Evidence for visual function of corneal interference filters

Evidence for visual function of corneal interference filters

r. Insect Physiol., 1971, Vol. 17, pp. 2287 to 2300. Pergamon Press. Printed in Great Britain EVIDENCE FOR VISUAL FUNCTION OF CORNEAL INTERFERENCE FI...

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r. Insect Physiol., 1971, Vol. 17, pp. 2287 to 2300. Pergamon Press. Printed in Great Britain

EVIDENCE FOR VISUAL FUNCTION OF CORNEAL INTERFERENCE FILTERS GARY D. BERNARD Department of Ophthalmology, Department of Engineering and Applied Science, Yale University, 333 Cedar Street, New Haven, Connecticut 06510 (Received 29 June 1971) Abstract-Corneal interference filters are more than just a device to influence the external appearance of the head by creating coloured eye patterns. They are also optical components of the compound eye. Measurements of reflectance spectra from cornea1 filters on single facets of living flies from four families are presented. They show that in localized regions of an eye that contains a mixture of filter colours, the reflectance characteristics often ‘fit’ in the sense that one type of filter is maximally reflecting at a wavelength where another type is minimally reflecting. In long-legged flies of the genus Condylostylus, which have alternating rows of two filter types, despite large variability in wavelength for maximal reflectance within a species the ‘fit’ of the two reflectance characteristics is preserved. Intersections between facets are often highly reflecting at a wavelength where the facet surface has low reflectance. They may serve to reduce glare caused by sources outside of the ommatidial visual field. These findings together with strong correlations between habitat and eye pattern support the hypothesis that cornea1 interference filters optically enhance contrast for coloured objects in a background of dissimilar colour, but do not exclude the possibility that they also could be part of a colour-vision system. INTRODUCTION

LAYERS in the

of insects often create structural colours that are an important factor in determining an insect’s appearance (MASON, 1927). Some flies have such colours on the corneal facets of their compound eyes (STEYSKAL,1957). For these insects, specialized cornea1 layers not only create coloured eye patterns but also tint the light reaching the retina, which raises the following questions: Can these specialized layers be performing visual functions for the eye that contains them ? And if so, what functions ? In an earlier paper on interference coloration of dipterous compound eyes (BERNARDand MILLER, 1968) we reported the ultrastructure of the specialized cornea1 layering that causes coloured eye patterns in some horseflies and deerflies (family T ab ani dae ) an d in a long-legged fly (family Dolichopodidae), demonstrated that the layers had alternating high and low refractive index, presented theoretical curves of reflectance vs. wavelength, and suggested that cornea1 filters may aid vision by optically enhancing contrast between objects of dissimilar colour. We presented no evidence to support this idea except for pointing out that the row-byrow detail of the eye pattern in some long-legged flies is so fine that it is probably not resolvable by another fly under ordinary circumstances. cuticle

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In an attempt to improve our understanding of cornea1 filters I began a comparative study of dipterous eyes that includes detailed observation of eye patterns of living flies, reflectance microspectrophotometry of single facets of living eyes, correlation of eye patterns to habitat and behaviour, and ultrastructural anatomy of the cornea and retina. The primary goal of this paper is to show that cornea1 interference filters are more than just a device to influence the external appearance of the head by creating coloured eye patterns; they also are optical components of the compound eye. Secondary goals are to present measurements of the spectral characteristics of filters in living flies from four families, to demonstrate that cornea1 colour can be quite variable among individuals of a given species, to show that the eye patterns of long-legged flies have features that are highly correlated to their habitat, and to discuss possibilities for visual function of cornea1 interference filters. The long-legged flies are particularly attractive for this study because many species have eyes with alternating rows of cornea1 facets of two different structural colours. Colour pictures of such eyes are shown in previous papers (BERNARDand MILLER, 1968, 1970). Since another fly probably cannot resolve this pattern for separation greater than a few millimetres, it seemed likely that the row-by-row pattern could be the consequence of a visual function rather than a display function. Furthermore, Dolichopodidae are very active, brilliantly coloured flies, and vision seems to be important to them. The males of many species have specialized antennae, legs, and heads that differ from their females, and contain ornaments that are employed in elaborate mating dances at close range and in plain sight of the female (VAN DUZEE et al., 1921; STEYSKAL, 1938, 1942). Also, males aggressively rush at one another and have tumbling aerial ‘dog-fights’ (STEYSKAL, 1947). Longlegged flies are predaceous, feeding on very small, fragile, soft-bodied animals such as midges and mosquito larvae (CREGAN, 1941). Vision is an important factor in locating and catching prey (BISHOP and HART, 1931). Another reason for studying Dolichopodidae is that many species are very local, that is, they are only found in a very particular habitat (CURRAN, 1934; ROBINSON, 1964; COLE, 1969). As VAN DUZEE (1921) put it, ‘The adults [of the genus Dolichopus] . . . are most abundant at the edge of water on mud; some species, however, are regularly found on foliage in half-shady places. Few are ever found in dry localities. They are very local, and even those species which have been collected in a dozen States are only to be found in just the right situation with regard to sunlight, moisture, and vegetation; a few feet away the search may be in vain.’ It seems reasonable that a fly’s visual system could have evolved to suit its particular environment and that correlations could exist between behaviour and cornea1 filter properties, especially for peculiarities of each.

MATERIALS

AND METHODS

Flies were captured with a hand net at various localities and placed in vials where they remained living until examined in the laboratory on the day of capture.

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Observation of eye patterns Individual flies were anaesthetized with CO,, dumped into a cyanide killing jar for several minutes, then mounted on a Leitz universal stage fitted to a Wild MS stereomicroscope equipped with the vertical illumination prism. Statements of eye patterns in a particular region of an eye assume vertical illumination and the eye positioned with ommatidia in that region oriented vertically, facing the illumination, so that the reflections originate from the middle of the corneal facets. Reflectance microspectrophotometry My instrument is based on Leitz equipment unless otherwise noted. It consists of a Model MPV microscope photometer mounted on an Ortholux stand equipped is a Photowith the special Pol-Opak illuminator. The photomultiplier-indicator volt 520 M. Narrow-band illumination was provided by a stabilized Bausch & Lomb quartz-iodide source and high-intensity monochrometer, with 350 to 800 nm grating, fitted with Leitz optics for imaging the monochrometer exit slit in the rear focal plane of the objective, and contained a Corning 3-74 filter to clear overlapping orders. The microscope was fitted with a fluorite (Pol.) 45 x jO.85 N.A. objective, with 10 x eyepiece. The aperture diaphragm was filled with light of less than 4 nm bandwidth, where the limiting wavelengths were somewhat greater than half intensity of the centre wavelength. The aperture stop was set so that the numerical aperture of the beam illuminating the cornea1 surface was O-25. Living flies were anaesthetized with CO,, decapitated, and mounted on the universal stage with Surgident periphery wax just above its melting point. The head was oriented so the facet to be measured was horizontal, and the field aperture adjusted so the illumination spot diameter was less than the facet diameter, usually excluding intersections. In most cases the objective was focused on the facet surface, but in cases where scattering from intersections was a problem the focus was set slightly below the surface to keep light reflected from intersections out of the measuring aperture. The MPV measuring aperture was adjusted to accept the disk of light reflected from the facet centre, which was usually between 8 and 10 E.L in dia. for facets 20 to 25 ,u in dia. Raw reflectance data were taken at 10 nm increments between limits of 410 and 700 nm. A Leitz 4 per cent reflection standard was used to obtain a standard baseline for the system to which the raw data were compared. Wavelength dependence of the standard reflectance, based on three calibration points supplied by Leitz, was taken to be 4 wavelength in nm 3300

>I

per cent.

The raw data were reduced and plotted with a PDPS/L computer teletype, digitized in units of 2 per cent. Reflectance curves were normalized to maximal reflectance, and the same normalization constant was applied to all curves in a given figure except for intersection’ reflectance curves. Only normalized reflectance

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characteristics are given in this paper because reliable absolute peak reflectames could not be obtained due to limited sensitivity of the Photovolt unit. RESULTS

I chose for detailed study a common local long-legged fly that is brightly coloured, very active, 4 mm long, and found on herbaceous leaves in the sun. Many flies were collected at the same time from a single large bush and brought to the laboratory for comparative study. Eye pattern in Condylostylus Both sexes usua~y had the following eye pattern: The ventral half of the eye contains alternating rows of bright yellow-green and red-orange facets with dim biue reflections where facets intersect. The dorsal area contains facets that are all red-orange, with just a hint of a row-by-row difference. Intersections are also blue, but brighter than thode in the ventral region. The transition between these two zones occurs near the equator of the eye. However, the region of the eye that faces forward and upward at an elevation of roughly 40 to 60” contains facets that are all deeper red than those in the extreme dorsal pole, and have no perceptible row-by-row difference. Although this was the most commonly observed pattern, considerable variability among individual flies was noticed. In order to characterize the variability I captured and studied the ventral region of eyes in a series of 25 flies, subjecting each to the same preparation. The individual at the long-wavelength limit of variability has alternating rows of yellow and deep red facets, while the one at the short-wavelength limit has blue-green and orange facets. The deep red facets occasionally display dim blue central reflections. Variability was also observed in the dorsal region, between limits of deep red and yellow-green. Despite the large variability from fly to fly, the left eye of a given individual always had the same coloration as its right eye. After making these observations I sent the series to G. C. Steyskal of the U.S. Department of Agriculture, an authority on Diptera, who reported that this series was not one species but two of a group of very closely related species (LOEW, 1864; ROBINSON, 1964) of the genus Co~y~o~~~~~, and that it is not possible to taxonomically d~ting~sh between females of the group. Males of the group are separated on the basis of only a single plastic character (LOEW, 1864). The males of my series were C. nipofemoratus (Walk.) and C. caudam (Wied.). Two other species of this group, C. inermis (Loew) and C. JEavipes(Aldrich), have since been taken in the same area, so I cannot-be certain that the females studied were one of the two species. However, on a later occasion I captured a series of over 20 such flies on the same bush over a time period of 10 min that were all determined to be C. nigrofemoratus. Examination revealed variability in both sexes that was about the same as in the first group, so it is clear that large variability in eye colour is characteristic of a single species. I also noticed large variability in body colours, a characteristic

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of the genus that was noted long ago by LOEW (1864) who wrote, ‘Of the (taxonomic) characters taken from the colouring, the most unreliable are those taken from the colouring of the head, thorax and abdomen.’

Measurements of Condylostylus Flies were selected that represent the average eye pattern and the limits of variability as determined with the stereomicroscope. Facets of both types were measured in the extreme ventral pole in three separate experiments. Normalized reflectance vs. wavelength for these flies is shown in Figs. la-c. Notice in all three graphs that the reflectance peak of one filter type is located very close to a reflectance minimum of the other filter type, and vice versa. Fig. 2 shows reflectance of two neighbouring facets in the dorsal pole of the eye of Fig. l(c). A facet intersection in this region was measured and its reflectance peak is also plotted in Fig. 2.

Measurements of a fruit jly A tiny fly of the family Tephritidae

that I found on the same bushes as C.

nigrofemoratw also has two types of filters, but in the frontal region of the eye in both sexes. However, the organizational pattern is not row-by-row but random. Fig. 3 shows measurements of two neighbouring facets for a female of this species, Euaresta bella. (Loew). The intersection between facets is highly reflective; its spectral characteristic is also shown in Fig. 3.

Eye pattern and measurements of horsejies Because the female horsefly Hybomitra ~~~phthalma (Macquart) was the fly of the most detailed study in our first paper (BERNARDand MILLER, 1968) I was very interested in measurements of the living fly. Only one such opportunity has arisen; the results are given in Fig. 4 for three facets near the middle of an eye. A facet in the top bright stripe was also measured and found to have a peak at 605 nm, with half-intensity points at 555 and 655 nm. Although I have not collected enough H. lasiophthalma to gauge variability, I was able to do so in another species of the same genus that has a similar eye pattern. Myrtle Bernard and I collected 73 females in the Olympic mountains of Washington State and studied the patterns of the living eyes with the stereomicroscope. We noticed marked differences in colours of the bright stripes, varying from orange to green. Another striking difference was large variation in widths of the two middle bright stripes. In the more common ‘thin’ variant these stripes are about 7 facets wide in their centre, whereas in the ‘thick’ variant they are about 12 facets wide. The table shows the 6 groups into which we divided this series of horseflies, as well as their stripe colours, width, and the number of females in each group. L. L. Pechuman, Curator of Insects at Cornell University and an authority on Tabanidae, determined every fly in this series to be a female Hybomitra atrobasti (McD.).

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Wavelength

nm

Wavelq?gth

FIG. l(a).

- nm

FIG. l(b).

-E -z .-N

E Wavelength FIG. l(c).

: nm

0

90 400

500

Wavelength

600

- nm

FIG. 2.

FIG. 1. Normalized reflectance characteristic for neighbouring facets in the extreme ventral region of the eye for three long-legged flies of the genus Condylostylus. The fly with most common eye colours is represented by Fig. l(b). Flies at the two limits of variability are represented by Figs. l(a) and l(c). FIG. 2. Normalized reflectance characteristics for facets in the extreme dorsal region of the Condylostylus eye of Fig. l(c), showing slight differences in the two types of facets (-O-Oand -O-O-). The peak of the intersection reflectance characteristic is plotted as (- qI- q-).

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Wavelength

400

0

- nm

500

Wavelength

FIG. 3. FIG. 3. Normalized intersection (-_i?--

600

- nm

FIG. 4. reflectance characteristic for two neighbouring facets and their q-) in the frontal region of the eye of a female fruit fly,

E. bella. FIG. 4. Normalized reflectance characteristics for three facets of a female horsefly H. Zasiophthalma. (- @-- e--), a facet in the second bright stripe from the top; (-o~o-), a facet in the thin row of facets bordering this bright stripe; (- AA-), a facet in the dark stripe immediately below the second bright stripe. All three facets have the same diameter and curvature; the same normalization constant is used for all three curves.

0” =o

t,

400

I.

I

I

I

500 600 Wavelength - nm

I

I

700

FIG. 5. Normalized reflectance characteristics for four types of facets in the eye of a female soldier fly, 0. Virgo. The eye pattern, diagrammed in the inset, has dull orange dorsal and ventral regions bordered by thick yellow bands. The central band is blue, bordered by thin red stripes.

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Measurements

of a soldier JEy

The female Odontomyia Virgo (Wied.) has an eye pattern caused by filters of four types organized in horizontal bands as shown in Fig. 5. Measurements of a representative facet of each of the four types are shown in Fig. 5. TABLE ~-VARIABILITY OF EYE-STRIPECOLOURAND WIDTH IN THE LIVING FEMALE HORSEFLYH. atrobasis Colour Colour Colour Colour

of first bright stripe of second bright stripe of third bright stripe of fourth bright stripe

Thickness of central bright stripes No. of females

0 0 0 0

0 G G 0

0 G G 0

0 G G G

0 G G G

G G G G

Thin 22

Thin 14

Thick 2

Thin 14

Thick 15

Thick 6

This group of 73 females is divided into 6 groups based on colours of the four bright stripes and the widths of the central bright stripes. O-orange; G-green. Measurement

of a june beetle

To improve my confidence that these reflectances were primarily determined by corneal surface reflectance, I measured the living eye of a june beetle, which lacks cornea1 filters. Its cornea1 reflectance, normalized to the reflectance of the standard, was constant to within + 3 per cent of its maximum reflectance over the entire wavelength band.

Ultrastructure

of the retina of long-legged j?ies

0. Trujillo-Cenoz and I studied the anatomical ultrastructure of the retina of the female Sympycnus lineatus Loew. This fly has an eye pattern similar to Condylostylus, but its alternating rows of yellow and red facets are found over a greater percentage of the eye’s surface, and the reflections are noticeably dimmer. The extreme dorsal area, roughly the top quarter of the eye’s surface, has a uniform orangish-red colour. Ventral facet coloration is also variable from female to female in this species, ranging from greenish-yellow and reddish-orange rows in some individuals to reddish-orange and deep red (or dim blue) rows in others. The male’s eye pattern is similar to the female’s, but I have not seen enough males to judge its variability. The most important finding of this anatomical study is that the retina of both Sympycnus and Condylostylus contains a row-by-row difference in anatomy that is exactly correlated with the row-by-row difference in cornea1 coloration. The superior central cells (SCC) and inferior central cells (ICC) in ommatidia with red corneas have rhabdomeric microvilli that are vertically oriented, while in the ommatidia with yellow corneas the SCC have horizontal microvilli and the ICC have vertical microvilli. This differs from muscoid flies such as Lucilia and

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Surcophaga where the rhabdomeric microvilli in all SCC are vertical and in all ICC are horizontal (MELAMEDand TRUJILLO-CEN~~Z, 1968). Details of this anatomical study are being published elsewhere (TRUJILLOCEN~Z and BERNARD,1972). DISCUSSION In order to study effects of cornea1 filters on vision one would like to know the transmittance measured at the distal end of the rhabdom. This could be approximated by isolating the cornea from the rest of the eye and making transmission measurements, but would suffer from artefacts introduced during preparation and mounting. To avoid such problems I chose to measure the reflectance of living, intact eyes. This approach has the disadvantage that the reflectance from the intact ommatidium is not exactly related to the desired transmittance. One difficulty is that reflections from within the eye contaminate the measurements somewhat but are probably unimportant except when the surface reflectance is very low. Another difficulty is that the transmission characteristic as seen by the rhabdom will not be exactly one-minus-measured-reflectance because of differences in ray paths and absorption, but the discrepancy should not be large (BERNARDand MILLER, 1968). The following discussion offers evidence in support of the hypothesis that cornea1 interference filters are optical components of the visual system, and suggests several possible functions.

Variability and ‘$t’ in Condylostylus Normalized cornea1 reflectance characteristics of long-legged flies, shown in Figs. 1 and 2, are quite narrow with half-intensity bandwidths of about 13 per cent, with deep minima and distinct secondary niaxima. These are qualities of a wellordered periodic layer system. W. H. Miller’s unpublished electron micrographs of sections of Condylostylus corneas demonstrate that the cornea1 filter contains 12 layers, 6 dense and 6 rare, where 11 are quarter-wavelength layers and where the anterior dense layer is three-quarter wavelengths thick. Fig. 1 demonstrates the large variability in ventral eye colours, with peak reflectance wavelength that varies at least 55 nm for the greenish facets and 80 nm for the reddish ones. Despite this large variability the two filter characteristics maintain their shapes and a precise relationship. For a given eye the two spectral characteristics ‘fit’ in the sense that one filter type is maximally reflecting at a wavelength where the second type of filter is minimally reflecting, and vice versa. Fig. 2 shows that in the dorsal pole there are two identifiable classes of filters, even though it is difficult to see much difference when examining the eye under the stereomicroscope with white-light illumination. It is remarkable that despite the large variability in filter colours from fly to fly of the same species, the peak of one filter characteristic fits a null of the other. Why ? Probably because the visual system is comparing what is viewed through

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one set of filters to what is viewed through the other set, and the ‘fit’ yields maximal difference for such a comparison. ‘Fit’ in other Diptera The fruit fly E. bella, a member of a different sub-order of flies, has brighter, narrower, and more saturated reflections than Condylostylus, with much brighter Although I have only measured a single individual, the results intersections. shown in Fig. 3 suggest that a ‘fit’ exists for two classes of filters in the anterior region of its eye. The horsefly of Fig. 4 has an eye with 12-layer filters but its reflectance characteristics are broader than for Condylostylus and have no distinct nulls or secondary peaks at shorter wavelengths. This suggests that the horsefly layer system is not as well-ordered as that of the long-legged fly. From our earlier work (BERNARD and MILLER, 1968; MILLER et al., 1968) I expected the maximal reflectance for a 1Zlayer blue facet to be the same as for a Q-layer orange one but was surprised to find the measured blue peak to be about 20 per cent of the orange peak, as shown in Fig. 4. This difference is even more striking in deer flies (Chrysops) where the peak reflectance in the blue is only 7 to 10 per cent of the peak orange-facet reflectance. The central bright stripes are bordered with a thin row of red facets (BERNARD and MILLER, 1968)which have reflectance characteristics that ‘fit’ those of the bright facets, as demonstrated in Fig. 4. The reflectance characteristics in the soldier fly 0. virgo of Fig. 5 are very broad and dim compared to the other species measured. However, note that the equatorial blue band, 7 facets wide, is bordered above and below by a thin red band, 1 or 2 facets wide. The maximal reflectance for blue filters is at 5 10 nm which is the wavelength for minimum reflectance of the filters in the thin red border. Also, the minimal reflectance for the blue filters is close to 610 nm which is a ‘fit’ for maximal reflectance of the yellow filters located above and below the central stripe. It appears that when a mixture of filter types occur in a localized region of an eye the filter characteristics ‘fit’ in the sense that one type of filter is maximally reflecting at a wavelength where another type is minimally reflecting. Variability

of cornea1 coloration

As demonstrated in Fig. 1 for Condylostylus and in the Table for Hybomitra, cornea1 colour is a highly variable character of some species. I have also found large variability in long-legged flies of the genera Campsicnemus, Chrysotus, However, large variability is not a Dolichopus, Gymnopternus, and Sympycnus. universal property of cornea1 colour. Plagioneurus univittatus Loew, Calyxochaetus frontalis (Loew), and Pelastoneurus vagans Loew show little variability among individuals that I have studied. Variability of structural eye colour is not limited to Diptera. Chrysopa carnea Stevens is a green lacewing of the order Neuroptera, family Chrysopidae, that has

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brilliant golden eyes. E. Macleod and J. K. Sheldon (manuscript in preparation) at the University of Illinois, Urbana, have reared a mutant that has green eyes. They have worked out the genetics of the two eye colours and found that gold mated with gold yields only gold progeny; green with gold yields proportions of one green to one gold; and green with green yields two green to one gold. When both parents are green-eyed there is an embryonic mortality of 2.5 per cent compared to almost zero in the other two types of crosses. They find that eye colour in the two groups is quite distinct although there is some variability within each group. The structural basis for eye colour of Neuroptera is different than in Diptera. The reflections originate only from facet intersections and the facet surface has very low reflectivity. Unpublished electron micrographs by W. H. Miller of corneas of Chrysopidae and Myrmeleontidae show regularly spaced cornea1 layers with relatively large difference in electron density in the regions where facets intersect. Neuropterous eye colour is probably caused by Bragg diffraction from these periodic layer sets. Retina of Sympycnus and Condylostylus MELAMEDand TRUJILLO-CEN~Z(1968) suggested that the two central retinular cells in muscoid flies such as Sarcophaga and Lucilia can function as an intraommatidial polarization analyser, based on the perpendicularity of microvilli and on the anatomical finding that the axons of the two central cells are enclosed in a glial sheath from the basement membrane all the way to the medulla and do not make connexions to the lamina. In the ventral region of the Sympycnus and Condylostylus eyes polarization differences for central cells have been eliminated in half of the ommatidia because their rhabdomeric microvilli are parallel. Therefore it is possible for the medulla to make spectral comparisons of central cells viewing the same point through different filter systems and not be influenced by polarization differences. Also, there are two spectral types of optical cartridges in the lamina because of the asymmetrical projection pattern of the six common photoreceptor axons onto the lamina, coupled with the occurrence of row-by-row alternation of filter types (TRUJILLO-CEN~Zand BERNARD,1972). Therefore there are row-by-row differences in the retina, lamina, and medulla that are exactly correlated to alternation of cornea1 filter coloration. This is direct evidence that cornea1 interference filters have a visual function. Correlation

of eye pattern

to habitat

Condylostylus species discussed here are found in the immediate neighbourhood of herbaceous foliage. They dash about on the surfaces of leaves, flit from one leaf to another, feed on smaller animals found on the leaves, engage in aerial dog-fights with each other, and are generally very busy but seldom fly far from the plant. Therefore, much of the time the ventral part of their eyes is viewing objects against a coloured background of green leaves.

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The cornea1 filter system could be acting as colour contrast filters improving the contrast of objects viewed against the green background (BERNARDand MILLER, 1968, 1970). For instance, a red object on a green leaf would appear darker relative to the leaf if viewed through a cornea1 filter that reflects red (negative contrast), but would appear brighter if viewed through a filter that reflects green (positive contrast). Comparison of the scene viewed through one filter set to that viewed through the other set would further enhance the contrast. If this is true then other species of Dolichopodidae found mostly on leaves and grass should also have a mixture of filters on the ventral half of their eyes, and those species of Dolichopodidae found on wet mud or water should be expected not to have a mixture of filters. I have collected over 50 species of long-legged flies distributed among 17 genera and have observed their eye patterns. The following genera are found on foliage and have a mixture of two filter types on the ventral half of their eyes (the number of different species I have observed is given in parentheses after each genus): Asyndetzcs (l), Chrysotus (6), Condylostylus (lo), Dolichopus (12), Gymnopternus (4), Sciapus (I), and Sympycnus (1). Exceptions are Asyndetus (1) and Diaphorus (2). The mixture of filter types is row-by-row in all of these genera except Asyndetus and Chrysotus where it is random. The following genera are found on wet mud and/or on water and do not have a mixture of filters but only a single filter type in a given local area of the eye, although the filter colour in the middle of the eye may differ from that in the rest of the eye: Campskemus (l), Hydrophorus (4), Liancalus (l), Pelastoneurus (2), PoZymedon (I), Rhaph~um (3), Tachytrechus (l), and ThinophiZw (1). Exceptions are PZugionewus (1) and Dolichopus (3). Although these are only preliminary results of a relatively small survey, a strong correlation does seem to exist between habitat and eye pattern, supporting the idea that a mixture of filter types aids vision by improving contrast of objects viewed against the green background. RefEections from facet intersections Diptera with cornea1 interference filters often have facet intersections that are highly reflective in a region of the spectrum where the cornea1 surface has low reflectivity. This is particularly true of the families Dolichopodidae, Tabanidae, and Tephritidae. As suggested by W. H. Miller (private communication), highly reflecting intersections may function to reduce glare in the spectral region where the retina is most sensitive. For long-legged flies that have reflective intersections, the intersections are usually brightest in the dorsal part of the eye. They could be effective in reducing glare caused by the sun’s bright disk. I know of two exceptional flies that are found on white sand in the sun, where glare is a particular problem. They have eyes with unusually bright and extensive intersection reflections. The first is a white horsefly Stenotabanus psammophilus (&ten Sacken) found on the beaches of eastern Florida, shown to me by L. L. Pechuman. The second is a long-legged fly, Asyndetus ammophilus Leow, that I

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found in central Florida. It has very bright intersection reflections over the entire eye which is unusual for dolichopodids, as is the habitat. Colour vision theory It is possible for a mixture of colour filters to endow an animal with colour vision. Such theories have long been popular for the coloured oil droplets found in some vertebrate retinas. WALLS and JUDD(1933) review theories for function of vertebrate intra-ocular filters and argue that oil droplets do not endow vertebrates with colour vision, they only modify it. In their view, oil droplets enhance colour contrasts and reduce glare according to a ‘multiplex’ system. Recent work by KING-SMITH (1969) h as revived the oil droplet theory of colour vision in the pigeon. The arguments of WALLS and JUDD(1933) ag ainst the oil-droplet colour vision theory do not apply to invertebrates. It is possible that mixtures of corneal interference filters endow flies with colour vision, but this possibility cannot be evaluated yet since behavioural, physiological, and microspectrophotometric studies of colour vision in flies with cornea1 filters have not been done. If filters were the only mechanism for ‘hue discrimination in CondyEostyZus it would be colour blind in the dorsal region of its eye and dichromatic in the ventral region, with a neutral point that exhibits large variability among individuals. In CaZZiphora, a fly that lacks cornea1 filters, LANGERand THORELL(1965) found that the central rhabdomeres had a microspectrophotometric extinction spectrum with peak at 470 nm while all six outer rhabdomeres had maxima at 515 nm, suggesting that dichromatic colour vision is possible in such flies. If in flies with cornealfilter mixtures there are also different extinction maxima in isolated central and outer rhabdomeres, a hybrid colour vision system with no neutral point is possible. Before these hypotheses for the functions of corneal interference filters can be elevated to scientific fact or dismissed, behavioural experiments plus retinal electrophysiology and microspectrophotometry are needed in addition to more information of the type presented here. Further effort should be worth while because it is now clear that cornea1 interference filters are an optical component of the compound eye. Acknowledgements-1 am indebted to my colleague W. H. MILLER for his encouragement, aid, and valuable suggestions, and to 0. TRUJILLO-CEN~Z, Instituto de Investigation de Ciencias Biologicas, Uruguay, for his important contribution to this work. I wish to express my appreciation to L. L. PECHUMAN,Cornell University, for the many times he has helped me with Tabanidae, and to G. C. STEYSKAL, U.S. Department of Agriculture, for determining many of my flies and for teaching me about behaviour and taxonomy of Diptera. I thank the American Museum of Natural History for use of their facilities at the Archbold Biological Station and the Southwest Research Station. This work is supported in part by research Grant EYOOO89 and research career development award EY48264 from the National Eye Institute, U.S.P.H.S., by N.S.F. Institutional Grant GU-2730, and by the Connecticut Lions Eye Research Foundation, Inc.

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GARY D. BERNARD REFERENCES

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