Single and Multiple Visual Systems in Arthropods

Extraction of two visual pigments from crayfish eyes prompted an electrophysiological examination of the role of visual pigments in the compound eyes of six arthropods. The intact animals were used; in crayfishes isolated eyestalks also. Thresholds were measured in terms of the absolute or relative numbers of photons per flash at various wavelengths needed to evoke a constant amplitude of electroretinogram, usually 50 µv. Two species of crayfish, as well as the green crab, possess blue- and red-sensitive receptors apparently arranged for color discrimination. In the northern crayfish, Orconectes virilis, the spectral sensitivity of the dark-adapted eye is maximal at about 550 mµ, and on adaptation to bright red or blue lights breaks into two functions with λ_max respectively at about 435 and 565 mµ, apparently emanating from different receptors. The swamp crayfish, Procambarus clarkii, displays a maximum sensitivity when dark-adapted at about 570 mµ, that breaks on color adaptation into blue- and red-sensitive functions with λ_max about 450 and 575 mµ, again involving different receptors. Similarly the green crab, Carcinides maenas, presents a dark-adapted sensitivity maximal at about 510 mµ that divides on color adaptation into sensitivity curves maximal near 425 and 565 mµ. Each of these organisms thus possesses an apparatus adequate for at least two-color vision, resembling that of human green-blinds (deuteranopes). The visual pigments of the red-sensitive systems have been extracted from the crayfish eyes. The horse-shoe crab, Limulus, and the lobster each possesses a single visual system, with λ_max respectively at 520 and 525 mµ. Each of these is invariant with color adaptation. In each case the visual pigment had already been identified in extracts. The spider crab, Libinia emarginata, presents another variation. It possesses two visual systems apparently differentiated, not for color discrimination but for use in dim and bright light, like vertebrate rods and cones. The spectral sensitivity of the dark-adapted eye is maximal at about 490 mµ and on light adaptation, whether to blue, red, or white light, is displaced toward shorter wavelengths in what is essentially a reverse Purkinje shift. In all these animals dark adaptation appears to involve two phases: a rapid, hyperbolic fall of log threshold associated probably with visual pigment regeneration, followed by a slow, almost linear fall of log threshold that may be associated with pigment migration.     

THE RATE OF CO2 ASSIMILATION BY PURPLE BACTERIA AT VARIOUS WAVE LENGTHS OF LIGHT

1. The relative absorption spectrum of the pigments in their natural state in the photosynthetic bacterium Spirillum rubrum is given from 400 to 900 mµ. The position of the absorption maxima in the live bacteria due to each of the pigments is: green pigment, 420, 590, 880; red pigment, 490, 510, 550. 2. The relative absorption spectrum of the green pigment in methyl alcohol has been determined from 400 to 900 mµ. Bands at 410, 605, and 770 mµ were found. 3. The wave length sensitivity curve of the photosynthetic mechanism has been determined and shows maxima at 590 and about 900 mµ. 4. It is concluded that the green bacteriochlorophyll alone and not the red pigment can act as a light absorber for photochemical CO₂ reduction.     

The photosensitive retinal pigments of fishes from relatively turbid coastal waters

Digitonin extracts have been prepared from the retinae of a dozen species of marine and euryhaline teleost fishes from turbid water habitats. Spectrophotometric analysis of the extracts shows that the photosensitive retinal pigments of these species have maximum absorption above 500 mµ. In nine species there are retinene₁ pigments with λ_max between 504 and 512 mµ. In the marine but euryhaline mullet, Mugil cephalus, there is a porphyropsin with λ_max 520 mµ. A mixture of rhodopsin and porphyropsin in an extract of a marine puffer, Sphoeroides annulatus, was disclosed by partial bleaching with colored light. In addition, one other species has a 508 mµ pigment, of which the nature of the chromophore was not determined. The habitats in which these fishes live are relatively turbid, with the water greenish or yellowish in color. The spectral transmission of such waters is probably maximal between 520 and 570 mµ. It is suggested that the fishes have become adapted to these conditions by small but significant shifts in spectral absorption of their retinal pigments. These pigments are decidedly more effective than rhodopsin in absorption of wavelengths above 500 mµ. This offers a possible interpretation of the confusing array of retinal pigments described from marine and euryhaline fishes.     

The Photosensitive Retinal Pigment System of Gekko gekko

Retinal extracts of Gekko gekko were found to contain two retinene₁ photopigments, one with maximum absorption at about 521 mµ, the second with a maximum in the region of 478 mµ. These pigments were assayed by the method of partial bleaching and their spectral characteristics studied by examining their difference spectra. The 478 mµ pigment was present in the extracts as 8 per cent of the total photopigment concentration. The two pigment systems were shown to be biochemically independent and to have different properties. Unlike the 521 mµ pigment, for example, the 478 mµ pigment was found to resist the action of NH₂OH and, within the cells, to be unaffected by sucrose solutions. These solutions destroyed or altered the 521 system so that extracts of sucrose-treated retinae were found to contain significantly less 521 photopigment. In digitonin solution the 521 pigment was unaffected by sucrose treatment. Both pigments were extracted from separated, washed outer segments and so are considered to be visual pigments. This dual system accounts for the spectral sensitivity of this gecko as determined by Denton. A search was made, but no evidence was secured for the presence of a photopigment absorbing at longer wavelengths. Electoretinographic data suggest, however, that an elevated sensitivity at longer wavelengths occurs in some geckos so that a continued search is justified for a third photopigment.     

Spectral Sensitivity of the Common Prawn, Palaemonetes vulgaris

The vision of Palaemonetes is of particular interest in view of extensive studies of the responses of its chromatophore systems and eye pigments to light. The spectral sensitivity is here examined under conditions of dark adaptation and adaptation to bright colored lights. In each case the relative number of photons per one-fiftieth sec flash needed to evoke a constant peak amplitude (usually 25 or 50 µv) in the electroretinogram (ERG) was measured at various wavelengths throughout the spectrum. The sensitivity is the reciprocal of this number. In dark-adapted animals the spectral sensitivity curve consists of a broad, almost symmetrical band, maximal at about 540 mµ, with a shoulder near 390 mµ. Adaptation to bright red or blue light, left on continuously throughout the measurements, depresses the 540 mµ peak without notably changing its shape or position, implying that only one visual pigment operates in this region. Adaptation to red light, however, spares a violet-sensitive system, so that a high, narrow peak at 390 mµ now dominates the spectral sensitivity function. The 540 and 390 mµ peaks are apparently associated with different visual pigments; and these seem to be segregated in different receptor systems, since the associated ERG's have markedly different time constants. It is suggested that these two sensitivity bands may represent the red- and violet-sensitive components of an apparatus for color differentiation.     

The nature of the lamprey visual pigment

From the retina of the land-locked population of the sea lamprey, Petromyzon marinus, a photolabile pigment was extracted which was identified spectrophotometrically as a member of the rhodopsin group of pigments. Using the absorption spectrum of a relatively pure solution and analysis by means of difference spectra, the peak of this pigment was placed at about 497 mµ. The method of selective bleaching by light of different wave lengths revealed no significant amounts of any other pigment in the extracts. A similar pigment was also detected in retinal extracts of the Pacific Coast lamprey, Entospenus tridentatus. These results are significant for two reasons: (a) the lamprey is shown to be an example of an animal which spawns in fresh water but which is characterized by the presence of rhodopsin, rather than porphyropsin, in the retina; (b) the primitive phylogenetic position of the lamprey suggests that rhodopsin was the visual pigment of the original vertebrates.     

A new photosensitive pigment of the euryhaline teleost,Gillichthys mirabilis

Retinal extracts have been prepared from dark-adapted mudsuckers by treatment of retinal tissue or of isolated outer segments of the visual cells with digitonin solution. The extracts were examined spectrophotometrically and found to absorb light maximally between the wave lengths of 488 and 510 mµ, depending on the proportion of yellow impurities and light-sensitive pigment present. This photosensitive pigment was shown to be homogeneous by partial bleaching of the extracts with monochromatic light of various wave lengths from 390 to 660 mµ. The mudsucker pigment was specifically demonstrated not to be a mixture of rhodopsin and porphyropsin; the adequacy of the method used to analyze such mixtures was shown by performing a control experiment with an artificial mixture of bullfrog rhodopsin and carp porphyropsin. Comparison of the hydroxylamine difference spectrum and of the absorption maximum of the purest retinal extract located the mudsucker photosensitive pigment maximum at 512 ± 1 mµ. Extraction of retinal tissue with a fat solvent after exposure to white light gave a preparation which after the addition of antimony chloride reagent developed the absorption band maximal near 664 mµ, which is characteristic of retinene₁. If an hour intervened between exposure of the retinal tissue to light and extraction of the carotenoid, the antimony trichloride test gave a color band maximal at 620 mµ, characteristic of vitamin A₁. No evidence of retinene₂ or vitamin A₂ was obtained. The euryhaline mudsucker has, therefore, a photosensitive retinal pigment with an absorption maximum halfway between the peaks of rhodopsins and of porphyropsins and belonging to the retinene₁ system characteristic of rhodopsins. The pigment is therefore named a retinene₁ pigment 512 of the mudsucker, Gillichthys mirabilis. It is uncertain whether this type of photosensitive pigment will be found in other euryhaline fishes.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.     

THE PHOTOTROPIC SENSITIVITY OF PHYCOMYCES AS RELATED TO WAVE-LENGTH

Under the circumstances of experimentation described, the sporangiophores of Phycomyces are found to be most sensitive to stimulation by light in the violet between 400 and 430 mµ. Toward the red, sensitivity falls to nearly zero near 580 mµ, while in the near ultra-violet around 370 mµ, sensitivity is still high. The previous experiments of Blaauw had placed the point of greatest sensitivity some 80 mµ nearer the red end of the spectrum. Because of the known presence in the sporangiophores of Phycomyces of "accessory" pigments, care must be taken in identifying such results with the absorption spectrum of the photosensitive substance.     

DERIVED PHOTOSENSITIVE PIGMENTS FROM INVERTEBRATE EYES

The red pigment in the eyes of the squid, blue crab, and horseshoe crab becomes photosensitive when treated with formalin, and bleaches in the light. The resulting change in density is approximately symmetrical around a maximum at 480 mµ in the blue green. This difference absorption spectrum is in rough agreement with the spectral sensitivity of the cephalopod eye and differs only slightly from the difference absorption spectrum of vertebrate visual purple. The formalin-sensitized pigment is not melanoid. Its bleaching in squid retinas releases large quantities of retinene. It is suggested that the light sensitivity of the normal squid photopigment may be independent of its light stability.     

Delayed light action spectra of several algae in visible and ultraviolet light

Action spectra for delayed light production by several algae were determined from 250 to 750 mµ incident light. In the visible portion of the spectrum the action spectra resemble those reported by previous workers for photosynthesis and light emission. Blue-green algae had a maximum at 620 mµ, red algae at 550 mµ, whereas green and brown algae have action spectra corresponding to chlorophyll and carotenoid absorption. In the ultraviolet portion of the spectrum delayed light is emitted by algae down to 250 mµ incident light. The action spectra of the different algae are not alike in the ultraviolet portion of the spectrum. This indicates that pigments other than chlorophyll must be sensitizing or shielding the algae in the ultraviolet region.     

The nature of the gecko visual pigment

Retinal extracts of the Australian gecko, Phyllurus milii (White), have revealed the presence of a photosensitive pigment, unusual for terrestrial animals, because of its absorption maximum at 524 mµ. This pigment has an absorption spectrum which is identical in form with that of other visual chromoproteins. It is not a porphyropsin, for bleaching revealed the presence, not of retinene₂, but of retinene₁ as a chromophore. Photolabile pigments with characteristics similar to those of the Phyllurus visual pigment were also detected in retinal extracts of six other species of nocturnal geckos. The presence of this retinal chromoprotein adequately accounts for the unusual visual sensitivity curve described by Denton for the nocturnal gecko. This pigment may have special biological significance in terms of the unique phylogenetic position of geckos as living representatives of nocturnal animals which retain some of the characteristics of their diurnal ancestors. The occurrence of this retinene₁ pigment, intermediate in spectral position between rhodopsin and iodopsin, is interpreted in support of the transmutation theory of Walls. The results and interpretation of this investigation point up the fact that, from a phylogenetic point of view, too great an emphasis on the duplicity theory may serve to detract attention from the evolutionary history of the retina and the essential unitarianism of the visual cells.     

MICROSPECTROPHOTOMETRY OF CYTOCHROMES IN THE SINGLE CELL AT ROOM AND LIQUID NITROGEN TEMPERATURES

By increasing further the sensitivity of microspectrophotometry, it is now possible to measure, under favorable conditions, the smaller absorption bands of the respiratory pigments of single cells in the visible region of the spectrum. A considerable aid in the distinction between cytochromes is afforded by liquid nitrogen microspectrophotometry. Under favorable conditions, the height of the peaks is increased over 8-fold at low temperatures. In diploid yeast, characteristically sharpened components not resolvable at room temperature are observed at low temperature; and in pentaploid yeast, a hitherto unrecognized pigment is observed at 583 mµ. These preliminary results indicate the feasibility and the value of low temperature microspectrophotometry of biological materials.     

THE EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

THE CHEMISTRY OF DAYLIGHT VISION

1. While several reports of photosensitive pigments from the retinas of animals possessing large numbers of cone cells have been published, the only study which could be confirmed was Wald''s discovery of iodopsin, a red-sensitive pigment from chicken eyes. 2. In its chemical properties, such as the range of pH stability and the effect of polar organic solvents, iodopsin resembles rhodopsin but is considerably more labile. 3. A partial purification from inert yellow impurities has been effected by prehardening the retinas in pH 4.9 acetate buffer before extraction by 2 per cent digitonin. Rhodopsin was an inevitable contaminant in most methods of extraction, but could be reduced to about 10 per cent of the absorption due to iodopsin by extraction of unhardened retinas with 4 per cent Merck''s saponin in ¾ saturated magnesium sulfate for about 1 hour. 4. The rate of bleaching of iodopsin was found to be first order and linear with respect to energy. 5. The bleaching effectiveness spectrum of iodopsin was determined with the aid of color filters of known energy transmission, and shows a maximum at 560 mµ in the yellow green with a lower plateau in the blue. The spectrum is in good agreement with the sensitivity of the human cones except for the wavelength of maximum bleaching effectiveness. The maximum sensitivity of the human cones is found at 540 mµ. 6. Previous reports of changes in pH and inorganic phosphate level of retinas due to bleaching could not be confirmed.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : II. THE ABSORPTION CURVES OF PHOTOSYNTHIN FROM SEVERAL SPECIES OF BACTERIA

Absorption curves have been obtained in the spectral region of 450 to 900 mµ for the water soluble cell juice of four species of photosynthetic bacteria, Spirillum rubrum (strain S1), Rhodovibrio sp. (strain Gaffron), Phaeomonas sp. (strain Delft), and Streptococcus varians (strains C11 and orig.). These curves all show maxima at 790 and 590 mµ due to bacteriochlorophyll, whose highest band, however, occurs at 875, 855, or 840 mµ depending on the species. The bacteria that appear red rather than brown have a band at 550 mµ due to a carotinoid pigment. An absolute absorption curve of bacteriophaeophytin has maxima at 530 and 750 mµ. The extraction of cell juice by supersonic vibration does not change the position of the absorption bands or of the light absorbing capacity of the pigment.     

Electroretinogram Characteristics and the Spectral Mechanisms of the Median Ocellus and the Lateral Eye in Limulus polyphemus

Electrical responses (ERG) to light flashes of various wavelengths and energies were obtained from the dorsal median ocellus and lateral compound eye of Limulus under dark and chromatic light adaptation. Spectral mechanisms were studied by analyzing (a) response waveforms, e.g. response area, rise, and fall times as functions of amplitude, (b) slopes of amplitude-energy functions, and (c) spectral sensitivity functions obtained by the criterion amplitude method. The data for a single spectral mechanism in the lateral eye are (a) response waveforms independent of wavelength, (b) same slope for response-energy functions at all wavelengths, (c) a spectral sensitivity function with a single maximum near 520 mµ, and (d) spectral sensitivity invariance in chromatic adaptation experiments. The data for two spectral mechanisms in the median ocellus are (a) two waveform characteristics depending on wavelength, (b) slopes of response-energy functions steeper for short than for long wavelengths, (c) two spectral sensitivity peaks (360 and 530–535 mµ) when dark-adapted, and (d) selective depression of either spectral sensitivity peak by appropriate chromatic adaptation. The ocellus is 200–320 times more sensitive to UV than to visible light. Both UV and green spectral sensitivity curves agree with Dartnall's nomogram. The hypothesis is favored that the ocellus contains two visual pigments each in a different type of receptor, rather than (a) various absorption bands of a single visual pigment, (b) single visual pigment and a chromatic mask, or (c) fluorescence. With long duration light stimuli a steady-state level followed the transient peak in the ERG from both types of eyes.     

THE VISIBILITY OF MONOCHROMATIC RADIATION AND THE ABSORPTION SPECTRUM OF VISUAL PURPLE

1. After a consideration of the existing data and of the sources of error involved, an arrangement of apparatus, free from these errors, is described for measuring the relative energy necessary in different portions of the spectrum in order to produce a colorless sensation in the eye. 2. Following certain reasoning, it is shown that the reciprocal of this relative energy at any wave-length is proportional to the absorption coefficient of a sensitive substance in the eye. The absorption spectrum of this substance is then mapped out. 3. The curve representing the visibility of the spectrum at very low intensities has exactly the same shape as that for the visibility at high intensities involving color vision. The only difference between them is their position in the spectrum, that at high intensities being 48 µµ farther toward the red. 4. The possibility is considered that the sensitive substances responsible for the two visibility curves are identical, and reasons are developed for the failure to demonstrate optically the presence of a colored substance in the cones. The shift of the high intensity visibility curve toward the red is explained in terms of Kundt''s rule for the progressive shift of the absorption maximum of a substance in solvents of increasing refractive index and density. 5. Assuming Kundt''s rule, it is deduced that the absorption spectrum of visual purple as measured directly in water solution should not coincide with its position in the rods, because of the greater density and refractive index of the rods. It is then shown that, measured by the position of the visibility curve at low intensities, this shift toward the red actually occurs, and is about 7 or 8 µµ in extent. Examination of the older data consistently confirms this difference of position between the curves representing visibility at low intensities and those representing the absorption spectrum of visual purple in water solution. 6. It is therefore held as a possible hypothesis, capable of direct, experimental verification, that the same substance—visual purple—whose absorption maximum in water solution is at 503 µµ, is dissolved in the rods where its absorption maximum is at 511 µµ, and in the cones where its maximum is at 554 µµ (or at 540 µµ, if macular absorption is taken into account, as indeed it must be).     

THE LASER AS A POTENTIAL TOOL FOR CELL RESEARCH

Freshly prepared hemoglobin solutions were successively irradiated up to five times with 1 MW (monochromatic wavelength) of green (530 mµ) laser power. Oxygenated hemoglobin showed no detectable change, but the spectral absorption of reduced hemoglobin showed a shift toward the characteristic curve for the oxygenated form. Intact human erythrocytes exposed to a power density of 110 MW/cm² of green laser radiation showed no appreciable change in diameter or mass, but they became transparent to a wavelength range from 400 to 600 mµ. A similar power density from a ruby laser failed to produce this bleaching effect. This response in the erythrocyte demonstrates a principle which suggests the laser as a tool for cell research: specific molecular components within a cell may be selectively altered by laser irradiation when an appropriate wavelength and a suitable power density are applied.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.