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Spectral transmission and short-wave absorbing pigments in the fish lens—I. Phylogenetic distribution and identity
0042-6989j93s6.00 + 0.00 Copyright CI 1993 Pergarmm Press Ltd
Vi&wa Res. Vol. 33, No. 3, pp. 289-300, 1993 Printed in %mt Britain. All rights reserved
Spectral Transmission and Short-Wave Absorbing Pigments in the Fish Lens-I. Phylogenetic Distribution and Identity A, THORPE,* Received
R. H. DQUGLAS,*t
R. J. W. TRUSCOTTI
22 May 1992
Fish lens transmission was found to vary depending on the type! and c~centration of short-wave absorbing compounds present within the lens. Pigments extracted from lenses of ten species were identified as mycosporine-like amino acids (mainly palythine, paiytbene and asterina-33$l_ s around 320-360 nm) which are also thought to be present in the majority of the 120 species examined here A mwel myycqm%Hik pigment with A,._% 385 nm was is&ted from the leas of the ffying fish, EXWXW~US okuuirosti& while lenses of several ck&y r&ted tropIca freshwater species were found to hare high concestratiuns of the tryptophan catabMte 34ydroxykyauremiae (A_ 370 nm). The type of lens pigment a species po~~sses and its concentration depends upcrnboth the animal’s phylogenetic group and its %ptical niche”. Fish
Lens Pigment rnycosporine 34ydroxykynurenine
Lens pigments in terrestrial vertebrates have been identified as tryptophan derivatives, such as 3-hydroxyLens pigments absorbing radiation in the regiun of kynurenine-0-B-glucoside found in the primate lens 300-400nm are commun in vertebrates (cooper $r. (van Heyningen, 1973a) and the N’acetyl derivative of Rubson, 1969a, b; Zigman, Paxhia & Waidron, 1985). 3-hydroxykynurenine in the lenses of the grey squirrel All animals possessing lenses with such short-wave (van Heyningen, 197330;Zigman & Paxhia, 1988). Lenses absorbing pigments inevitably suffer a loss in sensitivity of many fish also contain short-wave absorbing comand therefore tend to have a diurnal lifestyle (Walls & pounds (Heinermann, 1984, for review; Zigman, 1987; Judd, f933a, b>_It has been suggested that short-wave Douglas & McGuigitn, 1989) and although the specabsorbing lens pigments in surface dwelling fish serve to tral absorbance of the fish iens has been studied for increase the quality of the visual image (Muntz, 1972, decades (e.g+ Kennedy Br Milkman, 1956), the com1973) and may also protect the retina against U.V. pounds responsible for short-wave absorption have damage (Collier & Zigman, 1987). However, although been identified in only a few species. A smaI1 number the presence of fish lens pigments is commonly associ- of those fish lenses examined contain high concenated with high intensity radiation, pigments have also trations of tryptophan catabolites similar to those found been isolated from lenses of mesopekigic species in terrestrial vertebrates. Kynurenine, for example, has {McFall-Ngai, Crescitelli, Childress & Horwitz, 1986; been isolated from the lens of a single deep-sea species McFall-Ngai, Ding, Childress & Horwitz, 1988; Douglas (Thorpe, Tmscott & Douglas, 1992), and 3-hydroxy& Thorpe, 1992), which live in an environment where kynurenine is present in the lens of a gourami (Truscott, downwelling illumination is severely restricted in both Carver, Thorpe & Douglas, 1992). However‘, it is clear intensity arrd spectral composition (Jerlov, 1936). Lens that many tropical marine species have lenses containpigme& iti these deep-sea species are therefore unlikely ing mycosporine-like amino acids {Dunlap, Williams, to serve the same purpose as in surface-dwelling fish, and Chalker 8r Banaszak, 1989), which are quite unrelated other functions have been suggested, many relating to to tryptophan. I[n addition, mesopelagic fish have the detection of bioluminescence (Douglas & Thorpe, been shown to have a variety of largely uniden1992, for review). tidied short-wave absorbing compounds within the lens (Muntz, 1976a; McFall-Ngai eb ai_, 1988; Douglas & Thorpe, 1992). *Applied Vision Research Ckntre, Department of Optometry and Here we exzunine lenses from 120 species of both visual Science, City University, London ECIV 7DD, England. freshwater and marine fish, from temperate as well as ITo whom all correspondence should be addressed. tropical habitats, to determine how widespread shortfAustralian Cataract Research Fwndatian, University of Wollongong, New South W&s 2500, AustmEa. wave absorbing pigments are amung tefeosts and to INTRODUCTION
289
290
A. THORPE t’( id.
ascertain the distribution of the various classes of Lens pigments so far identified. MATERIALS AND METHODS Materials
Fish were obtained from several sources including the Plymouth Marine Laboratory, the Horniman Museum, the Scottish Marine Biological Association, cruises of R.R.S. Discovery and R.R.S. Calanus, and also local dealers. After initial identification (Wheeler, 1978; Nelson, 1984; Axelrod & Schultz, 1990), fish were killed by cervical transection followed by pithing. Eyes were enucleated and hemisected; on the rare occasions when lenses showed signs of opacity, they were discarded. Fish standard length and lens diameters were recorded. A number of frozen whole specimens were also obtained, these were allowed to thaw at room temperature before the lenses were treated exactly as for fresh animals. The effects of freezing on the whole lens transmission were found to be minimal over a 2 month period (Fig. 1). Lens transmission
Intact lens transmission (25&700 nm) was recorded within 10 min of death (or soon after thawing for frozen specimens), using a Shimadzu-UV240 spectrophotometer fitted with an integrating sphere, as detailed elsewhere (Douglas & McGuigan, 1989). Lenses were not placed in solution before or during scanning since pigments were found to leach readily when a lens was immersed in liquid. Absolute lens transmission values were measured for several species using the method of Bassi, Williams and Powers (1984). Readings were consistently 9699% for all lenses at 632.8 nm, agreeing well with initial recordings made on the spectrophotometer which displayed uniformly high transmission
100
-
50
-
s 6 ;ij
Pigment extraction
Lenses were thawed at room temperature and homogenized in l-2 ml distilled water. Samples were then centrifuged at 100,OOOg (4°C 30 min) and the supernatant further purified by ultrafiltration through YM2 (Amicon) membranes under nitrogen. Lens extracts were lyophilized prior to HPLC. HPLC
Lens pigment extracts were prepared for isocratic reversed-phase high performance liquid chromatography (HPLC) by redissolving the extract in 20% methanoi (HPLC grade). Samples were then injected onto a Whatman 10 GDS-3 ~mi-preparative column (250 x 9.4 mm i.d.) with a mobile phase of 20% methanol and 0.1% acetic acid as detailed by Dunlap et al. (1989), with a flow rate of 2 ml/min. Filtrate absorbance was recorded at 340 nm. Mycosporine-like amino acid standards extracted from tropical marine fish were supplied by Dr W. Dunlap from the Australian Institute of Marine Science. Pigment identification was carried out by injecting equal volumes of standard with the lens extract into the HPLC and monitoring any increase in absorbance at the retention time of the standard compounds. 3-Hydroxykynurenine was identified in a gourami (Truscott et al., 1992) and this sample was used to confirm, by HPLC, its presence in other lens extracts.
RESULTS
E z e l-
Lens spectral transmission and pigment absorbance
0 250
,
I
300
350
400
450
500
Wavelength tnml
FIGURE
above 500 nm. Spectra were therefore normalized to 100% at 700 nm for ease of comparison. After scanning, lenses were frozen for later pigment analysis Lens nucleus absorbance was also recorded for a number of fish from three species (Oreochromis niloticus, Merlangius merlangus and Carassius auratus) whose lenses contained palythene, After scanning, the whole lens was placed in 0.9% saline for 30 min after which the cortex coufd be easily slipped off the harder inner nucleus. The nucleus was then scanned in the spectrophotometer as for the whole lens. Using the nucleus and whole lens readings, the pigment concentration, based on the optical density at 360 nm (the I.,,,,, of palythene), in both the cortex and nucleus was calculated and expressed as absorbance units per mm pathlength.
I.
Spectral transmission of the right and left lenses of
Merlungius merlangus.
The
right
lens was scanned immediately after
death (solid line), whereas the left lens was scanned after the whole eye was frozen for 8 weeks (dashed line).
Intact lens transmission spectra showed large interspecies variability (Fig. 2). Of the 120 species examined 33 had spectra similar to that shown in Fig. 2(a), with a smooth, rapid fall in transmission at short wavelengths and 50% maximal transmission around 310-340 nm. No detectable amounts of pigment could be extracted from such lenses. Whole lens transmission spectra of the remaining 87 species displayed either an irregular decrease in transmission at short wavelengths [Fig. 2(b, c)] or a wavelength of 50%
291
PIGMENTS IN FKW LENSES-I
Xmax
Inm)
of
FKXJRE 3. Fr+!que~%cy histogram showing the distribution fish lens pigmenr &s_ Pigments were extracted from the lenses before measurzag spectral ~o~~~~~ W&a%?mi?re &an oz%eextractjon Was eati& values were averaged. Three major groups oflens o?It for a qXcksl A@@* pigment are repmsented, one in the region of 318-332 rim, one around 360 nm and one tit 370 nm.
r.M-7.33
6.04 3.69 5.45 4.50
I
6.&t
1
2.29
CM
31&w?
CM
335 334 325 3%
CM CM CM
CM
c
7
1.81~2.28
3%!-355
1 3
1.65 2.00-2.12
323 326-344
%
Tf:
-I-
324$360$ f
1
344
222 1.57 1.63-3.70 2.26 2.2X-2&3 I.81-4AM 1.12-4.82
315 326--X% 345 323-325 3twB2 318-391
4.60
370
CM
325, TX%
TF TF
292
A. THORPE TABLE
1 (continued)
Lens diameter
Lens 50% transmission
(mm)
(nm) 373 325
320
I
3.88 1.34
TF TF
I
1.11
318
_
TF
No. fish Serrasalmus nattereri Hyphessobrycon callisms
et al.
3
Pigment kIdi (nm)
Habitat*
Siluriformes Siluridae Kryptopterus bicirrhis
Salmoniformes Salmonidae Salmo gairdneri Osmeridae Osmerus eperlanus
16
3.22-6.20
322-334
_
CF
2
2.23-3.50
330-340
-
CM
7 6 4 33 8 9 8 1
5.01-8.60 4.67-9.00 5.75-8.31 3.12-6.90 5.36-10.13 3.32-5.80 3.37-5.84 2.39
398405 39245 394-396 383-397 396405 333-342 341-346 358
360 360 360 360 340,360 _ _
CM CM CM CM CM CM CM CM
5
2.53-7.55
32&341
_
CM
2
6.72-7.95
400
323,360
CM
1
3.31
391
330,360
TM
1.18-5.15 6.08-6.19 8.77
435442 390-392 340
385$ + _
CM CM CM
1.514.70
317-332
-
CM
1
0.81
315
_
TF
1
1.63
315
_
TF
Gadifonnes Gadidae Gadus morhua Pollachius virens Melanogrammus aeglejnus Merlangius merlangus Pollachius pollachius Trisoplerus minutus Trisopterus locus Ciliara muslela Merlucciidae Merluccius merluccius
Lophiiformes Lophiidae Lophius piscatorius Antennariidae Antennarius hispidus
Cyprinodontiformes Exocoetidae Exocoetus obtusirostris Hirundichthys a@nis Cypselurus cyanopterus Hemiramphidae Oxyporhampus micropterus Poeciliidae Poecilia reticulata
81 2
1 12
Atheriniformes Melanotaeniidae Melanotaenia parkinsoni
Gasterosteiformes Gasterosteidae Spinachia spinachia
5
1.39-1.56
320-321
_
CM
1 11 1 1
1.65 1.00-1.56 1.70 1.00
428 418423 421 412
+ 360 363 +
CM CM CM TM
1
2.41
+
CM
Dactylopteriformes Syngnathidae Syngnathus acus Syngnathus typhle Hippocampus ramulosus Hippocampus hudsonius
Scorpaeniformes Agonidae Agonus cataphractus Cottidae Taurulus lilljeborgi Taurulus bubalis Triglidae Trigla lucerna Aspitrigla cuculus Scorpaenidae Pierois volitans
395
3 4
4.5Wt.78
1.42-3.07
402408 393402
+ +
CM CM
I 8
3.05-5.76 2.82-5.44
402408 395404
328,360 325,360
CM CM
6
6.76-1.47
376401
322,360
TM
2.554.20 2.22
400404 398
325,360 +
CF CF
3.514.06
377-380
331
TM
Perciformes Centrarchidae Lepomis gibbossus Lepomis cyanellus Chaetodontidae Chaetodon spp
[continued opposite
293
PIGMENTS IN FISH LENSES-I TABLE 1 (continued)
Chaetodon frembli Chaetodon melanotus
Lens diameter (mm)
Lens 50% transmission (nm)
Pigment
No. fish
&u (am)
Habitat*
1 1
2.94 5.75
374 404
330 332
TM TM
1
3.35 0.866.81 0.98-3.08 1.43-3.34 1.94-2.03 1.50-3.57 2.01-2.09 1.41-3.41 3.01 3.10 2.88-3.69 1.10-1.34 1.50-2.38 1.82 2.82 2.58 1.94 2.745.51 1.75-2.08 1.65-1.86 1.58 0.85-2.06
338 339402 323-390 315-328 344360 327-394 325-339 335-390 358 392 381-394 348-355 371-385 351 348 400 417 425432 43e432 432437 440
+ 324,360 320,358 _
t
+ + + 323,360 + + 320,360 + + 320 321 + 325,360 324,360 327,362 370 370 370
TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF
1
3.93
407
326
TM
2 2 1 1
1.69-2.32 1.45-1.72 1.27 3.49
321 318-320 315 430
361
CM CM CM CM
1.663.31
364410
329,360
TF
1
2.63
430
+
TF
13 10 2 2
1.S2.55 1.01-1.54
1.09 2JW2.30
446-452 43943 435 385-392
370 370 370 327,360
TF TF TF TF
1
2.87
398
326,360
TF
1 8 5
3.73 2.18-3.98 2.70-3.70
404 378-397 388-410
331 + 330
TM TM TM
2
2.784.87
32&333
-
CM
1 1 1
4.12 4.27 2.51
402 406 395
331 330,360 327
TM TM TM
4
1.89-2.55
35&356
+
TM
1 2
1.48 2.43-2.49
407 397400
+ +
CM CM
14
1.54-3.70
372-386
321,359
CM
23
1.72-5.45
395402
327,359
CM
+ +
CM CM
Cichlidae Tilapia mariae Oreochromis niloticus Haplochromis tshmaeli Haplochromis argens Haplochromis piceatus Haplochromis sauvagei Haplochromis pyrrocephalus Haplochromis xenognathus Haplochromis nyererei Haplochromis “velvet black” Macropleurodus bicolor Astatorheochromis alluaudi Platytaeniodus degeni Lamprologus dahlia Lamprologus tetrocephalus Crenicichla lepidota Cichlasoma sevrum Astronotus ocellatus Herotilapia multispinosa Apistogramma curviceps Aequidens maronii Aequidens p&her
66 16
11 2 5 2 4 1 1 3 4 3 1 1 1 2 4 5 11 2 100
Plesiopidae Calloplesiops altevelis
Labridae Labrus bergylta Crenilabrus melops Ctenolabrus rupestris Thalassoma spp
Helostomatidae Helostoma temmincki
20
Anabantidae Ctenopoma oxyrhynchus
Belontiidae Trichogaster microlepis Trichogaster trichopterus Betta splen&ns Colisa fasciata
Osphronemidae Osphronemius goramy
Acanthuridae Acanthurus spp Zebrasoma Javescens Paracanthurus hepatus
Mullidae Mullus surmuletus Pomacanthidae Pygoplites diacanthus Holacanthus ciliaris
Centropyge heraldi Pomacentridae Amphiprion spp Bleniidae Parablennius gattorugine Liophrys pholis
Callionymiidae Callionymur lyra
Carangidae Trachus trachurus
Gobiidae Thorogobius ephippicutus Pomatoschistus microps
1 1
1.61 2.48
396 389
[continued overleaf
294
A. THORPE et ul. TABLE 1 (continued) No. fish
Lens diameter (mm)
Lens 50% transmission (nm)
Pigment %lill (nm)
Habitat*
Dicentrarchus labrax
4
2.64-3.99
403-406
+
CM
Scombridae Scomber seombrus Stichaeidae
6
4.88fi.69
360-402
325,360
CM
3
2.46-2.90
368-376
+
CM
5 18 6 1 4
2.87-5.61 0.73-5.75 1.86-4.67 3.79 2.26-5.42
396-410 399-408 399-407 405 333-389
323,360 331,360 328,360 + +
CM CM CM CM CM
2 2 2
2.21-2.67 l.&-1.64 3.02-3.41
324-325 320-324 328-332
-
CM
1
4.52
407
+
TM
1
7.90
301
321,360
TF
2
2.50-2.80
370-390
326
TM
Percichthyidae
Lumpenus lampretaeformis Pleuronectiformes
Pleuronectidae Platichthys flesus Pleuronectes platessa Li~nda ~imanda Microstomus kitt Hippoglossoides platessoides
Soleidae Microchirus variegatus Buglosidium luteum Solea solea
CM
CM
Te~a~oDti~o~~ Tetradontidae Arothron hipsidus
Diodontidae Diodon lystrix
Balistidae Cantherhines pufhis
*TM, Tropical marine; TF tropical freshwater; CM, cold marine;CF, cold freshwater. + , Pigment present but not extracted; -, no detectable pigment. tSpectra1 transmission not recorded. iPigment extract I,.. varies with age. (See Thorpe & Douglas, 1992 for details.) Species were classified after Nelson~(1984).
transmission above 400 nm [Fig. 2(d, e)]. All such lenses that were analysed contained short-wave absorbing pigments (Table 1). Lens pigments were extracted from 56 species and most fell into three main groups based on their wavelengths of maximum absorbance (AmaXs 320-330,360 and 370 nm) (Fig. 3). The lens of a single species (~xoc~e~~~ obtusirostris) contained a pigment with Amax 385 nm and another (Pollachius pollachius) contained a pigment absorbing maximally at 339 nm. The lenses of many species contained only one major lens pigment, but several had both 360 and 320-33Onm ,I,, pigments within the same lens (Table 1). Fish with spectral transmission as in Fig. 2(c), with a secondary peak around 320 nm, contained only ,I,,,,, 360nm pigment, whereas lenses with transmission as in Fig. 2(b) contained both 320-330 and 360 nm i,, pigments. A pigment with A,,, 370 nm isolated from lenses of several tropical freshwater species (Table 1) caused a secondary peak in the lens transmission at 310-320nm and 50% transmission wavelengths around 445 nm [Fig. 2(e)]. High concentrations of either 360 nm pigment alone, or high concentrations of both 320-330 and 360 nm pigments, resulted in smooth ~ansmission spectra as in Fig. 2(d).
three freshwater species (Cyprin~ carpio, 0. niluticus, Lepomis gibbosus) were found to contain predominantly palythene and palythine within their lens [e.g. Fig. 4(a, b)]. Whereas three tropical marine species (Chaetodon spp, Paracanthurus hepatus, Pygoplites diac~thus) only had significant concentrations of asterina330 within the lens [e.g. Fig. 4(c)]. One species of flying fish, E. obtusirostris, contained a Amax385 nm pigment which had a longer retention time than palythene, the most polar mycosporine compound previously examined [Fig. S(a)). However, this compound proved to be unstable in solution and degraded into three new compounds, one of which was palythine [Fig. V-41. Four mycosporine compounds were therefore found at high concentrations in fish lenses, each of which had a specific short-wave absorbance in the region of 3~~ nm (Fig. 6). Two tropical species, Aequidens p&her and Trichogaster trichopterus, had a ,I,,, 370 nm pigment within their lenses which did not co-elute with any of the mycosporine standards in HPLC (Fig. 7). This compound had three distinct absorbance peaks in the U.V. (Fig. 8), and was identified by NMR in T. trichopte~s as 3-hydroxykynurenine (Truscott et al., 1992).
Pigment identljication
Pigment distribution within the lens
Lens pigments from I4 species were identified by HPLC methods. Four marine species (~yngnath~ typhle, C&pea harengus, P. pollachius, Aspitrigla cuculus) and
Absorbance at 360 nm per mm pathlength in the nucleus and cortex from lenses of two species, 0. n~Iotic~ and M. merla~gus consistently showed
I 0
I
I
0 200
256
300
3SQ
400
450
A. THORPE rt t/l.
i I \
q
1’ \
LJ \ \ \ \ \
----A
I 200
I
I
I
I
0
5
IO
15
Time (man)
FIGURE 7. HPLC elution profile of the ,&,,, 370 nm pigment from the lens of A. p&her. The major peak eluted at 7.7 min. HPLC conditions as in Fig. 3.
higher absorbance in the cortex than in the nucleus (Table 2). However, the absorbances per mm for the nucleus and cortex of C. auratus were not significantly different (Table 2). DISCUSSION
Biochemical identity of lens pigments
Two distinct classes of pigment were isolated: mycosporine compounds with short-wave absorbance maxima at 320, 330, 360 or 385 nm (Fig. 6) and 3_hydroxykynurenine, a tryptophan derivative with 1max 370 nm (Fig. 8). Palythene and palythine were identified as the major pigments in S. typhle, C. harengus, P. pollachius, A. cuculus, C. carpio, 0. niloticus and L. gibbosus, while lenses of Chaetodon spp, P. hepatus and P. diacanthus were found to contain appreciable amounts of only asterina-330. The flying fish (E. obtusirostris) lens pigment, although unidentified, is also a mycosporine analogue. The tryptophan metabolite 3-hydroxykynurenine was identified in the lenses of A. pulcher and T. trichopterus. Even though the positive identification of lens pigments was carried out for relatively few species, the compounds present in lenses of other species could be
250
300
350
Wovelength
assessed from the spectral absorbance of their extracts. Lenses with only Amax360 nm pigment (several gadidae species and a single labridae species, see Table 1) could, based on the A,,,,, of the crude extract, be either palythene [Fig. 6(d)] or kynurenine, a tryptophan derivative which also absorbs at 360 nm (Thorpe et al., 1992). However, since these extracts had absorbance spectra with only a single peak at 360 nm, rather than the three peaks characteristic of tryptophan-related compounds (e.g. Fig. 8), the lenses probably contain palythene. In support of this, van Heyningen and Linklater (1976) similarly concluded that the lens pigments of several gadidae species were not tryptophan-related since the lenses did not metabolise tryptophan, and Dunlap et al. (1989) also found high concentrations of palythene in the lenses of labridae species. On the other hand, it is likely that all I,, 370 nm pigments (i.e. from lenses of three belontidae species and several cichlidae species, Table 1) are 3_hydroxykynurenine, as identified in T. trichopterus (Truscott et al., 1992) since all had complex short-wave absorbance spectra as in Fig. 8. Pigments absorbing in the 320-330 nm range presented a similar problem; these compounds could be either tryptophan metabolites (W-formylkynurenine absorbs maximally at 320 nm) or the mycosporine
No.
Nucleus (abs/mm) (SD)
Cortex (absimm) (SD)
P*
Oreochromis niloticus
3.094.11
8
0.19 (0.06)
0.52 (0.28)
0.001
Merlangius merlangus
3.894.86
5
2.61-3.92
12
0.31 (0.20) 0.09 (0.05)
0.1
Carassius auratus
0.16 (0.05) ‘0.13’ (0.07)
*Student’s r-test.
450
FIGURE 8. Spectral absorbance of the HPLC purified extract from the lens of A. p&her with imaxs around 230, 262 and 370 nm (solid line). The compound had a HPLC retention time and absorbance spectra identical to the lens pigment from T. trichopterus (dashed line), identified as 3-hydroxykynurenine (Truscott et al., 1992).
TABLE 2. Absorbance. at 360 nm per mm, for lens cortex and nucleus of three fish species
Lens dia
400
(nm)
NS
PIGMENTS
IN FISH LENSES-I
291
Pigment distributionwithin the lens compounds palythine, asterina-330 and palythinol, with maximal absorbance at 320, 330 and 331 nm Mycos~~ne related pigments and tryptophan derivarespectively. A~thou~ it has been suggested that N’- tives are all low molecular weight compounds. Both formylkynurenine is responsible for the absorbance classes of pigment are also easily extracted into water, of short-wave radiation in the lenses of some fish suggesting that they are not tightly bound to any large (Zigman, 1987), this compound has never been posi- protein moiety within the lens and are free to diffuse tively identified, and all pigments identified here throughout the lens nucleus and cortex. The data absorbing maximahy around 320-331 nm were mycoreported in Table 2 reveal that pigments are present in sporine compounds, mainly palythine and asterina-330. both nucleus and cortex of the lens, although the It is therefore thought that the majority of pigments concentration in each varies between species. Differabsorbing around 320-330 nm listed in Table 1 are ences in pigment concentration in the two lens parts have mycosporine-like amino acids. been observed in other species; Argyropelecus @inis, for Although mycosporine pigments have been pre- example, has a higher pigment concentration in the viously identified in lenses of tropical marine species cortex than the nucleus, causing the cortex to appear (Dunlap et al., 1989), they have not until now been visibly yellow whilst the nucleus appears colourless to identified in lenses of more temperate marine species human observers (McFall-Ngai et al., 1986; Yu, Cai, or freshwater fish. Mycosporine-like compounds are Lee, Kuck, McFall-Ngai & Horwitz, 1991; Douglas found in a range of tropical invertebrates (Nakamura, and Thorpe, 1992). Lens pigments in Argyropelecus are Kobayashi & Hirata, 1982) and the fact that many bound to lens ot-crystdlin and cannot diffuse freely temperate f&h have been found to have high con- throughout the lens. The reverse situation is observed centrations of mycosporine analogues in their eggs in Mulucosteus niger, another mesopelagic species, (Chioccara, Della Gala, De Rosa, Novellino & which has a yellow pigmented core within the nucleus Prota, 1980; Fraser, Grant, Middleton, Mitchell & (Douglas & Thorpe, 1992). The differential pigment Thomson, 1981) suggests that there is a readily avail- concentrations in the nucleus and cortex of the three able supply of mycosporine precursors in more species examined here suggests that their pigments may temperate waters. also be loosely associated with lens protein, as reported The lens pigment in one species of flying fish for a number of epipelagic species (Bon, Ruttenberg, (E. obtusirostris) appeared to have a longer A,, Dohrn & Batink, 1968). The fact that the pigments in (385 nm) than other mycosporine pigments (Fig. 7). two of these species appear to predominate in the Iens Since one of the degradation products of this pig- cortex suggests that they may be laid down later in ment was identical to palythine (,& 32Onm), the fly- hfe as a function of the animal’s age (see Thorpe 8t ing fish lens pigment is likely to be a more complex Douglas, 1993) or that the cortex is the site of pigment mycosporine analogue. The other two degradation accumulation, products also had I,,, s around 320 nm. Since the original pigment was so unstable, it was not possible to Phy~oge~et~cd~tr~b~t~onof lens pigments establish its identity by NMR or mass spectroscopy. Fish with short-wave absorbing patents in the ft is probable that t~ptophan-related pigments are of synthesized within the fish lens from free tryptophan, as Iens were represented in around three-quarters is the case in the human lens (van Heyningen, 1973b). the 49 families examined (Fig. 9), supporting previous evidence that the presence of short-wave absorbOther primates and squirrels also have lens pigments ing pigments in both freshwater and marine fish lenses derived from tryptophan catabolites in the same pathway (van Heyningen, 1973a, b; Zigman & Paxhia, 1988). is widespread (Muntz, 1973, 1976a, b; Dougias & 1989; Dunlap et al., 1989). Pigmented That a rneso~~g~c species (Thorpe et al., 1992) and a Morgan, number of freshwater species should have evolved lens lenses have primarily been described in fish which pigments closely related to those of higher terrestrial belong to the perciforme order or perciforme derived vertebrates is quite surprising. Specific enzymes are species (Muntz, 1973; Douglas & McGuigan, 1989), required for the reactions in this pathway and it could with the marked exception of the pigmented lenses be that they are only active in some species, possibly found in mesopelagic fish which belong to a range explaining why kynurenine compounds are not more of famihes (Muntz, f976a; Douglas & Thorpe, 1992). commonly found. The Syngnathidae (Pipefishes) examined here however, Two tryptophan-related compounds and four major are neither perciformes nor mesopelagic, yet they mycosporine compounds, along with a host of unidentoo have lenses with 50% transmission wavelengths of tified pigments from mesopelagic species (McFafl-Ngai up to 422 nm (Table l)? due to high concentrations et al., 1988; Douglas & Thorpe, 19921, have now been of pafythene in the lens. Lenses not appearing yellow; isolated from fish lenses. Lens pigmentation in fish but nevertheless having quite high concentrations of therefore seems to represent a good example of evol- short-wave absorbing compounds were also found in utionary convergence: a number of biochemically dis- several other non-perciforme families (e.g. gadidae and tinct compounds absorbing radiation in the same region pleuronectidae, Table 1). Perciforme species, however, seem to have evolved in fish lenses independently on a do show the greatest variety of lens pigments (Fig. 91, number of occasions. withy at feast six different types of pigment isolated, YR333-F-B
298
A. THORPE et al. ORDER
NUMBER OF SPECIES
ELASMOBRANCHII
E I
ANGUILUFORMES OSTEOGLOSSIFORMES CLUPEIFORMES _I
l- CYPRINIFORMES
I LA-
GAOIFORMES
CYPRlNODONTlFGRMES ATHERiNlFORMES LAMPRIFORMES GASTEROSlElFORMES DACTYLOPTERlFORMES SCORPAENIFORMES PERCIFORMES
PLEURONECllFORMES KTRAOGONllFORMES
KEY:
0
NO PIGMENT
m
360
m
320-330
I
320-350
PIGMENT
PIGMENT de 360
PlGMENTS
IIIM
370
PIGMENT
m
UNIDENTlflED PIGMENT
FIGURE 9. Phylogenetic distribution of lens pigments in fish. Pigments with similar near U.V.J._s have been grouped together for simplicity, though this does not necessarily mean that they are identica1 compounds.
probably reflecting the genetic diversity of this largest teleost order. Closely related species often have the same lens pigments For example, lenses of four members of the gadidae family: cod, saithe, whiting and haddock all have a &.,,_360 nm pigment (Table l), which is likely to be palythene. Other authors contirm the presence of a I,, 360 nm pigment in the lenses of several other species in this family (Bon et al., 1968; van Heyningen & Linklater, 1976). Few lenses had I,,, 370 nm pigment, but three belontidae species contained this compound (likely to be 3-hydroxykynurenine) and it was also found in three tropical freshwater cichlidae species (Table 1). Most cyprinids have lenses with no pigmentation (Fig. 9; Table l), and even those cyprinid species sometimes accumulating pigments (goldfish and carp), never have very high concentrations. Similarly, neither rajidae nor scyliorhynidae species ever show any evidence of lens pigmentation (Table 1, Fig. 9). Although the pigment composition within a family is often the same, concentrations can vary, with most whiting lenses for example having considerably lower pigment concentrations than lenses of both cod and haddock. This is also demonstrated by the large range in 50% transmission wavelengths of the cichlids (Table l), though many appear to contain both palythine and palythene pigments. Since the genetic make-up of congeneric species is likely to be quite similar, it may be that other factors, such as the animal’s environment, are
important for the determination levels in the lens.
of specific pigment
Ecological relationships and lens pigment function
Although phylogeny is important, the environment also appears to have a role to play in lens pigmentation, both in terms of adaptation to that environment and a direct effect of the environment on the lens. Many of the fish with pigmented lenses listed in Table 1 have a diurnal lifestyle, a factor often associated with highly pigmented lenses (Mu&, 1973). It has been suggested that some fish lens pigments may accumulate as a result of exposure to high levels of U.V. (Zigman & Gilbert, 1978). In support of this, although pleuronectidae and soleidae families are closely related and share the same environment, lenses of soleidae, which are nocturnal (Wheeler, 1978), have no pigment, whereas all pleuronectids which are generally diurnal, have pigmented lenses (Table 1). However, whilst it is true that many of the highly diurnal species, such as those living on coral reefs (e.g. pomacanthidae and pomacentridae familiesTable l), often do have highly pigmented lenses, it is difficult to believe in a direct role of sunlight when several fish occupying the same ecological niche can have quite different lens transmission characteristics. For example, although two species of flying fish (E. obtusirostris and Hirundichthys afinis) had high lens pigment concentrations (Table l), another species (Cypselurus cyanopterus) within the same family and
PIGMENTS p~s~&ly
sharing
&e
me
en@rom&
had
IN FISH LENSES-I
none
(Table 1). It has also proved impossible to induce fish lens pigment formation or affect pie-existing pigments by altering the lighting environment alone (Thorpe 8z Douglas, ~publish~ results). Various functions have been ascribed to lens pigments, including the protection of the retina from U.V. (Collier, Waldron & Zigman, 1989); increasing visual acuity by removing the wavelengths responsible for scatter, glare and chromatic aberration (Muntz, 1973, 1976b; Zigman & Gilbert, 1978); stabilizing lens protein (MeFall-Ngai et al., 1986) and as an aid in prey detection in mesopelagic species (Muntz, 197% Somiya, 1982; Douglas & Thorpe, 1992). Lens pigments in higher vertebrates have been shown to protect the retina from U.V. damage (Collier & Zigman, 1987; Collier et ai., 1989), and the fact that short-wave absorbing compounds have been identified in many highly diurnal species (Table 1; Douglas & McGuigan, 1989; Dunlap et al., 1989) also supports a role in U.V. protection in fish. However, fish living in very high light environments may also benefit from improvements in visual acuity offered by lens pigments, since effects of scatter, glare and chromatic aberration are more intense at short wavelengths (Muntz, 1972). It is therefore unlikely that all fish lens pigments perform the same function; a number of different lens pigments have evolved in both deep-sea and surface dwelling fish, presumably to serve a range of requirements.
REFERENCES Axelrcxl, H. R. & Schultz, L. P. (1990). ~~~~ of tropical ark Jishes (revised edn). New Jersey: TFH Publications.
Bassi, C. J., Williams, R. C. & Powers, M. K. (1984). Light transmittance by goldfish eyes of different sixes. Vision Research, 24, 1415-1419. Bon, W. F., Ruttenberg, G., Dobrn, A. & Batink, H. (1968). Comparative physicochemical investigations on the lens proteins of fishes. Ex~r~ntaI
Eye Research,
7, 603-610.
Chioccara, F., Della Gala, A., De Rosa, M., Novellino, E. & Prota, G. (1980). Mycosporine amino acids and related compounds from the eggs of fishes. Bulletin. So&W chimique air Belgique, 89, 1101-I 107. Collier, R. & Zigman, S. (1987). The gray squirrel lens protects the retina from near-W radiation damage. In Hollyfreid J. (Ed.), Degenerative retinai disorders. Clinicai and laboratory ~estigatio~
(pp. 571-585). New York: Liss. Collier, R. J., Waldron, W. R. & Zigman, S. (1989). Temporal sequence of changes to the gray squirrel retina after near-W exposure. Investigative Ophthalmology and Visual Science, 30, 631637.
Cooper, G. F. 8t Robson, J. G. (1%9a). The yellow colour of the lens of the 8rey squirrel (Sciurus c~oline~~ leucolir). Journal of Physiology, 203, 403410.
Cooper, G. F. & Robson, J. G. (1969b). The yellow colour of the lens of man and other primates. Journal of Physiology, 203, 41 l-417. Douglas, R. H. & McGuigan, C. M. (1989). The spectral transmission of freshwater teleost ocular media--an interspecific comparison and guide to potential ultraviolet sensitivity. Vision Research, 29, 871X79.
Douglas, R. H. t Thorpe, A. (1992). Short-wave absorbing pigments in the ocular lenses of deep-sea teleosts. Journal of the Marine Biological Association of the United Kingdom, 72, 93-l 12.
299
Dunlap, W. C+, Wiams, D. M., Chalker+ B. E. & Bauasxak, A. T. (1989). Biochemical photoadaptation in vision: UV-absorbing pigments in ilsh eye tissues. Comparative Biochemistry and Physiology, 93B, 601-607. Reinezrnann,P. H. (1984). Yellow iutraocular filters in fishes. ExperimentalBiology, 43, 127-147. van Heyningen, R. (1973a). Assay of fluorescent glucosides in the human lens. ExpdmentOrEye Research, IS, 121-126. van Heyningen, R. (1973b). The glucoside of 3-hydroxykynurenine and other fluorescent compounds in the htmmn lens. In The human lens in relation to cataract. Ciba Foundation symposium lP(pp. 151-171). Amsterdam: Elsevier. van Heyningen, R. & Linklater, J. (1976). Serine and threoniue ethanoi~ne phosphate diesters, and some other unusual compounds in the lens of the cod fish (Gad& morhua) and haddock (Gadus aegIe$nus). Experimental Eye Research, 23, 29-34. N. G. (1976). Marine optics. Amsterdam: Elsevier.
J er 1ov,
Kennedy, D. & Milkman, R. D. (1956). Selective light absorption by the lenses of lower vertebrates, and its influence on spectral ~n~ti~ty. Biology 3uIIet~, lil, 375-386. MeFall-Ngai, M., Crescitelli, F., Childress, J. & Horwitx, J. (1986). Patterns of pigmentation in the eye lens of the deep-sea hatchet&h Argyropelecus a$inis, Garman. Journal of Comparative Physiology A, 159, 791-800.
McFall-Ngai, M., Ding, L., Childress, J. & Horwitx, J. (1988). Biochemical characteristics of the pigmentation of mesopelagic tish lenses. Biofogy B~Iet~, f75, 397-402. Muntx, W. R. A. (1972). Inert absorbing and refhzting pigments. In Dartnall, H. J. A. (Ed.), Photochemistry of vision (IIanabooh of Sensory Physiology, Vol. VIIf 1) (pp. 529-565). Berlin: Springer. Muntx, W. R. A. (1973). Yellow filters and the absorption of light by the visual pigments of some Amazonian fishes. Vision Research, 13, 2235-2254.
Muntz, W. R. A. (1976a). On yellow lenses on mesopeIagic animals. Journal of the Marine Biologicaf Association of the United Kingdom, 56, 963-976,
Muntx, W. R. A. (1976b). The visual consequences of yellow filtering pigments in the eyes of fishes occupying different habitats. In Evans, G. C., Bainbridge R. & Rackham, 0. (Eds), Light as an ecologicaf factor ZZ(pp. 271-285). New York Halstead Press. Nakamura, H., Kobayashi, J. L Hirata, Y. (1982). Separation of mycosporine-like amino acids in marine organisms using reversed-phase high-performance liquid chromatography. Journal of Chromatography, 250, 113-I 18. Nelson, J. S. (1984). Fishes of the world (2nd edn). New York: Wiley. Plack, P. A., Fraser, N. W., Grant, P. T., Middleton, C., Mitchell, A. I. & Thomson, R. H. (1981) Gadusol, an enolic derivative of cyclohexan~l,3~ione present in the roes of cod and other marine fish. Biochemistry Journal, 199, 741-747. Somiya, H. (1982). ‘Yellow lens’ eyes of a stomiatoid deep-sea fish Malacosteus niger. Proceedings of the Royal Society of London B, 215, 481-489. Thorpe, A. & Douglas, R. H. (1993). Spectral transmission and
short-wave absorbing pigments in the fish lens-II. Effects of age. Vision Research, 33, 301-307. Thorpe, A., Truscott, R. 3. W. dt Douglas, R. H. (1992). Kynurenine identified as the short-wave absorbing lens pigment in the deep-sea fish Stylephorus chordatus. Experimental Eye Research, 5.5, 53-57. Truscott, R. J. W., Carver, J. A., Thorpe, A. & Douglas, R. H. (1992). The identification of 3-hydroxykyuurenine as the lens pigment in the gourami Trichogaster trichopterus. Ex~r~tal Eye Research, 54, 1015-1017.
Walls, G. L. BEJudd, H. D. (1933a). The intraocular colour filters of vertebrates. Britirh Journal of Ophthalmology, 14, 641675. Walls, G. L. & Judd, H. D. (1933b). The intraocular colour filters of vertebrates. British Joumai of Ophthalmology, 14, 705-725. Wheeler, A. (1978). Key to the Jishes of northern Europe. London: Frederick Wame. Yu, N.-T., cai, M.-Z., Lee, B.-S., I&k, J. F. R., McFa~-Ngai, M. Bt Horwitx, J. (1991). Resonance raman detection of a carotenoid in the lens of the deep-sea hatchetfish. Experimental Eye Research, 52, 415479.
300
A. THORPE et al.
Zigman, S. (1987). Tryptophan and ocular lens pigments. In Bender, D. A., Joseph, M. H. & Steinhart, H. (Eds), Progress in tryptophan and serotonin research 1986 (pp. 399403). Berlin: Gruyter & Co. Zigman, S. & Gilbert, P. W. (1978). Lens color in sharks. Experimental Eye Research, 12, 155-157.
Zigman, S. & Paxhia, T. (1988). The nature and properties of squirrel lens yellow pigment. Experimental Eye Research, 47, 819-824. Zigman, S., Paxhia, T. & Waldron, W. (1985). Biochemical features of the grey squire1 lens. Investigative Ophthalmology and Visual Science, 26, 1075-1082.
Acknowledgements-The authors would like to thank Dr G. McReid from the Homiman Museum, The Plymouth Marine Laboratory, Dr L. Ross, Professor J. Wagner and Professor J. Blaxter for generous supplies of fish; the masters and crews of R.R.S. Discovery and R.R.S. Calanus; Dr W. Dunlap of A.I.M.S. for mycosporine standards and herring lens pigment analysis; Dr J. Davenport for his help in the collection and identification of the flying fish; and Dr P. Herring for enabling RHD to take part in cruise 195 of R.R.S. Discovery. AT was supported whilst in Australia by a grant from the Department of Industry, Technology and Commerce (Australia).
Vi&wa Res. Vol. 33, No. 3, pp. 289-300, 1993 Printed in %mt Britain. All rights reserved
Spectral Transmission and Short-Wave Absorbing Pigments in the Fish Lens-I. Phylogenetic Distribution and Identity A, THORPE,* Received
R. H. DQUGLAS,*t
R. J. W. TRUSCOTTI
22 May 1992
Fish lens transmission was found to vary depending on the type! and c~centration of short-wave absorbing compounds present within the lens. Pigments extracted from lenses of ten species were identified as mycosporine-like amino acids (mainly palythine, paiytbene and asterina-33$l_ s around 320-360 nm) which are also thought to be present in the majority of the 120 species examined here A mwel myycqm%Hik pigment with A,._% 385 nm was is&ted from the leas of the ffying fish, EXWXW~US okuuirosti& while lenses of several ck&y r&ted tropIca freshwater species were found to hare high concestratiuns of the tryptophan catabMte 34ydroxykyauremiae (A_ 370 nm). The type of lens pigment a species po~~sses and its concentration depends upcrnboth the animal’s phylogenetic group and its %ptical niche”. Fish
Lens Pigment rnycosporine 34ydroxykynurenine
Lens pigments in terrestrial vertebrates have been identified as tryptophan derivatives, such as 3-hydroxyLens pigments absorbing radiation in the regiun of kynurenine-0-B-glucoside found in the primate lens 300-400nm are commun in vertebrates (cooper $r. (van Heyningen, 1973a) and the N’acetyl derivative of Rubson, 1969a, b; Zigman, Paxhia & Waidron, 1985). 3-hydroxykynurenine in the lenses of the grey squirrel All animals possessing lenses with such short-wave (van Heyningen, 197330;Zigman & Paxhia, 1988). Lenses absorbing pigments inevitably suffer a loss in sensitivity of many fish also contain short-wave absorbing comand therefore tend to have a diurnal lifestyle (Walls & pounds (Heinermann, 1984, for review; Zigman, 1987; Judd, f933a, b>_It has been suggested that short-wave Douglas & McGuigitn, 1989) and although the specabsorbing lens pigments in surface dwelling fish serve to tral absorbance of the fish iens has been studied for increase the quality of the visual image (Muntz, 1972, decades (e.g+ Kennedy Br Milkman, 1956), the com1973) and may also protect the retina against U.V. pounds responsible for short-wave absorption have damage (Collier & Zigman, 1987). However, although been identified in only a few species. A smaI1 number the presence of fish lens pigments is commonly associ- of those fish lenses examined contain high concenated with high intensity radiation, pigments have also trations of tryptophan catabolites similar to those found been isolated from lenses of mesopekigic species in terrestrial vertebrates. Kynurenine, for example, has {McFall-Ngai, Crescitelli, Childress & Horwitz, 1986; been isolated from the lens of a single deep-sea species McFall-Ngai, Ding, Childress & Horwitz, 1988; Douglas (Thorpe, Tmscott & Douglas, 1992), and 3-hydroxy& Thorpe, 1992), which live in an environment where kynurenine is present in the lens of a gourami (Truscott, downwelling illumination is severely restricted in both Carver, Thorpe & Douglas, 1992). However‘, it is clear intensity arrd spectral composition (Jerlov, 1936). Lens that many tropical marine species have lenses containpigme& iti these deep-sea species are therefore unlikely ing mycosporine-like amino acids {Dunlap, Williams, to serve the same purpose as in surface-dwelling fish, and Chalker 8r Banaszak, 1989), which are quite unrelated other functions have been suggested, many relating to to tryptophan. I[n addition, mesopelagic fish have the detection of bioluminescence (Douglas & Thorpe, been shown to have a variety of largely uniden1992, for review). tidied short-wave absorbing compounds within the lens (Muntz, 1976a; McFall-Ngai eb ai_, 1988; Douglas & Thorpe, 1992). *Applied Vision Research Ckntre, Department of Optometry and Here we exzunine lenses from 120 species of both visual Science, City University, London ECIV 7DD, England. freshwater and marine fish, from temperate as well as ITo whom all correspondence should be addressed. tropical habitats, to determine how widespread shortfAustralian Cataract Research Fwndatian, University of Wollongong, New South W&s 2500, AustmEa. wave absorbing pigments are amung tefeosts and to INTRODUCTION
289
290
A. THORPE t’( id.
ascertain the distribution of the various classes of Lens pigments so far identified. MATERIALS AND METHODS Materials
Fish were obtained from several sources including the Plymouth Marine Laboratory, the Horniman Museum, the Scottish Marine Biological Association, cruises of R.R.S. Discovery and R.R.S. Calanus, and also local dealers. After initial identification (Wheeler, 1978; Nelson, 1984; Axelrod & Schultz, 1990), fish were killed by cervical transection followed by pithing. Eyes were enucleated and hemisected; on the rare occasions when lenses showed signs of opacity, they were discarded. Fish standard length and lens diameters were recorded. A number of frozen whole specimens were also obtained, these were allowed to thaw at room temperature before the lenses were treated exactly as for fresh animals. The effects of freezing on the whole lens transmission were found to be minimal over a 2 month period (Fig. 1). Lens transmission
Intact lens transmission (25&700 nm) was recorded within 10 min of death (or soon after thawing for frozen specimens), using a Shimadzu-UV240 spectrophotometer fitted with an integrating sphere, as detailed elsewhere (Douglas & McGuigan, 1989). Lenses were not placed in solution before or during scanning since pigments were found to leach readily when a lens was immersed in liquid. Absolute lens transmission values were measured for several species using the method of Bassi, Williams and Powers (1984). Readings were consistently 9699% for all lenses at 632.8 nm, agreeing well with initial recordings made on the spectrophotometer which displayed uniformly high transmission
100
-
50
-
s 6 ;ij
Pigment extraction
Lenses were thawed at room temperature and homogenized in l-2 ml distilled water. Samples were then centrifuged at 100,OOOg (4°C 30 min) and the supernatant further purified by ultrafiltration through YM2 (Amicon) membranes under nitrogen. Lens extracts were lyophilized prior to HPLC. HPLC
Lens pigment extracts were prepared for isocratic reversed-phase high performance liquid chromatography (HPLC) by redissolving the extract in 20% methanoi (HPLC grade). Samples were then injected onto a Whatman 10 GDS-3 ~mi-preparative column (250 x 9.4 mm i.d.) with a mobile phase of 20% methanol and 0.1% acetic acid as detailed by Dunlap et al. (1989), with a flow rate of 2 ml/min. Filtrate absorbance was recorded at 340 nm. Mycosporine-like amino acid standards extracted from tropical marine fish were supplied by Dr W. Dunlap from the Australian Institute of Marine Science. Pigment identification was carried out by injecting equal volumes of standard with the lens extract into the HPLC and monitoring any increase in absorbance at the retention time of the standard compounds. 3-Hydroxykynurenine was identified in a gourami (Truscott et al., 1992) and this sample was used to confirm, by HPLC, its presence in other lens extracts.
RESULTS
E z e l-
Lens spectral transmission and pigment absorbance
0 250
,
I
300
350
400
450
500
Wavelength tnml
FIGURE
above 500 nm. Spectra were therefore normalized to 100% at 700 nm for ease of comparison. After scanning, lenses were frozen for later pigment analysis Lens nucleus absorbance was also recorded for a number of fish from three species (Oreochromis niloticus, Merlangius merlangus and Carassius auratus) whose lenses contained palythene, After scanning, the whole lens was placed in 0.9% saline for 30 min after which the cortex coufd be easily slipped off the harder inner nucleus. The nucleus was then scanned in the spectrophotometer as for the whole lens. Using the nucleus and whole lens readings, the pigment concentration, based on the optical density at 360 nm (the I.,,,,, of palythene), in both the cortex and nucleus was calculated and expressed as absorbance units per mm pathlength.
I.
Spectral transmission of the right and left lenses of
Merlungius merlangus.
The
right
lens was scanned immediately after
death (solid line), whereas the left lens was scanned after the whole eye was frozen for 8 weeks (dashed line).
Intact lens transmission spectra showed large interspecies variability (Fig. 2). Of the 120 species examined 33 had spectra similar to that shown in Fig. 2(a), with a smooth, rapid fall in transmission at short wavelengths and 50% maximal transmission around 310-340 nm. No detectable amounts of pigment could be extracted from such lenses. Whole lens transmission spectra of the remaining 87 species displayed either an irregular decrease in transmission at short wavelengths [Fig. 2(b, c)] or a wavelength of 50%
291
PIGMENTS IN FKW LENSES-I
Xmax
Inm)
of
FKXJRE 3. Fr+!que~%cy histogram showing the distribution fish lens pigmenr &s_ Pigments were extracted from the lenses before measurzag spectral ~o~~~~~ W&a%?mi?re &an oz%eextractjon Was eati& values were averaged. Three major groups oflens o?It for a qXcksl A@@* pigment are repmsented, one in the region of 318-332 rim, one around 360 nm and one tit 370 nm.
r.M-7.33
6.04 3.69 5.45 4.50
I
6.&t
1
2.29
CM
31&w?
CM
335 334 325 3%
CM CM CM
CM
c
7
1.81~2.28
3%!-355
1 3
1.65 2.00-2.12
323 326-344
%
Tf:
-I-
324$360$ f
1
344
222 1.57 1.63-3.70 2.26 2.2X-2&3 I.81-4AM 1.12-4.82
315 326--X% 345 323-325 3twB2 318-391
4.60
370
CM
325, TX%
TF TF
292
A. THORPE TABLE
1 (continued)
Lens diameter
Lens 50% transmission
(mm)
(nm) 373 325
320
I
3.88 1.34
TF TF
I
1.11
318
_
TF
No. fish Serrasalmus nattereri Hyphessobrycon callisms
et al.
3
Pigment kIdi (nm)
Habitat*
Siluriformes Siluridae Kryptopterus bicirrhis
Salmoniformes Salmonidae Salmo gairdneri Osmeridae Osmerus eperlanus
16
3.22-6.20
322-334
_
CF
2
2.23-3.50
330-340
-
CM
7 6 4 33 8 9 8 1
5.01-8.60 4.67-9.00 5.75-8.31 3.12-6.90 5.36-10.13 3.32-5.80 3.37-5.84 2.39
398405 39245 394-396 383-397 396405 333-342 341-346 358
360 360 360 360 340,360 _ _
CM CM CM CM CM CM CM CM
5
2.53-7.55
32&341
_
CM
2
6.72-7.95
400
323,360
CM
1
3.31
391
330,360
TM
1.18-5.15 6.08-6.19 8.77
435442 390-392 340
385$ + _
CM CM CM
1.514.70
317-332
-
CM
1
0.81
315
_
TF
1
1.63
315
_
TF
Gadifonnes Gadidae Gadus morhua Pollachius virens Melanogrammus aeglejnus Merlangius merlangus Pollachius pollachius Trisoplerus minutus Trisopterus locus Ciliara muslela Merlucciidae Merluccius merluccius
Lophiiformes Lophiidae Lophius piscatorius Antennariidae Antennarius hispidus
Cyprinodontiformes Exocoetidae Exocoetus obtusirostris Hirundichthys a@nis Cypselurus cyanopterus Hemiramphidae Oxyporhampus micropterus Poeciliidae Poecilia reticulata
81 2
1 12
Atheriniformes Melanotaeniidae Melanotaenia parkinsoni
Gasterosteiformes Gasterosteidae Spinachia spinachia
5
1.39-1.56
320-321
_
CM
1 11 1 1
1.65 1.00-1.56 1.70 1.00
428 418423 421 412
+ 360 363 +
CM CM CM TM
1
2.41
+
CM
Dactylopteriformes Syngnathidae Syngnathus acus Syngnathus typhle Hippocampus ramulosus Hippocampus hudsonius
Scorpaeniformes Agonidae Agonus cataphractus Cottidae Taurulus lilljeborgi Taurulus bubalis Triglidae Trigla lucerna Aspitrigla cuculus Scorpaenidae Pierois volitans
395
3 4
4.5Wt.78
1.42-3.07
402408 393402
+ +
CM CM
I 8
3.05-5.76 2.82-5.44
402408 395404
328,360 325,360
CM CM
6
6.76-1.47
376401
322,360
TM
2.554.20 2.22
400404 398
325,360 +
CF CF
3.514.06
377-380
331
TM
Perciformes Centrarchidae Lepomis gibbossus Lepomis cyanellus Chaetodontidae Chaetodon spp
[continued opposite
293
PIGMENTS IN FISH LENSES-I TABLE 1 (continued)
Chaetodon frembli Chaetodon melanotus
Lens diameter (mm)
Lens 50% transmission (nm)
Pigment
No. fish
&u (am)
Habitat*
1 1
2.94 5.75
374 404
330 332
TM TM
1
3.35 0.866.81 0.98-3.08 1.43-3.34 1.94-2.03 1.50-3.57 2.01-2.09 1.41-3.41 3.01 3.10 2.88-3.69 1.10-1.34 1.50-2.38 1.82 2.82 2.58 1.94 2.745.51 1.75-2.08 1.65-1.86 1.58 0.85-2.06
338 339402 323-390 315-328 344360 327-394 325-339 335-390 358 392 381-394 348-355 371-385 351 348 400 417 425432 43e432 432437 440
+ 324,360 320,358 _
t
+ + + 323,360 + + 320,360 + + 320 321 + 325,360 324,360 327,362 370 370 370
TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF TF
1
3.93
407
326
TM
2 2 1 1
1.69-2.32 1.45-1.72 1.27 3.49
321 318-320 315 430
361
CM CM CM CM
1.663.31
364410
329,360
TF
1
2.63
430
+
TF
13 10 2 2
1.S2.55 1.01-1.54
1.09 2JW2.30
446-452 43943 435 385-392
370 370 370 327,360
TF TF TF TF
1
2.87
398
326,360
TF
1 8 5
3.73 2.18-3.98 2.70-3.70
404 378-397 388-410
331 + 330
TM TM TM
2
2.784.87
32&333
-
CM
1 1 1
4.12 4.27 2.51
402 406 395
331 330,360 327
TM TM TM
4
1.89-2.55
35&356
+
TM
1 2
1.48 2.43-2.49
407 397400
+ +
CM CM
14
1.54-3.70
372-386
321,359
CM
23
1.72-5.45
395402
327,359
CM
+ +
CM CM
Cichlidae Tilapia mariae Oreochromis niloticus Haplochromis tshmaeli Haplochromis argens Haplochromis piceatus Haplochromis sauvagei Haplochromis pyrrocephalus Haplochromis xenognathus Haplochromis nyererei Haplochromis “velvet black” Macropleurodus bicolor Astatorheochromis alluaudi Platytaeniodus degeni Lamprologus dahlia Lamprologus tetrocephalus Crenicichla lepidota Cichlasoma sevrum Astronotus ocellatus Herotilapia multispinosa Apistogramma curviceps Aequidens maronii Aequidens p&her
66 16
11 2 5 2 4 1 1 3 4 3 1 1 1 2 4 5 11 2 100
Plesiopidae Calloplesiops altevelis
Labridae Labrus bergylta Crenilabrus melops Ctenolabrus rupestris Thalassoma spp
Helostomatidae Helostoma temmincki
20
Anabantidae Ctenopoma oxyrhynchus
Belontiidae Trichogaster microlepis Trichogaster trichopterus Betta splen&ns Colisa fasciata
Osphronemidae Osphronemius goramy
Acanthuridae Acanthurus spp Zebrasoma Javescens Paracanthurus hepatus
Mullidae Mullus surmuletus Pomacanthidae Pygoplites diacanthus Holacanthus ciliaris
Centropyge heraldi Pomacentridae Amphiprion spp Bleniidae Parablennius gattorugine Liophrys pholis
Callionymiidae Callionymur lyra
Carangidae Trachus trachurus
Gobiidae Thorogobius ephippicutus Pomatoschistus microps
1 1
1.61 2.48
396 389
[continued overleaf
294
A. THORPE et ul. TABLE 1 (continued) No. fish
Lens diameter (mm)
Lens 50% transmission (nm)
Pigment %lill (nm)
Habitat*
Dicentrarchus labrax
4
2.64-3.99
403-406
+
CM
Scombridae Scomber seombrus Stichaeidae
6
4.88fi.69
360-402
325,360
CM
3
2.46-2.90
368-376
+
CM
5 18 6 1 4
2.87-5.61 0.73-5.75 1.86-4.67 3.79 2.26-5.42
396-410 399-408 399-407 405 333-389
323,360 331,360 328,360 + +
CM CM CM CM CM
2 2 2
2.21-2.67 l.&-1.64 3.02-3.41
324-325 320-324 328-332
-
CM
1
4.52
407
+
TM
1
7.90
301
321,360
TF
2
2.50-2.80
370-390
326
TM
Percichthyidae
Lumpenus lampretaeformis Pleuronectiformes
Pleuronectidae Platichthys flesus Pleuronectes platessa Li~nda ~imanda Microstomus kitt Hippoglossoides platessoides
Soleidae Microchirus variegatus Buglosidium luteum Solea solea
CM
CM
Te~a~oDti~o~~ Tetradontidae Arothron hipsidus
Diodontidae Diodon lystrix
Balistidae Cantherhines pufhis
*TM, Tropical marine; TF tropical freshwater; CM, cold marine;CF, cold freshwater. + , Pigment present but not extracted; -, no detectable pigment. tSpectra1 transmission not recorded. iPigment extract I,.. varies with age. (See Thorpe & Douglas, 1992 for details.) Species were classified after Nelson~(1984).
transmission above 400 nm [Fig. 2(d, e)]. All such lenses that were analysed contained short-wave absorbing pigments (Table 1). Lens pigments were extracted from 56 species and most fell into three main groups based on their wavelengths of maximum absorbance (AmaXs 320-330,360 and 370 nm) (Fig. 3). The lens of a single species (~xoc~e~~~ obtusirostris) contained a pigment with Amax 385 nm and another (Pollachius pollachius) contained a pigment absorbing maximally at 339 nm. The lenses of many species contained only one major lens pigment, but several had both 360 and 320-33Onm ,I,, pigments within the same lens (Table 1). Fish with spectral transmission as in Fig. 2(c), with a secondary peak around 320 nm, contained only ,I,,,,, 360nm pigment, whereas lenses with transmission as in Fig. 2(b) contained both 320-330 and 360 nm i,, pigments. A pigment with A,,, 370 nm isolated from lenses of several tropical freshwater species (Table 1) caused a secondary peak in the lens transmission at 310-320nm and 50% transmission wavelengths around 445 nm [Fig. 2(e)]. High concentrations of either 360 nm pigment alone, or high concentrations of both 320-330 and 360 nm pigments, resulted in smooth ~ansmission spectra as in Fig. 2(d).
three freshwater species (Cyprin~ carpio, 0. niluticus, Lepomis gibbosus) were found to contain predominantly palythene and palythine within their lens [e.g. Fig. 4(a, b)]. Whereas three tropical marine species (Chaetodon spp, Paracanthurus hepatus, Pygoplites diac~thus) only had significant concentrations of asterina330 within the lens [e.g. Fig. 4(c)]. One species of flying fish, E. obtusirostris, contained a Amax385 nm pigment which had a longer retention time than palythene, the most polar mycosporine compound previously examined [Fig. S(a)). However, this compound proved to be unstable in solution and degraded into three new compounds, one of which was palythine [Fig. V-41. Four mycosporine compounds were therefore found at high concentrations in fish lenses, each of which had a specific short-wave absorbance in the region of 3~~ nm (Fig. 6). Two tropical species, Aequidens p&her and Trichogaster trichopterus, had a ,I,,, 370 nm pigment within their lenses which did not co-elute with any of the mycosporine standards in HPLC (Fig. 7). This compound had three distinct absorbance peaks in the U.V. (Fig. 8), and was identified by NMR in T. trichopte~s as 3-hydroxykynurenine (Truscott et al., 1992).
Pigment identljication
Pigment distribution within the lens
Lens pigments from I4 species were identified by HPLC methods. Four marine species (~yngnath~ typhle, C&pea harengus, P. pollachius, Aspitrigla cuculus) and
Absorbance at 360 nm per mm pathlength in the nucleus and cortex from lenses of two species, 0. n~Iotic~ and M. merla~gus consistently showed
I 0
I
I
0 200
256
300
3SQ
400
450
A. THORPE rt t/l.
i I \
q
1’ \
LJ \ \ \ \ \
----A
I 200
I
I
I
I
0
5
IO
15
Time (man)
FIGURE 7. HPLC elution profile of the ,&,,, 370 nm pigment from the lens of A. p&her. The major peak eluted at 7.7 min. HPLC conditions as in Fig. 3.
higher absorbance in the cortex than in the nucleus (Table 2). However, the absorbances per mm for the nucleus and cortex of C. auratus were not significantly different (Table 2). DISCUSSION
Biochemical identity of lens pigments
Two distinct classes of pigment were isolated: mycosporine compounds with short-wave absorbance maxima at 320, 330, 360 or 385 nm (Fig. 6) and 3_hydroxykynurenine, a tryptophan derivative with 1max 370 nm (Fig. 8). Palythene and palythine were identified as the major pigments in S. typhle, C. harengus, P. pollachius, A. cuculus, C. carpio, 0. niloticus and L. gibbosus, while lenses of Chaetodon spp, P. hepatus and P. diacanthus were found to contain appreciable amounts of only asterina-330. The flying fish (E. obtusirostris) lens pigment, although unidentified, is also a mycosporine analogue. The tryptophan metabolite 3-hydroxykynurenine was identified in the lenses of A. pulcher and T. trichopterus. Even though the positive identification of lens pigments was carried out for relatively few species, the compounds present in lenses of other species could be
250
300
350
Wovelength
assessed from the spectral absorbance of their extracts. Lenses with only Amax360 nm pigment (several gadidae species and a single labridae species, see Table 1) could, based on the A,,,,, of the crude extract, be either palythene [Fig. 6(d)] or kynurenine, a tryptophan derivative which also absorbs at 360 nm (Thorpe et al., 1992). However, since these extracts had absorbance spectra with only a single peak at 360 nm, rather than the three peaks characteristic of tryptophan-related compounds (e.g. Fig. 8), the lenses probably contain palythene. In support of this, van Heyningen and Linklater (1976) similarly concluded that the lens pigments of several gadidae species were not tryptophan-related since the lenses did not metabolise tryptophan, and Dunlap et al. (1989) also found high concentrations of palythene in the lenses of labridae species. On the other hand, it is likely that all I,, 370 nm pigments (i.e. from lenses of three belontidae species and several cichlidae species, Table 1) are 3_hydroxykynurenine, as identified in T. trichopterus (Truscott et al., 1992) since all had complex short-wave absorbance spectra as in Fig. 8. Pigments absorbing in the 320-330 nm range presented a similar problem; these compounds could be either tryptophan metabolites (W-formylkynurenine absorbs maximally at 320 nm) or the mycosporine
No.
Nucleus (abs/mm) (SD)
Cortex (absimm) (SD)
P*
Oreochromis niloticus
3.094.11
8
0.19 (0.06)
0.52 (0.28)
0.001
Merlangius merlangus
3.894.86
5
2.61-3.92
12
0.31 (0.20) 0.09 (0.05)
0.1
Carassius auratus
0.16 (0.05) ‘0.13’ (0.07)
*Student’s r-test.
450
FIGURE 8. Spectral absorbance of the HPLC purified extract from the lens of A. p&her with imaxs around 230, 262 and 370 nm (solid line). The compound had a HPLC retention time and absorbance spectra identical to the lens pigment from T. trichopterus (dashed line), identified as 3-hydroxykynurenine (Truscott et al., 1992).
TABLE 2. Absorbance. at 360 nm per mm, for lens cortex and nucleus of three fish species
Lens dia
400
(nm)
NS
PIGMENTS
IN FISH LENSES-I
291
Pigment distributionwithin the lens compounds palythine, asterina-330 and palythinol, with maximal absorbance at 320, 330 and 331 nm Mycos~~ne related pigments and tryptophan derivarespectively. A~thou~ it has been suggested that N’- tives are all low molecular weight compounds. Both formylkynurenine is responsible for the absorbance classes of pigment are also easily extracted into water, of short-wave radiation in the lenses of some fish suggesting that they are not tightly bound to any large (Zigman, 1987), this compound has never been posi- protein moiety within the lens and are free to diffuse tively identified, and all pigments identified here throughout the lens nucleus and cortex. The data absorbing maximahy around 320-331 nm were mycoreported in Table 2 reveal that pigments are present in sporine compounds, mainly palythine and asterina-330. both nucleus and cortex of the lens, although the It is therefore thought that the majority of pigments concentration in each varies between species. Differabsorbing around 320-330 nm listed in Table 1 are ences in pigment concentration in the two lens parts have mycosporine-like amino acids. been observed in other species; Argyropelecus @inis, for Although mycosporine pigments have been pre- example, has a higher pigment concentration in the viously identified in lenses of tropical marine species cortex than the nucleus, causing the cortex to appear (Dunlap et al., 1989), they have not until now been visibly yellow whilst the nucleus appears colourless to identified in lenses of more temperate marine species human observers (McFall-Ngai et al., 1986; Yu, Cai, or freshwater fish. Mycosporine-like compounds are Lee, Kuck, McFall-Ngai & Horwitz, 1991; Douglas found in a range of tropical invertebrates (Nakamura, and Thorpe, 1992). Lens pigments in Argyropelecus are Kobayashi & Hirata, 1982) and the fact that many bound to lens ot-crystdlin and cannot diffuse freely temperate f&h have been found to have high con- throughout the lens. The reverse situation is observed centrations of mycosporine analogues in their eggs in Mulucosteus niger, another mesopelagic species, (Chioccara, Della Gala, De Rosa, Novellino & which has a yellow pigmented core within the nucleus Prota, 1980; Fraser, Grant, Middleton, Mitchell & (Douglas & Thorpe, 1992). The differential pigment Thomson, 1981) suggests that there is a readily avail- concentrations in the nucleus and cortex of the three able supply of mycosporine precursors in more species examined here suggests that their pigments may temperate waters. also be loosely associated with lens protein, as reported The lens pigment in one species of flying fish for a number of epipelagic species (Bon, Ruttenberg, (E. obtusirostris) appeared to have a longer A,, Dohrn & Batink, 1968). The fact that the pigments in (385 nm) than other mycosporine pigments (Fig. 7). two of these species appear to predominate in the Iens Since one of the degradation products of this pig- cortex suggests that they may be laid down later in ment was identical to palythine (,& 32Onm), the fly- hfe as a function of the animal’s age (see Thorpe 8t ing fish lens pigment is likely to be a more complex Douglas, 1993) or that the cortex is the site of pigment mycosporine analogue. The other two degradation accumulation, products also had I,,, s around 320 nm. Since the original pigment was so unstable, it was not possible to Phy~oge~et~cd~tr~b~t~onof lens pigments establish its identity by NMR or mass spectroscopy. Fish with short-wave absorbing patents in the ft is probable that t~ptophan-related pigments are of synthesized within the fish lens from free tryptophan, as Iens were represented in around three-quarters is the case in the human lens (van Heyningen, 1973b). the 49 families examined (Fig. 9), supporting previous evidence that the presence of short-wave absorbOther primates and squirrels also have lens pigments ing pigments in both freshwater and marine fish lenses derived from tryptophan catabolites in the same pathway (van Heyningen, 1973a, b; Zigman & Paxhia, 1988). is widespread (Muntz, 1973, 1976a, b; Dougias & 1989; Dunlap et al., 1989). Pigmented That a rneso~~g~c species (Thorpe et al., 1992) and a Morgan, number of freshwater species should have evolved lens lenses have primarily been described in fish which pigments closely related to those of higher terrestrial belong to the perciforme order or perciforme derived vertebrates is quite surprising. Specific enzymes are species (Muntz, 1973; Douglas & McGuigan, 1989), required for the reactions in this pathway and it could with the marked exception of the pigmented lenses be that they are only active in some species, possibly found in mesopelagic fish which belong to a range explaining why kynurenine compounds are not more of famihes (Muntz, f976a; Douglas & Thorpe, 1992). commonly found. The Syngnathidae (Pipefishes) examined here however, Two tryptophan-related compounds and four major are neither perciformes nor mesopelagic, yet they mycosporine compounds, along with a host of unidentoo have lenses with 50% transmission wavelengths of tified pigments from mesopelagic species (McFafl-Ngai up to 422 nm (Table l)? due to high concentrations et al., 1988; Douglas & Thorpe, 19921, have now been of pafythene in the lens. Lenses not appearing yellow; isolated from fish lenses. Lens pigmentation in fish but nevertheless having quite high concentrations of therefore seems to represent a good example of evol- short-wave absorbing compounds were also found in utionary convergence: a number of biochemically dis- several other non-perciforme families (e.g. gadidae and tinct compounds absorbing radiation in the same region pleuronectidae, Table 1). Perciforme species, however, seem to have evolved in fish lenses independently on a do show the greatest variety of lens pigments (Fig. 91, number of occasions. withy at feast six different types of pigment isolated, YR333-F-B
298
A. THORPE et al. ORDER
NUMBER OF SPECIES
ELASMOBRANCHII
E I
ANGUILUFORMES OSTEOGLOSSIFORMES CLUPEIFORMES _I
l- CYPRINIFORMES
I LA-
GAOIFORMES
CYPRlNODONTlFGRMES ATHERiNlFORMES LAMPRIFORMES GASTEROSlElFORMES DACTYLOPTERlFORMES SCORPAENIFORMES PERCIFORMES
PLEURONECllFORMES KTRAOGONllFORMES
KEY:
0
NO PIGMENT
m
360
m
320-330
I
320-350
PIGMENT
PIGMENT de 360
PlGMENTS
IIIM
370
PIGMENT
m
UNIDENTlflED PIGMENT
FIGURE 9. Phylogenetic distribution of lens pigments in fish. Pigments with similar near U.V.J._s have been grouped together for simplicity, though this does not necessarily mean that they are identica1 compounds.
probably reflecting the genetic diversity of this largest teleost order. Closely related species often have the same lens pigments For example, lenses of four members of the gadidae family: cod, saithe, whiting and haddock all have a &.,,_360 nm pigment (Table l), which is likely to be palythene. Other authors contirm the presence of a I,, 360 nm pigment in the lenses of several other species in this family (Bon et al., 1968; van Heyningen & Linklater, 1976). Few lenses had I,,, 370 nm pigment, but three belontidae species contained this compound (likely to be 3-hydroxykynurenine) and it was also found in three tropical freshwater cichlidae species (Table 1). Most cyprinids have lenses with no pigmentation (Fig. 9; Table l), and even those cyprinid species sometimes accumulating pigments (goldfish and carp), never have very high concentrations. Similarly, neither rajidae nor scyliorhynidae species ever show any evidence of lens pigmentation (Table 1, Fig. 9). Although the pigment composition within a family is often the same, concentrations can vary, with most whiting lenses for example having considerably lower pigment concentrations than lenses of both cod and haddock. This is also demonstrated by the large range in 50% transmission wavelengths of the cichlids (Table l), though many appear to contain both palythine and palythene pigments. Since the genetic make-up of congeneric species is likely to be quite similar, it may be that other factors, such as the animal’s environment, are
important for the determination levels in the lens.
of specific pigment
Ecological relationships and lens pigment function
Although phylogeny is important, the environment also appears to have a role to play in lens pigmentation, both in terms of adaptation to that environment and a direct effect of the environment on the lens. Many of the fish with pigmented lenses listed in Table 1 have a diurnal lifestyle, a factor often associated with highly pigmented lenses (Mu&, 1973). It has been suggested that some fish lens pigments may accumulate as a result of exposure to high levels of U.V. (Zigman & Gilbert, 1978). In support of this, although pleuronectidae and soleidae families are closely related and share the same environment, lenses of soleidae, which are nocturnal (Wheeler, 1978), have no pigment, whereas all pleuronectids which are generally diurnal, have pigmented lenses (Table 1). However, whilst it is true that many of the highly diurnal species, such as those living on coral reefs (e.g. pomacanthidae and pomacentridae familiesTable l), often do have highly pigmented lenses, it is difficult to believe in a direct role of sunlight when several fish occupying the same ecological niche can have quite different lens transmission characteristics. For example, although two species of flying fish (E. obtusirostris and Hirundichthys afinis) had high lens pigment concentrations (Table l), another species (Cypselurus cyanopterus) within the same family and
PIGMENTS p~s~&ly
sharing
&e
me
en@rom&
had
IN FISH LENSES-I
none
(Table 1). It has also proved impossible to induce fish lens pigment formation or affect pie-existing pigments by altering the lighting environment alone (Thorpe 8z Douglas, ~publish~ results). Various functions have been ascribed to lens pigments, including the protection of the retina from U.V. (Collier, Waldron & Zigman, 1989); increasing visual acuity by removing the wavelengths responsible for scatter, glare and chromatic aberration (Muntz, 1973, 1976b; Zigman & Gilbert, 1978); stabilizing lens protein (MeFall-Ngai et al., 1986) and as an aid in prey detection in mesopelagic species (Muntz, 197% Somiya, 1982; Douglas & Thorpe, 1992). Lens pigments in higher vertebrates have been shown to protect the retina from U.V. damage (Collier & Zigman, 1987; Collier et ai., 1989), and the fact that short-wave absorbing compounds have been identified in many highly diurnal species (Table 1; Douglas & McGuigan, 1989; Dunlap et al., 1989) also supports a role in U.V. protection in fish. However, fish living in very high light environments may also benefit from improvements in visual acuity offered by lens pigments, since effects of scatter, glare and chromatic aberration are more intense at short wavelengths (Muntz, 1972). It is therefore unlikely that all fish lens pigments perform the same function; a number of different lens pigments have evolved in both deep-sea and surface dwelling fish, presumably to serve a range of requirements.
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Eye Research,
7, 603-610.
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(pp. 571-585). New York: Liss. Collier, R. J., Waldron, W. R. & Zigman, S. (1989). Temporal sequence of changes to the gray squirrel retina after near-W exposure. Investigative Ophthalmology and Visual Science, 30, 631637.
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J er 1ov,
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McFall-Ngai, M., Ding, L., Childress, J. & Horwitx, J. (1988). Biochemical characteristics of the pigmentation of mesopelagic tish lenses. Biofogy B~Iet~, f75, 397-402. Muntx, W. R. A. (1972). Inert absorbing and refhzting pigments. In Dartnall, H. J. A. (Ed.), Photochemistry of vision (IIanabooh of Sensory Physiology, Vol. VIIf 1) (pp. 529-565). Berlin: Springer. Muntx, W. R. A. (1973). Yellow filters and the absorption of light by the visual pigments of some Amazonian fishes. Vision Research, 13, 2235-2254.
Muntz, W. R. A. (1976a). On yellow lenses on mesopeIagic animals. Journal of the Marine Biologicaf Association of the United Kingdom, 56, 963-976,
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Acknowledgements-The authors would like to thank Dr G. McReid from the Homiman Museum, The Plymouth Marine Laboratory, Dr L. Ross, Professor J. Wagner and Professor J. Blaxter for generous supplies of fish; the masters and crews of R.R.S. Discovery and R.R.S. Calanus; Dr W. Dunlap of A.I.M.S. for mycosporine standards and herring lens pigment analysis; Dr J. Davenport for his help in the collection and identification of the flying fish; and Dr P. Herring for enabling RHD to take part in cruise 195 of R.R.S. Discovery. AT was supported whilst in Australia by a grant from the Department of Industry, Technology and Commerce (Australia).