Insect Visual Pigments

Insect Visual Pigments Richard H. White Biology Department, University of Massachusetts at Boston, Boston, Massachusetts, USA

1 Introduction 35 2 Extraction and measurement of insect visual pigments 38 3 Rhodopsin and metarhodopsin 4 0 4 Chromophore and photochemistry 47 5 Regeneration in insect visual systems 5 1 6 Insect color vision and ultraviolet sensitivity 53 7 The problem of the visual pigments of the higher flies 55 8 Transduction and adaptation 57 9 Insect photorgceptor membranes 60 10 Fmalcomments 62 Acknowledgements 62 References 62

1

Introduction

The work of the past decade has begun to outline the particular features of insect visual pigments. Until recently, we knew these photopigments only by inference from electrophysiological and behavioral measurements of spectral sensitivity. The photochemical interpretations of those physiological measurements were typically drawn in terms of the well-known characteristics of vertebrate photopigments. We have now come to realize, however, that extrapolation from vertebrate to invertebrate photoreceptors can be misleading in some important respects. With the characterization of insect visual pigments, the physiology of insect vision is at last being provided with a proper foundation. Although they are different in certain respects, insect visual pigments are similar in their basic features to the photopigments of vertebrates, and studies of insect visual pigments at the present time are necessarily comparative. Recent trends of research into the molecular basis of vertebrate vision, and the 35

36

RICHARD H. WHITE

biochemistry of photoreceptor membranes have been reviewed by Daemen (1973) and Ebrey and Honig (1975). These reviews should be taken as the citations for general statements about visual pigments in the present article. For background, I need only summarize the well-established characteristics of the vertebrate visual pigments that may be broadly designated “rhodopsins”.* Vertebrate rhodopsins consist of a glycoprotein, opsin, and a chromophore, retinaldehyde (more commonly retinal; formerly retinene), the aldehyde of vitamin A. Rhodopsins are hydrophobic proteins, whose natural environment is the specialized membrane of a photoreceptor cell. The chromophore is bound covalently to the opsin in a Schiff base linkage (-C=N-) between the carbonyl of the retinal and an &-aminogroup of a lysine in the protein. Direct spectra of purified vertebrate rhodopsins consist of a main band (the a-band) peaking in the visible, a secondary peak (the P-band) with much lower extinction in the near ultraviolet and a y-band at 280nm due to the absorbance of the aromatic amino acids of opsin (Fig. 1). The main absorption band of the free chromophore lies in the near ultraviolet, at about 380nm (Fig. 1). When it binds to an opsin, forming rhodopsin, the main absorption maximum (Amax) shifts into the visible region of the spectrum. The basis of this “bathochromatic shift” of chromophore absorbance to longer wavelengths is a central problem in current research on the molecular structure of visual pigments. Retinal is a polyene, characterized by a backbone of alternating double and single bonds (Fig. 1). Delocalization of the n-electron system of such a polyene would be expected to shift its absorbance to longer wavelengths. Such a modification of the chromophore’s electronic structure is thought to be accomplished by protonation of the Schiff base linkage between the opsin and the chromophore, and by additional poorly characterized interactions between the chromophore and the protein. The main absorption maxima of the visual pigments belonging broadly to the rhodopsin class range among the vertebrates from 430nm in the blue to 580nm in the yellow-orange. In these various rhodopsins, the chromophore is the same; only the opsins differ. Therefore, it is thought that the absorption maxima of different visual pigments are fine-tuned by the particular disposition of the charged opsin groups at the site of the chromophore. This is an inference, however, for the detailed protein structure of a visual pigment has not been determined as yet. Retinal, as a polyene, can exist as a number of geometric isomers. The chromophore of rhodopsin is specifically the 11-cis isomer of retinal, in which the polyene backbone is bent and twisted around carbon 11 (Fig. 1). The essential action of light absorbed by a molecule of rhodopsin is the photo*“Porphyropsins” form a second class of vertebrate visual pigments. They differ from rhodopsins only in their chromophore, which is 3-dehydroretinal. Porphyropsins are formed mainly in fresh water fishes and larval amphibians. Porphyropsin pigments have not been found in invertebrates.

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37

chemical isomerization of the chromophore from the bent 11-cis to the straight all-trans configuration. In vertebrates, the protein then undergoes conformational changes not requiring light that lead to the dissociation of the all-trans chromophore from the protein, leaving free retinal and opsin. Since rhodopsin absorbs in the visible, while free retinal and opsin absorb in the ultraviolet, which we cannot see, this process is called bleaching. When slowed at low temperature, or followed by fast photometric techniques, the bleaching process can be resolved into a series of spectrally distinct stages, the intermediates of ISOMERS OF RETINAL

E

Wavelength-nm Fig. 1. Absorbance spectra of retinal and cattle rhodopsin. (A,,,, all-trans and 1 1-cis isomers of retinal.

5 0 0 nm). The inset shows the

bleaching. A number of lines of evidence indicate that the stages of bleaching reflect the progressive “opening up” of the opsin’s structure, and the loosening of the association of the chromophore with it. For instance, intact rhodopsin is stable to a number of reagents that successfully attack opsin or retinal. All vertebrate visual pigments progress through similar bleaching stages, ending with the hydrolysis of the chromophore from the opsin. As rhodopsins are membrane bound proteins, they can be brought into solution without denaturation only in detergent extracts. Digitonin has long been the standard extractant; other detergents have been introduced in recent

38

RICHARD H. WHITE

years. Since vertebrate rhodopsins are bleached by light, difference spectra calculated by subtracting bleached from dark spectra accurately trace the abands of those rhodopsins whose Lax lie at wavelengths greater than 450 nm. In this way, vertebrate visual pigments can be measured in relatively crude extracts or in situ; in membrane suspensions, in whole retinas, in single photoreceptor cells by microspectrophotometry, and in intact functioning eyes by reflectance photometry. 2

Extraction and measurement of insect visual pigments

Well before arthropod rhodopsins were convincingly localized by direct measurement it had been inferred that they must be associated with arrays of microvilli elaborated from the plasma membranes of photoreceptor cells (see review by Win, 1972). The mass of microvilli of a particular receptor cell is called its rhabdomere. Where the rhabdomeres from several contiguous cells cluster together they are collectively termed a rhabdom [Fig. 3(c)l. The association of photopigments with rhabdomeres has been shown directly by microspectrophotometry (Langer and Thorell, 1966; Hays and Goldsmith, 1969; Brown and White, 1972). Like other visual pigments, insect rhodopsins can be brought into solution intact only in detergent micelles (Woken and Scheer, 1963; Gogola et al., 1970; Marak et al., 1970; Hamdorf et al., 1971b; Paulsen and Schwemer, 1972; Schwemer and Paulsen, 1973; Fernandez and Bishop, 1973; Ostroy et al., 1974). Therefore, they must be bound within the microvillus membranes of the rhabdomeres. Digitonin has generally been the detergent used for extracting insect rhodopsins in procedures that are modifications of the techniques developed for vertebrate and squid retinas. These standard procedures may be found in the review of Hubbard et al. (197 1). Vertebrate retinas are large, and photoreceptor membrane is contained within isolated cellular appendages, the outer segments of the receptor cells. Because of these features the outer segment membrane can be easily isolated in substantial amounts prior to rhodopsin extraction. Insect rhabdoms, however, are bound into the complex cellular fabric of small retinas, and consequently the extraction of insect rhodopsins is technically more dimcult. The accessory ommochrome and pteridine pigments densely contained within insect photoreceptors and contiguous cells are particularly bothersome in extraction procedures. They not only raise background extinction, but they may undergo pH or light-induced absorbance changes that can confuse photometric measurements (Bowness and Woken, 1959). Accessory pigments can be removed by repeated preliminary buffer washes of eye homogenates, or in flotation procedures that concentrate rhabdomere membrane prior to detergent extraction (Schwemer et al., 1971; Paulsen and

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39

Schwemer, 1972). Recently Weber and Zinkler (1974) have devised a method for isolating intact rhabdomeres from Culliphoru eyes. It has generally been found that the absorption spectra of visual pigments are not altered by digitonin extraction (however, see Bruno and Goldsmith, 1974, for a possible example of such an alteration). Detergents other than digitonin are more likely to lead to partial or complete denaturation (Daemen, 1973; Paulsen and Schwemer, 1973) and must be used with caution. Although the work of the past few years has convinced us that insect rhodopsins are orthodox hydrophobic visual pigment proteins bound within membranes, a buffer soluble protein with retinal chromophore was found in one of the earliest attempts to identify an insect photopigment (Goldsmith, 1958a,b). It was extracted from honeybee heads, accounting for half the retinal in the tissue. It behaved like a typical vertebrate rhodopsin, bleaching with the release of retinal to yield a difference spectrum with maximal absorbance change at about 440 nm. Although there is evidence from spectral sensitivity measurements that drone bees have 440 nm receptors, the extract was taken from a population of mostly worker bees in which units sensitive at 535 nm predominate. Thus Goldsmith (1972) and Bruno and Goldsmith (1974) subsequently suggested that the pigment he had extracted was in some respect an artifact. The question of the buffer soluble pigment has been reopened by Pepe et al. (1976). Extracts of bee retinas exposed to tritiated vitamin A were subjected to electrophoresis. The label migrated as a single peak among the faster buffer soluble proteins. The label introduced as vitamin A appeared to be associated with the protein in the form of retinal as in a rhodopsin. However, the retinalprotein complex has not been spectrally characterized, nor has its relationship to the photosensitive pigment extracted by Goldsmith been established. Pepe et ul. (1976) suggest that it might be a precursor of a membrane bound rhodopsin. Alternatively it might function in transport of retinaldehyde. A number of vitamin A binding proteins that act as carriers have been identilied in vertebrates (Heller and Bok, 1976). Microspectrophotometry (MSP), in which a preparation is mounted on the stage of a microscope inserted in the beam of a spectrophotometer, is another procedure that has been particularly useful for the analysis of insect visual pigments. With MSP-applications and limitationshave recently been reviewed by Liebman (1972)-photopigments can be measured in whole eyes, slices of retina, whole cells or in isolated rhabdoms. MSP offers the advantage of measuring photopigments in situ, and the possibility of localizing particular pigments within particular cells. Visual pigments are measured by MSP either in difference spectra, taken between dark and illuminated shples, as will be discussed in some detail below, or by subtracting the baseline spectrum of an adjacent cellular or extracellular region from the rhabdom spectrum (Langer and Thorell, 1966; Hays and Goldsmith, 1969).

RICHARD H. WHITE

40

Various complications, such as the orientation of rhodopsin molecules within their membranes, and optical effects associated with narrow, long or connected rhabdomeres, may alter absorption spectra measured in uiuo in comparison with measurements made on extracts under conditions in which the Beer-Lambert law may be rigorously applied (Snyder and Pask, 1973; Hamdorf and Schwemer, 1975). Such effects are of course important in the actual response of the photoreceptor, but a detailed discussion of them is beyond the scope of the present review. This important area has recently been covered in a review by Goldsmith and Bernard (1974) and by Snyder and Menzel (1975). It is particularly true for insects that the most convincing and informative studies have been those in which photopigments have been measured both in extracts and in situ by MSP. Other techniques that have been used to measure insect photopigments are reflectance photometry in a living butterfly (Stavenga, 1975), and measurement of a fast electrical response from the Drosophila eye (Pak and Lidington, 1974). The latter is similar to the early receptor potential (ERP) that has been recorded from vertebrate (Cone, 1967), squid (Hagins and McGaughy, 1967) and arthropod eyes (Brown et al., 1967; Minke et al., 1973). ERP responses arise directly from charge displacements during photopigment transitions, allowing direct measurements of visual pigments in intact eyes. The technique apparently has not been widely attempted with insects. 3

Rhodopsin and rnetarhodopsin

Squid rhodopsin was the first invertebrate photopigment to be well characterized (Hubbard and St. George, 1958). As in vertebrate rhodopsins, its chromophore is 11-cis retinal. It was found, however, that squid rhodopsin does not bleach. When light is absorbed the initial molecular events are similar to those described above for vertebrate rhodopsins: chromophore isomerization leads to conformational changes in squid opsin. But rather than proceeding to hydrolysis, the reaction culminates with the formation of a colored intermediate, metarhodopsin, with the chromophore in the all-trans configuration still attached to the opsin. Squid metarhodopsin was so named because of its biochemical similarity to the metarhodopsin I bleaching intermediate of vertebrate visual pigments. The difference is that squid metarhodopsin is stable at physiological temperature, whereas vertebrate metarhodopsins decay through additional intermediates that lead finally to hydrolysis. It has subsequently been found that metarhodopsin thermostability is a characteristic of invertebrate photopigments generally, and of insect visual pigments in $articular. Consequently, both the rhodopsin and metarhodopsin states of insect visual pigments are found in illuminated photoreceptors. The

INSECT VISUAL PIGMENTS

41

two thermostable states differ in isomeric form of the chromophore and configuration of the opsin, and generally differ in I,,, and molar extinction as well. A quantum of light absorbed by a molecule of insect rhodopsin converts it through isomerization of the chromophore to metarhodopsin; light absorbed by metarhodopsin re-isomerizes the chromophore regenerating rhodopsin. In continuous light of such spectral composition that it is absorbed by both rhodopsin and metarhodopsin, insect visual pigments flip back and forth between their two stable states, and a photoequilibrium is established. The

Fig. 2. MSP absorption spectra from a larval mosquito (Aedes aegypti) ocellus. (a) Spectra from an ocellus mounted in insect Ringer. Curves 1 and 2 were dark scans whose superposition shows baseline stability. Curve 3 was recorded after an intense flash of yellow light. The change in absorbance resulted from the photoconversion of some rhodopsin to metarhodopsin. (b) Spectra from the ocelli of a living animal. Curves 1 and 2 were measured in the dark; curve 3 was recorded after a yellow flash, and curve 4 was recorded after a subsequent blue flash. The yellow flash converted a portion of the rhodopsin to metarhodopsin; the blue flash reconverted some of the metarhodopsin to rhodopsin. (c) Difference spectrum calculated by subtracting curve 3 from curves 1 and 2 in (a).

concentrations of rhodopsin and metarhodopsin in such a photosteady state will depend on their respective I,,,, absorbance coefficients and quantum efficiencies, and on the spectral quality of the light. Some of the practical consequences of metarhodopsin thermostability for the measurement of insect photopigments are exemplified in a study of the larval mosquito ocellus (Brown and White, 1972). Larvae were grown in darkness in order to ensure that their ocelli would contain only rhodopsin and no metarhodopsin. They were then prepared for MSP in dim red light that hopefully would convert little or none of the rhodopsin to metarhodopsin. Figure 2(a)

42

RICHARD H. WHITE

shows measurements taken from an ocellus mounted in insect Ringer in the microspectrophotometer. Traces 1 and 2 are dark scans; curve 3 was recorded after irradiation with yellow light. As a result of irradiation, absorbance dropped at longer wavelengths and rose at somewhat shorter wavelengths with an isosbestic point at 500 nm. As will be later proven, the decline in absorbance was due to loss of rhodopsin, the increase resulted from the formation of a metarhodopsin photoproduct absorbing at shorter wavelengths, and curve 3 represents the photoequilibrium established by the yellow light. Figure 2(b) shows a similar experiment with an intact living animal. Once again, 1 and 2 are dark scans. Scan 3 followed yellow irradiation as in the preceding experiment. The animal was then illuminated with blue light that is absorbed more strongly by metarhodopsin, so that the photoequilibrium was shifted back somewhat towards rhodopsin (curve 4). The difference spectrum calculated by subtracting curve 3 from curves 1 and 2 in the experiment of Fig. 2(a) is shown in Fig. 2(c). Where the absorption spectra of rhodopsin and metarhodopsin overlap, they mutually subtract in difference spectra. Consequently, in Fig. 2(c) the rhodopsin spectrum to the right is cut off at shorter wavelengths whereas the metarhodopsin spectrum to the left is cut off on its long wavelength side. Measurements such as these show how the visual pigment behaves in situ but they do not accurately characterize either state of the pigment because their spectra interfere. This problem can be overcome, and the spectra of insect rhodopsins determined more accurately under conditions that promote hydrolysis of metarhodopsin. Illumination then results in bleaching, as with vertebrate photopigments. This is often the case when insect visual pigments are brought into solution; metarhodopsins tend to be less stable in digitonin extracts than in uiuo (Schwemer and Paulsen, 1973; Schwemer, personal communication). For MSP, fixation of receptor cells with glutaraldehyde, the histological fixative, generally preserves rhodopsins but renders metarhodopsin labile (Hays and Goldsmith, 1969; Brown and White, 1972). Glutaraldehyde fixation serves the additional purpose of stabilizing cellular structure by cross-linking proteins. It therefore helps to maintain constant spectral baselines and allows the use of other reagents that also promote hydrolysis such as hydroxylamine and potassium borohydride. The former reacts with retinal yielding retinaldehyde oxime, the latter reduces retinal to retinol (or reduces it on site with the same spectral result). To continue with the mosquito ocellus as an example, in the experiment of Fig. 3(a) a dark adapted larva was dissected into glutaraldehyde and mounted in the spectrophotometer in 0.1 M hydroxylamine. Curves 1 and 2 were recorded in the dark, curve 3 after the ocellus had been exposed to intense yellow light for 10 min. As a result, absorbance dropped in the spectral region around 5 15 nm, and rose at 360 nm. The large drop in absorbance at longer

INSECT VISUAL PIGMENTS

43

wavelengths presumably resulted from the bleaching of the main band of mosquito rhodopsin, the increase at 360nm from the formation of retinaldehyde oxime. The difference spectrum is plotted in Fig. 3(b). The rhodopsin 515 nm, is accurate at wavelengths longer than 450 nm. spectrum, A,, Below that its spectrum is obscured by that of the oxime.

Fig. 3. (a) Spectra from a ventral ocellus fixed in a glutaraldehyde and mounted in neutralized hydroxylamine. Curves 1 and 2 were recorded in the dark, curve 3 after the ocellus had been bleached for 10 min with yellow light. The difference spectrum plotted in (b) represents the true absorption spectrum of rhodopsin at wavelengths greater than 450 nm. The inset (c) is a photomicrograph of the ocellus that was measured as it appeared in the microspectrophotometer. For this study a white eye mutant was used that lacked screening pigment. The scalloped rosette at the center of the ocellus is its rhabdom. A 30 pn central area of the rhabdom was measured.

Several lines of evidence indicate that the dif'ference spectrum of Fig. 3(b) truly represents the spectrum of mosquito rhodopsin (R5 15). The most cogent reason for accepting it as a visual pigment is that it matches the spectral sensitivity of mosquito larvae measured from the electroretinogram (ERG) (Seldm et d., 1972). It is also matched well by the theoretical spectrum for a 515 nm rhodopsin calculated from Dartnall's nomogram (Dartnall,1953). Such a theoretical reGnance spectrum for retinal based visual pigments is a useful yardstick for judging the accuracy of measured spectra. The experiment in Fig. 3 also provides evidence that a rhodopsin with retinal chromophore has been measured rather than some other light sensitive substance in the cell. The formation of a photoproduct at about 360nm in the presence of hydroxyl-

44

RICHARD H WHITE

amine indicates that retinal was released by light to form retinaldehyde oxime. Finally, R5 15 was found only in the rhabdom, not in the bodies of the receptor cells. To summarize the general criteria for characterizing an insect rhodopsin: its spectra should reasonably match spectral sensitivity, there should be evidence that it has retinal for chromophore, and it should be localized to a rhabdom and/or shown to be membrane bound by its solubility characteristics in vitro. In measuring the spectra of insect visual pigments one must be aware that metarhodopsin stability can introduce serious distortions. Fig. 2(c) illustrates how difference spectra may fail to correspond closely with absorbance spectra when the spectrum of a photoequilibrium is subtracted from a dark spectrum. A more subtle artifact occurs when one starts with a mixture of rhodopsin and metarhodopsin. In the presence of hydroxylamine or under other conditions that promote bleaching, the difference spectrum will be the sum of the components. The result is a broadened spectrum whose peak lies somewhere between the A,, of the two pigments. The problem is the same when the spectrum of a rhabdomere is compared with a clear area of cytoplasm in MSP. An example is offered by the pioneering microspectrophotometric analysis of Calliphora visual pigment by Langer and Thorell (1966) done before insect metarhodopsin stability was recognized. These first measurements of the presumed absorption maximum of fly rhodopsin varied between 490 nm and 540 nm; most were around 515 nm. The spectra were broader than predicted by the Dartnall nomogram and did not match spectral sensitivity. Calliphora rhodopsin was subsequently found to lie at 490nm, its metarhodopsin at 575 nm (Hamdorf et al., 1973b; Stavenga et al., 1973). It is now clear that the early measurements were of various rhodopsin-metarhodopsin mixtures (see the comments of Langer following the paper of Stavenga et al., 1972). This problem can arise as the result of incomplete dark adaptation or because the MSP scanning beam is bright enough to convert measurable amounts of rhodopsin to metarhodopsin. For the measurements of mosquito rhodopsin described above we were fairly confident that we started with pure rhodopsin, since the larvae were hatched and reared in darkness and dissected in deep red light. In retrospect, however, we cannot be certain that even our best rhodopsin spectra were not contaminated with small amounts of metarhodopsin. It is evident from the foregoing discussion that it is easiest to deal with insect visual pigments whose rhodopsin and metarhodopsin spectra are widely separated. This is true, for instance, of the ultraviolet sensitive pigment of the Neuropteran Ascalaphus macaronius. The main absorption band of Ascalaphus rhodopsin lies at 345 nm, that of its metarhodopsin at 475 nm [Fig. 4(a)l. In this pigment system, light between 440nm and 600nm is absorbed only by M475. M475, like all visual pigments also absorbs light at

INSECT VISUAL PIGMENTS

45

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Fig. 4. Absorption spectra calculated from difference spectra for the rhodopsins and metarhodopsins of three insect species: (a) Ascalaphus macaronius, (b) Culliphora erythrocephala, (c) Deilephila elpenor. The rhodopsin spectra have been normalized to 1.0, and the metarhodopsin spectra have been calculated accordingly. The latter have relatively higher absorbances because their molar extinctions are higher. The curves labeled [R]represent the concentrations of rhodopsin in the photoequilibria that would be established by monochromatic irradiation assuming that the quantum efficiencies of rhodopsin and metarhodopsin are the same. The curves in (b) were recalculated by J. Schwemer for this paper.

TABLE 1

The absorption maxima (A, Order and species ORTHOPTERA

Perfplaneta americana

nm) of insect rhodopsins and their metarhodopsin photoproducts

Rhodopsin

Metarhodopsin

Reference Woken and Scheer (1963)

500

345 440 520 345 440 520 5 10 535

475 (acid) 380 (alkaline) 480 480 480 490 490 490 484 480

Gogala et al. (1970); Schwemer el al. (1971) Schwemer and Paulsen (1973); Hoglund et a/. (1973b)

Aedes aegvpti Drosophila melanogaster

515 480

480 580

Callfphora erythrocephala

490

575

Sarcophaga bullata Musca domestica

490 5 10 512 334

575

Brown and White (1972) Pak and Lidington (1974);Ostroy et a/. (1974) Hamdorfet a/. (1973b);Stavenga et al. (1972) Schwemer (personal communication) Marak et al. (1970) Murietal. (1976)

NEUROPTERA

Ascalaphus macaronius LEPIDOPTERA

Deilephila elpenor Manduca sexta" Galleria mellonella Aglais urticae

345

DIPTERA

HYMENOPTERA

Apis mellifera (drone)b

415 420

Brown and Schwemer (personal communication) Goldman et a/. (1975) Stavenga (1975)

Carlson and Philipson (1972)claimed to have measured four pigments in Manduca, A, 350,450,490,530.Their work has been criticized for poor technique (Goldsmith and Bernard, 1974). It is likely that they were confusing metarhodopsin with rhodopsin . These data are from an abstract; no spectra were published. A water soluble pigment (Amax, 440 nm) with retinal chromophore has also been extracted from honeybee eyes (Goldsmith, 1958a,b). a

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0

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%

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sz

2

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47

wavelengths shorter than where its main absorption band lies. Hence ultraviolet light up to 400 nm is absorbed strongly by R345 and to a lesser extent by M475 as well. Irradiation at 345 nm establishes a photoequilibrium consisting of about 1R:3M. Subsequent saturation of the system with blue light shifts the system back to 100 per cent R345 by photoregeneration (Schwemer et al., 1971;Hamdorf et al., 1973b). Figure 4(a) shows the spectra of Ascalaphus R345 and M475 calculated from difference spectra of digitonin extracts. Note that the extinction of metarhodopsin is higher than that of rhodopsin, presumably because the molar extinction of all frans-retinal is higher than that of the 11-cis isomer (Hubbard et al., 1971). From the relative absorbance of each pigment at a particular wavelength, one can calculate the photoequilibrium established by monochromatic irradiation at that wavelength, assuming that the two forms of the pigment have the same quantum efficiencies (Hamdorf et al., 1971a; Hamdorf et al., 1973b). The amount of rhodopsin in such theoretical equilibria is shown in Fig. 4(a). Experimental measurements of photoequilibrium concentrations of R345 and M475 in both extracts and by MSP measurements of Ascalaphus retinas agree well with the calculated photoequilibria. Similar photokinetic studies on the visual pigments of Lepidoptera and Diptera [Fig. 4(b), (c)] have given similar results: the relative concentrations of rhodopsin and metarhodopsin in a brightly illuminated photoreceptor appear to depend only upon the spectra and molar extinctions of the two states of the photopigment, and on the spectral quality of the light (Hamdorf et al., 1973b; Hoglund ef al., 1973a). The absorbance maxima of most insect metarhodopsins lie around 480 nm490 nm (Table 1). However, in certain pigment systems of the higher flies the metarhodopsins peak around 575nm, while the rhodopsins lie at about 490nm. Some cephalopods have similar pigment systems with long wavelength metarhodopsins (Brown and Brown, 1958; Schwemer, 1969; Hamdorf et al., 1972). Before leaving this general discussion of insect rhodopsins, I should mention that the molecular weight of Ascalaphus opsin has been measured by Paulsen and Schwemer (1973). From its relative mobility in sodium dodecylsulfate (SDS)polyacrylamide gel electrophoresis they estimated its molecular weight to be 35 OOO k 1800. This is similar to the molecular weights of frog and cattle opsins determined by the same method, but somewhat less than the molecular weights of cephalopod rhodopsins (Hagins, 1973; Paulsen and Schwemer, 1973). 4

Chromophore and photochemistry

The chromophore of insect visual pigments has been identified from both direct and indirect evidence as retinaldehyde (retinal). In the first successful bio-

48

RICHARD H WHITE

chemical study of insect vision (Goldsmith, 1958a,b), substantial amounts of retinal, identified by the standard reaction with antimony trichloride (CarrPrice procedure: Hubbard et al., 1971) were found in the heads of dark adapted honey bees. None was detected in their bodies. Retinal has subsequently been measured by the Carr-Price reaction in extracts from the heads, but not the bodies, of a number of insects including Orthoptera, Odonata, Lepidoptera, Coleoptera, Diptera and Hymenoptera (Wolken et al., 1960; Briggs, 1961; Wolken and Scheer, 1963). As far as we know, insects like other animals only obtain retinol (vitamin A) and retinal from their food, either directly or from other carotenoids. Retinol is not a vitamin with an essential systemic function for insects as it is for vertebrates. Consequently, insects can be grown, for generations if desired on carotenoid-free diets. Carotenoid deficiency produced ultrastructural changes in the photoreceptor cells of the mosquito (White and Jolie, 1966; Brammer and White, 1969). These morphological changes were accompanied by loss of visual sensitivity. The visual threshold of houseflies grown for several generations on a sterile defined diet without carotenoid rose at least four log units (Goldsmith et al., 1964; Goldsmith and Fernandez, 1966). Sensitivity returned to normal when they were fed fi-carotene. Thus even before insect visual pigments had been well characterized there was evidence pointing to retinal as the chromophore. More direct evidence was provided by Schwemer et al. (1971) and Brown and White (1972), when Ascalaphus and mosquito rhodopsins were shown to react with hydroxylamine and potassium borohydride. Only the chromophore of Ascalaphus rhodopsin (R345) has been well characterized. Retinal was measured in extracts of Ascalaphus eyes by the antimony trichloride method. It was bound exclusively to insoluble debris, presumably membrane fragments (Paulsen and Schwemer, 1972). Both the alltrans and the 11-cis isomers of retinal were identified by thin layer chromatography. Other isomers were not present in measurable amounts. The configuration of the chromophores of vertebrate and squid visual pigments have been identified by denaturing rhodopsin in the darkness, in order to release retinal while avoiding the isomerizing effect of light (Hubbard and Kropf, 1958). In a similar procedure, the chromophore of R345 was released after denaturation with Ag2+. Opsin prepared from bleached cattle retinas provides a sensitive and specific assay for the 11-cis isomer of retinal, since only this isomer promotes the regeneration of cattle rhodopsin, A,, 500 nm (Hubbard et al., 1971). When the chromophore released from denatured R345 was mixed with purified cattle opsin, cattle rhodopsin was regenerated. Therefore the chromophore of R345 is 11-cis retinal, like that of all other rhodopsins. Does light isomerize the chromophore to all-trans retinal as in other

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rhodopsins? Denaturation of a photoequilibrium mixture consisting of onethird R345 and two-thirds M475 released only a third as much 11-cisretinal as from 100 per cent R345. Since thin layer chromatography had demonstrated that the only retinal isomers present in the eye were 1 1 4 s and all-trans, the chromophore of M475 must be all-trans retinal. Hence the basic photochemistry of the Ascalaphus photopigment is similar to that of the visual pigments of other animals. Metarhodopsins, both vertebrate and invertebrate, typically act as pH indicators. The spectrum of Ascalaphus metarhodopsin also shifts with pH, changing from acid M475 to alkaline M380 with a pK of 9.2 (Schwemer et al., 1971; Hamdorf et al., 1973b). Obviously, M475 predominates at physiological pH; this was shown by MSP of Ascalaphus retinas. The pH sensitivity of metarhodopsins has been attributed to the reversible protonation of the Schiff base link between retinal and opsin. These observations indicate that the chromophore of Ascalaphus visual pigment is also bound as a Schiff base, at least in metarhodopsin. This is an important conclusion, since unlike the generality of rhodopsins the spectrum of retinal is shifted to shorter wavelengths when it is associated with its opsin. Speculations that this hypsochromatic spectral shift might result from a fundamentally different association of the chromophore and protein (cf. Goldsmith, 1972) now seem unlikely. Presumably the resonant structure of the chromophore is shortened by way of interactions with its opsin that are in addition to the covalent SchifT base link. This most interesting pigment system needs to be studied with the contemporary biophysical techniques, such as resonance Raman spectroscopy, that are now being applied to vertebrate visual pigments (Ebrey and Honig, 1975). After absorbing light visual pigments pass through a sequence of intermediate states. Interest in these intermediates is grounded in the notion that one of the transitions must lead to transduction, that is, to the generation of an electrical signal across the photoreceptor membrane. The intermediates of vertebrate and squid rhodopsins have been spectrally defined in low temperature experiments in which their rates of decay are slowed (Ebrey and Honig, 1975). The results of similar experiments with the visual pigments of Ascalaphus (Hamdorf et al., 1973b) are summarized in Fig. 5. At physiological temperature only the two thermostable states of the pigment, R345 and M475, can be measured with conventional slow speed spectrophotometry. Irradiation of R345 at -50° C produced a new intermediate, stable at that temperature, peaking at 375 nm. This was designated Lumirhodopsin (L375) in accordance with the terminology adopted for vertebrate visual pigments. When the temperature was subsequently raised to -15O C in darkness, L375 decayed to M475. These observations can be interpreted as follows. At -5OO C cis to

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trans chromophore isomerization occurs, but changes in opsin configuration presumably are prevented. The initial absorbance shift of 30nm to longer wavelengths is thought to reflect the change in chromophore conformation and the differing interactions of its cis and fruns forms with the unaltered protein. Raising the temperature then allows the protein to change conformation with an additional 100 nm shift. In other words, these data support the idea that the rhodopsin to metarhodopsin reaction includes conformational changes of opsin

1

Rhodopsin 345 nm ( 1 1 4 s )

Lumirhodopsin 375 nm (all-trans) Alkaline metarhodopsin 380 (dl-trans)

pK 9.2

f

Acid metarhodopsin 475 nm (al-trans)

Acid rnetarhodopsin 460 nm ( 1 1 4 s ) -15°C

Fig. 5. Intermediates of the ultraviolet sensitive visual pigment of Ascalaphus macaronius. Wavy lines represent photoreactions, straight lines, dark reactions.

provoked by the initial photoisomerization of the chromophore. In the opposite experiment, irradiation of M475 at -50 OC produced another intermediate at about 460 nm. When warmed in darkness that intermediate regenerated to R345. Since rhodopsin regenerates in darkness from the new intermediate, the chromophore of the intermediate must be 11-cis retinal. Moreover, the opsin presumably retains its metarhodopsin configuration in the new intermediate. Hence it has been designated 11-cis metarhodopsin. The large spectral shift to shorter wavelength that occurs when M460 regenerates to R345 is presumably due to thermal changes in the opsin that can take place after the chromophore has assumed the 1 1-cis configuration, and reflects the additional interactions between protein and chromophore that then becomes possible.

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The spectra and rates of decay of intermediates have been important data for understanding the molecular events that follow upon the absorption of light by rhodopsin. Photoreversal reactions, that is, photoregeneration of rhodopsin from intermediates via frans to cis isomerizations have also been studied in efforts to characterize the interactions of opsin and chromophore (Williams ef al., 1973). Ascalaphus 11-cis metarhodopsin is a remarkable intermediate of photoregeneration. Such metarhodopsins with cis chromophores have been only poorly characterized in vertebrate systems (see comments of Williams after the paper of Hamdorf et al., 1973). One further aspect of rhodopsin photochemistry that deserves comment: energy transfer from opsin to chromophore has been demonstrated in vertebrate rhodopsins. That is, light absorbed by the y-band at 280 nm can initiate bleaching (Kropf, 1967). When measurement of housefly spectral sensitivity was extended into the middle ultraviolet, a shoulder was found at 280nm (Goldsmith and Fernandez, 1968). The authors have suggested that this response at very short wavelengths is due to energy transfer from the opsin to the chromophore.

5

Regeneration in insect visual systems

The spectral sensitivity curves of insects and other invertebrates match rhodopsin but not metarhodopsin spectra (Hamdorf et al., 1973b), that is, only light absorbed by rhodopsin is transduced into a visual response. Therefore, there must be regenerative mechanisms for maintaining adequate concentrations of rhodopsin in illuminated eyes. From the preceding discussion it is clear that photoregeneration from stable metarhodopsin holds rhodopsin at a constant level in photoequilibria. Two features of insect photopigment systems tend to favor high rhodopsin levels in the photoequilibriaestablished by normal polychromatic daylight. Insect metarhodopsins generally have higher absorbance than their rhodopsins and most metarhodopsins peak in the blue-green near the emission maximum of daylight (Hamdorf et al., 1973b). For example, the daylight photoequilibrium of the ultraviolet sensitive system of Ascalaphus is 90 per cent rhodopsin (Hamdorf and Gogala, 1973; Hamdorfet al., 1971a). In other naturally illuminated insect photoreceptors, rhodopsin must be maintained well above the 50 per cent level (Hoglund et al., 1973a,b; Hamdorf and Schwemer, 1975). The predominant photopigment systems of the cyclorraphous flies with their blue-green sensitive rhodopsins and red absorbing metarhodopsins would seem perversely adapted to favor low rhodopsin levels. Nevertheless this is an exception that proves the rule. These flies have red accessory pigments that

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absorb the shorter visible wavelengths but leak red light (Goldsmith, 1965; Langer, 1967; Stavenga el al., 1973). Under normal conditions, their rhabdomeres are bathed in scattered red light absorbed selectively by metarhodopsin, and photoequilibria are pushed well over toward rhodopsin. Rhodopsin concentrations in photoequilibria as a function of wavelength are shown in Fig. 4. In vertebrates, regeneration is accomplished by an enzymically mediated visual cycle. All-trans retinal released from rhodopsin by bleaching is reduced to retinol, enzymatically isomerized, and reconverted to 1 1-cis retinal. The chromophore can then recombine with opsin to regenerate rhodopsin (Bridges, 1976). Since regeneration via the visual cycle does not depend on illumination-it occurs in both light and darkness-it is spoken of as “dark regeneration”. Does any sort of dark regeneration take place in insects? None was found in the mosquito ocellus where rhodopsin-metarhodopsin ratios set up by illumination did not change after more than an hour of subsequent darkness (Brown and White, 1972). Complete regeneration of rhodopsin in the moth Galleria required several days of darkness (Goldman ef al., 1975). In Drosophila the time constant of dark regeneration is about 6 h (Pak and Lidington, 1974), whereas in Calliphora it is 25 min (Stavenga et al., 1973). Stavenga (1975) has also reported a dark regeneration half time between 15 and 45 min in the butterfly Aglais. In no instance do we know the mechanism of dark regeneration, and the wide variation in the rates of regeneration among the few insects investigated suggests that there may be a variety of mechanisms. The only direct studies on the visual cycle in insects are contradictory. Taking as precedent the vertebrate visual cycle of bleaching and enzymatic regeneration, Goldsmith and Warner (1964) sought and found a retinal-retinol oxidation-reduction system in the head of the bee. As in the vertebrate retina, retinal predominated in darkness, retinol in light. They also found retinal reductase activity in the heads of bees. On the other hand, Paulsen and Schwemer (1972) found retinal in the eyes of Ascalaphus but no retinol in either light or dark adaptation. In light of our present understanding of insect visual pigments, their results are not surprising. There would be no reason to expect a light4ark retinal-retinol cycle in systems with stable metarhodopsins that do not bleach. I have mentioned the puzzling water soluble protein-retinal complexes that have been extracted from bees. The visual cycle characterized by Goldsmith and Warner is another interesting feature of the bee eye that demands further study. There are several obvious ways by which dark regeneration might be accomplished in insects. Enzymatic trans to cis isomerization of the free chromophore occurs in vertebrates, apparently within the outer segments (Bridges, 1976). An isomerase could account for those instances of relatively

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rapid dark regeneration in insects. It presumably would have to act upon retinal still attached to the metarhodopsin in the photoreceptor membrane. Very slow regeneration, as in Galleria (Goldman et al., 1975) might reflect turnover of visual pigment, that is, complete renewal of rhodopsin via protein synthesis and renewal of photoreceptor membrane. In this regard there is indirect evidence that some insect photoreceptor membranes may have high rates of turnover, rates sufficient to accomplish dark regeneration in a matter of hours (White and Lord, 1975). From the standpoint of physiological adaptation it is hard to see why insects-at least diurnal insects, in which some of the highest regeneration rates have been measured-should have any need to supplement photoregeneration with special mechanisms of dark regeneration. At best, a period of regeneration in darkness following illumination can increase absolute sensitivity by less than a factor of 2 because rhodopsin is already maintained at levels above 50 per cent in photoequilibria (Hoglund et al., 1973a,b). 6

Insect color vision and ultraviolet sensitivity

The existence in insect eyes of units sensitive to different regions of the spectrum is well known from electrophysiological measurements (Autrum and v. Zwehl, 1964; Burkhardt, 1964). Behavioral experiments, most notably with bees and Lepidoptera, have demonstrated true color vision based upon trichromatic perceptual systems (v. Frisch, 1965; Knoll, 1924). The only insects with demonstrated color vision whose visual pigments have been well characterized are the sphinx months Deilephila elpenor and Manduca sexta (Hamdorf et al., 1972a; 1973a; Schwemer and Paulsen, 1973; Schwemer and Brown, personal communication). Their visual systems are similar, and I will discuss only that of Deilephila. Three photopigments have been measured in intact retinas by MSP and in digitonin extracts. Their respective absorption maxima lie at about 345 nm (ultraviolet), 440 nm (blue) and 520 nm (green). Deilephila eyes contain four or five times as much R520 as R345 and R440, assuming that the molar extinctions of the three pigments are similar. The metarhodopsins of all three peak in the vicinity of 480 nm. Components of the Deilephila electroretinogram (ERG) corresponding to each of the three photopigments can be selectively light adapted, indicating that the pigments are localized in separate receptor cells (Hoglund et al., 1973a,b). Thus the essential basis for color perception is present: distinct visual pigments differentially sensitive across the spectrum housed in physiologically separate receptors. Hoglund et al. (1973a,b) have suggested that photoregeneration is important for maintaining this system of color discrimination in balance. Ambient sky light should tend to maintain the three pigments at similar relative concentrations in their respective photoequilibria since their metarhodopsins all

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have similar spectra centered on the wavelength of maximal daylight emission. In this way the relative sensitivities of the three types of receptor might be kept constant and color discrimination might simply be based on the comparison of the response amplitudes of the three receptors. The emission spectrum of the sun extends well into the ultraviolet, to about 300 nm, with a minor peak around 350 nm (Hamdorf el al., 1971a). It has been often pointed out that insects visually exploit these shorter wavelengths whereas vertebrates do not (Goldsmith and Bernard, 1974). Most vertebrates are blind below 400 nm because their lenses are yellow; they function as cut-off filters (Zigman, 1971). When their lenses are removed, vertebrates can see ultraviolet light via the P-band short wavelength absorbance of their visual pigments. I have discussed above the ultraviolet sensitive rhodopsin of Ascalaphus in which the main band is shifted to about 345 nm. It does not follow that all responses of insects to ultraviolet light must depend upon such pigments. As long as their lenses are transparent to ultraviolet light it will be absorbed by the minor bands of longer wavelength pigments. And if a visual pigment whose main band lies in the visible is confined within a very narrow rhabdomere, on the order 1 pm, its spectrum may be distorted so as to relatively increase its extinction at shorter wavelengths (Snyder and Miller, 1972; Snyder and Pask, 1973). There is a problem that must be particularly associated with ultraviolet sensitivity, one that has not yet been experimentally explored in insects: the pathological effect of light. It has been recognized in recent years that visible light can harm vertebrate photoreceptors even at moderate intensities. It is not certain what causes the damage, but photo-oxidation of membrane lipids may be one of a number of photochemical mechanisms. The antioxidant tocopherol (vitamin E) is a notable constituent of vertebrate photoreceptor membranes (Daemen, 1973). The action spectrum of retinal damage in vertebrates rises sharply at the shorter wavelength, higher energy part of the spectrum that for vertebrates ends at 400 nm (Ham et al., 1976). Thus insects, with their ultraviolet transparent eyes, would seem particularly vulnerable to photochemical pathology. While insects see at shorter wavelengths than do vertebrates, they have not extended their visual sensitivity as far into the red. Those vertebrate photopigments absorbing at the longest wavelengths (with A,, as far out as 620 nm) are porphyropsins whose chromophore is 3-dehydroretinal. This form of the chromophore, with its enhanced bathochromatic shift, is unknown among invertebrates. There are vertebrate rhodopsins whose spectra extend well into the red, with A,, to 580 nm. Spectral sensitivity measurements suggest that some butterflies may have red receptors peaking around 600nm (Swihart and Gordon, 1971). However, no attempt has been made to measure directly a visual pigment that might lie behind this long wavelength sensitivity.

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The problem of the visual pigments of the higher flies

The visual systems of the higher flies belonging to the cyclorraphous families are under particularly intense investigation, and it is worthwhile considering some of the unresolved problems having to do with their visual pigments. Each ommatidium of the fly compound eye is made up of 8 rhabdomeres, with those designated 1-6 standing in isolation in an outer ring around rhabdomeres 7 and 8 at the center. Rhabdomere 7 lies distal to 8 along the optical axis. Spectral sensitivity measurements on various fly species have shown large response maxima in the blue-green and ultraviolet, and in some instances lesser peaks in the blue, yellow-green and red (Autrum and Burkhardt, 1961; Burkhardt, 1962; Burkhardt and de la Motte, 1972; McCann and Arnett, 1972; Horridge and Mimura, 1975; Horridge et al., 1975; Rosner, 1975; Meffert and Smola, 1976). Rhabdomeres 1 - 6 have been identilled by intracellular electrophysiology as blue-green receptors, A,, 490 nm, in Calliphora (McCann and Arnett, 1972; Horridge and Mimura, 1975, Rosner, 1975). The sensitivity of the peripheral rhabdomeres is clearly due to R490, whose main band has been well characterized (Langer and Thorell, 1966; Hamdorf et al., 1972b; Stavenga et al., 1973). Receptors 1-6 also respond strongly to ultraviolet light (Burkhardt, 1962; McCann and Amett, 1972; Horridge and Mimura, 1975; Rosner, 1975). In fact they are as sensitive at 350 nm as at 490 nm. Although rhodopsin spectra extend into the ultraviolet, the extinction of the secondary short wavelength maximum @-band) of all visual pigments that have been adequately characterized-mainly vertebrate rhodopsins-is much lower than that of the main absorption band (a-band) in the visible (Fig. 1). Recently Horridge and Mimura (1975) and Rosner (1975) have sought to explain the peculiar action spectra of cells 1-6 by suggesting that their rhabdomere membranes carry two distinct photopigments, an ultraviolet sensitive rhodopsin as well as R490. Rosner argued from the adaptation characteristics of these cells. Horridge and Mimura found that the ultraviolet and blue-green responses are differentially sensitive to the plane of polarized light. The possibility of two rhodopsins in the same receptor is intriguing because so far no photoreceptor cell in any animal has been shown to synthesize more than one species of opsin. An alternative explanation of the high ultraviolet sensitivity of rhabdomeres 1-6 has been offered by Snyder and Miller (1972) and Snyder and Pask (1973). The rhabdomeres of flies are dielectric waveguides, whose geometric and optical properties should affect the absorption spectra of their visual pigments. Theoretically, confining a rhodopsin within a rhabdomere of small diameter would increase its ultraviolet absorbance relative to that of the a band at longer wavelengths. Finally, it should be pointed out that separate receptor cells with different

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sensitivities may be electrically coupled. In that event the location and absorption spectra of the resident photopigments cannot be deduced with certainty from action spectra, even those measured by intracellular recording (Shaw, 1969). In this regard, MSP absorption spectra of single peripheral rhabdomeres in Cafliphora show only a relatively small ultraviolet peak and give no suggestion of two pigments (Langer and Thorell, 1966). Absorption and action spectra do not match in the near ultraviolet. The high sensitivity of rhabdomeres 1-6 at the shorter wavelengths remains a fascinating puzzle. A new approach to identifying the spectral responses of particular cells has been taken by Harris et a f . (1976). They have examined Drosophifa mutants in which particular rhabdomeres either do not develop or degenerate. The spectral sensitivities and modes of adaptation of these mutant flies suggest that cells 1-6 are combined blue-green ultraviolet receptors, that cell 7 is an ultraviolet receptor and that cell 8 is a blue receptor. MSP measurements of Ostroy et a f . (1974) confirm that, as in Cafliphora,the peripheral rhabdomeres carry a bluegreen sensitive rhodopsin. Flies lose their blue-green sensitivity with the genetic elimination of rhabdomeres 1-6. They retain high ultraviolet sensitivity and relatively lower blue sensitivity. The response to ultraviolet light can be reduced by short wavelength adaptation, a result consistent with the assignment of high ultraviolet sensitivity to only one of the remaining central cells 7 or 8. Elimination of all rhabdomeres except number 8 renders the flies ultraviolet insensitive, leaving only a blue response that cannot be altered by adaptation. Hence, blue sensitivity must be associated with cell 8, ultraviolet sensitivity with cell 7. An ultraviolet sensitive pigment, Amax 370 nm was extracted by Harris et a f . (1976) from mutant Drosophifa retaining only rhabdomeres 7 and 8. Its presumed metarhodopsin lay at 470nm. However, as the pigment was measured in crude aqueous suspensions, further confirmation that it is truly a rhodopsin is desirable. Harris et af. (1976) were unable to isolate a blue sensitive rhodopsin from cell 8. It may have been measured by Langer and Thorell (1966) in an early study with MSP, but it has not been well characterized. Snyder and Pask ( 1973) suggested that the central rhabdomeres of fly retinulae might contain the same rhodopsin as that of rhabdomeres 1-6. They argued that the central rhabdomeres are narrow enough to suppress the a-band of R490 and shift its maximum into the blue, while enhancing the pband at 350nm. The results of Harris et al. (1976) do not support that suggestion. In Drosophifa the evidence suggests that there are separate ultraviolet and blue sensitive photopigments. However, the predicted effects of the physical properties of fly rhabdomeres upon the absorption spectra of their rhodopsins need to be experimentally tested by comparing their in vivo and in vitro spectra. The full characterization of the rhodopsins that reside in the small central rhabdomeres is a formidable challenge.

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Transduction and adaptation

The control of sensitivity in photoreceptor systems is a classical problem of vision physiology. Several factors are involved in setting sensitivity including as the amount of visual pigment that is present, pupil mechanisms-such migration of screening pigments in insect eyes-that regulate the amount of light entering a receptor, and mechanisms that have been designated electrical or neural adaptation. I will be concerned here with the relationship of rhodopsin concentration to sensitivity, but the particular complication of electrical adaptation needs to be dealt with first. Sensitivity changes of this sort reflect electrical modifications of membranes or of their ionic environments such that background illumination decreases the size of the electrical response evoked by a given stimulus. Electrical adaptation can have considerable effect at very low light levels. Vertebrate visual systems show several log units of change in threshold, that is, in the amount of light required to provoke a given receptor response, even at levels of background illumination that bleach only a few percent of the visual pigment (Weinstein et al., 1967). Dark recovery from such electrical adaptation takes place rapidly and does not depend upon pigment regeneration. When rhodopsin regeneration is prevented threshold drops quickly in darkness but only to a level that reflects the amount of photopigment remaining. Thus the relationship between rhodopsin concentration and the absolute sensitivity of a receptor is manifest after a short period of dark recovery that eliminates the larger effects of electrical adaptation, if during that period the amount of visual pigment remains constant. One might expect-indeed this was the early hypothesis-that absolute sensitivity would then be simply proportional to the amount of rhodopsin, to the quantum catching capacity of the receptor. This proved not to be the case in vertebrates, however; the relationship between rhodopsin concentration and sensitivity is logarithmic. In the rat, for instance, when 17 per cent of the rhodopsin is bleached, threshold increases by a factor of 10, a bleach of 34 per cent elevates threshold a hundredfold (Weinstein et al., 1967). This nonlinear relationship has not been explained. Similar experiments relating absolute sensitivity to rhodopsin concentration became possible in insects when their visual pigments could be reliably measured. The most appropriate insect photoreceptor systems for study are those with the greatest spectral separation between rhodopsin and metarhodopsin, in which the rhodopsin concentration can be widely varied. In such systems, photoequilibria containing different amounts of rhodopsin can be established by monochromatic light adaptation at different wavelengths, or different amounts of rhodopsin can be converted to metarhodopsin by varying either the intensity or duration of illumination. The latter procedure is comparable to the bleaching away of a vertebrate visual pigment except that a "

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minimal rhodopsin concentration is reached when the system has been driven to photoequilibrium. Since dark regeneration is negligible in insect eyes over short periods, rhodopsin concentration does not change over the few minutes of darkness required for recovery from electrical adaptation. During that short period of dark adaptation, sensitivity rises to a plateau whose level depends upon the rhodopsin concentration in the photoreceptor. The relationship between sensitivity and amount of rhodopsin has now been determined in several insects : Ascalaphus, Deilephila, Manduca and Calliphora (Hamdorf and Rosner, 1973; Hamdorf and Schwemer, 1975; Rosner, 1975). In contrast to the logarithmic relationship found in vertebrates, dark adapted sensitivity has been found in these insects to be a simple linear function of rhodopsin concentration. This has been particularly wellestablished for the blue-green sensitive system of Calliphoru, in which the concentration of R490 was varied from 100 per cent to 30 per cent, and receptor potentials were measured by both extracellular and intracellular methods. The most straightforward interpretation of the linear relationship is that each rhodopsin molecule activated by absorbing a quantum of light contributes equally to the generation of the receptor potential. An extensive theoretical discussion of visual pigment-absolute sensitivity relationships can be found in the article by Hamdorf and Schwemer (1975). In the experiments summarized above, metarhodopsin concentration necessarily varied in reciprocal relationship with the amount of rhodopsin. Hence the question arises of a possible role for metarhodopsin in setting sensitivity. With that possibility in mind, Razmjoo and Hamdorf (1976) studied carotenoid deficient Calliphora with reduced amounts of visual pigments in their photoreceptor membranes. The aim of these experiments was to assess the effect on sensitivity of lowered rhodopsin concentrations independent of altered rhodopsin-metarhodopsin ratios. Once again, an approximately linear proportionality between sensitivity and amount of rhodopsin was found. In another experiment, both carotenoid-rich and carotenoid-depleted flies were irradiated with blue light in order to set up similar photoequilibria with low proportions of rhodopsin. Although the relative change in rhodopsin concentration was the same, the carotenoid-rich flies suffered a relatively greater loss of sensitivity. The authors suggest that in photoreceptor membranes where visual pigment molecules are more densely packed, as in the carotenoid-rich Calliphora, metarhodopsin molecules may have some inhibitory influence on the membrane’s response to light activated rhodopsin. The preceeding discussion has introduced the fundamental question of how the absorption of light by a visual pigment provokes a potential change in the receptor cell membrane. In insects as in other invertebrates, the receptor membrane responds with depolarization, whereas vertebrate receptors hyper-

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polarize. Beyond that almost nothing is known about visual transduction specifically in insects. Compared with the visual cells of various vertebrates and other invertebrates, e.g. Limulus (Lisman and Brown, 1975), insect receptors have not seemed particularly favorable as experimental systems. In general, theories of visual transduction and adaptation involve changes in sodium conductance regulated by calcium ions or by cyclic nucleotides at sodium permeability sites (Ebrey and Honig, 1975). The hypothesis of Razmjoo and Hamdorf (1976) regarding an influence of metarhodopsin on transduction calls to mind a peculiar response of Drosophila photoreceptors to intense blue light that sets up a photoequilibrium favoring metarhodopsin. The receptors are thrown into a state of prolonged depolarization, so that they cannot respond to subsequent stimuli even though rhodopsin is still present. Although the cells recover only very slowly in darkness, they can be abruptly restored to normal function by red light that pushes the photoequilibrium back toward rhodopsin (Cosens and Briscoe, 1972; Minke et af., 1975; Stark and Zitzman, 1976). Thus accumulation of metarhodopsin in the membrane seems to be associated with its prolonged depolarization. However, the effect is apparently not tied directly to the presence of metarhodopsin, nor is the proportion of metarhodopsin to rhodopsin the significant parameter. Prolonged depolarization does not occur in Drosophila receptors whose total amount of visual pigment has been reduced by carotenoid deficiency (Stark and Zitzman, 1976). Stark and Zitzman suggest that there are a limited number of membrane sites, distinct from photopigment molecules, that control depolarization, that these sites are activated by photoconversion of rhodopsin to metarhodopsin, perhaps by way of a transmitter, and that such action persists for some time after stimulation. According to their hypothesis, prolonged depolarization occurs when these limited sites are fully activated by massive conversion of rhodopsin to metarhodopsin. Carotenoid deprivation is seen as lowering the ratio of visual pigment molecules to depolarization sites, so that the latter cannot be saturated. Drosophifuis also unique in offering mutants with impaired transduction for analysis. A group of such mutants on the x-chromosome, designated norp-A, is characterized by reduced or absent photoreceptor potentials, and differences in eye protein composition (Ostroy and Pak, 1974). The blue-green sensitive rhodopsin, R480, in one of these phototransduction mutants, norpAP'*, has been measured in extracts and by MSP (Ostroy et al., 1974). The spectra of R480 and its photoproduct, M580, were found to be similar in mutant and normal flies, but the mutant flies contained only a third as much photopigment. The reduced amount of visual pigment cannot in itself be advanced as an adequate explanation of the mutant phenotype, since the receptor

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potential is much more severely affected; it is either completely absent or nearly so. The primary cause of blocked transduction in these mutant flies remains unknown. 9

Insect photoreceptor membranes

The photoreceptor membranes of vertebrates are large flat sheets (outer segment disks), while those of insect and most other invertebrates are rolled into tight cylindrical microvilli ranging around 50 nm in diameter. Vertebrate rod outer segment disks are very fluid membranes, with the viscosity of a light oil, owing to their high content of unsaturated phospholipids and low content of cholesterol. The predominantly hydrophobic rhodopsin molecules are embedded in the membrane, but are oriented by hydrophilic interactions with the surrounding medium so that their chromophores are held parallel to the membrane plane. Otherwise, vertebrate rhodopsin molecules are free to rotate and move laterally within the plane of the fluid membrane (Ebrey and Honig, 1975).

Are invertebrate rhodopsins similarly mobile within their microviUus membranes? The question arises with particular pertinence when we consider the sensitivity of many insects and other arthropods to polarized light (Waterman, 1975). Perception of the plane of polarization is possible in the first place because retinal is a highly dichroic linear chromophore (Fig. 1). Vertebrates are generally insensitive to polarized light because the chromophores of their mobile rhodopsins are randomly oriented in the plane of the disk membrane perpendicular to the axis of incoming light. If such a flat membrane were rolled into a cylinder, however, it would be more sensitive to light polarized with the electric vector parallel to the long axis of the cylinder than to light polarized perpendicular to it (Hays and Goldsmith, 1969; Snyder and Laughlin, 1975; Laughlin et al., 1975). Since rhabdomeric photoreceptors are composed of cylindrical microvilli generally oriented perpendicular to the optical axis, they are inherently sensitive to polarized light. Invertebrates with rhabdomeric eyes are preadapted for the evolution of true polarized light perception in which the plane of polarization is behaviorally distinguishable from intensity variation (Waterman, 1975). If photopigments are organized in arthropod microvilli as they are in vertebrate disks, with their chromophores parallel to the plane of the membrane but otherwise oriented at random and free to rotate, the dichroism arising from microvillus geometry could provide dichroic absorbance ratios no greater than 2 : 1, in fact rather lower (Snyder and Laughlin, 1975). But much higher polarization sensitivities have been measured in arthropod eyes, e.g., 9 :1 in the bee (Menzel and Snyder, 1974). Hence it has been inferred that rhodopsin must

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be more rigidly oriented in arthropod microvilli-at least in animals with high polarization sensitivity-than in vertebrate disks. It has been argued that even if photopigments are mobile within the microvillus membrane they could be oriented by preferential axial streaming within the tightly curved phospholipid bilayer (Snyder and Laughlin, 1975). Direct measurements of chromophore orientation in rhabdomeres have been dimcult. The best data are from MSP measurements of crustacean rhabdoms. The earliest measurements recorded absorbance ratios no higher than 2 :1 with highest absorbance parallel to the axes of the microvilli (Waterman et al., 1969). These data seemed compatible with random photopigment orientation. More recently the ratio has been raised to 3 : l by better measurements (Goldsmith, 1975). These more recent data appear to demand nonrandom orientation. Another fruithl approach to the question of photoreceptor membrane organization is to measure rhodopsin mobility. Goldsmith and Wehner (1975); Wehner and Goldsmith (1975) have reported that the rhodopsin in crustacean rhabdomeres neither rotates nor moves along the microvillus axis. The data remain open to question, however, because formaldehyde was used to stabilize the preparations. Although this reagent does not impede the movement of rhodopsin in vertebrate disks, the possibility remains that it might crosslink rhodopsin in rhabdomeric membranes and so hinder normal mobility. Measurements of rhodopsin orientation with the same technical quality have not been made on insect rhabdomeres. Langer (1965) measured somewhat higher rhodopsin absorbances parallel to the axes of the rhabdomere microvilli in Culliphoru. Kirschfleld and Snyder (1975) have presented data suggesting that the chromophores may be oriented perpendicular to the microvillous axes in rhabdomeres 7 or 8 of the fly Muscu. Although rhodopsin mobility has not been directly measured in insect photoreceptors, some relevant inferences have been made from the lipid composition of their membranes. The proportions of phospholipids in Deilephilu and Asculuphus retinas were found to be similar to those in vertebrate disks (Zinkler, 1975). However, fatty acid components were less polyunsaturated, and there was twice as much cholesterol. It should be pointed out, however, that extracts of whole insect retinas are here being compared with purified vertebrate photoreceptor membrane. In any case, the lipid composition of these insect retinas is similar to that of squid (Benoken et ul., 1975) and Limulus (Mason et ul., 1973) rhabdoms. It has been argued from these data that the photoreceptor membranes of insects and other invertebrates are more viscous than vertebrate outer segment membranes. However, the relationship of lipid saturation and cholesterol content to membrane fluidity is not certain. There can be no substitute for direct measurements of the mobility and orientation of rhodopsin molecules and of the mobility of artificial probes introduced into

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rhabdomere membranes. To summarize, theory supported by some evidence suggests that in comparison with vertebrates, arthropod photoreceptor membranes are less fluid, and that photopigments are less mobile and more highly oriented. The structure of rhabdomere membranes has been examined directly by freeze-fracture electron microscopy. In this procedure membrane bilayers split and membrane proteins are revealed as bumps upon the separated phospholipid leaflets. Densely packed particles 70-90 A in diameter have been found associated with the cytoplasmic halves of rhabdomere membrane in the honey bee (Perrelet et ul., 1972), a crayfish (Fernandez and Nickel, 1976), and a snail (Brandenburger et ul., 1976). 10

Final comments

The recent studies that have begun to characterize insect visual pigments have given us few surprises. They appear to differ from vertebrate photopigments mainly in having metarhodopsins of greater thermostability, a characteristic shared by invertebrate photopigments in general. One insect visual pigment, however, is uniquely interesting. Asculuphus rhodopsin is the only ultraviolet sensitive rhodopsin that has been isolated. The shift of its chromophore’s absorbance to shorter wavelengths is an intriguing problem. The analysis of chromophore-opsin interaction in Asculuphus rhodopsin has begun with the low temperature characterization of its intermediates. A unique 1 1-cis metarhodopsin photoregeneration intermediate is a significant feature of its photochemistry. The ultraviolet sensitive rhodopsin of Asculuphus has joined the photopigments of vertebrates and cephalopods as one particularly suited to photochemical analysis. From it we may expect important insights into the molecular basis of photoreception. Acknowledgements

I thank Paul K. Brown, Reinhardt Paulsen and Joachim Schwemer for their helpful comments and criticism.

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