Plasticity of opsin gene expression in cichlids from Lake Malawi

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1 Molecular Ecology (21) 19, doi: /j X x Plasticity of opsin gene expression in cichlids from Lake Malawi CHRISTOPHER M. HOFMANN, KELLY E. O QUIN, ADAM R. SMITH and KAREN L. CARLETON Department of Biology, University of Maryland, College Park, MD 2742, USA Abstract Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild-caught fish to lab-reared F 1 that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild-caught fish were also expressed in lab-reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the (violet) opsin, which was always expressed at higher levels in lab-reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi. Keywords: cichlid, genetics, opsin, plasticity, speciation, vision Received 5 December 29; revision received 23 February 21; accepted 3 March 21 Introduction Correspondence: Christopher M. Hofmann, Fax: ; chofma1@umd.edu An organism s ability to adapt to its environment over the course of a lifetime has important consequences for its fitness. Crucial tasks such as finding food, evaluating mates and avoiding predation may change as a result of development or in response to variable environmental conditions. Sensory systems play an important role in meeting these requirements and phenotypic plasticity in these systems may help organisms adjust to changing conditions throughout life. Phenotypic plasticity also has important evolutionary consequences (West-Eberhard 1989; Price et al. 23; Pigliucci 25; Aubin-Horth & Renn 29). It may facilitate the colonization of novel environments and allow populations to adapt to new fitness optima (Price et al. 23). Furthermore, previous work in several taxonomic groups, including cichlids, has demonstrated that synergistic changes in environmental conditions and sensory systems can promote population differentiation and speciation (Endler 1991; Endler et al. 21; Seehausen et al. 28). The visual system has become a classic system for studying sensory adaptation. The fundamental unit of the visual system is the photoreceptor, which is composed of an opsin protein bound to a light sensitive chromophore (Wald 1968). Interactions between these two molecules influence the wavelengths at which the resulting visual pigment absorbs maximally (Yokoyama 2). Early studies of visual ecology linked shifts in photoreceptor sensitivity to differences in environmental light (Loew & Lythgoe 1978; Levine & MacNichol 1979; Lythgoe 1979; Bowmaker 1995) and subsequent studies have identified the specific amino acid substitutions in the opsin proteins that generate these shifts (Yokoyama 2, 22; Hunt et al. 21, 27). However, amino acid substitutions represent one of several different molecular mechanisms that can influence spectral sensitivity. Comparative studies suggest that changes in opsin gene expression can also be used to

2 CICHLID CONE OPSIN PLASTICITY 265 tune visual pigment sensitivity across species (Carleton & Kocher 21; Spady et al. 26; Hofmann et al. 29; see also Hofmann & Carleton 29). Changes in opsin gene expression may also be observed within a single species over the course of development (Beaudet & Hawryshyn 1999). For example, juvenile salmon and trout have UV sensitive cones, but during the process of smoltification (whereby they undergo physiological changes in preparation for a pelagic, marine lifestyle), they lose UV sensitivity (Hawryshyn et al. 1989; Bowmaker & Kunz 1987). In adulthood, they regain their UV sensitive cones before migrating back to their breeding grounds (Beaudet et al. 1997; Flamarique 2). Ontogenetic changes have also been observed in several other species of fish including tilapia, bream and eels (Archer et al. 1995; Carleton et al. 28; Shand et al. 28). Changes in opsin gene expression appear to play an important role in sensory adaptation (Hofmann & Carleton 29). Killifish provide a classic example of this phenomenon. Fuller et al. (23) observed differences in cone abundance between killifish living in clear springs and tannin stained waters and subsequent work demonstrated corresponding changes in cone opsin expression. Killifish from clear springs had higher levels of (UV) and (violet) opsin expression, while killifish from tannin stained waters had higher levels of (green) and (red) opsin expression (Fuller et al. 24). These differences could be recreated experimentally in greenhouses by raising fish in either clear water or water stained with LiptonÒ tea (Fuller et al. 25). More recently, similar differences were observed in sea bream that were raised under yellow filtered light (Shand et al. 28). The results of these two studies suggest that opsin expression may change in a manner that is concordant with environmental light, i.e. sensitivity is increased in regions of the spectrum where light is abundant (Hofmann & Carleton 29). Interestingly, a study in the New World cichlid Aequidens pulcher suggested that rearing fish under blue light conditions resulted in a reduction in the number of blue sensitive photoreceptors (Kröger et al. 1999; Wagner & Kroger 2). In this case, an excess of light in one region of the spectrum led to a reduction in sensitivity within that region. The authors interpreted this decrease as a compensatory mechanism that helps maintain colour constancy (Wagner & Kröger 25). East African cichlids are some of the most rapidly speciating animals on the planet. Lake Malawi boasts hundreds of closely related species that have evolved within the past 1 2 million years. Despite their recent evolutionary origin, cichlids from Lake Malawi exhibit diverse visual systems that span the visible and a portion of the ultraviolet (UV) region of the electromagnetic spectrum (Carleton 29; Hofmann et al. 29). This diversity is achieved by differentially expressing a subset (typically three to four) of seven ancestral cone opsin genes (Carleton & Kocher 21). These genes include the shorter-wavelength (UV), (violet) and (blue) genes, which are typically expressed within morphologically distinct single cones in the retina and the longer-wavelength (blue-green), a & b (both green), and (red) genes that are expressed in double cones (Fig. 1a) (Fernald 1981; Parry et al. 25; Carleton et al. 25, 28). The shifts in spectral sensitivity that result from differentially expressing these opsin genes represent some of the largest observed in vertebrates (Carleton 29; Hofmann & Carleton 29; Hofmann et al. 29). Two lines of evidence suggest that opsin expression is genetically controlled in African cichlids. First, captive reared Oreochromis niloticus (a riverine cichlid) undergo an ontogenetic progression from short- to long-wavelengths in a constant laboratory light environment (Spady et al. 26; Carleton et al. 28). Second, two genetic crosses between cichlids that express different subsets of opsins suggest that opsin gene expression is controlled by a few loci of large effect (Carleton et al. 21). However, since other studies have found both a genetic and an environmental component to opsin gene expression (e.g. Fuller et al. 25) and the degree of plasticity in African cichlids remains unknown, we sought to test whether UV minus, reduced intensity laboratory rearing conditions influenced opsin gene expression. In particular, we were interested in testing the hypothesis that (UV) opsin expression was genetically determined and maintained in the absence of UV light. Materials and methods Sample selection We sampled 1 individuals each from five different species during a field expedition to southern Lake Malawi in 28. All individuals were collected from clear, rocky reefs at shallow depths (<1 m) at Thumbi West ( S E) and Mbenji Islands ( S E). Four of these species fell into a short-wavelength visual group that is characterized by expression of the (UV) opsin in single cones (Carleton 29; Hofmann et al. 29). These species included Metriaclima mbenji, Metriaclima lombardoi, Melanochromis sp. black-white johanni and Labeotropheus trewavasae. One species, Melanochromis auratus, fell into a middle-wavelength group that is characterized by expression of the (violet) opsin in single cones. These groupings are based on cluster analyses of opsin

3 266 C. M. HOFMANN ET AL. a Relative irradience b Relative absorbance c 5 Relative quantum catch Wavelength (nm) Wavelength (nm) β β α Fig. 1 (a) Normalized irradiance spectra from Lake Malawi (blue) and our tropical aquaculture facility (red). (b) Spectral sensitivity of visual pigments expressing each of the seven cichlid cone opsins. Curves were generated using the equations of Govardovskii et al. (2). a is illustrated using a dotted line. (c) Calculated quantum catch that a visual pigment expressing each opsin would produce in the wild (solid bars) and in the lab (horizontal lines). Because a and b are functionally and genetically similar, only b was included in this calculation. expression as well as weighted estimates of single and double cone sensitivity (Carleton 29; Hofmann et al. 29). These species differed in their foraging modes. One species, L. trewavasae, is specialized for scraping algae off rocks (Albertson & Kocher 26; Ribbink et al. 28). Metriaclima tend to forage on zooplankton but are facultative omnivores, while Melanochromis forage on loose phytoplankton (McKaye & Marsh 1983; Ribbink et al. 1983; Konings 199; Hofmann et al. 29). We also collected fish from these same populations and bred them for one generation in our tropical aquaculture facility at the University of Maryland, College Park. The light environment in which these fish were raised lacked any UV component (Fig. 1a) and was reduced in overall intensity (25 5 lux) relative to shallow water conditions in the wild (which can be greater than 1 5 lux). We then sampled ten lab-reared F 1 from each species. M. auratus, L. trewavasae and M. mbenji were all sampled from a single brood. For Melanochromis sp. black-white johanni we sampled lab-reared fish from two possibly unrelated broods, five individuals per brood. For M. lombardoi we again sampled lab-reared fish from two possibly unrelated broods, seven from one and three from the other. All individuals were sampled after males from the brood began to display signs of nuptial coloration. Sampling was conducted randomly with respect to sex. All specimens were euthanized using an overdose of MS222 and the retinas were isolated and stored in RNAlater at )2 C. Quantifying opsin expression We used previously published methods to quantify opsin expression (Carleton & Kocher 21; Spady et al. 26). In brief, RNA from each specimen was extracted and reverse transcribed using commercially available kits and reagents (RNeasy, Qiagen and Superscript III, Invitrogen). Real-time, quantitative PCR reactions for each of the six cone opsins were run in parallel using opsin specific primers and probes. As in previous studies, the genetically and functionally similar a and b opsins were quantified together (Carleton et al. 25, 28; Spady et al. 26). A construct that contained a tandem array of segments from each opsin was used to calculate the PCR efficiency for each gene. We then used the critical cycle numbers and PCR efficiencies to calculate the relative expression of each opsin (see equations in Carleton & Kocher 21; Spady et al. 26). Reactions for all six opsins were run at least twice on separate reaction plates and an equal number of wild-caught and labreared samples were run on the same plate. A single master mix of primers and Taqman probes was used for each opsin gene for the entire experiment. Quantum catches We estimated the relative quantum catch (Q) that a visual pigment composed of each opsin protein would

4 CICHLID CONE OPSIN PLASTICITY 267 have under wild and laboratory conditions using the equation: Z Q ¼ IðkÞRðkÞdk where I(k) is the normalized irradiance spectrum (Fig. 1a) and R(k) is the photoreceptor absorption calculated using the equations of Govardovskii et al. (2) (Fig. 1b). Photoreceptor absorption curves were generated using k max values from heterologously expressed O. niloticus opsins (which are thought to be similar to those of the riverine ancestor that colonized Lake Malawi). We used a previously published calculation of the irradiance at 2 m depth off the coast of Thumbi West Island to represent wild conditions (Hofmann et al. 29). Irradiance in the lab was measured using an Ocean Optics USB4 spectrometer. Because we were interested in the relative performance of each visual pigment, we normalized the quantum catch of each visual pigment by the sum of the quantum catches from all visual pigments. Previous work suggests that ocular media are not limiting in cichlids from Lake Malawi (Hofmann et al. 21). Therefore, the potential influence of ocular media was not included in this estimate. Statistical tests We used t-tests to examine whether there were significant differences in mean opsin expression between wild-caught and lab-reared fish that came from a single brood. Before running these analyses, we examined whether there were any significant deviations from normality using a Shapiro Wilk test. We also examined whether wild and lab individuals differed in their variance using Bartlett s test. Most of the expression data did not differ significantly from a normal distribution, although a few genes did require ln transformation (Table 1). After these transformations, there were no significant differences in variance between wild and lab fish (Table 1). and expression in M. mbenji and expression in L. trewavasae could not be normalized. Wilcox tests were used for these three comparisons. We used generalized least squares to account for brood effects in the comparisons between lab-reared and wild-caught M. black-and-white johanni and M. lombardoi. This method can account for correlations between observations when estimating model parameters. We specified a correlation of.5 among members of each brood. All statistical tests were performed in R v2.8 (R Core Development Team 29). For all statistical analyses, we used a Bonferroni-corrected significance threshold of.1 to reduce the risk of false positives due to multiple tests (only five opsin genes were tested; no lab or wild individuals from any species expressed ). Results Expression differences We found significant differences in opsin expression between wild-caught and lab-reared individuals in all five species (Table 1). Melanochromis sp. black-white johanni and M. lombardoi each had two opsins that differed significantly between wild-caught and labreared fish, L. trewavasae and M. mbenji had three and M. auratus had four (Fig. 2, Table 1). was the only opsin to vary significantly across all species and in all cases expression was increased in labreared individuals (Fig. 2, Table 1). All four species that expressed appreciable levels of in the wild also expressed in the lab, although there were significant differences between lab and wild expression in three of these species (Fig. 2, Table 1). None of the species surveyed expressed appreciable levels of, either in wild-caught or lab-reared individuals (Fig. 2). While all species had significant differences in opsin expression between wild-caught and lab-reared individuals, the magnitude of these differences was greatest in Melanochromis sp. black-white johanni, M. lombardoi, and M. mbenji. Their expression decreased by 33%, 72% and 69%, respectively, while their expression increased by 14%, 8% and 7% (Fig. 2). These changes can be contrasted with those observed in L. trewavasae, which had only a 12% decrease in expression and a 2% increase in (Fig. 2). These findings suggest that plasticity varies across species. Single and double cone expression To examine further opsin expression in wild-caught and lab-reared individuals, we plotted the relative expression of single and double cone opsins in triangular space using a freely available Excel spreadsheet (Graham & Midgley 2). These plots illustrated the degree and the axes along which gene expression was changing within single and double cones. For example, although several opsin genes differed significantly between wild-caught and lab-reared L. trewavasae (e.g. and ), the magnitude of these differences was small and they overlapped considerably in single and double cone space (Fig. 3). For this species in particular, opsin expression appeared to be tightly clustered in both wild-caught and lab-reared individuals.

5 268 C. M. HOFMANN ET AL. Table 1 Summary of statistical tests. Values less than or equal to.1 were considered significant and are highlighted in bold Shapiro Wilks Transfromation applied Bartlett s Statistical test P Melanochromis auratus t.7 Melanochromis spp. black-white johanni.2 ln.456 GLS.48 Labeotropheus trewavasae t.15 Metriaclima lombardoi. ln.983 GLS.39 M. mbenji t.43 Melanochromis auratus t <.1 Melanochromis spp. black-white johanni GLS.28 Labeotropheus trewavasae t.3 Metriaclima lombardoi GLS.32 M. mbenji.5 Wilcox.684 Melanochromis auratus t.279 Melanochromis spp. black-white johanni GLS.783 Labeotropheus trewavasae t.3 Metriaclima lombardoi GLS.28 M. mbenji t.3 Melanochromis auratus t <.1 Melanochromis sp. black-white johanni.1 ln.95 GLS <.1 Labeotropheus trewavasae t.1 Metriaclima lombardoi GLS <.1 M. mbenji t.1 Melanochromis auratus.4 ln.23 t.7 Melanochromis spp. black-white johanni.772 ln.697 GLS.4 Labeotropheus trewavasae. Wilcox.684 Metriaclima lombardoi.1 ln.168 GLS <.1 M. mbenji.8 Wilcox <.1 Probability that the distribution sampled differs from normal. Probability that lab and wild individuals differ in their variance. Probability that mean opsin expression differs between lab and wild individuals. Although significant, overall expression was very low. Similarly, although there were statistically significant differences in expression between wild and lab M. auratus, plotting individuals in single cone space also suggested that these differences were small in magnitude (Fig. 3). Since M. auratus was the one species from the middle-wavelength group, this was likely because was already expressed predominantly in the single cones. We do note that one wild-caught M. auratus individual expressed the short-wavelength, rather than the middle-wavelength, gene set. This individual was a female that may have been misidentified in the field; therefore, she was excluded from the statistical analyses (although reperforming the analysis while including this individual did not influence which opsins were significantly different). Finally, comparing the single and double cone plots of both species of Metriaclima suggested that they responded in a similar fashion to changes in their rearing environment. Both species showed almost identical variation along the axis. Their double cone profiles also appeared quite similar, with both species falling near the vertex (due to high levels of expression) and slight variation along both the and axes (Fig. 3). Quantum catches Our estimated quantum catches suggest that visual pigments would perform differently under wild and laboratory conditions (Fig. 1c). In the wild, all visual pigments would perform relatively well. There was some variation, for example an -based visual pigment is predicted to have a lower quantum catch than an -based one. However, these differences were relatively minor, with a six-fold difference between the highest and lowest quantum catch. In the lab, the extent of these differences is increased by approximately an order of magnitude; the difference between the highest

6 CICHLID CONE OPSIN PLASTICITY 269 Relative expression (%) Relative expression (%) 8 M. auratus 8 M. sp "black-white johanni" M. lombardoi Relative expression (%) Relative expression (%) 8 M. mbenji Fig. 2 Mean opsin expression for each of the five species of Lake Malawi cichlids included in this study. Filled bars are wild-caught and open bars are labreared individuals. M. auratus (top left) has the middle-wavelength gene set, while the other four species have the short-wavelength set. Opsin genes that differed significantly in expression are marked with an asterisk. Error bars are ±SD. Note that expression was higher in lab-reared individuals from all species. Relative expression (%) 8 L. trewavasae and lowest quantum catch was over 6-fold. Because of the lack of UV illumination in the lab, an -based visual pigment would have virtually no quantum catch (Fig. 1c). Discussion Bounded plasticity We found that raising cichlids from Lake Malawi in a UV minus, reduced intensity light environment caused significant changes in opsin gene expression, although the magnitude of these changes varied across species. Our data also suggest that expression of the (UV) cone opsin does occur in the absence of UV light, albeit at lower levels in some species. In general, all of the opsins that were expressed in wild-caught fish were also expressed in lab-reared individuals, although several leaky genes that were expressed at low levels in the wild had much higher levels of expression in the lab. These results agree with recent work that found a significant genetic component to opsin expression in cichlids from Lake Malawi (Carleton et al. 21). Overall, our data suggest that there is some plasticity to opsin gene expression in cichlids from Lake Malawi, but this plasticity occurs within the framework of a genetically controlled expression profile. Several previous studies have demonstrated a direct relationship between photoreceptor abundance and gene expression (Fuller et al. 24; Carleton et al. 25, 28). Thus, our findings suggest that rearing conditions may influence overall retinal sensitivity. However, we do not yet know how the differences in gene expression that we observed are distributed within the retinal mosaic. could be co-expressed with in the same photoreceptors or visual pigments composed of these two opsins could form separate photoreceptors. These two different patterns of expression could generate very different sensory outcomes. If the two opsins are co-expressed, the sensitivity of individual photoreceptors would be broadened and shifted to longer wavelengths. Expression in separate photoreceptors would present the possibility of new opponent mechanisms and the potential to perceive additional colours. Spectral composition vs. intensity The wild and lab environments differ in intensity as well as spectral composition. In the wild, light levels at the water s surface can be upwards of 5 lux

7 27 C. M. HOFMANN ET AL. M. auratus Fig. 3 Triangle plots of opsin expression in single and double cone space. Filled circles represent wild-caught and open circles represent lab-reared individuals. Note that wild-caught and labreared M. auratus and L. trewavasae have little variation in single cone space, while both species of Metriaclima have considerable variation. M. sp. black-white johannii M. lombardoi M. mbenji L. trewavasae

8 CICHLID CONE OPSIN PLASTICITY 271 (although this decreases exponentially with depth), while light levels in our fish facility range from 25 to 5 lux. However, previous studies suggest that spectral composition (i.e. short- vs. long-wavelengths), rather than intensity, has a greater influence on photoreceptor abundance (reviewed in Wagner & Kröger 25). In addition, our estimates of visual pigment quantum catches support the role of spectral composition. An -based visual pigment is predicted to have virtually no quantum catch in the lab and lab-reared fish had lower levels of opsin expression. Although intensity differences cannot be ruled out, our findings suggest strongly that opsin expression corresponds to available light. Genetic inferences Our findings suggest that photoreceptor stimulation appears to play a role in opsin gene expression, although it remains to be seen whether the lack of stimulation or the relative increase in stimulation drives this change. We observed less opsin expression in an environment where an - based visual pigment would not be stimulated. An -based visual pigment would produce a greater quantum catch in the lab (approximately seven-fold higher than ) and lab-reared fish had higher levels of opsin expression. However, never appeared to be turned on, despite the fact that it would even further increase visual pigment quantum catch in the laboratory light environment (Fig. 1c). This absence of expression suggests the intriguing possibility that the ratio of : expression is controlled by a single genetic factor, and expression is controlled by a separate one. While speculative, these findings are supported by data from a genetic cross between species that express and (Carleton et al. 21). In this cross we observed transgressive expression of in the F 1 generation despite its absence in both parental species. Furthermore, and expression appear to vary inversely during the course of development. In several species, expression decreases and expression increases as a function of age (Carleton et al. 28). Future studies comparing wild-caught and labreared individuals from species that express will be particularly interesting. Previous work examining opsin expression in Dimidiochromis compressiceps, Tramitichromis intermedius and O. niloticus suggests that they all express high levels of, and, with little or no leaky expression of or in the UV minus lab light environment (Carleton et al. 28; Carleton 29). Gene expression in the lab agrees with expression of wild-caught individuals from D. compressiceps as well as Oreochromis spp. (Carleton 29). This lack of variation, combined with the fact that their lenses tend to block UV light (Hofmann et al. 21) suggests that species with this gene set may have less plasticity in their single cones or may not be as sensitive to UV plus and UV minus environments. Unfortunately, we were not able to include any species that express in our current study. No species from the rock dwelling (or Mbuna) clade in Lake Malawi sampled to date express (Hofmann et al. 29) and we did not collect members of the sand-dwelling clade, which express the long-wavelength gene set. Our findings also suggest that opsin expression may be more tightly regulated in some species (or genera) than others. We found differences in plasticity among the five species that we sampled. The two species from the genus Metriaclima, along with Melanochromis sp. black-white johanni, appeared to be more sensitive to environmental conditions than L. trewavasae. Although sampling errors, reduced genetic diversity among the F 1 siblings or family structure could play a role in this variation, we do not believe these factors alone could explain the pattern we observed among species for several reasons. First, several lab-reared F 1 fell outside the distribution of wild-caught parental phenotypes (Fig. 3). Second, we did not detect statistically significant differences in variance between wildcaught and lab-reared fish (Table 1). In fact, when multiple opsin genes are examined, our single and double cone plots suggest that lab-reared fish may have more variation in their expression profiles (Fig. 3). Finally, both plastic and non-plastic species came from single broods. A second alternative is that microhabitats of some species may more closely resemble the laboratory light conditions. Although we plan to test species from a greater range of depths and habitats in future studies, here we collected all species from shallow (<1 m) depths at similar clear, rocky locations. Therefore we feel this explanation alone is unlikely to explain our results. Whether these differences in plasticity arise from the genetic and regulatory architecture or whether there are specific feedback mechanisms that respond to environmental conditions remains to be seen. We are currently investigating the genetic basis of opsin expression by crossing species that express different sets of opsin genes and mapping expression in the F 2 (Carleton et al. 21). An intriguing avenue for future research would be to cross a less plastic species with a more plastic one, raise the offspring under UV plus and UV minus conditions and compare the locations of loci controlling plasticity and those controlling and expression.

9 272 C. M. HOFMANN ET AL. Evolutionary implications The plasticity that we observed in these five species may have important evolutionary implications. All individuals that were reared in the lab came from the same populations as our wild-caught fish. The fact that their offspring were plastic suggests that wild populations have the potential to adapt to novel environments, even if we do not observe much variation when we sample individuals from that population. This potential may have helped cichlids to colonize the rift lakes after they formed. A crucial question that remains is whether opsin expression can change throughout the lifetime of a single individual or whether it is set during a critical stage early in development. The plasticity that we observed could also have important implications for understanding sexual selection. Color has been demonstrated to play an important role in cichlid mate choice; therefore, changes in the visual pigment sensitivity of females (towards longer or shorter wavelengths) could exert a strong influence on male coloration (Endler 1992). Over time these changes may lead to divergence in phenotypes between populations that inhabit different light environments (e.g. Seehausen et al. 28). Furthermore, if female preferences are genetically coupled with male display traits (without direct or indirect benefits), the degree of plasticity present in female sensory systems could alter the trajectory of male elaboration (Fisher 193; Lande 1981). Our findings also have important implications for studies of mate choice that are conducted in laboratory settings. They suggest that fish raised under artificial light conditions may show different preferences from those in the wild due to plasticity in their sensory systems. Finally, plasticity in opsin expression may have facilitated trophic specialization in Lake Malawi. These trophic specializations are believed to have played an important role in cichlid diversification (Streelman & Danley 23; Jordan et al. 24) and previous work suggests that expression of is correlated with foraging mode (Hofmann et al. 29). Therefore, we feel it is worth noting that the species that exhibited the least plasticity in our study also has the most specialized feeding apparatus. Members of the genus Labeotropheus have subterminal mouths and teeth that are specialized for sheering epilithic (rock-associated) algae (Albertson & Kocher 26; Ribbink et al. 28). In contrast, species in the genus Metriaclima forage on plankton but are also facultative omnivores (McKaye & Marsh 1983; Ribbink et al. 1983). Although this remains an observation at present, examining plasticity across a broader range of species with divergent foraging habits may provide new insights into whether and how visual plasticity is adaptive. Future light manipulations We feel that differences between the light environment in Lake Malawi and our tropical aquaculture facility were responsible for the changes in opsin expression that we observed. However, we acknowledge that these wild and laboratory environments differ along a number of axes, including temperature, pressure, chemistry and diet. We plan to expand the breadth of our light manipulation experiments from general UV minus conditions to specific regions of the spectrum. We are particularly interested in examining conditions that mimic deep and turbid waters, as well as testing fish captured from these different environments. We would also like to use filters to examine how specific wavelengths of light influence opsin expression. Finally, we would like to see if we can rescue wild expression through the use of high intensity, full spectrum lights. These experiments will probe the limits of retinal plasticity. Conversely, they may also provide new insights into the limits of genetic regulation. Acknowledgements We would like to thank Thomas Kocher, Brian Dalton, Jennifer Ser, Ray Engeszer and Richard Zatha for help in collecting or measuring samples from Lake Malawi. We would also like to thank Bosco Rusuwa, the University of Malawi, and the staff at Lake Malawi National Park. The cichlid lab group at the University of Maryland provided valuable comments. Funding was provided by the National Science Foundation (IOB and IOS-84127) and the University of Maryland. References Albertson R, Kocher T (26) Genetic and developmental basis of cichlid trophic diversity. Heredity, 97, Archer S, Hope A, Partridge JC (1995) The molecular basis for the green-blue sensitivity shift in the rod visual pigments of the European Eel. Proceedings of Royal Society of London Series B, 262, Aubin-Horth N, Renn SCP (29) Genomic reaction norms: using integrative biology to understand molecular mechanisms of phenotypic plasticity. Molecular Ecology, 18, Beaudet L, Hawryshyn CW (1999) Ecological aspects of vertebrate visual ontogeny. In: Adaptive Mechanisms in the Ecology of Visio (eds Archer S, Djamgoz M, Loew E, Partridge J, Vallerga S). pp , Kluwer Academic Publishers, Cornwall. Beaudet L, Novales Flamarique I, Hawryshyn C (1997) Cone photoreceptor topography in the retina of sexually mature Pacific salmonid fishes. Journal of Comparative Neurology, 383, Bowmaker JK (1995) The visual pigments of fish. Progress in Retinal and Eye Research, 15, Bowmaker JK, Kunz YW (1987) Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the

10 CICHLID CONE OPSIN PLASTICITY 273 brown trout (Salmo trutta): age dependent changes. Vision Research, 27, Carleton K (29) Cichlid fish visual systems: mechanisms of spectral tuning. Integrative Zoology, 4, Carleton KL, Kocher TD (21) Cone opsin genes of African cichlid fishes: tuning spectral sensitivity by differential gene expression. Molecular Biology and Evolution, 18, Carleton KL, Parry JWL, Bowmaker JK, Hunt DM, Seehausen O (25) Colour vision and speciation in Lake Victoria cichlids of the genus Pundamilia. Molecular Ecology, 14, Carleton KL, Spady TC, Streelman JT et al. (28) Visual sensitivities tuned by heterochronic shifts in opsin gene expression. BMC Biology, 6:22, doi:1.1186/ Carleton KL, Hofmann CM, Klisz C et al. (21) Genetic basis of differential opsin gene expression in cichlid fishes. Journal of Evolutionary Biology, 23, Endler JA (1991) Variation in the appearance of guppy color patterns to guppies and their predators under different visual conditions. Vision Research, 31, Endler JA, Basolo A, Glowacki S, Zerr J (21) Variation in response to artificial selection for light sensitivity in guppies (Poecilia reticulata). American Naturalist, 158, Fernald RD (1981) Chromatic organization of a cichlid fish retina. Vision Research, 21, Fisher RA (193) The Genetical Theory of Natural Selection. Clarendon Press, Oxford. Flamarique IN (2) The ontogeny of ultraviolet sensitivity, cone disappearance and regeneration in the sockeye salmon Oncorhynchus nerka. The Journal of Experimental Biology, 23, Fuller RC, Fleishman LJ, Leal M, Travis J, Loew E (23) Intraspecific variation in retinal cone distribution in the bluefin killifish, Lucania goodei. Journal of Comparative Physiology A, 189, Fuller RC, Carleton KL, Fadool JM, Spady TC, Travis J (24) Population variation in opsin expression in the bluefin killifish, Lucania goodei: a real-time PCR study. Journal of Comparative Physiology A, 19, Fuller RC, Carleton KL, Fadool JM, Spady TC, Travis J (25) Genetic and environmental variation in the visual properties of bluefin killifish, Lucania goodei. Journal of Evolutionary Biology, 18, Govardovskii V, Fyhrquist N, Reuter T, Kuzmin D, Donner K (2) In search of the visual pigment template. Visual Neuroscience, 17, Graham DJ, Midgley NG (2) Graphical representation of particle shape using triangular diagrams: an Excel spreadsheet method. Earth Surface Processes and Landforms, 25, Hawryshyn CW, Arnold MG, Chaisson DJ, Martin PC (1989) The ontogeny of ultraviolet photosensitivity in rainbow trout (Salmo gairdneri). Visual Neuroscience, 2, Hofmann CM, Carleton KL (29) Gene duplication and differential gene expression play an important role in the diversification of visual pigments in fish. Journal of Integrative and Comparative Biology, 49, Hofmann CM, O Quin KE, Marshall NJ et al. (29) The eyes have it: regulatory and structural changes both underlie cichlid visual pigment diversity. PLoS Biology, 7, e1266. Hofmann CM, O Quin KE, Marshall NJ, Carleton KL (21) The relationship between lens transmission and opsin gene expression in cichlids from Lake Malawi. Vision Research, 5, Hunt D, Dulai K, Partridge J, Cottrill P, Bowmaker J (21) The molecular basis for spectral tuning of rod visual pigments in deep-sea fish. Journal of Experimental Biology, 24, Hunt DM, Carvalho LS, Cowing JA et al. (27) Spectral tuning of shortwave-sensitive visual pigments in vertebrates. Photochemical and Photobiological Sciences, 83, Jordan R, Howe D, Juanes F, Stauffer J Jr, Loew E (24) Ultraviolet radiation enhances zooplanktivory rate in ultraviolet sensitive cichlids. African Journal of Ecology, 42, Konings A (199) Cichlids and All the Other Fishes of Lake Malawi. TFH Publications, Neptune City. Kröger RHH, Bowmaker JK, Wagner HJ (1999) Morphological changes in the retina of Aequidens pulcher (Cichlidae) after rearing in monochromatic light. Vision Research, 39, Lande R (1981) Models of speciation by sexual selection on polygenic traits. Proceedings of the National Academy of Sciences, USA, 78, Levine J, MacNichol E (1979) Visual pigments in teleost fishes: effects of habitat, microhabitat and behavior on visual system evolution. Sensory Processes, 3, Loew ER, Lythgoe JN (1978) The ecology of cone pigments in teleost fishes. Vision Research, 18, Lythgoe J (1979) The Ecology of Vision. Clarendon Press, Oxford. McKaye KR, Marsh A (1983) Food switching by two specialized algae-scraping cichlid fishes in Lake Malawi, Africa. Oecologia, 56, Pigliucci M (25) Evolution of phenotypic plasticity: where are we going now? Trends in Ecology and Evolution, 2, Price TD, Qvarnstrom A, Irwin DE (23) The role of phenotypic plasticity in driving genetic evolution. Proceedings of Royal Society of London Series B, 27, R Development Core Team (29). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN , URL Ribbink A, Marsh A, Ribbink C, Sharp B (1983) A preliminary survey of the cichlid fishes of rocky habitats in Lake Malawi. South African Journal of Zoology, 18, Ribbink A, Marsh A, Marsh B, Sharp B (28) The zoogeography, ecology and taxonomy of the genus Labeotropheus Ahl, 1927, of Lake Malawi (Pisces: Gichlidae). Biological Journal of the Linnean Society, 79, Seehausen O, Terai Y, Magalhaes IS et al. (28) Speciation through sensory drive in cichlid fish. Nature, 455, Shand J, Davies WL, Thomas N et al. (28) The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri. Journal of Experimental Biology, 211, Spady TC, Parry JWL, Robinson PR et al. (26) Evolution of the cichlid visual palette through ontogenetic

11 274 C. M. HOFMANN ET AL. subfunctionalization of the opsin gene arrays. Molecular Biology and Evolution, 23, Streelman JT, Danley PD (23) The stages of vertebrate evolutionary radiation. Trends in Ecology and Evolution, 18, Wagner HJ, Kroger RHH (2) Effects of long-term spectral deprivation on the morphological organization of the outer retina of the blue acara (Aequidens pulcher). Philosophical Transactions of the Royal Society B: Biological Sciences, 355, Wagner H-J, Kröger RHH (25) Adaptive plasticity during the development of colour vision. Progress in Retinal and Eye Research, 24, Wald G (1968) The molecular basis of visual excitation. Nature, 219, West-Eberhard MJ (1989) Phenotypic plasticity and the origins of diversity. Annual Review of Ecology, Evolution, and Systematics, 2, Yokoyama S (2) Molecular evolution of vertebrate visual pigments. Progress in Retinal and Eye Research, 19, Yokoyama S (22) Molecular evolution of color vision in vertebrates. Gene, 3, C.H. is interested in the evolution of sensory systems and colour signals. K.O. is interested in the role of gene expression divergence in the evolution of adaptive phenotypes. A.S. is interested in integrative visual neuroscience and in exploring the relationship between genetic and neural components of colour vision. K.C. is interested in the evolution of visual systems and communication.