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6c. ADDRESS (Cty, State, and ZIP Co) 7b. ADDRESS (0ty, State, and ZIP Cod.)Dept. of Ophthalmology and Visual Science P. 0. B 12211P.O. Box 3333 Research Triangle Park, NC 27709-2211New Haven, CT 06510Ba. NAME OF FUNDING ISPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBERORGANIZATIONl Of appkal) @0 -6"

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11. TITLE (Include Sfcurty Cassfication)

Insect Compound Eyes: Design of Photoreceptor Arrays for Image Pre-processing

12. PERSONAL AUTHOR(S) GGary D. Bernard_______________________

13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT (Year, Month, Day) 115, PAGE COUNTFinal 1 FROM /8 TO12/8 1989, Februa 22 23

16. SUPPLEMENTARY NOTATION The view, opinions and/or findings contained in this report are those

of ihe auth(.Wand shuld not be construed a n fficial Department of the Army position,

17. COSATI CODES 18. SUBJECT TERMS (Contnue on reerse if necessary and identfv bv block number)

FIELD GROUP SUB-GROUP absorption spectra (visual pigment, rod, cone);

bleaching (visual pigments); butterfly (Copper,

19. AC IRAT tCan,,inue an rvers, if necesarv and idmntif by block number)

Insect eyes are constructed so that much of the destructionof irrelevant data and pre-processing of vital information isaccomplished by the receptor array itself, reducing substantiallythe computational load placed on the neural system.

One of the most important mechanisms for destruction ofirrelevant data is coarse spatial sampling. At long range,spectral properties of objects then become particularly importantas a source of signals conveying specific information aboutqualities of those objects.

Colored wing patterns of butterflies provide im-ortantsignals for recognition of species and sex, but little is knownabout butterfly color vision. This study analyzes data from

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Block 18 (cont):Metalmark, Monarch, Queen, Viceroy, Anartia, Apodemia, Bgassolis,Danaus, Euohydryas, Eurema, Limenitis, Lycaena, Vanessa, colorvision, wings, photoreceptors, visual pigments); camouflage;color vision (tetrachromatic, butterfly); compound eyes; image(neural, compound eye, pre-processing); insect eyes;metarhodopsin; mimicry; photochemistry (visual pigments);optophysiology; photoreceptor array; photodetector (array,pigments; spectral sensitivity, waveguide); physiology(intracellular optical); pupil (response, sensitization,butterfly); quantum catch; red (signals, receptors, visualpigments, vision); reflectance spectra (wings); retinaldensitometry; rhabdom; rhodopsin; signals (chromatic;ultraviolet, red, far-red); spectral sensitivity (butterfly,photoreceptors); ultraviolet (vision, webs, signals, receptors);visibility (wing, red, far-red, ultraviolet); visual pigments(P340, P350, P355, P360, P424, P450, P470, P450, P470, P505,P510, P515, P530, P550, P556, P560, P574, P600, nomogramtemplates, rod, cone, butterfly); waveguide (rhabdom,photodetector); wings (butterfly, reflectance spectra,visibility, far-red, orange, red, white);

Block 19 (cont):-optophysiological measurements of intact butterfly eyes. It

demonstrates great diversity in absorbance spectra of visualpigments and spectral sensitivity functions of photoreceptors,and characterizes quantitatively those functions. Reflectancespectra of wing patches are measured microspectrophotometrically.Calculated quantum catches for photoreceptors viewing illuminatedwing patches indicate which wing features have value as signals. ,

Some butterflies have red markings that are not detectableby most insects, but themselves possess specialized red-shiftedreceptors that are well stimulated by those red markings. Thetetrachromatic Apodemia mormo enjoys the widest visible spectrumand greatest far-red sensitivity of any terrestrial animal.

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INSECT COMPOUND EYES:

DESIGN OF PHOTORECEPTOR ARRAYS FOR IMAGE PRE-PROCESSING

FINAL REPORT

GARY D. BERNARD

FEBRUARY 1989

U. S. ARMY RESEARCH OFFICE

CONTRACT NUMBER: DAAL03-86-K-0008

ARO PROPOSAL NUMBER: 23451-LS

YALE UNIVERSITY SCHOOL OF MEDICINE

DEPARTMENT OF OPHTHALMOLOGY & VISUAL SCIENCE

APPROVED FOR PUBLIC RELEASE;

DISTRIBUTION UNLIMITED

S89 3 21! 073

THE VIEWS, OPINIONS, AND/OR FINDINGS CONTAINED IN THIS REPORT ARETHOSE OF THE AUTHOR AND SHOULD NOT BE CONSTPUED AS AN OFFICIALDEPARTMENT OF THE ARMY POSITION, POLICY, OR DECISION, UNLESS SODESIGNATED BY OTHER DOCUMENTATION.

I. TABLE OF CONTENTS

LIST OF ILLUSTRATIONS ......... .................... 3

BODY OF REPORT . . . . . . . . . . . . . . . . . . . . . . . 4STATEMENT OF THE PROBLEM STUDIED. ...... ............ 4BACKGROUND ........... ....................... 5

Visually Mediated Behavior of Butterflies. ....... 5Spectral Sensitivity ....... ............... 5Extent of Visible Spectrum ...... .......... 5Intracellular Optical Physiology .... ......... 6Retinal Densitometry, Photochemistry, and

Dark-Processes ...... ................ 7Optical Stimulation of a Receptor .... ......... 7

APPROACH TO THE PROBLEM ........ ................ 8Specific goals ......... .................. 8

SUMMARY OF THE MOST IMPORTANT RESULTS .............. 9Analysis of Absorbance Spectra of Visual Pigments 9Analysis of Pupillary Action Spectra ... ....... 9Results for N-Receptors. ....... ........... 10Two New Classes of Butterfly Visual Pigment .... 11Detectability of Red Wing Colors .. ......... . 12Communication Among Butterflies while Fooling

Birds ...................... 13A New Optical Principle for Camouflage of Beetles 13Visual Attraction of Insects by UV-Reflecting

Webs ........ ..................... . 14LIST OF ALL PUBLICATIONS ...................... 14LIST OF ALL PARTICIPATING SCIENTIFIC PERSONNEL ..... . 14

BIBLIOGRAPHY ......... ........................ 15

ILLUSTRATIONS ......... ........................ . 17

II. LIST OF ILLUSTRATIONS

Fig 1: Eighth-Order polynomial templates for vertebraterod and cone visual pigments ... .......... .17

Fig 2: Absorbance spectra of L-receptor visual pigments. 18

Fig 3: Spectral sensitivity functions for L-receptors. 19

Fig 4: Spectral sensitivity functions for M-receptors. 20

Fig 5: Tetrachromatic eye of Apodemia mormo, and itsfar-red sensitive visual pigment P600 ....... .21

Fig 6: Visibility of colored wing patches .. ....... .22 7iFig 7: Signal value of white wing markings withina mimicry complex ...... ................ .. 23

3 A '',° . . . .

NDs lob

Av ,

III. BODY OF REPORT

A. STATEMENT OF THE PROBLEM STUDIEDSuccessful insect species have developed strategies for

finding and acquiring food, detecting and avoiding predators,navigating, finding refuge, selecting a proper mate, and locatinga suitable site for egg-laying. Several sensory modes are usedto accomplish these tasks, but for many species vision iscrucially important. Visually specialized insects are interest-ing subjects for study because of the diversity in the opticaland physiological properties of their compound eyes, driven byevolutionary adaptation to the diversity of specialized behaviorsand lifestyles.

Compared to vertebrates, insects are very small. How is itthat such tiny eyes can be effective in meeting their visualneeds? The sheer quantity of visual data impinging upon an eyeis immense. The visual system must destroy most of that data,yet extract and process a small subset which contains informationvital to the species. The problem seems to be a severe one.

Should the optics and photodetector array acquire as muchdata as possible, then rely on the neural system to sift therelevant from the irrelevant? No. Insects have a much moresuccessful strategy. Their eyes are constructed so that much ofthe destruction of irrelevant data and pre-processing of realinformation is accomplished by the receptor array itself.This reduces substantially the computational load placed on theneural system.

One of the most important mechanisms for destruction ofirrelevant data is coarse spatial sampling. Spatial resolutionof insect eyes is seldom better than one cycle/degree, about 100times worse that of a human eye (rev: Wehner'). Thus, spatialdetail becomes important for an insect only at short range( < ten body-lengths). At long range, spectral and temporalproperties of objects become particularly important as a sourceof signals conveying specific information about qualities ofthose objects. Color vision is, therefore, a particularlyimportant mechanism fully developed by some insects.

Of all insect groups, the butterflies are best suited tostudies of color vision. They make most extensive use ofchromatic signals in mating, food-finding, and ovipositionbehaviors. Butterfly natural history and genetics are among thebest known of all insect groups.

Butterfly color vision is mediated by a photoreceptor arraythat contains from three to five distinct spectral types ofreceptor. My noninvasive physiological and photochemical studiesof the last decade have revealed great interspecific diversity inthe spectral properties of visual pigments and photoreceptors ofbutterflies. This project selects representative visuallyspecialized butterfly species, characterizes quantitatively thespectral properties of their photoreceptors, and compares thespectral design of the receptors to the spectral properties ofwings and host plants. The overall goal is to understand the

4

principles according to which the spectral properties of receptorarrays are selected.

B. BACKGROUND

Visually Mediated Behavior of ButterfliesButterflies lead very specialized lives. For example, it is

common for the caterpillars of a butterfly species to accept onlyone species of foodplant. Thus, gravid females must reliablylocate the proper foodplant. Vision is known to be important forhostplant detection, particularly at long range.

Courtship and mating must occur, of course, before eggs canbe laid. Males engage in visual searches for females, lookingfor females with wings that have the proper color contrasts,size, and type of wing movement (reviewed by Wehner';Silberglied ). At long range, the wing shape is not at allimportant, nor is the fine structure of color patterns. Presenceof bands or stripes of expected color against expected back-ground, however, may be very important. Ultraviolet (UV)components have been shown to be important in particular cases.

Spectral SensitivityCompound eyes of insects (reviews: Goldsmith & Bernard3;

Autrum 4 ; Bernard5) spatially sample through an array of lensespacked closely on the curved surface of the head. Underlyingeach lens is a cylindrical group of 8-12 photcreceptor cells thatare heterogeneous in their spectral, spatial, and polarizationalproperties. Each receptor cell contains a long, thin rhabdomerethat absorbs light owing to the presence of membrane-bound visualpigment. The primary event that leads to vision is thephotochemical change in a visual pigment molecule that is causedby absorption of a photon.

Wavelength affects the probability that the photon will beabsorbed by visual pigment. After a visual pigment moleculeabsorbs a photon and is isomerized, however, all informationabout the wavelength of that photon is lost. Thus, spectralanalysis must be accomplished by groups of receptors that havetwo or more distinct spectral sensitivity functions (rev.:Wyszecki & Stiles 6; Cornsweet ).

Following psychophysical notation, I define the S-receptoras the type with peak sensitivity at shortest wavelength,the M-receptor as the type with peak at intermediate wavelength,and the L-receptor as the type with peak sensitivity at longwavelength.

Extent of Visible SpectrumThe visible spectrum for humans extends from 400nm (deep

violet) to 700nm (deep red). Insects, on the other hand, areknown to see in the near-ultraviolet down to wavelengths as shortas 300nm. Prior to the late 1970's it was held that butterflieswere blind for wavelengths greater than about 620nm, while birdswere blind for wavelengths less than about 400nm. Thus, it was

5

thought that red/orange patches are visual signals for birds butnot butterflies, while violet or UV markings are signals forbutterflies but not birds. It is now known that both statementsare much too simplistic. Some butterfly species have speciallong-wavelength photoreceptors that allow them to see evenfurther into the red than humans (Bernardo). On the other hand,some bird species have special receptors that allow them to seein the near-UV (Chen et al). The behavioral, ecological, andevolutionary consequences of these two important findings are notyet understood.

Intracellular Optical PhysiologyPhotoreceptor cells of most insects contain intracellular

pigment granules that move in response to illumination of thecell's photodetector organelle, the rhabdomere. In thedark-adapted state, these granules are dispersed throughout thecytoplasm of the receptor cell. If the cell is well illuminated,the granules move centripetally, congregating next to therhabdomere where they are illuminated by the evanescent boundarylayer of light guided by the rhabdom waveguide. Absorption,reflection, and scattering by the granules decrease thetransmittance of the rhabdom, causing a loss in sensitivity ofthe receptor cell. This is called the pupillary response(Stavenga 0 ).

Pigment migration within a cell is wediated exclusively byphotoisomerization of visual pigment contained within that cell'srhabdomere. Thus, the granules in a cell can be used as anintracellular probe of the physiological response of that cell.Most insects have fused rhabdoms containing several spectraltypes of receptor, so the generalized pupillary response containscontributions from all receptor types.

Pupillary sensitization (Bernard"1 ), however, makes itpossible to isolate responses from only one receptor type andmeasure its spectral sensitivity function over most of thevisible spectrum. The trick is to design the spectral contentand intensity of the constant measuring beam so that it partiallylight-adapts the pupil of the desired spectral type, but leavesthe pupils of all other spectral types dark-adapted. Pupillaryresponses from the light-adapted pupil are considerably fasterthan those from dark-adapted pupils because the distance thegranules must move to exert an optical effect is smaller. Thus,the sensitivity of a moderately light-adapted pupil to shortflashes is much greater than the sensitivity of dark-adaptedpupils. This approach has been used to record from S-, M-, andL-receptors of bumblebees (Bernard & Stavenga12), honeybees(Bernard & Wehner13 ; Wehner & Bernard1

4 ), moths (Bernard et alis),and from specialized red-sensitive L-receptors of butterflies(Bernard).

This non-invasive method (Bernard 6 ), "Intracellular OpticalPhysiology", has the distinct advantages that: a) the animalunder study is completely intact and healthy with undisturbedoptics, retinal morphology, and physiology; b) responses are

6

stable and reproducible over very long periods of time -- aslong as five months in individuals of some butterfly species;c) the eye region that contributes to the response can beprecisely localized and bounded; and, d) both physiological andphotochemical measurements can be performed on the same receptorcells.

Retinal Densitometry, Photochemistry, and Dark-ProcessesEyeshine caused by tapetal reflectors makes the intact

butterfly eye an excellent preparation for spectroscopic study ofvisual pigments. Consider the optics: The visual pigments arecontained within a long, thin rhabdom waveguide that isterminated optically by a reflector. This spectroscopic sampleof photopigment is illuminated efficiently by a pair of lenses" .

The lens-waveguide-reflector unit is wrapped in shieldingpigment, protecting it from stray light. Performance of thissystem is unmatched. The level of stray light is about 0.05% andtwo-way photochemical changes of more than density 2.0 have beenmeasured (Bernard').

Like other insects, butterfly visual pigment is converted bylight to a photoproduct ("metarhodopsin") that has a differentabsorption spectrum than the native form of the visual pigment.Measurements of difference spectra can be analyzed to determinethe absorption spectra of both forms. Unlike other insects,butterfly metarhodopsin decays rapidly from the rhabdom in thedark. After discovering this fact, I exploited it to bleachvisual pigment from the rhabdom and to measure directly theabsorption spectrum of butterfly visual pigment (Bernard").Prior to my work on butterflies it was thought that insectmetarhodopsins were necessarily stable in the dark and thatbleaching of insect visual pigments was therefore impossible(Langer et a12°).

The kinetics of metarhodopsin's decay and visual pigment'sregeneration depend strongly on temperature (Bernard' ). Thisproperty, coupled with the fact that bleaching is possible inbutterflies, is very important for this project. It enablesquantitative measurement of both the absorbance spectrum andoverall density of each spectral type of visual pigment.Those results, together with the optically measured spectralsensitivity functions, enable very high-quality, quantitativecharacterization of the spectral properties of the photoreceptorarray.

Optical Stimulation of a ReceptorThe optical stimulation (quantum catch) of a receptor is

Q , the number of photons absorbed per unit time by thereceptor's visual pigment7'22 .

7

where Q0(X) is the quantum flux of daylight illumination6,R(X) is the wing's reflectance spectrum, andS( X) is the spectral sensitivity function for the photoreceptorcell.

Since the neural system can process only the informationprovided by the receptor cells, computations of stimulus valuesare a useful means of characterizing the neural image at thelevel of the receptor cells. The receptor potential is asaturating, hyperbolic function2 2 of Q.

C. APPROACH TO THE PROBLEMBetween 1973 and 1985 I acquired data from many species and

had qualitative evidence for diversity of color vision systems,but had not published because of ambiguities of possibleinterpretations of the experimental spectra. My ancient DECLAB-8/E computer limited severely the scope of experiments andallowed only rudimentary analysis.

The proposal for this three-year project, started in 1/86,included replacing the lab-computer system and installing apowerful workstation computer for scientific data analysis.This provided the tools necessary for accurate, quantitativecharacterization of the spectral properties of butterfly photo-receptor systems and those of the colored objects known to beimportant for species survival -- wings and hostplants.The overall goal is to understand the principles according towhich the spectral properties of receptor arrays are selected.

Specific goals of this three-year research project were to:a) measure the spectral properties of each receptor type,

including both physiological measurements of the spectralsensitivity function and photochemical measurements of visual-pigment absorption spectrum, in the same photoreceptor cells.

b) develop analytical methods to compare quantitatively toabsorbance nomogram-template functions derived from visualpigments of vertebrate rods and cones; extend the presentlyavailable templates to include the beta band as well as the longwavelength, log-linear tail of the alpha band.

c) Document the extent of the diversity of butterfly color-vision systems, particularly the ability to see at long wave-lengths (in the orange - red spectral region). Pay particularattention to closely related species that have very differentwing coloration and/or very different receptor sets.

d) Measure the reflectance spectra of colored wing patches,then characterize neural images at the level of the receptoraxons by computing the relative optical stimulation (quantumcatches) for each spectral type of receptor viewing eachdaylight-illuminated patch.

The original proposal included a section on polarizationvision. Reviewers of the proposal recommended that I drop thatsection and concentrate on the more interesting topic of colorvision. I did so.

-- - -- umm - - _ I m m m n ~ m U~ 8

D. SUMNRY OF THE MOST IMPORTANT RESULTS1. Analysis of Absorbance Spectra of Visual Pigments

Because my methods produce high quality absorbance andsensitivity spectra over a very wide spectral range (340nm -700nm), proper analysis of mixed responses requires accurateknowledge of the spectrum of a single receptor type, includingboth the beta band and the log-linear tail. Published nomogramtemplates are inadequate because they are limited to theneighborhood of the alpha peak, and predict neither the short-wavelength beta band nor the long-wavelength, log-linear tail ofthe absorbance spectrum (MacNichol23).

I created improved templates for absorbance as a function ofnormalized frequency-ratio, for both rod and cone visualpigments, by incorporating the best spectrophotometric data andmodern electrophysiological data (Bernard2 4). Eighth-orderpolynomial approximations are very accurate. See Figure 1.

My experimental absorption spectra for butterfly visualpigments were fit very well by the vertebrate-cone template, evenincluding the beta band. Figure 2A shows a least-squares fit ofthe cone absorbance-template to a partial bleach of the L-receptors of Brassolis. This absorbance spectrum is typical ofP530 visual piments found in the L-receptors of many insectsincluding bees 2 13 and moths's , as well as some otherbutterflieso'lo.

Figure 2 indicates the great diversity among butterflyspecies in the spectral properties of L-receptor visual-pigments.P510 of the buckeye (Fig. 2B) represents the short-wavelengthlimit for butterfly L-receptors. Note that absorption isnegligible for wavelengths > 600nm. Many butterflies such asheliconiids, pierids, danaids, and lycaenids have a visualpigment with lambda-max in the range 550nm - 560nm. An exarmple,P555 of the western checkerspot is shown in Fig. 2C; this pigmentabsorbs well in the orange-red (600nm-650nm) spectral band.

The long-wavelength limit for the lambda-max of vertebrate,retinal-based visual pigments is about 580nm26; Figure 2D showsthat the Copper butterfly, Lycaena nivalis, possesses P574, closeto that limit. This visual pigment is more sensitive at longwavelengths than the red-sensitive P565 visual pigment of thehuman red-sensitive cone.

2. Analysis of PuDillary Action SpectraEven though the generalized pupillary response of butter-

flies may contain contributions from several spectral types ofreceptor, it is possible to manipulate substantially thesummation rules for the mixed response through: i) pupillarysensitization to short flashes, ii) bleaching, or iii) use ofvery long, dim flashes.

Very tight least-squares fits of theoretical absorptancespectra to short-flash spectral sensitivity functions show thatonly a single receptor type contributes. Figure 3A shows aleast-squares fit of absorptance 25 (calculated from the coneabsorbance-template) to the short-flash spectral sensitivityfunction measured from the very same receptor cells of Brassolis

9

from which the absorbance spectrum Fig. 2A was obtained. Theprecision of the fit to the cone function is excellent, even asfar down on the long-wavelength tail as -3.5 log-units. Asimilar function based on the rod polynomial, which has ashallower tail than the cone polynomial, fits poorly. Thus,absorption spectra of butterfly visual pigments are essentiallyidentical to those of vertebrate cone visual pigments.

Figure 3B shows the short-flash, pupillary sensitivityfunction from L-receptors of the Buckeye, which is well fit by aP511(+-l) absorptance function. This is consistent with theSl0nm(+-l) estimate for the pigment absorbance function (Fig.2B).

Long-flash spectra obtained by eliciting small responsesfrom multiple receptor types are well fit by a linearly weightedsum of sensitivity functions of the contributing receptortypes 25 . Together with photochemical measurements, this providesstrong evidence for presence of minority receptor-types and ameans for characterizing their properties. An example for awestern Checkerspot E. chalcedona is shown in Fig. 3C.

In absence of photochemical data this would be a difficultspectrum to interpret because of the strange shape of the broadsecondary peak. However, Fig. 2C shows that this eye containsvisual pigment P556. The good fit of sensitivity functions R355and R556 to the pupillary action spectrum of Fig. 3C, in therange 350-580nm, is strong evidence for presence of S-receptorsR355. What about the poor fit for wavelengths > 580nm? Onepossible interpretation is, an artefact of stray-light leakagethrough reddish shielding pigment. Another possibility is,increased sensitivity owing to the presence of a minority, red-sensitive photoreceptor.

The latter possibility is probably correct, as shown in asimilar experiment with the closely related eastern CheckerspotE. phaeton. This butterfly possesses P560, but the sensitivityfunction of its L-receptors (Fig. 3D) departs dramatically atshort wavelengths from the absorptance spectrum expected forP560. In this eye the L-receptors are filtered by a photostable,blue-absorbing, red-transmitting filter. A precedent for thisoptical principle was first established in Sulphur butterflies byRib127.

3. Results for M-ReceptorsProperties of M-receptors (receptors having peak sensitivity

at intermediate wavelengths) can be studied after bleaching thevisual pigment of L-receptors. An example is shown in Fi's. 4A-Bfor the Florida Viceroy, whose L-receptors contain an unusualvisual pigment P515 (close relatives posses P530). Afterbleaching P515, the long-flash pupillary action spectrum (Fig.4B) is dominated by S-receptor R350 and M-receptor R450.Photochemical measurements (not shown) support the hypothesisthat M-receptors of the Viceroy contain visual pigment P450.

Similar experiments with the Sulphurs of genus Eurema (Fig.4D) show that their M-receptors contain visual pigment P424, asubstantial spectral shift compared to P450 of the Viceroy.

10

4. Two New Classes of Butterfly Visual PigmentMy early work on red-sensitivity in butterflies3 indicated

that the Metalmark Apodemia mormo possesses greater sensitivityin the far-red than any known insect. A new round of experimentswith both sexes of this butterfly was very productive:

a) Results enabled accurate determination of the absorbancespectrum of this far-red visual pigment (Fig. 5A), revealing analpha band with peak at 600nm having a shape identical to that ofan Al-based visual pigment of vertebrate cones. It was evenpossible to measure the beta band of P600. Very poor fits to A2absorption spectra indicate that the chromophore of P600 is not3-dehydroretinal, but is probably 3-hydroxyretinal.This P600 has a lambda-max that is at least 20nm greater than anyknown retinal-based visual pigment.

b) Rhabdoms in the frontal region of the male eye containdensity > 0.5 of visual pigment P600, in photoreceptor cells thatlack a pupillary response!

c) This presented a golden opportunity to follow in-vivophotoconversion of P600 to its photoproduct M508, using pure far-red light of wavelengths 700nm - 720nm. Results prove that P600is not simply a conventional visual pigment filtered byblue/green-absorbing photostable pigment as describe above forCheckerspot butterflies (Figs. 4C-D), but is a pure, unshieldedvisual pigment that has a lambda-max of 600nm, andcorrespondingly high sensitivity in the far red (Fig. 5A).

d) The eye of the female is not the same as the male,containing less than half of the male's titer of P600, but incells thit DO exhibit a pupillary response.

e) -.ction spectra at long wavelengths are very different inthe two halves of the female eye. The upper half (Fig. 5B,yellow beam) was dominated by a new class of receptor, havingpeak sensitivity at 505nm. This very clear isolation of receptorR505 was a surprise; There was no hint from the photochemicalstudies that visual pigment P505 was present, which is strongevidence that the photochemistry of P505 involves no spectralshift following photoisomerization. Switching from yellow towhite measuring beams caused substantial increase in pupillarysensitivity at short wavelengths, owing to contributions fromsensitized receptors R340 and R450.

f) Pupillary sensitivity of the lower half of the female eye(Fig. 5C) at long wavelengths is dominated by P600.Photochemical measurements supported the conclusion that P600 ispresent only in the ventral half of the eye.

g) In collaboration with J.K. Douglass and T.H. Goldsmith ofthe Yale Biology Department, we measured action spectra of theelectroretinogram (ERG) of the male eye, which showed that P600is indeed a visual pigment, contained in receptor cells thatcontribute to the ERG. Analysis of ERG action-spectra confirmedthe presence of P505 and P600, and revealed the presence of twoadditional spectral classes of receptor, R340 and R450. Thus,the butterfly Apodemia mormo is tetrachromatic (R340, R450, R505,and R600), and is a Guiness Book candidate for the widest visible

11

spectrum and greatest far-red sensitivity of any terrestrialanimal, and the greatest lambda-max of any retinal-based visualpigment.

h) Both wing surfaces of both sexes contain iridescentscales that reflect far-red light that is not visible to mostinvertebrates but is visible to P600 of Apodemia. These scalesmay have value as signals for species recognition.

5. Detectability of Red Wing ColorsOne possibility for the (Darwinian) adaptive significance of

the diversity of butterfly color-vision systems is tu maximizethe detectability of spectral signals flown by wings and hostplants. A widely accepted conclusion among entomologists is thatinsects, including butterflies, "can't see red" becausesensitivity of the typical invertebrate L-receptor R530 tomonochromatic red (approx. 650nm) is below 1% of that at 530nm.Results presented above show that some butterfly species havehigh sensitivity at 650nm. However, reflectance spectra fromwing colors are broadband, not narrowband. How bright is a redwing-patch, and how great is its contrast against the backgroundwhen viewed by an L-receptor? Consider the following example:

The nymphalid butterfly Anartia amathea has wings with whitespots and red bars on a dark-brown background. Reflectancespectra for white, red, and brown patches are shown in Fig. 6A.Reflectance of the red bars is very low for wavelengths less than550nm, rising to a peak at 700nm. Behavioral experimentsindicate that the red markings are important signals for matingbehavior .28 Compare quantum catches for specialized red-sensitive L-receptors of a male Anartia amathea (Bernardo), tothose for the typical invertebrate R530 L-receptor of anothernymphalid species, Vanessa cardui'9 . Both functions are shown inFig.6B together with the spectrum for quantum flux of daylightand the spectrum of the red patch. As described on page 7 theoptical stimulation (quantum catch Q) of a receptor is theintegral of the product of spectra for daylight, reflectance, andsensitivity. Bargraphs of Q, normalized to 100% for an idealflat-white reflectance, are shown in Fig. 6C for white, brown,and red patches. Because there is so little overlap between thered-patch spectrum and the R530 spectrum, the integral is only6% compared to 29% for R610 of Anartia. The contrast of the redpatch against the brown background is only 9% for Vanessacompared to 57% for Anartia. Therefore, the effect ofevolutionary adaptation's shifting from the ancestral R530receptor to the specialized R610 receptor is a dramatic increasein visibility of the red wing patches.

Figure 6D shows examples of reflectance spectra of orange,yellow and white wing patches from the Monarch and Viceroy. Theorange and yellow spectra have cutoff-spectra typical ofpigmentary colors. The whitish patches of Monarch and viceroyare spectrally quite different compared to one another.

12

6. Communication Among Butterflies while Fooling BirdsThe importance of red and orange wing patches is a

particularly interesting question. The orthodox explanation forthe "why" of red/orange wing coloration is to educate vertebratepredators that the butterfly is poisonous if eaten and thus toreduce predation on members of the mimicry complex . It is notat all necessary for butterflies to be able to see the red/orangepatches for mimicry to work. Little attention has been paid,therefore, to the question of visibility of wing markings tomembers of the mimicry complex. Are there significantdifferences in wing-reflectance spectra between mimic and model,and are the differences large enough to have value as a signalfor species recognition?

Butterflies of the Florida Queen / Florida Viceroy mimicrycomplex look very similar to avian predators and to us, but notto each other. Queens have receptors peaking at about 360nm,470nm, and 550nm. Viceroys, on the other hand, have receptorspeaking at about 350nm, 450nm, and 515nm (Fig. 4). Thus,Viceroys are much less sensitive at long wavelengths than Queens.The same statement is true for the celebrated Monarch/Viceroymimicry complex (data not shown).

Reflectance spectra for orange and white patches of FloridaQueen and Viceroy are shown in Fig. 7A. The spectra for theorange patches of the. two species are very similar, as expected.The spectra for white patches of the two species are quitedifferent at short wavelengths.

Calculated quantum catches (Figs. 7B-C) show very similarstimulation of L- and M-receptors for the Viceroy viewing eitherorange patch, and for the Queen viewing either orange patch. TheUV-sensitive S-receptor is stimulated more strongly by theViceroy orange, probably because of some UV-reflective scalesfound on the Viceroy wing but not on the Queen wing.

Comparison of quantum catches for white patches showsubstantial differences for eyes of both species. All threereceptor types have about the same stimulation when viewingViceroy-white. When viewing Queen-white the stimulation of S-receptors is 40% lower, and the stimulation of M-receptors isabout 17% lower than for Viceroy-white. Conclusion: thesewhitish spots may be important visual signals for speciesdiscrimination at long range.

This is curious, for it is now known that most bird eyescontain UV-sensitive photoreceptors 9. So birds have thecapability for detecting the short-wavelength differences inwhitish patches, but apparently ignore that data; Behavioralexperiments" show that birds recognize the similarity in orangecoloration of the dorsal wing surfaces and, having regurgitated aMonarch or Queen, are not willing to take a chance on a Viceroy.This, despite the fact that the bird had eaten many Viceroysbefore experiencing a Monarch or Queen.

7. A New QOtical Principle for Camouflaae of BeetlesA collaboration with Dr. T.D. Schultz of Arizona State

University, to understand the optics of beetle coloration, has

13

4

Lt

revealed an interesting new mechanism 30 for matching thereflectance spectrum of an insect's body to that of a background.The cuticle is sculptured with hexagonally packed, deep pits - 10microns diameter, that are coated with an 18-layer interferencefilter. The reflectance spectrum has substantial globalvariation over a 50 micron patch of surface (eg, a group of 9bright green pits surrounded by magenta pits). Because thesedimensions are well below the resolution limit of animal eyes, apredator sees a spectrum that is an average of the spatiallyinhomogeneous reflectance spectra, with peak intensity that iscontrolled by the depth of surface sculpturing. By varying layerthicknesses and sculpturing, different morphs of tiger beetlematch the different soils on which they occur, such as wet brownsand, reddish Tertiary sands, or grey basalt pebbles.

8. Visual Attraction of Insects by UV-Reflecting WebsA collaboration with Prof. C.L. Craig of the Yale Dept. of

Biology, to understand the optics of spider silks, has produced abreakthrough in the field of spider ecology & evolution.We learned that some spiders spin silk that selectively reflectsultraviolet light. Spiders use this silk to visually attractinsects. One type of UV-reflecting web is found in vegetationgaps illuminated by sunlight and skylight. Redirection of UVlight by the web highlights the gap and draws insects to it asthey fly from the interior of the vegetation towards open space.Another type of web is decorated with UV-reflecting bars,crosses, discs and bands that attract insects as they search forfood resources.

E. LIST OF ALL PUBLICATIONS

G.D. Bernard: Butterfly color vision - spectral propertiesof photoreceptors and wing patterns. J. Ot. Soc, Am. Series 2,MVl. 3, p44, (1986).

G.D. Bernard: Spectral characterization of butterfly L-receptors using extended Dartnall/MacNichol template functions.J. pt. Soc. Am. Series 2. 4(13), p123, (1987).

G.D. Bernard, J.K. Douglass and T.H. Goldsmith: Far-Redsensitive visual pigment of a metalmark butterfly.Investigative Ophthalmology 29 SUpol., p350, (1988).

T.D. Schultz and G.D. Bernard: Pointillistic mixing ofinterference colours in cryptic tiger beetles. Nature 337, 72-73, (1989).

F. LIST OF ALL PARTICIPATING SCIENTIFIC PERSONNEL

Gary D. Bernard

14

IV. BIBLIOGRAPHY

1) Wehner, R.: Spatial vision in arthropods. In: Handbook ofSensory Physiology, Vol. VII/6C, H. Autrum, ed., Springer,Heidelberg, pp 287-616, (1981).

2) Silberglied, R.E.: Communication in the lepidoptera. In:How Animals Communicate, T.A. Sebeok, ed., Indiana Univ.Press, (1981).

3) Goldsmith, T.H. and G.D. Bernard: The visual system ofinsects. In: The Physiology of Insecta, Vol. II, secondedition, M. Rockstein, ed., Academic Press, New York, pp165-272, (1974).

4) Autrum, H. (1981): Vision in Invertebrates. Handbook ofSensory Physiology, Vol. VII/6. Springer, Heidelberg.

5) Bernard, G.D.: A comparison of vertebrate and invertebratephotoreceptors. In: Vertebrate Photoreceotor Optics, J.M.Enoch & F.L. Tobey, Jr., eds. Springer Series in OpticalSciences, Vol. 23, Springer, Heidelberg, pp 433-463, 1981.

6) Wyszecki, G. and W.S. Stiles: Color Science, John Wiley,NY, (1967).

7) Cornsweet, T.N.: Visual Perception, Academic Press, NY,(1970).

8) Bernard, G.D.: Red-absorbing visual pigment of butterflies.Science 203, 1125-1127, (1979).

9) Chen, D.M., J.S. Collins and T.H. Goldsmith: Theultraviolet receptor of bird retinas. Science 225, 337-340,(1984).

10) Stavenga, D.G.: Pseudopupils of compound eyes. In:Handbook of Sensory Physiology, Vol. VII/6A, H. Autrum, Ed.,Springer, Heidelberg, pp357-439, (1979).

11) Bernard, G.D.: Optical physiology: a technique for isolationof pupillary responses from single types of insect photo-receptors. J. Ot. Soc.Am. 68, 1427, (1978).

12) Bernard, G.D. and D.G. Stavenga: Spectral sensitivities ofretinular cells measured in intact, living bumblebees by anoptical method. Naturwissenschaften 65, 442-443, (1978).

13) Bernard, G.D. and R. Wehner: Intracellular opticalphysiology of the bee's eye: I - spectral sensitivity. J.Comparative Physiology 137, 193-203, (1980).

14) Wehner, R. and G.D. Bernard: Intracellular opticalphysiology of the bee's eye: II - polarizationalsensitivity. J. Comparative Physiology 137, 205-214,(1980).

15) Bernard, G.D., E.D. Owens, and A.K. Hurley: IntracellularOptical Physiology of the Eye of the Pyralid Moth Amyelois,J. Experimental Zoology 229, 173-187, (1984).

16) Bernard, G.D.: Noninvasive optical techniques for probinginsect photoreceptors. In: Visual Pigments and PurpleMembranes, (Methods in Enzymology, Part H, Vol 81), L.Packer, ed., Academic Press, New York, pp 752-759, (1982).

17) Nilsson, D.-E., M.P. Land and J. Howard: Afocal AppositionOptics in Butterfly Eyes. Nature 312, 561-563 (1984).

15

18) Bernard, G.D.: Dark-Processes Following Photoconversion ofButterfly Rhodopsins, Biophysics of Structure and Mechanism9, 277-286, (1983).

19) Bernard, G.D.: Bleaching of rhabdoms in eyes of intactbutterflies. Science 219, 69-71, (1983).

20) Langer, H., P. Schlecht, and J. Schwemer: Microspectro-photometric investigation of insect visual pigments. In:Visual Pigments and Purple Membranes, (Methods inEnzymology. Part H. Vol 81), pp 729-741, (1982).

21) Bernard, G.D.: Dark-regeneration of butterfly rhodopsin atphysiological temperatures. Invest. ODhthalmol. 25 SUD2I.,60, (1984).

22) R.W. Rodieck: The Vertebrate Retina, W.H. Freeman, SanFrancisco, (1973).

23) MacNichol, E.F., Jr.: A unifying presentation of photo-pigment spectra. Vision Research 26, 1543-1556, (1986).

24) Bernard, G.D.: Spectral characterization of butterfly L-receptors using extended Dartnall/MacNichol templatefunctions. J. Opt, Soc. Am. Series 2. A(13), p123, 1987.

25) Goldsmith, T.H.: Interpreting trans-retinal recordings ofspectral sensitivity. J. Comparative Physiology 159, 481-487, (1986).

26) Honig, B., A.D. Greenberg, U. Dinur & T.G. Ebrey: Visual-pigment spectra: Implications of the protonation of theretinal Schiff Base. Biochemistry 15, 4593-4599, (1976).

27) Ribi, W.: Coloured screening pigments cause red eye glow inPierid butterflies. J. Comp. Physiol, 132, 1-9, (1979).

28) Emmel, T.C.: Mate selection and balanced polymorphism inthe tropical nymphalid butterfly Anartia fatima. Evolution2&, 96-107, (1972).

29) Brower, J.V.Z.: Experimental studies of mimicry in someNorth American butterflies, Part I. The monarch, Danausplexipus, and Viceroy, Limenitis archippus archippus.Evolution 12, 32-47, (1958). Ibid., Part III. Danausgilippus berenice and Limenitis archippus floridensis.Evolution 12, 273-285, (1958).

30) T.D. Schultz and G.D. Bernard: Pointillistic mixing ofinterference colours in cryptic tiger beetles. Nature 337,72-73, (1989).

16

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