Robert Arthu-r Costello B.Sc.A., University of Manitoba, 1965 M. Sc. , University of Manitoba, 1967. A THESIS SUBXITTED IN PARTIAL FULFILLMENT OF THE REQUIXE!.'IEI!IT'S FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biological Sciences a Robert Arthur Costello Simon Fraser University February, 1974 All rights reserved. This thesis may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Name : Robert Arthur Costello Degree : Doctor of Philosophy Title of Thesis: Effects of Environmental and Physiological Factors on the Acoustic Behavior of Aedes aegypti (L.) (Diptera: Culicidae). Examining Committee: Chairman: Dr. J. M. Webster P. Belton Senior Supervisor B.P. Beirne J.P. Rorden A.E.R. Downe External Examiner Professor Department of Entomology queen's University, Kingston, Ontario. 1. Date Approved: '3"c8,221-k 7 L! Author (dntc) ABSTRACT The study determines the effects of environmental and physioloqical factors on the acoustic behavior of the yellow fever mosquito, Aedes aeg~pti. The effect of the temperature of both larvae and adults and of the relative humidity, barometric pressure, and light in the environment of the adult were ex- amined. Physiological factors considered were age, matinq, feeding and oviposition in the female and mating and time of day as they affected males. The physiological and physical bases for observed changes in acoustic behavior are discussed and the potential of sound in mosquito control is considered. Wing-beat frequencies of females were measured by recording and subsequently analyzing the flight- tones of free flying mosquitoes with sonograms. The response of males to sound was measured by counting mosquitoes trapped with suction as they approached a point source of sound originating from a sine wave generator. Larval rearing temperature affected the acoustic behavior of both sexes. Increasing rearing temperature produced females with progressively higher wing-beat frequencies and males that responded optimally to higher frequencies. This was almost certainly the result of temperature-induced variation in adult size as manipulation of larval food ration and density had iii a similar, but less pronounced, effect on adult size and wing-beat frequency. As the ambient temperature increased, the wing-beat frequency of females increased and males were attracted optimally to higher frequencies. The Q10 values for the change in the female wing-beat frequency ranged from 1.10 to 1.15, indicating that the phenomenon had a physical rather than a biochemical basis, As both sexes reacted similarly to changes in temperature, acoustic synchrony was maintained both at different larval rearing and at different adult ambient temperatures. This was confirmed by observing the number of mating adults from various rearing temperatures and at different ambient temperatures. The soznd pressure level of the female flight-tone was largely unaffected by changes in ambient temperature, whereas females reared at different temperatures showed significant differences in flight-tone sound pressure level. The effect of relative humidity was dependent on ambient temperature. At 26 C, relative humidity had no influence on the female wing-beat frequency, but at 34 C there was an increase in rate of wing-beat with rising relative humidity. The female wing-beat frequency was unaffected by changes in atmospheric pressure or light within the limits of normal environmental variation. Of the physiological factors examined, age had the most marked effect on wing-beat frequency. The rate of wing-beat increased for two to three days after emergence from the pupal state, then remained more or less constant. Mating did not influence the female wing-beat frequency but did affect male acoustic behavior. Mated males showed decreased responsiveness to sound and increased flight activity. Females partaking of blood or sugar had slightly higher wing-beat frequencies after the meal than before. Conversely, oviposition caused a lowering in frequency. This was related to the load carried by the flying insect, as demonstrated by weighting females with externally attached loads. Male acoustic behavior showed no apparent changes over 24 hours in continuous light. ACKNOWLEDGEMENTS I would like to express sincere appreciation to my senior supervisor, Dr. Peter Relton, for his helpful suggestions, valued criticisms and guidance throughout the study and in the preparation of this thesis. Grateful acknowledgement is also extended to the other members of my supervisory committee, Dr. B. P. Beirne and Dr. H. L. Speer, for their advice and criticisms during thzse investigations. FUrther appreciation is extended to Miss Donna Vakenti, Laboratory Technician, for her assistance during the course of this study. TABLE OF CONTENTS Page Examining Committee Approval ............. ii Abstract ...................iii Acknowledgements .................... vi Table of Contents ................... vii List of Tables ..................... x List of Figures .................... xii Introduction ...................... 1 General Materials and Methods: (a) Rearing and Maintenance of Mosquitoes. .... 9 ( b) Determination of Faale Wing-Eeat Frequency. 10 (c) Determination of Male Response to.Sound. 15 (d) Spermathecal Examination to Assess Mating. .. 17 ( e) Statistical Analysis ............. 19 PART 1: Effects of Larval Rearing Temperature on the Acoustic Behavior of Aedes aegypti Adults . 20 Materials and Methods ............... 20 Results and Discussion: (a) Effect of larval rearing temperature on the female wing-beat frequency ......... 25 (b) Effect of larval rearing density and food ration on the female wing-beat frequency . 28 (c) Effect of larval rearing temperature on male acoustic behavior ........... 33 (d) Effect of larval rearing temperature on mating frequency. ................. 37 (e) Effect of larval rearing temperature on female flight-tone intensity ........ 40 (f) Differences in adult female weight between experiments. ................ 44 vii Page PART 2: Effects of Ambient Temperature on Acoustic Behavior of Aedes aegypti Adults ....... 47 Materials and Methods ............... 4-7 Results and Discussion ............... 53 (a) Effect of ambient temperature on the female wing-beat frequency ............. 53 (b) Effect of ambient temperature on male acoustic response ............. 58 (c) Effect of ambient temperature on flight activity and mating frequency ........ 62 (d) Effect of ambient temperature on the female flight-tone intensity ............ 64 PART 3: Effects of Relative Humidity. Barometric Pressure. and Light on the Wing-Beat Frequency of Aedes aegypti Females ................ 67 Materials and Methods: (a) Relative humidity .............. 67 (b) Barometric pressure ............. 69 (c) Light .................... 72 Results and Discussion: (a) Relativehumidity .............. 72 (b) Barometric pressure ............. 76 (c) Light .................... 79 PART 4: Effects of Physiological Factors on Acoustic Behavior of Aedes aegypti Adults ........ 82 Materials and Methods: (a) Age .................... 82 (b) Mati.ng ................... 83 (c) Feeding .................. 84 viii Page ( d) Oviposition ................. 86 (e) Periodicity of male response ........ 86 Results and Discussion (a) Age ..................... 87 (b) Mating .............. 90 (c) Feeding ..................+95 ( d) Oviposition ................. 98 ( e) Periodicity of male response ........ 100 Summary and Conclusions ................. 102 (a) Factors Effecting Acoustic Behavior ...... 102 (b) Mechanism of Changes in Acoustic Behavior ... 104 (c) Potential of Sound in Mosquito Control .... 107 ( d) Other Considerat ions ............. 111 Bibliography ...................... 113 LIST OF TABLES Table Page I Feeding schedule of 3 groups of A. aegypti larvae at 26 C and a density of 'll larval/ ml............ ......... Mean weight and wing-beat frequency (*std. error) of 50 A. aegypti females reared at various larvaT densities at 26 C. Females were weighed in groups of 50 ....... Mean weight and wing-beat frequency ( * S. E. ) of 50 A. aegypti females reared at different larval-food rations. Females were weighed in groups of 50 ................ Percentage of A. aegypti males reared at dif- ferent temperahres trapped on approaching a source of s0und.s of different frequencies. Tests were conducted at 26 C. Figures repre- sent the mean of 13 replicates of 25 males each .................... Percentage of females inseminated in one hour when 30 females, 10 from each rearing temperature of 16, 26, and 36 C, were caged with 10 males reared at one of these temp- eratures. Each figure represents the mean of12 replicates.. ............. Percentage of A. aegypti males responding to sounds of diffzrent frequencies and sound pressure levels. Percentage represent the mean of 10 replicates of 25 males each ... VII Effect of ambient temperature on the wing- beat frequency of A. aegypti females. Figures represent Them*S. E.) of 10 females ................... VIII Percentage of A. aegypti males responding to frequencies ranging from 350 to 650 Hz at different ambient temperatures. Each figure is the mean ( * S. E. ) of 35 replicates of 25 males each. ................ Percentage of females inseminated and flight activity at temperatures of 18, 23, 27, 31, and 24 C. Mating percentages are mean of 5 replicates of 10 females, and activity figures are mean of 10 females ........... 63 Table Page X Intensity of the female flight-tone