Photopigment spectral absorbance of Lake Malawˆi cichlids

Journal of Fish Biology (2006) 68, 1291 1299 doi:10.1111/j.1095-8649.2005.00992.x, available online at http://www.blackwell-synergy.com Photopigment spectral absorbance of Lake Malawˆi cichlids R. JORDAN*, K. K ELLOGG, D. H OWE, F. J UANES*, J. STAUFFER,JR{ AND E. LOEW** *Program in Organismic and Evolutionary Biology, University of Massachusetts, Amherst, MA 01003, U.S.A., Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, NJ 08901, U.S.A., Skidmore College, Environmental Studies Program, Harder Hall 192, Saratoga Springs, NY 12866, U.S.A., {The Pennsylvania State University, School of Forest Resources, 02C Ferguson Building, University Park, PA 16802, U.S.A. and **Cornell University, Department of Biomedical Sciences, T7-020 Veterinary Research Tower, Ithaca, NY 14853, U.S.A. (Received 29 April 2005, Accepted 7 October 2005) To predict spectral sensitivity, microspectrophotometry (MSP) was used to measure absorbance of photoreceptor cells from 15 species of Lake Malaŵi cichlids. Each fish had one rod and at least three cone pigments. UV-sensitive pigments were common, but spectral sensitivity did not clearly correlate with feeding mode or habitat. Journal compilation # 2006 The Fisheries Society of the British Isles Key words: Cichlidae; microspectrophotometry; ultraviolet vision; visual pigment. Approximately 2000 species of cichlids, most of which are endemic, inhabit the three great lakes of Africa (Barlow, 2000). Half of this diversity is found within Lake Malawˆi (Stauffer et al., 1997; Barlow, 2000). Two of the more notable theories generated to explain this astounding radiation are ecological speciation related to feeding mode and sexual selection through female choice (Danley & Kocher, 2001). Given the reliance of these theories on visually guided behaviours and that the cichlids of Lake Malaŵi exhibit both visually guided foraging and mate selection behaviour (Hert, 1991; McKaye et al., 1993; Kellogg, 1997; Couldridge, 2002; Jordan et al., 2003, 2004), a comparative understanding of visual physiology is necessary. The spectral limits of visual perception are determined in part by the visual pigments within the photoreceptors. The presence of several spectral classes of cone photoreceptors in cichlids suggests that colour vision plays a role in the above-mentioned behaviours, although luminosity information mediated by the Author to whom correspondence should be addressed at present address: Department of Ecology, Evolution and Natural Resources, Rutgers University, New Brunswick, NJ 08901, U.S.A. Tel.: þ1 732 932 8242; fax þ1 732 932 8746; email: rebeccacjordan@yahoo.com 1291 Journal compilation # 2006 The Fisheries Society of the British Isles

1292 R. JORDAN ET AL. rods cannot be ignored as a cue (Muntz, 1976; Levine & MacNichol, 1979; Carleton et al., 2000). Muntz (1976) examined only rod extracts and concluded that differences in the spectral position of maximum rod absorption (l max ) among species were not related to physical habitat differences, but might be correlated to differences in the light environment potentially driven by depth. Using microspectrophotometry (MSP), Levine & MacNichol (1979) examined cone sensitivities in Dimidiochromis (formerly Haplochromis) compressiceps (Boulenger) and Metriaclima (formerly Pseudotropheus) zebra (Boulenger), however, only data for the double cones were presented (mean S.D. short wavelength, SW: 536 4, long wavelength, LW: 569 6 and SW: 488 3, LW: 533 2 respectively). Carleton et al. (2000) reported an ultraviolet cone in a Lake Malawˆ i cichlid, M. zebra. While these studies were informative, a much broader analysis of the visual capabilities of Lake Malawˆ i cichlids is needed in order to understand the significance of spectral sensitivity to the ecology and evolution of this astoundingly diverse group. Here, MSP was used in a preliminary study to identify the visual pigments present in rods and cones from a variety of species of Lake Malawˆ i cichlids. The intention of this study was to identify possible gross differences in photoreception to serve as a baseline and impetus for deeper exploration of the correlation of spectral sensitivity and ecotypical features for these fishes. Prior to such an undertaking, a reasonable determination of presence or absence of pigments is appropriate. The housing of animals and methods used in this study were performed under the guidelines established by University of Massachusetts and Cornell University s Animal Care and Use Policy. The cichlid species (Table I) used were collected from the southern portion of Lake Malawˆi (14 S; 33 E), except for Copadichromis borleyi (Iles), Protomelas taeniolatus (Trewavas) and D. compressiceps. These three species were obtained as juveniles through the pet trade and probably bred in the U.S. Fish species were selected for their differences in habitat use and feeding mode. Phylogenetic diversity was maximized based on current interpretations of taxonomic relationships (Moran et al., 1994; Parker & Kornfield, 1997; Albertson et al., 1999). In the laboratory, each species was housed separately in 110 l tanks maintained at 24 28 C, ph 7 8 and 12L : 12D photoperiod. The fishes were fed a combination of flake and stick foods three times daily. Except for feeding, occasional care and tank maintenance, the fishes were not disturbed during the 3 months prior to experimentation. This preliminary survey of the photoreceptor types was performed by determining absorbance spectra from several photoreceptor cells from a single adult specimen of each species. Absorbance spectra were obtained for the rods and cones using a computer-controlled microspectrophotometer (Loew, 1994). The fishes were dark adapted for at least 3 h, anaesthetized using MS-222, sacrificed by decapitation, and enucleated under infra-red light using appropriate image converters. The eyecups were placed in standard phosphate buffer solution (ph 7.3) and the retina of one eye was teased away from the pigment epithelium. The retina was bisected, transferred to two cover slips, macerated using razor blades and insect pins, and overlaid with cover slips edged with silicone grease.

PHOTOPIGMENT SENSITIVITY IN CICHLIDS 1293 TABLE I. Ecological and taxonomic information on the 15 species surveyed for the MSP analysis. Habitat, depth and food data taken from Ribbink et al. (1983), Konings (1990) and J.R. Stauffer (unpubl. obs.). Species group refers to two major classifications of Lake Malawˆi fishes; species which are not members of either group are left blank Species Habitat Depth usually found Food Species group Copadichromis borleyi Rock M Open water plankton Utaka Cynotilapia afra Rock/sand interface M Plankton/algae Mbuna Melanochromis auratus Rock/sand interface S Algae/sediment Mbuna Metriaclima barlowi Rock/sand interface/sediment M Algae/plankton/sediment Mbuna Metriaclima benetos Rock S Algae/plankton Mbuna Metriaclima emmiltos Rock M Algae/plankton Mbuna Metriaclima livingstonii Sand with/shell cover D Algae/plankton/sediment Mbuna Metriaclima melabranchion Rock M Algae/plankton Mbuna Pseudotropheus t. tropheops Rock S Algae/plankton Mbuna Aulonocara hueseri Sand D Sand invertebrates Dimidiochromis compressiceps Vegetation S Fish Lethrinops parvidens Rock and sand D Zooplankton/algae Mylochromis lateristriga Sand/vegetation S Crustaceans Protomelas taeniolatus Rock M Algae Tyrannochromis macrostoma Rock D Fish S, 0 10 m; M, 0 30 m; D, <30 m.

1294 R. JORDAN ET AL. Each of the two preparations from a single fish was scanned in horizontal and vertical transects to isolate morphologically unique types of photoreceptor cells for measurement. Processing time for each preparation was c. 2 h. The baseline spectrum for absorbance measurements was obtained from a cell-free area of the preparation and continually checked for validity. Outer segments from rods and cones were identified and aligned with the measuring beam under infra-red light, after which an absorbance spectrum was obtained from 750 to 350 nm in interleaved down and up directions. The criteria for accepting the absorbance from ultraviolet cells as being true visual pigments and not a photoproduct were similar to those used by Loew (1994) and included outer segment dichroism and bleaching characteristics. Double cones were designated as such only if found as an intact pair. Absorbance spectra from c. 25% of cells measured were retained for analysis. Others were discarded because although pigment type could generally be discerned, the quality was insufficient for template fitting. Thus, sample sizes are effectively greater than reported. Similar sample sizes are common in studies of this type (Downing et al., 1986; Bowmaker & Kunz, 1987; Loew, 1994; McFarland & Loew, 1994; Losey et al., 2003). The Mansfield (1985) MacNichol (1986) method was used to estimate l max of the visual pigments using templates derived from Partridge & DeGrip (1991). This is a somewhat subjective process, but adequate for discerning differences among species. Absorbance maxima were averaged to obtain a single species-specific value for each photopigment type. The cone classes were divided into UVS [single cones with average l max <400 nm (ultraviolet)], SWS-S (single cones with average l max ranging from 409 to 425 nm), SWS-LS (single cones with average l max ranging from 447 to 453 nm), SWS-LD (double cones with average l max ranging from 472 to 492 nm), MWS (double cones with average l max ranging from 514 to 539 nm) and LWS (double cones with average l max ranging from 563 to 567 nm). All of the retinal preparations examined had at least three, and in one case, four cone spectral classes plus a single rod. Rod l max values ranged from 489 to 498 nm, with one exception at 510 nm (Table II). Template fitting identified the chromophore as vitamin A 1 -based. Typical absorbance spectra are shown in Fig. 1. Only one type of single cone, UVS, SWS-S or SWS-LS, was found in any preparation (Table II). Double cones of 12 species were SWS-LD/MWS pairs. In these cases, the l max of the medium wavelength sensitive cone was <540 nm. Three species had MWS/LWS doubles, with l max of the long wave sensitive cone >560 nm (Table II). One species had both types of double cones. Although it cannot be said with certainty that all types of cones present in these fishes were found, the data strongly suggest that there are differences in the photoreceptive pigments of these fishes that may be phylogenetically and ecologically significant. This conclusion is supported by the finding of only one single cone type in all 15 species, and only one species with two types of double cones. All members of the monophyletic rock-dwelling group of Lake Malawˆ i cichlids (i.e. the mbuna), except Melanochromis auratus (Boulenger), had a UVS cone. Non-mbuna species expressed an SWS-S pigment with the exception of D. compressiceps and P. taeniolatus, which expressed an SWS-LS.

PHOTOPIGMENT SENSITIVITY IN CICHLIDS 1295 TABLE II. Average l max (nm) by species and photoreceptor cell type. Where multiple cells were tested, S.D./number of cells are given in parentheses Species Rods UVS SWS-S single SWS-LS single SWS-LD double MWS double LWS double Copadichromis borleyi 494 424 (9/11) 478 (10/8) 539 (14/6) Cynotilapia afra 496 (5/2) 358 (12/6) 472 (5/6) 525 (2/7) Melanochromis auratus 497 (3/3) 414 (2/5) 482 (5/2) 525 (1/2) Metriaclima barlowi 491 366 (1/2) 483 (1/2) 523 (4/2) Metriaclima benetos 379 (13/3) 489 522 Metriaclima emmiltos 383 (11/12) 482 (1/3) 521 (4/4) Metriaclima livingstonii 495 (2/2) 364 (2/6) 473 (3/9) 526 (2/9) Metriaclima melabranchion 491 (5/17) 371(1/2) 477 (5/9) 515 (5/3) Pseudotropheus t. tropheops 510 371 (9/7) 492 (1/2) 531 (8/6) 565 Aulonocara hueseri 496 (2/3) 415 (2/2) 484 (12/2) 526 (1/3) Dimidiochromis compressiceps 498 (3/4) 447 (4/4) 533 (10/10) 567 (6/13) Lethrinops parvidens 489 426 (2/8) 514 (3/5) 563 (5/6) Mylochromis lateristriga 496 (8/7) 419 (3/7) 482 (3/7) 522 (6/8) Protomelas taeniolatus 496 (1/4) 453 (4/7) 527 (9/3) 566 (2/2) Tyrannochromis macrostoma 494 (6/3) 410 (4/5) 482 (2/13) 529 (5/12) UVS, single cones with lmax < 400 nm; SWS-S, single cones with lmax 409 425 nm; SWS-LS, single cones with lmax 447 453 nm; SWS-LD, double cones with l max 472 492 nm; MWS, double cones with l max 514 539 nm; LWS, double cones with l max 563 567 nm., measurement did not meet acceptance criteria. Data for Dimidiochromis compressiceps are used with permission from K. L. Carleton.

1296 R. JORDAN ET AL. Normalized optical density 1 (a) 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 350 400 450 500 550 600 650 700 750 1 (b) 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 350 400 450 500 550 600 650 700 750 1 (c) 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 350 400 450 500 550 600 650 700 750 Wavelength (nm) FIG. 1. Sample spectral absorbance curves from (a) a single cone receptor type UVS and from double cone receptor (b) type SWS-LD and (c) MWS type (see Table II) found in Metriaclima livingstonii. Each curve was normalized to the l max of the best fitting template curve indicated by the solid line. Furthermore, the absorbance maxima reported for double cones of D. compressiceps are within 3 nm of those reported in Levine & MacNichol (1979). Carleton & Kocher (2001) have investigated the molecular basis of spectral sensitivity in four species of Lake Malaŵi fishes including D. compressiceps. Interestingly, they report that these fishes share similar opsin sequences, and that the differences in spectral sensitivity seen among species are related to differences in gene expression. If the present methods systematically or randomly missed photopigment types, the suggestive pattern found would probably not have emerged. The spectral sensitivities of the closely related Lake Victoria and Lake Tanganyika cichlids have been studied in great detail (Fernald & Liebman, 1980; van der Meer & Bowmaker, 1995). Though these fishes had three cones

PHOTOPIGMENT SENSITIVITY IN CICHLIDS 1297 and one rod pigment, as did many in the present study, their photopigment cells tended to be sensitive to longer wavelengths. In the Lake Victoria fishes, rod l max tended to be >500 nm, single cone l max ranged from 455 to 464 nm and double cones had a 565 569 nm pigment paired with a 522 538 nm pigment. These values are very similar to those for the Lake Tanganyika cichlid Haplochromis burtoni Gu nther (rod l max ranged from 499 to 501 nm; cone average l max was 455 nm for single, 562 and 523 nm for double cone pairs; Fernald & Liebman, 1980). The longer wavelength-sensitivity reported in these cichlids correlates with the shallow and possibly more turbid habitats where these fish live (van der Meer & Bowmaker, 1995; Hofmann et al., 1999). There is a general expectation that freshwater fishes should have substantially longer (>500 nm) wavelength sensitivity due to the colour of most fresh waters. The absence of short-wavelength cones or a shift to the use of vitamin A 2 -based pigments, which absorb at longer wavelengths than their A 1 counterparts, support this belief (Bowmaker, 1990). Lake Malawˆ i is considered particularly clear (Muntz, 1976). Because of this clarity, ultraviolet light, along with the other short wavelengths, probably penetrates to at least 50 m in the lake (Loew & McFarland, 1990). Also important to note is that cichlids, as a family, are derived from a marine ancestor and are not strictly a freshwater group of fishes (Barlow, 2000). Spectral sensitivity may also be responding to the demands of species or mate recognition, interspecific competition or habitat use. For example, the two species of the sympatric Lake Victoria fishes described previously that were most similar in habitat use, differed greatly in spectral sensitivity, perhaps thereby reducing interspecific competition (van der Meer & Bowmaker, 1995). A similar argument was made for the blenny Blennius pholis L., and the goby Gobius paganelus L., which can inhabit the same small tidepool, but have different visual pigments and retinal organization (Loew & Lythgoe, 1978). The pigment differences among these Lake Malawˆ i species might also be driven by phylogenetic differences, because the mbuna are generally thought to be more closely related to each other and perhaps the deepwater species, than are the shallowwater sand dwellers. Furthermore, the mbuna live among rocks, and potential differences in spectral sensitivity could be attributed to the specific demands of such habitat. Davitz & McKaye (1978) found that individuals of the mbuna species Pseudotropheus macrophthalmus Ahl were able to discriminate between horizontal and vertical polarized light. This can be important for water column migration and orientation, needs that are especially strong in structural habitats. In conclusion, evidence suggests that differences in spectral sensitivity exist in Lake Malaŵi cichlids. Despite limited sample sizes, a baseline now exists from which predictions about the visual ecology of these fishes can be drawn. Important future directions include finer-scale retinal surveys of more taxa covering a greater diversity of habitats, and a more comprehensive examination of the visual environment of Lake Malawˆ i. We thank the Malawˆi government for allowing the collection of these fish. We also thank K. Carleton for her helpful discussion and for use of the D. compressiceps data. In addition, we thank three anonymous reviewers whose thoughtful comments have greatly

1298 R. JORDAN ET AL. improved this manuscript. This project was funded by the GAANN fellowship awarded to R.C. Jordan, the Jordan Endowment from the American Cichlid Association awarded to R.C. Jordan and K. A. Kellogg, a National Science Foundation Dissertation Improvement grant awarded to R.C. Jordan, and through a Healy Grant from the University of Massachusetts awarded to F. Juanes. Lastly, we acknowledge the University of Massachusetts and Cornell University where this work was completed. References Albertson, R. C., Markert, J. A., Danley, P. D. & Kocher, T. D. (1999). Phylogeny of a rapidly evolving clade: the cichlid fishes of Lake Malawˆi, East Africa. Proceedings of the National Academy of Sciences USA 96, 5107 5110. doi: 0027-8424/99/ 965107 4 Barlow, G. W. (2000). The Cichlid Fishes: Nature s Grand Experiment in Evolution. Cambridge: Perseus Publishing. Bowmaker, J. K. (1990). Visual pigments of fishes. In The Visual System of Fish (Douglas, R. H. & Djamgoz, M. B. A., eds), pp. 82 107. New York: Chapman & Hall. Bowmaker, J. K. & Kunz, Y. W. (1987). Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the brown trout (Salmo trutta): age-dependent changes. Vision Research 27, 2101 2108. doi: 10.1016/0042-6989(87)90124-6 Carleton, K. L. & Kocher, T. D. (2001). Cone opsin genes of African Cichlid fishes: tuning spectral sensitivity by differential gene expression. Molecular Biology and Evolution 18, 1540 1550. Carleton, K. L., Ha rosi, F. I. & Kocher, T. D. (2000). Visual pigments of African cichlid fishes: evidence for ultraviolet vision from microspectrophotometry and DNA sequences. Vision Research 40, 879 890. doi: 10.1016/Soo42-6989(99)00238-2 Couldridge, V. S. K (2002). Experimental manipulation of male eggspots demonstrates female preference for one large spot in Pseudotropheus lombardoi. Journal of Fish Biology 60, 726 730. doi: 10.1016/jfbo.2002.1896 Danley, P. & Kocher, T. (2001). Speciation in rapidly diverging systems: lessons from Lake Malawi. Molecular Ecology 10, 1075 1086. doi: 10.1046/j.1365-294X.2001.01283.x Davitz, M. A. & McKaye, K. R. (1978). Discrimination between horizontally and vertically polarized light by the cichlid fish, Pseudotropheus macrophthalmus. Copeia 1978, 333 334. Downing, E. G., Djamgoz, M. B. A. & Bowmaker, J. K. (1986). Photoreceptors of a cyprinid fish, the roach: morphological and spectral characteristics. Journal of Comparative Physiology A 159, 859 868. Fernald, R. D. & Liebman, P. A. (1980). Visual receptor pigments in the African cichlid fish, Haplochromis burtoni. Vision Research 20, 857 864. doi: 10.1016/0042-6989(80)90066-8 Hert, E. (1991). Female choice based on egg-spots in Pseudotropheus aurora Burgess 1976, a rock-dwelling cichlid of Lake Malawˆi, Africa. Journal of Fish Biology 38, 951 953. Hofmann, H. A., Benson, M. E. & Fernald, R. D. (1999). Social status regulates growth rate: Consequences for life-history strategies. Proceedings of the National Academy of Sciences USA 96, 14171 14176. Jordan, R. C., Kellogg, K. A., Juanes, F. & Stauffer, J. R. Jr. (2003). Evaluation of female mate choice cues in a group of Lake Malawˆi mbuna (Cichlidae). Copeia 2003, 181 186. doi: 10.1016/0042-6989(80)90066-8 Jordan, R. C., Howe, D. V., Juanes, F., Stauffer, J. R. Jr. & Loew, E. R. (2004). The role of UV in foraging in a group of Lake Malawˆi cichlids. African Journal of Ecology 42, 228 231. Kellogg, K. A. (1997). Lake Malawi cichlid mating systems: factors that influence mate selection. PhD Thesis, The Pennsylvania State University, Pennsylvania.

PHOTOPIGMENT SENSITIVITY IN CICHLIDS 1299 Konings, A. (1990). Ad Konings s Book of Cichlids and All the Other Fishes of Lake Malawˆ i. New Jersey: TFH Publishing. Levine, J. S. & MacNichol, E. F. (1979). Visual pigments in teleost fishes: effects of habitat, microhabitat, and behavior on visual system ecology. Sensory Processes 3, 95 131. Loew, E. R. (1994). A third, ultraviolet-sensitive, visual pigment in the Tokay Gecko (Gekko gekko). Vision Research 34, 1427 1431. doi: 10.1016/0042-6989(94)90143-0 Loew, E. R. & Lythgoe, J. N. (1978). The ecology of cone pigments in teleost fishes. Vision Research 18, 715 722. doi: 10.1016/0042-6989(78)90150-5 Loew, E. R. & McFarland, W. N. (1990). The underwater visual environment. In The Visual System of Fish (Douglas, R. H. & Djamgoz, M. B. A., eds), pp. 1 43. New York: Chapman & Hall. Losey, G. S., McFarland, W. N., Loew, E. R., Zamzow, J. P., Nelson, P. A. & Marshall, N. J. (2003). Visual biology of Hawaiian coral reef fishes. I. Ocular transmission and visual pigments. Copeia 2003, 433 454. MacNichol, E. F. (1986). A unifying presentation of photopigment spectra. Vision Research 26, 1543 1556. doi: 10.1016/0042-6989(86)90173-2 Mansfield, R. J. W. (1985). Primate photopigments and cone mechanisms. In The Visual System (Liss, A. & Fein, J. S. L, eds), pp. 89 106. New York: Chapman & Hall. McFarland, W. N. & Loew, E. R. (1994). Ultraviolet visual pigments in marine fishes of the family Pomacentridae. Vision Research 34(special uv issue), 1393 1396. McKaye, K. R., Howard, J. H., Stauffer, J. R. Jr., Morgan, R. P. II & Shonhiwa, F. (1993). Sexual selection and genetic relationships of a sibling species complex of bower building cichlids in Lake Malawˆi, Africa. Japan Journal of Ichthyology 40, 15 21. van der Meer, H. J. & Bowmaker, J. K. (1995). Interspecific variation of photoreceptors in four co-existing haplochromine cichlid fishes. Brain Behavior and Evolution 45, 232 240. Moran, P., Kornfield, I. & Reinthal, P. N. (1994). Molecular systematics and radiation of the Haplochromine cichlids (Teleostei: Perciformes) of Lake Malawˆi. Copeia 1994, 274 288. Muntz, W. R. A. (1976). Visual pigments of cichlid fishes from Malawˆi. Vision Research 16, 897 903. doi: 10.1016/0042-6989(76)90218-2 Parker, A. & Kornfield, I. (1997). Evolution of the mitochondrial DNA control region in the mbuna (Cichlidae) species flock of Lake Malawˆi, East Africa. Journal of Molecular Evolution 45, 70 83. Partridge, J. C. & DeGrip, W. J. A (1991). A new template for rhodopsin (vitamin A1-based) visual pigments. Vision Research 31, 619 630. doi: 10.1016/0042-6989(91)90002-M Ribbink, A. J., Marsh, B. A., Marsh, A. C., Ribbink, A. C. & Sharp, B. J. (1983). A preliminary survey of the cichlid fishes of rocky habitats in Lake Malawˆi. South African Journal of Zoology 18, 149 310. Stauffer, J. R. Jr., Bowers, N. J., Kellogg, K. A. & McKaye, K. R. (1997). A revision of the blue-back Pseudotropheus zebra (Teleostei: Cichlidae) complex from Lake Malawˆi, Africa, with description of a new genus and ten new species. Proceedings of the Academy of Natural Sciences of Philadelphia 148, 189 230.