S
AN ABSTRACT OF THE THESIS OF
Gary Boyd Pitman for the M. S. in ENTOMOLOGY (Degree) (Major)
Date thesis is presented y
Title SOME ENVIRONMENTAL FACTORS INFLUENCING REARING OF THE SPRUCE
BUDWORM, Choristoneura fumiferana (Clem.) (LEPIDOPTERA: TORTRICIDAE)
UNDER LABORATORY CONDITIONS.
Abstract approved , (Major Professor)
The purpose of this study was to determine the effects of controlled environmental factors upon the development of the spruce budworm (Choristoneura fumiferana Clem.) and to utilize the information for im- proving mass rearing procedures. A standard and a green form of the bud - worm occurring in the Pacific Northwest were compared morphologically and as to their suitability for mass rearing.
" An exploratory study demonstrated that both forms of the budworm could be reared in quantity in the laboratory under conditions outlined by Stehr, but that greater survival and efficiency of production would be needed for mass rearing purposes.
Further experimentation revealed that, by manipulating environmental factors during the rearing process, the number of budworm generations could be increased from one that occurs normally to nearly three per year. For the standard form of the budworm, procedures were developed for in- creasing laboratory stock twelvefold per generation. Productivity of the green form was much less, indicating that the standard form may be better suited for laboratory rearing in quantity.
Recommended rearing procedures consist of the following steps. Egg masses should be incubated at temperatures between 70 and 75 °F and a relative humidity near 77 percent. Under these conditions, embryo matur- ation and hibernacula site selection require approximately 8 to 9 days. The larvae should be left at incubation conditions for no longer than three weeks. A pre- conditioning temperature treatment, one week at 45°F, appears necessary before larvae in hibernacula are exposed to overwinter- ing conditions. The cold rest requirement is fulfilled partially by holding the larvae for 52 days at a constant temperature of 33 °F. This period appears to be optimal in terms of survival and minimum storage time. Completion of the resting period is brought about by constant ± illumination on a fluorescent light table at a temperature of 75 3 °F. Approximately 26 days is required for total larval issuance. 2
Newly issued larvae should be collected daily and confined in half - pint ice cream cartons containing newly prepared or freshly thawed, agar - coated Douglas -fir seedlings. The optimum number of larvae per carton has not been determined; however 20 per feeding container appears to be the maximum for uninhibited development. The first food change is governed by the rate of deterioration of food and is usually necessary 10 days after larval introduction. Two or three more food changes may be required; this will depend on the number of surviving larvae. When held at nearly 75°F, most larvae mature and pupate in approximately 30 days. Adult eclosion occurs within 7 to 8 days; absorbent substrate was found useful in aiding eclosion.
Satisfactory mass mating and oviposition can be obtained by using a large, walk -in cage containing young potted Douglas -fir trees. Egg masses can be concentrated on one or two trees at one corner of the cage by use of low intensity light at this corner.
Studies of photomicrographs of sectioned thoracic cuticles of 1 -day-
old pupae and empty pupae cases.. revealed groins dissimilarities between the two forms of the budworm. The outer region of the standard form's cuticle was markedly darker than the green form which suggests a differ- ence in exocuticle composition. However, no significant variation in total cuticle thickness or exocuticle thickness could be determined. No dif- ferences were found in the cuticle of mature larvae of the two forms.
Characteristics of the cuticle were noted in detail. The ventral portion of the thoracic cuticle is discontinuous. Each appendage is separated from internal structures by a membrane appearing to be an ex- tension of the outer cuticle. Integument rigidity is maintained through a bridle structure which is fastened to the outer cuticle of each forming appendage. The type of bridle associated with the forming wings was similar in both forms. The various bridle structures varied in size be- tween the two forms.
-+ SOME ENVIRONMENTAL FACTORS INFLUENCING REARING OF THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA (CLEM.) (LEPIDOPTERA: TORTRICIDAE) UNDER LABORATORY CONDITIONS
by
GARY BOYD PITMAN
A THESIS
submitted to
OREGON STATE UNIVERSITY
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
June 1963 APPROVED:
Associate Professor of Entomology
Chairman of Department of Entomology
Chairman of School raduate ommittee
Dean of Graduate School
Date thesis is presented 2,z /, /G 2-
Typed by Helen Sewell ACKNOWLEDGMENTS
Acknowledgment should first be made to the Pacific Northwest Forest and Range Experiment Station, U. S. Forest Service, and the Department of Entomology, Oregon State University. The cooperative agreement between the two organizations was the basis for this study. The author is grateful to several people whose guidance and assitance made the completion of this study possible. Specific acknowledgments are due, as follows:
To Dr. Julius A. Rudinsky, Professor of Entomology, Oregon State
University, for advice and criticism on all phases of this study.
To Dr. Paul O. Ritcher, Chairman, Department of Entomology, Oregon
State University, for assistance in planning this research.
To Mr. Valentine M. Carolin Jr., Entomologist, Pacific Northwest
Forest and Range Experiment Station, for his constant guidance and encouragement throughout the entire period of study.
To Mr. Robert L. Furniss, Chief, Division of Forest Insect Research,
Pacific Northwest Forest and Range Experiment Station, for constructive criticism of work plans and for reviewing the manuscript.
To Dr. Clarence G. Thompson, Insect Pathologist, Pacific Northwest
Forest and Range Experiment Station, for identification of pathogenic microorganisms encountered during the rearing program.
To Dr. Roger B. Ryan, Entomologist, Pacific Northwest Forest and
Range Experiment Station, for drawing Figure 5.
To Mr. Wallace C. Guy, Pacific Northwest Forest and Range Experiment
Station, for photographic assistance. To Mr. Thomas Onsdorff, Associate Professor of Food Technology,
Oregon State University, for making available the walk -in coolers used
in this research. TABLE OF CONTENTS
Page
INTRODUCTION 1
Status as a Forest Pest t , 1 Justification for Study 2 3 . Objectives. ., . . . . , .6
REVIEW OF LITERATURE . . r 4
Life History . . -, k. e 4
Diapause - 6
Rearing Techniques e ,...... 11
MATERIALS AND METHODS w Y 16
Obtaining Experimental Stock . 16 Laboratory Rearing Conditions 17
Mating and Oviposition . . , - 18 Walk -in cage 18
Gallon jars r 4 . .. ., 18
Plastic boxes _ . 19
Incubation and Pre -diapause .. . 21
Petri dishes - - 21
Ice cream cartons . 21
Light table , , ...... 23 Growth chambers 23
Diapause Is . , 24
Walk -in coolers i 24
Growth chamber modified for variable temperatures . 24
Post -diapause . . . 27
Handling procedure . . , 4 , 27
Natural food ...... 28
Modified food _ t - 29
Feeding procedure - 30
Preparation of Cuticle Sections . . . .. 32
Larvae and pupae ti M 32
Pupal case - 35 TABLE OF CONTENTS - Continued
Page
RESULTS 36
Preliminary Study 36 Environmental Studies 39
Fecundity and incubation , 39 Hibernacula selection 53 Incidence of non -diapause 54
Pre -diapause treatments ...... , . 55
Effects of a fluctuating . cold storage temperature . . 64 Role of temperature and light in terminating
diapause . . 66 Effect of a thermal shock treatment during diapause development 72
Modified food ,. .. ,r 75
Comparative Morphological Studies 4, 83
DISCUSSION - . . ., 93
98 CONCLUSIONS . . . - - LIST OF FIGURES
Figure Page
1. Large walk -in cage used for mass mating and
oviposition , 20
2. One gallon jar mating confinements 20
3. Stainless steel tray and individual plastic boxes used for individual mating confinements 22
4. Modified half -pint ice cream carton with egg -bearing needles cemented to lid 22
5. Diagrammatic view of the essential parts of the temperature programming device 26
6. Wooden rack used to hold ice -cream cartons during
feeding procedure , . 31
7. Relation of percent egg survival to relative humidity at three chamber temperatures as compared with standard rearing conditions 52
8. Effects of various pre -diapause treatments on larval issuance following 6, 8, 10, and 12 weeks cold rest
(Green form) - - 62
9. Summary of larval survival during diapause development after 4 different periods of cold rest, as determined by the mean percent survival for various pre -diapause
treatment groups. (Green form) ...... , . . . 63
10. Effect of fluorescent and incandescent illumination on diapause termination in larvae receiving
different periods of cold rest. (Standard form) . . . . 71
11. Photomicrograph of a 10 micron X- section (79.3X) through the posterior margin of the prothorax of
a 1 -day old pupa (Standard form) ' 85
12. Photomicrograph of a 10 micron X- section (16X) through the posterior margin of the prothorax
of a 1 -day pupa - old (Standard form) , 85
13. Photomicrograph of a 10 micron X- section (79.3X) through the posterior margin of the prothorax of a 1 -day old pupa (Green form) 86 LIST OF FIGURES - Continued
Page Figure
14. Photomicrograph of a 10 micron X- section (16X) through the posterior margin of the prothorax of
a 1 -day old pupa (Green form) . . . . 86
15. Photomicrographs of a 10 micron X- section (79.3X) of a portion of the pupal cuticle covering the mesothoracic wings after adult emergence.
A. Standard form. B. Green form ., 88
16. Photomicrograph of a 10 micron X- section (79.3X) through the thoracic region of an empty pupal case
(Standard form) ti ...... 88
17. Photomicrograph of a 10 micron X- section (79.3X) through the anterior margin of the first abdominal segment in a sixth stage larvae (Standard form) 90
18. Photomicrograph of a 10 micron X- section (79.3X) through the anterior margin of the first abdominal segment in a sixth stage larva (Bidwell) 90
19. Photomicrograph of a 10 micron X- section (79.3X) through the anterior margin of the first abdominal
segment in a sixth stage larva (Green form) ...... 91 LIST OF TABLES
Table Page
1. Daily record of oviposition by spruce budworm moths held in individual confinement (Green form) 40
2. Daily record of oviposition by spruce budworm moths held in individual confinement (Standard form) 43
3. Summary of embryo survival occurring in spruce budworm eggs when incubated at 75 ± 3°F. and a relative humidity of 55 -70% (Standard form) 47
4. Temperatures, humidities, and materials used for
treatment of the green form egg mass : : , . . 48
5. Survival of eggs given various temperature and humidity treatments compared with survival of
eggs reared under "standard conditions" (Green form) . . . 50
6. Summary of embryo survival occurring in spruce ± budworm eggs incubated at 75 3 °F. and a relative
humidity of 55 -70% (Green form) . . 51
7. Influence of two light sources on the rate at which newly emerged larvae select hibernacula sites
(standard form and green form) . 54
8. Summary of pre- diapause treatments ...... 57
9. Effects of various pre -diapause treatments on larval survival following 6, 8, 10, and 12 weeks
cold rest (Green form) . . . , , 59
10. Effects of various periods of fluctuating cold storage temperatures on overwintering mortality (Standard form) 67
11. Effects of different light sources on the reduction of diapause requirements in overwintering larvae subjected to varying lengths of cold storage
(Standard form) ...... 70
12. Effects of varying periods of cold rest on the reduction of diapause requirements in the green (A) and standard (B) forms of spruce budworm 73 LIST OF TABLES - Continued
Table Page
13 Summary of larval survival using natural Douglas -
fir seedlings (Standard form) .. 77
14. Summary of larval survival using modified
Douglas -fir seedlings (Standard form) ...... 79
15 Analysis of variance test on the number of larvae alive at the first food change when fed natural
Douglas -fir seedlings (Standard form) ...... 82
16. Summary of expected productivity when standard form spruce budworm are reared at optimum
laboratory conditions . 96 SOME ENVIRONMENTAL FACTORS INFLUENCING REARING OF THE SPRUCE BUDWORM, Choristoneura fumiferana (Clem.) (LEPIDOPTERA: TORTRICIDAE) UNDER LABORATORY CONDITIONS
INTRODUCTION
Status as a Forest Pest
The spruce budworm (Choristoneura fumiferana (Clem.)), a native insect, can be characterized as the most destructive forest defoliator in North America. Its recorded range is from Virginia north to
Labrador, west across Canada and the northern United States to the
Pacific coast, as far north as the 67th parallel in the Northwest
Territories, and south into northeastern California and southern New
Mexico. In western North America, the first recorded outbreak was in
1909 in British Columbia (39). The first record of this insect in western United States was in 1914 at Ashland, Oregon. In 1922 two separate outbreaks occurred in Idaho. Since then, many widespread and destructive infestations have been recorded in the Rocky Mountains and in the Pacific Northwest.
In the Pacific Northwest the first major control project was initiated against the spruce budworm in 1949, by aerial application of
DDT over 267,000 acres. In the subsequent 6 years, 3,583,000 acres of infestation were successfully treated by this method (68).
Aerial application of chemicals to forest lands is not restricted to insecticides, for such techniques have and are being used for disease and weed control, and to stimulate tree growth. For the most part, forest defoliator control heads the list of uses, with the past emphasis being directed towards the spruce budworm and gypsy moth 2
(Porthetria dispar (L.)). In the past, approximately 25 million acres have been treated in the United States and Canada in controlling these two defoliators (16).
Justification for Study
In the past, extensive losses of timber resources resulting from defoliation were averted by aerial applications of DDT. The use of this material has created problems, some of which may serve to limit the continued use of this insecticide. These problems, as they now exist, appear to be centered around a single consideration, the nonselective action of this material.
Considerable data have accumulated which considers the effects on
the forest fauna as a result of spruce budworm control projects. For example, numerous studies have shown that the mortality of forest
terrestrial and aquatic invertebrates was severe following some spruce budworm control programs. The results of a reduced aquatic invertebrate population are shown by a correspondingly high fish mortality.
The effect of DDT upon mammals, birds, amphibians, and reptiles appears to be less severe; however, the latter two groups show more
sensitivity than the former two. There is little direct injury to the
four groups, but at the same time insectivores would suffer as a result
of extensive killing of forest insects.
Public reaction is mounting against the widespread use of non-
selective pesticides. Public Law 85 -582 was passed, which directs the
Secretary of the Interior to undertake studies on the effects of pes-
ticides on wildlife populations. If chemical applications are to
continue as a tool for curbing forest pest outbreaks, more selective 3 insecticides must be developed. Solomon (53, p. 1146) summarizes by saying, "We need more insecticides but of a more selective action than many in general use. Then it will be easier to apply chemical control in a more discriminatory way, in a more realistic relationship to com- plex biological situations which it is our aim to modify."
Continuous availability of experimental animals is a prerequisite for developing selective insecticides. The purpose of this study is
to develop techniques for mass rearing of the spruce budworm so it may
be used effectively for insecticide screening. This insect serving as
an experimental animal appears to be a logical choice, especially when
past control efforts as well as its present destructive potentialities
are considered.
Development of satisfactory rearing techniques are not only neces-
sary for insecticide programs, but their consequence is apparent for
basic biological investigations of other kinds. The applicability of
such a rearing program could be useful in the field of biological con-
trol as well as studies on physiology, genetics, pathology and others.
Objectives
The objectives of this thesis may be summarized as:
A. To determine effects of environmental factors in the labora-
tory influencing success of mass rearing.
B. To compare two forms of the spruce ubdworm as to suitability
for mass rearing.
C. To compare morphological characteristics of two forms of the
spruce budworm. 4
REVIEW OF LITERATURE
Life History
The first critical description of the spruce budworm's life cycle
was completed in 1924, by Swaine, Craighead, and Bailey (57). Research
has since been completed which presents a detailed account of the
habits and reactions of this insect (3, 22, 24, 41, 60, 61, 62, 63, 64).
Under conditions found in the Northwest, the spruce budworm has 1 gen-
eration per year with the major portion of the life cycle being passed
as an overwintering second instar. During the spring, larvae emerging
from their overwintering sites exhibit a strong positive phototaxis and
of the tree. If crowding is not severe, feed- ' move toward the periphery
ing is usually initiated by mining the previous year's foliage. In
reference to the eastern population of spruce budworm, Atwood
(3, p. 64 -65) records the process of needle mining as follows: " - - -a
small web is spun connecting two needles and within this shelter a hole
is cut through the lower epidermis of the needle. The mine is then
continued between the upper and lower epidermis until the larva is
completely hidden." Atwood suggested this type of feeding behaviour
was atypical; however, McOugan (41, p. 441) was able to demonstrate a
similar mining pattern in the western form budworm. In most cases the
second stadium is completed within the needle; however, a portion of
the third stadium may also be completed before the needle is abandoned.
The remaining 3 instars develop at the expense of the current and pre-
vious year's foliage. 5
The spruce budworm larvae pass through their entire development on the host tree. Following a short prepupal period, transformation into obtect pupae typically takes place within the feeding shelter of the larvae. Under field conditions, the adult moths are usually ob- served to be ovipositing during the middle of July, however, this per- iod is variable and is governed by local climatic conditions. Mean fecundity is dependent on the degree of current defoliation; heavy defoliation may reduce the fecundity as much as 50 percent. The eggs are deposited on the under side of the needles in clusters which have a scale or shingle -like appearance. Incubation may be from 8 -12 days, depending on the thermal conditions.
After eclosion, the young larvae are subjected to physical factors which direct their efforts in selecting a hibernacula site. The se- quence of events leading to the establishment of larvae in their over - wintering sites has been studied by Wellington and Henson (60, p. 56 -82,
61, p. 168 -170) and their observations are summarized as follows:
A positive reaction to illumination leads the newly emerged larvae to
the needle tips on which the eggs have been laid. As a result of
crowding, many larvae drop down on silken threads to the next lower limb
or are caught in vertical air currents and transported to a new tree.
If the light is diffused, due to shading of the sun, the larvae will
move towards the periphery of the tree until contact is made with bark
flakes, lichens or debris of various sorts. Upon contacting such
material, the photopositive reaction is no longer operative and is re-
placed with a positive tactile response. On the other hand, if the sun
is not shaded, the larvae on nearing the periphery may be warmed above 6 their reversible temperature (approximately 86° F. (60, p. 168) by radiant heat so that they become temporarily photonegative and are driven away from the tree periphery. Once again on contact with lichens or other acceptable material, the larvae become tactile positive and seek out a spot to spin their hibernacula. When this protective silken structure is completed, a molt follows and they overwinter as dia- pausing second stage larvae.
Diapause
The occurrence of a predictable dormant period as part of the spruce budworm's normal life cycle warrants a closer examination. Throughout
its host range, most individuals terminate morphogenesis and prepare
for the onset of winter at a time when environmental conditions favour
continued development. Harvey (21, p. 1) appears to be the first worker to refer to this dormancy :s a true diapause. As a result of no available data indicating natural deviations from the described
overwintering habit, he chose to characterize the diapause of the spruce budworm as obligatory (22, p. 550). Harvey's usage of the term is in
accord with Shelford's (50, p. 153) definition of an obligatory dia-
pause. He states, "it is often universal resulting in a life cycle which is strictly univoltine with every individual in every generation
experiencing diapause, irrespective of any possible variations in the
environment."
The term diapause was first coined by Wheeler (65, p. 68), in 1893,
to describe a stage in the embryogenesis of the grasshopper, Xiphidium
ensifer Scudder. He recognized a stage between anatrepsis and kata-
trepsis, where the embryo remained in a poised or stationary position; 7
to this intermediate stage he gave the name diapause. Henneguy
(25, p. 424 -427) extended the term to include all growth arrestment,
sug- whether in the embryo, immature or imago. ' Shelford (50, p. 150)
diapause be restricted to cases where development ' gested that the term
is arrested "spontaneously" and does not respond to any ordinary
ameliorations of the external environment. All other types of inter-
rupted growth which is directly controlled by unfavorable conditions
could be referred to as quiescence. Lees (35, p. 3) criticizes
Shelford's interpretation, for in many cases the causes for the onset
of diapause can be traced back to the action of the environment.
The unmistakable presence of an obligatory diapause in the spruce
budworm is well documented, therefore, emphasis will be placed on fac-
tors which favour the resumption of development. However, DeWilde
(11, p. 5) reminds us of the fact that since Kogure's discovery of
photoperiodism in insects, the number of obligatory diapausing insects
has shrunk markedly. Two insects, Phalera bucephala (L.) (li, p. 5)
and Listroderes obliquus Klug. (12, p. 534 -535) are known to be 1
independent from the photoperiod. Continued research on insects which
pass the major portion of their life cycle, protected from natural
illumination, will undoubtedly show numerous species which are neutral
to the photoperiod. As an example, Ryan (48, p. 7 -8) discoi;..'.s the
photoperiod as the regulatory mechanism controlling the inception of
diapause in the Douglas -fir bark beetle (Dendrortonus pseudotsugae
Hopkins).
The inception and termination of diapause are governed by organs
of internal secretion, which are in turn responsive to certain definite 8 environmental stimuli (35, p. 2). Considerable progress has been made in elucidating the physiology of arthropod diapause (6, 7, 32, 35, 36,
is of 69, 70). In reference to diapause termination, temperature paramount importance. Lees (35, p. 53) states " - -- -this factor is by
far the most important environmental agency controlling the termination of diapause ...." Andrewartha (1, p. 52 -53) has presented a plausible
theory which aids in understanding how temperature exerts control over
diapause termination. Lees (35, p. 50) summarizes this theory by
be looked stating " - -- events preceding the resumption of growth can
upon as a process of gradual development which is influenced by tem-
perature in much the same fashion as morphogenesis." This process, for
which Andrewartha coined the term "diapause development" takes on the
connotation of a physiological development or physiogenesis which goes
on during the diapause stage, in preparation for the active resumption
of morphogenesis.
The temperature required for satisfactory diapause development,
varies from species to species; however, a relationship does exist
between the geographical distribution of a species and its optimum
thermal requirement. Species normally found in northern latitudes com-
monly require comparatively low temperatures. A temperature of 2 °C.
is most effective in terminating the embryonic diapause of the forest
tent caterpillar (Malacosoma disstria Hbn.) (27, P. 13). In the spruce
sawfly (Di rion polytoma (Htg.)), temperatures near -10 °C. are most
influential in terminating diapause. Ryan (49, p. 523) was able to
satisfy the diapause requirement of the Douglas -fir bark beetle with
a temperature of 6 °C. The spruce budworm is no exception; 9
Bergold (5, p. 18) reports that diapause could be terminated with a cold treatment of 1 °C. This relationship also exists for insects which are normally subjected to mild winters. The larvae of the carpet beetle (Anthrenus verbasci L.) have an optimum near 20 °C.
Characterizing a low temperature as an optimum thermal requirement for releasing an insect from diapause, may represent an over- simplifi- cation. This hypothesis is considered by Lees (35, p. 63 -64) in his statement "---- diapause development has so far been regarded as a
unitary process, it is unlikely that any simple chemical reaction would - proceed more rapidly at low than at moderately high temperatures within the biological range. The mechanism of diapause development may be pictured with a greater probability as depending on the interaction of two or more processes which have different positive temperature coeffi- cients and which compete for the same substrate." Lees cites Le Berre's work on incubation studies of Locusta migratoria Riley, in which 25 °C. represented the diapause development optimum for the first phase and
8 °C. for the second phase. In contrast, Hodson and Weinman (27, p. 5 -6) clearly demonstrated the presence of two thermal optimums for diapause development in the forest tent caterpillar; the initial stage required temperatures near 2 °C., whereas, the final stage could be completed at
25 °C.
Diapause development in the spruce budworm presents an interesting paradox. Data suggesting a dual thermal requirement is lacking; how- ever, after an initial cold requirement has been met, illumination will serve as the environmental stimulus in terminating diapause. Harvey
(24, p. 1205 -1206) tested the effect of different photoperiods on larvae lo which had been subjected to various periods of cold rest. He demon- strated that after various periods of cold storage, larvae responded to photoperiods both in varying emergence rates and percentage of larvae surviving. This relationship was most striking in larvae which had been stored for 6 weeks at 32 °F. When the overwintering larvae were exposed to photoperiods of 0, 12, 15, and 18 hours, 20 -50 percent of the larvae emerged in the first 10 days, while survival was never greater than 20 percent. With continuous illumination, relative few individuals emerged
in the initial period and the peak occurred near the 25th day with sur- vival reaching 65 percent. This general relationship was consistent up
to 14 weeks, after which the larvae showed reduced sensitivity to photo -
periods of 15, 18, and 24 hours. However, after 26 weeks cold rest,
photoperiods of 0 and 12 hours still showed marked inhibitory effects
on issuing larvae.
In light of Harvey's data, it is interesting to note certain insects
with facultative diapause that are capable of resuming growth when ex-
posed to long -day photoperiods. Baker (4, p. 152) records several
species of treehole- inhabiting mosquito larvae that appear to respond
to long photoperiods by renewing development.
Other agents appear to be responsible for the termination of arrest-
ed growth. The role of photoperiod has been discussed above. In many
insects water may play a dominant role in diapause termination. Dia-
pause is broken in the grasshopper, Melanoplus differentiolia Thomas,
when the eggs are treated with various fat solvents and then supplied
with water. Resumption of growth appears to be a factor of rehydration,
made possible through the action of the solvents rendering the hydropyle 11 permeable to water.
With other insects, water is sometimes mistaken as the agency that
terminates diapause. According to the literature, there are cases where
the physiological process of diapause is completed, but post -diapause development cannot begin until moisture is taken up from the surround-
ing environment. As pointed out by Lees (36, p. 7), "When water is
lacking, diapause is often prolonged by a period of quiescence."
Conclusive data are lacking which indicates moisture as a prerequisite
for the emergence of overwintering spruce budworm larvae.
Rearing Techniques
Prior to 1951, data were wanting which described conditions and
techniques for rearing the spruce budworm. For the most part earlier workers (3, 60, 61, 62, 63, 64) confined their studies to field -col-
lected material and no effort was made to develop a mass rearing program.
Bergold (5, p. 17 -23), while studying a polyhedral disease, briefly
described a few conditions under which he was successful in rearing
3 generations of spruce budworm. Diapause was satisfied by keeping the
larvae at 6 °C. for 1 week with a subsequent storage at 1 °C for 4 -6
months. Development was initiated under a post -diapause environment
of 22 °C. and a relative humidity of 60 -80 percent. Depending on the
season, fresh or deep frozen young buds of balsam fir (Abies balsamea
(L.) Mill.) served as the food for the developing larvae. Precise
efficiency figures are not available from Bergold's work but he does
state that out of 17,056 overwintering larvae, approximately 57.5 per-
cent developed successfully. 12
Investigations into the genetics of the spruce budworm prompted
Stehr (55, p. 423 -428) to initiate a study on laboratory rearing pro -
cedures. The spruce budworm is not easily adapted to a laboratory ' rearing program, due primarily to 3 innate characteristics. They may be listed as, (a) the presence of an extended dormant period, approxi- mately 10 months under natural conditions, (b) an apparent strict food requirement, and (c) a high incidence of mortality. As a result of these features, Stehr (55, p. 423) outlined 3 objectives necessary for efficiently utilizing this insect: "(a) an increase in the number of generations that can be reared in a given period; (b) independence from the seasonal limitations in food supply, and (c) a reduction in mortal- ity." Techniques employed by Stehr which are incorporated in this thesis will not be covered in this discussion, therefore, only the salient points of his objectives will be considered.
Stehr's efforts in developing a rearing program were not oriented towards a comprehensive study of diapause requirements; however, he was able to define pre -diapause conditions which favoured overwintering survival. For a period of 3 -5 weeks after eclosion, the young larvae are left at rearing room temperature (21.5 t 1 °C.). This period appears to be necessary for certain physiological processes to be completed in preparation for the subsequent cold storage, however, the significance and optimal duration of this period were not established. Artificial overwintering conditions were imposed by storing the dormant larvae in either of two cold rooms. The temperatures were 0 °C. and 5 °C., with a variable exposure period from 12 -26 weeks. A breakdown of diapause development mortality is not available but 25 -30 percent survival 13 figures are indicated. These values represent an additive figure of mortality occurring during incubation, diapause and establishment of larvae on their food.
Independence from field -collected food was obtained by utilizing the feeding techniques developed by Bergold (5). Freshly opened balsam fir buds were tightly packed into dixie cups, sealed with wax and deep - frozen at -70 °F. for several hours. This material was then stored at
-5 °C. and taken from cold storage as the rearing program demanded.
Stehr indicates freshly- thawed buds retarded larval development but fecundity and viability were apparently unaffected. Of the larvae that became established on this type of food, 60 -80 percent survived to maturity.
Stehr evaluates his rearing techniques as follows: from 20 mating pairs, assuming a mean fecundity of 140 and a mating success of 60 -65 percent, 250 adults may be expected in the next generation. This represents a six fold increase per generation and apparently was con- sistent through the F4 generation. As indicated by his results, no perceptible deleterious effects occurred as a result of rearing tech- niques. As acknowledged by Stehr, overwintering conditions appear to be the factor responsible for the major portion of rearing mortality and further increases in survival would be realized if these conditions could be improved.
Following Stehr's (55) publication, Harvey (23) reported the results of a study on the diapause requirements of the spruce budworm.
This work was concerned . in part, with the effects of different lengths 14 of cold storage on the ability of the larvae to survive and develop to maturity.
To determine the effects of different lengths of cold rest, Harvey placed a large number of fairly uniform overwintering larvae in a 32°F.
cold room. Groups of larvae were removed to rearing room temperature
(71 °F.) at intervals of 3 to 4 weeks until treatments ranging up to
42 weeks were obtained. Mortality was assessed at 4 different stages;
overwintering, pre -emergence, establishment on food and individuals
reaching adulthood. The results of this study show negligible mortality
during cold storage for periods up to 30 or 35 weeks. Pre -emergence
mortality was high for the shorter storage times and progressively
decreased to a low of 4 percent by the 9th week of cold treatment; after
which the mortality gradually increased to near 100 percent by the 40th
week. A similar cold storage -mortality relationship was noted with
larvae becoming established on their food; however, establishment
mortality was generally considerably larger than pre - emergence mortality.
A direct relationship existed between individuals reaching adulthood
and the length of cold storage; as the exposure period increases, mor-
tality decreases. As a result of these data, Harvey concluded that 10
weeks is the effective minimum storage time for maximum rearing perfor-
mance. Under the conditions of this study, a rearing efficiency of
approximately 35 percent was obtained.
Efforts to effectively rear the spruce budworm have been hindered
by the obligatory requirement of an extended cold rest as part of the
normal life cycle. In 1954, Harvey (21, p. 1) reported that a few
laboratory- reared larvae were able to continue development without a 15
cold rest period. Observations on laboratory- reared stock indicated
that a few individuals deviated from the normal pattern of behavior.
These larvae reacted in the typical manner, except for the fact that
the second instars did not terminate activity immediately following the molt, but instead vacated their hibernacula and wandered on the gauze.
Harvey was able to isolate 43 of these larvae and successfully reared
18 to adults. From these adults, 5 fertile matings were obtained and
the same behaviour was noted in the next generation. To this behav-
ioural expression, Harvey appropriately gave the name non -diapause.
In a later paper (22) Harvey reported that he was able to select
for this behaviour, until by the 12th generation the requirement for a
cold rest was apparent in only a small percentage of the insects.
Harvey was able to significantly increase the efficiency of spruce bud -
worm rearing. In the second N -D generation productivity attained values
as high as eightfold with a period as short as 2 months for the comple-
tion of the life cycle. Although the proportion of N -D individuals was
greater in subsequent generations, the productivity remained steady.
All increases resulting from the higher proportion of N -D individuals
were offset by the increased failures of mating, incubation and survi-
val of the first instars. Accompanying these changes was a progressive
increase in development time. Harvey attributes these deficiencies to
continued laboratory selection. 16
MATERIALS AND METHODS
Obtaining Experimental Stock
There are two native forms of spruce budworm in the Pacific North- west that appear to be quite distinct. One form is endemic to south- eastern Oregon and parts of northeastern California, with the heaviest population occurring in the Fremont National Forest. This form is typified by being green in the later instars and early pupal stage and will be referred to as the green form in subsequent notation. The second native form is less restricted and is found in many areas of
Washington and Oregon. The later instars and pupae of this form are not green and henceforth will be designated as the standard form.
Collections were made from three different localities, as follows:
July 1960 -- full -grown larvae and pupae from Lakeview, Oregon, and Hat
Point, Oregon; October 1960 -- overwintering second -stage larvae from
Goldendale, Washington and Lakeview, Oregon; June 1961 -- partly grown larvae from Lakeview, Oregon.
The overwintering larvae obtained from the first summer's collect- ing were placed in a cold room that had been set at too low a tempera- ture. Complete mortality resulted which required a second collection to be made in October and the following summer.
The locality for the second collection of standard form budworm was
Simcoe Ridge, approximately 400 yards east of Kaiser Guard Station, near Goldendale, Washington. Host material was three Douglas -fir
(Pseudotsuga menziesii (Mirb) Franco.) trees, from which limb and bole sections were taken to obtain larvae in hibernation. A total of 17
17 five -gallon ice cream containers containing limb sections and
4 large cardboard boxes with bole sections were obtained.
The locality for the second collection of green form budworm was in the Warner Ranger District, Fremont National Forest, near Summit
Prairie, Oregon, which is located about 14 miles north of the junctions of U. S. Highway 395 and Oregon Highway 66. Limb terminals of white fir containing the larvae and pupae were clipped off and placed in quart ice -cream containers. These containers were used for transporting the insects and later served as feeding confinements for the developing budworm.
Laboratory Rearing Conditions
The forest insect laboratory at Oregon State University contains a large, well insulated room which was used for the major portion of this study. Adequate control of environmental conditions such as temperature, humidity and illumination was easily obtained.
Throughout these investigations, the rearing room was maintained at
75 t 3°F. Such control was achieved by the use of a thermostatic con-
.1 trolled floor heater and a wall type air conditioning unit. The air conditioner was allowed to run continuously, while the heater ran only intermittently. Accurate temperature regulation was achieved in this fashion but greater control could probably be obtained with a more sen- sitive heating thermostat.
Very little air stratification occurred as a result of this type of temperature control. The relative humidity was maintained between 55 and 70 percent. Some difficulty was encountered in maintaining the proper relative humidity during the summer months. The desired control 18 was obtained by periodically wetting the rearing room floor. All tem- perature and relative humidity readings were taken from a Foxboro
hygrothermograph. Temperature calibration of the recording instruments was completed using standard mercury- filled glass thermometers; no
humidity calibration was attempted.
The absence of windows afforded a nearly light -tight rearing room,
so little difficulty was encountered during photoperiod studies. Some
diffused light entered beneath the door, but this source was eliminated
by tacking a heavy strip of tar paper on the base of the door. The
rearing room was maintained as aseptic as possible, by periodically
washing the ceiling, walls and floor with a concentrated solution of
sodium hypochlorite.
Mating and Oviposition
Walk -in cage. Mass mating and oviposition was satisfactorily
achieved by the use of a large walk -in cage (Figure 1) containing
6 potted 3 -year old Douglas -fir seedlings. Trays lined with an absorb-
ent material, containing freshly moulted pupae, were placed on the floor
of the cage. Stehr (55, p. 427) has pointed out the importance of an
absorbent substrate during pupal eclosion. This material is important
for soaking up the meconium, discharged soon after emergence, and it
also offers a rough surface which aids the moth in freeing itself from
the pupal case.
Gallon jars. During the course of this study, it became necessary
to aggregate large quantities of eggs of known age. This objective was
reached by confining 10 pairs of budworm adults in each of 16 one -gallon
jars. The jars were placed over Douglas -fir boughs which had been 19 inserted into small holes drilled in a pied: of plywood. Cans filled with water were arranged beneath the plywood stage so that each bough
received a supply of water. (Figure 2).
Plastic boxes. Two experiments required the separation of individ- ual pairs so mating and oviposition behaviour could be followed. Con-
finement was obtained by using small plastic boxes, 3 -1/4 inches by
- 2 -3/4 inches by 1 -1/4 inches, each of which contained a single Douglas
fir branch terminal. Ventilation was afforded by drilling three
1/2 -inch holes in each side of the boxes. Two stainless steel trays,
32 inches by 3 -1/4 inches by 2 -1/2 inches, each capable of holding 24
boxes, were utilized as a water reservoir for maintaining the Douglas -
fir foliage (Figure 3). Each tray was fitted with a 1/2 -inch shoulder,
1 -1/2 inches below the top of the tray. This shoulder permitted the
boxes to be suspended above the water level and prevented the accumu-
lation of moisture in the plastic boxes. 20
1
Figure 1.- -Large walk -in cage used for mass mating and oviposition.
!,3 CI - , awfm. an=n NW, t -
1
Figure 2. --One gallon jar mating confinements. 21
Incubation and Pre -diapause
Petri dishes -- Egg- bearing Douglas -fir needles, collected from the various mating confinements, were glued to the bottom of 16 x 100 mm
Petri dishes with water -soluble cement. Acceptable hibernacula sites were provided by pressing a double layer of 2 x 2 inch surgical gauze onto a piece of translucent parafilm. 1/ The parafilm is then placed over the dish rim so that the gauze surface is directly above the egg masses. After replacing the Petri dish top, the dish is inserted into a light tight envelope constructed of black paper with a 1 -inch circular
hole on one side. The closed light mask is then placed on a light table.
All light is excluded from the dish except for the illumination from
below which serves to attract the newly emerged larvae to the gauze mesh.
After a short searching period, the young larvae commence to spin their
silken hibernacula between the threads of the surgical gauze.
Ice cream cartons. Each carton (Figure 4) was modified by covering
a 2 -inch hole cut in the bottom with a piece of transparent cellulose
acetate, which was secured in place with a mixture of paraffin and bees- wax. Such a mixture proved necessary to prevent the paraffin from be-
coming brittle during subsequent cold storage. Time required to modify
the ice cream cartons was relatively minor. As the second instar larvae
evacuate their hibernacula, upon satisfaction of the diapause require-
ment, a positive phototaxis is exhibited. In order to establish the
larvae on their food, the gauze containing larvae is removed from the
carton bottom and glued to the carton lid.
1/ Scientific Supplies Co., Division of Van Waters & Rogers Inc., 600 Spokane St., Seattle 4, Washington. 22
Figure 3. -- Stainless steel tray and individual plastic boxes used for individual mating confinements.
Figure 4. -- Modified half -pint ice cream carton with egg -bearing
' needles cemented to lid. 23
Light table. The first stage larvae were established in the gauze mesh by placing the ice cream cartons on a light table. The light table actually consisted of four smaller partitioned units, each approximately
47 inches by 18 inches, two of which contained a bank of 2 forty -watt
"Cool White" fluorescent lights and the other 2 units contained 2 seven and one half -watt frosted white incandescent lights. The "low inten-
sity light" units were covered with a piece of single- thickness trans- parent glass, with the distance from glass to lights being approximately
15 inches. The distance was slightly greater for the "high intensity
light" units. Considerable heat was produced in these units and to
avoid subjecting the larvae to high temperatures, two pieces of single -
thickness glass, separated by 1/4 inch plywood strips were used. A fan was used to blow a continuous stream of air between the two pieces of
glass. At no time did the upper glass surface exceed 1 °C. of ambient.
A 24 -hour day -night clock switch was used to provide the desired
photoperiod. The light table wiring was designed so each photoperiod
consisted of combinations of both high and low intensity light. Such
an arrangement proved useful in studies on hibernacula site selection
and diapause termination.
Growth chambers. Investigations concerned with various environ-
mental effects (i.e., temperature, humidity and photoperiod) on egg
incubation and pre -diapause larvae, necessitated the utilization of
4 National Appliance 1/ growth chambers (3 -model 6610 and 1 -model 6710).
The desired temperatures and photoperiods were achieved through each
1/ National Appliance Co., 7634 W. Capital Highway, Portland 19, Oregon. 24 chamber's self- contained controlling system. Maintenance of proper humidities required the addition of special materials consisting of gallon desiccator jars and various solutions of chemicals and distilled water.
Temperature regulation in the model 6610 chambers was extremely precise and did not vary .5 °F. from the desired control. The 6710 model chamber was unable to maintain the temperature with such exact- ness, and fluctuations of = 3 °F. were often noted. Illumination in the 3 large chambers was provided by eight 20 -watt "Cool White" fluor- escent lamps, controlled by a 24 -hour photoperiod timer. One 15 -watt
"Cool White" fluorescent lamp served as the source of light in the smaller chamber.
Diapause
Walk -in coolers. The requirement of a cold period as part of the spruce budworm's normal life cycle was fulfilled by storing the over - wintering larvae in a walk -in cooler maintained by the Department of
Food and Dairy Technology. The temperature and relative humidity im- posed upon the diapausing larvae were 33 ±1 °F. and 94±4 %. A temperature of 46=1 °F. and relative humidity of 82í2% was used as a pre -conditioning treatment in preparation for the 33 °F. cold room and was obtained by the walk -in cooler in the forest insect laboratory.
Growth chamber modified for variable temperatures. One experiment was designed to test the effect of a variable temperature on diapausing spruce budworm. The temperature treatment consisted of a temperature fluctuation, continuously changing between a maximum of 51 °F. and a minimum of 33±.5°F. and required 24 hours for the completion of one 25 cycle.
The system controlling the heating and cooling of a National Appli- ance growth chamber (model 6610), was modified to accommodate a temper- ature programming device. This device, constructed at the forest
insect laboratory, is designed to program harmonic temperature fluctua-
tion in any unit containing integral heating and cooling systems (47).
A diagrammatic view of the essential parts of the device is shown
in Figure 5. The drive unit is a synchronous motor (A) geared to make
one revoluation per day. The motor from an Intermatic time switch was
used, although any comparable motor would have been suitable. A cir-
cular steel plate 7 -1/4 inches in diameter and 1/4 inch thick is fas-
tened to the time switch unit and is used as the generating disc (B).
It contains tapped holes, each located at a progressively greater
distance from the center of the disc. A telescoping arm (C) is fas-
tened to the generating disc at any one of the holes by a bolt. The
telescoping arm adjusts the setting of a Cenco -Dekotinsky bimetallic
thermoregulator (catalog number 99012) through rack (D) and pinion (E)
gears as the generating disc is turned by the motor. The rack gear
runs in a wooden guide Lubricated with powdered graphite.
Although only one temperature fluctuation was used, the device is
capable of numerous temperature programs. The amplitude of the tem-
perature fluctuations is determined by the hole into which the teles-
coping arm is bolted. The mean about which the temperature fluctuates
is adjusted by disengaging the rack and pinion gears and turning the
pinion gear to the desired position. Additional small adjustments in
the mean may be made by changing the length of the telescoping arm. 26a
Figure 5.-- Diagrammatic view of the essential parts of the temperature
programming device. l!1v -- - F
E
1 If r - Q
C
CD D
B
A - SYNCHRONOUS MOTOR B - GENERATING DISC C - TELESCOPING ARM D - RACK GEAR E - PINION GEAR F - THERMOREGULATOR A .,.-Nrig- - 27
The generating disc contains 11 holes, the outermost being 3 -1/4
inch inches from the center; succeeding holes are 1 inch apart and 1/2
3.5 closer to the center of the disc. The maximum fluctuation is $ °F.
By drilling additional holes at various distances from the center of the disc an almost infinite range of fluctuations can be achieved.
The versatility of this instrument is increased when a time switch is used to control the power of the driver motor. By stopping the gener- ating disc at predetermined intervals, it is a simple matter to couple
fluctuating temperatures with constant temperatures.
Advantages associated with a programming device of this design are worthy of mention. The major merits of this control unit are the
simplicity of construction, low cost of construction and ease of dupli-
cating previous temperature fluctuations. These advantages, as indi-
cated by the literature (2, 14, 15, 29, 43, 56, 66, 67), are not found
in any single temperature controlling instrument known to the writer.
Post -diapause
Handling procedure. Termination of diapause and hibernacula evac-
uation were obtained by removing the overwintering larvae from cold
storage directly to the light table. Upon successful emergence, larvae
are once again photopositive and drop to the bottom of the cartons on
silken threads. Periodic examination of the emergence containers
allows the larvae to be collected and transferred to separate feeding
containers. Direct handling of newly emerged larvae should only be
attempted with a camels hair brush. When the larvae are disturbed,
they normally react by rapidly withdrawing from the source of stimulus
and et the same time they spin a silken thread. The silken thread can 28
be used to transport the larva, therefore, all direct body contact is
avoided.
Natural food. Douglas -fir foliage served as food for all labora-
tory- reared stock, however, field -collected immature instara were
allowed to terminate feeding on the host from which they were collected.
Three basic types of foliage were tested and their applicability
assessed for feeding developing larvae. The foliage types consisted
of quick- frozen buds, shade foliage of the current year's growth and
5 -month old seedlings. The seedling foliage was used in its natural
form and later served as the basic ingredient for a modified food.
In the spring of 1960, newly ruptured buds were collected and
quick - frozen using 95% ethanol and dry ice. Such a mixture reduced
. the alcohol temperature to -70 °F. The buds were packed in a perfor-
ated container and quickly immersed in the alcohol solution. After
sufficient time had elapsed, approximately 2 minutes, the frozen buds
were removed, quickly sealed in pint frozen food cartons and stored at
-10 °F. in an upright type deep freeze. A certain amount of hazard
exists when using such a freezing method and serious damage will result
if excessive amounts of super- cooled alcohol reach the skin.
An ample supply of Douglas -fir seeds was provided by the School of
Forestry, Oregon State University. Prior to planting, all seeds were
given a -germination treatment which consisted of the following.
The seeds were soaked in water for approximately 24 hours after which
the seeds were drained, wrapped in wet paper towels and stored in a
35 °F. cold room. The cold room storage is necessary for seed stratifi-
cation and lasted for a period of approximately 14 days. 29
Preparation of the seed flats requires certain precautions against the occurrence of damping -off organisms. Satisfactory control was obtained by soil sterilization, use of a material termed "Perlite
Plaster" and the application of recommended dosages of a fungicide marketed by the trade name of "fermocide ". 1/ A 2 -inch layer of perlite plaster is used to cover the soil and serves as a substratum for the germinating seeds. This material is useful because it retards the spread of the damping -off organisms and reduces water loss from the sur- face of the flats.
Modified food. Douglas -fir seedlings served as the foliage factor for the modified food and were prepared by cutting off the root systems at ground level. This material was thoroughly rinsed in tap water in an attempt to remove adhering dirt and debris. The next step required a 15 minute rinse in a 2% solution of sodium hypochlorite; this treat- ment was directed toward surface sterilization of the young trees. The sodium hypochlorite solution was drained off and the foliage rinsed in tap water and finally in distilled water.
Ten grams of Difico -Bacto agar were dissolved in 275 milliliters of boiling distilled water. After the agar had completely dissolved, the solution was permitted to cool to 80° C. at which time 150 milligrams of propionic acid(3odium salt), dissolved in 25 milliliters distilled water, was added. A pilot study using various concentrations of fungi- cide, indicated 500 parts per million was a sufficient level to prevent rapid contamination from microorganisms. The choice of propionic acid
1/ Active ingredient is fermate. 30 as the fungicide was based on its relatively low mammalian toxicity.
After continued cooling had reduced the agar solution to 45 °C., one -half liter of prepared seedlings was added. For adequate mixing, vigorous stirring was necessary. The foliage was removed after per- mitting the excess agar to drain off and then quickly spread out on a small, clean, porcelain tray. To assure proper agar coverage, the tray was placed in a refrigerator causing the agar to solidify quickly.
Poor foliage coverage will result if too much agar is permitted to drain off or if the tray is not immediately placed in the refrigerator.
When the modified food is to be used within 20 days, the tray can be covered with a piece of parafilm and kept at 35 °F. If a longer storage period is required, the modified food should be sealed in an air tight container, quick frozen and stored at -10 °F.
Feeding procedure. The feeding container normally employed was unaltered ice cream cartons, however, surveillance studies on feeding larvae required the use of Petri dishes. Transfer of newly emerged larvae to their food was accomplished by a technique previously describ- ed under the section on "Post -diapause handling procedure ". Light was excluded from the Petri dishes by enclosing each dish in an envelope made of black drawing paper. The ice cream containers were stored in a wooden rack, containing 8 removeable shelves, each capable of hold- ing 36 cartons (Figure 6). The larvae were protected from the light by placing a heavy, black cloth hood over the rack. 31
Figure 6. -- Wooden rack used to hold ice -cream cartons during feeding procedure. 32
Pupae were removed from the feeding containers prior to eclosion and placed on trays (see page 18) or individually in screw neck vials, each of which contained a piece of absorbent paper. Pupae were handled by cupping the cremaster with forceps, thereby avoiding the risk of puncturing the integument. When necessary, pupae could be sexed by
counting the number of obdominal segments behind the wing pads; females
have 4 distinct segments, males have five.
Preparation of Cuticle Sections
Larvae and pupae. Sixth instar larvae and newly molted pupae were
prepared for microscopic examination in the following manner.
Embedding and cutting procedure:
1. Live specimens immersed in cold water, temperature raised
to 70oC.
2. Specimens removed and immersed in cold water.
3. Specimens cut into desired sections.
4. Sections fixed in "Zenkers fixing fluid" for 4 hours.
Fixative consisted of:
Potassium dichromate 2.5 gr.
Mercuric chloride 5 gr.
Distilled water 100 cc.
Glacial acetic acid 5 cc. (added just before fixing)
5. Fixed tissue washed in running water for 24 hours.
6. Tissue treated with dioxane- iodine solution,
4 changes, 30 minutes each. 33
7. Tissue dehydrated in pure dioxane, 4 changes,
30 minutes each, 5th change for 12 hours.
8, Tissue rinsed in xylol for 2 minutes.
9. Tissue infiltrated with paraffin (melting point 56- 58 °C.),
4 changes, 30 minutes each.
10. Embedded in small paper boxes containing molten paraffin,
rapidly cooled in ice water.
During the final embedding step care should be taken to prevent the ice water from coming in direct contact with the cooling paraffin. If this precaution is not observed the paraffin blocks will split, with the result being occasional damage to the embedded tissue.
After the paraffin blocks had cooled for a sufficient time to assure complete solidification, they were made fast to a small block of wood.
For a detailed description of this step see . Guyer (19, p. 37). The blocks were secured in the head of an automatic rotary microtome and sectioned at the desired thickness. For complete sectioning techniques see Guyer (19, p. 37 -40).
Mounting and staining procedure:
The paraffin sections were mounted on slides by the aid of Mayer's albumen fixative by following the procedure outlined by Guyer
(19, p. 40 -41). The slides were permitted to dry on a warming table, set at 45 °C. for approximately 12 hours. At the end of this period the slides were ready to be stained. The stain used was Heidenhain's hematoxylin. However, this stain is not a specific for cuticular material, but it served nicely to differentiate noncellular and cellular 34 tissue. The sequence for staining is as follows:
1. Xylol rinse 1 for 2 minutes.
2. Xylol rinse II for 2 minutes.
3. Absolute ethyl alcohol rinse.
4. 95% ethyl alcohol rinse for 2 minutes.
5. 50% ethyl alcohol rinse for 2 minutes.
6. Distilled water rinse for 3 minutes.
7. Fresh solution of ferric ammonium sulfate for 5 minutes.
Solution was made up from:
Clear ferric ammonium sulfate crystals - 1.15 gm.
Distilled water - 50 cc.
8. Rinse in tap water.
9. Quick rinse in distilled water.
10. Iron hematoxylin for 10 minutes. (Fresher the stain the longer the time)
11. Tap water until stain turns blue, approximately 5 minutes.
12. Destained with saturated aqueous solution of picric acid
for approximately 5 minutes. The time is variable and the
destaining process should be followed with a microscope.
13. Washed in tap water, 6 changes, each for 5 minutes.
14. 50% ethyl alcohol rinse for 2 minutes.
15. 95% ethyl alcohol rinse for 2 minutes.
16. Absolute ethyl alcohol rinse.
17. Cleaning xylol rinse for 3 minutes.
18. Cover glass, using balsam as a mounting media.
19. Warming table for 2 days. 35
Pupal case. Preparation of empty pupal cases for microscopic examination required a slightly different technique. The tissue fixa- tive step was omitted and replaced with a treatment designed to soften the cuticle. This treatment involved soaking the cases for approxi- mately 3 days in a solution of 70% ethyl alcohol and concentrated nitric acid, 97 parts to 3 parts by volume respectively. This phase of pre- paration was followed by 3 dioxane rinses, each lasting for 30 minutes.
The paraffin embedding was similar to the method previously described as was the sectioning and mounting. The pupal cases were examined with- out the aid of stains. 36
RESULTS
Preliminary Study
Prior to initiating major experimentation, a limited study on rear-
ing techniques was undertaken. This study was performed in order to
develop or anticipate methods and equipment which would be necessary
in subsequent studies on mass rearing procedures. As a result of
Stehr's (55) work on rearing techniques, laboratory conditions used in
rearing all active stages were maintained at a temperature of 75 $ 3 °F.
and a relative humidity of 55 -70% and hereafter will be referred to as
standard conditions.
The first trial rearing was started in late spring of 1961. Small
lots of overwintering larvae, which had been stored in 5- gallon ice
cream containers at 33 °F. since October of 1960, were removed to the
rearing room. The method for collecting emerging larvae was similar to
the one used by Fellin (13, p. 28 -31). As the larvae vacated their
hibernacula, they were attracted to the rearing room light and accumu-
lated in shell vials which had been inserted into the ice cream con-
tainers.
In the first feeding trial newly emerged larvae were transferred to
Petri dishes, each of which contained several freshly thawed Douglas -
fir buds. Each dish was sealed with a piece of parafilm and enclosed
in a light -tight envelope (55). At the end of 5 days, the dishes were
examined for the presence of feeding larvae. The results of this feed-
ing trial were completely negative, for in all dishes 100 percent mor-
tality had occurred. 37
The cause for this high mortality was not immediately apparent, however, later observations indicated the food was toxic to the young
larvae. After a short period of contact with the foliage, the larvae
became immobile and within a day or two were observed to be in a mori-
bund condition. A strong odor of turpentine was detected in each dish
indicating the alcohol- freezing treatment had some effect on the suita-
bility of the Douglas -fir buds.
Subsequent feeding trials proved that Douglas -fir shade foliage of
the current year's growth would suffice as a food source for developing
larvae. However, seasonal limitations precluded the utilization of
this type foliage for year -round rearing programs. The requisite of a
continuous rearing program required the availability of suitable food,
regardless of the season. Dependence upon field- collected food was
eliminated by growing Douglas -fir seedlings in the forest insect lab-
oratory.
During the preliminary feeding study one fact became clear, that
glass Petri dishes were not suitable for a mass rearing program.
Breakage, coupled with the high initial cost, resulted in the adapta-
tion of disposable half -pint ice cream cartons. The cartons were found
to be satisfactory and replaced the Petri dishes as feeding cages. One
difficulty encountered with the use of this type of container was the
inability to quickly assess the condition of developing larvae. The
author is of the opinion that still a third type of container, not
tested in this study, would be more suitable for a mass- rearing program. 38
Falcon Plastics 1/, producers of sterile disposable plastic labware, markets a Petri dish that might suffice for spruce budworm rearing pro-
grams. Each dish is designed to be used only once and retails for
approximately 5 cents, depending on the quantity purchased.
Another important result obtained from this preliminary study was
the importance of illumination on developing larvae. Newly emerged
larvae can be characterized by their limited food reserves and their
strong positive phototaxis. As a result of the foregoing factors, ex-
cessive mortality is avoided by the elimination of all light during the
initial feeding period (12, p. 426). Later observation indicated an
enhancement of survival when light was excluded throughout the feeding
period.
Stehr (55, p. 425 -426) has outlined a method for mating the male
and female moths; however, his techniques would impose certain restric-
tions on a mass- rearing program. His procedure requires prior sexing
of each pupa as well as individual confinement for each pair. To in-
crease the efficiency of Stehr's technique, a large walk -in cage, con-
taining young potted Douglas -fir trees, was tested for its suitability
as a mass mating enclosure.
Effective utilization of the Douglas -fir needles as oviposition
sites would be seriously impaired if mated females could not reach the
potted seedlings. Under field conditions, Wellington and Henson
(60, p. 170) found that fully gravid females were incapable of active
1/ Division of B -D Laboratories Inc., 5500 West 83rd Street,
Los Angeles 55, Calif. 39
Flight until one or two egg masses were dropped. Their observations were not corroborated under the conditions of this study. Mated fe- males easily took flight and only a few egg masses were found on the
trays, floor and sides of the cage.
Daily collections of needle- bearing egg masses were made by removing
each potted tree from the cage. Due to the similarity in foliage and
young embryo color, considerable time was required for each collection.
Time necessary for egg mass accumulation can be reduced by placing a
low intensity light at one corner of the cage; moths were attracted to
the light, which resulted in the concentration of eggs on one tree.
Environmental Studies
Fecundity and incubation. By utilizing small plasic boxes, each
containing a single Douglas -fir branch terminal, individual familes
were set up and an accurate count of egg deposition obtained. Forty -
nine such boxes were used for determining the fecundity of the green
form spruce budworm. Fecundity values for the standard form were re-
corded from 34 families. Both forms were taken from the Fi laboratory
generation which had been reared under similar conditions using Doug-
las-fir shade foliage as the food source.
The duration of egg laying for both forms was quite similar, with
oviposition continuing for as long as 12 days in the green form and 11
days 111 the standard form. In the green form, by the end of 6, 8, and
10 days, 33, 52, and 97 percent, respectively, of the 46 moths laying
(3 failed to oviposit) had completed oviposition (Table 1). Table 1.- -Daily record of oviposition by spruce budworm moths held in individual confinement (Green form)
Family : , Days after mating (numbers of eggs)
No. . 1 : 2 . 3 : 4 : 5 . 6 . 7 : 8 . 9 . 10 . 11 . 12 : Total
1 11 46 63 16 136
2 . 101 21 15 137
3 3 49 47 4 34 - 11 2 150 4 8 8 5 1 23 5 22 21 72
6 6 113 ' 22 141 7 10 61 29 10 4 114 8 50 5 5 27 11 98 9 67 37 18 36 25 183 10 53 36 10 99 11 11 7 18 12 9 9 1 8 21 58 106 13 13 116 17 15 161 14 71 10 42 8 131 15 6 72 49 127 16 88 11 25 5 129 17 3 12 5 7 73 100
18 32 80 46 38 18 . 214 19 109 37 30 18 7 201 20 130 42 17 189
21 117 . 9 17 143 22 6 8 9 15 6 33 77 23 101 33 24 8 23 6 195 24 3 4 19 4 12 21 8 71
25 29 ' 29 26 134 24 35 4 197 Table 1.-- (Continued)
Family : Days after mating (numbers of eggs)
: : . . . : No. . 1 . 2 3 : 4 . 5 . 6 . : 7 : 8 . 9 : 10 11 : 12 Total
27 97 26 25 7 155 28 0 29 2 4 4 10 30 3 134 26 163 31 87 20 6 113
32 ' 4 83 87 33 90 24 5 7 126 34 5 8 23 13 62 3 114 35 1 8 105 13 127 36 33 3 36 37 0 38 79 8 87 39 83 47 27 4 12 173 40 3 106 25 134
' 41 4 9 33 46 42 2 89 40 40 5 176 43 4 4 44 123 11 134
' 45 0 46 85 95 6 186 47 1 7 14 22 8 52 48 89 89 49 23 10 33
Totals 0 175 1262 1162 1240 452 106 206 287 245 50 86 Accumulative totals 0 175 1437 2599 3839 4291 4397 4603 4890 5135 5185 5271 Accumulative percent 0 3.3 27.3 49.3 72.8 81.4 83.4 87.3 92.8 97.4 97.4 100.0 42
In the standard form, by the end of 6 days, 16 percent of the 31 moths ovipositing (3 failed) had completed oviposition, by 8 days 52 percent and 10 days 90 percent (Table 2). Accumulative totals of eggs laid gave a somewhat different picture of egg production for these periods.
The totals for the green form moths during the 3 periods were:
6 days - --81 percent, 8 days - --87 percent and 10 days - --97 percent.
The number of eggs laid ranged from 0 -201, averaging 107.6. During the same three periods, the standard form moths totals were 78, 93, and
99 percent. The average number of eggs laid was 141.3 with a range of
0 -281. Table 2.- -Daily record of oviposition by spruce budworm moths held in individual confinement (Standard form)
Family .
...... No. : 1 : 2 : 3 . 4 . 5 : 6 : 7 : 8 . 9 10 . 11 : 12 ;: . 13 Total
1 3 112 66 47 22 15 7 272 2 119 43 26 188 3 29 29 4 1 1 4 4 5 - 31 46 5 - 6 57 39 20 14 11 141 7 3 84 - 34 28 - - 149 8 16 19 13 18 27 20 7 31 151 9 - 10 53 52 54 27 17 - - 203 11 7 5 6 15 8 15 19 55 17 - - 147 12 113 18 24 - 14 - 2 171 13 4 9 5 18 14 76 34 18 52 7 187 15 5 19 38 9 7 15 93 16 101 5 13 18 5 142 17 88 120 31 42 281 18 21 37 12 38 27 21 9 19 184 19 5 4 3 1 13 20 56 29 42 8 18 6 159 21 2 40 89 31 31 6 199 22 38 5 43 23 3 90 20 105 13 231
r Table 2. -- (Continued)
Family :
No. : 1 : 2 : 3 : 4 : 5 . 6 : 7 . 8 : 9 : 10 : 11 : 12 : 13 : Total
24 5 4 21 38 4 4 76 25 130 18 9 157 26 81 22 37 2 142 27 - 28 9 82 - 29 28 148 29 67 18 - 5 6 33 129 30 42 43 18 15 7 12 137 31 113 38 42 193 32 5 19 81 30 19 29 4 187 33 4 6 6 16 34 32 80 18 12 9 151
Totals 0 15 1160 1322 424 526 434 208 96 148 50 Accumulative totals 0 15 1175 2497 2921 3447 3881 4089 4185 4333 4383 Accumulative percent 0 3.4 26.5 57.0 66.6 78.6 88.5 93.3 95.5 98.8 100 45
In order to determine the incubation period for both spruce budworm forms, egg masses of known age were collected from various individual pairs. Based on 1077 eggs, incubated at standard conditions (Temp.
73 ± 5 °F. and R. H. 55 -70 %) eclosion of the green form larvae took place approximately 169.1 hours (7.04 days) after oviposition. The head cap- sules were visible through the chorion approximately 1.2 days prior to eclosion. A total of 292 eggs, incubated under similar conditions, were used for determining eclosion in the standard form. No significant dif- ferences in incubation periods could be detected between the two forms.
The head capsules were visible after 149.9 hours (6.24 days) and eclo-
sion followed within 27.3 hours. Stairs (54, p. 152) reports that the
incubation period of the spruce budworm was 6.25 days, however, his
rearing temperature (77 °F.) was slightly higher which probably accounted
for the accelerated rate of embryo development.
Another phase of the spruce budworm embryogenesis was studied in
order to determine the conditions which best favoured survival. Before
testing the effect of various environmental factors on embryo survival,
it appeared necessary to assess the mortality occurring under standard
rearing conditions. Once again, eggs produced from the moths held in
individual confinement were used for initiating this study.
Families 15 through 25 from Table 2 were arbitrarily chosen as repre-
sentatives of the standard form. Table 3 summarizes the results; out of
1578 eggs a mean survival of 81.7 was obtained. The survival values,
except for families 19, 22, and 24 are rather uniform and appear to be
more indicative of rearing mortality. The high mortality occurring in 46
the eggs from family 19 is characteristic of females which are only
partially successful in their attempt to oviposit. The female moths of
families 22 and 24, each laid one egg mass which was atypical. Normally
eggs are deposited in single ranks of 2 or 3 but occasionally a female
will lay a two layered egg mass. Often the emerging larvae on the
bottom layer will destroy the embryo immediately above when it chews
through the chorion. The egg mass of family 22 deposited on the 3rd day
Cb after mating and the egg mass of family 24 deposited on the 8th day;
both sustained high mortality as a result of this factor. The incidence
11 of this type of atypical oviposition was low, however, Carolin states
that rarely are layered egg masses encountered under natural conditions.
A summary of embryo survival is given in Table 3.
To determine the effects of various temperatures and humidities on
embryo survival, eggs were obtained by placing 10 pairs of green form
adults in each of 16 one gallon jars, each of which contained a bough of
Douglas -fir. This small confinement afforded an accurate determination
of the age of each egg mass. Enough eggs were obtained in this fashion
to initiate the last treatments within two days of the first.
Using growth chambers, four temperatures, 68 f 1 °F., 70.5 = .5 °F., ± 75 .5°F., and 86.5 ± .5 °F. were compared at four relative humidities
which varied slightly according to the temperature used. Table 4 shows
the exact treatment for each chamber. Anhydrous CaC12 was used to
obtain zero percent relative humidity and saturated sand for 100 percent
relative humidity. Different salts in saturated solution, with an ex-
cess of salt were used to obtain intermediate humidities.
1/ Personal communication, 1962. 47
Table 3.-- Summary of embryo survival occurring in spruce budworm eggs when incubated at 75 ± 3°F. and a relative humidity of 55 -70% (Standard form).
Family Total No. of Survival No. observations (Percent)
15 93 84.9
16 142 92.9
17 281 95.7
18 184 89.1
19 13 15.3
20 159 88.0
21 199 93.9
22 43 81.3
23 231 93.0
24 76 75.0
25 157 90.4
Total eggs observed 1578
Average survival 81.7 48
Table 4.-- Temperatures, humidities and materials used for treatment of the green form egg masses
Sequence : Temperature : - -- Humidity - --
Chamber : Treatment : degrees Percent
number : assigned : (Fahrenheit): Material : Relative humidity
1 A 70.5±.5 CaC12 0
B " NaNO3 77.7
C " Ca(NO3) 56.0 (55.8)1/
D " Sand 100
2 A 68±.5 Ca(NO3)2 56
B " NaC1 77.5
C " CaC12 0
D " Sand 100
3 A 75.5t.5 NaC1 76.5 (75.5,75.0)1/
B " CaC12 0
C " NaBr 57
D " Sand 100
4 A 86.5±.5 NaC1 75.9 (75.5,75.1)1/
B " Sand 100
C It CaNO3 51.0 (47.0)1/
D it CaC12 0
1/ Tabular values differ, depending on source of reference. 49
The egg masses collected on the first day were pooled and used to
start chambers 1 and 2 and their corresponding controls. Egg masses were assigned to the treatments and controls by randomization. The egg masses assigned to the controls were incubated under standard conditions which also included a 24 hour photophase. 1/ On the second day, treat- ments and controls were started in chamber 3, and on the third day in
chamber 4, with the controls for both chambers being assigned and
treated as previously described. Data from chamber 2 was not usable because of the complete mortality that resulted during the treatments.
The chamber was losing its refrigerant during the experiment and mortal-
ity apparently resulted from the freon gas escaping into the chamber.
Tables 5 and 6 show the data obtained from treatments in chamber 1,
3, and 4. Preliminary analysis indicates differences in survival oc- curring between treated eggs and eggs reared at standard conditions for dates on which treatments were initiated. As a result of variation in percent survival between the standards of July 20 and July 22, survival
figures for standard conditions may not be reliable. Conclusive quali- tative interpretations of the data are precluded due to the standard mortality variation and the lack of replications within each treatment.
1/ Refers to the light phase of the photoperiod. Taken from a paper
entitled, Photoperiodism and Insect Development, by S. D. Beck,
presented at the Biological Colloquium, Oregon State University,
1962. Table 5. =- Survival of eggs given various temperature and humidity treatments compared with survival of eggs reared under "standard conditions" (Green form)
Date treatment : Treatments - Number : Survival : Level of significance
initiated : Temperature : Humidity : observations :(Percent) : X2 : 5% 17%
July 20, 1961 - Standard - 446 81.9 - (Chamber 1) 70.5° 0 281 49.2 12.990 Yes Yes It 77.7 213. 87.4 3.659 No No " 56.0 227 89.5 7.267 Yes Yes It 100 187 83.0 1.072 No No
July 21, 1961 - Standard - 443 76.5 -
(Chamber 3) 75.5° . 76.5 140 90.0 12.044 Yes Yes
" 0 84 ' 65.5 4.472 Yes No " 57 108 66.7 4.348 Yes No " 100 149 87.3 7.858 Yes Yes
July 22, 1961 - Standard - 402 73.5 - (Chamber 4) 86.5 75.9 100 64.0 3.662 No No " 100 129 82.9 4.630 Yes No it 51 161 82.0 4.395 Yes No " 0 127 48.9 27.162 Yes Yes 51
Table 6. -- Summary of embryo survival occurrin in spruce budworm eggs incubated at 75 t 3°F. and a relative humidity of 55 -70% (Green form).
Sample Total No. of Survival No. observations (Percent)
1 446 81.9
2 443 76.5
3 402 73.5
Total eggs observed 1291
Average percent survival 77.3
The general survival between relative humidity and egg survival at different temperatures is shown in Figure 7. As might be expected, zero percent humidity was the most severe at all temperatures tested.
Regardless of the incubation temperature, 100% relative humidity had little effect on embryo success. These results were surprising because excessive mycelial growth was observed on all egg masses which were reared in a saturated atmosphere. When compared against the mean sur- vival at standard conditions, the best incubation treatment can be represented by a temperature of 70 -75 °F. and a relative humidity from
56 to 100%. 52a
Figure 7.-- Relation of percent egg survival to relative humidity at
three chamber temperatures as compared with standard rear-
ing conditions. PERCENTAGE SURVIVAL 100 40- 60- 70- 90 50- 80- < 0 56 70.5 °F. 78 100 PERCENT 0 TEMPERATURE RELATIVE 57 75.5 °F. 77 HUMIDITY 100 T. STANDARD STD.- 0 51 86.5 °F. 76 100 CONDITIONS 55 75 IX ó -70 ±3 53
Hibernacula selection. Emerging larvae of both forms were subjected to incandescent and fluorescent illumination and no striking differences were detected in the rate at which hibernacula sites were selected.
Each form of the spruce budworm was treated by assigning 8 Petri dishes, each of which contained a single egg mass, to the appropriate section on the light table. Hibernacula selections were followed by gridding off the part of the dish immediately adjacent to the parafilm and gauze.
The period required for total eclosion was sufficiently short so that the time for selection was based on the first larva emerging. The site was considered selected once the larva commenced spinning. Observations were taken every two hours after eclosion, except for the time between
11 p.m. and 7 a.m.
Table 7 shows the summary of results obtained in this experiment.
The average time for selection was based on the period necessary for
total selection; however, in one dish 3 larvae failed to select a site and eventually died. Individual egg mass variation was no greater than
4 hours from the calculated mean, therefore, only the summarized data is
presented in tabular form. Generally, larvae failed to select a site within the first 2 -3 hours after emerging, regardless of the illumina-
tion to which they were exposed. 54
Table 7.-- Influence of two light sources on the rate at which newly emerged
1 larvae select hibernacula sites (standard form and green form)
Number of Light : Average time for
Form observations intensity : selection (hrs.)
á/ Standard 278 High 35.0 Standard 222 Low b/ 39.4
Green 178 High 36.1
Green , 164 Low 41.0
a/ Fluorescent
b/ Incandescent
Incidence of non -diapause. The increase in laboratory rearing pro- ductivity obtained by Harvey (22, 13), using N -D spruce budworm prompted a series of studies designed to develop a N -D stock. Before any de- tailed experimentation was undertaken, several hundred untreated, stand- ard form eggs were followed from the time of eclosion up to one day be- fore the larvae were placed in the cold room. As a result of this close surveillance, 63 larvae which had apparently vacated their hibernacula and were wandering on the gauze were collected and placed on Douglas -fir seedlings. Out of the original 63, 5 commenced feeding and the remainder either spun a new hibernacula or eventually died. Only one of the five larvae reached adulthood. A mate was not available, thereby, precluding the perpetuation of the apparent N -D genotype.
Harvey (22, p. 563 -564) determined that a 24 hour photophase during the latter half of the first stadium was the most effective treatment 55
for inducing the N -D larvae to vacate their hibernacula. In order to
assess the incidence of N -D individuals in the F2 generation of the
standard form spruce budworm, 1108 eggs were divided among 12 Petri
dishes. The dishes were placed on the fluorescent section of the light
table and given a 24 hour photophase. Each dish was checked twice
daily for 45 days. The results of this study were inconclusive. A
total of 12 larvae were found wandering and were placed on food for
continued morphogenesis. All 12 larvae died without a single one ini-
tiating feeding. It appears doubtful that these larvae were N -D indi-
viduals for earlier observations have shown that larvae occasionally
abandon their hibernacula to search out a new overwintering site.
Pre -diapause treatments. The results obtained by Harvey (22)
definitely supports the conclusion that inception of the N -D behaviour,
' in the spruce budworm was genetically controlled. However, even N -D
individuals require the proper environmental stimulus (24 hour photo -
phase) before the behaviour can be effectively manifested. Therefore,
it was hypothesized that if the diapause in the western population of
spruce budworm was not an "all or none" condition (8, p. 415) a reduction
in the cold rest requirement might be brought about through various pre -
diapause treatments.
Green form larvae obtained from the incubation study were used to
test this hypothesis. To minimize any effect humidity may have had on
the viability of successful embryos, only eggs reared at the intermediate
humidities were used. Prior to initiating this experiment a rough esti-
mation was made as to the number of larvae needed to satisfactorily fill 56 each proposed treatment. It immediately became apparent that the number of eggs receiving an intermediate humidity treatment were insufficient.
Additional eggs were collected from the gallon jar mating confinements and incubated at intermediate humidities.
Using growth chambers, 2433 second stage larvae were given pre -dia- ± pause treatments consisting of 3 experimental temperatures, 70.5 .5,
75.5 i .5 and 86.5 : .5 °F. with each chamber programming a different photophase of 20, 18 and 16 hours respectively. The larvae were distri- buted among 40 Petri dishes with each treatment receiving 4 dishes. Pre - diapause treatment assignments were accomplished in the following manner.
Larvae obtained from eggs incubated at one of the three experimental temperatures were maintained at their respective temperature or removed to the standard temperature (75 t 3 °F.). Larvae with a standard temper- ature incubation history were either subjected to one of the experimen- tal treatments or remained at the standard temperature. Therefore, the
3 possible combinations of treatments for each temperature and photo - period were (1) eggs incubated under experimental conditions, resulting larvae untreated, (2) eggs incubated under experimental conditions, re- sulting larvae treated, (3) eggs incubated under standard conditions, resulting larvae treated. The control for this experiment is repre- sented by individuals which were held under standard conditions during both egg and larval stages. Treatment sequences are summarized in
Table 8.
Three weeks after eclosion, all pre -diapause treatments were termi- nated by placing the larvae in a walk -in cooler. To ascertain the 57
Table 8.-- Summary of pre- diapause treatments
. Treatment Sequence
Treatment : : Temperature : Humidity : Photophase
No. : Stage :(degrees F.) :(relative %): (hours) ± 1 egg 70.5 .5 77.7 0 " larva 75 f 3 J 55 -70 á/ 24 a/
2 egg 70.5 t .5 56.0 0 larva 70.5 f .5 75 t 6 20
3 egg 75 t 3 55 -70 24 larva 70.5 t .5 75 f 6 20
4 egg 75.5 t .5 76.5 0 larva 75 ± 3 55 -70 24
5 egg 75.5 ± .5 57.0 0 larva 75.5 t .5 75 f 6 18
6 egg 75 ± 3 55 -70 24 larva 75.5 t .5 75 t 6 18
7 egg 86.5 t .5 75.9 0 larva 75 $ 3 55.70 24
8 egg 86.5 t .5 51.0 0 larva 86.5 t .5 75 t 6 16
9 egg 75 ± 3 55 -70 0 larva 86.5 t .5 75 t 6 16
± 10 egg 75 3 55 -70 24 larva 75 t 3 55 -70 24 a/ Represent standard conditions 58 influence of various pre -diapause treatments, on diapause requirements, one dish from each of the ten treatments was removed after 6, 8, 10, and
12 weeks cold storage. Overwintering mortality was determined 2 days after removal from cold storage by gently touching each larva with the tip of a dissecting needle. All larvae that responded to this stimulus had obviously survived the cold storage treatment. Daily records of larval issuance were kept until 40 days after cold storage; larvae that failed to evacuate their hibernacula by the end of this period were considered to be dead. From these observations, pre -issuance mortality was calculated. The results of this experiment are shown in Table 9 and figures 8 and 9.
When the overwintering and pre -issuance survival, for each cold rest period is considered independent of pre -diapause treatments, a general trend can be established. Overwintering mortality decreased from a high of 38 percent after 6 weeks cold rest to a low of 15 percent after 12 weeks cold rest. During the same periods, pre -issuance mortality de- clined from 81 percent to 39 percent. These general relationships are only in fair agreement with Harvey (22). He found that overwintering mortality maintained a steady low level (less than 5 %) up to 22 weeks cold storage. However, he showed a similar relationship as found in this study between pre -issuance mortality and the duration of cold rest periods. When individual treatments within each cold rest period are considered independently, a somewhat different picture of survival is obtained. Table 9.-- Effects of various pre -diapause treatments on larval survival following 6, 8, 10 and 12 weeks cold rest (Green form)
. Percent mortality during . : No. of diapause development Larvae issuing
Treatment a/ : observations :overwintering : Pre -issuance :number : percent : mean day: Range
6 weeks 1 64 7.7 42.1 37 57.8 28 19 -32
2 67 5.9 22.3 5' 52 77.6 27 21 -37 3 43 41.7 100 - - - - 4 14 100 100 - - No - 5 38 29.0 100 - - - - 6 46 2.2 100 - MD - WO 7 43 53.5 81.3 8 18.6 24.1 23 -25 8 31 100 100 - - - 9 48 44.0 100 - - - - 10 127 3.9 70.8 37 29.1 26.8 22 -30
8 weeks
1 77 32.0 100 - - - - - 2 40 0 100 - - - - 3 49 1.0 100 - - - - 4 61 13.1 83.6 10 16.4 20 15 -22 5 51 3.9 100 - - - - 6 42 50.0 100 - - - - 7 51 17.6 100 - - - - 8 46 30.4 100 - - - - 9 76 32.9 100 - - - - 10 77 13.1 93.4 5 6.4 16 15 -17 a/ For explanation of treatments see Table 10. Table 9.-- (Continued)
Percent mortality during . No. of diapause development Larvae issuing
Treatment á/ : observations :overwintering : Pre -issuance :number : percent : mean day: Range
10 weeks 1 73 0 16.4 61 83.5 21.5 15 -34 2 82 2.0 10.9 73 89.0 19.4 11 -28 3 53 16.1 84.9 8 15.0 24.6 24 -25 4 45 24.5 86.6 6 13.3 13.5 10 -15 5 81 1.1 7.4 75 92.5 21.9 16 -30 6 65 4.6 18.4 53 81.5 21.7 16 -31
7 45 2.2 100 . - - - - 8 31 100 100 - - - - 9 58 34.4 86.2 8 13.7 25.2 24 -30 10 61 19.7 52.4 29 47.5 22.2 15 -29
12 weeks 1 130 2.1 17.6 107 82.3 14.5 9 -27 2 38 0 0 38 100 14.0 9 -34 3 143 0 34.2 94 65.7 19.9 8 -33 4 51 19.6 58.2 21 41.1 12.4 10 -17 5 35 9.5 40.0 21 60.0 20.9 13 -31 6 64 2.0 7.8 59 92.1 19.3 9 -34 7 61 4.9 9.8 55 90.1 12.9 9 -24 8 47 53.2 100 - - - - 9 74 43.3 100 - - - - 10 105 17.1 27.6 76 72.3 17.5 11 -30
0 61
Figure 3 illustrates the relationship between pre -diapause treat- ments and cold rest periods in terms of days required to obtain mean lar- val issuance. Irrespective of the cold rest period, treatments 1, 2, and 10 were consistently more effective in terminating diapause as com- pared with the other treatments. However, larvae subjected to treat- ments 1 and 2 failed to emerge after 8 weeks cold rest. Due to the graphic method by which the various treatments are shown, treatment 4 appears to be effective, particularly after 8 and 10 weeks cold storage.
These data are not sensitive to numbers of larvae vacating their hiber- nacula, and only 16 individuals successfully resumed activity after these two periods of cold rest. Treatment 4 can hardly be considered significant.
The general complete failure of larvae to terminate diapause in all treatments, after 3 weeks cold storage cannot be explained. As evident from Table 11, overwintering conditions were not responsible for this high level of mortality and records of laboratory rearing conditions failed to show any contributing factor.
Pre -diapause treatments 8 and 9 failed to enhance the reduction of diapause requirements. Total mortality resulted in all larvae subjected to these treatments. This observation was constant except for larvae receiving a 10 week cold storage, however, survival in treatment 9 was only 13.7 percent.
The effect of various pre -diapause treatments on larval survival following different lengths of cold rest is shown in Figure 9. For simplification of illustration, larvae surviving treatments 1 through 9, I-
30 6 WKS. COLD REST 30 10 WKS. COLD REST
20 20
10 10 i 4 r O 4 O
U o 4 ' z 4 o 30- 8 COLD REST WKS. REST r. H 30- 12 WKS. COLD
4 z 4 7 i 20- 20-
10- 10-
1 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 9 10 PRE -DIAPAUSE TREATMENT PRE -DIAPAUSE TREATMENT
Figure 8. -- Effects of various pre- diapause treatments on larval issuance following 6, 8, 10 and 12 weeks cold rest. (Green form) 63a
Figure 9. Summary of larval survival during diapause development after
4 different periods of cold rest, as determined by the mean
percent survival for various pre -diapause treatment groups.
Values for standard conditions do not represent a mean.
(Green form).
OP PERCENTAGE SURVIVAL 100 20-1 30- 40- 50- 60- 70- 80- 90- 10- - 1-3 1 4-6 6 ET 7-9 STD. 10 I 1-3 4 -6 PRE NO. I 7 8 -DIAPAUSE -9 WEEKS STD. 10 COLD TREATMENT 1-3 REST 4 -6 10 7 -9 STD.- STD. 10 STANDARD 1-3 4 -6 CONDITIONS 12 7 -9 STD. 10 64 for each of the four cold rest periods were grouped according to the 3 possible combinations. Therefore, mean survival values were determined for treatments 1 -3, 4 -6, and 7 -9. Larvae surviving standard conditions after each cold rest period do not represent a mean value.
The average conditions for treatments 1, 2, 3, and 10 appears to favour survival, regardless of the cold rest period, however, treatments
4, 5, and 6 show an increased effectiveness at the two longer cold rest periods. The mean for treatments 1 through 3 would be higher, if not for the latter treatment. Except for 8 weeks cold rest, percent survi- val for treatment 3 is significantly lower after all periods of cold storage.
The results of this experiment indicates that most larvae cannot t resume morphogenesis after being held at 86.5 .5, as apparent from treatments 8 and 9. The apparent anomaly with treatment 7 suggests that a similar high incubation temperature, affects subsequent larval survi- val. The reason for this carry -over effect is not apparent.
Effects of a fluctuating cold storage temperature. The literature provides several reports on the difference between the effects of con- stant and varying temperatures. Uvarov (58, p. 35 -38) cites several examples where development was markedly affected by varying temperatures.
With the beetle Popillia japonica Newm., development was retarded if the optimum temperature was alternated with one above the optimum range, but an accelerating effect was noted when the optimum alternated with a lower temperature. Development was increased in the cutworm Porosagrotis orthogonia Morr., by the use of a variable temperature. The embryos of 65 the grasshopper Melanoplus mexicanus Sauss., developed at the fastest rate when an effective temperature alternated with a temperature devel- opmental zero. However, with the grain aphid, Toxoptera graminum Pond., a constant temperature was more effective; the wax moth, Galleria melonella L., showed no differences in response to constant or variable temperatures. Arias (2, p. 38) while studying the biology of the garden
at temperatures slug, found that food - consumption was higher fluctuating but no differences occurred in growth. He attributed this to an in- crease in metabolic rate.
Data have accumulated that suggest fluctuating temperatures have a marked effect on development rates of the egg and overwintering larva of the spruce budworm. Under natural fluctuating conditions McGugan
(41, p. 440) found the incubation period was approximately 2400 units
(degree hours above 42° F.), whereas, under a constant 71 -72°F. Harvey
(22, p. 552) reported the rate to be 5290 units. Both Fellin (13, p. 54) and Miller (42, p. 410 -411) observed that survival and emergence was
greater when larvae were stored under natural fluctuating conditions than
-Alen stored at a constant 32 °F.
To test the hypothesis that a fluctuating cold storage temperature would be more effective in terminating diapause, 1092 standard form dia- pausing larvae were subjected to a temperature fluctuating between 51 °F. ± and 33 .5 °F. The temperature programming device and a National
Appliance growth chamber were used to obtain this artificial winter con-
dition. Groups of larvae were removed to the rearing room, at various
lengths of cold rest starting on the 29th day and continuing through the 66
80th day. Three dishes of larvae served as controls and were given a constant temperature during cold storage. The sequence of treatments is given in Table 10. Overwintering mortality was determined 2 days after removal to rearing conditions.
The results of this study were very striking; complete pre -issuance mortality occurred in all treatments, irrespective of temperature or duration. As evident from Table 10, overwintering mortality was not severe in larvae exposed to fluctuating or constant temperatures. From these data, it was surmised that fluctuating temperatures were not re- sponsible for the total pre -emergence mortality. The rearing records were checked and they showed that the larvae inadvertently had been left on the light table for an additional three weeks after eclosion. Stehr
(55, p. 425) found that leaving larvae at rearing conditions for periods
longer than six weeks after oviposition was decidedly harmful in terms of pre -emergence survival. These results certainly substantiate his
conclusions.
Role of temperature and light in terminating diapause. The impor-
tance of illumination in terms of diapause termination was definitely
established by Harvey (24). After shorter periods of cold rest (6 -14 weeks), a 24 hour photophase was most effective in terminating diapause.
Harvey's (22) work with a nearly homogeneous population of non -diapause
stock also demonstrated the importance of continuous light for the opti- mal expression of this behaviour. Thus it seemed important to determine
if different light intensities would influence survival and emergence of
diapausing larvae. Table 10. -- Effects of various periods of fluctuating cold storage temperatures on overwintering mortality (Standard form)
: Pre -diapause : Days in .
Sample : No. of . photophase : Temperature : cooler : Percentage overwintering
No. : observations : (hrs.) treatment : (33 °F.) 1 mortality
1 114 24 F á/ 29 12.5
2 82 . ,' 24 F 29 11.0 3 84 24 F 35 14.4 4 80 24 F 35 25.0 5 105 24 F 41 7.5 6 67 24 F 41 24.0
7 44 - 24 F 47 20.5 8 129 24 F 47 14.3 9 109 24 C b/ 41* 9.2 10 71 24 C 41* 15.5 11 113 24 C 80* 11.9 12 94 24 F 80 19.2
a/ Fluctuating temperature b/ Constant temperature
* Values do not include 7 days at 45 °F. 68
A total of 2894 overwintering larvae of the standard form stock, divided between 22 Petri dishes were used to determine the effect of varying lengths of cold periods and different light intensities on per- cent survival and rate of hibernacula evacuation. When this experiment was started, the rearing program was not of sufficient scale to accommo- date the production of several hundred larvae within a limited period of time. During April, May and June of 1961, various numbers of dishes were placed in a walk -in cooler. On July 26, the dishes were removed from cold storage and placed on the appropriate section of the light table.
In order to arrive at a quantitative expression of the two light sources (incandescent and fluorescent), a Kintel 1/ electronic galvano- meter, model 204A was used to measure the amount of light in each treat- ment. The following readings represent the amount of energy reaching a central position directly above each light source and are as follows: fluorescent- -868.4 micro watts per square centimeter, incandescent- -
719.2 micro watts per square centimeter. No effort was made to deter- mine the range of intensities as governed by the position of the dishes on the light table.
1/ A Division of Coho Electronics Inc., 5725 Kearny Villa Rd.,
San Diego 2, California. 69
Daily observations of the dishes allowed for an accurate determina- tion of the time required for complete issuance of second stage larvae.
At the end of a 30 day period, following removal from cold storage, a count was made of all larvae still in their hibernacula and percent survival of larvae calculated. For analysis, periods of cold treatment were arbitrarily divided into (1) 42 -45 days, (2) 51 -58 days, and
(3) 71 -90 days. Table 11 and Figure 10 shows the results obtained.
The shortest periods of cold treatment (42 -45 days) showed a differ- ence in survival between high and low intensities. At 42 days, influor- escent was only partially effectual in breaking diapause, whereas, under the influence of the low intensity incandescent, larvae were unable to resume activity. By the end of 45 days, high intensity illumination was capable of terminating diapause in about half of the overwintering lar- vae while low intensity illumination successfully stimulated activity in only 2.5 percent of the overwintering larvae. Treatment periods of
51 days or longer showed little differences in survival resulting from
the light intensities used and a generally high survival of about 91 percent.
Evident by Table 11 and Figure 10 the period required for evacuation of hibernacula varied indirectly with number of days cold rest and ranged from 11 to 28 days. When each light source is considered inde- pendently, starting with the 51 -day of cold rest and continuing through
the 90 -day, a somewhat different picture of issuance performance is ob-
tained. Over the various cold rest periods tested, fluorescent illumi- nation is consistently more influential in stimulating the resumption
of morphogenesis. Table 11. -- Effects of different light sources on the reduction of diapause requirements in overwintering larvae subjected to varying lengths of cold storage (Standard form).
Days in : Dish : Light : No. of larvae : Percentage : No. of days for : Total No. of days cooler : No. : source :overwintering :surviving: survival : total issuance : to break diapause áf 42 1 High 77 34 44.1 27 69 b/ 42 15 Low 83 0 0 45 6 High 21 12 57.1 27 72 45 17 Low 77 2 2.5 51 4 High 48 48 100 24 75 51 22 Low 66 65 98.4 27 78
52 ' 10 High 89 82 92.1 24 76 52 19 Low 105 97 , 92.3 28 80 53 7 High 89 84 94.3 23 76
53 16 Low 132 88 66.6 ' 21 74 55 3 High 50 40 80 23 78 55 12 Low 52 40 76.9 26 81 58 9 High 118 117 99.1 24 72
58 14 Low 363 ' 330 90.9 22 80
- 71 5 High 93 88 94.6 20 91 71 18 Low 109 101 92.6 20 91 82 11 High 575 546 94.9 18 100 82 21 Low 96 54 56.2 30 112
85 . 8 High 410 391 - 95.3 14 99
85 13 Low 131 125 _ 95.4 19 104 90 2 High 41 41 100 1'. 101 90 20 Low 69 66 95.6 17 107 a/ Fluorescent V b/ Incandescent o Figure 10. > w Z î W O 4 u O -J cx Ñ o -- Jr lol NUMBER DAYS COLD REST 42 90 42 45 45 52 90 52 53 53 55 55 58 58 82 82 85 85 Effect 51 51 71 71 of termination diapause 111111 Mil .: MIMI= .CEI ..m 1180 ...... m ...... m ...... 11..Nm od et (Standard rest cold 27 of - 0 27 ...... m fluorescent BE (b) - 24 NUMBER MI. 27 FLUORESCENT II 24 MN 28 in OF and 23 form). DAYS larvae m. 21 incandescent 23 FOR 26 m eevn different receiving TOTAL 24 91 22 INCANDESCENT ® 20 ISSUANCE Earn illumination 20 18 m 30 II 14 M 19 . II periods 11 on 17 69 75 74 78 81 82 80 91 91 100 112 99 104 101 107 72 78 76 80 76 _ 71 Ó 4 z 3: m cc Ó 0 4 4., O m w 4 Y E 4 a 4 w c a.bl TOTAL NUMBER OF DAYS TO BREAK DIAPAUSE 72
Table 12 was constructed to show the survival and emergence perform- /
ance of the two spruce budworm forms which had received nearly identical
periods of cold rest, as well as similar treatments after cold rest.
The values for the green form were obtained from Table 9 and represent
the larvae treated under standard conditions. Data for the standard
form was extracted from Table 11.
The green form apparently failed to show the same general pattern of
response to the duration of cold storage as observed in the standard
form. Larvae receiving 84 days cold rest required the same amount of
time for total issuance as did the larvae receiving 42 days cold rest.
The larvae subjected to 56 days cold rest showed a much lower issuance
time; however, due to the low number of larvae surviving, the value must
be considered as unweighted. Survival trends, in terms of the amount of
cold treatment received suggests that overwintering mortality is reduced
as the length of cold storage is increased. However, when the two forms
are compared, survival after all lengths of cold storage is significant-
ly higher in the standard form. If overwintering survival can be taken
as a measure of vigor, the standard form represents the superior test
animal.
Effect of a thermal shock treatment during diapause development.
The use of a shock treatment for terminating diapause is not a new con-
cept in diapause studies. Pepper (45, p. 380) successfully broke the
diapause in the sugar beet webworm (Loxostege sticlicalis L.) by im-
mersing the quiescent stage in various chemicals. Hinton (26, p. 285)
and Lees (35, p. 66) mention that diapause can be brought to an end in Table 12. -- Effects of varying periods of cold rest on the reduction of diapause requirements in the green (A) and standard (B) forms of spruce budworm.
(A)
Days in : Dish : No. of larvae No. of days a/ :Percentage: Total No. of days
cooler . No. : overwintering : surviving :for total issuance: survival : to break diapause
42 1 127 37 30 29.1 72
56 2 76 5 18 6.5 74
70 3 61 29 29 47.5 99
84 4 105 76 30 72.3 114
(B)
Days in : Dish : No. of larvae No. of days á/ :Percentage: Total No. of days
cooler : No. : overwintering : surviving :for total issuance: survival : to break diapause
42 1 77 34 27 44.1 69
55 3 ,' 50 40 23 80 78
71 5 93 88 20 94.6 91
85 8 410 391 14 95.3 99 a/ Number of days on fluorescent light table. 74 the silkworm embryo by a wide variety of shock treatments. These in- clude exposure to high temperatures, temporary immersion in acids and mechanical and electrical shocks. Cole (9, p. 5 and 14) working with an
Idaho population of spruce budworm, obtained data that indicated the diapause could be broken with a thermal shock treatment. His treatment consisted of storing 4, 15 -inch long Douglas -fir billets, containing overwintering larvae at 32 -40 °F. for 1 week, then at -25 °F. for 8 hours.
Then the billets were removed to rearing conditions (72- 78 °F.) for hibernacula evacuation. Cole obtained 105 larvae, from which he con- cluded that diapause had been broken.
To ascertain if this technique would be applicable for terminating diapause in the green form spruce budworm, 462 larvae distributed among
11 Petri dishes were subjected to the following thermal shock treatment: ± (1) three weeks after egg eclosion at 75 3 °F., (2) 1 week at 43- 45 °F.,
(3) 5 weeks at 33 °F., (4) 3.3 hours at minus 21 °F. and (5) 4.7 hours at minus 25 °F. The dishes were then removed to the fluorescent section of the light table and held at standard conditions for 16 days.
Two days after removal to the rearing room, each larvae was gently touched with the tip of a dissecting needle. This first examination
indicated that total mortality had occurred in all dishes; however, it ' was theorized that larvae may have still been under the influence of the cold treatment and were unable to react to the stimulus. Thus, similar checks were periodically made for the next 14 days.
These examinations failed to show that inactivity was due to a cold stupor and in actuality all larvae had succumbed to the shock treatment. 75
As a result of these observations, it was concluded that a thermal shock, as used in this experiment was extremely deleterious and incapable of terminating diapause in the green form spruce budworm.
Modified food. One disadvantage associated with feeding young
Douglas -fir seedlings to developing larvae was the rapidity at which the foliage dried. As a result of this rapid drying, maintenance of food acceptability necessitated that the seedlings be changed at least every
4 days. This factor resulted in frequent food changes, which especially during the initial feeding period, appeared to result in high larval mortality. These conditions prompted the development of a suitable food which was more resistant to dehydration.
No effort was made to develop an artificial media, such as used for rearing the sugar cane borer (Diatraea saccharolis (Feb.)) and lepi- dopterous corn insects (73). A brief account of a modified food was described by Wellington (59, p. 1). She prepared the medium by combin- ing a foliage -water suspension mixed in a Waring blender with hot agar:
Mycoban was used as the fungicide. Preparation procedures were very briefly discussed and no ingredient ratios were given. Apparently some success was obtained in rearing spruce budworm with this media for he mentions that the food had no effect on growth or fecundity.
Based on this approach, a pilot study was initiated to determine the suitability of this type food. Douglas -fir seedlings were mixed with hot agar, after first being masticated in a Waring blender. The agar - foliage mixture was poured into Petri dishes and allowed to solidify.
Five nearly mature larvae were placed in each of five dishes and their 76 feeding habits observed. In 4 dishes, larvae wandered on the surface of the agar and eventually died. However, in the remaining dish one larva was observed to have fed on a partially protruding needle. Based on this observation, it was surmised that larvae required an irregular sur- face to stimulate feeding. A new batch of food was prepared, (see
Material and Equipment) but this time intact seedlings, lightly covered with agar were used. When nearly mature larvae were introduced to this type of food, feeding immediately commenced and all larvae successfully completed development.
Second stage larvae, of the standard form F3 generation were used to determine the suitability of this modified food. A total of 200 larvae were equally distributed between 10 petri dishes containing modified food.
A similar number of larvae were placed on 10 small bouquets of Douglas - fir seedlings. Longevity of the seedlings was increased by enclosing the root systems, of each bouquet, in a plastic bag of wet peat moss. The bouquets were placed in quart mason jars and covered with a piece of plastic screen. The results of this study are shown in Tables 13 and 14. Table 13.-- Summary of larval survival using natural Douglas -fir seedlings (Standard form)
Experiment started 1/8/62 Food :Feeding:No. of larvae:
: No. larvae : Date : No. larvae :period : reaching : No. of : Mean fecundity
Jar No.: used in test : changed : alive : o : d: adulthood : pairs mated : of matings
1 20 1 -29 12 34 30 8 4 149.7 2 -5 9 2 -10 8
2 20 1 -29 7 34 30 7 3 136.4 2 -6 7 2 -9 7
3 20 1 -29 14 35 31 11 5 167.7 2 -4 12 * 3A 2 -10 6 * 3B 2 -11 5
4 20 1 -29 9 32 30 3 1 117 2 -5 3 2 -7 3
5 20 1 -29 9 34 31 4 3 153.9 2 -7 2 -9 Table 13.-- (Continued)
: Food : Feeding :No. of larvae: .
. No. larvae : Date : No. larvae : period : reaching No. of :Mean fecundity
Jar No.: used in test : changed : alive : o : or : adulthood : pairs mated : of matings
6 20 1-29 9 33 29 7 2 130.2 2-4 7 2-8 7
7 20 1 -29 10 35 30 5 2 159.9 2 -7 5 2 -11 5
8 20 1 -29 5 33 30 5 2 -4 5 2 -7 5
9 20 1 -29 4 32 31 3 2 125 2 -6 3
10 20 1 -29 8 34 - 3 3** 138.2 2 -6 4 2 -9
* Due to crowding, larvae were divided between two jars.
** oo used from jar 8 for mating oo Table 14.-- Summary of larval survival using modified Douglas -fir seedlings (Standard form)
Experiment started 1/7/62
Food : Feeding :No. of larvae:
Dish : No. larvae : Date : No. larvae : period reaching No. of :Mean fecundity
: : : alive : : o" : adulthood : pairs mated : of matings No. used in test changed 9
1 20 1 -17 10 33 29 6 2 112.9 1 -25 8 1 -31 7 2 -3 7 2 -9 7
2 20 1 -17 7 32 29 5 1 143 1 -25 7 2 -1 5 2 -7 4
3 20 1 -17 9 34 30 7 3 137.7 1 -25 9 1 -30 7 2 -4 7 2 -10 7 i. Table 14.-- (Continued)
Food : Feeding :No. of larvae:
Dish : No. larvae . Date : No. larvae : period : reaching . No. of :Mean fecundity
No. : used in test : changed : alive : o : oT : adulthood : pairs mated : of matings
4 20 1 -17 2 - 27 1 1 -25 1 2 -1 1
5 20 1 -17 6 , 29 4 1 -25 4 2 -3 4 2 -5 4
6 20 1 -17 7 33 27 5 3* 121.8 1 -25 6 2 -1 5 2 -9 5
7 20 1 -17 9 33 29 6 3 131.5 1 -25 8 2 -1 6 2 -5 6 2 -8 6
oCO Table 14.-- (Continued)
. Food : Feeding :No. of larvae: :
Dish : No. larvae : : : Date No. larvae period : reaching No. of :Mean fecundity
: No. used in test : changed : alive : o : d' : adulthood : : t pairs mated of matings
8 20 1 -17 6 31 29 4 2 145.6 1 -25 5 2 -2 5 2 -6 5
9 20 1 -17 7 32 30 5 2 127.1 1 -25 6 2 -2 5 2 -7 5
10 20 1 -17 0
* 1 o used from dish No. 5 82
A preliminary analysis of data failed to show any significant dif- ferences between the fecundity of moths fed modified food. Based on results obtained in this experiment as well as earlier feeding traits, the highest percent of feeding mortality occurred when larvae were becom- ing established on their food. Therefore, it seemed necessary to analyze for possible differences between the two types of food in terms of num- bers of larvae surviving the first food change. An analysis of variance test was applied and the results are given in Table 15.
Table 15.-- Analysis of variance test on the number of larvae alive at the first food change when fed natural Douglas -fir seedlings and modified AI Douglas -fir seedlings (Standard form).
Source of : Degrees Mean
variation : of freedom square F
Replication 9 13.12 2.09 NS b/
Treatments 1 22.05 3.52 NS (food)
Error 9 6.27
Total 19
a/ See page 73 for description of modified food.
b/ NS: No significant differences at 1 or 5% level.
As evident from this table, no significant differences were detected between the two food types. These results are not based on larvae of similar ages. When the establishment survival was assessed, larvae feed- ing on natural food were 11 days older than larvae feeding on modified food. 83
It was concluded from this experiment that dehydration could be re- tarded by covering the seedlings with a layer of agar. The presence of extraneous material, such as agar and fungicide appeared to have no effect on survival or fecundity. The data obtained under the conditions of this study supports the conclusion that a modified food will suffice for rearing standard form spruce budworm.
Comparative Morphological Studies
During the course of rearing both forms of spruce budworm, it became increasingly apparent that the green form pupa was extremely fragile.
Invariably, when young pupae were roughly handled, an integument rupture occurred on the venter of the thorax. This fragility was less notice- able as the pupae matured; however, judicious handling was required throughout the stage, if excessive mortality was to be prevented. In contrast, the standard form pupa within a few days after pupation was extremely tough and rigid and mortality seldom occurred as a result of laboratory handling. In the green form, the haemolymph was visible through the nearly translucent pupal cuticle during the major part of maturation. Shortly after pupation of the standard form, sclerotin was apparent in the pupal case. This material increased in density until just prior to adult eclosion the pupal cuticle was completely opaque.
These observations strongly indicated that cuticle composition varied between the two forms. Marked differences in visible sclerotin were apparent; however, a variation in cuticle thickness would also help ex- plain obvious differences between the two forms. In order to further elucidate inter -cuticle differences, photomicrographs were taken from serial thoracic sections of 1 -day old pupae. Preliminary examination 84
of the serial sections revealed little variation in cuticle thickness
between the thoracic regions; therefore, a 10 micron section was taken
through the posterior region of the prothorax (Figures 11, 12, 13, and
14.).
A measurement taken from point A (Figures 11 and 13) disclosed both
forms to have a cuticle thickness of 12 microns. The exocuticle (Exo.
Figures 11 and 13) did not vary significantly and was determined to be
6 microns in the green form and 5 microns in the standard form.
Further examination of photomicrographs disclosed cuticle character-
istics that were not apparent at the onset of this study. The pupal
cuticle was discontinous. Each appendage (Cx1 and Ant.) was separated
from internal structures by a membrane (Mb.) appearing to be an exten-
sion of the outer endocuticle. Integument rigidity was maintained
through a bridle structure which firmly coupled the outer cuticle of
each forming appendage (Bdl).
A similar internal bridle structure (Bd2,) associated with the form-
ing wing was found in both forms. Although not evident from the green
form photomicrographs, the large bridle structure (Bd2) is more closely
associated with the metathoracic wings (W2,) (Figures 11 and 12). A
lesser type structure is seen separating the mesothoracic wings (Bd3.).
The relative position of each appendage and its associated bridle is
shown in Figures 12 and 14.
L 85
36,1- gdi . yetExo A
CxT rC x1
,rBd2
__ _.a.- f, L r, r " - 3 Mb
_
Figure 11. Photomicrograph of a 10 micron X- section (79.3X) through the posterior margin of the prothorax of a 1 day old pupa (Standard form). A, point of cuticle measurement; Ant, antennae; Boll, bridle one; Bd2, bridle two; Bd3, bridle three; '' Cx1, coxae one; Exo, exocuticle; Mb, membrane; W3, mesothoracicuesot wing.
, Bd Cx ÿ4 c. Ant ti , '`o M b_ r33 W Bd ` 2y , W3 V _
111/ lib.' moor R '`/ L L
Figure 12. Photomicrograph of a 10 micron section (16X) through the posterior margin of the prothorax of a 1 day old pupa (Standard form). W2, metathoracic wing.
1.1 86
36.»- N Bd 1 ,-A_ \ --
V' -, CX1. . . ; d r . Mb .rr , Cr-
Figure 13. Photomicrograph of a 10 micron X- section (79.3X) through the posterior margin of the prothorax of a 1 day old pupa (Green form).
. A, point of cuticle measurement; Boil, bridle one; Bd2, bridle two; Cxl, coxa one; Exo,
exocuticle; Mb, membrane. '
Bd1 Cx s + . w M b O t O ` 2 Bd2 '. - ')t,r ;444404 . . 6;!: l Jj ;L' e ,No- . _ i ru a._t1.a _.. '. _ %tii . _ - - '.-
Figure 14. Photomicrograph of a 10 micron X- section (16X) through the posterior margin of the prothorax of a 1 day old pupa (Green form). W2, metathoracic wing; W3, mesothoracic wing. 87
After considering the available evidence, the fragile nature of the
green form pupae does not appear to be a factor of cuticle thickness.
Instead the exocuticle composition is probably responsible for differ-
ences in cuticle durability. The general reduced size of the various
bridle structures in green form pupae may help to account for the frail
integument, however, the significance of these structures has yet to be
determined.
Ten micron sections were also prepared from empty pupal cases, in
order to arrive at a precise stage of development for each spruce bud - worm cuticle. The cuticle covering the mesothoracic wings was chosen
for the comparative study. No measurable difference in exocuticle or
total cuticle thickness was found as compared with 1 -day old pupae.
However, considerable variation in exocuticle composition can be noted between the two forms (Exo. Figure 15 A and B).
After observing numerous adult moths, as they emerged from their pupal cases, it was surmised that the various bridle structures might represent a type of ecdysial apparatus. Adult appendage release would be facilitated if during eclosion, a rupture occurred at each point at which the cuticle was coupled.
To test this theory, a close examination was made of all sectioned empty pupal cases. The evidence did not show that a cuticle rupture occurred between each appendage, for several bridle structures were observed to be intact (Figure 16). 88
36jí. 36JÁ
t .. Exo El o
\Endo Endo
A. B.
Figure 15. Photomicrographs of a 10 micron X- section (79.3X) of a portion of the pupal cuticle covering the mesothoracic wings after adult emergence. A. Standard form. B. Green form. End, endo- cuticle; Exo, exocuticle.
36»
Bd
Figure 16. Photomicrograph of a 10 micron X- section (79.3X) through the thoracic region of an empty pupal case (Standard form). Bd, bridle; Mb, membrane. 89
A series of 6th instar larvae were sectioned in order to ascertain
if integument dissimilarities could be detected. Ten micron sections of the first abdominal segment were taken from the green form and stand-
ard form. Spruce budworm larvae collected at Bidwell, California, in
1954 were also examined for integument variations. The dorsal vessel was used as a point of orientation and all cuticle measurements were
taken above this organ.
The cuticle thickness for both the standard form larvae and Bidwell
larvae was approximately 15 microns (Figures 17 and 18). Precise meas-
urements were precluded due to the cuticlar processes (52, p. 56) which
gave the outer cuticle a three -dimensional appearance. Wittig (72,
p. 408) working with mature larvae of Choristoneura murinana (Hb.) re-
ported the prothoracic shield to be approximately 15 microns thick.
The cuticle in green form larvae appeared to deviate from the standard
form and Bidwell material, for a comparative measurement showed a
thickness of only 10 microns (Figure 19).
Based on the specimens examined, exocuticle appears to be nearly
deficient in the larval skin. This is in contrast to the pupa, where
50 percent of the cuticle is made up of hard exocuticle. These obser-
vations are in accord with Wittig (71, p. 553 and 558). She found that
in the larva and imago of Perla abdominalis Burm., exocuticle was con-
fined to the larval cuticular processes; however, adult exocuticle was
less restricted and found throughout the outer regions of the integument. 90
R,1
3 Oy . A l r I .:`-*4;,-. , .., ^ - - - - A . , a ~ y..& 111.1111.11 M-111101.4!'111!11111111Mrl rWilr` - _, . .: .. . c DV, ti -.
. "';r'!` - . rt rY 116grqiPU' 4 V%i ,J4 .1 % q -4-.41P -. _..._.'.A
r, l :-
Figure 17. Photomicrograph of a 10 micron X- section (79.3X) Through the anterior margin of the first abdominal segment in a sixth stage larva (Standard form). A, point of cuticle measure- ment; DV, dorsal vessel.
rt5 r i
3 Op
A A 041e6444.,_.r!lie- 1 '' 44 044 I 4' i .1.
,MÉ - r .- . 04, 7144/ ,t Via /
..0111111 -
Figure 18. Photomicrograph of a 10 micron X- section (79.3X) through the anterior margin of the first abdominal segment in a sixth stage larva (Bidwell). A, point of cuticle measurement; DV, dorsal vessel. 91
30ji iiO4,416) i - ; " "`.,N a. . - rilmAsfr,-:. ...a .- - `M ». .1 rj ,.
47-
Figure 19. Photomicrograph of a 10 micron (79.3X) through the anterior margin of the first abdominal segment in a sixth stage larva (Green form). A, point of cuticle measurement; DV, dorsal vessel. 92
After careful consideration of all data concerned with comparative studies on the two spruce budworm forms, it was concluded that the green form is a subspecies of Choristoneura fumiferana (Clem.). Environmental studies on factors affecting laboratory rearing revealed significant differences between the two forms. Mean fecundity was lower in the green form (107 eggs) as compared to the standard form (141 eggs).
Both forms had similar laboratory life histories and were reared to maturity on Douglas -fir shade foliage. Probably, the most marked dis- similarity, in terms of response to laboratory conditions was the low survival of green form larvae during diapause development. After 84 days cold rest, 72 percent of the green form larvae successfully vacated their hibernacula. However, 94 percent of the standard form larvae, responded to only 71 days cold rest.
Designating the green form as a subspecies is " supported by the morphological studies. Disparities in exocuticle composition were in- dicated. The various bridle structures examined showed differences between the two forms. Both bridle 1 and 2 in the green form are re- duced as compared to the standard form. Another obvious distinction, apart from morphological characteristics is the difference in haemolymph color. The blood in the green form, during later larval in3tars and early pupal stages is green, whereas, the blood in the standard form at similar stages of development is amber colored.
The green form appears to be geographically isolated. The popula- tion is endemic to the eastern parts of southern Oregon and northern
California. This restricted distribution partially satisfies the re- quirement for proposing a subspecies category (40). 93
DISCUSSION
The preliminary study demonstrated that both standard form and green form spruce budworm can be reared under the laboratory conditions out- lined by Stehr. However, subsequent studies on environmental factors affecting laboratory productivity, definitely established that Stehr's techniques and conditions are not necessarily optimal for rearing spruce budworm native to the Pacific Northwest.
Although various techniques have been developed which augment the rearing efficiency, significant increases in productivity appear to be a result of reduction in time necessary for diapause development and a generally high (90- 100%) overwintering survival. This high overwinter- ing survival was not obtained with the green form stock, indicating the standard form can better be adapted to a laboratory rearing program.
This conclusion finds support from other data including mean fecundity, general over -all vigor, and ease of handling.
The low diapause development mortality occurring in the standard form is in agreement with the data obtained by Harvey (23); however, he subjected larvae to a 2 -3 week longer cold rest period. Differences in survival might easily be explained on the basis of variation in vigor between the two laboratory stocks of spruce budworm. Within one year
1/ Carolin found a threefold increase in the wild population from which the standard form nucleus was collected.
1/ Personal communication. 1962. 94
In light of Harvey's work on a non -diapause strain of spruce budworm
plus various other studies on insects showing photo- induced responses,
the conditions favouring diapause termination are of special interest.
Harvey (22, p. 562) tested high intensity fluorescent and low intensity
tungsten light and obtained differences in response for the inception of
the N -D behaviour. He attributed these variations to an illumination
threshold factor and not to the spectral differences of the two light
sourced. These conclusions agree with other workers (12, 34, 36).
After a threshold for induction is surpassed, intensity is no longer a
factor in inducing a particular response. However, a response such as
the inception of diapause shows an action spectrum above the threshold
intensity.
Lees (34, p. 460 -461) found maximum sensitivity for the evocation of
diapause in the mite, Netatranychus ulmi Koch, to be 430 -460 millimi-
crons. Dickson (12, p. 523) induced diapause in only a few oriental
fruit moths with wave lengths above 600 millimicrons. DeWilde (11,
p. 12 -13) states that the general action spectrum for nearly all insects
showing a photo- induced diapause, lies between the ultraviolet
(365 millimicrons) and blue green (500 millimicrons). The maximum sen-
sitivity is normally in the blue, with wave lengths longer than 530
millimicrons being inactive.
In order to explain the different responses obtained with "Cool
White" fluorescent and incandescent illumination it seems tenable to
suggest an action spectrum for the termination of diapause. Standard
form larvae receiving 42 days cold storage were unable to evacuate their 95 hibernacula under the influence of incandescent illumination. After a similar cold rest period, 44 percent of the larvae responded to fluores- cent illumination. With an extended cold rest (51 -90 days) both light sources were equally effective in stimulating hibernacula evacuation; however, larvae under the influence of fluorescent illumination con- tinued to issue at a faster rate.
A marked difference in spectral characteristics exists between the
two light sources. . "Cool White" fluorescent has an energy peak around
570 millimicrons, with a secondary peak at 470 millimicrons. Tungsten
incandescent peaks in short wave infrared and is very deficient in blue and green. It is suggested, based on the data and conditions of this
study that standard form spruce budworm larvae show an action spectrum
for diapause termination similar to that required by certain facultative
insects for the inception of diapause.
Table 16 was constructed to show the productivity which might be ex- pected from the standard form spruce budworm when reared at optimum
conditions. These values were extracted from the environmental condi-
tions studied and represent the maximum survival obtained. 96
Table 16.-- Summary of expected productivity when standard form spruce budworm are reared at optimum laboratory conditions.
Survival
Stage : Apparent (percent) Real (percent)
Incubation 81.7 81.7
Pre -diapause 90.0 73.5
Diapause development 96.0 70.5
Establishment on food 31.5 22.2
Completion of rearing 92.0 20.4
If a rearing program were initiated with 1000 eggs, approximately
204 larvae would successfully pupate and of this number 17 adults would fail to transform. Based on a 1/1 sex ratio, 9 percent mating failure and a mean fecundity of 141, an approximate twelvefold increase may be expected in the next generation. Under the conditions of this study, a total of 2.7 generations per year were obtained.
Based on the experience gained to date, a potential of 5 generations per year would be possible, providing the rearing program was carefully scheduled and the aid of two full -time technicians was available.
The low establishment survival, 31.5 %, remains the major factor responsible for reduction of rearing stock. These results indicate that refinement of the modified food is needed. Although hibernacuation evacuation was used as the criterion for distinguishing between favour- able and unfavourable diapause development conditions, it cannot be overlooked that cold storage may effect the establishment of issuing 97
larvae. Any conditions which tend to enhance larvae becoming estab-
lished on their food will lead to significant increases in rearing pro-
ductivity.
Cannibalism was observed to occur in feeding confinements where the
ratio of larvae to available food was high. This behaviour was undoubt-
edly responsible for a part of the mortality occurring prior to the
first food change. Stehr (55, p. 426) reported a similar behaviour in
his laboratory stock of spruce budworm. Harris (20, p. 328), working with the black cutworm, (Agrotis ypsilon (Rott.), found that cannibal-
ism was severe when crowding became excessive.
The standard form budworm has been reared through 3 consecutive
generations, without any serious mortality from microorganisms. However,
in the F3 generation, the presence of pathogenic yeast was detected.
This microorganism accounted for a few losses but its identification
and virulence potential was not determined. Four genera of fungi,
Hirsutella, Empusa, Beauveria and Isaria, contain known pathogenic
species on spruce budworm (37). The green form was comparatively free
of pathogens; however, during the first laboratory generation, a virus,
tenatively determined as a granulosis was observed. Nearly all infected
larvae survived, consequently, it was not considered a significant fac-
tor in rearing productivity. 98
CONCLUSIONS
The spruce budworm is not easily adapted to laboratory study. The
requirement of an extended cold rest, seasonal limitations in food
supply and high rearing mortality has discouraged efforts to rear this
insect as a test animal.
Under the conditions of this tudy, a significant reduction in dia-
pause development time has been obtained. As a result of this reduction
the number of generations per year has been increased to nearly 3. The
development of a modified food has served two purposes, (1) independence
from a natural food supply and (2) decrease in number of food changes
required to maintain food acceptability. Laboratory survival has been
enhanced whereby an approximate twelvefold increase per generation may
be expected.
These results permit the following rearing recommendations to be
made. Egg masses should be incubated at temperatures between 70 and
75 °F. and a relative humidity near 77 percent. Embryo maturation and
hibernacula site selection will require approximately 8 to 9 days. The
larvae should be left at incubation conditions for a period no longer
than 3 weeks. A pre -conditioning temperature treatment consisting of
1 week at 45 °F. appears necessary before the quiescent larvae are ex-
posed to overwintering conditions. The requirement of a cold rest is
partially fulfilled by subjecting the larvae to 52 days of artificial winter (33 °F.) conditions. This duration appears to be optimal in
terms of survival and minimum storage time. Approximately 26 days - -
99
After removal to the fluorescent light table total larval issuance can
be expected to be complete.
Newly emerged larvae should be collected daily and confined in half -
pint ice cream cartons containing newly prepared or freshly thawed,
agar- coated Douglas -fir seedlings. The optimum number of larvae per
carton has not been determined; however, 20 per feeding cage appears to
be the maximum. The first food change will be governed by the rate of
deterioration of food and will probably be necessary 10 days after lar-
val introduction. Two or three more food changes may be required; how-
ever, this will depend on the number of feeding larvae. Most larvae
will mature and pupate in approximately 30 days.
Pupae should not be handled for at least 2 days after pupation.
Forceps can be used to grip their cremaster thereby reducing integument
punctures. Adult eclosion will occur within 7 to 8 days and should take
place on a rough, absorbent substrate.
Satisfactory mass mating and oviposition can be accomplished in a
large walk -in cage containing young potted Douglas -fir trees. Egg
masses can be concentrated on 1 or 2 trees by placing a low intensity
light at one corner of the cage. Egg mass collections can be made on
the fifth and tenth days after mating, thereby avoiding additional time
and labour required by daily collections.
f 100
BIBLIOGRAPHY
1. Andrewartha, H. G. Diapause in relation to the ecology of insects. Biological Reviews of the Cambridge Philosophical Society 27:50 -107.
2. Arias, R. O. The biology of the grey garden slug (Deroceras reticulatum) (Muller) under laboratory conditions. Master's thesis. Corvallis, Oregon State University, 1960. 64 numb. leaves.
3. Atwood, C. E. The feeding habits of young spruce budworm larvae. The Canadian Entomologist 76:64 -66. 1944.
4. Baker, F. C. The effect of photoperiodism on resting treehole mosquito larvae. The Canadian Entomologist 67:149 -153. 1935.
5. Bergold, C. H. The polyhedral disease of the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae) Canadian Journal of Zoology 29 :17 -23. 1951.
6. Bodine, J. H. Hibernation and diapause in certain Orthoptera II. Response to temperature during hibernation and diapause. Physio- logical Zoology 5:538 -548. 1932.
7. Hibernation and diapause in certain Orthoptera III. Diapause - -A theory of its mechanism. Physiological Zoology 5:549 -554. 1932.
8. Bucher, G. E. Winter rearing of tent caterpillars, Malacosoma spp. (Lepidoptera: Lasiocampidae). The Canadian Entomologist 91 :411 -446. 1959.
9. Cole, W. E. Spruce budworm population- damage study. (Progress Report) Boise, Idaho. U. S. Dept. of Agric., Forest Service, Intermountain Forest and Range Experiment Station, Boise Research Center, 1957. 14 p. (Processed).
10. Cottam, C. and E. Higgins. DDT: Its effects on fish and wildlife. 1946. 14 p. (U. S. Fish and Wildlife Service. Circular 11).
11. DeWilde, J. Photoperiodism in insects and mites. Annual Review of Entomology 7:1 -26. 1962.
12. Dickson, R. C. Factors governing the induction of diapause in the oriental fruit moth. Annals of the Entomological Society of America 42 :411 -437. 1949. 101
13. Fellin, D. G. The effect of cold temperatures on the length of diapause of the spruce budworm (Choristoneura fumiferana (Clem.)). Master's thesis. Corvallis, Oregon State University, 1960. 80 numb. leaves.
14. Fliters, N. E. and P. S. Messenger. Bioclimatic studies of oriental fruit fly in Hawaii. Journal of Economic Entomology 46:401 -403. 1953.
15. Bioclimatic cabinets used in studies on the Mexican fruit fly and the pink boll worm. 1956. 12 p. (U. S. Dept. of Agri- culture, Agricultural Research Service. ARS 33 -33).
16. Furniss, R. L. Answers to some criticism of aerial spraying in forest management. Journal of Forestry 57 :260 -262. 1959.
17. George, J. L. Effects on fish and wildlife of chemical treat- ments of large areas. Journal of Forestry 56:250 -254. 1959.
18. Possible effects of widespread use of forest chemicals on wildlife populations. In: The use of chemicals in southern forests, Proceedings of the 9th Annual Forestry Symposium, Louisiana State University, 1960. Baton Rouge, 1960. p 152.
19, Guyer, M. F. Animal micrology. Chicago, University of Chicago Press, 1953. 327 p.
20. Harris, C. R. A laboratory method of mass rearing the black cut- worm, Agrotis ypsilon (Rott), for insecticide tests. The Canadian Entomologist 90:328-331. 1958.
21. Harvey, G. T. Absence of diapause in rearing of the spruce bud- worm. Canada. Department of Agriculture. Forest Biology Division. Bi- monthly Progress Report 10(4):1 -4. 1954.
22. The occurrence and nature of diapause -free development in the spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Canadian Journal of Zoology 35:549 -572, 1957.
23. Studies of the diapause requirements of the spruce budworm. Paper read at the annual meeting of the Entomological Society of Canada, Lethbridge, Alberta, Oct., 1957.
24. A relationship between photoperiod and cold -storage treat- ment in the spruce budworm. Science 128:1205 -1206. 1958.
25. Henneguy, F. L. Les insects: Morphologie, reproduction, embryo - g &nie. Paris, Masson, 1904. 804 p. 102
26. Hinton, H. E. The initiation, maintenance and rupture of diapause: a new theory. Entomologist 86:279 -291. 1953.
27. Hodson, A. C. and C. J. Weinman. Factors affecting recovery from diapause and hatching of eggs of the forest tent caterpillar, Malacosoma disstria Hbn. St. Paul, 1945. 31 p. (Minnesota Agri- cultural Experiment Station. Technical Bulletin 170).
28. Hoffmann, C. H. and E. P. Merkel. Fluctuations in insect popula- tions associated with aerial applications of DDT to forests. Jour- nal of Economic Entomology 41:464 -473. 1948.
29. Howe, R. W. A method for obtaining a controlled daily temperature cycle. Annals of Applied Biology 44:188 -194. 1956.
30. Kerswill, C. J. and P. F. Elson. Preliminary observations on effects of 1954 DDT spraying on Miramichi stocks. Progress Reports of the Atlantic Coast Stations of Fisheries Research Board of Canada. 62 :17 -24. 1955.
31. Langford, R. R. Forest spraying and some effects of DDT. Toronto, 1949. 174 p. (Ontario Dept. Land and Forests. Biological Bulle-
tin 2) .
32. Lees, A. D. The physiology of diapause. Science Progress 38:735 -742. 1950.
33. _ Diapause. Science Progress 40:306 -312. 1952.
34. Environmental factors controlling the evocation and ter- mination of diapause in the fruit tree red spider mite, Metatranychus ulmi Koch (Acarina: Tetranychidae). Annals of Applied Biology 40:449 -486. 1953.
35. The physiology of diapause in arthropods. London, Cam- bridge University Press, 1955. 150 p.
36. The physiology and biochemistry of diapause. Annual Review of Entomology 1:1 -16. 1956.
37. MacLeod, D. M. Fungi associated with the spruce budworm. Canadian Department of Agriculture Forest Biology Division Bi- monthly Progress Report 5(1) :1 -4. 1948.
38. Marples, E. C. Significances of fish mortality in forest spraying operations. In: Proceedings of the 48th Western Forestry and Conservation Association 1957. p. 16 -17.
39. Mathers, W. G. The spruce budworm in British Columbia. Forestry Chronicle 8:154 -157. 1932. 103
40. Mayr, E., E. G. Linsley, and R. L. Usinger. Methods and prin- ciples of systematic zoology. New York, McGraw -Hill, 1953. 336 p.
41. McGugan, B. M. Needle mining habits and larval instars of the spruce budworm. The Canadian Entomologist 86:439 -453. 1954.
42. Miller, C. A. The measurement of spruce budworm population and mortality during the first and second larval instars. Canadian Journal of Zoology 36:409 -422. 1958.
43. Munger, F. An adaptation of a thermograph to regulate variable temperature. Journal of Economic Entomology 37:554. 1944.
44. Packard, A. S. Insects injurious to forest and shade trees. U. S. Entomological Commission, Fifth Report, 1890. 957 p. (At head of title: U. S. Dept. of Agriculture).
45. Pepper, J. H. Breaking the dormancy in the sugarbeet webworm L. sticticalis L., by means of chemicals. Journal of Economic
Entomology 30:380. 1937. '
46. Petersen, Björn. On the evolution of Pieris rapi L. Evolution 3: 269 -278. 1949.
47. Pitman, G. B. and R. B. Ryan. A simple device for producing har- monic temperature fluctuations. The Canadian Entomologist (In Press).
48. Ryan, R. B. Some environmental factors affecting development and emergence of the Douglas -fir beetle Dendroctonus pseudotsugae Hopkins (Coleoptera: Scolytidae), in the laboratory. Master's thesis. Corvallis, Oregon State University, 1959. 62 numb. leaves.
49. Termination of diapause in the Douglas -fir beetle, Dendroctonus pseudotsugae Hopkins (Coleoptera: Scolytidae), as an aid to continuous laboratory rearing. The Canadian Entomologist 91:520 -525. 1959.
50. Shelford, V. E. Laboratory and field ecology. Baltimore, Williams and Wilkins, 1929. 608 p.
51. Slifer, E. H. The effects of xylol and other solvents on diapause In the grasshopper egg, together with a possible explanation for the action of these agents. Journal Experimental Zoology 102 -333 -356. 1946.
52. Snodgrass, R. E. Principles of insect morphology. New York, McGraw -Hill, 1935. 667 p. 104
53. Solomon, M. E. Insect population balance and chemical control of pests. Chemistry and Industry, 1953, p. 1143 -1147.
54. Stairs, G. R. On the embryology of the spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera). The Canadian Entomologist 92:147 -154. 1960.
55. Stehr, G. A laboratory method for rearing the spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). The Canadian Entomologist 86:423 -528. 1954. 'r 56. Stone, W. E. An instrument for the reproduction, regulation and control of variable temperature. Journal of the Washington
Academy of Sciences 29:410 -415. 1939. .
57. Swaine, J. M., F. C. Craighead, and I. W. Bailey. Studies on the spruce budworm (Cacoecia fumiferana Clem.) Ottawa, 1924. 91 p. (Canada. Dept. of Agriculture. Entomological Branch Bulletin, new ser. 37).
58. Uvarov, B. P. Insects and climate. Transactions of the Ento- mological Society of London 79:1 -247. 1931.
59. Wellington, E. F. Artificial medium for larval rearing. Canada Department of Agriculture Forest Biology Division Bi- monthly Progress Report 5(1) :1 -4. 1949.
60. Wellington, W. G. and W. R. Henson. Notes on the effects of phy- sical factors on the spruce budworm Choristoneura fumiferana (Clem.). The Canadian Entomologist 79:168 -170, 195. 1947.
61. Wellington, W. G. The light reaction of the spruce budworm. The Canadian Entomologist 80 :46 -82. 1948.
62. The effects of temperature and moisture upon the behav- iour of the spruce budworm, Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae) I. The relative importance of graded temperature and rates of evaporation in producing aggregations of larvae. Scientific Agriculture 29:201 -215. 1949.
63. The effects of temperature and moisture upon the behav- iour of the spruce budworm, Choristoneura fumiferana Clemens ( Lepidoptera: Tortricidae) II. The responses of larvae to gradients of evaporation. Scientific Agriculture 29:216 -229. 1949. 105
64. Wellington, W. G. Variation in silk spinning and locomotor activ- ities of larvae of the spruce budworm, Choristoneura fumiferana Clemens at different rates of evaporation. Proceedings and Trans- actions of the Royal Society of Canada 44(5):89 -101. 1950.
65. Wheeler, W. M. A contribution to insect embryology. Journal of Morphology 8:1 -160. 1893.
66. White, E. B. and P. Debach. A wide -range variable temperature cabinet for bio- ecological studies. Journal of Economic Entomol- ogy 53:1030 -1034. 1960.
67. Wishart, G. An adaptation of a standard bi- metallic thermoregu- lator to control variable temperature. The Canadian Entomologist 72 :78 -31. 1940.
68. Whiteside, J. M. Spruce budworm control in Oregon, Washington, 1949 -1956. In: Proceedings of Tenth International Congress of Entomology Montreal, 1956. Vol. 4. Ottawa, Mortimer, Limited, 1958. p. 291 -302.
69. Williams, C. H. Physiology of insect diapause. The role of the brain in the production and termination of pupal dormancy in the grant silkworm Platysamia cecropia. Biological Bulletin
90 :234 -243. .
70. Physiology of insect diapause. An endocrine mechanism for the influence of temperature on the diapause pupa of the cecropia silkworm. Biological Bulletin 110:201 -218. 1956.
71. Wittig, G. Untersuchungen am Thorax von Perla abdominalis BURN. (Larvae and Imago) unter besonderer Berücksichtigung des peripheren Nervensystems und der Sinnesorgane. Zoologische Jahrbücher 74:491 -570. 1955.
72. Morphologie und Entwicklung der Raupen des Tannentrieb - wicklers Choristoneura murinana (HB.) (Lepidopt., Tortricidae) I. Die Unterscheidung der Larvenstadien). Zeitschrift Fur Pflanzen- krankheiten 66 :401 -409. 1959.
73. Yung -Song, Pan. Diets for rearing the sugarcane borer. Journal of Economic Entomology 54:257 -261. 1961.
r.