Bull Mar Sci. 93(2):339–353. 2017 research paper https://doi.org/10.5343/bms.2016.1044
Species-specific development of retinal architecture in elopomorph fishes: adaptations for harvesting light in the dark
1 Department of Biological Michael S Grace 1 * Sciences, Florida Institute of 2 Technology, 150 West University Scott M Taylor Blvd., Melbourne, Florida 32901. 2 Department of Biology, University of West Florida, 11000 ABSTRACT.—Elopomorph fishes are distinguished in University Pkwy, Pensacola, part by a shared leptocephalus larval form. These glass- Florida 32514. clear, ribbon-like larvae are remarkably similar across the * Corresponding author email: elopomorpha, yet they mature to radically different forms as
Because there is substantially less light available for vision, life at night poses unique challenges for the visual system of animals, particularly for species that are heavily reliant upon vision for predation, predator avoidance, and/or conspecific interactions including courtship and reproduction. The difference in light levels between day and night may span more than 10 orders of magnitude, which is far greater than the sensitivity range of a given class of vertebrate retinal photoreceptor
Bulletin of Marine Science 339 © 2017 Rosenstiel School of Marine & Atmospheric Science of the University of Miami 340 Bulletin of Marine Science. Vol 93, No 2. 2017 cells. In fact, even as fish move between light and deep shadow in the daytime, or between darkness and locations illuminated by artificial light at night, the changes in illumination level may require adjustments of the retina to maintain visual function. The outer retinas of vertebrate animals contain two morphologically and functionally distinct types of light-detecting photoreceptor cells. Rod photoreceptors are highly-sensitive light detectors that allow monochromatic vision in low-light conditions. Cone photoreceptors have much lower absolute sensitivity to light such that they are functional only at higher light conditions, and because many species possess multiple types of cones with distinct spectral sensitivities, cones often provide color vision. Therefore, a highly rod-dominated retina, that is, a retina with a high rod:cone ratio, is better adapted for low-light or nocturnal vision than a retina with a lower rod:cone ratio. The rod:cone ratio is only one of a variety of vertebrate retinal features that may be subject to selection pressure, allowing different species (or different life stages within a species) to effectively occupy distinct temporal and light-level niches. The superorder Elopomorpha comprises a group of bony fishes that possess an unusual larval form known as the leptocephalus. These highly elongated and laterally-flattened larvae have very small heads relative to the much larger, glass- clear, ribbon-like body, yet their heads contain well-developed and highly-pigmented eyes that are highly rod dominated for vision under dim light conditions (Taylor and Grace 2005, Taylor et al. 2011a,b, 2015), compared with the larval-stage retinas of most other teleost species, which contain only cones (Evans and Fernald 1993, Negishi and Wagner 1995, Shand 1997, Helvik et al. 2001, Lara 2001). In a comprehensive analysis of leptocephalus visual development, Taylor, Grace and colleagues discovered that as elopomorphs develop through a series of life stages, their retinas change in remarkable ways, and the retinal development trajectory can be dramatically different among elopomorph taxa (Taylor and Grace 2005, Taylor et al. 2011a,b, 2015). For example, in their adult forms, the Atlantic tarpon (Megalops atlanticus Valenciennes, 1847) and ladyfish (Elops saurus Linnaeus, 1766) are large, fast-swimming piscivorous fishes that are frequently observed feeding at or near the water surface during the day, during crepuscular phases, and at night. However, their retinas change dramatically from the larval stage to the adult stage, and they change in ways that are very different from that of the diurnal, high-light dwelling bonefish [Albula vulpes (Linnaeus, 1758)]. The purpose of the work presented here is to assess some of the post-larval specializations that develop in the retinas of different members of the Elopomorpha with respect to their abilities to efficiently detect light for visual processes under different lighting conditions. This work is discussed in the context of fish behavior and the conservation of species that have significant ecological and economic values.
Materials and Methods
Animals and Tissue Preparation.—All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Florida Institute of Technology. Settlement-stage M. atlanticus, E. saurus, and A. vulpes were cap- tured by plankton net during night-time flood tides at Sebastian Inlet, Florida, as described previously (Shenker et al. 1993, Taylor and Grace 2005), immediately euthanized in 100 mg L−1 tricane methanesulphonate (MS-222), and stored in Grace and Taylor: Adaptations of the elopomorph retina 341 paraformaldehyde-based fixative (see Taylor and Grace 2005). For light-adapted ju- venile E. saurus and M. atlanticus, fish were collected during bright daylight hours from impounded mangrove marshes adjacent to the Indian River Lagoon in Brevard County, Florida, by either seine net or hook-and-line fishing. Fish were immediately euthanized with 500 mg L−1 tricane methanesulphonate (MS-222) and eyes removed and placed in fixative solution. For dark-adapted juvenile E. saurus and M. atlan- ticus, fish were collected at dusk and held for 2 hrs in complete darkness prior to enucleation under dim red light. Juvenile A. vulpes were collected during bright day- light hours by seine net from 1-m depth clear water at Key Biscayne and Naples, Florida. Adult M. atlanticus and E. saurus were collected at night by hook-and-line at Sebastian Inlet, Florida. Adult A. vulpes were collected by hook-and-line in waters <2 m depth off Key Biscayne, Florida. Specimen preparation and histological methods were as described (Taylor et al. 2011a,b). Briefly, tissues were embedded in Durcupan ACM epoxy resin (Electron Microscopy Sciences, Hatfield, PA) for light microscopy (LM) and transmission elec- tron microscopy (TEM). Sections were cut at 70 nm (TEM) or 1.0 µm (LM) on a Leica Ultracut UC6 ultramicrotome (Leica Biosystems, Buffalo Grove, Illinois). For op- sin immunofluorescence, tissues were infiltrated in 20% sucrose, embedded in OCT compound, and frozen sections cut at 16–18 µm on a Leica CM1850 cryostat (Leica Biosystems, Buffalo Grove, Illinois). Immunofluorescence.—Frozen sections were incubated for 8 hrs at room tem- perature with the mouse monoclonal anti-rhodopsin MAB5316 (Millipore) at 1:500 dilution. Antibody dilutions and labeling procedures were performed as previously described (Taylor et al. 2011a). This antibody (RRID AB_2156055) was raised by hyper-immunization of mice with rat rod outer segments; it specifically labels rod photoreceptor outer segments in mammals (Hicks and Barnstable 1987, Xu and Tian 2008) and fish (Taylor et al. 2011a). Primary antibody was omitted as a control (which produced no labeling). Transmission Electron and Light Microscopy.—TEM and light microscopy were performed as previously described (Taylor et al. 2011a). Briefly, for TEM, sec- tions were mounted on formvar-coated copper mesh grids and stained in a CO2-free environment for 1 min with 0.5% aqueous lead citrate, rinsed with 0.01 m NaOH and then stained for 1–2 min in 2% aqueous uranyl acetate. To enhance contrast, the tis- sue was rinsed again with CO2-free water and then stained for one additional minute in fresh 0.5% lead citrate. Imaging was performed on a Zeiss EM900 TEM (Carl Zeiss Inc., Peabody, Massachusetts) at 80 kV. Photoreceptor outer segments lengths and widths were digitally measured from the longest/widest profiles in a single section using AnalySIS 5/iTEM imaging software (Olympus Soft Imaging Solutions Corp, Münster, Germany). Individual photoreceptors were randomly selected for measure- ment and sampled throughout all retinal regions (dorsal, ventral, nasal, temporal). Obviously oblique OS were excluded from analysis and lengths were measured only when the entire outer segment length was visible. Photoreceptor cell counts were performed as previously described (Taylor et al. 2011a) and mean rod:cone ratios cal- culated across the retina and for specific retinal regions (dorsal, ventral, nasal, tem- poral). Cells were quantified in 1.0 μm–thick radial cross-sections using standard light microscopy and digital imaging. Cells were quantified in the dorsal, ventral, 342 Bulletin of Marine Science. Vol 93, No 2. 2017 nasal, and temporal regions, within four 0.05-mm strips in each region. Standard errors were calculated for all cells quantified across these retinal regions.
Results
Rod Densities and Rod-Cone Ratios.—Photoreceptor densities and rod:cone ratios were calculated from data in previously published papers (Taylor et al. 2011b, 2015). The adult speckled worm eel, Myrophis punctatus Lütken, 1852, a strictly noc- turnal species, has a rod density of 105 rods 0.05 mm−1 and a rod:cone ratio of 28:1 (Taylor et al. 2015). In comparison, adult M. atlanticus has a rod density of 152 rods 0.05 mm−1 and rod:cone ratio of 21:1, while adult E. saurus has 100 rods 0.05 mm−1 and a rod:cone ratio of 17:1. Both of these species are often observed feeding both diurnally and nocturnally, as well as during crepuscular periods (SM Taylor pers obs). Finally, adult A. vulpes, primarily a diurnal feeder in very shallow water, has the lowest rod density (76 rods 0.05 mm−1) and the lowest rod:cone ratio (13:1). Results are summarized for the entire retina in Table 1 and rod:cone ratios for specific retinal regions (dorsal, ventral, nasal, temporal) are summarized in Table 2. Functional Morphology of Photoreceptor Outer Segments.— Morphological analysis of the photoreceptor layer in adult M. atlanticus reveals a retina specialized for light capture in low-light environments (Fig. 1). By both light and transmission electron microscopy, it is readily apparent that rod outer segments are highly organized into bundles. These bundled outer segments are stacked, but parallel with each other, and appear to exclude other cellular components (i.e., cone outer segments and retinal pigmented epithelium processes). By contrast, the diur- nal A. vulpes, which occupies brightly lit, shallow coastal waters, lacks rod bundles. Albula rods are thin, highly elongated, and arranged in multiple, overlapping layers (Fig. 1). Light Reflecting Tapetum Lucidum.—At the earliest stages of development, the retinas of all elopomorphs studied lack visible evidence of a light-reflecting ta- petum lucidum (M. atlanticus, E. saurus, A. vulpes, and a variety of eel species from five different families; Taylor et al. 2011b). However, by 65 d after settlement in -in shore juvenile habitat, E. saurus exhibits the development of a retinal tapetum in conjunction with changes in the structure and organization of rod outer segments (Fig. 2). By adulthood, both M. atlanticus (Figs. 1A–C, 3) and E. saurus exhibit a well- developed tapetum lucidum that completely surrounds rod outer segment bundles in both light-adapted and dark-adapted conditions, and surrounds extended cone outer segments in the dark. In contrast, the diurnal bonefish (A. vulpes) retina completely lacks the tapetum lucidum throughout all developmental stages (adult shown in Fig. 1D–F). Development of Retinal Architecture.—The light-reflecting tapetal layer begins to develop during metamorphosis of the leptocephalus larvae. In early post- metamorphic E. saurus, there is a significant amount of melanin beyond and between photoreceptor outer segments (Fig. 2), but the tapetum is poorly, if at all, developed. However, within the first 2 mo beyond the start of metamorphosis, melanin levels appear significantly reduced, and by the same time the formation of the tapetum has begun (Fig. 2). Grace and Taylor: Adaptations of the elopomorph retina 343 3 1 n 1 1 Y Y Y Y Stacked rods Y Y N N Grouped rods Y Y N N Tapetum 2.95 (0.25) 2.20 (0.66) 1.90 (0.43) 2.68 (0.23) Rod OS width 5.96 (0.14) 5.83 (0.21) 16.65 (0.37) 21.12 (2.27) Rod OS length 28:1 21:1 17:1 13:1 Rod:cone 76 (2.3) 105 (4.2) 129 (3. 3) 100 (4.3) 3 cells per retina). See Taylor et al. (2011b, 2015) for detailed methods and additional information. Errors represent information. methods and additional 2015) for detailed et al. (2011b, Taylor See ≥ 3 cells per retina). Rod density Both Both Diurnal Nocturnal Nocturnal/diurnal
Myrophis punctatus Myrophis Megalops atlanticus Elops saurus Albula vulpes Table 1. Retinal specializations for low-light vision in four ecologically-distinct elopomorph species at the adult stage. Rod densities are measured in cells per in cells measured are Rod densities stage. adult the at species elopomorph vision in four ecologically-distinct for low-light specializations 1. Retinal Table are in μ m ( n 0.05 mm and rod measurements standard error of the mean (in parentheses) for all cells quantified across retinal regions. OS = outer segment. 344 Bulletin of Marine Science. Vol 93, No 2. 2017
Table 2. Comparison among species of rod:cone ratios across different retinal regions at the adult stage. See Taylor et al. (2011b, 2015) for detailed methods and additional information.
Rod:cone Rod:cone Rod:cone Rod:cone dorsal ventral nasal temporal n Megalops atlanticus 18:1 24:1 18:1 25:1 1 Elops saurus 22:1 21:1 17:1 17:1 1 Albula vulpes 12:1 14:1 14:1 14:1 1 Myrophis punctatus 35:1 31:1 28:1 30:1 3
Photoreceptor outer segment morphology begins to change over the same course of time. Early in the metamorphic process, rods are shorter and thicker compared to 2 mo later when they are relatively longer and thinner, and appear to already be grouping into bundles separated by tapetal material (Fig. 2). In M. atlanticus and E. saurus, rod outer segments become smaller over the course of development, during the same time period when rod densities dramatically increase (Fig. 2). At settlement (metamorphosis), M. atlanticus rod OS are 18.3 mm in length and 4.9 mm in diameter,
Figure 1. Retinal specializations in elopomorph fishes associated with light habitat. (A–C) In the diurnally/nocturnally active elopomorph M. atlanticus, rod outer segments (arrowheads) are small in size, clustered into tight bundles and stacked into multiple layers, evident with (A) anti-rhodopsin immunohistochemistry, (B) transmission light microscopy, and (C) transmission electron microscopy; also, light colored, light-reflecting tapetal crystals (t) are clearly evident be- tween the rod bundles. In comparison, (D–F) in the diurnal species A. vulpes, rod outer segments (arrowheads) are much larger, are not bundled into groups and there is only melanin pigment (m) but no tapetum. However, as in all elopomorphs studied so far, rods are stacked into multiple layers. Scale bars: A, B, D, E = 25 μm; C, F = 2.5 μm. Grace and Taylor: Adaptations of the elopomorph retina 345
Figure 2. Ontogenic development of retinal specializations for low-light vision. (A) In the diur- nally/nocturnally active elopomorph E. saurus, early post-metamorphic fish have a single layer of cones (c) and rods (r) and only have melanin pigment (m) with no reflective tapetum. (B) However, 65 d later, rods (r) have become smaller and more numerous, are starting to form dis- tinct groups, are surrounded by lighter-appearing tapetal material (t), and are extended farther out (sclerally) beyond the cones (c). Scale bar = 25 μm. but at the juvenile and adult stages, these have been reduced to 9.9 mm length, 2.5 mm diameter, and then 6.0 mm length, 2.2 mm diameter, respectively (Taylor et al. 2011a). Using identical methods, rod OS lengths and diameters were measured here in E. saurus, A. vulpes, and M. punctatus. From the settlement to juvenile to adult stage in E. saurus, rod OS lengths decrease from 15.1 to 11.6 to 5.8 mm, respectively, and OS diameters changed from 4.4 to 1.6 to 1.9 mm, respectively. In comparison, rod outer segments in the diurnally-active species A. vulpes remain about the same size throughout development (mean length 21.6 mm, diameter 2.6 mm), while in the noc- turnal worm eel, M. punctatus, rod OS size only decreases moderately from settle- ment (length 23.7 mm, diameter 3.7 mm) to adult (length 16.7 mm, diameter 3.1 mm). Retinomotor Movement.—In larval and early post-metamorphic elopomorphs, rod and cone photoreceptor outer segments occupy the same positions in the retina throughout the daily light-dark cycle (data not shown), but later in the juvenile stage, substantial retinomotor movements develop in M. atlanticus (Fig. 3) and E. saurus. Under light-adapted conditions (i.e., daytime; Fig. 3A–C), cone outer segments were observed retracted toward the inner margin of the retina, nearer the incoming light, while rod bundles were extended distal to the photoreceptor soma, farther from the incoming light. Conversely, in the dark-adapted retina (i.e., nighttime; Fig. 3D–F), cone outer segments were extended distally toward the back of the eye, while rod outer segment bundles were observed retracted toward the inner margin of the reti- na, closer to the incoming light. It should be noted that melanin granules within the retinal pigmented epithelium, which are known to exhibit retinomotor movement in other species (see Burnside et al. 1982, Burnside and Basinger 1983), appeared not to move according to lighting conditions in the species studied here. In both M. atlan- ticus and E. saurus, melanin granules form aggregations between or “capping” cone outer segments in the light-adapted retina, and formed dense aggregations between neighboring rod outer segment bundles in the dark-adapted retina (Fig. 3). 346 Bulletin of Marine Science. Vol 93, No 2. 2017
Figure 3. Specialized retinomotor movements in juvenile M. atlanticus and E. saurus. (M. atlan- ticus shown; E. saurus is identical with respect to the results shown). (A–C) In the light-adapted retina, entire groups of rods (r) extend sclerally and are completely surrounded by reflective tapetal material (t); cones are retracted vitreally, exposed to incoming bright light, surrounded on the scleral side by melanin pigment (m) and tapetum. Rods and cones are shown at higher magnification in (B) and (C), respectively. (D–F) In the dark-adapted retina, cones extend scler- ally and are surrounded by reflective tapetal material (t); Entire groups of stacked rods (r) are retracted vitreally, exposed to incoming dim light, surrounded on the scleral side by reflective tapetum and flanked by melanin pigment (m). Rods and cones are shown at higher magnification in (E) and (F), respectively. Scale bars: A,B,C,D,E = 25 μm; F = 10 μm.
Discussion
Rod and Cone Cell Populations.—The larvae of elopomorph fishes (leptoceph- ali) are highly rod-dominated (Taylor et al. 2011a,b, 2015), in stark contrast to the pure-cone or highly cone-dominated retina in the larvae of most teleost fish species. This suggests that elopomorph leptocephalus larvae are adapted for vision in low- light conditions. This observation is consistent with the diurnal vertical migrations of leptocephalus larvae, deeper during the day and shallower at night (Miller 2009), that result in a perpetual dim-light existence prior to settlement. As leptocephali un- dergo settlement, generally moving into shallower coastal or estuarine habitats, they undergo metamorphosis into the juvenile stage. During metamorphosis, both rod and cone photoreceptors are rapidly added to the retina (Taylor et al. 2011a,b, 2015). The retinas of all elopomorphs remain rod-dominated into adulthood, but rod densities and rod:cone ratios can be excellent predictors of the degrees of diurnality Grace and Taylor: Adaptations of the elopomorph retina 347 and nocturnality exhibited by particular species (Pankhurst 1989, Fishelson et al. 2004). The results presented here clearly distinguish the benthic, nocturnal speckled worm eel (M. punctatus), and the crepuscular and nocturnal tarpon (M. atlanti- cus) and ladyfish E.( saurus) from the strictly diurnal bonefish (A. vulpes). The for- mer three species all have greater rod densities and higher rod:cone ratios than A. vulpes, consistent with effective visual function in very distinct light environments. Interestingly, M. atlanticus rod density was higher than even the benthic M. puncta- tus, but dramatic difference in eye size alone suggests that the Atlantic tarpon relies much more heavily upon vision than does the speckled worm eel, which may rely more heavily upon chemical cues for guidance of behavior (Fishelson 1995, Barreto et al. 2010). Comparing rod:cone ratios among different retinal regions can also sug- gest differences in specific visual abilities and behaviors. For example, in M. atlanti- cus, substantially higher rod:cone ratios in the ventral and temporal regions suggest better low-light sensitivity in the forward and upward-looking directions in this sur- face-feeding, nocturnal predator. In comparison, the comparatively high rod:cone ratio in the dorsal retina of M. punctatus suggests greater low-light sensitivity in the downward-looking direction in this nocturnal bottom-feeder. Tapetum Lucidum and Photon Harvesting.—The tapetum lucidum (TL) is a layer in the back of the eye that reflects light back onto the photoreceptors, increasing the amount of light available for vision and thus increasing low-light visual sensitiv- ity (Wagner et al. 1998, Schwab and others 2002, Ollivier et al. 2004, de Busserolles et al. 2014, Francke et al. 2014). A reflective TL can be detected in a particular spe- cies by shining a flashlight into the eye and observing that the eye “shines” from light reflected back through the pupil. Many species of fishes possess a TL, which is strongly associated with nocturnality and with species living in deep water or high turbidity (Somiya 1980, Takei and Somiya 2002, Francke et al. 2014). Fishes may pos- sess either a choroidal TL with reflective material behind the retina, or a retinal TL in which reflective material—often guanine crystals—is present in the cytoplasm of retinal pigmented epithelial (RPE) cells (Schwab et al. 2002, Ollivier et al. 2004). RPE cells create a layer behind (and sometimes extending between) the photorecep- tors that can contain light-absorbing melanin pigment and/or light-reflecting tapetal material. Both M. atlanticus and E. saurus have a substantial retinal TL (Figs. 1, 2; see also Taylor et al. 2011a, 2015). In E. saurus, the TL develops shortly after metamorpho- sis, around the time that feeding behavior begins (Fig. 2; Taylor and Grace 2005). The common snook, Centropomus undecimalis (Bloch, 1792), is not closely related but is ecologically similar to M. atlanticus and E. saurus, and also possesses a TL (Eckelbarger et al. 1980), suggesting that the TL is ecologically beneficial for large, crepuscular/nocturnal predatory fishes. In contrast, the diurnal, shallow-water elo- pomorph A. vulpes (Fig. 1D–F) and the strictly nocturnal worm eel M. punctatus do not have a TL (Taylor et al. 2015). Instead, the RPE contains high amounts of melanin pigment, indicating that it absorbs light rather than reflecting it back onto the pho- toreceptors. Diurnal species typically lack a TL because reflection and scattering of light can decrease daytime visual acuity, resulting in a blurred image (Beynon 1985). This further supports the conclusion that the A. vulpes retina is specialized primarily for daytime vision. Conversely, M. punctatus, like many other anguilliform eel spe- cies (Elopomorpha: Anguilliformes), is a nocturnal species that burrows in sediment 348 Bulletin of Marine Science. Vol 93, No 2. 2017 during the day. There are also no tapeta lucida in the few other eel species in which retinal morphology has been studied (Braekevelt 1988a,b, Wang et al. 2011), suggest- ing that anguilliform eels generally may lack the TL, possibly because many rely on olfaction rather than vision to detect food, predators, and conspecifics (Fishelson 1995, Barreto et al. 2010). Rod Photoreceptors: Size Trade-Off, Stacking, and Bundling.—The sensi- tivity of individual photoreceptors has been modeled to increase along with increas- ing outer segment dimensions (de Busserolles et al. 2014). Therefore, one strategy for increasing visual sensitivity in dim light is to increase OS length and diameter. This is readily apparent in some deepwater fishes (de Busserolles et al. 2014). There is a trade-off, however, because increasing the size of individual photoreceptors decreas- es photoreceptor density within the same lateral space. Thus, while larger photore- ceptors are individually more likely to be activated by photons, fewer photoreceptors will be activated per unit retinal area. This will result in reduced convergence of light information onto higher order neurons and, therefore, decreased image brightness. In M. atlanticus and E. saurus, rod photoreceptor outer segments become smaller over the course of development while their densities dramatically increase. This sug- gests that in these species, convergence of light information may increase over the course of development, along with image brightness and low-light visual sensitivity. Several ecologically distinct elopomorph species have a multibank retina, meaning that rod inner and outer segments are stacked into multiple layers. Examples include the large predators M. atlanticus (Taylor and Grace 2005, Taylor et al. 2011a), E. sau- rus (Taylor and Grace 2005, Taylor et al. 2015), and A. vulpes (Taylor and Grace 2005, Taylor et al. 2015), the nocturnal worm eel M. punctatus (Taylor et al. 2015) and the catadromous Japanese eel, Anguilla japonica Temminck and Schlegel, 1846 (Omura et al. 2003). A multibank retina is common in deepwater fish species that live in a constant dim-light environment (Wagner et al. 1998, de Busserolles et al. 2014), but is uncommon in coastal/shallow water species, such as the elopomorphs described here. The stacking of rods, in theory, increases visual sensitivity by allowing a higher rod density to be maintained in the retina, increasing convergence of light infor- mation onto higher order neurons and increasing the likelihood that photons will stimulate rods before passing completely through the retina. M. atlanticus and E. saurus rod inner and outer segments are grouped into tight clusters in addition to being stacked (Fig. 1) (Taylor and Grace 2005, Taylor et al. 2011a, 2015). This specialization is thought to pool light information from many rods to increase visual sensitivity in low-light conditions, and may be a specialization to enhance contrast and movement detection in water with little light or poor optical quality (Pusch et al. 2013, Francke et al. 2014). In most cases, grouped photoreceptors are associated with a reflective tapetum as seen here, where photoreceptor bundles are interdigitated with wedges of RPE containing reflective tapetal crystals (Somiya 1980, Francke et al. 2014). This arrangement effectively produces reflective “cups” around the rod clusters, trapping and scattering the light within the cups and dra- matically increasing the amount of light available for absorption by the rods (Francke et al. 2014). Together, the presence of stacked and bundled rod outer segments, high rod densi- ties, high rod:cone ratios, and the presence of a well-developed tapetum likely pro- vide M. atlanticus and E. saurus with exceptionally good low-light/nighttime visual Grace and Taylor: Adaptations of the elopomorph retina 349 sensitivity compared to many other coastal teleost species that lack this combination of features. A. vulpes—another elopomorph—provides an excellent contrast. This shallow-water, high-light, diurnal species lacks all of these adaptations for efficient low-light visual function. Retinomotor Movements.—If too much light falls on the retinal photorecep- tors, particularly the highly sensitive rod cells, they quickly become overstimulated and nonfunctional. The mammalian eye adjusts the amount of light falling on the retina by constricting or dilating the pupil, but aside from a few known exceptions (Douglas et al. 1998), pupil diameter is fixed in teleost eyes (Schmitz and Wainwright 2011). To adjust light capture by photoreceptors, the retinas of fish (and some other taxa) may utilize retinomotor movements in which photoreceptor outer segments and melanin pigment granules move within the retina to optimally position accord- ing to time of day (Burnside et al. 1982, Burnside and Basinger 1983, Taylor and Grace 2005). In bright light, cone outer segments and melanin pigment granules of some species move vitreally within the retina, toward the front of the eye. At the same time, rod outer segments move toward the sclera at the rear of the eye, behind the melanin, which decreases the amount of light reaching rods (Fig. 2B). This places cones, which function optimally in bright light, directly in the light path while pro- tecting rods from over-stimulation. Opposite movements occur in the dark: rods move vitreally, while cones and melanin pigment move sclerally, allowing rods maxi- mum exposure to available light. Retinomotor movements, therefore, modulate reti- nal architecture for optimal photon capture according to lighting conditions. The capacity for retinomotor movement changes over the course of development. In at least some species, the retina becomes capable of retinomotor movements either during or shortly after metamorphosis from the larval to the juvenile stage (present study, Taylor and Grace 2005, Mukai et al. 2012). In E. saurus, the distance the rods extend (in bright light) increases more than 2-fold within the first 65 d after meta- morphosis (Fig. 2) and continues to increase through the juvenile and adult stages (Taylor and Grace 2005). These observations suggest that, in at least some species, the ability of the retina to adjust to light level increases through ontogeny. While retinomotor movements have been identified in many teleost species, inter- specific comparison of the characteristics and extent of these movements is lacking. In the elopomorphs M. atlanticus and E. saurus (but not A. vulpes), dense groups containing exclusively rods or cones are maintained in both light and dark condi- tions, and these entire clusters undergo retinomotor movements (this report; Taylor and Grace 2005). In bright light, cone clusters surrounded by RPE cells containing reflective tapetal material contract vitreally toward the outer limiting membrane. This results in the formation of tapetal “cups,” where reflective tapetal material sur- rounds the cone clusters except on the side facing the incoming light. Meanwhile, rod bundles extend sclerally and are completely surrounded by RPE/tapetum, preventing bright light from reaching rod outer segments. In the dark, the opposite movements occur, causing rod clusters to be exposed to incoming light, surrounded by reflective tapetum on the remaining sides (Taylor and Grace 2005). Similar exclusive photo- receptor groupings and associated retinomotor movements have been described in a small number of species, the majority of which live in low-light conditions or high turbidity (Francke et al. 2014) and where increased light sensitivity and/or contrast detection should be important. 350 Bulletin of Marine Science. Vol 93, No 2. 2017
Implications for Conservation.—Anthropogenic disturbances including coastal construction runoff, waste and nutrient influx, and other forms of pollution can substantially increase turbidity and eutrophication, significantly altering optical qualities of coastal and estuarine waters (Horodysky et al. 2010). When these influ- ences are combined with biological, environmental, and meteorological events, opti- cal quality can change abruptly and dramatically (Koltes and Opishinski 2009). Both experimental and theoretical studies indicate that increased turbidity can harm feeding abilities in some fishes (e.g., large, visually-guided predators), while enhanc- ing feeding abilities in others (e.g., small fish larvae) (Utne-Palm 2002, Horodysky et al. 2010, Ranåker et al. 2012). These differential effects might exert selective pres- sures on community structures. How particular species and/or developmental stages are affected by changes in optical qualities of the water depends upon specific feed- ing behaviors and visual capabilities, dictated in part by retinal morphology and function. Turbidity and eutrophication reduce light transmission through the water, result- ing in reduction of the total amount of light available for vision (Omar and Matjafri 2009). Overall light intensity is also reduced if there is a reduction in light avail- ability or an increase in water depth. It is therefore common for species that live in high turbidity, are nocturnal, or that live in deep water to have retinal specializa- tions that improve low-light visual sensitivity. Consistent with this, the elopomorph species M. atlanticus and E. saurus, both of which live at least part of their lives in highly-turbid estuaries and are both nocturnally and diurnally active, have excep- tionally high rod:cone ratios, a tapetum lucidum, grouped/stacked rods, and special- ized retinomotor movements. All of these characteristics have also been described in deepwater species (Wagner et al. 1998), and in the nocturnally active mormyrid fish, Gnathonemus petersii (Günther, 1862) that inhabits highly turbid rivers and streams (Pusch et al. 2013, Francke et al. 2014). It therefore might be expected that in nocturnal species with retinae adapted for low-light visual sensitivity (e.g., M. at- lanticus), vision will be less affected by changes in water clarity than in species that have retinas adapted for high-light/daytime vision (e.g., A. vulpes). However, given the low light levels and challenges that nighttime imposes on the visual system, it is unknown how further reductions in available light will affect the abilities of noctur- nal species to feed, find predators and locate mates. In summary, the extremely low availability of light at night makes vision challeng- ing for any nocturnal animal. The challenge is even more important in water, where clear water by itself reflects, scatters, and absorbs light, and dissolved and particulate matter reduce light availability even more. Nocturnally active fishes can have a va- riety of retinal specializations that improve sensitivity to light. Comparative studies utilizing elopomorph fishes, which begin life as similar larvae with similar retinas and later diverge into radically different forms, have improved our understanding of retinal specializations for nocturnal vision. The retina, however, is only one part— the starting point—of the visual system, and many of the functional attributes of the retina and brain associated with nocturnal vision are still poorly understood. For example, modifications of the biochemical phototransduction apparatus in pho- toreceptor outer segments, and neural processing within the retina and in visual centers in the brain could each (or all) be modified to affect visual sensitivity. To fully understand adaptations of visual systems to life at night, future studies must focus Grace and Taylor: Adaptations of the elopomorph retina 351 also on photoreceptor biochemistry and neural information processing. Ultimately, increasing our understanding of retinal adaptations will better define how fishes -in teract with their environments and how they might be affected by human-induced and natural habitat disturbance.
Acknowledgments
The authors thank M Scripter, Florida Institute of Technology, and M Larkin, University of Miami RSMAS, for help with collection of juvenile and adult specimens.
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