THE EFFECTS OF ATMOSPHERIC IONIZATION ON INSECTS MEASURED WITH A STATIONARY FLIGHT APPARATUS by JACOB HILDEBRANDT B.A., University of British Columbia, 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of PHYSICS We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, I960: I n 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 t h e r e q u i r e m e n t s f o r an advanced degree 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 agree 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 agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department 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 not 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 . The U n i v e r s i t y o f B r i t i s h Columbia, Vancouver $, Canada. ABSTRACT In recent years a* number of publications have shown a renewed interest in the possible effects of atmospheric electricity on bio^ logical systems. The work here presented is an attempt to measure the changes in the behaviour, or the power output, of certain insects; during stationary flight in a unipolar atmosphere. Since several published references indicate the existence of a "weather sense" in insects, which has been attributed to sensitivity to atmospheric space charge fluctuations, various species of f l i e s were chosen for this study. Various studies have been carried out on insect flight endurance with flight mills. For this work a new type of closed insect flight, apparatus has been developed, in which environmental conditions can be altered, and the pull and wing beat frequency of the f l y under test recorded simultaneously.. Insects were attached to a mechano-electrical transducer either directly or indirectly. The transducer output was resolved into an AC component, whose frequency corresponds to the wing beat frequency, and a DC component whose magnitude corresponds to the force exerted by the insect in flight. Both components were traced on graph-ic a l recorders. In a modified apparatus two transducers were used for measuring the pull and wing beat frequency independently. Even under identical physical conditions insects were found to behave in an erratic manner, so that possible small effects due to variations of the ion concentration were di f f i c u l t to establish.. i i i The spiracles of some f l i e s were dilated by increasing the ambient CO^ concentration up to 15$ causing various changes in the flight patterns without rendering them more regular. From a statistical analysis of the energies produced, some evidence can be obtained that individuals of Muscina stabulans and Lucilia sericata under a negative ionization excess display greater activity than those under normal conditions or under positive ion excess. More tests w i l l be necessary to substantiate the results. The use of the apparatus as an entomological olfactometer or for the test of other insect responses is suggested; several insect repellents were tested on the same apparatus. i v TABLE OF CONTENTS 1. INVESTIGATIONS INTO THE BIOLOGICAL EFFECTS OF AIR IONIZATION A. General remarks 1 B. Atmospheric i o n i z a t i o n . . . ( i ) A i r ions and c o n d u c t i v i t y measurements 2 ( i i ) P r o d u c t i o n of a i r ions 4: ( i i i ) P h y s i o l o g i c a l s t u d i e s 5> ( i v ) Mechanism of i o n a c t i o n 8 C. Insect s t u d i e s 8 D. Method of i n v e s t i g a t i o n ( i ) F l i g h t m i l l s 9 ( i i ) S t a t i o n a r y f l i g h t apparatus 10 2. EXPERIMENTAL DEVELOPMENT OF A STATIONARY FLIGHT APPARATUS A. P r e l i m i n a r y s t u d i e s on ions produced by a t r i t i u m source .11 B. Transducer operated s t a t i o n a r y f l i g h t apparatus . . . . . ( i ) Mechano-electronic transducer 12 (j.'O. . ( i i ) c l n s e c t i mounting . . . . . . . 1 2 - ' ( i i i ) Feeding 13: ( i v ) F l i g h t s t i m u l a t i o n . . . . . . . . . 1 4 (v) T o r s i o n wire . 1 5 ( v i ) Transducer c a l i b r a t i o n . . . . 16 ( v i i ) Wing beat frequency determination and r e c o r d i n g . . . 16 ( v i i i ) Time of f l i g h t i n t e g r a t i o n . . . . . 1 7 ( i x ) P u l l r e c o r d i n g . . . 1 8 V C. A p p l i c a t i o n of the sta t i o n a r y f l i g h t apparatus ( i ) Experiments i n io n i z e d a i r (a) Ion Sources . . . • • . • • • 1 9 (b.) Grounding the insect . . . . . . 1 9 (c) Experimental, controls 20 ( i i ) A d d ition of carbon dioxide to io n i z e d a i r 21 ( i i i ) Use of insect r e p e l l e n t s 22 D. Insect rearing , 22 3. RESULTS OF AIR-IONIZATION STUDIES A. Observations on in s e c t f l i g h t 24 B. Insect f l i g h t i n an ioniz e d atmosphere. . . . \ ( i ) Description of f l i g h t records. . . . . . . . . . . . 2 6 ( i i ) Duration of f l i g h t . 2 7 ( i i i ) Power and energy of insects i n f l i g h t 27 ( i v ) Forward p u l l and speed of i n s e c t s i n f l i g h t 30 (• ' :(v)'jEffectaof(;cafbonJ.d'ioxide^and'insectlfepeitlents . . . 31 C. F i n a l Discussion . . . . . 32 APPENDICES A, An estimate of the mean distance through which ions d i f f u s e along a tracheole of radius IOJA. . 34 B. Improved experimental arrangement f o r fu r t h e r i n v e s t i g a t i o n 37 BIBLIOGRAPHY 39 i LIST OF ILLUSTRATIONS, TABLES, AND PLATES F i g . 1 Determination of i o n current d e n s i t y 44' F i g . 2: Ion c u r r e n t d e n s i t y from a 50 m i l l i c u r i e t r i t i u m source? as a f u n c t i o n of d i s t a n c e and p o t e n t i a l 44 F i g . 3 Method f o r measuring e f f e c t of metal g r i d s between source and c o l l e c t o r probe 45 F i g . 4 Currents produced by the arrangement shown i n F i g . 3 . . . 46 F i g . 5 Diagram of RCA 5734 transducer and e x t e n s i o n , . 47 F i g . 6 B a s i c bridge c i r c u i t f o r transducer . . . . 47 F i g . 11 Method of a t t a c h i n g i n s e c t . . . . . . 4 8 F i g . 8 View of t o r s i o n wire mounting shown i n P l a t e 3 48 F i g . 9 Method of c a l i b r a t i n g displacement and output of the transducer f o r a g i v e n a p p l i e d f o r c e 49 F i g . 10 C a l i b r a t i o n curves f o r transducer . . 4 9 Fig.11 Method of o b t a i n i n g constant flow of COg 50 Fig.12. COg l e v e l s i n the wind tunnel as a f u n c t i o n of gas . . . . pressure at the o r i f i c e . . . . . . . . . 50 Fig.13 P o i n t s i n the wind tunne l at which COg l e v e l s were sampled 51 Fig.14 Rate of i n c r e a s e of CO,, at p o i n t s shown i n F i g . 13 51 Fig.15 Sample forward f o r c e r e c o r d i n g obtained from an i n s e c t i n the s t a t i o n a r y f l i g h t apparatus 52 Fig.16 Wing beat frequency r e c o r d accompanying F i g . 15. . . . . . 53 Fig.17 Transformation of Voltage versus Time graphs shown i n F i g . 15 i n t o Voltage 3/2 versus Time p l o t s . . . . . . . . 54 Fig.18 P u l l changes due to the presence of two commercial r e p e l l e n t s . 54 Fig.19 Block diagram of f l i g h t apparatus and. automatic r e c o r d i n g system 55 Fig.20 D e t a i l e d schematic diagram . . . . . . . 5 6 v i i Table 1 Tabulations of. duration of insect flight 57 Table 2. Energies of f l i e s measured in the flight apparatus under normal conditions and under ionization. . . . . . 59 Table 5. Summary of results of ionization study. . . . 61 Plate 1 Experimental arrangement. 62! Plate 2 Flight apparatus and graphical recorders. . . . . . . . 63 Plate 3 Detail of flight apparatus 64 v i i i ACKNOWLEDGEMENT This work was carried out with the assistance of a National Research Council of Canada Bursary and Studentship to the writer during the period September, 1958, to May, I960, and a National Research Council of Canada Research Grant to Dr. Otto Bluh» Thanks are expressed to the National Research Council of Canada for their financial support, and to Dr. 0. Bluh for the sug-gestion of the research topic and for placing his research funds at my disposal. I am.grateful to Mr. G. J. Spencer, Professor Emeritus of the Zoology Department, for frequent advice regarding entomological problems, and to Mr. Earl Price, of the Physics Department, for his help in technical matters during the construction of the apparatus. 1 1. INVESTIGATION INTO THE BIOLOGICAL EFFECTS OF AIR-IONIZATION A,. General remarks Throughout history claims have been advanced for possible weather effects on living organisms. It was recognized that physiolog-i c a l and even psychological states of man were affected by certain atmospheric conditions, e.g., thunderstorms, or wind currents variously known as Fohn, Mistral, Solano, Chinook, etc. Various- efforts have been made to isolate the known physical variables, such as temperature, pressure, humidity, atmospheric pollutants, etc., and to measure their effects. In the present century a considerable number of investiga-tions have centred around such additional geophysical variables as atmospheric electricity and radioactivity, geomagnetism, sunspots, cosmic radiation, gravitational fields, and ozone concentration. These studies indicated that atmospheric ionization is one of the electric-a l factors in our environment having certain specific physiological effects, and, to some extent affecting man's general state of ^ e l l -being'. The mechanism by which extremely small numbers of unipolar charges can influence an organism has not, so far, been elucidated and experiments are s t i l l under way in several countries in order to clarify the matter. Considerable scientific interest has been attached to the f i e l d of biometeorology, or the study of the influence of climate, weather, altitude, etc., on animals and plants and in particular on 2 man's health. This border-field of medicine and meteorology has produced a certain amount of inconclusive and even conflicting publica-tions. In the past decade attempts have been made to review c r i t i c a l l y a l l previous work, to isolate the many variables in weather which affect living organisms, and to make controlled quantitative laboratory experiments. By isolating the variables one may, however, be eliminating the natural conditions which might consist of a combination of factors. Interest in the therapeutic uses of ions has stimulated successful research into c l i n i c a l applications, for example, in the reli e f of air-borne allergies, asthma, and high blood pressure. B. Atmospheric ionization (i) Air ions and conductivity measurements Coulomb had noticed the electric conductivity of air in 1795 > but i t was not until 1899 that Elster and Geitel discovered the existence of air ions, i.e., gaseous particles of molecular size in the atmosphere, carrying positive or negative charges. The ionizing process almost always occurs by the removal from Og or Ng of one electron which then may become attached to neutral Og forming 0~, or much less frequently, recombine with the positive ion. None of the electrons become attached to Ng and Ng, ions are not observed (Martin 1954). Thus the ultimate result of ionization is the production of negative molecular ions of oxygen, and positive molecular ions of nitrogen and oxygen, with the predominating. Ion clusters are formed 3 when dipolar water molecules and perhaps also polarized oxygen molecules attach themselves to the ions, or when the ions become bound to atmos-pheric impurities. Free air ions have been classified on the basis of their size and mobility into three categories: small, large, and intermediate. The small ions have mobilities ) between 1 and 2 cm/sec. per volt/cm., (v v= velocity, £"= f i e l d strength). It has generally been found that negative ions have slightly greater mobilities than positive ions (Chalmers 1957, p. 55)» although the values depend on humidity, tempera-ture, pressure, age, the presence of 0^, or of other substances, etc. The small ion is thought to consist of roughly 4 to 12; Og, or HgO molecules: clustered around the charged particle. The much larger Langevin ions (1905) have mobilities in the order of l/500 that of the small ions and are probably charged atmos-pheric pollution particles or condensation nuclei (evaporated sea salt, dust, smoke, acid, etc.) The third or intermediate group discovered by Pollack (1915) with/-between 0.1 and 0.01 CGS is believed to consist of HgSO^ particles comprising some 2000 molecules each (Chalmers, p. 57). Since 1905 mobilities and also ionic concentrations have been measured by the cylindrical condenser method of Gerdien (Chalmers, p. 128). A later version with parallel plates using essentially the same principle has been described (Hicks and Beckett, 1957/), while on a different principle, Montel (l939» 1944, 1956) utilized the transit time between two AC grids* 4 A very simple and convenient means of o b t a i n i n g a measure of io n d e n s i t i e s employs a micromicroammeter and the Beckett i o n current probe (Krueger, Hicks, Beckett 1958).- The number N of i o n s s t r i k i n g the target, per u n i t area and per u n i t time, i s determined from the equation N=l/qA, where I = c u r r e n t , qi = e l e c t r o n i c charge, and A area of the probe. ( i i ) P r o d u c t i o n of a i r ions The p r i n c i p a l n a t u r a l agents g i v i n g r i s e to ions i n the atmos-phere above land are (Frey 1956): ( l ) the r a d i o a c t i v i t y of f r e e a i r g i v i n g r i s e to 4.9 i o n p a i r s per cc per s e c ; (2?) the r a d i o a c t i v i t y of the e a r t h : 3.1 i o n p a i r s / e c / s e c ; and (3} space r a d i a t i o n : 1.5 i o n p a i r s / c c / s e c ; that i s , a t o t a l of 9.5 i o n p a i r s / c c / s e c r e s u l t i n g i n an average ambient i o n c o n c e n t r a t i o n of approximately 700 i o n p a i r s / c c . The l a t t e r f i g u r e may vary w i t h weather c o n d i t i o n s from 40 to 80,000 i o n p a i r s / c c . Ever s i n c e the p o s s i b l e t h e r a p e u t i c value of a i r ions was suggested, e f f o r t s have been made to produce them a r t i f i c i a l l y i n d e s i r e d p r o p o r t i o n s f o r c o n t r o l l e d experiments without having to r e l y on the v i c i s s i t u d e s of the weather. Dessauer (1931) charged submicroscopic p a r t i c l e s of p h a r m a c o l o g i c a l l y i n e r t MgO i n a flame, and T c h i j e v s k y (19.39) produced ions by corona d i s c h a r g e s from sharp p o i n t s with high v o l t a g e s generated by an e l e c t r o s t a t i c machine. Although i t was found that d e l e t e r i o u s n i t r o g e n oxides and 0^ accompany any high voltage d i s c h a r g e , t h i s type of i o n generator, now manufactured 5 by the Philco Corporation is being successfully used in allergy treatment (Kornblueh, Piersol, and Speicher 1956, Locke I960). X-rays and ultraviolet rays have been known for some time to produce a "clean" ionization, but the equipment was elaborate and costly, while the efficiency of ion production was relatively low. The Westinghouse "Sterilamp" (Knowles and Reuter 1940;) was expected to overcome some of the difficulties encountered in UWionization. Apparently i t is s t i l l under development (Science News Letter, 75(12), p. 185, 21 Mar. 1959). In 1952; the Wesix Electric Heater Company instituted a research project to study and design radioactive sources. Polonium: 210, emitting short range alphafe was replaced in 1956 by the longer-lived tritium, adsorbed on titanium. The tritium f o i l is in the centre of a plastic holder; i t emits soft beta rays (l5 KEV), which ionize the surrounding air. The plus and minus ions may be separated electrostatically. This type of ion generator is now most frequently employed, and has been used in the present investigation.* ( i i i ) Physiological studies; The literature on ionized air can be traced back to 1903 when Sokoloff f i r s t pointed towards natural ionization as a biological factor. The favourable effects of negative ions on blood pressure, * Thanks are expressed to Mr. Wesley W. Hicks, President of the Wesix Electric Heater Company, San Fransisco, who provided us with three ion sources. 6 respiration, and general fatigue reported by Dessauer (l93l), ?after 10 years of study, have generally been supported by subsequent investigators (Yaglou et a l . 1933, Okada 1938, etc.).. The Russian Tchijevsky published at least 20 papers between 193.4. and 1941 in which he attributed remarkable curative powers and life-sustaining qualities to ions.. After 1946 a trend developed to discount almost a l l of the earlier statements. Awareness of the lack of uniform experimental conditions, and of the production of ozone and nitrous oxides in the electrical discharge methods of ion generation are responsible for the lack of faith sometimes found in discussing the subject.. For many years people have tried to determine what weather-factors can be correlated with the observed periodic changes during illnesses, of accident rates, death rates, birth rates, moods, etc. As such changes occurred indoors as well as in the open, air electrical factors were examined which penetrate buildings without appreciable losses, and when air ion densities measured indoors were found to follow closely those measured outdoors (Israel 195l), this discovery supported the theory that ionization could be one of the factors involved (Frey 1951, 1952, 1955, Gutmann 1957i von Deschwanden and Miller 1952, Michalowicz 1958, Dubos; 1959, to mention but a few). The results of such investigations are in general inconclusive. The difficulties and dangers of making hasty correlations with single meteorological factors have been emphasized by Israel (l95l))» Berg (19.55)'., Hartweger (1956), and Buettner (1957). At the present time most investigators are using a r t i f i c i a l climates under laboratory conditions, isolating one variable at a time. 7 The main successes have been achieved in experiments on man and certain mammals. The results to date are quite extensive and only a few of the major ones can. here be mentioned, Krueger and his associates have been studying the effects of gaseous air ions on the c i l i a r y rates of the mammalian trachea (Krueger and Smith 1957, 1958, 1959). Their conclusion is that 0~ ions accelerate the rate, while CO^ ions are responsible for the adverse effects such as reduced cil i a r y activity, contracture of tracheal smooth muscle, pronounced mucosal ischemia, and enhanced vulnerability to trauma due to exaggerated l a b i l i t y of the mucosal vessels (Krueger, Hildebrand, and Meyers 1959). However, Schorer (1952), after more than ten years study, reported that whenever the number of negative ions exceeded the positive ions, the feeling of well-being was disturbed. Kornblueh has attempted to resolve some of the discrepancies (Licke I960); by postulating that positive charges may be beneficial only for heart ailments (in those 60$ of the population who are not entirely immune to ions), whereas negative charges are generally to be preferred. c Studies have also been made relating ion activity to glandular activity (Nielson and Harper 1954), growth (Danforth 1952-, Worden and Thompson 1956), pollinosis (Kornblueh and Gr i f f i n 1955), tumor., regression (Eddy et a l . 195l)> ulcers (Cupcea et a l . 1959)» rheumatism (Tchijevsky 1940:, Verdu 1955), and other physiological functions and diseases* 8. (iv) Mechanism of ion action Rohrer (1952) concluded from studies on dissolved protein that positive ions could cause the stimulating action of vagotonal regulation by electron withdrawal, or oxidation. Krueger and Smith (195-9.X offer an explanation on an enzymatic basis for the acceleration in ciliary rate by negative ions and the concomitant beneficial effects to the respiratory system through the observation that negative ions increase the rate of reduction of cytochrome oxidase* Cupcea, Deleanu, and Frits (1959) summarize three possible mechanisms thus far proposed (mainly by East-European researchers): (l); a pulmo-gastric or tracheo-gastric reflex, (2.) a humoral pathway, through change in colloid stability of serum protein, or (3) by charges taken up which exert direct effects on the brain stem. It is of course also possible that a combination of these pathways may be operating. Discrepancies s t i l l persist in the literature, even though measuring techniques and ion control have greatly improved. The absence of any well-founded theory about the physico-chemical mechanism of ion action makes i t s e l f felt in the continuing controversial nature of statement on the subject* C. Insect Studies Entomologists have observed behavioral changes among certain insects in association with storm activity; for example Uvarov (l93l) refers to a book by Marchal (1912) in which the statement is found that 9 i n s e c t s are more a c t i v e j u s t p r i o r to thunderstorms. Schua (1954) observed an i n c r e a s e d i r r i t a b i l i t y and an a l t e r e d food uptake among honey bees i n f l u c t u a t i n g f i e l d s corresponding to those of thunderstorms (quoted by W e l l i n g t o n , 1957).. Praenkel and Gunn (l940) and Andrewartha (1954) a l s o mention weather e f f e c t s on i n s e c t s . In view of these o b s e r v a t i o n s and statements, i t seemed p r o f i t a b l e to undertake a l a b o r a t o r y study of the metabolic a c t i v i t y of i n s e c t s as a f u n c t i o n of a i r i o n c o n c e n t r a t i o n . As m a n i f e s t a t i o n of i n s e c t a c t i v i t y , p u l l and wing beat frequency during s t a t i o n a r y f l i g h t were measured. D. Method of I n v e s t i g a t i o n (i) F l i g h t m i l l s Metabolic s t u d i e s concerned with f l i g h t endurance and energy consumption have been made with v a r i o u s forms of roundabouts i n which the i n s e c t i s c o n s t r a i n e d to f l y i n a c i r c u l a r path by being a t t a c h e d to an arm f r e e to r o t a t e i n a h o r i z o n t a l plane. In t h i s way Krogh and Weis-Fogh (1952:)' flew a number of l o c u s t s simultaneously* and Hocking (1953) made extensive s t u d i e s on the b i t i n g f l i e s of NorthemSanada. A s i m i l a r f l i g h t - m i l l was b u i l t on suggestion of Dr. Otto Bluh and used by a r e s e a r c h group i n the F e d e r a l Entomological Laboratory at Kamloops, B.C., as e a r l y as 1952^ (unpublished). Krogh ( l 9 5 l ) ; determined the Og consumption of l o c u s t s i n s t a t i o n a r y f l i g h t while s t i m u l a t i n g the sensory h a i r patches on the f r o n s and v e r t e x w i t h an a i r stream (Weis-Fogh 1949). 10 (ii.)' Stationary flight apparatus Tor our purposes flight-mills or roundabouts were thought unsuitable since observations and measurements are much more readily made on a stationary insect, and furthermore, the large volume of air required could not be kept uniformly ionized at a high ion density. That stationary flight in an air stream was in most respects equivalent to free flight was shown by Hollick (1940), provided the velocity of the air stream was equal to the speed of flight of the insect. It was accordingly decided to attach the specimen under test to a sensitive mechano-electrical transducer in a wind tunnel to obtain an instantaneous measure of the pull exerted by the insect. Of the five methods of obtaining the wing beat frequency described by Hocking (1953), the stroboscopic technique was by far the simplest, although, as wil l be described later, i t proved inadequate for our purposes. Both parameters were found to be subject to fa i r l y rapid fluctuations so i t was essential that they be recorded simultaneously for future comparative studies. 11 2. EXPERIMENTAL DEVELOPMENT OF A STATIONARY FLIGHT APPARATUS A. P r e l i m i n a r y s t u d i e s on ions produced by a t r i t i u m source U n i p o l a r i o n d e n s i t y measurements were made i n the l a b o r a t o r y by means of the arrangement shown i n F i g . 1. The c o l l e c t o r probe c o n s i s t e d of a c i r c u l a r b rass d i s c of r a d i u s 1.3 cm surrounded by a grounded guard r i n g to reduce d i s t o r t i o n of the e l e c t r o s t a t i c f i e l d i n the v i c i n i t y of the probe. A K e i t h l e y Model 200 Electrometer equipped w i t h a decade shunt enabled current measurements down to -13 approximately 10: amp. The dependence of the current d e n s i t y on a c c e l e r a t i n g p o t e n t i a l and on e l e c t r o d e s e p a r a t i o n i s shown i n F i g . 2. Wire g r i d s with a 7 mm mesh were then p l a c e d i n the i o n stream as shown i n F i g . 3. By v a r y i n g the r e l a t i v e DC p o t e n t i a l s of the elements and the d i s t a n c e between elements i t was p o s s i b l e to o b t a i n a s e r i e s o f curves s i m i l a r to vacuum tube c h a r a c t e r i s t i c s , r e l a t i n g g r i d and c o l l e c t o r c u r r e n t s , v o l t a g e s , and i n t e r e l e c t r o d e spacing ( F i g . 4.). I t was assumed that any a p p r e c i a b l e d i f f e r e n c e s i n the m o b i l i t i e s of the v a r i o u s a i r ions g i v i n g r i s e to the i o n current would appear as i r r e g u l a r i t i e s of the i o n - c u r r e n t c h a r a c t e r i s t i c s . Since none were observed i t could be concluded that a l l ions of the same p o l a r i t y have approximately equal m o b i l i t i e s , i . e . , that they were a l l l i g h t ions and that the c o n c e n t r a t i o n of p o l l u t a n t s i n the l a b o r a t o r y , which may g i v e r i s e to heavy i o n s , was n e g l i g i b l y s m a l l . 12 Bi Transducer operated stationary flight apparatus: (i) Mechano-electronic transducer For converting a small mechanical force or displacement into an electrical impulse (cf. for literature Lion 1959) a transducer tube, type RCA 5734, was selected. As illustrated in Fig. 5, i t is essentially a triode with the grid and cathode assembly held in a fixed position, while the anode, supported by a rod, is capable of angular displacement through the centre of a thin flexible metal diaphragm, thus altering the plate current. An extension of the l/8" plate-pin by means of a thin glass rod of 4" or 5" length increased the sensitivity enough to enable a blowfly, exerting a force of 30 mg for example, to produce a voltage change of approximately 1 volt in the bridge circuit shown in Fig. 6. To minimize dr i f t , care must be taken to provide constant voltages for both the filament and plate. (Thus one must allow for a time of about 8 hours after charging a lead storage ce l l to permit the voltage to become stable.) It was found that the variable balancing potentiometer should be small relative to the fixed resistances so that irregularities at the sliding contact will not appreciably affect the stable balance (Fig. 6). The output from point A may then be amplified for display or recording. ( i i ) Insect mounting The specimen may be immobilized for mounting by ether, etc., but for insects anaesthesia with COg has been found most convenient. 133 A short exposure for 1 min. is usually sufficient for 30 sec anaesthesia during which time the insect may be attached to its mounting f o i l (Fig. 7). It was found that a narrow strip of aluminum takes up most of the unwanted complex-vibrations set up by the wing motion, leaving only a sine wave for each wing beat. Presumably in order for the click mechanism to function, the mesonotum moves dorso-ventrally while the wings take up bistable positions (Pringle 1958). The thin f o i l thus serves also to allow almost free mesonotal movement in the vertical plane. For attaching the insects to the f o i l Hocking has suggested a mixture of beeswax and resin, although beeswax alone seems to serve the purpose quite well. A droplet of wax is melted onto the tip of the f o i l with a 5 watt nichrome wire heater and quickly applied to the middle of the mesonotum in the region devoid of macrochaetae anterior to the scutoscutellar suture. (Care is taken not to seal the suture because the scutellar sclerite must remain free to articulate during wing movements.) The droplet which solidifies immediately on contact is kept as small as possible consistent with firm attachment. ( i i i ) Feeding Hocking's study revealed that many adult insects when flown to exhaustion require only carbohydrates and a brief rest while feeding before continuing again in prolonged flight. Consequently before each test-flight i t is necessary only to feed the insect a few mg of 20-25$ sugar solution. For precise metabolic studies Hocking coated the bore of his micropipettes with wax: to prevent adhesion of sugar solution, and then recalibrated them. 14 For our purposes i t was s u f f i c i e n t to feed the specimen w i t h an o r d i n a r y 1Q;X (l.0A= 0.001 cc) haemocytometer red c e l l d i l u t i n g p i p e t t e , c o n t r o l l i n g the flow by o r a l s u c t i o n through a l e n g t h of s m a l l rubber tubing. Most specimens r e a d i l y a p p l i e d the p r o b o s c i s to the tapered t i p of the p i p e t t e and withdrew the measured q u a n t i t y of s o l u t i o n . (iv)' F l i g h t s t i m u l a t i o n References u s u a l l y s t a t e that l o s s of t a r s a l contact i s s u f -f i c i e n t stimulus to i n i t i a t e f l i g h t movements. T h i s was found to be t r u e , but i t i s not s u f f i c i e n t to maintain f l i g h t , i n most cases f o r more than a few seconds. (This problem i s not so s e r i o u s i n the r o t a t i n g f l i g h t - m i l l s , although even there with the a d d i t i o n a l s t i m u l i of a i r f l o w and the moving background Hocking had d i f f i c u l t y i n d u c i n g Apis m e l l i f e r a , D r o s o p h i l a melanogaster, and Aedes communis to f l y . ) Of the s p e c i e s a v a i l a b l e around the u n i v e r s i t y campus, gardens, and nearby a g r i c u l t u r a l areas, only L u c i l i a s e r i c a t a and Muscina stabulans could be induced to f l y f o r lengthy p e r i o d s , provided they were s t i m u l a t e d by an a u x i l i a r y a i r j e t d i r e c t e d at the f r o n t of the i n s e c t . Removing the t a r s i , an expedient, which proved s u c c e s s f u l on S c h i s t o c e r c a (Krogh and Weis-Fogh 195l) and Phormia (Friedman 1959), had a p p a r e n t l y l i t t l e e f f e c t on the s p e c i e s used here. Although a few t e s t f l i e s flew without i n t e r r u p t i o n from the time the a i r j e t was turned on u n t i l they had depleted t h e i r energy r e s e r v e s , the m a j o r i t y stopped t h e i r f l i g h t a f t e r 10-20 minutes f o r no apparent reason, the stops becoming more frequent as the Insect neared ex-h a u s t i o n . Once at r e s t i n the a i r stream, an e x t e r n a l stimulus such 15 as bodily contact, quickly bringing an object nearby, a loud noise, or shutting off the air stream would be required to reinitiate flight* By using the amplified output of the transducer to operate a shutter in the air stream i t was possible to obtain flights to exhaustion within a period of about one hour without further assistance from the operator. Hocking (1953) describes how his f l i e s alighted on a launching pad to rest for brief periods and.then once again resumed flight. A landing strip in the form of a rotating roller was placed below an insect'; in the stationary mill. The f l y was expected to resume flight each time i t tried to land and found no firm footing. A 5 / 8 " OD rubber hose served as a cylinder, mounted on a horizontal axle belt-driven by a.small reversible electric motor, while the insect was held about 1 / 8 " from the cylinder surface. When the roller was put into motion, the insect began to f l y immediately, but very soon tried to land. After a few thwarted attempts i t would stop flying with its legs drawn up away from the roller. Apparently the air stream is required as a constant stimulus. (v) Torsion wire To facilitate insect mounting and calibration of the DC amplifier, and for wider versatility, the direct mounting (Fig. 5 and Fig. 7 ) was replaced by the torsion wire arrangement shown in Fig. 8 and illustrated in the plates. Strains on the transducer produced while attaching the f o i l to the lever system were prevented by disconnecting at point B of Fig. 8 . The effective length of the extension to the transducer illustrated in the figure was 10/3 x 5, or 16.5 cm. By 16 adjusting the position of point C the mechanical advantage may be altered to suit the size and strength of the insect. (vi); Transducer calibration The characteristics of the 5734 were checked for linearity by applying a force horizontally to the extension rod (Fig. 9), and measuring the output voltage with VTVM and the displacement on the micrometer scale of a microscope. The results are shown in Fig. 10. Since a l l later experimental measurements were in the 0 to 2 volt range, i t is seen that in this interval both output and displacement are directly proportional to applied force. (vii) Wing beat frequency determination and recording The frequency of the wing beats was at f i r s t determined by the standard stroboscopic method at fixed time intervals of 20 sec and then plotted graphically. Because of the unpredictable variation in frequency i t soon became apparent that a more rapid means of recording would be required i f the test were to have any validity. By trying a number of different glass extensions to the transducer i t was possible to select a size and weight such that the resonant frequency of the rod (or a multiple thereof) was in the neighbourhood of the observed wing beat frequency. A flying insect then developed an AC voltage of:''0^ 05 volt or greater at the transducer, sufficiently large to be displayed on an oscilloscope. By comparing the sine wave from the transducer with the signal from an audio oscillator by means of the Tektronix Dual Trace Unit 53C* one could measure the 17 , frequency c o n t i n u o u s l y . To o b t a i n the r e s u l t s i n g r a p h i c a l form, a potentiometer was mounted c o a x i a l l y with the frequency c o n t r o l of the ; audio o s c i l l a t o r i n the c i r c u i t shown i n F i g . 19 and the output d i s p l a y e d on an E s t e r l i n e - A n g u s r e c o r d e r . ( v i i i ) Time of f l i g h t i n t e g r a t i o n I t was thought that the e f f e c t of ions might be to i n c r e a s e or decrease the a c t i v i t y of i n s e c t s , as i n d i c a t e d by the statements on t h e i r "weather-sense". T h i s e f f e c t should then be r e f l e c t e d i n the t o t a l f l y i n g time o b t a i n a b l e from a g i v e n q u a n t i t y of carbohydrate. F o r example, i f a c e r t a i n type of i o n exerted a s t i m u l a t i n g e f f e c t , the p u l l and wing beat frequency should i n c r e a s e , w i t h the necessary consequence that f u e l would be expended at a g r e a t e r r a t e . The t o t a l f l y i n g time thus might serve as an independent check on a c t i v i t y r a t e s . A stop watch proved u n s a t i s f a c t o r y , p a r t i c u l a r l y where the number of stops was frequent, as i t r e q u i r e d the continuous c l o s e o b s e r v a t i o n of the t e s t i n s e c t while the operator had a l s o to note the p u l l and wing beat frequency. By u s i n g the transducer output to operate an e l e c t r i c c l o c k , a very p r e c i s e measure of the f l i g h t time was obtained. With the T r i g g e r i n g Mode of the T e k t r o n i x 531 on AC Slow, the T r i g g e r i n g Slope c o n t r o l could be so a d j u s t e d that both t r a c e s from the Dual Trace Unit 53C disappeared whenever one of them f e l l below an a r b i t r a r y minimum amplitude. Thus a s i g n a l appeared at the V e r t i c a l Out t e r m i n a l o n l y as long as the i n s e c t was f l y i n g . T h i s output was a m p l i f i e d and r e c t i f i e d to actuate a DC r e l a y which i n t u r n c o n t r o l l e d the P r e c i s i o n Time-It c l o c k . 18 The same r e l a y c o u ld be u t i l i z e d to operate the a i r stream s h u t t e r when f l i g h t ceased. Rather than t u r n o f f the blower motor i t s e l f , i t was found p r e f e r a b l e to shut o f f the a i r stream a b r u p t l y w i t h a s h u t t e r about 2! cm i n f r o n t of the mounted i n s e c t ( F i g . 18 and P l a t e 3 ) . The sudden movement of the nearby s h u t t e r was an a d d i t i o n a l stimulus to resume f l i g h t . L a t e r the r e c o r d e r - d r i v e motors were a l s o c o n t r o l l e d by t h i s r e l a y , thus e l i m i n a t i n g the gaps i n the graphs corresponding to the r e s t p e r i o d s of the i n s e c t . ( i x ) P u l l r e c o r d i n g The f o r c e measured by means of the VTVM of F i g . 6 was p l o t t e d versus time, but here, even more so than i n the case of the wing beat frequency, l a r g e random v a r i a b i l i t y n e c e s s i t a t e d continuous g r a p h i c a l r e c o r d i n g to enable an examination of the p u l l p a t t e r n s under v a r i o u s c o n d i t i o n s . A simple two stage DC a m p l i f i e r enabled the transducer output of approximately 1 v o l t to be recorded on a second E s t e r l i n e - A n g u s g r a p h i c a l r e c o r d e r . One could a l s o have used the ready-made K e i t h l e y Model 210 e l e c t r o m e t e r which has a b u i l t - i n h i g h input impedance DC a m p l i f i e r designed f o r use d i r e c t l y w i t h an E s t e r l i n e - A n g u s . For very low input a p p l i c a t i o n s , P r a g l i n and Brecher (1955) have designed a s t a b l e and l i n e a r high g a i n a m p l i f i e r s p e c i f i c a l l y f o r the 5734 transducer. 1$ G. Application of the stationary flight apparatus (i) Experiments in ionized air (a) Ion sources - Two tritium ion sources were positioned on either side of the air jet in front of the insect and the Beckett probe was placed about 1 cm behind. This arrangement gave a probe current of approximately 5xl0~ 1 0 simp. Turning on the air jet did not alter this value appreciably, indeed sometimes even increasing the ion current, indicating entrainment of ions by the air stream. The ion velocity was estimated from mobility data to be at least twice that of the air jet generated by a hair-dryer type blower and motor. Ionized molecules could therefore drift across and thoroughly mix with a slower air current. (b) Grounding the insect - An insect suspended on an insulator in a unipolar atmosphere builds up a static electrical charge and thus limits the number of ions which can reach its surface. The aluminum f o i l attached to the animal was therefore grounded by means of a very fine copper wire loosely strung from the roof of the wind tunnel. Its presence had negligible effect on forces measured by the transducer. The electrical resistance of the integument and of the drop of beeswax was then determined by charging a 0.01juf condenser to l/e of the charging potential. RC time constants of approximately 100 seconds or longer indicated a resistance greater than 10^ ohms. An ion current of 10 amp would then produce at least a 10 volt drop. Although this figure was small relative to the 500 volt potential of the ion source, to ensure optimum ion current at a l l times the integument 20; of the insect was electrically grounded by bridging the beeswax with inorganic silver micropaint (SCP 12, Micro-Circuits Company, New Buffalo, Mich.). This left in the circuit only a resistance of less than 10 ohms due to the exoskeleton. The aluminum f o i l s themselves were coated with beeswax to reduce the number of ions bypassing the insect via a low resistance path to ground. Total ion currents through the insect i t s e l f were between 3x10*" P and 6xl0 : 7 1 0 amp. (c) Experimental controls - When preliminary flights had revealed the marked differences in behaviour between similar individuals, internal control seemed most likely to offer the best means of detecting small magnitude changes. A standard deviation of the measured quantities was therefore determined for each individual insect from a number of control runs; the runs under the desired conditions were then compared only with the insect's own control obser-vations. Each test run in ionized air was usually preceded by two control runs and followed by one in unionized air. It proved to be dif f i c u l t to find specimens which would f l y well enough for several successive measurements, and many flight-runs were therefore lef t unfinished. For this reason the number of complete recordings remained relatively small. No selections as to size, sex:, and age of the f l i e s were made. Although insects could be made to resume flight immediately after feeding, some evidence of fatigue persisted in the succeeding run. 21. Consequently after feeding 5 mg. of 20$ sucrose solution, specimens were allowed to rest for a half hour period before the next experimental run. A number of t r i a l s were made in which the insect was not flown to exhaustion before feeding. As these did not reveal any obvious new features, and since quantitative comparisons are more d i f f i c u l t , they are not included in the subsequent calculations. ( i i ) Addition of carbon dioxide to ionized air In an effort to smooth out some of the fluctuations in the recorded quantities the concentration of COg in the tunnel was increased up to 15$. Above this level activity ceased altogether. The possibility existed that with higher concentrations of blood COg the strong peaks of activity and the subsequent minima would not appear so pronounced and thus enable a better evaluation of the ion study. Carbon dioxide was metered into the system by the method illustrated in Fig. 11. A constant flow rate was maintained by keeping a fixed pressure at an orifice leading to the blower. A water manometer gave an expanded scale when the mercury manometer read below 40 mm. Concentrations up to 15$ were readily achieved, but above this level the gas diffused out of the tunnel almost as fast as i t entered (Fig. 12). To obtain an estimate of the time required for the COg concentration to reach a steady state, and also to see i f appreciable concentration gradients existed in the tunnel, a small sample was withdrawn and analyzed at one minute intervals after the CO^ valve was opened. After obtaining a concentration-time curve at one point in the tunnel, i t was flushed with fresh air and the process repeated at 22: s e v e r a l other p o i n t s ( F i g . 13). F i g . 14 shows that at the p a r t i c u l a r pressure of 35 cm. HgO the COg l e v e l remained e s s e n t i a l l y constant a f t e r 4 minutes throughout the whole volume of the apparatus. ( i i i ) Use of i n s e c t r e p e l l e n t s * By i n t r o d u c i n g v a r i o u s r e p e l l e n t s and a t t r a c t a n t s i n t o the wind tunnel the f l i g h t m i l l c o u l d be u t i l i z e d as an entomological o l f a c t o m e t e r . Since the apparatus d i d not r e c i r c u l a t e a i r i n a c l o s e d system, the chemicals were introduced i n t o the f r e s h a i r stream near the blower before reaching the j e t n o z z l e . A i r from a compressor was sa t u r a t e d by passing i t through a f l a s k c o n t a i n i n g the l i q u i d r e p e l l e n t , and thence to the blower. In t h i s way the v e l o c i t y of the j e t remained u n a l t e r e d , while the c o n c e n t r a t i o n of r e p e l l e n t was highest at the s i t e of the i n s e c t . D. Insect r e a r i n g During the summer months a s u f f i c i e n t number of L u c i l i a and Muscina can be caught near the n a t u r a l breeding grounds adjacent to d a i r y and p o u l t r y barns. A week's supply may be taken w i t h an i n s e c t net at one excur s i o n and t r a n s f e r r e d to cages. Water must be made a v a i l a b l e immediately as d e s s i c a t i o n can r e s u l t w i t h i n a few hours i n hot and dry weather. S e v e r a l days' supply of water may be introduced at one time by i n v e r t i n g a f i l l e d g l a s s j a r onto f i l t e r paper i n a * Dr. Robert Wright, B r i t i s h Columbia Research C o u n c i l , suggested t h i s experiment and k i n d l y p r o v i d e d the r e p e l l e n t s . 23 petri dish. If eggs are required for continued breeding, scraps of fresh liver, meal, fish, etc., may be placed on the moist paper daily and the eggs removed (Peterson 1953). Insects not needed for egg-laying do not require a protein diet and may be maintained satisfactorily for several days on carbohydrate alone. Wicks of dental gause dipping in 5$ sugar solution are recommended for feeding but sugar cubes lying in the cage seem to serve the purpose satisfactorily. Eggs recovered from the breeding cages are placed on a larval medium consisting of ground meat or a mixture of yeast, milk and agar boiled in water. Active maggots are best kept at a temperature of 23-25°C; and a humidity of 70$. To reduce the disagreeable odour, the protein content of the culture medium is kept low. When fully grown, the larvae crawl into loose dry sand or sawdust to pupate and the adults emerge in approximately two weeks time. In the f a l l and winter months these insects tend to deposit fat in preparation for hibernation rather than produce 'eggs (Galtsoff 1937). Fresh liver must therefore be available before and during the c r i t i c a l few days that the young female will oviposit. 24 3. RESULTS OF AIR-IONIZATION STUDIES A. Observations on i n s e c t f l i g h t H o l l i c k (1940), working w i t h Muscina stabu l a n s , demonstrated that wing movements i n f r e e f l i g h t d i f f e r e d s i g n i f i c a n t l y from those of the same i n s e c t h e l d s t a t i o n a r y i n s t i l l a i r . Since the f a s t e n e d i n s e c t i s c o n t i n u a l l y t r y i n g to a c c e l e r a t e and t w i s t i t s e l f f r e e of the p o i n t of attachment, t h i s c o u l d i n part e x p l a i n the i r r e g u l a r p u l l observed. I t i s noteworthy however that the wing beat frequency remained r e l a t i v e l y s t a b l e u n t i l near exhaustion when i t f e l l o f f r a t h e r s h a r p l y to a minimum near 2/3 that of c r u i s i n g speed. T h i s s t a b i l i t y i s con-s i s t e n t with the g e n e r a l l y accepted view that the frequency i s governed l a r g e l y by the mechanical resonance of the wing system ( P r i n g l e 1957) which enables a c t i v e muscle c o n t r a c t i o n s to take p l a c e at such a r a p i d r a t e that tetanus would r e s u l t i f one nerve impulse had to be t r a n s m i t t e d per beat ( B o e t t i g e r 1 9 5 l ) . In t h i s connection Hocking (p. 230) p o i n t s out that P r i n g l e found i t necessary to p o s t u l a t e an a n t a g o n i s t i c a c t i o n between the two s e t s of f l i g h t muscles which each contract a c t i v e l y i n response to being s t r e t c h e d by the other p a i r . Hocking o f f e r s the a l t e r n a t i v e e x p l a n a t i o n that only the s t r o k e immediately f o l l o w i n g the a c t i o n p o t e n t i a l i s powered b i o l o g i c a l l y , and so p r e d i c t s a s l i g h t l y decreasing amplitude between su c c e s s i v e nerve s t i m u l i . . No such p e r i o d i c v a r i a t i o n s i n amplitude appeared i n the t r a c e s on our o s c i l l o s c o p e . With a very slow sweep speed, about 50-100 suc c e s s i v e wing-beats were d i s p l a y e d at once. During t h i s i n t e r v a l approximately 5 nerve pu l s e s should have 25 arrived, and one would therefore expect a step in amplitude every 10 - 20 beats. The uniform amplitude of the wing beats tends to support Pringle's theory of myogenic contraction. In tethered flight insects have been observed to attempt, in varying degrees and in various ways, to free themselves from the point of attachment. These efforts may take the form of twisting by increasing the amplitude of one wing beat or, as in our observations, by periods of extraordinarily strong flight. If one examines Hocking's data on parasite drag as a function of velocity (pp. 66-267) i t is apparent that the horizontal force at maximum speed is almost equal to the weight of the insect. Our measurements showed however that an insect weighing 20^ -25 mg frequently recorded forces of 50-60 dynes (twice its weight) for short durations, followed by periods of relatively weak flight. One could reason that the insect overexerts i t s e l f for a short time, then slows down to recover. As already noted, greater pull is brought about mainly by a change in the angle of attack of the wings and in the amplitude of the wing beat, rather than by an increased beat frequency.. When interpreting the results i t is necessary to remember that the insect acquires different characteristics after some time on the mill. Flight periods generally become longer, but they are weaker, more erratic and show more numerous rest periods. It seems that although certain insects have an extremely efficient metabolic system requiring only water and carbohydrate and almost no mineral, l i p i d or protein replacement, s t i l l after prolonged muscular activity catabolic degeneration does become evident. 26 In those cases where a single run appears to be longer or more energetic than the others, one cannot rule out the possibility of energy carry-over from the previous run. For his work on flight endurance, Hocking arbitrarily defined exhaustion as failure to resume flight after three successive stimulations. We found however that further stimulation sometimes induced up to four minutes more flight. In any case i t is impossible to t e l l precisely when the insect has depleted a l l its reserves. Atmospheric temperature, pressure, and humidity were recorded but no corrections were made to the results. Conditions in the laboratory were fa i r l y uniform over the period during which any given insect was being flown. B. Insect flight in an ionized atmosphere (i) Description of flight records Sample pull and wing beat frequency recordings are shown in Fig. 15 and Fig. 16 respectively. The pull recordings generally show strong flight for the f i r s t few minutes after the commencement of a run, followed by an alternation of weaker and stronger activity. The average forward force diminishes gradually as the energy reserves are expended. Superimposed on the major features are rapid fluctuations having short periods of several seconds to half a minute. Consistent patterns, characteristic of flight under a particular set of conditions, were not observed on superficial inspection. 27 The wing beat frequency is relatively much more regular. A rested Lucilia began flying at a rate of 200-240 beats per second, dropping off to about 180 per second after 15-30 minutes of flight. At the end of a run the frequency f e l l f a i r l y rapidly to around 140-160, when flight ceased altogether. As in the case of the pull recordings each run showed novel variations and no features common to particular conditions could be established. Of the thirty sets of records obtained, seven were sufficiently complete to be used in the subsequent quantitative analysis. ( i i ) Duration of flight Flight times for those runs in which measured amounts of sugar solution were fed are shown in Table 1. Standard deviations for each specimen were calculated using the relation 0" 3 jr^T IL It is seen from the summary in Table 3 that two flights with positive ions and two with negative ions f e l l outside the 2o~limits (5$ probability), but since these times are not consistently longer or shorter they cannot be regarded as significant, and could be attributed to sudden and un-predictable changes in the character of the insect% behaviour. ( i i i ) Power and energy of insects in flight The total power required by an insect in free flight is the sum of two terms: the power needed to maintain the insect in the air, and the power needed for forward movement (Hocking p. 269). A semi-empirical expression for the total power i s : P = ^ + \ p CD S V3 where the f i r s t term describes the power to remain aloft, and the second 28 term r e l a t e s to the motive power, and where b = a constant f o r each i n s e c t , & = the amplitude of the wing beat i n r a d i a n s , n = the wing beat frequency, P = the a i r d e n s i t y , C = the dimensionless drag c o e f f i c i e n t , ranging from 1.1 to 2.3, S = the c r o s s s e c t i o n a l area of the thorax, V = the f l i g h t v e l o c i t y . The f o r c e r e q u i r e d to maintain a g i v e n v e l o c i t y , known as p a r a s i t e drag d p , i s represented by the i d e a l hydrodynamic formula ( c f . Hocking p. 268)': JLP = l - ^ C p S V * In the present apparatus the f l i g h t v e l o c i t y of the i n s e c t i s not known by d i r e c t measurement,' However, a vo l t a g e v p r o p o r t i o n a l to dp i s recorded: d P= cv, where c i s a constant, i n our case 20 dynes/volt found from the c a l i b r a t i o n curve ( F i g . 10). By e l i m i n a t i n g V from the above two equations and s u b s t i t u t i n g f o r dp one obtains an equation connecting P d i r e c t l y with v: I t may be assumed that s i n c e f o r sm a l l i n s e c t s the power r e q u i r e d to remain a i r b o r n e i s much l e s s than the power f o r forward movement at h i g h speeds (e.g. see Hocking p. 257 and p. 270 f o r t y p i c a l v a l u e s ) , one may ne g l e c t the e f f e c t of v a r i a t i o n s i n the term b/(9n on the t o t a l power requirement. I t i s probable that i n f i x e d f l i g h t b/9n i s n e a r l y constant. In the ensuing d i s c u s s i o n , t h e r e f o r e , only the second term Kv w i l l be c o n s i d e r e d . 29 For purposes of comparing insect responses under different atmospheric conditions, i t is desirable to determine the total energy (rather than the power at any instant) expended by an insect after being fed a measured quantity of sugar solution. Since Energy = Power x Time = (Kv z)(t), one may find a quantity proportional to energy by integrating a plot of v3//*versus t, K being unknown. Relative energies are then treated by statistical methods to find i f significant changes due to the presence of ions have occurred. The information from the graphical recorder gives only the voltage v versus time t, so in order to get the v^-t curve one has to regraph each point, raised to the 3/2 power. The raw records were f i r s t marked off in one minute intervals. An average value of v was chosen in the interval, then raised to the 3/2 power by slide rule, and plotted without intermediate tabulation. Wherever large or rapid changes occurred, intervals smaller than one minute were chosen. A sample transformation is shown in Fig. 17. Each graph was integrated three times by means of a planimeter, and the average value of the area tabulated (Table 2). The standard deviations were computed separately for each insect from the control runs in ionized air using the expression 0~ _i The results of these calculations are shown in Table 2 and in Table 3. Six out of the nine runs taken with negative ion excess show energies larger than the mean energies (obtained from control runs) by more than one standard deviation an a i r d e n s i t y 0 of 0.00120 gm/cc, and a mean t h o r a c i c c r o s s s e c t i o n a l area of 8 mm*", one o b t a i n s f o r P = ( " j ? ^ 0 ^ ) * ( c v ) ^ X 31 a value of 4700 erg/sec. Using the graph of power versus a i r speed given by Hocking (p. 270), one f i n d s that t h i s power produces a v e l o c i t y of about 490 cia/sec, or n e a r l y 10 mph. T h i s f i g u r e i s i n reasonable agreement with Hockings experimental data on f l i g h t speeds (pp. 301-302). I t should be noted that f o r short time i n t e r v a l s these s p e c i e s were capable of producing d e f l e c t i o n s of up to 1.5 v o l t s , t h i s being equivalent to almost double t h e i r normal a i r speed. The v e l o c i t y may a l s o be c a l c u l a t e d from the equation r e l a t i n g p a r a s i t e drag.and a i r speed dp = • ^ • ^ ^ B ^ V = cv. S u b s t i t u t i n g the above values f o r the parameters i n t o t h i s equation and s o l v i n g f o r V one o b t a i n s : V = ~ --%• = 472 cm/sec. i n c l o s e agreement with the f i g u r e f C o S of 490 cm/sec obtained above. One f u r t h e r check can be made from Hocking's graph of p a r a s i t e drag versus a i r speed (p. 266). Our c a l i b r a t i o n data show that 0.5 v o l t from the transducer i s produced by a f o r c e of 10 dynes. On Hocking's graph t h i s corresponds to a v e l o c i t y of approximately 500 cm/sec, again i n good agreement with the observed speeds. (v) E f f e c t of Carbon d i o x i d e and i n s e c t r e p e l l e n t s A p r o g r e s s i v e decrease i n the s t r e n g t h of f l i g h t became evident as the COg c o n c e n t r a t i o n was i n c r e a s e d , without e s s e n t i a l l y changing the b a s i c p a t t e r n . F l i g h t movements were extremely weak from 10-15$ COg, and above t h i s l e v e l ceased a l t o g e t h e r . The data used i n the s t a t i s t i c a l d i s c u s s i o n o r i g i n a t e d i n runs i n which the COg c o n c e n t r a t i o n was normal. 3 2 The effects of two commercial insect repellents are illustrated by the sample recording of Fig. 18. Although insects generally flew more strongly when these chemicals were f i r s t introduced, the pull returned to normal after a few minutes. This may be due to exhaustion, or to inurement of the chemoreceptors which become less,sensitive under prolonged stimulation (cf. mammalian olfactory sensation). The study of the effects of insect repellents was not pursued in greater detail.. C. Final discussion In these investigations attention should be given to the mechanism by which the ions could exert their effects on the insects. The sites which seem most logical are the tracheal system, the external sensory system, or the external integument i t s e l f . Krueger's work on the mammalian trachea, in which negative ions increase the ciliary rate and positive ions decrease the rate and reduce mucous flow, definitely shows that the lining of the upper respiratory tract is affected by ions. The tracheal system of insects differs however from the mammalian respiratory system in several important respects: i t is lined with a dry wax-covered layer of cuticle (Roeder 1953) instead of a mucous membrane well-supplied with circulatory and nervous elements; and the diameters of the tracheoles are much smaller than those of lung-breathing animals. Although the fine branches of the ultra-tracheolar network are f i l l e d with liquid, this tracheolar fluid is known to be retracted during activity to allow increased^ventilation. From a knowledge of diffusion rates, kinetic theory, and recombination coefficients i t can 33 be estimated that a large proportion of the ions entering the spiracles is possibly discharged before the ions reach the terminal cells (see Appendix A). Since i t is known that almost no diffusion takes place between the air tubules and the blood (Williams 1953, Snodgrass 1956), ions, such as Og, would have to travel the whole length of the tracheole before reaching an enzyme system at the terminal c e l l . There i s , therefore, some reason to believe that the tracheolar system is not the only channel through which ions may affect insects, i f they do so. The olfactory organs of insects (the sensilla lasiconica, the sensilla coelonica and ampulacea, and the sensilla placodea) are located externally around the mouth parts and antennae. The exoskeleton in these regions is perforated by pore ducts, beneath which are situated the olfactory nerves covered only by a very thin membrane allowing diffusion of odorous substances (Frisch 1954). As well as the sensory pores, a network of fine helical pore canals permeates the integument (Roeder 1953) enabling part of the Og uptake and 2 5 $ of the COg output to be exchanged by cutaneous diffusion (Weber 1954). It is conceivable that air ions could reach the insect's system by these pathways as easily as through the tracheoles. With the limited number of complete t r i a l runs, statements on the effect of air-ionization on insect metabolism can only be attempted with caution. The analysis seems to indicate that an excess of negative air ions, in particular, results in an enhanced insect activity. It appears probable that our laboratory findings give substance to the previous qualitative observations on activities and air-electrical weather factors; a continuation of the experiments along the same lines may contribute to a clarification of the question of "weather sense" in insects. 34 APPENDIX A An estimate of the mean distance through which ions diffuse along a tracheole of radius 10/t. The procedure wil l be to calculate the average number of collisions per second that each ion in a gas undergoes with the walls of the container; then to determine the lifetime of an ion from recombin-ation data and the collision frequency; and finally to estimate the distance the ion diffuses or is propelled by respiratory movements during this lifetime. (i) Collision frequency with the wall It is shown from Kinetic theory (eg. Moore, 1955, p. 167) that the number of molecules striking unit area per second is l/4 nc, where n is the number of molecules per cc and c is their mean speed, o 19 At 20 C, n = 2.5x10 per cc. For an ion consisting of a cluster of five Og molecules c = 200 m/sec at room temperature. The total number of collisions with the walls of area A is l/4ncA per second, or l/4y-cA, where N is the number of molecules in the box of volume V. The number of collisions per second per molecule is then -^-'^ycA= . For a long thin cylinder with radius R «the length L, this becomes c_JjL^L - c o r e x p l i c i t i y xoo m//u^ = 1 G7 gee"1 f o r a tubule tfir K L Z n 2 < 10*10 m of radius 10^ u.. ( i i ) Average lifetime of an ion Nolan and de Sactay (192?) have derived recombination equations for small ions and large nuclei, which, in a unipolar atmosphere with no rate of production, reduce to - ^ j r ~ 1. r>' ^ >z $ r>t » w n e r e \ is the recombination coefficient, n^ is the concentration of small ions, and N^ is the number of uncharged large nuclei. Since each collision with the wall may as an approximation be considered equivalent to a collision with a large nucleus, one can use the above equation to find the number of collisions an ion makes before losing its charge. Consider one condensation nucleus of radius 0.2/* and one ion in 1 cc of a gas. Then the total number of collisions which the nucleus undergoes with the ion is c/U I£L2*« • ^ z a.S yito'^ptr *c However i f n, = 2xl0 6, N. = 1, Y)= 5xl0f 7, then ^n,= 1 per second, that i s , one ion decays per second. But this number of 6 -5 ions undergoes 2x10 x 2.5 x 10. = 50 collisions with the condensation nucleus. One can therefore conclude that after 50 collisions one ion will lose its charge to a nucleus. In a tracheole with a radius of 10yxthe average lifetime is therefore JQ^= 5X10 ^sec. ( i i i ) Diffusion distance The distance which an Og molecule diffuses during a time a t may be found from Einstein's diffusion equation Ax = 2Dt. Using D=0.173 — Ax - .[ * « o.i7S K T « I O ' 1 " : I.13*/OC™. T h i s i s o n i y about sec, v l/lOO the length of a tracheole. Respiration is however aided by active pumping movements of the abdominal segments so that the tracheal trunks behave like bellows. If the speed of the air streaming into the 36 tracheole were as high as 20m/sec, the distance travelled in 5xl0~^sec is s t i l l only 10~ cm, about l/lO the length of a tracheole. It should be noted that the tracheole tapers to a diameter of O^^&t its inner extremity, thus further increasing the probability of decay. (iv) Estimate of distance which an ion travels in the human respiratory tract Consider the nasal passage as a tube of cross sectional area 2 1 cm . During a deep breath about 2 lit r e s of air may be inspired in half a second. Thus the velocity of the air in the tube is 40 m/sec. The number of collisions an ion makes with the tube wall is a. R or 17,700 per sec. Assuming again that an ion can survive an average 50 x 10~^ -3 of 50 collisions, the mean lifetime w i l l be ^. „ „ — = 2.8 x 10 sec, - L . i I 3 -3 and i t wi l l travel a distance of 4 x 10 x 2.8 x 10 — 11 cm. This is approximately the length of the nasal passage and takes the ion into the trachea where the aforementioned specific effects have been observed. 37 APPENDIX B Improved experimental arrangement for further investigation So that natural conditions be reproduced as closely as possible i t seems essential that the speed of the a i r moving past a tethered insect must at a l l times be equal to the speed the insect would have i f i t were i n free f l i g h t . H o l l i c k ( 1 9 4 0 ) observed that wing movements of Muscina stabulans i n stationary f l i g h t were i d e n t i c a l with those in free f l i g h t only when the drag of the a i r stream just balanced the measured forward p u l l . This means that i n our f l i g h t m i l l the output of the transducer must be made zero at a l l times. One could acheive this condition most simply by manually adjusting the speed of an exhaust blower, or better perhaps by l e t t i n g the transducer operate a s e l f -regulated error-actuated servomeonanism which controls the speed of the blower. The f l i g h t speed of the f l y i s then equal to the a i r speed i n the wind tunnel and may be recorded by means of a calibrated hot wire anemometer. (To reduce turbulence the ion sources may be removed from their holders and placed some distance ahead and to the sides of the insect.) From a second transducer nearby on which is mounted a dead insect one obtains a voltage proportional to parasite drag dp. The power i s found simply by e l e c t r o n i c a l l y multiplying the anemometer voltage by the parasite drag. (P=dpV=Force x d i f : a n c e ^ Energy/time=Power * u xme The quantity most often required, the energy, i s then obtained by planimetric or electronic integration of the power-time relation without the intermediate inaccuracies involved in the tedious task of r a i s i n g 38 the force graph to the 3/2 pov/er. Also, absolute values of energy could be obtained and not merely relative values. ; 39 BIBLIOGRAPHY ANDREWARTHA, H.G., and BIRCH, L.C., (1954) "The D i s t r i b u t i o n and Abundance of Animals", (Book) U n i v e r s i t y of Chicago P r e s s , Chicago. BERG, H., (1955) "Widersprechende Aussagen i n der medizienische Meteorologie", Munchenera Medizienische Wochenschrift, .27(23), 10: June 1955, pp. 749-752. BOETTIGER, E.G., ( l 9 5 l ) " S t i m u l a t i o n of the f l i g h t muscles of the f l y " , Anatomical Record, v o l . I l l , 1951, p. 443. 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(1956) "Die atmosptiarishe Radioaktivitat als biologischer Reiz", Medizienische Klinik,' j j l , 1956, pp. 577-583. 1 FRIEDMAN, S., (l959) "Sustained f l i g h t i n Phormia (by a new method) and i t s effects on blood pH", Journal of Insect Physiology, 2(2), May 1959, PP. 118-9. von FRISCH, K., (1954) "The Dancing Bees", (Book), Methuen, London, English Edition 1954. GALTSOFF, LUTZ, WELCH and NEEDHAM, (l937) "Culture methods for invertebrate animals", (Book), Comstock, Ithaca N.Y., 1937, pp. 414-417. GUTMANN, M.J., (l957) "The influence of sorocco weather on bronchial asthma i n Jerusalem", Acta Medica Orientalia, 16(ll-12), 1957, pp. 255-261. HARTWEGER, E.W.S., (1956) "Zur Therapie der Wetterfuhligkeit und Wetterempfindlichkeit ('Fohnerkrankungen*)", Medizienische K l i n i k , £1(12), 23 Mar. 56, pp. 474-6. HICKS, W.W., and BECKETT, J.C., (1957) "Control of a i r ionization and i t s b i o l o g i c a l e f f e c t s ", AIEE Transactions, 7 6 ( 3 0 ) , Pt. 1, 1957 pp. 108-112. 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KSRNBLUEH, IvH., PIERSOL, G.M. and SPEICHER, F.P., (1956) "Relief from air-borne a l l e r g i e s " , Report to Philco Corporation, 1 Oct. 1956. 1 41 KROGH, A., and WEIS-FOGH, T., (l95l) MThe respiratory exchange of the desert locust (Schistocerca gregaria Forskal) before, during, and after f l i g h t " , Journal of Experimental Biology, 2g, 1951, pp. 344-357. (1952) "A roundabout for studying sustained f l i g h t of locusts", Journal of Experimental Biology, 2_9, 1952, pp. 211-219. KRUEGER, A.P., HICKS, W.W., and BECKETT, J.C., (1958) "Effects of unipolar a i r ions on microorganisms", Journal of the Franklin Institute, 26j5(l) July 1958, pp. 9-19. KRUEGER, A.P. and SMITH, R.F., (l957) "Effects of a i r ions on isolated rabbit trachea", Proc. Soc. Exp. B i o l . Med., 96., 1957, pp. 807-9. (1958) "The effects of a i r ions on the l i n i n g of mammalian trachea", Journal of General Physiology, 42_(l), 20 Sept. 1958, pp. 69-82. —• (l959 ) "Parameters of gaseous ion effects on the mammalian trachel", Jor. Gen Physiol., 42(5), 20 May 1959, pp. 959-969. (l959 b). "Enzymatic basis for the acceleration of c i l i a r y a c t i v i t y by negative a i r ions", Nature, 183(4671), 9 May 1959, p. 1332. KRUEGER, A.P., SMITH, R.F., HILDEGRAND, G.J., and MYERS, C.E., (1959) "Further studies of gaseous ion action on trachea", Proceedings of the Society of Experimental Biology and Medicine, 102(2), Nov. 1959, pp. 355-7. LION, K.S., (1959) "Instrumentation i n S c i e n t i f i c Research, E l e c t r i c a l Input Transducers", McGraw-Hill Book Co. Inc., N.Y., 1959. LOCKE, J.K., (i960) "Ionized a i r and human health", Popular Electronics, 13.(3), Sept. 1960. 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YAGLOU, CP., BENJAMIN, L.C, and BRANDT, A.D., (1933) "Physiological changes due to exposure to ionized air", Journal of Heating, Piping, and Air Conditioning, _5_, 1933, pp. 422-430. 600v i i Tritium source { =* \ Beckett probe and guard ring Ti Ion current Reversing switch rrrj Electrometer rtn FIG. I Determination of ion current density FIG. 2 Ion current density from a 50 millicurie tritium source as a function of distance and potential. rf VTVMi 1, Electrometer it 71 Source Gt Probe n 1 J ; 600v J /7" 7 7 Electrometer 300v ! / 7 7 7 Electrometer fTTl VTVM /7T7 FIG. 3 Method of measuring the effect of metal grids between source and collector probe. ¥6 L5T V£ Potentiol of G 4 relative to source for various distances to the probe x. FIG. 4 Currents produced by arrangement shown in FIG. 3 . V| fixed a t - 4 2 5 v , V$ = -560v, distance source to G, 3cm , G, to G 2 I cm . The currents are a function of the variables V 2 and of x (distance between G z and the collector probe) Fixed cathode and grid Movable plate 6.9 mm. O.D. Pinholder from miniature tube base 125" pin, .040" 0D. 5" glass rod, .040" I.D. FIG. 5 Diagram of 5734 transducer and extension. 6v Battery FIG. 6 Basic bridge circuit for transducer FIG. 7 Method of attaching insect. Pull 10 Wax B cm jp+S cm vertical glass rod attached to transducer. Direction of resultant force. Vertical torsion wir< N 3cm -Aluminum damping vane immersed in oil. FIG. 8 View of torsion wire mounting shown in Plate 3. Gloss rod extension FIG. K) Calibration curves for transducer FIG. II Method of obtaining constant flow of CO Percent CO* FIG. 12. C0 2 levels in the wind tunnel as a function of gas pressure at the orifice. To C0 2 analyzer C 0 2 and -air from blower. FIG. 13 R)ints in the wind tunnel at which CO a levels were sampled. Analyzer intake 1.6 in. from floor. CC\ (%) Orifice pressure 35 cm H 2 0 2 4 6 8 Time (min) after start of C 0 2 flow. 10 FIG. 14 Rote of increase of C0 2 at points shown in FIG. 13. FIG. 15 Sample forward force recording obtained from an insect in the stationary flight apparatus. FIG. 16 Wing beat frequency record accompanying FIG. 15. 0.0 0.2 0.4 0.6 Volts 0.8 1.0 1.2 1.5 2 4 0 220 2 0 0 180 VYing beat frequency (pa/sec) 160 .52 Time min.) 10 15 20 Paper speed I21 per hour. 25 30 0 Specimen No. 21 5 10 15 July 2 2 , 1959. 20 25 30 35 Lob. temp. 27.5°C 0 5 10 Fed 6.0 mg. 2 0 % sugar solution 15 20 25 30 35 40 45 50 FIG. 17 (above) Transformation of Voltage versus Time graphs shown in FIG. 15 into Voltage^* versus Time plots. FIG. 18 (right) Pull changes due to the presence of two commercial insect repellents. BLOWER J b GAS ANALYSER R E P E L L E N T VAPORIZER BLOWER iTOOv DC REGULATED r~| POWER SUPPLY DC AMPLIFIER TRANSDUCER No.1 i TRANSDUCER No. 2 AUTOMATIC AIR S T R E A M S H U T T E R ELECTROMETER IMPEDANCE MEASURING CIRC. ELECTROMETER AUDIO O S C L L A T O R P O T E N T I O M E T E R WING BEAT FREQUENCY RECORDER o CRO D U A L T R A C E A _ 4»B OUT HOv A C I2» O C ^ ) — T s r " AC R E L A Y B L O C K DIAGRAM O F FLIGHT A P P A R A T U S A N D A U T O M A T I C R E C O R D I N G S Y S T E M D E T A I L E D S C H E M A T I C D I A G R A M TABLE I Tabu l a t i o n s of d u r a t i o n of i n s e c t f l i g h t under normal c o n d i t i o n s and under i o n i z a t i o n . Specimen number Normal P o s i t i v e Negative 2 L 85.3 Min 80.0 84.0 83.2+2.8 81.3 Min 3 L 64.2 74.3 81.7 73.3+8.8 66.3 Min 87.3 4 L 105.7 86.5 82.2 90.0 104.3 90.8+6.9 77.3 94.0 5 L 48.3 56.8 61.7 -58.3 48.-7 54.5 51.5 54.7+5.3 56.7 58.8 10 M 47.3 52.1 59.1 81 .3 59.9+15.0 63.0 60.53 11 M 40.6 39.5 41.7 45.7 40.6+3.8 54.4 14 M 101.0 78.1 64.8 16 M 44.8 39.5 46.7 38. Ii 44.5 38.6 37.8 35.0 4T7T+3.8 3T7T 59.3 TABLE I (Continued) Specimen Number Normal P o s i t i v e Negative 18 M 54.6 38.3 54.0 49.0+9.2 56.1 19 M 34.0 39.2: 50.9 36.4 40.1+7.5 50.9 46.4 20 M 29.5 28.9 31.6 35.0 27.7 38.2 24.8 31.8+4.0 31.9 41.1 21 M 32.0 50.0 41..0+13 3 6..53 L i n d i c a t e s L u c i l i a s e r i c a t a M i n d i c a t e s Muscina stabulans TABLE II Energies of f l i e s measured in the flight apparatus under normal conditions and under ionization. (Arbitrary units proportional to energy.) Specimen Number Normal Positive Negative 10 M 73.9 85.6 67.7 67.4 131.4 64.8 84.4+10.0 11 M 78.7 84.2 90.0 88.4 56.5 73.5 88.2 54.0 73.5+15.4 16; IM 62.5 66.5 74.-9 53.8 50.2 41.2 40.3 48.4 60.4 68.2 53.5+10.3 19 M 50.3 66.1 71-6 48.8 53.8 67.2 55.2+8.4 20 L 76.7 79.5 67.4 55.2 61.6 106.1 69.2 69.8 82.9 76.6+17.3 21 M 68.9 77.2 73.2 71.1+3.4 TABLE II (Continued) Specimen Number Normal P o s i t i v e Negative 23 L 87.9 83.1 101.9 78.9 83.4+6.4 61 TABLE III Summary of results of ionization study Time of flight Energy Output Positive Negative Positive Negative Within Mean + M+0" 4 35 2 4 < M-C 3 1 0 1 > M+2'0~ 1 2 0: 2 < M-2.0- 1 0 0 0 Outside M+3