and Thomas, 1965).

Nematodes suspended in water and sprayed on trees killed 60

percent of larvae seeking cocooning sites (Dutky, 1959).

The major problem associated with the use of this nematode

against L. pomonella is the rapid desiccation of the

organisms in anything other than moist conditions (Poinar,

1971).

Strains of the bacteria Bacillus cereus Frankland and

Frankland and Bacillus thuringiensis Berlinger were isolated

from and tested against codling moth larvae. Applications

of 13. cereus did not protect apples from damage (Philips

et al., 195 3; Stephens, 1957). Dissemination of B.

thuringiensis reduced damage to apples but not to an

acceptable level (Dolphin et al., 1967; Jaques, 1961; 7

McEwen et al. , 1960; Oatman, 1965; Roehrich, 19 6 4). How­ ever, there is some promise in using B. thuringiensis with chemical insecticides to protect apples (Dolphin et al.,

1967).

C. Autocidal Control

The possibility of using sterile moth releases as a means of suppressing codling moth populations was investi­ gated (Butt et al., 1970; Proverbs et al., 1966, 1969).

The success of sterile moth releases depends on the over­ flooding of native populations with sexually sterile males that mate with females resulting in the production of infertile eggs (Proverbs, 1964). Populations of egg para­ sitoids (Trichogramma spp.) may also be more effective because of the increase in egg production from more matings

(Nagy, 1973). Chemicals are still necessary to reduce codling moth populations to a level where sterile male releases are successful (Proverbs, 19 6 4). The major disadvantage of this control technique is the high cost of rearing, sterilization, and release of males (Hoyt and

Burts, 19 74).

D. Pheromone Traps

In the past ten years some attention has been directed towards using traps baited with live females or female sex pheromones to attract males and reduce populations.

Proverbs et al. (1966) used live virgin females in a trap to monitor males in a sterile moth release program. Butt and Hathaway (19 66) found that extracts from the abdominal tips of virgin females attracted male moths. Later,

McDonough et al. (19 69) isolated the pheromone and Roelofs et al. (19 71) characterized it as trans-8 , trans-10- dodecadien-l-ol. Studies using pheromone traps to suppress populations have not reduced fruit damage to acceptable levels (Proverbs et al., 1975; Willson and Trammel, 1975^).

However, the capture of male moths by traps baited with live virgin females or synthetic pheromone has proven useful in monitoring populations and predicting infestation levels

(Bastiste, 19 72; Bethell et a l . , 19 72; Madsen and Davis,

1971; Madsen and Vakenti, 1972, 1973; Madsen et al., 1974;

Riedl and Croft, 1974).

XII. A Granulosis Virus of L. pomonella

A granulosis virus of L. pomonella is typical of all granulosis viruses (genus Baculovirus) in that the rod­ shaped, DNA, virus particle is occluded within a capsule of crystalline protein (Stairs et al., 1966). The capsules of the granulosis virus of JL. pomonella are about 300 mp by 400 mp in dimensions although some are cuboidal and much larger measuring up to 5 pm (Stairs et al., 1966; Barefield

^H. R. Willson and K. Trammel. 19 75. Factors limiting control of tortricid (Lepidoptera: Tortricidae)apple pests by pheromone-baited traps. Paper presented at annual meeting of E.S.A. Nov. 30 - Dec. 4, 1975. New Orleans. and Stairs, 1970). The rods are SO mu wide and 300 mp long

(Tanada, 196 4).

In the laboratory, the virus causes death in five to twelve days after ingestion of capsules (Tanada, 1964).

Larvae of the oriental fruit moth, Grapholitha molesta

(Busck) , a close relative of Ij. pomonella, are also susceptible to the virus (Falcon et al., 1968). As the disease progresses larvae become sluggish and paler in color. At death, larvae are flaccid and eventually disin­ tegrate .

Virus rods and capsules were found in the fat body, hypodermis, tracheal matrix, and Malpighian tubules in a study of the histopathology of the disease (Tanada and

Leutenegger, 1968). Tests were performed to determine if virus was excreted from the Malpighian tubules but the results were inconclusive.

Centrifugation techniques were used to study the infectious components of the virus (Barefield and Stairs,

19 70). An infectious form was found that was smaller than virus rods. The structure of this component could not be determined, however, it contained all the genetic informa­ tion necessary to cause granulosis in first instar larvae.

Studies were conducted on nucleic acid metabolism in infected larvae using histochemical and autoradiographic techniques (Benz and Wager, 1971; Wager and Benz, 1971).

Results of the autoradiographic studies indicated that the 10

development of the virus begins in the nucleus and con­

tinues in a restricted area of the cell after rupture of

the nuclear membrane. Rates of DNA and RNA synthesis at

various times during infection were also determined.

The time sequence of morphological and histochemical

changes were estimated from the histochemical studies

(Wager and Benz, 19 71). Ten different pathogenic stages

were defined. The major stages include first cytopatho-

logical changes at 18 to 24 hours after infection during

the second stage, appearance of virogenic stroma at stage

3, disappearance of nuclear membrane during stage 4, and

the first appearance of mature granulosis capsules 66 to

72 hours after infection during stage 8.

Resvatova (19 72) and Keller (19 73) studied the effects

of temperature on the incidence of granulosis in larvae.

Resvatova found that, with a concentration of 9.8X108 capsules/ml, 8 3.9 percent of first and second instar larvae died from granulosis at 16 °C, whereas, 41.9 percent died at 2 8 °C. The opposite effect occured with fifth instar larvae for 3.2 percent developed granulosis at 16 °C and

15.4 percent at 28 °C. Keller reported no significant difference in mortality for first instar larvae at these temperature extremes using concentrations of 7.7X104,

7.7X108 , and 7.7X108 capsules/ml. However, at the lowest concentration significantly less larvae developed granu­ losis at 34 °C than at 30 °C with an incidence of 22.4 11 percent and 5 3.2 percent, respectively.

The pathogenicity and incubation period of the virus

in first instar larvae has been studied (Keller, 1973).

First instar larvae were fed different concentrations of

virus sprayed on the surface of apples. Some of the suspen­

sions used in the experiment contained a wetting agent

(Etalfix). The LDcn and LD__ values for first instar larvae ou 9 0 were about 30 and 220 capsules, respectively with the

wetting agent. Corresponding LT^q values with an LD^q and

an LDgg were 6.5 and 4.5 days, respectively. There was no

significant difference in the LD^g values for suspensions

without a wetting agent, however, the LDgg value of 1,800

capsules was much higher.

Transstadial transmission of the virus has been demon­

strated (Etzel and Falcon, 1976). Fifth instar larvae were

inoculated per os with intact capsules and some of the

pupae were infected with granulosis. Transmission from

the pupal to the adult stage occured only after virus rods were injected into the hemocoel of the pupa. Tests were

conducted to demonstrate transovum transmission of virus but the results were inconclusive.

Several people have tested the virus in the field as

a possible control agent for L.. pomonella. The virus was

evaluated as a control measure in a California orchard

from 1966 to 1968 (Falcon, 1971; Falcon et al., 1968),

Virus suspensions containing 0.0 3 percent of Triton‘S(K) 12

X-100 wetting agent were sprayed on trees with a handgun

from a portable orchard sprayer at a rate of four to five

gallons/tree. The concentrations used in 1966, 1967, and

1968 were 3.4X10-^, 1.2X10^^-, and 9.5X10^ capsules/gallon,

respectively. Five applications were sprayed in 1966,

seven in 196 7, and twelve in 196 8. The total number of

capsules received by each tree in 1966, 196 7, and 196 8

were 8.5X101 2 , 4.2X10-1-2, and 5.7X10-^, respectively. The

best results were obtained in 1968 when a vacated entry or

larva were found in only 2.3 percent of the harvested

apples compared to 45 to 70 percent of apples on untreated

trees. Ten percent of the apples were damaged in 1966 and

five percent in 196 7. Thus, the best results were obtained with more frequent applications even though fewer capsules were used.

There was no evidence that virus became established as a natural mortality factor in populations. However,

virus diseased larvae were found in apples from trees bordering trees sprayed with virus in 1966. Diseased

larvae were also found in bands around the border trees as well as in control trees located seven rows north of the treated trees. In 196 7, diseased larvae were found in some of the fruit from border trees and trees sprayed with virus in 1966.

Similar studies were conducted in Ohio (Bode, 1970).

Two apple trees were sprayed three times in 19 6 7 with an 13 aqueous suspension of virus at the rate of 24 gallons/tree with an air blast sprayer. The concentration used in the applications on August 3, August 15, and August 24, were

1.33X1010, 1. 33X10-^, and 1019 capsules/gallon, respectively.

Therefore, each tree was sprayed with a total of 1 .7X10-^ capsules for the season. The virus did not reduce feeding on apples but killed 20 percent of the larvae after entry and another 7 3 percent after emergence from apples. The virus apparently did not spread and increase among the population.

The virus was tested in New Zealand as part of an integrated control program in apple orchards CC. H. Wearing, personal communication). An aqueous suspension of virus at a concentration of about 7 . 6 X 1 0 capsules/gallon was applied nine times using a Turbomist machine from 19 70 to

1971. In 1974 to 1975, trees were sprayed six times by a

Konig air blast sprayer set for dilute spraying at a concen­ tration of 7.6X109 capsules/gallon. A proprietary ultra­ violet light-screen material (IMC) was added to the spray at a concentration of 50 grams/100 liters.

More larval mortality occured in the virus treated trees than in the control trees in both experiments. The virus failed to protect fruit from infestations (41 percent), however, larval mortality in the treated trees was high.

In 19 70 to 19 71, 85 percent of the larvae died before emerging from apples on treated trees as compared to 23.4 m

percent in control trees, and in 19 74 to 1975 there was

9 4.7 percent mortality in apples on treated trees and 32.1

percent in the controls. Thus, with fewer applications and

a lower concentration, mortality was higher in 19 74 to 19 75

suggesting that the IMC additive enhanced the properties

of the spray in some manner. There was some evidence of

spread of virus in the orchard since infected larvae were

found in apples from control trees in 19 75.

Virus was disseminated in apple orchards in Switzerland

(Keller, 19 73). Trees were sprayed with suspensions of

virus containing one percent powdered milk using an air

blast sprayer. Each of 254 trees received 9.5X10-*-^ capsules

(about two larval equivalents = two L.E.) per application.

Trees were treated four times at intervals of two weeks so

each tree received a total of 3.8X10*^'L capsules for the

season. Deep entries in harvested apples were reduced by

95 to 99 percent and 80 percent of the larvae died without

causing damage.

The effects of sunlight, temperature, and rainfall on

virus activity were also determined (Keller, 1973), Trees were sprayed in two areas with 1 L.E., 10 L.E., and 100

L.E. of virus. Apples were sampled at 1, 3, 7, 15, 31, and 6 3 days after treatment and assayed with first instar

larvae for retention of virus activity in spray deposits.

By 15 days after treatment more than half of the virus was

inactivated at each dosage and nearly all of it was lost 15 after 6 3 days. Experiments showed that 9 0 percent of the virus inactivation was caused by sunlight and the other 10 percent by direct or indirect effects of temperature.

Rainfall had no significant effect on loss of virus activity in spray deposits.

Various spray additives were tested for protection of virus from inactivation (Keller, 1973). The materials and concentrations used were as follows: powdered skim milk,

1.0 percent; Leucophor BCF, 0.2 percent; and PVA-Binder,

1.0 percent. The additives were mixed with virus suspen­ sions and sprayed on trees at the rate of one larval equivalent/tree. Apples were sampled at 2, 6 , 14, and 30 days after treatment and assayed with first instar larvae for retention of virus activity in spray deposits. The results showed that powdered skim milk was the best of the three additives. Fifty percent of virus from spray deposits containing skim milk remained active 13 days compared to 4, 5, and 5 days for spray deposits containing no additives, Leuchphor BCF, and PVA-Binder, respectively. METHODS AND MATERIALS

I. Codling Moth Rearing Program

A continuous culture of the codling moth was started in January, 1974. Eggs were obtained from Dr. W. M. Bode,

Pennsylvania State University Laboratory in Biglerville.

The eggs were placed in six ounce paper cups and covered with plastic lids. They were incubated at 2 9 to 30 °C and

18 hours photophase until larvae hatched four to five days later.

Neonate larvae were removed with a number one artist's brush and placed in individual three-quarter ounce plastic cups (Fig. 1) filled one-third full with an artificial diet.

The following ingredients were used in the diet:

Ingredient Amount

baby lima beans 450 g sucrose 90 g apple seeds 60 g agar 30 g methyl-p-hydroxybenzoate 9 g ascorbic acid 9 g raw linseed oil 20 ml formaldehyde (37% solution) 3 ml water 2 ,300 ml

(Bode, 1970). The diet was prepared by first soaking lima beans for 18 to 2 4 hours in 1,400 ml of water. All of the

16 17

Figure 1. Plastic food cups and oviposition container used in the rearing program. 18 ingredients except the agar were poured into a gallon

container and mixed in a Waring® blender for five minutes.

The agar was added to 900 ml of boiling water. This solu­

tion was added to the rest of the ingredients and blended

for two minutes. The diet was dispensed into the cups

with a plastic squeeze bottle. The cups of food were left

to cool for at least 30 minutes before they were used or

stored in a refrigerator. The amounts used in the recipe yielded between 700 and 800 cups of food.

The larval, pupal, and adult stages were reared in the

food cups at a temperature of 29 to 30 °C and 18 hours photophase. Each generation was about 30 days in length.

Egg, larval, and pupal development were completed in 5, 16, and 9 days, respectively.

The food cups were checked daily for newly emerged adults which were placed into plastic containers (Fig. 1) lined with fluted wax paper for egg deposition. Twenty- five adults were placed in each of the circular containers

(7.6 cm high and 2 0.3 cm in diameter) and kept at 29 to

30 °C and 18 hours photophase. After oviposition was completed the adults were removed and the eggs returned to the incubator. Neonate larvae were placed in the food cups within 2 4 hours after hatching.

The culture was continously reared for 18 generations until September, 19 75 when fecundity became very low. New eggs were obtained in June, 19 7 5 from Biglerville, Pennsylvania and reared for five generations.

II. Virus Production

Granulosis virus was obtained from Dr. J. F. Howell,

Yakima Agricultural Research Laboratory; Yakima, Washing­ ton. Virus was mass produced in the laboratory by feeding fifth instar larvae virus on food. Larvae were removed from the food cups when they were from 12 to 15-days old and placed in a 4 cc cavity of a plastic depression plate filled one-third full with artificial food (Fig. 2). The food was coated with a suspension of virus. The virus suspension was prepared by macerating two infected, fifth instar larvae in a sintered glass tissue grinder and mixing the contents with ten ml of distilled water. A drop of the suspension was deposited on the food using a Pasteur pipette. After larvae were placed on the food the entire plate was covered with Parafiln®, Later, acetate sheeting was sealed on the plates with a hot iron because many of the larvae had escaped through the Parafiln®. The plates with larvae were kept at room temperature (22 °C) for six days and then stored at -75 °C. Later, when the infected larvae were used in the preparation of virus suspensions they were removed from the food while still frozen. 20

Figure 2. Depression plate used in infection of larvae for mass propagation of virus. Note the openings in the ParafilnE) where larvae have escap ed. 21

III. Orchard Population Studies

A, Study Site

Experiments with L. pomonella were conducted from 19 73

to 19 75 in Overlook Orchard, a University Experimental Farm,

located near Carroll, Ohio. The trees used in the studies were in various sections of the orchard. Section A contained 16 rows of semi-dwarf trees planted as two-year olds in 1971 (Fig. 3). Each row consisted of 25 to 32 trees of one of the following varietiesj Red Delicious,

Golden Delicious, or Jonathan. The different varieties were planted in alternating rows. The rootstock for all of the trees was Malling-Merton 106.

The trees were not sprayed until 19 7 3 when they received Cypre?^ in pre-bloom and Captan® in all post­ bloom treatments. In 19 74 the trees received the same fungicide treatments. At the half-inch green stage all of trees were sprayed with dormant oil and Systo The majority received Imidar^ in the petal fall and remaining seven cover sprays. The rest were sprayed with other insecticides or not at all in experiments conducted by Dr.

R. P. Holdsworth of the Department of Entomology at Ohio

State University. Cypre^^ and Captan® were used again in

19 75 on all of the trees. All of the Red Delicious and

Golden Delicious trees except for those in rows B and E received Phosphamidon® at the tight cluster stage and

Imidan® for the petal fall and six cover sprays. The 22

28© ® © 27© ® © c (20 m) 2 6 0 ® ©

2 5 © ® © © ® ®

2 4 © ® © © ® © IV 23© & © ® © IX VII 22© © © ® © B 21© © © © VI 20© '® © © ® IX 19© 8 0 © ® IV II 18© ® © ® II VIII 17© ® © © ® VIII 16© ® © © 4 © VII III 15© ® © © ® © A 14© ® © © ® © 13© ® 0 0 ® ©

12© & 0 © l£j) © VIII 11© ® ® © u ® 1 ® 0 10© ® © ®-Golden Delicious IX VI 9© ® © © ® ©-Red Delicious I 8© ® © ® © -Jonathan III VII 7© ® © © ® ■^11,111,1 V—IO applications VI V ,V I,V II,V I1 1—14 applications 6® ® © © IV IV I,V-107 capsules 5® © ® ® © ® II,VI-107capsules with skim V 4® ® © © © milk and charcoal VII V 3® ® ® © ® © III,VII-109 capsules

2 © ® 0 © IV,VIII-109capsules with skim milk and charcoal 1® ® 0 © IX -C ontrol FED C Scale-lcm=6m

Figure 3. Hap of sections A (rows A through F) and B of Overlook Orchard showing the experimental design used in 1975. 23

same insecticides were applied to the Jonathan trees with

the addition of Karathane®.

The trees in section B (Fig. 3) were standard sized

trees planted 35 years ago. They were regularly sprayed with fungicides and insecticides until 19 73 when no insec-

ticides and only the fungicide

Section C (Fig. 4) of the orchard was located about

20 meters north of section B and consisted of 106 Melrose and 14 Golden Delicious semi-dwarf trees. The Melrose trees were a hybrid from Red Delicious and Jonathan varieties developed at the Ohio Agricultural Research and Development

Center in Wooster, Ohio. The trees were planted in 19 6 7 and some in the western portion of the orchard were replanted further east in 196 8 . Insecticides and fungi­ cides were regularly applied through 19 73. In 19 74 no pesticides were sprayed and in 19 75 the trees were treated with the fungicide Cypres^.

B . Study of Population Increases

From 19 7 3 to 19 75 populations in section B were moni­ tored to determine increases in the absence of insecticides.

During each growing season the fruit on the trees with good yield were examined for codling moth feeding. One hundred fruit were randomly chosen and examined on a tree about once every two weeks. Feeding was classified as a success­ ful (larval tunnel) or unsuccessful entry (sting). ~4cT 4c ® 4b ® 4a 1b ® 3b 2a ® 3a ® ® ® la ®

3f

2f ® C 2e ® If ® 1e ® Id 3d ®

1c ® 2d •-M elrose ®-Golden Delicious a-10 capsules b-Control tree for a c-10 capsules 4 d-Control tree fore e-109capsules f-Control tree for e Scale-lcm =6m

Figure Map of section C of Overlook Orchard showing the experimental design used in 1974. 25

C. Virus Dissemination

Three different concentrations of virus were sprayed on selected trees in section C to determine the effects of relatively low concentrations of virus on the second gener­ ation in 19 74. Because there was so much variability in the size of the trees and fruit set, it was decided to conduct three separate experiments with three different concentrations of virus. The following concentrations were r e rj used: 10 , 10 , and 10 7 capsules/ml. Four trees were sprayed with each concentration of virus and a treated tree was matched with an untreated one of similar size and fruit set (Fig 4). The four pairs of trees used in an experiment were chosen by assigning every pair of trees a number from a random number table and allocating four successive, numbered pairs to each of the experiments.

The spray solutions were prepared from infected, fifth instar larvae kept at -75 °C for at least one year in plastic depression plates (Fig. 2). From 35 to 75 larvae were removed from the plates while still frozen and dropped into a 50 ml test tube. Five ml of distilled water were added and the larvae were crushed with a glass rod. The contents minus cuticular material were siphoned from the tube with a Pasteur pipette and placed into two 15 ml centrifuge tubes. The suspension was spun in a Sorvall

RC2-B automatic, refrigerated centrifuge at 5,000 g for thirty minutes using a SS34 angle head rotor. The 26

supernatant was discarded and the pellet resuspended in

three ml of distilled water. Half of the suspension was

layered on each of two 20-60 (w/w) stepwise sucrose gra­

dients in 15 ml centrifuge tubes and spun at 5,000 g for

t+5 minutes in a HB-4 swinging bucket rotor. The layer

containing the granulosis virus was removed with a Pasteur

pipette and suspended in five ml of distilled water. The

suspension was spun at 5,000 g for 30 minutes using the

angle head rotor. The supernatant was discarded and the

pellet resuspended. This procedure was repeated three

times to remove the sucrose. Following the washing steps

the suspension was sonicated for one minute with a Lab-

Line Ultratip Labsonic System. Stock suspensions were

standardized with an American Optical phase-contrast micro­

scope (Phasestar model) and a Petroff-Hauser bacterial

counting chamber. The capsules in each of 20 squares were

counted from two samples and averaged. The yield of virus 9 in prepared by these methods ranged from 1.7X10 to 5.5X10XU capsules/larva. Stock suspensions were stored at 4 °C for

later use.

A Solo® Junior 410 mistblower (Fig. 5) was used to apply the aqueous suspensions of virus at a rate of 0.5

liters/minute. Ten applications of each virus concentration were made at biweekly intervals from July 29 to August 31 in the early evening hours between 7:00 and 9:00 P.M. One hundred ml of liquid were applied from a position about two Application of virus with Solc@ Junior 410 mistblower. 28 meters from the tree on the down wind side with the spray directed towards the apples (Fig. 5). The different dosages 7 8 of virus sprayed with 100 ml of liquid/tree were 10 , 10 , and 10^ capsules. During spray applications the control trees were avoided as much as possible.

The effect of dosage on larval populations was assessed by a number of methods. All of the apples were examined for feeding sites once a week from July 21 to September 17 when the apples were harvested. In the laboratory the harvested apples were examined for feeding sites. Apples with successful entries were dissected. Larvae found in the apples were placed in food cups and reared at 29 to

30 °C and 18 hours photophase until they died or developed to the adult stage. Tissue smears from dead larvae were examined with a phase-contrast microscope. Granulosis was detected by the presence of many capsules which appear as small, ovoid structures.

Six inch wide burlap bands were placed around the trunk of each tree on July 9 to serve as cocooning sites for mature larvae from which the relative size of the population was estimated. The bands were examined regularly for larvae and pupae which were removed on September 2 7. Larvae were reared in food cups kept at 29 to 30 °C and 18 hours photo­ phase until they died or developed to the adult stage.

Tissue smears from dead larvae were examined with a phase- contrast microscope. 29

The trees in section C were not sprayed with virus in

19 75. Larvae in apples and bands were sampled periodically in 19 75 to determine if the virus had persisted since 19 74.

To determine the effects of frequent applications of low virus dosages on populations, two relatively low concentrations of virus were disseminated in section A during 19 75. Powdered skim milk and powdered, activated charcoal were added to some of the formulations to deter­ mine their ability to protect capsules from inactivation.

The best time to spray virus during the season was also investigated.

In 19 75, rows B and E were treated with virus. The experimental design was a randomized complete block with four replications of each treatment (Fig. 3), The two C concentrations of virus used in the experiment were 10 n and 10 capsules/ml. Powdered charcoal (1% w/v) and skim milk (1% w/v) were added to some of the formulations. In each block there were eight different treatments and an untreated control. Four of the trees received 10 applica­ tions of virus and the other four received 14. Of the four trees receiving either 10 or 14 applications, one was n sprayed with a total of 10 capsules plus additives, one 7 9 with 10 capsules alone, one with 10 capsules plus additives, and one with 10^ capsules alone (Fig. 3). The virus suspensions were prepared and standardized as in

1974. 30

The trees were sprayed with a Solc^ Junior 410 mist- blower at a rate of 0.5 liters/minute. Each of the treated

trees received 100 ml of material. The trees receiving 107

capsules with no additives were sprayed first followed by 7 9 trees receiving 10 capsules with additives, 10 capsules with no additives, and 10^ capsules with additives.

Half of the treated trees were sprayed each week from

June 16 to September 3 for a total of 14 applications. The other half were sprayed only when needed based on the recovery of adult males from a female-baited trap and the proportion of fruit infested. As a result, these trees were sprayed at the same time as the other trees except for four applications from July 3 to July 2 4. Trees were treated either in the morning or early evening when the fruit was dry and the air was calm.

The female-baited trap was similar to one described by

Proverbs et al. (1966). The trap was made from a round, plastic container 11.4 cm high and 15.8 cm in diameter.

Half of the bottom was removed to permit the free flow of air (Fig. 6 ). The inner surface was coated with Stikenl® to capture adult males attracted to the females in the trap. The holding container for the females was similar to one described by Bastiste (19 70) except that the water­ ing vial was not permanently fastened but made so it could be snapped in and out. The trap was hung by a wire in a tree of section B on May 2. Two newly emerged adult 31

Figure 6 . Female-baited trap used to monitor flight periods of adults in 1975. 32

females from the laboratory culture were placed in the holding container each week through September 13. The trap was checked at least twice a week for adult males which were removed along with other insects caught by the Stikenf^.

The trap was replaced on June 12, June 26, July 2 4, and

August 21 because of loss of sticking power or an accumu­ lation of insects.

The effects of different treatments on codling moth populations were assessed in a number of different ways.

A random sample of 100 fruit was examined on each tree for larval feeding sites every week from June to September.

On September 15, 50 apples were picked from each tree by random selection around the perimeter of the tree. The apples were inspected for codling moth and other infesta­ tions. Apples with successful entries by L,. pomonella larvae were dissected. All larvae found were reared in food cups at 29 to 30 °C and 18 hours photophase until they died or developed to adults. Tissue smears of dead larvae were examined with a phase-contrast microscope.

Six inch wide burlap bands were placed around the trunk of each tree on June 9. The bands were examined for larvae and pupae every week through the end of August. On

September 2 2 the larvae and pupae were taken from the bands and brought back to the laboratory to complete development.

Tissue smears from dead larvae were examined with a phase- contrast microscope. 33

A bioassay was performed on samples of apples sprayed

with virus to determine the effects of the environment on

retention of virus activity in spray deposits. Four apples

were randomly picked from each of 15 trees at one, three,

seven, and twelve days after spray application. Three of 7 the 15 trees were untreated, three were sprayed with 10 7 capsules plus additives, three with 10 capsules alone, 9 9 three with 10 capsules plus additives, and three with 10

capsules alone. Apples were held by the stem and placed

in individual plastic bags that were transported to the

laboratory in a styrofoam container. Four neonate larvae were placed on randomly selected positions on each apple.

Half of a gelatin capsule measuring eight mm in diameter and 0.55 cc in volume was placed over each larva (Fig. 7).

The capsules were held in place by fitting them into a circular incision made in the apple with a cork borer of the same diameter. The moisture of the fruit softened the end of the capsule resulting in a secure bond. The fruit with larvae and gelatin capsules were kept in subdued

light and 2 2 °C. After ten days the capsule was broken off and the area examined for the larva. If the larva was not found after a thorough search it was recorded as dead.

Tissues of dead larvae were examined with a phase-contrast microscope. Mortality figures were corrected with Abbott's formula (Abbott, 1925), 34

Figure 7. Apples with gelatin capsules used to contain larvae in specific areas for bioassay of virus activity in spray deposits. 35

Spray coverage was determined by placing glass slides in trees to monitor spray material. Glass slides, 2.5 cm by 7.6 cm in dimensions, were fastened to limbs with clothes pins (Fig. 8) at heights of one, two, and three meters in a tree. One slide was placed at each height in four trees sprayed with suspensions containing additives. Spray deposits on the slides were allowed to dry for 30 minutes before being removed and placed in slide boxes. Care was taken to handle the slides by the edges.

In the laboratory, samples of droplets were counted and measured. The slides were examined with a phase- contrast microscope at 100 times magnification. Ten random samples of spray deposits on each side of a slide were observed by using the area of view under the micro­ scope as the sample area and randomly moving the slide across the stage and stopping at 10 different locations. o The area taken in by each sample was 1.13 mm . The number and diameter of the droplets in a sample area were measured and recorded. An ocular micrometer was used to measure the diameter of the droplets to the nearest pm.

A study was undertaken to determine the physical relationship among virus capsules, charcoal particles, and skim milk using a scanning electron microscope. This was done to gain insight into the protective abilities of char­ coal particles and skim milk for virus capsules against inactivation by ultraviolet light. The scanning electron 36

Figure 8. Glass slide fastened in a tree with a clothes pin to monitor a spray application. 37 microscope used was a Cambridge Stereoscan, model S4-10.

Pieces of apple skin and glass cover slips with spray deposits were examined. Material was coated with gold at approximately 20 0 angstroms in thickness in a Denton vacuum evaporator. The beam was set at 2 0 kilovolts and material was scanned at angles of 35, 45, and 60 degrees to the beam at magnifications ranging from 4,800 to 22,000 times.

Micrographs were taken of selected areas.

IV. Susceptibility of Larvae to Virus

The susceptibility of first and fifth instar larvae to virus was tested. First instar larvae were tested for susceptibility to virus applied on the surface of apples and artificial food. Virus was given to fifth instar larvae on discs of food and through per os inoculations, with a fine glass needle. Data from all of the experiments were analyzed by probit analysis (Finney, 19 71),

The virus suspensions were prepared in the same manner as the material used in field experiments and were standard­ ized using a method developed by Williams and Backus (1949).

The concentration of a virus suspension was estimated by comparing counts of capsules to counts of latex spheres of a known concentration using an electron microscope. The q concentration of the spheres was 9.7X10 /ml and each sphere was approximately 200 mu in diameter. Equal proportions of a virus suspension and the spheres were mixed together and 38 sprayed on 200 mesh grids coated with Formvai®. Counts of spheres and capsules were made on ten squares in each of two grids with an RCA EtlU 3G electron microscope.

Serial dilutions of a standard preparation of virus were tested against first instar larvae in food cups used in the rearing program. The surface of the food was sterilized under an ultraviolet light source for 15 minutes. One drop of suspension (0.00 3 ml) from a sterile

Pasteur pipette was deposited in each cup and spread across the surface of the food with the tip of a glass rod. A neonate larva was put into each cup after the suspension dried for five minutes and covered with a plastic lid.

Larvae were transferred from the oviposition containers with a number one artist's brush which was frequently dipped in a one percent solution of sodium hypochloride and dryed on clean tissue paper. The experiment was repeated once. Untreated food cups were used as controls.

The larvae were kept at 29 to 30 °C and 18 hours photo­ phase.

The larvae were examined each day after treatment.

Tissues from dead larvae were examined under a phase- contrast microscope. The number dying from granulosis and time to death were recorded. The surface area of food consumed was estimated in mm and recorded for HO of the larvae dying from granulosis. 39

In the test of susceptibility of first instar larvae to virus on apples, Golden Delicious apples from untreated trees in section B were used. The technique of using a gelatin capsule to confine a larva to a specific area on an apple was employed in this experiment. Three serial dilutions of virus were tested. In addition, charcoal and skim milk were added to virus suspensions in a separate experiment. A suspension was spread on the surface of an apple with a number five camel hair brush. Each stroke of the brush spread 0.00 5 ml of liquid over an area of 2 800 mm . One larva and capsule were placed over each area.

Four larvae were tested on an apple and four apples were used for each treatment and control. The experiments were replicated three times. The untreated surface of the apple was one control. Charcoal and skim milk mixed with dis­ tilled water were spread on apples for the other control.

Apples with larvae were kept at 22 °C in subdued light.

The apples were examined for signs of larval activity at ten days post-treatment, The number of larvae not consuming food was recorded. Larval tunnels were carefully examined for larvae in the other cases.

Different ages of fifth instar larvae were tested for virus susceptibility using virus applied to small discs of artificial food and through per os inoculations. Larvae were 11, 12, 13, and 14-days old at 29 to 30 °C rearing temperature and 18 hours photophase. The discs of food IJ I

‘to were five mm high and five mm in diameter and each was placed in a three-quarter ounce plastic cup. Three serial

dilutions of a standard preparation of virus were tested.

A one ml pipette was used to drop 0.0 5 ml of material on each disc of food. After five minutes one larva was placed in each cup and covered with a plastic lid. Control larvae were fed untreated food. Larvae were kept at 29 to 30 °C and 18 hours photophase. Ten larvae from each age group were tested. The experiment was repeated using 15 larvae/ age group/dilution. Additional tests were performed on

12-day-old larvae using five serial dilutions of a standard preparation and ten larvae/dilution. The experiment was repeated three times.

At the end of two days the amount of food eaten by each larva was estimated and recorded. Additional food was placed in the cup if all of the food was eaten. The test animals were examined every day after treatment and records were kept of survival and development. Tissue smears of dead larvae and pupae were examined with a phase-contrast microscope to determine cause of death.

Per os inoculations were performed with an Isco microinjector, a one ml syringe, and a blunted glass needle mounted on a rubber cork (Fig. 9), Inocula were two micro­ liters in volume. Three serial dilutions of a standard preparation were tested. Larvae were anaesthetized with carbon dioxide for 45 seconds before inoculation with virus. 41

Figure 9. A. Material used in per os inoculations of fifth instar larvae. The stereo-microscope at the left was used to view a larva during inoculation with virus by the Isco microinjector at the right. B. Enlargement of a portion of A showing the glass needle mounted on the rubber cork at the end of a one ml syringe. 42

Larvae were viewed under a stereo-dissecting scope while the needle was placed about halfway into the alimentary canal to reach the midgut area. After inoculation with virus they were returned to three-quarter ounce, plastic cups with no food and kept at 29 to 30 °C and 18 hours photophase. Ten larvae from every age group were tested with each dilution. Ten control larvae were inoculated with distilled water. The experiment ttfas replicated once.

The same type of data gathered in the experiment using discs of food was taken in this experiment. RESULTS

I. Population Increases in the Absence of Insecticides

In 19 73, insecticides were not applied in section B

and populations increased to infest 5 percent of the apples by July and further increased to 23 percent in September

(Fig. 10). In the last sample taken on September 21, from

9 to 40 percent of the apples on individual trees showed

feeding by larvae.

The following year, populations increased earlier than

in 19 73 with 5 percent of the apples infested by June (Fig.

10). By mid-July larvae had entered about 12 percent of the apples and no further increase was observed until

September when 2 5 percent of the fruit were infested.

In 19 75, infestations were higher than the previous two years. Although the mean percent damage was only slightly higher in the months of June and July the range of damage among the trees was more extensive ranging from

0 to 30 percent as compared to 0 to 18 percent in 19 74 and 0 to 10 percent in 1973 (Fig. 10). Final infestation

levels were higher reaching 30 percent of the fruit in

September.

43 Figure 10. Proportion of apples infested from 197 from infested 197 3 to apples of 5 Proportion 10. Figure

% Fruit Infested - 0 4 20 - 0 3 - 0 3 20 - 0 3 20 0 1 10 10 - - - - - n=5 1974 n=l 1 n=l 1975 n = 13 1973 range. (section B), The dotted lines indicate the indicate lines dotted The B), (section June

X ul etmber Septem ly Ju onth M X August X

us

II. Effects of Virus Dissemination

A. 19 7U Studies

Each of the virus dosages used in 197U (10^, 10 8, and o 10 capsules/tree) caused granulosis disease in larval

populations. All of the larvae in the harvested apples were

reared in the laboratory until they died or developed to

the adult stage. Larvae from sprayed and unsprayed apples

developed granulosis showing that the virus spread to the

unsprayed or control trees (Table 1). There were, however,

a larger proportion of diseased individuals from trees

sprayed with virus (Fig. 11).

Reductions in codling moth infestations and populations in trees sprayed with virus were estimated by comparing

levels in sprayed trees to those in control trees. Since virus spread to the control trees the magnitude of effect of the virus on populations and infestations was probably higher than estimates from comparisons between sprayed and unsprayed trees.

Larvae from bands on sprayed and control trees also died from granulosis (Table 2). In addition, larvae were infected with bacteria (Bacillus sp.). More larvae from the bands were found with Bacillus sp. than from the apples

(Tables 1 and 2). Also, a small number of larvae from the bands were parasitized by nematodes and hymenopterous parasitoids (Table 2). Table 1. Fate of larvae removed from apples harvested in 1974 and subsequently reared in the laboratory.

Died prematurely (cause)______Survived to Granulosis % Bacillus sp. Unknown adult stage Total n 10 capsules/tree 16 29.6 0 15 23 54

Control 8 15.7 1 18 24 51

g 10 capsules/tree 15 34.9 0 14 14 43

Control 10 25.6 0 17 12 39

109 capsules/tree 13 44.8 0 4 12 29

Control 14 30.4 3 5 24 46 iue 1 Gauoi otlt f ave eoe from removed larvae of mortality Granulosis 11.Figure

% of Total ah oun s h ttl ubro lra found larvae of number total the is column each reared subsequently and 1974 in harvested apples in the laboratory. The number at the top of top theat number The laboratory. thein n h apples.in the O Cnrl 0 nrl 0 Control 10 ontrol C 10 Control lO

1+7

Table 2. Fate of larvae removed from bands in 1974 and subsequently reared in the laboratory.

Died prematurely (cause) Hymenopterous Survived to Granulosis Bacillus sp. Nematodes parasitoids Unknown adult stage Total

10 capsules/ tree 13 11 32

Control 10 23

10® capsules/ tree 1 0 0 14 14 30

Control 12 5 1 18 10 47

10 capsules/ tree 4 1 4 23 24 63

Control 2 0 4 31 43 85

-p 00 49

Trapping of mature larvae in burlap bands was an

efficient method of estimating population levels in trees.

At harvest, the number of larvae in the bands on the control

trees was correlated to the number of vacated larval entries

in apples suggesting that the number of larvae in a band

was a reliable indicator of the number of larvae leaving

the apples from a tree. Analysis showed a correlation

coefficient of 0.669 which is significant at the 0.05

level (Table 3).

An analysis of the band data revealed that virus 8 9 dosages of 10 and 10 capsules/tree applied twice a week

from July 30 to August 31 caused significant mortality and

reduction of second generation populations, whereas, a n lower dosage (10 capsules/tree) did not affect seasonal

population trends (Table 4 and Fig. 12). Population

reduction was observed in late August and reached a maxi- Q mum of about 42 and 34 percent in trees sprayed with 10 g and 10 capsules , respectively.

Similar population trends were observed when successful

entries by larvae were used as an index of population 7 levels (Fig. 13). Virus treatment with dosages above 10

capsules/tree reduced larval populations. The effects were first observed in the latter half of August and

reached a maximum during September. The maximum reduction 8 9 was 19 and 2 8 percent in trees sprayed with 10 and 10

capsules, respectively. Reduction on trees sprayed with 50

Table 3. Correlation of vacated entries in control apples harvested in 1974 to larvae collected from the bands. The correlation coefficient (0.669) is significant at the 0.05 level (F = 7.07).

Tree Vacated Entries Larvae in Band

2b 34 14

3b 3 7

4b 17 22

Id 48 15

2d 5 29

4d 3 4

If 60 53

2f 60 31

3f 20 10

4f 8 6 51

Table 4. Comparison of the increase of second generation larvae and pupae in bands from treated and control trees in 1974.

No, of larvae No. of larvae and pupae and pupae Tree (increase) Tree (increase)

1 0 7 capsules/tree Control

la 19 2b 14

2a 19 3b 7

3a 6 4b 22

Total 44 a Total 43

Chi2 = 0.02 00 o 1 —t

capsules/tree Control

lc 5 Id 13

2c 9 2d 26

3c 11 4d 4

Total 25 Total 43

Chi2 = 7.53^

1 0 9 capsules/tree Control

le 24 If 39

2e 23 2f 29

3e 4 3f 7

4e 0 4 f 5

Total 51 Total 80

Chi2 = 10.5c a A chi-square test was applied to the total increase . of larvae and pupae in each experiment. w P = 0.01 C P = 0.005 Figure Figure

Mean Increase/Band 10 10 10 15- 15-| 2 1 Ices f eod eeain ave n pupae and larvae generation second of Increase . atrd nbns uig 1974. during bands in captured • Control • capsules IO • • Control • l capsules lO • • Control • 1 capsules 10 • ugust A th n o M pe er b eptem S 52

Figure 13, Increase of successful entries during 1974. during entries successful of Increase 13,Figure Cumulative % Increase 0 4 20 0 4 20 20 0 4 0 6 0 6 0 6 h iiil ubro nre ws estimated was entries of number initial The rmto r-ramn counts. pre-treatment twofrom • Control • * Control * l capsules lO • • Control • 1 capsules 10 • O capsules lO August

onth M etmber Septem

5*1 Table 5. Comparison of the increase of successful entries in treated and control trees in 1974. The initial number of entries was estimated from two pre-treatment counts.

Increase in Increase In Successful Successful Entries Entries Tree % 0 \Zp~a Tree % 0 \/p 10 7 capsules/tree Control la 47.2 43.45 lb 60.0 50.77 2a 72.3 58.24 2b 63.9 53.07 3a 55.1 47.93 3b 25.9 30.59 4a 25.4 30.26 4b 39.9 39.17 Mean 50.0 Mean 47.4 t = 0.26

108 capsules/tree Control 1c 57.9 49.54 Id 74.8 59.87 2c 65.4 53.57 2d 64.0 53.13 3c 47.1 43.34 3d 68.1 55.61 4c 27.2 31.44 4d 63.9 53.07 Mean 49.4 Mean 67.7 -■ C\J OJ 3 II t - 109 capsules/tree Control le 43.8 41.44 If 66.9 54.88 2e 41.8 40.28 2f 68.9 56.11 3e 38.0 38.06 3f 53.7 47.12 4e 37.2 37.58 4f 82.1 64.97 Mean 40.2 Mean 67.9 t = 4.20b

a A paired t-test was applied to an arcsin trans­ formation (0 nTp") of the data.

b p = 0.025. 55 q 10 capsules was significant at the 0.025 level (Table 5).

There was a steady loss of fruit from the trees rang- • p ing from a total of 42.7 percent in trees sprayed with 10

capsules to 5 7.9 percent in the control trees of the same

experiment (Fig. 14). Between August 2 7 and September 4

a large proportion of the apples containing successful

entries dropped from the trees (Table 6 ). This appears to

account for the reduction in the rate of successful entries

during this period (see arrows in Fig. 13). Therefore, the

total proportion of fruit with successful entries was

probably higher than estimated.

Feeding sites were reduced on trees sprayed with a Q dosage of 10 capsules. Feeding sites were found on 71 and 51 percent of the fruit on the control and sprayed trees, respectively (Table 7). Apparently 21 percent of

the larvae were killed by virus before they caused detect- 9 able feeding damage. Dosages lower than 10 did not cause Q significant differences although 10 capsules/tree reduced

feeding damage by about 14 percent (Table 7).

The actual mortality caused by virus was estimated by

comparing the number of mature larvae in the bands. On o g trees sprayed with 10 and 10 capsules the total mortality caused by virus was estimated at 42 and 34 percent, respect­ ive ly.

Pre-entry and post-entry mortality were estimated on g trees sprayed with 10 capsules. About 2 0 percent of the 56 -• 107capsules 5 0 •• Control

4 0 //s f 3 0

2 0 // lO

-•1 0 capsules 5 0 -• Control / 4 0

3 0

2 0 /■/ y

10

T -• lO capsules 5 0 ■• Control

40-

3 0

20 -

10- /•

Ju ly August Septem ber M onth

Increase of fruit loss from trees during 1974. 57

Table 6 . Fruit drop from trees from August 27 to September 4, 19 74.

Estimated fruit drop Successful No entry % entry % Total n 10 capsules/ tree 22 24.4 68 75.6 90

Control 2 6 . 9 27 9 3 .1 29

q 10 capsules/ tree 10 31.3 22 68.7 32

Control 4 6.1 62 93.9 66

109 capsules/ tree 5 6.6 65 92.9 70

Control 4 5.2 73 94.8 77 58 Table 7. Comparison of the increase of infestations in treated and control trees in 1974. The initial number of feeding sites was estimated from two pre-treatment counts.

Increase in Increase in Infestations Infestations Tree % 0 \ / j ? % 0 \/p 7 10 cansules/tree Control la 51.9 46.09 lb 61.7 51.77 2a 80 .9 64.08 2b 70.3 56.98 3a 71.8 57.92 3b 25.9 30.59 4a 49 .4 44.66 4b 41.4 40. 05 Mean 63.5 Me an 49.9 t = 1.21 Q 10 capsules/tree Control lc 62. 7 52.36 Id 77.8 61. 39 2c 72.5 58.37 2d 64.0 53.13 3c 59.6 50.53 3d 72 .1 58.12 4c 37.2 37.58 4d 73.0 58.69 Mean 58.0 Mean 71.7 t - 1-53 9 10 capsules/tree Control le 48.1 43.91 If 68.0 55.55 2e 45.8 42.59 2f 71.2 57.54 3e 55.4 48.10 3f 63.3 52.71 4e 53-9 47.24 4f 82.5 65.27 Mean 50. 8 Mean 71.2 t = 4.27b

a A paired t-test was applied to an arcsin trans­ formation (jzf \7p) of the data.

b p = 0.025. 59

larvae died by virus before entering fruit and another 14

percent died after entry (Table 8 ). Of the post-entry

mortality, about seven percent died by virus while attempt­

ing to enter fruit and another seven percent died after establishing feeding tunnels (Table 8). These data show

that virus can cause death in larvae at any stage of development.

Apparently, virus did not persist at an infectious level from 1974 to 1975. Only one larva of the 73 sampled in 19 75 was infected with granulosis.

Less than one percent of the harvested fruit were without insect feeding or oviposition sites. Feeding or oviposition by fruitworms (Noctuidae); the plum curculio,

Conotrachelus nenuphar (Herbst); and the codling moth accounted for most of the infestations. The majority of the codling moth feeding was in the form of vacated larval entries ranging from 34 to 59 percent of the apples among the different experiments (Fig. 15). Larvae were found in many of the fruit ranging from 13 to 30 percent. There was one larva/apple in every case and these were in various stages of development. Ten percent or less of the fruit had unsuccessful entries and the proportion of apples with unsuccessful entries was greater on the sprayed trees than on the control trees (Fig. 15). 60

Table 8 . Estimated mortality of larvae caused by virus in trees sprayed with 1 0 ° and 10 ^ capsules.

% Mortality caused by virus

o g 10 capsules/tree 10 capsules/tree

I. Pre-entrya - 20

II. Post-entry - m

a. Unsuccessful*5 entry (sting) - 7

b. In fruit and post-emergence - 7

Totalc 42 34

aCalculated by subtracting % infestations in fruit from sprayed trees from % infestations in fruit from control trees.

kCalculated by subtracting % unsuccessful entries in control trees from % unsuccessful entries in sprayed trees.

cCalculated from formula % M = x 100 where A = the number of larvae in bands on control trees and B = the number of larvae in bands on sprayed trees. iue 5 Cdigmt edn i ape hretd in harvested apples in feeding moth Codling 15, Figure

% % of Total lOOn 20 - 0 4 - 0 6 - 0 8 - f | No feeding |No f iiu. the total number of apples. of number total the 94 Te ubra te o o ahclm is column each of top the at number The 1974. 7Control 0 1 •* . v 2 248 227 Z O ontrol C lO 3 15 7 248 276 155 236 , '«> i,h w ) E aae entry Vacated rva O ontrol C lO SB

61

62

1975 Studies

There were two distinct flight periods of adult codling moths in 19 75(Fig. 16B). The number of captured males rose sharply at the end of May to 2 4/week and gradu­ ally dropped to none by the end of June. The second flight period was about twice as long as the first extend­ ing from the beginning of July to the middle of September.

A high of 30 captured males/week was reached in the middle of August which was slightly higher than the peak of the first flight period (Fig. 16B) .

Feeding damage was low in the experimental blocks in section A until about three weeks after the beginning of the second flight period (Fig. 16). Feeding sites were found on less than one percent of the fruit through June and July but in block 2 the damage quickly rose at the beginning of August (see arrow in Fig. 16A) and reached a maximum of 21,7 percent at harvest. The number of infested apples in block 1 began to increase in mid-August (see arrow in Fig. 16A) and reached a maximum of 7.3 percent.

In the two blocks (3 and 4) furthest away from section B there was no increase in infested fruit until the end of

August (see arrow in Fig. 16A). At the end of two weeks the proportion of infested fruit increased to five percent.

Table 9 shows the number of successful entries in the apples harvested from the four blocks in section A. There doesn’t appear to be any treatment effect on the proportion iue 6 Cmaio faut lgt eid t subsequent to periods flight adult of Comparison 16. Figure

No. Adult Males % Fruit Infested 0 2 - 0 3 10 0 2 10 -i 5 2 15- - - - - June y a M . nettos . dl ae cpue i the in captured males B. Adult Infestations A. eaebie trap. female-baited 1975. during B and A sections in infestations onth M uly Ju Aug fB •Al *A2

6 4 Table 9, Successful entries at harvest in 1975. Data were analyzed by using a two-way analysis of variance.

% Successful entries Block

Treatmenta la 2b 3a 4a Mean

10 spray applications 7 b 1 0 ' capsules/tree- 2 22 0 8 8.0

107 capsules/tree,+e 2 28 4 2 9.0 q 10 capsules/tree,- 10 6 0 4 5.0 Q 10 capsules/tree,+ 10 10 0 4 6.0

14 spray applications n 10 capsules/tree,- 2 14 4 2 5.5 n 10 capsules/tree,+ 2 28 4 2 9.0

1 0 9 capsules/tree,- 2 14 0 2 4.5

1 0 9 capsules/tree,+ 6 12 4 6 7.0

Control 2 34 8 0 1 1 . 0

f* No significant difference at the 0,05 level, k- = No additives ^ = With skim milk and charcoal Blocks followed by the same letter are not significantly different from one another at the 0.05 level. 65 of successful entries (F = 0.345), however, the proportion of successful entries in block 2 was significantly differ­ ent from the other blocks (F = 12.5 3).

A closer analysis of the data from block 2 shows that virus dosage had a significant effect on the proportion of successful entries (Table 10). The addition of skim milk and charcoal to the spray suspensions and the four additional applications apparently had no effect on the proportion of successful entries (Table 10).

The effect of virus treatment was seen at the end of

July. More successful entries were found in apples on trees 7 9 sprayed with 10 capsules than on trees sprayed with 10 capsules (Fig. 17). The magnitude of difference was less than two percent through August and increased to 11.5 percent in September, Also, at harvest there was a difference of 12 percent in the proportion of successful n entries on the control tree and trees sprayed with 10 capsules suggesting that this dosage also may have caused a reduction in populations. q Treatment of trees with 10 capsules also reduced the proportion of feeding sites. Significantly more infested 7 apples were found on trees sprayed with 10 capsules them q on trees sprayed with 10 capsules (Table 11). The differ­ ence of 9 percent in the proportion of infestions is an estimate of the increased amount of pre-entry mortality 9 occuring in trees sprayed with 10 capsules. 6 6 Table 10. Effects of virus dosage, spray additives, and number of applications on successful entries in 1975 (block 2).

Successful Successful Entries Entries Tree% 0 Vp a Tree % 0 Vp 7 10 capsules/tree 10 ^ capsules/tree

16B 22 27.97 15B 6 14.18 18B 28 31. 95 17B 12 20.27 20B 24 29 . 33 22B 14 21.97 21B 14 21. 97 23B 10 18.44 Mean 22 .0 Mean 1 0 .5 t = 3 . 37b

With additives No additives 17B 12 20.27 15B 6 14.18 18B 28 31.95 16B 22 27 .97 2 0B 24 29 . 33 21B 14 21.97 23B 10 18.44 22B 14 21.97 Mean 18. 5 Mean 14. 0 ll ri­ o .80

10 spray applications 14 spray applicat ions

15B 6 14.18 17B 12 20.27 16B 22 27.97 20B 24 29.33 18B 28 31.95 2 IB 14 21.97 23B 10 18.44 22B 14 21.97 Mean 16. 5 Mean 16 .0

t = 0.05

a A t-test was applied to an arcsin transformation Cj*\7p-) of "the data.

b p = 0.02. iue 7 Ices o scesu etis n 95 bok 2). (block 1975 in entries successful of Increase 17. Figure

Cumulative % Increase -i 0 4 20 - 0 3 io- - y ueAugust lyJune u J

—® f , —/ ®— ® ft,.— Month ,— ember b m te p e S ®1Q(4) f ®Control (1) 67 68

Table 11. Effect of virus dosage on feeding sites in 1975 (block 2).

Infestations Infestations

Tree______%_ 0 v/pa _____ Tree______%_ 0 \7p~ 7 9 10 capsules/tree 10 capsules/tree

16B 24 29.33 15B 18 25.10

18B 28 31.95 17B 14 21.97

2 OB 30 33.21 2 2 B 18 25.10

21B 16 23.58 23B 12 20.27

Mean 24.5 Mean 15.5

t = 2.61b

a A t-test was applied to an arcsin transformation igs \7p) of the data.

b p = 0.05. 69

Statistically reliable comparisons could not be made between the control tree and trees sprayed with virus in block 2 , however, infestation levels on all of the trees sprayed with virus were lower than the level observed on 7 the control tree. A dosage of 10 capsules/tree reduced feeding sites by 32 percent and a 5 7 percent reduction 9 was recorded for trees sprayed wxth 10 capsules.

The largest number of individuals found in the bands in section A was nine (five pupae and four larvae) taken on the last sample date. Two of the larvae died from unknown causes and one from a fungal pathogen of unknown identity.

A total of 5 7 larvae were recovered in apples harvested from section A (Table 12). Seventy-two percent of the larvae developed to the adult stage in the laboratory.

Seven percent of the mortality was caused by granulosis; 7 all in larvae taken from apples on trees sprayed with 10 capsules.

About 80 percent of the apples harvested in block 2 were not damaged by codling moth larvae (Fig. 18). The largest proportion of the infested apples contained larvae.

Twenty-five percent of the larvae feeding on apples harvested from trees sprayed with 10 capsules died during entry, however, only ten and five percent of larvae from trees sprayed with 10 capsules and the control tree, respectively died while trying to enter fruit. Table 12. Fate of larvae removed from apples harvested in 1975 and subsequently reared in the laboratory.

Died prematurely (cause) ______Survived to Granulosis Bacillus sp. Unknown adult stage Total

10^ capsules/tree 4 0 5 17 26

10^ capsules/tree 0 0 6 18 24

Control 0 1 0 6 7 71

| | No loading \/\ With larva

|^[ Umutteiiful antry ^ Vacated entry

lOO-i ------

8 0 -

"5 6 0 - O I- <*> o 5? 4 0 -

Control lO7 io9 (1) 14) (4)

Figure 18. Codling moth feeding in apples harvested in 1975 (block 2). Fifty fruit were harvested from each tree . 72

Other organisms fed or oviposited on less than five percent of the apples during the growing season. Plum curculio and fruitworm infestations were observed early in the season followed by bird; tussock moth, Henerocampa sp.; and unknown feeding later.

Ill. Analysis of Spray Deposits

A. Spray Coverage

The methods and materials used in spraying fruit apparently gave adequate coverage. Spray material was found on all of the glass slides placed in trees and from

88.8 to 98.8 percent of the sample areas contained at least one droplet of spray deposit (Table 13). In some cases, a side was completely coated with spray material.

The diameter of spray deposits ranged from 6 3 to 1,321 pm with an overall mean of 22 3±2 pm. The mean diameter at different heights in trees ranged from 192il7 pm at three meters to 2 4lil3 pm at two meters with one meter inter­ mediate at 212i9 pm. There was no significant difference in the diameters of spray deposits at different heights in trees at the 0.05 level (F = 1.734).

B. Retention of Virus Activity on Apples in the Orchard

Virus activity was detected in spray deposits on apples sampled at one, three, and seven days post-treatment.

In bioassay tests performed in the laboratory, larvae infected with granulosis were found in apples from trees 73

Table 13. Measurements and analyses of spray deposits on glass slides in trees during treatment with virus. Virus suspensions contained charcoal and skim milk. Four slides were fastened at each height.

______Height of Slides in Trees_____

One Meter Two Meters Three Meters

No. of droplets 295 205 120

Mean diameter of droplets (pm)a 212 ±9.2 2i*l ± 13.0 192 ±17.5

No. of droplet^/ 2 sample area (1.13 mm ) 4.9 3.4 3.0

No. of sides coated with spray

Samples with at least one droplet (%) 98.8 88 . 8 98.8

aNo significant difference at the 0.05 level (F = 1.734). 74 Q sprayed with 10 capsules through the seventh day after n application and in apples from trees sprayed with 10 capsules on one and three days post-treatment (Table 14).

From the estimates of mortality caused by virus, more active 9 virus was detected on apples from trees sprayed with 10 capsules with additives on one, three, and seven days post­ treatment than on apples sprayed with the other treatments

(Fig. 19).

A large proportion of the larvae died within 2 4 hours after being placed on apples and without consuming food.

Out of 48 larvae used in each test as many as 2 5 died within 24 hours (Table 15). There was a significant difference in the number of dead larvae among the sample times at the 0.05 level (F = 20.95) and no difference among the treatments (F = 0.647) suggesting that virus had no effect on larvae unless detectable feeding occured.

A high proportion of the larvae on untreated apples died after tunneling in the fruit. Mortality ranged from

29.7 percent on the third day after application to 56.2 percent on the seventh day (Table 14). In a few cases, the entrance area was infected with a fungus of unknown identity which may have killed some of the larvae, but most of them died from unknown causes. Table 14. Results of bioassay of spray deposits of virus on apples sampled at one, three, seven and twelve days after application.

Mortality of test larvae on X days after application

1 3 7 12 Aa C*> ACAC A C

Control 33.3 - 29.7 - 56.2 - 23.8 -

1G7 capsules with no additives 27.5 0.0 51.1 (if 30.4 41.6 0.0 32.4 11.2 7 10 capsules with additives 50.0 Cl) 25.0 48.6 (1 ) 26.8 50.0 0.0 28.6 6.2 q 10 capsules with no additives 39.1 (3) 8.7 38.4 (2) 12,4 48.6 0.0 23.3 0.0

10^ capsules with additives 64.2 (4) 46.3 53.8 (2) 34.3 64.2 (2)18.2 23.8 0.0

aA = actual mortality; does not include larvae that died within 24 hours.

= corrected mortality; calculated from the formula: C = A-B X 100 where B= control mortality (%) (Abbott, 1925). 100-B

c 50.0 (1) = granulosis positively identified in one larva. Figure 19. Relative activity of virus in the orchard the in virus of activity Relative 19. Figure

Index of Virus Activity 10 2 - 4 - 3 - 5 6 8 - 9 - 7 - - - nape uignoae frt ntr larvae. instar first neonate, using apples on niomn bsdo bosa f pa deposits spray of bioassay a on based environment as Post-Treatment Days ®109capsules-additives • lO9 capsules+ additives lO9 • capsules+ ®107capsules + additives ®107capsules 1 capsules-additives ®10

76

77

Table 15. Number of larvae dying after 24 hours in experiment of bioassay of spray deposits. Forty-eight larvae were used in each test.

Days Post -Treatment

Treatment a la 3b 7b 12b Total

Control 21 11 16 6 54

n 10 capsules with no additives 19 3 12 11 45

10^ capsules with additives 20 11 12 6 49 q 10 capsules with no additives 25 9 11 5 50

q 10 capsules with additives 20 9 6 6 41

Total 105 43 57 34 239

aNo significant differences at the 0.05 level (F = 0,647). Days post-treatment followed by the same letter are not significantly different from one another at the 0.05 level. 78

C. Microscopic Examination

A scanning electron micrograph of capsules on a glass cover slip (Fig. 20) was compared to a micrograph of cap­ sules on the surface of an apple (Fig. 21) and a micro­ graph of capsules and additives on a cover slip (Fig. 22).

The contour of the apple was very uneven compared to the relatively smooth surface of the glass cover slip. How­ ever, the undulating surface of the apple did not appear to shield capsules from sunlight since they were not hard to locate in the micrograph (Fig. 21). The appearance of charcoal particles and skim milk were determined by com­ paring Figure 20 with Figure 22. During the electron scanning of the cover slip with virus plus additives, some capsules were sighted in clusters of charcoal parti­ cles but many were seen without protection by skim milk or charcoal (Fig. 22). Although exact numbers could not be determined more than 50 percent of the capsules sighted were exposed. This does not take into consideration capsules hidden from view under charcoal particles.

IV. Susceptibility of Larvae to Virus

A. First Instar

Neonate, first instar larvae were apparently more susceptible to virus on artificial food than on apples.

Using probit methods it was calculated that a dosage of five capsules was required to kill 50 percent of the 79

Figure 20. Scanning electron micrograph of capsules on a glass cover slip. Bar represents 1 pm. 80

Figure 21. Scanning electron micrograph of capsules (arrows) on the surface of an apple. Capsules were suspended in water and sprayed on apples in the orchard. Concentration of the suspension was 107 capsules/ml. Bar represents 2 ym. 81

Figure 22. Scanning electron micrograph of capsules (arrows), skim milk (SM), and charcoal particles (C) on a glass cover slip. Note the two capsules mixed with the charcoal particles at the end of the black arrow and the exposed capsule at the end of the white arrow. Bar represents 1 pm. 82 larvae on artificial food, whereas, on apples a dosage of about 400 capsules/larva was required to cause 50 percent mortality. This is an 80-fold difference at the LE>5Q dosage. The slopes of the two dosage-mortality lines were similar (1.350 on artificial food and 0.877 on apples)

(Table 16 and Fig. 23). Thus, larvae responded to virus on both surfaces in a predictable manner, and the differ­ ence in LD,.q values was probably the result of differences in the properties of the two surfaces, e.g., the artificial food was highly absorbant, whereas the apple surface was not.

Larvae were more variable in their response to virus on apples with additives than to virus alone. This was shown by the decided decrease in the slope of the dosage- mortality line. The slope for the virus-additive mixture was 0.36 7 while that for virus alone was 0.8 77 (Table 16 and Fig. 2 3). The LD^g value of 844 capsules for larvae to virus on apples with additives was within the 9 5 percent confidence interval of the LD^g value for virus on apples with no additives. Apparently, larvae were more susceptible to virus dosages below the LD5Q level if it was consumed with the additives (Fig. 23). The additives may have an independent effect because 2 3.3 percent of the larvae died on control apples with additives only, whereas, 14.3 percent of the larvae died on the untreated controls (Table 18). Table 16. The LD50 values and 95% confidence intervals for first instar larvae treated with virus on artificial food and apples.

95% Confidence ______Interval______LD50 Lower Upper Test (capsules/larva) (cap sule s/larva) (capsule s/larva) Slope

Virus on artificial food 5 3 8 1.350

Virus on apples with no additives HOI 122 1.0HX103 0.877

Virus on apples with additives 84H 90 5.20X107 0.367

00 OJ iue 3 Dsg-otlt rsos o lra following larvae of response Dosage-mortality 23. Figure

% % Mortality 5 - - 0 5 - 0 3 - 0 7 - 0 9 10 - 5 9 1 0.1 - - -3 4 - -6 ecn mortality. percent diie. ent, is isa lra were larvae instar first with Neonate, apples CC) and additives. additives, no with apples as fe tetet Arw niae 100 indicates Arrow read were treatment. results after the days and tests the in used neto o iu o () riiil od CB) food, artificial (A) on virus of ingestion a. oe lg scale) (log Dose O lO IO io

10

IO 84

85

Table 17, Mortality of first instar larvae following ingestion of virus on artificial food. Forty larvae were used in each test.

% Mortality____ Approx. no. No. larvae Unknown capsules/larva in testa Granulosis causes o o

Control 40 • 5.0

3 38 34.2 7.9

14 34 73.5 5.9

28 38 92. 2 2.6

140 36 91.7 8.3

280 36 100.0 0.0

2 x surface consumed = 5.9 mm

Does not include larvae that died within 24 hours from unknown causes. 86

Table 18. Mortality of first instar larvae following ingestion of virus on apples. Forty-eight larvae were used in each test.

Approx. no. No. larvae Corrected capsules/larva in test5 % Mortality % mortality

Virus with no additives

Control 35 14. 3 —

1 34 14.7 0.4

102 45 40.0 ( 2 ) c 29.9

104 42 90.4 (5) 88 , 8

surface consumed = 3.7 mm2

Virus with charcoal and skim milk

Control 43 23.3 -

1 35 34.3 14. 3

10 41 41.5 23.7

103 40 62 .5 (4) 51.1

surface consumed = 3,6 mm2

aDoes not include larvae that died within 24 hours from unknown causes. ^Calculated from the formulai c = A-B x ^0 0 where c = corrected mortality (%), A =100-B actual mortality (%), and B = control mortality (%) (Abbott, 1925). c40,0 (2) = Granulosis positively identified in two larvae. 87

In the bioassays on apples, only a few of the larvae

located in the entrance tunnels were infected with

granulosis. In apples treated with virus alone two larvae 0 h ingesting 10 capsules and five ingesting 10 capsules were

infected. Four infected larvae were diagnosed from apples

treated with the highest virus dosage with additives.

Other larvae dying from granulosis disintegrated before the

larval tunnels were examined at ten days post-treatment.

The incubation periods for granulosis in neonate,

first instar larvae at different virus dosages on artifi­

cial food were studied. At the LDg^ level (140 capsules/

larva) there appeared to be a second response to virus by

larvae after 80 percent of the individuals were dead as

shown by a sudden change in the slope of the time-

mortality line (Fig. 24). This bimodal response also

occured in larvae ingesting 2 80 capsules and to a lesser

extent in larvae consuming 2 8 capsules. At the two

lowest dosages (3 and 14 capsules/larva) the upper ends

of the time-mortality lines were also truncated, but in

these cases it was due to a cessation of response (Fig.

24). The first response was used to calculate the time- mortality lines for 3 to 140 capsules/larva using probit methods. For 2 80 capsules/larva a line was drawn between the first mortality at four days and zero mortal­

ity (approximately 2 probits) at three days to estimate

the LTgQ value. The lines for the second responses to 88 *3 capsules/ larva 014 ®28 9 9 •14 0 ■7 0 280

95 .0® 9 0

70

5 0 5 -5 1J

30-

IO

5 o

1-

T------1------1---- 1------1--- 1--- 1---1--- 1 4 5 6 7 8 9 IO 11 12

Days (log scale)

24 Time-mortality response of neonate, first instar larvae to various doses of virus ingested with artificial food. Arrow indicates 100 percent mortality. 89

Table 19. The LTgg values and 95% confidence intervals for first instar larvae treated with virus on artificial food.

95% Confidence interval_____ Approx. no. ^Tgg Lower Upper capsules/larva (days) (days) (days) Slope

3 9.68 8.30 12.76 4.674

14 8.07 7.51 8.77 6.006

28 5.69 5.40 5.99 8.960

140 5.00 4.76 5.30 11.371

280 3.7 - - 22.3 90

yirus were fitted to the points by eye.

For the first response, the length of time for granu­

losis development was inversely proportional to virus

dosage. LT_rt values decreased with increased dosage b 0 ranging from 9.7 days for 3 capsules/larva to 3.7 days

for 280 capsules/larva (Table 19). Generally, there was

less variability in the length of the incubation periods

with increased virus dosage as shown by the progressive

increase in the slopes of the time-mortality lines from

4.6 74 for 3 capsules/larva to 22.3 for 2 80 capsules/

larva (Table 19 and Fig. 2 4).

B. Fifth Instar

Young, fifth instar larvae were not as susceptible to virus as neonate larvae treated with virus. The LD,.rt 50 value for fifth instar larvae (12-days old) on artificial

food was 49 capsules, whereas, for younger larvae on the same media the LDC~ value was five capsules. This is a ou ten-fold difference in susceptibility. Fifth instar larvae were also more variable in their response to virus as shown by the decreased slope of the dosage-mortality line

(Fig. 25). The slope of the line for fifth instar larvae was 0.348, whereas, for younger larvae it was 1.35 0.

There was more than a 60,000-fold difference in suscepti­ bility of fifth instar larvae to virus, whereas, in first instar larvae there was only a 300-fold difference. iue 5 Dsg-otlt rsos f A noae first neonate, (A) of response Dosage-mortality 25, Figure

% % Mortality - 0 3 - 0 9 5 0 - -5 -a -5 - 0 5 - 9 9 - 0 7 10 - 5 9 - 5 - -6 -7 -4 -3 ave o iu o riiil od Arrow food. artificial on virus to larvae ntrad B ffh ntr 1-as old) (12-days instar fifth (B) and instar indicates indicates IO oe lg scale) (log Dose 100 percent mortality. percent IO IO

91

92

Table 20. Mortality of fifth instar larvae (12-days old) following ingestion of virus on artificial food. Thirty larvae were used in each test.

_____ % Mortality____ Approx. no No. larvae Unknown capsules/larva in testa Granulosis causes

Control 24 0 4.1

6 26 38 . 5 0 . 0

60 24 50 . 0 0.0

600 25 68.0 0.0

6X10 3 23 69.6 0.0

6X104 27 88 . 9 0.0

proportion of food discs consumed = 80.0%

^oes not include larvae that died within 4 8 hours. 93

The pattern of time-mortality responses of fifth instar larvae to different doses of virus were similar to those of first instar larvae. At each dosage level there was a change in the slope of the time-mortality line at about seven days post-treatment (Fig. 26). With increas­ ing dosage the magnitude of change in slope or response by larvae to virus decreased (Fig. 26). Accordingly, these data were analyzed in the same manner as corresponding data for first instar larvae.

For the first response, the length of time for granu­ losis development was inversely proportional to virus dosage. The LT^q values ranged from 8.1 days for 6 capsules/larva to S.9 days for 6X10^ capsules/larva

(Table 21). Generally, there was less variability in the length of the incubation periods with increased virus dosage as shown by the progressive increase in the slopes of the time-mortality lines ranging from 7.183 for 60 capsules/larva to 11.964 for 6X10^ capsules/larva (Table

21 and Fig. 26).

The amount of virus consumed by fifth instar larvae was estimated by recording the proportion of the food discs eaten by each individual and averaging the results. Dosage levels were then calculated from known amounts of virus placed on the food discs. Fifth instar larvae that were

12-days old consumed an average of 80 percent of the food.

All of the larvae used in the test consumed food. Figure

% % Mortality - 0 9 - 0 7 50- - 0 3 - 5 9 - 9 9 O- lO 26. Time-mortality response of fifth instar larvae instar fifth of response Time-mortality 26. - 5 1 - r-8 4 - -6 -7 netdwt riiil food. artificial with ingested ( 12 * o V) dy od t dfeet oe o virus of doses different to old) -days 5 i as lg scale) (log Days 6 Q6X104 ®6X103 600 ® 0 6 0 • 6 capsules / larva capsules 6 • \

7 i 8

~ 10 9 i

I “ 14 94

95

Table 21. The LT50 and LD^n values with 95% confidence intervals for fifth instar larvae C12-days old) treated with virus on artificial food.

95% Confidence interval rox. n o , LT50 Lower Upper ules/larva (days) (days) (days) Slope

6 8.16 7.13 11.2 9 7.735

60 8 . 65 7. 32 14.09 7 .183

600 7.62 7.02 8,65 8.099

6X10 3 6 , 82 6.32 7,74 10.090

6X104 5.95 5.66 6.35 11.964

l E>50 (capsules/larva)

48 . 99 2 .75 236.98 0.348 96

A number of the larvae died from unknown causes within 48 hours of the experiment and were not included in the analysis of the results (Table 20). The only other unknown mortality occured to one of the control larvae after seven days.

Developmental and behavioral differences were observed in fifth instar larvae of different ages. By eleven days after development at 2 8 to 30 °C and 18 hours photophase most larvae had molted to the last or fifth instar. About

20 percent of the larvae at 13 days began to spin a cocoon for pupal development and at 14 days nearly 90 percent had completed the process. Approximately 50 percent of the larvae had completed pupation after 15 days.

Young, fifth instar larvae (11- and 12-days old) were more susceptible to virus than larvae actively spinning a cocoon or beginning pupation (13- and 14-days old) (Fig.

27B and Table 22). Generally, mortality of larvae, treated at 11 or 12 days with virus on artificial food, progressively increased with increased virus dosage.

Percent mortality ranged from 12.5 to 90.9 for 11-day-old larvae and from 11.1 to 9 5.0 for 12-day-old larvae.

Mortality of 13-day-old larvae fluctuated from 16.7 per- 2 cent at 3 capsules/larva to 7.1 percent at 3.3X10 capsules/ larva and then increased to 60.0 percent at 3.3X104 capsules/larva. Larvae inoculated at 14 days only responded to the two highest dosage levels with 11.8 and 19.0 iue 7 Gauoi mraiy f ifrn ae o fifth of ages different of mortality Granulosis 27.Figure % Mortality lOOn Y/ riiil food. artificial with virus of ingestion (B) and virus of 112“days old 112“days ntrlra floig A pro inoculations os (A) per following larvae instar 11-days old 11-days 0 1 X 2 6 - 3 oe (capsules/larva) Dose 5- 0 1 X -6 .5 2 10 X 2 | 114-days old 114-days | 13-days old 13-days 5- 0 1 X -6 .5 2 2X10

97

98

Table 22. Mortality of different ages of fifth instar larvae following ingestion of virus with artificial food. Twenty-five larvae were used in each test.

% Mortality Approx. no. N o . larvae Unknown capsules/larva in testa Granulosis causes

11-days old

Control 24 0.0 0.0 6 2 24 12.5 0.0 6X10^7 24 87.5 0.0 6X10 22 90.9 0.0

12-days old

Control 22 0.0 0 . 0 5 2 18 11.1 0,0 5X10 20 95.0 0.0 5X10^ 19 84.2 0.0

13-days old

Control 16 0.0 6.3 3 2 18 16 . 7 0.0 3.3X10 14 7.1 7.1 3.3X10 20 60.0 0.0

14-days old

Control 20 0.0 0.0 3 2 18 0.0 0.0 2.5X10 17 11.8 0.0 2.5X10 21 19. 0 0.0

aDoes no include larvae that died within 48 hours or those that did not consume food. 99

Table 23. Mortality of different ages of fifth instar larvae following per os inoculation of virus. Twenty larvae were used in each test.

% Mortality Approx. no. N o . larvae Unknown capsules/larva in testa Granulosis causes

11-days old

Control 16 0.0 25.0 2X10 ^ 17 64.7 29.4 2X104 10 80.0 10.0 2X106 9 88.8 11.2

12-days old

Control 18 0.0 16 .7 2X102 16 25.0 12,5 2X10^ 14 92. 9 0 . 0 2X10® 7 100. 0 0.0

13-days old

Control 16 0.0 0.0 2X102 19 5.3 10.5 2X10^ 18 5.6 22.2 2X10 12 16.7 0.0

14-days old

Control 18 0.0 0.0 2X10?: 19 0.0 5.3 2X10^ 18 0.0 16.7 2X10 13 30.8 0.0

aDoes not include larvae that died within 48 hours. 1 0 0 percent mortality.

The amount of virus ingested by larvae was dependent on the stage of development. Young, fifth instar larvae

(11- and 12-days old) consumed more of the food discs than older larvae (13- and 14-days old). The proportion of the food discs eaten by larvae of different ages were as follows: 11-days old, 90 percent; 12-days old, 79 percent;

13-days old, 50 percent; and 14-days old, 40 percent. Thus, with increasing age progressively less virus was ingested.

Virus dosages for each age interval are recorded in Table

22.

Not all of the larvae consumed food and these individ­ uals were not included in the results. The proportion of larvae not consuming food ranged from 7 percent of the larvae treated at 11 days to 29 percent of the larvae treated at 14 days.

Different ages of fifth instar larvae were given virus through per os inoculations. This was done so that all of the larvae received the same amount of virus regardless of age. Larvae inoculated per os responded in a similar manner to virus as larvae ingesting virus with food (Fig.

27). At each dosage level, larvae inoculated at 11 or 12 days were much more susceptible to virus than larvae inoculated at 13 or 14 days (Table 2 3).

Time-mortality responses of larvae to different doses of virus were plotted. The lines for 11- and 12-day-old 1 0 1

larvae were similar for both methods of administering virus

(Figs. 2 8 and 29). Although the LT&0 values differed at

some dosage levels, the slopes of the lines were similar

(Tables 24 and 25). For example, the for 11-day-old

larvae inoculated per os with 2X10 capsules was 9.4 days and that for 12-day-old larvae was 2 6,2 days. However, the slopes of 3.85 and 2.45 of the time-mortality lines for

larvae inoculated at 11 and 12 days, respectively were not significantly different.

The LTcri values for larvae treated with virus at 13 or o u 14 days were somewhat higher than those for larvae treated at 11 or 12 days (Tables 24 and 25). The LT^q value for C 14-day-old larvae inoculated per os with 2X10 capsules was

11.1 days as compared to 5.1 days for larvae inoculated at

11 days and 5.8 days for larvae inoculated at 12 days. The

LTgg for 13-day-old larvae treated with virus on food was

6.9 days, whereas, corresponding values for 11- and 12-day- old larvae were 5.1 and 5.8 days, respectively.

Some of the larvae died within 48 hours after treat­ ment in both experiments. Most of the early mortality occured to 11- and 12-day-old larvae inoculated per os

(Table 2 3). Presumably, the process of inserting the glass needle into the alimentary canal had a deleterious effect on younger larvae because of their smaller size but this does not explain why more of the larvae died at the highest dosage level (Table 23). Figure

% % Mortality 2 O- lO 5 - - 0 5 - 5 9 lO- 8 Tm-otlt rsos o dfeet gs of ages different of response Time-mortality . -3 -7 -3 netdwt atfca food. virus of doses artificial with different to ingested larvae instar fifth 012 ® 13 • 11-days old 11-days •

3- 0 1 X -6 .3 3

-X1 capsules/larva 10 5-6X as lg scale) (log Days IO 0 1 2

iue 9 Tm-otlt rsos o dfeet gs of ages different of response Time-mortality 29. Figure

% % Mortality 5 - - 0 5 5 0 - -5 - 0 5 - 5 9 0 5 O- lO io- - 5 9 TO- - 5 9 -7 -7 _7 ro idcts 0 pret mortality. percent 100 indicates Arrow

diitrdtruh e s inoculations. os per through administered it isa lra t dfeet oe o virus of doses different to larvae instar fifth 012 ll-rday&old • ®14 t*

as lg scale) Clog Days 2X10’ 2X10 X0 capsules/larva 2X10 15 10 3 18

10 4

Table 24. The LT50 values with 95% confidence intervals for different ages of fifth instar larvae treated with virus on artificial food.

95% Confidence interval____ Age of larvae LT50 Lower Upper (days) (days) (days) (days) Slope

2 5 to 6X10 capsules/larva

11 7.18 6.19 9.31 11.361

12 6.45 6.19 6.80 21.354

3.3 to 6X10^ capsules/larva

11 5.05 4.85 5.30 20.989

12 5.88 5.55 6.33 12.510

13 6.90 6.35 7.78 7.474 105

Table 25. The LTcn values with 95% confidence intervals for different ages of fifth instar larvae inoculated per os with different doses of virus.

95% Confidence interval____ Age of larvae LT50 Lower Upper (days) (days) (days) (days) Slope

2X10^ capsules/larva

11 9.47 6.67 31.32 3.85

12 26.20 15.70 179.41 2.45

2X10^ capsules/larva

11 6.07 5.55 6.81 9.400

12 6.35 5.85 6.96 10.379

2X106 capsules/larva

11 5.10 4.58 5,82 8.897

12 5,82 5.28 6.44 10.760

14 1 1 . 08 9.10 42.52 7.595 106

Larvae surviving doses of virus administered by either

method appeared to have about the same chance to pupate and

emerge as adults as control larvae. Granulosis was detected

in some of the pupae but the causes for the rest of the pupal mortality are unknown (Tables 26 and 27). Adult emer­

gence in control larvae ranged from 50,0 to 87.5 percent

(Tables 26 and 2 7). In most cases, percent emergence of control adults was similar to emergence of adults that were given virus as larvae. Two exceptions were individuals fed virus with food at 12 and 14 days. For larvae treated at

12 days, percent emergence of control adults was 6 3.6 as compared to 37.5 for individuals consuming five capsules.

Progressively fewer adults emerged with increasing virus dosage for larvae treated at 14 days ranging from 70.0 percent of the control adults to 47.1 percent of the if individuals consuming 2.5X10 capsules. 10 7

Table 26, Pupal development and adult emergence of different ages of fifth instar larvae surviving after consuming virus with artificial food.

Pupae Approx, no, No, larvae With % Adult capsules/larva in test No. % granulosis Emergence

11-days old

Control 24 24 100 .0 0 50.0 6 2 24 21 87.5 0 47 .6 6X10 24 3 12 .5 0 0 . 0 6X104 22 2 9.1 0 100, 0

12 -days old

Control 22 22 100,0 0 63.6 5 2 18 16 88,9 0 37.5 5X10 2 20 1 5.0 0 100.0 5X104 19 3 15.8 0 100.0

13 -days old

Control 16 15 93 . 8 1 60 . 0 3 18 15 83,3 0 73.3 3,3X102 14 12 85,7 0 58.3 3,3X10h 20 8 40,0 1 75,0

14 -days old

Control 20 20 100.0 0 70.0 3 2 18 18 100,0 0 61.1 2.5X102 17 15 88.2 1 53 . 3 2,5X10 21 17 80.1 0 47.1

^ o e s not include larvae that died within 48 hours or those that did not consume food. 108

Table 27. Pupal development and adult emergence of different ages of fifth instar larvae surviving per os inoculations of virus.

Pupae Approx. no. No. larvae With % Adult cap s ule s/larva in testa No. % granulosis emergence

11-days old

Control 16 12 75.0 0 50.0 2X102 17 1 5.9 0 100.0 2X10^ 10 1 10,0 0 0.0 2X10 9 0 0,0 0 -

12-days old

Control 18 15 83.3 0 80.0 2X10, 16 10 62 . 5 1 40.0 2X10^ 14 1 7.1 0 100. 0 2X10 7 0 0.0 0 -

13-days old

Control 16 16 100.0 0 87.5 2X10 2 19 16 84.2 1 62.5 2X10^ 18 13 72.2 0 69.2 2X10 12 10 83.3 3 60.0

14 -days old

Control 18 18 100.0 0 77.8 2X10 2 19 18 94.7 0 66.7 2X10^ 18 15 83,3 0 64.3 2X10 13 9 69.2 1 66.7

aDoes not include larvae that died within 48 hours. DISCUSSION

Dissemination of virus during 1974 and 19 75 demon­

strated that relatively low levels of virus cause granulosis

disease in larvae and reduction of populations and infesta-

tions. With a dosage level of 10 capsules/tree in 1974

there was no reduction in populations or infestations.

With an increased dosage of 10® capsules/tree there was

a noticeable reduction in populations and 10® capsules/tree reduced both populations and infestations (Table 28). Bode

(19 70) applied almost 45 times more virus than the highest

dosage disseminated in this study against the second

generation in the same orchard in 196 7. Populations were reduced by 93 percent, however, no reduction of infesta­ tions was observed. Apparently, higher dosages were more effective in reducing populations but not necessarily in reducing infestations.

Virus disseminated periodically throughout the entire growing season (19 75) was more effective in reducing infes­ tations than virus sprayed during the second generation only (1974) (Table 28). When the first application of virus was made in 19 75 no larval infestations were observed in section A, whereas, in 19 74 as many as 16

109 Table 28. Data from disseminations of virus in apple orchards by various researchers.

% Reduction Dosage No. of Total no. Researcher (capsules/tree) applications capsules/tree lb pc

Sheppard 1974 10 (32 days) 4X108a 0 0 (Ohio, USA) 10108° 10 (32 days) 4X109a 0 42 109 10 (32 days) 4X1010a 29 34

1975 l°g 10 (89 days) 4X108a 32 - 10y 10 (89 days) 4X1010a 57

Bode (1970) varied 3 (21 days) 1.7X1012 0 93 (Ohio, USA)

Falcon et al. 1966 1.7X1012 5 (107 days) 8.5X1012 87 (1968) (Calif. , 1967 6X1011 7 (85 days) 4.2X1012 33 81 USA)

Keller (1973) 9.5X1010 4 (42 days) 3.8X1011 73 - (Switzerland)

^Multiplied by a factor of four to correct for smaller size of dwarf trees. I = Infestations CP = Population I l l percent of the apples on a tree had been damaged by larvae when virus was first disseminated. Thus, populations were already high in 1974 when virus was introduced and low dosage levels were not very effective in reducing infesta­ tions. The first generation of larvae were probably just beginning to hatch in 19 75 and mortality of these individ­ uals may have reduced the number of potential reproductive adults thereby decreasing the number of second generation larvae. 8 12 A wide range of doses (10 to 10 capsules/tree) applied over the entire growing season in this and other studies had similar effects on the reduction of infesta­ tions, however, with increasing dosage there was a progressively larger reduction of successful entries. 8 10 Applications of about 10 and 10 capsules/tree reduced infestations by 32 and 5 7 percent, respectively, whereas, in California, dissemination of 100 times more virus than 12 the highest dosage used in this study or 10 capsules/ tree reduced infestations by only 33 percent (Falcon et al. , 1968). Applications of 1 0 ^ capsules/tree in Switzer­ land reduced infestations by 7 3 percent, however, the codling moth is univoltine in Switzerland and less than

15 percent of the apples on control trees were damaged

(Keller, 1973), Infestations include both successful

(entrance tunnels) and unsuccessful entries (stings). A closer examination of the data reveals that with dosages 1 1 2 of 10 ^, 101 0 , 10^^, and 10***^ capsules/tree successful entries were reduced by 35, 6 8 , 90, and 95 percent, respec­ tively. Thus, similar proportions of the larval popula­ tions in all of the studies were feeding on apples but progressively more died during the initial stages of entry with increased virus dosage.

Reduced infestations or feeding damage may have been caused by a number of factors. Virus disseminated over the entire growing season reduced larval populations thus adult populations may have been lowered. Therefore, the produc­ tion of second generation larvae was diminished which caused a corresponding reduction in infestations. However, other factors may be involved since applications of virus directed towards only the second generation caused a 21 percent reduction of infestations. Some larvae may have died from virus infections before entering apples. It has been shown that neonate larvae placed on leaves sprayed with a q concentration of 10 capsules/ml consume enough leaf material to receive an infectious dose of virus (Keller,

1973). Larvae may also have ingested virus as they hatched from eggs contaminated with virus from spray applications.

Some larvae may have consumed lethal doses of virus after they had entered apples. First instar larvae tested in the laboratory with virus died from granulosis within ten days post-treatment suggesting that older larvae dying in apples probably ingested virus at some time after they had 113

entered since larval development in the orchard usually

takes from 14 to 25 days (Cutright, 1964). Small amounts

of virus may have been washed into entrance tunnels during

spray applications. Also, rainfall may have spread virus

from diseased larvae into tunnels. Some individuals were

probably susceptible to low doses of virus acquired in

this manner since 38 percent of young, fifth instar larvae

tested in the laboratory died from a dose of only six

capsules.

The extent to which virus activity was retained in

spray deposits on apples was directly dependent on dosage.

Most of the virus was inactivated within seven days at a q dosage of 10 capsules/tree, however, Keller (19 73) used

7.5X10^, 7.5X10'*'1 , and 7.5X10*^ capsules/tree and virus was effective longer with increased dosage. After 6 3

days about 2 5 percent of the virus from the highest dosage was still active. Further tests indicated that about 90

percent of the virus inactivation was caused by sunlight

and the other 10 percent by the action of temperature

(Keller, 1973). Rainfall did not appear to have any effect.

Known wavelengths of ultraviolet radiation inactivated a

granulosis virus of Pieris brassicae (David, 19 69)

suggesting that ultraviolet wavelengths of sunlight

probably caused most of the inactivation of virus in the

field. Charcoal and skim milk seem to be more effective in protecting virus when added to concentrated suspensions of virus than dilute suspensions. There was a two-fold exten­ sion of virus activity when skim milk and charcoal were

n added to suspensions containing 10 capsules/ml. Keller

(19 73) tested skim milk and other protectants in suspen­ sions containing 7,500 times more capsules and found that virus remained active three times longer with the addition of skim milk. The addition of skim milk and charcoal to concentrated suspensions of a granulosis virus of Pieris rapae and a nuclear polyhedrosis virus of Trichoplusia ni also resulted in a three-fold extension of activity

(Jaques, 19 72). At higher concentrations there is more likelihood for virus to lodge under charcoal particles or be covered by skim milk simply because of increased numbers of inclusion bodies.

The addition of skim milk or similar agents to suspen­ sions may be important in obtaining proper coverage of apples with virus. Laboratory tests showed that the addition of skim milk and charcoal to suspensions of virus results in increased mortality of larvae on apples. The skin of an apple is somewhat hydrophobic since it is covered with a waxy material. The addition of skim milk to suspensions acts as a spreader-sticker (Angus and Luthy,

19 71) resulting in a more even distribution of virus when brushed on apples as compared to virus in water alone. 115

As a result, larvae on apples with virus plus additives had

an increased chance of consuming lethal doses of virus than

larvae on apples with virus alone.

In the orchard studies, coverage of fruit was appar­

ently adequate but better coverage was probably obtained

with suspensions containing additives. Droplets of fine

mist produced by mistblowers dry soon after contact, but with the addition of a spreader-sticker such as skim milk

individual droplets will spread over a larger area result­

ing in better coverage (Furmidge, 1959). This effect was probably not detected in the orchard because of low infesta­ tions in 19 75.

Charcoal may increase the susceptibility of larvae to virus as well as cause larval mortality independently. Dr.

W. M. Bode (personal communication) found that spraying of apple trees with suspensions of the granulosis virus of the red-banded leafroller mixed with charcoal reduced infesta­ tions of both codling moth and red-banded leafroller suggesting that charcoal caused mortality in the codling moth larval populations. The mode of action of charcoal is not known. Histopathological and dosage-mortality studies of the effects of virus plus charcoal and charcoal alone on larvae are needed before definite conclusions can be made.

Truncation of the upper ends of the time-nortality lines for laboratory populations of first and fifth instar larvae was partially the result of the experimental method 116

used but most of it was probably due to resistance to virus

among individuals. Larvae did not begin to feed on the

virus-treated food at the same time and some individuals

may not have ingested a lethal dose of virus until three or

four days after treatment. As a result, the slopes of the

time-mortality lines would gradually decrease due to the

delayed death of these larvae. However, the slopes of most of the time-mortality lines either abruptly decreased or there was a complete cessation of response showing that some individuals were very resistant to granulosis infec­ tion. With increasing virus dosage more of these individ­ uals were infected with granulosis and less time was required to cause death. This was shown by the progressive increase of the slopes of the second response lines. These results show the importance of careful analysis of time- mortality data in addition to evaluation of dosage-mortality responses.

Fifth instar larvae were resistant to a wider range of virus dosages than first instar larvae. Other authors have reported decreased susceptibility to virus among older larvaeCe.g., Magnoler, 19 75; Stairs, 1965a) but a decided difference in the range of response between larval instars of the same species has not been observed. Populations of fifth instar larvae probably required larger amounts of virus to cause disease than first instar larvae because of their increased mass. If there were no physiological 117

differences between first and fifth instar larvae, the

quantal responses to virus should have been similar.

Apparently, there were significant physiological differ­

ences which affected susceptibility to virus.

Larvae approaching pupation were less susceptible and

more unpredictable in their response to virus than younger

larvae. Some of this resistance was because of the

decreased rate of feeding by older larvae, but when inoc­

ulated per os with the same virus dosages as younger larvae

they were still more resistant to viral infections. This may be the result of changes during metamorphosis when the midgut epithelium of insect larvae is replaced by embry­ onic regenerative cells. Studies with nuclear polyhedrosis viruses in prepupae of insects have shown that these cells are resistant to virus attack (Bird, 1953; Stairs, 1965b).

Apparently, granulosis viruses invade the midgut region

(Summers, 19 69) and larvae nearing pupation were probably less susceptible to virus because of cellular changes in this gut area.

Virus doses did not seem to have a significant effect on postlarval stages. In another study, there was an

"occasional11 transmission of virus to pupae after larvae were inoculated per os (Etzel and Falcon, 1976). It is doubtful that very many pupae become infected in the orchard because in the oresent study only three pupae became infected after ingesting virus as larvae. Low 118

levels of infection in pupae cannot be diagnosed with

certainty using a phase-contrast microscope because of the

small size of capsules. Nevertheless, larvae surviving

treatment with virus appeared to develop normally to the

adult stage.

Virus may be spread from tree to tree by rain and wind. Virus disseminated in 19 74 spread to control trees

and infected larvae in apples and under burlap bands.

Spray suspensions were always directed away from control

trees during application but some may have contaminated

trees by drifting in the wind. However, the proportions of

larvae from untreated fruit that died from granulosis were

relatively high ranging from 8/51 (15.7%) to 14/46 (30,4%),

These levels were probably too high to be caused by drift­

ing of spray alone. Some virus from infected larvae may have spread from tree to tree by rain and wind between

applications. Bird (19 61) found that nuclear polyhedrosis

viruses of sawflies were spread from infections at high

levels in a tree to low levels through the action of rain­

fall. Capsules from disintegrated larvae could be spread

in a tree in the same manner and combinations of wind and

rain could carry virus to other trees.

Virus may also be transported by animals moving from tree to tree including the movement of adult codling moths.

Studies have shown that adults may be very active; sometimes

travelling as far as one and a half miles (Howell and Clift, 119

19 74; Steiner, 1940; Van Leeuwen, 19 40; White et al., 19 73;

Wood, 1965). Females in particular may be effective trans­ mitters of virus to neonate, larval populations by contami­ nating apples with virus as they oviposit. Apparently, this is how the nuclear polyhedrosis virus of the European spruce sawfly, Diprion hercyniae (Hartig), is transmitted from one generation to the next (Nei.lson and Elgee, 1968).

Apparently, not enough virus persists from year to year to cause significant mortality of larvae. Only one case of granulosis was diagnosed from 7 3 larvae sampled one year after spraying. Similar results have been obtained by other authors. From the 19 6 6 experiment conducted by Falcon et al. (196 8) 4/20 (5.0%) of the larvae from apples and 15/130 (11.5%) of the larvae from bands sampled in 196 7 were infected with granulosis. However, when virus sprays were discontinued in 1970, populations of

L. pomonella surged to levels obtained with removal of insecticides (Falcon, 1973). Bode (1970) sampled a total of 89 3 larvae in bands one year after he disseminated virus and none were infected with granulosis.

Ij. pomonella is probably not a specific host for this virus since apparently few opportunities exist for trans­ mission. Virus may persist in the soil but there does not appear to be any mechanism for transmission of it to the food source of larvae as there is with granulosis viruses of Trichoplusia ni and Pieris rapae where capsules 120

^re splashed onto the low lying cabbage plants from the

soil by rainfall (Jaques, 19 70). Larvae feed individually

on fruit and spin cocoons apart from one another so very

little if any virus is spread by contact of diseased and

healthy individuals. Transovum transmission of virus

evidently does not occur in this virus-host system either

(Etzel and Falcon, 19 76).

Apparently, repeated applications of virus are neces­

sary to reduce feeding on apples in Ohio and other loca­

tions where the codling moth is nultivoltine. Better

reductions of infestations were obtained in areas where the

codling moth is univoltine (Keller, 1973; Wearing, personal

communication). However, fruit was still not acceptable

for certain markets since as many as 2 0 percent contained blemishes or stings from unsuccessful entries by larvae.

When chemical insecticides were not used in an orchard

other insect species fed on apples and caused damage. Only about one percent of the apples harvested in 19 74 were free

from damage by fruitworms, the plum curculio, the codling moth, etc. In other areas such as Wisconsin, the apple maggot, Fhagoletis pomonella (Walsh); the red-banded leaf-

foller, Argyrotaenia velutinana (Walker); and the eye- spotted bud moth, Spilonota ocellana (Denis and Schiffer- muller) caused considerable damage to apples when insecti­ cides were not used (Oatman, et al . , 1966). In contrast,

feeding damage by other organisms was not excessive in an 1 2 1 orchard in California where the virus was used and chemical insecticides excluded (Falcon et al. , 1968).

Thus, the exclusive use of the virus to protect fruit from damage shows promise in some areas. In locations where other insects are of economic importance it may be used with chemical insecticides or other control methods such as ster­ ile moth releases in an integrated control program. Results 7 of this study show that a virus dosage as low as 10 cap­ sules or 1/500 of an L.E./tree can reduce infestations.

More than enough virus can be easily propagated for use in orchards at this dosage in an integrated control program.

The methods used in this study for rearing larvae and propa­ gating virus were simple and required inexpensive equip­ ment. In addition, keeping virus in situ at -75 °C for as long as two years did not affect the virulency of the virus.

Thus, virus can be stored effectively until needed.

Further research should be directed towards determining the best combination of time, dosage, and formulation for dissemination of virus. This will probably have to be determined for each major locality where the virus is used and will also depend on other control methods used in the program. Of these three variables, formulation should receive the most emphasis since it is probably the cheapest way to reduce costs. Proper formulation will result in better distribution and protection of virus reducing the number of applications and amount of virus needed. SUMMARY

Relatively low dosages of a granulosis virus of the

codling moth, Laspeyresia pomonella (L. ) were disseminated

in an Ohio apple orchard in 19 74 and 19 75 to determine the

effects of low concentrations on populations. Activated,

powdered charcoal (1% w/v) and powdered skim milk (1% w/v)

were evaluated for their ability to protect and spread

virus. In addition, the optimum time to apply virus was

determined.

Low doses of virus caused some reduction of populations

and infestations. Dosages of 10 (1/500 larval equivalent),

108 (1/50 larval equivalent), and 108 (1/5 larval equiva­

lent) capsules/tree directed towards the second generation reduced population levels by 0, 42, and 34 percent, respec­ tively. Infestations were also reduced by 20 percent with 9 a dosage of 10 capsules/tree. However, disseminations of

7 9 • 10 and 10 capsules/tree were more effective when applied over the entire season reducing infestations by 32 and 5 7 percent, respectively.

Virus mortality seemed to occur before larvae entered apples. neonate larvae may have consumed lethal doses of virus as they hatched from eggs or as they fed on leaves.

122 12 3

The extent to which virus activity was retained in spray deposits on apples was directly dependent on virus dosage. Virus activity was detected up to seven days post- g treatment on apples from trees sprayed with 10 capsules and for only three from trees sprayed with 10 capsules.

According to bioassays, charcoal and skim milk were only slightly effective in protecting virus from inactiva­ tion. There was no significant effect of the additives in reducing infestations, however, laboratory studies showed that charcoal may have increased the susceptibility of larvae to virus and caused larval mortality independently.

In addition, skim milk apparently acted as a spreader- sticker and insured that virus was evenly distributed over the waxy surface of apples.

Different larval instars were also studied in the laboratory for their susceptibility to virus. Neonate, first instar larvae were highly susceptible to virus. The

LDgg was only five capsules per larva, however, there was a 300-fold difference in susceptibility among these young larvae. Results of time-mortality studies indicated that about 20 percent of the neonate larvae were very resistant to virus infections.

Although the I-*D for the last or fifth instar was 10 w U times higher than that for first instar larvae, 35 percent died from a dose of only 6 capsules/larva and 89 percent from a dose of 6 X 1 0 capsules/larva. This was a 60 ,000-fold 12 4 difference in susceptibility to virus among these older larvae. These data showed that fifth instar larvae were much more variable in their response to virus than first instar larvae. This is the first report of a decided difference in variability of response to virus by two larval instars of the same species.

As larvae neared pupation they were less susceptible and more unpredictable in their response to virus. A small proportion of pupae (<1%) were infected when inoculated per os with virus as larvae but, generally, individuals surviving a dose of virus developed normally to the adult stage.

From 15 to 30 percent of larvae in apples harvested from unsprayed trees were infected with granulosis. Virus may have been spread by rain and wind or animals moving from tree to tree. Capsules were probably washed into larval tunnels by the action of rainfall.

Repeated applications of virus are apparently necessary to reduce populations since virus did not persist at an infectious level from 19 74 to 19 75 and epizootics of virus disease were not observed. CONCLUSIONS

1. Dissemination of relatively low doses of virus

caused granulosis disease in larvae and reduction of popu­

lations and infestations.

2. Virus sprayed periodically during the entire grow­

ing season was more effective than virus directed towards

only the second generation. 8 12 3. A wide range of doses (10 to 10 capsules/tree)

disseminated periodically over the entire growing season in

this and other studies had similar effects on the reduction

of infestations. However, with increasing dosage there was a progressively larger reduction of successful entries.

4. Evidence from this study suggests that some virus mortality occured before larvae entered apples. Neonate

larvae may have consumed a lethal dose of virus as they hatched from eggs of as they fed on leaves.

5. The extent to which virus activity was retained in spray deposits on apples was directly dependent on virus dosage,

6 . Charcoal and skim milk were more effective in protecting virus from inactivation when added to the more concentrated suspensions of virus.

12 5 12 6

7. Charcoal may have increased the susceptibility of

larvae to virus and caused larval mortality independently.

8. The addition of a spreader-sticker such as skim

milk to suspensions insured that virus was distributed

evenly over the waxy surface of apples.

9. About 80 percent of the individuals in larval popu­

lations (first and fifth instar) appeared to be much more

susceptible to virus than the remaining 20 percent.

10. The LDg0 for neonate, first instar larvae was only

five capsules/larva, however, there was a 30 0-fold differ­

ence in susceptibility to virus among these larvae.

11. Fifth instar larvae were about 200 times more

variable in their response to virus than first instar

larvae.

12. This is the first report of a decided difference

in variability of response to virus by two larval instars

of the same species.

13. As larvae approached pupation they were less

susceptible and more unpredictable in their response to

virus.

14. Fifth instar larvae surviving a dose of virus

usually developed to the adult stage in a normal manner.

Virus did not have a significant effect on post-larval

stages.

15. Larvae may have ingested infectious doses of virus after they had entered apples. Capsules were 127 probably washed into entrance tunnels during spray applica­ tions. Also, rainfall may have spread capsules from infected larvae into tunnels.

16. Virus may have been spread by rain and wind or animals moving from tree to tree. However, epizootics of virus disease did not occur and the virus did not persist at an infectious level from year to year.

17. The use of the virus to reduce populations and infestations shows more promise in areas where L. pomonella is univoltine or where damage to fruit by other insect species is minimal.

18. Virus may be integrated with other control measures such as chemical insecticides or sterile moth releases to reduce populations and infestations. BIBLIOGRAPHY

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Laboratory and Orchard Records

136 137

Table 29. Record of rearing program from January, 1974 to December, 1975.

No. surviving Generation to adult stage % Total

1 522 69. 6 750 2 401 63.1 636 3 390 61. 3 636 4 106 34.9 304 5 298 67.0 445 5 132 61.7 214 7 201 67.0 300 8 222 45.3 490 9 254 61.8 411 10 153 56.0 273 ii 131 50.8 258 12 213 72,4 294 13 141 62.4 226 14 125 57.6 217 15 133 57.6 231 16 169 44.9 376 17 131 53.0 247 18 a 150 35.1 427

1^ 228 63.9 357 2 205 70.2 292 3 191 52.6 363 4 330 76.2 433 5 143 63.3 226

Culture discarded after this generation because of low survival.

^First generation from new eggs obtained in June, 197 5. 138 Table 30. Condition of apples from trees sprayed with 107 capsules and control trees in 1974.

______Condition of apples______Unsuccessful Successful Date Mo entry______entry____ entry Total

7 10 capsules/tree

7/21 430 0 25 455

7/29 418 0 31 449

8/6 397 2 27 426

8/14 365 5 38 408

8/20 306 5 73 384

8/27 186 6 179 371

9/4 170 0 111 281

9/17 58 21 148 a 227

Control trees

7/21 530 0 9 539

7/29 517 0 11 528

8/6 442 2 10 454

8/14 417 2 32 451

8/20 363 2 68 433

8/27 245 2 138 385

9/4 263 0 111 374

9/17 104 8 136 k 248

a5 5 with larvae ^51 with larvae 139 Q Table 31. Condition of apples from trees sprayed with 10 capsules and control trees in 1974.

______Condition of apples______Unsuccessful Successful Date No entry entry entry Total g 10 capsules/tree

7/21 375 0 38 413

7/29 369 0 35 404

8/6 372 1 27 400

8/14 323 3 40 366

8/20 241 6 75 322

8/27 189 4 137 330

9/4 182 0 112 294

9/17 78 20 138 a 236

Control trees

7/21 340 0 28 368

7/29 324 0 37 361

8/6 280 2 39 321

8/14 266 7 68 341

8/20 172 5 123 300

8/27 98 0 159 257

9/4 94 0 91 185

9/17 30 6 119b 155

a5 5 wxth larvae 4 6 with larvae 140

Table 32, Condition of apples from trees sprayed with 109 capsules and control trees in 1974,

______Condition of apples______Unsuccessful Successful Date No entry_____ entry____ entry Total g 10 capsules/tree

7/21 420 0 78 498

7/29 403 0 91 494

8/6 362 0 72 434

8/14 377 9 86 472

8/20 305 8 146 459

8/27 183 4 209 396

9/4 182 0 144 326

9/17 89 29 158a 276

Control trees

7/21 417 0 53 470

7/29 413 0 51 464

8/6 391 2 37 430

8/14' 371 12 62 445

8/20 256 5 156 417

8/27 117 4 212 333

9/4 126 0 139 265

9/17 44 8 196b 248

^37 with larvae 48 with larvae Table 33. Number of larvae and pupae recovered from burlap bands on trees in 1974.

10^ Capsules 10® Capsules 10® Capsules Date per tree Control per tree Control per tree Control

7/29 2.3, 1.3a 0.3, 0.3 2 .6, 2.3 0 .6, 3.6 3.0, 4.5 4.5, 8.2

8/6 0.6, 1.6 1.3, 0 1.3, 2.6 2 .0, 0.6 2 .2, 3.5 1.5, 6.7

8/20 2 .0, 0 0.0, 0.3 1 .6, 0.3 1 .0, 0.6 2.7, 0 5.0, 0

8/27 2 .0, 0.3 0 .6, 0 2 .0, 0 3.6, 0 4.2, 0 7.7, 0

9/4 7.6, 0 3.3, 0 5.0, 0 13.0, 0 8.5, 0 13.2, 0

9/27 16.6, 0 14.3, 0 10.3, 0 16.0, 0 16.0, 0 25.0, 0

alarvae, pupae 141 142 Table 34. Number of unsuccessful and successful entries observed in 100 randomly selected apples from trees in block 2 during 1975,

No. of unsuccessful and successful entries/ ______100 apples______

Date I II III IV V VI VII VIII IX

6/10 0 , 0 a 0,0 1 0,0 0,0 0,0 0,0 0,0 0,0 0,1

6/19 0,1 0,1 0,0 0,0 0,0 0,0 0,0 0,0 0,1

6/26 0,1 0,2 0,1 0,0 0,0 0,0 1,1 0,0 0,2

7/3 0,1 0,2 0,1 0,0 0,1 0,0 0,0 0,0 0,1

7/9 0,1 1,3 1,0 0,0 0,0 0,0 0,0 0,0 0,3

7/18 0,0 0,1 2,0 0,0 0,0 2,0 0,0 0,0 0,3

7/24 0,0 0,3 2,1 0,0 0,0 0,0 0,0 1,0 0,3

7/31 0,1 0,4 1,1 0,2 0,4 1,2 0,1 0,0 0,4

8/7 1,0 0,5 2,3 1,0 0,5 1,2 0,1 1,2 0,5

8/12 1,0 0,6 0,3 1,1 0,2 1,2 0,0 1,2 3,2

8/18 2,1 0,6 2,2 1,1 1,4 1,4 0,1 0,2 5,4

8/27 2,6 1,8 0,9 0,4 0,8 2,11 0,1 1,2 2 ,11

9/3 1,9 0,16 4,6 0,6 0,9 1,9 0,5 1 , 4 0,16 •P o 9/15b 1,11 H 6,3 1,5 1,7 3 ,12 2,7 0,7 1,17

^Unsuccessful entries, successful entries From 50 harvested apples