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Rearing studies of the Douglas-fir cone moth, Barbara colfaxiana (Kearfott) (Lepidoptera: Tortricidae) Ebata, Timothy Masao 1986

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REARING STUDIES OF THE DOUGLAS-FIR CONE MOTH, BARBARA COLFAXIANA (KEARFOTT)(LEPIDOPTERA:  TORTRICIDAE).  by T i m o t h y Masao E b a t a B.Sc. Z o o l o g y , The U n i v e r s i t y o f B r i t i s h C o l u m b i a  A Thesis Submitted i n P a r t i a l F u l f i l l m e n t of The R e q u i r e m e n t s f o r t h e Degree o f Master of Science in The F a c u l t y o f Graduate S t u d i e s (Department o f F o r e s t r y , F o r e s t Entomology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to t h e r e q u i r e d s t a n d a r d  The U n i v e r s i t y o f B r i t i s h Columbia O c t o b e r 1986 (c) T i m o t h y Masao E b a t a  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l f u l f i l m e n t of the requirements  for  an advanced degree at t h e U n i v e r s t i y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g or p u b l i c a t i o n this thesis for financial  DEPARTMENT OF FORESTRY  Date:  October  1986  of  g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n  permission.  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  I  Columbia  - ii  -  ABSTRACT  Several attempts were made at improving an a r t i f i c i a l  rearing  method developed by Ruth and Hedlin (1969) f o r the D o u g l a s - f i r cone moth from 1983 to 1985.  During 1983 and 1984, d i f f e r e n t container types,  establishment methods and d i e t formulations were tested r e s u l t i n g in the production of a method that reduced both l a r v a l handling and media moisture l o s s .  However, unexpected high m o r t a l i t y occurred during the  l a t e L4 stage when the insect constructed a cocoon within or adjacent to the media, and at pupation when developmental problems became obvious. These symptoms suggested the presence of a disease, however, no pathogens were observed with the l i g h t microscope.  These forms of m o r t a l i t y had  not been reported by Ruth and Hedlin (1969).  It was postulated that  media nutrient degradation was one cause since the method described by Ruth and Hedlin (1969) allowed the larvae to feed continuously on r e l a t i v e l y fresh (nutrient r i c h ) media while the larvae fed on unchanged media throughout the l a r v a l feeding period with my method. In 1985, two spruce budworm were replaced with f r e s h l y made media of the same formulation two weeks a f t e r establishment with eggs.  Although pupal s u r v i v a l  differed  considerably between the two d i e t s , no s i g n i f i c a n t improvements in l a r v a l or pupal s u r v i v a l were obtained by changing media.  In a second experiment, the i n i t i a l concentrations of l i n o l e n i c and L-ascorbic acid contained in Robertson's (1981) d i e t were varied to produce 12 d i f f e r e n t d i e t s . a r t i f i c i a l diets.  Both nutrients were reported to degrade in  Larval s u r v i v a l and pupal production were d i r e c t l y  r e l a t e d to the d i e t a r y concentration of l i n o l e n i c but not L-ascorbic acid.  The best o v e r a l l pre-winter s u r v i v a l of 53.9% and post-winter  s u r v i v a l of 37.8% were obtained on Roberston's (1981) d i e t with four times and two times the normal concentration of l i n o l e n i c a c i d , respectively.  L i n o l e n i c acid appeared to be an e s s e n t i a l nutrient f o r  the cone moth since i t s absence produced the worst pre- and post-winter (5)  survival.  Bioserv -reared pupae and pupae r e s u l t i n g from e i t h e r of  these "best" d i e t s were not s i g n i f i c a n t l y d i f f e r e n t in weight from n a t u r a l l y - r e a r e d pupae.  Adult emergence success was not affected by  increasing q u a n t i t i e s of d i e t a r y l i n o l e n i c acid above the normal concentration. The incidence of 2YD appeared r e l a t e d to rearing temperature. Prolonged diapause incidences were s i g n i f i c a n t l y d i f f e r e n t between a r t i f i c i a l l y - and n a t u r a l l y - r e a r e d pharate adults f o r each year of a r t i f i c i a l rearing (1983 through 1985). A sample of cone moths from 40 trees revealed that w i t h i n stand v a r i a t i o n of 2YD incidences was r e l a t i v e l y narrow.  This r e s u l t suggested  - iv -  that the proportion of cone moths entering 2YD may be regulated by factor(s) that uniformaly affect the stand, such as temperature, rather than as a response to the individual tree's reproductive potential in the year following larval feeding.  This relationship and the improvement of  the 1985 rearing method requires further investigation.  - V -  TABLE OF CONTENTS Page TITLE PAGE ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS 1.0 INTRODUCTION 1.1 Bionomics of the Douglas-fir cone moth 1.2 Possible Mechanisms for the Insect/Host Synchrony 1.3 Artificial Rearing 1.4 Objectives 2.0 GENERAL METHODS FOR REARING STUDIES 2.1 Source, Storage and Handling of Eggs 2.2 Egg Establishment on Media 2.3 Media Preparation 2.4 Rearing Environment 2.5 Analyses 3.0 ARTIFICIAL REARING 3.1 Introduction 3.2 Materials and Methods, 1983 3.2.1 Diet Formulation and Processing 3.2.2 Experimental Design 3.2.3 Media Establishment Procedures Eggs Larvae 3.2.4 Inspections and Pupal Handling 3.2.5 Adult Emergence and Overwinter Survival 3.3 Results, 1983 3.3.1 Media Condition 3.3.2 Survival 3.3.3 Pupal Weights 3.3.4 Incidence of Prolonged Diapause 3.3.5 Pharate Adult Survival and Adult Emergence 3.4 Discussion and Conclusions, 1983 3.5 Introduction, 1984 3.6 Materials and Methods, 1984 3.6.1 Rearing Method 3.6.2 Experimental Design 3.6.3 Diet Inspections 3.6.4 Pupal Extraction and Overwinter Storage 3.6.5 Disease Analysis  i ii v vii ix xi 1 3 8 11 13 14 14 14 16 17 17 19 19 19 19 21 22 22 22 22 23 24 24 25 28 32 32 33 37 37 37 38 40 42 42  - vi 3.7 Results, 1984 3.7.1 Media Condition 3.7.2 Survival 3.7.3 Larval Behaviour and Development 3.7.4 Pupal Weights 3.7.5 Incidence of Prolonged Diapause 3.7.6 Disease Analysis 3.7.7 Other Experiments 3.8 Discussion and Recommendations, 1984 3.9 Introduction, 1985 3.10 Materials and Methods, 1985 3.10.1 Rearing Method 3.10.2 Experimental Design 3.10.3 Diet Preparation and Inspections 3.10.4 Pupal Extraction and Storage 3.10.5 Disease Analysis 3.11 Results, 1985 3.11.1 Media Condition 3.11.2 Survival 3.11.3 Pupal Weights 3.11.4 Incidence of Prolonged Diapause Relationship With Diet Relationship With Rearing Temperature 3.11.5 Disease Analysis 3.12 Discussion, 1985 4.0 FIELD STUDY 4.1 Introduction 4.2 Materials and Methods 4.3 Results 4.4 Discussion 5.0 REFERENCES APPENDIX I ARTIFICIAL DIET FORMULAE AND MIXING INSTRUCTIONS A Robertson's (1979) Western Spruce Budworm Diet B Ingredients and Mixing Procedures for Bioserv® Diets C Robertson's (1981) Western Spruce Budworm Diet APPENDIX II Correspondences from Insect Pathologists A Dr. John Cunningham re: 1984 Diagnosis B Mr. Gerard Thomas re: 1985 Diagnosis APPENDIX III Fixing and Staining Procedures (Clark 1981)  43 43 43 51 52 53 53 55 56 63 64 64 64 66 68 71 72 72 73 87 96 96 96 101 101 109 109 110 112 112 117 123 123 124 125 127 128 139 130  - vii LIST OF TABLES Table  Page  Mortality of artificially reared B_. colfaxiana by life stage and rearing condition (environment chamber reared insects only). May 1983 to April 1984.  27  A comparison of mean pupal weights of cone moths reared on artificial medium contained in four creamer types maintained in an environment chamber and naturally reared pupae extracted from cones collected near Keremeos, B.C.  29  A comparison of mean pupal weights of cone moths reared artificially in 1966 and 1983 (all chamber reared) and naturally in 1983. Comparisons are stratified by sex and diapause condition.  31  IV Summary of survival through each life-stage of Q. coifaxiana reared on three Bioserv® diets and several variations of diet concentration and establishment method. May 1984 to 21 April 1985.  44  I  II  Ill  V Summary of mortality through the L4 stage of B. colfaxiana reared on three Bioserv® diets and several variations of diet concentration and establishment method. May 1984 to 21 April 1985 VI  VII  46  Summary of mortality through the pupal (charate adult) stage of B. colfaxiana reared on three Bioserv® diets and several varTations of diet concentration and establishment method. May 1984 to 21 April 1985.  49  A comparison of mean pupal weights of B. colfaxiana reared on Bioserv^s Spruce Budworm diet (BW)^ by a) diapause condition and sex, and b) by sex (1YD and 2YD data combined). May to October 1984.  54  VIII Comparison of mean pupal weights of B. colfaxiana reared naturally at four densities. Insects were extracted from cones collected from Keremeos, B.C., 25 August 1984.  57  IX Summary of survival through each life stage of B. colfaxiana reared on 14 diets and two media conditions TExperiments 1 and 2), 6 May 1985 to 8 April 1986.  74  X Summary of mortality through the fourth instar of B. colfaxiana reared on 14 diets and two media conditons ^Experiments 1 and 2), 6 May to 16 October 1985.  77  - vi i i XI  Summary of m o r t a l i t y through the pupal (pharate adult) stage of Q. c o l f a x i a n a reared on 14 d i e t s and two media conditions (Experiments 1 and 2 ) , 6 May to 16 October 1985.  79  XII  Summary of m o r t a l i t y through the pharate adult stage of B. c o l f a x i a n a reared on 14 d i e t s and two media conditions TExperiments 1 and 2 ) , 17 October 1985 to 8 A p r i l 1986.  85  XIII  Comparison of mean pupal weights of 1YD and 2YD J3. c o l f a x i a n a reared on s i x a r t i f i c i a l d i e t s May to 16 October 1985.  88  XIV  Comparison of mean pupal weights of female and male J3. c o l f a x i a n a (1YD and 2YD data combined) reared on s i x a r t i f i c i a l d i e t s , and reared n a t u r a l l y on cones, May to 16 October 1985.  89  Comparison of mean pupal weights of female and male IB. c o l f a x i a n a (1YD and 2YD data combined) reared on changed and unchanged BIO and RN d i e t s , May to 16 October 1985.  90  XVI  Comparison of mean pupal weights of J3. c o l f a x i a n a reared on a r t i f i c i a l and natural d i e t s . Comparison A includes a l l d i e t s with greater than 12 female and 11 male pupae. Comparison B includes a l l d i e t s with greater than 22 female and 28 male pupae.  93  XVII  Comparison of mean pupal weights of B. c o l f a x i a n a reared on three Spruce Budworm d i e t s in 1966, 1983, 1984 and 1985, and pupal weights of cone moths extracted from cones c o l l e c t e d from cones c o l l e c t e d from Keremeos, B.C. in 1983 and 1984.  95  XV  - ix LIST OF FIGURES Figure  Page  1  L i f e cycle of the D o u g l a s - f i r cone moth (from Hedlin et a l . 1980).  4  2  The r e l a t i o n s h i p between the incidence of prolonged diapause in J3. c o l f a x i a n a and the s i z e of the cone crop maturing in the year a f t e r l a r v a l feeding (Spearman's rank c o r r e l a t i o n c o e f f i c i e n t r ) ( f r o m Hedlin et a l . 1982).  7  3  a) Location of Keremeos study s i t e s in B r i t i s h Columbia; b) The approximate l o c a t i o n of "Dahlgard's", "Outward Bound", and "Ashnola" study s i t e s about 20 km west of Keremeos, B.C.; c) Photograph of "Outward Bound" showing i n t e r i o r Douglas-fir stand s t r u c t u r e and s i t e topography.  15  4  Two 7 dram v i a l s containing Budworm d i e t (BW) showing l a r v a l feeding a c t i v i t y ( r i g h t ) and pre-pupal cocoon ( l e f t ) of J3. c o i f a x i a n a .  41  5  Larval and pupal abnormalities, a) Dead L4 in cocoon;b) "marbly" L4; c) deformed pupa; and d) incomplete pupa.  47  6  Pupal production and s u r v i v a l to November 1984 of B. c o l f a x i a n a reared on three commercially prepared diets.  50  7  I l l u s t r a t i o n s of various forms of pupal m o r t a l i t y and adult emergence observed 28 February to 8 A p r i l 1986 a) Normal pupa; b) p a r t i a l l y emerged pharate adult; c) shrunken or collapsed pupa or pharate a d u l t ; d) dead incomplete pupa; e) normal adult f u l l y eclosed; f-g) adult f u l l y eclosed with deformed wings. (Drawings by L. F r i s k i e ) .  71  8  Pupal production and pre-winter (to 16 October 1985) s u r v i v a l of B. c o l f a x i a n a reared on varying concentrations of l i n o l e n i c acicT.  81  9  Pupal production and pre-winter (to 16 October 1985) s u r v i v a l of B. c o l f a x i a n a reared on a r t i f i c i a l d i e t containing varying concentration of L-ascorbic a c i d .  82  Pupae with unusual pigmentation on abdomen accompanied with s p l i t t h o r a c i c sutures. a)close-up of bands and s p l i t sutures l i n e s ; b) comparison with a healthy pupa (upper).  83  s  10  - X -  11  Mean pupal weights of EL coifaxiana reared on artificial diets containing varying concentrations of linolenic acid. Also included are mean pupal weights of 1983 naturally-reared and 1985 Bioserv® Budworm (unchanged) reared cone moths.  92  12  Prolonged diapause incidence of J3. coifaxiana pharate adults reared on Robertson's (1981) Budworm diet (RN) containing three different concentrations of linolenic acid. RNU1 & RNU2 = unchanged RN, vials No. 1-100 and 101-200, respectively.  97  13  Prolonged diapause incidence of J3. coifaxiana pharate adults reared on Robertson's (1981) Budworm diet (RN) containing four different concentrations of L-ascorbic acid. RNU1 & RNU2 = unchanged RN, vials No. 1-100 and 101-200, respectively.  98  14  Prolonged diapause incidence of Q. coif axiana pharate 100 adults reared on four Spruce Budworm diets at seven different constant temperatures (regression line); and for naturally reared pharated adults collected from 1982 to 1985. Data for 1982 naturally reared insects was obtained from Sahota et a l . (1985). All artificially reared insect data except for 1983-85) were obtained from Hedlin et aj_. (1982)  15  The proportion of cone moths in prolonged diapause and proportion of female buds on 25 1985 twigs collected from a single upper crown branch from the three Keremeos study sites analysed separately (a-c) and together (d).  113  16  The proportion of pharate adults in 2YD and proportion of female buds per 25 1985 twigs collected from a single upper crown branch from individual trees withi the three Keremeos area study sites. Only trees with > 50 (triangles) and > 40 (circles) live pupae were plotted.  114  - xi -  ACKNOWLEDGEMENTS  I thank my committee members Drs. G.E. Miller, B. van der Kamp, J.A. McLean for their direction and assistance throughout my research.  I am  deeply indebted to Mr. D.S. Ruth whose experience and wisdom proved invaluable to me.  I also thank the staff of the Pacific Forestry Centre  who assisted me in this thesis.  I particulary thank Drs. H.S. Whitney,  T.S. Sahota and I. Otvos, and for their technical assistance, Ms. D. Chu, Mr. A. Ibaraki, Mrs. L. Manning, Ms. M. Senecal, Ms. P. Hanson and Mr. C. Alexander. I thank Ms. L. Friskie for her assistance at UBC in assessing adult emergence and for her superb illustrations depicting the same.  Finally,  I give my deepest appreciation to my wife, Cheryl, whose patience and dedication guided me through "the rough spots". The 1985 research was partially funded by a CFS Block Grant award. Summer wages were provided by the Pacific Forestry Centre in conjunction with the federal student employment programs COSEP (1983-1984) and Challenge '85 (1985).  - \ 1. INTRODUCTION  The Douglas-fir cone moth, Barbara colfaxiana (Kearfott) (Lepidoptera: Tortricidae), is one of the most destructive cone and seed insects of both coastal and interior varieties of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco, var. menziesii and glauca (Beissn.) Franco, respectively).  In coastal seed orchards and natural  stands, B>. colfaxiana damage intensity varies sporadically and does not relate to size of the cone crops in previous years.  In contrast, in  interior Douglas-fir stands the damage occurs more consistently and the heaviest damage usually appears in years immediately following abundant cone crops (Miller et a l . 1984). Severe infestations have resulted in complete seed crop losses (Hedlin 1960) and, as recorded in 1981 at Keremeos by Miller et a l . (1984), a severe infestation coupled with a very light cone crop can lead to complete larval mortality resulting from the premature death of all cones due to heavy cone moth larval feeding.  Fortunately for B_.  colfaxiana, and for many other species of cone and seed insects, a variable proportion of their populations each year will remain in prolonged or extended diapause for two or more winters thus protecting that portion of the population from severe intraspecific competition during poor cone crop years.  Douglas-fir cone crops vary in size widely  between years and in no recognizable pattern (Dobbs et a l . 1976) except that two heavy cone crops rarely occur in consecutive years.  - 2Prolonged diapause is believed to be a life strategy adapted by insects utilizing sporadically abundant habitats (Powell 1974). B_. colfaxiana appears to have refined this adaptation one step further. Hedlin et al. (1982) reported a significant negative rank correlation between prolonged diapause and the size of the cone crop maturing the year after larval feeding. This result suggests that the cone moth is in synchrony with its host through some unknown mechanism. Such an adaptation would allow the cone moth population to avoid intraspecific competition during poor crop years and also permits it to maintain a large attacking population to utilize infrequent heavy cone crops. Prolonged diapause may also serve to reduce interspecific competition.  Annila (1981) hypothesized that spruce seed destroying  insects use prolonged diapause to avoid years of heavy predation by cone destroying insects by taking advantage of years when the cone destroyer populations are at their lowest.  This usually occurs after one or more  years of poor cone crops since the cone destroyers identified in Annila's 1981 report did not undergo any form of host synchronized prolonged diapause.  B. colfaxiana may be utilizing prolonged diapause  like the spruce seed destroyers since its main insect competitor in the Keremeos study area appears to be another cone destroyer, the f i r coneworm, Dioryctria abietivorella Grote (Lepidoptera: Pyralidae). Coneworms feed indiscriminantly on all structures within a Douglas-fir cone, including B. colfaxiana larvae and pupae, however their damage, which reflects their population size, appears to be less consistent than  - 3that of the cone moth.  Perhaps J3. colfaxiana has adopted a close  synchrony with its host as a result of selection pressures from intraand/or interspecific competition. Regardless of its origin, prolonged diapause is an important factor in the population dynamics of the cone moth.  A thorough understanding  of the population dynamics of a pest species in relation to its damage intensity is a necessary requisite for pest management (Miller 1984). In addition, research on prolonged diapause and its relationship with factors governing cone crop sizes (i.e. through larval food quality or environmental conditions - see section 1.3) may elucidate the physiological processes that control reproductive bud differentiation.  1.1 Bionomics of the Douglas-fir cone moth B. colfaxiana has a univoltine life cycle (Fig. 1).  Mottled adults  of B. colfaxiana emerge from pitch-coated cocoons near the axis of cones in late April, one to three weeks prior to reproductive bud burst (G.E. Miller^, pers. comm.)  Mating occurs in the tree crowns shortly after  emergence and when temperatures reach the threshold temperature for activity.  Male moths are attracted by females with a mating pheromone.  Combinations of L-9-dodecenol and 15-50% Z-9-dodecenyl acetate are strong sex attractants and are probably similar to the pheromone (Hedlin et a l . 1983).  1  Program Director, Pacific Forestry Centre, Canadian Forestry Service, Victoria, B.C.  - 4-  Figure 1.  Life cycle of the Douglas-fir cone moth (from Hedlin et al. 1980).  - 5Eggs are cemented to bracts while the megastrobili are upright and open for pollination (Hedlin 1960).  Usually one to three eggs are  deposited per conelet but up to 44 eggs on one conelet have been recorded (Hedlin 1960).  Hatching occurs within two weeks after  oviposition and the larvae immediately burrow into the bract and scales of the now closing conelet.  Larvae that are unable to find a suitable  entrance point soon die of starvation (Hedlin 1960).  Pitching out is  another major form of mortality at this stage (Hedlin 1960).  The larvae  develop through four instars while they indiscriminantly mine through bracts, scales and developing ovules. around the cone axis.  Tunnels are usually concentrated  A single larva may destroy 65% of the seed while  two or more may destroy 100% (Hedlin 1960).  The larvae are  cannibalistic which likely limits the absolute number of larvae that may successfully survive within a cone of a given size.  As mentioned  earlier, extremely high egg densities per conelet may lead to abortion of the conelet before larval development is completed. Pupation occurs near the cone axis within a thickly woven cocoon and the population has usually completed pupation by mid-July. Adult apolysis (i.e. the separation of pupal and adult cuticles) occurs within 24 hours of pupation, thus the cone moth overwinters as a pharate adult (i.e. an adult concealed within the pupal skin) which is considered to be an unusual feature of this insect's life history (Sahota et al. 1982).  Pharate adults undergo an obligatory diapause that appears to be  initiated by early-October and can only be terminated after exposure to  - 6 -  a chilling period no shorter than three or four months (G.E. Miller^, pers. comm.).  Once diapause is terminated, adult development continues  even at 0°C which results in rapid adult emergence shortly after environmental conditions become favourable (Sahota et a l . 1982). Not all of the pharate adults will emerge after one winter; these individuals are known to be in prolonged diapause and will emerge only after exposure to another summer and winter.  Keen (1958) noted that  some specimens remained in prolonged diapause for three seasons. The proportion of cone moths in prolonged diapause varies considerably each year and appeared to be negatively correlated with the size of the cone crop in the year following larval feeding (Fig. 2).  Other researchers  have shown similar results (Saksons 1973; Bakke 1971; and Annila, cited in Miller and Hedlin 1984). Sahota et a l . (1985) discovered that one year diapause (1 YD) and prolonged diapause (2YD) pharate adults were morphologically distinct by mid-October, six months before Douglas-fir megastrobili are available for oviposition.  1YD pharate adults are distinguished from 2YD insects  by their more advanced state of pigmentation and scale development which is readily visible through the transparent pupal cuticle (Sahota et a l . 1985). This pre-winter diapause differentiation and its synchronous relationship with the cone crop in the following spring suggests that the pre-adult developmental period may be the critical time in which environmental cues (including host plant information) may influence the induction of prolonged diapause.  - 7 -  .Figure 2. The relationship between the incidence of prolonged diapause in j3. colfaxiana and the size of the cone crop maturing in the year after larval feeding (Spearman's rank correlation coefficient r )(from Hedlin et al. 1982). s  - 81.2 Possible Mechanisms for the Insect/Host Synchrony B. colfaxiana's apparent synchrony with its host infers that a mechanism exists by which the insect is able to "determine" the size of the cone crop six months before the crop is present.  Two hypotheses  were presented by Hedlin et a l . (1982) to explain this phenomenon. The first hypothesis involves the insect's ability to relate to the climatic factors that have been suggested to favour production of large cone crops - warm, dry, sunny summers 14 months before cone crop maturation (this period coincides with the period of lateral bud differentiation (Allen and Owens 1972) and the larval feeding period of B. coifaxiana) (Lowry 1966; van Vredenburch and La Bastide 1969; and Eis 1973). The apparent above average temperature requirement during the summer prior to cone maturation may also influence the induction of prolonged diapause in B. coifaxi ana. A correlation analysis performed by Hedlin et a l . (1982) between several weather variables and the incidence of prolonged diapause failed to show any significant relationships at the 95% level of confidence. In the same report, cone moths reared at a range of constant temperatures (18 to 28°C) on an artificial diet had a significant negative correlation between prolonged diapause incidence and rearing temperature which confirmed that above average summer temperatures favour 1YD. Failure of the analysis of weather data to show a similar relationship may be attributed to the means of recording and reporting the data. Temperature data may have been more meaningful had they  - 9 -  been o b t a i n e d from w i t h i n cones and had been c o l l e c t e d c o n t i n u o u s l y and e x p r e s s e d as degree-day a c c u m u l a t i o n s r a t h e r than u s i n g the summarized d a i l y and monthly t e m p e r a t u r e s s u p p l i e d by Environment Canada.  The  stands s t u d i e d by H e d l i n e t a l . (1982) were remote so t h e weather s t a t i o n d a t a p r o b a b l y d i d not equate c o m p l e t e l y t o c o n d i t i o n s at t h e study s i t e  ( M i l l e r ^ , p e r s . comm.).  The second h y p o t h e s i s p r e s e n t e d by H e d l i n e t a l . (1982) proposed t h a t cone moth/cone c r o p synchrony may be r e g u l a t e d by t h e c o m p o s i t i o n of the i n s e c t ' s l a r v a l d i e t . physiological  A l t h o u g h not y e t f u l l y u n d e r s t o o d , t h e  p r o c e s s e s t h a t govern t h e d i f f e r e n t i a t i o n o f  reproductive  buds c o u l d a l s o d i r e c t l y o r i n d i r e c t l y i n f l u e n c e t h e i n d u c t i o n o f p r o l o n g e d d i a p a u s e i n J3. c o l f a x i a n a . Some e v i d e n c e s u g g e s t s t h a t growth hormones may be i n v o l v e d r e p r o d u c t i v e bud d i f f e r e n t i a t i o n .  F o l i a r a p p l i c a t i o n s of several  in plant  growth r e g u l a t o r s have been a s s o c i a t e d w i t h improvements i n cone production in several conifer species  ( P u r i t c h 1972).  o f t h e s e s u b s t a n c e s were t h e n o n - p o l a r g i b b e r e l l i c which promoted m e g a s t r o b i l i Pharis 1976).  The most n o t a b l e  a c i d s GA^ and GA^  p r o d u c t i o n i n immature D o u g l a s - f i r  An a n a l y s i s o f c o n e - b e a r i n g and n o n - p r o d u c i n g  c l o n e s found no s i g n i f i c a n t d i f f e r e n c e s  (Ross and  Douglas-fir  i n the l e v e l s o f any endogenous  growth r e g u l a t o r s , however, McMullen (1980) suggested t h a t her  results  were i n c o n c l u s i v e s i n c e t h e d i f f e r e n c e s may e x i s t a t l e v e l s t h a t were u n d e t e c t a b l e w i t h her methods. gibberellic  Differences  i n endogenous l e v e l s  a c i d i n Pinus c a r i b b a e a (Rawal and Agrawal 1982) and  of  - 10 gibberel1in-1ike substances in Norway spruce (Ivonis et a l . 1982) were reported between flowering and non-flowering trees.  This latter  evidence suggests that minute differences in endogenous growth regulator titres may exist between flowering and non-flowering Douglas-fir. These differences may be distinct enough for cone moth larvae to determine their host's reproductive condition and then induce single year or prolonged diapause to f i t the pattern of cone availability. Plant growth hormones have been shown to affect other physiological processes in another insect.  A feeding study by Visscher Newman (1982) revealed  that the addition of three plant growth hormones, gibberellic acid (GA-j), auxin (indole-3-acetic acid, IAA), and kinetin to the diet of paired adult grasshoppers raised their fecundity and viable egg production compared to controls.  This study supports the hypothesis  that the observed seasonal correlation of growth and reproduction of the insect is influenced significantly by the changing concentrations of plant growth hormones. Genetic variability likely plays a major role in the interpretation of these hypothetical cues since a single cone may contain both 1 YD and 2YD pharate adults (D.S. Ruth , pers. comm.).  This observation was  also reported for C_. strobillela by Bakke (1963).  Thus, for a given set  of stimuli, some individuals are more sensitive than others.  A simple  heritability study by Hedlin et a l . (1982) showed that the prolonged diapause trait could not not be directly selected for.  2  Technician, Pacific Forestry Centre, Canadian Forestry Service, Victoria, B.C.  - 11 1.3 Artificial Rearing Development of an artificial rearing method is a multi-faceted problem which must accommodate both the physical (i.e. rearing temperature and relative humidity, media texture, shape, moisture content, etc.) and chemical (i.e. nutrient content, nutrient balance, token stimuli and phagostimulants, etc.) requirements of the organism (Singh 1977).  Artificial rearing of cone and seed insects must also  consider their tunnelling habit thereby stressing the importance of media texture and moisture content.  Very little is known about the  nutritional requirements of cone and seed insects since very few species have been successfully reared on artificial diets and complete analyses have not been performed on the nutritional composition of cone scales and bracts. Current research by Y. Prevost  (pers. comm.) on black spruce  cones in eastern Canada and a study by Katayose (1979) on Abies sachalinensis are two studies that specifically examined the change in crude chemical components,of the whole cone during the periods of fertilization to seed maturity. only been performed on seeds.  For Douglas-fir, similar studies have Unfortunately, these studies examined  only the changes in chemical components during the ripening and germinating periods and do not describe the changes in nutrition during the cone moth larval feeding period (Ching 1963).  Without this  information it is difficult to produce a diet that simulates the  3  PhD. Candidate, University of Guelph, Ontario.  - 12 changing compostion of the host.  Instead of attempting to mimic the  host exactly, artificial rearing studies of cone and seed insects (and many other insects) have relied on the adaptation of successful artificial diets developed for other insects (not necessarily for conifer feeding species).  These diets were then modified to suit the  specific requirements of the species being reared. Southern pine cone and seed insects were some of the first cone and seed insects to be successfully reared. the rearing of the following species:  The most notable examples are Eucosoma sp. (Barras and Norris  Jr. 1965; Ebel and Yates III 1973), Dioryctria abietella (Ebel 1959; Fatzinger 1980) Dioryctria amatella (Williams and Fatzinger 1980). Ebel (1959) and Ebel and Yates III (1973) used waxed first-year pine cones as the food source while the others used purely artificial diets without host tissues.  As an example of the success of one of these  rearing methods, by 1980, 39 succesive generations of Dioryctria amatella had been reared on an artificial diet (Williams and Fatzinger 1980). In B.C., the spruce seedworm, Cydia (=Laspeyresia) youngana, and the Douglas-fir cone moth, B_. colfaxiana, had been reared for the first time on McMorran's (1965) eastern spruce budworm diet (Hedlin 1965). Ruth and Hedlin (1969) later described their method to complete the entire B. colfaxiana life-cycle in the laboratory.  They identified  rapid media moisture loss as the main mortality factor due to entrapment of young larvae in condensation formed on the media and the vial walls, and mortality resulting from handling during frequent transfers of  - 13 larvae from dried out media to fresh media.  Survival using this rearing  method averaged 30% although one replicate achieved 61% survival. 1.4 Objectives  I initially proposed to test the diet/diapause hypothesis by first improving an existing rearing method by increasing early larval survival and then, using this improved method, test the effects of various metabolites associated with cone production on the induction of prolonged diapause.  However, due to problems encountered in rearing the  cone moth, the main objective of the thesis focussed on producing an efficient rearing method. Rearing methods were evaluated so as to a.  improve early instar survival by reducing handling, moisture loss, and condensation, and  b.  improve late L4 and pupal survival by altering diets and their constituents.  In addition, it was realized that no attempt had ever been made to determine if the insects were indeed reacting to the reproductive condition of the host trees.  A field sampling study was undertaken in  mid-August, 1985 to determine the relationship between the incidence of prolonged diapause in pharate adult Douglas-fir cone moths and the degree of cone crop initiation in interior Douglas-fir in the Keremeos area.  - 14 2.0 GENERAL METHODS FOR REARING STUDIES 2.1 Source, Storage and Handling of Eggs  In all three years of artificial rearing experiments (1983-85), eggs were obtained from three study sites approximately 20 km west of Keremeos, B.C. (Fig. 3).  Megastrobi1i, with eggs, were collected from  pole-pruned Douglas-fir branches during the last week of April and placed in plastic bags.  The megastrobili remained refrigerated at  approximately 4°C for up to 4 days prior to transport to the Pacific Forestry Centre (PFC) in Victoria, B.C. where they were stored at 0°C in a walk-in cold storage room.  Length of 0°C storage varied from  year to year depending on the rate of media establishment.  Dramatic  decreases in hatching rate were observed in eggs stored over three weeks under these conditions.  2.2 Egg Establishment on Media  Bracts with attached fertile (visible embryo and/or head capsule) and undamaged eggs were plucked from megastrobili, stored on moistened filter paper placed in petri dishes, kept refrigerated at 4°C until required, and then sterilized for 10 minutes in 10% formalin (with 2-3 drops of Tween 20®), rinsed in distilled water for 10 minutes, and air dried on filter paper disks.  The air drying process in 1984 and 1985  occurred under a flow of sterilized air in a laminar flow hood. The sterilization procedure was adapted from Robertson's (1979) method  - 15 -  Figure 3. a) Location of Keremeos study sites in British Columbia; b) the approximate location of "Dahlgard's", "Outward Bound (O.B.), and "Ashnola" study sites about 20 km west of Keremeos, B.C. c) Photograph of "Outward Bound" showing interior Douglas-fir stand structure and site topography.  - 16 developed for western spruce budworm eggs.  Sterilized bracts were  firmly wedged distal-end down into a triangular crevice made in the media's upper surface.  Care was taken to avoid direct contact with the  egg and the media as such contact appeared to hamper successful hatching.  In the 1983 study, bracts were simply secured to the media  with minimal disturbance to the egg but with no particular orientation. In 1984 and 1985, bracts were oriented so the eggs were readily visible through the clear 7 dram vial walls.  This made for more rapid checking  and avoided exposure to contaminants as occurred when the vial or creamer lids were removed.  2.3 Media Preparation  General procedures common to all years are as follows. The agar/water mixture was autoclaved for 20 minutes at 104°C and was allowed to cool to 65°C before adding the dry mix in order to prevent degeneration of heat labile components. nor cooled specifically to 65°C in 1983.  Liquid agar was not autoclaved Dry mixtures were added to  measured volumes of liquid agar and were mixed in a domestic size Osterizer® blender (2 L) in small batches to facilitate rapid pouring into individual containers (i.e. vials or creamers) before the media became too viscous.  After the media hardened sufficiently, triangular  crevices were excavated from the centre of the upper surface of the media with a flame sterilized scalpel blade.  Completed containers were  placed in new plastic bags secured with a twist tie or elastic band and stored at 0°C.  - 17 2.4 Rearing Environment Containers established with eggs or first instar larvae were then placed into an environment chamber with timer-controlled light and temperature regimes.  Developmental progress and media condition of each  container were assessed every week (bi-weekly in 1985) during the larval feeding period.  Pupae were removed from their media, weighed, assessed  for diapause condition and returned to their individual rearing containers minus the media.  Pupae (pharate adults) were overwintered in  either these vials or gelatin capsules placed outdoors in Vancouver. Adult emergence success was assessed by bringing the pharate adults indoors during March.  2.5 Analyses  Comparisons of rearing methods and diets were based on the number of pupae produced, pupal weights, and the proportion of surviving pharate adults in prolonged diapause as assessed in October in the year of rearing.  Adult emergence success was assessed in 1983 and 1985.  Analyses of pupal weights were performed on the statistical packages MIDAS (Fox and Guire 1976) and UBC ANOVAR (Greig and Osterlin 1978). Comparisons of paired means with t-tests were calculated on MIDAS. Multiple range tests used to compare several means were Scheffe's test for means with unequal sample sizes (MIDAS), and Student-Newman-Keuls test for means with equal sample sizes (UBC ANOVAR).  Simple linear  - 18 regression was analyzed with MIDAS. obtained with TELLAGRAF.  Computer generated figures were  - 19 3.0 ARTIFICIAL REARING 3.1 Introduction, 1983  Based on the results of Ruth and Hedlin (1969), it was believed that marked improvements in survival could be obtained by reducing the rate of media moisture loss through media waxing or the use of containers with less water permeable lids than the non-absorbant cotton plugs used by Ruth and Hedlin,(1969).  Thus, in May 1983, two container types, two  rearing environments, two media coatings (waxed and non-waxed), and two methods of diet establishment were tested to determine if a "better" rearing method could be found.  The following is a description of the  artificial rearing research in 1983 and the two years of follow-up research that were performed to obtain that "better" method. The rationale behind the design of the 1984 and 1985 experiments developed from the results of the 1983 rearing attempts.  3.2 Materials and Methods, 1983  3.2.1 Diet Formulation and Processing  A modified version of the McMorran (1965) eastern spruce budworm diet was produced following the procedure outlined by Roberston (1979) (Appendix la). Several ingredients for the Roberston formulation were not available and were substituted or altered as per recommendations of  - 20 M. Tolman (pers. comm.^).  These substitutions were:  (1) wheat germ instead of wheat embryo (2) sorbic acid for potassium sorbate (3) citric acid in place of L-ascorbic acid (4) pure linolenic acid instead of a 40% solution (5) Bioserv's prepared vitamin mixture (#742) in place of Nutritional Biochemical Corp.'s vitamin diet fortification mixture. An important alteration in Roberston's procedure was made during the blending of the dry and liquid diet components.  Hot water (boiled in a  electric kettle) was added to the diet to maintain a particular viscosity to facilitate dispensing.  The volume of water added varied with each  batch but all of the resulting diet had water contents that exceeded the specifications given by Roberston (1979).  The excess moisture produced a  lot of condensation inside the rearing containers and this in turn was a major cause of LI entrapment mortality. The procedure for mixing the diet was as described in the general methods in Chapter 2 except for the following steps.  Diet was not poured  under the laminar flow hood nor was it protected from contamination during cooling.  Diet was poured directly into plastic creamer cups and  into 9 X 9 X 1 cm plastic petri dishes.  After solidifying, the diet and  containers were placed into plastic bags and stored at 4°C until required.  Plugs were made by punching out disks of diet contained in the  petri dishes using a 1.5 cm diameter cork borer sterilized by soaking in  Technician, Pacific Forestry Centre, Canadian Forestry Service, Victoria, B.C.  - 21 a bath of 70% ethanol.  Once established with eggs or L i ' s , plugs were  placed in 4 dram snap-cap plastic vials (Premo Plastics Ltd., Victoria, B.C.) and sealed with the unmodified lids.  The cylindrical plugs  contained approximately 2.5 mL of media while the creamers held approximately 3.5 mL of media.  This quantity of diet was sufficient to  complete larval development; no larva completely consumed the contents of either a plug or a creamer.  A waxed coating was prepared by repeatedly (R)  dipping plugs into liquid Parowax  "fondu-style" until a sturdy coating  was achieved, and for creamers, liquid wax was poured onto the surface of the media until it was completely sealed. 3.2.2 Experimental Design  A 2^ factorial experiment was used to compare the effects of four treatments designed to avoid media shrinkage and larval handling (i.e. temperature, waxing, media form, and establishment method).  Half of the  1600 insects established were placed in an environment chamber and the other half in a shade house at ambient (Victoria, B.C.) atmospheric conditions.  The chamber was kept at a constant 20°C and an average  relative humidity of 65% (humidity changed with the weather conditions) while the shade house temperature and humidity fluctuated widely as a function of daily cycles and changing weather patterns.  In each  temperature regime, two types of media forms (plugs and creamers) were either waxed or unwaxed and either established with eggs or larvae. Equal sample sizes of 100 insects were set up in each treatment.  - 22 3.2.3 Media Establishment Procedure Eggs  The method was described previously in section 2.1.2. Larvae Larval establishment consisted of transferring Li's from conelets or hatching trays (i.e. eggs on bracts layed on moistened filter paper and left at room temperature in a covered petri dish) into a 1 mm dia. x 1 cm deep hole cut into the diets upper surface.  Larvae were gently lowered  into the hole while being suspended from a fine wire probe by a thread of insect-produced silk. the larvae.  This was the only form of handling experienced by  Ruth and Hedlin (1969) used this method to establish all of  their larvae.  3.2.4 Inspections and Pupal Handling  Inspections were made on an irregular basis; time between observations ranged from 2 to 4 weeks.  The surface of the media was  inspected for signs of larval feeding or for signs of mortality or escape.  At no time were larvae excavated from their media unless it had  dried severely thus requiring the transfer of the insects to fresh media.  The cone moth larvae rarely fed on the outside of the media so  observations of their development and behaviour were based on infrequent  - 23 glimpses of the larvae whenever they could be seen from the outside. Media was not dissected as larval disturbance was believed to be harmful, especially during moulting. Pupae were extracted from a shell of hardened media.  Great care was  necessary to avoid damaging the pupae while the media was chipped away. Successfully extracted pupae were weighed on a Sartorius 1207 MP2 electronic balance on 18 August 1983 and weights were recorded to the nearest 0.1 mg. Pupae were stored individually in labelled gelatin capsules, each containing a small wad of non-absorbant cotton to prevent the pupae from sticking to the capsule if accidentally moistened. Capsules were stored outdoors but under cover in Vancouver. Naturally reared pupae extracted from cones collected in late August 1983 and used for comparison with the artificially reared insects, were weighed on 11 September 1983 and stored similarily.  Identification of pharate adults  in prolonged diapause was verified at PFC, Victoria on 20 October 1983. Pharate adults in prolonged diapause have lighter coloured wingpads with less scale and pigment development than those in one-year diapause (Sahota et al. 1985).  An assessment of survival following extraction  from either media or cones was made on 21 October 1983.  3.2.5 Adult Emergence and Overwinter Survival  Pharate adults were brought indoors on 1 March 1984 to determine the overwintering survival and the success of adult emergence.  In addition,  - 24 the method used to identify 1YD and 2YD pharate adults was also assessed.  Adult eclosion occurred within the gelatin capsules.  Emergence was checked daily from 4 March to 2 April 1984, although some adults eclosed between 1-2 March.  Adults were classified as: 1) normal  2) normal but dead (possibly due to the cramped conditions within the capsule) and 3) incompletely eclosed due to failure of the pupal and adult cuticles to separate.  Pharate adults that were alive but did not  eclose were considered to be in 2YD. They were kept indoors for the simulation of another spring/summer cycle but they all died from dessication during the summer months (survival was assessed in September, 1984). 3.3 Results, 1983  3.3.1 Media Condition  During the eight weeks of larval feeding, media condition appeared to remain quite suitable for larval feeding based on the pliability of the diet and the intensity of the frass production both of which were very subjective measures.  Media shrinkage due to moisture loss occurred  gradually and was reduced by the waxed coating unless the wax had cracked or chipped away which resulted in a drying rate very similar to unwaxed media.  By early August, the media hardened and pupae had to be chipped  out of the diet.  Creamers were kept upside down so the media often  separated from the container and slumped down onto the l i d . Fortunately,  - 25 the larvae were usually tunnelling inside the diet and were unharmed. During the first three weeks in the chamber, condensation formed on the walls of the vials containing the unwaxed plugs and caused LI mortality. Only 8 of the 800 chamber reared containers were severely contaminated with mold despite the lack of any extra effort to keep the media relatively sterile.  The diet contained anti-microbial agents that  appeared to provide good protection from contamination.  3.3.2 Survival  Overall larval survival was extremely poor.  The majority of the  insects kept in the shade house at ambient temperatures drowned in the heavy condensation that collected at the bottom of both types of containers.  Unexpected high temperatures (28°C) in May and the sealed  nature of the containers were blamed for the severe mortality.  Because  of the poor survival, only chamber-reared insect data will be presented. Of the 800 chamber-reared insects, only 96 (12.0%) developed into pupae.  Egg to pupa development was rapid.  The first pupa was observed  on 24 June 1983, 43 days after being established as an LI.  However, two  L4's failed to pupate and were s t i l l feeding by 18 August 1983 well after the majority of the artificially reared insects had pupated.  These two  larvae eventually died without moulting into pupae. Ninety-six pupae were extracted by 25 July 1983 from the artificial media, and by 24 October 1983, 35 had survived. Of the four creamer  - 26 types, the unwaxed creamers with larval establishment (UL) appeared to have the lowest pupal mortality through this period (i.e. 29.4%) followed in ascending order of mortality by UE, WL, and WE (data not shown). Pupal mortality was apparently due mainly to damage during extraction and possibly to the compounded effects of an unknown disease and/or poor larval nutrition.  Seventy-seven percent of the wild pupae survived  extraction from cones and one month of storage. Survival by life stages for each rearing method are summarized in Table I. data.  Two periods of high mortality were evident from the inspection  The first period occurred shortly after establishment.  Mortality  of eggs and LI larvae ranged from 27 to 77% of the total number established.  The major causes of death were hatching failure and larval  entrapment on the media surface due to moisture.  An unexpected second  period of high mortality occurred during the final stages of larval development just prior to pupation.  Four to 26% of the insects  established died as L4's inside their cocoons.  The L4 corpses were soft  and fluid filled which suggested either an advanced stage of decomposition or the presence of a microbial pathogen. A non-statistical comparison of establishment methods shows an interesting difference between mortality of Li's (Table I).  For plugs  and creamers, larval established media had higher overall LI mortality (54.0%) than the egg established diets (27.1%). Furthermore, the combined egg and LI mortality of the egg established media was s t i l l significantly lower than the total LI mortality on the larval established  - 27 Table I.  Mortality of artificially reared B_. colfaxiana by life stage and rearing condition (environment chamber reared insects only), May 1983 to 2 April 1984. Number of insects dying in each life stage  Container: Life  Plugs  Surface:  Waxed  Creamers Unwaxed  Waxed  Stage Establishment:  Eggs  LI  Eggs  LI  Eggs  EGG  50  N/A@  23  N/A  14  LI % ( mortality*)  24 (48)  73 (73)  23 (29.9)  77 (77)  13 (15.1)  Egg + LI  74  46  Unwaxed LI N/A  33 (33)  27  Eggs 16 13 (15.1)  LI N/A 33 (33)  29  L2  1  5  6  4  1  4  1  2  L3  2  1  5  3  0  4  4  7  L4 (normal)  4  2  8  3  2  0  1  1  L4 (in cocoon)  7  4  16  7  24  20  26  16  Unknown or escaped  9  11  12  2  16  23  15  19  Pupa (incomplete)  0  0  0  1  0  0  3  0  Pupae+Pharate Adults (normal)^: 24 October 1983 2 April 1984  2 2  3 3  6 6  3 3  23 25  8 9  10 10  11 13  Total Dead PostWinter  99  99  99  100  95  93  89  91  Pre/Post-winter Survival (%)  2.0/ 2.0  .0/ .0  1.3/ 1.3  8.1/ 5.8  8.0/ 7.0  13.1/ 13.1  4(0)/ 1  5(2)/ 8(5)/ 5(1)/ 2 3 4  #  Adults Emerged (normal)/in 2YD @  * t  0/1  0/1  K D  0/ 0  -  N/A Not applicable Expressed as a percentage of Li's either hatched or established. Pre/post-winter survival assessed 24 October/ 2 April 1984 Total number of dead pupae and pharate adults on inspection date.  11.0/ 9.0  - 28 media (44.0 vs. 54.0%, respectively).  Hatching failure totals were increased  by an inconsistency in the method of egg establishment used for the waxed plugs.  Nineteen eggs were removed from their bracts and placed directly on  the media.  None of these eggs hatched which suggested that either the direct  handling of the eggs or the contact with the media was responsible for the cessation of egg development and hatching.  Eggs appear to be sensitive to  disturbance and can not be removed undamaged from the substrate on which they were deposited on. Overall, there were no notable differences in survival (assessed post-winter) between establishment methods or coatings, however, between container types, survival on plugs (0.9%) was lower than on creamers (8.7%). Unfortunately, the creamers were considered unsuitable containers since the larvae were able to chew through the plastic wall and escape. Twenty-seven larvae were known to have escaped this way.  3.3.3 Pupal Weights  Male and female wild pupae were significantly heavier than the artificially reared pupae (Table II).  Within the creamer types, the UL  (unwaxed creamers established with larvae) produced significantly heavier pupae for both males and females than the remaining three creamer types, and these others were not significantly different in weight.  Female pupae reared  on WL (waxed creamers established with larvae) were the only case where pupae reared on UL (unwaxed creamers established with larvae) were not significantly heavier.  - 29 Table II.  A comparison of mean pupal weights of cone moths reared on artificial medium contained in four creamer types maintained in an environment chamber and naturally reared pupae extracted from cones collected near Keremeos, B.C.  X + S.D. mg (n)  @  Rearing Condition  Male  Female  UE  6.4 + 1.9  (17) c  7.2 + 2.1  UL  9.6 + 1.4  (7)  b  11.0 + 2.8  WE  7.5 + 1.8  (14) c  WL  6.4 + 1.9  (9)  Natural L  Male  14.0 + 2.3  vs. Female* (10) c  N.S.D.  (8)  b  N.S.D.  7.5 + 2.8  (15) c  N.S.D.  c  7.7 + 1.4  (8)  (52) a  15.9 + 2.6  be  (45) a  N.S.D. **  Means within each rearing condition followed by the same letter, not significantly different, Scheffe's test, P< 0.05.  *  Student's t-test comparison between sexes for each rearing condition. ** = P< 0.01. N.S.D. = Not significantly different, P< 0.05.  UE UL WE WL  = = = =  unwaxed creamers established with eggs unwaxed creamers established with Li's waxed creamers established with eggs waxed creamers established with Li's  - 30 Comparison of mean pupal weights of males and females for each rearing regime revealed that, of the five rearing regimes tested, female pupae were significantly heavier only for the naturally reared insects (Table II). A comparison of mean pupal weights of one-year (1YD) and prolonged diapausing (2YD) cone moths revealed no significant differences (Table III).  Thus, the hypothesis that pharate adults in 2YD should be heavier  than those in 1YD, due to the assumption that 2YD insects require greater energy reserves than those in 1YD, must be rejected. Comparisons of mean pupal weights by sex and diapause status revealed that all artificially reared insects were significantly lighter than those naturally reared (Table III).  Pupal weight and diapause data  from 1966 artificial rearings of cone moths at 21°C on McMorran's (1965) diet (Ruth unpubl. data) were compared with 1983 data (Table III).  There were no significant differences in weight of the 1966  artificially reared insects between sexes or diapause condition (Table III).  However, there were significant differences between 1966 and 1983  artificially reared cone moths for each corresponding sex and diapause category.  In all cases, 1966 reared insects were heavier.  Naturally  occurring pupae in 1983 were not significantly different in weight than 1966 artificially reared cone moth pupae (Table III).  - 31 Table III.  A comparison of mean pupal weights of cone moths reared artificially in 1966 and 1983 (all chamber reared) and collected from cones in 1983. Comparisons are stratified by sex and diapause condition.  Mean Pupal Weight + S.D. mg (n) Rearing Condition Sex and Diapause Status  Artificial 1983  12.9 + 3.4(10)a#  9.0 + 2.9(20)b  16.1 + 2.8(16)a  15.2 + 2.2(9) a  7.1 + 2.2(5) a  14.8 + 1.5(23)a  12.3 + 1.7(9) a  7.9 + 2.2(20)b  12.8 + 1.7(ll)a  12.9 + 2.3(9) a  6.9 + 2.6(10)b  13.8 + 1.9(22)a  @  Females 1 YD 2 YD Males 1 YD  Natural 1983  Artificial 1966  48.7  2 YD  27.0  60.0  Overall % in 2YD: @ Source: *  D.S. Ruth , unpublished data. 2  Means within each sex and diapause status row followed by the same letter are not significantly different, Student's t-test, P< 0.05.  - 32 3.3.4  Incidence of Prolonged Diapause  Twenty-seven percent of the 1983 artificially reared cone moths were in prolonged diapause (33% male, 20% female) from a total of 55 surviving insects (Table III).  The proportion of naturally occuring insects in 2YD  was 60% (67% male, 59% female) based on a sample of 62 pharate adults (Table III).  Similar proportions were also attained from samples of the  natural population collected concurrently from the same area (D.S. Ruth and A. Ibaraki , pers. comm.).  Thus, approximately 50% more  pharate adults were in prolonged diapause in the field reared sample than those reared in the controlled environment.  Thirty-seven cone moths  reared in 1966 at 21°C (vs. 20°C in 1983) had 48.7% in 2YD (50% male, 42.1% female) (Table III).  3.3.5 Pharate Adult Survival and Adult Emergence  Thirty-five of the 40 pharate adults overwintered survived until at least 2 April 1984.  Of these survivors, 20 emerged, 10 as normal adults,  4 with deformed wings, and 6 had severe difficulties eclosing. The remaining 12 survivors were in 2YD. When compared by establishment method, there appeared to be no difference in the emergence statistics (Table I).  All of the pharate adults in 2YD were correctly identified in  October, 1983, thereby verifying the accuracy of characterizing 2YD pharate adults by the absence of pigmentation and scale development.  5  Technician, Pacific Forestry Centre, Canadian Forestry Service, Victoria, B.C.  - 33 3.4 Discussion and Conclusions, 1983 1983 s rearing results were unsatisfactory. 1  Overall survival was  extremely low and pupal weights were significantly lower than both 1966 artificially reared and 1983 "wild" or naturally occuring cone moths. Early instar mortality resulting from entrapment by condensation on the media surface was a major mortality factor but was much less of a problem than in Ruth and Hedlin's (1969) rearings.  However, unusually high L4  mortality just prior to pupation, which was not observed in the 1969 study, occurred during the 1983 rearings.  Ruth and Hedlin (1969) found  that survival was quite good and uneventful after the LI stage. A literature review also revealed no reports of similar mortality occurring in rearing studies for other cone and seed insect species. Identification of the cause(s) of this mortality was considered to be a main objective of future rearing efforts.  Positive results of 1983's  rearing study were quite significant in terms of improving the rearing method.  Early instar survival from LI to L3 was excellent, especially on  media established with eggs on bracts.  Establishing media in this way  has several advantages: 1) eggs and bracts may be sterilized whereas Li's cannot; 2) bracts may provide Li's with essential micro-nutrients or initial phagostimulants that are not available from the artificial diet; and 3) bracts act as a platform for initial bract and artificial diet feeding during which time the media surface moisture may lessen and reduce the hazards of entrapment.  The use of bracts as an initial food  - 34 source was not a detriment to this study since the main objective was to produce the most efficient method of rearing the cone moth. Container types were important for larval survival.  Plugs in sealed  vials were not appropriate due to the greater risk of condensation on the vial and media surfaces and to the instability of the plug which rocked whenever the vial was handled during inspections.  This rocking of the  media plug often crushed larvae feeding underneath i t .  Creamers were  more suitable for cone moth rearing due to their larger media volume which probably maintained media moisture content at acceptable levels longer than the plugs.  Creamers also had cardboard lids which "breathed"  and usually prevented condensation formation except in extreme temperatures like those experienced in the shade house.  However, larvae  escaped by boring through the creamer walls and thus creamers were judged unsuitable.  A pilot study begun in early June, 1983 demonstrated that  clear plastic 7 dram vials (Premo Plastics Ltd., Victoria, B.C.) containing approximately 10 mL of diet were suitable for rearing the cone moth.  A modification of the lid to include a cardboard covered hole for  improved gas exchange was suggested.  This container type would then  combine the media mass and permeable lid of the creamers with the security offered by the 5 dram vials. Although waxing appeared to slow media moisture loss (vs. unwaxed media), it appeared unnecessary since both containers tested adequately maintained media moisture throughout the larval feeding period.  - 35 -  Pupal damage during extraction could be avoided completely by maintaining a closer vigil on the insects' development and extracting pupae when the media was still soft and pliable.  Pupal storage in the  gelatin capsules was later not recommended since earlier experiences showed that adult eclosion and wing expansion was hampered by the confines of the capsule (G.E. Miller^, pers. comm.).  Instead, pharate  adults should be stored in larger containers which allow more room for eclosion and wing expansion.  The 7 dram vials used to rear the insects  were suitable for this purpose. The proportion of pharate adults in prolonged diapause corresponded well with the results of Hedlin et al. (1982) for the rearing temperature of 20°C.  Thus, temperature appeared to be a consistent factor  influencing the induction of prolonged diapause.  This statement is  reinforced by the obvious difference in prolonged diapause incidences between the natural and artificially reared cone moth populations. The difference may have been caused by different temperatures at which the larvae were exposed to during the most critical phase when the induction of prolonged diapause was determined.  In addition, stress on the larvae  from the artificial rearing environment and unnatural selective mortality may have affected the incidence of prolonged diapause. The success of adult eclosion is the usual criterion to assess an artificial diet and rearing method.  The poor, adult eclosion experienced  in this study may be the direct result of several factors - the most important being: 1) nutritional deficiency or imbalance and 2) disease.  - 36 It was not clear whether the differences in diet formulations outlined in section 3.2.1 significantly altered the nutritional quality of the diet, nor is it known if disease was responsible for the unusual L4 mortality and poor adult eclosion success.  The spruce budworm diet was chosen for  use in this rearing experiment simply because it had provided satisfactory results in past rearing attempts and not because it was the "best" diet.  Perhaps L4 survival and adult eclosion success would  improve if a better or specifically tailored diet were used. To avoid possible microbial contamination (such as might have been responsible for the high L4 mortality) more stringent sterile conditions could be used.  If high L4 mortality persisted, then it would be likely  that factors other than microbial pathogens were responsible for L4 mortality unless the pathogens were transmitted trans-ovarially. The major differences between the methods used in 1983's cone moth rearing and Ruth and Hedlin's (1969) were, respectively: 1) the larvae were reared on one portion of media vs. several portions of media over the larval feeding period; 2) diet formulation alterations and substitutions were made as compared to strict adherence to McMorran's (1965) recipe; and 3) half of the media were established with eggs on bracts vs. all of the media with L i ' s .  One factor or a combination of  these major differences were considered the possible causes of the high L4 mortality.  - 37 3.5 Introduction, 1984 1983's results revealed that improving the rearing method for the Douglas-fir cone moth would not be an easy task.  Several questions were  raised concerning the containers used, the diet's formulation, and the possibility that a microbial pathogen may have contaminated the insects. Thus, the main objectives of 1984 s research were: 1  1) to evaluate a refined rearing method, developed from 1983's rearing experiences, by using 7 dram vials with modified lids and to establish the insects as eggs on bracts; 2) to determine if other artificial diets were more suitable for rearing the cone moth; and 3) to reduce the chance of pathogenic microbial contamination of the insects by using sterile techniques.  3.6 Materials and Methods, 1984  3.6.1 Rearing Method  As described previously in sections 2.1.2 and 3.4, 1984's rearing methods involved the use of only surface sterilized eggs on bracts to establish 7 dram vials that contained about 10 mL of artificial diet (except for the test of eggs layed on waxpaper).  Snap-cap lids were  modified by cutting a 2.5 cm diameter hole into the lid's centre.  Lids  were washed in soap and water and dried in an oven and then sterilized in  - 38 70% ethanol.  Cardboard inserts were cut out by hand with ethanol  sterilized scissors from cup lids which were plastic coated on one side. The lids with inserts were prepared on the laminar flow bench. The chamber used for rearing the cone moth larvae was also used to culture the seed pathogen Sirococcus sp.  A daily temperature and light cycle of  16 hrs at 20°C (light) and 8 hrs at 14.4°C (dark) (average temperature was 18.5°C) was employed. Light condensation formed briefly on the vial walls during the transition periods between cooling and heating.  Temperature and humidity were recorded with a  hygrothermograph.  Temperature regimes were very stable and accurate  throughout the rearing period but relative humidity varied from 45 to 60% depending on the ambient atmospheric conditions. maintained throughout the rearing process.  Sterile conditions were  Diet pouring, cooling, hole  excavation, egg and bract sterilization, media establishment, lid sterilization and insert preparation, and inspections took place under a laminar flow of sterilized air. clean with 70% ethanol.  Instruments and work surfaces were wiped  Plastic gloves that were wiped with 70% ethanol  were used whenever the media was handled.  3.6.2 Experimental Design  Three commercially available artificial Tortricid diets, developed for rearing the Nantucket pine tip moth, Rhyacionia frustrana (Comstock), the codling moth, Cyckia (=Laspeyresia) pomonella (L.) and the spruce budworm, Choristoneura fumiferana (Clemens), were tested.  These diets  - 39 are referred to as "Tip Moth" (TM), "Codling Moth" (CM) and "Budworm" (BW) diets and were specifically Bioserv® (Bioserv Inc., Frenchtown, New Jersey, USA) diet numbers 9675, 9370 and 976L9, respectively (a list of diet ingredients and instructions for mixing are presented in Appendix lb).  The Bioserv® spruce budworm diet was selected instead of the  Robertson (1979) diet used in 1983 since its formulation was nearly identical to McMorran's (1965) diet (used by Ruth and Hedlin 1969) and because of its convenience.  All three diets consisted only of an agar  (wet) mix that was liquified in the autoclave and then blended into a pre-mixed dry portion (Appendix lb). A minimum of 100 eggs were established on each of these diets.  Double concentration spruce budworm  diet (2XBW) and tip moth diet (2XTM) were accidentally mixed when only half of the required agar was added.  These diets were added to the  previously planned diet tests. Effects of the host material on larval development and pupal production was tested in two ways.  First, 16 g of freeze-dried coastal  Douglas-fir megastrobili were added to the dry mix of one batch of normal concentration BW diet (BWS = BW with strobili powder); 114 vials containing this mixture were established with eggs.  The megastrobili  powder was added to see if it acted as a phagostimulant thereby increasing the feeding rate and ultimately increasing the size of the pupae.  Unfortunately, the diet was not mixed thoroughly enough to evenly  distribute the dry mix components resulting in the stratification by weight of the various ingredients. This anomaly was likely the main cause of the resulting poor larval survival (when compared to survival on  - 40 normal BW). Second, fertile eggs laid on waxpaper were established on 56 normal BW (BWW) and 58 normal TM (TMW) vials. These eggs originated from a waxpaper drum cage containing newly emerged adult cone moths from 1983 Keremeos collected cones.  Eggs on bract-sized pieces of waxpaper were  surface sterilized as per the eggs on bracts.  This experiment was  designed to determine the effect of exclusion of all traces of the host from the larval diet. In total, 730 vials were established in a comparison of seven different diets and two establishment methods.  The entire media  preparation and establishment procedure was completed on 11 May 1984 for egg-on-bract established diets.  Media established with eggs-on-waxpaper  were completed on 11 June 1984.  An example of an established vial with  visible larval activity is shown in Figure 4. 3.6.3 Diet Inspections  Each vial was inspected weekly from the time of establishment until the last signs of larval activity were recorded.  Unhatched eggs were  replaced after one week with eggs from the same source that had been stored at 0°C. Developmental progress was monitored carefully but the vials remained unopened unless the larva either showed no signs of new feeding for at least 3 weeks after hatching or if it had burrowed into the media, normally as an L4, and had not been actively feeding for at least 2 weeks as evidenced by frass production.  In both cases, the plug  of diet was removed from the vial and then carefully split open under the  Figure 4.  Two seven dram vials containing Budworm diet (BW) showing larval feeding activity (right) and pre-pupal cocoon (left) of B. colfaxiana.  - 42 dissecting microscope.  If the larva was s t i l l active, the diet was  gently reassembled and placed carefully back into the vial or the insect was transferred to a fresh diet (i.e. a vial poured at the same time as the original vial and stored at 0°C). 3.6.4 Pupal Extraction and Overwinter Storage  When living pupae were discovered during the inspections they were transferred into wedges of corregated cardboard to facilitate normal adult emergence (by simulating the tunnels within a cone) and then were placed back into their labelled rearing vials. 31 October 1984 and the survivors weighed.  Pupae were inspected on  The last inspection occured  on 5 November 1984 and the survivors were placed outdoors but under shelter in Vancouver to overwinter.  The cone moths were brought indoors  on 25 April 1985 to assess adult emergence.  3.6.5 Disease Analysis  Samples of apparently healthy, moribund, and dead L3's, L4's and pupae were sent to the Canadian Forestry Service's Forest Pest Management Institute in Sault Ste. Marie, Ontario on 16 July 1984 to be inspected by Dr. John Cunningham. Insect samples were packaged in their original media and vials and were shipped by courier.  The samples were assessed  immediately by dissection under a light microscope.  - 43 3.7 Results, 1984 3.7.1 Media Condition  Feeding appeared unhindered throughout the entire larval developmental period by the apparent decrease in diet moisture content. Media contamination by micro-organisms was infrequent and could be traced to incomplete surface sterilization of the bracts due to the adherence of air bubbles on the bract surfaces.  Once mold fungi became established,  the diet became completely engulfed.  3.7.2 Survival  Overall survival in 1984's rearing experiments was again very low. Of the eggs which hatched and developed into pharate adults, pre-winter survival ranged from 0 to 20.2%.  Best survival was obtained on the  normal BW diet (20.2%) followed in decreasing order by 2xBW (20%), BWC (17%), TM (10%) and BWS (7.6%). and TMW diets.  No survivors were found on CM, 2xTM, BWW  The number dying in each life stage was summarized in  Table IV. Hatching success on all egg-on-bract established diets was high, ranging from 83.8 to 100% (average=94.4%).  Diets established with  eggs-on-waxpaper had poorer hatching success (i.e. BWW 75.0% and TMW 46.6%).  LI to L3 survival was relatively uniform on all BW and normal TM  diets established with eggs-on-bracts. 82.8%.  Survival ranged from 63.8 to  Both eggs-on-waxpaper established diets and 2xTM LI  - 44 Table IV.  Summary of survival through each life-stage of B. colfaxiana reared on three Bioserv^ diets and several variations of diet concentration and establishment method. May 1984 to 21 April 1985.  Diet and Treatment  % Surviving through each life-stage  Overall Survival Pupa+ (Survivors/ Pharate # Hatched) Adult (Pre/Post)*?  Egg  LI  L2  L3  L4  Budworm, Normal (BW)  98.0  89.9  93.2  98.8  61.0  40.0  20.2/1.0%  Budworm, with megastsrobi1i (BWS) Budworm, Double concentration (2xBW)  93.0  83.0  99.9  93.7  65.3  12.2  5.7/0%  87.2  89.6  93.0  82.5  33.3  20.0/0%  95.6  76.9  84.0  97.6  73.2  36.7  17.0/0%  Budworm, eggs on 75.0 waxpaper (BWW)  71.4  73.3  81.3  33.3  0  0/0%  Codling Moth (CM) 83.8  22.7  60.0  41.7  20.0  0  0/0%  Nantucket pine tip moth, normal (TM)  97.3  83.7  91.3  92.9  76.9  20.0  10.9/0%  Nantucket pine 93.3 tip moth, double concentration (2xTM)  80.4  75.6  70.6  54.2  0  0/0%  Nantucket pine 46.6 tip moth, eggs on waxpaper (TMW)  74.1  75.0  80.0  33.3  0  0/0%  Budworm, Coastal eggs (BWC)  100  @ Pre/Post = Pre-winter assessment (5 November 1984) vs. Post-winter assessment (21 April 1985)  - 45 to L3 survival ranged from 42.8 to 44.4%.  The worst survival during  these stages was experienced on CM diet (5.7%).  Most causes of LI to L3  mortality could not be explained except for entrapment of very small larvae on media and vial surface moisture.  A small proportion of Li's  escaped from the vials (12/648 or 1.8%) through small gaps between the vial and the cardboard insert.  Gaps resulted from imperfectly cut  cardboard disks that did not seal the entire l i d . L4 mortality was again unexpectedly high but varied widely between diets (Table V). Lowest mortality during this stage was on 2xBW diet (17.5%) followed in ascending order by BWW (18.2%), TM (23.1%), BWC (26.8%), BWS (34.7%), BW (39%), 2xTM (45.8%), BWW and NTW (66.6%), and CM (80%).  The most common symptom of L4 mortality was the dead apparently  normal larvae in the pre-pupal cocoon (Fig. 5a).  Not as common were dead  L4's with "marbly" and bloated bodies (Fig. 5b). The marbled appearance arose from the black or darkened haemolymph contrasting with the white fat body. Survival through the pupal stage appeared to be extremely poor, especially on diets which had previously shown promising larval survival.  Pupal survival ranged from 0% (CM,2xTM,BWW,TMW) to 40%  (BW)(Table VI and Fig. 6).  Figure 6 graphically shows the drop in pupal  numbers during the period between extraction from the diet t i l l 31 August 1984.  The most prominent cause of mortality was dessication during this  period through to the pre-winter inspection on 6 November 1984. During this time, the pupae were stored in their vials at room temperature. Mortality resulting from incomplete moulting, typified by the retention  - 46 Table V. Summary of mortality through the L4 stage of IB. colfaxiana reared on three Bioserv^ diets and several variations of diet concentration and establishment method. May 1984 to 21 April 1985.  Diet and Treatment  % of L4's dying with each symptom (n)@ None*  In Cocoon^  Marbly&  Overall L4 Mortality (%)  3.7(3)  26.8(22)  8.5(7)  39.0  Budworm, with 18.7(14) megastsrobi1i (BWS)  16.0(12)  -  34.7  Budworm, Double 7.5(3) concentration (2xBW)  10.0(4)  -  17.5  Budworm, Coastal eggs (BWC)  9.8(4)  17.1(7)  -  26.8  Budworm, eggs on waxpaper (BWW)  44.4(8)  22.2(4)  -  66.6  Codling Moth (CM)  80.0(4)  -  -  80.0  Nantucket pine tip 15.4(12) moth, normal (TM)  7.7(6)  -  23.1  Nantucket pine tip 41.7(10) moth, double concentration (2xTM)  4.2(1)  -  45.8  Nantucket pine tip 66.7(8) moth, eggs on waxpaper (TMW)  -  -  66.7  Budworm, Normal (BW)  as a % of the number of insects entering the L4 stage; n= number of L4's dying in each category * Unknown cause of death with no unusual external symptoms ^ Died within pre-pupal cocoon of unknown causes & Died outside of cocoon with darkened haemolymph (Fig. 5b), cause unknown L  Figure 5. Larval and pupal abnormalities, a) dead L4 in cocoon dead "marbly" L4; c) deformed pupa; and d) incomplete pupa.  - 48 -  - 49 Table VI. Summary of mortality through the pupal (pharate adult) stage of B_. colfaxiana reared on three Bioserv® diets and several variations of diet concentration and establishment method. May 1984 to 21 April 1985.  Diet and Treatment  Budworm, Normal (BW)  % of Pupae dying with each symptom (n)@ None*  44.0(22)  Incomplete Moult 12.0(6)  Deformed  Survivors)^ 4.0(2)  Budworm, with 46.9(23) megastsrobi1i (BWS)  38.8(19)  Budworm, Double 27.3(9) concentration (2xBW)  9.1(3)  6.1(2  16.7(5)  3.3(1  Budworm, Coastal eggs (BWC)  43.3(13)  Budworm, eggs on waxpaper (BWW)  83.3(5)  2.0(1)  16.7(1  Codling Moth (CM)  Other^  Overall % Pupal Mortality (# of  100(1)  24.2(8)  60.0 (11/9) 87.8 (3/3) 66.7 (6/5) 63.3 (4/7) 100 100  Nantucket pine tip 61.7(37) moth, normal (TM)  16.7(10)  1.7(1  Nantucket pine tip 53.9(7) moth, double concentration (2xTM)  38.4(5)  7.7(1  100  Nantucket pine tip 25.0(1) moth, eggs on waxpaper (TMW)  25.0(1)  50.0(1  100  80.0 (7/5)  @ as a % of the number of insects entering the pupal stage; n= number of pupae dying in each category * Normal appearance with no visible symptoms ^ pupae killed by mites prior to 5 November 1984 or lost during inspection & female/male survivors to 5 November 1984. One normal adult male emerged on 21 April 1985. No other adults or 2YD pharate adults survived the winter.  - 50 -  Legend ZZ) SURVIVING PUPAE • i TOTAL PUPAE PRODUCED  BIOSERV NANTUCKET PINE TIP MOTH BIOSERV CODLING MOTH  DIET TYPE  Figure 6. Pupal production and pre-winter survival of B. colfaxiana reared on three commercially prepared diets.  - 51 of larval head capsules and legs while the abdomen developed normally (Fig. 5c), was found consistently on all diets except CM (none survived to pupate).  Less frequently observed were deformed pupae that died  during moulting or shortly thereafter from dessication. Deformation resulted after the pupae failed to shed the L4 cuticle, most commonly around the thorax and head capsule, thereby trapping the newly formed pupae while the sclerites continued to harden (Fig. 5d).  Pre-winter  mortality (specifically, from 31 October to 6 November 1984) was severe due to predation by scavenger mites that are believed to have originated from cones that were being dissected near the stored pupae.  During the  overwintering period, dessication and mite predation continued and only one pupae survived to yield a normal adult male that emerged on 26 April 1985. 3.7.3 Larval Behaviour and Developmental Rate  Immediately after hatching, the Li's began feeding on the bract and usually followed the bract into the media crevice where they remained through the L2 stage.  L2 and L3 feeding was mainly on the bract but  changed to artificial diet as the bract was depleted.  During this phase,  the larvae would often venture out of the crevice and wander around the side of the media plug, feeding occasionally on the media.  Some of the  larvae would return to the crevice while others continued feeding on the side of the media and eventually burrowed into the plug.  Moulting to the  next stadium was almost always preceeded by the construction of a lightly  - 52 woven cocoon.  L4's tunneled and fed voraciously on the diet and  eventually constructed a thickly woven cocoon either inside or on the outer surface of the media plug within which they pupated.  None of the  cocoons were constructed on the l i d . Durations of each stadium were approximately the same for all diets except CM which had greatly extended L2, L3 and L4 stages due to starvation.  LI and L2 durations were difficult to assess since the  larvae were not disturbed when they were not observable through the vial sides or from above when the lid was removed.  Hatching began within one  day after establishment and was completed in one week.  The estimated  average duration of the LI stage was 12 days; the L2 stage began about 12 days after establishment and lasted for approximately 18 days.  L3's were  first noted 23 days after establishment and the peak numbers were recorded 7 days later. days.  The L3 stage was estimated to last for about 12  Observations of L4's began 30 days after establishment, peaked 12  days later, and was estimated to have lasted an average of 27 days. The first pupa was discovered 42 days after establishment while the majority of pupae were found 22 days later.  Thus, the estimated average duration  of the cone moth's larval development from egg to pupae was 64 days or about 9 weeks after establishment.  3.7.4 Pupal Weights  Few pupae survived to 6 November 1984 (weighed on 31 October 1984) which made comparisons of diets with their mean pupal weights almost  - 53 meaningless.  Fortunately, an additional 30 pupae were obtained from an  another experiment where 140 normal BW diets were established with eggs-on-bracts. 49.  The total number of pupae reared on BW diet was then  Overall pupal weight comparisons between naturally and artificially  reared^ cone moths in 1983 and 1984 and pupae reared artificially in 1966 and 1985 are made in section 3.11.3.  For 1984 BW-reared pupae alone, 1YD  and 2YD pupal weights were not significantly different while female pupae were significantly heavier than male pupae (Table VII).  3.7.5 Incidence of Prolonged Diapause  Only BW diet produced sufficient numbers of pharate adults to adequately assess the proportions in prolonged diapause.  Of the 49  pharate adults produced, 68% were in 2YD.  3.7.6 Disease Analysis  Results of the analysis of samples of healthy, sick, and dead cone moth larvae and pupae sent to FPMI on 16 July 1984 are presented in Appendix Ila.  No pathogenic microorganisms could be found under the  light microscope inspections.  - 54 Table VII. A comparison of mean pupal weights of B. colfaxiana reared on Bioserv®^s Spruce Budworm diet (BW)@ by aj diapause condition and sex, and b) by sex (1YD and 2YD data combined). May to Oct. 1984.  Diapause Status  Mean Pupal Weight + S.D. mg (n) Female  Male  t-test  #  Results  1 YD  9.5 + 1.6(11)  8.5 + 1.3(4)  N.S.D.  2YD  8.6 + 2.4(18)  8.4 + 2.8(22)  N.S.D.  b) Combined  8.9 + 3.7(29)  8.4 + 6.8(20)  N.S.D.  a)  @  #  Includes 20 females and 11 males from a supplementary rearing experiment reared under identical conditions as BW. Student's t--test, P< 0.05.  N.S.D. =  Not significantly different.  - 55 3.7.7 Other Experiments In addition to these main experiments, several other experiments were performed to f u l f i l l my curiosity about some unexplored aspects of the insect's biology under natural and artificial conditions. Unfortunately, the results of most of these side experiments were inconclusive for several reasons so they will only be mentioned here for interest's sake. The efficacy of a waxpaper drum oviposition cage, first described by Knott et a l . (1966) and used for J3. colfaxiana by Ruth and Hedlin (1969), was tested and an attempt was made to determine the optimum adult density and sex ratio for maximizing fertile egg production. Due to the low adult emergence rate, too few adults were available to test more than one replicate of each combination of density and sex ratio so the results were inconclusive. However, the drum cages were quite successful at inducing fertile egg oviposition on the waxpaper.  This cage, for some  unknown reason, has promoted inconsistent egg production over the several years it has been used (D.S. Ruth , pers. comm.). Another experiment examined the effects of larval density on mean pupal weight of cone moths reared on the artificial diet and in cones. Fifty vials containing budworm diet were established with five eggs and bracts inserted into a star-shaped hole in the media. None of the larvae survived past the L4 stage however observations of territoriality and cannibalism, which usually resulted in the survival of only the most dominant larva, confirmed Ruth and Hedlin's (1969) observations that B>. colfaxiana must be reared individually beyond the L2 stage.  - 56 -  The hypothesis that increased competition within cones would reduce the average pupal weight was tested by collecting cones from the Keremeos area and carefully dissecting them to determine the final pupal density within each cone.  The density was estimated based on the count of live,  dead and traces of predated pupae observed.  Live pupae were segregated  into their estimated density classes, sex, and diapause condition and then were weighed individually and the mean pupal weights for each density class were compared with Scheffe's test.  For female pupae in 2YD  and male pupae in 1 YD, pupae reared alone were significantly heavier than pupae reared at a density of three and four pupae per cone (Table VIII). This result suggests that pupal weight decreases with an increase in intraspecific competition, therefore, weights of naturally reared pupae should be stratified by rearing density before they are compared to pupae that were reared singly on artificial media. In another experiment, cone moth eggs collected from coastal Douglas-fir in the town of Hope, B.C were reared under identical conditions as the Keremeos collected eggs placed on spruce budworm diet (BWC).  The objective was to determine if the coastal cone moths were  easier to rear.  Table IV shows that coastal cone moths do not have  noticeably different survival rates than the interior cone moths. 3.8 Discussion and Recommendations, 1984  BW diet appeared to be the best of the three commercially prepared diets for rearing B. colfaxiana.  Although both BW and TM diets were  - 57 Table V I I I . Comparison of mean pupal weights of J3. c o l f a x i a n a reared n a t u r a l l y at four d e n s i t i e s . Insects were extracted from cones c o l l e c t e d from Keremeos, B.C., 25 August 1984.  Rearing Density (Pupae/ Cone)  Female 1 YD  Mean Pupal Weight + S.D. (n)  2YD  1 YD  Male 2YD  One  11.9 + 2.3(10)a@  14.1 + 1.8(6)a  Two  11.7 + 2.1(27)a  12.2 + 2.4(10)ab 10.5 + 1.6(23)ab 10.8 + 2.4(16)a  Three  11.5 + 2.0(18)a  11.4 + 2.2(9)b  Four  10.7 + 2.4(9)a  10.9 + 1.6(7)b  11.3 + 2.6(6)a  9.3 + 1.9(13)b  11.5 + 1.4(15)a  10.3 + 2.1(13)a  @ Mean pupal weights followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t , Scheffe's t e s t , P< 0.05.  - 58 casein and toasted wheat germ based, the poorer survival on the TM diet may be explained by the differences in diet compositions.  TM has  fructose, dextrose, cholesterol, sorbic acid, Brewer's yeast, and linseed oil which BW does not.  Conversely, ingredients included in BW and not in  TM are methyl para-hydroxybenzoate, 4 M KOH, Aureomycin and Formaldehyde (all included as anti-microbial agents).  TM reared larval survival was  approximately the same as all of the BW diets (except BWW) until the pupal stage, thereafter, pupal mortality exceeded that of normal BW diet.  The differences in diet composition likely affected the  development of normal pupal cuticles as witnessed by the higher rate of dessication of pupae on TM than on BW diets. Doubling the concentration of either BW or TM diets did not improve overall survival nor did inclusion of freeze-dried megastrobili (though the latter result is inconclusive based on the poorer than normal BW survival for BWS for reasons that were explained in section 3.5). Codling moth diet appeared to be unpalatable to the cone moth larvae as most larvae completely consumed the bract without feeding on the diet and subsequently died of starvation.  CM diet was based on finely ground Lima  beans with ground (not toasted) wheat germ; casein and several other ingredients found in the BW diet were absent in the CM diet.  Creswell et  al. (1971) found that a soybean based diet was unsuitable for rearing the Nantucket pine tip moth while a wheat germ diet gave higher survival. Bract feeding appears to be very important for improving early instar (LI and L2) survival.  Though not essential for larval survival  (eg. BWW and NTW larvae survived on artificial diet alone), bracts  - 59 probably provide the young larvae with much of the essential nutrients required for development into later instars and maybe also into the pupal stage.  It appeared that artificial diet feeding occurred after large  portions or all of the bract had been consumed.  Prior to this switch,  artificial diet was consumed sporadically along with the bract.  It is  important to note that larvae on cones do not actively feed on bracts but bore through them to feed on the scale tissue and developing ovules (Hedlin 1960). Poorer hatching success of eggs established on waxpaper may have resulted from two possible problems.  The first involved the absorbance  of the 10% formaldehyde by the waxpaper which may not have washed off and also may have come in contact with the underside of the egg. exposure to the formaldehyde may have killed the eggs.  Extended  The second  problem may have stemmed from the unknown fertilization success of adults mated in drum oviposition cages.  It is possible that infertile eggs  layed by unmated females may also have been established on the media. Few eggs were available for establishment so all but the damaged and opaque white eggs were established even though some showed little signs of embryo development.  Therefore, the large hatching success differences  between waxpaper and bract substrates may be artificial.  As early larval  survival was better on egg-on-bract established diets, it is recommended that this method be adopted. Future studies should examine the effects of establishing eggs laid on waxpaper along side a previously frozen bract.  Such a study would  confirm whether or not the complete life-cycle could be efficiently  - 60 reared year-round without the requirement of fresh megastrobili for oviposition and early larval feeding (provided that mating and ovipostion in the waxpaper drum cages are successful).  Ideally, megastrobili could  be collected in large quantities when available and deep frozen until required.  Without this flexibility, the maintanence of a laboratory  colony of cone moths would be extremely inefficient, that is, larger numbers of eggs would be required from drum cages than would be necessary if they were established with bracts to obtain sufficient numbers of adults.  A major objective of developing a mass-rearing technique is to  attempt to shorten the cone moth life-cycle to facilitate pre-season testing of pheromones and host attractants.  Unless a substitute for  fresh bracts is found, maintaining a large laboratory colony of cone moths would be extremely difficult and would not f u l f i l l this objective. Comparisons of survival between diets was strictly qualitative since replications were not made for each diet tested.  Regardless of the final  survival obtained, it was clear from the observations of mortality in the L4 and pupal stages that some aspect of the method or diet was preventing their proper development.  Ruth and Hedlin (1969) did not observe this  unusual L4 mortality or the imperfect pupae.  Their method differed in  two major aspects: 1) the larvae were frequently transferred to fresh media whenever the media plug dried severely; and 2) L4's were removed from media when pupation was believed to be imminent, therefore, the larvae did not pupate within the media as was done in 1983 and 1984. Artificial rearing literature abounds with nutritional studies that have examined the effects of addition and/or deletion of particular  - 61 nutrients.  Dadd (1973), in his review of these studies, reported that  the deletion of several different nutrients produced poor larval survival and pupal development in a wide range of phytophagous lepidopteran species (eg. folic acid, L-ascorbic acid, choline chloride, polyunsaturated fatty acids, etc.).  Perhaps Robertson's (1979) diet and  Bioser\P's spruce budworm diet were initially deficient in one or more of these essential nutrients.  Conversely, perhaps essential nutrients  were depleted or oxidized over the 8 to 10 week larval feeding period. Two nutrients that are known to oxidize readily under typical artificial rearing conditions, and have been identified as essential for larval and pupal development of several lepidopteran species, are L-ascorbic acid and the fatty acids linoleic acid (CI8:2) and Linolenic acid (CI8:3). Rapid degradation of L-ascorbic acid in an aphid diet was reported by Dadd et a l . (1967).  Linolenic acid, the more bioactive of the two C18  polyunsaturates, degrades to a peroxide which is toxic to insects (Vanderzant et a l . 1959). Dietary deficiencies, in general, may explain the unexpected L4 mortality, pupal deformaties, and abnormal adult emergence observed in 1983 and 1984.  These deficiencies may affect the development of pupae  and adults by disrupting the balance of hormones, particularily ecdysone and juvenile hormone.  The incompletely moulted pupae, also observed by  Creswell et a l . (1971), exhibit a condition termed by Wigglesworth (1959) as "metathetely" defined as "the retention of larval characters in the adult stage". Metathetely was believed to be caused by an abnormally high titre of juvenile hormone (Creswell et a l . 1971).  Dadd (1973) noted that  - 62 the presence of f a t t y acids with vitamin E may f a c i l i t a t e the absorption and u t i l i z a t i o n of other n u t r i e n t s .  Thus, f a t t y acid or vitamin E  d e f i c i e n c y may r e s u l t in the inadequate absorption of hormone precursors, f o r example s t e r o l s , which are the precursors f o r ecdysone.  I t was  recommended that future research should concentrate on t e s t i n g the hypothesis that n u t r i t i o n a l d e f i c i e n c i e s are the cause of the poor L4 and pupal s u r v i v a l experienced in 1983 and 1984.  In a d d i t i o n , future rearing  studies should i n v e s t i g a t e the e f f e c t of removing L4's from the d i e t j u s t before pupation and should develop a method of protecting overwintering pharate adults from scavenger mite predation and severe d e s s i c a t i o n . To summarize 1984's research, i t appeared that the f i r s t two objectives o u t l i n e d in the i n t r o d u c t i o n (section 3.5) were met.  First,  the "new and improved" method of e s t a b l i s h i n g eggs on bracts in sealed 7 dr p l a s t i c v i a l s was successful both at maintaining adequate media moisture during the l a r v a l feeding period and at preventing a l l but a small f r a c t i o n of the larvae from escaping (those escaping did not tunnel through the v i a l w a l l s ) .  Second, the normal spruce budworm d i e t appeared  to be the best of the three d i e t s tested f o r rearing the cone moth although the,poor L4 and pupal s u r v i v a l were unexplained.  The t h i r d  o b j e c t i v e , to eliminate media contamination, p a r t i c u l a r l y of pathogenic microorganisms, also appeared to have been met as evidenced by the rare occurrence of contaminated media and infrequent discovery of apparently diseased larvae ( i . e . the "marbly" L 4 ' s ) .  The professional diagnosis of  these "marbly" larvae revealed no obvious signs of pathogens, t h e r e f o r e , the cause of t h i s condition i s probably a n u t r i t i o n a l  deficiency.  - 63 3.9 Introduction, 1985  The prime objective of 1985's research was to solve the mystery of the poor L4 and pupal s u r v i v a l experienced in 1983's and 1984's which did not occur in 1966's a r t i f i c i a l rearing attempts.  The recommendations f o r  future research o u t l i n e d in section 3.8 were used in 1985 to develop two main experiments.  The f i r s t experiment examined two aspects: a) the (5)  differences between Bioserv ' s Spruce Budworm d i e t and Robertson's (1981) western spruce budworm d i e t , and b) the e f f e c t s of r e p l a c i n g the old media with f r e s h l y made media two weeks a f t e r establishment.  The  l a t t e r part of t h i s experiment tested the hypothesis that d i e t freshness was important to l a r v a l s u r v i v a l and successful pupation.  The second  experiment proposed that i n i t i a l concentrations of the nutrients L-ascorbic acid and l i n o l e n i c acid were i n s u f f i c i e n t to support cone moth larvae through the e n t i r e rearing period, t h e r e f o r e , d e l e t i n g and increasing the i n i t i a l concentration of these two n u t r i e n t s in unchanged Roberston (1981) d i e t would t e s t f o r corresponding decreases and increases in cone moth s u r v i v a l and adult development.  The experiment  would also show i f e i t h e r n u t r i e n t was e s s e n t i a l f o r the cone moth as has been reported f o r several other insect species. Although the o r i g i n a l diet/diapause hypothesis was not tested in 1985, the expected r e s u l t s of 1985's rearing experiments were hoped to f i n a l l y resolve the a r t i f i c i a l r e a r i n g problems and thus c l e a r the path f o r future research r e q u i r i n g a r t i f i c i a l l y reared J3. c o l f a x i a n a .  - 64 3.10 Materials and Methods, 1985 3.10.1 Rearing Method  Egg collection and storage methods, rearing containers, lid modification, and establishment procedures (for eggs-on-bracts only) were identical to those used in 1984.  Environment chamber conditions were  held at a constant 20°C with a 16hr daylight cycle.  Relative humidity  varied with the daily ambient conditions but varied only from 65 to 75% over the entire rearing period (1 May to 16 October 1985).  Sterile  methods used in 1985 differed slightly from 1984. All instruments used to dissect and probe the media were flame sterilized rather than soaked in 70% ethanol.  Diet mixing, pouring and establishment were done under a  laminar flow of sterile air as in 1984.  Cardboard inserts were prepared  with a hollow drill on a drill press and, just prior to insertion into the lids, were sterilized by immersion in 70% ethanol which was allowed to evaporate in the laminar flow hood. Modified snap-cap plastic lids, some recycled from 1984, were washed in mild chlorine bleach for 10 minutes, rinsed with tap water and oven dried.  3.10.2 Experimental Design  Spruce Budworm diet and 410 vials of normal concentration Robertson (1981) diet (RN) were divided into two groups.  The first group contained 207 BIO and 200 RN  - 65 vials which had two week-old media removed and replaced with freshly made (within 2 days) media of the same formulation.  The remaining unchanged  vials were not disturbed except when necessary during the bi-weekly inspections.  BIO and RN vials were changed 10 to 14 days and 12 to 16  days, respectively, after establishment.  The larvae were in or near the  L3 stage and were transferred by coaxing them onto the new media or by hoisting them to their new vial by their silk (as done in 1983 for larval established diets, see section  Transfer was delayed until the  majority of larvae reached the L3 stage because it was believed that unacceptably high mortality would result from transferring Li's and L2's.  A second transfer to fresh media two weeks later was not done  since some of the larvae had begun to pupate. In Experiment 2, the RN diet was modified as shown in Appendix Ic and resulted in the production of 12 diets differing in their L-ascorbic acid (AA) and 55% linolenic acid (LA) contents.  AA and LA concentrations  varied from "none" (eg. RN-LA-AA, RN-LA+AA, etc.) to 4 times the concentration found in RN (eg. RN+3XLA, RN+3XAA, RN+3XLA+3XAA) (Appendix Ic).  Diets were not completely free of L-ascorbic acid since the ICN  vitamin fortification mixture (ICN Biomedicals Inc., Cleveland, Ohio, USA) contained AA and supplied the diet with AA at the rate of ,54g/100g of diet.  Linolenic acid was present in all diets since it constitutes .3  to .6% of the dry weight of wheat germ (Kent 1975) which was a basic ingredient in all of the diets. eggs for each diet.  At least 100 vials were established with  The final diet established on 17 May 1985  (RN+3XLA+3XAA) was established with eggs collected two weeks  - 66 -  (8 May 1985) after the initial egg collection on 25 April 1985, thus, the vials were established with well developed eggs.  These eggs did not  hatch as successfully, possibly the result of greater sensitivity to sudden cooling or to cold storage. One of the 12 diets tested was a batch of RN that was supplemented with pure Cholesterol (USP) crystals at the rate of .05g/100 g of diet. Cholesterol is an excellent dietary source of sterols which act as precursors for ecdysone synthesis (T.S. Sahota , pers. comm.). 6  3.10.3 Diet Preparation and Inspections  Diets were prepared as in 1984 except mixing of ingredients occurred at the high speed setting on an Osterizer domestic blender for the durations and in the sequences listed by Robertson (1981)(Appendix lc). Diets prepared for Experiment 2 are listed in Appendix lc. The basic Robertson (1981) diet was altered by deleting or adding the normal compliment of either AA or LA. Some diets contained up to 4 times the normal concentration of AA and/or LA recommended by Robertson (1981). Each batch produced about 105 vials although variation occurred because of hand pouring of unequal volumes into each vial and because of delays in pouring that allowed the media to cool and stick to the dispensing  Research Scientist, Canadian Forestry Service, Pacific Forestry Centre, Victoria, B.C.  - 67 beaker.  All diets were poured no more than one week before they were  established with eggs on bracts. A total of eight inspections were made bi-weekly which did not permit an accurate estimate of developmental rate as LI, L2 and a portion of the L3 stages were completed within the two week period.  Inspections  were made with a dissecting microscope under the laminar flow hood (as in 1984).  Instead of leaving the non-observable larvae undisturbed as was  the procedure in 1984, media plugs were probed or removed from their vials and dissected with forceps and a scalpel.  This thorough inspection  provided more precise information on the time of death of larvae, especially L4's that died within their cocoons (Fig. 5a). Moribund "marbly" L4's that were found were removed from their media, returned to their vials minus the diet, set aside on the flowbench and were monitored daily to determine if they would eventually pupate or die. None of these larvae pupated before dying, however, some of these larvae were included in a disease analysis before they died. Because mold contamination of diets was linked to the presence of incompletely surface sterilized bracts, 1985's inspections included removal of bracts as soon as they showed signs of mold growth (i.e. visible hyphae covering the bract). replaced.  Contaminated bracts were not  Mold was inhibited by the anti-microbial ingredients in the  diet, however, after 6 weeks (also observed in 1984), the mold overcame the diet's defenses and eventually engulfed the media. Most anti-microbial agents are effective only in a narrow pH range (Funke 1983), therefore, diet degradation may have changed the pH sufficiently to reduce their potency.  - 68 During the first inspection, eggs that had not hatched by that time were replaced with eggs on bracts recently plucked from stored megastrobili collected originally on 8 May 1985.  The eggs had been  stored for over three weeks at 0°C and the hatching success was low. Before surface sterilization, replacement eggs were carefully selected by the following criteria: a) the head capsule had to be visible, b) it was undamaged, and c) it had to show visible signs of life (eg. moving mandibles, pumping dorsal vessel or movement of any sort).  Fortunately,  each diet tested required, on average, only nine eggs to be replaced.  3.10.4 Pupal Extraction and Storage  Whenever a pupa was discovered it was immediately removed from the media, the media was discarded and the pupa was returned to its rearing vial and returned to the environment chamber.  The pupal environment in  the rearing chamber was gradually cooled to simulate the transition from summer to f a l l .  Temperatures were changed from a constant 20°C to  20°C for 12 hrs: 15°C for 12 hrs on 12 August 1985.  The temperature  was reduced again on 30 August 1985 to a constant 10°C. Relative humidity throughout the cooling period remained constant at about 75%. Pupae were inspected bi-weekly in a continuation of the larval inspections and were weighed on 16 August 1985.  Pupae were inspected for  a final time on 16 October 1985 before they were moved to UBC for winter storage.  Pupal weight analysis used the August 15-16th weights but only  for the October 16th survivors.  Prior to storage, the lids used during  - 69 rearing were replaced with new, unmodified lids to prevent mite predation and reduce moisture loss.  The labelled cardboard inserts were removed  from the lids and placed inside the vials before they were resealed. Pupae were overwintered outdoors in a sheltered enclosure at the South Campus field house at UBC. Pupae were brought indoors on 25 February 1986 to assess overwinter survival and the success of adult emergence over a two week period. Adult success was evaluated with an adaptation of Dadd's (1983) "emergence index".  The conditions of living moths were recorded under  the following categories: (1) normal, (2) wings deformed, (3) partially emerged, and (4) pharate adults in 2YD (Fig 7a-g).  Category (2), wings  deformed, included moths that were likely able to mate but were unable to fly (Fig. 7f and g).  Category (3), partially emerged adults, included  all adults that had split the pupal integument but had only a small portion of the adult extruded (Fig. 7b).  All pharate adults that showed  movement on 9 April 1986 were considered to be in prolonged diapause (Category 4).  Dadd's emergence index served as a "summary performance  value which would take account of the degree of imperfections in emergence" for each treatment he tested.  Three categories were used to  describe adult emergence and each were given a value depending on their degree of viability.  Normal adults were assigned a value of one, adults  with warped wings were .75, adults with deformed wings were .5 while severely deformed and partially emerged adults were assigned zeros. Pharate adults in 2YD were omitted from the calculation of the indices since, at the time of writing, there was no way of predicting next  - 70 -  Figure 7.  Illustrations of various forms of pupal mortality and adult emergence observed 28 February to 8 April 1986. a) Normal pupa, b) partially emerged pharate adult, c) shrunken or collapsed pupa or pharate adult, d) dead incomplete pupa, e) Normal adult fully eclosed, f - g) adult fully eclosed with deformed wings. (Drawings by L. Friskie).  - 71 spring's emergence success of these pharate adults.  These values were  then summed, and the sum expressed as a percentage of the total numbers of individuals to reach adulthood in the treatment (Dadd 1983).  3.10.5 Disease Analysis  A more concerted effort was made in 1985 to determine whether "marbly" L4's were actually showing the symptoms of a disease which may not have been detected by Dr. Cunningham in 1984 (Appendix Ila). On 28 June 1985, two groups of samples containing 15 dead L4's in cocoons and 15 dead "marbly" L4's were sent by courier to Mr. Gerard M. Thomas (Consulting Diagnostic Services, Berkley, CA, USA) for diagnosis. The results and procedure used for this diagnosis are given in Appendix l i b . In addition to this diagnosis, 7 living cone moths (2 "normal L4's collected from Mesachie Lk., B.C., 3 Moribund "marbly" or spotted L4's, and 2 incompletely moulted pupae) were fixed in Carnoy's solution, embedded in Paraplast, cut into 4 u sections, mounted, and stained with Hamm's Azan stain to determine if polyhedrosis or granulosis inclusion body-forming viruses were present (Clark 1981)(Appendix III). Unfortunately, the process of mounting the sections onto the microscope slides was done incorrectly so the sections floated free during staining. microscope.  A few sections were stained and were examined under a compound  - 72 3.11 Results, 1985 3.11.1 Media Condition  As in 1984, an assumed decrease in media moisture content did not appear to interfere with feeding over the entire larval feeding period. Although a higher rearing temperature (20°C) was used as compared to 18.5°C in 1984, the media shrank radially at about the same rate but the larvae completed their development earlier than in 1984.  A noticable  absence of condensation on the vial walls was attributed to the use of the constant rearing temperature.  Deleting or supplementing LA or AA did  not appear to affect media drying rate or any other physical feature of the media. Contamination by bacteria and mold fungi was minimal thereby confirming the effectiveness of the sterile techniques used during diet preparation, media establishment, and inspections.  Only six of the 2,068  vials established were completely engulfed by mold (likely Aspergi11 us sp. or PeniciIlium sp., H.S. Whitney^, pers. comm.).  L4's were able to  feed on moldy diet without obvious ill-effects, however, when transferred to new media, the mold rapidly colonized the media. When the L4's pupated, the mold hyphae penetrated the cocoons and began growing on the pupal cuticle resulting in the death of the pupae.  Two dead pupae were  found in mold engulfed media; both pupae had soft cuticles and were partially decomposed.  Research Scientist, Canadian Forestry Service, Pacific Forestry Centre, Victoria, B.C.  - 73 3.11.2 Survival Detailed observations made during 1985's inspections were summarized for all life stages (Table IX) for cone moths reared on each diet and treatment.  Overall, the best survival was obtained on RN+LA and RN+3xLA  (37.8 and 36.3%, respectively); the worst survival was on all three RN diets without LA (1.0 to 2.0%)(Table IX). The following is a detailed description of each mortality category for each of the life stages. A. Eggs Hatching rate prior to egg replacement averaged 92.3% and ranged from 65.1% to 97.1%; after replacement, the proportion hatched increased to an average of 96.1% (range 77.4 to 100%). An average of 8.75 eggs per diet and treatment were replaced.  Eggs collected on 8 May 1985 had the  poorest hatching rate (77.4%). B. LI LI mortality was classified into six categories: 1) entrapment by moisture on bracts, 2) entrapment by moisture on diet, 3) engulfed by mold on the bract, 4) unknown cause of death (i.e. normal appearance) with no signs of feeding, 5) unknown cause of death with feeding on bract only, and 6) unknown cause of death with feeding on bract and diet. Overall LI survival on all diets and treatments averaged 94.4% (range 61.0 to 100%).  The most frequently observed form of mortality on all  diets and treatments except RN+3xLA+3xAA was the unknown cause of death  - 74 Table IX. Summary of survival through each life-stage of B. coifaxiana reared on 14 diets and two media conditions (Experiments 1 and 2), 6 May 1985 to 8 April 1986.  Diet and Treatment  % Surviving through each life-stage* Egg  0  LI  L2  L3  L4  Overall Survival Pupa+ (Survivors/ Adult # Hatched) (Pre/Post)* (Pre/Post) 5  Experiment 1: BIO Changed Unchanged  96.1 99.5  95.5 97.1  96.8 98.5  97.8 98.5  37.2 58.9  56.7/31.3 36.3/13.3  19.1/10.6% 20.1/8.8%  RN Changed Unchanged  97.5 98.1  95.9 98.5  98.4 97.5  98.4 98.0  42.0 34.0  77.6/51.3 78.8/54.5  30.3/20.0% 25.2/17.5%  Experiment 2: RN-LA-AA RN-LA RN-LA+AA  95.3 97.2 98.0  97.0 99.0 100  96.9 98.1 99.0  89.5 96.1 96.9  27.1 32.7 40.0  13.0/8.7 18.8/2.8 5.3/3.1  3.0/2.0% 5.7/1.0% 2.0/1.0%  RN-AA RN+AA RN+3xAA RN+Cholesterol  99.1 97.2 95.0 99.0  95.5 100 92.7 93.9  100 99.0 95.5 92.5  95.2 100 97.6 96.5  36.0 44.7 55.4 50.6  63.9/50.0 20.9/16.4% 65.2/47.8 28.8/21.2% 65.2/47.8 31.3/22.9% 71.4/52.4 30.3/22.2%  RN+LA-AA RN+LA RN+LA+AA  92.0 98.0 100  98.9 94.9 95.1  98.9 97.8 99.0  97.8 98.9 100  53.4 57.8 53.6  76.6/59.6 39.1/30.4% 92.3/71.2 49.0/37.8% 84.6/59.6 42.7/30.1%  RN+3xLA RN+3xLA+3xAA  98.1 77.4  95.1 61.0  97.9 86.0  97.9 90.7  73.1 59.0  80.9/54.4 53.9/36.3% 78.3/34.8 22.0/9.8%  BIO RN AA LA @ * ^  Bioserv® Spruce Budworm diet Robertson's (1981) Western Spruce Budworm diet L-ascorbic acid 55% linolenic acid Survival after replacement as a % of the numbers entering each life-stage Pre/Post = Pre-winter assessment vs. Post-winter assessment  - 75 following feeding on bracts and diet.  RN+3xLA+3xAA had 32 dead Li's of  which 12 died without feeding and another 12 died after becoming entrapped on the diet or bract surface.  For the majority of diets and  treatments, LI mortality was third in importance next to pupal and L4 mortality. C. L2 Survival through the L2 stage was excellent.  Seven mortality  classes were identified: 1) entrapment on diet or vial surface moisture, 2) bloated or swollen abdomens (a possible symptom of a disease), 3) incomplete moult to L3, 4) injury during inspection, 5) unknown cause of death following feeding on bract and diet, and 7) engulfed by mold. Each category was rarely, if ever, found for each diet and treatment.  The  most frequent form of mortality was the unknown cause of death following bract and diet feeding. Overall average L2 survival was 97.0% (range 86.0 to 100%).  L2 survival was considered to be approximately equal for  all diets (i.e. none of the diets suffered severe losses in the L2 stage). D. L3 L3 survival, like the L2 survival, was excellent for all diets and treatments (average 96.9% with a range of 89.5 to 100%)(Table IX). Mortality was categorized similarly to L2 mortality but with three additional categories (i.e. unknown cause of death accompanied by a shrunken abdomen, unknown cause of death with little or no feeding after L3 eclosion, and drowning at the base of the vial in condensation). The worst L3 survival occurred on RN-LA-AA and was due mainly to unknown factors, however, two L3's were accidentally killed (four L2's were also  - 76 killed).  In a mass rearing situation, detailed inspections would not be  required, and therefore, handling mortality would be eliminated. Mortality through the L2 and L3 stages appeared random. E. L4 Generally, survival through the L4 stage, as noted in 1983 and 1984, was markedly lower than in the LI through L3 stages.  Five categories  were used to assess L4 mortality: 1) "escapees", those larvae which were known to have escaped via gaps between the plastic lid and the cardboard insert (larvae may have escaped in any instar but for convenience all "escapees" were classified as L4's since their numbers were both low and nearly consistent for all diets/treatments),  2) unknown cause of death  sometimes accompanied with small dark spots on the abdomen, 3) multiple moults (up to L7), 4) apparently normal pre-pupal larvae within their cocoons (Fig. 5a), and 5) larvae with the "marbly" symptom (with or without cocoons) (Fig. 5b)(Table X). Only one L4 was killed accidentally during an inspection and was classified as "escaped" in Table X. Overall survival through the L4 stage for all diets and treatments ranged from 27.1 to 73.1% and averaged 47.2%.  In Experiment 1, BIO  unchanged media had greater L4 survival than changed media (i.e. 58.9% vs. 37.2%) but for RN unchanged L4's had slightly poorer survival than changed larvae (i.e. 34.0% vs. 42.0%).  For Experiment 2, diets without  LA had lower average L4 survival than diets with LA (i.e. 33.3% vs. 50.0%).  L4 survival appeared to increase slightly with increasing  concentrations of LA but no trend was obvious for the various AA concentrations.  - 77 Table X.  Summary of mortality through the fourth instar of B. coifaxiana reared on 14 diets and two media conditions (Experiments 1 and 2), May 6 to October 16, 1985.  % of L4's dying with each symptom^  Diet and Treatment  None*  Experiment 1: BIO Changed Unchanged  8.3 9.4  1.1 2.1  26.7 17.2  22.8 10.9  3.9 1.6  62.8 41.1  RN Changed Unchanged  7.2 9.3  2.2 0.5  37.0 50.0  8.3 4.1  2.8 2.0  57.5 65.9  Experiment 2: RN-LA-AA RN-LA RN-LA+AA  16.5 9.2 8.4  17.6 22.4 17.9  32.9 31.6 27.4  3.6 1.0 6.3  2.4 3.1 0  72.9 67.3 60.0  RN-AA RN+AA RN+3xAA RN+Cholesterol  9.0 1.9 7.2 4.8  12.0 1.9 1.2 8.4  39.0 37.9 24.1 31.3  2.0 12.6 8.4 2.4  2.0 1.0 3.6 2.4  64.0 55.3 44.6 49.4  RN+LA-AA RN+LA RN+LA+AA  5.7 2.2 3.1  4.5 1.1 3.1  34.1 27.8 33.0  2.3 10.0 6.2  0 1.1 1.0  46.6 42.2 46.4  0 10.3  4.3 2.6  21.5 17.9  0 0  1.1 10.3  73.1 41.0  RN+3xLA RN+3xLA+3xAA BIO RN AA LA @ *  Multiple Moults  In Marbly* Cocoon*  Escaped  Bioserv® Spruce Budworm diet Robertson's (1981) Western Spruce Budworm diet L-ascorbic acid 55% linolenic acid as a % of the numbers of insects entering the L4 stadium Unknown causes of mortality  Overal1 % Mortality through L4  - 78 A comparison of diets and treatments by each L4 mortality category revealed some interesting trends.  The number of "escapees" varied  randomly throughout Experiment 1 and 2 and averaged 1.8 larvae per 100 vials established.  Dead L4's showing no symptoms were the most frequent  on RN-LA-AA (16.5%) and the least on RN+3xLA (0%), however, no obvious trend could be observed for either LA or AA concentrations.  RN and BIO  changed and unchanged diets were virtually identical in this category. "Multiple moult" deaths were most frequent in diets without LA (i.e. 17.6 to 22.4% vs. an average of 3.5% for all other diets including BIO). The absence of LA appeared to affect the transformation from larva to pupa. No trend in L4 mortality was evident for changing AA concentrations.  "In  cocoon" mortality was the most common symptom for all diets and treatments and ranged from 17.2 to 50.0% (average 30.6%).  No obvious  trends for "in cocoon" mortality were observed with respect to LA or AA concentrations, however, RN changed and unchanged media had greater "in cocoon" mortality than BIO changed and unchanged media.  "Marbly" L4's  occurred sporadically on all but two diets and treatments.  Changed BIO  had the highest incidence (22.8%) while both RN+3xLA and RN+3xLA+3xAA had no "marbly" L4's. For all RN-type diets, the average incidence of "marbly" L4's was 4.8% (range 0 to 12.6%) compared with an average of 16.8% (range 10.9 to 22.8%) for the two BIO treatments. F. Pupae (Pharate Adults) (i) Pre-Winter Survival The most remarkable differences in survival between diets and treatments were observed in the pupal/pharate adult stage (Table XI; Fig.  - 79 Table XI.  Summary of mortality through the pupal (pharate adult) stage of B. coifaxi ana reared on 14 diets and two media conditions jExperiments 1 and 2), 6 May to 16 October 1985.  Diet and Treatment  % of pupae dying with each symptom^ Incomplete Moult  Deformed Spots & Split Bands Suture  EXPERIMENT 1: BIO Changed Unchanged  28.4 18.6  3. 0 8. 8  6.0 18.6  RN Changed Unchanged  11.8 9.1  2. 6 4. 5  0 1.6  EXPERIMENT 2: RN-LA-AA RN-LA RN-LA+AA  43.5 43.8 34.2  4. 3 3. 1 15. 8  30.4 28.3 23.7  RN-AA RN+AA RN+3xAA RN+Cholesterol  16.7 26.1 8.7 14.3  5. 6 6. 5 2. 2 2. 4  8.5 5.8 1.9 2.9 17.4  RN+LA-AA RN+LA RN+LA+AA RN+3xLA RN+3xLA+3xAA BIO RN AA LA ® *  3. 0 15. 0  Overall % MortWhite Other* ality Belly through Pupa  0 2.7  3. 0 0  43 .3 63 .7  0 0  0 3.0  7. 9 3. 0  21 .0 21 .2  4. 3 3. 1 0  0 0 0  4. 3 3. 1 21. 0  87 .0 71 .9 94 .7  0 0 0 0  0 0 0 0  0 0 0 0  13. 9 2. 2 23. 9 11. 9  36 .1 34 .8 34 .8 28 .6  2. 1 1. 9 5. 8  0 0 0  2. 1 0 0  0 0 0  8. 5 0 7. 7  23 .4 7.7 15 .4  1. 5 0  1.5 0  0 0  1.5 0  11. 8 4. 3  19 .1 21 .7  Bioserv^ Spruce Budworm diet Robertson's (1981) Western Spruce Budworm diet L-ascorbic acid 55% linolenic acid as a % of the number of insects entering the pupal stage normal appearance, no known cause of death  - 80 8 and 9).  Figures 8 and 9 represent pupal survival as a percentage of  the total number hatched thus indicating the overall survival on the diet and treatment.  In addition, these figures depict pre-overwintering pupal  survival from pupal eclosion until 16 October 1985. In Experiment 1, BIO produced more pupae but had considerably poorer survival than RN. Changed media survival was greater than unchanged media for BIO only; RN changed and unchanged media were almost equal in pupal survival (Table XI). Experiment 2 demonstrated that LA was an important nutrient for pupal survival (Fig. 8) whereas AA was not (Fig. 9).  Diets without LA  had the poorest pupal survival whereas the production and survival of pupae increased with the increase in LA concentration (Fig. 8). Pupal mortality was divided into six categories (Table XI). Unusual dark pigmented bands and spots were observed on the abdomens of some pupae, primarily those reared on BIO (changed and unchanged), RN-LA-AA, RN-LA, and RN-LA+AA (Fig. 10a and b).  These same diets, particularly  BIO, also produced pupae with split suture lines in the pupal cuticle that followed the antennal, leg and wing-pad margins.  All pupae that  showed these bands, spots or split sutures were dead by 16 October 1985. These pupae were often dessicated. Incompletely moulted or developed pupae (Fig. 5c), possibly related to hormonal imbalances, were nearly always the largest source of pupal mortality for all diets and treatments.  LA supplemented diets produced  pupae with fewer developmental abnormalities and the frequency of incompletely developed pupae was inversely related to the dietary level of LA (Table XI).  - 81 -  Legend IZZI SURVIVING PUPAE • i TOTAL PUPAE PRODUCED  NORMAL [LA]  M i l 1  4XLA  •  ***** DIET TYPE  Figure 8. Pupal production and pre-winter (to 16 October 1985) survival of EL colfaxiana reared on artificial diet containing varying concentrations of linolenic acid.  - 82 -  Legend EZ) SURVIVING PUPAE • i TOTAL PUPAE PRODUCED  NORMAL [AA]  •  y  HI  DIET TYPE  Figure 9. Pupal production and pre-winter survival (to 16 October 1985) of B. colfaxiana reared on artificial diet containing varying concentrations of L-ascorbic acid.  Figure 10. Pupae with unusual pigmentation on abdomen accompanied with split thoracic sutures, a) close-up of bands and split suture lines; b) comparison with a healthy pupa (upper).  - 84 The incidence of pupal deformities caused by moulting difficulties (Fig. 5d), appeared to be unrelated to dietary LA or AA concentrations although this condition was generally rare (i.e. an average of two deformed pupae per diet/treatment). (ii) Post-Winter Survival Assessment and Adult Emergence Overall overwinter survival (survivors included normal adults and adults with deformed wings, partially emerged adults, and pharate adults in 2YD) was relatively constant for all RN diets with LA (normal to quadruple concentrations) except RN+3xLA+3xAA (where average survival was the highest at 77%).  Overwinter survival of pharate adults reared on  changed BIO, which was slightly lower than on all RN with LA diets, was markedly greater than that of cone moths reared on unchanged BIO (63.2 vs. 36.6%).  This difference in survival between changed and unchanged  media was not observed on RN diet.  For RN diets without LA, one to two  pharate adults per diet survived through the winter (note that very few survived through the pre-winter stage).  Of the pharate adults that died  without attempting to eclose, those that died with visible collapse and shrinkage of the abdomen were classified as "dead with pupal exuviae shrunken" (Table XII). dessication. unknown.  These insects were assumed to have died through  The cause of death of apparently normal pharate adults was  No association could be made between diet and treatment and the  number dying in the "shrunken" or "normal" categories. No errors were made in October 1985 in identifying pharate adults in prolonged diapause. 100%.  Survival of 2YD pharate adults ranged from 0 to  For both BIO and RN diets, 2YD pharate adults reared on unchanged  - 85 -  Table X I I .  Summary of m o r t a l i t y through the pharate adult stage of B. c o l f a x i a n a reared on 14 d i e t s and two media c o n d i t i o n s (Experiments 1 and ? ) , 17 October 1985 to 8 A p r i l 1986.  Post-winter  Number Diet and Treatment  Number Overwintered (1YD/2YD)  Experiment 1: 610 Changed Unchanged  Alive  Number Dead  Emerged Deformed Normal Wings  in 2YD  Died Pupal Pupal E c l o s i n g Exuviae Exuviae Normal Shrunken  Overwinter survival (%)c  Emergence Index  21 /17 35/6  12(57.1)9 9(25.7)  1 1  10(58.8)*> 0(0)  2 5  8 7  5 19  63.2 36.6  82.1 63.3  45/15 42/10  24(53.4) 20(47.6)  1 4  10(66.7) 5(50.0)  6 9  11 12  8 2  71.9 73.1  79.0 66.7  Experiment 2: RN-LA-AA RN-LA RN-LA+AA  1/2 3/3 1/1  0(0) 1(33.3) 0(0)  0 0 0  1(50.0) 0(0) 0(0)  1 0 t  1 2 0  0 3 1  66.7 33.3 50.0  RN-AA RN+AA RN+3xAA RN+Cholesterol  16/7 27/3 27/3 23/7  12(75.0) 12(44.4) 15(55.6) 11(47.8)  0 2 2 1  3(42.9) 3(100) 2(66.7) 4(57.1)  4 5 3 6  1 2 6 7  3 6 2 1  82.6 73.3 73.3 73.3  75.0 68.4 80.0 63.9  RN+LA-AA RN+LA RN+LA+AA  24/12 38/10 32/11  20(83.3) 26(68.4) 21(65.6)  0 3 3  5(41.7) 7(70.0) 7(63.6)  3 3 4  2 7 7  6 2 1  77.8 81.3 81.4  87.0 85.9 80.4  RN+3xLA RN+3xLA+3xAA  36/19 12/6  20(55.6) 5(41.7)  1 2  16(84.2) 1(16.7)  8 0  3 4  7 6  81.8 44.4  70.7 85.9  RN  Changed Unchanged  BIO B i o s e r v Spruce Budworm d i e t RN Robertson's (1981) Western Spruce Budworm d i e t AA L-ascorbic acid LA 55% l i n o l e n i c a c i d % s u r v i v a l of overwintered 1YD pharate adults emerging as normal ° % s u r v i v a l of overwintered 2YD pharate adults % s u r v i v a l of a l l overwintered pharate adults R  a  c  adults  0 100 0  - 86 media had poorer overwintering survival than those reared on changed media.  This difference may be insignificant. The 2YD.pharate adult  survival for RN+3xLA+3xAA was 16.7% while survival for the remaining diets with LA ranged from 41.7 to 100%.  There was no obvious  relationship between dietary LA (normal or higher concentrations only) or AA concentrations and the overwinter survival of 2YD pharate adults. Overall performance of the diets and treatments tested (assessed as the percentage of all hatched insects that survived through the winter) appeared to be related to the dietary concentration of LA (Table IX). Diets containing double and quadruple the normal concentration of LA were ranked the highest followed by normal concentration RN-type diets, then BIO and finally RN-LA diets.  Vials containing media replaced after two  weeks of rearing had slightly higher overall survival than unchanged media for RN and BIO. For all diets except those without LA, emergence index values ranged from 63.3 to 85.9 and varied between diets with no discernable pattern associated with the dietary concentration of LA or AA.  This demonstrated  that the diets or rearing method s t i l l may be inadequate for the production of normal adults regardless of the concentration of LA above the normal RN concentration. To summarize the survival results, increasing concentrations of LA appeared to proportionately increase the production and pre-winter survival of normal pupae but did not improve overwintering survival. Thus, partial solutions to the poor survival problem have been obtained by increasing dietary levels of LA.  - 87 Changing media two weeks after they were established with eggs produced only marginal improvements in survival.  The rearing method  and/or diet formulation requires improvement, particularly at increasing pharate adult survival before and during the overwintering stage and increasing the proportion of normal adult emergence, before being considered acceptable for a mass rearing program.  3.11.3 Pupal Weights  The t-test results listed on Table XIII indicated that there were no significant differences in pupal weight between 1YD and 2YD cone moths except for females reared on unchanged RN. Thus, pupal weight comparisons by diet/treatment and sex maximized all of the available samples by pooling 1YD and 2YD pupal weights.  T-tests comparing female  and male pupal weights for several diets and treatments showed that females were significantly heavier than males (alpha=.05) for all diets tested except RN+LA and RN+LA+AA (although the latter two diets were significant at alpha=.10) (Table XIV). For pupal weights recorded in Experiment 1, t-tests were used to determine if the transfer to fresh diets affected pupal weight and to determine if BlO-reared pupae were significantly different from RN-reared pupae (Table XV). For BIO, changing diets had no effect on pupal weight while for RN, unchanged pupae were significantly heavier than changed pupae (alpha=.05).  Within each sex and treatment category, BIO pupae  were significantly heavier than RN pupae except for females reared on unchanged media which were not significantly different.  - 88 Table XIII.  Comparison of mean pupal weights of 1YD and 2YD B_. colfaxiana reared on six artificial diets, May to October 16, 1985.  Mean Pupal Weight + S.D. mg (n)  Diet Type  Sex  1 YD  Female Male  13.9 + 3.2(28) 12.3 T 2.6(26)  14.5 + 3.4(6) 12.4 + 2.7(18)  N.S.D. N.S.D.  Female Male  11.7 + 3.4(21) 9.7 + 2.7(23)  10.7 + 4.2(4) 9.4 + 1.8(11)  N.S.D. N.S.D.  Female Male  13.8 + 2.9(23) 12.0 2.5(17)  9.5 + 4.3(6) 11.2 2.4(3)  N.S.D.  Female Male  11.2 + 4.0(11) 7.5 T 2.3(13)  9.0 + 2.5(5) 6.7 T 3.9(7)  N.S.D. N.S.D.  RN+LA  Female Male  11.7 + 3.6(14) 10.1 + 3.1(13)  10.3 + 2.2(4)  9.2 + 3.4(7)  N.S.D. N.S.D.  RN+LA+AA  Female Male  11.7 + 3.9(19) 10.5 3.1(13)  11.0 + 3.8(5) 8.3 2.6(6)  N.S.D. N.S.D.  RN+3XLA  Female Male  11.2 + 3.3(17) 8.7 + 3.4(18)  11.8 + 2.6(6) 10.0 T 1.3(11)  N.S.D. N.S.D.  Experiment 1: BIO Changed + Unchanged RN Changed^: Unchanged Experiment 2: RN+LA-AA  2YD  t-test Results  *  RN Changed male and female mean pupal weights were significantly less than Unchanged mean male and female pupal weights (Table XV). N.S.D. = Not significantly different, P< 0.05, * = significantly different at P< 0.05.  - 89 Table XIV.  Diet Type  Comparison of mean pupal weights of female and male J3. colfaxiana (1YD and 2YD data combined) reared on six artificial diets, May to 16 October 1985, and reared naturally in cones, October 1983.  Mean Pupal Weight + S.D. mg (n) Female  Male  t-test Results  Experiment 1: BIO Changed + Unchanged  14.0 + 3.2(34)  12.3 + 2.6(44)  **  RN Changed^ Unchanged  11.4 + 3.4(26) 13.1 T 3.7(30)  9.5 + 2.5(35) 12.0 T 2.6(22)  ** N.S.D  Experiment 2: RN+LA-AA  10.5 + 3.7(16)  7.2 + 2.9(20)  **  RN+LA  11.4 + 3.4(18)  9.9 + 3.2(30)  RN+LA+AA  11.5 + 3.8(24)  9.8 + 3.0(19)  *  RN+3xLA  11.4 + 3.2(23)  9.2 + 2.8(29)  **  Natural, 1983  15.9 + 2.6(45)  14.0 + 2.3(52)  **  N.S.D  @  RN Changed male and female mean pupal weights were significantly less than Unchanged male and female mean pupal weights (Table XV).  #  N.S.D. = Not significantly different, * = significantly different at P< 0.05, ** = significantly different at P< 0.01.  - 90 Table XV.  Mean Pupal Weight + S.D. mg (n)  Diet Type Experiment 1: BIO Female  RN  Comparison of mean pupal weights of female and male j5. colfaxiana (1YD and 2YD data combined) reared on changed and unchanged BIO and RN diets, May to 16 October 1985.  Unchanged  Changed  t-test Results  Significance  14.4 + 4.0(15)a  14.5 + 2.5(19)a  N.S.D.*  .51  Male  12.1 + 2.3(23)a'  13.2 + 2.9(22)a'  N.S.D.  .84  Female  11.6 + 3.4(25)b  13.3 + 3.5(28)a  **  .99  11.6 + 2.7(23)b'  *  .96  Male  @  9.6 + 2.4(35)b'  Means within each column followed by a different letter are significantly different, Student's t-test, P< 0.05. a,b = Females, a',b' = Males. N.S.D. = Not significantly different, * = significantly different at P< 0.05, ** = significantly different at P< 0.01.  - 91 Figure 11 graphically shows the relationship between mean pupal weight and all diets tested in 1985.  No obvious relationship could be  seen, mainly due to the wide differences in sample size from which each mean was derived from.  The effects on pupal weight of varying  concentrations of LA and AA were assessed using Student-Newman-Keuls (SNK) multiple range test for selected diets (including 1983 naturally-reared pupae) and for each sex (1YD and 2YD data were pooled)(Table XVIa and b).  Because of the highly variable survival on  each diet type and the equal or near-equal sample size requirement for the SNK test (Winer 1971), sample sizes for each diet were reduced to a minimum number equal to the smallest sample size of the diets included in the comparison.  Samples were randomly eliminated from each data set  until the desired minimum was attained.  The comparisons of mean pupal  weights from the diets tested in experiment 2 were done twice, the first comparison included nine diets (including unchanged BIO and 1983 naturally-reared pupae) with sample sizes of 13 and 12 for female and male pupae, respectively (Table XVIa).  The second comparison was the  same as the first except it included only four of the most productive diets resulting in samples sizes of 23 for females and 29 for males (Table XVIb).  The results of the first analysis were, for females, that  all diets except RN+LA-AA, RN+Cholesterol and RN+3XAA were not significantly different in weight than naturally-reared (1983) pupae; and, for males, five of the nine diets produced pupae that were significantly different in weight than naturally-reared pupae: RN+3XLA, RN+LA+AA, RN+AA, RN+LA-AA and RN+3XAA. The second analysis revealed  - 92 -  Legend  E2 FEMALES • i MALES  a> E x  o m —j  2  CL Z < LLI  2  DIET  TYPE  F i g u r e 1 1 . Mean pupal w e i g h t s o f B. c o l f a x i a n a r e a r e d on a r t i f i c i a l d i e t s c o n t a i n i n g v a r y i n g concentrations of l i n o l e n i c a c i d . A l s o i n c l u d e d are mean pupal w e i g h t s o f 1983 n a t u r a l l y - r e a r e d and 1985 Bioserv® Budworm (unchanged) r e a r e d cone moths.  - 93 Table XVI. Comparison of mean pupal weights of B. coifaxi ana reared on artificial and natural diets. Comparison A includes all diets with greater than 12 female and 11 male pupae. Comparison B includes all diets with greater than 22 female and 28 male pupae. Mean Pupal Weight + S.D. mg (n) Diet Type  Female^  Male@  Comparison A: Natural, 1983  15.2 + 2. 5 13)a*  13.6 + 1.8 12)a  BIO Unchanged  14.1 + 2. 6 13)ab  12.5 + 2.5 (12)ab  RN Unchanged  13.0 + 3. 3 13)ab  11.8 + 2.3( 12)ab  RN+3xLA  12.3 + 3. 3 13)ab  10.1 + 2.3 J2)bc  RN+LA  12.1 + 3. 6 13)ab  10.8 + 3.6( 12)abc  RN+LA+AA  11.8 + 3. 7 13)ab  9.5 + 2.9 12)bc  RN+AA  11 .3 + 2. 9 13)ab  7.8 + 1.7( 12)bc  RN+LA-AA  10.7 + 3. 8 13)bc  7.7 + 3.4 [12)c  RN+Cholesterol  10.7 + 1. 7 13)bc  10.8 + 2.6( 12)abc  9.7 + 2. 1 13)c  7.8 + 1.7 [12)c  Comparison B: Natural, 1983  15.2 + 1. 9 23)a  13.4 + 2.0 [29)a  BIO Unchanged  14.3 + 2. 9 23)ab  13.1 + 2.8 (29)a  RN Unchanged*  12.2 + 3. 9 23)bc  10.4 + 2.7 [29)b  RN+LA+AA  12.1 + 3. 8 23)bc  RN+3xLA  11.7 + 3. 1 23)c  RN+3xAA  RN+AA @  * i  9.7 + 2.8 [29)b 10.6 + 3.1 [29)b  1 YD and 2YD data combined six males from changed RN were added to get n=29 Means followed by a different letter are significantly different, Student-Newman-Keuls test, P< 0.05.  - 94 that, for both sexes, RN-based diets produced significantly lighter pupae than those reared on cones.  Female BlO-reared pupae were significantly  heavier than RN+3XLA-reared pupae and male BlO-reared pupae were significantly heavier than those reared on RN, RN+3XLA and RN+LA. Thus, even though BIO survival was inferior to RN-based diets with LA (especially RN+3XLA), pupal weight analyses showed that BIO produced pupae were most similar to those collected from cones.  Within the RN  diets compared in both analyses, only two (i.e. RN+LA-AA and RN+3XAA) were significantly different in weight. The final pupal weight comparison summarized the results of four years of artificial rearing on normal concentration, unchanged Budworm diets (i.e. 1966-McMorran s, 1984 and 85-Bioserv*s, and 1983 and 1  85-Robertson's)(Table XVII).  For female pupae, 1983 naturally-reared  pupae were the heaviest followed by 1966 and 1985 artificially-reared pupae and 1984 naturally-reared pupae.  1983 and 1984 artificially-reared  pupae were not significantly different from each other but were significantly lighter than pupae from all other rearing conditions. Male pupae were similar to females except 1966 and 1985 (BIO) artificiallyreared pupae were not significantly different from 1983 naturally-reared pupae.  Thus, 1985's RN-reared pupae were equivalent in weight to 1966's  artificially-reared pupae and to 1984's naturally-reared pupae. Pupae extracted from cones in 1983 were, for some unknown reason, significantly heavier than those collected in 1984.  - 95 Table XVII. Comparison of mean pupal weights of B. colfaxiana reared on three Spruce Budworm diets in 1966, 1983, 1984 and 1985, and pupal weights of cone moths extracted from cones collected from Keremeos, B.C. in 1983 and 1984.  Mean Pupal Weight + S.D. mg (n) Rearing Regime Formulation/Year  Female  Budworm Diet: McMorran 's (1965)/1966 Robertson's (1979)/!983  Male  14.0 + 3.1(19)b#  12.5 + 2.0(17)ab  8.7 + 2.9(19)c  8.1 + 2.5(17)c  9.0 + 2.3(19)c  8.7 + 3.0(17)c  Robertson's (1981)/1985  13.6 + 3.4(19)b  11.7 + 2.3(17)b  Bioserv® /1985^  14.0 + 3.3(19)b  12.6 + 2.3(17)ab  Cones: 1983  16.4 + 2.2(19)a  13.7 + 1.4(17)a  1984  12.6 + 1.9(19)b  10.6 + 1.8(17)b  @  Bioserv® /1984  Source: D.S. Ruth, unpublished data. Means followed by a different letter are significantly different, Student-Newman-Keuls test, P< 0.05. Unchanged media only.  - 96 3.11.4 Incidence of Prolonged Diapause Relationship With Diet  In Experiment 1, replacement of media appeared to have little effect on the incidence of 2YD for either RN or BIO diets although the latter had a larger difference between changed and unchanged treatments (Table XII).  For example, pharate adults resulting from changed media had a  higher incidence of 2YD than those reared on unchanged media (i.e. 44.7% vs. 14.6% for BIO and 25.0% vs. 19.2% for RN). Individually, these differences were not significant, however, when the data for both diets was combined, the differences between changed and unchanged media was significant (i.e. 33.7% for changed media, n=98, vs. 17.2% in 2YD, n=93, for unchanged media) at the 95% level of confidence (comparison of two proportions, Walpole 1974). Experiment 2 resulted in a fairly narrow range of 2YD incidences over the 11 RN-based diets examined (i.e. 12 to 33% in 2YD). Figures 12 and 13 showed that no distinct relationships were obvious between the dietary concentration of either LA or AA and the incidence of 2YD. Relationship With Rearing Temperature  Because of the relatively narrow range of prolonged diapause incidences noted above, all of the diapause data from 1985 s artificial 1  rearings were pooled to obtain an overall average of 24.9 + 8.7% in 2YD  - 97 -  Figure 12. Prolonged diapause incidence of B. colfaxiana pharate adults reared on Robertson's (1981) Budworm diet (RN) containing three concentrations of linolenic acid. RNU1 and RNU2 = unchanged RN diet, vials no. 1-100 and 101-200, respectively.  - 98 -  HI CO  D  < <  18  36 N=23rz  3044  QL Q  Q LU  CD 2  20-  O  _l  o  cc 0.  z  30  10'A V/A  -AA  NORMAL [AA] DIET  30  ca  DOUBLE QUADRUPLE  T Y P E  Figure 13. Prolonged diapause incidence of B. c o l f a x i a n a pharate adults reared on Robertson's (1981) Budworm d i e t (RN) containing four concentrations of L-ascorbic a c i d . RNU1 and RNU2 = unchanged RN d i e t , v i a l s no. 1-100 and 101-200, respectively.  - 99 (n=499).  Hedlin et a l . (1982) presented results which demonstrated a  significant inverse relationship between artificial rearing temperature and the incidence of 2YD. To determine if 1983 to 1985's artificial rearing results agreed with these earlier results, diapause data from 1983 and 1984 artificial rearings, along with 1985's overall average, were included into Hedlin et al.'s (1982) data set and a simple linear regression was run on it using MIDAS (Fox and Guire 1976).  Data from  Hedlin et al.'s (1982) test of five photoperiods were summed and included as the data point for 23°C.  As observed in the graph (Fig. 14), the  new data appears to f i t well with the earlier data and the relationship remained significant (r=-0.85) although the correlation coefficient in Hedlin et al.'s (1982) analysis was r=-0.94. In contrast, the incidence of prolonged diapause of naturally reared pupae collected during the same years (1983 to 85) differed considerably from the artificially reared pupae (Fig. 14).  Due to sample size  limitations (i.e. the total number of samples must exceed 50)(Walpole 1974), statistical analysis of the differences between the 2YD incidences of naturally and artificially reared cone moths was only possible for 1985's data which showed that a significant difference occurred between the two proportions (Walpole, 1974).  Thus, it would appear that  temperature significantly affects the induction of 2YD.  - 100 -  100 - i  Figure 14. Prolonged diapause incidence of EL colfaxiana pharate adults reared on four Spruce Budworm diets at seven different constant rearing temperatures (regression line); and for naturally reared pharate adults collected from 1982 to 1985. Data for 1982 naturally-reared insects was obtained from Sahota et a l . (1985). All artificially-reared insect data (except for 1983 to 1985) was obtained from Hedlin et a l . (1982).  - 101 3.11.6 Disease Analysis Results of the disease analysis performed by Mr. Gerard Thomas (Consulting Diagnostic Services, Berkley, CA) reported that no primary pathogens were detected on either dead "marbly" or "in cocoon" L4's. Only secondary, saprophytic bacteria in insignificantly small numbers were discovered (Appendix lib). Sections of two "healthy" L4's (collected at Mesachie Lake, B.C.) and two "marbly" L4's were examined for the presence of polyhedrosis inclusion bodies using Hamm's stain.  No inclusion bodies (which stain  red with Hamm's stain) were found. Dr. Cunningham's original diagnosis in 1984 was confirmed in 1985 samples, namely that no pathological organism was responsible for either the "marbly" or "in cocoon" L4 mortality symptoms.  Thus, pathogens are  an improbable cause of mortality and these unusual symptoms of larval mortality are probably related to diet and/or environmental conditions.  3.12 Discussion, 1985  The rearing method and.all diets tested in 1985 provided excellent survival through the egg to the L4 stage.  However, L4 survival was poor  as noted in my earlier rearing attempts, and appeared to be related directly to the dietary concentration of LA. Pupal production and survival also appeared to be directly related to the concentration of LA contained in the media, although little additional improvements in  - 102 post-winter survival and adult emergence success were observed beyond the LA concentration found in RN diet.  The best survival obtained in 1985  (37.8%) may still be considered too low to rate the current rearing method as acceptable (survival of +75% was desired), thus, it appears that improvements in either diet or methods are required to obtain higher overall survival, particularly during the pre-winter stage.  Although  increased levels of LA in the diet promoted L4 survival, adult emergence indices did not exceed 86.  Dadd (1983) obtained an emergence index of  100 for greater wax moths reared on diets containing LA. Thus, some aspect of the rearing method (diet other than LA concentration or rearing environment) may be responsible for the sub-optimal adult emergence. Further rearing work should examine these aspects in greater detail and should include an assessment of adult emergence index of naturally-reared cone moths extracted from cones and overwintered under the same conditions as the artificially-reared insects.  This latter study would  provide a comparison for rating the success of rearing methods but a means of avoiding mite predation must be found to reduce overwinter mortality. The demonstrated bio-activity of LA in Experiment 2 provided another example to a growing list of phytophagous insects that require LA for successful adult eclosion.  Improved survival or development of larvae  and pupae by the addition of LA (observed in Experiment 2) has been reported less frequently than for its effects on adults (Dadd 1983). Polyunsaturated fatty acids (PUFA) are believed to be incorporated into the insect integument (Navon et a l . 1985).  Therefore, PUFA  - 103 deficiencies result in defects in the integument which may lead to dessication and moulting problems.  Pupal survival in particular is  likely very dependent on the production of normal integument. LA may also be important in the production of juvenile hormone. Supernumerary L4's and incompletely developed pupae that were reared on LA deficient diets may have developed from conditions that upset the normal juvenile hormone levels prior to pupation.  This relationship  between dietary LA and pupal developmental problems has not been explored in the literature, therefore, an explanation of LA's role in development is not available. Creswell et a l . (1971) noted incompletely moulted pupae in sub-optimal diets but did not identify LA or other PUFA's as the deficient nutrient(s). This thesis has demonstrated that the cone moth requires unpurified linolenic acid for normal development.  Future experiments should  determine the precise requirements of the cone moth for PUFA's, both in terms of concentration and composition.  Purified linolenic acid,  linoleic acid, and other PUFA's should be tested at varying concentrations and combinations with experiments similar to those reported by Dadd (1983). The absence of any effect of altering dietary AA may have resulted from the complete oxidation of AA within the first two weeks of rearing in all diets tested.  Dadd et a l . (1967) recorded rapid rates of AA  degradation from a liquid diet; these rates may also apply to the agar-based spruce budworm diets.  AA may s t i l l be an essential nutrient  - 104 for the cone moth but may be required only at very small quantities (this minimum may have been exceeded by the quantity contained in the vitamin mix alone), and/or the minimum quantity required to complete development was obtained during initial feeding on the diet or the bract. The product of AA oxidation, dehydroascorbic acid, also has vitamin C activity which is lost when the lactone ring of dehydroascorbic acid is hydrolyzed (Lehninger 1979).  Thus, vitamin C activity may not be lost in  artificial diets that have lost all AA through oxidation.  The exact  physiological function of AA in some animals requiring it is unknown, including insects (Lehninger 1979).  Navon et a l . (1985) described  various symptoms of AA deficiency of noctuid and sphingid larvae and pupae.  Larval symptoms included bloating and a decline in tensile  strength of the cuticle that were associated with disorders in food assimilation and alteration of cuticle composition. AA is believed to be required as a cofactor for normal collagen production and also by fatty acid and hydrocarbon hydroxylases of the insect integument (Navon et a l . 1985).  Bloated larvae were occasionally found during 1985's cone moth  rearing but could not be related to initial dietary AA concentration. Pupal symptoms of AA deficiency included a failure for the complete extension of the "sclerotized regions of the wing pockets such that a portion of the untanned abdomen was visible" (Navon et a l . 1985).  This  symptom was observed in pupae reared on unchanged BIO an RN diets and was classified as "white belly" (Table XI), however without analysis  - 105 of AA concentrations in the diet, the cause of this symptom remains unknown.  Conversely, a requirement for AA for the cone moth cannot be  ruled out from my results. Pre-pupal mortality of L4's within cocoons was nearly consistent for all diets and treatments.  This independence from diet composition  suggests that a physical problem may have killed these larvae.  Dead L4's  in cocoons appeared to be smothered by the wet webbing that clung to them.  Perhaps the webbing did not adhere to the walls of the tunnels  within the media as it does in cones.  Consequently, struggling caused by  the L4 may have bound the webbing tighter and death may have resulted from exhaustion and/or asphyxiation.  There are two solutions to this  putative problem. The first, would be to transfer the late L4 larvae to a rigid, non-shrinking surface that webbing readily adheres to (eg. cardboard).  Cocoon construction would then be safe from excess moisture  and would prevent the cocoons from collapsing as a result of detached webbing.  In addition, safe extraction of pupae would be facilitated by  this method. A constant vigil is required to time the transfer of each L4 after feeding had ceased, therefore, this process would involve a substantial amount of labour over a short period. Alternatively, all L4's could be transferred when the first cocoon is constructed and fresh media supplied (in addition to the sterilized cardboard substrate) to larvae that had not finished feeding.  Dead L4's in cocoons were not  observed by Ruth and Hedlin (1969) who removed the L4's just before pupation and placed them on a fresh block of media; this procedure prevented them from pupating within the media.  Thus, a second solution  - 106 to L4 mortality within cocoons may be to adopt Ruth and Hedlin's (1969) method since it appears the most practical and has been shown to be effective. Pupal weights did not provide the best means of ranking the most suitable diet.  For example, BIO had one of the poorest overall survival  rates but had mean male and female pupal weights that were the closest to naturally occurring pupae.  Conversely, cone moths reared on RN+3xLA,  which had the best overall pre-winter survival, had male and female mean pupal weights that were significantly lighter than pupae extracted from cones.  The unexpected highly significant difference between changed and  unchanged RN-reared pupae (i.e. changed pupae were lighter than unchanged pupae) could not be explained. Although no relationship between diet and the incidence of prolonged diapause has been reported in the literature, some evidence suggested that normal insect diapause induction is regulated by food quality (Visscher Newman 1982).  No relationships between levels of dietary LA or  AA and the incidence of 2YD were noted in this thesis.  Similarly,  Vanderzant and Reiser (1956) found that changes in dietary protein, carbohydrate, and fat contents of artificial media did not affect the number of pink bollworms entering diapause. The significant differences between naturally and artificially reared cone moth 2YD incidences in 1983 to 1985 strongly suggests that the rearing environment (including the diet) affects the induction of 2YD.  Hedlin et a l . (1982) reported a significant inverse correlation of  constant rearing temperature to the proportion of cone moths that entered  - 107 2YD which was also supported by my artificial rearing data from 1983 through 1985.  Thus, temperature may be the most important factor  regulating prolonged diapause induction, assuming no interactions exist between diet and rearing temperature.  To test this assumption,  comparisons should be made using cone moths reared on cones at the same temperatures used by Hedlin et al. (1982).  Performing this experiment,  however, may be difficult because of problems in maintaining fresh host material (mold growth would be uncontrolled) and handling-related mortality may be prohibitively high due to regular transfer of larvae to fresh cones.  An examination of the effect of rearing temperature of each  life stage on 2YD induction should be done to determine the most temperature sensitive period.  Insights into the effects of temperature  on the differentiation of lateral buds may be gained by identifying the hosts phenology that corresponds to the cone moth's temperature sensitive developmental stage. Yet unanswered is the question of whether plant growth regulators affect the incidence of 2YD in cone moths.  With the rearing method  developed in this thesis, a simple experiment could be performed to determine the effects of adding increasing quantities or different types of plant growth regulators to the artificial media on the proportion of pharate adults that enter prolonged diapause. I believe that a satisfactory rearing method and diet have been developed in this thesis.  Further improvements may be obtained by slight  modifications in the L4 rearing procedure, and by determination of the missing nutrients or physical factors that may have limited pharate adult  - 108 survival.  Adult behaviour, particularly in the production and response  to semio-chemicals (eg. host odours and sex pheromones), should be added to the existing parameters used to determine the success of a particular diet or rearing method.  Once these improvements are made, the cone moth  may be reared with less labour than was used in this thesis and by Ruth and Hedlin (1969).  The only labour intensive periods would be during  diet establishment, L4 transfer, and pupal storage.  r  109 -  4.0 FIELD STUDY 4.1 Introduction  The relationship between the incidence of prolonged diapause and cone crop size described by Hedlin et a l . (1982) was based on cone crop and cone moth data sampled throughout the study sites and did not examine the relationship within individual trees.  Other researchers have also  described this phenomenon for other cone and seed insect species with stand-based samples (Bakke 1963; Annila 1982 cited in Miller and Hedlin 1984; Saksons 1973). Two hypotheses were developed by Hedlin et a l . (1982) to explain the mechanism behind the observed synchrony between B_. colfaxiana and its host.  The first, as described earlier for the justification of the  artificial rearing study, was the existence of an ability for cone moth larvae to "read" various metabolic clues contained in the larval food allowing them to "forecast" the host's reproductive condition in the following year.  Thus, cone moth populations within individual trees  would have different proportions of pharate adults in prolonged diapause depending on the size of each host tree's cone crop in the year following larval feeding.  The alternative hypothesis was that prolonged diapause  incidence is controlled by a factor that is independent of the host. This host-independent hypothesis was supported by Hedlin et al.'s (1982) artificial rearing study which showed a significant inverse correlation between constant rearing temperature and the incidence of prolonged  - no diapause.  If the latter hypothesis is correct, individual tree sampling  should reveal little variation of prolonged diapause incidences between trees since the regulating factor would influence the cone moth populations on each tree equally for a given area. The objective of this field study was to determine which of the two hypotheses could be rejected by collecting both infested 1985 cones containing pharate adult cone moths and 1985 twiglets with 1986's reproductive buds from individual trees to obtain estimates of diapause status and 1986 cone crop estimates on an individual tree basis.  4.2 Materials and Methods  Forty-six interior Douglas-firs were sampled from the three Keremeos-area study sites described in section 2.1.1  and Figure 3 on 19 -  23 August 1985 (21 trees were sampled from "Outward Bound", 15 from "Dahlgard's", and 10 from Ashnola River).  From each tree, at least 50  infested cones (i.e. cones exuding frass from larval tunnels) were collected randomly from the middle and lower thirds of the crown and placed into labelled paper bags that were stapled closed.  In addition to  the cones, 25 1985 twiglets were randomly sampled from a single south-facing upper crown branch that was cut down with a telescoping pole-pruner.  To obtain consistency in sampling the upper crown, the  fifth to seventh whorl was sampled.  However, variation in tree height  and age probably produced major differences in sampling location within the upper crown.  These twigs were stored in labelled paper bags. An  - Ill estimate of height (+ 1 m) and a rough estimate of 1985's cone crop (light, moderate, and heavy) were made for each tree. tree vigour were also recorded.  Observations of  Each tree was then flagged and a tree  number was written on the flagging to aid in relocating the trees.  Trees  were selected using two criteria: 1) the upper crown must be within the reach of the pole pruner thereby limiting maximum tree height to about 16 m, and 2) the tree must have at least 50 cones that were easily collected with the pole pruner.  Trees with broken tops were not sampled.  Lateral buds were assessed in the lab within two weeks of sampling. Each lateral bud was carefully examined and dissected under the dissecting microscope and were classified as female (megastrobili), male (megasporangia), and non-reproductive buds (Allen and Owens 1972). Non-reproductive buds were not classified further as vegetative, latent or aborted buds. Total numbers and percentages of female, male and non-reproductive buds per tree were tabulated. Cones were stored at room temperature at UBC from 24 August to 30 September 1985.  Cone dissection was delayed until all pharate adults  should have differentiated into 1 YD or 2YD (Sahota et a l . 1985). Cone dissection and pupal extraction began on 1 October and was completed on 15 November 1985. 2,115 living pupae.  A total of 2,575 cones were dissected which yielded Each pupa was sexed and classified as in 1YD or 2YD  with the method described by Sahota et a l . (1985). Untransformed diapause percentages and bud counts were plotted and correlation coefficients were calculated for individual sites and for all sites combined.  - 112 4.3 Results Six of the 46 trees sampled were omitted since too few live pupae (i.e. fewer than 26 pupae) were extracted to obtain an acceptable estimate of prolonged diapause incidence.  Correlation coefficients for  individual sites (Fig. 15 a-c) and all sites combined (Fig 15d) were all insignificant.  In Figure 16, the number of outlying points decreased and  the consistency of the prolonged diapause condition between trees became more apparent as the number of observations for each diapause assessment increased.  These data suggested that the incidence of prolonged diapause  was not related to the proportion of female buds per 25 twiglets sampled for each tree.  Furthermore, the narrow range of points about an average  of 56.1 + 7.6% in 2YD (for trees with > 50 pupae, n=15) revealed that the inter-tree prolonged diapause variation was low (Fig. 16).  4.4 Discussion  There appears to be little evidence to support the hypothesis that cone moths adjust their diapause condition in response to the reproductive condition of the individual tree.  However, difficulties  exist in accepting the statistical validity of the cone crop estimate used in this sampling study.  Cone crop estimates for an entire tree can  not be adequately made from a single branch sample due to the extreme branch-to-branch variation that exists within and between individual  - 113 -  90  a) Outward Bound  80  A  70 H 60  b) Dahlgard's A  A A  50  A A ^ A A  A  A  AA  A A  A  A  r=.300 n=13  30  A  90  i i i i i i i i i i  80 70  ^  A  r=-.143 n=19  40  A  A A  c) Ashnola River A A  60 50 40  r=.472 n=8  r=.005 n=40  30 20  A T-T i i i i i l i i i i i i i i i  0 1 2 3 4 5 6 7 8 91011121314  1 i i i i i i i i i i i i i i i i  0 1 2 3 4 5 6 7 8 91011121314  % FEMALE BUDS PER 25 TWIGS  Figure 15. The proportion of cone moths in prolonged diapause and the proportion of the total number of lateral buds that were female on 25 1985 twigs (sampled from a single upper crown branch collected from individual trees) from the three Keremeos study sites and analyzed separately (a-c) and together (d).  - 114 -  Legend 80  A  >40 Pupae/Tree  X  >50 Pupae/Tree  X X  70  A  X 60  o  QC  OL  X x X X XX  50  A For > 5 0 pupae/tree: r=0.472, n=15 For > 4 0 pupae/tree: r=0.500, n=22  X A  40-  A 30  H 0  1  2  1  4  1  6  1  8  1  10  % FEMALE BUDS PER 25 TWIGS  Figure 16. The proportion of cone moths in prolonged diapause and the proportion of the total number of lateral buds that were female on 25 1985 twigs (sampled from a single upper crown branch collected from individual trees) from the three Keremeos study sites. Only data from trees with > 50 (crosses) and > 40 (triangles) live pupae were plotted.  - 115 whorls (G.E. M i l l e r \ pers. comm.; Schenk et al. 1972). Similar problems were encountered by Campbell et al. (1984) in estimating western spruce budworm larval densities per crown strata or per tree based on individual branch samples.  Another problem with my cone crop estimation  method was there was no means of determining if the range of buds counted corresponded to a wide range of cone crops.  The maximum proportion of  female buds in any sample was only 14.8% and this is considered to be a "light" crop (G.E. Miller^, pers. comm.). Regardless of the accuracy of the relative cone crop estimates in this study, the data revealed that a relatively narrow range of 2YD incidences exists within the stands sampled.  Confidence limits could  have been calculated about each prolonged diapause estimate for each tree (based on a sample of n- pupae) if an estimate of the total number of cone moths per tree (N^) had also been made.  The cone moth population  per tree could be calculated by 1) estimating the number of cones per tree, 2) obtaining the average number of live pupae per cone from samples from all trees, and 3) multiplying the number of cones per tree by the average number of pupae per cone.  Thus, if n.. approached FT (which  produces a narrowing of the confidence limits about the estimate), the 2YD percentage obtained from trees with low numbers of live pupae (i.e. less than 25 live pupae per tree) would be included into a correlation analysis.  Program Director, Pacific Forestry Centre, Canadian Forestry Service, Victoria, B.C.  - 116 Temperature is likely the universal factor controlling the induction of 2YD, as was concluded from Hedlin et a l . ' s (1982) rearing work. Further field studies should compare the within stand variation of 2YD incidences from sites that are spaced far enough from each other so they will differ climatically.  Such a study may reveal an increase in  variation in 2YD incidences as climatic conditions during the larval period become either cooler or warmer than at the Keremeos study site. Miller et a l . (1984) reported that for coastal Douglas-fir, there was no relationship between cone crop size (per stand) and the incidence of prolonged diapause.  This may indicate that the coastal cone moth  populations have not evolved a method of synchrony with their host because of lower selection pressure to do so, or perhaps early summer weather on the coast, which can be more variable than in the interior, may not be suitable for the insects to "predict" the size of the cone crop in the following year.  Future studies should also examine the  within tree variation of 2YD incidence which may lead to stratification of crowns for future prolonged diapause research involving individual tree cone moth sampling. The influence of crop size on 2YD on an individual tree level should be re-examined with a more precise method of cone crop estimation either using reproductive bud counts or by assessing the same trees in the following summer. Without this information, uncertainty remains regarding the role of the host in the induction of 2YD in cone moths.  - 117 5.0 REFERENCES Allen, G.S. and J.N. Owens. 1972. The life history of Douglas-fir. Environment Canada. For. Serv., Ottawa. 139pp. Annila, E. 1981. Fluctuations in cone crop and seed insect populations in Norway Spruce. Comm. Instituti Forest. Fenn. 101. Annila, E. 1982. Diapause and population flutuations in Megastigmus specularis Whalley and Megastigmus spermatrophus Wachtl (Hymenoptera:Torymidae). Ann. Ent. Fenn. 48: 35-56. (cited in Miller and Hedlin 1984). Bakke, A. 1963. Studies on the spruce cone insects Laspeyresia strobi1 leia(L.)(Lepidoptera: Tortricidae), Kaltenbachiola strobi (Winn.)(Diptera:Itonidae) and their parasites (Hymenoptera) in Norway. Medd. Norska Skogforsokev. J_9: 1-151. Bakke, A. 1971. Distribution of prolonged diapausing larvae in populations of Laspeyresia strobilei la L. (Lep., Tortricidae) from spruce cones. Norsk. Ent. Tidsskr. ]8: 89-93. (cited in Miller and Hedlin 1984). Barras, S.J. and D.M. Norris Jr. 1965. In vitro establishment and development of Eucosoma sp. larvae from cones of Pinus resinosa on an artificial nutrient medium. J. Econ. Entomol. 58(5): 1083-1084. Bell, R.A., C D . Owens, M. Shapiro and J.R. Tardif. 1981. Chapter 6.5. Mass rearing and virus production. In: The Gypsy Moth: research towards integrated pest management. (C.C. Doane and M.C. McManns, eds.).USDA For. Serv. Science and Education Agency. Tech. Bull. 1584. 757pp. Campbell, R.W., N. Srivastava, T.R. Torgenson and R.C. Beckwith. 1984. Patterns of occurence of the Western Spruce Budworm (Lepidoptera: Tortricidae): larvae, pupae and pupal exuviae, and egg masses. Environ.Entomol. J_3: 522-530. Ching, Te May. 1963. Fat utilization in germinating Douglas-fir seed. Plant Physiol. 3 8 ( 6 ) : 722-728. Creswell, M.J., E.E. Sturgeon and R.D. Eikenbary. 1971. Laboratory rearing of the Nantucket pine tip moth, Rhyacionia frustrana, on an artificial diet. Ann. Ent. Soc. Amer. j54: 1 159-1 163. Clark, G. 1981. Staining Procedures. Wilkins, London, U.K. 512 pp.  Fourth Edition.  Williams and  - 118 Dadd, R.H. 1973. Insect nutrition: current developments and metabolic implications. Ann. Rev. Entomol. U5: 381-420. Dadd, R.H. 1983. Long-chain polyenoics and the essential fatty acid requirements of the waxmoth, Galleria mellonella. J. Insect Physiol. 29(10): 779-786. Dadd, R.H., D.L. Krieger and T.E. Mittler. 1967.' Studies on the artificial feeding of the aphid Myzus persicae (Sulzer) - IV. Requirements for water soluble vitamins and ascorbic acid. J. Insect Physiol. U: 249-272. Dobbs, R.D., D.G.W. Edwards, J. Konishi and D. Wallinger. 1976. Guidelines to collecting cones of B.C. conifers. B.C. For. Serv./Can. For. Serv. (Victoria) Joint Rep. 3. 98pp. Ebel, B.H. 1959. Laboratory rearing of a pine cone insect, Dioryctria abietella (D. and S.). J. Econ. Entomol. 52(4): 561-564. Ebel, B.H. and H.O. Yates III. 1973. Rearing and biological observations of a Southern pine cone insect, Eucosoma cocana (Lepidoptera: Olethreutidae). Ann. Ent. Soc. Amer. 66(1): 88-92. Eis, S. 1973. Cone production of Douglas-fir and Grand Fir and its climatic requirements. Can. J. For. Res. 3^: 61-70. Fatzinger, C.W. 1970. Rearing succesive generations of Dioryctria abietella (Lepidoptera: Pyralidae(Phyctidae)) on artificial media. Ann. tntomol. Soc. Amer. 63(3): 327-352. Fox, D.J. and K.E. Guire. 1976. Documentation for MIDAS (3rd Ed.). Statistical Research Laboratory, The University of Michigan. 203 pp. Funke, B.R. 1983. Mold control for insect-rearing media. Soc. Amer. (Summer 1983):41-44.  Bull. Ent.  Greig, M. and D. Osterlin. 1978. UBC ANOVAR. Analysis of variance and co-variance. UBC Computing Centre. 69 pp. Hedlin, A.F. 1960. On the life history of the Douglas-fir cone moth, Barbara colfaxiana (Lepidoptera: Olethretidae) and one of its parasites, Glypta eyetriae Cush. (Hymenoptera: Ichneumonidae). Can. Entomol. 92: 826-834T" Hedlin, A.F. 1965. Rearing cone insects in the laboratory on an artificial diet. Can. Dept. of Forestry. Bi-month. Prog. Rept. 21_(2):4.  - 119 Hedlin, A.F., G.E. Miller and D.S. Ruth. 1982. Induction of prolonged diapause in Barbara colfaxiana (Lepidoptera: Olethreutidae): correlations with cone crops and weather. Can. Entomol. 114(6): 465-471. Hedlin, A.F., J. Weatherston, D.S. Ruth and G.E. Miller. 1983. Chemical lure for male Douglas-fir cone moth, Barbara colfaxiana (Lepidoptera: Olethreutidae). Environ. Entomol. 12(6): 1751-1753. Hedlin, A.F., H.O. Yates III, D.C. Tovar, T.W. Koerber, and E.P. Merkel. 1980. Cone and seed insects of North American conifers. Can. For. Ser./U.S.D.A. For. Serv./Secret. Agric. Recur. Hidraul., Mexico. Victoria, B.C. 122pp. Ivonis, I. Yu, L.V. Kylina, V.B. Litvinova, and E.V. Khokhlina. 1982. Annual dynamics of gibberel1in-1ike substances in needles of Norway spruce clones of different sexes. Soviet Plant Physiol. 28(6,11): 915-920. (Cited in Forestry Abstracts 44(2)). Katayose, T. 1979. Seasonal changes in chemical components of Abies sachlinensis cones during the periods of fertilization and maturity. Research Bull., College Expt. Forests, Hokkaido Univ. 36(3): 633-662. (Cited in Forestry Abstracts). Keen, F.P. 1958. Cone and seed insects of western forest trees. USDA Tech. Bull. No. 1169. Kent, N.L. 1975. Technology of cereals, second Ed., Pergamon Press, Toronto, 306 pp. Knott, C M . , F.R. Lawson, and J.M. Hobgood, Jr. 1966. Oviposition cage for the Tobacco Budworm and the Corn Earworm. J. Econ. Ent. 59: 1290. ~ Lehninger, A.L. 1979. Biochemistry. York, N.Y. 1104 pp.  Second Ed. Worth Publ. Co., New  Lowry, W.P. 1966. Apparent meteorological requirements for abundant cone crop in Douglas-fir. For. Sci. 2/. 382-332. McMorran, A. 1965. A synthetic diet for the spruce budworm, Choristoneura fumiferana (Clem.)(Lepidoptera: Torticidae). Can. Entomol. 9_7: McMullen, E.E. 1980. Effect of applied growth regulators on cone production in Douglas-fir, and relation of endogenous growth regulators to cone production capacity. Can. J. For. Res. 10: 405-414.  - 120 Miller, G.E. 1984. Pest management in Douglas-fir seed orchards in British Columbia. In: Proc. cone and seed insects working party conf. IUFR0., Athens, Geo., Aug. 1983. Miller, G.E. and A.F. Hedlin. 1984. Douglas-fir cone moth and cone gall midge: relation of damage and prolonged diapause to seed cone abundance in British Columbia. In: Proc. cone and seed insects working party conf. IUFRO, Athens, Geo., Aug. 1983. Miller, G.E., A.F. Hedlin and D.S. Ruth. 1984. Damage by two Douglas-fir cone and seed insects: correlation with cone crop size. J. Entomol. Soc. B.C. 8]_: 46-50. Navon, A., J. Nesbitt, W. Henzell and H. Lipke. 1985. Effects of ascorbic acid deficiency on growth and cuticle composition of Manduca sexta and Spodoptera littoral is. Insect Biochem. 15(2): 285-291. Powell, J.A. 1974. Occurance of prolonged diapause in ethmiid moths. Pan-Pacific Ent. 50: 220-225. Puritch, G.S. 1972. Rept. BC-X-65.  Cone production in conifers.  Can. For. Serv. Inf.  Rawal, S.K. and D.C. Agrawal. 1982. Gibberellins in flowering and nonflowering trees of Pinus caribaea. Indian Forester 108(1): 62-65. (Cited in Forestry Abstracts 44(6)). Ross, S.D. and R.P. Pharis. 1976. Promotion of flowering in the Pinaceae by Gibberellins. I. Sexually mature, non-flowering grafts of Douglas-fir. Physiol. Plant. 36: 182-186. Robertson, J.L. 1979. Rearing the Western Spruce Budworm. Canada/USA Spruce Budworm program, USDA For. Serv., Washington, D.C. 18pp. Robertson, J.L. 1981. Unpublished revision to 1979 formula and preparation procedure for the Western Spruce Budworm diet. Ruth, D.S. and A.F. Hedlin. 1969. Rearing the Douglas-fir cone moth, Barbara colfaxiana (Kearfott), on an artificial diet in the 1aboratoTyl J. Ent. Soc. B.C. 66: 22-25. Sahota, T.S., D.S. Ruth, A. Ibaraki, S.H. Farris and F.G. Peet. 1982. Diapause in the pharate adult stage of insect development. Can. Entomol. 114: 1179-1183. Sahota, T.S., S.H. Farris and A. Ibaraki. 1983. Timing of initiation of pharate adult development in Barbara colfaxiana (Kft.)(Lepidoptera: Olethreutidae). Can. J. Zool. 61: 2305-2306.  - 121 Sahota, T.S., A. Ibaraki and S.H. Farris. 1985. Pharate adult diapause of Barbara colfaxiana (Kft.): differentiation of 1- and 2-year dormancy. Can. Entomol. 117(7): 873-876. Saksons, J.L. 1973. Insect fauna of the generative organs of pine (Pinus sylvestris L.) and spruce (Picea abies Karst.) in the Latvian S.S.R. Zaschita lesa: 29-52^ [Translation by Canadian Secretary of State-Multilingual Serv. Div., August 1, 1984], Schenk, J.A., D.D. Everson and J.R. Gosz. 1972. Sampling cone production in Douglas-fir stands for insect population studies. Forest, wildlife and range Exper. Stn., Moscow, Idaho. Stn. Paper No. 10. 10 pp. Singh, P. 1977. Artificial diets for insects, mites and spiders. IFI/Plenum Publ. Corp. New York, N.Y. 594pp. Turenen, S. 1974. Polyunsaturated fatty acids in the nutrition of Pieris brassicae (Lepidoptera). Ann. Zool. Fennici. _]_]_: 300-303. Van Vredenburch, C.L.H. and J.G.A. LaBastide. 1969. The influence of meteorolgical factors on the cone crop of Douglas-fir in the Netherlands. Silvae Genetica J_8: 182-186. Vanderzant, E.S., D. Kerur and R. Reiser. 1957. The role of dietary fatty acids in the development of the pink bollworm. J. Econ. Ent. 50(5): 606-608. Vanderzant, E.S. and R. Reiser. 1956. Aseptic rearing of the Pink Bollworm on synthetic media. J. Econ. Ent. 49(1): 7-10. Visscher Newmann, S. 1982. Plant growth hormones affect grasshopper growth and reproduction. L i : Proc. 5th int. symp. insect-plant relationships, Wageningen, 1982. (J.H. Visser and A.K. Minks, Eds.). 464pp. Walpole, R.E. 1974. Introduction to statistics. Publ. Co. Inc., New York. 340pp.  Second Ed. MacMillan  Wigglesworth, V.B. 1959. The control of growth and form: a study of the epidermal cell in an insect. Cornell Univ. Press, Ithaca, N.Y. 140pp. Williams, I.L. and C.W. Fatzinger. 1980. Duration of the larval, pupal, and moth stages of the southern pine coneworm, Dioryctria amatella (Hulst.). USDA For. Serv. Res. Note. SE-295. S.E. For. Expt. Stn., Asheville, N.C.  - 122 Winer, B.J. 1971. Statistical principles in experimental design. McGraw-Hill Book Co. Toronto. 907 pp.  - 123 APPENDIX I Diet Formulae and Mixing Procedures A - Robertson's (1979) Western Spruce Budworm Diet Group A: Distilled water Difco Bacto Agar Vitamin-free Casein Alphacel Salt mixture W Ground Wheat embryo  930 mL 40.5 g 58.5 g 9.0 g 15.0 g 49.5 g  Group B: Sucrose 43.5 g Distilled water 250 mL 4 M Potassium Hydroxide 10.0 mL 55% Linolenic acid 3.0 mL ICN Vitamin Fortification mixture 18.0 g Ascorbic acid 6.0 g Methyl parahydroxybenzoate 2.55g Potassium sorbate 1.8 g Aureomycin (14.1% chlorotetracycline) 1.4 g 37% Formalin .9 mL Yield:  1500 g of diet  Procedure (paraphrased from Robertson 1979): 1. Autoclave all ingredients in Group A together at 15 psi for 50 minutes at the liquid setting. 2.  While ingredients in group A are being autoclaved, measure those in group B and mix in the blender.  3.  Remove mixture A from the autoclave, pour it in the mixer bowl and stir with the flat beater attachment (Hobart mixer).  4.  When the temperature cools to 60° C, add the ingredients from group B.  5.  Stir this mixture at low speed until it cools to from 43° C to 45° C, then pour it into the desired containers. Do not pour the diet at tempertures above 45° C or the wheat embryo will settle during cooling. Below 43° C, the agar forms gelatinous lumps.  6.  The diet is ready to cut and use about 1 hour after it has been poured. It may be stored in the refrigerator.  - 124 B - Ingredients and Mixing Procedures for Bioserv® Diets Spruce Budworm Diet #9769 Ingredients (from the Bioserv®1985 catalog): Media-Gel, casein, salts W, fiber, wheat germ (toasted), MPH, aureomycin, ascorbic acid, choline chloride, sucrose, vitamin Mix #9624, 4 M KOH, formaldehyde. Quantities of each ingredient as listed in McMorran (1965) and Grisdale (1973) as cited in the catalog. Bioserv® would not reveal the exact quantities of ingredients used in their diets (trade secret). Codling Moth Diet #9370 Ingredients: Media-Gel, lima beans (finely ground), Brewer's yeast, sucrose, methyl paraben, ascorbic acid, linseed o i l , ground raw wheat germ, formaldehyde. Quantities of each ingredient as listed in Navon and Moore (1971)(and others) cited in the catalog. Nantucket Pine Tip Moth Diet #9624 Ingredients: Media-Gel, casein, fructose, cholesterol (USP), sorbic acid, choline chloride, wheat germ (toasted), alphacel, Brewer's yeast, ascorbic acid, linseed o i l , vitamin mix #9624. Quantities of each ingredient as listed in Creswell et a l . (1972) as cited in the catalog. Procedure (from package instructions): 1. Mix contents of agar pack into the recommended quantity of water. Bring to a boil and add entire contents of the dry mix package. 2.  Mix thoroughly for 2 minutes and dispense immediately.  - 125 C - Robertson's (1981) Western Spruce Budworm Diet Ingredients: Quantities used in Experiment 1 Group 1: Difco Bacto Agar Distilled water  101.5 g 2000 mL  Group 2: Distilled water 4 M KOH Kretschmer® wheat germ 55% Linolenic acid Group 3: ICN Vitamin mixture L-Ascorbic acid Group 4: Methyl para-benzoic acid Sorbic acid, Potassium salt Aureomycin (5.5% Chlorotetracycline) 37% formalin Group 5: Distilled water Vitamin-free Casein Alphacel Sucrose Salt mix W Yield:(approximate)  Quantities used in Experiment 2  32.4 g 640 mL  625 22.5 127.5 30  mL mL g mL  200 mL 7.2 mL 39.6 g -LA=0, LA=2.4, 2xLA= 4.8, 4xLA=9.6 g  45 15  g g  14.4 g -AA=0, AA=4.8, 2xAA= 9.6, 4xAA=19.2 g  6.4 g 4.5 g  2.0 g 1-4 g  4.5 g 2.3 mL  1-4 g .7 mL  325 146.2 22.5 108.7 37.5  mL g 9 g g  3750 g of diet = 350 vials/ batch (1.5 batches mixed)  104 mL 46.8 g 7.2 g 34.8 g 12.0 g 1200 g of diet = 105 vials/ batch (12 batches mixed)  - 126 Procedure (paraphrased from Robertson's 1981 unpublished procedure): 1.  Autoclave water and agar mixture (Group 1) for 40 minutes on the slow exhaust setting. Leave in the autoclave for 15 minutes with the door shut. Take the containers with the molten agar out and let them cool to 60° C. Test the temperature every 15 minutes.  2.  While Group 1 is autoclaving, blend the ingredients in Group 2 in the blender for 2 minutes at high speed.  3.  Add Group 3 to the blended Group 2 mixture and blend for 1 minute at high speed.  4.  Add Group 4 to the Groups 2+3 mixture and blend for 1 minute at high speed.  5.  Place Group 5 in the mixer with Groups 2+3+4. Mix until Group 1 has reached 60° C. Pour Group 1 into the mixer and mix the entire mixture for 10 minutes.  6.  Pour the diet into the desired containers and let cool for 1 hour before use.  - 127 APPENDIX II Correspondences From Insect Pathologists A - Dr. John Cunningham re: 1984 Diagnosis B - Mr. Gerard M. Thomas re: 1985 Diagnosis  - 130 APPENDIX III Fixing and Staining Procedure (Clark 1981)  A Modified Azan Staining Technic for Insects I latum  (l%6)  Designed to show: Polyhcdrosis and granulosis inclusion body viruses in lepidopteran insect larval tissues. Fixation: Alcoholic Bouin's. Picric acid I gm. glacial acetic acid—15 ml, formalin—60 nil. 80'* ethyl alcohol - 1 5 0 ml. Embedded In: Paraffin.  Preparation of solutions: A . Dissolve 0.1 gm of azocarmine G in 100 ml of distilled water and boil the solution for 5 min. Allow to cool and add 2 ml glacial acetic acid. Filter before use. B.  Dissolve in 1(K) ml of distilled water, 1.0 gm phosphotungstic acid, 0.1 gm aniline blue (water soluble), 0.5 gm orange G , and 0.2 gm fast green F C F .  Procedure: 1. Toluene via alcohols to water. 2. Transfer to 50% acetic acid for 5 min. 3. Rinse in distilled water for 2 min. 4. 5. 6. 7. 8. 9. 10. 11. 12.  Slain to Solution A for 15 min. Rinse in distilled water for 5 sec. Transfer to aniline {\'< in 95% ethanol) for 30 sec. Rinse in distilled water for 5 sec. Counterstain in Solution B for 15 min. Transfer to 50% ethanol for 10 sec. Transfer to absolute alcohol, 2 changes, 30 sec each. Transfer to toluene. 2 changes. Mount in neutral, synthetic mounting medium. c  Results: Virus inclusion bodies—red; epiculicle—red; endocuticle—blue; muscle—light blue to blue green; epidermal cells—yellowish-green; fat body cells yellowish green with dark green nuclei: nerve tissue—light blue; silk gland—green, contents red or blue; midgut epithelium—green and blue.  


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