I I
71-17,960
BODE, William Morris, 1943- THE CODLING MOTH, LASPEYRESIA POMONELLA (LEPIDOPTERA: OLETHREUTIDAE): EFFECTS OF AN INTRODUCED GRANULOSIS VIRUS ON A FIELD POPULATION AND LABORATORY REARING ON ARTIFICIAL DIETS.
The Ohio State University, Ph.D., 19 70 Entomology University Microfilms, A XEROX Company , Ann Arbor, Michigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED THE CODLING MOTH, LASPEYRESIA POMONBLLA (LEPIDOPTERAs OLETHREUTIDAE)s
EFFECTS OF AN INTRODUCED GRANULOSIS VIRUS ON A FIELD POPULATION
AND LABORATORY REARING ON ARTIFICIAL DIETS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
William Morris Bode, B.A.
******
The Ohio State University 1970
Approved by
Adviser Academic Faculty of Entomology ACKNOWLEDGMENTS
I thank my adviser, Dr, G. R. Stairs, for his assistance and advice given so generously throughout the period of this research;
Dr, R. P. Holdsworth, Jr., for employing me as a research assistant and giving me the opportunity to do research in the field; and my wife Becky for typing this manuscript and being so patient while I completed my graduate studies,
I thank The Graduate School of The Ohio State University for awarding me a Dissertation Year Fellowship,
Research funds and stipend were provided by the Ohio
Agricultural Research and Development Center, Wooster, Ohio,
ii VITA
March 18, 19^3 . . . . Born - Wooster, Ohio
1965 ...... B.A., The College of Wooster, Wooster, Ohio
I966-I969...... Research Assistant, Ohio Agricultural Research and Development Center, Wooster, Ohio
1969-1970 Fellow, The Graduate School, The Ohio State University, Columbus, Ohio
1970 ...... Appointed Assistant Professor of Entomology at the Pennsylvania State University Fruit Research Laboratory, Arendtsville, Pennsylvania
FIELDS OF STUDY
Major Field: Entomology
Studies in Integrated Control of Insect Pests, Dr, Robert P, Holdsworth, Jr.
Studies in Insect Virology. Dr. Gordon R. Stairs
iii TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS...... ii
VITA ...... iii
LIST OF T A B L E S ...... vi
LIST OF FIGURES...... viii
INTRODUCTION...... 1
REVIEW OF LITERATURE ...... 3
I. The codling moth, Laspeyresia pomonella ( L . ) ...... 3
History and distribution...... 3 Host plants...... 3 Classification ...... ^ Morphology...... 5 Behavior ...... 7
II. Natural control of L. pomonella . . , ...... 13
Parasites and predators ...... 13 Pathogens...... 17
III. A history of applied control of L. pomonella...... 21
Early methods ...... 21 Modern insecticides ...... 23 Autocidal control ...... 26 Modified spray programs ...... 30
IV. Laboratory rearing of L. pomonella...... 3^
Rearing on apples ...... 3^ Artificial d i e t s ...... 36 Disease prevention...... 39
iv Page
V. Insect v i r u s e s ...... ^2
General descriptions...... ^2 Granulosis virus diseases...... ^3 Invasion of insect ti s s u e s ...... ^5 Histopathology , ...... ^6 Disease symptoms...... **8 Diagnosis...... ^9 Non-inclusion viruses ...... 50 Use of viruses for insect control...... 51 A granulosis virus of L. pomonella...... 5^
INTRODUCTION OF A GRANULOSIS VIRUS INTO A FIELD POPULATION OF L. POMONELLA...... 57
I. Materials and methods ...... 57
II. Results ...... 63
III. Discussion...... 72
LABORATORY REARING OF L. POMONELLA ON ARTIFICIAL DIETS .... 76
I. Materials and methods...... 76
II. Results...... 83
III. Discussion...... 98
SUMMARY...... 102
BIBLIOGRAPHY...... 10^
APPENDIX A. Supplies for Rearing L. pomonella in the Laboratory...... 117
APPENDIX B. Records of Development of L. pomonella on Artificial Diets ...... 118
v LIST OF TABLES
Table Page
1. Estimated mortality of L. pomonella larvae in fruit and during entire larval period on virus-treated and untreated apple tre e s ...... 64
2. Accumulation of injury from second-generation L. pomonella larvae in samples of 500 apples examined on one virus-treated tree and four control trees. 196? ...... 65
3. Accumulation of mature L. pomonella larvae under trunk bands on one virus-treated tree and four control trees. 196? ...... 68
4. Incidence of microorganisms found in association with dead L, pomonella larvae in 1968 ...... 70
5. Survival of L. pomonella reared on different artificial diets ...... 84
6. Periods of development for L. pomonella reared on different artificial diets ...... 85
7. Oviposition by individual L. pomonella females reared on three different artificial d i e t s ...... 89
8. Average weights of female and male pupae of L. pomonella reared on three different diets...... 96
9, Mortality in the immature stages of a generation of L. pomonella reared in the laboratory on the modified Patana d i e t ...... 97
10, Development of L. pomonella on the Redfem diet, 24 April to 20 August, 1968 ...... 119
11. Development of L. pomonella on the Redfem diet, 8 May to 6 August, 1969 ...... 120
vi Table Page
12. Development of first generation of Washington strain of L. pomonella on the Redfem diet, 19 May to 2 *4- June7l959 ...... 121
13. Development of second generation of Washington strain of L. pomonella on the Redfem diet, 16 July to 28 August, 1969 ...... 122
lh. Development of L. pomonella on the Redfem diet, 17 October, 1969, to 8 January, 1970...... 123
15. Development of L. pomonella on the Redfem diet, 5 December, 1969, to 4 January, 1970...... 12^
16. Development of L. pomonella on Patana diet, 16 December, 1969, to 15 January, 1970 ...... 125
17. Development of L, pomonella on Patana diet, 25 January to 25 February, 1970 ...... 126
18. Development of L. pomonella on modified Patana diet, 1 March to ^ April, 1970 ...... 127
vii LIST OF FIGURES
Figure Page
1. Diagram of the location of apple trees comprising the North Block of Overlook Orchard ...... 58
2. Relative increments in populations of second- generati„n L. pomonella larvae injuring fruit on virus-treated and untreated trees in 1967 67
3. Accumulation of mature second-generation L. pomonella larvae under ti'unk bands...... 69
4. Rates of development of four populations of L. pomonella reared on artificial diets ...... 86
5. Distribution of number of eggs laid and proportion which hatched for nine female L. pomonella reared on the Redfern wheat germ diet, July, 1969 90
6. Distribution of number of eggs laid and proportion which hatched for 23 female L. pomonella of Washington strain reared on the Redfem wheat germ diet, May to August, 1969 ...... 91
7. Distribution of number of eggs laid and proportion which hatched for 24 female L. pomonella reared on the Patana lima I'd an diet, January, 1970 ...... 92
8. Distribution of number of eggs laid and proportion which hatched for 33 female L. pomonella reared on the Patana lima bean diet, March, 1970 ...... 93
9. Distribution of number of eggs laid and proportion which hatched for 42 female L. pomonella reared on the Patana diet modified by the addition of sucrose, apple seeds, and linseed oil. April, 1970 94
viii INTRODUCTION
The codling moth, Laspeyresia pomonella (L.) (Lepidopteras
Olethreutidae), is an important pest of apple and other pome fruits in nearly every region of the world where they are cultivated. Fre quent applications of insecticides are often required to protect fruit from injury.
A granulosis virus pathogenic for L. pomonella was recovered from dead larvae found in Mexico. In laboratory studies the virus when ingested in sufficient quantity was found to readily cause the death of larvae. The virus is receiving the attention of entomologists who are looking for microbial insecticides for insect pests. The virus will probably infect only a small number of closely related species. In that respect it might be used as a "selective" insecticide that would control L. pomonella but not affect other animals in the treated area.
If it can be used as a selective insecticide, predators and parasites can be preserved to provide natural regulation of pests.
The granulosis virus was introduced into an orchard in south- central Ohio so that its effects on L. pomonella could be studied. No information concerning the virus-host interaction in an orchard situ ation was available at that time. A small amount of virus in aqueous suspension was applied to two apple trees with an orchard sprayer.
Treated and control populations of L. pomonella were examined in order
1 2 to determine the effect of the virus. The introduced virus apparently did cause mortality in the populations on treated trees. This was, to my knowledge, the first study of this granulosis virus in an orchard in the north-central region of the United States.
The rearing of L. pomonella on artificial diets was investigated for the purpose of increasing the production of the insect for labora tory study and virus propagation. A good artificial diet for labora tory rearing would permit the production of L. pomonella for investiga tions of the biology, physiology, genetics, pathology, and other char acteristics of the species. The granulosis virus pathogen can only be multiplied in the living host.
The artificial diets were evaluated by measuring survival, dura tion of development, and oviposition of L. pomonella. A lima bean diet which was used for the first time to rear this species produced immedi ate improvements in production. Survival was increased and development time was reduced from that obtained with the standard rearing medium. REVIEW OF LITERATURE
THE CODLING MOTH, LASPEYRESIA POMONELLA (L.)
History and distribution
From its probable origin in the Euro-Siberian region
(Balachowsky, 1966), L. pomonella has spread to nearly every place throughout the world where its primary hosts, plants of the genus
Malus, are cultivated. It occurs in all of Europe, Russia, the Middle
East, North Africa, India, China, and Japan (Balachowsky, 1966). Cod ling moth depredation was first observed in the United States (New
England and New York) at the middle of the nineteenth century. It may have been introduced to Nova Scotia from France quite early because apples were grown there in I685 (Putman, 1963). It was reported in
Tasmania in 1855» in New Zealand in I865, and had invaded parts of
Australia by 1885 (Frogatt, 1901, cited in Balachowsky, 1966). L. pomonella has been known in South Africa since I885 and South America in the last half of the nineteenth century (Balachowsky, 1966). It appeared in California in 1873 (Slingerland, 1898, cited in Balachowsky,
1966) but not until 1905 in British Columbia (Putman, 1963).
Host plants
The primary hosts of L. pomonella are the pomaceous fruits
3 apple, pear, and quince. It is a conspicuous pest when the production of fruit is concentrated in orchards. Secondary hosts of economic importance are apricots and walnuts and sometimes peaches, plums, and almonds (Balachowsky, 1966).
Classification
Until very recently the codling moth was known to North American entomologists as Carpocapsa pomonella (Linnaeus). In his revision of the classification of North American Olethreutidae, Heinrich (1926) placed Carpocapsa in the subfamily Laspeyresiinae. Heinrich indicated that there was not much to distinguish the adult C. pomonella from mem bers of the genus Laspeyresia Httbner. He included 36 North American species in the genus Laspeyresia while retaining pomonella as the sin gle Carpocapsa species. Mackay (1959) in describing the larvae of the
North American Olethreutidae related C. pomonella more closely to the
Laspeyresia Httbner than to Carpocapsa saltitans Westwood, the only other North American species.
Linnaeus named the codling moth Phalaena Tinea pomonella and described it in the Tenth Edition of his Systema Naturae (Vol. 1, p. 538) in 1758. Treitschke in 1830 (Schmet. Eur,, Vol. 8, p. 160) placed this insect in the genus Carpocapsa but spelled the species name as pomonana. Walsingham in 1897 (Proc. Zool. Soc. London, p. 130) included pomonella in the genus Cydia. The synonym Cydia pomonella
(L.) has frequently been used by European writers. 5
Laspeyresia pomonella (Linnaeus)
Synonomyi Phalaena Tinea pomonella Linnaeus, 1758
Carpocapsa pomonana Treitschke, 1830
Carpocapsa pomonella (Linnaeus)
Cydia pomonella Walsingham, 1897
Morphology
The life stages of L, pomonella were described by Tadic" (1957).
Forbes (1923) gave a description of the adult including a figure of the wing venation, and a map of the seta of the larva, Heinrich (1926) included drawings of the male and female genitalia, A technical des cription of the last-instar larva is given by Mackay (1959). The des criptions which follow are taken from these authors.
The adults are small, gray moths. The body is about 10 mm long, and the wingspread is about 20 mm. The forewings are marked with fine alternating light and dark gray striations. The distinguishing feature of the moth is a brown field near the tip of the forewing which is nearly enclosed by two bronze-colored bands. This area is marginned proximally by a black band. The hind wings are light gray to brown, without markings. The body of the moth is covered with gray scales.
Male and female can be distinguished by the external genitalia of the terminal abdominal segments. Paired, spoon-like claspers (harpes) are located at the posterior end of the male's abdomen. A small fleshy area without scales is seen underneath the tip of the female's abdomen.
This is the egg exit (oviporus). The copulatory pore (vulva) is usu ally visible on the posterior edge of the eighth abdominal stemite. 6
The male also bears long hairs along the vein Cu of the hind wing.
Eggs of L. pomonella are laid singly and are cemented to a sur face, usually a leaf or fruit. The egg is creamy-white in color, and is flattened. It is somewhat elliptical, in shape, and its greatest diameter is 1.0 to 1.2 mm. The surface of the egg is reticulated when seen through a microscope. The development of the embryo can be seen through the partially transparent chorion. The embryo develops cen trally, surrounded by yolk cells. A transitory "red ring" stage occurs when the yolk turns reddish. A "black spot" stage occurs when the head capsule of the larva darkens, about one day before hatching.
When it hatches, the first-instar larva is about 2 mm long. Its head is black and wider than its body. The body is creamy white or pale yellow in color. There are normally five larval instars. The last-stage larva is 18 to 20 mm long. The head capsule is brown and the prothoracic shield is nearly colorless. The dorsal side of the full-grown larva is usually pink, ranging from a p>ale shade to deep rose. The ventral side is creamy-white to yellowish. The codling moth larva is distinguished from larvae of the oriental fruit moth and les ser apple worm by the absence of an anal comb.
The sexes of individual larvae can be distinguished in the fifth instar. The testes of male larvae can be seen through the dorsal integument of the fifth abdominal segment, opposite the third pair of prolegs. They appear as two black spots along the midline} one lobe lies on either side of the heart. The two black spots, of course, are not present in the female fifth-instar larva.
The mature larvae transform into pupae. Pupae are 10 to 12 mm 7
long. The cuticle tans to brown within a day following pupation. As
the emergence of the adult nears, the darker pigmentation of the wing
scales can be seen through the pupal cuticle.
Although the pupae of males and females are very similar, they can be distinguished by characters of the abdominal cuticle (Tadi£,
1957). Ventrally, the eighth abdominal segment of the female is con
stricted at the midline, and there is an impressed line running between the anterior and posterior margins. The male is distinguished by the presence of two small elevated spots on either side of the ventral mid line on the ninth abdominal segment. The eighth abdominal segment of the male is not constricted ventrally as in the female. Another method for the sex determination of codling moth pupae is given by Peterson
(1965)* Counting caudally on the ventral side from the tip of the wing pads, males have four dark intersegmental bands and females have only three.
Behavior
The seasonal activity of L. pomonella begins in the spring, when diapause is broken. Warming temperatures following the cold of winter signal the return of activity. Pupation occurs in the cocoon where the fifth-instar larva spent the winter. Just before eclosion the pupa pushes itself about halfway free of the cocoon and exit tube. Propul sion is provided by movements of the abdomen and is aided by transverse rows of teeth on the dorsum of abdominal segments which prevent back ward movement (Tadic, 1957). When transformation is completed, pres sure from the moth splits the pupal skin longitudinally down the dorsal 8 surface of the thorax and transversely behind the head. The moth frees its head and legs and then pulls its body from the pupal case. The wings are shorter than the abdomen when the moth first appears, but after several minutes they expand to their normal length (Tadic, 1957)•
During daylight hours, the moths remain inactive and are con cealed under leaves in the tops of trees (Tadic, 1957)* After sunset and when the temperature is above 60° F., the moths become active and take flight (Tadic, 1957)* Male and female moths may mate within a day after eclosion, and the female may begin oviposition about two days after emergence, Oviposition occurs in twilight when the temperature is above 60° F., the air is calm, and there is no rain (Isely, 1938)*
Van Leeuwen (1940) using marked moths and bait traps found that the average distance traveled by released moths was 143 feet, Steiner
(1940) in five years of tests found that marked moths flew an average of 200 feet, but he recaptured some up to 2,000 feet from point of release.
The female lays eggs singly, usually on the upper surfaces of leaves of fruit clusters. The female moths have shown a slight prefer ence for depositing eggs in the tops of trees rather than in the lower portions, resulting in a larger proportion of damaged fruit in the tops of trees (Richardson and DuChanois, 1950a* 1950b; Woodside, 1944;
Summerland and Steiner, 1943). MacLellan (1962) states that eggs in thoroughly examined trees were seldom found more than six inches from fruit. In Ohio females laid the greatest number of eggs three or four nights after emergence (Outright, 1964), Isely (1938) observing caged moths determined that in midsummer a female lays 90 per cent of her 9
eggs during the first seven days of the oviposition period.
The number of eggs laid by a single female may be higher than
300. List and Yetter (1927, cited in Putman, 1963) found more than 300
eggs and oftcytes in ovaries of dissected moths. The largest number of
eggs reported laid by a single caged moth was 345 (Isely, 1938). Caged
moths in laboratories or insectaries are commonly reported to lay an
average of 50 to 60 eggs per female. Putman (1963) thought that this
was considerably fewer than were normally laid in an orchard.
Larvae hatch during a period of 8 to 13 days following oviposi
tion, depending upon temperature (Cutright, 196*0. The tiny larvae
must find food within a day or less, or they will starve or desiccate.
Larvae crawl, seemingly without direction, until they happen upon a
fruit. Steiner (1939) reported observing larvae entering apples as far
as 10 feet from the site of oviposition. When a fruit is reached, the
larva next searches for a place to enter. The depressions of the calyx
or stem provide good protected places for the larvae to enter the
fruit. Entry may be made on the side of a fruit where the surface is
roughened by some other form of damage. The tiny larva can quickly
penetrate the skin (cuticle) of the fruit and when insidj will cover
the entrance with silk to conceal itself (Putman, 1963).
The development of the larva takes place entirely within the
fruit. As it feeds, the larva is tunnelling more or less directly to the center of the fruit. Larval development in a fruit requires 16 to
25 days in Ohio (Cutright, 1964). Near the end of this period the
larva reaches the seeds and feeds on these to get nutrients which are apparently essential to the completion of its development (Tadic, 10
1957)* When the larva has finished its feeding in an apple, it enlarges the tunnel by which it entered the apple, or cuts a new tunnel by which it emerges from the fruit (Putman, 1963).
MacLellan (i960) states that most larvae leave fruit during the night. They crawl down branches from fruit on the tree, or sometimes lower themselves to the ground on threads of silk that they spin. The fully grown larvae of the first summer generation search for places in which to transform to adults. Larvae usually find concealment tinder bark scales, or in cracks or crevices on larger limbs and the trunk.
In a protected place the larva constructs around itself a cocoon from the silk it elaborates. Cocoons of non-diapause larvae are elongate and have rather thin walls. There is usually an attached exit tube which is closed by a circular partition at the inner end (Putman,
1963). The individual may spend 14 to 20 days in the cocoon (Cutright,
1964) before emerging as an adult.
In Ohio, moths of the first summer generation lay eggs as those of the preceding generation did except that more of the eggs may be laid directly on apples, now that the fruit are larger. Development is the same as in the preceding generation, but the duration may be shorter because of higher temperatures. In Ohio, the second generation is the last complete one of the year (Cutright, 19&5)• The shortening daylight periods in August and September induce a state of diapause in the fifth-instar larvae. Full-grown larvae leave the fruit and conceal themselves as did the larvae of the preceding generation. However, development temporarily ceases and the larva remains in its cocoon (or hibemaculum) through the winter (Tadic, 1957). 11
The most important factor producing diapause is the decreasing length of daylight as the summer season draws to an end. Dickson
(19^9) described the role of the daily photoperiod in the induction of diapause in the codling moth. Peterson and Hamner (1968) have deter mined that diapause results when the length of the photoperiod to which larvae are exposed is 13.5 hours or less. Hanson and Harwood (1968) demonstrated some changes that characterize diapausing larvaej (i) testes are reduced to half normal size; (ii) gametogenesis ceases;
(iii) spermatids degenerate; (iv) oxygen consumption is reduced; (v) the amount of cytoplasm in the midgut epithelium decreases; (vi) mitochon drial activity in fat body cells is reduced; and (vii) the larva con structs a thick-walled circular cocoon.
Nearly all of the overwintering larvae are found on the trees on which they developed, even though a large proportion may have first dropped to the ground (Gould and Geissler, 19^1). Loose bark scales provide cocooning sites for many larvae, while cracks, cavities, prun ing scars, and ends of broken branches are shelter for others. Headlee
(1929a, 1929b) found in a New Jersey orchard that 91 P®r cent of the larvae on six trees overwintered on the trunk and large branches covered with rough bark, 9 per cent on smaller branches, and none on the ground. Other authors have found varying proportions of the larvae hibernating in the soil or among debris on the ground (Chandler, 1928;
Steiner, 1929J Youthers and Carlson, 19^1). In essence, overwintering larvae are most abundant where suitable shelter for cocooning is avail able. Larvae which remain on the ground may be consumed by predators
(MacLellan, i960). 12
Diapause is broken in the spring when rising temperatures permit
the resumption of development. Peterson and Hamner (1968) found that
after chilling diapausing larvae at ^.5° C. for 12 days or longer a
large proportion terminated diapause after being returned to a tempera
ture of 24° C. Also, they reported that diapausing larvae broke dia
pause after 28 days at 2b° C. and a 16-hour photoperiod without any
exposure to cold. Hanson and Harwood (1968) found that the breaking of diapause was accompanied by the resumption of gametogenesis, increased
oxygen consumption, increased activity of mitochondria in fat body cells, and the remodeling or reconstruction of the cocoon. According to Putman (1963), prior to pupation the overwintered larva cuts a hole in its cocoon and constructs an exit tube with a partition at the inner end. NATURAL CONTROL OF L. POMONELLA
Parasites and predators
Many parasites have been recovered from L. pomonella in North
America but only two species, Ascogaster quadridentata Wean, and
Trichogramma minutum Riley, occur commonly (Putman, 1963). Ascogaster quadridentata Wesm. (= A. carpocapsae Viereck) (Hymenoptera: Braconidae) deposits its egg in the cytoplasm of a host egg. The parasitism of L.
pomonella by A. quadridentata was described by Cox (1932). The para
site larva feeds inside the fifth-instar host larva and greatly stunts
its growth. The fourth-instar parasite larva feeds externally and com pletes the destruction of the host. The parasite spins its cocoon inside the cocoon of the host. The parasite will overwinter as a first-instar larva in the body cavity of the diapausing host larva and resume development along with the host in the spring,
A, quadridentata parasitism of mature L. pomonella larvae under trunk bands was as high as 41 per cent on unsprayed trees and 19 per cent on sprayed trees in western New York in 1931 (Cox, 1932). In
Georgia, 21.3 per cent of the larvae examined in 1936 were found to be parasitized by A. quadridentata (Webb, 1937). Average parasitism over five years by A. quadridentata in an unsprayed Ontario orchard was 21 per cent (Garlick, 1948). A. quadridentata was found to be responsible for 94 per cent of the 5.7 per cent larval parasitism during a
13 14 three-year study in West Virginia (Jaynes and Marucci, 1947), A. quadridentata was reported to have killed 9 per cent of the hibernating larvae during the winter 1959-60 in Nova Scotia (MacLellan, 1962).
In most situations, the effectiveness of A. quadridentata is limited by hyperparasitism. In Georgia, 37 per cent of the A. quadridentata found in L. pomonella larvae were parasitized by
Perilampus spp. (Hymenopteraj Perilampidae) (Webb, 1937). Perilampus capitatus Smulyan was found to parasitize A. quadridentata in West
Virginia (Jaynes and Marucci, 1947). A. quadridentata in codling moth larvae was parasitized by Perilampus tristis Mayr, P. fulvicomis
Ashmead, and Dibrachys cavus (Walker) (Hymenopteraj Pteromalidae) in
Ontario (Boyce, 1948). In western New York Dibrachys boucheanus Ratz
[= D. cavus (Walker)] was reared from L. pomonella larvae parasitized by A. quadridentata (Cox, 1932).
Trichogramma minutum Riley (Hymenopteraj Trichogrammatidae) is a common parasite of L. pomonella eggs. Parasitism prevents development of the host embryo (Sweetman, 1958). In an unsprayed orchard in
Indiana in 1938, 32 per cent of the first-brood eggs and 43 per cent of the second-brood eggs were parasitized by T. minutum (Summerland and
Steiner, 1943). In unsprayed and heavily infested orchards in north central Washington during July and August, 1934, 80 to 84 per cent of the codling moth eggs were parasitized by T. minutum (Yothers et al.,
1935)• Up to 28 per cent of the eggs observed in an experimental orchard in West Virginia were parasitized by T. minutum (Jaynes and
Marucci, 1947). In an integrated control orchard in Nova Scotia, 2.4 per cent of the eggs examined in I960 were killed by T. minutum 15
(MacLellan, 1962).
Flanders (1930) described the mass production T. minutum for field release and biological control of L. pomonella. In 1927, 100,000
Trichogramma released on six walnut trees produced an average of 36.4 per cent egg parasitism. When T. minutum was released on several apple and pear trees, the highest artificial parasitism was 72.9 per cent, while natural parasitism was as high as 45.7 per cent (Flanders, 1930).
In the opinion of Dolphin and Cleveland (1966), mass rearing and inun dative release of T. minutum generally were not economical or effective enough to compete with chemical control methods.
Other parasites have been recovered from L. pomonella larvae, but their controlling effects were considered insignificant (Webb,
1937s Jaynes and Marucci, 1947? Boyce, 1948). In Georgia, Pristomerus agalis (Cresson) accounted for 2.7 per cent of larval parasitism, and
Phanerotoma tibialis (Hald.) (Hymenopteraj Braconidae) accounted for
1.4 per cent (Webb, 1937). In West Virginia, 16 Hymenoptera and one
Diptera (tachinid) were reared from parasitized larvae, but altogether these were responsible for only 6 per cent of the total parasitism
(Jaynes and Marucci, 1947). Fifteen species of minor parasites found in Canada were listed by Boyce (1948).
Small numbers of four species of parasites found in Europe were introduced into some orchards in the Niagara Peninsula of Ontario,
Canada (Boyce, 1948). The species were Ephialtes caudatus Ratz.
(Ichneumonidae) and Cryptus sexannulatus Grav. from France, and Elodia tragica (Meig.) and Pristomerus vulnerator Panz. from England. However, these parasites did not become established and were not recovered in 16 later years (Putman, 1963).
Populations of L. pomonella eggs and larvae may be reduced by predators. In an Indiana orchard during 1938, 19 per cent of the eggs examined were destroyed by predators, predominantly larval chpysopids
(aphis lions) (Summerland and Steiner, 19*4-3)• Larvae and adults of
Tenebroides corticalis Melsheimer (Coleopteras Ostomidae) were found feeding on larvae under burlap bands (Woodside, 19*4-2). In California, larvae of Cymatodera ovipennis (LecCont) and Cymatodera sp. (Coleopteras
Cleridae), and a larval raphidiid were observed feeding on larvae
(Lloyd, 19*4*0.
Jaynes and Marucci (19*4-7) observed predation on L. pomonella in
West Virginia orchards during 1938, 1939, and 19*4-0, and reported the following information. Thrips, Leptothrips mali (Fitch) (Thysanoptera:
Phloeothripidae), were observed feeding on eggs. Adult coccinelids
Hippodamia convergens Guerin, Anatis quindecimpunctata (Olivier), and
Coccinella novemnotata Hbst. consumed eggs when confined with them.
Predation of larvae under trunk bands in an unsprayed orchard was as high as 1*4-.*4- per cent. Ants, especially Solenopsis molesta (Say), were the most numerous predators of larvae under bands. Adult Tenebroides corticalis Melsheimer (Coleopteras Ostomidae) were also seen feeding on larvae under bands. Ants appeared to be important predators of larvae seeking cocooning sites, for 90 per cent of the larvae released on the ground in tests were killed by Formica fusea var. subsericea Say. Ants were also observed feeding on pupae in cocoons.
Studies of L. pomonella in Nova Scotia revealed another set of predators. Four mirids (Hemiptera) Diaphnidea sp. (probably pellucida 1?
Uhler), Hyaliodes harti Knight, Philophorous perplexus D, and S., and
Blepharidopterous angulatus (Fall.) were predators of eggs and young larvae. In 1961, 19.7 per cent of the eggs examined were destroyed by predators. The mirid Phytocoris sp. (probably dimidiata) and the mite
Anystis agilis Banks were common predators of young larvae (MacLellan,
1962, 1963). For a period of seven years, woodpeckers destroyed an average of 52 per cent of the larvae overwintering on tree trunks. The species responsible were the hairy woodpecker, Dendrocopos villosus villosus (Linnaeus), and the northern downy woodpecker, Dendrocopos pubescens medianus (Swainson) (MacLellan, 1958).
Pathogens
Several pathogenic organisms have been found to produce limited mortality in natural codling moth populations. Pathogenic fungi, bac teria, viruses, and protozoa have been isolated from diseased field- collected codling moth larvae.
As much as 40 per cent of the population of codling moth larvae in a Virginia orchard were killed by a fungus, probably Hirsute11a subulata Petch (Charles, 1941). Another pathogenic fungus, Beauveria bassiana (Balsamo, Vuillemin), was found to produce a low level of lar val mortality among codling moths in West Virginia orchards (Jaynes and
Marucci, 1947). Artificial application of spores in sprays and dusts increased larval mortality considerably. These authors also noted that the incidence of B. bassiana disease was higher in cool, wet seasons.
B. bassiana was also observed to cause mortality of larvae overwinter ing under the bark of Persian walnut trees in California (Michelbacher 18
et al., 1950). Several fungus species were tested to determine their
pathogenicity (Arkhipova, 1965)* Beauveria globulifera Pic, and
Metarrhizum anisopliae (Metsch.) Sor, were found to be more pathogenic
than Beauveria bassiana. Spicaria farinosa Fr. and Hirsute11a subulata
Petch were only slightly pathogenic.
Strains of the bacteria Bacillus cereus Frankland and Frankland
have been isolated from diseased L, pomonella larvae in the United
States and Canada. These bacteria are moderately pathogenic when fed
or injected into the hemocoel (Stephens, 1952). When artificially
established populations of larvae were sprayed with suspensions of B.
cereus spores, survival was significantly lower than in an untreated
population (Phillips, Bucher, and Stephens, 1953). Applications of B.
cereus spores to trees at 2.7 x 10^ spores per square millimeter did
not protect apples from damage (Stephens, 1957).
Commercial insecticide preparations of the bacteria Bacillus
thuringiensis Berliner have been tested for codling moth control. In a
New York orchard damage was reduced by 50 per cent in one season, but
protection was not adequate for commercial production (McEwen et al.,
I960). Fewer codling moth entries were noted on fruit sprayed with
commercial preparations of B. thuringiensis (Jaques, 1961). A rather
high dosage of B. thuringiensis was needed to kill codling moth larvae
as compared to some other lepidopterous pests. Thirty per cent of the
larvae on treated apples completed development (Roerich, 1964). Appli
cations of B. thuringiensis to apple trees in Wisconsin reduced codling moth damage by 50 per cent in a year when the population was low, but
in a year when the population was high the reduction was only from 19
95.5 per cent to 91.3 per cent damage (Oatman, 1965). European red
mite populations increased in test plots where B. thuringiensis was
used (Oatman, 1966). Dolphin et al. (196?) tested a B. thuringiensis
preparation, Thuricide 90 T S ® , containing 30 x 10^ spores per gram,
which was mixed at the rate of 21 pounds per 100 gallons of spray.
After trees were sprayed with 10 gallons of this mixture, mortalities
of 6 0 , 54, and 42 per cent were produced when treated fruit was
infested with larvae in the laboratory. The authors suggested that B.
thuringiensis might be used to selectively reduce codling moth popula
tions in biological or integrated control programs.
A nematode (Rhabditida; Steinemematidae) was recovered from
diseased codling moth larvae found in a Virginia orchard (Dutky and
Hough, 1955). It was discovered that the nematode, called DD-I3 6 , was
the vector of a bacterial pathogen which killed larvae (Dutky, 1959).
The bacterium was described as a new species, Achromobacter
nematophilus, by Poinar and Thomas (1965). The taxonomic position of
the DD-I36 nematode was investigated, and it was found to be conspe-
cific with Neoaplectana carpocapsae Weiser isolated from L. pomonella
larvae in Czechoslovakia (Poinar, 1967).
When suspended in water and sprayed on the trunks and larger
branches of apple trees, the nematodes produced at least 60 per cent mortality among larvae seeking cocooning sites (Dutky, 1959). The nematode was propagated in larvae of the wax moth, Galleria mellonella
L. (Dutky, 1959) or on pork kidney on peptone glucose agar slants
(Dutky et al., 1964). Infection is initiated when third-stage juvenile nematodes enclosed in the cuticle of the second stage are ingested by 20 the insect. They emerge from the second-stage cuticle soon after reaching the insect's crop or midgut and work their way through the midgut epithelium into the hemocoel. Cells of the bacterium
Achromobacter nematophilus Poinar and Thomas are released from the nematode's intestine into the insect's hemocoel (Poinar and Himsworth,
1967). The bacterium multiplies quickly in the hemocoel and causes death within 48 hours (Poinar and Thomas, 196?). The third-stage nema todes invade the fat body, Malpighian tubules, silk glands, and muscles of the host before its death (Poinar and Himsworth, 19&7)• Developing nematodes eat the bacteria as well as tissues of the dead host (Poinar,
1966), and they cannot reproduce unless A. nematophilus is in the hemo coel of the host (Poinar and Thomas, 1966). Eggs hatch within the female and larval nematodes consume most of her tissue before escaping into the. host's hemocoel (Welch, 1963). Three or four generations may develop in one host (Dutky, 1959). As many as 200,000 ensheathed infective-stage nematodes may be produced from one wax moth larva
(Poinar and Thomas, 1966).
Until a new host is found, the infectious third juvenile stage will survive at moderate to low temperatures if kept moist. It does not feed, and the bacterium is retained in its intestine (Welch, 1961).
The successful use of the nematode for insect control requires applica tion in a rather moist environment. It can probably be used most suc cessfully against aquatic and soil insects (Welch, 1961). When sprayed onto foliage, survival time is limited by desiccation. A HISTORY OF APPLIED CONTROL OF L. PQMONELLA
Early methods
In the nineteenth century before the advent of chemical control measures, most apple production was in relatively small farm orchards.
There was not much that the farmers could do to protect their fruit from the ravages of L. pomonella. The Ohio State Horticultural Society in 1867 recommended that dropped fruit be fed to hogs or be boiled in water to kill the larvae (Cutright, 196*0. Bands made from twisted hay were wrapped around tree trunks to catch mature larvae. The bands were removed and burned at weekly intervals (Cutright, 1964). The losses of apples due to codling moth damage in the last quarter of the nineteenth century were often estimated to be as high as 50 to 75 per cent
(Cutright, 1964).
Chemicals were first used in Ohio about 1880. Paris green and london purple were used against L. pomonella and the plum curculio
(Cutright, 1964). The first orchard-spraying machines were used in
Ohio around 1887, and the first Ohio Spray Schedule with recommenda tions for insect and disease control was distributed in 1897 (Cutright,
1964). Lead arsenate replaced other chemicals for codling moth control after the beginning of the present century. Lead arsenate was more persistent, less phytotoxic, and was compatible with the lime sulfur that was required to combat the recently introduced San Josi" scale
21 22
(Putman, 1963). Lead arsenate was widely used and provided good con trol in Ohio up to 1930 (Cutright, 1964).
Although it provided good control when first introduced, lead arsenate gradually became less effective. In an apple orchard in southern Ohio 80 per cent of the fruits were damaged in 1944 despite a petal-fall spray and six cover sprays of lead arsenate (Cutright, 1954).
Hough (1928, 1929, 1943) demonstrated that selection for the more resistant individuals was creating more resistant populations of cod ling moths in orchards in Virginia where lead arsenate was used.
Steiner et al. (1944a) studied, in Indiana, an orchard sprayed with lead arsenate and an orchard which had not been sprayed for five years.
Nearly all of the fruit in both orchards was injured. When codling moths from these two orchards were examined for strain differences, it was observed that larvae of the strain less susceptible to lead arse nate crawled over sprayed fruit for shorter distances and spent less time in entering fruit. The authors suggested that the mechanism of resistance was behavioral rather than physiological. The diminishing effectiveness of lead arsenate was a general occurrence in United
States orchards, and a search for new ways to control the codling moth was initiated.
The banding of trees to capture and kill mature larvae made a brief reappearance. Siegler et al. (1927) suggested the use of cloth bands impregnated with a poison, beta-naphthol, for supplementary con trol of the codling moth. Worthley (1932, 1934) reported on the test ing of chemically treated bands in orchards in Pennsylvania, The use of beta-naphthol-treated corrugated paper bands in conjunction with 23 thorough scraping of the bark to eliminate other cocooning sites killed many codling moth larvae (Newcomer et al., 1933* Steiner and Ackerman,
19365 Baker, 1944). Worthley (1932) reported that on five thoroughly scraped, unsprayed trees, 96.84 per cent of the larvae of one genera tion on the trees were trapped and killed under corrugated strawboard bands treated with an emulsion of beta-naphthol and lubricating oil.
However, the reduction of populations by this method was not great enough to prevent economic damage to fruit, and considerable labor had to be expended to remove potential codling moth cocooning sites from the trees each season.
Modern insecticides
The first tests of DDT in Ontario in 1944 gave encouraging results (Putman, 1963). For Ohio in 1946 the general use of DDT pro duced the lowest level of injury in many years (Cutright, 1964).
Steiner et al. (1944) in field tests in Indiana found DDT more effec tive than the standard lead arsenate program. Three per cent of the
DDT-treated apples were infested, while 25 per cent of the lead- arsenate-treated apples were infested. Harmon (1945) found in western
New York, where lead arsenate resistance was not yet a problem, that even at the low concentration of 0.8 pound per 100 gallons, DDT was as effective as the standard lead arsenate program. Newcomer and Dean
(1953) tested DDT for seven years at Yakima, Washington. They found that three or four applications of DDT (2 and 1 pounds per 100 gallons respectively) gave much better control than six or seven applications of lead arsenate. 2k
Cutright (195*0 found that DDT was much more effective than lead
arsenate in 19**5 in a heavily infested orchard in southern Ohio. Total
injury was 51 per cent on trees treated with lead arsenate and 2.3 P«r cent on trees treated with DDT (five cover sprays each). A DDT spray program was immediately adopted in this orchard. However, in 1951 ade quate control of codling moth was not obtained with DDT (Cutright,
195*0 • Use of parathion in the program in 1952 improved control. In another test in the same orchard in 1953 about 1*+ per cent of the apples sprayed with lead arsenate were infested, while about 95 per cent of those sprayed with DDT were infested. Cutright (195*0 sug gested that this strain of codling moth had become resistant to DDT.
Hamilton (1956) tested first-instar larvae from this strain in the laboratory and concluded that they, as well as larvae from two
Washington strains, were definitely resistant when compared with larvae of 13 other strains. Glass and Fiori (1955) and Barnes (1958) con firmed the existence of DDT-resistant strains in New York and
California, respectively.
However, the use of DDT was accompanied by increasing damage from previously minor apple pests. Populations of red-banded leaf roller, plum curculio, eye-spotted budmoth, apple aphid, and orchard mites increased when DDT was used. These pests were not controlled by
DDT, and their natural predators and parasites were suppressed when DDT was used (Hough and Hill, 195**; Oatman and Libby, 1965; Oatman, 1966),
Steiner et al. (19****) observed increasing populations of the European red mite, Panonychus ulml (Koch), and the two-spotted spider mite,
Tetranychus urticae (Linnaeus), and the absence of the mite predator 25
Stethorus punctum (LeConte) (Coleoptera: Coccinellidae) on trees sprayed with DDT.
Other chemicals were being tested for codling moth control, even while DDT was effective in most orchards. The organo-phosphate insec ticide parathion was used in some Ohio orchards where L. pomonella was suspected to be resistant to DDT (Cutright, 1964). Carbaryl, a carbam ate insecticide, was tested in 1955 and found very effective (Cutright,
1964). Azinphosmethyl was also found to be an excellent insecticide for control. Carbaryl and azinphosmethyl were first used commercially in Ohio in 1958* and through their use excellent control was maintained into the 1960's (Cutright, 1964).
In 1956 and 1957 Madsen and Hoyt (1958) tested some of the new insecticides in Washington. Ryania (a botanical), carbaryl, and azin phosmethyl gave control comparable to the standard DDT applications.
They also tested materials in an orchard where codling moths were not controlled with DDT. Trithion, ethion, carbaryl, and ryania provided satisfactory control of the heavy infestation, Hamilton and Cleveland
(1957) tested ryania and found that when used at 6 pounds per 100 gal lons of water, it compared favorably with DDT used at 2 pounds of 50 per cent wettable powder per 100 gallons. They also found that nico tine and oil combined with 3 to 4 pounds of ryania (95*8 per cent wet- table powdei) per 100 gallons gave improved control, especially in reducing the number of larval stings on fruit.
Chiswell (1962) in England also found that azinphosmethyl, car baryl, and ryania were the best new chemicals for codling moth control.
Chiswell observed that populations of the European red mite increased 26 considerably when carbaryl was applied, and to a lesser extent when diazinon and ryania were used. He also found that azinphosmethyl pro vided long-term control of the European red mite. Oatman and Libby
(1965) determined in Wisconsin that azinphosmethyl, diazinon, and a lead-arsenate-DDT combination were the most effective chemicals for the control of codling moth as well as the other important insect pests.
Autocidal control
Proverbs (1964) described autocidal control as "the sustained overflooding of the native population with sexually sterile males."
The release of reproductively sterile moths into natural populations for the purpose of regulating or eradicating the codling moth has been for the past 10 years the primary research of two projects, one in
Washington state and the other in British Columbia.
Proverbs and Newton (1962a) tested gamma radiation as a means of sterilizing codling moths. After analyzing the effects of various dos ages of gamma radiation on eggs, larvae, pupae, and adults, it was determined that the greatest reduction in fertility without undesirable side effects was produced by exposing mature pupae or newly emerged adults to a dosage of 40 krad of gamma radiation. A cobalt-60 radi ation source was used. This dosage of gamma radiation produced domi nant lethality in at least 98 per centcf the sperm without affecting adult emergence, mating behavior, or adult longevity. Irradiation of other stages (eggs, mature larvae, or young pupae) caused too much mortality. However, sperm from irradiated males were not so competi tive as those from normal males (Proverbs and Newton, 1962b). In cage tests when irradiated male moths were released at a ratio of 10 to one normal male and one normal female, there was a reduction of 75 per cent in the number of progeny. Additional caged release tests were reported by Proverbs and Newton (1962c). When they released moths on caged apple trees in a ratio of 10 irradiated males to one normal male and one normal female, the resulting reduction in mature larval progeny was about 85 per cent. When the experimental ratio was 20 irradiated males to one normal male and one normal female, the reduction was nearly 98 per cent. When an equal proportion of irradiated female moths was released with irradiated males in a ratio of 20:20s1:1, the reduction in mature larval offspring was slightly less, 94 per cent.
Proverbs and Newton (1962c) observed that irradiated males mated satisfactorily with normal females, and that the mated females laid a normal complement of eggs, but the embryos usually died at or before the red ring stage.
Hathaway (1966) also experimented with gamma radiation for cod ling moth sterilization. He too found that 40 krad produced sterility in 98 per cent of the males treated, and that females treated with 20 krad deposited no viable eggs. He reported somewhat smaller reductions in numbers of progeny in field cage tests than did Proverbs and Newton
(1962c). I irradiated males crossed at a 20:1:1 ratio with untreated males and females reduced the progeny by 84 per cent. When both irra diated males and females were crossed with normal males and females at a 20:20:1:1 ratio, the reduction of offspring was 76 per cent.
Hathaway (1966) suggested that a field release of sterile males in a ratio of 30 or 40 to one wild male could substantially reduce the 28 natural population.
Proverbs and Newton (1962c) theorized* “When the ratio of irra diated males to normal males is 20»1, the irradiated insects outnumber the normal males so greatly that each female almost invariably mates first with an irradiated male and lays most of her complement of eggs before she copulates with a normal male."
For three years sterile males were released during the peak of emergence of the first of two seasonal broods in an abandoned 20-tree apple orchard in British Columbia (Proverbs et al., 1966). In 1962,
1963, and 1964, 21,300, 67,500, and 89,200 irradiated male moths were released, respectively. The ratios of sterile males to natural males determined by light trap collections were 8*1 in 1962, 21*1 in 1963» and 715*1 in 1964. The damage to apples by second-brood larvae was reduced from about 5 per cent to 0.05 per cent in this period.
The investigators in British Columbia later tried to increase the efficiency of the sterilization procedure by irradiating moths instead of pupae, and releasing sterile female as well as male moths in the field (Proverbs et al., 1967). In the laboratory the larval progeny was reduced 80 per cent when treated moths were caged with untreated moths at the ratio of 15 irradiated males and 15 irradiated females to one untreated male and one untreated female. Irradiated male and female moths were released in a 2-hectare abandoned apple orchard* 271»000 in 1964 and 478,000 in 1965. The proportion of har vested apples injured by codling moths was reduced from 60 per cent in
1963 to 1.6 per cent in 1964 and 0.3 per cent in 1965.
Field trials of sterile moth release were conducted at Yakima, 29
Washington, in 1966 (White et al., 1969). The test orchard was com prised of 24 apple and five pear trees which had never received insec ticide applications. An average of 500 irradiated moths of both sexes was released per day six days each week from mid-May through mid-
September, for a total of about 60,000 moths. Capture of moths in light and sex-lure traps indicated an average ratio of 23 sterile males to one native male moth. One pre-release application of parathion was made to reduce the native population to a density which could be treated by the number of sterile moths available. The proportion of fruit injured by codling moths was reduced from about 50 per cent in
1965 to 1.57 per cent in 1966.
The sterile moth release technique was tested in a commercial orchard in British Columbia in 1966, 1967, and 1968 (Proverbs et al.,
1969). Moths of both sexes (one day or less of age) were exposed to
50 krad of gamma radiation. They were released three times per week throughout the season of moth activity. Control was more than adequate when the ratio of sterile males to native males was about 20:1 or higher. The occurrence of larval entries when the ratio was allowed to fall to 10:1 or below demonstrated the necessity for maintaining a high ratio. At one time (in 1968) the accidental release of incom pletely sterilized females required emergency control measures and par tially upset the results of the test. In 1966 and 1967* 341,200 and
24,900 sterile moths were released per hectare, and the resulting aver age ratios of sterile to native males were 280:1 and 33*1 respectively.
Damage to fruit at harvest was 0.003 per cent in 1966 and 0.095 per cent in 1967. In 1968, 37,600 sterile moths were released for 30 first-brood control and 167,600 for second-brood control. Resulting average ratios of sterile to native males were 17»1 and 5*+*l for first brood and second brood, respectively. The resulting proportion of apples damaged was 0.71 per cent, of which 0.5*+ per cent was thought to have been produced by progeny of inadequately sterilized female moths that were accidentally released.
Modified spray programs
Several authors have demonstrated that insecticides hinder the natural control of L. pomonella. In western New York during 1931 parasitism of larvae by Ascogaster quadridentata Wesm. varied from 10 per cent in orchards sprayed with a lead arsenate-lime sulfur schedule to *+0 per cent in unsprayed orchards (Cox and Daniel, 1935), It was shown that female parasites exposed to lead arsenate-sprayed foliage lived half as long and parasitized half as many eggs as those not exposed (Cox and Daniel, 1935), Comparison of A. quadridentata para sitism of larvae tinder various spray treatments in New Jersey showed l*+,8 per cent parasitism with lead arsenate, 2k,9 per cent with fixed nicotine and oil, and 33»*+ per cent with fixed nicotine alone (Driggers and O'Neill, 1938), In an orchard sprayed with lead arsenate and lime,
5.1 and 2.6 per cent of the eggs were parasitized by Trichogramma, while in an unsprayed orchard 55.3 end 6*+.5 per cent of eggs were para sitized (Driggers and Pepper, 1936). Parasitism of second generation larvae was much higher in an unsprayed orchard (71 per cent) than in sprayed orchards (7*5 and 16.6 per cent) (Driggers and Pepper, 1936).
Egg parasitism by Trichogramma was reduced about 21 per cent when 31
foliage was sprayed with lead arsenate, and about 48 per cent when lead
arsenate and lime sulfur were used (Boyce, 194-3).
Egg predators Haplothrips faueri Hood and Leptothrips mail
(Fitch) were suppressed by sulfur fungicide use in Nova Scotia
orchards. The predatory mite Anystis agilis Banks was eliminated by
lead arsenate sprays, while the effectiveness of the larval parasite
Ascogaster quadridentata Wesm. was reduced (Pickett and Patterson,
1953). Ryania was first used in Nova Scotia in 1953 and showed consid
erable promise as a selective insecticide (Pickett and Patterson,
1953).
In Nova Scotia 25 pesticides were tested in orchards to deter
mine their effects on predators and parasites. Glyodin and captan
(fungicides) and Ovotran, ryania, nicotine sulfate, and lead arsenate
were selected as relatively harmless chemicals to use in integrated
control orchards (MacPhee and Sanford, 1956). The need for selective
insecticides for codling moth control was emphasized by Clancy and
McAlister (1956) to enhance the natural control of such pests as
orchard mites, red-banded leaf roller, plum curculio, and scale
insects. A ryania program was as effective as the standard commercial
DDT program for control of codling moth in West Virginia in 1952, 1953»
and 1954 (Clancy and McAlister, 1956). DDT eliminated predaceous mites
(Typhlodromus spp.), and without their controlling influence European
red mite populations grew rapidly (Clancy and McAlister, 1956). The
fungicide glyodin was noted to have a mite-suppressing effect (Clancy
and McAlister, 1956).
Ryania also gave good control of the codling moth in tests 32 conducted in New York and Indiana (Hamilton and Cleveland, 1957)* The addition of nicotine in oil helped reduce stings permitted by ryania alone. Ryania, however, did not control plum curculio, red-banded leaf roller, or Forbes scale. In the first season after the use of DDT was discontinued in a New Jersey orchard, codling moth and most other phy tophagous insects were controlled by lead arsenate and ryania, while predators controlled the European red mite (Thomas, Specht, and
Driggers, 1959).
Native predators and parasites have successfully provided eco nomic control of L. pomonella (less than 2 per cent of the crop dam aged) in integrated control programs in Nova Scotia (Wood, 1965). In
Nova Scotia there is only one codling moth generation each year. Dam age to fruit in neglected orchards over a 12-year period ranged between
1 and 12 per cent (MacLellan, 1963). Populations apparently were regu lated in the egg and first-instar larval stages by general predators
(MacLellan, 1963). The use of ryania when needed for control of L. pomonella in Nova Scotia has allowed the survival of predators and parasites which control the eye-spotted bud moth, the gray-banded leaf roller, and the European red mite (Madsen, 1968).
In British Columbia it is hoped that control of the codling moth by the radiation-induced sterility method will eliminate the necessity of using broad-spectrum insecticides which destroy predators and para sites (Madsen, 1968).
In central Ohio, the European red mite was controlled by preda tors when insecticides not harmful to them were used. However, ryania mixed at the rate of 6 pounds of 50 per cent wettable powder per 100 33 gallons of water and applied at the rate of 20 gallons per tree did not give satisfactory control of codling moth, plum curculio, or apple mag got (Holdsworth, 1968; 1970)* The use of azinphosmethyl at the recom mended commercial dosage for control of codling moth and other insects destroyed the European red mite predator complex (Holdsworth, 1968).
The codling moth in integrated control orchards in Washington was ade quately controlled by lower than standard dosages of azinphosmethyl,
Imidan®, and DDT plus parathion which allowed a phytophagous mite,
Tetranychus mcdanieli, to be controlled by a predatory mite,
Typhlodromus occidentalis (Hoyt, 1969). In south central Pennsylvania reduced concentrations of azinphosmethyl controlled the codling moth and allowed effective European red mite predators to survive (Asquith,
1970). LABORATORY REARING OF L. POMONELLA
The most common way to obtain codling moth larvae for laboratory use is to collect larvae that form cocoons under trap bands placed around the trunks of apple trees. Larvae in diapause are first sub jected to cold refrigerator temperatures to break diapause and then stored at 50° F. until needed in the laboratory (Farrar and Flint,
1930)* Larvae can be reared on apples in the laboratory during summer and fall, but storage techniques do not permit a year-round food sup ply. The availability of larvae in orchards decreased as organic insecticides were introduced and better care was taken of orchards
(Hamilton and Hathaway, 1966).
Rearing on apples
The suitability of apple fruit and foliage for L. pomonella nutrition was studied by Heriot and Waddell (19^2). The rates of development and survival of larvae fed on diets of immature seeds, immature pulp, mature seeds, mature pulp, leaves, and normal fruit were detemined. Development was most rapid (an average of 3^ days from neonate larva to adult) on a diet of immature seeds alone. Survival was better on pulp of immature apples (31.^ per cent versus 25.9 per cent on immature seeds). No larvae completed development on mature seeds. Few (2 per cent) of the larvae which were fed pulp of mature
34 35 apples survived to adulthood. Three and six-tenths per cent of the larvae that were fed apple leaves became adults, but no eggs were laid.
Best survival, 51*7 per cent, was achieved on whole immature fruit.
Oviposition was increased significantly when ripe apples were placed in oviposition cages (Van Leeuwen, 19^7).
Continuous rearing was greatly aided by the explanation of the role of photoperiod in the control of diapause (Dickson, 19^9).
Dickson et al. (1952) reported continuous rearing of the codling moth on thinning apples under constant illumination at 84° F, and 35 per cent relative humidity. Adults were mated in celluloid cylinders, and eggs were laid on waxed paper lining the cylinders. Twenty eggs per female was considered good production, and about 60 per cent of these were viable. To begin the rearing, eggs were distributed over green apples in trays. Strips of corrugated cardboard were provided for the cocooning of full-grown larvae. The proportion of the population sur viving from eggs to adults was about 10 per cent.
Apples used for rearing purposes should be protected in the orchard by fungicides and insecticides. This creates a problem of harmful chemical residues on the apples (Hamilton and Hathaway, 1966).
Sprayed apples should weather in the orchard for three to four weeks, or as an alternative they could be washed with an aqueous trisodium phosphate solution before being artificially infested with larvae.
Most apple varieties could be stored at 3^° to 38° F. for up to one year (Hamilton and Hathaway, 1966),
At Yakima, Washington, L. pomonella was mass-reared on thinning apples (Hamilton and Hathaway, 1966). Larvae were reared at 82° to 36
86° F. and 60 to 70 per cent relative humidity under a 16-hour photo period, Larvae reached maturity in about 14 days, and adults emerged after a pupal stage lasting about eight days. One hundred to 125 adults were mated in a cylindrical plastic cage 11 inches long and 5 inches in diameter. Eggs were laid on pleated waxed paper lining the cylinder. About 80 eggs per female were laid during the first five days of oviposition. Ten thousand moths were produced daily at this laboratory during the summer of 1965 (Hamilton and Hathaway, 1966).
Artificial diets
The first successful use of an artificial diet for mass-rearing
L. pomonella was at the USDA laboratory at Vincennes, Indiana (Hamilton and Hathaway, 1966), There, an artificial diet developed by Vanderzant and Davich (1958) for rearing the boll weevil, Anthonomus grandis
Boheman, was modified by Redfem (1964) for rearing the codling moth.
The addition of ascorbic acid was important for the production of nor mal adults (Redfem, 1964). The medium was dispensed into plastic jelly cups, and two newly hatched larvae were put in each cup. Up to
July, 1964, 35 generations of codling moth had been reared on this diet
(Hamilton and Hathaway, 1966). Survival from larvae to adults was about 60 per cent (Redfem, 1964).
At the USDA laboratory at Yakima, Washington, L, pomonella was successfully reared on a modification of the diet developed by Ignoffo
(1963) for the cabbage looper, Trichoplusia ni (Htibner), which, in turn, was based upon the diet used by Vanderzant and Reiser (1956) to rear the pink bollworm, Pectinophora gossypiella (Saunders). 37
These two artificial diet formulations combined apple pulp, apple seeds, and ascorbic acid with mixtures containing plant or milk proteins, amino acids, fatty acids, sterols, mineral salts, vitamins, wheat germ, sugar, cellulose, and mold inhibitors, all of which was solidified with agar.
A rather unusual departure from the casein-wheat germ diet of
Ignoffo (1963) was the modification of Brinton et al, (1969) used for mass-rearing L. pomonella. Agar, the original solidifying agent, was replaced with wood sawdust, wheat flour, and wood pulp. Media contain ing agar desiccated and cracked too readily. In addition, purified agar was more expensive. Wheat bran was added to improve the physical consistency. Mold growth was inhibited with sorbic acid and aureomy- cin, and by lowering the pH of the medium to 3*5 with citric acid. The medium was poured into trays 30 by 46 by 2.5 cm deep. The surface was scarified with a fork and coated lightly with a spray of melted paraf fin (Howell, 1967), After surface sterilization with a 3 per cent aqueous solution of sodium hypochlorite, eggs were placed on the food still attached to the paper on which they were laid. Larvae were reared at 27+1° C. and 60 to 65 per cent relative humidity under a 17- hour photoperiod. All larvae were mature by the 18th day. The maximum number of adults was obtained on the 28th day. Survival from eggs to adults was 52 per cent. The oviposition cage developed for this mass- rearing program was described by Proverbs and Logan (1970).
Ascorbic acid is required for the development of the codling moth to the adult stage. The minimum requirement was determined to be between 0.4 and 0.8 g per 100 g of diet (Rock, 1967). Linoleic or 38 linolenic acid was also found to be an indispensible dietary constitu ent (Rock, 1967). These were the fatty acids found most effective in promoting adult emergence.
The amino acid content of fifth-instar codling moth larvae was quantitatively determined by Rock and King (196?). The analysis pro vided the proportions of 17 L-amino acids which were substituted for the protein casein in the preparation of a chemically-defined artifi cial diet.
Rock (1967) used a casein diet for nutritional studies and a wheat germ diet, modified from that for the bollworm (Vanderzant et al., 1962), for laboratory rearing. Five successive generations were reared on the wheat germ diet with 55 to 65 per cent of the larvae developing into normal adults. On the casein diet survival of two generations was 79 and 75 per cent, and average development time at
81° F. was about 28 days.
A basic lima bean diet was used by Shorey (1963) to rear the cabbage looper, Trichoplusia ni (HUbner) (Lepidopterai Noctuidae). He used lima beans (Henderson bush variety) and agar as the solidifier.
Shorey and Hale (1965) substituted less expensive pinto beans for lima beans and reported the successful rearing of eight species of Noctuidae
(Lepidoptera) in addition to the cabbage looper.
The pinto bean diet of Shorey and Hale (1965) was utilized in a slightly modified form for the rearing of the hickory shuckworm,
Laspeyresia caryana (Fitch) (Lepidopterai Olethreutidae) by Schroeder and O s b u m (1969). Seven generations were reared with from 6l to 85 per cent of the larvae becoming adults. The codling moth is now 39 considered to be of the same genus as the hickory shuckworm, which is an important pest of pecan.
In North America L. pomonella is being mass-reared in the labo ratory for two programs evaluating the release of sterilized moths for population control in orchards. At Yakima, Washington, codling moths are reared on thinning apples (White et al., 1969). At Summerland,
British Columbia, Canada, about 20 per cent of the nearly one million moths released were reared on the artificial diet of Brinton et al.
(1969), while the majority were reared on green apples.
Disease prevention
Microbial disease of insects or contamination of food in a labo ratory colony is a constant threat to production (Greenberg, 1970).
When large numbers of an insect species are reared in the same enclo sure, one diseased individual might initiate an epizootic which can wipe out the entire population.. The growth of fungus, yeast, or bac teria on the food can interfere with the production of insects. Tech niques for disease prevention in insect rearing have recently been reviewed by Greenberg (1970).
In the mass-rearing of insects careful attention must be paid to sanitation. The rearing of lepidopterous larvae usually begins with surface sterilization of the eggs. Newly hatching larvae may receive pathogens from the surface of contaminated eggs. Eggs of the European c o m borer, Ostrinla nubilalis (Httbner) (Lepidopterai Pyralidae), were surface sterilized by a 10-minute immersion in an aqueous solution of
2 per cent sodium hydroxide and 2 per cent formaldehyde (Beck and 40
Stauffer, 1950). Eggs of the cabbage looper, Trichoplusia ni, were
washed five minutes in a 0.3 per cent aqueous solution of sodium hypo
chlorite (Ignoffo, 1963). The sodium hypochlorite was neutralized by
washing the eggs with a 10 per cent aqueous solution of sodium thio-
sulfite, followed by several rinses with sterile distilled water. A
0.3 per cent aqueous solution of sodium hypochlorite was also used by
Patana (1969) for the surface sterilization of eggs of six species of
Lepidoptera reared on artificial food in the laboratory. Schroeder and
Osburn (1969) used 0,15 per cent sodium hypochlorite to sterilize the
eggs of the hickory shuckworm, Laspeyresla caryana.
Rock (1967) disinfected codling moth eggs by washing them for 30
minutes in a 6.5 per cent aqueous formaldehyde solution and rinsing
them three times with sterile distilled water. Proverbs et al. (1969)
washed codling moth eggs with a 3 per cent aqueous solution of sodium
hypochlorite plus 0.1 per cent wetting agent (Triton x-100®) for three
minutes. The eggs were then rinsed with water.
The effectiveness of two popular egg surface sterilants was
demonstrated by Vail et al. (1968). When eggs of the cabbage looper,
Trichoplusia ni, contaminated with inclusion bodies of a nuclear poly-
hedrosis virus were washed with 0.3 per cent sodium hypochlorite or
10 per cent formaldehyde in aqueous solutions, none of the resulting
larvae were diseased, while mortality of larvae from unwashed eggs was nearly total. However, a somewhat smaller proportion of the sodium hypochlorite eggs hatched, presumably because removal of the egg cho
rion by this treatment permits excess water loss. Larvae from eggs treated with the sodium hypochlorite solution also took longer to 41 develop than those treated with the formaldehyde solution or the untreated controls (Vail et al., 1968). The addition of 0.04 per cent formaldehyde solution (formalin) to the rearing medium prevented dis ease of larvae hatched from polyhedrosis-virus-contaminated eggs, while all check larvae became diseased (Vail et al., 1968).
The growth of certain fungi on the surface of wheat germ diets for L. pomonella and the European pine shoot moth, Rhyacionia buoliana
(Schiffernrilller) (Lepidopterai Olethreutidae) were controlled by sur face treatments with sodium hypochlorite, methyl parahydroxybenzoate, or sorbic acid solutions (Chawla et al., 1967). Each was effective when a weak solution was sprayed on the food surface when fungal con tamination first appeared. This method avoided incorporation of the inhibitors with the diet which might have affected larval development
(Chawla et al., 1967). INSECT VIRUSES
General descriptions
The viruses causing diseases of insects are divided into two categories. The viruses which have inclusion bodies are the most numerous and best known of the insect viruses now identified. Ignoffo
(1968) states that viral diseases have been found in about 300 agri cultural insect pests and about 95 per cent of these are caused by occluded viruses. The other major group is comprised of the non inclusion viruses. These have not been studied intensively and only about 30 such diseases of insects are known (Vago, 1968).
The inclusion-type viruses cause the formation of proteinaceous crystalline inclusion bodies within infected cells (Stairs, 1968). The inclusion bodies are not infectious, but the virions contained within them are. There are three types of viruses with inclusion bodiesi
(i) the nuclear-polyhedroses, (ii) the cytoplasmic polyhedroses, and
(iii) the granuloses. The names of these types are derived from their shapes and the part of the cell in which they are produced. Nuclear- polyhedrosis viruses comprise about 44 per cent of described viral dis eases, and cytoplasmic-polyhedrosis viruses, about 36 per cent.
The two polyhedroses have relatively large many-sided protein inclusion bodies, called polyhedra, which contain many virions. Ten to
42 one hundred virions (infectious particles) are embedded, singly or in bundles, in each nuclear-polyhedrosis virus polyhedron. One hundred to one thousand spherical virions are embedded in each cytoplasmic- polyhedrosis virus polyhedron. They also differ in other important respects. The nuclear polyhedroses are produced primarily in the nuclei of infected cells, and the virions are rod shaped and composed of DNA. The virions along with two enveloping membranes generally measure 20 to 50 mp in diameter and 200 to ^00 nyi in length (Bergold,
1963). The cytoplasmic polyhedroses are formed in the cytoplasm of infected cells, and the virions are spherical and composed of RNA.
Virions of cytoplasmic-polyhedrosis virus lack membranes and measure
50 to 90 mp in diameter (Stairs, 1968). In addition, the tissues infected by these two types differ. Both are diseases of various lepi- dopterous larvae, but nuclear-polyhedrosis viruses infect cells of the fat body, trachael epithelium, epidermis, and blood, while cytoplasmic- polyhedrosis viruses infect only cells of the midgut epithelium.
Nuclear-polyhedrosis viruses also infect the midgut epithelium of some sawfly larvae (Order Hymenoptera),
Granulosis virus diseases
The third inclusion-type of insect virus is the granulosis virus. These viruses have an ovo-cylindrical protein inclusion body which is sometimes described as a "capsule" (Huger, 1963). Usually only one rod-shaped virion is contained in each inclusion body. Infec tion is primarily restricted to fat body cells of lepidopterous larvae, but in some cases the blood cells and epidermis are also primary sites 44 of infection (Stairs, 1968).
The first granulosis virus disease to be described was found in the larva of the large white butterfly, Pieris brassicae Linnaeus, by
Paillot in France in 1926 (Smith, 1967). The virus nature of granu loses was confirmed by Steinhaus in 1947 for a disease of the vari egated cutworm, Peridroma margaritosa Haworth, and by Bergold in 1948 for the fir-shoot roller, Choristoneura murinana Htlbner (Smith, 1967).
The granulosis diseases are most commonly known from lepidopterous larvae of the families Phalaenidae, Pierldae, Olethreutidae, and
Tortricidae (Stairs, 1968).
The size range of granulosis capsules from several insect spe cies as reported by Huger (1963) was 300 to 5 H mp in length and 119 to 300 mp in width. The size of the virus rods, or virions, also varies from species to species. The range of sizes reported by Huger
(1963) was 36 to 80 mp wide by 245 to 411 mp long.
Electron micrographs of thin sections of granulosis virus cap sules show a macromolecular paracrystalline lattice of protein mole cules (Smith, 1967), or as Bergold (1959) states, the protein molecules are in a cubic arrangement. The structures of inclusion bodies are similar for nuclear-polyhedrosis virus and cytoplasmic-polyhedrosis virus (Stairs, 1968). The molecular weight of the protein of the granulosis virus capsule of Choristoneura murinana (L.) is 300,000
(Bergold, 1963). The protein of the C. murinana granulosis virus has a normal complement of amino acids (Wellington, 195^)•
The virus rod (or virion) is enclosed by an inner "intimate” membrane and an outer membrane (Huger, 1963). Detailed chemical 45
analyses of the rod-shaped virions show that the core as well as the membranes have a nearly complete complement of amino acids (Wellington,
1954). In addition, the membranes contain ether-soluble lipids and the
core contains about 8 per cent DNA, Analysis of the base composition
of DNA from a number of nuclear-polyhedrosis and granulosis viruses
show distinct specific differences between virions from the two groups
(Wyatt, 1952; Bergold, 1963). Protein in the virus rod is different from inclusion body protein (Stairs, 1968).
A classification of the insect viruses according to the base composition of their nucleic acids was considered by Bellett (1969).
He suggested that nuclear-polyhedrosis viruses and granulosis viruses comprise a single group of genetically related viruses, and that the cytoplasmic-polyhedrosis viruses are completely unrelated to that group. Data on serological cross-relations, amino acid composition, and nucleic acid base composition indicated three subgroupsj (i) the nuclear-polyhedrosis viruses of Hymenoptera; (ii) the granuloses of
Choristoneura; and (iii) the nuclear-polyhedrosis viruses of the other
Lepidoptera plus the granuloses of Recurvaria, Lamphygma, and Pieris.
Bellett (1969) suggests that "a granulosis is merely a nuclear poly- hedrosis in which there happens to be only one virus particle per bun dle and only one bundle per inclusion body,"
Invasion of insect tissues
Electron microscopic studies made by Summers (1969) of the inva
sion of the granulosis virus of Trichoplusia ni indicated that envel oped virions gain entry into the susceptible cells by a process of 46
phagocytosis or by fusion of opposing lipoprotein membranes of the
virus and host cell. Summers (1969) suggested that the following
events occurred during the infection process. In the midgut the cap
sules were disrupted, and the virus rods complete with membranes were
released. Dissociation of the protein capsule was probably effected by
th® high pH of the midgut contents. Within two hours post infection many virus rods were found in association with microvilli of midgut
cells. Virions entered midgut cells, leaving the outer membranes out
side the cells. Then some virions became associated end-on with nuclear pores, and some empty intimate viral membranes were seen associated with nuclear pores. The viral genome may have been injected into the cell nucleus (Summers, 1969). Six to ten hours post infection there were changes in the nucleus suggesting eclipse-phase activity.
By 24 hours post infection viral progeny were observed. The nuclear membrane broke down and its contents including new virions were mixed with the cytoplasm. Virions did not become occluded in the gut cells
(Summers, 1969). How the virions were released into the hemocoel was not determined. Virions in vesicles might have passed through the basement membrane of the midgut epithelium (Summers, 1969).
Hi stopathology
The manner in which granulosis viruses enter susceptible cells has not yet been demonstrated. Information regarding virus replication was summarized by Huger (1963). The first change observed in infected fat body tissue is the "mitotic proliferation" of cells producing an increase in volume. Then nuclei of infected cells are seen to enlarge, and the chromatin material condenses into distinct strands. Soon nuclei disappear while chromatin strands fragment, disperse, and dis appear. A network which gives a positive Feulgen reaction for nucleic acid then forms in the infected cell. Huger and Krieg (1961) suggested with electron-microscopy that the virus rods might arise from the
Feulgen-positive network. The network becomes Feulgen negative as the cell becomes filled with virus capsules. Capsules are formed by the progressive deposition or crystallization of protein around the virus rods (Huger, 1963). The final stage is the release of capsules into the hemolymph as infected cells lyse.
"Long virus rods" were found associated with granuloses in lar vae of 12 species examined by Smith and Brown (1965b). Long virus rods were branched and massed together in bundles. It was suggested that the long virus rods might represent an alternate replication cycle and that the branching might result of a disturbance of the replicative mechanism. Smith and Brown (1965a) suggested the following "alternate replication cyclej" (i) virus rod is extruded from protein capsule;
(ii) rod lengthens and its diameter decreases; (iii) threads of virus material branch; (iv) threads break into short rods; (v) short rods are enveloped with outer membrane; and (vi) the protein capsule is depos ited around the virion.
An electron microscopic study of the granulosis virus disease of the indian meal moth, Plodia Interpuncte11a (Httbner), indicated that virus rods are produced in association with a stroma in the cytoplasm of infected fatbody cells (Amott and Smith, 1968a). Virus rods became associated with the smooth endoplasmic reticulum. The authors 48
suggested that the outer membrane of the virus rod is derived from the
endoplasmic reticulum. The intimate membrane appeared to form after
the outer membrane was in place. However, the authors stated that the
intimate membrane is not a unit membrane. Long branching rods were not
found in association with this granulosis virus as they were in other
granuloses investigated (Smith et al., 1964; Smith and Brown, 1965b).
Further study of the granulosis virus of Plodia interpunctella
revealed the presence of atypical protein capsules (Amott and Smith,
1968b). The authors found cubic capsules, giant capsules, compound
capsules, capsules with two to six virus rods, and large aggregations
of crystalline capsule material, in addition to normal capsules. Cubic
crystals were found associated with the granulosis virus disease of
Choristoneura fumiferana by Stairs (1964). He determined that this was
a strain of virus that produced only cubic inclusion bodies. Stairs
(1966) found giant cubic inclusion bodies and long virus rods along
with typical capsules in the granulosis virus disease of L. pomonella
larvae. Elongate capsules were observed in electron-microscope studies
of the granulosis of Pygaera anastomosis (Lepidoptera: Notodontidae)
(Sidor and Krstic, 1969).
Disease symptoms
Symptoms of granulosis disease in Lepidoptera were described by
Huger (1963). Loss of appetite and cessation of feeding are noted
first. Larvae then progressively become weaker and more sluggish.
Body color pales because of the accumulation of virus capsules. Hemo-
lymph becomes turbid (sometimes milky) as cells lyse and release 49 capsules into hemocoel. Moribund larvae are usually flaccid and dis tended. After death, larvae darken and turn black.
The duration of the disease may depend upon the developmental stage of the larva, the temperature, the dose of virus, and the viru lence of the inoculum. Death usually occurs in the larval stage, but sometimes not until the pupal stage is reached (Huger, 1963). Tanada
(1953) stated that the hypodemis (epidermis) as well as the fat body of the imported cabbage worm, Pieris rapae (L.), are infected. After death the internal tissues become liquefied and the integument is very easily ruptured. Death usually occurs four to eight days after infec tion. In the cabbage looper, Trichoplusia ni, the fat body is the pri mary site of infection. The integument remains tough even after death.
Median lethal time for fourth-instar cabbage looper larvae fed large doses of capsules was about 17 days (Hamm and Paschke, 1963).
Diagnosis
When viewed with a microscope with phase contrast optics and darkfield illumination, virus capsules in the hemolymph are highly refractive and move rapidly with Brownian motion. Heavy concentrations of capsules appear bluish. Spherical vesicles containing many strongly vibrating capsules are frequently discharged from diseased fatbody cells. The most conclusive proof of diagnosis is the demonstration of occluded virus rods with an electron microscope (Huger, 1963).
To isolate capsules, virus-killed larvae are suspended in water and kept at room temperature until the tissues have putrified and the capsules have sedimented to the bottom of the container in a whitish 50
layer (Huger, 1963). Capsules can be purified by differential cen
trifugation or by rate-zonal centrifugation on a density gradient col
umn. Virus rods are liberated from capsules by dissolving the protein
capsule in weak alkali. The alkali solution recommended is
0.004-0.03 M Na2C03 or KgCO^ in 0.05 M Nacl or KC1 (Huger, 1963;
Bergold, 1964).
Non-inclusion viruses
This group of insect viruses is distinguished by the fact that
the virions (infectious nucleo-protein particles) are not occluded in
a protective protein crystal or polyhedron. More than 30 such viruses
have been described (Vago, 1968). Most are spherical or icosahedral
(20-sided) and range from 35 to 160 mja in diameter (Ignoffo, 1968).
Among the non-inclusion viruses are the iridescent viruses:
Tipula iridescent virus (TIV) of the larva of the crane fly Tipula
paludosa Meigen (Diptera); Seriscesthis iridescent virus (SIV) of
Seriscesthis pruinosa Dalman (Coleoptera); and the iridescent virus
of Chilo suppressalis Walker (Lepidoptera) (Vago, 1968). These three viruses contain DNA. An iridescent blue color is produced by high con
centrations of these viruses in diseased larvae. Iridescent viruses have also been found in larvae of several species of Aedes mosquitoes
(Vago, 1968). TIV has been transmitted experimentally to some larvae
of Lepidoptera and Coleoptera (Smith, 1967). The site of replication for the iridescent viruses studied is the cytoplasm of fatbody cells
(Smith, 1967). The particles are probably icosahedrons (20-sided
solids) (Smith, 1967). SIV has also been artificially transmitted to 51
some larvae of Diptera and Lepidoptera (Smith, 1967).
Two non-inclusion viruses have been isolated from adult honey
bees (Apis mellfera Linnaeus) affected by paralysis (Smith, 1967).
One is acute bee paralysis virus (ABPV) which produces isometric par
ticles about 28 mp in diameter. The other is chronic bee paralysis
virus (CBFV) which produces ovoid particles about 27 by njp.
One non-inclusion virus which replicates in the nucleus of fat-
body cells is the densonucleosis of Galleria mellonella Linnaeus lar
vae. This is an icosahedral virus, 21 to 23 mp in diameter, containing
DNA (Vago, 1968).
Virus diseases were first associated with mites when the
European red mite, Panonychus ulmi Koch, and the citrus red mite,
Panonychus citri McGregor, were found to be infected with non-inclusion
viruses (Vago, 1968).
The first virus disease of an orthopteran was a non-inclusion
type found in the cricket Gryllus bimaculatus Geer. An icosahedral
virus about 30 mp in diameter causes a paralysis disease (Vago, 1968).
The use of viruses for insect control
Polyhedrosis and granulosis viruses have been considered for
applied insect control because they are relatively specific, fairly
fast-acting, persistent, and not harmful to other animals (Ignoffo,
1968). Viruses have been most effective against forest-defoliating
pests such as the European pine sawfly, Neodiprion eertlfer (Geoffroy), and the European spruce sawfly, Diprion hercynae (Hartig) (Tanada,
1967). A nuclear-polyhedrosis virus was introduced into Newfoundland 52 by Balch in 1946 to control the European spruce sawfly (Hall, 1964).
A nuclear-polyhedrosis virus of the European pine sawfly, obtained from
Sweden, controlled that insect in Ontario (Bird, 1953) and in the
United States (Hall, 1964). The virus is still recommended for control of this insect (Heimpel, 1968).
Artificial application of the nuclear-polyhedrosis virus of the alfalfa caterpillar, Colias eurytheme Boisduval, in California with conventional spray equipment was successful in reducing damage to alfalfa by that insect (Steinhaus and Thompson, 1949).
A nuclear-polyhedrosis virus of the cabbage looper, Trichoplusia ni (Httbner), produced encouraging controlling effects when sprayed on cabbage, cauliflower, and broccoli in New York (McEwen and Hervey,
1958). Relatively low dosages of the virus (0.94 diseased larvae per acre) initiated artificial epizootics which greatly reduced looper populations. A granulosis virus also has been tested for control of
T. ni but was found not to be as effective as the nuclear-polyhedrosis virus (Jaques, 1970). It was demonstrated that the nuclear- polyhedrosis virus of T. ni persisting in the soil could be responsible for reinitiating epizootics the following year without reapplication
(Jaques, 1970).
The success of a nuclear-polyhedrosis virus in controlling
Heliothis species has encouraged development of pilot programs for com mercial production (Allen, 1968). Six Heliothis species are known to be susceptible to the virus (Allen, 1968). The virus is being examined most extensively for control of Heliothis zea (Boddie) on cotton, com, and tomatoes, and Heliothis virescens (Fabricius) on tobacco (Ignoffo, 53
1968).
Tanada (1956) tested a granulosis virus of the imported cabbage worm, Pieris rapae (L.)t for control of that insect in Hawaii. The virus used had been stored for one to two years under refrigeration without loss of virulence. Mortality in the field was high following the application of sprays containing four and eight macerated diseased larvae per gallon of water.
The persistence of the activity of a granulosis virus of Pieris brassicae (L.) has been studied by David (1965) and David and Gardiner
(1966, 1967). They found that virus activity would persist in soil for at least one year and on cabbage leaves in the field for at least four months. The activity was not destroyed rapidly over the normal range of temperature, but activity did decrease with time. Considerable virulence remained after three years of storage in a refrigerator.
When testing the Heliothis nuclear-polyhedrosis virus, Allen
(1968) found that its activity was reduced about 65 per cent after six hours of exposure to sunlight on foliage. Activity was completely lost after one day of exposure in the field. The granulosis virus of Pieris brassicae was inactivated within a few days after application to foli age exposed to sunlight, but virus not exposed to direct sunlight in the field may persist longer (David et al., 1968).
The effectiveness of a granulosis virus of the red-banded leaf roller, Argyrotaenia velutinana (Walker), for apple protection was examined by Glass (1958). One application of 5» 50, or 100 diseased and triturated larvae per 100 gallons of water was sprayed on apple trees when first-brood eggs were hatching. Most of the larvae were killed, but not before apples were damaged. However, the percentage of fruit injured was reduced by 14 to 26 per cent when compared with untreated trees which had an average of 54 per cent of the fruit dam aged by the leaf roller. The extent of injury was also less on virus- treated apples. It was concluded that the virus kills too slowly to prevent damage to apples, but that it might be used to reduce popula tions so that subsequent generations would not be so injurious,
A granulosis virus of L. pomonella
A granulosis virus infection of L. pomonella was first detected in larvae collected from apple and pear trees in Mexico (Tanada, 1964),
The granulosis virus has since occurred in laboratory cultures of the codling moth at Summerland, British Columbia} Yakima, Washington; and
Vincennes, Indiana (Falcon et al., 1968), In the laboratory the virus disease caused the death of codling moth larvae in 5 to 12 days. Symp toms of the disease are similar to those described for other granuloses of Lepidoptera.
Virus capsules measured by Tanada (1964) had average dimensions and standard errors of 393.9+4.29 mp by 207.7+9.76 191. Virus rods, including membranes, measured 35.5+8.02 mp by 50.7+0.30 mp. Atypical cubic capsules as large as 5 p were found along with typical capsules in diseased larvae by Stairs et al, (1966). Each capsule usually con tains only one virus rod, and each virus rod is enveloped by two mem branes. Long strands of virus-like material were also observed in infected cells (Stairs et al,, 1966).
The histopathology of the disease was described by Tanada and 55
Leutenegger (1968). The virus infects the fat body, epidermis, tra
cheal matrix, and perhaps the Malpighian tubules. Within two days fol
lowing the ingestion of virus the fat-body cells exhibited characteris
tic pathology. The disease apparently progressed as Huger (1963) des
cribed. Epidermal cells began to show signs of infection on the third
day. With the electron microscope, capsules and free virus rods were
observed in cells of the fat body, epidermis, tracheal matrix, and
Malpighian tubules.
High-speed centrifugation of viral products from diseased cod
ling moth larvae revealed the existence of an infectious component
which was smaller than the rod-shaped virus particle (Barefield and
Stairs, 1970). This unidentified material must have contained the
entire viral genome, for it produced the disease and granules charac
teristic of the granulosis virus.
There has been some interest in the use of this virus for
applied control of L. pomonella. Cover sprays of the virus have been
evaluated in a California orchard (Falcon et al,, 1968). Laboratory
studies indicated that most of the first-instar larvae died soon after
chewing through the skin of apples sprayed with granulosis virus. The virus for field use was propagated in codling moth larvae reared on an
artificial diet. The average yield of virus per larva was 6.6 x 10^
Q capsules in 1966 and 1.9 x 10 capsules in 196?. Sprays applied to
apple trees contained about 3.^ x 1 0 ^ capsules per gallon in 1966 and
1.2 x 1 0 ^ capsules per gallon in 1967. Four to five gallons of virus
suspension were sprayed on each tree for each application.
The virus was applied to coincide with periods of high 56
egg-laying activity. Samples of apples from the treatments were exam
ined to determine the amount of codling moth damage. Trunk bands were
used to estimate the number of full-grown larvae seeking cocooning
sites (Falcon et al ., 1968).
Four trees (5 per cent of the orchard) were sprayed with virus
in 1966 and 28 trees (35 per cent of the orchard) in 1967. Five virus
applications were made in 1966 from May 2 to August 17. Seven appli
cations were made in 1967 from June 20 to September 13. Few larvae
survived in fruit of virus-treated trees, and most larvae produced only
stings. Too long an interval between two sprays (three weeks instead
of two) in 1967 resulted in a period of slightly increased larval dam
age. Ten per cent of the harvested apples were destroyed in 1966 and
5 per cent in 1967 (Falcon et al., 1968).
In 1966 an average of 30 larvae was found in trunk bands on unsprayed trees, while the average for virus-treated trees was 4, rep
resenting a reduction of 87 per cent. In 1967 an average of 14.7 lar vae was found in trunk bands on check trees, and the average was 3.2 for virus-treated trees, or a reduction of 78.5 per cent. Virus-killed larvae found on trees treated the previous year indicated that some virus activity persisted from one year to the next (Falcon et al,,
1968), The experiment was continued in 1968 with 12 sprays, each con- 9 taining 9.5 x 10 virus capsules per gallon, being applied. At harvest only 2.3 per cent of the fruit was economically damaged. INTRODUCTION OF A GRANULOSIS VIRUS
INTO A FIELD POPULATION OF L. POMONELLA
I. MATERIALS AND METHODS
In the summer of 1967, a granulosis virus pathogen of the cod
ling moth, Laspeyresia pomonella (L.), was applied to two apple trees
in the orchard of the Overlook Farm of the Ohio State University near
Carroll, Ohio. A study was initiated in order to determine what effect
the virus would have on L. pomonella in a field situation in south-
central Ohio. The experiment was intended to determine! (i) whether
the virus when applied to apple trees would infect and kill larvae;
(ii) whether the treated population would be reduced by virus disease;
(iii) whether damage to apple fruits would be reduced by the treatment;
and (iv) whether the virus disease would spread and increase among cod
ling moths in this orchard.
The study was confined to the Jonathan cultivar of apple and to
trees in a semi-isolated block (Fig. 1) which for the preceding three years had been sprayed only with a fungicide (Captan®). Insecticides
had not been applied in this isolated area of the orchard for the past
three years. For the experiment two trees were sprayed with the virus
preparation on three occasions during the period when the second L.
pomonella generation was developing. Populations on four untreated
trees were monitored as a control. These trees were selected because
57 58
n o o o ft ooo o 0 o o °o 0 0 o © o oo o ooo 8 oo o © ooo oo ooooooooo0 0 © 0 0 0 0 ©0
Figure 1. Diagram of the location of apple trees comprising the North Block of Overlook Orchard. "V" designates the two trees to which granulosis virus was applied in 1967. "C" indicates the four trees used as controls. 59
they bore higher numbers of fruit than most other Jonathan trees in the
block.
Examination of 62 mature L, pomonella larvae from the first gen
eration in 1967 revealed no occurrence of granulosis virus disease.
The larvae were collected from trunk bands on 20 trees in the experi mental block and 20 trees in the main part of the orchard. These trees were not sprayed with insecticides in 1967.
The timing of the virus applications was determined by the pres
ence and abundance of first-generation moths in the orchard. Bait traps containing a fermenting brown sugar solution and sassafras oil were suspended in six widely separated trees which had not been sprayed with insecticides. The bait was made by dissolving two pounds of brown
sugar in two gallons of water and adding one teaspoon of sassafras oil.
Each bait can was suspended from branches in the center of the tree by a cord on pulleys so that it could be lowered for examination. The bait was replaced about every two weeks. The traps were examined every three or four days from July 7 to September 9. Adults were found in traps on all but three occasions during that period. It was impossible to separate the flight periods of the first and second seasonal genera tions, but the peak of first-generation flight appeared to come during the last week of July (25th to 31st). On that basis, the first appli cation of virus was made as soon after the beginning of August as was possible.
The granulosis virus was propagated in the laboratory by feeding contaminated food to laboratory-reared fifth-instar codling moth lar vae. An aqueous suspension of granulosis virus material obtained from 60 larvae previously killed by the virus was painted on the surface of artificial food. The larvae ingested the virus with the food and in four to ten days were killed by the granulosis virus disease. Bodies
Q of diseased larvae contained millions of virus capsules (about 8 x 107 by particle count). Virus-killed larvae were refrigerated at 40° F. until used.
For the first application of virus in the orchard on August 3»
1967, the body contents of 80 virus-killed larvae was dispersed in
125 ml of sterile distilled water. The dead larvae had been stored in a refrigerator for one to eight days. A particle count made with a phase-contrast microscope and a bacteria counting chamber gave an esti mate of 6.65 x 1011 virus capsules in this preparation.
At the orchard 125 al °f the virus suspension was added to 50 gallons of water in an air-blast orchard sprayer. Prior to this the sprayer had been flushed with clean water. The two apple trees were sprayed late in the afternoon to avoid the detrimental effects of sun light and wind. The sprayer was operated as for standard chemical insecticide application. Foliage and fruit were thoroughly wetted with the spray. Each tree received approximately 24 gallons of the aqueous virus suspension.
The second application of virus was made in the same manner 12 days later (August 15). The virus that was used came from 80 diseased fifth-instar larvae which had been refrigerated for one to twelve days after death. The third virus spray was applied nine days later, on
August 24. This final spray contained the virus from 60 diseased lar vae which had been kept in a refrigerator for one to twenty-one days. 61
The final preparation thus contained about three-fourths of the virus
used in the two preceding sprays.
Twice weekly, from July 28 to September 1^, $00 apples were
examined in place on one virus-treated tree and on each of the four
control trees. The number of apples with larval stings (shallow feed
ing sites) and entries was noted. Three counts from each tree before
the first virus application provided an estimate of the initial popu
lations of larvae per tree. Once each week during the test period the
burlap bands on the trunks of the trees were examined for the presence
of mature larvae. At harvest, all the apples were picked and counted,
and the number of apples with stings and entries was recorded. Each
damaged apple was dissected in order to determine the fate of the larva
that had entered. Remains of dead larvae were examined with a phase-
contrast microscope to decide whether the granulosis virus disease was
the cause of death. A drop of sterile distilled water was added to
larval tissue on a glass slide and a cover glass was pressed over it
to make a squash preparation for microscopic examination. The presence
of masses of characteristic bluish iridescent virus capsules in fat body tissue was the criterion for diagnosing granulosis virus disease.
During the 1968 growing season burlap bands on the trunks of 38
trees in the experimental block were examined for the presence of dead
larvae. On June 7 bands were checked for overwintered larvae of the
second generation of 1967. Larvae of the first 1968 generation were
examined on August 8. Second-generation larvae under bands were exam
ined on August 27 and again on September 12. Harvest samples of 200
apples each were picked from one tree sprayed with virus in 1967 and 62 from three untreated trees in the block. Apples that were stung and wormy were dissected; and, if dead larvae were found, they were exam ined with the phase-contrast microscope for the presence of granulosis disease or other pathogens. INTRODUCTION OF A GRANULOSIS VIRUS
INTO A FIELD POPULATION OF L. POMONELLA
II. RESULTS
The dissemination of granulosis virus into a field population of L. pomonella was successful. Larval populations on treated trees were reduced, as shown by the lower number of mature larvae found under trunk bands (Table 1). A total of 26 virus-diseased larvae was found in harvested apples, and five were found under trunk bands on treated trees. As estimated from injury to harvested apples, populations of larvae within apples were reduced by 36 per cent on treated trees and
16.3 per cent on control trees (Table 1). The difference indicated that about 19.7 per cent of the larvae on the treated tree apparently were killed by the virus while still in apples. When numbers of larvae found under trunk bands on treated and control trees were compared, it was apparent that the virus reduced populations of larvae by about 93 per cent (Table 1).
Initial larval populations were different on the treated and control trees (Table 2)j and in order to compare the effects of the virus, later population levels were adjusted relative to the initial populations. When this adjustment was made, it became apparent that the virus-treated population did not increase as much as the control population during the second to sixth weeks after the first virus
63 Table 1. Estimated mortality of L. pomonella larvae in fruit and during entire larval period on virus-treated and untreated apple trees. Initial populations were estimated from the total number of injured apples per treatment (A), and populations which completed development in fruit were estimated from the total number of apples with deep entries (B). The difference between numbers of larvae found under trunk bands on treated and untreated trees gave the estimate of total larval mortality caused by virus.
# mortality Harvested apples in fruit Average no. x 100 larvae under Treatment (A) total injured (B) wormy A trunk bands
Virus3 286.5 183.5 36# 3.0
Control*3 198,7 166.3 16.3# in fruit all larvae Reduction by virus: 19.7# 93# £ Counts from 2 trees were averaged. Counts from **■ trees were averaged. 65 Table 2. Accumulation of injury from second-generation L. pomonella larvae in samples of 500 apples examined on one virus-treated tree and four control trees. 1967. Treated tree Control trees No. apples No. apples in.jured/500 examined injured/ Avg. for Date 500 examined £1 #2 #2 #4 controls 7/28/67 3 2 0 l 0 0.75 7/31/67 9 2 0 2 0 1.00 8/3/67 8 4 4 0 1 2.25 8/7/67 8 3 3 2 1 2; 25 8/10/67 19 15 5 0 3 5.75 8/14/67 22 8 14 1 2 6.25 8/17/67 21 6 8 24 8 10.50 8/21/67 33 10 23 14 8 13.75 8/24/67 26 10 22 18 11 15.25 8/28/67 35 10 29 19 17 18.75 8/31/67 36 7 28 14 14 15.75 9/5/67 29 10 14 20 15 14.75 9/8/67 24 9 18 28 14 17.25 9/11/67 24 9 19 31 15 18.50 9/14/67 25 10 20 26 14 17.50 66 application (Fig. 2). During the fifth, sixth, and seventh weeks the population on the treated tree was about 50 per cent lower than the control population. Another set of data, based upon the numbers of larvae found under trunk bands, showed that the population of mature larvae was much lower on the treated tree (Table 3). An average of 41.5 larvae was trapped on the control trees, while three were found on the treated tree. This data indicated that the virus caused about 93 per cent mortality of larvae. Graphical analysis of the trunk band samples (Fig. 3) revealed that the population of mature larvae on the treated tree did not increase after the fifth week following the initial virus application. The trapped population on the control trees accumulated steadily from the fifth to the tenth week at the average rate of about five per week. These data probably reflect the effect of the virus on larval populations more accurately than the indirect estimates made from damaged fruit. During the summer of 1968 no additional virus nor insecticide was applied in the experimental block, but the population of L. pomonella larvae was periodically sampled in an effort to determine whether the granulosis virus applied in 1967 was still effective. On June 7, 11 dead larvae and 1 dead pupa were found along with 5 living larvae and 39 pupae or pupal cases under trunk bands in the test block. Three of the dead overwintered larvae appeared to be infected by nema todes and six by fungus (Table 4), Virus, bacteria, or other pathogens were not detected in the remaining dead individuals. Mortality among the mature first-generation larvae under trunk bands was checked on L. pomonella larvae injuring fruit on virus-treated and untreated trees in 1967. Index of codling moth population iue2 RelativeincrementsFigure 2.in populations of second-generation 8 1 2 3 5 6 7 virus application Weeks untreated virus-treated 6? 66 Table 3. Accumulation of mature L. pomonella larvae under trunk bands on one virus-treated tree and four control treesa. 1967. Ntimber of larvae accumulated under trunk bands virus- control control control control avg. for Date treated il £2 £2 #4 controls 7/31/67 0 0 0 0 0 0.00 8/7/67 0 0 0 0 0 0.00 8/14/67 0 0 0 0 0 0.00 8/21/67 1 0 0 0 0 0.00 8/28/67 2 4 2 0 1 1.75 9/5/67 2 14 20 1 8 10.75 9/11/67 3 22 29 6 15 18.00 9/18/67 3 27 42 19 31 29.75 9/25/67 3 28 44 34 41 36.75 10/2/67 3 29 45 37 46 39.25 10/9/67 3 29 ^5 39 50 40.75 10/20/67 3 32 45 39 50 41.50 a The larvae were removed from the tree at each examination of the band, and the number found was added to the total from the previous examinations. virus-treated treeand theaverage number found onfour control trees. larvae Shownunder trunk here arebands.the number found on one No. larvae -under trunk bands iue3 AccumulationFigure of3* mature second-generation L. pomonella 10 20 virusapplications Weeks 10 virus-treated .•— 11 untreated 69 70 Table 4. Incidence of microorganisms found in association with dead L. pomonella larvae in 1968. No. of No. of larvae found individuals found with microorganism Place found and generation alive dead nematode fungus bacteria Under bands overwintered 44 12a 3 8 0 1st generation 238° 19 3 1 4 2nd generation 779 26 7 4 11 In apples (9/20/68) both generations (b) 17 1 9 7 a Number includes one dead pupa. k Most of the surviving larvae had matured and left apples by this time. Seventeen dead larvae were found in 139 injured apples from sample of 800. c Live pupae and pupal cases from which adults had emerged are included in this number. 71 August 8. Under 38 bands were found live larvae, 37 live pupae, and 156 pupal cases of emerged adults. There were also 11 dead larvae and 8 dead pupae. Microscopic examination of the dead individuals revealed one fungus infection, three nematode infections, and four bacterial infections (Table 4). Again, no virus-infected individuals were found. Trunk bands were examined on August 27 and September 12 to determine the mortality of mature second-generation larvae. A total of 779 live larvae and 26 dead larvae was found under bands on 32 trees. Among the dead larvae there were signs that seven had nematode infec tions, eleven had bacterial infections, and four had fungus infections (Table k). No virus-infected individuals were found. On September 20, a sample of 200 apples was picked from each of four trees including one which had been sprayed with virus in 1967. The 139 apples that bore codling moth injury were dissected and 17 dead larvae were found. One of the dead larvae appeared to have a nematode infection, seven had bacterial infections, and nine had fungus infec tions (Table k ). No virus-infected larvae were found in the apples. INTRODUCTION OF A GRANULOSIS VIRUS INTO A FIELD POPULATION OF L. POMONELLA III. DISCUSSION The test of the granulosis virus in an apple orchard demon strated that L. pomonella larvae can be killed when a virus suspension is applied to trees with an air-blast sprayer. Fewer larvae survived on the treated tree than on the control trees (Table lj Fig. 3). The number of larvae reaching trunk bands on virus-treated trees indicated a large reduction of larval populations on virus-treated trees. Analy sis of larval mortality showed that perhaps 20 per cent of the intital larval population died from virus while in the fruit and another 73 per cent died after they vacated fruit. In this test the granulosis virus was estimated to have killed at least 93 per cent of the original lar val population. The virus did not reduce injury of apples (Table 2), but it did increase larval mortality and thereby would reduce popula tion density in the next generation. When used in place of nonselec- tive insecticides for regulating L. pomonella populations, this virus would allow native predators and parasites to exert more control on L. pomonella and other pests. Most of the insecticides and miticides used in orchards today eliminate the beneficial species as well as the pests. The use of reduced dosages or more selective pesticides might preserve the activity of effective natural control agents (Asquith, 1970). 72 73 There were no signs of the granulosis virus in the test block in 1968. Other pathogens were found causing a low level of mortality among mature larvae under trunk bands and developing larvae in apples (Table 4). Apparently the introduced virus did not become associated with the codling moth population. Also, the infectious virus was not present where larvae of the succeeding generations would ingest it, or at least was not present in effective concentrations. One can speculate that this granulosis virus is not a specific parasite of L. pomonella. Only one natural occurrence of the disease has been reported (Tanada, 1964). If the parasite-host relationship were evolved, there would be mechanisms of dispersal and reinfection that would insure survival of the virus from generation to generation of the host. If such a virus were introduced into a noninfected host population, one would expect the disease to become established and be perpetuated in that population. Obviously, L. pomonella is not a good host for a virus. The opportunities for transmission of the virus would be few. L. pomonella larvae are solitary) thus, the disease is not likely to be transmitted from one individual to another. Also, the larva feeds internally, away from contaminated material, for most of its life. The only time that virus-contaminated food material might be ingested is when the first- instar larvae chew through the skin of the apple. Virus would have to thoroughly coat the surface of each apple in order to assure being ingested. Since fruit and foliage fall from the tree each year, there seems to be little opportunity for recontamination of food material. Granulosis viruses of Trichoplusia ni and Pieris rapae are effective 74 because they persist in the soil and are splashed onto the host plant by rain (Jaques, 1970). Parasites and predators could transmit virus from diseased to healthy larvae, but few are associated with L. pomonella (Putman, 1963). Transovum transmission of the pathogen has not been indicated nor demonstrated. Effective use of the virus as a microbial insecticide probably depends upon periodic reintroduction to the surface of fruit. Effec tive virus must be present where the larva chews through the skin of the fruit. Virus activity is probably terminated after a brief expo sure to high temperature and ultraviolet-light. Reapplication is therefore necessary to keep active virus on the fruit surface. Falcon et al. (1968) found that five to seven applications of the virus during the season reduced L. pomonella populations and fruit injury consid erably. I found that three applications of about the same amount of virus per tree reduced survival of the second generation considerably, but did not prevent injury to apples. Of course, many conditions dif fered in the two test situations. More facts need to be learned about the granulosis virus-L. pomonella interaction in order to determine how to utilize the virus in an orchard. Laboratory and field bioassays should be conducted to determine the effective dosage required to kill first-instar larvae. From that information the concentration of virus in the spray could be calculated. In order to know how often to apply the virus, the length of its activity under field conditions will have to be determined. Or one might investigate how to time virus applications so that they coin cide with the periods when larvae enter fruit. Also important to 75 studying the virus-host relationship would be development of better methods for sampling populations of L. pomonella. LABORATORY REARING OF L. POMONELLA ON ARTIFICIAL DIETS I. MATERIALS AND METHODS L. pomonella was reared in the laboratory on an artificial nutrient medium developed by Redfem (1964). The original diet was slightly modified. Walnut meats and whole apple seeds were not included, and wheat germ and a small amount of tannic acid were added. The ingredients of this artificial diet are listed below. Ingredient Amount Group #lt dried apple slices 120 g apple seeds 60 g boiling distilled water 1,000 ml Group #2: wheat germ 108 g sucrose 120 g soybean protein 120 g Wesson salt mixture 25 g glycine 2.50 g cysteine 1.25 g cholesterol 1.25 g tannic acid 0,025 g dry active yeast 25 g Group #3» alphacel 60 g 10# aqueous KOH 75 ml raw linseed oil 20 ml mold inhibitor (20 g sorbic acid, 15 g methyl-p-hydroxybenzoate, 170 ml 95# ethyl alcohol) 45 ml Group #4» agar 40 g boiling distilled water 1,200 ml 76 77 Ingredient Amount Group #5: ascorbic acid (dissolve in 250 ml sterile distilled water) 20 g vitamin solution (5 g Vanderzant's Vitamin compound dissolved in 100 ml sterile dis tilled water) 25 ml The sources of ingredients for this diet and of other materials used in rearing are listed in the Appendix. The ingredients were combined in a gallon-size Waring® blendor in the order which follows: (i) apple slices, apple seeds, and boiling water were put into the container and mixed at high speed for about 90 seconds; (ii) dry ingredients of Group No. 2 were added and mixed at low speed for about 45 seconds; (iii) alphacel and liquid ingredients of Group No, 3 were added and mixed at low speed for 30 seconds; (iv) the agar was then dissolved in boiling water and combined with the previous mixture by blending at low speed for approximately 15 seconds; and (v) the ascorbic acid and vitamin solution were added last and the mixture was blended at low speed for 15 seconds. The warm liquid mixture was dispensed into five-eighths-ounce plastic jelly cups from a one-liter plastic squeeze bottle. Each cup was filled to about two-thirds capacity. Filled cups, on the surface of a table, were covered with clean, autoclaved cotton toweling, and the food was allowed to cool for about two hours before it was used or stored in a refrigerator. A second artificial diet was used later for rearing L. pomonella. This diet had been used by Patana (19&9) to rear six species of 78 Lepidoptera and two species of Coleoptera which feed on cotton. Food yeast was substituted for brewer's yeast and agar for Gelcarin which Patana had used. The ingredients of the artificial diet are as followsi Ingredient Amount baby lima beans 450 g dried food yeast 90 g methyl-p-hydroxybenzoate 9 g ascorbic acid 9 g formaldehyde (37$ solution) 3 ml agar 45 g water 2,300 ml For preparation of the artificial diet, baby lima beans were covered with 1,400 ml of water in a two-liter beaker and allowed to soak for 18 to 24 hours. The beans and the water in which they were soaked were poured into the gallon container of the blendor. The yeast, methyl-p-hydroxybenzoate, ascorbic acid, and formaldehyde were added to the container. These ingredients were blended for five min utes, or until the temperature of the mixture was about 43° C. The agar was added to 900 ml of boiling water in a beaker and stirred until dissolved. The melted agar was then added to the blendor container and blended briefly at low speed until mixed. This warm liquid mixture was dispensed into five-eighths-ounce plastic jelly cups from a one-liter plastic squeeze bottle. Six to seven ml or one-third of the capacity of the cup was an adequate amount of food. The cups of food were covered with clean, autoclaved cotton toweling, and the food was allowed to cool before it was used or stored in a refrigerator. The recipe provided nearly three liters of food or enough to prepare about 475 cups of food. 79 When a second generation of L, pomonella was reared on the lima bean diet, a decline in the production of moths was evident. In an effort to increase survival, some extra ingredients were added to the medium, and this was fed to the third and succeeding generations. Sucrose (60 g), apple seeds (60 g), and raw linseed oil (20 ml) were added to the medium already described. Neonate larvae were transferred with a No. 1 artist's brush from oviposition containers to the surface of artificial food in plastic jelly cups. One larva was placed in each cup. A plastic-lined paper cap was used to close each cup after a larva was put on the food. The brush used for transferring larvae was frequently dipped into 0.2 per cent sodium hypochlorite to sterilize and wet it. Cups containing lar vae were stacked in plastic crisper boxes, and filled boxes were placed in an incubation cabinet. The temperature for rearing was 80+2° F. A daily light period of 15 hours was provided by fluorescent lamps con trolled by an automatic time switch. Development from first-instar larva to adult was completed in one closed cup of food. Cups in the incubator were examined near the time when emergence (eclosion) of adults was expected to occur. Cups containing adults were removed from the incubator every day or two. Adults were removed from the rearing cups and put into eight-ounce plastic-lined paper cups for mating and oviposition. A piece of waxed paper measuring about 2§ by 12 inches was fluted and placed around the inside of each cup. Two holes were made in a plastic snap-on cup lid with a No. 4 cork borer. Plastic tubing about two feet in length, one piece of three-sixteenths-inch inside diameter and another piece of 80 one-fourth-inch inside diameter, were pushed into the holes in the lid to make an aspirator from the cup. One end of the smaller-diameter tubing was covered with a few layers of cheesecloth before it was pushed into the hole in the cap. This was the suction tube of the aspirator, and the cheesecloth was used as a filter to prevent sucking moths and scales into the mouth. Moths were removed from the rearing cups with the paper-cup aspirator. Up to 20 moths were put into each paper cup. When the plastic tubes were removed from the cover, the holes were covered with masking tape. The cups, containing adults, were placed in the incuba tor at 80° F. Humidity was often increased for mating and oviposition by putting the cups into a plastic crisper box with a moistened cotton towel in the bottom. The top was placed on the box to maintain an ele vated humidity. Oviposition usually commenced on the second day after mating and continued for several days. After they had been in the cup for five days, the moths were removed and discarded. Occasionally the inside of the cup and the waxed paper on which the eggs were laid were washed with a 0.2 per cent aqueous solution of sodium hypochlorite for five minutes, and then were rinsed with three changes of sterile distilled water. This surface sterilization of eggs was not done regularly as long as fungal contamination in rearing containers was not a problem, because it seemed to be detrimental to egg hatching. After the adults were removed, the plastic cap was replaced on the cup which contained the eggs. The tape was removed from the two holes in the cap, and a piece of plastic food wrap was put over the top 81 of the cup and held there with a rubber band. The cup with eggs was placed in a plastic crisper box with a moistened cotton towel in the bottom and was returned to the 80° F. incubator. Eggs hatched about five days after oviposition when incubated at 80° F. Neonate larvae for continued rearing were transferred to food within one day of hatching. The oviposition of individual female moths was studied to deter mine whether it differed for moths reared on different diets, A single unmated female moth and two males of about the same age (usually one to two days post eclosion) were placed together in an envelope made from a 6-by-10-inch sheet of Parafilm®. The envelope was sealed by the pres sure of a stylet drawn over the overlapped edges. The envelopes which contained moths were kept in an 80° F. incubator with a 15-hour light period for mating, oviposition, and incubation of eggs. Mating occurred in the envelope, and oviposition usually began one or two days later. Eggs could be counted through the transparent envelope as they were laid. A record was made of the total number of eggs laid by each individual female. Sterile, unhatched eggs could be distinguished from hatched eggs because the latter were clear with an opalescent chorion remaining and the former were cloudy, yellowish, or brown. An evaluation of the lima bean diets was made by comparing weights of pupae reared on these with weights of pupae reared on thin ning apples. One group of pupae which was weighed was reared on the original Patana diet; the second population was reared on the Patana diet to which sucrose, apple seeds, and linseed oil were added; and the third group of pupae was reared on thinning apples at the USDA 82 Entomology Research Division laboratory at Yakima, Washington. Larvae reared on thinning apples were received from Dr. B. A. Butt on April 11, 1970. All pupae were weighed within 2k hours after pupation. Male and female pupae were identified, and all pupae were weighed indi vidually on an electronic semi-micro laboratory balance (Sartorious®). LABORATORY REARING OF L. POMONELLA ON ARTIFICIAL DIETS II. RESULTS All artificial diets were sufficiently complete to sustain the development of L. pomonella (Table 5) • Survival of six generations on the Redfem diet ranged from 5.2 to 33.1 per cent with a mean of 15.5 per cent. Survival was more than three times higher on the Pa tana diet. It ranged from **8.2 to 7**.8 per cent with the highest occurring when this diet was supplemented with sucrose, apple seeds, and linseed oil. The rate of development on the Patana diet was higher than on the Redfem diet (Table 6). For three consecutive generations reared on the Patana diet, 50 per cent adult eclosion occurred on days 33, 31, and 29, respectively. For six generations reared on the Redfem diet, 50 per cent eclosion occurred no earlier than day 55 (Fig. 3, C), and in one generation 50 per cent eclosion did not occur until day 75 (Fig. 3, B). The period of adult eclosion was nearly twice as long when lar vae were reared on the Redfem diet as when they were reared on the Patana diet (Table 6 ). For one population reared on the Redfem diet, the eclosion period lasted 75 days. The first adult emerged on day 35 and the last on day 110 (Fig. 3*B). On the other hand, for one 83 84 Table 5. Survival of L. pomonella reared on different artificial diets*. No. neonate Group Diet larvae No. adults # survival lb Redfem 1.755 581 33.1 2 Redfem 875 77 8.8 3° Redfem 2,423 127 5.2 4 Redfem 411 76 18.5 5 Redfem 862 154 17.9 6 Redfem 554 51 9.2 7 Patana 1,441 743 51.6 8 Patana 3,075 1,481 48.2 9 Patana, 2,337 1,747 74.8 modified a Groups 3 and 4; 5 and 65 and 7, 8 , and 9 are samples from consecu tive generations. b Group No. 1 was reared at ambient room temperature. All others were reared at 80+2° F. c These are progeny of codling moths reared on thinning apples at the Entomology Research Division laboratory, Yakima, Washington, and received from B. A. Butt on March 19, 1969. 85 Table 6, Periods of development for L. pomonella reared on different artificial diets4. Avg. no. days Range days Group Diet No. adults to eclosion to eclosion i 0 0 lb Redfem 581 63 VO 2 Redfem 77 55 45 - 76 3C Redfem 127 56 42 - 75 0 0 o 4 Redfem 76 62 1 5 Redfem 154 75 35 - 110 6 Redfem 51 55 42 - 63 7 Patana 743 33 26-48 8 Patana 1,481 31 25-44 9 Patana, 1,747 29 23-46 modified a Groups 3 and 4; 5 and 6 | and 7, 8 , and 9 are samples from consecu tive generations. b Group No. 1 was reared at ambient room temperature. All others were reared at 80+2° F. c These are progeny of codling moths reared on thinning apples at the Entomology Research Division laboratory, Yakima, Washington, and received from B. A. Butt on March 19, 1969. 86 100 c o •H(0 o H o bO 120 Days to adult eclosion Figure 4-, Rates of development of four populations of L. pomonella reared on artificial diets. Populations A, B, and C were reared on Redfem diet. Population D was reared on Patana diet. (A was reared from 11 March to 20 August, 1968. B was reared from 27 August, 1969» to 8 January, 1970. C was composed of progeny of L. pomonella from Yakima, Washington, reared on thinning apples, and was reared from 1 April to 24- June, 1969. D was reared on Patana diet from 16 December, 1969, to 23 February, 1970. D was composed of progeny of population B.) 87 population reared on the Patana diet which was supplemented with sucrose, apple seeds, and linseed oil, the eclosion period was only 19 days; the first adult emerged on day 25 and the last on day 44, A population comprised of progeny of individuals which were reared on thinning apples at Yakima, Washington, was reared on the Redfem diet (Group No, 3 in Tables 5 and 6 ). Development was com pleted in a relatively short time in comparison with other populations reared on the Redfem diet. The development period ranged from 42 to 75 days with 50 per cent eclosion occurring on day 56 (Table 65 Fig. 3i C), However, only 5*2 per cent of the neonate larvae became adults (Table 5)1 compared with a 21.1 per cent average survival rate for the strain adapted to the Redfem diet over a period of several genera tions, The rate of survival for the following generation of the Washington strain reared on the Redfem diet increased to 18.5 per cent (Table 5, Group No, 4), The first population reared on the Patana lima bean diet had a survival rate of 51.6 per cent (Table 5* Group No. 7) and a development period of 26 to 48 days with 50 per cent adult emergence on day 33 (Fig. 3» D). The parental generation had been reared on the Redfem diet with 9.2 per cent survival (Table 5» Group No. 6 ). The develop ment period had ranged from 42 to 63 days and 50 per cent eclosion had occurred on day 55. More complete data pertaining to the development of L. pomonella populations on the artificial diets are presented in Tables 10 - 18 of the Appendix. Females reared on the Patana lima bean diet laid a higher 88 average number of eggs than did females reared on the Redfem diet (Table 7). Distribution of the number of eggs laid by individual females and the proportion hatched are shown for five populations in Figures 5-9. The average numbers of eggs laid by two groups of females reared on the Redfem diet were 54.7 and 59.2 (Table 7). From those two groups of eggs 63 and 77 per cent, respectively, hatched. Females from two consecutive generations reared on the Patana diet laid averages of 83 and 51.5 eggs per female (Table 7). For these two groups the percentages of eggs which hatched were 58.6 and 45.8, respectively. A decrease in average oviposition and percentage of eggs hatched occurred in the second generation (Fig. 8 ). When the Patana diet was "fortified" with sucrose, apple seeds, and linseed oil, ovi position by the next generation was increased to an average of nearly 118 eggs per female, and 51.3 per cent of all eggs hatched (Table 7). For females reared on the Redfem diet, hatching was relatively high (above 65 per cent) for the individuals laying higher numbers of eggs (Fig. 6 ). Although average oviposition was high for females reared on the modified Patana diet, the individual variation in oviposition and hatching seemed greater than when the Redfem diet was used (Fig. 9). The largest number of eggs laid by a female reared on the Redfem diet was 14-3, of which 130 or 91 per cent hatched. The highest number of eggs laid by any individual in all tests was 185. The female which laid that number of eggs was reared on the modified Patana diet. One hundred twenty-five or 67.6 per cent of those eggs hatched (Fig. 9). The modified Patana diet apparently enabled more individuals to lay larger numbers of eggs, but the proportion of eggs that did 89 Table 7. Oviposition by individual L. pomonella females reared on three different artificial diets. Avg. no. Range of Avg. no. Range of % of No. eggs per eggs per eggs hatched eggs hatched all eggs Diet females female female per female per female hatched Redfem 9 5b,7 5-90 32.4 0-66 63 1 Redfem 23a 59.2 0 £ 46.1 0 - 130 77 Patana 24 83.0 0 - 162 48.5 0 - 130 58.6 1 0 Patana 33 51.5 0 - 12b 23.6 00 45.8 Patana, 42 117.9 11 - 185 60.4 0 - 143 51.3 modified a Progeny of codling moths reared on thinning apples at the Entomology Research Division laboratory, Yakima, Washington, and received from B. A. Butt on March 19, 1969. diet, July, 1969.hatched for nine femaleL, pomonella reared wheat ongerm the Redfem iue5 DistributionFigure 5.of number ofeggs laid and proportion which # of eggs hatched 100 10 20 60 80 30 50 70 90 0 -«t 20 k k 0 Numberofeggs laid per female 0 0 0 10 14-0 120 100 80 60 160 8 200 180 90 Redfem wheat germ diet,Redfem May to August,1969. hatched for23 female L. pomonella of Washingtonstrain reared on the Figure $ of eggs hatched 100 80 10 20 6 Distribution. of number of eggslaid and proportion which 20 Number of eggslaid by individual female 0 10 4 10 8 200 180 160 140 120 100 91 hatched for 24 female L. pomonella reared on thePatana lima bean diet, January, 1970. iue7 DistributionFigureof 7. number of eggslaid and proportion which $> of eggs hatched 100 100 r 40 10 20 60 80 30 50 70 90 0 - * ---- 20 1 * — 40 Number of eggs laid by individualfemale -- 1 -- —, *— 60 --- 80 1 100 1 120 1 140 1 160 1 180 1 — [ — 200 92 March, 1970. hatched for iue8 DistributionFigureof 8. number of eggslaid and proportion which % of eggs hatched 100 40 10 80 20 30 60 50 70 90 0 33 20 female L.pomonella reared on the Patana lima bean diet, Number of eggslaid byindividual female 80 0 10 4 10 8 200 180 160 140 120 100 93 by theaddition ofsucrose, apple seeds,hatched for and April, linseed42female 1970. oil, L. pomonella reared on the Patana diet modified iue9 DistributionFigure of9. number of eggslaid and proportion which % of eggs hatched 100 20 10 80|— 20 Number ofeggs laid by individual female 100 120 140 160 180 200 94 95 hatch was smaller than with the Redfern diet (Table 7; Fig. 9). The weights of pupae of L. pomonella reared, on (i) the original Patana diet, (ii) the modified Patana diet, and (iii) thinning apples were compared (Table 8 ). The average weights of male and female pupae from the original Patana diet were lowest of all. The average weights of pupae reared on the modified Patana diet were closer to those of pupae reared on thinning apples. The average weight of female pupae reared on the modified Patana diet was 6.1 mg more than that of female pupae reared on the original Patana diet, and 2.4 mg less than the average weight of female pupae reared on thinning apples. The average weight of male pupae reared on the modified Patana diet was 5*9 mg more than that of male pupae reared on the original Patana diet, and 0.2 mg less than the average weight of male pupae reared on thinning apples. The greatest ranges of weights were found within the samples of female pupae from all three groups. The ranges of weights for female pupae from the original Patana diet, the modified Patana diet, and thinning apples were 19.3 mg, 30.3 mg, and 28.4 mg, respectively. A final observation on the rearing of L. pomonella in the labo ratory is an analysis of the mortality occurring in the developmental stages of a population. A record of mortality was kept for a popula tion that was reared on the modified Patana diet. The results of the study are shown in Table 9. Most of the mortality occurred in the first larval stage. It was observed that many of the first-instar lar vae which died did not commence feeding. A large number of individuals also died as pupae. In many such cases of death the completely formed adult could be seen through the pupal cuticle. 96 Table 8 . Average weights of female and male pupae of L. pomonella reared on three different dietsa. Female pupae Male pupae Avg. wt. Range Avg. wt. Range Diet No. (mg) (mg) No. (mg) (mg) Patana 53 32.8 24.6 - 43.9 41 25.1 18.7 - 34.8 Patana, modified 53 38.7 17.9 - 48.2 52 31.0 19.2 - 39.5 Thinning apples 25 41.1 27.9 - 56.3 33 31.2 19.6 - 40.6 a Pupae were weighed within 24 hours after pupation. 97 Table 9. Mortality in the immature stages of a generation of L. pomonella reared in the laboratory on the modified Patana dieta . Larval instars Life stages; I II III IV V Pupa Total Number dying; 312 9 8 9 29 208 570 $> mortality; 13.90 0.1? 0.39 0.38 1.29 8.90 29.93 1o survival; 86,60 86.93 86.09 85.71 89.97 75.57 75.57 a The generation began with 2,337 neonate larvae; 12 were killed by fungal contamination of the diet; 1,79-7 became adults. LABORATORY REARING OF L. POMONELLA ON ARTIFICIAL DIETS III. DISCUSSION In a study of artificial diets used to rear L. pomonella in the laboratory, it was found that development on the wheat germ diet (Redfem, 196*0 was abnormal. Survival from neonate larvae to adults averaged only 15.5 per cent (Table 5) and the average development time to adult emergence ranged from 55 to 75 days for six generations (Table 6). Rock (1967) reported 90 per cent survival and an average of 28 to 29 days to adult emergence for L. pomonella reared on apples; and 75 to 79 per cent survival and 28 days to emergence for rearing on an artificial ("casein") diet, Brinton et al. (1969) obtained 60 per cent survival and development from neonate larvae to adults in an average of 28 days for rearing on a casein-wheat germ diet after Ignoffo (1963). Oviposition by females reared on the Redfem diet was not high in comparison with results obtained by others. Moths reared on apples laid about 80 eggs per female (Hamilton and Hathaway, 1966), Brinton et aL (1969) obtained 1*4-5 eggs per female from moths reared on a casein-wheat germ diet, and an average of 87 per cent hatched. Two groups of moths reared on the Redfem diet in this study laid an aver age of about 55 and 60 eggs per female, and the average percentage that 98 99 hatched was 63 and 77 > respectively (Table 7). When rearing was switched from the Redfern diet to the lima bean diet of Patana (1969), the average development period was almost halved (Table 6), and the survival rate was more than tripled (Table 5). Average oviposition was increased to 83 eggs per female, but the pro portion hatching decreased to 58.6 per cent (Table 7). This was the first known use of the Patana lima bean diet for rearing L. pomonella. Apparently, there were nutritional inadequacies in the Redfern diet that was used. It is possible that some ingredients had decom posed during storage. With a diet this complex it would be hard to determine which components were inadequate. The second generation reared on the Patana diet did not develop as well as the first. Survival decreased slightly, and average ovipo sition was down by about 32 eggs per female. It was apparent that this diet was not completely adequate. Consequently, the diet of the next generation was supplemented with sucrose, apple seeds, and linseed oil. Sucrose is said to be a feeding stimulant; the linolenic and linoleic acids of linseed oil are required for pupation and metamorphosis; and apple seeds might provide essential amino acids. For the population reared on this modified diet, survival rose to nearly 75 per cent (Table 5), the average development period was down to 29 days (Table 6); and oviposition rose to about 118 eggs per female (Table 7); but egg hatch was only 5113 per cent. The modified Patana diet was superior in most respects to the Redfern or normal Patana diets. Analysis of mortality in developmental stages of L. pomonella reared on the modified Patana diet showed that most of the deaths occurred in 100 the egg stage (Table 7)» the first larval stage, and the pupal stage (Table 9). The diet may lack nutrients which favor survival in these stages. Additional sucrose or another feeding stimulant might increase survival of first-instar larvae. When progeny of L. pomonella reared on thinning apples were reared on the Redfem diet, the survival was only 5.2 per cent (Table 5) • This effect might indicate a genetic response with only 5*2 per cent of the initial population being capable of utilizing and developing to maturity on the artificial food. Survival for the succeeding genera tion of that strain on the Redfem diet was 18.5 per cent, further indicating genetic selection and adaptation of the strain to a new food. When a strain was switched from the Redfem diet to the Patana diet, survival increased by more than five times. This might demon strate a physiological response with more genotypes of the strain able to utilize the lima bean medium and develop to maturity. Other evi dence for survival of more genotypes is the wider variation in ovipo sition and egg hatch (Table 7? Figures 7 - 9)» and in pupal weights (Table 8). The fact that development time was short and without much variation indicates the diet provides good nutrition for the rapid development of surviving genotypes. Besides nutritional benefits, the lima bean diet was easier to prepare and was less expensive to use because of the small number of easily obtainable ingredients. The Redfem diet had been broken down into a large number of simple nutritional components, some of which were only available from specialized manufacturers at high cost. If the artificial diet is to be used for rearing and not for nutritional 101 studies, the lima bean medium seems to offer many advantages. Whether the modified Patana diet would be satisfactory for the long-term rearing of L, pomonella can only be determined by continued rearing. Perhaps in further testing it would not prove adequate, and more additions or modifications would have to be made. Certainly an adequate diet as simple as this one is would be an asset to the mass- rearing of L. pomonella and should merit further use and testing. SUMMARY A granulosis virus pathogenic for the codling moth, Laspeyresia pomonella (L.) (Lepidopterai Olethreutidae), was introduced into an orchard block in south-central Ohio, and the effect upon the host popu lation was evaluated. The virus was applied as an aqueous suspension using a standard air-blast orchard sprayer. Apples were examined to see if injury to fruit was reduced on treated trees. Larvae in fruit and trapped in bands on tree trunks were examined to determine whether the introduced virus was causing mortality. The L. pomonella popula tion was studied the following year to learn whether the virus was effective upon succeeding generations. Following the application of virus, dead virus-diseased larvae were found on the treated trees. It was estimated that about 20 per cent of the larvae on treated trees died from the virus disease while still developing in fruit, and that a total of 93 per cent of all lar vae on treated trees died from the disease before the pupal stage was reached. The virus as applied reduced survival of treated populations to a considerable extent but did not reduce economic injury to fruit. The virus did not spread to any appreciable degree from the sites of intro duction during the generation in which it was effective. The pathogen did not persist in the test area to cause mortality the following 102 103 season. This granulosis virus can effectively reduce L. pomonella populations if it is reintroduced when the first-instar larvae of each generation are beginning to feed. One should not expect the disease to become enzootic. When laboratory rearing of L. pomonella was examined, it was found that the insect was not developing normally on the Redfern wheat germ diet. Survival from neonate larvae to adults was an average of only 15.5 per cent over six generations. The average period required for development was extended nearly two-and-one-half times the normal. The lima bean diet of Patana was evaluated as a rearing medium for L. pomonella. Survival of the first generation reared on this new diet increased to 51.6 per cent, and development was completed in the near-normal period of 33 days. Results were even better when the Patana diet was supplemented with sucrose, apple seeds, and linseed oil. Survival increased to nearly 75 per cent, and the average devel opment period dropped to 29 days. Also of importance was the fact that oviposition more than doubled to an average of nearly 118 eggs per female. If this diet proves to be an adequate medium for continuous rearing, it may provide maximum production in a minimum of time. The modified Patana diet has many fewer ingredients than the Redfem diet and thus is cheaper to use. It apparently supports a wider variety of genotypes than did the Redfem diet. Where mass-production of L. pomonella is desired, it may prove to be a superior rearing medium. BIBLIOGRAPHY Allen, G, E, 1968. Evaluation of the Heliothis nuclear polyhedrosis virus as a microbial insecticide. Proceedings of the Joint United States-Japan Seminar on Microbial Control Agents of Insects. Fukuoka, Japan, 1967. p. 37-41. Arkhipova, V. D. 1965. Fungous diseases of the codling moth, Carpocapsa pomonella L. (Lepidoptera, Tortricidae). Entomol. Rev. 44: 48-54. Amott, H. J. and K. M. Smith. 1968a. 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M. 1944, Codling moth infestations at different heights in apple trees. Va. Agric. Expt. Sta. Bull. 36O. 10 p. Worthley, H. N. 1932. Chemically treated bands in Pennsylvania. J. Econ. Entomol. 25s 1133-1143. Worthley, H. N. 1934. A second report on codling moth bands in Pennsylvania. J. Scon. Entomol. 27s 346-352. Wyatt, G. R. 1952. The nucleic acids of some insect viruses. J. Gen. Physiol. 36: 201-205. Yothers, M. A,, P. B. Allen, and P. M. Scheffer. 1935. High percent age of parasitization of codling moth eggs by Trichogramma minutum Riley in Wenatchee, Washington District. J. Econ. Entomol. 28: 247-248. Yothers, M, A. and F, W, Carlson. 1941. Orchard observations of the emergence of codling moths from two-year old larvae. J. Econ. Entomol. 34: 109-110. APPENDIX A SUPPLIES FOR REARING L. PQMONELLA IN THE LABORATORY Jelly Cups (No. 2310) and Lids (No. 2347)» premium Plastics, Inc., 465 West Cermak Road, Chicago, Illinois 60616 Plastic Crisper (No. 395F)» Tri-State Plastic Molding Co., Henderson, Kentucky Parafilm "M," Marathon Division, American Can Co., Neenah, Wisconsin Dried Apple Slices, Vacu Dry Co., Emeryville, California 94608 Apple Seeds, Herbst Brothers Seedmen, Inc., 100 North Main Street, Brewster, New York Soybean Protein, Worthington Foods, Inc., 900 Proprietors Road, Worthington, Ohio 43085 Wheat Embryo (germ), Nu-Age Biorganic Products of Canada Limited, 101 Shorncliff Road, Toronto 18, Ontario, Canada Alphacel, Nutritional Biochemicals Corp., 26201 Miles Road, Cleveland, Ohio 44128 Vanderzant Modification Vitamin Mixture for Insect Diet, Nutritional Biochemicals Corp., 26201 Miles Road, Cleveland, Ohio 44128 Salt Mixture - Wesson, General Biochemicals, 925 Laboratory Park, Chagrin Falls, Ohio 44022 Bacto-Agar, Difco Laboratories, Detroit, Michigan 117 APPENDIX B RECORDS OF DEVELOPMENT OF L. POMONELLA ON ARTIFICIAL DIETS 118 119 Table 10. Development of L. pomonella on the Redfem diet, 24 April to 20 August, 1968. The initial population was 1,755 neonate larvae, and the survival was 33.1 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths $ of total emergence moths j> of total of adult emerged emerged of adult emerged emerged 44 4 0.68 66 10 65.22 45 1 0.85 67 18 68.31 46 2 1.19 68 37 74.67 48 4 1,87 69 24 78.80 49 13 4.10 70 10 80.52 50 10 5.82 71 14 82.92 51 17 8.74 72 35 88.94 52 17 11.66 73 8 90.31 53 20 15.10 74 10 92.03 54 12 17.16 75 10 93.75 55 12 19.22 76 4 94.43 56 25 25.52 77 8 95.80 57 23 29.47 78 5 96.66 58 25 33.77 81 3 97.17 59 36 39.96 82 5 98.03 60 20 43.40 83 1 98.20 61 14 45.80 85 6 99.23 62 14 48.20 86 3 99.7^ 63 26 52.67 88 1 99.91 64 13 54.90 89 1 100.00 65 50 63.50 total 581 120 Table 11. Development of L. pomonella on the Redfem diet, 8 May to 6 August, 1969. The initial population was 875 neonate larvae, and the survival was 8.8 per cent. Days to So. of Cumulative Days to No. of Cumulative emergence moths i of total emergence moths $ of total of adult emerged emerged of adult emerged emerged 45 1 1.29 58 1 70.12 46 2 3.89 59 6 77.92 48 12 19.48 60 4 83.11 49 5 25.97 61 1 84.41 50 1 27.27 62 1 85.71 51 2 29.87 63 3 89.61 52 10 42.85 64 2 92.20 53 2 45.45 65 2 94.80 54 5 51.94 72 1 96.10 55 8 62.33 73 1 97.40 56 3 66.23 76 _2 100.00 57 2 68.83 total 77 121 Table 12. Development of first generation of Washington strain of L. pomonella on the Redfem diet, 19 May to 2*+ June, 1969. The paren tal generation had been reared on thinning apples. The initial popula tion was 2,423 neonate larvae, and the survival was 5*2 per cent. Days to No. of Cumulative Days to Mo. of Cumulative emergence moths % of total emergence moths $> of total of adult emerged emerged of adult emerged emerged 42 1 0.80 59 1 65.22 4 3 3 3.21 60 4 68.44 44 2 4.82 61 4 71.66 46 8 11.27 62 3 74.07 47 3 13.68 63 10 82.13 48 14 24.97 64 3 84.54 49 9 32.22 65 1 85.34 50 4 35.44 60 3 8 7.75 51 3 37.85 67 6 92.58 52 2 39.46 69 2 94.19 53 6 44.29 70 1 94.99 54 3 46.70 71 1 95.79 55 4 49.92 72 2 97.40 56 9 57.17 73 1 98.20 57 7 62.81 74 1 99.00 58 2 64.42 1 100.00 1 1 total 124 122 Table 13. Development of second generation of Washington strain of L. pomonella on the Redfem diet, 16 July to 28 August, 1969. The initial population was 411 neonate larvae, and the survival was 18.5 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths % of total emergence moths $ of total of adult emerged emerged of adult emerged emerged 50 1 1.31 65 2 70.99 52 5 7.88 66 2 73.62 54 2 10.51 67 1 74.93 55 2 13.14 68 1 76.24 56 3 17.08 69 1 77.55 57 3 21.02 70 3 81.49 58 6 28.91 72 4 86.75 59 6 36.80 73 1 88.06 60 6 44.69 75 1 89.37 61 3 48.63 76 3 93.31 62 7 57.84 79 2 95.94 63 2 60.47 81 1 97.25 64 6 68.36 85 _2 100.00 total 76 123 Table 14. Development of L. pomonella on the Redfern diet, 17 October, 1969, to 8 January, 1970. The initial population was 862 neonate larvae, and the survival was 17.86 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths $> of total emergence moths f> of total of adult emerged emerged of adult emerged emerged 35 4 2.59 71 4 41.97 37 1 3.23 72 4 4^.56 38 1 3.87 73 3 46.50 39 5 7.11 75 10 52.99 41 3 9.05 76 1 53.63 42 2 10.34 77 5 56.87 43 5 13.58 78 6 60.76 45 2 14.87 79 12 68.55 46 2 16.16 80 4 71.14 47 1 16.80 81 1 71.78 49 1 17.44 82 5 75.02 51 5 20.68 83 3 76.96 52 2 21.97 84 1 77.60 53 1 22.61 85 7 82.14 55 1 23.25 87 2 83.43 56 1 23.89 88 6 87.32 57 3 25.83 90 3 89.26 58 3 27.77 91 2 90.55 59 1 28.41 92 6 94.4* 60 3 30.35 93 2 95.73 63 2 31.64 95 1 96.37 65 1 32.28 96 1 97.01 66 1 32.92 102 1 97.65 67 2 34.21 104 1 98.29 68 3 36.15 108 1 98.93 69 2 37.44 110 1 100.00 70 3 39.38 total 155 124 Table 15. Development of L. pomonella on the Redfem diet, 5 member, 1969» to 4 January, 1970. The initial population was 5> neonate larvae, and the survival was 9.20 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths $ of total emergence moths # of total of adult emerged emerged of adult emerged emerged 42 2 3.92 54 4 47.04 43 1 5.88 55 8 62.72 45 1 7.84 56 2 66,64 48 1 9.80 57 3 72.52 49 4 17.64 58 6 84.28 50 3 23.52 59 2 88.20 51 2 27.44 60 2 92.12 52 4 35.28 62 1 94.08 53 2 39.20 § 1 _J 100.00 total 51 125 Table 16. Development of L. pomonella on Patana diet, 16 December, 1969, to 15 January, 1970. The initial population was 1,441 neonate larvae, and the survival was 51*6 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths # of total emergence moths jo of total of adult emerged emerged of adult emerged emerged 26 1 0.13 37 36 90.14 27 5 0.82 38 32 94.61 28 21 3.75 39 8 95.72 29 39 9.20 40 9 96.97 30 45 15.49 41 8 98.08 31 89 27.93 42 2 98.35 32 132 46.39 43 1 98.48 33 96 59.81 44 5 99.17 34 83 71.41 45 3 99.58 35 67 80.78 47 1 99.71 36 31 85.11 48 1 100.00 total 715 126 Table 17. Development of L. pomonella on Patana diet, 25 January to 25 February, 1970. The initial population was 1,549 neonate larvae, and the survival was 48.2 per cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths ia of total emergence moths % of total of adult emerged emerged of adult emerged emerged 25 7 0.93 35 42 86.80 26 12 2.53 36 29 90.68 27 22 5.^7 37 19 93.22 28 61 13.64 38 17 95.49 29 109 28.25 39 10 96.83 30 107 42.59 40 10 98.17 31 86 54.11 41 4 98.70 32 106 67.51 42 2 98.96 33 66 76.35 43 6 99.76 34 36 81.17 44- 1 100.00 total 746 127 Table 18. Development of L. pomonella on modified Patana diet, 1 March to 4 April, 1970. The initial population was 2,119 neonate larvae, and the survival was 74.8 pier cent. Days to No. of Cumulative Days to No. of Cumulative emergence moths % of total emergence moths jb of total of adult emerged emerged of adult emerged emerged 23 2 0.12 34 27 95.98 24 8 0.62 35 26 97.61 25 60 4-. 39 36 10 98.23 26 148 13.70 38 7 98.6 7 27 251 29.49 39 7 99.11 28 281 47.17 40 5 99.42 29 270 64.16 41 4 99.67 30 207 77.18 43 1 99.73 31 128 85.23 44 1 99.79 32 86 90.64 45 1 99.85 33 58 94.29 46 1 100.00 total 1,589