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Rhodopsin of the larval mosquito.

Larvae of the mosquito Aedes aegypti have a cluster of four ocelli on each side of the head. The visual pigment of each ocellus of mosquitoes reared in darkness was characterized by microspectrophotometry, and found to be the same. Larval mosquito rhodopsin (lambda(max) = 515 nm) upon short irradiation bleaches to a stable photoequilibrium with metarhodopsin (lambda(max) = 480 nm). On long irradiation of glutaraldehyde-fixed tissues or in the presence of potassium borohydride, bleaching goes further, and potassium borohydride reduces the product, retinal, to retinol (vitamin A(1)). In the presence of hydroxylamine, the rhodopsin bleaches rapidly, with conversion of the chromophore to retinaldehyde oxime (lambda(max) about 365 nm).


INTRODUCTION
Most insects that undergo complete metamorphosis have, at various stages of their life cycles, three different organs of photoreception. A simple visual system, consisting of a few lateral ocelli, functions in the larva, whereas compound eyes and dorsal ocelli differentiate at metamorphosis to the adult (Goldsmith, 1964). Most of our knowledge of the physiology of insect vision has come from behavioral or electrophysiological experiments. Only a few attempts have been made to measure insect visual pigments directly: in extracts of heads of adult honeybees (Goldsmith, 1958 a, b) and cockroaches (Wolken and Sheer, 1963), in extracts of the compound eye of a neuropteran, Ascalaphus macaronius (Gogala et al., 1970), and by microspectrophotometry of the compound eye of the flesh fly Calliphora (Langer, 1966(Langer, , 1967. The visual pigments of ocelli have never before been examined directly.
In this paper we describe the visual pigment in lateral ocelli of larvae of the yellow fever mosquito Aedes aegypti L. Our particular interest in the biochemistry of the mosquito ocellus springs from earlier morphological studies. The configuration of photoreceptor membranes and other features of receptor cell ultrastructure are altered by light (White and Sundeen, 1967;White, 1967White, , 1968. In order to begin making physiological sense of these morphological effects of light, we need to investigate the visual cycle and the nature of the visual pigment. The larva has a cluster of four ocelli on each side of the head (Figs. I and 2).
THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 59,1972 pages 401-414

401
Each ocellus is a rosette of several photoreceptor cells, which in the light microscope are seen to bear a prominent lobed structure (Figs. 2 and 5 c) called the rhabdom. The dorsal and ventral ocelli contain 11-13 cells each and the central ocellus made up of two fused rosettes has approximately 26 cells. At the ultrastructural level, the rhabdom is shown to be a mass of microvilli which sprout from the distal surfaces of the receptor cells. The visual pigment is presumably localized in the microvillous membranes. In these features, the mosquito ocellus is organized as a typical arthropod photoreceptor. We measured the visual pigment of the ocelli with a microspectrophotometer. Dense accessory pigmentation makes microspectrophotometry of normal ocelli difficult. However, animals of a white-eye mutant proved to be very suitable, since their ocelli, lacking all masking pigments, are transparent ( Fig.  2 a). Indeed, the head is so transparent (Fig. ) that the measurements can be made in living larvae. We anticipate that the entire visual cycle will be accessible to analysis in living animals. Moreover, we have been able to complement and extend our spectrophotometric experiments with measurements of the electrical activity (electroretinogram) of the ocelli (Seldin et al., 1972).

METHODS
The microspectrophotometer used in the following experiments is essentially that described by Brown (1961). A Zeiss WL microscope is mounted in the sample beam of a Cary 14 recording spectrophotometer (Cary Instruments, Monrovia, Calif.). The Zeiss Ultrafluor (ultraviolet transmitting) optics includes a X 100, 1.25 NA objective, a X 10 ocular, and a 0.8 NA condenser. The image of the measured field is delimited by an adjustable diaphragm at the entrance lens of the phototube housing. The reference beam is adjusted to match the size and energy of the sample beam by means of a diaphragm and neutral density screens. Only the sample beam passes through the specimen, and the base line is zeroed using the multipot system of the Cary spectrophotometer.
Ocelli for spectrophotometric measurement were mounted in an appropriate medium between quartz cover slips sealed with Vaseline. This microcell was mounted in the spectrophotometer, and a carefully selected 30 /im field within the specimen was aligned on the cross hair of a X 20 viewing ocular. Under these conditions, either a single ocellus or a group of ocelli could be scanned, depending on their orientation. Spectra were recorded from 650 to 300 nm at 25 A/sec at room temperature, with no measurable bleaching of the visual pigment.
Bleaching flashes were from two sequentially flashed M3 flashbulbs, each having a 20 msec flash duration. For yellow flashes, a Corning 3484 filter (cut-off 525 nm; Coming Glass Works, Corning, N.Y.) was used. For blue flashes, a Wratten 47 filter (maximal transmission 440 nm; Eastman Kodak Co., Rochester, N.Y.) was used. For ultraviolet flashes, a Wratten 18A filter (maximal transmission 360 nm) was used. These filters were all coupled with a Jena KG3 heat filter (Jenaer Glas Werke, Schott und Genoden, Jena, Germany). For long irradiations, a microscope lamp with a tungsten bulb was used, with appropriate filters.
The mosquitoes (Aedes aegypti) used in this study were grown from eggs kindly supplied by Prof. George B. Craig, Jr., Department of Biology, University of Notre Dame. White-eye mutants (Bhalla, 1968) were generally used, but we also measured the visual pigment of wild-type animals of the Rockefeller strain. Larvae were hatched and reared in darkness to the fourth (final) instar. Larvae grown in darkness have much larger rhabdoms than those grown in light (White, 1967). Consequently we were able to use a relatively large scanning field. Growing animals in darkness also avoids the possible complication of having metarhodopsin present from a previous illumination. The metarhodopsin of these animals is very stable and possibly may persist even after a long period of dark adaptation. Whole heads, or whole animals, were mounted in the microcell so that they were oriented as in Fig. 1. In this position, the ocelli are superimposed so that two or more are measured simultaneously. In most such preparations, the head was left intact, so that the medium bathing the ocelli was normal hemolymph. Alternatively, one side of the head capsule was cut away and mounted in the microcell with ocelli oriented as in Fig. 2 so that each ocellus could be measured individually. In such preparations, the ocelli were immersed in various media in the microcell.  Unfixed ocelli were mounted in insect Ringer (NaC1 6.5, KCI 1.4, CaC2, 0.12, NaHCO3 0.1, and Na 2 HPO 4 0.01 g/liter) adjusted to an appropriate pH with phosphate buffer. In other experiments, the ocelli were fixed for 1 hr in 5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), and then washed in buffer. They were either used immediately or refrigerated in buffer for future use. All manipulations were carried out in dim red light of wavelengths longer than 690 nm. Fig. 3 a illustrates an experiment in which a region of an ocellus that included the rhabdom (Field 1, curve 1), and then a more proximal region outside the rhabdom (Field 2, curve 2) were separately scanned. The difference between these spectra (Fig. 2 b) represents approximately the absorption spectrum of the rhabdom. It shows two major peaks, one with maximal absorbance (2max) at 515 nm, and the other with Xmax at 345 nm. The 515 nm band suggests the presence of a rhodopsin. There is also a minor band at 450 nm. The 345 nm substance, whose absorbance is several times greater than that of the rhodopsin, was always present in fresh preparations, but absent or greatly diminished in glutaraldehyde-fixed material. We do not know what it represents. However, we found no evidence that it changes in the light or is a visual pigment.

RESULTS
The spectrum from a central ocellus in pH 7.2 Ringer, recorded in the dark, is shown in Fig. 4 a (curves 1 and 2). After exposure to two flashes of yellow light the spectrum changed to curve 3. The difference spectrum of this photoconversion, plotted in Fig. 4 c, displays a maximal decrease in absorbance at about 550 nm, owing to the bleaching of rhodopsin, and a maximal increase at about 465 nm, owing to the formation of metarhodopsin. Similar results were obtained when ocelli were initially bleached with blue or ultraviolet flashes.
A second experiment of this kind is shown in Fig. 4 b. Curves 1 and 2 are dark scans from the ocelli of a living animal; curve 3 was recorded following two yellow flashes. The ocelli were then given two flashes of blue light and curve 4 was recorded. A large portion of the metarhodopsin produced by the yellow flashes was reconverted to rhodopsin by the blue flashes. We were able repeatedly to bleach and photoregenerate rhodopsin in these ocelli with either flashes or long irradiations. Hence, the product of each of our bleaches was a photoequilibrium, yellow light shifting it toward metarhodopsin, blue light toward rhodopsin.
Such photoequilibria were very stable. When the ocelli of living animals were flashed with sufficient yellow light to produce photoequilibrium and then left in darkness, the equilibrium initially established between rhodopsin and metarhodopsin remained stable for at least an hour, i.e., there was no measurable regeneration of rhodopsin in darkness during that period. Similar photoequilibria were established when glutaraldehyde-fixed ocelli were flashed with yellow light, and some photoregeneration could be obtained following a subsequent flash with blue light. However, glutaraldehyde-fixed ocelli are irreversibly bleached by several minutes of irradiation with yellow light. We Recordings of absorption spectra of a central ocellus, mounted in insect Ringer at pH 7.2, measured in the dark. Spectrum curve 1 is that of a 30 um field centered on the rhabdom, and spectrum curve 2 that of a field centered on a clear adjacent area. The inset diagram shows the position and relative size of the fields that were scanned. (b) Spectrum obtained by subtracting curve 2 from curve 1. It represents mainly the absorption spectrum of the rhabdom, including the rhodopsin at 515 nm, a small peak at 450 nm, and a large peak at 345 nm. The last two represent unknown substances, presumably not visual pigments.
have found that glutaraldehyde fixation increases the rate of photobleaching of mosquito rhodopsin, as was observed in the rhabdoms of the spider crab (Hays and Goldsmith, 1969), crayfish (Waterman et al., 1969), and lobster (Bruno, 1971).
Such difference spectra as are shown in Fig. 4 c do not represent the true absorption spectra of either rhodopsin or metarhodopsin since the spectra of the two pigments overlap broadly. Bleaching ocelli in the presence of hydroxylamine, however, accurately determines the spectrum of the rhodopsin. Hydroxylamine speeds the hydrolysis of the metarhodopsin by irreversibly combining with its chromophore retinal, to form retinaldehyde oxime (Wald and Brown, 1950;1953).
Such an experiment is presented in Fig. 5. A glutaraldehyde-fixed ocellus (shown in Fig. 5 c) was mounted in 0.1 M neutralized hydroxylamine. Dark spectra were recorded (curves 1 and 2). The rhodopsin was then bleached for 10 min with yellow light (curve 3). The difference spectrum (curves I and 2 minus 3; Fig. 5 b) shows rhodopsin with X,ma = 515 nm, and retinaldehyde oxime (upside down) with Xmx about 365 nm. 1 Since the oxime does not absorb light above 450 nm this difference spectrum accurately represents the true absorption spectrum of the rhodopsin above 450 nm. Glutaraldehyde fixation was helpful in these experiments, since it resulted in more stable base lines. However, it was not essential; similar spectra were obtained when fresh ocelli were bleached in the presence of hydroxylamine.
The true difference spectrum of metarhodopsin is more difficult to determine. For this we used an indirect method, illustrated in Fig. 6, to estimate its spectrum from such steady-state difference spectra as are shown in Fig. 4 c. Such a difference spectrum is the algebraic sum of absorbance changes due to both the bleaching of rhodopsin and the formation of metarhodopsin. Hence, subtracting the absorption spectrum of the rhodopsin bleached from the difference spectrum should yield the spectrum of metarhodopsin. In order to determine the amount of rhodopsin bleached in any particular experiment, we assumed that the absorbance change at 600 nm (asterisk in Fig. 6, curve 1) represents only the bleaching of rhodopsin. From the absorbance of rhodopsin at 600 nm (Fig. 7) we calculated the total rhodopsin bleached (Fig. 6, curve 2). Subtracting curve 2 from curve 1 yields curve 3, the spectrum of metarhodopsin (X,, = 480 nm).
Difference spectra of the photoproduct were also obtained from experiments in which glutaraldehyde-fixed ocelli were flash bleached in the presence of hydroxylamine. In such experiments, the photoproduct initially produced gradually hydrolyzed in the dark. Difference spectra so obtained were similar to the calculated metarhodopsin spectra except that their Xmax lay at a slightly shorter wavelength (about 470 nm). This result is consistent with the unpublished observation of P. K. Brown, that a bleaching intermediate (glutaralde- All spectra are arbitrarily brought to the same height. The rhodopsin spectrum is the average of seven difference spectra measured in the presence of hydroxylamine; the metarhodopsin spectrum is the average of three calculated spectra, the retinaldehyde oxime spectrum is the average of seven spectra, and the retinol spectrum is one difference spectrum. The triangles show the Dartnall nomogram of a hypothetical rhodopsin with Xma = 515 nm. hyde intermediate) at slightly shorter wavelengths than vertebrate metarhodopsin I is found in glutaraldehyde-fixed vertebrate retinas. Difference spectra from most of these experiments are summarized in Fig. 7. The rhodopsin and retinaldehyde oxime spectra are averages of seven difference spectra, and the metarhodopsin spectrum is the average of three calculated spectra. The spectrum of mosquito rhodopsin resembles a hypothetical rhodopsin with Xn,, = 515 nm (triangles in Fig. 7) computed from Dartnall's nomogram (Dartnall, 1953). Though white-eye mutants were used for the bulk of our experiments, we also obtained similar results with wild-type animals, whose eyes have normal accessory pigments. Fig. 7 also shows a difference spectrum of retinol (vitamin A 1 ) obtained when ocelli were illuminated for longer times in the presence of potassium borohydride. This speeds the hydrolysis of metarhodopsin, and reduces the product, retinal, to vitamin A (Wald and Brown, 1956). The potassium borohydride and hydroxylamine experiments, in which retinoll and retinaldehydel oxime are formed, respectively, clearly demonstrate that the chromophore of mosquito rhodopsin and metarhodopsin is retinal 1 .
Bleaching experiments in freshly dissected heads at pH 5, 6, 7, and 9, and intact larvae, gave results similar to those described above. At alkaline pH, however, base lines tended to be unstable, especially near 345 nm.

DISCUSSION
Analysis of insect visual pigments in extracts has proved difficult because of contamination with accessory pigments, and because the concentration of visual pigments is relatively low. Goldsmith (1958 a, b) extracted a pigment with max. about 440 nm from 20,000 honeybee heads with neutral buffer solution. This is as yet the only visual pigment that does not display the insolubility in water of typical membrane proteins. This pigment bleached to a 380 nm photoproduct, presumably retinal. Although Bowness and Wolken (1959) reported a similar buffer extraction of a pigment from housefly compound eyes, it is likely that the extract contained an accessory pigment, not a visual pigment (Goldsmith, 1964). Wolken and Sheer (1963) used digitonin to extract a 500 nm pigment from the cockroach (Blatta orientalis). It bleached like a rhodopsin, presumably to retinal, but its half-band width (67 nm) was unusually narrow, and did not fit the Dartnall nomogram. As both the honeybee and cockroach extracts were made from whole heads, the extracted pigments could have come from either the compound eyes or dorsal ocelli. Langer, 1966Langer, , 1967 measured with a microspectrophotometer the visual pigments in the compound eye of a white-eyed mutant of the blowfly (Calliphora erythrocephala). They found two pigments (Xmax = 510 and 470 nm) segregated in separate rhabdomeres. Gogala et al. (1970) have recently reported the digitonin extraction, as well as microspectrophotometric measurement, of a 345 nm visual pigment from the compound eye of a neuropteran, Ascalaphus macaronius. When irradiated with ultraviolet light, the pigment was converted to a 480 nm metarhodopsin. On long-wavelength irradiation the original 345 nm pigment was regenerated. Curiously, we also find a 345 nm substance associated with the mosquito rhabdom, but neither our spectrophotometric nor spectral sensitivity data (Seldin et al., 1972) indicate that it is a visual pigment. Mosquito rhodopsin resembles the lobster rhodopsin measured in digitonin extracts by Wald and Hubbard (1957) and in isolated lobster rhabdoms (Bruno, 1971). Lobster rhodopsin has Xmax = 515 nm, and bleaches to a relatively stable metarhodopsin with Xm,, = 490 nm. Both lobster and mosquito metarhodopsin appear to remain unchanged over a range of pH. Invertebrate metarhodopsins, unlike vertebrate, tend to be stable, at least in vitro (Brown and Brown, 1958;Hubbard and St. George, 1958;Mathews et al., 1963;Hays and Goldsmith, 1969). We have shown that mosquito metarhodopsin is stable in vivo as well. Again in contrast to vertebrate visual pigments, mosquito rhodopsin did not regenerate in darkness, suggesting that photoregeneration may be an important mechanism of rhodopsin renewal in the insect photoreceptor.