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A study of some factors influencing the orientation behaviour of the ambrosia bettle Trypodendron lineatum… Chan, Vernon Bruce 1967

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A STUDY OF SOME FACTORS INFLUENCING THE ORIENTATION BEHAVIOUR OF THE AMBROSIA BEETLE TRYPODENDRON LINEATUM (OLIVIER) (COLEOPTERA: SGOLYTIDAE) by VERNON BRUCE CHAN B.Sc, U n i v e r s i t y of V i c t o r i a , 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science i n the Department of Zoology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1967 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t 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 , I ag r ee t h a t t h e 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 . I f u r t h e r ag r ee 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 o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depa r tmen t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f 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 V a n c o u v e r 8, Canada i i ABSTRACT The behaviour of the ambrosia beetle Trypodendron llneatum ( O l i v i e r ) has been considered i n respect to i l l u m i n a t i o n , body moisture and host f a c t o r s . The i n v e s t i g a t i o n was designed to study p o t e n t i a l uses of t h i s insect as a t e s t instrument f o r chemical studies of host wood a t t r a c t a n t s . A preliminary study of host a t t r a c t a n t s was al s o conducted using a newly-proposed bioassay technique. Monochromatic l i g h t at the wavelength 543 millimicrons was found to be the sole peak of stimulation to t h i s i n s e c t i n the v i s i b l e spectrum. Beetles displayed a p o s i t i v e photic response by walking toward the source of l i g h t . A decrease i n s e n s i t i v i t y occurred on e i t h e r side of t h i s peak, and i n the longer wavelengths the s e n s i t i v i t y to l i g h t diminished at 735 mi l l i m i c r o n s . Evidence to date indicated a second peak of s e n s i t i v i t y i n the u l t r a v i o l e t region of the spectrum; the l a t t e r appearing to be much greater than the peak i n the v i s i b l e spectrum. The i n t e n s i t y of any wavelength was a l s o found to be a l i m i t i n g f a c t o r i n a f f e c t i n g beetle response, although i n the longer wave-lengths the s e n s i t i v i t y appeared to be a function of wavelength alone. Monochromatic l i g h t as a standard has been proposed f o r future bioassay techniques to act i n opposition to odour s t i m u l i . Red i l l u m i n a t i o n was found to be e f f e c t i v e i n simulating darkness to T. lineatum. •Green 1 u n a t t r a c t i v e sapwood shavings of Douglas-fir a f t e r placement under oxygen d e f i c i e n t conditions became a t t r a c t i v e to T. lineatum. Maximum attractiveness, was indicated i n wood placed under anaerobiosis f o r 20 to 26 hours. Beyond 30 hours, l i t t l e sign of attra c t a n t s was noted. Control wood ser i e s d i d not undergo any t r a n s i t i o n , t h i s leading to the conclusion that a s i g n i f i c a n t change occurred i n wood as a r e s u l t of the anaerobic treatment. The implications of t h i s r e s u l t have been discussed. The successful use of i i i wood shavings has made possible further studies on the nature of origin of attractants. The use of the anemotactic behaviour of beetles of both sexes to an airstream carrying host odour was found to be a highly efficient technique of analysis. The role of greater quantities of light in attracting insects away from a source of olfactory stimulation became increasingly apparent from this study. Moisture loss of the insect apparently did not alter their response to white light. iv ACKNOWLEDGMENTS The author wishes to express his deep gratitude to Dr. Kenneth Graham, for his many helpful suggestions and for his endless encouragement throughout the conduct and preparation of this thesis. Special acknowledgment is extended to Dr. G. G. E. Scudder for his reading of the manuscript and for the use of various pieces of laboratory equipment, and to Miss D. Lee for her endless patience and typing skills during the preparation of this thesis. Thanks are also extended to Dr. J. T, McFadden for his suggestions on the statistical analyses. This study was conducted with the aid of grants to Dr. K. Graham from the National Research Council of Canada. Special financial assistance is gratefully acknowledged from the University of British Columbia (student laboratory assistantships), Later Chemicals Co., Ltd., and the British Columbia Loggers' Association. Acknowledgement is also extended to the Department of Zoology, University of British Columbia, for the use of laboratory space and facilities. V TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 5 1. The experimental i n s e c t : i t s source and method of h a n d l i n g . . . 5 2. Study of responses to monochromatic l i g h t 6 (a) Source of l i g h t 6 (b) Condit ions of wavelength and i n t e n s i t y 8 i Varying wavelengths at a constant i n t e n s i t y 10 i i Vary ing both wavelength and i n t e n s i t y 11 3. Study of the e f f e c t of water l o s s on phot ic r e sponse . . . 12 (a) Dehydration o f beet les 12 (b) Tes t ing of phot ic response. 13 4. The e f f e c t of subject ing 'g reen ' wood to anaerobic condi t ions • 15 (a) Preparat ion of the wood 15 (b) Bioassay techniques 20 EXPERIMENTAL RESULTS 29 1. Study of responses to monochromatic l i g h t 29 (a) Varying wavelengths at a constant i n t e n s i t y 29 (b) Vary ing both wavelength and i n t e n s i t y 33 Z. Study of the e f f e c t of water l oss on phot ic response . . 36 3. The e f f ec t of subject ing ' g reen ' wood to anaerobic DISCUSSION 52 I The e f f e c t s of var ious phys i ca l f ac to rs on the' responses of Trypodendron l ineatum (O l i v i e r ) 52 1« MODOClirOni£L"biC Xi§[lltja » « # o 0 « a < t 0 0 0 0 * » « o 0 0 o o o © 0 0 f l 0 O 0 o i > 0 0 o « 0 « 0 o $2 a . General COI1S— d@I*8>i/_L0nS o » 0 0 « o 0 0 0 « 0 0 0 0 0 0 0 » 0 0 0 0 0 0 0 0 « 0 0 0 » » » 52 v i TABLE OF CONTENTS (continued) Page b. New information f o r the bioassay technique 56 2. Water l o s s versus photic responses and i t s implications to the bioassay technique 58 II The modification and bioassay of 'green' wood using the newly-proposed technique of a n a l y s i s . ••• 60 1. The wood fa c t o r s 60 2. The bioassay technique 66 CONCLUS TONS • ••«*«»«»«*e««*o«o»oo0»««e«««e»«oo«»0««o«*»««««o««««««*****« 9^ LITERATURE C I T E D • • • « « « o o o « o « « o « « « « ••••••«•••••••«••••••••••••••«•• 71 APPENDICES 77 v i i LIST OF TABLES Table No. Page 1. Summarized r e s u l t s of beet le responses to var ious wavelengths o f monochromatic l i g h t at an equal i n t e n s i t y 30 2. Summarized s t a t i s t i c s of pos i t i v e phot ic responses o f Trypo-dendron adul ts to var ious wavelengths of monochromatic l i g h t 3. Var iance ana l y s i s r e s u l t s tested at the 5% l e v e l o f s i g n i f i -cance showing the d i f f e rences i n pos i t i v e phot ic responses between wavelengths tested at an equal i n t e n s i t y 34 4. Table of values from the monochromator c a l i b r a t i o n graphs (appendix 2) showing the percentage of pos i t i v e phot ic responses f o r 10 per cent i n t e r va l s ranging from 0 to 60 per cent (appendix l ) and the corresponding requi red energy output o f monochromatic l i g h t f o r the wavelengths tes ted 35 5. Comparison of the su r v i v a l times of the two groups of bee t l e s , one i n the 'humid environment' ( cont ro l ) and the other on the ' d r y ' environment as measured at the e a r l i e s t time a f t e r death. 37 6. Comparison of weight losses by Trypodendron i n the ' d r y ' and 'humid' environments, the f i n a l f i g u r e was recorded at the e a r l i e s t poss ib le measurement a f t e r death 37 7. Summarized i n d i v i d u a l comparisons o f d i f f e rences i n phot ic responses with water loss fo r the ' r e f r a c t o r y ' and ' r e a c t i o n ' times as d isp layed by adu l t Trypodendron to white l i g h t at the \% l e v e l of s i g n i f i c a n c e . . . . . . . . . . . . . . . . . . . . . . . . . 39 8. Tabulated r e s u l t s of the var ious phot ic responses of adu l t Trypodendron to white l i g h t with increas ing water l o s s 40 9. Summarized data of responses from bioassay t e s t s o f wood samples subjected to vary ing times under anaerobic cond i t i ons . Responses are computed as percentages i n 4 hour i n t e r v a l s f o r the three d i f f e r e n t l i g h t i n g s i t ua t i ons • 47 10. Summarized data and response percentages of Trypodendron adu l t s tested to var ious wood samples and c o n t r o l s . . . . . . 50 v i i i LIST OF ILLUSTRATIONS F igure No. Page 1. Shimadzu spectrophotometer Model QV-50 used as a source o f monochromatic l i g h t to t e s t the e f f e c t s of var ious wavelengths and i n t e n s i t i e s on the phot ic responses o f f l i gh t- inexper i enced T , l ineatum adu l ts 7 2a, Apparatus used f o r studying the phot ic responses of T , l ineatum adu l ts to monochromatic l i g h t 9 2b. Monochromatic l i g h t ex i t hole placed i n pos i t i on to pro ject a beam of l i g h t across the t es t arena s u r f a c e . . . . . 9 3. Beet le-laden aluminum containers placed i n one of the two Pe t r i d i sh environments. P l a s t i c screen was placed beneath the containers to prevent any contact with the f i l t e r paper (e i ther L. Apparatus used to t es t the phot ic responses o f f l i g h t - i n e x p e r -ienced T . l ineatum to white l i g h t fo l low ing vary ing per iods of de s i c c a t i on . The Reichert i l l um ina to r with the Bausch and Lomb heat f i l t e r was used as the source of l i g h t . . . . . L4 5. Double samples o f wood chips i n 250 ml . Erlenmeyer f l a s k s placed i n the ' anaerob ic ' chamber. 18 6. ' Ae rob i c ' con t ro l se r i es of wood chips i n 250 ml. Erlenmeyer f l a sk s and placed in to a large p l a s t i c bag and sealed 19 7. Apparatus used f o r the bioassay of wood us ing monochromatic l i g h t (735 mu), t h i s s imulat ing darkness to T„ l ineatum. Wood f a c to r s were presented i n double se r i es v i a a f i n e g lass nozzle located a t the r i g h t of the t e s t a r e n a . . . . . 23 8a . Beet les pos i t ioned on entomological cardboard points with glue by the pronotum. As a precaut ion aga inst water l o s s , points were mounted over a medium of water 24-8b. Female T . l ineatum adul t mounted f o r capt ive f l i g h t - e x e r c i s e . Specimen i s seen r e t r a c t i n g i t s wings a f t e r f l i g h t , . . . ZL 9. Apparatus used to t e s t wood i n the 'oppos ing ' l i g h t s i t u a t i o n . The Reichert i l l um ina to r with a Bausch and Lomb heat f i l t e r was used to produce a broad d i f f u s e beam of l i g h t . The same apparatus as descr ibed fo r the 'dark ' s i t u a t i o n was used to 10. Apparatus used to t es t wood with the overhead c e i l i n g l i g h t s produced by four f lourescent lamps located about 2 meters above the t es t stage. The apparatus f o r the presentat ion of wood f ac to rs was the same as that descr ibed f o r the 'dark ' and i x LIST OF ILLUSTRATIONS (continued) Figure No. Page 11. Graphs of t o t a l l e d p o s i t i v e and near p o s i t i v e (?<•) responses of Trypodendron to various wavelengths of monochromatic l i g h t at an equal i n t e n s i t y 31 12. Graphs of the cumulative percentage weight losses of i n d i v i d u a l Trypodendron adults with increased exposure time i n the two environments. a. desiccated group (dry environment) b. non-desiccated group (humid environment) 38 13. Line tracings of paths taken by two f l i g h t - i n e x p e r i e n c e d female Trypodendron adults to white l i g h t while being subjected to varying periods of de s i c c a t i o n . a. Responses of a beetle from the desiccated group. b. Responses of a beetle from the non-desiccated group 41 14. Line tracings of the paths taken by two flight-experienced female Trypodendron adults i n the three d i f f e r e n t l i g h t i n g s i t u a t i o n s and tested to various types of wood. a. a t t r a c t i v e Douglas-fir sapwood c o n t r o l b. 'anaerobic' sample (23.5 hours) c. 'aerobic' c o n t r o l (23.5 hours) d. a i r only e. 'green' c o n t r o l f . a t t r a c t i v e Douglas-fir c o n t r o l again.... 43 15. Line tracings of the paths taken by two male and one female flight-experienced Trypodendron adults tested i n the 'dark' and 'opposing' l i g h t s i t u a t i o n s . Wood fa c t o r s include? a. a t t r a c t i v e Douglas-fir sapwood control b. 'anaerobic' sample (21 hours) c. 'aerobic' c o n t r o l (21 hours)..... ........... 44 16. Point graph of t o t a l l e d percentage p o s i t i v e and near p o s i t i v e (?+•) responses to wood shavings placed under anaerobic conditions f o r varying periods (2 to 46.5 hours). Three sets of points have been included f o r the three d i f f e r e n t l i g h t i n g s i t u a t i o n s (dark, opposing, overhead) 46 17. Frequency histograms of t o t a l l e d percentage p o s i t i v e and near p o s i t i v e responses of Trypodendron to 'anaerobic' samples divided i n t o 4 hour treatment classes and tested under the three l i g h t i n g s i t u a t i o n s % a. beetles tested In 'dark' b. beetles tested i n 'overhead' l i g h t s i t u a t i o n c. beetles tested i n 'opposing' l i g h t s i t u a t i o n . . . . . . . . . . . . . 48 INTRODUCTION 1 The o r i e n t a t i o n reactions of the ambrosia beetle Trypodendron lineatum ( O l i v i e r ) are a subject of considerable i n t e r e s t and importance to i n v e s t i g a -tors of t h i s i n sect, as w e l l as to students of S c o l y t i d behaviour i n general. It i s desired to understand the f a c t o r s and behavioural mechanisms which influence t h i s species i n i t s discovery of host logs both as to taxonomic i d e n t i t y and state of morbidity. A. thorough knowledge of the subject w i l l contribute to an understanding of important features of the ecology and host r e l a t i o n s of t h i s i n s e c t , as w e l l as provide information f o r using the beetles as bioassay instruments i n the furth e r study of chemical a t t r a c t a n t s or rep e l l e n t s . Considerable information has been gained on t h i s subject by various i n v e s t i g a t o r s working i n both f i e l d and laboratory, but some important aspects remain to be explored. It i s known that the following f a c t o r s influence the reactions of T. lineatum i n respect to host tree f a c t o r s : the p h y s i o l o g i c a l state of the insect, pre-conditioning of the insect, host tree f a c t o r s , and ph y s i c a l f a c t o r s of the environment at the moment of observation. The photic responses are among the most compelling f a c t o r s i n the behaviour and ecology of T. lineatum. A photonegative behaviour evidently causes the newly-emerged adults i n summer to seek shade and enter a long dormancy i n the s o i l (Dyer and Kinghorn, 1961). In spring, temperature a c t i v a t i o n and a photopositive response cause emergence and d i s p e r s a l . The photopositive response i s however modified by ambient temperature, such that a maximum proportion of a population sample becomes photopositive at about 26 to 28 degrees Centigrade. Thereafter a photonegative response appears u n t i l a l l i n d i v i d u a l s avoid the l i g h t at AO degrees Centigrade (Francia, 1965). The photopositive response tends to override a latent capacity to respond to host factors u n t i l f l i g h t has occurred (Graham, 1959). 2 The swallowing of a i r during f l i g h t (Chapman, 1956) evidently i s the i n i t i a t i n g mechanism f o r the change of r e l a t i v e response thresholds to odour and l i g h t (Graham, 1961). As l i t t l e as f i v e minutes of f l i g h t may be s u f f i c i e n t to change the responses i n at least some i n d i v i d u a l s (Francia, 1965). The mechanism by which the swallowed a i r acts i s evidently not pressure as such (Francia, 1965). Now that the importance of the photic response i s better understood, i t i s possible to study o l f a c t o r y responses i n T. lineatum without interference from photic e f f e c t s . Olfactory t e s t s require e i t h e r that the beetles be pre-conditioned by f l i g h t so that they can respond to odour even i n the presence of l i g h t , or that the beetles be placed i n t o t a l darkness during an olf a c t o r y t e s t , or that observations be c a r r i e d out under a wavelength of l i g h t which i s i n v i s i b l e to the beetle, yet v i s i b l e to the human observer. Various c r i t e r i a of response have been devised. One i s manifest i n the beetles boring i n t o wood containing inherent or applied a t t r a c t a n t s (Graham and Werner, 1956). Another i s manifest i n an up-wind anemotactic response which i s prompted by the presence of odour i n the a i r stream (Francia, 1965). A t h i r d i s manifest i n the tendency of the beetles to r e t a i n t h e i r p o s i t i o n within a substratum f i e l d of odour. In t e s t s employing pre-flown beetles, a beam of l i g h t provides a c r i t i c a l t e s t of the apparent response to odour. I f odour i s absent or i n e f f e c t i v e , a photopositive r e a c t i o n i s manifest. Therefore a l i g h t source i s no longer an obstacle to the o l f a c t o r y study but a u s e f u l f a c t o r i n i t . The studies of photic responses of t h i s insect and i t s o l f a c t o r y responses i n the presence of a l i g h t source have heretofore depended on an a r b i t r a r i l y chosen i l l u m i n a t i o n standard. The e f f e c t of l i g h t on photic responses alone i s known only i n general terms, and there remains to be 3 determined the e f f e c t of wavelength and i n t e n s i t y . Knowledge of t h i s kind w i l l be not only of s c i e n t i f i c i n t e r e s t , but w i l l permit the d e f i n i t i o n of reference standards i n the bioassay of host a t t r a c t a n t s . Accordingly, one of the objectives of the present i n v e s t i g a t i o n was the study of photic responses to l i g h t of d i f f e r e n t wavelengths and i n t e n s i t i e s . Another question concerning o r i e n t a t i o n responses i n T. lineatum pertains to the e f f e c t s of moisture loss. E a r l i e r observations by Graham and Werner ( 1 9 5 6 ) , using these beetles i n darkened arena t e s t s , showed that they f a i l e d to display t h e i r usual aggregative tendencies about odour sources when the humidity was very low. The general v i t a l l i m i t s f o r moisture loss were known from work of N i j o l t and Chapman ( I 9 6 4 ) . T. lineatum may survive losses of between 1 0 and 2 5 per cent. Since o r i e n t a t i o n studies sometimes expose the beetles to moderate laboratory humidities of about 50% R.H. f o r up to s i x hours, i t was important to know i f t o t a l moisture loss within v i t a l l i m i t s a f f e c t s responses to l i g h t and odour. Another problem i n the study of f a c t o r s i n f l u e n c i n g the responses of ambrosia beetles to t h e i r host trees concerns the nature and o r i g i n of the chemical a t t r a c t a n t s . Graham ( 1 9 6 2 ) had demonstrated that fresh sapwood subjected to anaerobic conditions f o r 2 4 hours becomes a t t r a c t i v e to T. lineatum. whereas the usual span of time required f o r attractiveness to develop i n a log may be weeks or months. Fresh sapwood kept under aerobic conditions f o r 2 4 hours remained unattractive. The discovery of e f f e c t s of anaerobiosis on sapwood i s s i g n i f i c a n t f o r studies aimed at both understanding and c o n t r o l l i n g the a t t r a c t a n t -formation process. The adoption of an a c c e l e r a t i n g process affords an opportunity of speeding up the search f o r metabolic i n h i b i t o r s of a t t r a c t a n t formation. The time f a c t o r required f o r the chemical changes under anaerobic conditions was known only on an empirical basis. It remains to be discovered how long a period i s required to produce a detectable change, and how long i s required to produce a maximum change This problem was thus another objective of the study. MATERIALS AND METHODS 1. The experimental insects i t s source and method of handling. Experimental i n s e c t s were the ambrosia beetle Trypodendron lineatum ( O l i v i e r ) , gathered from t h e i r overwintering quarters i n f o r e s t duff and under the bark of old Douglas-fir trees near Cowichan Lake on Vancouver Island, B r i t i s h Columbia i n early A p r i l of 1965, just p r i o r to t h e i r normal emergence and attack. The beetles contained i n bark and f o r e s t l i t t e r were stored i n f i v e g a l l o n metal U.S. army water cans and placed i n a r e f r i g e r a t o r at a temperature of 0-5 degrees Centigrade. Some of these beetles were adults that had survived the previous seasons attack, but the majority were newly-developed adults that had never undergone any attack on host breeding material. Whenever t e s t specimens were required, a handful of beetle-containing medium was placed i n a shallow pan and f l o a t e d over warm water at a temperature of approximately 30 degrees Centigrade. Beetles on warming up emerged to the surface of the duff, from which they were immediately removed to standard 5 inch P e t r i dishes containing a piece of moistened f i l t e r paper, u n t i l they were required f o r t e s t i n g . Extreme care was taken i n handling beetles since mutilated i n d i v i d u a l s are unsuitable f o r t e s t s of behaviour. Those with e i t h e r broken or amputated legs showed d i f f i c u l t y i n walking, i n many cases r o l l i n g over and moving t h e i r legs f r a n t i c a l l y i n an unsuccessful attempt to recover a standing p o s i t i o n . Gara (1963) has stated that the l o s s of an antenna can a l s o render an i n d i v i d u a l unsuitable f o r t e s t purposes since these structures contain many of the sensory organs necessary f o r insect o r i e n t a t i o n . He has correlated a 50 per cent reduction i n performance with the l o s s of an antenna. 6 2. Study of responses to monochromatic l i g h t , (a.) Source of l i g h t . This phase of the present study was to investigate the e f f e c t s of varying wavelengths and i n t e n s i t i e s of monochromatic l i g h t on the photic responses of Trypodendron. The source of t h i s l i g h t was supplied by a Shimadzu Spectrophotometer Model QV-50 (Fig. 1). This instrument was equipped with a hydrogen discharge lamp as w e l l as a tungsten lamp, the l a t t e r having an output rangingfrom 350 to 1200 millimicrons. The hydrogen discharge lamp was designed to produce an output beyond 350 mu in t o the shorter wavelengths of the u l t r a v i o l e t . Preliminary t e s t s indicated that the output i n t o the shorter wavelengths of the u l t r a v i o l e t with the hydrogen lamp were not s u f f i c i e n t f o r the experiments at hand. Tests described hereafter were therefore l i m i t e d to the wavelengths produced by the tungsten filament, namely 299 to 880 millimicrons.* Designated wavelengths were obtained by a simple turn of a c a l i b r a t e d d i a l , the l i g h t produced being emitted through a one inch diameter hole at one end of the instrument housing. The i n t e n s i t y of l i g h t was c o n t r o l l e d by varying the width of the collimator s l i t i n the spectrophotometer."'" The quantity of l i g h t emitted was measured by means of the photomultiplier located i n a separate housing u n i t next to the l i g h t emission hole. This * .values appear to be 'odd' because the spectrophotometer was r e c a l i b r a t e d a f t e r t h i s experimental seri e s t Expanding the collimator s l i t not only changes the quantity of l i g h t emitted but also the q u a l i t y , since expanding the s l i t allows a larger area of the sp e c t r a l band to be emitted, thereby increasing the i n t e n s i t y . This however would also tend to increase the 'fringe' wavelengths. Most experiments however, u t i l i z e d very small s l i t widths thereby increasing' greatly the 'purity' of the transmitted l i g h t . Figure 1. Shimadzu spectrophotometer Model QV-50 used as a source of monochromatic light to test the effects of various wavelengths and intensities on the photic responses of flight-inexperienced T. lineatum adults. 8 photometer could e a s i l y be removed from the remainder of the instrument when required. I t d i d not however give readings i n the standard l i g h t measurement u n i t s since i t was not o r i g i n a l l y designed f o r such a study, but rather i n u n i t s of a percentage transmission/absorption of l i g h t . This value however was found to s u f f i c e f o r the purposes of the present study, since a l l that was required was a standard f o r which to measure the energy emission of the various t e s t wavelengths and t h e i r corresponding i n t e n s i t i e s . An approximate standard l i g h t measure was obtained by use of a Photovolt photometer Model 200. Most i n t e n s i t i e s were estimated to range between a minimum of 0.02 foot-candles and a maximum of 6.00 foot-candles. (b) Conditions of wavelength and i n t e n s i t y . For the purposes of t e s t i n g , a l l photocells were removed from obstructing the path of the beam of l i g h t emitted by the previously c a l i b r a t e d spectrophotometer. This beam was projected across a l e v e l arena platform, the l a t t e r covered with a piece of white 8£" x 11" w r i t i n g paper; t h i s serving as a substrate on which the experimental beetles could meander. At the proximal edge of the paper, the width of the beam was approximately 12.0 mm., while at i t s d i s t a l edge i t was approximately 32.0 mm. Preliminary t e s t i n g with the col l i m a t o r s l i t of the monochromator indicated that expanding and contracting of the s l i t did not a l t e r the dimensions of the beam of monochromatic l i g h t . Figures 2a and b i l l u s t r a t e the apparatus and set-up used. Teste were conducted on fli g h t - i n e x p e r i e n c e d beetle's by p l a c i n g an i n d i v i d u a l specimen i n the centre of the te s t f i e l d with i t s head f a c i n g towards the source of monochromatic l i g h t . A l l t e s t s were conducted i n a darkened room. However, an opposing white l i g h t source was pla'aed i n F i g . 2a. Apparatus used f o r studying the photic responses of T. lineatum adults to monochromatic l i g h t . The beam of l i g h t was projected across the tes t arena from the l e f t . During re-alignment periods the t e s t f i e l d was illuminated by the opposing Reichert i l l u m i n a t o r with the Bausch and Lomb heat f i l t e r interposed. F i g . 2b. Monochromatic l i g h t e x i t hole placed i n p o s i t i o n to project a beam of l i g h t across the t e s t arena surface. The c e n t r a l s t a r t i n g point of the arena i s marked with a cross (+). 10 opposition to the source of monochromatic l i g h t , t h i s serving to il l u m i n a t e the t e s t f i e l d during the beetle r e - a l i g n i n g periods. Responses of beetles were merely recorded as either p o s i t i v e or negative; p o s i t i v e responses being those i n which i n d i v i d u a l s walked towards the source of monochromatic l i g h t , e ither d i r e c t l y or i n d i r e c t l y from t h e i r s t a r t i n g p o s i t i o n . Negative responses were those i n which beetles showed no tendency to orient towards the l i g h t and displayed otherwise random movements. Two separate methods were adopted i n t h i s study to t e s t the ef f e c t s of the various wavelengths and t h e i r respective i n t e n s i t i e s . i Varying wavelengths at a constant i n t e n s i t y . The f i r s t was the simpler of the two methods, i t i n v o l v i n g only 9 d i f f e r e n t wavelengths (34-8 mu to 735 mu), a l l at a s i m i l a r i n t e n s i t y . The responses of beetles at the constant i n t e n s i t y would therefore depend on the 'stimulative e f f i c i e n c y 1 of each i n d i v i d u a l wavelength. A measure of the att r a c t i v e n e s s of each wavelength could therefore be expressed as a percentage of the p o s i t i v e responses exhibited by the t o t a l complement of t e s t beetles; the higher the percentage the greater the 'stimulative e f f i c i e n c y ' . In t h i s s e r i e s , 29 fli g h t - i n e x p e r i e n c e d Trypodendron adults were tested, of which 20 were females. Each i n d i v i d u a l was given 10 t r i a l s to each wavelength but some however were not tested to a l l of the 9 wavelengths. Also 7 females and 7 males were discarded because they showed no apparent capacity to respond at a l l to any wavelength of monochromatic l i g h t . Chapman (1955b) has described s i m i l a r i n s e c t s i n an apparently normal population which showed no i n c l i n a t i o n to respond l i k e the majority of the i n d i v i d u a l s . Results are therefore from a sample population of i n d i v i d u a l s showing at l e a s t some a c t i v e capacity to respond to the monochromatic l i g h t . They are al s o confined to in s e c t s i n the light-adapted state. 11 i i Varying both wavelength and i n t e n s i t y . The second method of t e s t i n g was somewhat more complicated than the f i r s t , but was s i m i l a r to i t i n many respects. Some a d d i t i o n a l wavelengths were tested t h i s time together with those previously described, there'by g r e a t l y expanding the experimental s p e c t r a l range. Also, i n t e n s i t i e s over a wide and varied range were tested on the beetles, which were examined s i n g l y under pedestrian s i t u a t i o n s . In t h i s schedule of events f o r each of the wavelengths tested (299 to 880 mu), the i n t e n s i t y was manipulated such that t e s t beetles responded p o s i t i v e l y to the monochromatic l i g h t approximately 80 to 100 per cent of the time at the minimum i n t e n s i t y which gave maximum response while at the lowest i n t e n s i t y there was no furth e r p o s i t i v e response (Appendix.1). This lowest maximum i n t e n s i t y would therefore be the l i m i t i n g threshold of the insects} t h i s threshold varying with the 'stimulative e f f i c i e n c y ' of the wavelength. The r e s u l t s of t h i s t e s t i n g procedure would therefore y i e l d a d i r e c t p o s i t i v e c o r r e l a t i o n between beetle response and i n t e n s i t y . By using a graph of previously c a l i b r a t e d energy output readings f o r each wavelength examined (Appendix 2), i t was possible to d i r e c t l y f i n d the quantity of energy necessary to produce any percentage of p o s i t i v e responses. For the purpose of t h i s i n v e s t i g a t i o n , 7 percentages of p o s i t i v e response were analyzed f o r each of the wavelengths; these ranging from a zero percentage (threshold) to an approximate 60 per cent p o s i t i v e point, with intermediate values at 10 per cent i n t e r v a l s (Table 4). Resultant energy output values at each of the response l e v e l s were expected to vary according to the 'stimulative e f f i c i e n c y ' of each wavelength. That i s to say, at an equal percentage p o s i t i v e response l e v e l , wavelengths with a higher 'stimulative e f f i c i e n c y ' would require l e s s energy ( l i g h t ) to invoke a p o s i t i v e 1 2 response than those wavelengths having a lower 'stimulative e f f i c i e n c y ' . 3. Study of the e f f e c t of water los s on photic response, (a) Dehydration of beetles. The experiment to examine the e f f e c t s of water l o s s on the photic responses of Trypodendron was conducted only with newly-emerged post-diapaused i n d i v i d u a l s , thereby being f l i g h t - i n e x p e r i e n c e d . This precaution was adopted f o r t h i s p a r t i c u l a r study since v a r i a t i o n s i n photic responses are known to occur as a r e s u l t of the a d d i t i o n a l f a c t o r of f l i g h t - e x e r c i s e (Francia, 1965). For weighing the beetles, small aluminum f o i l containers approximately 1.5 cm. i n diameter and 1.5 cm. i n height were constructed. Each was perforated with small pinholes near i t s base and al s o equipped with a t i g h t - f i t t i n g l i d , a l s o of aluminum f o i l . Containers were f i r s t weighed on a Mettler Microgrammatic Balance Model M5 with an accuracy i n the o p t i c a l range of _ 0.002 mg. Beetles of both sexes were placed i n d i v i d u a l l y i n each container and the accompanying l i d attached so as to prevent any possible escape. Beetle-laden containers and l i d s were again weighed, thus e s t a b l i s h i n g a weight f o r each beetle. A s i m i l a r procedure has been described by N i j o l t and Chapman (1964.) i n t h e i r drinking experiments with Trypodendron. Two 5 inch P e t r i plates with l i d s were then used to house the beetle-laden containers. One plate contained a piece of moistened f i l t e r while the other was l e f t dry. Three pieces of 0.3 cm. mesh p l a s t i c screening were then placed over the f i l t e r papers to prevent any contact by the aluminum containers when these were added to the P e t r i dishes ( F i g . 3). The dry container represented a normal des i c c a t i o n chamber but t h i s time using only room temperatures and humidities. The moistened container served as a c o n t r o l . 13 Both groups were then placed i n t o a darkened container and l e f t f o r an alloted period of time afterwhich beetles were again re-weighed and tested to the white l i g h t . (b) Testing of photic response. Immediately after weighing, each beetle was tested for photic responses to white l i g h t . Again the arena-type s i t u a t i o n was used along with a Reichert microscope i l l u m i n a t o r Model 'Lux FNI' operating through a v a r i a b l e r e s i s t o r . An output s e t t i n g of 3.0 amperes was adopted, and a l s o to avoid any thermal s i d e - e f f e c t s a Bausch and Lomb heat f i l t e r was interposed ( F i g . 4). A Photovolt photometer measuring, the output of the lamp showed i t to be approximately 14 foot-candles at the centre of the t e s t field. Beetles were allowed 8 i n d i v i d u a l t r i a l s , each commencing with a beetle aligned towards the source of l i g h t . Responses were recorded using a t h i n p e n c i l l i n e made by a p e n c i l following approximately 4.0 cm. behind the beetle. Mot only was the path of beetles recorded from the c e n t r a l s t a r t i n g point but a l s o the time required f o r each beetle to walk to the nearest edge of the test surface. In a d d i t i o n to measuring this 'reaction' time, a second time measure was included. This was designated the 'refractory' period and was the duration of time between placing a beetle on the c e n t r a l s t a r t i n g point to the commencement of the 'reaction' time. The importance of studying t h i s time is quickly realized when i t i s considered that i n t e r n a l changes of the insect can be demonstrated merely from studying i t s responses to various external f a c t o r s . Water l o s s by the i n s e c t may appear i n the form of slower movements of the appendages i n response to l i g h t . During the 'refractory' period a beetle when placed on the t e s t surface normally had I t s legs t i g h t l y folded against the body, the antennae were u F i g . 3. Beetle-laden containers placed i n one of the two P e t r i d i s h environments. P l a s t i c screen was placed beneath the containers to prevent any contact with the f i l t e r paper (either wet or dry). Three of the l i d s have been removed from the capsules to show the beetles. F i g . L. Apparatus used to t e s t the photic responses of f l i g h t - i n e x p e r -ienced T. lineatum to white l i g h t following varying periods of d e s i c c a t i o n . The Reichert Illuminator with the Bausch and Lomb heat f i l t e r was used as the source of l i g h t . held t i g h t l y against the head, and the e n t i r e insect appeared motionless at t h i s time. Gradually the antennae began to v i b r a t e strongly. S l i g h t rocking side movements of the e n t i r e i n s e c t soon followed, t h i s being due to the gradual unfolding of the legs beneath. Then the i n s e c t rose quickly to i t s f e e t and began to walk. When reacting to the l i g h t , the a n t e r i o r portion of the body became elevated s l i g h t l y higher than the p o s t e r i o r , while the sensory antennae continued to v i b r a t e a c t i v e l y . A f t e r t e s t i n g , each beetle was returned to i t s appropriate environment and t h i s procedure was continued u n t i l a l l the beetles eventually died. Results were analyzed to show whether or not any changes had occurred i n the responses to l i g h t . Also two variance analyses were performed f o r each beetle, one f o r the 'refractory' periods and the other f o r the 'reaction' times. In a l l , fourteen Trypodendron specimens were used f o r t h i s experiment. In some cases however, a beetle had died before i t was tested more than once, therefore no variance analyses of the d i f f e r e n c e s between groups of t r i a l s could be performed. However, f o r the remaining beetles, the number of times each was subjected to i t s s e r i e s of t r i a l s v aried from 2 to 7, depending on the s u r v i v a l time of each. Variance analyses could therefore be attempted; these showing either s i g n i f i c a n t or non-significant d i f f e r e n c e s f o r the groups of t r i a l s f o r eaeh beetle. Individual comparisons f o r each group of t r i a l s by each beetle were then made using the method of Tukey's w. These r e s u l t s were tested at the 1% l e v e l of s i g n i f i c a n c e . 4. The e f f e c t of subjecting 'green' wood to anaerobic conditions, (a) Preparation of the wood. The wood material used f o r t h i s Btudy of a t t r a c t a n t formation consisted of slabs of Douglas-fir (Pseudotsuga menziesii Mirb. Franco) sapwood taken 16 from f r e s h l y - f e l l e d t r e e s . The bark was removed to reduce p o t e n t i a l contamination by micro-organisms from the bark surface. The slabs were stored at -18°C u n t i l required f o r experiments. A choice had to be made as to the further preparation of the wood. Preliminary studies by Graham (1962) showed that s o l i d sections of wood could be modified i n respect to attractancy by withdrawing the a i r under vacuum, or by d i s p l a c i n g the a i r with l i q u i d under vacuum. Attractancy developed thereafter under anaerobic conditions. I t was a l s o found that s o l i d blocks were unsuitable f o r the study of b i o l o g i c a l l y a c t i v e chemicals on processes i n the wood because the dissolved chemicals d i d not n e c e s s a r i l y move i n with the aqueous phase. The development of bioassay techniques using o r i e n t a t i o n responses (Francia, 1965) instead of boring-in a c t i v i t y , has made i t possible to use wood i n a more f i n e l y divided state such as sawdust or shavings i n which perfusion with solutions presents no serious obstacle. Three a l t e r n a t i v e s presented themselves f o r the d i v i s i o n of wood substance i n t o a dimensionally reduced state. One consisted of grinding, another of chipping and the t h i r d of shaving. Chipping was eliminated because of the non-uniformity of s i z e of the resultant wood. Shaving was chosen over grinding beoause i t appeared to o f f e r the desired degree of uniformity of d i v i s i o n with a minimum of damage to the ray c e l l s i n which i t i s presumed the a t t r a c t a n t forming processes reside. The wood was planed along r a d i a l faces with a carpenters' planer to produce shavings approximately 3.5 centimeters i n width and 0.5 millimeters i n thickness. Immediately a f t e r planing, the shavings were cut i n t o small rectangular sections which were then placed i n t o a s e r i e s of 250 m i l l i l i t e r Erlenmeyer f l a s k s f o r treatment. Enough chips to reach the neck of each f l a s k were used i n each instance. Care was a l s o taken to avoid excessive drying of the chips as t h i s could render them unsuitable f o r the experiments at hand. Although several d i f f e r e n t treatments were applied to the unattractive 'green 1 wood chips, the majority of samples were subjected to placement under oxygen d e f i c i e n t conditions f o r varying periods of time ranging from 2 to 47 hours. These untreated wood chips were placed i n t o a vacuum chamber ( F i g . 5) capable of creating a vacuum to 30 inches of mercury. In a l l cases, the chamber was pumped f r e e of a i r to near capacity (generally between 26-28 inches of mercury) f o r 20 minutes and slowly r e f i l l e d with nitrogen gas. This condition was not however s t r i c t l y anaerobicj, since mixed with the nitrogen gas was oxygen at a concentration of about one-half of one per cent. The a d d i t i o n of the nitrogen was not designed to modify the changes i n wood i n any way, but merely to equalize the pressure between wood chips and the surrounding medium. An unequal pressure between the i n t e r n a l environment of the wood chips and the surrounding vacuum would tend to draw out any a t t r a c t a n t formed i n the wood from the l a t t e r , thus r e s u l t i n g i n a much reduced estimate of the a c t u a l degree of a t t r a c t i v e n e s s of the treated wood. Preliminary experiments without nitrogen y i e l d e d wood which l o s t i t s a t t r a c t i v e properties very quickly. Also to increase the possible quantity of a t t r a c t a n t formed, thereby increasing the time a v a i l a b l e f o r bioassay of the material, two b o t t l e s of each treatment were tested simultaneously by interconnecting them by a length of rubber tubing. A comparable control s e r i e s of doubled b o t t l e s was designed to p a r a l l e l the 'anaerobic' s e r i e s . This s e r i e s , designated the 'aerobic' control involved the placing of b o t t l e s containing untreated 'green' wood chips i n t o a large sealed p l a s t i c bag (Fig. 6) and l e f t under t h i s condition f o r the required period of time. Both 'anaerobic' and 'aerobic' series were removed at the same time from t h e i r respective environments when t e s t i n g with beetles F i g . 5 . Double samples of wood chips i n 250 ml Erlenmeyer f l a s k s placed i n the 'anaerobic' chamber. A l l gases were pumped out f o r 20 minutes and nitrogen gas was then r e f i l l e d i nto the chamber. Samples remained i n the chamber f o r periods ranging from 2 to 4-6.5 hours. 19 Fig. 6. 'Aerobic' control series of wood chips in 250 ml Erlenmeyer flasks and placed into a large plastic bag and sealed. These controls were designed to parallel the treated 'anaerobic' samples. 20 demanded them. In contrast to the 'anaerobic' s e r i e s , l i t t l e or no change was expected from the 'aerobic' controls. An additional control of 'green' untreated wood was a l s o placed i n t o a freezer and was removed approximately one hour before a bioassay was to be conducted. This c o n t r o l was not always used i n every period of t e s t i n g but regular periodic examination of t h i s 'green' wood was undertaken to r e - e s t a b l i s h that t h i s o r i g i n a l wood was indeed u n a t t r a c t i v e . Also as an a d d i t i o n a l c o n t r o l , a series of a t t r a c t i v e Douglas-f i r sapwood samples was used; t h i s to ensure that test, specimens were reacting s a t i s f a c t o r i l y to s u i t a b l e host material. This Douglas-fir was taken from two o l d , but s t i l l h ighly a t t r a c t i v e samples gathered at Cowichan Lake i n May of 1961 and stored i n a freeze*. Preparation of t h i s wood f o r t e s t i n g was s i m i l a r to that followed f o r the 'green' wood c o n t r o l . An attempt was a l s o made to fu r t h e r modify the 'green' wood, t h i s time by the a d d i t i o n of d i s t i l l e d water to the samples. This endeavour i s prompted by the f a c t that i f chemical modifiers are i n future to be added to the wood shavings, these are to be added i n l i q u i d form. I t i s therefore necessary to examine the e f f e c t s of water alone i n a l t e r i n g the processes involved i n the t r a n s i t i o n of wood from 'green' to ' r i p e ' . Preliminary studies were conducted on various s i z e s of Douglas-fir sapwood blocks to obtain a 100$ saturation l e v e l . This gave a quantitative estimate f o r which to add d i s t i l l e d water to the ,test chips? however i n most cases somewhat more water was required to saturate them since much water c o l l e c t e d on the sides of the containers. Extreme care was also taken to avoid any leaching of the chemical solutes. Samples were again divided i n t o 'anaerobic' and 'aerobic' groups and given s i m i l a r treatments as outlined f o r the 'unwetted' s e r i e s . (b) Bioassay techniques. For the bioassay of wood samples, two a l t e r n a t i v e s presented themselves, 21 both proposed by Francia (1965). One depended upon the c e n t r i p e t a l response of beetles i n a f i e l d of odour, while the other depended upon the anemo-olfactory o r i e n t a t i o n and locomotion of beetles i n an odour-laden airstream. For the c e n t r i p e t a l response, the length of a meandering path or the duration of contact with the odour f i e l d a r i s i n g from below the substratum, would provide a measure of at t r a c t i v e n e s s of any wood sample. The anemo-olfactory response on the other hand, would depend upon d i r e c t i n g an odour-laden airstream towards fl i g h t - i n e x p e r i e n c e d beetles In darkness or towards flight=.experienced beetles i n the presence of an opposing l i g h t source. In t h i s t e s t , the strength of o l f a c t o r y a t t r a c t i o n would be measured i n terms of the strength of l i g h t necessary to i n h i b i t the o l f a c t o r y influence. Preliminary studies with wood samples of varying degrees of attractiveness y i e l d e d r e s u l t s favouring the anemo-olfactory t e s t over the c e n t r i p e t a l t e s t . The main reason was that i t appeared to be more s e n s i t i v e than the other i n d i s t i n g u i s h i n g d i f f e r e n c e s i n att r a c t i v e n e s s of wood. The f a c t that an insect displays a t r a v e r s a l , f i r s t towards, then f i n a l l y reaches the source of o l f a c t o r y influence y i e l d s a c l e a r l y indisputable r e s u l t of a t t r a c t i v e properties i n the source. I t would a l s o tend to be l e s s time-consuming to use the anemo-olfactory technique although r e p l i c a t i n g of t e s t s would reduce t h i s advantage. To produce t h i s odour-stream, a i r was direc t e d through the double s e r i e s of b o t t l e s , the supply of a i r a r i s i n g from a standard laboratory o u t l e t . The airstream was presented to the t e s t arena by a short length of rubber tubing with a f i n e glass nozzle at the end. V e l o c i t y of flow was measured with a 'Wallac-Thermex' thermo-anemometer and set at 0.5 meters per second, t h i s being regulated with a small adjustable clamp placed across the a i r supply hose. 22 The t e s t arena consisted e s s e n t i a l l y of a piece of cardboard with 8^" x 11" sheets of white w r i t i n g paper mounted on top. . This white w r i t i n g paper served as a medium on which to mark the r e s u l t s , these being i n the form of l i n e tracings of the paths taken by beetles i n response to the various t e s t f a c t o r s . From the c e n t r a l s t a r t i n g point on the t e s t surface, a t h i n p e n c i l l i n e was used to follow the path of a beetle to one of the edges of the t e s t arena. The bioassay as such was performed using three d i f f e r e n t l i g h t i n g s i t u a t i o n s , each designed to act i n opposition to the source of odour. The f i r s t was a seri e s whereby l i g h t was produced by the monochromator, previously c a l i b r a t e d and tested to produce a source of red l i g h t of the wavelength 735 mu at such an i n t e n s i t y as to simulate darkness to T. lineatum ( F i g . 7 ) . T h i s however l e f t the t e s t arena s u f f i c i e n t l y illuminated such that the experimenter could s t i l l c l e a r l y view the responses of the i n s e c t s . However i n t h i s case, since no photic s t i m u l i were sensed by the beetles, any a t t r a c t i v e wood samples should a t t r a c t the i n s e c t s , both non-flown and flown. Beetles when attracted displayed an anemotactic behaviour, walking against the gradient of odour to reach the source. I f not at t r a c t e d , they wandered aimlessly about the t e s t arena. For the present in v e s t i g a t i o n s a l l beetles tested were flight-experienced, the period of f l i g h t - e x e r c i s e ranging from approximately 10 to 35 minutes. This was accomplished by mounting beetles with 'Permount' glue by the pronotum on standard entomological cardboard points mounted on pins and pl a c i n g these over a dis h of water (Figs. 8a & b ) . This l a t t e r precaution was taken to minimize any water l o s s by the experimental i n s e c t s . Beetles i n t h i s mounted p o s i t i o n expanded and beat t h e i r wings i n an attempt to f l y . Most continued t h i s f l i g h t - e x e r c i s e f o r the whole period of time a l l o t e d but some however displayed a f l i g h t behaviour 23 Fig. 7. Apparatus used for the bioassay of wood using monochromatic light (735 mu), this simulating darkness to T. lineatum. Wood factors were presented in double series via a fine glass nozzle located at the right of the test arena. A controlled velocity of air from a standard laboratory outlet was used to produce the odour-laden airstream. 2A F i g . 8b. Female T. lineatum specimen mounted f o r captive f l i g h t -exercise. Specimen i s seen r e t r a c t i n g i t s wings a f t e r f l i g h t . 25 with many intermittent stops. Whenever beetles were required for t e s t i n g , they were c a r e f u l l y removed from the points and stored i n covered P e t r i dishes containing moistened f i l t e r paper. The order of presentation of wood samples was quite uniform during the course of the experiments, each s e r i e s commencing with an a t t r a c t i v e Douglas-fir c o n t r o l , followed by the 'anaerobic' samples, then the 'aerobic' samples. Depending on i t s necessity however, a re-bioassay of the various other samples was conducted only from time to timej these being the 'green' wood and a i r alone as a c o n t r o l . The number of t r i a l s given each sample and each beetle was also quite v a r i a b l e because i n most cases the time required to t e s t the many samples and beetles was a l i m i t i n g f a c t o r . The second stage of bioassay involved e s s e n t i a l l y the same procedure as i n the 'dark' s i t u a t i o n except that t h i s time an opposing white l i g h t source was substituted i n place of the red monochromatic l i g h t ( F i g . 9), while a l l other extraneous l i g h t s remained eliminated. The source of t h i s white l i g h t was from the Reichert microscope i l l u m i n a t o r previously described. This lamp, set at 3.0 amperes was adjusted to produce a broad d i f f u s e f i e l d of l i g h t across one end of the t e s t arena. The Photovolt photometer measuring the output of t h i s lamp showed i t to be approximately 14 f o o t -candles at the centre of the t e s t f i e l d . To eliminate the e f f e c t s of heat emitted from t h i s tungsten lampy a Bausch and Lomb heat f i l t e r was placed i n the path of the beam of l i g h t . Beetles tested previously i n the 'dark' s i t u a t i o n of the bioassay were again tested with the wood samples, but i n those cases where a considerable duration of time had elapsed, beetles were re-flown to ensure that they were thoroughly f l i g h t - e x e r c i s e d . Whenever a t t r a c t i v e wood was presented to a beetle, i t was expected that the l a t t e r would walk against the odour-bearing airstream to reach the 26 source. Samples that were highly a t t r a c t i v e should a t t r a c t an insect most of the,time, while those samples not as a t t r a c t i v e should not a t t r a c t an insect at a l l , i n t h i s case as with weakly a t t r a c t i v e samples, beetles were expected t o either be attracted to the opposing l i g h t or d i s p l a y an i n d i f f e r e n t behaviour, that i s not being attracted to either a l t e r n a t i v e . In t h i s bioassay s i t u a t i o n , having the standard source of opposing i l l u m i n a t i o n , i t would therefore be possible to t e s t the strengths of attractiveness of any number of samples ranging from highly a t t r a c t i v e to u n a t t r a c t i v e . In the t h i r d stage of the bioassay, the l i g h t i n g was again modified ( F i g . 10). This time l i g h t originated from the overhead c e i l i n g lamps, co n s i s t i n g of four Cool-ray flourescent lamps located about 2 meters above the t e s t stage. Measurements with the Photovolt photometer indicated an output r a t i n g from these flourescent lamps of approximately 40 foot-candles at the source and approximately 10-12 foot-candles at the tes t arena. Using the same procedure for the presentation of wood odour and preparation of beetles as described for the other two l i g h t i n g s i t u a t i o n s , i t was again possible to t e s t the degree of attractiveness of the samples. Those a t t r a c t i v e should again a t t r a c t the experimental specimens while those u n a t t r a c t i v e again should not do so. In t h i s case, beetle o r i e n t a t i o n would again be expected to be random. The r e s u l t s of the wood modification study are based on 50 i n d i v i d u a l s e r i e s of t r i a l s and u t i l i z i n g a t o t a l of 90 Trypodendron adults. I t should be mentioned here that not a l l of the three bioassay l i g h t i n g s i t u a t i o n s described e a r l i e r were used i n these 50 serie s of t r i a l s , i n most cases t h i s v a r i a t i o n was again due to the lack of time required f o r the immediate t e s t i n g of the treated m a t e r i a l . Also the r e s u l t s from t h i s wood study, o r i g i n a l l y i n the form of beetle Fig. 9. Apparatus used to test wood in the 'opposing' light situation. The Reichert illuminator with a Bausch and Lomb heat fi l t e r was used to produce a broad diffuse beam of light. The same apparatus as described for the 'dark' situation was used to present the wood factors. Fig. 10. Apparatus used to test wood with the overhead ceiling lights produced by four flourescent lamps located about 2 meters above the test stage. The apparatus for the presentation of wood factors was the same as that described for the 'dark' and 'opposing' light situations. 28 tr a c i n g s , were computed and summarized i n ta b l e form. R e l a t i v e differences were examined between treated and untreated samples, the l a t t e r Including both a t t r a c t i v e and un a t t r a c t i v e controls„ Much emphasis was placed on analyzing the e f f e c t s of varying periods of anaerobic treatment on the attractiveness of wood chips as indicated by the beetle responses. Experiments i n the present study were conducted from October, 1965 to J u l y , 1966. EXPERIMENTAL RESULTS 29 1. Study of responses to monochromatic light. (a) . Varying wavelengths at a constant intensity. An analysis of the data for the 29 specimens and 2,577 individual trials yielded several interesting and highly significant results. Of the 9 wavelengths tested from 348 mu to 735 mu at an equal intensity, the greatest positive attraction to light occurred with the wavelength 543 mu in the electromagnetic spectrum. Positive responses decreased gradually on either side of this peak, but somewhat more rapidly towards the longer wavelengths (Fig. 11 and Table l ) . Of a l l the 29 male and female specimens tested at this most stimulating wavelength, the mean positive response was 82.4 per cent of individuals tested, and ranging from 77.8 to 87.0 per cent at the 0.05 confidence level. In the longer wavelengths, 686 mu had the lowest •stimulative efficiency' recorded, while in the shorter wavelengths a similar result occurred at 397 mu. At the extreme limit of testing with one constant intensity of light an unexpected phenomenon was recorded in the shorter or ultraviolet wavelength of 348 mu. Responses of beetles were recorded as approximately 40.3 per cent, which was about a two-fold increase over the low adjacent wavelength of 397 mu (18.6 per cent)(Table l ) . Further examination Into the ultraviolet wavelengths below 348 mu with this method of equal intensities was not possible with the limited emissive capacity of the monochromator. Variance analyses on the responses of the 29 beetles tested showed conclusively that the 'stimulative efficiency' of a given wavelength was similar between individuals (Table 2). Males and females appeared to respond equally well to any given wavelength (Fig. 11), although the variability for Table 1. Summarized r e su l t s of beet le responses to var ious wavelengths of monochromatic l i g h t at an equal i n t e n s i t y . Wavelength (nap.) T o t a l females + Per cent pos i t i v e (females) T o t a l males + Per cent pos i t i v e (males) T o t a l females & males + Grand t o t a l Per cent p o s i t i v e (females & males) 348 mu u l t r a v i o l e t 83 117 41.5$ 34 56 37.8$ 117 173 290 40.3$ 397 mu u l t r a v i o l e t 34 166 17.0% 20 70 22.2$ 54 236 290 18.6$ 446 mu v i o l e t 56 144 28.0$ 24 66 26.7$ 80 2l0 290 27.6$ 495 mu green 88 112 44.0$ 36 54 40.0$ 124 166 290 42.6$ 543 mu yellow-green 172 28 86.0$ 67 23 74.0$ 239 51 290 82.4$ 590 mji yel low 132 68 66.0$ 51 39 56.7$ 183 107 290 63.1$ 642 mu red 65 131 33.2$ 32 58 35.6$ 97 189 286 33.9$ 686 mu red 20 170 10.5$ 19 71 21.1$ 39 241 280 13.9$ 735 mu red 1 179 0.6$ 3 87 3.3$ 4 266 270 1.5$ Figure 11. Graphs of t o t a l l e d percentage p o s i t i v e and near p o s i t i v e (?+) responses of Trypodendron t o various wavelengths of monochromatic l i g h t at an equal i n t e n s i t y . 32 Table 2. Summarized s t a t i s t i c s of pos i t i v e phot ic responses of Trypodendron adul ts to var ious wavelengths of monochromatic l i g h t at an equal i n t e n s i t y , abbrev ia t ions ! S^ - va r i ance , S - standard d e v i a t i o n , S j - standard error of the mean Wave-length Beet les S2 % S % % Mean % Upper l i m i t (.05) Lower l i m i t (,05) 348 mu females males summed 66.05 74.45 67.74 8.13 8.63 8.23 1.82 2.88 1.53 41.5 37.8 40.3 45.3 44.4 43.4 37.7 31.2 37.2 397 mu females males summed 85.26 169.45 112.32 9.23 13.02 10.60 2.06 , 4.34 1.97 17.0 22.2 18.6 21.3 32.2 22.6 12.7 12.2 14.6 446 mu females males summed 101.05 100.00 97.54 10.05 10.00 9.88 2.25 3.33 1.84 28'. 0 26.7 27.6 32.7 34.4 31.4 23.3 19.0 23.8 495 ngi females males summed 141.05 125.00 134.98 11.88 11.18 11.62 2.66 3.73 2.16 44.0 40.0 42.8 49.6 48.6 47.2 38.4 31.4 38.4 543 mn females males summed 120.00 127.78 147.54 10.95 11.30 12.15 2.45 3.76 2.26 86.0 74.0 82.0 91.4 82.7 87.0 80.6 65.3 77.8 590 mu females males summed 172.63 100.00 165.03 13.14 10.00 12.85 2.94 3.33 2.39 66.0 56.7 63.1 72.2 64.4 68.0 59.8 49.0 58.2 642 mu females males summed 103.10 127.79 113.00 10.15 11.30 10.63 2.27 3.76 1.97 33.2 35.6 33.9 37.9 44.3 37.9 28.5 . 26.9 29.9 686 mu females males summed 60.82 161.11 113.63 7.80 12.69 10.66 1.79 4.23 2.02 10.5 21.1 13.9 14.3 30.8 18.0 6.7 11.4 9.8 735 mu females males summed approx. 0 n -- - - -33 males alone was greater than that f o r females. This apparent d i f f e r e n c e between the sexes however may have been due to the l i m i t e d number of males examined. Also i n the majority of beetles tested, specimens appeared to respond somewhat more quickly to the more stimulating as opposed to the l e s s e r stimulating wavelengths. Table 3 summarizes the resultant variance analyses between the various wavelengths t e s t e d . (b) Varying both wavelength and i n t e n s i t y . Photic responses of beetles tested using t h i s method are based on 7 females and 3 males, with a t o t a l of 1,843 i n d i v i d u a l beetle t r i a l s . Responses, although much more v a r i a b l e than those described e a r l i e r i n the method employing a constant i n t e n s i t y , were i n most cases quite s i m i l a r for the range of wavelengths from 348 mu to 735 mu. Several t r i a l s with Trypodendron i h the more extreme u l t r a v i o l e t range again substantiated the f i n d i n g derived by the f i r s t methodj that i s , the wavelengths 348 mu, 319 mu and 299 mu are i n f a c t quite stimulating to T. lineatum. L i t t l e d i f f e r e n c e occurred i n responses to 348 mu and 319 mu r e s p e c t i v e l y , but a highly s i g n i f i c a n t d i f f e r e n c e existed between these and 397 mu as e a r l i e r described. A wavelength of 299 mu was extremely stimulating to Trypodendron, being perhaps 3 to 4 times greater than any other wavelength tested, i n c l u d i n g 543 mu i n the v i s i b l e range (Table 4 ) . In e s t a b l i s h i n g the extreme l i m i t of stimulation to Trypodendron i n the longer wavelengths of the electromagnetic spectrum, a region i n the red portion of the spectrum was reached whereby the author was able to perceive without much d i f f i c u l t y , a l l actions taken by T. lineatum i n response to the red monochromatic l i g h t . Beetles however d i d not respond to t h i s red l i g h t (735 mu), t h i s i n d i c a t i n g a t o t a l i n s e n s i t i v i t y to t h i s wavelength at the Table 3. Var iance analys is r e su l t s tested at the 5% l e v e l of s i g n i f i c a n c e showing the d i f f e r ences i h Trypodendron pos i t i ve phot ic responses between wavelengths tested at an equal i n t e n s i t y . Values greater than 8.6 are s i g n i f i c a n t ( * ) . 1.5 (735 mu) 13.9 (686 mu) 33.9 (642 mu) 63.1 (590 mu) 82.6 (543 mu) 42.6 (495 mn) 27.6 (446 mu) 18.6 (397 mu] 40.3 (348 mn) 1.5 (735 mu) - 12 .4* 32.4 * 6 1 . 6 * 8 1 . 1 * 4 1 . 1 * 26 .1* 1 7 . 1 * 3 8 . 8 * 13.9 (686 mu) 12.4 * - 20.0 * 4 9 . 2 * 68.7 * 28.7 * 1 3 . 7 * 4 .7 ' 26 .4 * 33.9 (642 mu) * 32.4 20.0 * -29.2 K 48.7 8 . 7 * 6 .3 15.3 6.2 63.1 (590 mu) 61.6 * 49.2 * 29.2 * -19.5 * 20.5 * 35.5 * 4 4 . 5 * 2 2 . 8 * 82.6 (543 mu) 8 1 . 1 * 6 8 . 7 * 48.7 * 1 9 . 5 * -40.0 5 5 . 5 * 6 2 . 0 * 42.3 * 42.6 (495 mu) 41.1 * 28.7 * 8 . 7 " 2 0 . 5 * 4 0 . 0 * - * 15.0 * 24.0 2.3 27 ;6 (446 mu) 26.1 13.7 * 6.3 3 5 . 5 * 55.0 * 15.0 * -9 . 0 * 1 2 . 7 * 18.6 (397 mu) 1 7 . 1 * 4.7 1 5 . 3 * 4 4 . 5 * 6 2 . 0 * 2 4 . 0 * 9 . 0 * - 2 1 . 7 * 40.3 (348 mu) 38.8 * 2 6 . 4 * 6.2 2 2 . 8 * 4 2 . 3 * 2.3 12.7 * 21 .7* -35 Table A, Table of values from the monochromator c a l i b r a t i o n graphs (appendix 2) showing the percentage of p o s i t i v e responses f o r 10 per cent i n t e r v a l s ranging from 0 to 60 per cent (appendix l ) and the corresponding required energy output of monochromatic l i g h t f o r the wavelengths tested. abbreviations: M.S.= monochromator s l i t width trans. = energy output of any given wavelength Wavelengths 0$ M.S. trans. 10$ M.S. trans. Posit. 20$ M.S. trans. .ve response 1 30$ M.S. trans. evels 4 0 $ M.S. trans. 50$ M.S. trans. 6 0 $ M.S. trans. 299 mu ultraviolet 1.70 3.0$ 1.92 3.9$ - -. - - -319 mu ultraviolet 1.00 32.0$ 1.23 48.0$ 1.45 65.0$ 1.70 88.0$ 1.95 117.0$ - -348 mu ultraviolet 0.32 25.0$ 0.41 43.0$ 0.49 60.0$ 0.62 95.0$ 0.73 135.0$ 0.85 180.0$ 0.99 243.0$ 397 mu ultraviolet 0.14 23.0$ 0.26 83.0$ 0.38 175.0$ 0.53 295.0$ 0.70 505.0$ 0.88 765.0$ 1.09 1065.0$ 446 mu violet 0.10 35.0$ 0.18 110.0$ 0.28 260.0$ 0.40 440.0$ 0.53 645.0$ 0.66 840.0$ 0.84 1140.0$ 495 mu green 0.08 42.0$ 0.09 50.0$ 0.10 65.0$ 0.12 90.0$ 0.15 145.0$ 0.18 210.0$ 0.24 340.0$ 543 mu yellow-green 0.04 10.0$ 0.05 17.0$ 0.06 25.0$ 0.07 37.0$ 0.09 65.0$ 0.10 82.0$ 0.13 140.0$ 590 rap. yellow 0.07 23.0$ 0.075 27.0$ 0.08 32.0$ 0.09 43.0$ 0.11 62.0$ 0.13 88.0$ 0.17 160.0$ 642 mu red 0.16 22.0$ 0.19 35.0$ 0.25 60.0$ 0.32 93.0$ 0.40 145.0$ 0.49 210.0$ 0.62 325.0$ 686 mu red 0.40 50.0$ 0.47 65.0$ 0.57 95.0$ 0.68 135.0$ 0.82 185.0$ 0.95 240.0$ 1.15 335.0$ 735 mu red 1.55 175.0$ 1.60 188.0$ 1.68 210.0$ 1.80 240.0$ 1.98 300.0$ -i n t e n s i t y employed. Further t e s t s with the method using several i n t e n s i t i e s of l i g h t showed quite c l e a r l y that f o r most of the wavelengths tested i n the shorter wavelengths of the red region of the spectrum, the i n a b i l i t y of Trypodendron to respond to various wavelengths i s a function not only of the wavelength i t s e l f , but also the i n t e n s i t y (Table 4 ) . This i s true f o r the whole range of wavelengths examined. A region of no stimulation was reached at about 780 mu and beyond i n t o the longer wavelengths. No further t e s t i n g was attempted beyond 880 mu since the monochromator energy output was decreasing quite r a p i d l y beyond t h i s point. As f o r the effectiveness of the opposing re-alignment l i g h t , no a t t r a c t i o n f o r Trypodendron was observed. The presence of t h i s source of white i l l u m i n a t i o n therefore d i d not appear to a f f e c t i n any way the beetle responses to monochromatic l i g h t . 2. Study of the e f f e c t of water l o s s on photic response. Of the 14 beetles exposed to t h i s treatment, none l i v e d longer than 93 hours. Pronounced differences i n longevity occurred between the two groups te s t e d j those i n the humid environment l i v i n g approximately twice as long as those i n the dry environment (Table 5). E s s e n t i a l l y no d i f f e r e n c e i n the s u r v i v a l times existed between males and females i n the dry environment. However a d i f f e r e n c e appeared i n some but not a l l of the i n d i v i d u a l s i n the moist environment group, the females surviving somewhat longer than the males. As f o r the weight l o s s of the beetles, the data suggest that there i s e s s e n t i a l l y no proportionate d i f f e r e n c e i n weight losses between males, and females i n t h e i r respective environments. However there was a s i g n i f i c a n t d i f f e r e n c e between the beetles i n the two groups, the moist environment group l o s i n g between one-sixth to one-half of that l o s t by beetles i n the dry 37 Table 5. Comparison of the s u r v i v a l times of the two groups of b e e t l e s , one i n the 'humid' environment and the other i n the ' d r y ' environment as measured at the e a r l i e s t time a f t e r death . The time i n t e r va l s i n which beet les died are shown. Humid environment (time i n hours) Dry environment ' (time i n hours) (females) (males) (females) (males) 29.0 - 44.5 27.5 - 31.5 1.0 - 5.5 7.5 - 18.5 29.0 - 44.5 22.5 - 27.5 7.5 - 18.5 7.5 - 18.5 52.5 - 68.5 29.0 - 44.5 7.5 - 18.5 79 .0 - 93.0 29.0 - 44.5 7.5 - 18.5 Table 6. Comparison of weight l osses by Trypodendron i n the ' d r y ' and 'humid' environments, the f i n a l f i g u r e was recorded at the e a r l i e s t poss ib l e measurement a f t e r death. Humid environment {% of o r i g i n a l weight) Dry environment (% of o r i g i n a l weight) (females) (males) (females) (males) 8.53 7.56 40.22 31.08 18.37 18.19 41.30 48.93 15.43 \5 .69 + 34.98 17.05 4.53 + 32.92 "^tested at d i f f e r e n t time 38 Figure 12. Graphs of the cumulative percentage weight losses of individual Trypodendron adults with increased exposure time in the two environments. a. Desiccated group (dry environment) b. Non-desiccated group (humid environment) Time (hours) Table 7. Summarized" i n d i v i d u a l comparisons of dif f e r e n c e s i n photic responses with water l o s s f o r the 'refractory' and 'reaction' times as displayed by adult Trypodendron to white l i g h t at the 1% l e v e l of s i g n i f i c a n c e . abbreviations: N.S. - non-significant comparisons S. - s i g n i f i c a n t comparisons Beetle No. Refractory Period Reaction Time 1 1 N.S. 1 N.S. 2 - -3 1 N.S. 1 N.S. 4 2 N.S. 2 N.S.. 1 S. 1 S. 5 1 N.S. 1 N.S. 6 1..N.S. 1 N.S. 7 3 S. 3 N.S. 8 10 N.S. 10 N.S. 9 3 N.S. 1 N.S. 2 S. 10 21 N.S. 21 N.S. 11 - -12 -13 3 N.S. 2 N.S. 1 s. 14 3 N.S. 3 N.S. Tot a l s 46 N.S. 4 S. 50 46 N.S. 4 S. 50 40 Table 8. Tabulated results of the various photic responses of adult Trypodendron to white light with increasing water loss. abbreviations: m = male f = female Beetle No. & Sex Responses Responses + Responses • Responses t Responses • Responses + Responses f 0 hours 6.0 hrs. 18.0 hrs. 25.0 hrs. oup 1 (f) 7 1 8 0 -cated gr 2 (f) 3 (f) 7 1 8 0 8 0 Desic 4 (f) 5 (m) 6 (m) 8 0 8 0 8 0 o o o to to to 7 1 0 hours 18.0 hrs. 29.0 hrs. 47.0 h r s . 53.0 hrs. 67.0 hrs. 77.0 hrs. 7 (f) 8 0 8 0 8 0 -8 (f) 8 0 8 0 8 0 8 0 7 1 -ft 9 (f) 8 0 8 0 6 2 -ated gro 10 (f) 11 (m) 8 0 8 0 8 0 8 0 8 0 8 0 8 0 6 2 o o •H CQ 12 (m) 2 6 -CD T3 1 a 0 hours 7.0 hrs. 22.0 hrs. 27.0 h r s . o a • 13 (m)1* 14 (m)f 7 1 8 0 7 1 8 0 7 1 8 0 — -tested at d i f f e r e n t time a Figure 13. l i n e tracings of paths taken by two flight-inexperienced female Trypodendron adults to white light while being subjected to varying periods of desiccation. a. Responses of a beetle from the desiccated group 1 4:15 p.m. May 2 i i 9:45 p.m. May 2 i i i 9:40 a.m. May 3 b. Responses of a beetle from the non-desiccated group. i 5:10 p.m. May 2 i i 11:00 a.m. May 3 i i i 9:40 p.m. May 3 abbreviations: RP = refractory period RT = reaction time (measured in one-hundredth of a minute intervals) Fig. 1 3 a i . Desiccated group Beetle No. 4 (female) Time: 4:15 p.m. May 2 Trial RP RT Response 1 49 44 + 2 52 73 + 3 44 109 + 4 40 48 + 5 36 6 2 + 6 72 54 t 7 40 47 + 8 30 55 + F i g . 1 3 a i i . Des iccated group Beet le No. 4 (female) Time: 9:45 p.m . May 2 T r i a l HP RT Response 1 144 38 2 60 55 3 52 105 + 4 112 58 + 5 48 48 + 6 16 32 + 7 30 42 + 8 35 35 Fig. 13aiii. Desiccated group Beet le No. 4 (female) Time: 9:40 a.m. May 3 T r i a l RP RT Response 1 2 109 • 2 7 150 + 3 17 90 • 4 14 116 + 5 18 72 + 6 12 55 + 7 17 188 -8 8 62 + 13bi. Non-desiccated group Beetle No. 9 (female) Time: 5:10 p.m. May 2 Trial RP RT Response 1 31 12 + 2 22 28 + 3 39 18 + 4 32 30 + 5 24 18 • 6 37 21 + 7 18 22 + 8 12 15 • Fig. 13bii. Non-desiccated group Beetle No. 9 (female) Time: 11:00 ajm. May 3 Trial RP RT Response 1 3 17 + 2 4 14 + 3 7 15 + 4 14 19 + 5 1 6 24 + 6 5 18 + 7 31 21 + 8 38 22 + f CD O F-i O CO •a F i g . 1 3 b i i l . Non-desiccated group Beet le No. 9 (female) Time: 9:40 p.m. May 3 T r i a l RP RT Response 1 125 122 • 2 19 54 + 3 27 276 4 32 269 + 5 18 84 + 6 21 27 -7 45 114 • 8 40 90 + environment group' (Table 6 ) . Cumulative weight l o ss data (Appendix 3) were plotted against titae for males and females i n both experimental groups (Figs. 12a and b). Variance analyses f o r the ' r e f r a c t o r y ' and ' r e a c t i o n ' times y i e lded a total of 50 individual comparisons at the 1% l e v e l of s i gn i f i c ance (Table 7 ) . As summarized in Table 8 and i l l u s t r a t e d i n F igures 13a and b fo r des iccated and non-desiccated groups, no s i g n i f i c a n t d i f f e r ence i n phot ic responses to white light appeared i n these f l i gh t- inexper i enced bee t l e s . A comparison between the number of d i r e c t versus i n d i r e c t p o s i t i v e responses of Trypodendron between the groups of t r i a l s was not attempted, as the d is tance covered by a beetle is i m p l i c i t i n the ' r e a c t i o n ' times a lready analyzed. In a few i s o l a t e d inc idents beet le responses d id not fo l low a cons is tent pa t t e rn . Th i s type of behaviour accounted fo r the s i g n i f i c a n t d i f f e r ences found i n Table 7 . 3. The effect of subject ing 'g reen ' wood to anaerobic cond i t i ons . Modified 'green' wood presented to Trypodendron adul ts prompted varying degrees of response. These responses, recorded as beet le path tracings (Figs. 14 and 15) were summarized and c l a s s i f i e d under the following three headings. (1) p o s i t i v e a t t r a c t i on to wood samples (+) (2) negative a t t r a c t i on to wood samples (-) (3) a t t r a c t i o n to wood samples that appeared to be pos i t i v e but were in most cases incomplete (?+) The degree of a t t rac t i veness of anaerob ica l l y t rea ted wood i n i t s normally moist s ta te was r e l a t ed to the durat ion of subject ion to oxygen deficient cond i t i ons . For the f i r s t 4 hours of anaerob ios i s , no t r a n s i t i o n t The final weight was es tab l i shed at the time of the e a r l i e s t weighing a f t e r death. A3 Figure 14. Line tracings of the paths taken by .two flight-experienced female Trypodendron adults in the three different lighting situations and tested to the various types of wood. a. attractive Douglas-fir sapwood control b. 'anaerobic1 sample ( 2 3 . 5 hours) c. 'aerobic' control ( 2 3 . 5 hours) d. air only e. 'green' control f. attractive Douglas-fir control again abbreviations: I - beetle no. I II = beetle no. II 1 = tested In 'dark' 2 = tested in 'opposing' light situation 3 = tested in 'overhead' light situation a = first t r i a l b = second t r i a l c = third t r i a l I2 i jpposing and red l i g h t F i g . 14b. - 'anaerobic' sample (23.5 hours I l a t l i b -I l i a + I l l b + I2a -12b + II2a + 112b * I3a -13b -II3a • II3b + l i b I3b 135 L3a I2h->odour II2b I l i a 44 Figure 15. Line tracings of the paths taken by two male and one female flight-experienced Trypodendron adults tested in the 'dark' and 'opposing' light situations. Wood factors include: a. attractive Douglas-fir sapwood control b. 'anaerobic* sample (21 hours) c. 'aerobic' control (21 hours) abbreviations: I beetle no. I (female) II beetle no. II (male) III beetle ho. I l l (male) 1 tested in 'dark' 2 tested in 'opposing' light situation a first t r i a l b second t r i a l V _oposing and red l i g h t F i g . 15a. - a t t r a c t i v e Douglas-fir Beetle No. I - flown 20 min. Beetle No. II - flown 20 min. Beetle No. I l l - flown 20 min. (intermittent) I l a + I2a + l i b + I2b -I l i a + II2a -I l l b - II2b + I l l l a + III2a ?+ I l l l b + III2b + I l l b F i g . 15b. - 'anaerobic' sample (21 hours) I l a + I2a + l i b + I2b -I l i a + II2a ?+ I l l b - II2b -I l l l a + III2a + I l l l b + III2b + I2b 4-5 from the o r i g i n a l 'green' state was noted. However the attractiveness, as exemplified by beetle responses gradually increased i n the i n t e r v a l from approximately 4 to 20 hours, and appeared to reach i t s peak of attractiveness between 20 to 26 hours. Subjection of the wood to longer periods of anaerobiosis resulted i n a rapid f a l l - o f f i n a t t r a c t i v e n e s s . A 30 hour treatment rendered the wood una t t r a c t i v e ( F i g . 16 and Table 9). Some v a r i a b i l i t y occurred i n the time i n t e r v a l to produce i n i t i a l and peak attractiveness as w e l l as f a l l - o f f ( F i g . 16). Both males and females appeared to respond equglly w e l l to t h i s treated wood (Figs. 14b and 15b). A t t r a c t i v e Douglas-fir sapwood as a c o n t r o l also attracted Trypodendron beetles (Figs. 14a, f j 15a). Beetles responded extremely w e l l to t h i s c o n t r o l i n a l l of the three separate t e s t s i t u a t i o n s (Table 10). Of p a r t i c u l a r s i g n i f i c a n c e with regard to t h i s c o n t r o l when compared with the anaerobically treated wood i s that i n a l l three s i t u a t i o n s , the l a t t e r as judged by the per cent of p o s i t i v e responses was never as a t t r a c t i v e as the Douglas-fir c o n t r o l . Also 'anaerobic' wood tested i n darkness appeared to be more a t t r a c t i v e than that tested i n either of the two l i g h t e d s i t u a t i o n s ( F i g s . 17a, b, o). A s i m i l a r r e s u l t occurred with the a t t r a c t i v e Douglas-fir c o n t r o l . With 'anaerobic' wood i n a l l time i n t e r v a l s a s l i g h t l y higher p o s i t i v e o l f a c t o r y response wgs indicated when the c e i l i n g l i g h t s were used, as compared with the t e s t i n which an opposing l i g h t was used as a reference ( F i g s . 17b and c ) . The 'green' sap-jood controls showed no a t t r a c t i v e properties ( F i g . 14e and Table 10). S i m i l a r l y the c o n t r o l s e r i e s l e f t i n normal aerobic conditions d i d not d i s p l a y any a t t r a c t i v e properties (Figs. 14c, 15c and Table 10) when presented to T. lineatum. This r e s u l t was common to a l l time i n t e r v a l s tested ranging from 2 to 46.5 hours. Leaving wood under 46 Figure 16. Point graph of t o t a l l e d percentage pos i t i v e and near positive'"(?*•) responses t o wood shavings placed under anaerobic condi t ions f o r vary ing per iods of time (2 to 4-6.5 hours ) . Three sets of points have been inc luded f o r the three d i f f e r e n t l i g h t i n g s i tua t i ons ( dark, opposing and overhead). 100 90 -80 • 5* 70 a 13 60 a CQ <r> * 50 to CO H 1 40 £> tn O CD 30 s 20 -10 -aO o • • o o » o 0 0 1 10 OA Oboo o • • o o o o cP • Beetles tested in 'dark' 0 Beetles tested in 'opposing' light situation O Beetles tested in 'overhead' light situation — r -15 — r 20 ^> - q O r~ 25 • O • • o * o o o o» o» O 1 30 35 40 45 i 50 Duration of exposure to anaerobic treatment (hours) 47 Table 9. Summarized data of Trypodendron responses from bioassay tests of wood samples subjected to varying times under anaerobic conditions. Responses are computed as percentages in 4 hour intervals for the three different lighting situations. abbreviations: dk = tested in 'dark' ov - tested in 'overhead' light situation op = tested in 'opposing' light situation Tot. = Total Time Tot. Tot. Tot. Grand % % i interval t ? + - total + ?+ - + & ? + 1-4 dk. 0 0 10 10 0 0 100.0 0 OV. 0 0 10 10 0 0 100.0 0 op. 0 0 14 14 0 0 100.0 0 dk. 0 0 11 11 0 0 100.0 0 4.5-8 ov. 0 1 10 11 - 0 9.1 90.9 9.1 op. 0 1 14 15 0 6.7 93.3 6.7 dk. 2 5 11 18 11.1 27.8 61.1 38.9 8.5-12 ov. 7 0 15 22 31.8 0 68.2 31.8 op. 0 0 20 20 0 0 100.0 0 dk. 8 0 7 15 53.3 0 46.7 53.3 12.5-16 ov. 4 3 16 23 17.4 13.0 69.6 30.4 op. 0 1 18 19 0 5.3 94.7 5.3 dk. 5 3 12 20 25.0 15.0 60.0 40.0 16.5-20 ov. 5 2 13 20 25.0 10.0 65.0 35.0 op. 1 3 16 20 5.0 15.0 80.0 20.0 dk. 33 5 22 60 55.0 8.3 36.7 63.3 20.5-24 ov. 14 1 16 31 45.2 3.2 51.6 48.4 op. 21 6 20 47 44.7 12.8 42.5 57.5 dk.. 6 7 29 42 14.3 16.7 69.0 31.0 24.5-28 ov. 5 7 35 47 10.6 14.9 74.5 25.5 op. 1 3 28 32 3.1 9.4 87.5 12.5 dk. 0 0 10 10 0 0 100.0 0 28.5-32 ov. 0 1 9 10 0 10.0 90.0 10.0 op. 0 0 10 10 0 0 100.0 0 dk. 0 1 4 5 0 20.0t 80.0 20.0f 32.5-36 ov. 0 0 5 5 , 0 0 100.0 0 op. 0 0 10 10 0 0 100.0 0 dk. 0 1 12 13 0 7.7 92.3 7.7 36.5-40 ov. 1 0 s-3 4 25.5+ 0' 75.0 25.0+ op. 0 0 12 12 0 0 100.0 0 dk. 0 0 4 4 0 0 100.0 0 40.5-44 ov. 0 0 4 4 0 0 100.0 0 op. 0 0 4 4 0 0 100.0 0 dk. 0 0 7 7 0 0 100.0 0 44.5-48 ov. 0 0 0 - - - - -op. 0 0 0 — — +insufficient data 48 Figure 17. Frequency histograms of t o t a l l e d percentage p o s i t i v e and near p o s i t i v e responses of Trypodendron to 'anaerobic' samples divided i n t o 4 hour treatment classes and tested under the three l i g h t i n g s i t u a t i o n s : a. beetles tested i n 'dark' b. beetles tested i n 'overhead' l i g h t s i t u a t i o n c. beetles tested i n 'opposing' l i g h t s i t u a t i o n a. Beetles tested i n 'dark' 60 -w c respondi 50 -40 , • beetles 30 " beetles mber of 20 " & 10 • 0 4 8 12 16 20 24 28 32 36 4 0 44 Duration of exposure to anaerobic treatment (hours) 70 -60 -•50 40 30 20 10 b. Beet les tes ted i n 'overhead' l i g h t s i t ua t i on 0 4 8 12 16 20 24 28 32 36 40 44 Durat ion of exposure to anaerobic treatment (hours) c . Beet les tested i n 'oppos ing ' l i g h t s i t u a t i o n T — — i r 0 4 8 12 16 20 24 28 32 36 40 44 Durat ion of exposure to anaerobic treatment (hours) laboratory conditions for these periods of time therefore has no noticeable effects on 'green' wood, so long as a satisfactory moisture level was retained as i t was with chips kept in plastic bags. Anaerobically treated wood in the vacuum chamber also appeared to be at normal moisture levels after treatment, and no condensation was seen on the wall surfaces of the chamber. Air alone as a control also did not induce any responses by T. lineatum (Fig. 14d and Table 10). An interesting difference between the 'anaerobic' and 'aerobic' groups was immediately noticed on opening either the vacuum chamber or the plastic bag. A rather sweet and highly fragrant woody odour was released from the 'anaerobic' chamber, this probably associated with the attractiveness of wood. This fragrance was similar to the human sense as was the odour from Douglas-fir sapwood which had become attractive under field conditions. No similar detectable fragrance was noticed from the 'aerobic' controls, these possessing only the odour of fresh wood as found in 'green' wood. Results of the few tests involving the addition of water to 'green' wood are inconclusive at the present time. Wood that was moistened and then placed under 'anaerobic' conditions for 20-24 hours did not become as attractive as wood not moistened and otherwise treated similarly. However, wetted wood placed only in 'aerobic' conditions also became somewhat attractive to Trypodendron (Table 10). As for the behaviour of the beetles themselves, a definite positive attraction to the attractive samples and controls was observed. In these instances, both male and female Trypodendron walked either directly or, more often indirectly towards the source of odour. Direct responses were those in which beetles followed along the gradient of odour, seldom deviating from odour-stream (Figs. 14a, b, f j 15a, b). Indirect responses in most cases Table 10. Summarized data and response percentages of Trypodendron adults t o various wood samples and c o n t r o l s . abbreviations: dk. - tested i n 'dark' s i t u a t i o n ov. - tested i n 'overhead' l i g h t s i t u a t i o n op. - tested i n 'opposing' l i g h t s i t u a t i o n Wood sample or control T o t a l T o t a l T o t a l Grand % % % + ? + - t o t a l + ?+ — + & ? + 'aerobic' controls dk. 0 3 80 83 0 3.6 96.4 3.6 ( a l l time i n t e r v a l s ) ov. 0^ 0 51 51 0 0 100.0 0 op. o ,1 53 54 0 i i ? 98.1 1.9 . . .. dk. 82 22 28 132 62.1 16.7 21.2 78.8 a t t r a c t i v e Douglas-fir ov. 66 9 42 117 56.4 7.7 35.9 64.1 op. 70 20 49 139 50.3 14.4 35.3 64,7 'anaerobic' wetted dk. 2 2 13 17 11.8 11.8 76.4 23.6 samples ov. 7 0 10 17 41.2 0 58.8 41.2 op. 0 1 18 19 0 5.3 94.7 5.3 dk. 0 5 8 13 0 38.5 61.5 38.5 'aerobic' wetted ov. 5 0 15 20 25.0 0 75.0 25.0 c o n t r o l op. 1 1 10 12 8.3 8.3 83.3 16.6 dk. 0 1 33 34 0 2.9 97.1 2.9 'green' c o n t r o l 6v. 1 2 20 23 4.3 8.7 87.0 13.0 op. 0 0 26 26 o 0 100.0 0 dk. 0 1 19 20 0 5.0 95.0 5.0 a i r alone 6v. 0 0 16 16 . 0 0 100.0 0 op. 0 0 31 31 0 0 100.0 0 51 were those i n which beetles either walked i n somewhat c i r c u l a r meandering paths to reach the source or walked away, then towards the source, generally at an angle to i t ( F i g . 14a). Whenever a beetle crossed the path of the odour-stream, i t s head would a l i g n d i r e c t l y towards the source as though the insect was r e - e s t a b l i s h i n g i t s bearings r e l a t i v e to the source. In the odour-stream i t s e l f , a strong i n v e s t i g a t i v e behaviour was displayed as a conspicuous sideward movement of the beetles' head and f r a n t i c a l l y v i b r a t i n g antennae. On reaching the source however, the beetles i n most cases touched t h e i r heads to the glass nozzle, c i r c l e d a few times, but eventually walked away, either to approach the source of 'opposing' l i g h t or s t r i c t l y meandering about the t e s t arena, depending on the s i t u a t i o n . When p o s i t i v e responses were incomplete (recorded as ?+), a beetle merely responded to the host wood by walking against the gradient of odour but never a c t u a l l y reaching the source ( F i g . 14a). Reduction of the a i r speed s l i g h t l y created no apparent change of response. When unatt r a c t i v e samples were presented, i n the majority of cases the a t t r a c t i o n by the beetles to the 'opposing' source of i l l u m i n a t i o n was quite d i r e c t (Figs. 14c, d, e| 15o), while f o r the 'overhead' and 'dark' s i t u a t i o n s , the responses appeared as meandering paths or s t r a i g h t paths towards the edges of the t e s t arena (Figs. 14c, d, ej 15c). DISCUSSION I The effects of various physical factors on the responses of Trypodendron lineatum (Olivier). 52 1. Monochromatic light, a. General considerations. The data from this phase of the study show that the criterion for relative strength of response to the photic stimulus was the percentage of individuals responding in a test population, the stronger the stimulative effect, the greater the number of individuals responding. Post-diapause, flight-inexperienced adult Trypodendron. both male and female were most strongly attracted to yellow-green monochromatic light at the wavelength of 54-3 millimicrons. This region of peak response resembles that recorded by other investigators for certain other insects. It should be noted here that this wavelength was one of the points chosen for experimental convenience, thereby not truly demonstrating precisely that 543 mu is the most stimulating wavelength in the visible spectrum to Trypodendron. Wigglesworth (1965) has described for the honeybee Apis mellifera Linn., the most efficient part of the visible spectrum in attracting the bee is the yellow-green at about 543 mu or 530 mu. Also the larvae of the mealworm Tenebrio have been found to be readily aroused by the light of 535 mu (Bertholf, 1931). This peak of greatest stimulation also varies greatly between insects however, for in the visible spectrum the cockroach Periplaneta has a peak response at 500 mu (green)(Walther, 1958); and for the ground beetle Carabus, 430 mu (Hasselmann, 1962). Other insects have two peaks of stimulation, such as the blowfly Calllphora at 507 mu (green) and 630 mji (red)(Walther and Dodt, 1957). Burkhardt (1962) describes only one peak in the visible spectrum at 490 mu for Calllphora. The single peak of stimulation in the visible spectrum however 53 appears to be most common i n insec ts (Wigglesworth, 1965). The gradual decrease i n ' s t imu l a t i v e e f f i c i e n c y ' from the peak to the v i o l e t on the one hand and the red wavelengths on the other i nd i ca te c l e a r l y that Trypodendron exh ib i t s strong pos i t i v e responses only to the one reg ion i n the v i s i b l e spectrum. Th is decrease i s more gradual toward the shorter than the longer wavelengths. Th is form of luminos i ty curve i s s im i l a r to that d iscovered by Jahn (1946) fo r the grasshopper Melanoplus, which has a peak s e n s i t i v i t y i n the blue-green at about 500 mu. The l eas t s t imu la t ing wavelength of 397 mu c l o se l y approximates that descr ibed by Burkhardt (1962) f o r the b lowf ly C a l l l p h o r a (400 mu). For the longer wavelengths, not only d id the ' s t imu l a t i v e e f f i c i e n c y ' f a l l more abrupt ly from the peak, but a lso i n t h i s range of the spectrum the r o l e of the i n t e n s i t y has been found to be a primary va r i ab l e i n l i m i t i n g the e f fec t i veness of the wavelength. In the red range p a r t i c u l a r l y , the wavelength i s not the only determinant of beet le responsiveness , but a lso the i n t e n s i t y from the emit t ing source. Wavelengths which i n i t i a l l y appeared to be l i m i t i n g to T . l ineatum could induce a response i f the i n t e n s i t y was ra i sed to a s a t i s f a c t o r y l e v e l . Mazokhin-Porshnyakov (1964) descr ibes a s im i l a r phenomenon to ex i s t fo r other i n s e c t s . However, eventua l ly a point (780 mu) i s reached whereby there i s no fur ther response i n the red port ion of the electromagnetic spectrum. With these longer wavelengths i t i s reasonable to assume that the l i m i t ind ica ted fo r wavelength s e n s i t i v i t y i s t r u l y a func t ion of wavelength rather than i n t e n s i t y , s ince an increase of i n t e n s i t y d i d not provoke a response. Wigglesworth (1965) s tates that most insec ts seem to be i n s e n s i t i v e to the deeper shades of r e d . The honeybee fo r ins tance , does not respond to wavelengths beyond 650 mu (von F r i s c h , 1914J Kuhn, 1927). Wasps (Vespa) 54 a f t e r being t r a i n e d to v i s i t a black surface can be divided equally by black or red (Schremmer, 1941). S i m i l a r l y i n the blowfly C a l l i p h o r a , the v i s i b l e spectrum extends as f a r as 730 mu (Autrum and Stumpf, 1953). On the other hand other i n s e c t s such as the b u t t e r f l i e s P i e r i s brassicae (Linn.) and Vanessa u r t i c a e Linn, have an undoubted perception f o r red ( U s e , 1928), and when presented with red paper models or flowers, even show a preference f o r these objects. At the other end of the electromagnetic spectrum towards the u l t r a v i o l e t , responses appeared to increase a f t e r reaching a low response at 397 mu. This increase was examined only up to 299 mu, but f o r t h i s short i n t e r v a l i t rose abruptly. Although the evidence to date i s s t i l l l i m i t e d , immediate i n d i c a t i o n s are that Trypodendron responds exceptionally w e l l i n the u l t r a v i o l e t wavelengths, possibly much more so than to the extremely stimulating wavelengths of the ordinary dispersion spectrum. This conclusion has been put f o r t h by several other authors f o r other i n s e c t s i n c l u d i n g Bertholf (1931) and Lutz (1924). The l a t t e r have found that i n the honeybee Apis m e l l i f e r a Linn, and the f r u i t f l y Drosophila, a second peak of 'stimulative e f f i c i e n c y 1 four or f i v e times as high as the peak i n the v i s i b l e part of the spectrum has been observed at 365 mu. Walther and Dodt (1957) have described the peak at about 340 to 350 mu f o r Periplaneta and f o r C a l l i p h o r a . Cameron (1938) has found also that i n the housefly Musea no peak of stimulation could be detected i n the v i s i b l e range of the spectrum but recorded a very high peak at about 365 mu and another even.further i n t o the u l t r a v i o l e t at 302 mu. No t r a c e of the maximum at 365 mu has been described by Sander (1933) and Wigglesworth (1965). The weak em i s s i v i t y of the monochromator f o r the shorter wavelengths prohibited the establishment of the ultimate wavelength, l i m i t f o r the s e n s i t i v i t y of Trypodendron to u l t r a v i o l e t . Whether or not there i s any v a r i a b i l i t y i n s e n s i t i v i t y i n either of the two parts^ of the Trypodendron eye has yet to be ascertained. In Periplaneta the high s e n s i t i v i t y to u l t r a v i o l e t i s l i m i t e d to the d o r s a l h a l f of the eye while the green,sensitive receptors are found i n a l l parts (Walther, 1958). Hertz (1938) suggested that the u l t r a v i o l e t region i s probably perceived as a true colour to i n s e c t s , even though i t i s i n v i s i b l e to man. Ozone i n the upper atmosphere f i l t e r s out a l l the f a r u l t r a v i o l e t , and l i t t l e shorter than 300 mu reaches the earth (Goldsmith, 1961). Because of t h i s Mazokhin-Porshnyakov (1964) concludes that i n nature u l t r a v i o l e t r a d i a t i o n plays a minor r o l e i n the reactions of i n s e c t s . Tests by Chapman and Kinghorn (1958) using traps i n v o l v i n g u l t r a v i o l e t l i g h t although a t t r a c t i v e to Trypodendron (Chapman and Kinghorn, 1955), f a i l e d to catch large numbers, probably because of low evening temperatures which were not s u f f i c i e n t to stimulate Trypodendron to f l y . Also the use of coloured cardboard traps d i d not a t t r a c t T. lineatum even during daylight hours when s i g n i f i c a n t numbers of these insects were observed i n f l i g h t . I t i s probable that other external stimulating f a c t o r s such as a t t r a c t i v e host odour were at t h i s time determining beetle responses since beetles were i n t h e i r normal post-hibernating attack condition. In t h i s laboratory however, monochromatic l i g h t was the only f a c t o r presented. Three types of receptor c e l l s have been described by Burkhardt (1962) f o r the blowfly C a l l i p h o r a . The green s e n s i t i v e receptors however account f o r almost 75 per cent of the t o t a l complement, while blue s e n s i t i v e (470 mp.) and yellow-green (520 mp) c e l l s comprise the remainder. I t i s suggested that the greatest stimulating wavelength (490 mu) i s a d i r e c t r e s u l t of the predominance of the green type receptor c e l l . In Trypodendron, i t i s Trypodendron has divided eyes. 56 possible that the majority of c e l l s are of the yellow-green type: t h i s could account for the peak of stimulation at 543 mu i n the v i s i b l e spectrum. Burkhardt (1962) described also that a l l 3 receptor types show a peak of stimulation at 350 mu i n the u l t r a v i o l e t . This phenomenon i s also possible i n the case of T. lineatum. Walther and Dodt (1959) however described only two types of receptor systems, one s p e c i a l i z e d to the u l t r a v i o l e t wavelengths while the other i s s p e c i a l i z e d to the v i s i b l e spectrum. Although the beetles were c o n t i n u a l l y being subjected to white l i g h t during the r e - a l i g n i n g periods of t h i s study, no apparent changes i n response through beetles becoming dark adapted were noted. Dolley (1929) has found that dark adaption f o r one hour increased the s e n s i t i v i t y of the eye of the hoverfly E r 1 s t a l l s tenax (Linn.) by 21 times. b. New information for the bioassay technique. Future bioassay of experimental wood samples w i l l depend on c e r t a i n e s s e n t i a l standards as w e l l as r e f i n e d techniques. The r e s u l t s of the present study of the action of monochromatic l i g h t on the photic responses of Trypodendron make possible the d e f i n i t i o n of photic standards of reference f o r the bioassay of o l f a c t o r y s t i m u l i . The f i n d i n g s demonstrate the importance of wavelength as f a c t o r s i n a t t r a c t i o n of beetles to l i g h t . The r e l a t i v e •stimulative e f f i c i e n c y ' of an odour can therefore be ascertained only i f i t i s tested against a l i g h t which i s standardized i n respect to these parameters. Previous studies by F r a n c i a (1965) depended on an a r b i t r a r i l y chosen standard of white l i g h t as a photic reference for photic responses. The i n v e s t i g a t i o n on monochromatic l i g h t has established what would be s a t i s f a c t o r i l y considered a constant l i g h t source to be placed i n opposition to t e s t host materials. Since the wavelength 543 mu has been thoroughly tested and found to be extremely e f f i c i e n t i n a t t r a c t i n g T. lineatum even at low i n t e n s i t y l e v e l s , i t appears to be an i d e a l source of standard l i g h t . Being i n the v i s i b l e range of the spectrum also adds to the advantages f o r the in v e s t i g a t o r . Two measures of host attractiveness can be attained using a standard source of l i g h t , one involving a constant i n t e n s i t y while the other uses varying i n t e n s i t i e s . With constant i n t e n s i t y the response of beetles would depend on the attractiveness of d i f f e r e n t wood samples, the more a t t r a c t i v e the sample, the greater the number of p o s i t i v e responses to i t . This method would give a s o l i d measure of attractiveness of any number of wood samples of varying degrees of att r a c t i v e n e s s . This has i n f a c t been the method of bioassay adopted i n the present i n v e s t i g a t i o n on the modification of 'green' wood although three d i f f e r e n t constant i l l u m i n a t i o n sources were used. When i n t e n s i t y of the standard l i g h t i s varied however, l i g h t i n t e n s i t y becomes an i n d i r e c t but possibly more precise measure of host wood at t r a c t i v e n e s s . The quantity of l i g h t required to equalize responses between l i g h t and wood could be used f o r Trypodendron; therefore with a constant wavelength of l i g h t , a sample which i s extremely a t t r a c t i v e would require more energy to equalize the responses of the insects to an approximate one-to-one r a t i o than a weakly unattractive sample. Any combination of r a t i o s between l i g h t and wood could be used, including the minimum quantity of energy required to a t t r a c t a l l the insects away from the wood source. I t must be expected however, that considerable v a r i a t i o n i n responses of d i f f e r e n t i n d i v i d u a l s w i l l appear. V a r i a t i o n w i l l be the r e s u l t of i n t r i n s i c d i f f e r e n c e s i n the r e a c t i v i t y of d i f f e r e n t i n d i v i d u a l s , as w e l l as of d i f f e r e n t i a l s u s c e p t i b i l i t y to modification by f l i g h t experience. The study has also established a standard of red monochromatic l i g h t at the wavelength of 7 3 5 mu to simulate darkness to the beetles, while leaving 58 the test arena sufficiently visible to the investigator. Previous studies of Francia (1965) and Wright (1966) used red cellophane over a source of light to reduce the photic stimulus. Since red monochromatic light of specific wave-lengths and intensities has been found to be non-stimulating to Trypodendron, this affords an opportunity to study the effects of olfactory stimuli alone in determining beetle responses. 2. Water loss versus photic responses and its implications to the bioassay  technique. The study on the effects of water loss on the photic responses of Trypodendron to white light showed conclusively that l i t t l e or no change occurred in flight-inexperienced individuals. Although this phase of the study was a purely artifical situation as such, a number of important implications can be derived from i t for the bioassay technique. Assuming that the principle of water loss applies to attraction to the odour factors as well, i t is possible that for the bioassay itself, flight-inexperienced or unflown T. lineatum adults may therefore be tested in darkness or under dark red illumination without any apparent side-effects arising from this water loss. Although this study did not cover a comparable situation for flight-exercised or flown beetles, indications from the bioassay of the present investigation are that no observable changes had occurred. Specimens in the bioassay however were kept in moistened containers to minimize any possible loss, but in most cases they were tested for extensive periods of time under laboratory conditions. In these instances, a beetle was continually being tested and retested for periods of up to six hours without any apparent modifications in the orientation to either light or host material. This result was most easily observed in situations where attractive Douglas-fir controls were offered to Trypodendron at the commencement and then at the 59 conc lus ion o f a s e r i e s of t e s t s . It i s s t i l l poss ib l e however that other undetected changes i n bee t l e responses may have r e s u l t e d , e spec i a l l y s ince f l i gh t-exper i ence i t s e l f i s a great modi f ie r of phot ic o r i en ta t ion (F ranc ia , 1965). The f a c t that Trypodendron l o s t water cont inuously under laboratory temperatures and humidi t ies s i g n i f i e s the importance of mainta in ing adequate moisture l e v e l s . The maximum water l o s s of 25 per cent before death r e s u l t s , as descr ibed by N i j o l t and Chapman (1964) approximates qu i te c l o se l y the values obtained from t h i s i n v e s t i g a t i o n . An i n t e r e s t i n g observat ion regarding the s u r v i v a l times and water l osses i n the two bee t l e groups was noted where those i n the humid environment surv ived longer but l o s t much l e s s water than those i n the dry environment, as measured a f t e r death . Th is poss ib l y s i g n i f i e s that water i s not the only f ac to r which determines the s u r v i v a l of the i n s e c t s . Food has been discounted by Chapman (1955a). Temperature however i s probably another important f a c to r a f f e c t i n g the s u r v i v a l of the i n s e c t s . D i rec t contact wi th con t ro l l ed quan t i t i e s of water are a l so p re requ i s i t es to t h e i r s u r v i v a l (Chapman, 1955a). The few minor d i f f e rences i n some of the r e s u l t s fo r the ' r e f r a c t o r y ' and ' r e a c t i o n ' times can most r e a d i l y be explained as a random behaviour of the insec t i n ques t ion . S ince each group at any per iod of t e s t i n g had only 8 t r i a l s , even one extremely prolonged response by a bee t l e could e a s i l y upset the o v e r a l l group response. These extreme responses were i n many cases so great that they genera l l y exceeded the combined t o t a l fo r the remainder of the t r i a l s i n a group, e i ther f o r the ' r e f r a c t o r y ' or ' r e a c t i o n ' times (Appendix 4 - bee t l e Ho. 9 and No. 10 ) . In these instances any given beet le d id not respond in the ' r e f r a c t o r y ' per iod t e s t s | i t exh ib i ted l i t t l e or no movement fo r a prolonged per iod of time as i f not responding to any externa l 60 s t i m u l i . In the 'reaction' t e s t s , beetles exhibited either frequent prolonged stops or generally long meandering paths of response, thus,extending greatly the recorded time of response. The majority of groups of 8 t r i a l s however were f a i r l y consistent f o r each beetle under consideration. Beetle e x h i b i t i n g e i t h e r short or long 'refractory' or 'reaction' times tended to reproduce t h i s type of behaviour as many times as each was tested. In t h i s way analyses of data y i e l d e d r e s u l t s that were not s i g n i f i c a n t l y d i f f e r e n t f o r each of the times a beetle was t e s t e d . I I The modification and bioassay of 'green' wood using the newly-proposed  technique of a n a l y s i s . 1. The wood f a c t o r s . This study shows conclusively that the subjection of 'green' una t t r a c t i v e Douglas-fir sapwood to oxygen d e f i c i e n t conditions induced attractiveness f o r the ambrosia beetle Trypodendron lineatum ( O l i v i e r ) . I t was also shown that the degree of attractiveness of t h i s wood depended l a r g e l y on the duration of subjection to anaerobic conditions. Wood not placed under these conditions f o r s i m i l a r periods of time d i d not become a t t r a c t i v e . O r i g i n a l l y 'green', a sample of host material became s l i g h t l y a t t r a c t i v e a f t e r L hours under 'anaerobic' conditions and gradually rose i n attractiveness with f u r t h e r periods of anaerobiosis. For these experiments, the t r a n s i t i o n from the 'green' u n a t t r a c t i v e to the ' r i p e 1 a t t r a c t i v e condition reached i t s optimum between the time i n t e r v a l from 20 to 26 hours. After t h i s peak of attractiveness the wood l o s t i t s attractiveness a f t e r about 30 hours of oxygen d e f i c i e n t conditions. Some v a r i a b i l i t y d i d occur however between wood samples given s i m i l a r periods of treatment. This could possibly have been a r e s u l t of the t e s t beetles themselves i n addi t i o n to the wood. In the f i e l d , 61 Chapman (1962) and Dyer and Chapman (1965) have recorded a considerable degree of v a r i a b i l i t y between logs of s i m i l a r f e l l i n g dates. The reason f o r t h i s apparent d i s p a r i t y remains unknown at the present time. Wood placed under anaerobic conditions f o r periods ranging from 30 to 46.5 hours also yielded r e s u l t s i n d i c a t i n g that l i t t l e i f any a t t r a c t i v e properties remained. A s i g n i f i c a n t change i n 'green' wood can therefore be at t r i b u t e d to the anaerobic treatment. Wood i n the o r i g i n a l 'green' u n a t t r a c t i v e state had a s l i g h t l y pungent fragrance while that i n the a t t r a c t i v e 'ripe' condition possessed a sweet f r u i t y fragrance. Unattractive wood i n the 'spent' condition had a rather f l a t woody odour, d i s t i n c t l y separable from the other two conditions. In some i n -stances, a s l i g h t l y sour odour could be detected i n those samples l e f t f o r the longer periods of time. Because of i t s highly t r a n s i e n t nature, these odours were found to d i s s i p a t e quickly during or a f t e r laboratory t e s t i n g . The experimental treatment appears to have induced i n 20 hours, odour changes which i n the f i e l d require several weeks or months (Dyer and Chapman, 1965} Gaumann, 1930} Hadorn, 1933} Mathers, 1935} Prebble and Graham, 1957). The foregoing findings which strongly i n d i c a t e the involvement of anaerobic processes, provide a possible explanation f o r some of the f i e l d observations reported by various authors. Several authors attempted to c o r r e l a t e suscepti-b i l i t y with moisture content. Kinghorn (1956) used moisture content as an in d i c a t o r of attack density but merely found that the apparent l a c k of moisture may have l i m i t e d the density of attacks, but an excess of moisture did not i n any way increase l o g at t r a c t i v e n e s s . Johnson (1961) i n western Washington has shown that sections of logs of western hemlock with branches remaining were not attacked while the remaining portions of the bole suffered considerable attacks. In comparison, adjacent tops with branches removed sustained f a i r l y 62 heavy a t t acks . A lso the por t ion of the bole with tops in tac t were found to have a s i g n i f i c a n t l y lower moisture content . Later s tudies a lso showed that t rees with crowns i n t ac t susta ined no Trypodendron attacks whi le t rees without branches sustained heavy a t t acks . Moisture samples of the t rees showed the l e v e l to have dropped to approximately AO per cent from we l l over 100 per cent i n those t rees wi th tops i n t ac t (Johnson, 1964). Contrary to the r e s u l t s of Kinghorn (1957) and Dyer (1963), i t has been suggested that the rate of dry ing has i n h i b i t e d the formation of a t t rac tant substances. B l e t ch l y (1961)j C h r i s t i a n (1932) and F i she r and Thompson (1952) a l so be l i e ve moisture to be an e s s e n t i a l f a c to r a f f e c t i n g beet le a t tack . We are now confronted wi th the quest ion as to why moisture content could be important, and whether i t i s important per se or merely an i n c i d e n t a l co r r e l a t e which accompanies the t rue i n f l uence . When the anaerobic experiments were undertaken, they were based on the hypothesis of Graham (1962) that one of the accompaniments to s t ress or death i n a t ree i s the reduct ion or cessation, of sap movement. Th is i n turn would a r res t the intake of d i sso l ved oxygen with s o i l mois ture . It i s assumed that the l i v i n g c e l l s of the sapwood, namely the wood cambium and the ray c e l l s (MacDougal et a l , 1929), normal ly depend on d i sso l ved oxygen i n the sap, or f r ee oxygen i n the i n t r a - and i n t e r c e l l u l a r spaces f o r normal metabolism. General knowledge of intermediary metabolism would i nd i ca t e that depr i va t ion of oxygen would r e s u l t i n g l y c o l y t i c of* fermentat ive metabolism which y i e l d s fermentat ive end products (Meyer et a l , 1960). Th is may expla in the nature of o r i g i n of a t t rac tan ts i n wood i f oxygen r e a l l y plays a s i g n i f i c a n t r o l e i n normal metabolism of cambial and ray c e l l s , and i f oxygen does f a l l below a c r i t i c a l concentrat ion i n a f e l l e d or dying t r e e . At present , d i r e c t data are l a ck ing on changes i n the gas composit ion wi th in a t r e e . The repor ts on moisture changes i n logs may however have 63 significance. When moisture is lost from a tree, air must replace i t , thereby-providing a substitute form of aeration which would delay the onset of fermen-tative metabolism and thus prevent attractants from forming. The work of several authors on autoclaving and starch content also offer possible bases for theories on the processes occurring in felled trees or logs. Kinghorn and Chapman (1957) have studied the effects of autoclaving and aging in Douglas-fir blocks. Sections were obtained from freshly felled trees, treated, then stored until flight and attack by T. lineatum. It was found that autoclaving and aging together rendered them unsusceptible to attack. This result could have arisen through either the prevention or the depletion of any attractant substances, the effect was in most likelihood due to the heat treatment rather than the aging. This could possibly indicate that an enzymic process may be involved in the attractant formation process. Busgen (1929) contends that the starch level is a good indicator of changes in the physiology of living trees. Chapman et a l (1963) studied the effects of starch content as a determinant of log attractiveness. They concluded however that a high starch content was correlated with a low density of attacks but also that a. low starch content did not induce attack. High starch contents are prevalent in standing trees and freshly cut timber, these have been shown to be un-attractive to Trypodendron (Chapman, L959, 1961; Dyer, 1964} Dyer and Chapman, 1965? Francia, 1965} Hadorn, 1933; Kinghorn and Chapman, 1957} Novak, I960} Prebble and Graham, 1957). Jones (i960) in West Africa however studied various aspects of ambrosia beetle biology and found no correlation to exist between the degree of susceptibility to either starch or moisture content. Meyer et al (I960) described the possible outcome in higher plants, normally aerobic which have been exposed for varying periods of time to anaerqbic environments. At least two possible injurious effects have been described as resulting from this process. One is that in metabolically active tissues especially, the curtailed rate of energy release is probably inadequate for the normal maintenance of cell processes and deleterious effects are soon engendered within the cells. Another possible result of fermentation is the accumulation of substances which exert toxic effects on the protoplasm. Ethyl alcohol and other more or less toxic compounds are known to be accumulated in the cells. The presence of volatile substances falls closely in line with the pattern of events found in susceptible host wood. Browne (1952) has reported that the injection of alcohol into trees causes attraction of some ambrosia beetles, even after wood has become unattractive through seasoning. The process of metabolic fermentation has also been postulated by other investi-gators studying host selection including Baker (1956), Champlain and Kirk (1926), Chapman (1956), Ohnesorge (1953) and Person (1931). Binion (1962) reported attraction by T. lineatum to fermenting mixtures in beer dregs. MacDougal et a l (1929) and Boberg and Juhlin-Dannfelt (1926, 1928) investigated the changes in cut logs and found conclusively that fermentative and other changes occur in the living cells of sapwood leading to changes in the composition of gas. Carbon dioxide is always present in amounts much greater than in the atmosphere (sometimes 60 times as great) while the amount of oxygen is much less, and the sum of oxygen and carbon dioxide is less than in air. Scarth (1930) and Scarth and Gibbs (1930) testing wood from floated logs found very high concentrations of carbon dioxide. Fermentation inside floated logs of balsam and spruce has been described by Scarth and Jahn (1930). Attacks by Trypodendron have been studied in floated water-soaked logs by Dyer and Chapman (1962). The results obtained from the phase of the study with the addition of water to the wood shavings need further clarification. A possible explanation as to why both 'anaerobic' and 'aerobic' samples became attractive to Trypodendron may be that water may somehow be blocking the normal function of gas exchange associated with the woody tissues, thus again providing another a r t i f i c i a l anaerobic situation. Dyer and Chapman (1962) concluded that water is not a deterrent to the formation of attractant substances. Essentially a l l authors today studying the mechanism of origin of host attractant suggest that the phenomenon is purely metabolic in nature rather than the result of extraneous biological factors such as micro-organisms. It is not impossible however that the production of attractants is under some influence of bacteria, yeasts or even fungi. There is no doubt that micro-organisms are present on Douglas-fir sapwood and are probably functioning at or near the sites of cellular activity (Bier, 1966). Their effect on the production of attractants however remains an open question. Yite and Gara (1962) have ruled out the role of yeasts and other micro-organisms as a possible source of attractants but this applies only to those associated directly with the insect. Person (1931) provides three possible alternatives regarding the nature of this attractant formation. The first that he suggests is that of micro-organisms solelyj the second is the cellular activity of wood alone, and the third is a combination of both micro-organisms and wood factors. It is noteworthy that of a l l the various treatments tested, none produced the level of attractiveness of naturally 'ripened' wood in logs. This phenome-non may signify that much of the attractant substance produced anaerobically from 'green' wood could have dissipated from the shavings before they were tested. The use of interconnected double series of bottles however appeared to minimize this effect. The fact that T. lineatum when attracted to the source are not retained at this site has been described earlier by Francia (1965). A considerable degree of variability in the period of retention was observed between beetles. It is most probable that other stimuli must be involved in retaining the insects at the source. Rudinsky (1966) has postulated a visual cue to be necessary for the Douglas-fir beetle Dendroctonus psQudotsugae Hopkins once this insect has entered the perimeter of attraction. Tactile stimuli must also become a part of the host selection requirements once the beetles have reached the source of attraction (Rudinsky and Daterman, 1964). In any event, the importance of the 'anaerobic' hypothesis must be recognized such that i t can now play a significant role in the formulation of a working hypothesis to further investigate the manner in which wood becomes susceptible to beetle attack. Perhaps the next line of pursuit would be to test for oxygen levels in both trees and logs (Graham, 1966). 2. The bioaseay^technique. As for the bioassay technique used in the present investigation, several important comments should be made in reference to its effectiveness. It has become apparent that the anemo-olfactory technique offers a satisfactory degree of sensitivity when distinguishing host factors of various degrees of attractiveness. Pencil tracings have been found to be appropriate for recording responses, mainly because they are permanent records of the exact paths taken by a beetle. However when this type of record is not required, i t is possible to record only the mere presence or absence of a traversal by a beetle (Wood and Bushing, 1963). In future studies, traversal times would prove useful i f critical measurements of differences are required (Anderson and Fisher, I960; Pertunnen, 1957). It is the aim of any investigator to accomplish as much as possible with a limited supply of experimental equipment in the shortest possible time. It 67 would therefore seem more logical to use more experimental beetles, a l l tested at the same time to give a sample population response to one specific host treatment. This contrasts with the present undertaking in which individual beetles were used. Wellington et al (1954) have briefly mentioned this very subject and have stated i t to be for one, dependent on its convenience to the observer. With Trypodendron. at least for the present time, i t is easier to handle individual specimens. Results are eventually discussed in terms of groups irrespective of the method of study. Although the study of wood susceptibility is related to the whole beetle population, i t is also important to study the variations in individual behaviour. This has been repeatedly demonstrated in the experiments where much variability was encountered with this Trypodendron population. It was found that certain beetles consistently gave poor responses while others gave highly favourable responses. Unless individual beetles could be identified within a group, this particular behavioural pattern would probably go undetected. Even with the three lighting situations of this study, i t became clearly established that the intensity of the opposing light can be used in future studies as a measure of attractiveness. The data obtained on wood attraction when overhead light was used did not differ as much from that with the opposing light, but i t was strikingly different when the dark red light situation was compared to the other two situations. This reflects clearly the role of light in attracting insects away from any source of olfactory stimulation. A slightly higher positive response to wood however appeared lti the 'overhead' light situation, the latter having an approximate light intensity of 10 to 12 foot-candles at the arena test field. The 'opposing' light was approximately 14 foot-candles on the test field,, this higher intensity would therefore draw more beetles away from the host wood source. The implications for future 68 bioassay studies of this finding have been discussed earlier in this text with reference to the use of monochromatic light as a possible source of standard opposing illumination. CONCLUSIONS 69 1. Of the wavelengths of monochromatic l i g h t tested using the two methods of a n a l y s i s , a peak of 'stimulative e f f i c i e n c y ' occurred at 54-3 mu. On either side of t h i s maximum, the 'stimulative e f f i c i e n c y ' decreased, t h i s decrease was more gradual towards the longer wavelengths of the u l t r a v i o l e t . 2. A threshold of stimulation was reached i n the longer wavelengths of the red portion of the spectrum at which time and beyond which T. lineatum was not capable of detecting any source of t h i s i l l u m i n a t i o n , while the author was s t i l l capable of observing the beetles' responses. 3. Towards the u l t r a v i o l e t range of the spectrum, i n d i c a t i o n s from the r e s u l t s using the two methods of t e s t i n g are that an increase i n 'stimulative e f f i c i e n c y ' appears from approximately 4-00 mu and continues i n t o the u l t r a v i o l e t wavelengths. Using the method of varying i n t e n s i t i e s , r e s u l t s suggest that the 'stimulative e f f i c i e n c y ' i s highest i n t h i s region, perhaps 3 to 4 times greater than the v i s i b l e wavelength 543 mu. 4. From the study of monochromatic l i g h t , i t was shown that the wavelength i s not the only f a c t o r determining the photic o r i e n t a t i o n of T. lineatum, but a l s o the i n t e n s i t y of any given wavelength. 5. Male and female T. lineatum appear to respond equally well to monochro-matic i l l u m i n a t i o n . • 6. The use of monochromatic l i g h t i n the bioassay technique can now be j u s t i f i e d . Not only has a threshold of response been found at 735 mu to simulate darkness to T. lineatum, but a l s o highly stimulating wave-lengths are proposed as possible standard sources of i l l u m i n a t i o n . This i m p l i c a t i o n has been discussed i n the t e x t . 7. The use of the anemo-olfactory behaviour of T. lineatum can a l s o now be j u s t i f i e d as a method of bioassay. As pedestrians, t h i s i n s e c t i s quite amenable to use as a t e s t instrument f o r determining the attractiveness of any number of wood samples, provided proper environmental conditions are maintained and beetles are i n t h e i r proper p h y s i o l o g i c a l stage. 8. 'Green' u n a t t r a c t i v e Douglas-fir sapwood can be made to undergo a chemical t r a n s i t i o n to an a t t r a c t i v e ' r i p e ' state and a f u r t h e r 'spent' una t t r a c t i v e state as indicated by Trypodendron responses, by placement of such wood under oxygen d e f i c i e n t conditions f o r varying periods of time. 9. For wood shavings of Douglas-fir sapwood, an optimum of attractiveness developed f o r periods of 20 to 26 hours of anaerobic treatment; t h i s a t t r a c t i v e n e s s beginning at approximately 4 hours and reaching an unattractive or 'spent' state at approximately 30 hours. However, some v a r i a b i l i t y d i d r e s u l t between samples of s i m i l a r treatment times. 10. No change occurred i n the 'aerobic' co n t r o l wood shavings l e f t under laboratory conditions. A s i g n i f i c a n t change to wood can therefore be a t t r i b u t e d to the anaerobic treatment. 70 11. 'Green' wood was also unattractive to T. lineatum. this result agreeing with the findings of other investigators.. 12. No significant difference was found to occur in the photic responses of T. lineatum to white light as a result of increasing water loss. This not only applied to the ratio of positive to negative responses, but also to the reactivity of both males and females. 13. Trypodendron males and females did not appear to be able to withstand water loss while being subjected to laboratory conditions (44-4-6 per cent R.H., 23-26 degrees Centigrade). Extreme care in storage and handling must be taken to prolong the survival time of these insects. LITERATURE CITED 71 Anderson, J . "Mr and K . C F i she r , I960. The response of the white pine weev i l to na tu ra l l y occurr ing r e p e l l e n t s . -Can. J . Zoology 54-7-564. Autrum, H. and H. Stumpf. 1953. E lek t rophys io log ische Untersuchungen uber das Farbensehen von C a l l i p h o r a . Z. v e r g l . Phys i o l . j_5: 71-104-. Baker, J\ M. 1956. Investigations on" the oak pinhole bore r , Platypus cy l lndrus Fab. A progress r epo r t . B. W. P. A. Annual Convention, 1956. Be r tho l f , L.M. 1931. The d i s t r i b u t i o n of s t imula t i ve e f f i c i e n c y i n the u l t r a -v i o l e t spectrum fo r the honeybee. J . Agr . Research __2* 379-4-19. B i e r , J . E, 1966. Personal communication. B i n i on , W. 1962. A t t r a c t i o n of the ambrosia beet le Trypodendron by beer dregs . Proc . Ent . Soc. B. C. __h 53. B l e t ch l y , J . D, 1961. A review of f ac to rs a f f e c t i n g ambrosia bee t le at tack i n t rees and f e l l e d l ogs . The Empire Fores t ry Review LO ( l ) (103): 13-18. Boberg, S. and M. Juh l in-Dannfe l t . 1926._ Viktsundersukningar a f l o t tgods (weight of dr i ven l o g s ) . Skogsvardsfor . T i d s k r . 2_: 262-282. (See a l s o B i o l . A b s t r . 1: 7400, 1927.) , 1928, On f l y tba rhe ten hos fu ru f lo t togdds (the buoyancy of pine l o g s ) . Skogsvardsfor. T i d s k r . 26: 1-38. (See a l s o B i o l . Abs t r . 2: 15148, 1929.) Browne, F. G. 1952. Suggestions f o r fu ture research i n the con t ro l of ambrosia bee t l e s . Malayan Forester 1_»: 197-206. Burkhardt, D. 1962. Spec t ra l s e n s i t i v i t y and other response c h a r a c t e r i s t i c s of s ing le v i s u a l c e l l s i n the arthropod eye. Soc. Exp. B i o l . Symp. 16: 86-109. Busgen, M, 1929. The s t ruc ture and l i f e o f f o r e s t t r ee s . (3rd e d . , rev . E. Munch, t r ans . T . Thompson), Chapman and H a l l , London. Cameron, J . W."M. 1938. "The l i g h t reac t ions of the housef l y Musca domestica L i n n , to l i g h t of d i f f e r e n t wavelengths. Can. J . Research, D, 16: 307-342. Champlain, B.C. and H. B. K i r k . 1926. Ba i t pan i n s e c t s . Ent . News J 2 : 2 8 8 = 291. Chapman, J . A. 1955a. Su r v i v a l of Trypodendron. Bi-monthly Progress Report , Canada, Dept. of Fores t ry 11 (2 ) : 3=4. 1955b. Phys io log i ca l and b i o l o g i c a l s tudies on the ambrosia beet le Trypodendron l ineatum (O l i v . ) and the Doug las- f i r beet le Dendroctonus pseudotsugae Hopk. Inter im Report, Canada, Dept. of A g r i c u l t u r e , Forest B io logy Laboratory, V i c t o r i a , B.C. 72 Chapman, J. A. 1956. Physiological and biological studies on the ambrosia beetle Trypodendron lineatum (Oliv.). Interim Report 1955-2, Canada, Dept. of Agriculture, Forest Biology Division, Victoria, B.C. 1959. Forced attacks by the ambrosia beetle Trypodendron. Bi-monthly Progress Report, Canada, Dept. of Agriculture, Forest Biology Division 15. (5) : 3. 1961. A note on felling date in relation to log attack by the ambrosia beetle Trypodendron. Bi-monthly Progress Report, Canada, Dept. of Forestry 17 (5)s 3-4. 1962. Field studies on attack flight and log selection by the ambrosia beetle Trypodendron lineatum (Oliv.)(Coleopteras Scolytidae) The Can. Ent. 2 4 ( 1 ) : 74-92. 1966. The effect of attack by the ambrosia beetle Trypodendron lineatum (Olivier) on log attractiveness. The Can. Ent. 9JS (l) : 50-59. and J. M. Kinghorn. 1955. Flight trap studies of forest coleoptera and other insects with special reference to Trypodendron  lineatum (Oliv.). Interim Report 1954-4, Forest Biology Laboratory, Canada, Dept. of Agriculture, Victoria, B.C. ; 1958. Studies of flight and attack activity of the ambrosia beetle Trypodendron lineatum (Oliv.) and other scoly-tids. The Can. Ent. 9J) (6)s 362-372. Chapman, J.A., S. H„ Farris and J. M. Kinghorn. 1963. Douglas-fir"sapwood starch in relation to log attack by the ambrosia beetle Trypodendron. Forest Science 9_ (4 ) : 430-439. Christian, M. B. 1939. Experiments on the prevention of ambrosia beetle damage in hardwoods. Southern Lumberman 159 (2009)s 110-112. Dolley, W. L. 1929. Dark adaptation in the eye of Erlstalis tenax. Physiol. Zool. 2s 483-489. Dyer, E. D. A. 1963. Attack and brood production of ambrosia beetles in logging debris. The Can. Ent. 9J> (6) : 624-631. 1964. Studies of ambrosia beetles in relation to felling date of"trees. Annual Report, Canada, Dept. of Forestry, Forest Entomology and Pathology Branch, March, p. 130. and J. A. Chapman. 1962. Brood productivity of ambrosia beetles in water-soaked logs. Bi-monthly Progress Report, Canada, Dept. of Forestry 18 (5)s3. 1965. Flight and attack of the ambrosia beetle Trypodendron lineatum (Oliv.) in relation to felling date of logs. The Can. Ent. 9J7 (1): 42=57. 73 Dyer, E. D„ A. and J. M. Klnghorn. 1961. Factors influencing the distribution of overwintering ambrosia beetles, Trypodendron lineatum (Oliv.). The Can. Ent. 9J. (9 ) : 74-6-759. Fisher, R. 0. and G. H. Thompson. 1952. Recent developments in the prevention of attack by ambrosia (pinhole bprer) beetles in standing trees and logs.' Sixth British Commonwealth Forestry Conference, Canada, August, 16 pp. Francia, F. C. 1965. Studies of some aspects of behaviour in the ambrosia beetle Trypodendron lineatum (Olivier). Doctor of Philosophy Thesis, University of British Columbia. -Frisch, K. von. 1914. Der Farbensinn und Formansinn der Biene. Zool. Jahrb., Physiol. 25_: 1-188. Gara, R. I. 1963. Studies on the flight behaviour of Tps confusus (Lec.) (Coleoptera: Scolytidae) in response to attractive material. Contr. Boyce Thompson Inst. 22s 51-66. Gaumann, E. 1930. Untersuchungen uber den Einfluss der Fallungszeit auf die Eigenschaften des Fichten-und Tannenholzes. II. Teil. Einfluss der Fallungszeit auf die'Dauerhaftigkeit des Flchten-und Tannenholzes. Beihefte zu den Zeitschriften des Schweiz. Forstvereins 6s 26-32. Buchler and Co., Bern. Goldsmith, T. H. 1961, The color vision of insects. In Light and Life. (V. D. McElroy and B. Glass, edd.) John Hopkins Prates, pp. 771-794-. Graham, K. 1959. Release by flight <exercise"of a chemotroplc response from photopositive domination in a scolytid beetle. Nature 184-s 283-284-. 1961. Air-swallowing: a mechanism of photic reversal of the beetle Trypodendron. Nature 19_i (4-787) s 519-520. 1962. Unpublished data. 1966. Personal communication. and A. E." Werner. 1956. Chemical aspects:"of log selection by ambrosia"beetles. Unpublished Interim Report, Canada, Dept. of Agriculture, Forest Biology Division, March, 25 pp. Hadorn, C. 1933. Recherches sur la morphologie, les stades evolutifs et l'hivernage du bostryche lisere (Xyloterus lineatus Oliv.). Supplement aux organes de la Societe forestiere suisse. No. 11. Hasselmann, E. M. 1962, Uberdie relative spektrale Empfindlichkeit von Kafer-und Schmetterlingsaugen bei verschiedenen Helligkeiten. Zool. Jahrb., Physiol. 69s 537-576. Hertz, M. 1938. New experiments on colour vision in bees. J . Exp. Biol. 16: 1-8. Use, D. 1928. Farbensinn der Tagfalter. ,Z. vergl. Physiol. 8s 658-692. Jahn, T. L. 1946. The electroretinogram as a measure of wavelength sensitivity to light. J. N.Y. Ent. Soc. 54? 1-8. Johnson, N. E. 1961. Ambrosia"beetle attacks in young-growth western hemlock. Bi-monthly Progress Report, Canada, Dept. of Forestry 17 (5)s 3. ' 1964. Effects of different drying rates and two insecticides on beetle attacks in felled Douglas-fir and western hemlock. Weyerhauser Company Research Note No. 58 s, 16 pp. Jones, T. I960. Ambrosia beetle research in West Africa. Seventh Common-wealth Ent. Conference Report, pp. 88-97. Kinghorn, J. M. 1956. Sapwood moisture in relation to Trypodendron attacks. Bi-monthly Progress Report, Canada, Dept. of Agriculture, Forest Biology Division 12 (5)s 3-4. •"' 1957. An induced differential bark-beetle attack. Bi-monthly Progress Report, Canada, Dept. of Agriculture, Forest Biology Division _3 (2)s 3-4. and J . A. Chapman. 1957. The effect of Douglas-fir log age oh attack by the ambrosia beetle Trypodendron lineatum (Oliv.). Ent. Soc. of B.C. Proc. __: 46-49. Kuhn, A. 1927. Farbensinn der Bienen. Z. vergl. Physiol. 5s 762-800. Lutz, F . T E . I924. Apparent non-selective characters and combinations of characters" The colors of flowers and the vision of insects, with special reference to ultraviolet. Ann. N.Y. Acad. Sci. 29s 233-283. MacDougal, D. T., J. B. Overton and G. M. Smith". 1929. The hydrostatic-' pneumatic system of certain trees? Movement of liquids and gases. Carnegie Institution of Washington, Washington. Mathers, W. G. 1935. Time df felling In relation to Injury from ambrosia beetles or pinworms. B.C. Lumberman 12 (8)s 14. Mazokhin-Porshnyakov, G. A. 1964. Color vision in insects - study methods and the present state of our knowledge. Ent. Rev. 42 (3)s 257-266. Meyer, B. S., D. B. Anderson and R. L. Bohning. I960. Introduction to plant physiology. D. van Nostrand Company, Inc. Toronto. Nijolt, W. W. and J . A. Chapman. 1964. Uptake of water by the beetle Trypodendron following desiccation. Bi-monthly Progress Report, Canada, Dept. of Forestry 20 (6)s 3-4. Novak, V. I960. The striped wood-boring^beetle (ambrosia beetle) and the fight against i t . Statnf Zemelelske Nakladatelstvf, Prague. 75 Ohnesorge," B. 1953. Der Einfluss von Geruchs; und Geschmackstoffen auf die Wahl der Frasspflanzen biem grossen braunen Riisselkafer Hylobius  abietis L. Bietrage zur Entomologie 3_s 437-468. Person, H. L . 1931. Theory in explanation of the selection of certain trees by the western pine beetle. J. Forestry 29696-699. Pertunnen, V. 1957. Reactions of two bark beetle species Hylurgops palliatus Gyll". and Hylastes ater Payk. (Coleopteras Scolytidae) to the terpene oC -pinene. Ann. Ent. Fenn. 23s 101-110. Prebble, M. L. and K. Graham! 1957. Studies of attack by ambrosia beetles"in softwood logs on Vancouver Island, British Columbia. Forest Science . 2: 90-112. Rudinsky, J7 A. 1966." Host selection and invasion by the Douglas-fir beetle Dendroctonus pseudotsugae Hopkins in coastal Douglas-fir forests. The Can. Ent. |8 (1); 98-111. ' and G. E, Daterman, 1964. Field studies on flight patterns and olfactory responses of ambrosia beetles in Douglas-fir forests of Western Oregon. The Can. Ent. 26 (10)s 1339-1352. Sander, W. 1933. Phototaktische Reaktionen der Bienen auf Llchter verschei-dener Wellenlange. Z. vergl. Physiol. 20s 267-286. Scarth, G. W. 1930. Sinkage studies. IV. Themechanism of absorption of water by wood blocks. Can. J. Research 3_t 107-114. and E. C. Jahn. 1930. Sinkage studies. I. The mode of penetra-tion of water in logs: preliminary field experiments. Can. J. Research 2: 409-424. and R. D„ Bibbs. 1930. Sinkage studies. III. Changes in the water-gas system in 16gs during seasoning and flotation. Can J. Research 3_: 80-93. Schremmer, F. 1941. Zum Nachweis der Rot-blindheit von Vespa. Z. vergl. Physiol. 28: 457-466. Vite, J. P. and R. I. Gara. 1962. Volatile attractants from ponderosa pine by bark beetles (Coleopteras Scolytidae). Confer. Boyce Thompson Inst. 21 (5): 251-274. Walther, J. B. 1958. Changes induced in spectral sensitivity and form of retinal action potentials of the cockroach eye by selective adaptation. J. Insect Physiol. 2: 142-151. • and E. Dodt. 1957. Elektrophysiologische Untersuchungen liber die Ultraviolettempfindlichkeit von Insektenaugen. Experientia J3 s 333-334. 76 Walther, J. B. and E. Dodt. 1959. "Die Spektralsehsitivitat von Ihsektenkom-plexaugen im Ultraviolett bis 290 mu. Elektrophysiologische Untersuchungen an Calllphora und Periplaneta. Z. Naturf. 1J^> 273-278. Wellington, W. G., C. R. Sullivan and W. R. Hensen. 1954. Tne light reactions of larvae of the spotless Fall webworm"Hyphantria textor Harr. (Lepidiptera: Arctiidae). The Can. Ent. 86; 529-542. Wigglesworth, V. B. 1965. The Principles of Insect Physiology. 4th ed. Methuen and Co.Limited., 741 PP. Wood, D. L. and R. W. Bushing."1963. The olfactory response of Ips confusus (Leconte) (Col.; Scolytidae) to the secondary attractant in the laboratory. The Can. Ent. 25 (10): 1066-1077. Wright, R. H. 1966. An insect olfactometer. The Can. Ent. 22 (3 ) : 282-285. APPENDICES 77 Appendix No. Page 1. Graphs of responses of T. lineatum to varying wavelengths and intensities of monochromatic light. From these graphs, figures were derived at 10 per cent intervals for the range from 0 to 60 per cent positive response; these to be then correlated to an energy output of the monochromator 78 Example graphs includes i 319 mu i i 348 mu i i i 543 mu 2. Graphs derived by extrapolation of calibrations for the monochromator yielding relative energy output ratios by the tungsten filament for various wavelengths ............ 79 Example graphs includes i 319 mu i i 348 mji i i i 543 mu 3. Cumulative weight loss data of Trypodendron adults with time and expressed as a percentage of the original weight .... SO Abbreviations: dry wt. = oven dry weight 4. Summarized data of responses of two flight-inexperienced Trypodendron adults (No. 9 & 10) to the humid environment and tested for 'refractory' and 'reaction' times. Positive and negative responses have been included %l Abbreviations: RP = refractory period RT = reaction time (measured in one-hundredth of a minute intervals) 78 Appendix 1 i . 319 mu 0.9 I . — — i • — r — 1 1 — T - 1 1 r 0 10 20 30 40 50 60 70 80 90 100 Number of beetles responding (%) 0 10 20 30 40 50 60- 70- 80 90 100 Number of beetles responding- (%) Appendix 1 i i i . 543 Number of beetles responding {%) 79 Appendix 2 i . 319 mu Monochromator.energy transmission values (%) Appendix 2 i i . 348 mu 1.00 0.90 0.80 0.70 CQ Xi •p —I •rl -p 0.60 ca u o •s s 5 0.50 o o c o s 0.32 - 25.0$ 0.41 - 43.0$ 0.40 0.30 0.20 50 100 150 200 Monochromator energy transmission values ($) 250 543 mu Monochromator energy transmission values (%) Per cent beetle,weight change from original weight with cumulative time (hours) Beetle No, 5.5 7.5 18.5 25.5 30.0 46.5 54.5 70.5 78 .5 92.0 dry wt. P. o CD O O • H in CD Q 1 (if) 2 (f) 3 (f) 4 (f) 5 (m) 6 (m) -9.48 -41.30+ -9.14 -11.15 -7.93 -20.50 -11.75 -50.20 -12.20: -13.85 -11.17 -24.24 -40.22t -65.57 -34-98+ -32.92 1" -31.08* -48.93 + -62.83 -66.06 -58.62 -47.26 -46.66 -60.10 -67.82 -65.81 -65.26 -55.05 -54.72 -66.10 -69.01 ^65.46 -66.46 -70.35 -66.17 -68.80 -69.36 -65.92 -67.18 -70.98 -66.46 -68.46 -69.98 -66.34 -67.38 -71.35 -67.12 -69.04 0 o u tto "8 o o •H 01 CD 13 I n 7 (f) 8 (f) 9 (f) 10 .(f) 11 (m) 12 (m) -0.19 -0.63 -4.20 -1.27 -0.00 -3.73 +3.73 +1.65 -3.81 -1.55 -0.73 -4.69 -1.79 -2.66 -6.70 -3.68 -2.47 -8.60 -3.30 -2.78 -7.66 -5.89 -5.52 -14.31 -7.53 -3.76 -13.84 -8.19 -6.56 -15.94 -8.53* -7.21 -15.43 1 -8.38 -7 .56 f -18.19 + -9.52 -12.34 -19.56 -9.02 -7.56 -18.31 -13.55 -18.37* -22.80 -14.70 -9.17 -22.27 -17.08 -19.24 -22.80 -15.50 -9.44 -24.37 -17.05 7.5 22.5 27.5 31.5 13 (m ) * 14 (m)* -2.51 -0.38 •3.05 -2.54 -4.48 -41531 -5.69' tweight established at first weighing after death of beetle 'N'data for two male beetles tested at a different time Appendix L, Non-desiccated group (humid environment) Beetle No* 9 5:10 p.m. May 2 11:00 a.m. .May 3 9:40 p.m. May 3 Trial RP RT Response RP RT Response RP RT Response 1 31 12 3 17 + 125 122 2 22 28 4 14 + 19 54 3 39 18 7 15 27 276 -4 n 18 t 14 19 t 32 269 5 32 36 16 24 + 18 84 6 37 21 + 5 18 21 27 -7 16 22 t 31 21 + 45 114 + 6 12 15 + 38 22 40 90 Total 213 164 118 150 327 1036 Beetle No. 10 5:15 p.m. May 2 11:10 a.m .May 3 10:00 p.m /May ; Trial RP RT Response RP RT Response RP RT Response 1 100 12 f 4 12 f 30 15 + 2 5 12 + 7 19 + 5 20 + 3 3 17 9 24 •f 5 28 + 4 5 16 2 14 + 1 95 5 A 28 1 24 + 1 12 + 6 8 18 i 2 33 + 1 23 7 4 26 • 1 25 3 31 + 8 4 145 1 28 1 35 + Total 133 274 27 179 47 259 Beetle No. 10 3:35 p.m. May 4 10:00 p. ,m. May 4 12:00 a.m. May : Trial RP RT Response RP RT Response RP RT Response 1 1 22 4 36 + 1 50 2 11 15 3 69 + 2 27 + 3 2 28 2 30 2 26 + 4 11 18 + 2 23 7 35 5 8 29 + 2 23 + 2 36 + 6 16 26 + $ 20 • 4 7 30 + 7 20 24 + 1 118 7 32 + 8 8 37 + 8 31 + 3 29 •f Total 77 199 25 350 31 265 Beetle No. 10 9:30 p.m. May 5 Trial RP RT Response 1 3 47 -2 4 119 3 10 154 + 4 7 154 5 24 110 + 6 10 34 + 7 3 76 + 8 8 71 -Total 69 634 

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