A comparison of spectral sensitivities of caudal photoreceptors of epigeal and cavernicolous crayfish

Comp. Biochem. Physiol., 1966, Vol. 19, pp. 409 to 415. Pergamon Press Ltd. Printed in Great Britain

A COMPARISON OF SPECTRAL SENSITIVITIES OF CAUDAL PHOTORECEPTORS OF EPIGEAL AND CAVERNICOLOUS CRAYFISH* J A M E S L. L A R I M E R , D A N I E L L. T R E V I N O and E B E R T A. A S H B Y t Department of Zoology, University of Texas, Austin (Received 25 April 1966) A b s t r a c t - - 1 . Spectral sensitivity data are presented for the caudal photoreceptors of four species of epigeal crayfishes including Procambarus clarkii, Procambarus simulans, Orconectes virilis and Cambarus sciotensis. In addition, data are given for the single blind cavernicolous species, Orconectes peUucidus australis.

2. A Amax of 502 m/z was obtained for the receptors of all the epigeal forms. Although some variations are seen among these curves, they probably reflect the function of identical pigments. 3. The curves obtained on receptors of the cavernicolous form, O. peUucidus australis, show a slight shift toward the blue when compared with the data from the epigeal animals. The Areax is estimated to be 497 m/~. 4. Comparison of spectral sensitivities of the caudal photoreceptors with those of the compound eyes of P. clarkii and O. virilis indicates that the caudal receptor pigment is different. Since the compound eyes contain a dichromatic system, it is assumed that at least three independent genes control the carotenoidprotein visual pigments of crayfishes. INTRODUCTION THE sixth abdominal ganglion of crayfishes contains light-sensitive neurons (Prosser, 1934) which are capable of mediating a locomotor reflex of the pereiopods (Welsh, 1934). Although the basic neurophysiology of the receptors has been investigated extensively (Kennedy, 1958a, b, 1963; Kennedy & Preston, 1960; H e r m a n n & Stark, 1963a, b), few comparative studies have been made. Since only two neurons are involved (Kennedy, 1963), it is not feasible with present techniques to isolate and characterize by direct chemical means the pigments in these receptors. Bruno & K e n n e d y (1962) have shown, however, that spectral sensitivity data may be obtained by electrophysiological techniques. T h e present work stemmed from a n u m b e r of interesting observations and questions concerning comparative data on the system. T h e initial spectral sensitivity measurements of Bruno & K e n n e d y showed that, in the crayfish, Procambarus clarkii, the dark-adapted caudal neurons have different wavelength sensitivity from those of the dark-adapted eye (Kennedy * Supported by Grant NB-05423 (J. L. L.) and Training Grant 5T1 GM-836 (E. A. A.) from the National Institutes of Health. t Present address: Department of Anatomy, Southwestern Medical School, Dallas, Texas. 409

410

JAMES L. LARIMER, DANIEL L. TREVINO AND EBERT A. ASHBY

& Bruno, 1961), with the caudal photoreceptor giving a maximum sensitivity at about 500 mk~ while the eye gave a Areax at 570 m/x. Recent measurements by Wald (1963a), also based on ERG techniques and using red and blue adapting light, revealed that the eyes of P. clarkii possess two pigments, with both still clearly displaced from the maximum of the caudal photoreceptor. Evidence is also available which indicates that different species of crayfish have evolved different pigments in the compound eyes (Wald, 1962, 1963@ We have therefore compared spectral sensitivities of the caudal photoreceptors of five species of crayfish in an effort to determine if a similar evolution has occurred in the second system. We have included among the species examined Orconectes virilis and Procambarus clarkii since data are available for comparing the response of the compound eye. In addition, we have also examined the spectral response of the sixth ganglion photoreceptors of a blind cavernicolous crayfish, Orconectes pellucidus australis, since it has been shown that at least one other blind species, Cambarus setosus, possesses a functional caudal photoreceptor (Larimer, 1966). Finally, data on Procambarus simulans and Cambarus sciotensis are also reported to extend the comparisons to related species and genera. MATERIALS AND METHODS The data are based upon energy measurements of monochromatic light required to induce near threshold discharges of 1-5 spikes from the nerve following 10 sec stimuli (Bruno & Kennedy, 1962). The cords were isolated and stored at 5°C in crayfish physiological saline (Van Harreveld, 1936) for periods up to 20 hr to minimize interfering spontaneous discharges from unrelated neurons. During the course of the experiments, each preparation was maintained at constant temperature, preferably in the range of 13-17°C, which further limited spontaneous discharges and also raised the threshold to easily measurable values. The signals were first passed to an AC-coupled preamplifier then observed from an oscilloscope with a parallel audio monitor. Each preparation was dark-adapted for 20-30 rain before beginning the tests. Approximately 2-3 min of dark adaptation was provided between stimuli. The additional 3-5 min period of dark adaptation was allowed following any accidental exposure to stimuli substantially above threshold. Most of the observations were made from a single 2-3 connective to assure that the response was from a single photoreceptor neuron. The photic stimulator was a modification of that used by Kampa et al. (1963). The parallel beam from a 500 W projection lamp (General Electric CZX) was directed through second-order interference filters (Bausch & Lomb) and Coming blocking filters which effectively transmitted only the desired band. The detailed transmission characteristics of the combined filters were determined using a Cary Model 14 spectrophotometer. The half-band widths of the filters varied from 8 mtL to 14 m/~. The beam was attenuated in steps using standard neutral density filters (Bausch & Lomb), while continuous intermediate attenuation was achieved by means of a Kodak circular neutral density wedge (A from 0 to 1.0). The beam was chopped with a large-aperture Compur shutter (tachistoscope shutter mod.

SPECTRAL SENSITIVITIES OF CAUDAL PHOTORECEPTORS

411

VS-1, Lafayette Instr. Co.). Stimulus duration was measured by means of a phototube output connected to the second beam of the oscilloscope. The lamp, shutter and a wheel carrying the interference filters were mounted in a standard projector housing from which the condensing lenses had been removed. The power for the lamp was maintained constant by means of an electronic voltage regulator (Sorensen Mod. 1001). Energies were obtained from a selenium photocell (International Rectifier Mod. DP-3) which had been calibrated in the system against a calibrated thermopile. The beam was directed either to the preparation or to the detector by means of a front surface mirror which was rotated through 90 °. The entire apparatus was aligned on a standard optical bench. Since the preparations were small (about 1 mm dia. for the sixth ganglion) in comparison to the illuminated area (about 3 cm), the beam was scanned for uniformity using a pinhole photocell. The variation in intensity across the field was found to be no greater than 4 per cent. During the actual measurements, the preparations were placed as nearly as possible at the beam center. The animals were kept in the laboratory for periods up to 2 weeks. They were maintained at constant temperature of either 24°C in the case of the epigeal species or at 17°C in the case of the cave form. They were fed once weekly. P. clarkii and P. simulans were obtained locally in central Texas. Specimens of C. sciotensis were collected from streams in the vicinity of Blacksburg, Virginia, while 0. virilis was purchased from a biological supply house. 0. p. australis was collected from Shelta Cave, Madison Co., Alabama. Authoritative identifications were obtained for each species. RESULTS In order to facilitate comparisons, the spectral sensitivity data have been reduced to relative values as follows. The energies (E) which produced threshold responses were calculated first in terms of quanta/sec/cm ~ for each wavelength, then converted to the reciprocal values (I/E). The highest reciprocal energy value was set equal to one, and the remaining values were then calculated as fractions thereof. The plots presented below are therefore in terms of relative values of 1/E on the ordinate versus wavelength in m/z on the abscissa. From 5 to 10 experiments were run on the cords of each species. From these, an average curve was plotted along with standard errors for each point. The spectral sensitivity function for P. clarkii (Fig. 1A) agrees with that presented by Bruno & Kennedy (1962) with the minor difference that the curve from the present data is somewhat steeper. The wavelength of maximum sensitivity is estimated to be in the region of 500-505 n~. The caudal photoreceptors of P. simulans and O. virilis appear to possess identical sensitivities when compared with that of P. clarkii. The data from these three species therefore have been averaged for comparison with those obtained for O. p. australis and C. sciotensis (Fig. 1B). Data from the blind species O. p. australis appear to deviate most from the remaining curves. Although this difference is small, there is a uniform shift of the spectral sensitivity toward shorter wavelengths, placing the peak sensitivity

412

JAMES L. LARIMER, DANIEL L. TREVINO AND EBERT A. ASHBY

at about 497 m/, as compared to 502 m/~ for the other species. T h e data for C. sciotensis are identical with those of the average curve, particularly with respect to the wavelength of peak sensitivity; however, the curve is not as steep.

A

B a8

10 -

~' c/ork// (N=6)

>

",,

, /

\

K) -

>-

Average Curve

7- o.( z uJ

08 -

~

0.8

co - LC

w O~ >

04

06 -

,

_J

/

,~

~

~-Rimu/on/(N,9)

o~-

~

-

~'L,

0£

/,

~ o2

- IC '

,_ 04

~¢

o.; O~

~J

f

\ ~ "v\ o~'~,o~',,, ou~ro/is(N:S)

-C.sc~.s,s(N=~) \

Y,,

- 0.'

t

i

I

450

i

i

t

t

[

t

t

,

=

500 WAVELENGTH

m/=

I i 550

h

t

J

,

J

I

4~o

i

I

t

I

i

5oo WAVELENGTH

*

l

mp.

I

i

i

i

1

55o

FIG. 1. Relative wavelength sensitivities of the caudal photoreceptors of crayfish. The continuous curves in (A) and (B) were obtained by averaging (N) individual curves. The vertical bars are standard errors for each point. The upper dashed line in (B) is an average of the three curves in (A). The vertical mark in the curve for 0. p. australis indicates the deviation of peak sensitivity from the remaining curves. It is apparent that the caudal photoreceptors of the species examined have very similar wavelength sensitivity functions. Those of P. darkii, P. simulam, O. virilis and perhaps C. sciotensis are probably identical. T h e response of the receptor of O. p. australis is certainly not greatly different from the others since the standard error lines overlap with the average curve at some of the longer wavelengths. DISCUSSION It seems unlikely, from the arguments of Wald (1958, 1963b, 1964) concerning the origins and the evolution of visual pigments in general, that the caudal photoreceptor possesses a class of pigments other than carotenoid proteins. It has already been suggested (Bruno & Kennedy, 1962) that the spectral sensitivity data indicate that the caudal photoreceptors probably contain a pigment similar to most other

SPECTRAL SENSITIVITIES OF CAUDAL PHOTORECEPTORS

413

retinal rhodopsins. This seems reasonable since the typical rhodopsins, e.g. of the terrestrial vertebrates and the shallow-water marine fishes, exhibit absorption maxima in this region. It is assumed therefore that the crayfishes possess two independent photoreceptor systems in the cephalic eye and one pigment in the caudal photoreceptor, all of which utilize carotenoid-protein photosensitive pigments. The retinal pigments of the eyes of Procambarus clarkii and of Orconectes virilis are known to be dichromatic. Using red and blue adapting illumination and electroretinogram amplitude criteria, P. clarkii yielded ~max 575 m/~ and 445 m/~ while O. virilis showed maximum sensitivities at 570 m/~ and 435 m/~ (Wald, 1963a). Clearly, none of these values corresponds to the maximum sensitivity of 502 m/~ found for the caudal photoreceptors of the same species. Two pigments have been extracted from the eyes of O. virilis. Both contain the chromophore retinal 1, and exhibit Amax at 562 n ~ and 508 m/~ (Wald, 1962). Pigment 508 may correspond to that found in the sixth ganglion photoreceptor; but this is not yet truly established. Direct comparison of spectral sensitivity data obtained from compound eyes with those of single neuron receptors, or even with difference spectra, is complicated by a number of factors, the most notable being the presence of screening pigments, reflecting substances and perhaps even the fluorescence of certain components such as the pteridines which have been shown to be present in crustacean eyes (Viscontini et al., 1955; Kleinholz, 1955). For example, the difference spectrum of isolated lobster rhodopsin is maximum at 515 m/~ (Wald & Hubbard, 1957) while spectral sensitivity curves by ERG measurements yield J~max at 525 m~ (Kennedy & Bruno, 1961). Similarly, the presence of a blueabsorbing (and red-transmitting) pigment in the eyes of certain insects has created considerable confusion concerning their red-light sensitivity (Goldsmith, 1965). It appears that at least three genes control the carotenoid proteins of the crayfishes. Those responsible for the dichromatic systems of the eyes have evolved some relatively minor differences among some species, presumably associated with changes in the opsins. The indications are that the caudal photoreceptor pigment is controlled by a third and independent gene which has not shown major variation, at least in the epigeal forms examined. Spectral sensitivity data from the eyes of marine crustaceans (Wald, 1963a; Kennedy, 1964) commonly also show dual peaks. In certain insects with ocelli in addition to complex and divided compound eyes, as many as five wavelength-sensitivity peaks are seen (Ruck, 1965). The presence of three independent pigments is therefore not unusual in arthropods. Despite the considerable behavioral and neurophysiological work on the caudal photoreceptors, the significance of their light sensitivity remains somewhat obscure. Even less is understood concerning the functions of the receptors in blind carvernicolous crayfish. In the two blind species examined thus far, Cambarus setosus (Latimer, 1966) and O. pellucidus australis both have retained the lightsensitive photoreceptors in spite of an apparently complete regressive evolution of the visual elements of the cephalic eyes (Parker, 1890; Fingerman et al., 1964). Evidence was presented by Wells (1959) that the blind crayfish, C. setosus and C. ayersii, do not show the locomotory reflex when illuminated caudally, but do 14

414

JAMES L. LARIMER,DANIEL L. TREVlNO AND EBERT A. ASHBY

r e s p o n d to illumination of the head, p r e s u m a b l y in response to dermal or C N S light reception. T h e possibility remains, therefore, that the caudal receptor, a l t h o u g h still light-sensitive, has lost its functional connexions with the m o t o n e u r o n s that drive the pereiopods. It has been speculated (Larimer, 1966) that the tactile integrative functions of the p h o t o r e c e p t o r ( K e n n e d y , 1963) have been retained in the blind animals for their positive adaptive value, w i t h o u t loss of the photosensitive properties. Acknowledgements--The authors wish to thank Dr. Jack Myers for his assistance in the design and calibration of the photic stimulator. We are grateful to Dr. Horton Hobbs and Dr. Perry C. Holt for identification of specimens. Thanks are extended to Dr. Thomas C. Barr for helping to find sources of cave crayfish, and to Mr. Stewart B. Peck for collecting them. We thank Mr. Fred D. Henson for collecting C. sciotensis and Mr. Gary Shelton for his technical assistance throughout the work. Finally, we appreciate the efforts of Dr. Donald Kennedy who critically read the manuscript. REFERENCES BRUNO M. S. & KENNEDY D. (1962) Spectral sensitivity of photoreceptor neurons in the sixth ganglion of the crayfish. Comp. Bioehem. Physiol. 6, 41-46. FINGERMAN M., OGURO C., MIYAWAKIM. & McKINNELL R. (1964) The neurosecretory system in the head of the blind cave crayfish Cambarus setosus Faxon. Am. Midl. Nat. 71,415-421. GOLDSMITHT. (1965) Do flies have a red receptor ? J. gen. Physiol. 49, 265-287. HERMANN H. (1964) Stochastic properties in the negative phototropic behavior of the crayfish. J. exp. Zool. 155, 381-402. HERMANN H. & STARK L. (1963a) Prerequisites for a photoreceptor structure in the crayfish tail ganglion. Anat. Rec. 147, 209-217. HERMANN H. & STARKL. (1963b) Single unit responses in a primitive photoreceptor organ. ft. Neurophysiol. 26, 215-228. KAMPA E. M., ABBOTT B. C. & BODEN B. P. (1963) Some aspects of vision in the lobster, Homarus vulgaris, in relation to the structure of its eye. ft. mar. Biol. Ass. U.K. 43, 683-699. KENNEDY D. (1958a) Responses from the crayfish caudal photoreceptor. Am. ft. Ophthal. 46, 19-26. KENNEDY D. (1958b) Electrical activity of a "primitive" photoreceptor. Ann. N . Y . Acad. Sei. 74, 329-336. KENNEDY D. (1963) Physiology of photoreceptor neurons in the abdominal nerve cord of the crayfish, ft. gen. Physiol. 46, 551-572. KENNEDY D. (1964) The photoreceptor processes in lower animals. In Photobiology, Vol. II (Edited by GIESE A. C.), Chap. 14, pp. 79-121. Academic Press, New York. KENNEDY D. 86 BRUNO M. S. (1961) The spectral sensitivity of crayfish and lobster vision. J. gen. Physiol. 44, 1089-1102. KENNEDY D. & PRESTONJ. B. (1960) Activity patterns of interneurons in the caudal ganglion of the crayfish, ft. gen. Physiol. 43, 655-670. KLEINHOLZL. (1955) The nature of the reflecting pigment in the arthropod eye. Biol. Bull., Wood's Hole 109, 362. LARIMER J. L. (1966) The presence of a functional caudal photoreceptor in blind cavernicolous crayfish. Nature, Lond. 210, 204-205. PARKERG. H. (1890) The eyes in blind crayfishes. Bull. Mus. comp. Zool. 20, 153-162. PROSSERC. L. (1934) Action potentials in the nervous system of the crayfish--II. Responses to illumination of the eye and caudal ganglion. J. cell. comp. Physiol. 4, 363-377.

SPECTRAL SENSITIVITIES OF CAUDAL PHOTORECEPTORS

415

RUCK P. (1965) The components of the visual system of the dragonfly, ft. gen. Physiol. 49, 289-307. VISCONTINI M., SCHMID H. & HADOaN E. (1955) Isolierung fluoreszierender Stoffe aus Astacus fluviatilis. Experientia 11, 390-392. WALD G. (1958) Photochemical aspects of visual excitation. Expl Cell Res. Suppl. 5, 389--410. WALD G. (1962) Visual pigments of the freshwater crayfish. Fed. Proc. 21, 344. WALD G. (1963a) Single and multiple visual systems in arthropods. Fed. Proc. 22, 519. WALD G. (1963b) Phylogeny and ontogeny at the molecular level. In Evolutionary Biochemistry (Edited by OPAaIN A. I.), Proc. Fifth int. Congr. Biochem., Moscow, 1961, pp. 12-51. Pergamon Press, London. WALD G. (1964) The origins of life. Proc. natn. Acad. Sci. U.S.A. 52, 595-611. WALD G. & HUBBARD R. (1957) Visual pigment of a decapod crustacean: the lobster. Nature, Lond. 180, 278-280. WELLS P. H. (1959) Responses to light by cave crayfishes. Occ. Pap. natn. speleol. Soc. 4, 3-15. WELSH J. H. (1934) The caudal photoreceptor and responses of the crayfish to light. J. cell. comp. Physiol. 4, 379-388. VAN HARaEVELDA. (1936) A physiological solution for fresh-water crustacea. Proc. Soc. exp. Biol. Med. 34, 428-432.