Four eyes are better than two: how proteins show evolutionary adaptation in a four-eyed fish

By Lian Guo (Scripps College) [Edited by Lars Schmitz, as part of BIOL 167 “Sensory Evolution”, an upper division class at the W.M. Keck Science Department. Written for educational purposes only.]

Two eyes are usually considered the norm, probably because most of the organisms we come in contact with only have two. Why would you ever need more than two? If an organism remains in the same type of environment throughout their lifespan, one set of eyes is probably sufficient for survival. However, there are organisms that live at the direct interface of two habitats, which results in selection favoring morphologies adapted to both environments. While the idea of a “four-eyed” fish may seem quite comical at first, this fish is an example of adaptation at its finest. Anableps anableps is a small freshwater fish that has evolved dual optical systems within its eyes (Figure 1). As it sits just under the water’s surface, the upper regions of its eyes can see above the waterline while its body and lower part of its eyes remain underwater. Isn’t that cool?

Figure 1. a) A picture of Anableps anableps sitting just below the waterline (By H. Zell (Own work), via Wikimedia Commons). [http://commons.wikimedia.org/wiki/File:Anableps_anableps_01.jpg]

Figure 1. a) A picture of Anableps anableps sitting just below the waterline (By H. Zell (Own work), via Wikimedia Commons). [http://commons.wikimedia.org/wiki/File:Anableps_anableps_01.jpg]

Figure 1. b) A drawing of Anableps anableps from 1909 (By Unknown (Reptiles, amphibia, fishes and lower chordata) [Public domain], via Wikimedia Commons). [http://commons.wikimedia.org/wiki/File:Anableps_anableps2.jpg]

Figure 1. b) A drawing of Anableps anableps from 1909 (By Unknown (Reptiles, amphibia, fishes and lower chordata) [Public domain], via Wikimedia Commons). [http://commons.wikimedia.org/wiki/File:Anableps_anableps2.jpg]

Each eye has two distinct pupil apertures (entrance of light), corneas (refraction of light), and hemiretinas (reception of light), but only one optic nerve to deliver visual information to the brain (Oliveira et al., 2006). Interestingly, the lines of sight for aerial and aquatic vision cross one another, meaning that the dorsal (back) hemiretina and ventral (front) cornea receive aquatic light, while the ventral hemiretina and dorsal cornea receive aerial light (Figure 2). Anableps anableps eyes have a single ovoid lens, which is unordinary for freshwater fish which typically have spherical lenses. It is thought that this ovoid lens allows for aerial and aquatic light to simultaneously come into focus.

Figure 2. A schematic drawing of A. anableps eye. (AeL- aerial line-of-sight; AqL - aquatic line-of-sight; L - lens; ON - optic nerve; DT - dorsal tip of the retina; D - dorsal retina; M - medial retina; V - ventral retina). [Figure from Owens et al. (2012)].

Figure 2. A schematic drawing of A. anableps eye. (AeL- aerial line-of-sight; AqL – aquatic line-of-sight; L – lens; ON – optic nerve; DT – dorsal tip of the retina; D – dorsal retina; M – medial retina; V – ventral retina). [Figure from Owens et al. (2012)].

Although this extreme morphology was notable enough to contribute to A. anableps’ common name (Largescale foureyes), it was not well-supported experimentally what purpose the dual system held. Presumably, one region of the eye was for aerial sight while the other region was for aquatic sight. However, this was not confirmed at a molecular level. Current research has focused on associating genotypes of opsins with observed phenotypes, and describing both in the context of ecological factors. In order to explore molecular differences between the dorsal and ventral eye regions, Owens et al. (2012) examined the opsins expressed in both A. anableps retinas and compared their findings to a “normal morphology” freshwater fish from a sister genus, Jenynsia onca. All vertebrate vision is dependent on five families of opsin proteins: sws1 and sws2 (short wavelength sensitive), rh2-1 and rh2-2 (middle wavelength sensitive), and lws (long wavelength sensitive) (Owens et al., 2012). In order to map the type and concentration of each type of opsin, the authors used in situ hybridization with opsin riboprobes to localize opsin DNA sequences in both A. anableps and J. onca.

Both species exhibited a normal distribution of cones with sws1, sws2b, and rh2-2 opsins; neither exhibited sws2a opsins (Figure 3). While J. onca had uniform expression of rh2-1 expression across its retina, adult A. anableps had a large number of ventral cone cells containing rh2-1, but rh2-1 was only present in a small patch of cones in the dorsal eye retina. In contrast, lws opsins were found to have the opposite distribution in A. anableps, only being detected in the dorsal half of the retina. Cones expressing lws were limited to the dorsal region of the retina of two J. onca specimens.

Figure 3. Expression domains in dorsal and ventral retinas by opsin type and wavelength sensitivity. [Figure from Owens et al. (2012)].

Figure 3. Expression domains in dorsal and ventral retinas by opsin type and wavelength sensitivity. [Figure from Owens et al. (2012)].

For A. anableps, the striking difference in lws and rh2-1 distribution may have evolved with their unusual eye morphology. These observations indicate that wavelength sensitivity differs in dorsal and ventral regions of the retina. Owens et al. (2012) hypothesized that the reduction in rh2-1 transcripts in the dorsal region could be a trade-off that allows for increased lws expression. More lws expression would mean enhanced sensitivity to long wavelength light (543-576 nm, yellow colors). In the brackish waters that A. anableps lives in, there is often dissolved organic matter which will shift light abundance to longer wavelengths; the prevalence of longer wavelengths underwater matches the increased expression of lws in the aquatic retina of the A. anableps eye. Having rh2-1 opsins in the ventral eye retina closely matches opsin distribution of other surface-dwelling fish like J. onca. rh2-1 is sensitive to 492-539 nm wavelengths (green color). Since downwelling light is most prevalent at 500 nm, and upwelling light peaks around 580 nm, it appears that A. anableps eyes are well-adapted to their respective fields of view.

While no other studies look specifically at the difference in A. anableps opsins between the dorsal and ventral hemiretinas, there are several others that examine other morphological and molecular differences between eye regions. A. anableps dorsal corneas were found to have a lower density of cells in comparison to ventral corneas, potentially due to osmotic pressure differences between air and water (Simmich et al., 2012). Swamynathan et al. (2003) also found the dorsal cornea to be thicker, flatter, and contain 15x more glycogen than the ventral cornea. The authors of this paper proposed this specialization protected the dorsal cornea against UV irradiation and desiccation. In general, previous findings support the specialized adaptation of the dorsal and ventral sections of A. anableps eyes, correlating to each region’s field of view.

While the evidence is building for specialized adaptation in this species, there is still more to be explored. Owens et al. (2012) stated that behavioral tests on wavelength sensitivity and discrimination in both A. anableps fields of view should be conducted in order to understand how differences in expression domains influence vision. This experiment also had a relatively small sample size (2 adult and 2 juvenile A. anableps) which presumably came from the same population. If the authors can duplicate the study for another population of A. anableps, it would reinforce that this is not a localized adaptation. Furthermore, this experiment could be conducted with additional species that are closely related to Anableps and Jenynsia, allowing to more carefully trace evolutionary modifications in response to ecological transitions in this group of fishes. One should also repeat this kind of analysis for other species of fish that live at the surface of water to see if this opsin expression pattern exists elsewhere. It would also be interesting to gain a better understanding of how information from both eye regions combine to send one message to the brain. In all, Owens et al. 2012 provides molecular-based support for adaptation in A. anableps eyes, adding to the growing literature concerning specialized structures within a single sensory system. In particular this multi-region optical system seems to have evolved in response to physical constraints that result from living at the interface of water and air.

References

Oliveira, F. G., Coimbra, J. P., Yamada, E. S., Montag, L. F. A., Nascimento, F. L., Oliveira, V. A., Da Mota, D. L., Bittencourt, A. M., Da Silva, V. L., Da Costa, B. L. D. S. A. 2006 Tophographic analysis of the ganglion cell layer in the retina of the four-eyed fish Anableps anableps. Vis Neurosci 23, 879-886. (DOI: 10.10170S0952523806230232)

Owens, G. L., Rennison, D. J., Allison, W. T., Taylor, J. S. 2012 In the four-eyed fish (Anableps anableps), the regions of the retina exposed to aquatic and aerial light do not express the same set of opsin genes. Biol Lett 8, 86-89. (DOI:10.1098/rsbl.2011.0582)

Simmich, J., Temple, S. E., Collin, S. P. 2012 A fish eye out of water: epithelial surface projections on aerial and aquatic corneas of the ‘four-eyed fish’ Anableps anableps. Clin Exp Optom 95, 140-145 (DOI:10.1111/j.1444-0938.2011.00701.x)

Swamynathan, S. K., Crawford, M. A., Robison, G., Kanungo, J., Piatigorsky, K. 2003 Adaptive differences in the structure and macromolecular compositions of the air and water corneas of the “four-eyed” fish (Anableps anableps). FASEB Journal 17, 1996-2005. (DOI: 10.1096/fj.03-0122com)

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