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Analysis of visual pigment absorbance and luminescence emission spectra in marine ostracodes (Crustacea: Ostracoda)
Camp. Biochem. fhysiol. Vol. 104A,No. 2,pp. 333-338,1993
0300-9629/93 $6.00+ 0.00
Printed in Great Britain
0 1993Pergamon Press Ltd
ANALYSIS OF VISUAL PIGMENT ABSORBANCE AND LUMINESCENCE EMISSION SPECTRA IN MARINE OSTRACODES (CRUSTACEA: OSTRACODA) ANDREA L. HUVARD* Department of Biology, University of California, Los Angeles, CA 90024, U.S.A. (Tel. 805 493-3341) (Received
30 April 1992; accepted 5 June 1992)
Abstract-l. The luminescence emission spectra and the visual pigment absorbance spectra in digitonin extracts were examined and compared for marine luminescent ostracodes. 2. The rhodopsin absorbance spectrum of one non-luminescent species was determined for comparison. 3. For all species examined (Vargula tsujii, V. graminicola and Skogsbergia lerneri), the difference spectra for visual pigment absorbance peaked at 460nm. 4. The luminescence emission spectra were measured by means of a computer controlled optical multi-channel analyser (OMA). 5. For the two luminescent species examined (V. graminicola and V. shulmanae), the A,,, of the emitted light was 473 nm with full width at half maxima (FWHM) of about 80 nm. 6. The luminescence emission spectra were also determined for male/female, adult/juvenile and dried/live pairs of V. graminicola. 7. All had a A,,,,, of 473 nm and in all cases the curves were unimodal with the FWHM of 80 nm. 8. V. graminicola and V. shulmanae are found in Panama in the Caribbean and the males emit their luminescence in complex display patterns (signals) for purposes of attracting receptive females. 9. Since the emission spectra for these two signalling species were similar, it would appear that species recognition is not based on wavelength but rather some other aspect of the luminescent display.
INTRODUCTION Early studies of visual pigments of marine organisms examined the hypothesis that the maximum spectral absorbance of a photopigment is an adaptation for increased visual sensitivity to the light available in a particular environment (Sensitivity Hypothesis, reviewed in Lythgoe, 1972). These early studies were primarily concerned with the visual pigments of organisms that live in light-limited environments (i.e. the deep sea). This Sensitivity Hypothesis has since been tested in several species of marine fishes, mammals and invertebrates from a variety of marine habitats (e.g. McFarland, 1971; Goldsmith, 1972; Fernandez, 1978; Crescitelli et al., 1985; Cronin and Forward, 1988; Forward et al., 1988; Grober, 1990). There are well documented cases that meet the conditions of the Sensitivity Hypothesis. For example, in deep sea fishes (i.e. Munz, 1958; Fernandez, 1978; Crescitelli et al., 1985) the A,,, of the visual pigments matches the ambient downwelling light (,l,,, of In contrast, the approximately 460-480 nm). spectral sensitivity of shallow water species can often exceed a A,,,,, of 500 nm. Many deep sea and midwater organisms are bioluminescent and the emitted luminescence can match the wavelength of the ambient downwelling light (Denton et aI., 1985; Herring, 1985). The match *Present address: Department of Biology, California Lutheran University, Thousand Oaks, CA 91360, U.S.A.
between bioluminescence and visual pigment absorbance spectra has been documented for several species of deep sea fishes and invertebrates (O’Day and Fernandez, 1974; Warner et al., 1978; Young and Mencher, 1980; Crescitelli et al., 1985; Denton et al., 1985). A unique example of matching has been demonstrated for some deep sea fishes that emit red luminescence; these fishes also have visual pigments sensitive to longer (red) wavelengths of light (O’Day and Fernandez, 1974). Two recent studies of intertidal and estuarine invertebrates (Cronin and Forward, 1988; Forward et al., 1988) examine the photopigment spectra of animals living in variable light conditions. With the use of microspectrophotometry, Cronin and Forward (1988) showed that several species of intertidal to subtidal decapods have maximum spectra that range from 473-515 nm. Considering that the 1,, for bioluminescence is usually coastal between 490-520 nm (Morin, 1983) it seems likely that many shallow water organisms may have photopigments that are adapted to match both the broad spectrum of ambient light and bioluminescence. Ostracodes are small crustaceans (usually <3 mm long) that are widely distributed in most freshwater and marine habitats. Ostracodes in the family Cypridinidae are abundant in shallow marine waters (< 30 m) and some species are bioluminescent. In the Caribbean, male ostracodes in the nominal genus Vurgulu emit their bioluminescence in complex 333
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temporal and spatial patterns that act as intraspecific courtship signals which attract receptive females (Morin, 1986; Cohen and Morin, 1990). Since the compound eye most probably acts as the primary photoreceptor of these luminescent signals (Huvard, 1990a), it was of interest to determine whether their visual pigment absorbance matches the luminescence emission spectrum. There are a few published studies on the structure of the ostracode visual system (Andersson, 1979; Andersson and Nilsson, 1981; Land and Nilsson, 1990; Huvard, 1990a,b). There are, however, no studies on the spectral characteristics of the visual pigments of any ostracode. Further, there are only a few studies on the ostracode luminescence emission spectra (Johnson, 1967; Shimomuro and Johnson, 1970; Tsuji et al., 1975; Herring, 1983; Widder et al., 1983). The purpose of my study was to compare the spectral characteristics of the ostracode luminescence with the visual pigments. Since many species of marine luminescent ostracodes use bioluminescence in conjuction with complex behavior, the study also aimed to determine whether the emission spectra were different between (1) species, (2) males and females of the same species, (3) juveniles and adults of the same species, and (4) live and dried specimens.
MATERIALS
AND METHODS
Collection and maintenance of ostracodes
All ostracodes were collected following the methods of Cohen and Morin (1986) whereby the animals were attracted to baited “cone traps” or by discrete collections during their luminescent displays. Because these ostracodes are most active at night, the traps were always set just before sundown and retrieved and rebaited periodically throughout the night. The following species were collected from the the following areas: (1) I’urgulu tsujii, from Habitat Reef and other shallow water reefs (10-20 m depth) adjacent to the Catalina Marine Science Center, Santa Catalina Island, CA (33.5”N: 117.5”W); (2) Vargula graminicola and (3) Skogsbergia lerneri (a non-bioluminescent species) in turtle grass beds (3-10m depth) off the San Bias Island of Panama (9.55”N: 78.92”W), (4) Vurgula shulmanae in areas of the San Blas Islands of Panama from shallow (12-14m depth) reef slopes primarily among gorgonians. All ostracodes were maintained in the laboratory at UCLA in 1 1beakers (18°C for tropical species and 15°C for temperate water species) and fed small pieces of frozen fish until experiments could be performed. Visual pigment analysis
To determine the characteristics of the absorbance spectra of the visual pigments, the eyes of three ostracode species (V. tsujii, V. graminicola and S. Zerneri) were dissected and the pigments were extracted
with digitonin. The extracts were examined by conventional spectrophotometry. Since the compound eyes of these ostracodes are about 250 pm in diameter, at least 400 individuals of each species (800 compound eyes) were used. Prior to dissection, the animals were placed in the dark for at least 3 hr and then were transferred to 4% potassium alum in sea-water. The eyes and anterior body regions were subsequently dissected away from the rest of the bodies under dim red light. In all cases, the medial eyes were not separated from the compound eyes. If there was lag time between dissection and extraction, the eyes were stored in potassium alum at 4°C; the bodies were also kept in potassium alum for later use as control tissue. The anterior body regions including the eyes were then washed in distilled water, followed by a 0.1 M phosphate buffer wash (pH 7.3). The pigments were extracted in 2% digitonin in the same buffer and the extracts were subsequently analysed spectrophotometrically. The samples were then treated with hydroxylamine and since hydroxylamine can potentially destroy the pigment (Stavenga and Schwemer, 1984) two experiments were always performed; one with hydroxylamine and one without. No difference was ever detected between the extracts treated with this reagent and it was concluded that in this case, hydroxylamine has no effect on the pigment. To positively identify the pigment as a retinal, sodium cyanide (NaCN) was added. This treatment converts the retinal to retinoic acid, which can be identified by its characteristic spectrum (Crescitelh and Karalvy, 1989). The difference spectra were obtained with the use of photometric curves. The ostracode bodies were treated in the same manner and used as control tissue. Luminescence emission spectra
The luminescence emission spectra of two species, graminicola and V. shulmanae, were analysed. To determine if any differences existed between adult males, females and juveniles (4th instar), and live or dried animals, one of each of these categories was examined (V. graminicolu). One adult V. shulmanae was examined. The luminescence spectra were measured and corrected with the same computer controlled optical multi-channel analyser (OMA) with which dried V. tsujii and V. hilgendorfi were previously tested (Widder et al., 1983). A detailed description of this technique is given by Widder et al. (1983). We used a slit width of 1 mm with no other collection optics. To induce luminescence in live animals, the ostracodes were placed in a small chamber (5 mm in diameter) on a microscope slide in a drop of sea-water and were stimulated with a single low voltage D.C. pulse (threshold for luminesence is about 60 V for 10 msec). Dried animals were placed in a small drop of fresh water to induce luminescence. The luminescence spectra are presented here as normalized curves of relative intensity where 100% represents the maximum emission. Vargula
Ostracod
visual pigments
335
anterior region of the bodies. For the two species examined (V. graminicola and S. lerneri), the posterior portions of the animals always produced in difference spectra that had multiple maxima; the difference spectra are very different from those obtained from the anterior regions of the ostracodes (Figs 1B and C). Therefore, it was concluded that the photopigment that produced a difference spectrum maximum of 460 nm (Fig. 1) is included in the tissues of the anterior portion of the ostracodes (i.e. the eyes).
::I: fi_____... A L
I
/
I
I
Luminescence emission spectra
I
400
I
600
500 Wavelength
700
(nm)
Fig. 1. Difference spectra of the visual pigments of the luminescent ostracodes Vargulatsujii(A) and V. graminicola (B), and that of the non-luminescent ostracode, Skogsbergia lerneri. Solid lines represent the difference spectra of the eyes and the anterior end of the body (I.,,, = 460 nm) and the dashed lines show those of the control tissue (posterior portion of the body excluding eye tissue).
For the two species (V. graminicola and V. shulmanae) analysed for their luminescence emission spectra, the L,,, was 473 nm and the full width at half maximum (FWHM) was 78-80 nm; the signal to noise ratio was high (> 183) (Figs 3A, B). In all cases the emission spectra curves were unimodal. In addition, the emission spectra for the male/female and adult/juvenile pairs of V. graminicola also peaked at 473 nm and had very similar shapes to those shown in Figs 3A and B. The dried individual of V. graminicola also had an emission A,,,, of 473 nm. There is a close correlation between the luminescence emission spectra and the visual pigment absorbance difference spectra for an adult male of V. graminicola (Fig. 4). The emission spectra peaked at 473 nm while the pigment absorbance spectra peaked at about 460 nm.
RESULTS
Visual pigment absorbance spectra For all three species of ostracodes whose visual pigment absorbance spectra were examined (shown as difference spectra), the A,,,,, was about 460 nm (Fig. 1). Because the absorbance maxima occur within the blue-green region of the visible spectrum, the photopigments are presumably rhodopsin. With the addition of sodium cyanide the pigment was converted to a retinoic acid (characteristic A,,,,, of 360-380 nm, Fig. 2) which identifies it as a retinal. The controls were tested to ensure that the pigments yielding the spectra were in fact located in the 00
A
r
I
500 Wavelength
600
700
(nm)
Fig. 2. Difference spectra of V. graminicolu for pigment extracts after treatment with NaCN (converts retinal to retinoic acid). &,_ = 380 nm.
Fig. 3. Normalized curve of the luminescence emission spectra for V. graminicola(A) and V. shulmanae(B) where 100% represents maximum emission. Iz,, is 473 nm in both cases.
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Wavelength
(nml
Fig. 4. Normalized spectra of eye pigment absorbance spectra (line a) and luminescence emission spectra (line b) for V. graminicola.
DISCUSSION
The term rhodopsin has traditionally been used as an umbrella term that describes any retinal based visual pigment with absorbance maxima falling within blue-green wavelengths (Goldsmith and Bernard, 1985). However, color descriptive names are now being used to describe the pigments that fall under this broad category; rose colored pigments are rhodopsins and golden pigments are chrysopsins (Crescitelli et al., 1985). Using these definitions, the ostracode pigment would be classified as a chrysopsin because it appears as a golden-brown pigment. In freshwater crayfish and some insects, multiple visual pigment systems have been demonstrated which implies they have the basis for color vision (Schwermer 1989). However, for most marine crustaceans, only one pigment system has been described (Scott and Mote, 1974). The present study found that only one pigment is present in the cypridinid ostracode eye (Fig. 1). To examine the characteristics of visual pigments, three methods have been traditionally employed (Stavenga and Schwemer, 1984; Cronin, 1986): (1) spectrophotometry of digitonin extracted pigments, (2) microspectrophotometry (MSP) of receptor cells or portions of those cells and (3) extracellular or intracellular measurements of electrical responses (electroretinogram = ERG) of intact eyes. The experimental procedures employed in this study were the measurement of the absorbance spectra of digitonin extracted pigments and thus translated into difference spectra. Since spectral sensitivity is directly related to the absorbance spectra (Cronin, 1986), it therefore can be used to produce the spectral sensitivity of visual pigments. For marine invertebrates, specifically crustaceans, all three methods have been employed (e.g. ERG, Frank, 1986; Grober, 1990; digitonin extractions of visual pigments, Hiller-Adams et al., 1988; MSP, Cronin and Forward, 1988; Widder et al., 1987). Spectral differences have been found when more than one method was used on the same species. For example, in one case of a marine crab (Scott and Mote, 1974), the A,,,,, as measured from spectrophotometry of digitonin extractions was about 20nm
shorter than the results obtained from MSP or ERG methods. For marine organisms, the visual pigment absorbance spectral range often matches the light regime of the surrounding water (McFarland, 1986). Marine invertebrates tend to have a relatively narrow spectral sensitivity range between 450 and 550nm whereas in marine fishes the A,,,,, can exceed 600 nm (Cronin, 1986). In crustaceans, the absorbance maxima lies between 450 and 535 nm (Schwemer, 1989). The present study on marine cypridinid ostracodes shows that the ostracode visual pigment absorbance spectra maxima falls near the low end of this range at 460 nm. With the exception of some deep-sea bioluminescence, which can be red, most marine bioluminescence falls within blue to green wavelengths. Widder et al. (1983) examined the luminescence emission spectra of 70 different species of organisms from various habitats. Their study included two species of dried ostracodes in the genus Vurgula (V. tsujii and V. hilgendor$i) and the luminescence emission spectra peaked between 465 and 469 nm. The emission spectra were nearly identical for both of these species. Other studies of emission spectra of V. higendorfi yield results ranging from 459 to 465 nm (460 nm, Herring, 1983; 460-465 nm, Johnson, 1967; 465 nm, Shimomura and Johnson, 1970; 459 nm, Tsuji et al., 1975). These results differ from the results of the present study (L,,, of 473 nm for all individuals examined) by as little as 4 nm and as much as 14 nm. Variation in emission maxima within a particular taxon is not unusual (Herring, 1983) however, the general characteristics of the curves have never been shown to be different between species of the same taxon for any marine organism (e.g. all euphausiids have the same general emission spectra characteristics). For ostracodes, the emission spectra for all of the above mentioned studies have the same general characteristics; they are all unimodal and have FWHM of about 80 nm. Most of the studies that examined the relationship between bioluminesence and vision in marine organisms (e.g. O’Day and Fernandez, 1974; Nicol, 1978; Stearns and Forward, 1984; Buskey and Swift, 1985; Herring, 1985) have been behavioral in nature and, considering the number of luminescent marine organisms with well developed visual systems, the number of comparative physiological studies is very small. The ostracodes in the present study are nocturnally active. During the day, they live within the interstice of the substrate and at night, they move off the bottom for purposes of mating (Morin, 1986; Cohen and Morin, 1989). Three of the four species studied are bioluminescent (V. graminicola, V. shulmanae and V. tsujii; S. lerneri is not bioluminescent). Two of these, V. graminicola and V. shulmanae, are restricted to the Caribbean and, males emit their luminescence in species-specific patterns during courtship at night (Morin, 1986; Cohen and Morin, 1989) in order to
Ostracod visual pigments attract receptive conspecific females. This mating behavior seems to be restricted to Caribbean species and to date, complex signaling has not been observed in ostracodes from other areas (Cohen and Morin, 1989). One of the purposes of this study was to correlate the visual sensitivity with the spectra1 quality of luminescence; thus indicating whether or not an ostracode might be cueing not only on the pattern of emission but also on the wavelength of the luminescence. It was found, however, that all species examined emitted light at approximately the same wavelength and, furthermore, the visual pigments of all species examined, including the non-luminescent species, have identical absorbance spectra. Therefore, in conclusion it can be stated that the wavelength of light is probably not important in species recognition among conspecifics. Instead, other variables (such as the temporal and spatial signaling patterns) are probably acted on in this complex behavior. Further, the peaks for the luminescence and pigment absorbance spectra did not match exactly (473 and 460nm, respectively), however, it may be that some aspect of the luminescence spectra other than the peak may be important for perception of the light. Acknowledgements-I would like to thank the late Dr F. Crescitelli for assistance with the visual pigment extraction experiments and Dr E. Widder for allowing me to use the OMA. 1 am also indebted to the staff of the Catalina Marine Science Center and Dr J. G. Morin for providing animals. The manuscript was greatly improved by the critical readings of Drs A. Cohen, F. Crescitelli, M. S. Grober, J. G. Morin and L. Muscatine. This study was supported by a NSF Grant (# BSR-89-05931 to J. G. Morin).
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Cohen A. C. and Morin J. G. (1990) Morphological relationships of bioluminescent Caribbean soecies of Vargala (Myodocopa). Proc. Tenth Internatioial Symposium on Ostracoda, Aberystwyth, 1988. Crescitelli F., McFall-Ngai M. and Horwitz J. (1985) The visual pigment sensitivity hypothesis: further evidence from fishes of varying 157. _ _ habitats. J. coma. _ Phvsiol. _ 323-333.
Crescitelli F. and Karvaly B. (1989) The visual pigment cyanide effect. Photochem. Photobiol. 50, 785-791.
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Cronin T. W. (1986) Photoreception in marine invertebrates. Am. Zool. 26, 403-415. Cronin T. W. and Forward R. B. (1988) The visual pigments of crabs I. Spectral characteristics. J. camp. Physiol. A, 162, 463-478.
Denton, E. J., Gilpin-Brown J. B. and Wright P. G. (1985) The angular distribution of the light produced by some mesopelagic fish in relation to their camoflage. Proc. R. Sot. Lond. B 182, 145-158. Fernandez H. R. (1978) Visual pigments of bioluminescent and nonbioluminescent deep-sea fishes. Vision Res. 19, 589-592.
Forward R. B., Cronin T. W. and Douglas J. K. (1988) The visual pigments of crabs. II. Environmental adaptations. J. camp. Physiol. A 162, 479-490. Frank T. M. (1986) Visual spectral sensitivity of deep-sea decapod crustaceans. Am. 2001. 26, 35A. Goldsmith T. H. (1972) The natural history of invertebrate visual pigments. In Photochemistry of Vision Handbook of Sensory Physiology (Edited by Dartnall H. J. A.), Vol. VII, pp. 685-719. Springer, New York. Goldsmith T. H. and Bernard G. D. (1985) Visual pigments of invertebrates. Photochem. Photobiol. 42, 805-809. Grober M. S. (1990) Luminescent flash avoidance in the nocturnal crab Portunus xantusii. II. Cardiac and visual responses to variations in stimulated luminescent flashes. J. exp. Biol. 148, 42748. Herring P. J. (1983) The spectral characteristics of luminous marine organisms. Proc. R. Sot. (B) 220, 183-217. Herring P. J. (1985) How to survive in the dark: bioluminescence in the deep sea. Symp. Sot. exp. Biol. 39, 323-350. Hiller-Adams P., Widder E. A. and Case J. F. (1988) The visual pigments of four deep-sea crustacean species. J. camp. Physiol. A 163, 63-72.
Huvard A. L. (1990a) The ultrastructure of the compound eyes of two species of marine ostracodes (Crustacea: Ostracoda: Cypridinidae). Acta Zoo/. 71, 217-223. Huvard A. L. (1990b) Ultrastructural study of the naupliar eye of the ostracode Vargula graminicola (Crustacea: Ostracoda). Zoomorphology 110, 47-51. Johnson F. H. (1967) Bioluminescence. In Comprehensive Biochemistry (Edited by Florkin M. and Stotz E. H.), Vol. 27, 79-136. Land M. F. and Nilsson D.-E. (1990) Observations on the compound eyes of the deep-sea ostracode Macrocypridina castanea. J. exp. Biol., in press. Lithgoe J. N. (1972) The adaptation of visual pigments to the photic environment. In Photochemistry of Vision, Handbook of Sensory Physiology (Edited by Dartnall H. J. A.), Vol. VII/l; pp. 566603. Springer, New York. McFarland W. N. (1971) Cetacean visual oiaments. Vision -1 Res. 11, 1065-1076. McFarland W. N. (1986) Light in the sea-correlation with behaviors of fishes and invertebrates. Am. Zool. 26, 389-401.
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Munz F. W. (1958) Photosensitive pigments from the retinae of deep-sea fishes. J. Physiol. 140, 22C~235. Nicol L. A. C. (1978) Bioluminescence and vision. In Bioluminescence in Action (Edited by Herring P. J.), pp. 367-398. Academic Press, New York. O’Day W. T. and Femandez H. R. (1974) Aristostomias scintellans (Malacosteidae): a deep-sea fish with visual pigments apparently adapted to its own bioluminescence. Vision Res. 14, 545-550.
Schwermer J. (1989) Visual pigments of compound eyes. Structure photochemistry and regeneration. In Facets of Vision (Edited by Stavenga D. G. and Hardie R. C.), pp. 112-133. Springer, New York.
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0300-9629/93 $6.00+ 0.00
Printed in Great Britain
0 1993Pergamon Press Ltd
ANALYSIS OF VISUAL PIGMENT ABSORBANCE AND LUMINESCENCE EMISSION SPECTRA IN MARINE OSTRACODES (CRUSTACEA: OSTRACODA) ANDREA L. HUVARD* Department of Biology, University of California, Los Angeles, CA 90024, U.S.A. (Tel. 805 493-3341) (Received
30 April 1992; accepted 5 June 1992)
Abstract-l. The luminescence emission spectra and the visual pigment absorbance spectra in digitonin extracts were examined and compared for marine luminescent ostracodes. 2. The rhodopsin absorbance spectrum of one non-luminescent species was determined for comparison. 3. For all species examined (Vargula tsujii, V. graminicola and Skogsbergia lerneri), the difference spectra for visual pigment absorbance peaked at 460nm. 4. The luminescence emission spectra were measured by means of a computer controlled optical multi-channel analyser (OMA). 5. For the two luminescent species examined (V. graminicola and V. shulmanae), the A,,, of the emitted light was 473 nm with full width at half maxima (FWHM) of about 80 nm. 6. The luminescence emission spectra were also determined for male/female, adult/juvenile and dried/live pairs of V. graminicola. 7. All had a A,,,,, of 473 nm and in all cases the curves were unimodal with the FWHM of 80 nm. 8. V. graminicola and V. shulmanae are found in Panama in the Caribbean and the males emit their luminescence in complex display patterns (signals) for purposes of attracting receptive females. 9. Since the emission spectra for these two signalling species were similar, it would appear that species recognition is not based on wavelength but rather some other aspect of the luminescent display.
INTRODUCTION Early studies of visual pigments of marine organisms examined the hypothesis that the maximum spectral absorbance of a photopigment is an adaptation for increased visual sensitivity to the light available in a particular environment (Sensitivity Hypothesis, reviewed in Lythgoe, 1972). These early studies were primarily concerned with the visual pigments of organisms that live in light-limited environments (i.e. the deep sea). This Sensitivity Hypothesis has since been tested in several species of marine fishes, mammals and invertebrates from a variety of marine habitats (e.g. McFarland, 1971; Goldsmith, 1972; Fernandez, 1978; Crescitelli et al., 1985; Cronin and Forward, 1988; Forward et al., 1988; Grober, 1990). There are well documented cases that meet the conditions of the Sensitivity Hypothesis. For example, in deep sea fishes (i.e. Munz, 1958; Fernandez, 1978; Crescitelli et al., 1985) the A,,, of the visual pigments matches the ambient downwelling light (,l,,, of In contrast, the approximately 460-480 nm). spectral sensitivity of shallow water species can often exceed a A,,,,, of 500 nm. Many deep sea and midwater organisms are bioluminescent and the emitted luminescence can match the wavelength of the ambient downwelling light (Denton et aI., 1985; Herring, 1985). The match *Present address: Department of Biology, California Lutheran University, Thousand Oaks, CA 91360, U.S.A.
between bioluminescence and visual pigment absorbance spectra has been documented for several species of deep sea fishes and invertebrates (O’Day and Fernandez, 1974; Warner et al., 1978; Young and Mencher, 1980; Crescitelli et al., 1985; Denton et al., 1985). A unique example of matching has been demonstrated for some deep sea fishes that emit red luminescence; these fishes also have visual pigments sensitive to longer (red) wavelengths of light (O’Day and Fernandez, 1974). Two recent studies of intertidal and estuarine invertebrates (Cronin and Forward, 1988; Forward et al., 1988) examine the photopigment spectra of animals living in variable light conditions. With the use of microspectrophotometry, Cronin and Forward (1988) showed that several species of intertidal to subtidal decapods have maximum spectra that range from 473-515 nm. Considering that the 1,, for bioluminescence is usually coastal between 490-520 nm (Morin, 1983) it seems likely that many shallow water organisms may have photopigments that are adapted to match both the broad spectrum of ambient light and bioluminescence. Ostracodes are small crustaceans (usually <3 mm long) that are widely distributed in most freshwater and marine habitats. Ostracodes in the family Cypridinidae are abundant in shallow marine waters (< 30 m) and some species are bioluminescent. In the Caribbean, male ostracodes in the nominal genus Vurgulu emit their bioluminescence in complex 333
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temporal and spatial patterns that act as intraspecific courtship signals which attract receptive females (Morin, 1986; Cohen and Morin, 1990). Since the compound eye most probably acts as the primary photoreceptor of these luminescent signals (Huvard, 1990a), it was of interest to determine whether their visual pigment absorbance matches the luminescence emission spectrum. There are a few published studies on the structure of the ostracode visual system (Andersson, 1979; Andersson and Nilsson, 1981; Land and Nilsson, 1990; Huvard, 1990a,b). There are, however, no studies on the spectral characteristics of the visual pigments of any ostracode. Further, there are only a few studies on the ostracode luminescence emission spectra (Johnson, 1967; Shimomuro and Johnson, 1970; Tsuji et al., 1975; Herring, 1983; Widder et al., 1983). The purpose of my study was to compare the spectral characteristics of the ostracode luminescence with the visual pigments. Since many species of marine luminescent ostracodes use bioluminescence in conjuction with complex behavior, the study also aimed to determine whether the emission spectra were different between (1) species, (2) males and females of the same species, (3) juveniles and adults of the same species, and (4) live and dried specimens.
MATERIALS
AND METHODS
Collection and maintenance of ostracodes
All ostracodes were collected following the methods of Cohen and Morin (1986) whereby the animals were attracted to baited “cone traps” or by discrete collections during their luminescent displays. Because these ostracodes are most active at night, the traps were always set just before sundown and retrieved and rebaited periodically throughout the night. The following species were collected from the the following areas: (1) I’urgulu tsujii, from Habitat Reef and other shallow water reefs (10-20 m depth) adjacent to the Catalina Marine Science Center, Santa Catalina Island, CA (33.5”N: 117.5”W); (2) Vargula graminicola and (3) Skogsbergia lerneri (a non-bioluminescent species) in turtle grass beds (3-10m depth) off the San Bias Island of Panama (9.55”N: 78.92”W), (4) Vurgula shulmanae in areas of the San Blas Islands of Panama from shallow (12-14m depth) reef slopes primarily among gorgonians. All ostracodes were maintained in the laboratory at UCLA in 1 1beakers (18°C for tropical species and 15°C for temperate water species) and fed small pieces of frozen fish until experiments could be performed. Visual pigment analysis
To determine the characteristics of the absorbance spectra of the visual pigments, the eyes of three ostracode species (V. tsujii, V. graminicola and S. Zerneri) were dissected and the pigments were extracted
with digitonin. The extracts were examined by conventional spectrophotometry. Since the compound eyes of these ostracodes are about 250 pm in diameter, at least 400 individuals of each species (800 compound eyes) were used. Prior to dissection, the animals were placed in the dark for at least 3 hr and then were transferred to 4% potassium alum in sea-water. The eyes and anterior body regions were subsequently dissected away from the rest of the bodies under dim red light. In all cases, the medial eyes were not separated from the compound eyes. If there was lag time between dissection and extraction, the eyes were stored in potassium alum at 4°C; the bodies were also kept in potassium alum for later use as control tissue. The anterior body regions including the eyes were then washed in distilled water, followed by a 0.1 M phosphate buffer wash (pH 7.3). The pigments were extracted in 2% digitonin in the same buffer and the extracts were subsequently analysed spectrophotometrically. The samples were then treated with hydroxylamine and since hydroxylamine can potentially destroy the pigment (Stavenga and Schwemer, 1984) two experiments were always performed; one with hydroxylamine and one without. No difference was ever detected between the extracts treated with this reagent and it was concluded that in this case, hydroxylamine has no effect on the pigment. To positively identify the pigment as a retinal, sodium cyanide (NaCN) was added. This treatment converts the retinal to retinoic acid, which can be identified by its characteristic spectrum (Crescitelh and Karalvy, 1989). The difference spectra were obtained with the use of photometric curves. The ostracode bodies were treated in the same manner and used as control tissue. Luminescence emission spectra
The luminescence emission spectra of two species, graminicola and V. shulmanae, were analysed. To determine if any differences existed between adult males, females and juveniles (4th instar), and live or dried animals, one of each of these categories was examined (V. graminicolu). One adult V. shulmanae was examined. The luminescence spectra were measured and corrected with the same computer controlled optical multi-channel analyser (OMA) with which dried V. tsujii and V. hilgendorfi were previously tested (Widder et al., 1983). A detailed description of this technique is given by Widder et al. (1983). We used a slit width of 1 mm with no other collection optics. To induce luminescence in live animals, the ostracodes were placed in a small chamber (5 mm in diameter) on a microscope slide in a drop of sea-water and were stimulated with a single low voltage D.C. pulse (threshold for luminesence is about 60 V for 10 msec). Dried animals were placed in a small drop of fresh water to induce luminescence. The luminescence spectra are presented here as normalized curves of relative intensity where 100% represents the maximum emission. Vargula
Ostracod
visual pigments
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anterior region of the bodies. For the two species examined (V. graminicola and S. lerneri), the posterior portions of the animals always produced in difference spectra that had multiple maxima; the difference spectra are very different from those obtained from the anterior regions of the ostracodes (Figs 1B and C). Therefore, it was concluded that the photopigment that produced a difference spectrum maximum of 460 nm (Fig. 1) is included in the tissues of the anterior portion of the ostracodes (i.e. the eyes).
::I: fi_____... A L
I
/
I
I
Luminescence emission spectra
I
400
I
600
500 Wavelength
700
(nm)
Fig. 1. Difference spectra of the visual pigments of the luminescent ostracodes Vargulatsujii(A) and V. graminicola (B), and that of the non-luminescent ostracode, Skogsbergia lerneri. Solid lines represent the difference spectra of the eyes and the anterior end of the body (I.,,, = 460 nm) and the dashed lines show those of the control tissue (posterior portion of the body excluding eye tissue).
For the two species (V. graminicola and V. shulmanae) analysed for their luminescence emission spectra, the L,,, was 473 nm and the full width at half maximum (FWHM) was 78-80 nm; the signal to noise ratio was high (> 183) (Figs 3A, B). In all cases the emission spectra curves were unimodal. In addition, the emission spectra for the male/female and adult/juvenile pairs of V. graminicola also peaked at 473 nm and had very similar shapes to those shown in Figs 3A and B. The dried individual of V. graminicola also had an emission A,,,, of 473 nm. There is a close correlation between the luminescence emission spectra and the visual pigment absorbance difference spectra for an adult male of V. graminicola (Fig. 4). The emission spectra peaked at 473 nm while the pigment absorbance spectra peaked at about 460 nm.
RESULTS
Visual pigment absorbance spectra For all three species of ostracodes whose visual pigment absorbance spectra were examined (shown as difference spectra), the A,,,,, was about 460 nm (Fig. 1). Because the absorbance maxima occur within the blue-green region of the visible spectrum, the photopigments are presumably rhodopsin. With the addition of sodium cyanide the pigment was converted to a retinoic acid (characteristic A,,,,, of 360-380 nm, Fig. 2) which identifies it as a retinal. The controls were tested to ensure that the pigments yielding the spectra were in fact located in the 00
A
r
I
500 Wavelength
600
700
(nm)
Fig. 2. Difference spectra of V. graminicolu for pigment extracts after treatment with NaCN (converts retinal to retinoic acid). &,_ = 380 nm.
Fig. 3. Normalized curve of the luminescence emission spectra for V. graminicola(A) and V. shulmanae(B) where 100% represents maximum emission. Iz,, is 473 nm in both cases.
ANDREA L. HWARD
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Wavelength
(nml
Fig. 4. Normalized spectra of eye pigment absorbance spectra (line a) and luminescence emission spectra (line b) for V. graminicola.
DISCUSSION
The term rhodopsin has traditionally been used as an umbrella term that describes any retinal based visual pigment with absorbance maxima falling within blue-green wavelengths (Goldsmith and Bernard, 1985). However, color descriptive names are now being used to describe the pigments that fall under this broad category; rose colored pigments are rhodopsins and golden pigments are chrysopsins (Crescitelli et al., 1985). Using these definitions, the ostracode pigment would be classified as a chrysopsin because it appears as a golden-brown pigment. In freshwater crayfish and some insects, multiple visual pigment systems have been demonstrated which implies they have the basis for color vision (Schwermer 1989). However, for most marine crustaceans, only one pigment system has been described (Scott and Mote, 1974). The present study found that only one pigment is present in the cypridinid ostracode eye (Fig. 1). To examine the characteristics of visual pigments, three methods have been traditionally employed (Stavenga and Schwemer, 1984; Cronin, 1986): (1) spectrophotometry of digitonin extracted pigments, (2) microspectrophotometry (MSP) of receptor cells or portions of those cells and (3) extracellular or intracellular measurements of electrical responses (electroretinogram = ERG) of intact eyes. The experimental procedures employed in this study were the measurement of the absorbance spectra of digitonin extracted pigments and thus translated into difference spectra. Since spectral sensitivity is directly related to the absorbance spectra (Cronin, 1986), it therefore can be used to produce the spectral sensitivity of visual pigments. For marine invertebrates, specifically crustaceans, all three methods have been employed (e.g. ERG, Frank, 1986; Grober, 1990; digitonin extractions of visual pigments, Hiller-Adams et al., 1988; MSP, Cronin and Forward, 1988; Widder et al., 1987). Spectral differences have been found when more than one method was used on the same species. For example, in one case of a marine crab (Scott and Mote, 1974), the A,,,,, as measured from spectrophotometry of digitonin extractions was about 20nm
shorter than the results obtained from MSP or ERG methods. For marine organisms, the visual pigment absorbance spectral range often matches the light regime of the surrounding water (McFarland, 1986). Marine invertebrates tend to have a relatively narrow spectral sensitivity range between 450 and 550nm whereas in marine fishes the A,,,,, can exceed 600 nm (Cronin, 1986). In crustaceans, the absorbance maxima lies between 450 and 535 nm (Schwemer, 1989). The present study on marine cypridinid ostracodes shows that the ostracode visual pigment absorbance spectra maxima falls near the low end of this range at 460 nm. With the exception of some deep-sea bioluminescence, which can be red, most marine bioluminescence falls within blue to green wavelengths. Widder et al. (1983) examined the luminescence emission spectra of 70 different species of organisms from various habitats. Their study included two species of dried ostracodes in the genus Vurgula (V. tsujii and V. hilgendor$i) and the luminescence emission spectra peaked between 465 and 469 nm. The emission spectra were nearly identical for both of these species. Other studies of emission spectra of V. higendorfi yield results ranging from 459 to 465 nm (460 nm, Herring, 1983; 460-465 nm, Johnson, 1967; 465 nm, Shimomura and Johnson, 1970; 459 nm, Tsuji et al., 1975). These results differ from the results of the present study (L,,, of 473 nm for all individuals examined) by as little as 4 nm and as much as 14 nm. Variation in emission maxima within a particular taxon is not unusual (Herring, 1983) however, the general characteristics of the curves have never been shown to be different between species of the same taxon for any marine organism (e.g. all euphausiids have the same general emission spectra characteristics). For ostracodes, the emission spectra for all of the above mentioned studies have the same general characteristics; they are all unimodal and have FWHM of about 80 nm. Most of the studies that examined the relationship between bioluminesence and vision in marine organisms (e.g. O’Day and Fernandez, 1974; Nicol, 1978; Stearns and Forward, 1984; Buskey and Swift, 1985; Herring, 1985) have been behavioral in nature and, considering the number of luminescent marine organisms with well developed visual systems, the number of comparative physiological studies is very small. The ostracodes in the present study are nocturnally active. During the day, they live within the interstice of the substrate and at night, they move off the bottom for purposes of mating (Morin, 1986; Cohen and Morin, 1989). Three of the four species studied are bioluminescent (V. graminicola, V. shulmanae and V. tsujii; S. lerneri is not bioluminescent). Two of these, V. graminicola and V. shulmanae, are restricted to the Caribbean and, males emit their luminescence in species-specific patterns during courtship at night (Morin, 1986; Cohen and Morin, 1989) in order to
Ostracod visual pigments attract receptive conspecific females. This mating behavior seems to be restricted to Caribbean species and to date, complex signaling has not been observed in ostracodes from other areas (Cohen and Morin, 1989). One of the purposes of this study was to correlate the visual sensitivity with the spectra1 quality of luminescence; thus indicating whether or not an ostracode might be cueing not only on the pattern of emission but also on the wavelength of the luminescence. It was found, however, that all species examined emitted light at approximately the same wavelength and, furthermore, the visual pigments of all species examined, including the non-luminescent species, have identical absorbance spectra. Therefore, in conclusion it can be stated that the wavelength of light is probably not important in species recognition among conspecifics. Instead, other variables (such as the temporal and spatial signaling patterns) are probably acted on in this complex behavior. Further, the peaks for the luminescence and pigment absorbance spectra did not match exactly (473 and 460nm, respectively), however, it may be that some aspect of the luminescence spectra other than the peak may be important for perception of the light. Acknowledgements-I would like to thank the late Dr F. Crescitelli for assistance with the visual pigment extraction experiments and Dr E. Widder for allowing me to use the OMA. 1 am also indebted to the staff of the Catalina Marine Science Center and Dr J. G. Morin for providing animals. The manuscript was greatly improved by the critical readings of Drs A. Cohen, F. Crescitelli, M. S. Grober, J. G. Morin and L. Muscatine. This study was supported by a NSF Grant (# BSR-89-05931 to J. G. Morin).
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