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The electroretinogram and spectral sensitivity of the compound eye of Aedes aegypti

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Title:
The electroretinogram and spectral sensitivity of the compound eye of Aedes aegypti
Creator:
Goldman, Leonard J., 1943-
Publication Date:
Language:
English
Physical Description:
76 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Compound eyes ( jstor )
Dark adaptation ( jstor )
Eyes ( jstor )
Insects ( jstor )
Pigments ( jstor )
Receptors ( jstor )
Retinal pigments ( jstor )
Sand sheets ( jstor )
Spectral sensitivity ( jstor )
Wavelengths ( jstor )
Aedes aegypti ( lcsh )
Compound eye ( lcsh )
Dissertations, Academic -- Entomology and Nematology -- UF
Electrophysiology ( lcsh )
Entomology and Nematology thesis Ph. D
Insects -- Physiology ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1971.
Bibliography:
Includes bibliographical references (leaves 72-75).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Leonard J. Goldman.

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University of Florida
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Th-e Fc tr r t i nogran and Spectral Sens it vi tyy of the Compound Eye of Aedes aeyti












By

LEO!ARD J. GOLOMAN











A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE- REUI REMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY









UNIVERSiTY OF FLORIDA 1971















ACKNOWLEDGEMENTS


I wish to thank Dr. H. Cromroy for his support, patience, and guidance without which this research would not have been possible. I am also indebted to Dr. M. S. Meyer for the use of his equipment and space and also for his advice and suggestions throughout the course of this work. I also wish to thank Dr. W. Dawson for his invaluable help and suggestions.

During the course of this work I received support from an Office of Civil Defense Grant - DAHC-20-69-C-0291, Modification P648-1.
































ii















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . .. . . . . ii

LIST OF FIGURES ................... . . . . . iv

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . v

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . I

LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . 3

MATERIALS AND METHODS . . ....... ....... . . . 14

Insects and Equipment . ....... ......... . 14

Experiments . . . . . .. . . . . . . . . . . . . . . .. . . 25

RESULTS AND DISCUSSION ... ... . .......... . . . 33

CONCLUSIONS . .......................... . . . . . . . 70

LITERATURE CITED .................. . . . . . . 72

BIOGRAPHICAL SKETCH ... .. ...... ...... .. . 76














LIST OF FIGURES


Figure Page

i. Schematic diagram of an ommatidium of
A. egypti . . . . . . . . . . . . . . . . . . .. . . 6

2. Schematic diagram of the equipment used for recording the ERG of the mosquito . . . .. ... 19

3. Equipment used for recording the ERG of the mosquito . . . . . . . . . . . . . . . . . .. . . 21

4. A typical recording of an ERG from the dark adapted eye of A. aegypti . . . . . . . . . . . 35

5. Time-course of dark adaptation following exposure to room lighting. .. .... ..... . 38

6. Energy response curves based on ERG's recorded for the saturation level
experiment ......... . . . . . . . . . . 41

7. Time course of dark adaptation following five minutes of li ht adaptation with
2.2 x 106 ergs cm- sec-1 of white light . ..... 49 8. Light adaptation . .. . . ........ ... . 52

9. Spectral sensitivity curve for the visible region of the spectrum . . . . .... . . . . . 55

10. Spectral sensitivity curve for the UV
region of the spectrum ... . . . . .......... 58

11. The effect of selective adaptation on
the spectral sensitivity curve. . . . . . . .... 61

12. Influence of long wavelength adapting
light on the a.iplitude of the response to
a constant energy UV stimulus . .. . . . . . . . . 64







iv








Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

THE ELECTRORETINOGRAM AND SPECTRAL SENSITIVITY
OF THE COMPOUND EYE OF Aedes aegypti By

Leonard J. Goldman

August, 1971

Chairman: Dr. Harvey L. Cromroy
Major Department: Entomology and Nematology

Electrophysiological techniques were used to study the physiology of the compound eye of the mosquito, Aedes aegypti. The electroretinogram (ERG) was a monophasic negative potential wave. There was no indication that wavelength information was encoded in the ERG waveform. The time course of dark adaptation was determined for both dark-eye and white-eye mosquitoes. Following prolonged exposure to room lights the mosquitoes required 30-40 minutes to dark adapt. The time course of light adaptation was studied using three intensities of adapting light. Light adaptation was completed within approximately 50 sec. The method of equal response was used to determine spectral sensitivity of the eye. The eye was maximally sensitive to 540 nm light with secondary peaks of sensitivity at 360 nm and 330 nm in the UV region of the spectrum. The results of selective adaptation experiments indicated that vision in A. aegypti was mediated by a single visual pigment. The UV sensitivity was attributed to cis band absorption by the visiial pigment and UV absorption by a UV excited, fluorescent material in the eye.






v















INTRODUCTION


In Christophers' (1960) book on Aedes aypti L. only three

pages are devoted to "Reaction to Light and Visual Response." Six pages are devoted to a description of the structure of the compound eye. If Christophers' book were to be published today, the length of these sections would not change perceptibly.

Aedes aegypti, which is generally known as a daytime biter

(Chr.istophers, 1960), is most often found in shaded areas or resting on dark surfaces. It is apparent that visual cues, photoperiod and ambient light levels play an important part in this mosquito's behavior. In addition, much of our knowledge about the numbers and distribution of various mosquito species is based on light-trap studies. With light playing such an important role in mosquito research, it is surprising that there has been virtually no work done on the physiology of the compound eyes of mosquitoes. Behaviora! studies have been done on the response of mosquitoes to various colors but these are largely uninterpretable in terms of the physiology of the eye.

In recent years electrophysiological techniques and equipment have been developed which make it possible to directly measure the responses of the eye. These techniques have been successfully applied in studies on the eyes of many insects. Extensive electrophysiological studies have not been performed on mosquito eyes. The results of the little electrophysiology that has been done on mosquito eyes

I







2

do not significantly contribute to an understanding of the physiology of mosquito vision.

The goal of this research was to investigate the physiology of the compound eye of Aedes aeg i using electrophysiological techniques. The electroretinogram (ERG), dark and light adaptation, and the spectral sensitivity of the eye were studied.















LITERATURE REVIEW


Several behavioral experiments designed to test the influence of colored surfaces and light on the attraction of Aedes apgypti L. have been reported. The results of these early experiments have not been consistent.

Brett (19338) tested the attractiveness of different colored cloths to Aedes aeqyjpt. Each color was tested against either a black or white standard. The order of attractiveness, when tested

against black, was: black> red >brown>white >blue> green> yellow. When tested against white the order was: red> black >brown> green > blue , yel low> whi te.

In another study Gilbert and Gouck (1957) studied the influence of surface color on the landing rates of caged Aedes aegypti mosquitoes on colored filter paper disks. A count of the number of

mosquitoes resting on the disk was made thirty seconds after they had been disturbed. The amount of light reflected was measured with an exposure meter. When disks reflecting 40 foot-candles were tested the color preference was as follows: yellow >orange, red >green, violet, black, blue, white. With disks reflecting 20 foot-candles the results wv2re as follows: orange, red> violet, black, green, blue> white. Yellow was not included in the 20 foot-candle tests.

These experiments cannot be interpreted in terms of the spectral

sensitivity of the mosquito eye since the spectral quality and insity df the various colors were not reported.

3








In another study Ludwig (1954) determined the influence of

colored light on the attraction of Aedes aegypti to a hand. He used broad-band colored glass filters within the spectral range of 365950 nm. Tests were conducted at three light intensities. Ludwig obtained maximum attraction in the ultraviolet (UV) region and in the visible region between 515 and 605 nm.

Interpretation of the results obtained by Ludwig (1954) are

complicated by the broad-band nature of the stimuli and the lack of any data on the energy of the stimuli. In any case, behavioral tests alone are not adequate for determining the spectral sensitivity of the eye. Only by knowing the spectral sensitivity beforehand could it be proven that the result of a behavioral test is a true measure of the spectral sensitivity of the insect eye.

The structure of the compound eye of Aedes aegypti has been described in detail by Brammer (1970) (Figure I). The dioptric apparatus is composed of a corneal layer, a corneal lens and four cone cells. The cone cells give rise to six cytoplasmic extensions which pass proximally into the receptor region of the ommatidium. Below the region of the rhabdoimi these cytoplasmic extensions enlarge and form pigment filled sacs. Surrounding the cone cells are two primary pignent cells. The cell bodies of an undetermined number of secondary pigr;ent cells surround the primary pigment cells. These secondary cells have elongated portions which pass proximally between

adjacent ommatidia to the basement membrane.

Immediately proximal to the cone cells is the receptor region of the eye. The receptor layer is composed of eight heavily pigmented retinula cells. Six of these (designated RI-R6) are situated

































Figure 1. Schematic diagram of an ommatidium of A. aegypti. L
corneal lens, C - cone cells, CE - cytoplasmic extension of cone cells, S - pigment filled sacs at the end of the
cytoplasmic extensions of the cone cells, R - retinula
cells R1-R6, R - peripherally located retinula cell,
R8 - centrally located retinula cell.
























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* S















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\\

__________________ S K'


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- U







7

peripherally around a central retinula cell (!R). The retinula cell designated R7 lies outside the ring formed by R -R The relative position of this cell remains constant over large portions of the eye. The rhabdomere of R7 is on a projection that extends into the

center of the ring for ed by R -R6. The rhabdomere microvilli of R7 seem to be stacked parallel to and directly above (distad) the microvilli of the R8 rhabdomere.

The organization of the mosquito ommatidium is similar to that

of Dipterans of the suborder Cyclorrapha, with the exception that the rhabdom is of the fused type while the Cyclorraphans have an open rhabdom; i.e., the rhabdomeres project into an open intracellular space.

Electrophysiological techniques have been used extensively in investigations on the functioning of the compound eyes of insects. There are several review articles covering this subject (Burkhardt, 1964; Goldsmith, 1964) and a book by Mazokhin-Porshnyakov (1969) which treats the entire subject of insect vision, There are no

references to experiments performed on mosquito eyes in any of these works. There are only two reports of the results of electrophysiological techniques applied to mosquito eyes. Brammer and White (1968) show the wave-Form of the electroretinogram (ERG) of Aedes aegypti while Yinon (1970) presents a recording of the ERG of Culex pipiens L. In both cases the mosquito ERG was a simple monophasic negative potential,

It would be valuable at this point to review some of the work

that has been done on other insects. This would provide a background against which the results obtained from experiments on Aedes aegypti can be compared.







8

The mechanisms underlying dark and light adaptation of compound eyes are poorly understood. The three main factors involved in adaptation are: a) resynthesis of visual pigment, b) recovery of active membranes, and c) changes in the position of the accessory pigments or in the photoreceptors themselves (Goldsmith, 1964).

The time course of dark adaptation in diurnal and nocturnal Lepidoptera was studied by Bernhard and Ottoson (1960a, 1960b). They measured the sensitivity changes associated with adaptation by determining the light energy necessary to elicit an ERG of a given magnitude. They found that in diurnal Lepidoptera the sensitivity of the eye increased rapidly during the first few minutes of dark adaptation and then leveled off. The eye was over 90% adapted after 10 minutes and completely adapted after 20 minutes. Dark adaptation in nocturnal species was a more complex phenomenon. The initial changes in sensitivity wvere simi lar to those found in the diurnal species. After several minutes, however, a second phase of adaptation appeared during which there was an additional increase in sensitivity which continued over a 20-30 minute period. Coincident with this second phase was the onset of migration of the accessory pigments in the eye. No pigment migration occurred in the eyes of the diurnal insects. These authors believed the rapid dark adaptation of diurnal Lepidoptera and the initial phases of adaption in nocturnal Lepidoptera, were the results of resynthesis of the visual

pi gments.

Goldsmith (1963) studied the course of dark and light adaptation in the worker honeybee. Dark adaptation in this insect had the same time course as in the diurnal Lepidoptera. Initially, the rate







9

of dark adaptation was very rapid, the greatest increase in sensitivity occurring within the first few minutes of darkness. The eye was fully dark adapted within 10-15 minutes.

Dark adaptation in four species of insects was investigated by

Ruck (1958). In A isL m!j.ifera L., Pachy/~ljIax longpennis Burmeister, and Phormia rgina Meigen, dark adaptation was generally complete within 15 minutes. In Periolaneta americana L. adaptation required a longer but undetermined time.

In Calliphora erythrocephala Meigen both light and dark adaptation have been studied. Using intra-cellular recording techniques Washizu (1964) found that during light adaptation the sensitivity of the eye decreased to 20 percent of its dark adapted level within 13 seconds of the onset of illumination. withinn 50 seconds the sensitivity was down to 10 percent. Dark adaptation was achieved in 30-40 seconds.

Goldsmith (1963) subjected the dark adapted compound eyes of Apis melifera to a repetitive test flash and recorded the resultant ERG's. When an adapting light was turned on the base line shifted and the test flash response decreased in magnitude, Within a few seconds the eye had reached a steady level of adaptation. Subsequent presentations of the test flash superimposed upon the adapting light

elicited equal size responses.

In the moth Galleria mellonella L. light adaptation ran a faster time course than pigment migration and was usually complete within several seconds. Pigment migration, from the dark to the light adapted state, required up to 30 minutes to reach completion (Post and Goldsmith, 1965).







10

Light and dark adaptation is sometimes characterized by more than visual pigment regeneration and accessory pigment migration. In some insects, adaptation is accompanied by a change in the distance between the rhabdom and the proximal end of the crystalline cone.

Walcott (1969) described this phenomenon in the eye of Dytiscus marqinalis L. When the eye was light adapted the distance between the rhabdom and cone was as great as 120 urn. The crystalline tract that traverses the distance between the cone and the rhabdom was drawn out into a thin thread. During dark adaptation, the rhabdom moved distally to within 10 um of the cone. The crystalline tract became correspondingly short and thick. A similar phenomenon was found to take place in a giant water bug, Lethocerus americanus Leidy.

The movement of retinula cells is also known to occur in craneflies, mosquitoes (Culex and Anopholes), and the water boatmen (Notonecta). In these insects there is no known crystalline thread and the distance traversed by the retinula cells is on the order of several microns at most (Walcott, 1969; Mazokhin-Porshnyakov, 1969).

Electrophysiological studies on the spectral sensitivity of the compound eyes have been done on only a very small number of insects.

The spectral sensitivity of Apis mellifera has been studied by Goldsiith (1960). Using the method of equal response and selective

adaptation, he found that the compound eye of the worker bee had a two receptor visual system with the maximal sensitivity at 535 nm and a shoulder or minor peak of sensitivity at 345 nm. Using the method of equal response but not selective adaptation on the drone Goldsmith (1961) found that the location of the peak of the spectral sensitivity









curve did not remain constant. This was apparently caused by the contributions of several receptors. He concluded that there were

probably three receptors present with peaks at 535 nm, 440 nm, and 345 nm.

Both the dorsal and ventral eyes of the Whirligig beetle,

Dinfutes ciliatus Forsb. had peaks of sensitivity at 520 nm with a shoulder in the UV region (Bennett, 1967). Selective adaptation experiments indicated that a two receptor system was present. The fact that the energy-response curves for wavelengths in the UV and visible regions were not parallel supported the two receptor hypothesis,

The compound eyes of dragonflies have different spectral sensitivities in the dorsal and ventral regions. In Libellula luctuosa Burimerster, sensitivity to wavelengths shorter than 380 nm was common to all regions. In the visible region, however, the dorsal part of the eye had a second peak at 420 nm while the peak in the ventral part was at 550 nm (Ruck, 1965). In Anax junius Drury the dorsal region had 380 nm receptors while the ventral region had several receptors with peaks in the region of 420-520 umrn. The dorsal region of Libellula needhami Westfall was maximally sensitive to 410 nm while the ventral region had several receptors with peaks in the region of 450-550 nm (Horridge, 1969).

Bennett et al. (1967), working on the eye of Locusta migratoria L., used single cell recording techniques along with the method of equal response. All the cells from which they recorded had major peaks at 430 nm and a peak of variable height at 515 nm. In this region of the spectrum the degree of sensitivity ranged between 15% and I00/o of the sensitivity to 430 nm wavelength light.







12

Three species of backswimmer, Notonecta irrorata Uhl., N.

insulate Kirby, and N. undulata Say were all found to have three receptors maximally sensitive to 520-530 nm, 370 nm and 475 nm (Bennett and Ruck, 1970).

Thie ye oF the cockroach, P-rip.laneta aiericana L.,was investigated by Mote and Goldsmith (1970). They recorded from single retinula cells while presenting equal photon stimuli to the eye. Two receptor systems were found with maximal sensitivities at 365 nm and 500-510 nm.

The eye of Calliphora er3_ythrocha has been studied with intracellular techniques and equal quanta stimuli (Autrum and Burkhardt, 1961; Burkhardt, 1962). All the retinula cells in the dorsal part of the eye were found to be most sensitive to 1489 nm light with a second peak of sensitivity at 345 nm. The ventral part of the eye contained three types of retinula cells. Those were maximally sensitive at 463 nm, 491 nm and 524 nm. All had second peaks at 345 nm.

Spectral absorption of single rhabdomeres was measured with a microspectromneter by Langer and Thorell (1966). Fresh sections of the eye of the chalky mutant of Calliphora erythrocephla were mounted between quartz glass cover slips. The normal arrangement of the rhabdomeres was preserved enabling them to relate the absorption curves to specific cells. The six outer rhabdomeres had two absorption peaks, one at about 500 nm (main peak) and a second peak between 350 and 380 nm. In the central rhabdomere R 7, the main peak was about 460 nm, the second peak was between 350 and 380 nm. In all the rhabdomeres the main peak decreased as bleaching occurred while the UV peak increased in size relative to the main peak.






13

The literature presented in this section represents only a small portion of the research which has been done on insect eyes. Additional literature will be cited in the Results and Discussion section so that the results obtained from the experiments performed on the mosquito eye can be compared to those obtained in similar experiments performed on other insects.















MATERIALS AND METHODS


Insects and Equipment


The majority of the insects used in these experiments were fourday-old adult female mosquitoes, Aedes aeqypti. These were obtained daily from the colony maintained at the Insects Affecting Man and Animals Research Laboratory of the United States Department of Agriculture in Gainesville, Florida. The mosquitoes in this colony have deeply pigmented eyes. The adult female white-eye mutants of Aedes aegypti that were used in some experiments were obtained on an irregular basis from Dr. J. Seawright of the Insects Affecting Man and Animal Laboratory, Gainesville, Florida. Bhalla (1968) described the genetics and biochemistry which result in the whiteeye trait.

The colony was maintained at 28-300C and 60-70% relative humidity. The photoperiod was 8.5 hours light and 15.5 hours dark with the light period starting at 8:00 A.M. The mosquitoes were transported from the rearing room to the laboratory in a small holding cage made from a 20 cm long piece of 7.5 cm diameter plexiglass tubing with removable screening at the end. In the laboratory the mosquitoes were transferred to a larger holding cage.

To record the ERG complete immoblization of the mosquito was accomplished in the following manner: Microscope slides cut into

1.5 cm squares were coated with wax (Tempilstiks, 450C melting point), 14







15

which, after melting, would remain liquid at room temperatures until disturbed. Mosquitoes were chilled to facilitate handling and placed venter up on the slides. The proboscis was bent downward into the liquid wax to hold the head steady. The wax solidified on contact, holding the mosquito firmly in place. The mosquitoes would remain alive for many hours following this treatment.

In preliminary attempts to record the mosquito ERG, both glass capillary and chlorided silver wire electrodes were tried. All attempts with the glass electrodes were unsuccessful while the silver

wire electrodes gave consistently good results. Silver wires, 2.5 cm long, were electro-etched to a tip diameter of approximately 12 um. The electrolyte used for etching was a solution of 30% NaOH saturated with NaCN. The best etching was achieved when using a range of 2-8 volts A.C. The electrodes were chlorided in a 3M KCI solution just prior to being used. Fresh electrodes were used for each preparation.

The mosquito preparation was placed on a small platform located between two Leitz micromanipulators. The platform was an inverted plastic pill vial which had been cut to a height of about 2 cm. The cut was made at a slight angle so that, when in position on the platform, the mosquito preparation was tilted towards the stimulus source. The preparation was held onto the platform with a small piece of clay. A binocular microscope was suspended directly over the platform.

The mounted mosquito was aligned so that the ommatidia of the

central region of the eye were pointing directly at the stimulus source.

Hypodermic needles (20-gauge) mounted on banana plugs were used







16

to hold the electrodes. These plugs were fastened to short wooden dowels which were clamped securely in the instrument holders of the micromanipulators. Electrical connections between the electrodes and the amplifier were made through the banana plugs. The electrode placement was observed through the microscope. The ground electrode was inserted into the abdomen by forcing it through one of the intersegmental membranes of the venter. The recording electrode was centered in the compound eye facing the stimulus source. The eye deformed under pressure of the electrode making penetration of the corneal layer difficult. After penetration the electrode was withdrawn so that only the tip remained in the eye.

The electophysiological responses of the eye were amplified with a Bioelectric Instruments, Inc., NFI Neutralized Capacity Amplifier. Except where noted the amplifier was always used at the minimum amplification (2X). With this setting the input impedance was greater than 10 ohms and the band pass, which was set on the "maximum" position,extended from DC to approximately 1 MHZ. Capacity neutralization was not used since a 5 mV square wave was amplified without distortion. The output from the amplifier was directed simultaneously into a Tektronix 502A oscilloscope and a Moseley 7000A X-Y recorder. The recorder had a slewing speed of 20"/sec and an accuracy of 0.2 percent of the full scale reading.

Monochromatic light was obtained with a Farrand Foci-Flex monochromator with a fixed slit width of 10 nm bandpass. Its spectral range was from 200-785 nm. For all the experiments,in which wavelengths greater than 400 nm was used, the light source for this instrument was a Bausch and Lomb microscope lamp with a 6v tungsten







17

filament bulb. A 9X microscope ocular was used as a focusing lens at the exit slit of the monochromator (Figures 2 and 3). When UV light was required, a Bausch and Lomb high intensity UV monochromator, equipped with a super pressure mercury vapor light source, was substituted for the tungsten filament light source. The spectral output of mercury vapor lamps is continuous from 275 nm to the infrared region of the spectrum. Within the UV region characteristic emission lines occur at 302, 313, 334, 365, 390 and 398 nm (Jagger, 1967).

The exit slit of the UV monochromator was placed in line with, and approximately 4 cm away from, the entrance slit of the Foci-Flex monochromator. Transmission of stray light through the Foci-Flex was minimized by filtering the entrance and exit slit of the FociFlex with a Corning CS 7-54 colored glass filter (7lmax = 325 nm, \1,1/2 max = 250 nm and 390 nm). The focusing lens was not used in conjunction with the UV light source.

The mercury vapor lamp tended to vary in output; the beam undergoing sudden changes in energy level at irregular intervals. A photocell was used in an attempt to monitor these changes. The photocell was placed in a beam of stray light emitted from the base of the lamp housing. The output of the photocell was fed into a storage Oscilloscope. Shifts in the oscilloscope trace coincided with flickering of the light source. An attempt was made to run all tests while the energy was stable.

The energy of the monochromatic light beam was measured with a YSI Model 65 radiometer equipped with a YSI Model 6551 radiometer probe. Equipped in this manner the radiometer had a linear response
































Figure 2. Schematic diagram of the equipment used for recording
the ERG of the mosquito. A-light source, B-monochromator, C-shutter, D-mirror mounted on track, E-slide
wires with neutral density filters, F-radiometer probe,
G-mosquito preparation, H-micromanipulator, I-amplifier,
J-oscilloscope, K- X-Y recorder.





19






r
B- A













H K>' H-l



I




J
(,)
























Figure 3. Equipment used for recording the ERG of the mosquito. A-light source, 8-monochromator,
C-shutter, D-mirror mounted on track, E-slide wires with neutral density filters,
F-radiometer probe, G-mosquito preparation, H-micromanipulator.




































F










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z
r .I-r
c br~ ZI

Ir


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22

AtOhroughout the range of 280 to 2600 nm. Within these wavelength limits energy measurements could be made within the range of 0-2.5xi06 ergs cm-2 - sec-1 with an accuracy of + 5% of the full scale reading. The probe of the radiometer was mounted in a fixed position normal to the monochromatic light beam. To obtain a measurement of the light energy a small back-surfaced mirror was moved along a track into the light beam, reflecting the light into the probe. A small front-surfaced mirror was substituted when UV light was being measured. The optical path length was the same for the radiometer probe and the mosquito eye.

A shutter was used to obtain light stimuli of known duration. It was made from a piece of black cardboard mounted on the end of a 14 cm length of 2 mm diameter wood dowel. The dowel was mounted in the shaft of a Brush Instruments penmotor which was driven with a Grass Instruments S4 Stimulator. In the off position the black cardboard shutter was in front of the monochromator lens. A monophasic square wave from the stimulator drove the shutter upward, allowing the monochromatic light to pass for the duration of the square wave pulse. Either single or repetitive light stimuli of known duration could be obtained in this manner.

Within the range of 400-650 nm, the energy of the monochromatic light was controlled with Kodak Wratten gelatin neutral density filters (No. 96). Filters of 0.30 optical density (50%/ transmission) were cut into 2.5 cm squares. These were mounted on three separate slide wires so that they could be moved normal to the direction of the monochromatic light beam. The slide wires had one, two and three filters, respectively,so that when used singly or in combination six levels of transmission could be obtained between 50%/ and 1.56%.







23

The neutral density filters were not used in the UV region. Instead, a series of wire screens were placed singly between the B & L UV monochromator and the entrance slit of the Foci-Flex monochromator. The plane of the screen was normal to the light beam. The four screens used each had different mesh sizes and thus, differing percentages of transmission. The screens were designated 1, 2, 3, and 4 according to the transmission. All the transmission values were determined empirically

Screen Percent Transmission

1 65
2 49 3 36 4 31 1+4 20

by measuring the energy of the monochromatic light with and without the filter in place. This was done at 20 nm intervals within the range of 300-400 nm.

In some experiments the stimulus source was a high intensity

Xenon Flash (Strobelume Type No. 1532-B, General Radio Co., Cambridge, Mass.). The flash was triggered with the Grass Stimulator. A

Tektronics 549 storage oscilloscope was used to observe the ERG responses to the flash. A Viewlex projector equipped with a 750 W tungsten filament bulb was used when high intensity light was required. To increase the stimulus intensity the light from the projector was focused on a Fresnel condensing lens. The beam diameter was about

5 cm at the focal point of the lens.

Various experiments were performed in which colored glass filters, neutral density filters, interference filters or an infra-red (IR) blocking filter, were used. The colored glass filters (manufactured by the Corning Glass Works) were:







24

Filters Description

CS 7-51 Maximum transmission = 365 nm; 1/2 transmission at
330 nm & 395 nm.

CS 3-72 Sharp Cut Filter. Transmission>80% from 541 nm
750 nm. Transmission = 37% between 436 466 nm.
Transmission <0.5% below 411-nm.

CS 3-76 Maximum transmission = 730 nm. 1/2 transmission = 575 nm.
No transmission below 505 nm.

The neutral density filters were manufactured by the Corning Glassworks and Optics Technology, Inc. The optical densities and percents transmission are listed below.

Optical Densities Percent Transmission

.3 50 .5 31 .6" 25
.9" 12.5
1.0"" 10
1.2" 6.2
2" 1
3"" .1

Corning Glass Works.
.Optics Technology,Inc.

The two interference filters (Bausch and Lomb) had second order peaks at 660 nm and 605 nm. The band widths at the 1/2 transmission points were equal to 22 nm and 16 nm, respectively. The 660 nm filter had a first order peak at 350 nm (as determined with a Bausch and Lomb spectrophotometer). By using the appropriate colored glass filter (CS 3-72 or CS 7-51) either the 660 nm or 350 nm peak could be filtered out independently of the other. The 553 nm filter was always used along with the CS 3-72 filter to eliminate any UV peak which may otherwise have been present.

The IR blocking filter (Baird-Atomic) had 80% transmission between 375 nm and 575 nm. The half--transmission points were at 315 nm and 680 nm.






25

Experiments

Two series of experiments were performed to determine the time required for the eye to dark adapt. In the first experiment, the main objective was to determine the time course of dark adaptation following exposure to the overhead lights in the laboratory. This was important since nearly all subsequent experiments were to be performed on dark adapted eyes. For this experiment the monochromotor was set for 500 nm and a 25% transmission neutral density filter combination was moved into the beam. The energy incident upon the
mosqu to ee wa 0.4 103-2
mosquito eye was 0.4 x 103 erg cm-2 sec -1. One-second stimuli were presented at set intervals after the room lights were extinguished.

The amplitudes of the recorded ERG's were determined with all measurements made from the baseline to the peak along the rising side of the response. The occurrence of two or more successive responses of the same amplitude indicated that the sensitivity of the eye had stabilized. The eye was then considered dark adapted. White-eye mutants were tested in an identical manner to determine the influence of the screening pigments on the time-course of dark adaptation.

The intensity of the room light was relatively low compared to the light intensities normally encountered by the mosquito in its natural environment. The room lights delivered only 0.5 - 0.8 x 103 erg cm-2 sec -1 to the eye. The dark adaptation curve obtained in these experiments would only represent a portion of the curve of total adaptation since the eye was not fully light adapted at the beginning of the experiment. In order to completely light adapt the eye it was necessary to first determine the stimulus intensity re-







26

quired to achieve saturation. At saturation, the response of the eye is at its maximum.

Stimuli which saturate the eye supply the receptors with as many quanta as they can "use." Increasing the stimulus intensity beyond the saturation level would not increase the response amplitude. Exposure of the eye to the saturation level intensity would insure

that the eye was fully light adapted at the onset of the dark adaptation experiments.

The following experiment was performed to determine the saturation level of the mosquito eye. The mosquito eye was set at the focal point of the Fresnel Lens-Viewlex projector combination. The mosquito preparation was shielded by a black cardboard housing which reduced the amount of stray light incident on the mosquito. The

stray light came from the projector housing and from reflections off the surface of the Fresnel Lens. Two small apertures were made in the sides of the housing to accommodate the electrode holders, and one (2.5 x 2.5 cm) at the front of the housing for the passage of the light stimulus. The stimulus duration was controlled with the

shutter which completely covered the front opening in the housing. The stimulus intensity was controlled with glass neutral density filters. To measure the stimulus energy the probe of the radiometer was placed in the housing in place of the preparation. The IR blocking filter was mounted in the front opening of the housing while the energy was measured. The limits of transmission of this filter roughly coincide with the limits of the determined spectral sensitivity of the mosquito eye. Non-excitatory wavelengths were therefore eliminated from the energy measurement. Glass neutral density filters







27

(optical density = 0.3 or 0.6) were used to reduce the intensity to within the measurable limits of the radiometer.

The mosquito eye was allowed to dark adapt for 30-40 minutes

(based on the results of the previous dark adaptation experiments). The eye was then stimulated with a 0.1 sec. flash of each of several intensity levels of light. Glass neutral density filters were used to obtain the various test intensities. The log of the stimulus energy was plotted against the response amplitude to obtain an energyresponse curve. With stimulus energies below saturation, the energyresponse curve should be linear. As the saturation energy is approached the slope of the curve should decrease, reaching zero as the saturation energy is surpassed. In the saturation level experiments, several stimuli were presented at each energy level. The interval between successive stimuli was decreased in steps from 30 secs to 2.5 sec. The magnitude of the response decreased when the interval between stimuli was less than the minimum time required by the eye to recover from the previous stimulus. This procedure was repeated with all energy levels tested. The minimum time required for the eye to recover from stimuli of different intensities was determined in this manner.

The procedure for determining the time course of dark adaptation from a saturated or near saturated condition was similar to the previous dark adaptation experiment. The equipment and its physical arrangement was the same as in the saturation level experiment. Mosquito eye preparations were light adapted with the Viewlex projector. Adaptation times varied between 1 min and 10 min. Several adapting energies were tried. These energies were obtained with







28

neutral density filters used with and without the IR blocking filter. The test stimulus was a .1 sec flash of white light from a tungsten filament microscope lamp. As with the adapting light, several stimuli of varying energy levels were tried. When the IR blocking

filter was used during the light adaptation phase of the experiment it was also retained in place during the dark adaptation phase.

To determine the time course of light adaptation, the mosquito preparation was dark adapted from room light conditions for 30-40 min. The eye was then exposed to a repetitive stimulus of 500 nm light (approximately 1.5 x 103 erg cm2 sec-1) presented in 0.1 sec bursts at I sec intervals. After 10 minutes a tungsten filament adapting light was turned on. The energy of the unfiltered adapting light was 2xl0 erg cm-2 sec- at the preparation. Neutral density filters with 1%, 10%, and 31% transmission were used to obtain different adapting light intensities. Changes in the magnitude of the repetitive ERG would coincide with the changes in sensitivity brought on by the adapting light. Since the time course of adaptation was known to be very rapid in other insects, the close spacing of test flashes was considered necessary so that transient events might be detected. Short duration test flashes (0.1 sec) were used so that their adaptive effect on the eye would be minimized. The test flash energy level was large enough to elicit measurable ERG amplitudes even when the highest intensity adapting light was used.

The method of equal response was used to determine the spectral sensitivity of the eye. For this procedure the preparation was allowed to dark adapt for at least 30 min. In the visible region of the spectrum the wavelengths of monochromatic light tested were







29

at 20 nm intervals between 400 and 600 nm. In some of the first tests 650 nm light was also used. The test wavelengths were presented in order starting at either end of the range. Because of the time required to complete a test only one run was possible on each mosquito. Each test wavelength was presented at seven different energy levels obtained with gelatin neutral density filters. The duration of each stimulus was I second with a 30 second dark interval between successive stimuli of the same wavelength. This procedure was modified for the UV tests. The stimulus duration was decreased to 0.5 sec with 15 sec elapsing between successive stimuli. Decreasing by 50%* the time required to test each wavelength improved the chances of completing a test between energy changes in the UV source. When obvious energy changes occurred during a wavelength test the energy level was remeasured and the test was repeated. Sometimes these changes were so transient in nature that they went unnoticed during the tests but became apparent when the data was analyzed. Tests in the UV were conducted over 300-380 nm. Stimulus energy was controlled with the wire screen filters.

For some of the tests the energy of the unfiltered monochromatic light was measured either just prior to or immediately after each test. In most cases, however, energy was measured both before and after the test with the average value being used for the calculations. The difference between the two measurements was usually no more than

0.1 x 103 erg cm-2 sec-1 regardless of the total energy measured. This variation is porbably inherent in the radiometer rather than in the light source except as noted previously for the UV source. The differences stemmed from drift in the instrument and rounding errors when the meter was read.







30

Plots of the magnitude of the ERG as a function of the stimulus energy for each test wavelength resulted in a series of energyresponse curves for each preparation. A level of equal response of the ERG within the range of the energy-response curves was chosen for each preparation. The equal response level was always chosen to transect the most linear region of the family of energy-response curves. The same level of equal response could not be used on each mosquito eye preparation due to the differences in sensitivity of individual preparations. Equal response energies for each test wavelength were then determined graphically. The equal response energy for any test wavelength was the stimulus energy required to elicit an ERG equal in amplitude to the chosen level of equal response. The spectral sensitivity curve was obtained by plotting the reciprocals of the equal response energies, normalized to the 540 nm value (360 nm in the UV tests), as a function of wavelength.

Selective adaptation was used in an attempt to determine the number of receptor systems contributing to the spectral sensitivity curve. A tungsten filament microscope lamp was mounted on a ring stand and directed at the preparation. The light was slightly above and in front of the preparation. Interference filters (605 nm or 660 nm) were used to limit the spectral output of the adapting light. The intensity of the adapting light was controlled with a variable transformer. The mosquito eye preparation was first dark adapted.

The eye was then repeatedly stimulated in the region of 490-500 nm. The intensity of the adapting light was slowly increased until a decrease in the amplitude of the ERG's (those resulting from the 490-500 nm stimuli) was observed. This procedure was adopted in the







31

hope that an adapting energy could be found which would have a minimal effect on any short wavelength receptor and a maximal effect on a long wavelength receptor.

The spectral sensitivity curve of the eye under the continuous

influence of the adapting light was then determined. Two preparations,

one adapted with the 605 nm interference filter and the other adapted with the 660 nm interference filter, were tested at 20 nm intervals between 400-600 nm. Three preparations were tested at 10 nm intervals between 460 and 560 nm while dark adapted and then retested while under the influence of the 660 nm adapting light.

Based on the results of the spectral sensitivity experiments it seemed necessary to check the possibility that the eye had separate UV and visible light receptor systems. If separate systems were present, the response of the eye to a constant energy UV stimulus would remain constant while the eye was adapted to wavelengths in the visible region of the spectrum. Conversely, if the receptor systems were "linked" or one in the same then they would not be independently adaptable. In this experiment the UV stimuli were obtained by using the Xenon Flash in conjunction with the 660 nm interference filter and the CS 7-51 colored glass filter. This filter combination allows transmission only in the region of the first-order transmission peak of the interference filter ( N max = 350 nm). The emission of Xenon is continuous throughout the visible and UV regions of the spectrum down to 300 nm. There is essentially no emission below 300 nm (Wyszecki and Stiles, 1967). The adapting light was a tungsten filament bulb (Ziess microscope lamp) filtered with the CS 3-72 cutoff filter. This filter only transmits wave-







32

lengths longer than about 420 nm. For this experiment it was desirable to have both the stimulus and the adapting light reach the eye on the same optical axis. To achieve this a 0.3 neutral density filter was used as a beam splitter. The strobe light was positioned directly in line with the eye. The neutral density filter was mounted on this line at a 450 angle. A 30 cm long glass fiber optic light pipe was used to conduct the adapting light to the preparation side of the neutral density filter. By adjusting all the angles it was possible to have both lights reach the eye on the same optical axis. The UV was transmitted through the neutral density filter while the adapting light was reflected off its surface. The strobe light was collimated with a cone made of several layers of heavy brown paper. The base of the cone fit snuggly over the strobe light. The apex was cut off leaving an opening of 5 cm. The 660 nm and CS 7-51 filters were mounted on the beam splitter support. When

the apex of the cone was set flush against these filters there was no evidence of stray light. The intensity of the adapting light was controlled with neutral density filters placed between the light source and the fiber optic light pipe. The mosquito was allowed to dark adapt for at least 30 min. The UV stimuli were presented at first with no adapting light and then with increasing intensities of adapting light. The responses were observed and measured with the storage scope. The amplification was changed for each preparation so that the responses appeared large enough on the screen of the oscilloscope to be easily measured.















RESULTS AND DISCUSSION


The response of the eye to a short duration light stimulus was a rapid monophasic negative-going potential which tended towards a steady.state as the response maximum was neared. At the end of the light stimulus, the return of the potential to its prestimulus level was accomplished in two phases -- a rapid positive-going potential followed by a slowly decaying after-potential. The ERG recording in Figure 4 was typical of the response to a 1 sec stimulus in all regions of the spectrum tested. There was no evidence to suggest that wavelength information is in any way encoded in the ERG waveform. In some insects, such as the worker honeybee (Goldsmith, 1960), the whirligig beetle, Dineutes ciliatus (Bennett, 1967) and the butterfly, Heliconius erato hydara Hewitson (Swihart, 1963) the waveform of the ERG varies depending on the wavelength of the stimulus. The sources of these variations are multiple receptor systems with different spectral sensitivities. The uniformity of the ERG waveform in the mosquito suggests that only one receptor system is present.

At the start of the dark adaptation experiments, with the preparation exposed to constant fluorescent room lighting, the potential of the eye remained constant. When the room lights were turned off, the response was a positive-going potential the waveform of which was very similar to the light-off portion of the ERG. The maximum positive potential was reached within the first 15 sec of dark adapta33
































Figure 4. A typical recording of an ERG from the dark adapted eye
of A. aegypti. The stimulus duration was 1 sec. The
energy was adjusted to give a lOmV response. The waveform was similar for all stimulus wavelengths between
300 nm and 650 nm.






35





















5 tmrV







ON OFF







36

tion. The extent of the positive deflection varied from one preparation to another with a range of 5-11 mV. Several factors contributed to these differences in sensitivity, the most important of which is probably the damage done on insertion of the electrode into the eye. Electrode depth and angle of penetration may also be involved. Physiological differences between mosquitoes are another possible factor.

As the eye dark adapted, the amplitude of the responses to the test flashes (0.4 x 103 erg cm-2 sec-l, 500 nm, sec duration) increased, reaching a maximum in about 30-40 minutes. For each preparation, the amplitude of the ERG's were measured and normalized to the amplitude of the maximum response. In Figure 5 (a) the means of these values for five preparations are plotted as a function of total elapsed time. The curve is smooth and continuous with no indication of a second phase of adaptation as found in certain nocturnal Lepidoptera with migratory screening pigments. The results obtained from the experiments on the white-eye mutants are presented in Figure 5 (b). The similarity of the dark adaptation curves of the dark-and white-eyed mosquitoes may indicate one of two things; either screening pigments do not play a significant role when the eye dark adapts following exposure to room lighting (overhead fluorescent lighting; total energy at the insect eye was 0.5 - 0.8 x 103 erg cm-2 sec-1) or there is no discernible latancy between the onset of darkness and the initiation of screening pigment migration. The latter explanation seems the more likely since the axial migration of screening pigment is known to occur in the primary pigment cells of mosquitos (Mazokhin-Porshnyakov, 1969). The effect of the pigment






























Figure 5. Time-course of dark adaptation following exposure to room
lighting (0.5 - 0.8 x 103 erg cm-2 sec-i). The maximum
response was obtained when the eye was fully dark adapted.
a) Dark adaptation curve for five dark-eyed mosquitoes.
b) Dark adaptation curve for two white-eyed preparations.








38


100- L..----- -'s






0







c 60 o
u o I I


o




0 10 20 30 40 50 Elapsed time - minutes






10 0 � .- -_c-- ---------"

0Q





E

E X0 I E b
6 0 -P
U






0 10 20 30 40 50 Elapsed time - minutes







39

might be to limit the rise time of the adaptation curve. The whiteeye mosquitoes seem to achieve a greater percent maximum response sooner, after lights out, than the dark-eye mosquitoes.

The results of the saturation level experiment are shown in Figure 6. The linearity of the energy-response curves indicated that saturation was not achieved. The highest energy level used was

5.5 x 106 erg cm-2 sec-1. Since the stimulus energy was measured through the IR blocking filter, only those wavelengths which could effectively stimulate the eye contributed to the total energy measurement. This was based on spectral sensitivity curve for A. aeqypti which will be presented later in this section. When the stimulus energy was 5.5 x 106 erg cm-2 sec-1 the illumination incident upon the eye was 2.6 x 106 lux. This measurement was made with a Salford Electronic Instruments Photometer. The illumination on a clear sunny day is only 105 lux (LeGrand, 1957). Under even the brightest of normal conditions the eye of A. aegypti would not be saturated.

Curves of the spectral distribution of the energy eminating

from tungsten filament bulbs at various color temperatures are readily available. A polar planimeter (K and E) was used to measure the area under a 3,0000 K color temperature spectral distribution curve. Only the area between 315 nm and 680 nm was considered since these are the transmission limits of the IR blocking filter. The wavelength at the midpoint of this area was 557 nm. By assuniing that all the energy was at 557 nm, the number of quanta in the stimulus can be calculated.
































Figure 6. Energy response curves based on ERGs recorded for the
saturation level experiment. Upper curve (closed circle) and middle (open squares) curve are based on ERG recordings from the center of the eyes of two different preparations. Lower curve (closed squares) is based on ERG's
recorded from peripheral ommatidia of the same preparation
as used for the middle curve.







41














2(



25





II 0
24












-, 21 O
4J


E 2. 0















16
III








.3 .4 .5 .6 .7 .8 .9 1.0 2.0 3.0 4.0 5.0 6.0 7.0


Stimulus energy x 10 erg cm-2 sec-1






42

The energy of a single quantum can be determined with the equation:

E = h, [1] where E is the energy in ergs, h is Plancks constant (h = 6.625 x 10-27 erg sec), C is the speed of light (2.99 x 1010 cm sec-1) and n is the wavelength of the quantum in centimeters. By substituting the values into equation I with A = 557 x 10-7 cm, the energy was determined to be 3.51 x 10-12 ergs quanta-l. The highest energy stimulus was 5.5 x 106 ergs cm-2 sec-1. Therefore the quantal flux at the-surface of the mosquito eye was:

5.5 x 106 ergs cm-2 sec-1 1.54 x 1018 quanta cm-2 sec-1
3,51 x 10-12 ergs quanta'l

During the 0.1 sec stimulus, the eye was exposed to 1.54 x 1017 quanta cm-2. The average area of a facet of the compound eye in female A. aelPti is 4741,2 (Christophers, 1960). Each facet oriented perpendicular to the direction of the stimulus received

7.3 x 1011 quanta.

In houseflies and cockroaches the visual pigment present in the rhabdomeres has been extracted, characterized and quantified. Houseflies were found to have 3.7 x 107 molecules of retinal per rhabdomere, the cockroach (Periplaneta americana) had 4.3 x 107 molecules of rhodopsin per rhabdomere. The visual pigment concentrations in insect rhabdomeres compare quite well with the range of 10 - 109 rhodopsin molecules found in vertebrate rods (Wolken, 1971). In view of the fact that the number of visual pigment molecules per receptor cell is quite comparable for even very diverse forms, it would be reasonable to estimate that a mosquito has about 107 visual pigment molecules per rhabdomere.







43

The total number of visual pigment molecules per rhabdom would be 8 times this or approximately 108 molecules. This is of interest since the eye did not saturate when stimulated with approximately

7.3 x 1011 quanta per ommatidium. The quanta apparently outnumbered the visual pigment molecules by a factor of 103. The insect eye is generally considered to be a one-quantum receptor which means that the absorption of one quantum of light by one visual pigment molecule is enough to induce a physiological event in the receptor (Reichardt, 1965; Scholes, 1965). The capacity to respond to a single quantum has also been demonstrated in the compound eye of Limulus (Fourtes and Yeandle, 1964). There is, at present, no evidence to suggest that single quantum sensitivity is not also characteristic of the mosquito visual system. If so, then the efficiency of the mosquito eye is considerably less than 100%. That is, only a small portion of the quanta incident on the cornea ever interact with the visual pigments in the rhabdom. Quantal losses can occur in several ways.

The corneal layer of the eye accounts for only a negligible loss. Spectral transmission studies on the corneal layer of the housefly (Goldsmith and Fernandez, 1968) and a dragonfly, Aeschna cyanea Mull. (Kolb et al., 1969),indicate that with wavelengths longer than about 350 nm the transmission is around 95%. Many insects, particularly nocturnal forms, have corneal nipples which increase the transmission by reducing the amount of surface reflection (Bernhard ct al., 1965). The housefly does not have corneal nipples. It may be assumed that the dragonflies also do not have corneal nipples since these are usually not present in strictly diurnal forms. The mosquito however does have corneal nipples which should increase the






44

transmission to more than the 95%, characteristic of the dragonfly and housefly. Clearly, the corneal layer is unlikely to be responsible for any appreciable loss of quanta. Within each ommatidium the rhabdom is sheathed in cells containing screening pigments. Even the retinula cells, outside of the rhabdomere area, contain pigment granules. The presence of these screening pigments suggests that some of the light passing through the corneal lens is not focused on the rhabdom but passes into the receptor layer of the eye as stray light which is absorbed by the pigment granules. The amount of stray light emanating from the corneal lens and crystalline cone of the tobacco hornworm has been estimated to be 20% of the light normally incident on the lens (Miller et al., 1968). A similar situation may exist in the mosquito. In addition, the cone cells of the mosquito have cytoplasmic extensions which terminate in pigment filled sacs located just proximal to the rhabdom. It is tempting to compare these to the crystalline tracts of other insects (Doving and Miller, 1969). If the termination of the cone cell extensions in light absorbing pigment filled sacs is analogous to the termination of the crystalline tracts at the rhabdom, then the function of the cytoplasmic extensions might be to control the amount of light reaching the rhabdom under bright light conditions by siphoning a portion of the light out of the cone cell.

This hypothesis may not be valid since the crystalline tracts of Lepidoptera are 4-10 u in diameter (Miller et al., 1968), while the cytoplasmic extensions of the mosquito cone cells are only .3-.4 u in diameter (Brammer, 1970). Pending an analysis of the optics of the mosquito eye, particularly measurements of the refractive index







45

of the cone cells and their extensions, it is not possible to do any more than conjecture about their possible function.

It is apparent that many quanta never reach the rhabdom of the eye even when they arrive parallel to the ommatidial axis. The quantal loss is even greater when the light is at an angle to the ommatidial axis. Doving and Miller (1969) list half-power beam widths for a number of insects as reported by various authors. As a point source is moved off the ommatidial axis, the sensitivity of the ommatidium drops. The half-power beam width is the angular displacement which corresponds to half sensitivity. The values listed for several insects with apposition eyes fall at about 60-70. Again while there is no mosquito data available it seems reasonable to suppose that a similar condition exists in the mosquito eye.

In the saturation level experiments the mosquito was aligned so that the stimulus would be normal to the centrally located ommatidia. The axes of the peripheral ommatidia (at the medial margin of the eye) were about 500 out of alignment with the source. This greatly exceeds the presumed half-power beam width. Yet, when the electrode was placed at the periphery, the responses were of nearly the same amplitude as those recorded with the electrode centrally located in the same eye (Figure 6). Because of the limited angle of acceptance, it is doubtful that sufficient quanta entered the peripheral ommatidia via the corneal lens to account for the amplitude of the responses. Two factors may have contributed to the peripherally recorded ERG. First, the screening pigments may have allowed sufficient leakage of stray light within the receptor layer of the eye, and perhaps even within the head capsule, to stimulate







46

peripheral ommatidia which were not subject to direct illumination. Second, the insect ERG is the mass response of many ommatidia, not only those located in the immediate vicinity of the electrode. The

amplitude of the peripherally recorded ERGs was related to the amount of light leakage in the eye and the electrotonic spread of potential from the centrally located ommatidia. Due to the potential spread it might be difficult to detect saturation in the central region of the eye while large numbers of off-axis ommatidia were not saturated. The contribution to the centrally recorded ERG, from the unsaturated peripheral region of the eye, would increase as the stimulus energy increased.

The final point regarding the loss of quanta is that the visual pigment does not have the same probability of interaction with quanta of different wavelengths. Since the actual stimulus was of a broad band nature, many of the quanta that do enter the rhabdom pass through without being absorbed.

Scholes (1965) recording from single retinula cells in the

locust eye found that the voltage response saturated at light intensities which would induce the photolysis of only 105 visual pigment molecules. It is not possible to infer from Scholes' data what quantal flux was required at the corneal surface to saturate the receptor. It is interesting to note that in connection with another experiment reported in the same paper, he states that "the disparity between available quanta and yield of responses is discouragingly high." This would indicate that very few visual pigment molecules were bleached in relation to the number of quanta arriving at the corneal surface. In studies with the locust eye Shaw (1968) reported







47

that the most sensitive retinula cell measured had an efficiency of 8%. Other cells measured were as little as one-tenth as sensitive.

It was not possible to determine the time-course of dark adaption from saturation since the saturation level of the eye was not found. Prolonged exposure of the eye to the highest intensities used in the saturation experiment resulted in death of the preparation, apparently the result of heating. Out of approximately 10 attempts made at light adapting with slightly lower energies, only one was successful. In this experiment, the mosquito was adapted for 5 minutes with 2.2 x 10 erg cm-2 sec-1 of light transmitted through the IR blocking filter. The intensity of this adapting light was greater than the room light intensity by a factor of roughly 103. The IR blocking filter was used to eliminate the IR radiation which was thought to be responsible for the heating of the other preparations. The test stimulus was 2.8 x 10 erg cm-2 sec-I of white light emanating from a tungsten filament bulb and delivered to the preparation through the Fresnel lens and the IR blocking filter. The dark adaptation curve was similar to those previously described and provided no new insight into the problem of screening pigment migration (Figure 7).

The minimum time required for the mosquito eye to recover from

0.1 sec flashes of different intensities of light was determined. The recovery time following the lower energy stimuli (0.35-1.38 x 10 erg cm-2 sec-1) was only five seconds. Following the most energetic stimuli (2.75-5.5 x 106 erg cm-2 sec-1) the recovery time increased to 10-15 sec. The effect on the eye of relatively short light flashes was qualitatively different from the effect of longer






























Figure 7. Time course of dark adaptation following five minutes of
light adaptation with 2.2 x 106 ergs cm-2 sec-1 of white
light.






























100. . -------o-oo
800 a 6






L











0 10 20 30 40 sO

Elapsed time - minutes







50

exposures to light. Exposure to room lighting for several hours induced a more stable change in the sensitivity of the eye than exposure to short duration flashes of light 103 times more energetic. The eye required 30-40 minutes to dark adapt following room light exposure, while recovery from short flashes required only seconds. The specific mechanisms underlying these differences are not known. Screening pigment migration and changes in the position of the rhabdom may be involved in the recovery following long exposures to light.

The results of the light adaptation experiments differ from

those obtained by Goldsmith for the honeybee (1963). In the mosquito preparation the ERGs resulting from the repetitive stimulation were,

under conditions of constant darkness, all of the same amplitude. The response to the onset of the adapting light was a rapid negative shift in the baseline accompanied by a reduction in amplitude of the ERG responses. The baseline shift and the sensitivity decrease occurred within the first second of light adaptation. The amplitude of both effects was proportional to the adapting light intensity (Figure 8). After the maximum negative deflection had been reached the baseline started to shift in a positive direction and continued to shift for approximately 50 sec. During this period the amplitude of the test ERG increased slightly. While the adapting light was on, the baseline remained negatively displaced with respect to its dark adapted level. In the honeybee (Goldsmith, 1963) when the adapting light was turned on, the baseline shifted negatively but did not undergo any subsequent positive shift. Also, the amplitude of the ERG responses to the test stimuli did not increase following the






















Figure 8. Light adaptation. The onset of the adapting light caused a negative deflection of the
baseline and a decrease in the amplitude of the responses to the constant energy test stimuli. Adapting light energies were: a) 0.2 x 103 ergs cm-2 sec-1, b) 2 x 103 ergs
cm-2 sec-1, c) 6.2 x 103 erg cm-2 sec-1.













N 0






Wi A41 fitL! j~ ,.
*.,..Sr. ,,,4


















00i Iiii







53

initial suppression under the influence of the adapting light. It is tempting to attribute the positive baseline shift to migration of the screening pigment into a light adapted position. This explanation is weakened,if not entirely negated, by the fact that the same effects were seen in a white-eye mutant. Thus, the role of the migratory screening pigments in light adaptation remains obscure.

The spectral sensitivity curve for the visible portion of the spectrum is presented in Figure 9. The curve is based on the spectral sensitivity curves of 7 mosquitoes. The spectral sensitivity curve peaks at 540 nm. There is a shoulder centered around 500 nm. The standard error is greatest in the region of the shoulder. The shoulder and the large standard error in this region are explained by the fact that most of the spectral sensitivity curves for single preparations had a minor peak or shoulder in the region of 480-500 nm. The sensitivity at 650 nm was so low that the amplitude of the ERGs never achieved the equal response level for the preparation. When compared to the sensitivity at 540 nm, the sensitivity at 650 nm was less by a factor of about 10-2. For this reason, 650 nm stimuli

were not routinely tested.

In general, the data obtained in the UV spectral sensitivity

experiments were not wholly satisfactory due to the energy fluctuation of the UV source. Evidence of this was seen in the energy-response curves where unnoticed transient energy changes during the testing resulted in a scatter of points rather than the linear array normally expected. In these instances, the energy-response curve was drawn on the basis of the "best fit" for a line parallel to the rest of the energy-response curves. The "best fit" line was one























Figure 9. Spectral sensitivity curve for the visible region of the spectrum. The curve is based
on the spectral sensitivity of seven mosquitoes. The vertical lines indicate the
standard error of the mean for each point.













-so




I IC > 6040








20
!i


!ii

i







400 40 0 520 S 00

Wavelength (nm)







56

intersecting the most points in the scatter. This problem made it difficult to determine the validity of the data with respect to the actual spectral sensitivity of the mosquito. Even if poorly representative, the data still show that the mosquito eye is sensitive to UV radiation with the peak occurring somewhere within the range of 330-360 nm. At best, the data indicate that there are two peaks in the UV at 330 nm and 369 nm (Figure 10). All the preparations had these two peaks, so they may be characteristic of the true UV sensitivity curve. The occurrence of a single peak of sensitivity in the UV is well known for many insects. Two peaks, however, have not previously been reported. This is not to say that there is no basis for assuming the validity of the two peak outcome of these experiments; there is evidence that two separate UV receptor systems may be present in the mosquito eye (Kay, 1969). Further discussion of this will be presented later in this section.

The 360 nm and 540 nm energy-response curves were used to

determine the energy that would be required to evoke a 9mV ERG with stimuli in these regions of the spectrum. The 9mV responses would have required 0.23 x 103 erg cm-2 sec-1 at 360 nm but only 0.1 x 103 erg cm-2 sec-I at 540 nm. The eye was approximately 50% as sensitive at 360 nm as it was at 540 nm.

The shoulder located at 500 nm suggested that two receptor systems with different absorption maxima were present in the eye. Selective adaptation experiments were undertaken to test this hypothesis. Two preparations were tested at 20 nm intervals while under the influence of a red adapting light (605 nm and 660 nm, respectively). The spectual sensitivity curves for these preparations had a peak at 520 nm.and a shoulder in the 560-580 region of the curve.































Figure 10. Spectral sensitivity curve for the UV region of the
spectrum. The curve is based on the spectral sensitivity
of four mosquitoes. The vertical lines indicate the
standard error of the mean for each point.






58










100 - O 1)








.






40





20






300 320 340 360 380

Wavelength (nm)







59

Three dark adapted preparations which had been run at 10 nm intervals between 460 and 560 nm were retested under the influence of a 660 nm adapting light. The data for these tests is presented in Figure 11. The major effect of the adapting light was to depress the height of the entire curve. There was also a slight change in the relative heights of the peaks. Both peaks are the same height in the 660 nm adapted state while,in the dark adapted state, the 540 nm peak was greater than the 510 nm peak.

The results of these selective adaptation experiments were not consistent with the expected results of a two receptor system with widely separated )\ max. Other investigators using selective adaptation have obtained dramatic changes in the shape of the spectral sensitivity curve which clearly indicated the presence of independent receptor systems with different X max. The successful application of this technique has been reported by Bennett (1967), Bennett and Ruck (1970) and Goldsmith (1960). These examples serve to illustrate the usefulness and power of selective adaptation in the identification of multiple receptor systems in the eye.

The experimental results of selective adaptation of the mosquito eye indicate that a single receptor system is responsible for the spectral sensitivity curve in the visible region of the spectrum. It might be argued that the spectral distributions of the adapting lights were such that with two receptor systems present, both would be effected and selective adaptation would not be achieved. This does not appear to be the case. Assuming that these hypothetical receptors had absorption maxima at 500 nm (general region of the shoulder) and 540 nm (peak of the spectral sensitivity curve), it
































Figure 11. The effect of selective adaptation on the spectral sensitivity curve. a) Spectral sensitivity curve of three
dark adapted preparations. b) Spectral sensitivity curve of the same three preparations light adapted with 660 nm
light. The preparations were actually much less sensitive in b than in a.







61







10
/ / /
o so- C





60'-L




>.

> 460 500 540 SSO30


4-J
U
C




S. 100- o. e..Q..-.~l- ----o
/



















60 500 540 5S0


Wavelength (nm)







62

is difficult to visualize an effect of the adapting lights on both receptors, especially when the 660 nm interference filter was used. For this filter, the half transmission point on the short wavelength side of the peak was at 644 nm. The transmission was approximately 1% at 600 nm. It is not likely that this adapting would effect both of the hypothetical receptor systems to such an extent that selective adaptation would not be achieved.

In most of the insects which have been studied, selective adaptation or single cell recording techniques have revealed that the UV and visible receptor systems are independently adaptable. An apparent exception is Calliphora erythrocephala. In this insect the spectral sensitivity curves of single retinula cells have two peaks, one in the visible and another in the UV. These peaks are not independently adaptable (Burkhardt, 1964).

The situation in A. aegypti is similar to that found in Calliphora in that adaptation with visible light resulted in suppression of the UV sensitivity. This was demonstrated in the series of experiments in which the eye was stimulated with constant energy UV stimuli while the intensity of the purely visible adapting light was increased. The relative amplitude of the UV stimulated ERGs are shown plotted in Figure 12 as a function of the adapting light energy. It is very clear that the UV sensitivity decreased as the adapting light intensity increased. This effect could not have been due to any UV in adapting light since this was filtered with a CS 3-72 cutoff filter which blocks all light with wavelengths shorter than around 420 nm. The purity of the UV stimulus was ensured by using the 660 nm interference filters along with the CS 7-51 colored glass
























Figure 12. Influence of long wavelength adapting light on the amplitude of the response to a
constant energy UV stimulus. The response amplitude is expressed as a percentage
of the UV response of the fully dark adapted preparation. The results of two
experiments are presented.
















UV response amplitude


O O 0 O O




















r0 0 Oi
rt





0 , ,
-h 0 a, 00 nO












+-Q
00


0 o0













t9







65

filter. The peak of transmission for this filter combination was at 350 nm. There was no transmission of wavelengths longer than 405 nm. The decrease in UV sensitivity could only have been due to the adaptation with visible light. It might be hypothesized that the decrease in UV sensitivity was due to some sort of inhibitory mechanism. This would mean the adaptation of one receptor system would inhibit the response of a second receptor system - in this case, a visible receptor inhibiting a UV receptor. If it is valid to generalize from the results of experiments performed on migratory locusts, inhibition does not explain the results obtained with mosquitoes. In the locust eye no interaction between ommatidia was found (Tunstall and Horridge, 1967; Shaw, 1968). In addition, it was shown that retinula cells within a single ommatidia function independently of each other (Shaw, 1968). This latter point is supported by two independent studies on the cockroach eye. Walther (1958) used selective adaptation to reveal two independent receptor systems with peaks in the UV and visible. This was confirmed by Mote and Goldsmith (1970) who then showed that the UV and visible receptors occurred in different retinula cells within the same ommatidium (Mote and Goldsmith, 1971). If inhibitive interaction between retinula cells were the rule in insect compound eyes then Walther would have obtained results comparable to those obtained with the mosquito.

The characteristics of the mosquito's visual pigment could

explain the results of the spectral sensitivity and selective adaptation experiments. All the visual pigments which have been chemically extracted and characterized are composed of a retinal (Vitamin A






66

aldehyde) prosthetic group associated with an opsin protein. The retinal may be either of two types; retinall which is derived from Vitamin A or retinal which is derived from Vitamin A . Retinal
2 2 2 differs from retinal in that it has an extra double bond in the terminal 6 carbon ring (Dartnall, 1962). Any opsin may be conjugated with either retinal or retinal2. The 17max of a retinal based visual pigment is at a longer wavelength than if the same opsin were associated with a retinall prosthetic group. The spectral position of the /, max is also affected by the opsin. The range of ) max for retinall based pigments extends from 430 nm to 560 nm while the range for retinal2 based pigments extends from 510 nm to 620 nm (Dartnall, 1962). The spread of -)max is due to the differences in opsins from different animals and from different visual receptors within the same animal.

The visual pigments of only a few insects have been extracted and characterized. Visual pigments with A max in the visible region of the spectrum have been extracted from the honeybee, housefly and cockroach (Wolken, 1971). Retinall was found in all three insects while retinal2 was found only in the housefly (Wolken, 1971). A UV sensitive pigment with ;)max at 345 nm has been extracted from the eye Ascalaphus macaronius Scop. (Gogala et al., 1970). The prosthetic group of this pigment was identified only as retinal with no mention made of whether it was retinall or retinal2. The major peak of the mosquito spectral sensitivity curve is at 540 nin which is within the spectral range of both retinall and retinal2 based visual pigments. This fact, together with the occurrence of both retinal and retinal in dipterous insects, makes it virtually impossible to determine,







67

from the spectral sensitivity curve, which retinal is involved in

the mosquito visual pigment.

Brammer and White (1968) raised A. aegypti on a vitamin A and

B-carotene deficient diet. They reported a reduction in the amplitude of the ERG but not its complete elimination indicating that some vitamin A was still available to the mosquitoes. Brammer and Whites' experiment supports the hypothesis that the mosquito has a retinal based visual pigment. Their study sheds no light on the problem of whether retinall or retinal2 is the visual pigment prosthetic group in A. aegypti.

All known retinal based visual pigments have the same shape absorption spectra in the visible region of the spectrum (Dartnall, 1962). The shape of the absorption curves of retinal2 pigments are slightly broader than the curves of the retinall pigments but otherwise are very similar (Munz and Schwanzara, 1967). The shape of the mosquito spectral sensitivity curve can not be duplicated with absorption curve constructs based on the Dartnall nomogram (Dartnall, 1962) or the nomogram for retinal2 based visual pigments (Munz and Schwanzara, 1967). The visual pigments have smooth absorption curves while the mosquito spectral sensitivity curve has a shoulder centered around 500 nm. This shoulder is apparently due to absorption by the screening pigments in the mosquito eye. These pigments are ommochromes and pterins (Bhalla, 1968). In general, these types of pigments have absorption bands in the visible region of the spectrum with > max in the region of 500-550 nm (Goldsmith, 1964; Hoglund et al., 1970). The spectral sensitivity curve would be depressed in the region of the screening pigment absorption bands, when compared to the shape







68

of the absorption curves of the visual pigments. Therefore the peak of the spectral sensitivity curve may not coincide with the absorption peak of the visual pigment. The )max of the visual pigment may be at some wavelength shorter than 540 nm, perhaps around 520 nm530 nm.

All retinal based visual pigments with absorption bands in the visible region have secondary absorption bands in the UV region of the spectrum. These bands are called cis bands or cis peaks. The cis peak height is 20%-30% of the height of the main absorption band. Absorption of light in the cis peak region is effective in bleaching the visual pigment (Dartnall, 1962). In many insects the UV and visible peaks of spectral sensitivity are due to separate pigment systems. In the mosquito eye there is apparently only one visual pigment. The UV sensitivity must be due, at least in part, to cis band absorption. The fact that the UV and visible peaks in the mosquito eye cannot be independently adopted supports this hypothesis. The ability of aphakic humans (without lens in the eye) to see UV light has been well documented (Wald, 1945; Dodt and Walther, 1958; Burian and Ziv, 1959). This ability demonstrates that cis band absorption can result in meaningful visual responses. The expected

separation between the )max of the main absorption peak and the Smax of the cis peak is 9,700 + 400 wave numbers (Dartnall, 1962). Assuai;ng that the major absorption peak of the mosquito visual pigment is located at 530 nm, then the cis peak should occur at 350 nm. The nearest peak is at 360 nm. The relative sensitivity at 350 nm is approximately 50%r of the 360 nm maximum or 25% of the 540 nm maximum. This last figure is based on the fact that the sensitivity






69

at 360 nm is only about 50% of the 540 nm sensitivity. Dartnall (1962) stated that cis peak absorption was 20%-30% of the main peak absorption. The mosquito eye sensitivity to 350 nm agrees well with

this estimate.

In general, the absorption curves of cis bands are broad and rather flat. The shape of the mosquito UV spectral sensitivity curve is not typical in this respect. This may be the result of the existence of non-retinal UV sensitive material within the eye. Kay (1969) found UV absorbing fluorescent material in the eyes of all the insects he examined. This material was present within the retinula cells. Kay presented the excitation emmission spectra of these materials from a number of different insects. Extracts from

the mosquito eye (Culex) were maximally excited at 292 nm and 364 nm. The emission peak of the fluorescent material was at 450 nm. Kay's hypothesis was that this non-retinal system augments the UV sensitivity of the eye. The energy absorbed in the UV region is transferred to the retinal based visual pigment via the fluorescence at 450 nm. If the system does in fact operate in this manner, then the UV vision in the mosquito eye would be mediated by both cis peak absorption and this fluorescent material. This would explain why the UV and visible sensitivity peaks were not independently adaptable. Both modes of UV sensitivity are ultimately due to the single retinal based visual pigment. The 360 nm peak in the spectral sensitivity curve is in close agreement with 364 nm excitation peak in extracts from Culex eyes. No excitation peak was found in these extracts which would correspond to the 330 peak in A. aegypti. However, this does not preclude the existence of an appropriate fluorescent material in A. 2egyti eyes.















CONCLUSIONS


1) The ERG of A. aegypti was a monophasic negative wave of

potential. At the onset of a light stimulus the baseline underwent a rapid negative shift. Following the stimulus the return to the baseline was accomplished in two phases; a rapid positive-going phase followed by a slowly decaying after potential. There was no indication that wavelength information was in any way encoded in the ERG waveform.

2) The dark adaptation curves for dark-eye and white-eye mosquitoes were very similar. Approximately 30-40 minutes were required for the eye to dark adapt following exposure to room lights (0.5-0.5 x 103 erg cm-2 sec-1). The migratory screening pigments do not appreciably affect the time-course of dark adaptation.

3) The saturation level of the eye was not found. With the highest intensity stimuli the eye received 7.3 x 1011 quanta per ommatidium. Since each ommatidium contains only 108 visual pigment molecules, the efficiency of the eye must be very low. Quanta losses could have occurred at all levels along the optical pathway within the ommatidia.

4) The eye required only 10-15 sec to recover from 0.1 sec

high intensity flashes of light (energy = 5.5 x 106 erg cm-2 sec-1). When this is compared with the 30-40 minute period required for the eye to dark adapt following prolonged exposure to room lights, it



70






71

becomes apparent that different mechanisms are involved in the generation of the ERG and light adaptation.

5) Light adaptation occurred in two phases. When the adapting light came on the baseline shifted rapidly in a negative direction, at the same time the sensitivity of the eye decreased. These effects occurred within the first second of light adaptation. During the next 50 sec, the baseline shifted in a positive direction and the amplitude of the ERG responses to the test stimuli increased slightly.

6) The spectral sensitivity curve for A. aegypti had a major

peak at 540 nm. Two smaller peaks were located at 360 nm and 330 nm. A single retinal based visual pigment mediates vision in A. aegypti. The 'max of the visual pigment probably does not coincide with the 540 peak of the spectral sensitivity curve due to the filtering effect of the screening pigments. The UV sensitivity is a result of cis band absorption by the retinal based pigment and absorption by a UV excited fluorescent material in the retinula cells.














LITERATURE CITED


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Bernhard, C. G., W. H. Miller, and A. R. Moller. 1965. The insect
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Bernhard, C. G., and D. Ottoson. 1960a. Comparative studies on dark
adaptation in the compound eyes of nocturnal and diurnal
Lepidoptera. J. Gen. Physiol. 44:195-203.

Bernhard, C. G., and D. Ottoson. 1960b. Studies on the relation
between the pigment migration and the sensitivity changes during
dark adaptation in diurnal and nocturnal Lepidoptera. J. Gen.
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Bhalla, S. C. 1968. White eye, a new sex-linked mutant of Aedes
aegypti. Mosquito News 28:380-385.

Brammer, J. D. 1970. The ultrastructure of the compound eye of a
mosquito Aedes aegypti L. J. Exp. Zool. 175:181-196.

Brammer, J. D., and R. H. White. 1968. Vitamin A deficiency:
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Brett, G. A. 1938. On the relative attractiveness to Aedes aegypti
of certain coloured cloths. Roy. Soc. Trop. Med. Hyg., Trans.
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Burian, H. M., and B. Ziv. 1959. Electric response of the phakic and
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Arch. Ophthalmol. 61:347-351.




72







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Burkhardt, D. 1962. Spectral sensitivity and other response
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Dartnall, H. J. A. 1962. The photobiology of visual processes,
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Dodt, E., and J. B. Walther. 1958. Fluorescence of the crystalline
lens and electroretinographic sensitivity determinations.
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Doving, K. B., and W. H. Miller. 1969. Function of insect compound
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Fourtes, M. G. F., and S. Yeandle. 1964. Probability of occurrence
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74

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BIOGRAPHICAL SKETCH


Leonard J. Goldman was born July 29, 1943, in Brooklyn, New

York. In June of 1960 he was graduated from Erasmus Hall High School. He received a Bachelor of Arts degree from Brooklyn College in June of 1964. He was enrolled as a graduate student at the University of Miami, Coral Gables, Florida, from September, 1964 to June, 1966. In July, 1966, he enrolled in the Department of Entomology of the University of Florida where, until the present time, he has pursued his work toward the degree of Doctor of Philosophy.

Leonard J. Goldman is married to the former Brenda Leigh Baldwin

and is the father of one child.




























76








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Harve 7 . Cronroy, Chair an Profes r of Entomology nd Nematology


I certify that I have read this study arnd that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Thomas J. Walker
Professor of Entomology and Nematology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Marion S. Mayer
Courtesy Assistant Professor of Entomology and Nematology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




Pilio/S. Callahan
Courtesy Professor of Entomology and Nematology








I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.




William W. Dawson
Professor of Ophthalmology



This dissertation was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

August, 1971





.,De , College of griculture





Dean, Graduate School




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PAGE 1

The i'lectroretlnograni and Spectral Sensitivity of the Compound Eye of Aedes ae gypt i By LEONARD J, GOLDMAN A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REfiUi REMEHTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1971

PAGE 2

ACKNOWLEDGEMENTS I vj\sh to thank Dr, H. Cromroy for his support, patience, and guidance without which this research would not have been possible. I am also indebted to Or, M, S, Meyer for the use of his equipment and space and also for his advice and suggestions throughout the course of this \i/ork. I also wish to thank Dr, W. Dawson for his invaluable help and suggestions. During the course of this work I received support from an Office of Civil Defense Grant DAHC-20-69-C-0291 , Modification P6'48-l. « * I I

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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LiST OF FIGURES iv ABSTRACT v INTRODUCTION | LITERATURE REVIEW 3 MATERIALS AND METHODS ]k Insects and Equipment \k Experiments 25 RESULTS AND DISCUSSION 33 CONCLUSIONS 70 LITERATURE CITED 72 BIOGRAPHICAL SKETCH 76 i i i

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t LIST OF FIGURES Figure Page 1. Schematic diagram of an ommatidium of A^. aegypt i . 6 2. Schematic diagram of the equipment used for recording the ERG of the mosquito 19 3. Equipment used for recording the ERG of • the mosquito 21 A. A typical recording of an ERG from the dark adapted eye of A. aegypt i 35 S' Time-course of darl< adaptation following exposure to room lighting 38 6. Energy response curves based on ERG's recorded for the saturation level experiment . Ij] 7. Time course of dark adaptation following five minutes of light adaptation with 2.2 X 10" ergs cm"^ sec"^ of white light ^9 8. Light adaptation 52 9. Spectral sensitivity curve for the visible region of the spectrum 55 10. Spectral sensitivity curve for the UV region of the spectrum . 58 11. The effect of selective adaptation on the spectral sensitivity curve 61 12. Influence of long v/avelength adapting lifjht on the amplitude of the response to a constant energy UV stimulus 64 iv

PAGE 5

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ELECTRORETINOGRAM AND SPECTRAL SENSITIVITY OF THE COMPOUND EYE OF Aedes aegypti -y By Leonard J. Goldman August, 1971 Chairman: Dr. Harvey L. Cromroy Major Department: Entomology and Nematology Electrophysiological techniques were used to study the physiology of the compound eye of the mosquito, Aedes aegypti. The electroretinogram (ERG) was a monophasic negative potential wave. There was no indication that wavelength information was encoded in the ERG waveform. The time course of dark adaptation wasdetermi ned for both dark-eye and white-eye mosquitoes. Following prolonged exposure to room lights the mosquitoes required 30-^0 minutes to dark adapt. The time course of light adaptation was studied using three intensities of adapting light. Light adaptation was completed within approximately 50 sec. The method of equal response was used to determine spectral sensitivity of the eye. The eye was maximally sensitive to 5^0 nm light with secondary peaks of sensitivity at 360 nm and 330 nm in the UV region of the spectrum. The results of selective adaptation experiments indicated that vision in A. aegypti was mediated by a single visual pigment. The UV sensitivity was attributed to cis band absorption by the visual pigment and UV absorption by a UV excited, fluorescent material in the eye. V

PAGE 6

INTRODUCTION In Christophers' (I96O) book on Aede s aegypti L. only three pages are devoted to "Reaction to Light and Visual Response." Six pages are devoted to a description of the structure of the compound eye. If Christophers' book were to be published today, the length of these sections vjould not change perceptibly. Aedes aegypti . which is generally known as a daytime biter (Christophers, I96O), is most often found in shaded areas or resting on dark surfaces. It is apparent that visual cues, photoperiod and ambient light levels play an important part in this mosquito's behavior. In addition, much of our knowledge about the numbers and distribution of various mosquito species is based on light-trap studies. V/ith light playing such an important role in mosquito research, it is surprising that tliere has been virtually no work done on the physiolofjy of the compound eyes of mosquitoes. Behavioral studies have been done on the response of mosquitoes to various colors but these are largely uni nterpretable in terms of the physiology of the eye. v • • ' • j In recent years electrophysiological techniques and equipment have been developed vjhich make it possible to directly rreasure the responses of the eye. These techniques have been successfully applied in studies on the eyes of m.any insects. Extensive electrophysiological studies have not been performed on mosquito eyes. The results of the little electrophysiology that has been done on mosquito eyes I

PAGE 7

2 do not significantly contribute to an understanding of the physiology of mosqui to vi sion. The goal of this research was to investigate the physiology of the compound eye of Aedes aegyp ti using electrophysiological techniques. The electroreti nogram (ERG), dark and light adaptation, and the spectral sensitivity of the eye vjere studied.

PAGE 8

LITf^RATURE REVIEW Several behavioral experiments designed to test the influence of colored surfaces and light on the attraction of Aedes a egypti L. have been reported. The results of these early experiments have not been cons i stent. Brett (1938) tested the attractiveness of different colored cloths to Aedes aegypti . Each color was tested against either a black or white standard. The order of attractiveness, when tested against black, was: bl ack > red > brown > whi te > bl ue > green > ye 1 1 ow. When tested against white the order was: red > bl ack >brown > green > b 1 ue ^ ye i 1 ow > v^/h i te . " ' ; In another. study Gilbert and Gouck (1957) studied the influence of surface color on the landing rates of caged Aedes aegypti mosquitoes on colored filter paper disks. A count of the number of mosquitoes resting on the disk v;as made thirty seconds after they had been disturbed. The amount of light reflected was measured with an exposure meter. Wlien disks reflecting ^0 foot-candles were tested the color preference was as follows: yellow >orange, red >green, violet, black, blue, white. With disks reflecting 20 foot-candles the results v;ere as follows: orange, red > violet, black, green, blue>white. Yal low v;as not included in the 20 foot-candle tests. These experiments cannot be Interpreted in terms of the spectral sensitivity of the mosquito eye since the spectral quality and i nsity of the various colors were not reported. 3

PAGE 9

In another study Ludwig determined the influence of colored light on the attraction of Aedes a egypti to a hand. He used broad-band colored glass filters within the spectral range of 365950 nm. Tests were conducted at three light intensities. Ludwig obtained maximum attraction in the ultraviolet (UV) region and in the visible region between 515 and 605 nm. Interpretation of the results obtained by Ludwig {]35^) are complicated by the broad-band nature of the stimuli and the lack of any data on the energy of the stimuli. In any case, behavioral tests alone are not adequate for determining the spectral sensitivity of the eye. Only by kna-/ing the spectral sensitivity beforehand could it be proven that the result of a behavioral test is a true measure of the spectral sensitivity of the insect eye. The structure of the compound eye of Aedes aegypti has been described in detail by Brammer (1970) (f-igure 1). The dioptric apparatus is composed of a corneal layer, a corneal lens and four cone cells. The cone cells give rise to six cytoplasmic extensions v/hich pass proximal ly into the receptor region of the ommatidium. Below the region of the rhabdom these cytoplasmic extensions enlarge and form pigment filled sacs. Surrounding the cone cells are two primary pigment cells. The cell bodies of an undetermined number of secondary pigment cells surround the primary pigment cells. These secondary cells have elongated portions v/hich pass proximally between adjacent ommatidia to the basement membrane. Immediately proximal to the cone cells is the receptor region of the eye. The receptor layer is composed of eight heavily pigmented retinula cells. Six of these (designated R,-R/-) are situated

PAGE 10

Figure 1. Schematic diagram of an ommaticlium of A. ae gy p t i . L corneal lens, C cone cells, CE cytoplasmic extension of cone cells, S pigment filled sacs at the end of the cytoplasmic extensions of the cone cells, R retinula cells R^-Rg, R peripherally located retinula cell, Rn "" cen tra 1 1 y located retinula cell.

PAGE 12

7 peripherally around a central retinula cell (Rg) . The retinula cell designated lies outside the ring Formed by Rj'R^The relative position of this cell remains constant over large portions of the eye. The rhabdomere of is on a projection that extends into the center of the ring formed by R,-R-. The rhabdomere microvilli of R^ seem to be stacked parallel to and directly above (distad) the microvilli of the Rg rhabdomere. The organisation of the mosquito ornmatidium is similar to that of Dipterons of the suborder Cyclorrapha, with the exception that the rhabdom is of the fused type while the Cyc 1 orraphans have an open rhabdom; i.e.j the rhabdomeres project into an open intracellular space. Electrophysiological techniques have been used extensively in investigations on the functioning of the compound eyes of insects. There are several review articles covering this subject (Burkhardt, 196'+; Goldsmith, ]36k) and a book by Mazokhi n-Porshnyakov (I969) wiiich treats the entire subject of insect vision. There are no references to experiments performed on mosquito eyes in any of these works. There are only two reports of the results of electrophysiolog ical techniques applied to mosquito eyes. Brammer and White (1968) show the wave-form of the electroreti nogram (ERG) of Aedes ae gypti while Yinon (1970) presents a recording of the ERG of Cule x pi piens L In both cases the mosquito ERG v;as a simple monophasic negative potential. it would be valuable at this point to review some of the work that has been done on other insects. This would provide a background against which the results obtained from experiments on Aedes aeq ypti can be compared.

PAGE 13

8 The mechanisms underlying dark and light adaptation of compound eyes are poorly understood. The three main factors involved in adaptation are: a) resynthesis of visual pigment, b) recovery of active membranes, and c) changes in the position of the accessory pigments or In the photoreceptors themselves (Goldsmith, 196^). The time course of dark adaptation in diurnal and nocturnal Lepidoptera was studied by Bernhard and Ottoson (1960a, 1960b). They measured the sensitivity changes associated with adaptation by determining the light energy necessary to elicit an ERG of a given magnitude. They found that in diurnal Lepidoptera the sensitivity of the eye increased rapidly during the first Few minutes of dark adaptation and then leveled off. The eye was over 307o adapted after 10 minutes and completely adapted after 20 minutes. Dark adaptation In nocturnal species was a more complex phenomenon. The initial changes in sensitivity Vvere similar to those found in the diurnal species. After several minutes, hov^Jever, a second phase of adaptation appeared during which there was an additional Increase in sensitivity which continued over a 20-30 minute period. Coincident with this second phase was the onset of migration of the accessory pigments in the eye. Mo pigment migration occurred in the eyes of the diurnal insects. These authors believed the rapid dark adaptation of diurnal Lepidoptera and the initial phases of adaption in nocturnal Lepidoptera, were the results of resynthesis of the visual pigments. . Goldsmith (1963) studied the course of dark and light adaptation in the worker honeybee. Dark adaptation in this insect had the same time course as in the diurnal Lepidoptera, Initially, the rate

PAGE 14

9 of dark adaptation was very rapid, the greatest increase in sensitivity occurring within the first few minutes of darkness. The eye was fully dark adapted within 10-15 minutes. Dark adaptation in four species of insects was investigated by Ruck (1958). In Api s me]] i fera L. , Pach ydiplax l onqipennis Burm.eister, and Phormia reqina Meigen, dark' adaptation was generally complete within 15 minutes. In Periplaneta a mericana L. adaptation required a longer but undetermined time. In Calliphora er y throcepha 1 a Meigen both light and dark adaptation have been studied. Using i ntra-cel 1 ul ar recording techniques V/ashizu {\36k) found that during light adaptation the sensitivity of the eye decreased to 20 percent of its dark adapted level within 13 seconds of the onset of illumination. Within 50 seconds the sensitivity was down to 10 percent. Dark adaptation was achieved in 30-40 seconds. Goldsmith (1963) subjected the dark adapted compound eyes of Api s me I i fera to a repetitive test flash and recorded the resultant ERG's. When an adapting light v,/as turned on the base line shifted and the test flash response decreased in magnitude. Within a fev/ seconds the eye had reached a steady level of adaptation. Subsequent presentations of the test flash superimposed upon the adapting light elicited equal size responses. In the moth G al leri a mel lonel la L, light adaptation ran a faster time course than pigment migration and was usually complete within several seconds. Pigment migration, from the dark to the light adapted state, required up to 30 minutes to reach completion (Post and Goldsmith, 1965).

PAGE 15

10 Light and dark adaptation is sometimes characterized by more than visual pigment regeneration and accessory pigment migration. In some insects, adaptation is accompanied by a change in the distance between the rhabdom and the proximal end of the crystalline cone. V/alcott (1969) described this phenomenon in the eye of D y ti scus marqi nali s L. When the eye vjas light adapted the distance between the rhabdom and cone was as great as 120 urn. The crystalline tract that traverses the distance between the cone and the rhabdom was drawn out into a thin thread. During dark adaptation, the rhabdom moved dis tally to within 10 um of the cone. The crystalline tract became correspondingly short and thick. A similar phenomenon vias found to take place in a giant water bug, L ethocerus americanus Leidy. The movement of retinula cells is also known to occur in craneflies, mosquitoes (Cu 1 ex and Anopholes) , and the v/ater boatmen (Notonecta) . In these insects there is no known crystalline thread and the distance traversed by the retinula cells is on the order of several microns at most (Walcott, 19^9; Mazokh i n~Porshnyakov , I969). Electrophysiological studies on the spectral sensitivity of the compound eyes have been done on only a very small number of insects. The spectral sensitivity of A pi s me 1 1 i fe ra has been studied by Goldsi.iith (i960). Using the method of equal response and selective adaptation, he found that the compound eye of the v/orker bee had a two receptor visual system with the maximal sensitivity at 535 nm and a shoulder or minor peak of sensitivity at 3^+5 nm. Using the method of equal response but not selective adaptation on the drone Goldsmith (1961) found that the location of the peak of the spectral sensitivity

PAGE 16

1 1 curve did not remain constant. This was apparently caused by the contributions of several receptors. He concluded that there were probably three receptors present with peaks at 535 nm, '+^(0 nm, and 3^5 nm. • Both the dorsal and ventral eyes of the Whirligig beetle, Dingutes ciliatus Forsb. had peaks of sensitivity at 520 nm wi th a shoulder in the UV region (Bennett, 1967). Selective adaptation experiments indicated that a two receptor system was present. The fact that the energy-response curves for wavelengths in the UV and visible regions were not parallel supported the two receptor hypothesis. ' • The compound eyes of dragonflies have different spectral sensitivities in the dorsal and ventral regions. In Li be 1 1 ul a 1 uc tuosa Burimerster, sensitivity to wavelengths shorter than 380 nm was common to all regioiis. In the visible region, however, the dorsal part of the eye had a second peak at ^20 nm while the peak In the ventral part was at 550 nm (Ruck, I965). In Anax J uni us Drury the dorsa! region had 38O nm receptors vjhlle the ventral region had several receptors v/i th peaks In the region of ^^20-520 urn. The dorsal region of Libel lula necdhami Westfall was maximally sensitive to 4I0 nm while the ventral region had several receptors with peaks in the region of ^t50-550 nm (Horridge, I969). Bennett e_t aj_. (I967), working on the eye of Locusta miqratoria L. , used single cell recording techniques along with the method of equal response. All the cells from which they recorded had major peaks at '•1-30 nm and a peak of variable height at 515 nm. In this region of the spectrum tiie degree of sensitivity ranged between 15% and 100% of the sensitivity to ^+30 nm wavelength light.

PAGE 17

12 Three species of backswimmer, Noton scta j prorata Uhl,, N. ins ulata Kirby, and N. undulata Say were all found to have three receptors maximally sensitive to 520-530 rim, 370 nm and hlS nm (Bennett and Ruck, 1970). The eye of the cockroach, Perip laneta ame ricana L.,was investigated by .Mote and Goldsmith (1970). They recorded from single retinula cells v^hile presenting equal photon stimuli to the eye. Two receptor systems were found with maxiraal sensitivities at 365 nm and 500-510 nm. The eye of Calliphora erythrocephla, has been studied with intracellular techniques and equal quanta stimuli (Autrum and Burkhardt, I96I; Burkhardt, I962). All the reti nula ceil s in the dorsal part of the eye were found to be most sensitive to '189 nm light with a second peak of sensitivity at "^hS nm. The ventral part of the eye contained three types of retinula cells. Those were maximally sensitive at .'+63 nm, '61 nm and 52^ nm. All had second peaks at 3^5 nm. Spectral absorption of single rhabdomeres was measured with a mi crospectrometer by Langer and Thorell (I966). Fresh sections of the eye of the chalky mutant of Cal l iphora erythrocephla were mounted between quartz glass cover slips. The normal arrangement of the rhabdomeres was preserved enabling them to relate the absorption curves to specific cells. The six outer rhabdomeres had two absorption peaks, one at about 500 nm (main peak) and a second peak betvyeen 350 and 380 nm. in the central rhabdomere , the main peak was about ^60 nm, the second peak was between 350 and 38O nm. In all the rhabdomeres the main peak decreased as bleaching occurred while the UV peak increased in size relative to the main peak.

PAGE 18

13 The literature presented in this section represents only a small portion of the research which has been done on insect eyes. Additional literature will be cited in the Results and Discussion section so that the results obtained from the experiments performed on the mosquito eye can be compared to those obtained in similar experiments performed on other insects.

PAGE 19

MATERIALS AND METHODS Insects and Equipment The majority of the insects used in these experiments v;ere fourday-old adult female mosquitoes, Aedes aegypti . These were obtained daily from the colony maintained at the insects Affecting Man and Animals Research Laboratory of the United States Department of Agriculture in Gainesville, Florida. The mosquitoes in this colony have deeply pigmented eyes. The adult female white-eye mutants of Aedes aeqypti that were used in some experiments were obtained on an irregular basis from Dr. J. Seawright of the Insects Affecting Man and Animal Laboratory, Gainesville, Florida. Bhalla (I968) described the genetics and biochemistry which result in the whiteeye trait. The colony was maintained at 28-30°C and 60-70% relative humidity. The photoperiod v/as 8,5 hours light and 15.5 hours dark vjith the light period starting at 8:00 A.M. The mosquitoes were transported from the rearing room to the laboratory in a small holding cage made from a 20 cm long piece of 7.5 cm diameter plexiglass tubing with removable screening at the end. In the laboratory the mosquitoes were transferred to a larjer holding cage. To record the ERG complete immobl i zation of the mosquito was accomplished in the following manner: Microscope sli-des cut into 1.5 cm squares were coated with wax (Tempi I s t i ks , ^5°C melting point).

PAGE 20

15 which, after melting, would remain liquid at room temperatures until disturbed. Mosquitoes were chilled to facilitate handling and placed venter up on the slides. The proboscis was bent downward into .the liquid wax to hold the head steady. The wax solidified on contact, holding the mosquito firmly in place. The mosquitoes would remain alive for many hours following this treatment. In preliminary attempts to record the mosquito ERG, both glass capillary and chlorided silver wire electrodes were tried. All attempts with the glass electrodes were unsuccessful while the silver wire electrodes gave consistently good results. Silver vjires, 2.5 cm long, were electro-etched to a tip diameter of approximately 12 urn. The electrolyte used for etching was a solution of 30% NaOH saturated with NaCN. The best etching was achieved when using a range of 2-8 volts A.C. The electrodes were chlorided in a 3M KCl solution just prior to being used. Fresh electrodes were used for each preparation. The mosquito preparation was placed on a small platform located between two Leitz micromanipulators. The platform was an inverted plastic pill vial which had been cut to a height of about 2 cm. The cut was made at a slight angle so that, when in position on the platform, the mosquito preparation was tilted towards the stimulus source. The preparation was held onto the platform with a small piece of clay, A binocular microscope was suspended directly over the platform. The mounted mosquito was aligned so that the ommatidia of the central region of the eye were pointing directly at the stimulus source. Hypodermic needles (20-gauge) mounted on banana plugs were used

PAGE 21

16 to hold the electrodes. These plugs were fastened to short wooden dowels which were clamped securely in the instrument holders of the micromanipulators. Electrical connections between the electrodes and the amplifier were made through the banana plugs. The electrode placement was observed through the microscope. The ground electrode was inserted into the abdomen by forcing it through one of the intersegmental membranes of the venter. The recording electrode was centered in the compound eye facing the stimulus source. The eye deformed under pressure of the electrode making penetration of the corneal layer difficult. After penetration the electrode was withdrawn so that only the tip remained in the eye. The electophysiologi cal responses of the eye vjere amplified with a Bioelectric Instruments, Inc., NFl Neutralized Capaci ty Ampl i f ier. Except where noted the amplifier was always used at the minimum amplification (2X), With this setting the input impedance was greater than lo'' ohms and the band pass, which was set on the "maximum" posi tion, extended from DC to approximately 1 MHZ, Capacity neutral iza tion vjas not used since a 5 niV square wave was amplified without distortion. The output from the amplifier was directed simultaneously into a Tektronix 502A oscilloscope and a Moseley 7000A X-Y recorder. The recorder had a slewing speed of 20"/sec and an accuracy of 0.2 percent of the full scale reading. Monochromatic light was obtained with a Farrand Foci-Flex monochromator with a fixed si it width of 10 nm bandpass. Its spectral range was from 200-785 nm. For all the experiments, in \-yhich wavelengths greater than ^fOO nm was used, the light source for this instrument v^as a Bausch and Lomb microscope lamp with a 6v tungsten

PAGE 22

17 filament bulb. A 9X microscope ocular was used as a focusing lens at the exit slit of the monochromator (Figures 2 and 3). When UV light was required, a Bausch and Lomb high intensity UV monochromator, equipped vj\ th a super pressure mercury vapor light source, was substituted for the tungsten filament light source. The spectral output of mercury vapor lamps is continuous from 275 nm to the infrared region of the spectrum. Within the UV region characteristic emission lines occur at 302, 313, 33^, 365, 390 and 398 nm (Jagger, 1967). ' The exit slit of the UV monochromator was placed in line with, and approximately h cm away from, the entrance slit of the Foci-Flex monochromator. Transmission of stray light through the Foci -Flex was minimized by filtering the entrance and exit slit of the Foci Flex with a Corning CS 7-5^ colored glass filter ( 7l max = 325 nm, Xl/2 max = 250 nm and 390 nm) . The focusing lens vjas not used in conjunction with the UV light source. The mercury vapor lamp tended to vary in output; the beam undergoing sudden changes in energy level at irregular intervals. A photocell was used in an attempt to monitor these changes. The photocell was placed in a beam of stray light emitted from the base of the lamp housing. The output of the photocell was fed into a storage Oscilloscope. Shifts in the oscilloscope trace coincided with flicl
PAGE 23

Figure 2. Schematic diagram of the equipment used for recording the ERG of the mosquito. A-light source, B-monochromator, C-shutter, D-mirror mounted on track, E-slide wires with neutral density filters, F-radiometer probe, G-mosquito preparation, H-micromanipulator , l-amplifier, J-osci 1 loscope, KX-Y recorder.

PAGE 24

19

PAGE 25

1. o 4-1 (0 B O « IU) JZ U O 0) O c — I OQ >o c I. o M1 LU +-> O m L. o ^ 03 O Q. LU c O o 4-> OD c o 3 (U CT •o W c o 3 i u o 0) £ CD t. I. O lU o IjQ Mu O i_ •u 'i IX U) 1 in Q !_ (U +J s_ c o E Cl •o x: 1_ rr 1 Lu O 3 u.

PAGE 27

22 ftroughout the range of 280 to 2600 nm. Within these wavelength 6 limits energy measurements could be made within the range of 0-2.5x10 9-1 + ergs cm"'^ sec with an accuracy of _ 5% of the full scale reading. The probe of the radiometer was mounted in a fixed position normal to the monochromatic light beam. To obtain a measurement of the light energy a small back-surfaced mirror was moved along a track into the light beam, reflecting the light into the probe, A small front-surfaced mirror was substituted when UV light was being measured. The optical path length vias the same for the radiometer probe and the mosqu i to eye , A shutter was used to obtain light stimuli of known duration. It was made from a piece of black cardboard mounted on the end of a ]k cm length of 2 mm diameter wood dowel. The dowel was mounted in the shaft of a Brush Instruments penmotor which was driven with a Grass instruments Sk Stimulator. In the off position the black cardboard shutter was in front of the monochromator lens. A monophasic square wave from the stimulator drove the shutter upward, allowing the monochromatic light to pass for the duration of the square wave pulse. Either single or repetitive light stimuli of known duration could be obtained in this manner. Within the range of kOO-G^O nm, the energy of the monochromatic light was controlled with Kodak Wratten gelatin neutral density filters (No. 96). Filters of O.30 optical density (50% transmission) were cut into 2.5 cm squares. These were mounted on three separate slide wires so that they could be moved normal to the direction of the monochromatic light beam. The slide wires had one, two and three filters, respectively, so that when used singly or in combination six levels of transmission could be obtained between 50% and 1,56%.

PAGE 28

23 The neutr<3l density filters were not used in the UV region. Instead, a series of wire screens were placed singly between the B SL UV monochromator and the entrance slit of the Foci -Flex monochromator. The plane of the screen was normal to the light beam. The four screens used each had different mesh sizes and thus, differing percentages of trarismi ssi on. The screens were designated 1,2," 3, and k according to the transmission. All the transmission values were determined empirically Screen Percent Transmission 1 65 2 k9 t 36 % 31 1+^+ 20 by measuring the energy of the monochromatic light with and without the filter in place. This was done at 20 nm intervals within the range of 300-ifOO nm. In some experiments the stimulus source was a high intensity Xenon Flash (Strobelume Type No. 1532-B, General Radio Co., Cambridge, Mass.). The flash was triggered with the Grass Stimulator. A Tel
PAGE 29

2k Fi ] ters Descri ption CS 7-51 Maximum transmission = 365 nm; 1/2 transmission at 330 nm & 395 nm. CS 3-72 Sharp Cut Filter, Transmi ss i on > 80% from 5^1 nm 750 nm. Transmission = 37% between k3S kSS nm, Transmi ss i on < 0, 5% below ^11 nm. CS 3-76 Maximum transmission = 730 nm. 1/2 transmission = 575 rim. No transmission below 505 nm. The neutral density filters were manufactured by the Corning Glassworks and Optics Technology, Inc. The optical densities and percents transmission are listed below. Opti cal Densi ties Percent Transmission •3l 50 .5?" 31 .6""' 25 .9": 12.5 1 . 07"' 1 0 1.2""' 6,2 3.1 " Corning Glass V/orks. ""Optics Technol ogy J nc. The two interference filters (Bausch and Lomb) had second order peaks at 66O nm and 6O5 nm. The band widths at the 1/2 transmission points were equal to 22 nm and 16 nm, respectively. The 66O nm filter had a first order peak at 35O nm (as determined with a Bausch and Lomb spectrophotometer). By using the appropriate colored glass filter (CS 3-72 or CS 7-50 either the 66O nm or 350 nm peak could be filtered out independently of the other. The 553 nm filter was always used along with tie CS 3-/2 filter to eliminate any UV peak which may otherwise have been present. The IR blocking filter (Bai rd-Atomi c) had 80% transmission between 375 nm and 575 nm. The ha 1 f-transmi ssion points were at 3I5 nm and 680 nm.

PAGE 30

25 Experiments Two series of experiments were performed to determine the time required for the eye to dark adapt. In the first experiment, the main objective was to determine the time course of dark adaptation following exposure to the overhead lights in the laboratory. This was important since nearly all subsequent experiments were to be performed on dark adapted eyes. For this experiment the monochromotor was set for 5OO nm and a 25% transmission neutral density filter combination was moved into the beam. The energy incident upon the mosquito eye was O.'f x 10-^ erg cm sec . One-second stimuli were presented at set intervals after the room lights were extinguished. The omplitudes of the recorded ERG's were determined with all measurements made from the baseline to the peak along the rising side of the response. The occurrence of two or more successive responses of the same amplitude indicated that the sensitivity of the eye had stabilized. The eye was then considered dark adapted. White-eye mutants were tested in an identical manner to determine the influence of the screening pigments on the time-course of dark adaptation. The intensity of the room light was relatively low compared to the light intensities normally encountered by the mosquito in its natural environment. The room lights delivered only 0,5 0.8 x 10^ 5 1 erg cm ^ sec to the eye. The dark adaptation curve obtained in these experiments would only represent a portion of the curve of total adaptation since the eye was not fully light adapted at the beginning of the experiment. In order to completely light adapt the eye it was necessary to first determine the stimulus intensity re-

PAGE 31

26 quired to achieve saturation. At saturation, the response of the eye is at its maximum. Stimuli which saturate the eye supply the receptors with as many quanta as they can "use," Increasing the stimulus intensity beyond the saturation level would not increase the response amplitude. Exposure of the eye to the saturation level intensity vjould insure that the eye was fully light adapted at the onset of tte dark adaptation experiments. The following experiment was performed to determine the saturation level of the mosquito eye. The mosquito eye was set at the focal point of the Fresnel Lens-Viewlex projector combination. The mosquito preparation was shielded by a black cardboard housing which reduced the amount of stray light incident on the mosquito. The stray light came from the projector housing and from reflections off the surface of the Fresnel Lens. Two small apertures were made in the sides of the housing to accommodate the electrode holders, and one (2.5 X 2.5 cm) at the front of the housing for the passage of the light stimulus. The stimulus duration was controlled with the shutter which completely covered the front opening in the housing. The stimulus intensity was controlled with glass neutral density filters. To measure the stimulus energy the probe of the radiometer was placed in the housing in place of the preparation. The IR blocking filter was mounted in the front opening of the housing while the energy was measured. The limits of transmission of this filter roughly coincide with the limits of the determined spectral sensitivity of the mosquito eye. Non-excitatory wavelengths were therefore eliminated from the energy measurement. Glass neutral density filters

PAGE 32

27 (optical density = 0.3 or 0,6) were used to reduce the intensity to within the measurable limits of the radiometer. The mosquito eye was allowed to dark adapt for 30-^0 minutes (based on the results of the previous dark adaptation experiments). The eye vjas then stimulated with a 0.1 sec flash of each of several intensity levels of light. Glass neutral density filters were used to obtain the various test intensities. The log of the stimulus energy was plotted against the response amplitude to obtain an energyresponse curve. With stimulus energies below saturation, the energyresponse curve should be linear. As the saturation energy is approached the slope of the curve should decrease, reaching zero as the saturation energy is surpassed. In the saturation level experiments, several stimuli were presented at each energy level. The interval between successive stimuli was decreased In steps from 30 sees to 2.5 sec. The magnitude of the response decreased when the interval between stimuli was less than the minimum time required by the eye to recover from the previous stimulus. This procedure was repeated with all energy levels tested. The minimum time required for the eye to recover from stimuli of different intensities was determined in this manner. The procedure for determining the time course of dark adaptation from a saturated or near saturated condition was similar to the previous dark adaptation experiment. The equipment and its physical arrangement was the same c^s in the saturation level experiment. Mosquito eye preparations were light adapted with the Viewlex projector. Adaptation times varied between 1 min and 10 min. Several adapting energies were tried. These energies were obtained with

PAGE 33

28 neutral density filters used with and without the IR blocking filter. The test stimulus was a ,1 sec flash of white light from a tungsten filament microscope lamp. As with the adapting light, several stimuli of varying energy levels were tried. When the IR blocking filter was used during the light adaptation phase of the experiment it was also retained in place during the dark adaptation phase. To determine the time course of light adaptation, the mosquito preparation was dark adapted from room light conditions for 30-40 min. The eye was then exposed to a repetitive stimulus of 500 nm light (approximately 1.5 x 10^ erg cm~^ sec"') presented in 0.1 sec bursts at 1 sec intervals. After 10 minutes a tungsten filament adapting light was turned on. The energy of the unfiltered adapting light was 2x10^ erg cm"^ sec"' at the preparation. Neutral density filters with 1%, 10%, and 31% transmission were used to obtain different adapting light intensities. Changes in the magnitude of the repetitive ERG would coincide with the changes in sensitivity brought on by the adapting light. Since the time course of adaptation was known to be very rapid in other insects, the close spacing of test flashes was considered necessary so that transient events might be detected. Short duration test flashes (O.I sec) were used so that their adaptive effect on the eye would be minimized. The test flash energy level was large enough to elicit measurable ERG amplitudes even when the highest intensity adapting light was used. The method of equal -esponse was used to determine the spectral sensitivity of the eye. For this procedure the preparation was allowed to dark adapt for at least 30 min. In the visible region of the spectrum the wavelengths of monochromatic light tested were

PAGE 34

29 at 20 nm intervals between ^00 and 600 nm. In sonie of the first tests 650 nm light was also used. The test wavelengths were presented in order starting at either end of the range. Because of the time required to complete a test only one run was possible on each mosquito. Each test wavelength was presented at seven different energy levels obtained with gelatin neutral density filters. The duration of each stimulus was 1 second with a 30 second dark interval betv;een successive stimuli of the same wavelength. This procedure was modified for the UV tests. The stimulus duration was decreased to 0,5 sec with 15 sec elapsing between successive stimuli. Decreasing by 50% the time required to test each wavelength improved the chances of completing a test between energy changes in the UV source. When obvious energy changes occurred during a wavelength test the energy level was remeasured and the test was repeated. Sometimes these changes were so transient in nature that they went unnoticed during the tests but became apparent when the data was analyzed. Tests in the UV were conducted over 3OO-38O nm. Stimulus energy was controlled with the wire screen filters. For some of the tests the energy of the unfiltered monochromatic light was measured either just prior to or immediately after each test. In most cases, however, energy was measured both before and after the test with the average value being used for the calculations. The difference between the two measurements was usually no more than 0.1 X 10^ erg cm"^ sec"' regardless of the total energy measured. This variation is porbably inherent in the radiometer rather than in the light source except as noted previously for the UV source. The differences stemmed from drift in the instrument and rounding errors when the meter was read.

PAGE 35

30 Plots of the magnitude of the ERG as a function of the stimulus energy for each test v/avelength resulted In a series of energyresponse curves for each preparation, A level of equal response of the ERG v;ithin the range of the energy-response curves was chosen for each preparation. The equal response level vjas always chosen to transect the most linear region of the family of energy-response curves. The same level of equal response could not be used on each mosquito eye preparation due to the differences in sensitivity of individual preparations. Equal response energies for each test wavelength were then determined graphically. The equal response energy for any test wavelength was the stimulus energy required to elicit an ERG equal in amplitude to the chosen level of equal response The spectral sensitivity curve was obtained by plotting the reciprocal of the equal response energies, normalized to the 5^+0 nm value (360 nm in the UV tests), as a function of wavelength. Selective adaptation was used in an attempt to determine the number of receptor systems contributing to the spectral sensitivity curve, A tungsten filament microscope lamp was mounted on a ring stand and directed at the preparation. The light was slightly above and in front of the preparation. Interference filters (605 nm or 660 nm) were used to limit the spectral output of the adapting light. The intensity of the adapting light was controlled with a variable transformer. The mosquito eye preparation v^as first dark adapted. The eye was then repeatedly stimulated in the region of 490-500 nm. The intensity of the adapting light was slowly increased until a decrease in the amplitude of the ERG's (those resulting from the 490-500 nm stimuli) was observed. This procedure was adopted in the

PAGE 36

31 hope that an adapting energy could be found which would have a minimal effect on any short wavelength receptor and a maximal effect on a long wavelength receptor. The spectral sensitivity curve of the eye under the continuous influence of the adapting light was then determined. Two preparations, one adapted with the 605 nm interference filter and the other adapted with the 660 nm interference filter, were tested at 20 nm intervals between ^00-600 nm. Three preparations were tested at 10 nm intervals between 460 and 56O nm while dark adapted and then retested while under the influence of the 660 nm adapting light. Based on the results of the spectral sensitivity experiments it seemed necessary to check the possibility that the eye had separate UV and visible light receptor systems. If separate systems were present, the response of the eye to a constant energy UV stimulus would remain constant while the eye was adapted to wavelengths in the visible region of the spectrum. Conversely, if the receptor systems were "linked" or one in the same then they would not be independently adaptable. In this experiment the UV stimuli were obtained by using the Xenon Flash in conjunction with the 66O nm interference filter and the CS 7-51 colored glass filter. This filter combination allows transmission only in the region of the first-order transmission peak of the interference filter ( A max = 350 nm) . The emission of Xenon is continuous throughout the visible and UV regions of the spectrum down to 300 nm. There is essentially no emission below 300 nm (V/yszecki and Stiles, I967). The adapting light was a tungsten filament bulb (Ziess microscope lamp) filtered with the CS 3-72 cutoff filter. This filter only transmits wave-

PAGE 37

' 32 lengths longer than about ^1-20 nm. For this experiment it was desirable to have both the stimulus and the adapting light reach the eye on the same optica] axis. To achieve this a 0.3 neutral density filter was used as a beam splitter. The strobe light was positioned directly in line with the eye. The neutral density filter was mounted on this line at a '45° angle. A 30 cm long glass fiber optic light pipe was used to conduct the adapting light to the preparation side of the neutral density filter. By adjusting all the angles it was possible to have both lights reach the eye on the same optica] axis. The UV was transmitted through the neutral density filter while the adapting light was reflected off its surface. The strobe light was colli mated with a cone made of several layers of heavy brown paper. The base of the cone fit snuggly over the strobe light. The apex was cut off leaving an opening of 5 cm. The 66O nm and CS 7-5' filters were mounted on the beam splitter support. V/hen the apex of the cone was set flush against these filters there was no evidence of stray light. The intensity of the adapting light was controlled with neutral density filters placed between the light source and the fiber optic light pipe. The mosquito was allowed to dark adapt for at least 30 min. The UV stimuli were presented at first with no adapting light and then with increasing intensities of adapting light. The responses were observed and measured with the storage scope. The amplification was changed for each preparation so that the responses appeared large enough on the screen of the oscilloscope to be easily measured.

PAGE 38

RESULTS AND DISCUSSION The response of the eye to a short duration light stimulus was a rapid monophasic negative-going potential which tended towards a steady state as the response maximum was neared. At the end of the light stimulus, the return of the potential to its prestimulus level was accomplished in tvjo phases — a rapid positive-going potential followed by a slowly decay i ng after-potenti al . The ERG recording in Figure 4 was typical of the response to a 1 sec stimulus in all regions of the spectrum tested. There was no evidence to suggest that wavelength information is in any way encoded in the ERG waveform. In some insects, such as the worker honeybee (Goldsmith, I960), the whirligig beetle, Dineutes ciliatus (Bennett, I967) '^nd the butterfly, H eliconius erato hydara Hewi tson (Swihart, 19^3) the waveform of the ERG varies depending on the wavelength of the stimulus. The sources of these variations are multiple receptor systems with different spectral sensitivities. The uniformity of the ERG waveform in the mosquito suggests that only one receptor system is present. At the start of the dark adaptation experiments, with the preparation exposed to constant fluorescent room lighting, the potential of the eye remained constant. When the room lights were turned off, the response was a positive-going potential the waveform of which was very similar to the light-off portion of the ERG, The maximum positive potential was reached within the first 15 sec of dark adapta33

PAGE 39

Figure k. A typical recording of an ERG from the dark adapted eye of A. aeqypti . The stimulus duration was 1 sec. The energy was adjusted to give a lOniV response. The v;aveform was similar for all stimulus wavelengths between300 nm and 65O nm.

PAGE 40

35 ON OFF

PAGE 41

36 tion. The extent of the positive deflection varied from one preparation to another with a range of 5-11 mV. Several factors contributed to these differences in sensitivity, the most important of which is probably the damage done on insertion of the electrode into the eye. Electrode depth and angle of penetration may also be involved. Physiological differences between mosquitoes are another possible factor. As the eye dark adapted, the amplitude of the responses to the test flashes {Q.k x lO-' erg cm sec , 500 nm, 1 sec duration) increased, reaching a maximum in about 30-^0 minutes. For each preparation, the amplitude of the ERG's were measured and normalized to the amplitude of the maximum response. In Figure 5 tlie means of these values for five preparations are plotted as a function of total elapsed time. The curve is smooth and continuous with no indication of a second phase of adaptation as found in certain nocturnal Lepidoptera with migratory screening pigments. The results obtained from the experiments on the white-eye mutants are presented in Figure 5 (b) , The similarity of the dark adaptation curves of the darkand white-eyed mosquitoes may indicate one of two things; either screening pigments do not play a significant role when the eye dark adapts following exposure to room lighting (overhead fluorescent lighting; total energy at the insect eye was 0.5 0,8 x 10^ erg cm"^ sec"') or there is no discernible latancy between the onset of darkness and the initiation of screening pigment migration. The latter explanation seems the more likely since the axial migration of screening pigment is known to occur in the primary pigment cells of mosquitos (Mazokhi n-Porshnyakov, 1969). The effect of the pigment

PAGE 42

Figure 5, Time-course of dark adaptation followinq exposure to room lighting (0.5 0,8 x 10^ erg cm-2 sec"'). The maximum response was obtained when the eye was fully dark adapted. a) Dark adaptation curve for five dark-eyed mosquitoes. b) Dark adaptation curve for two white-eyed preparations.

PAGE 43

38 Elapsed time minutes

PAGE 44

39 might be to limit the rise time of the adaptation curve. The vjhiteeye mosquitoes seem to achieve a greater percent maximum response sooner, after lights out, than the dark-eye mosquitoes. The results of the saturation level experiment are shown in Figure 6. The linearity of the energy-response curves indicated that saturation was not achieved. The highest energy level used was 5.5 X 10^ erg cm"^ sec"'. Since the stimulus energy was measured through the IR blocking filter, only those wavelengths which could effectively stimulate the eye contributed to the total energy measurement. This was based on spectral sensitivity curve for A. aeqypti which will be presented later in this section. When the 6 9-1 stimulus energy v;as 5,5 x 10 erg cm"'' sec the illumination incident upon the eye was 2,6 x 10^ lux. This measurement was made with a Salford Electronic Instruments Photometer. The illumination on a clear sunny day is only 10^ lux (LeGrand, 1957). Under even the brightest of normal conditions the eye of A, aeqypti would not be saturated. . , Curves of the spectral distribution of the energy eminating from tungsten filament bulbs at various color temperatures are readily available. A polar planimeter (K and E) was used to measure the area under a 3,000° K color temperature spectral distribution curve. Only the area between 315 nm and 680 nm was considered since these are the transmission limits of the IR blocking filter. The wavelength at the midpoint of this area was 557 nm. By assum'ing thar all the energy was at 557 nm, the number of quanta in the stimulus can be calcul a ted.

PAGE 45

Figure 6. Energy response curves based on ERGs recorded for the saturation ievel experiment. Upper curve (closed circle) and middle (open squares) curve are based on ERG recordings from the center of the eyes of two different preparations. Lower curve (closed squares) is based on ERG's recorded from peripheral ommatidia of the same preparation as used for the middle curve.

PAGE 46

1

PAGE 47

, k2 The energy of a single quantum can be determined with the equation: , , where E is the energy in ergs, h is Plancks constant (h = 6,625 x 10"27 e,-g sec), C is the speed of light (2,99 x lo'° cm sec'M and A is the K'ave 1 ength of the quantum in centimeters. By substituting the values into equation 1 wi th ?\ = 557 x lO"^ cm, the energy was determined to be 3,51 x 10"^ ^ ergs quanta"'. The highest energy stimulus was 5,5 x 10 ergs cm"'' sec" ' , Therefore the quanta! flux at the. surface of the mosquito eye was: _5^_x 10^ ejqs cm-2 see"' ^ ] , 54 x lo'^ quanta cm"^ sec'K 3,51 X 10-'2 ergs quanta"! During the 0,1 sec stimulus, the eye was exposed to 1 . 54 x 10^^ quanta cm , The average area of a facet of the compound eye in 2 female A, aeqypti is hjk/-'(Christophers, I960). Each facet oriented perpendicular to the direction of the stimulus received 7.3 X 10' ' quanta. In houseflies and cockroaches the visual pigment present in the rhabdomeres has been extracted, characterized and quantified. Houseflies were found to have 3.7 x 10^ molecules of retinal per rhabdomere, the cockroach (P eriplaneta americana ) had 4.3 x 10^ molecules of rhodopsin per rhabdomere. The visual pigment concentrations in insect rhabdomeres compare quite well with the range of 10^ 10^ rhodopsin molecules found in vertebrate rods (Wolken, 1971) In view of the fact that the number of visual pigment molecules per receptor cell is quite comparable for even very diverse forms, it would be reasonable to estimate that a mosquito has about 10^ visual pigment molecules per rhabdomere.

PAGE 48

The total number of visual pigment molecules per rhabdom v;ould o be 8 times this or approximately 10 molecules. This Is of interest since the eye did not saturate when stimulated with approximately 7-3 X lo'' quanta per ommatldium. The quanta apparently outnumbered the visual pigment molecules by a factor of 10^. The insect eye is generally considered to be a one-quantum receptor which means that the absorption of one quantum of light by one visual pigment molecule Is enough to Induce a physiological event In the receptor (Reichardt, 1965; Scholes, 1965). The capacity to respond to a single quantum has also been demonstrated in the compound eye of Limulus (Fourtes and Yeandle, \S6k) . There is, at present, no evidence to suggest that single quantum sensitivity Is not also characteristic of the mosquito visual system. If so, then the efficiency of the mosquito eye Is considerably less than 100%. That is, only a small portion of the quanta incident on the cornea ever interact with the visual pigments In the rhabdom. Quanta! losses can occur In several ways. The corneal layer of the eye accounts for only a negligible loss. Spectral transmission studies on the corneal layer of the housefly (Goldsmith and Fernandez, I968) and a dragonfly, Aeschna cyanea Mull, (Kolb et £]_. , 1969) , indicate that with wavelengths longer than about 350 nm the transmission Is around 95%. Many Insects, particularly nocturnal forms, have corneal nipples which increase the transmission by reducing the amount of surface reflection (Bernhard £1.11-. 1965). The housefly does not have corneal nipples. It may be assumed that the dragonflies also do not have corneal nipples since these are usually not present In strictly diurnal forms. The mosquito however does have cornea] nipples which should increase the

PAGE 49

transmission to more than the S5%, characteristic of the dragonfly and housefly. Clearly, the corneal layer is unlikely to be responsible for any appreciable loss of quanta. V/ithin each ommatidium the rhabdom is sheathed in cells containing screening pigments. Even the retinula cells, outside of the rhabdomere area, contain pigment granules. The presence of these screening pigments suggests that some of the light passing through the corneal lens is not focused on the rhabdom but passes into the receptor layer of the eye as stray light v/hich is absorbed by the pigment granules. The amount of stray light emanating from the corneal lens and crystalline cone of the tobacco hornv/orm has been estimated to be 20?^ of the light normally incident on the lens (Miller et^ aj_, , I968). A similar situation may exist in the mosquito. In addition, the cone cells of the mosquito have cytoplasmic extensions which terminate in pigment filled sacs located just proximal to the rhabdom. It is tempting to compare these to the crystalline tracts of other insects (Doving and Miller, I969) . If the termination of the cone cell extensions in light absorbing pigment filled sacs is analogous to the termination of the crystalline tracts at the rhabdom, then the function of the cytoplasmic extensions might be to control the amount of light reaching the rhabdom under bright light conditions by siphoning a portion of the light out of the cone cell. This hypothesis may not be valid since the crystalline tracts of Lepidoptera are h-]0 u In diameter (Miller et_ aj_. , I968), while the cytoplasmic extensions of the mosquito cone cells are only .3-. 4 u in diameter (Brammer, 1970). Pending an analysis of the optics of the mosquito eye, particularly measurements of the refractive index

PAGE 50

of the cone cells and their extensions, it is not possible to do any more than conjecture about their possible function. It is apparent that many quanta never reach the rhabdom of the eye even when they arrive parallel to the ommatidial axis. The quantal loss is even greater when the light is at an angle to the ommatidial axis. Doving and Miller (I969) list half-power beam widths for a number of insects as reported by various authors. As a point source is moved off the ommatidial axis, the sensitivity of the ommatldium drops. The half-power beam width is the angular displacement which corresponds to half sensitivity. The values listed for several insects with apposition eyes fall at about 6°-7°. Again while there is no mosquito data available it seems reasonable to suppose that a similar condition exists in the mosquito eye. In the saturation level experiments the mosquito v/as aligned so that the stimulus would be normal to the centrally located oiTimatidia. The axes of the peripheral ommatidia (at the medial margin of the eye) were about 50° out of alignment with the source. This giaatly exceeds the presumed half-power beam width. Yet, when the electrode was placed at the periphery, the responses were of nearly the same amplitude as those recorded with the electrode centrally located in the same eye (Figure 6). Because of the limited angle of acceptance, it is doubtful that sufficient quanta entered the peripheral ommatidia via the corneal lens to account for the amplitude of the responses. Two factors may have contributed to the peripherally recorded ERG. First, the screening pigments may have allowed sufficient leakage of stray light within the receptor layer of the eye, and perhaps oven within the head capsule, to stimulate

PAGE 51

46 peripheral ommatidia which were not subject to direct illumination. Second, the insect ERG is the mass response of many ommatidia, not only those located in the immediate vicinity of the electrode. The amplitude of the peripherally recorded ERGs was related to the amount of light leakage in the eye and the electrotonic spread of potential from the centrally located ommatidia. Due to the potential spread it might be difficult to detect saturation in the central region of the eye vihWe large numbers of off-axis ommatidia were not saturated. The contribution to the centrally recorded ERG, from the unsaturated peripheral region of the eye, would increase as the stimulus energy increased. The final point regarding the loss of quanta is that the visual pigment does not have the same probability of interaction with quanta of different wavelengths. Since the actual stimulus was of a broad band nature, many of the quanta that do enter the rhabdom pass through without being absorbed. Scholes (1965) recording from single retinula cells in the locust eye found that the voltage response saturated at light intensities which would induce the photolysis of only 10^ visual pigment molecules. It is not possible to infer from Scholes' data what quanta! flux was required at the corneal surface to saturate the receptor. It is interesting to note thatjn connection with another experiment reported in the same paper, he states that "the disparity between available quanta and yield of responses' is di scouragi ngly high," This would indicate that very few visual pigment molecules were bleached in relation to the number of quanta arriving at the corneal surface. In studies with the locust eye Shaw (I968) reported

PAGE 52

^7 that the most sensitive retinula cell measured had an efficiency of 8%. Other cells measured v;ere as little as one-tenth as sensitive. It was not possible to determine the time-course of dark adaption from saturation since the saturation level of the eye was not found. Prolonged exposure of the eye to the highest intensities used in the saturation experiment resulted in death of the preparation, apparently the result of heating. Out of approximately 10 attempts made at light adapting with slightly lower energies, only one was successful. In this experiment, the mosquito was adapted 6 ? 1 for 5 minutes with 2.2 x 10 erg cm~^ sec"' of light transmitted through the IR blocking filter. The intensity of this adapting light was greater than the room light intensity by a factor of roughly lO^, The IR blocking filter was used to eliminate the IR radiation which was thought to be responsible for the heating of the other preparations. The test stimulus was 2.8 x 10^ erg cm~^ sec"' of white light emanating from a tungsten filament bulb and delivered to the preparation through the Fresnel lens and the IR blocking filter. The dark adaptation curve vjas similar to those previously described and provided no new insight into the problem of screening pigment migration (Figure 7). The minimum time required for the mosquito eye to recover from 0.1 sec flashes of different intensities of light was determined. The recovery time following the lower energy stimuli (0.35-1.38 x 10 erg cm sec"') was only five seconds. Following the most energetic stimuli (2,75-5,5 x 10^ erg cm"^ sec"') the recovery time increased to 10-15 sec. The effect on the eye of relatively short light flashes was qualitatively different from the effect of longer

PAGE 53

Figure 7. Time course of dark adaptation fol lowing five minutes of light adaptation with 2.2 x 10 ergs cm"2 sec"' of v/h i te light.

PAGE 54

^9 10 20 30 40 SO Elapsed time minutes

PAGE 55

50 exposures to light. Exposure to room lighting for several hours induced a more stable change in the sensitivity of the eye than 3 exposure to short duration flashes of light 10 times more energetic. The eye required 30-^0 minutes to dark adapt following room light exposure, v;hile recovery from short flashes required only seconds. The specific mechanisms underlying these differences are not i
PAGE 56

0) Jl M cn +J in u 0) 0) 4J o :^ o c cn o i_ (U X u c u Q) CM 0) i-J c -Q 0) (TJ a 4-1 in. — c 1 > o O o s WCM (D OJ Ol V C 1
PAGE 57

52

PAGE 58

53 initial suppression under the influence of the adapting light. It is tempting to attribute the positive baseline shift to migration of the screening pigment into a light adapted position. This explanation is weakened, if not entirely negated, by the fact that the same effects vjere seen in a white-eye mutant. Thus, the role of the migratory screening pigments in light adaptation remains obscure. The spectra] sensitivity curve For the visible portion of the spectrum is presented in Figure 9. The curve is based on the spectral sensitivity curves of 7 mosquitoes. The spectral sensitivity curve peai
PAGE 59

9 lit a J3 0) M a > M 3 u o D 0) c JZ I— l/l (U I. E i_ o 0) (J Q. (/I rt (0 (U JC > 4-* Mx: o fC o in CD 0) (U o 1+J in • cr +j j3 in c O e o Q. > c jC > o t-l in (U 1_ 4o o O M>uc > l_ > U +-> 0) >in jr c +j (U > in o M OJ U w i_ o c M u (U o 01
PAGE 60

55

PAGE 61

56 intersecting the most points in the scatter. This problem made it difficult to determine the validity of the data with respect to the actual spectral sensitivity of the mosquito. Even if poorly representative, the data still show that the mosquito eye is sensitive to UV radiation v-jith the peak occurring somewhere within tlie range of 330-360 nm. At best, the data indicate that there are two peaks in the UV at 330 nm and 369 nm (Figure 10). All the preparations had these two peaks, so they may be characteristic of the true UV sensitivity curve. The occurrence of a single peak of sensitivity in the UV is v;ell known for many Insects. Two peaks, hov/ever, have not previously been reported. This is not to say that there is no basis for assuming the validity of the two peak outcome of these experiments; there is evidence that two separate UV receptor systems may be present in the mosquito eye (Kay, I969). Further discussion of this will be presented later in this section. The 360 nm and 5^0 nm energy-response curves were used to determine the energy that would be required to evoke a 9mV ERG with stimuli in these regions of the spectrum. The 9mV responses would have required 0.23 x 10^ erg cm"^ sec"' at 360 nm but only 0,1 x 10^ erg cm sec at 5*10 nm. The eye was approximately 50^ as sensitive at 360 nm as it v/as at 5^0 nm. ' The shoulder located at 500 nm suggested that two receptor systems with different absorption maxima were present in the eye. Selective adaptation experiments were undertaken to test this hypothesis. Two preparations were tested at 20 nm intervals while under the influence of a red adapting light (6O5 nm and 66O nm, respectively). The spectual sensitivity curves for these preparations had a peak at 520 nm. and a shoulder in the 56O-58O region of the curve.

PAGE 62

Figure 10, Spectral sensitivity curve for the UV region of the spectrum. The curve is based on the spectral sensitivity of four mosquitoes. The vertical lines indicate the .•: standard error of the mean for each point.

PAGE 63

58 Wavelength (nni)

PAGE 64

59 Three dark adapted preparations vjhich had been run at 10 nm intervals between kSO and 56O nm were retested under the influence of a 660 nm adapting light. The data for these tests is presented in Figure ]]. The major effect of the adapting light was to depress the height of the entire curve. There was also a slight change in the relative heights of the peaks. Both peaks are the same height in the 660 nm adapted state while, in the dark adapted state, the 5^0 nm peak was greater than the 5IO nm peak. The results of these selective adaptation experiments were not consistent with the expected results of a two receptor system with widely separated >\ max. Other investigators using selective adaptation have obtained dramatic changes in the shape of the spectral sensitivity curve which clearly indicated the presence of independent receptor systems with different A max. The successful application of this technique has been reported by Bennett (I967), Bennett and Ruck (1970) and Goldsmith (I96O). These examples serve to illustrate the usefulness and power of selective adaptation in the identification of multiple receptor systems in the eye. The experimental results of selective adaptation of the mosquito eye indicate that a single receptor system is responsible for the spectral sensitivity curve in the visible region of the spectrum. It might be argued that the spectral distributions of the adapting lights were such that with two receptor systems present, both would be effected and selective adaptation would not be achieved. This does not appear to be the case. Assuming that these hypothetical receptors had absorption maxima at 500 nm (general region of the shoulder) and 5^0 nm (peak of the spectral sensitivity curve), it

PAGE 65

-= 1 Figure 11. The effect of selective adaptation on the spectral sensitivity curve, a) Spectral sensitivity curve of three dark adapted preparations, b) Spectral sensitivity curve of the same three preparations light adapted v;i th 660 hm light. The preparations were actually much less sensitive in b than in a. '

PAGE 66

Wavelength (nm)

PAGE 67

62 is difficult to visualize an effect of the adapting lights on both receptors, especially v/hen the 660 nm interference filter vvas used. For this filter, the half transmission point on the short wavelength side of the peak was at Skk nm. The transmission was approximately 1% at 600 nm. It is not likely that thi s adapti ng would effect both of the hypothetical receptor systems to such an extent that selective adaptation vjould not be achieved. in most of the insects which have been studied, selective adaptation or single cell recording techniques have revealed that the UV and visible receptor systems are independently adaptable. An apparent exception is Calliphora ery throcephal a . In this insect the spectral sensitivity curves of single retinula cells have two peaks, one in the visible and another in the UV. These peaks are not independently adaptable (Burkhardt, 196^+).. .. The situation in A. aegypti is similar to that found in C al 1 i phora in that adaptation with visible light resulted in suppression of the UV sensitivity. This was demonstrated in the series of experiments in which the eye was stimulated with constant energy UV stimuli while the intensity of the purely visible adapting light vjas increased. The relative amplitude of the UV stimulated ERGs are shown plotted in Figure 12 as a function of the adapting light energy. It is very clear that the UV sensitivity decreased as the adapting light intensity increased. This effect could not liave been due to any UV in adapting light since this was filtered with a CS 3-72 cutoff filter wiiich blocks all light with wavelengths shorter than around ^20 nm. The purity of the UV stimulus was ensured by using the 660 nm interference filters along with the CS 7-5I colored glass

PAGE 68

C o a) u) 1/1 U j-i c i_ o tu ^Q. CL O in lU 03 in L. 4-" l/l . — (U 13 _c (/) 4-> -c (U jr M MlU 10 o (/I 3 (U > 1_ Ol =3 Q. c c O >o (U > c o 4-1 0) c c G 0) (1) 4-J -C 11/1 4J OJ <+c Q. c o '4X u o (!)

PAGE 69

gprn ! [dLUe osuodsaj /\n

PAGE 70

filter. The peak of transmission for this filter combination was at 350 nm. There was no transmission of wavelengths longer than 405 nm. The decrease in UV sensitivity could only have been due to the adaptation vyith visible light. It might be hypothesized that the decrease in UV sensitivity v;as due to some sort of inhibitory mechanism. This would mean the adaptation of one receptor system would inhibit the response of a second receptor system in this case, a visible receptor inhibiting a UV receptor. If it is valid to generalize from the results of experiments performed on migratory locusts, inhibition does not explain the results obtained vj\th mosquitoes. In the locust eye no Interaction between ommatidia was found (Tunstall and Horridge, 1967; Shaw, I968). In addition, it vyas shown that retlnula cells within a single ommatidia function independently of each other (Shaw, I968). This latter point is supported by two independent studies on the cockroach eye. Walther (1958) used selective adaptation to reveal two independent receptor systems with peaks In the UV and visible. This was confirmed by Mote and Goldsmith (1970) who then showed that the UV and visible receptors occurred in different retlnula cells within the same ommatldlum (Mote and Goldsmith, 1971). If inhlbitive interaction between retlnula cells were the rule In Insect compound eyes then V/alther would have obtained results comparable to those obtained with the mosquito. The characteristics of the mosquito's visual pigment could explain the results of the spectral sensitivity and selective adaptation experiments. All the visual pigments which have been chemically extracted and characterized are composed of a retinal (Vitamin A

PAGE 71

66 aldehyde) prosthetic group associated v;ith an opsin protein. The retinal may be either of two types; retinal^ v/hich is derived from Vitamin A or retinal which is derived from Vitamin A . Retinal 1 2 2 2 differs from retinal^ in that it has an extra double bond in the terminal 6 carbon ring (Dartnall, 1962). Any opsin may be conjugated with either retinal or retinal . The ^ max of a retinal based ' 2 visual pigment is at a longer v^avelength than if the same opsin were associated with a retinal^ prosthetic group. The spectral position of the }) max is also affected by the opsin. The range of max for retinal I based pigments extends from 430 nm to 560 nm v;hile the range for retinal^ based pigments extends from 510 nm to 620 nm (Dartnall, 1962). The spread of /j max is due to the differences in opsins from different animals and from different visual receptors within the same animal. The visual pigments of only a few insects have been extracted and characterized. Visual pigments with /) max in the visible region of the spectrum have been extracted from the honeybee, housefly and cockroach (V/olken, 1971). Retinal^ was found in all three insects while retinal^ was found only in the housefly (Wolken, 1971). A UV sensitive pigment with /)max at Z^S nm has been extracted from the eye Ascalaphu s macaroni us Scop. (Goga 1 a et^ aj_. , 1970). The prosthetic group of this pigment was identified only as retinal with no mention made of whether it was retinal ^ or retinal^. The major peak of the mosqui to spectral sensitivity curve is at S'^O nm which :s within the spectral range of both retinal^ and retinal^ based visual pigments. This fact, together with the occurrence of both retinal, and retinal 1 2 in dipterous insects, makes it virtually impossible to determine,

PAGE 72

67 from the spectral sensitivity curve, which retinal is involved in the mosquito visual pigment. B rammer and White (1968) raised A, aegypti on a vitamin A and B-carotene deficient diet. They reported a reduction in the amplitude of the ERG but not its complete elimination indicating that some vitamin A was still available to the mosquitoes. Brammer and Whites' experiment supports the hypothesis that the m.osquito has a retinal based visual pigment. Their study sheds no light on the problem of whether retinal j or retinal2 is the visual pigment prosthetic group in A, aegypti . . ' ' ' All knovjn retinal ^ based visual pigments have the same shape absorption spectra in the visible region of the spectrum (Dartnall, 1962). The shape of the absorption curves of retinal^ pigments are slightly broader than the curves of the retinal ^ pigments but otherwise are very similar (Munz and Schwanzara, I967). The shape of the mosquito spectral sensitivity curve can not be duplicated with absorption curve constructs based on the Dartnall nomogram (Dartnall, 1962) or the nomogram for retinal^ based visual pigments (Munz and Schwanzara, I967). The visual pigments have smooth absorption curves v;hile the mosquito spectral sensitivity curve has a shoulder centered around 5OO nm. This shoulder is apparently due to absorption by the screening pigments in the mosquito eye. These pigments are ommochromes and pterins (Bhalla, I968). In general, these types of pigments have absorption bands in the visible region of the spectrum with > max in the region of 500-550 nm (Goldsmith, 1964; Hoglund et al., I970). The spectral sensitivity curve would be depressed in the region of the screening pigment absorption bands, when compared to the shape

PAGE 73

68 of the absorption curves of the visual pigments. Therefore the peak of the spectral sensitivity curve may not coincide with the absorption peak of the visual pigment. The ^ max of the visual pigment may be at some wavelength shorter than 5^0 nm, perhaps around 520 nm530 nm. All retinal based visual pigments with absorption bands in the visible region have secondary absorption bands in the UV region of the spectrum. These bands are called cis bands or cis peaks. The CIS peak height is 20%-30% of the height of the main absorption band. Absorption of light in the cis peak region is effective in bleaching the visual pigment (Dartnall, 1962). in many insects the UV and visible peaks of spectral sensitivity are due to separate pigment systems. In the mosquito eye there is apparently only one visual pigment. The UV sensitivity must be due, at least in part, to cis band absorption. The fact that the UV and visible peaks in the mosquito eye cannot be independently adopted supports this hypothesis The ability of aphakic humans (without lens in the eye) to see UV light has been well documented (Wald, 19^+5; Dodt and Walther, 1958; Burian and Ziv, 1959). This ability demonstrates that cis band absorption can result in meaningful visual responses. The expected separation between the '/Vmax of the main absorption peak and the ^ max of the cis peak is 9,700 t ^00 wave numbers (Dartnall, 1962). Assuming that the major absorption peak of the mosquito visual pigment is located at 530 nm, then the cis peak should occur at 350 nm. The nearest peak is at 36O nm. The relative sensitivity at 350 nm is approximately 50% of the 36O nm maximum or 25% of the 5^0 nm maximum. This last figure is based on the fact that the sensitivity

PAGE 74

69 at 360 nm is only about 50% of the 5^+0 nm sensitivity. Dartnall (1962) stated that cis peak absorption was 2070-30% of the main peak absorption. The mosqui to eye sensitivity to 350 nm agrees we] 1 wi th this estimate. In general, the absorption curves of cis bands are broad and rather flat. The shape of the mosquito UV spectral sensitivity curve is not typical in this respect. This may be the result of the existence of non-retinal UV sensitive material within the eye. Kay (1969) found UV absorbing fluorescent material in the eyes of all the insects he examined. This material was present within the retinula cells. Kay presented the excitation emmission spectra of these materials from a number of different insects. Extracts from the mosquito eye ( Culex ) were maximally excited at 292 nm and 364 nm. The emission peak of the fluorescent material was at ^+50 nm, Kay's hypothesis was that this non-retinal system augments the UV sensitivity of the eye. The energy absorbed in the UV region is transferred to the retinal based visual pigment via the fluorescence at nm. if the system does in fact operate in this manner, then the UV vision in the mosquito eye v/ould be mediated by both cis peak absorption and this fluorescent material. This would explain why the UV and visible sensitivity peaks \-iere not independently adaptable. Both modes of UV sensitivity are ultimately due to the single retinal based visual pigment. The 36O nm peak in the spectral sensitivity curve is in close agreem.'^'nt with 364 nm excitation peak in extracts from Culex eyes. Mo excitation peak was found in these extracts which would correspond to the 33O peak in A. aeqypti . However, this does not preclude the existence of an appropriate fluorescent material in A, aeqypti eyes.

PAGE 75

/ ; • . CONCLUSIONS 1) The ERG of A. aegypti was a monophasic negative viave of potential. At the onset of a light stimulus the baseline underwent a rapid negative shift. Following the stimulus the return to the baseline was accomplished in two phases; a rapid positive-going phase followed by a slov/ly decaying after potential. There vjas no indication that wavelength information v/as in any way encoded in the ERG waveform. ' ; 2) The dark adaptation curves for darl<-eye and white-eye mosquitoes were very similar. Approximately 30-^0 minutes were required for the eye to dark adapt following exposure to room lights (0.5-0.5 X 10 erg cm"'^ sec" ) . The migratory screening pigments do not appreciably affect the time-course of dark adaptation. 3) The saturation level of the eye was not found. V/ith the highest intensity stimuli the eye received 7-3 x lo'' quanta per 8 cmmatidlum. Since each ommatidium contains only 10 visual pigment molecules, the efficiency of the eye must be very low. Quanta losses could have occurred at all levels along the optical pathv^ay within the ommat i d i a. 4) The eye required only 10-15 sec to recover from 0.1 sec high intensity flashes of light (energy = 5.5 x 10^ erg cm"^ sec"'). When this is compared with the 30-'40 minute period required for the eye to dark adapt following prolonged exposure to room lights, it 70

PAGE 76

71 becomes apparent that different mechanisms are involved in the generation of the ERG and light adaptation. 5) Light adaptation occurred in two phases. When the adapting light came on the baseline shifted rapidly in a negative direction, at the same time the sensitivity of the eye decreased. These effects occurred within the first second of light adaptation. During the next 50 sec, the baseline shifted in a positive direction and the amplitude of the ERG responses to the test stimuli increased slightly. 6) The spectral sensitivity curve for A. aegypti had a major peak at S^fO nm. Two smaller peaks were located at 36O nm and 330 nm. A single retinal based visual pigment mediates vision in A. aegypti . The 7\m3x of the visual pigment probably does not coincide with the 5^+0 peak of the spectral sensitivity curve due to the filtering effect of the screening pigments. The UV sensitivity is a result of cis band absorption by the retinal based pigment and absorption by a UV excited fluorescent material in the retinula cells.

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LITERATURE CITED Autrum, H., and D. Burkhardt. I96I. Spectral sensitivity of single visual ce lis. Nature 190:639. Bennett, R. R. I967. Spectral sensitivity studies on the win i rl i gi g beetle, Dineutes ci 1 i atus . J. Insect Pliysiol. 13:621-633. Bennett, R. R. , and P. Ruck. 1970. Spectral sensitivities of darkand 1 i glit-adapted Notonecta compound eyes. J. Insect Physiol. 16:83-88. ' • Bennett, R. R. , J. Tunstall, and G. A. Horridge. I967. Spectral sensitivity of single retinula cells of the locust. Z. vergl. Physiol. 55:195-206. Bernhard, C. G. , W. H. Miller, and A. R. Moller. I965. The insect corneal nipple array. Acta Physiol. Scand. 63(Suppl. 2^3): 1-79. Bernhard, C. G. , and D, Ottoson. 1960a. Comparative studies on dark adaptation in the compound eyes of nocturnal and diurnal l.epidoptera. J. Gen. Physiol. kk:]33-203. Bernhard, C. G., and D. Ottoson. 1960b. Studies on the relation between the pigment migration and the sensitivity changes during dark adaptation in diurnal and nocturnal Lepidoptera. J. Gen. Physiol. W:205-215. Bhalla, S. C. I968. V/h i te eye, a new sex-i inked mutant of Aedes ae gypti . Mosquito News 28:380-385. Brammer, J. D. 1970. The ul trastructure of the compound eye of a mosquito Aedes aegypti L. J. Exp. Zool. 175:181-196. Brammer, J. D. , and R. H. White. I968. Vitamin A deficiency: effect on mosquito eye ul trastructure. Science 163:821-823. Brett, G. A. 1938. On the relative attractiveness to Aedes aegypti of certain coloured cloths. Roy. Soc. Trop. Med. Hyg., Trans. 32:n3-12i». Burian, H. M. , and B. Ziv. 1959Electric response of the phakic and aphakic human eye to stimulation with near ultraviolet. A.M. A. Arch. Ophthalmol. 61:3^7-351. 72

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73 Burkhardt, D. 1962. Spectral sensitivity and other response characteristics of single visual cells in the arthropod eye. Syniposia Soc. Exp. Biol. 16:86-109. Burkhardt, D. 196'l. Colour discrimination in insects. Advances in Insect Physiol. 2:131-173. Christophers, S. R. I96O. Aedes aegypti (L.) The yellow fever mosquito. Cambridge Univ. Press, London. 733 p. Dartnall, H. J. A. I962. The photobiology of visual processes, Vol. 2, p. 323-533. In H. Davson (ed.) The eye. Academic Press , New York. Dodt, E., and J. B. Walther. 1958. Fluorescence of the crystalline lens and el ectroreti nograph i c sensitivity determinations. Nature 181:286-287. Doving, K. 3., and W. H. Miller. I969. Function of insect compound eyes containing crystalline tracts. J. Gen. Physiol. 5t:250-267. Fourtes, M. G. F. , and S. Yeandle. 1964. Probability of occurrence of discrete potential waves in the eye of Li mu 1 us . J. Gen. Physiol. 47:443-'i63. Gilbert, I. H., and H. K. Gouck. 1957Influence of surface color on mosquito landing rates. J. Econ. Entomol. 50:678-680. Gogala, M. , K. Hamdorf, and J. Schwermer. 1970. UV-Sehf arbs toff bei Insekten. Z. vergl. Physiol. 70:^110-413. Goldsmith, T. H. I96O. The nature of the retinal action potential and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. J. Gen. Physiol. 43:775-799. Goldsmith, T. H. I96I. The physiological basis of v/avelength discrimination in the eye of the honeybee, p. 357-375In V/. H. Roseblith (ed.) Sensory communication. M. I .T. Press, Cambridge, Mass. Goldsmith, h. H. I963. The course of light and dark adaptation in the compound eye of the honeybee. Comp. Biochem. Physiol. 10: 227-237. Goldsmith, T. H. 1964. The visual system of insects. Vol. I, p. 397462. In M. Rockstein (ed.) The physiology of insecta. Academic Press , New York. Goldsmith, T. H., and H. R. Fernandez. I968. The sensitivity of housefly photoreceptors in the mid-ultraviolet and the limits of the visible spectrum. J. Exp. Biol. 49:669-677.

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7^1 Hoglund, G, , H. Langer, G. Struwe, and B. Thorell, 1970. Spectral absorption by screening pigment granules in the compound eyes of a moth and a v;asp. Z, vergi, Physiol. 67:238-2if2. Horridge, G, A, I969. Unit studies on the retina of dragonflies. Z. vergl. Physiol. 62:1-37. Jagger, J. 1967. Introduction to research in ultraviolet photobiology. Prentice-Hall, Inc., Englevjood Cliffs, New Jersey. ]6k p. Kay, R. E, I969. Fluorescent materials in insect eyes and their possible relationship to ultra-violet sensitivity. J. Insect Physiol. 15:2021-2038. Kolb, G., H. Autrum, and E. Eguchi. 1969. Die spektrale Transmission des dioptri schen Apparates von Aeschna cyanea Mull. Z. vergl. Physiol. 63:^3^^39. Langer, H. , and B. Thorell. I966. Mi crospectrophotometry of single rhabdomeres in the insect eye. Exp. Cell, Res, ^1:673-677. LeGrand, Y. 1957Light, colour and vision. Chapman and Hall Ltd., London. 5' 2 p. Ludwig, P. D. , Jr. 195^. Spectral studies on the yellovj fever mosquito, Aede s aeqypti (L.). Ph.D. Thesis, Ohio State Uni versi ty, 98 p. Mazokhi 11-Porshnyakov, G. A. I969. Insect vision. Plenum Press, New York. 306 p. Miller, W. H. , G. D. Bernard, and J. L. Allen. I968, The optics of insect compound eyes. Science 162:760-767. Mote, M. I., and T. H. Goldsmith. 1970. Spectral sensitivities of color receptors in the compound eye of the cockroach Per i pi aneta . J, Exp. Zool. 173(2) : 137-1^5. Mote, M. I,, and T. H. Goldsmith. 1971. Compound eyes: localization of two color receptors in the same ommatidium. Science 171:12541255. Munz, F, W. , and S. A. Schwanzara. I967. A nomogram for retinene2based visual pigments. Vis. Res. 7:111-120. Post, C, T, , Jr., and T. H. Goldsmith. I965. pigment migration and light-adaptation in the eye of the moth, Galleria mel lone 11a . Biol. Bull. 128:if73-'^87. Reichardt, W. E. I965. Q,uantum sensitivity of light receptors in the compound eye of the fly Musca . Cold Spring Harbor Symp. Quant. Biol. 30:^93-515.

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75 Ruck, P. 1958. A comparison of the electrical responses of compound eyes and dorsal ocelli in four insect species. J. insect Physiol, 2:261-274. Ruck, P. 1965. The components of the visual system of a dragonfly. J. Gen. Physiol. ^9:289-307. Scholes, J. 1965, Discontinuity of the excitation process in locust visual cells. Cold Spring Harbor Symp. Q.uant. Biol. . 30:517-527.. ... ... . . .. ........ Shaw, S. R. 1968. Organization of the locust retina, Symp. Zool . Soc, London 23: 135-163. . Swihart, S. L. I963. The electroreti nogram of Heliconius e rato (Lepidoptera) and its possible relation to established behavior patterns. Zoologica i+8('+) : 1 55" 165. Tunstall, J,, and G. A. Horridge, I967. Electrophysiological investigations of the optics of the locust retina. Z. vergl. Physiol . 55: I67-I82. Walcott, B. 1969, Movement of retinula cells in insect eyes on light adaptation. Nature 223:971-972. Wald, G, 19^5. Human vision and the spectrum. Science 101:653-658. Walther, J, 8, 1958, Changes induced in spectral sensitivity and form of retinal action potential of the cockroach eye by selective adaptation, J, Insect Physiol. . Washizu, Y. 196^1. Electrical activity of single retinula cells in the compound eye of the blowfly Calliphora erythrocephal a . Comp. Biochem. Physiol. 12:369-387. Wolken, J. J. 1971. Invertebrate photoreceptors. Academic Press, New York. 179 p. Wyszecki , G. , and W. S. Stiles. I967. Color science. John Wiley and Sons, Inc., New York, 628 p. Yinon, U. 1970. Similarity of the electroreti nogram in insects. J. Insect Physiol. 16:221-225.

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BIOGRAPHICAL SKETCH Leonard J, Goldman v^as born July 29, 19^+3, in Brooklyn, Nevi; York. In June of I96O he vjas graduated from Erasmus Hall High School. He received a Bachelor of Arts degree from Brooklyn College in June of I96U. He vjas enrolled as a graduate student at the University of Miami, Coral Gables, Florida, from September, 196^ to June, I966. In July, 1966, he enrolled in the Department of Entomology of the University of Florida where, until the present time, he has pursued his vjork toward the degree of Doctor of Philosophy. Leonard J. Goldman is married to the former Brenda Leigh Baldwin and is the father of one child. 76

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Harvey^ Croifiroy, Chairman Professor of Entomology ^nd Nematology I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Thomas J. Vla]kpfr Professor of Entomology and Hematology I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 0. Marion S. Mayer Courtesy Assistant Professor of Entomology and hematology I certify that ! have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Pli i 1 i p/S . Callahan Courtesy Professor of Entomology and Nematol ogy

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and Is fully adequate, in ?cope and quality, as a dissertation for the degree of Doctor of Philosophy. Wi 1 1 i am W. Dawson ' Professor of Ophthalmology This dissertation was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August, 1S71 Dean, Graduate School