Effects of Environmental and Physiological Factors on the Acoustic Behavior of Aedes Aegypti (L.) (Diptera: Culicidae)

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

Biological Sciences

a Robert Arthur Costello Simon Fraser University

February, 1974

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 examined. 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

(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 different temperahres trapped on approaching a source of s0und.s of different frequencies. Tests were conducted at 26 C. Figures represent 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 temperatures. 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 at different temperatures. Data represents mean (I, S. E.) of 10 females ...... XI Mean flight-tone frequency (& S. E.) of 10 A. aegypti females at different barometric pressurzs ...... XI1 Wing-beat frequency of A. aegypti females aTter 15 minutes under right and dark conditions. Each figure is the mean (I, S. E.) of 25 females......

XI11 Effect of mating on wing-beat frequency of A. aegypti females. Data are the mean Tkmof10 females...... XIV Percentage of virgin and mated males responding to frequencies of 475 and 525 Hz. Data are the percentage ( S. E.) of 25 replicates of 100 males each...... xv Flight activity of virgin and mated males over one hour. Data are mean ( + S. E. ) of 10 females ......

XVI Effect of feeding on wing-beat frequency of -A. aegyptifemales ...... A: mean wtng-beat frequency (*S. E.) of groups of 10 females

B: #of individual females showin positive (+), negative (-), or no 70) change in wing-beat frequency after feeding.

XVI I Effect of oviposition on wing-beat frequency of -A. aegypti females......

A: mean wing-beat frequency ( I, S. E. ) of groups of 10 females

B: # of individual females sho~in~~positive (+), negative (-), or no change in wing- beat frequency with oviposition. Figure Page Recording chamber ( disassembled) and microphone for recording ths flight- tone of free flying mosquitoes...... 12 Sonogram of flight-tone of Aedes aegypti female in free flight. Scale 3/4 actual. educed to 3/4 linear size) ...... 14 Diagram of apparatus used to test the response of Aedes aegypti males to sounds of various frequencies...... 18 Wing-beat frequency and weight of A. aegypti females reared at different temperatures. Each bar represents the mean of 50 females. 26

Percentage of A. aegypti males reared at different tempzratures responding to various frequencies...... 36 Flight-tone sound pressure level (db) at 2.54 crn and weight of A. aegypti females reared at different temperatures...... 41 Change in wing- beat frequency and corresponding Q1, values with changing ambient temperature. Points rzpresent the mean ( & S. E.) of 10 females...... 54 Apparatus used to study effect of barometric pressure on wing-beat frequency of -A. aegypti females...... 70 Wing-beat frequency of A. aegypti females at different relative humizities and temperatures. Points represent mean (& S. E.) of 10 females. 73 -A. aegypti female weighted with nylon line glued to the ventral abdominal surface ....85

Mean wing-beat frequency of 13 A. aegypti females during 5 days after aduTt emergence. . 88 Mean $ of A. aegypti responding to different frequencies at one hour intervals over a 24 hour period. Data is mean ( & S. E. ) of 10 replicates of 20 males...... I01

xii The mating behavior of mosquitoes has been the subject of numerous studies. A descriptjon of mosquito mating behavior was recorded as early as 1760 by G. Rivelle, who observed mosquitoes copulating in his cabin aboard ship in the Indian Ocean (cited by Horsfall, 1955). Since that time, and particularly in this century, numerous studies of the mating habits of mosquitoes have been conducted. A focal point of many investigations has been the mechanism serving to bring the sexes together. Vision appears to be of limited or no importance in the sexual orientation of most mosquitoes (~ownes,1969). However in sabethine mosquitoes it has been suggested vision plays a major role. The males are brightly colored, do not swarm, and have non-plumose antennae a add ow and Corbet, 1961). The importance of visual elements in the mating of these mosquitoes is further emphasized by movements in Sabethes chloropterus that have been interpreted as a courtship dance (~alindo,1958). Although the term 11 pheromone" had not been coined when many of the studies were conducted, it is apparent such chemicals are involved as sex attractants in some species. Opifex fuscus males mate with females before the latter have completely cleared the pupal case o irk, 1923; Marks, 1958). Clearly sound plays no role in the orientation of males of this species. Observations on the mating behavior of Deinorcerites cancer (~aegerand Fhinezee, l959), Sabethes chloropterus ( ~alindo,1958), Mansonia uniformis ( ~ayewickrerne, 1953; Laurence, 1960) and Culiseta i-nornata (~eesand Onishi,

1951; Kliewer --et al., 1966) indicate that females of these species also produce odors that act as attractants to males. In these species males lack the long fibrillae which character- ize antennae of mosquitoes that utilize sound as a sex attractant. The presence of a male pheromone that attracts females was reported by Gjullin --et al. (1967) in Culex quinquefasciatus, -C. tarsalis, and -C. pipiens. However these species also mate in flight, males apparently orienting to the female 11 flight-tone" , the sound produced by the movement of the wings during flight. Perhaps the male pheromone serves to bring females in close proximity to males which then sense and respond to the female flight sound. The lack of involvement of a short range attractive sex pheromone for Aedes aegypti was demonstrated by Roth (1948) and by Nijhout and Craig (1971). Both studies demonstrated that tethered females, suspended in a cage of males, were attractive to males only when the wings were in motion. Wing- less females and females with the wings held down were ignored. Roth also commented on the lack of interest of males for resting females in the same cagc.

The use of sound by mosquitoes as a means of communication between the sexes consists of the perception by males of the female flight-tone and their subsequent rapid orientation to its source where copulation is initiated. The production of sound by females and the orientation to sound by males is referred to as acoustic behavior. The antennae are the organs of sound reception in the male (~oth,1948), and the female flight-tone frzquency is depzndent on the rate of wing-beat. The mosquitoes of most concern to man employ sound as a sex attractant. Anopheles spp,, some of which are vectors of malaria, have been found to use sound to bring the sexes together (~ates,1941; Roth, 1948; Kahn and Offenhauser, 1949 a,b; Tischner, 1953). Similarly, mosquitoes of the genus Culex, most notoriously the vectors of encephalitis, use sound (~agiand Taguti, 1941; Roth, 1948). The role of sound in the mating of Aedes spp., particularly the yellow fever mosquito

-A. aegypti (L.), has been the subject of numerous studies (~oth, 1948; Roth --et al., 1952; Tischner, 1953; Wishart and Riordan, 1959; Wishart --et al., 1962; Belton, 1973). In species that utilize sound as a sex attractant the antenna1 fibrillae of males are longer than those of females, increasing the surface area of the male flagellum to approximately ten times that of the female (~lements,1963). At emergence male fibrillae are recumbent, lying flattened against the shaft of the flagellum, but become more or less perpendi- cular to the shaft after two days. The fibrillae remain permanently erected in -A. aegypti whereas in most other species, particularly Canadian species, they are erect only during periods of activity, usually dusk and dawn, and remain recumbent during most of the day (~oth,1948; Nielsen and Nielsen, 1962). The male antenna.1 flagellae act as displacement receptors which resonate in a sound field at a given frequency while remaining

more or less at rest at other frequencies. In this way they serve as a band-pass filter, vibrating in response to a narrow band of frequencies which normally includes the fundamental of the female flight-tone. Sounds made by other males and the majority of other environmental sounds are thus filtered out.

Vibration of the flagellum apparently causes displacement of' the terminal filaments of the scolophores of the Johnston's organ of the second antennal segment, thereby producing potential changes leading to the formation of action potentials in the antennal nerve fibers. Aedes aegypti was used in many studies of mosquito mating because of its constant mating state and the ease in which it can be maintained in th3 laboratory. Clements ( 1963), reviewing mosquito reproductive behavior, divided the mating response of -A. aegypti males to a flying female into four stages: 1) taking to flight, 2) orientation to the source of the sound stimulus, 3) seizing the female, and 4) clasping the female genitalia. The second stage, orientation to the sound stimulus, is the area of investigation of this study. The orientation of male mosquitoes to the female flight- tone was first conclusively demonstrated by Roth (1948).

Since his study other workers have examined the acoustic behavior of mosquitoes, particularly -A. aegypti. One product of these studies is that a wide range of wing-heat frequencies has been attributed to this species. Female wing-beat frequencies of 385 ';O Hz (cycles per second) (~ischnerand Schief, l954), 427 Hz (~hristophsrs,1960), 449 - 603 with a mean of 493 Hz

(wishart and Kiordan, 1959), 600 Hz (~ahnand Offenhauser,

1949 b) , and 730 Hz (offenhauser and Kahn, 1949) have been reported. It is known that ternperatur2 affects wing-beat frequency (~otavalta,1947), but it is unlikely that this factor alone would account for such wide variation. Although the environmental temperature was not reported in many of the above examples, it is likely that these measurements were made at similar laboratory temperatures, as extreme conditions, even in tropical areas, would probably have been reported. Hence, unless these investigators used faulty techniques for frequency measurements there must be other factors affecting wing-beat and possibly other aspects of mosquito acoustic behavior. Past efforts to identify environmental and physiological factors affecting acoustic behavior of mosquitoes were incomplete. Some of these factors have been investigated but there has been no comprehensive study made on a single species. In some cases the results of different workers have been contradictory, as exemplified by the range of wing-beat frequencies reported for -A. aegypti described above. Environmental factors other than temperature, such as relative humidity, light, and barometric pressure have been given little attention. Significant contri- butions to the effects of environmental factors have been made by Chadwick and Williams (1949) and Sotavalta (1947, 1954). However, those studies were somewhat inconclusive on the effects of RH and barometric pressure on mosquito acoustic behavior, and did not consid~rlight. Sotavalta (l947), dzscribing his results on subjecting -A. aegypti to sub-atmospheric pressures, states that "because of the behavior or these insects during experiments it is best not to draw any exact conclusions. I1 Chadwick and 'iliilliams ( 1949) found the wing-beat frequency of Drosophila changed with atmospheric pressure, but Chadwick (1953), in discussing those results, stated "the variations in air density normally met with in the life of an insect . . . are of negligible importance insofar as their effect on rate of wing-beat is concerned. 11

The role of relative humidity is more clearly established.

~otavalta(1954) found RH affected the wing-beat frequency of insects at temperatures above 30 C, although -A. aeapti was not studied. No reference could be found on effects of light on the acoustic behavior of mosqu-itoes. The present study attempts to resolve the effects of relative humidity, barometric pressure, and light on the acoustic behavior of -A. aegypti. Temperature has been shown to affect the wing-beat frequency of some insects ( Sotavalta, 1947; Farnworth, 1972a, b) . However no comprehensive studies have been conducted on the influence of temperature on the acoustic behavior of mosquitoes which is an integral part of the mating process. Studies have been made on the influence of physiological factors on acoustic behavior. The change in acoustic beha,vior with age has been given some attention (wishart and Riordan, 1959) but the effects of mating, oviposition, and the possibility that there may be periodic changes in acoustic behavior have been given littlz or no scrutiny. The change in the female wing-beat frequency after a blood-meal has been examined with different results. Nuttal and Shipley ( 1902) claimed an increase in the rate of the wing-beat after a blood-meal rendered the female more attractive to males. However, Roth (1948) felt that the change in frequency was insufficient to effect its attractiveness. In past studies, difficulties arose because of the use of techniques that required subjective evaluation, such as in the determination of wing-beat frequency, and also in some cases because of a lack of knowledge of variables such as the possible influence of rearing conditions and the ambient temperature at analysis. In the present study the methods employed required no judgement by the experimenter, and apparent variables were considered and controlled. Previous attempts to use sound as a mosquito control technique have, for various reasons, been largely unsuccessful.

It is hoped the present study of factors influencing acoustic

behavior will provide insight into the basic controlling mechanisms of this phenomenon, which in turn will suggest methods of controlling mosquitoes by manipulating acoust,ic behavior. Perhaps by interfering with male to female orienta- . tion the frequency of mating can be lowered, which could result in smaller subsequent populations. The objective of the present study was to identify environmental and physiological Tac tors affecting the acoustic behavLor of Aedes --aegypti adults and, in the light of the experimental results, to consider the mechanisms involved in acoustic behavior and the potential of sound in mosquito control. GENERAT, MATERIALS AND METJ-TODS

Materials and methods common to all or most experiments are described in this section. Specific techniques are described in the appropriate section.

(a) Rearing and maintenance of mosquitoes: An undetermined strain of Aedes aegypti, maintained in the insectary of Simon Praser University since 1966, was used. The eggs were from females blood-fed on guinea pigs. For most experiments larvae were reared in uncovered 29 x 18 x 4.5 cm white enamel pans. 500 ml of distilled water was poured into each pan, resulting in a depth of approximately 1 cm. Finely ground Purina dog chow was provided as food according to the schedule outlined by Gerberg (1970) (full ration, Table I). Larval densj-ty varied from 0.2 to 0.5 larvae per ml water, depending on the number of adults required.

Pupae were handled with a commercial medicine dropper which had the open end filed off to make a wide mouth. Pupae were sexed by size and placed in separate cages to obtain virgin adults. Adult emergence and maintenance cages were 18 cm by 12 cm by 16 ern high. The floor, ceiling, and two 16 x 12 cm sides were wood. One of the 16 x 18 cm sides was nylon mesh, while the opposit? side was of clear plastic having a central hole fitted with a #3 rubber stopper. The plastic served as an easily removed door. Adults were provided with absorbent cotton rolls soaked in a 2@ sucrose solution as food. Relative humidity in the cage was maintained at 60 to 65% with a paper towel wick in a beaker of water. Adults, removed wi-th an aspirator when needed, were kept alive and in apparent good health for up to six weeks in these cages.

( b) Determination of female wing-beat frequency: A number of methods have been employed to measure the wing- beat frequency of mosquitoes and other insects. In an early study, Nuttal and Shipley (1902) used the acoustic method, comparing the sound emitted by flying mosquitoes to a known pitch produced by tuning forks. This technique has been criticized as being subjective and was largely abandoned except by Sotavalta (1947, 1952, 1953) who had a musician's training and an ear for absolute pitch. Less talented workers have utilized electronic equipment to obtain objective determinations of wing-beat frequency. The stroboscopic method, involving synchronizing the frequency of short light flashes with the insect wing-beat, has been used extensively hadwi wick, 1939; Chadwick and Williams, 1949; Williams and Galambos, 1950; Yurkiewicz and Smyth, 19G5) but has the disadvantage that the specimen must be tethered, which as is described later, can affect the rate of wing-beat of some insects. The most satisractory methods have involved placing a microphone in a flight chamber and either directly analyzing the wing-beat frequency of the free flying insect or recording the flight-tone for future analysis. The cathode ray oscilloscope has been the most often uscd analytic device (~illiams and Galambos, 1950; Sotavalta, 195';; Tischner, 1953). A permanent record can be obtainzd by photographing the oscilloscope screen, but this can be costly and time consuming. Pen recorders, which display frequencies directly on paper have been employed by Farnworth (1972, a, b) and Sotavalta (1953), but because of their inertia are much less accurate than other methods. A technique dzveloped by Boettiger and Furshpan (1952) involved electrostatically charging an insect either by induction or through contact with a charged object and potential changes, recorded oscillographically, were observed when the wings moved back and forth near an input amplifier lead.

In the present study, the flight-tones of flying mosquitoes were recorded and the frequency determined on a sound spectrum analyzer. Kahn and Offenhauser (1949 a, b) first' used the sound spectrograph for mosquito wing-beat analysis, and this device has been used extensively to study other insect sounds

(~umortier,1963; Walker and Gurney, 1972; Spooner, 1973). The recording chamber for most experiments consisted of a thick-walled glass jar lined with a layer of paper towelling to minimize resonance and fitted at the mouth with a rubber stopper. A hole through the stopper permitted insertion of' a

Sony F 96 Dynarnic microphone (l?igure 1). The microphone was connected to the microphone input of a Heathkit Solid State amplifier, which fed the signal to a Sony Model 250 tape Figure 1: Recording chamber (diassembled) and microphone for recording the flight-tone of free flying mosquitoes. recorder. IIeadphones plugged into the amplifier allowed monitoring of the mosquito in the recording chamber. When the mosquito was in flight, usually induced by tapping the recording chamber, the flight-tone was recorded and later transferred to the sound spectrum analyzer (~aySona-Graph, model 6061 B) and a sonogram produced.

A typical sonogram is shown in Figure 2. Frequencies present in the sound are shown as lines of variable darkness corresponding with sound. pressure. The ordinate is frequency and the abscissa time. Mosquito flight-tones consist of a fundamental tone (A) representing the wing- beat frequency, usually around 500 Hz, and numerous harmonics ( B) . One or more calibration tones (c) were introduced, producing marks at

500 Hz intervals. The wing-beat frequency was determined from the sonogram in two ways. First, a direct reading was made of the fundamental frequency relative to the calibration tone. Secondly, and more accurately, the fundamental and harmonics of the mosquito flight-tone sonagram were compared to the fundamental and harmonics of a sonogram resulting from an electronically produced sound of known frequency recorded in the same manner as the flight-tone. This procedure permitted confident determination of the wing-beat frequency within & 10 Hz. As well as comparing experimental sonograms to those of known frequencies, sonograms of a mosquito under different conditions could be directly compared. Figure 2: Sonogram of flight-tone of Aedes aegypti female in free flight. Scale 314 actual. A: fundun~ental B:harmon ics C:calibration tone The technique of recording the fl-ight-tone for subsequent analysis on a sound spectrum analyzer was employed for several reasons. The flight-tone of many females could be recorded over

a short period and analyzed later, permitting replicates of individuals of the same age and physiological state at similar environmental conditions. An objective and accurate det ermina-

tion of wing-beat frequency was readily achieved and a permanent record was obtained for future reference. The flight-tons of free-flying rather than tethered mosquitoes was analyzed when a preliminary experiment indicated

tethering modified the f light-tone. The mean wing- beat fre-

quencies ( & S. E. ) of 10 females in free flight, and subsequently

tethered to a rotating and non-rotating flight mill were 488 i

6, 502 k 7, and 518 i 6 Hz respectively. The wing-beat frequencies under the three treatments were significantly different (t-test, p < .05) , apparently due to the load imposed by tethering, and therefore, although fastened specimens were more easily recorded, only flight-tones produced by free-flying mosquitoes were analyzed. (c) Determination of male response to sound: An early study of the response of male mosquitoes (~ulex

sp.) to sound was conducted by Mayer (1874). Using a microscope he observed and measured vibration of the antenna1 fibrillac responding to tuning forks and concluded 512 Hz

produced the most vibration. A similar study by Yaki and Taguti (1941) on Culex pipiens males mdicated that 217 Hz, produced by an 'I elcctro-acoustic machine" caused greatest vibration. Roth (1940) dzmonstrated that -A. aefrypti males flew towards sounds produced liy tuning forks and an audio oscillator, but he used as his criterion of response the 1' seizing and clasping1' reaction rather than orientation to the sound source. Some workers have studied the response of male mosquitoes to recordings of female flight sounds both in the field (~ahnand

Offenhauser, 1949; Offcnhauser and Kahn, 1949) and the laboratory (~ovak,1966). Tischner (1953) and Wishart --et al. (15~62)~ using an electrophysiological approach, inserted probes into the male Johnston's organ and measured on an oscilloscope the electrical activity in response to artificial sounds. This time consuming and t ethnically demanding method, although demonstra- ting the frequencies to which the antennae resonated, did not necessarily determine the most attractive frequencies. High speed photography has been used to study the orientation of mosquitoes to sound. This is an excellent method for studies involving few mosquitoes but becomes impractical for larger numbers. The most objective and convenient method for measuring acoustic response of large numbers of males, and the model for the method used in the present study, was devzloped by Wishart and Riordan (1959). Males were drawn with suction into a counting chamber as they approached a source of sound. No judgement had to be applied in deciding whether a mosquito had responded, and large numbers were readily studied. A diagram of' the apparatus used in the present study is shown in Figure 3. A known number of ma1.e mosquitoes were placed in the test cage (A) and any approaching the sound source were suckzd in the counting chamber (C) . In this way the percentage of males in the tcst cage responding to a particular frequency was determined. The desired frequencies were obtained from a sine wave generator, and a five second sound presentation period, found by Wisha-rt and Riordan (1959) to be most effective for practical purposes, was achieved by inserting an automatic timer in the circuit. A sound pressure level of 65 db at 2.54 cm from the sound source was maintained in all frequency tests where sound pressure was not being investigated. An electric fan was ada,pted to provide suction with a relatively low level of background noise. The fan speed was rheostatically controlled to produce suction that preliminary tests demonstrated would draw in only the mosquitoes approaching the sound source. Fewer than 0.5% of males induced to fly by agitation of the cage were trapped when no sound was used. As the experiments were designed to assess the response of mosquitoes in flight, the sides of the test cages were brushed just prior to sound presentation to induce flight. Trapped mosquitoes, once counted, were returned to the test cage by detaching the suction fan and gently blowing on the line.

(d) - Sphermathecal examination to assess mating: The rate of mating was determined when necessary by examining the female spermathecae for sperm after a period of Figure 3: Diagram of apparatus used to test the response of Aedes aegypti males to sounds of various frequencies. A, test cage; B, tygon tubing, 12 rrm I. D.; C, clear plastic counting chamber, 50 x 22 rmn; D, sound source ( electromagnetic miniature earphone) ; E, automatic timer; F, sine wave generator (~eathkit,model lg-18).

exposure to males. Specimens were stunned Tor dissection by

drawing them into an aspirator, then expelling them forcefully against a screened surface and removing the legs and wings from the dazed mosquitoes. This mzthod proved quicker than using ether or C02 as anaesthetics. Also there was no danger of immobilizing the sperm, thereby making them difficult to observe, as occurred in preliminary tests using ether. The spermathecae were removed under a dissecting microscope using fine-pointed forceps to hold the abdomen and pull on the external genitalia. Once removed, the internal repro-

ductive organs including the spermathecae were placed in a drop

of -A. aeapti saline solution (~ayes,1953) on a microscope

slide. After removing fat body and other tissue to isolate the three spermathecae, a cover slip was applied and they were examined under phase contrast for the presence of sperm. ( e) Statistical analysis The statistical significance of the difference between means was tested to the .05 probability level by t-tests. Changes in direction rather than differences between means were analyzed in some experiments, and a binomial expansion was used to determine significance in these instances. Effects of Larval Rearing " >mn~ratureon the Acoustic Behavior of Aedes aecxpti Adults

An examination of the literature indicates that the wing- beat frequsncy of mosquitoes nay be inversely related to the size of the mosquito. Females of Aedes punctor, -A. campestris,

-A. cornmunis,- and -A. impiger, described by Carpenter and La Casse (1955) as medium to large species, have respective wing-beat frequencies of 247 - 311 Hz. (~otavalta,1947), 311 - 332 Hz,

213 - 230 Hz, and 305 - 380 Ilz (~ocking,1953). All of these are lower than the frequencies reported for females of -A. aegypti, a small species. Females of the very small species Uranotaenia lowii, ha,ve been found to have a wing- beat frequency of approximately 1,000 Hz elto ton, unpublished data), which is higher than that of the larger -A. aegypti females. This study was conducted to determine the influence on acoustic behavior of adult size variation induced by manipulation of larval rearing conditions, particularly temperature. Aspects of the relationship of acoustic beha,vior and larval rearing conditions examined in this study were (1) frequency and sound pressure level of the female flight-tone, (2) response of males to different frequencies, and (3) the frequency of mating.

Materials and Methods Adults of different sizes were obtained by rearing larvae at 16, 26, and 36 C: in incubators accuratz to 1 C. A

concentration of 1 larva per rnl water was maintained at all temperatures and all larvae were fed according to the schedule described earlier. Emzrgence time was noted, and adult age was known within 12 hours. Adults were maintained in an unmated state. Because thermal stress can cause suppression of male characteristics of mosquitoes o or sf all and Anderson, 1961), thereby influencing mating frequency and possibly acoustic behavior, males produced at 36 C were examined and those dis- playing intersex genitalia were discarded. Variation in adult size was also obtained through manipulation of larval rearing density. Larvae were reared in pans containing 300 ml of water, adjusted daily to compensate for loss through evaporation. Three hundred, 750, and 1500 first instar larvae were placed in separate pans, resulting in densi-

ties of 1, 2.5, and 5 larvae/ml. Temperature was maintained at 26 C, and the food ration per larvae remained constant at

all densities.

A third method of obtaining adults of different sizes was by variation of larval food ration. Three pans or 300 ml water and 300 larvae were fed according to the schedule described in

Table I, each pan receiving a different ration. Rearing

temperature for each group was 26 C. Larva2 receiving the "full ration" were considered to be on the optimal diet (~orlan--et al.

1963) while those on half and fifth ratios were on sub-optimal diets. TABLE I

Feeding schedule of 3 groups of A. aegypti larvae at 26 C and a density of 1-larva /ml.

DAI1,Y I~OOIIRATION (mg/Larva)

FULL RATION 1/2 RATION 1/5 RATION

DAY O* 0,2 0.1 0.04

DAY 1 0.3 0.15 0.06

DAY 2 0.4 0.2 0.08

DAY 3 on 0.6 0.3 0.12

4t DAY OF HATCHING Pupae from thz temperature, density and food rationing treatments were sexed and placsd in emergence cages at 26 C.

Emergence time was not,ed within 12 hours. The wing-beat freque~iciesof 3 to 4 day old females from each treatment were determined as described in Chapter 2, and they were then killed and weighed as a group. To study the acoustic behavior of males from different larval rearing temperatures, 3 to 4 day old males were presented with 13 frequenci~sranging from 350 to 650 Hz at 50 Hz intervals. The percentage response of males from each temperature was recorded using the apparatus described earlier. Thirteen replicates, each of 25 males, were taken from each rearing temperature. To avoid possible bias due to pre- conditioning or habituation, cage one was first presented with the lowest frequency and then with progressively higher frequencies. Cage two was first exposed to the second lowest frequency, and so on for subsequent cages. Thus each frequency was first tested on a "fresh" cage, then on a cage that had been subjected to a frequency once previously, and so forth. Thirty minutes were allowed between tests. Assuming the acoustic behavior of one or both sexes is influenced by larval rearing temperature through its effect on adult size, the orientation of male to female mosquitoes would be affected, and a change in mating frequency could occur between adults reared at different temperatures. This was investigated by caging together, at 26 C, males and females from the same and different rearing temperatures and determining by spermathecal examination the frequency of mating. Virgin males and females, 3 to 4 days old, were used. Each cage

contained 10 males from one of the lG, 26, or 36 C larval

rearing temperatu-res, and 30 females, 10 from each of the rearing temperatures. Thus, each male was presented with females from his own and two other rearing temperatures. To distinguish females from each rearing temperature, they were dusted with the Dupont fluorescent marker dyes rhodamine and uranine. Females from two rearing temperatures were marked with different dyes while those from the third were left un- marked. Preliminary experiments indicated the dust had no effect on the female flight-tone. The sexes were left together

for one hour during which the cages were agitated at 5 minute / intervals to induce flight. The female spermathecae were then examined for sperm as evidence of mating.

As well as examining the effect of rearing temperature on the frequency of the female flight-tone, the effect on the sound pressure level was also studied. Females from larval

rearing temperatures of 16, 26, and 36 C were tethered by the dorsal thoracic surface and situated laterally one inch in front of a Brcel and Kjaer Impulse Precision Sound Level Meter, Type

2204. Tethering consisting of gluing fenlales by the ventral

surface of the thorax to the head of a vertical size 0 insect pin, using silicone sealent as an adhesive. After determining the flight-tone sound pressure levels, the females from each rearing temperature were weighed as a group.

The sensitivity of males to differences in sound pressure level was examined by przsentine them with sine generator produced frequencies of 450, 500, and 550 Hz, each at 48, 53, and 57 db at 2.54 cm. These represent the range of female flight-tone sound pressure levels found in the previous experiment. Ten cages of 25 males per cage were tested at each of the 9 treatments at half hour intervals. On approaching the sound source, males were trapped as described earlier and counted.

Results and Discussion

(a) Effect of La,rval Reari-ng Temperature on the Female Wing- beat Freauencv

The female wing-beat frequency increased and female size decreased with jncreasing larval rearing temperature (~igure

4). Females reared at 16, 26, and 36 C had mean weights of 2,5, 2.0, and 1.2 mg and significantly different (t-test, p < .05) mean wing-beat frequencies of 430, 505, and 565 Hz respectively. The relationship of wing-beat frequency to weight is non-linear, as can be seen in Figure 4 by comparing wing- beat frequencies to the llweight values. Because the mosquitoes were weighed as groups rather than

individually, no tests could be applied to demonstrate statistical differences in the weights of the groups from dif-

ferent temperatures. However the differences appear to be

sufficiently large to permit the assumption that the rearing Figure 4: Wing-beat frequency and weight of -A. aegypti females reared at different temperatures. Each bar represents the mean of 50 females. WING BEAT FREQUENCY

4 WEIGHT

16 26 36 REARING TEMPERATURE (" C) temperatures used did in fact result in females of different sizes. The relationship of the adult size of mosquitoes to the temperature to which the immature stages are exposed is a well known phrtnoinmon, with adult size decreasing with increasing temperature as occurred in the present study (~lements, 1963) . These results indicate that in reporting mosquito wing- beat frequencies it is necessary to state the rearing temperature. This presents no difficulty for laboratory reared specimens, but often can only be estimated for field collected insects. Powever, in ths absence of temperature information, the weight of agults analyzed could be reported to make wing-beat frequency more meaningful. It seems probable that, had rearing temperature or weight of females analyzed in the past been cited, much of the wide range of reported frequencies could be explained. Studies of insect flight indicate that the change in wing- beat frequency with rearing temperature may be due to differences in wing size associated with adult size variation. Sotavalta

( 1952) demonstrated that the wing-beat frequency of insects is susceptible to changes in the mechanical load on the wing. He loaded wings with colloidion and "sub-loaded" wings by cutting off pieces, and found that the wing-beat frequency was approximately inversely proportional to the cube root of the moment of inertia of the wings. Loaded wings resulted in a lower wing- beat frequency and sub-loaded wings beat at a higher frequency. The same investigator studying other aspects of insect flight found that his data could be fitted to the equation

F = 0.3A - 1.3B - 0.75C -t- H + Q where 17 = log wing-beat frequency; A = log mass of insect

in milligrams; B = log wing length in millimeters; C = log

stroke amplitude in degrees; H and Q are 11 constants" which encompass some residual variation. The relationship states in effect that wing-beat frequency varies directly with 0.3 power of the weight, and inversely with the 1.3 and 0.75 powers of wing length and stroke amplitude. Hence, wing-beat frequency is influenced to a greater degree by wing length and related stroke amplitude than by the weight of the mosquito, explaining the increased rate of wing-beat with decreasing body size and accompanying decreased wing size, despite a smaller flight muscle mass.

(b) Effect of Larval Rearing Density and Food Ration on the Female Wing-Beat Frequency

Varying the larval rearing density by a factor of 5 had no apparent effect on the size and no significant effect (t- test, P > .05) on the wing-beat frequency of adult' females (Table 11). It was anticipated, on the basis of previous studies, that increasing larval density would influence adult size.

Barbosa --et al. (1972) reared -A. aegypti at concentrations of 0.5, 2.0, 6.5, 8.0, 12.0, and 16.0 larvae per ml and found pupal weights of .75, .45, and .15 mg respectively at the three lowzst densities, the weights remaining constant at the other densities. Mean weight and wing-beat frequency ( i std. error) of 50 A. aegypti females reared at various larval densitiFs at 26 C, Females were weighed in groups of 50.

ADULT FENALE FEMALE FLIGHT WEIGHT (mg. ) TONE (EZ).

REARING DENSITY ':Jada (1965) observed a slight but definite dccrease in thorax and wing length in -A. aegypti females reared at densities of .01, .16, .64, and 2.5 larvae per ml. However both studies involved considerably fewer actual larvae than the present study, which used 300, 750, and 1500 larvae in 300 ml to obtain the three density levels (Table 11). In Barbosa's experiment the numbers ranged from 40 larvae at the lowest density to 1280 at the highest, and in Wadals study the range was 1 to 250 larvae. Greenough --et al. (1971), however, found variation in the weight of adults resulting from larvae reared at the same density but at different absolute numbers. It is therefore not always valid to compare results on the basis of density alone without considering absolute numbers. The fact that, in the present study no variation in adult size occurred at the two lowest densities indicates the larvae were already 11 overcrowd.edl'. This is supported by the relatively small size of the females at all three density levels, comparable in size to females reared at high temperatures (Figure 4), and by Barbosa's study in which the adults from the treatments with the higher larval numbers were relatively constant in size compared to the changing adult size with rearing density at lower larval numbers.

As shown in Table 111, larval food ration apparently influenced adult female size and significantly influenced wing- beat frequency. Females became progressively smaller and their TABLE 111

Mean weight and wing-beat frequency ( S. E. ) of 50 A. aegypti fmales rearzd, at 26 C, at different larval food rations. Females weighed in groups of 50.

ADULT FEMALE ADULT FEbPALE FOOD RATION" WEIGIIT ( rng . ) FLIGHT- TONE ( HZ )

FULL RATION

1/2 RATION

1/5 RATION 1.4 519 4 29

* SEE TABLE I wing-bzat frequency higher ads the food ration per larva was decreased. The group of females given the least food were only about half the weight of those given the most. Statistical tests indicate adults resulting from larvae on a "full" ration had significantly (t-test, p < .05) lower wing-beat frequencies

than those resulting frorn larvae reared on a "1/5" ration. No reports of previous studies on the influence of larval food on adult wing-beat frequency could be found, but investigations have demonstrated the relationship of larval feeding and adult

size. Weilding (1928) produced small -A. aegypti adults from starved or undsrfed larvae. Brust (1968), feeding Aedes vexans

and -A. aegyp$i on starvation diets, observed reduced larval and pupal weights. The relationship of size to wing-beat frequency has already been discussed. The changes in wing-beat frequency and adult size produced by manipulation of larval food ration were greater than those produced by varying larval density. This is in agreement with the findings of Moore and Whitacre (1972) who, working with -A, aegypti, concluded that larval nutrition, but not density, affects production of growth retardant factor that had been shown by Ikeshoji and Mulla (1970) to result in reduced larval and pupal size. Hence, in rearing adults for acoustic studies, large numbers of larvae may be reared without causing

variability in acoustic behavior if an adequate food supply is maintained. (c) Effcct of' Tarval Rearing 'Temperature on Male Acoustic Behavior Adult -A. aezypti males reared at different temperatures showed variation in acoustic behavior. Males from different temperatures, when presented with frequencies ranging from

350 to 650 Hz, showed differences in their degree of response and were optimally attracted by different frequencies. Table

IV shows the percentage of males reared at 16, 26, and 36 C responding to each frequency. Males responded optimally to higher frequencies when the rearing temperature was increased. The optimally attractive frequency for males reared at 16, 26, and 36 C was 450, 525, and 550 Hz respectively. As reported previously (~igure4), the mean wing-beat frequencies of females reared at 16, 26, and 36 C were 430 i 26, 505 & 25, and 565 & 28 respectively. The frequencies found to be most attractive to males and the frequencies of the f ernalesf wing-beat do not coincide exactly, but do agree within the limits of significance delineated by standard error. This suggests that acoustic synchrony is main - tained between the female wing-beat frequency and the optimally attractive frequencies to males, both increasing with rearing temperature. If the acoustic behavior of one sex changed while the other remained constant, acoustic synchrony would be broken and a decline in mating would almost certainly result.

Rearing temperature also influenced total response. As shown by the mean percentage response values for each rearing Percentage of' A. aegypti males reared at drirferent temperatures trapped on approaching a source of sounds of different frequencies. Tests were conducted at 26 C. Figures represent the mean of 13 replicates of 25 males each.

Larval Rearing Temperatures (C) -16 26 -36 FREQUENCY ( HZ) $ MA~sRESPONDING 350 375 400 425 450 475 500 525 550 575 600 625 650

MEAN ( k S. E.) 11.9 & 1.4 19.6 2.0 22.3 * 2.4 - HIGHEST $ RESPONSE FOR A RERRING TEMPERATURE temperature, males reared at 26 and 36 C were more responsive to sound than those reared at 16 C (t-test, p < .05).

The bimodal pattern to male response that is apparent when the data in Table IV are graphed (~igure5) may be significant. The two-peaked curves correspond well with the curves that Belton (unpublished data) found for the vibration of male antennae in response to sounds of different frequencies. The frequencies represented by the peaks are not multiples, and therefore not harmonics, of a common fundamental or of one another. Belton's work indicates the male antennae are less sensitive to the frequencies between the peaks. Further work is needed to resolve this phenomenon. As was found with females, pales reared at different temperatures varied in size. Mean weights of 50 males from rearing temperatures of 16, 26, and 36 C were 1.2, 0.8, and 0.7 mg, respectively. It is known that the temperature experienced by the larva affects the body proportions of the adult. The length of the thorax, wings, and leg segments are affected by rearing temperature (~lements,1963) and, although no reports of changes in antenna1 dimensions with rearing temperature were found, it is reasonable to assume they are similarly affected.

This indicates that differences in physical properties of the male antennae may be responsible for changes in numbers responding to sound. The larger fibrils on the antennae of males reared at the lower temperature would probably require more energy to cause the same amplitude of vibration. This would Figure 5: Percentage of A, aegypti males reared at different temperatures rzsponding to various frequencies. explain th? higher responsiveness of smaller males. The greatest difference in size was between males reared at 16 and 26 C, and these also displayed the greatest difference in responsiveness able IV). Males reared at 26 and 36 C were similar in size and also responsiveness.

The upward shift in the optimally attractive frequency with size is more difficult to explain. It is probably related to the smaller size of the Johnstonf s organ, although no measurements were made of the cuticular parts, sensory cells, or fluid volume, all of which could be involved. No reports of previous studies on changes in male acoustic behavior with rearing temperature could be found, and perhaps neuro-physiological studies would aid in understanding this phenomenon.

( d) Effect of Larval Rearing Temperature on Fiating Frequency When presented with females from various larval rearing temperatures, -A. aegypti males mated more often over a one hour period with females from their own rearing temperature than from different temperatures. Table V shows that males reared at 16 C inseminated more females reared at 16 C than at 26 or 36 C, the

36 C reared females having the lowest rate of insemination. Likewise, males reared at 26 and 36 C inseminated more females from their own rearing temperature than from others. This supports the theory that the female wing-beat frequency and male response change synchronously.

Table V also shows that males reared at 26 C inseminated more femal-es, 65s from all three rearing temperatures, than the TABLE V Percentage of females inseminated in one hour when 30 females, lo rrorn each rearing temperature of 16, 26, and 36 C, were caged with 10 males reared at one of these ternp~~atures.Each figure represents the mean of 12 replicates.

$ INSZMINATED FENALES MALE REARING REARED AT CAGE TEMPERATURE 16 c 26 c 36 c

- MEAN 57 3 p

-15 % FEMALES INSEMINATED DURING 24 HOUR EXPOSURE TO MALES ( CONTROL) 16 and 36 C reared males which inseminated 39 and 54$ respective-

ly. This is probably due to the overlapping of the 26 C reared

females wing-beat frequ:ncy into the attractive range of the 16 and 36 C reared males. This is supported by the two lowest rates of insemination, 18 and 336, which occurred where the male and

female rearing tempera,ture were most separated, 16 C males with 36 C females and 36 C males with 16 C females, respectively. To ensure that the genitalia of the mosquitoes of different sizes were not physically incompatible, thereby affecting mating, males and females from each rearing temperature were caged together for 24 hours and the percentage of inseminated females

determined. In all cases, as shown by the bracketed figures in

Table V, over 90% insemination occurred indicating mating was not hindered by genitalia size differences. The differences in the frequency of mating could not have

resulted from possible effects of rearing temperature on the

flight activity of adults as the cages were agitated at regular intervals to induce flight. Also, as previously reported, there is no evidence that attractant pheromones are involved in the mating of -A. aegypti, so it is improbable that the differences in mating are a consequence of rearing temperature influencing the

quantity or character of such a pheromone. These results indicate that the larval rearing temperature

influences acoustic behaviour, and therefore must be a considera- tion in rearing mosquitoes for acoustic or mating studies, or for

release into natural populations. (e) Ef'fect of' 1,arvzl Iicaring 'i'emperature on Female Fli-ght-Tone Sound Pressure Level

It was considered probable that adul-L size differences resulting from different rearing temperatures influenced the sound pressure level as well as the frequency of the female flight-tone. Figure 6 shows this is the case, with the sound pressure level increasing with size. Females reared at 36 C had significantly lower (t-test, p < .05) flight-tone sound pressure levels and were significantly smaller (p < .05) than those reared at 16 or 26 C. Significant differences did not occur between rearing temperatures of 16 and 26 C (t-test, p < .05). Christophers (1960) reported that the wing length and area increase proportionally with adult weight. Although no references could be found to changes in wing-beat amplitude with increasing wing length, it seems probable that the angle of wing displacement from horizontal would remain constant with changing adult size, and hence that the amplitude measured at the top of the longer wing would be greater. This greater amplitude, combined with the larger wing area, would displace a greater mass of air with each stroke resulting in a more intense flight-tone. The results in Figure 6 show that as rearing temperature became higher adults became smaller and the flight-tone sound pressure level decreased. A 20 C reduction in temperature caused a halving of both weight and sound pressure. A separate study, to be reported later, demonstrated that the differences in the frequency of the wing-beat of adults at different ambient tempera- -4 la-

Figure 6: Flight-tone sound pressure level (db) at 2.54 cm and weight of A. aegypti females reared at different t emperaturzs . MEAN WEIGHT OF 10 FEMALES

16 26 36

REAR ING TEMPERATURE OC tures has little if any influence on the flight-tone sound pressure level. Having determined that the female flight-tone sound pressure varies with size, an experiment was conducted to study the effect of this variation on the attractiveness of such flight- tones to males. Table VI shows there was no difference in the percentage of males responding to the different sound pressures, although a difference in response to frequencies occurred with more males responding to 500 and 550 Hz than to 450 HZ. This is in agreement with the observations of Wishart and Riordan (1959) who found the soumd pressure of a sound could vary as much as

6 db without appreciably affecting its attractiveness to -A. aegypti males. Belton (lg67b) reported an optimal sound pressure range of 50 - 70 db in attracting maleAedes stimulans to a sound placed below a swarm. The sensitivity of male mosquitoes to a wide range of sound pressure levels suggests the female flight-tone can attract males over some distance despite diminishing intensity. For example, Wishart and Riordan (1959) observed males changing their flight path and darting towards tethered females from up to 10 inches away where the female flight-tone sound pressure level was approximately 20 db. It should be remembered the present study was conducted in cages where the distances between the sexes were small. If a flying female is considered a point source of sound, there is an area around bounded by a particular sound pressure that is di- TABLE VI

Percentage of A. ae~yptimales responding to sound waves of diff $?Tent frequencies and sound pressure levels. Percentages represent mean of 10 replicates of 25 males each.

$ IMLES RESPONDING Frequency (HZ) Sound Pressure Level (db) 450 -500 -550 MEAN&. E. 48 37 * 3.1 44 k3.5 44 't 3.7 42 ~r 2.2 rectly proportional -to the intensity of the point source. The greater the area, the greater the probability a male will en- counter it. This is obviously important in the field where the distance between males and females is usually greater than that between caged mosquitoes. Hence, while differences in the sound pressure level of the flight-tone resulting from differences in adult size do not qualitative1.y influence its attractiveness, the louder flight-tone of larger females can probably be perceived over a greater distance. Once perceived, however, the attractiveness of the flight-tone of mosquitoes of different sizes appears to depend on frequency and is independent of sound pressure.

( f) Differences in adult female we_i.ght between experiments The mean weights of females reared at the "standard" conditions described in the Materials and Methods section showed differences between experiments. Female mean weights ranged from 1.3 (p. 41) to 2.6 mg (p. 31) despite simj.lar rearing conditions of temperature, nutrition, and density. Hence, unless other unknown and uncontrolled aspects of rearing conditions play a role in determining adult size, these differences must be owing to factors other than rearing conditions. The differences in female size observed between experiments in this study could have been caused by two factors. First, adults used were raised from eggs obtained from an insectary over a 8 month period. During this time the conditions, particularly temperature, in the insectary varied and adults of different sizes were undoubtedly produced in the culture. No reports on the influence of the size of the female parent on egg size could bc found, but this must be considered a possibility. The difference in egg size might then be manifested in differences in adult size. The only related study that could be found lends support to this hypothesis. Oelhafen (1961, quoted by Clements, 1963) found that females of Culex pipiens molestus from undernourished larvae lay not only fewer but also smaller eggs than usual. This is an example of the rearing condi.tions experienced by the female parent influencing egg size.

A second probable cause of differences in female size between experiments is that they were taken from different parts of the adult emergence period. It was observed throughout the study that the earlier emerging females were noticeably smaller than later emerging ones. In experiments where the weights of females were compared, all females used had emerged within 24 hours of one another. However, as it was felt no direct com- parisons of female size between experiments was required, females used in different experiments were often from differmt parts of the emergence period. Hence, in one experiment females may have been early emergers, while in another study medium or late emerging females might have been used, depending on their availability at the time the experiment was conducted.

It is probable the two factors discussed above account for much, if not all, of the size variation in females from one -4 6- experiment to another despite similar rearing conditions. PART 2 Effects of Ambient Temperature on Acoustic Behavior of

Aedes aegypti Adults

The efyect or ambient temperature on the wing-beat frequency of insects was first studied by Sotavalta (1947). He reported directly proportional changes in wing-beat frequency over a range of ambient temperatures in Diptera such as Drosophila spp., Musca domestics and -M. autumnalis, Tipula sp., some unidentified culicids, and a moth, Hemaris sp. More recently Farnworth (1972b) reported the same effect in

Periplaneta spp. In most of these insects these changes seem to be of little behavioral consequence other than possibly influencing flight speed, but in mosquitoes where the sound produced by the wing of the female in flight attracts and guides the male, a change in wing-beat frequency could influence mating behavior. The present study was conducted to determine the influence of ambient temperature on the acoustic behavior of -A. aegypti adults of both sexes and to determine the effect of any changes in acoustic behavior on mating frequency.

Materials and Methods

The flight-tone of -A. aempti females in free flight were recorded at temperatures of 20, 24, 27, 33, and 35 C and subsequently analyzed to determine the wing-beat frequency using the method described earlier. All females, rearzd at 24 C, were 4 to 5 day old virgins when used. Temperature regimes were obtained by manipulating the temperature of the laboratory with a ther~r~ostatically controlled furnace. The temperature of the recording chamber itself was monitored by observing a thermometer inserted through the rubber stopper. Temperature fluctuation was not a problem as each recording took less than a minute. Females were analyzed at each ambient temperature at 45 minute intervals. Fifteen minutes were allowed for the mosquito to adapt to the new temperature, although Chadwickls (1939) work suggests a shorter adaptation period would have been sufficient. Relative humidity in the laboratory fluctuated less than lo$ from the lowest to highest temperature and hence was considered to be of little consequence. This assumption was borne out in subsequent experiments (part 3). Ten females were examined at each ambient t ernperature. The effect of ambient temperature on the acoustic response of males was studied by presenting a range of frequencies to them at different temperatures, and determining the most attractive frequency at each temperature. Frequencies were presented at 50 132 intervals over a range of 350 to 650 Hz, at temperatures of 18, 23, 27, 31, and 34 C.

All males were reared at 24 & 1 C and at similar densities and food rations, as previous experience indicated rearing conditions affected adult acoustic behavior (part 1). Roth (1948) demonstrated that the "mating state" influmced male response, so pupae were sexed by size and the ma,les placed in separate cages to insure virginity. Adult emergence time was noted and males were not tested until they were 4 to 5 days old, as

Wishart and Riordan (1959) found that male acoustic response changes with age up to 4 or 5 days and then remains relatively constant. Thirty-five cages of twenty-five males each were employed, each cage being first exposed to a different tzmperature-frequency combination (35 combinations were used). Therefore, cage 1 was first tested with 350 Hz at 18 C, and cage 2 with 400 Hz at 18

C. Likewise cage 8 was first tested with 350 Hz at 23 C, the next highest temperature, and cage 35 with 650 Hz and 35 C, the greatest frequency-temperature combination. Subsequent tests for each cage were at successively higher frequencies within each temperature before proceeding to the next temperature. This avoided possible bias due to conditioning or habituation through previous exposure to sound. Each frequency was presented for 5 seconds and, as only males in flight respond to sound, the cages were agitated just prior to the sound presentation. Frequencies were presented at 15 minute intervals unless a change in temperature was involved, in which case one hour was allowed for temperature manipulation and adaptation of the mosquito. For example, there was a one hour period between testing 650 Hz at 18 C and 350 Hz at 23 C. The males attracted to the source of sound were drawn by suction into a collecting chamber where they were counted and subsequently returned to the source cage as described earlier.

The influence of amhient temperature on the frequency of mating of -A. aegypti adults was studied by caging together, at various temperat~zres, virgin males and females and examining the female spermathecae for sperm. Ten males and females were caged together at each of the test temperatures. Temperatures of 18, 23, 27, 31, and 34 * 1 C were used. After a half-hour, during which the cages were subjected to as little disturbance as possible to avoid inducing flight, the female spermathecae were examined for sperm as evidence of mating. Five cages were used at each temperature.

Flight is a prerequisite to mating in -A. aegypti (~iordan,

1965). Therefore, in order to interpret the results of the effect of ambient temperature on mating frequency, it was necessary to determine the influence of ambient temperature on flight activity. The activity of 4 day old virgin females was monitored for one hour at the same temperatures used to determine the frequency of mting. Various techniques have been devised to record mosquito activity automatically. Powell --et al. (1966) used a wire grid with alternate strands connected to positive and negative poles. The electric current chamged each time the mosquito moved and the event was recorded by a pen-chart recorder. However, this measured locomotor rather than flight activity and therefore was not appropriate for the present study.

Jones (1964) described a method of recording mosquito flight activity utilizing an electronic apparatus that converted the sound of a flying mosquito into an electric impulse that trig- gered a pen on a chart recorder. A similar apparatus was designed for the present study but was abandoned because of difficulties encountered. A primary problem was the elimination of background noise and vibration in a laboratory shared with other workers on independent projects while maintaining sufficient acoustic sensitivity to pick up the flight sound of a single mosquito. It was also difftcult to obtain a precise measurement of flight duration from the chart rezdout. Although these problems could have been overcome, it was decided that, as the experimmt required a few hours to complete, direct observations and measurements of activity would be made. The primary concern in using this method was that the mosquito under observation might be stimulated into flight by movements or sound made by the observer. This was avoided by separating the mosquito from the observer with black plastic sheeting having a small clear plastic observation window. The mosquitoes were illuminated by a 25 watt incandescent light bulb placed 4 feet above the activity chamber, while the observor was in darkness and presumably invisible to the mosquitoes. This presumption appeared to be val3.d as numerous unsuccessful attempts were made in preliminary tests to induce flight with vigorous movements and noise making by the observer. The activity chambers consisted of 480 ml glass jars fitted with rubber stoppers. The chamber itself undoubtedly provided a degree of soundproofing. A thermometer was inserted through the stopper to monitor the chamber temperature. The number of flights and duration of each, as measured by a stop watch, was recorded. The activity of ten females was observed at all five ambient temperatures.

All tests were conducted between 9 A. M. and 3 P. M., and were completed in five consecutive days. Each day a different temperature was tested first, followed by the other temperatures

in sequence. Hence, any differences in flight activity could be attributed to temperature rather than possible circadian activity rhythms.

A second parameter, after frequency, of the female flight- tone that could be affected by ambient temperature is sound pressure level. To examine this, females were tethered in a fixed position 2.54 cm from the microphone of a Brfiel and Kjaer Impulse Precision Sound Level Meter, Type 2204. Flight-tone

sound pressure was then measured at temperatures of 23, 29, and

34 C. The females, 4 day old virgins, were glued by the ventral thoracic surface to the head of an upright pin. Preliminary

experiments indicated the position of the mosquito relative to the microphone influenced the sound pressure measurement, so

all readings were taken with the insect facing the microphone. Short puffs of air were used to stimulate the mounted specimens to beat their wings. Background noise in the experimental area,

a room especially designed for acoustic studies, was 32 deci-

bels. Ten females were examined at the three temperatures and,

as a control, 10 females were examined three times at one temperature, 29 C, at the same intervals as those exposed to different temperatures. Hence, if difyerences in flight-tone sound pressure levels were observed in control as well as treatment indi-

viduals, it would be due to factors other than ternperatu~e, perhaps fatigue.

Results and Discussion

(a) Effect of Ambient Temperature on the Female Wing-beat Frequency

As shown in Figure 7, the wing-beat frequency of the -A. aegypti females examined increased with an increase in the ambient temperature (t-test, p < .05 for each temperature incre-

ment). The rate of change in frequency, as measured at 3 C

intervals, was more or less constant from 20 to 32 C, increasing

at 6.3 - 6.7 Hzldegree C able VII). From 32 to 35 C the rate of increase was slightly but not significantly (t-test, p > .05)

less, 5.3 Hzldegree C.

The increase in wing-beat caused by a 10 C rise in tempera-

ture, designated &I,, was significantly smaller (t-test, p < .05)

for the 32 to 35 C range than that for the 20 to 23 C range, indicating that the influence of temperature on wing-beat fre-

quency decreases as temperature increases.

The Qlo values indicate that the temperature effect is

physical. Hoar (1966) states that &lo values give some clues

to the nature of processes influenced by temperature. Ql0 values less than 1.5 are usually associated with physical pro- Figure 7: Change in wing-beat frequency and corresponding Ql values with changin ambient temperature. Points represent the mean ?+ S. E.) of 10 females. WING BEAT FREQUENCY STD, ERROR /u

20 23 26 29 32

AMBIENT TEMPERATURE (OC) TABLE VSI Effect of ambient t emperature on the wing- beat frzquency of A. acgypti females. Figures represent the meamE. ) of 10 females.

A AI4131ENT A 1,TING- REAT HZ/OC TEMPERATURE FREQUEITCY ( HZ) INCREASE -Ql0 ccsses while those for thermochemical (enzymic) reactions range from 2 to 3. The physical changes probably occur in muscular or skeletal tissues rather than in the nervous system, as flight in mosquitoes, as in all holometabolous insects except the dragonflies (~donata), is not under direct nervous control. Pringle (19119) found that increasing the intensity and/or number of nerve impulses to the flight muscles did not effect the rate of contraction. He stated that a critical number of impulses are required to maintain excitation of muscle fibres which causes the contractile elements to become sensitive to stretching. On being stretched the fibres respond with a twitch-like contraction and in so doing stretch the antagonistic muscle fibres, setting up a myogenic rhythm.

If fewer than the critical number of nerve impulses reach the muscle fibres, the contractions cease rather than decline in number, while more impulses than required to maintain excitation do not increase the frequency of contractions.

Hence it appears unlikely nervous tissue is involved in temperature- induced changes in wing-beat frequency. More probably the changes are myogenic in origin, orginating perhaps in temperature effects on sarcoplasmic viscosity, myofilament length, or membrane conductivity. The latter was studied by Kerkut --et al. (1961), who found that temperature changes affected the resting potential of insect muscle fibres. Temp- erature fluctuations my also affect the flexibility of the thoracic cuticle which in turn could modify wing-beat frequency. The flexing movem~ntsrequired of the tl~oracicskeletal elemcnts, particularly the tergites and pleurites, during insect flight have been described by Chadwick (1953). Beament (1958) demonstrated that temperature affects the molecular arrangement of the insect cuticle, thereby influencing its permeability. This molecular rearrangement could also influence the flexibility of the thoracic sclerites. Relative inflexibility at low temperatures would result in a rate of wing-beat lower than at high temperatures where flexibility is greater. Resilin, a rubber-like protein found in certain extracellu- lar cuticular structures in insects, may also be affected by temperature changes. This protein provides a reversible elasticity to the exoskeleton, and is apparently important in imparting flexibility to such structures as the prealar arm and wing-hinge ligament (Weis-~ogh, 1960). The elastic properties (force and modulus) of these structures vary directly with absolute temperature (~ndersonand Weis-Fogh, 1964), and this would probably influence the rate of wing-beat.

The application of &lo values to changes in insect wing- beat frequency with ambient temperature has been criticized by Farnworth ( l972b), who claimed Richards ( 1963) and Yurkiewicz and Smyth (1965) erred in expressing the temperature induced increase in wing-beat frequency of the American cockroach, -P. americana and the sheep blowfly -P. sericata, respectively in &lo values. Farnworth claims this operation is invalid because Qlo is an exponential function and should be applied only to exponential incl-ease, and su-ggests that for linear results the rate

of increase should be expressed as Hz/dcgree. The value of QI, will of course change with temperature if the relationship is

linear, but it is clearly valid to calculate QI, if the tempera-

ture range is given.

(b) Effect of Ambient Temperature on Male Acoustic Response Males responded optimally to progressively higher frequencies as the ambient temperature increased. This trend can be seen in

Table VIII, with 350 Hz the optimally attractive frequency at the

low test temperature of 18 C and 550 Hz the most attractive at

34 C, the highest test temperature. At some of the temperatures the optimally attractive frequency probably fell between the

frequencies tested. For example, at 23 C the most attractive

frequency probably lay between 400 and 450 Hz, as the response

to these frequencies was not significantly different. This almost certainly applies to the response at 27 C, where the percentage of males responding to 450 and 500 Hz differed by

only 0.2%. The optimally attractive frequency at this temperature was probably closer to 475 than 450 or 500 Hz. The trend of changing response with temperature serves to maintain synchrony between male and female acoustic behavior at

different temperatures. If male response remained constant with changing temperature while the female wing-beat frequency fluctu-

ated, a decline in mating would result as the temperature changed from that where the female wing-beat frequency coincided with

the most attractive frequency to males. However this is avoided TABLE VIII

Percentage of A. azgypti males responding to frequencies rangi& from 350 to 650 Hz at different ambient temperatures. Each figure is the mean ( i S. E. ) of 35 replicates of 25 males each.

Goo 0.7 i .2 5.6 * .8 9.7 i 1.7 18.1g.1 21.1 i 2.2

highest $ response for a temperature by the sychronous response of ma1.e and female acoustic behavior to temperature.

It can be argued that mating at high temperatures would be a waste of energy if such temperatures adversely affected factors such as sperm survival. or adult longevity. However, experiments discussed later in this section indicate that the temperature range used in this study does not influencz sperm survival.

There is evidence that mosquito survival is influenced by temperature (~ar-zeev,1957)) but it is evidently an advantage to have mating take place as the females may survive long enough to oviposit, or, if the adverse temperature is temporary the lifetime of the female may not be reduced. In addition to influencing the frequency to which males were most attracted, increasing ambient temperature also resulted in a greater overall responsiveness. The percentage of males responding to all frequencies ranged from a low of 1.7% at 18 C to a high of 19.1% at 31 C able VIII). This is probably a reflection of the positive influence of temperature on flight activity able IX). Although the cages were agitated to induce flight prior to the sound presentation, it is likely that at the lower temperature some of the malessettled immediately while males at higher temperacures more probably continued flying throughout the test and hence were more likely to be attracted. The change in male response with temperature appears to have a physical basis. Tischner and Schief (1954)) observing the resonance frequency of the antennae of -A. aegypti males, reported that "the funicles of the males remain more or less accurately attuned to the primary note of the female flight- noise regardless of temperat,ure". This indicates, as resonance is a physical phenomenon, that temperature in some way effects the mechani.ca1 properties of the antenna. The importance of the components of the antennae on acoustic behavior was shown by Roth (1948). He performed various antennal operations, including removal of 9 of 13 segments and removal of practically all the fibrillae leaving a bare shaft, and observed a considerable decrease in response. He also shellacked the base of the antennae to irnrnobilize it and found males so treat<-d ignored acoustic stimuli. This indicates that rigidity may affect response. Temperature may affect the rigidity with which segment 3 of the antennal shaft articulates with the Johnstonf s organ of segment 2. An increase in temperature may decrease this rigidity, allowing the antennal shaft to vibrate more rapidly. The articulation of the fibrils with the antennal shaft may be similarly involved. The physical properties of the sensory cells of the Johnston's organ may also be influenced by temperature. It is unlikely that temperature significantly affects the dimensions of the fibrils and other antennal components, as the greater length with increasing temperature would result in a lower rather than higher optimal frequency, which is the opposite of the observed results. (c) Effect of Ambient Temperature on Flight Activity and Mating Frequency

Flight is a necessary prerequisite to mating in -A. aeapti (~iordan,1965)) and hence factors influenring flight activity also influence mating. Table IX shows flight activity and the number of females successfully mated ( inseminated) at different ambient temperatures. Each temperature increment from 18 to 31

C resulted in significant (t-test, p < .05) increases in both mating and flight activity. Significant differences did not

occur between 31 and 34 C. The percentage of females inseminated increased with ambient temperature from 0% at 18 C to 53% at 31 C. These differences were undoubtedly caused, not by the males and

females being I1 out of tune" at different temperatures but by

the effect of temperature on flight activity. Therefore, although the rate of mating varied with temperature, the postulation that

synchrony is maintained in male and female acoustic behavior at

changing temperature is not refuted. The relationship of temperature to insect flight activity has been studied by a number of workers. Rudolfs (1924) stated that "activity of mosquitoes increases with the increase in temperature unless the temperature becomes too high".

Christophers (1960) suggested that temperature influenced the biting rate of mosquitoes through a general influence upon the

activity of the organism. Haufe (1964) observed an increase in

take-off frequency and duration of free flight (as opposed to tethered flight) of -A. aegypti females with increasing tempera- TABLE IX $ of females inseminated and female flight activity at tempcraturzs of 18, 23, 27, 31, and 34 C. Mating percentages are mean of 5 replicates of 10 females, and activity figures are mean of 10 females.

FLIGHT- TIME % FEMALES MEAN NO. MUN DURATION ( SECS) 3; AMBIENT INSEMINATED OF FLIGHTS OF EXCH FLIGHT PER FEMALE TENP (c) in 30 minutes in one hour (SECS) in one hour

3:- mean # of flights x mean duration per flight ture to 311 C where it declined, which is similar. to the activity trend of the present study. The flight speed of sheep blowfly adults was measured on a turnabout or flight-mill by Yurkiewicz and Swth (1965), and was found to increase with temperature over a range of 15 to 30 C. Wright and Knight (1966) related mosquito activity to number caught in various types of traps, and found that of 3098 Aedes trivittatus caught, 9576 were taken between 18 and 28 C over a total temperature range of 14 to 30 C. Rowley and Graham (1968) flew -A. aegypti females to exhaustion on flight mills and found they flew longest at 27 C and least below 15 C and above 32 C. This experiment provided a measure of the effect of temperature on flight endurancs rather than spontaneous flight activity, and hence the optimal temperature of 27 C observed by Rowley and Graham is not directly comparable with the optimal temperature of 31 C observed in the present study. These experiments, while differing in detail, demonstrate flight activity increases with temperature to an optimal level.

(d) Effect of Ambient Temperature on the Female Flight-Tone Sound Pressure Level

The sound pressure level of the female flight-tone was not significantly (t-test, p > .05) influenced by changes in ambient temperature co able x). It was anticipated that the resistance of the air to a vibrating wing would probably increase at higher wing-beat frequencies and that for a given amplitude, the sound pressure level would increase with frequency (~eranek,1962). However this study indicates the frequency change (490 - 580 HZ) TABLE X

Sound pressure level of the female flight-tone at different temperatures. Data represents mean (+ S. E.) of 10 females.

Sound pressure Sound pressure level (db) at level (db) at Temp. (c) 2. 511. cm Temp. (c) 2.54 cm over a 23 to 34 C temperature range is insufficient to result in measurable intensity differences.

Other workers examining the loudness of the flight noise of mosquitoes made their measurements at a single temperature

(wishart and Riordan, 1959; Jones, 1964). The present study demonstrates that the sound pressure level does not vary measurably with temperature and therefore, of the two parameters of the female flight-tone, frequency and sound pressure level, only the formel- influences the acoustic attractiveness of females to males at different temperatures. PART 3 Eff'ec ts of Relative IIumidity, naromctric Pressure, and Light on the Wjng-beat Fveyuency of Aedes aepjpti Females

Numerous studies have been made on the effect of relative humidity, light, and to a lesser extent, barometric pressure on the behavior of mosquitoes. Field studies such as those conducted in recent years by Ridlingmayer (1967, 1971)) Dow and Gerrish (l970), and. ldiura and Reed (1970) have related flight activity, as measured by trap catches, to relative humidity and phases of the moon. Other behavioral aspects examined in the field include biting activlty right and Knight, 1966) and oviposition pe el ton, 1967 a). Studies in the laboratory have largely concentrated on the influence of relative humidity, light, and in some cases barometric pressure, on flight activity and performance (~aufe,1964; Jones --et al. 1966, 1967; Rowley and Graham, 19G8), host orientation (~ar-Zeev, 1960; Platt --et al., l957), biting activity (~in~scoteand Francis, 1954; Lumsden, 1947), and swarming (~ates,1941; Nielsen and Nielsen, 1962). However, little attention has been given to the effect of the environmental factors mentioned on mosquito acoustic behavior. Previous studies and the need for further research in this area were discussed in the Introduction.

Materials and Methods (a) Relative humidity

The wing-beat frequency of free flying -A. aegypti females was analyzed at relative humidities of 5, 50, and 9575, each at temperatures of 26 and 34 C. The experimental chambers were 355 ml wide mouthed glass jars fitted with a rubber stopper. Holes in the stoppcr permitted insertion of a microphone and a thermometer to monitor temperature. The 5% R H regime was achieved by placing a 2 cm

, > layer of calcium chloride on the floor of the chamber. Ninety- five per cent R H was attained by lining the jar with moistened paper towels. For the 50$ R H treatment the chamber was left at ambient laboratory conditions as the experiment was conducted only when laboratory Ii H was within & 5% of 50% R H. No instru- ments small enough to measure R H within the recording chamber were a.vailable, so a larger chamber (12 x 16 x 16 cm) of clear perspzx was used to determine the ability of calcium chloride and moist paper towelling to create 5 and 95% relative humidities. Relative humidities were measured by a membrane hygrometer (~erdexmodel, Bacharach Industrial Instrument Co.), accurate to

1.5% R H. A 1 cm layer of calcium chloride resulted in 5% R H within 25 minutes with no further decrease over two hours of further monitoring. Ninety-five per cent R H was achieved with moist paper towelling within 20 minutes and increased to 97% after two more hours.

The experimental temperatures of 26 and 34 C were attained within a degree by maintaining the laboratory at the lower temperature and using a water bath for the higher. Ten females, four to five days old and virgin, were tested in each of the 6 envjronments. Females 1 to 5 were exposed to increasing R H and temperature, so they were first tested at 5s

R H and 26 C and last at 957; R H and 34 C. Females 6 to 10 were exposed to decreasing R H and temperature, so 9576 R H at

34 C was the first and 5$ R 11 at 26 C the last treatment,. At each treatment a ten minute adaptation period was allowed prior to recording the flight-tone. The females were often reluctant to fly at 95% R H, so it was usua,lly necessary to shake the recording chamber to induce flight. The recording and analysis technique is described earlier. (b) Barometric pressure The wing-beat frequency of free-flying females was determined at 3 levels of barometric pressure. Four to five day old virgin females were exposed to pressures of ambient, ambient plus 10 cm and ambient minus 10 cm mercury. The pressure extremes were achieved by evacuating or compressing the air in the treatment chamber, a 355 ml glass jar fitted with a rubber stopper. Holes in the stopper allowed for insertion of a microphone and two lines of tygon tubing. One line led to a pump, the other to a mercury manometer. The stopper was taped tightly in place and stop-cock grease applied to all joints to render the chamber air-tight . Figure 8 illustratzs this apparatus. The pressure within the chamber was manipulated by attaching the pressure line to the appropriate nozzle of the double oil- less diaphragm compressor. The line was attached to the inlet nozzle to lower the pressure and the outlet nozzle to increase Figure 8: Apparatus used to study effect of barometric pressure on wing-beat frequency of -A. aegypti females. it. When the deslred pressure was attained, as indicated by the mercury manometer, the valve on the pressure lins was closed. To achieve ambient pressure the line was left unattached to the compressor.

Preliminary tests were conducted to determine if pressure changes affected the recording microphone. A sound of 500 Hz emitted by a small speaker placed in the recording chamber was recorded at the three experimental pressures. No differences in frequency or intensity were observed.

A second preliminary experiment was conducted to determine if the temperature change in the gas of the chamber caused by rapid adiabatic pressure changes was large enough to influence wing-beat frequency. The temperature, monitored via a thermometer inserted into the chamber, was found to increase approximately 1 C from the low to high pressure. When air was evacuated from the chamber at ambient pressure, a drop of slightly less than 0.5 C was observed. This fluctuation is similar to that observed by Sotavalta (1947) who found that the temperature in a chamber was lowered by 0.5 to 1.0 C when the air was evacuated to 0.1 atmosphere. In the present study the thermometer was found to return to the ambient laboratory temperature within 5 to 7 minutes of the pressure change. Ten females were analyzed, five at increasing and five at decreasing pressure. Another ten females, used as controls, were analyzed with the treatment females but only at ambient pressure. A ten minute adaptation and temperature equilibrium period was allowed prior to analysis after each change in pressure. (c) Light The wing-bzat frequency of' twenty-five -A. aegypti females was determined under light and dark conditions. Virgin females, four to five days old, were placed singly into a 355 ml transparent glass jar and their flight-tone recorded. Under light conditions the light intensity at the top of the jar, approximately 1.2 m below a standard fluorescent light fixture, was 700 lux. After recording the flight-tone the mosquito was then transferred to a similar jar which had the outer surface coated with three layers of black vinyl electrical tape (scotch brand). The effectiveness of the light-proofing was checked by placing inside a 24 watt microscope bulb with the transformer at maximum intensity. In a darkened room no light could be observed

through the tape coating. Both jars were fitted with a rubber stopper that had a central hole for insertion of a microphone. The flight-tone was recorded 15 minutes after each female was placed in the chambers. Temperature was maintained at 25 5 1 C.

Results and Discussion (a) Relative humidity

The wing-beat frequencies of -A. aegypti females at different relative humidities and temperatures are shown in Figure 9.

At 26 C the mean wing-beat frequencies of 499, 502 and 501

Hz at 5, 50, and 95% R H respectively were not significantly dif- Figure 9: Wing-beat frequency of A. aegypti females at different relative humidities and-emperatures. Points represent mean (5S. E.) of 10 females. 7 STD. ERROR

5 O/o 50 '10 RELATIVE HUMIDITY ferent (t-test, p > .05). However at 34 C significant differences (t-test, p < .05) between 5 and 95% relative humidities did occur. The mean wing-beat frequency of ten females was 547, 557, and 562 Hz at 5, 50 and 95% R H respectively. The results are similar to those observed on other inszcts in previous studies. So.tavalta ( 1954) found that at temperatures greater than 30 C the wing-beat frequency of Drosophila melanogaster was higher at 100% R H than at 50 to 60$ R H.

Similarly, Yarnworth ( l9'72b) observed significant differences in the rate of wing-beat of Periplaneta americana males between 50 and 95% R H at temperatures above 27 C with higher frequencies at the higher relative humidity. Below 27 C he found no significant differences in wing-beat frequency between 50 and 95%

R H. The effect of relative humidity on wing-beat frequency appears to be the result of changes in the internal temperature of the insect caused by evaporative cooling. This hypothesis is supported by Ramseyl s (1935) and Beament' s (1945, 1958) findings that increased water loss through the insect cuticle occurred at temperatures above 30 C owing to molecular changes in the epicuticular wax. If water loss caused thoracic cooling, wing-beat frequency, which has been shown to decrease with a decrease in temperature, would be lower at lower relative humidities where evaporation is greatest. F'urther support to the evaporative cooling theory was provided by Farnworth (1972a), who measured the internal temperature of -P. americana at different ambient conditions and found the internal thoracic temperature of cockroaches both at rest and in tethered flight were higher at 95 than at 50$ R H. The effect of relative humidity on the efficiency of heat transfer between the mosquito and the znvironment must also be considzred. Heat transfzr would increase with increasing R H, as water vapour has a greater specific heat than air. Mosquitoes, like other insects, are poikilotherms and therefore their body temperature is essentially that of the surrounding environment. However, due to the heat generated by muscular activity, the temperature of insects in flight is usually a few degrees higher than ambient (~igglesworth,1966). A flying insect with no evaporative cooling would therefore lose heat at a rate proportional to the environmental relative humidity. This, in turn, would cause a similar decrease in the wing-beat frequency with increasing R H, and would occur independently of ambient temperature. In the present study wing-beat frequency increased with increasing R H, and this occurred only at 34 C. At 26 C no differences in frequency were observed. These results are not consistent with those expected from the effects of heat loss through conductivity. Therefore, while the influence of relative humidity on the efficiency of heat transfer cannot be discounted, it appears that differences in heat loss resulting from different rates of evaporation at various relative humidities have a greater effect on body temperature and hence wing-beat frequency.

The effect of R H at temperatures above 30 C on the wing- beat frequency of -A. acpjpti females is unlikely to infl-uence the attractiveness of the female flight-tone to males. In thi-s study a relative humidity range of 907; from 5 to 95% R 1-1 resul-ted in a frequency change of only 15 Hz. It is unlikely that mosquitoes would encoumter such extremes, particularly the lower relative humidities, in the field. If such conditions did occur, mating would ~videntlybe precluded by an inhibitory effect on flight. Thompson ( 1938) and Rowley and Graham (1968) found that flight activity decreased below 3@ R H and above 89 to 90% R W. Additional evidence that changes in the female wing-beat frequency caused by relative humidity would be unlikely to effect its attractiveness is indicated by the present author's study of male response to sounds of different frequencies. This shows that a 15 Hz change in wing-beat frequency is insufficient to influence the attractiveness of the flight-tone. Relative humidity does not appear to be an important factor in the acoustic behavior of mosquitoes. Therefore, in conducting experiments on this aspect of mosquito biology it is not necessary to maintain a precise relative humdity if extreme conditions are avoided. ( b) Barometric pressure The wing-beat frequency of -A. aegypti females was found to be not significantly (t-test, p > .05) influenced by changes in barometric pressure. Table XI shows there was no difference in wing- beat frequency at pressures above, below, and at ambient atmospheric pressure. TABLE XI

Mean flight-tone frequency (*S. E.) of ten Aedes aegypti females at different barometric pressures

FLIGHT- TONE ( HZ )

CHANGING PRESSURE

AMBIENT MINUS AMBIENT PLUS 10 cm Hg 10 cm Hg

501 4 7

CONSTANT PRESSURE

AMBIENT AMBIENT The small fluctuations in wing- beat frequency that occurred with changing pressure may have been ths results of a slight variation in temperature due to adiabatic changes of' the air.

However, if this wers the case wing-beat frequency would have been highest at the greater-than-ambient pressure situation, which did not occur. More probably the small frequency differences resulted from a combination of factors associated with pressure change such as temperature, relative humidity, and perhaps oxygen and C02 concentration, or possibly represented random experimental variation. Sotavalta (1947) subjected -A. aegypti to pressures as low as 0.02 to 0.05 atmospheres but was unable to demonstrate conclusively any effect on wing-beat frequency. Chadwick and Williams ( l949), however., found that the wing-beaJ-, frequency of Drosophila spp. was reduced as the density of the surrounding air was altered, The frequency decreased by 17% as the pressure was increased five fold from 760 mm, and decreased 10% as pressure was decreased eight fold from 760 m. The pressure differences are much greater than those used in the present study, and it is unlikely such variation would be encountered in natural conditions. The lack of effect on wing-beat frequency of the range of atmospheric pressures examined in this study, which probably represents the limits of normal barometric pressure fluctuation, indicates that Chadwick (1953) was correct in stating he variation in air density normally met with in the life of an insect, except occasional.1.y in mountainous regions where small species were readily blown to great heights, are of negligible importance insofar as their rate of wing-beat is concerned. 11 The resuLt,s of this experiment lend support to the generally accepted theory which was outlined earlier for the mechanism of wing-beat control in mosquitoes. If the wing-beat could be altered by nervous control to allow for different flight conditions, flight speed would' probably be maintained by lowering the wing-beat frequency in dense air and increasing it in thin air. The fact that this did not occur indicates that the indirect flight-muscles contract at a constant rate regardless of air density. (c) Light The possible influence of light on wing-beat frequency was examined because previous studies, as described earlier, have shown this environmental factor to influence other aspects of mosquito behavior. The present study found that wing-beat frequency was not significantly different (t-test, p 7 .05) under light and dark conditions. The results of this study are shown in Table XII. The effect of light on behavior is often the result of hormanally induced changes in the physiological state of an organism. However, as wing- beat frequency in mosquitoes is evidently under myogenic control and has not been demonstrated to be influenced by the endocrine system, the lack of effect of light on the rate of wing-beat is not unexpected. The function TAULE XI1

Wing-beat frcquency of A. azgypti females after light al?d dark conditions. Each 15 minutes undei, \ - figure :ls the mean ( I, S. E. ) of 25 females.

WING-BEAT FREQ,UENCY

0 lux 491 & 5 I-Iz of a situation where the rate of wing-beat fluctuates with intensity is difficult to imagine as light can change hourly

such as on a day with a cloud-filled sky. Effects of Physiolo::ical Factors on Acoustic Behavior of Aedes aegypti Adults

In an early study Nuttal and Shipley (7902) observed an increase in thz rate of wing-heat of mosquitoes after a blood meal and claimed thi.s rendered the female more attractive to males. A change in wing-beat frequency of mosquitoes and other insects with age has been observed by Chadwick (1953)) Farnworth (1972 b) and Roth (1948). Roth (1948) and Wishart and Riordan (1959) 'also found that male acoustic behavior varied with age and "mating state". The present study examines the influence of age, mating, feeding on blood and sugar, oviposition, and diurnal rhythm on the acoustic behavior of Aedes aegypti adults and attempts to determine the basis for, and biological significance of, any observed variation.

Materials and Methods

Age: The flight-tone of free-flying females was recorded and analyzed for frequency daily over five days from adult emergence.

Thirteen virgin females reared at 26 C were analyzed. The first analysis was made as soon after emergence as flight could be induced, which in all but 3 females was within 12 hours. The second analysis was made when the mosquitoes were 13 to 24 hours old, and subsequent determinations were made at 24 hour intervals so the females were 25 to 48, 49 to 72, 73 to 96, and 97 to 120 hours old. Mating: Twenty virgin females, 3 to 4 days 01-d, were divided into two equal groups. One group remained unmat ed throughout the four day experimental period while the other group was mated on the second day. The wing-beat frequency of the females in each

group was measured as described earlier. At the end of the experiment mating was conf irrned by spermathecal examinat ion.

Ambient temperature was maintained at 26 1 C. To study the effect of mating on male responsiveness recently mated (within 5 minutes) and virgin males were presented with two frequencies simultaneously. Simultaneous presentat ion was used as this was considered the most valid way of determining the relative attractiveness of two frequencies. The suction apparatus described earlier for quantifying male response was modified by replacing the single line from the suction fan with

two lines. Each tube had a collecting chamber and a point source speaker connected to separate sine wave generators. The speakers were situated at the open end of each of the tubes which were inserted into opposite sides of the test cage, so they were approximately 12 cm apart. The onset and 5 second duration of the signal from each sine wave generator was controlled by the same automatic timing device, hence the two signals were presented simultaneously. One generator produced 475 Hz and the other 525 Hz, both at sound pressure level of 55 db at 2.54 cm.

The experiment consisted of 10 cages of virgin and 10 cages of mated males, with 25 mal-es per cage. Each cage was tested ten times. The rearing and test temperature was 26 C. The cages were agitated irnrnedlately prior to sound presentation to induce flight.

The effect of mating on the activity of males was examined by placing virgin and recently mated (within 5 minutes) males singly in glass chambers and observing the number and duration of flights. A one hour observation period for each male was used, and a stop watch was employed to obtain flight data. Test temperature was approximately 27 C, and virgin and mated males were tested at the same time so both were exposed to similar environmental fluctuations. The activity of 10 virgin and 10 mates males was monitored.

Feeding: Thirty virgin females, 3 to 4 days old, were divided into three equal groups. One group was maintained on water alone for the three day experimental period, a second group was given sugar on the second day, and the third group was allowed a blood- meal on the second day. The wing-beat frequency of each female was determined, in free-flight, on day one (3 to 4 days after emergence), day two before feeding and as soon after feeding as flight could be induced by tapping the recording chamber (1 to

4 hours), and day three 24 hours after feeding. Another group of 10 females was loaded (weighted) externally by glueing, with Dow silicone sealant, a 1.5 rnm length of nylon fishing line (~upont8 lb test) to the ventral abdomen (~igure Figure 10: -A. aegyyptti female weighted with nylon line glued to the ventral abdominal surface.

10). The flight-tone was recorded before and after loading to

determine any effect on w-ing-beat frequency.

The weight of the 1.5 mm length of line was found to be about 1 mg. Weight gained through feeding was determined by weighing females before and after the meal.

Oviposit ion: The effect of oviposition on the female wing-beat frequency was determined by measuring it before and after egg laying. Two groups of ten mated females each were used. One group was allowed to feed without disturbance on human blood while the other group was not blood fed. The wing-beat frequency of both

groups was analyzed daily commencing the day before each blood

fed female had oviposited. The laboratory was maintained at about 27 C and 45 - 55% K H. Periodicity of Male Response: The effect of time of day on male acoustic behavior was

studied by presenting males with sounds at 4 hour intervals,

commencing at 2 A M, over a 24 hour period and observing response.

Frequencies of 450, 500, and 550 Hz were presented at each of the six test times. The males were 4 day old virgins. The tempera-

ture was 28 C and light intensity was maintained at 700 lux at the top of the test cage throughout the experiment. Ten cages of 20 males were used, and each was exposed to each frequency at each time. Cages were agitated before each test to induce flight so acoustic response rather than flight activity was

being measured. Resu1.t~and Discussion Age:

Changes in the wing-beat frequency with age of -A. aegypti females were studied by Tischner and Schief (1954), while Wishar t and Yiordan (1959) examined changes in the response of' males of different ages to sound. Wing-beat frequency was re- examined in the present study using mosquitoes in free flight, a more accurate technique than the analysis of tethered females done by Tischner and Schief, Also, the wing-beat frequencies observed during the present study were higher than those measured in the earlier study. Male response was not studied because the technique used to examine this factor was modelled after that used by Wishart and Riordan and the optimally attractive frequencies they rzported are similar to those I determined in

Part 1.

Figure 11 illustrates the change in wing-beat frequency of -A. aegypti females for five days from adult emergence.

This curve is very similar to that found for males and females of this species by Tischner and Schief (1954). However the frequencies they observed were somewhat lower which may have resulted from lower rearing and ambient test temperatures. It is unlikely to be the result of tethering, as this raises the rate of wing-beat (page 15).

A similar increase in wing-beat frequency in the first days after adult emergence has been observed in cockroaches (~arnworth,

1972 b) and Drosophila hadwi wick, 1953) . Levenbook and Williams Figure 11: Mean wing-beat frequency of 13 A. aegypti females during 5 days after adult emergTnce. 24 48 72 96 120 HOURS AFTER ADULT EMERGENCE ( 1955) found that the wing-bzat frequmcy and the rnitochondr-la1 cytochrome c of the flight muscles of the blowfly Phorr~liaregina showed a corresponding increase during the first Tour days after adult emergence from the pupal state, and suggested these factors were correlated. Rowley and Graham (1968) related the effect of glycogen utili-zation with age to the flight performance of' -A. aegypti females, but this study examined flight activity and endurance rather than wing-bsat frequency.

Changes in male acoustic behavior with age have also been reported. Roth (1948) found males began to respond to tuning forks 15 hours after emergmce but responded to an increasingly greater range of frequencies for at least the first fifty hows after emergence. Wishart and Riordan ( 1959) showed -A. aspapti males responded optimally to increasingly higher frequencies for up to 4 or 5 days after emergence. Roth related the change in response with age to the increasing extension of the antenna1 fibrillae for the first 48 hours after emergence. The change in acoustic behavior with age is part of a general sexual maturation of -A. aegypti adults. Neither sex can successfully mate for the first one or two days after emergence from the pupal state. In newly emerged males the genitalia are essentially upside-down and must rotate 180 degrees before copulation can occur ( ~lements,1963). This usu.ally takes about

15 to 20 hours, but males do not usually copulate for another 4 or 5 hours (Roth, 1948). It has recently been determined that although -A. aegypti females will mate soon after emergence no sperm are transferred until the female is 1 to 2 days old. This post-emergence ref'raciory period is under hormonal control and is manifested by the female actively preventing firm genital union by retraction of its genitalia (~wadzand Craig, 1968; Gwadz --et al., 1971; Lea, 1968; Spielman --et al., 1969). By the time adults have achieved a physiological mating state, acoustic behavior has developed to unite the sexes. The female wing-beat frequency has increased to the lower part of the range of sounds attractive to males which are showing optimal response to frequencies lower than those they will respond to later. Male and f erna1.e acoustic behavior remains synchronous as they age, with the female wing-beat frequency and the frequency to which most vales respond increasing and then reaching a plateau. Mating:

The data in Table XI11 indicate that mating doe's not affect the rate of female wing-beat (t-test, p > .05). Daily fluctuations occurred in both mated and virgin (control) females, and, as they were similar in size and direction, were probably the result of small temperature differences. Although mating had no influence on female acoustic behavior, rated and virgin males showed differences in acoustic behavior and flight activity (~ablesXIV and XV).

The increased flight activity of males after mating (t-test, p < .05) is in agreement with the results of Nijhout and Craig (1972), although they measured the flight duration of males Effect of mating on wing-beat frequency of A. aegypti fzmales. Data are the mean (5 S. E.) for lg females.

DAYS AFTER WING- BEAT FREQUENCY ( HZ) EMmGENCE GROUP A GROUPB

512 * 9 MATING 510 * 7

GROUP A: VIRGIN THROUGHOUT

GROUP B: MATED ON DAY 5 TABLE XI:V

Percentage of virgin and mated males responding to frequzncizs of 475 and 525 Hz. Data are the $ & S. E. of 25 replicates of 100 males each.

$ MALES RESPONDING

TOTAL RATIO 475: 525 VIRGIN 1.5:1

).rATED 3:

" Tests conducted within 5 minutes of mating. TABLE XV

Flight activity of virgin and mated males over one hour. Data are mean + S. E. for 10 males.

# of Duration Total flights of flights (sees) flight-tirnz

VIRGIN

MATED induced to fly by cage agitation while the present study was concerned with spontaneous flight activity. Nijhout and Craig attribute the increased activity to a non-volatil e sex phzromone that males receive on contact with females, and they suggest; this improves the chance for a male to find a second fema,le after one successful mating.

The fact that, as shown in Table XIV, mated males have a decreased acoustic response (t-test, p < .05) indicates a male having just mated is unlilcely to be again attracted to the female he separated from. The increased activity would then serve to move the mated male to a different area where other, possibly unmated, females would be encountered. This prevents wasteful expenditure of energy in pursuing a mated female which in -A. aegypti can only be successfully inseminated once (~raig,1967). The greater acoustic responsiveness of virgin males suggests that a female is more likely to be mated by a virgin male than one that had previously mated, resulting in greater genetic heteroxygosity in the gene pool of the population. Although the degree of responsiveness was affected by mating, the proportion of males attracted by frequencies was unchanged. For both virgin and mates males, 475 Hz was more attractive than 525 Hz. As shown in Table XIV, the ratio of males attracted by 475 Hz to those attracted by 525 Hz was very similar for mated and unmated males, 1.6: 1 for the former and 1.5: 1 for the latter. Therefore, although less responsive, males remain tuned to the female wing- beat frequency which is unaf'f ect~dby mating. Feeding:

Partaking of a blood or sugar meal results in a small but

definite increase in the female wing-beat frequency. The data

in Table XVI-A, which represents the mean wing-beat frequencies

of groups of 10 females, indicates that althou-gh the mean wing-

beat frequency increased, it was not a statistically significant

change (t-test, p > .05). However, as shown in Table XVI-B, when the wing-beat frequency of individual females within each

group is considered it becomes apparent and statistically demonstrable (binomial expansion, p < .05) that a significant number of females had an increased rate of wing-beat after

feeding. Nuttal and Shipley (1902) reported that feeding increased the rate of wing-beat of both male and female mosquitoes. However, they did not determine the frequency before and after feeding but rather categorized females according to distension

of the abdomen by food and then compared the flight-tone. This approach assumes all the females tested had the same wing-beat

frequency prior to feeding, but my experience suggests this is an invalid assumption. Hinds (~oth,1948, p. 285) observed that partly or fully fed -A. aegypti females seemed more attractive to males than unfed females. However Roth found that

11 starved females were no less attractive, durTng flight, than females the abdomens of which were distended with honey solution;

that individuals the abdomens still distended with blood, the TABLE XVI

Effect of feeding on wing-beat frequency of -A. aegypti females. A: MEAN WING-BEAT FREQUENCY f S. E. OF GROUPS OF 10 FEMALES

MEAN WING- HEAT FREQUENCY ( HZ) DAY 1 DAY 2 TREATMENT DAY 3 DAY 4

GROUP A 493 ?C 10 492 + 11 Nil 490 i 11 492 * 12

GROUP 13 477 & 11 477 i 11 Sugar 488 i 11 480 * 10

GROUP C 483 * 8 481 & 8 Blood 487 *8 484 *9

B. NO. OF INDIVIDUAL FEYILES SHOWING POSITIVE (+), NEGA FIVE ( - ) , or NO ( 0) CHANGE IN WING-BEAT FREQUENCY AFTER FEEDING. DIRECTION OF W. B. FREQUENCY CHANGE IN 10 FEMALES DAY 1 -+ DRY 2 DAY 2 + BAY 3 DAY 3 + UAY 4

GROUP A: 2 2 6 1 2 7 2 2 6

GROUP B: 2 2 6 ;c 8 1 1 2 7 1

GROUP C: 1 2 7 4t-x 8 2 2 7 1

3"5 BLOOD MEAI, meal having bezn talcen after 12 hours prior to the females being exposed to males, also were no more attractive than

starved females". He concluded that Hinds was probably observing a difference in attraction due to a difference in age of the females. The present study supports Rothts conclu-

sions that changes in wing-beat frequency resulting from feeding are too small to affect its attractiveness. Weighting or loading females with lengths of nylon thread weighing approximately 1 mg caused a significant increase ( t-test, p < -05) in the wing-beat frequency. The mean ( * S. E. ) rate of wing-beat of 10 females changed from 537 i 10 to 564 i 10 Hz with loading, while that of control females changed insignifi-

cantly from 533 k 9 to 536 5 9 Hz (t-test, p > .05).

It is difficult to understand why an external load of 1 mg causes a much greater increase in wing-beat frequency than that resulting from a blood meal, found to average 0.75 mg. Perhaps the external load causes an unnatural flight posture which the more evenly distributed internal. load does not produce. The abdomen of the loaded females seemed to 11 droop" slightly during flight while this was not observed in the fed mosquitoes.

This study suggests feeding is unlikely to affect acoustic behavior of -A. aegypti adults, as feeding induced changes in wing-beat frequency are small. Even if large changes were caused, it would very likely be of little significance in the mating of the species. Newly fed individuals are usually inactive and do not resume flight until much of the meal has been excreted, which occurs in a relatively short time. Evidence indicates tha-t mosquitoes may excrete clear fluids equal to nearly onz-half the weight of the blood meal in the first two hours after feeding (~oorman,1960). The relatively rapid return of the wing- beat frequency to the pre-fesding level is shown in the data in

Table XVI, A' and B. Hence, females may be flying within hours of feeding with undiminished attractiveness to males. Oviposition:

The data in Table XVII, A and B, indicates oviposition has a slight effect on the female wing-beat frequency. The mean wing-beat frequency of 10 females decreased from 527 Hz before to 522 Hz after oviposition in the Group B females while increasing from 530 to 531 Hz in the non-ovipositing Group A females. The differences in the means are not statistically different (t-test, p > .05), but when the change in direction of the flight-tone is considered it becomes apparent that oviposition had a significant effect on the wing-beat frequency (binomial expansion, p 4 .05). This effect may be attributable to a decrease in the load carried by the female after oviposition, the inverse of the small increase in frequency caused by an increase in weight through feeding. As might be expected, the change in frequency does not reverse after oviposition as it does one day after feeding. This lowering in frequency by release of eggs is undoubtedly too small to influence the attractiveness of the female flight-tone. TABLE XVII

Effect of oviposition on wing-beat frequency of -A, azgypti females

A: MEPAN WING-BUT FREQUENCY k S. E. OF GROUPS OF 10 PEMAT,ES

WING-BEAT FREQ.UENCY ( HZ)

DAY 1 DAY 2 DAY 3 DAY 4 NO GROUP A: 531 k 8 530 k 8 OVIPOSITION 531 k 9 528 + 9

GROUP B: 526 & 8 527 k 7 OVIPOSITION 522 & 7 523 * 6

B: NO. OF INDIVIDUAL FENALES SHOWING POSITIVE (+) , NEGATIVE ( - ) , OR NO ( 0) CHANGE IN WING- BEAT FREQUENCY WITH OVIPOSITION.

DIRECTION OF W. B. FREQUENCY CHANGE ( 10 FEMALES)

DAY 1 -I, DAY 2 DAY 2 + DAY 3 DAY 3 + DAY 4

4- - 0 + - 0 + - 0 ------GROUP A: 1 3 6 1 9 2 8

GROUP B: 2 17 1 7 2 1 2 7

GROUP A: NO OVIPOSITION

GROUP B: OVIPOSITION ON DAY 2 + DAY 3 Periodicity of Nale R~spons~ The time of day had no effect (t-test, p > .05) on male acoustic response when illumination was maintained at a constant

intensity over the 24 hour test period (~igure12). The per-

centage of males responding to all frequencies, and the relative attractiveness of zach frequency, remained constant over a 24 hour period. Although this experiment demonstrated thatacoustic behavior is not periodic, other aspects of mosquito behavior have been

found to be influenced by the time of day. There is evidence that swarming, biting, and oviposition are controlled by diurnal rhythms (~lements,1963). Figure 12: Mean % of A. aegypti males responding to different frequencieT at one hour intervals over a 24 hour period. Data is mean ( + S. E. ) of 10 replicates of 20 males. MEAN RESPONSE TO ALL 3 FREQUENCIES

RESPONSE TO 450 HZ.

0 RESPONSE TO 500 HZ.

RESPONSE TO 550 HZ.

2 AM 6 AM 10AM 2PM 6 PM 10 PM TIME OF DAY SUi\%@JlY AND CONCLUSIONS

This final scction of the thesis is devoted to a summation

of the experimental results and the mechanisms thought to be involved in acoustic behavior, as indicated by these resu.lts. The potential of usi-ng sound as a means of mosqulto control, and the relation of this study to other aspects of mosquito biology are also considered. (a) Factors effecting acoustic behavior: Temperature is the environmental component having the greatest influence on mosquito behavior. Larval rearing temperature effects the acoustic behavior of both sexes, increasing rearing temperature producing females with progressively higher wing- beat frequencies and males that respond optimally to higher frequencies. Ambient temperature has a similar effect on adults.

As the temp'erature rises the female wing-beat frequency increases and males are attracted optimally to higher frequencies. Hence, as both sexes react similarly, acoustic synchrony between males and females is maintalned with both different larval rearing and adult ambient temperatures. This was confirmed by mating aclul'ts from various rearing temperatures. The sound pressure level of the female flight-tone was 1argel.y unaffected by changes in ambient temperature whereas females reared at different temperatures showed a consistent decrease in flight-tone sound pressure level with increasing temperature (3 db for lo0 c). Thz effect of relative humidity was dependent on ambient

temperature. At low temperatures, relative humidity had no

effect on the female wing-beat frequency, but at higher tempera-

tures there was significant increase in rate of' wingbcat wit11

rising relative humidity.

The female wing-beat frequency was unaffected by changes

in atmospheric pressure or short term changes in light intensity.

Of physiological factors examined, age had the most marked effect on the female 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, while not influencing the female wing-beat frequency, affected 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. On the other hand, oviposition caused a lowering in frequency. Male acoustic behavior did not appear to be under the

influence of a diurnal rhythm. It was apparent that, despite having similar rearing conditions, physiological states and ambient conditions at analysis, mosquitoes showed variation in acoustic behavior. This was particularly evident for females, as individuals reared and tested

together could have wing-beat frequencies differing by 25 Hz

and, in a few instances, up to 50 Hz. It is probable that such

differences relate to variation in the size of the insects resulting from gm~ticdifferences. Although the genetic factors involved in growth regulation are complex and poorly understood, this seems th: most plausible explanadtion for differences in acoustic behavior when other factors are constant.

Aedes aegypti, the spccies used in this study, does not occur naturally in Canada. Canadian mosquitoes, unlike -A. aegypti, tend to be active only during a certain part of the day or night, usually at dusk or dawn. In males of most such species, the antenna1 fibrillae rernain recumbent most of the day and are erected only during the periods of activity. The fibrillae of -A. aegypti mal2s, on the other hand, remain permanently erected (~oth,1948; Nielson and Rielson, 1962). This indicates that environmental or endogenous factors, perhaps rhythmic, influence the acoustic behavior of Canadian species and this is an area requiring further research if sound is to be considered as a means of mosquito control. Other aspects of the biology of -A. aegypti, while often d-iffering in detail, are sufficiently similar to Canadian species to draw fairly general conclusions. (b) Mechanism of changes in acoustic behavior: The effect of rearing temperature on the female wing-beat frequency is almost certainly a result temperature induced size variation. Manipulation of food ration and larval density had a similar but smaller effect on size and wing-beat frequency.

The decreasing rate of wing-beat with increasing size is probably caused by the greater wing mass of the large mosquitoes. The variation in acoustic response of males reared at different temperatures may be the result of' temperature induced size differences. Lower rearing temperatures produced bigger males with correspondingly larger antennal fibrils that require slightly greab~renergy to vibrate. This may account for the lower level of acoustic responsiveness of large males relative to smaller ones, but is not an entirely satisfactory explanation as the larger fibrils present a greater 11 collecting" area and therefore should cause increased sensitivity to sound. Further information on the role of antenna1 size on male response could be obtained by fabricating antennae-like structures of different sizes and observing their acou.s,tic resonance characteristics. The basis of the shift in acoustic response of males of decreasing size to hlgher frequ-encies is uncertain but may be explained in physical terms such as the size of the Johnstonsf organ. The Q1, values for the change in female wing-beat frequency with ambient temperature ranged from 1.10. to 1.15, indicating the phenomenon has a physical rather than bio-chemical basis. Modification by temperature of muscle tissue characteristics such as sarcoplasmic viscosity, myofilament length, or membrane conductivity, or temperature induced changes in cuticular flexibility or the elasticity of resilin may be involved. The changing response of males with ambient temperature probably results from physical alteration of the antennae. Tempera- ture may affect the rigidity of' the articulation of the fibrils with the antenna1 shaft, and of segment 3 with the Johnstonst organ, thereby influencing the vibration of these structures. The interaction of' rel-ative humidity and high temperature to modify the female wing-beat frequency appears to be a result of evaporative cooling changing the insects intemal temperature. Molecular changes in epicuticular wax at high temperatures permits water loss at a rate directly related to ambient relative humidity, so at low relative humidities there is greater water lost than at high ones. Consequently the greater internal cooling causes the wing-hea,t frequency to be less at low rela- t ive humidity.

The basis of the increase in female wing-beat frequency during the first days after adult emergence is 1argel.y undetermined, but it is probably related to an increase in mitochondria1 cytochrome c of the flight muscles which in other Diptera parallels the change in rate of wing-beat with time.

The decreased acoustic response and increased flight activity of males subsequent to mating is probably owing to a pheromone received on contact with females during copulation (~ijhout and Craig, 1972). This is almost certainly the cause of the rise in flight activit,~,and at present is the only explanation for the decline in response to sound. Modifications of wing-beat frequency caused by feeding and oviposition appear to be the result of changes in the load carried by the flying insect. Blood and sugar meals caused a slight and transient increase in wing- beat frequency . Wing- beat frequency was also increased by externally attached loads. Oviposition, on the other hand, brought about a slight but long term decrease. This indicates that the wing-beat frequency changes in order to compensate for changes in load, allowing a more-or-less constant flight speed to bz maintained.

Natural changes in speed and direction of flying mosquitoes, although not fully understood, apparently do not involve changes in the frequency of the wing-beat. Bsssler (1958) found that flight speed of -A. aegypti females is regulated in part by changes in the amplitude of the wing-beat. Gillett (1971), in a review of mosquito flight, concluded that the speed and direction of flight is controlled by alterations in the wing-angle brought about by direct flight muscles.

(c) Potential of sound in mosquito control: The obvious utilization of sound for controlling mosquitoes is to attract males to a trap or killing device, thereby depleting breeding stock. This approach has been attempted, with only limited success, by Kahn and Offenhauser (1949 a) who played disc recordings of the flight sounds of female Anopheles albimanus to lure males to an electric grid. However in attempting to "call" males from a distance they found the sound intensity rendered it repellent to the approaching mosquito. This was confirmed in the laboratory by Wishart and Riordan (l959), who then recommended, but did not field test, traps emitting a sequence of sounds of gradually decreasing intensities to attract mosquitoes from a distance without repelling them on approach. Another technique to be considered is the use of large numbers of traps emitting an attractive sound of constant but unrepellent intensity, effective over short distances. If placed strategically and timed propsrly, a series of such traps could conceivably have an appreciable diminishing effect on the numher of males in a population. Males generally emerge before females and, hence, sound traps placed near mosquito breeding sites could reduce mating and thereby lower subsequent population levels.

Traps could also be located in proximity to previously identified swarming sites, thereby providing large concentrations of males in flight. Traps could be made relatively inexpensively and, if portable, could be moved from area to area. It is clear from the work of Roth (1948) and Kahn and Offenhauser (1949 a) that resting males cannot be attracted to sound, and thus the traps need only be operated at those times when rcosquitoes are most active, usually dusk and dawn. The development of a simple and inexpensive sound attractant trap, particularly for use in relatively small and isolated areas, should be investigated further.

The results of this study indicate that a reduction in mating could be brought about by manipulating acoustic behavior. Al- though various environmental factors, particularly temperature, were found to influence acoustic behavior, it is difficult to envisage how these could be applie~to control field populations. Perhaps rather than attempting to manipulate acoustic behavior through modifications of environmental components, the mechanism of environmentally induced alterations should be investigated fully and, if possible, exploited through the development of chemicals that duplicate the effect. As the basic mechanisms of female acoustic behavior is better understood than that of males, substances designed to act on the former could be more readily developed. Juvenile hormones may be worth exploring as they have been Sound to induce flight-muscle degeneration in the bark beetles -Ips confusus (Dorden and Slater, 1968) and are suspected to act similarly on other insects (~ohnson,1957;

Stewee --et al., 1963). Perhaps a similar result could be achieved in mosquitoes, and, if so, minimal levels, important for economic and environniental reasons, could be applied to induce only partial degeneration and hence render the female flight-tone less attractive. Chemicals affecting mitochondria might be similarly employed, particularly if they could be made selective. The flight mechanics of insects involves flexing of the thoracic skeleton by indirect flight muscles. Wing-beat frequency would therefore be affected by changes in the elasticity of the exoskeleton, and perhaps substances can be identified that either soften or harden this structure, perhaps by acting on the peptide bonds of resilin, a protein known to be one of the elements imparting elasticity to the exoskeleton. Chemicals that effect the cuticle in this way could also alter male response to sound by influencing the articulation or the antenna1 fibrils and shaft thereby altering its resonant properties. Such chemicals as discussed above would have the advantage of affecting only insects or, at worst, invertebrates. Information gained in this study may be of value in other areas of mosquito control. For example, the influence of larval rearing temperature on adult acoustic behavior indicates this is of prime importance in the release of laboratory reared populations to mate with wild populations. Hence, in rearing mosquitoes for a sterile male release programme it would be

desirable to have the released males and wild females as close to acoustic synchrony as possible. If synchrony is not achieved, wild females would be more attractive to wild than released rfiales, resulting in a less effective programme as it relies on the females being mated by released sterile males before wild fertile males. As adu1.t acoustic behavior is influenced by the larval environment, laboratory populations should be reared under con-

ditions similar to wild populations. However, the difficulties in determining and duplicating the temperatures and. nutritional and density levels of wild populations are obvious, and an alter- native would be desirable. Considering the relationship of size to acoustic behavior, a relatively easy yet accurate method of duplicating the acoustic characteristics of a wild population would be to determine the

size of the wild adults, and then manipulate the rearing conditions of the laboratory population to attain adults of the same size. This study has demonstrated that the larval rearing

I temperature has a greater influence on adult size than does either I I larval food ration or density. In additton, t'empzrature would be easier to manipulat,e under mass rearing conditions than either larval food ration or density. Adult size variation over a range of rearing temperatures could be determined so that when adults of a particular size were desired, that specific temperature could be used.

The role of sound In mosquito mating is important and, with further study may be another weapon that could be used by man tc control mosquitoes.

( d) Other considerations: Clements (1963) discussed the role of acoustic behavior as a species isolating mechanism and concluded that 1 I the female flight-tones of different species of mosquitoes show insufficient diversity to act as an isolating mechanism". The results of this study support Clements, as males were found to respond, to some degree, to a wide range of frequencies which could in many cases include the wing-beat frequency of females of other species. Where the distribution of species with a similar flight-tone overlaps, it is probable that there are additional isolating mechanisms to prevent mating. One of these could be the use of distinct swarm sites where males of many dipterous insects assemble (~ownes,1958). The function of swarming of mosquitoes is poorly understood but it is generally agreed that swarms consist solely of males. In some species females have been observed entering swarms, but in others females have seldom been observed. Does a swarm of males serve a reproductive function by attracting females in some manner? If there is an attractive mechanism, it is improbable that sound is involved. Atternpts were made during the present study to attract -A. aegypti females to sounds of different frequencies and intensities, but no apparent response

could be elicited. It seems, therefore, that if swarming males

do in some way attract females, sound is not involved and that

other possi-ble mechanisms such as long distance pheromones should be considered and examined.

Other insects besides mosquitoes appear to use sound in the

same way as that described for -A. aegypti (Culicidae). These in- elude biting midges (~erato~onidae), swarming midges (Chirono- midae) and probably also gall midges (Cecidomyidae) where the

flight sound of the sexes differs as it does in mosquitoes, and

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