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The organization of the visual system in the bonnethead shark (Sphyrna tiburo)

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Title:
The organization of the visual system in the bonnethead shark (Sphyrna tiburo)
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English
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Osmon, Amy L
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University of South Florida
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Subjects / Keywords:
vision
ganglion
shark
Bonnethead
retina
Dissertations, Academic -- Psychology -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The goal of this project was to examine the visual system of the bonnethead shark (Sphyrna tiburo). The eyes of this shark are located at the extreme lateral ends of a broad, elongated cephalofoil. Better understanding of their visual system may aid in determining the adaptive benefits of their usual head shape. The proposed project examined one specific aspect of their visual system: the organization of retinal ganglion cells and identification of areas of increased resolution. Two experiments were conducted to realize these aims: (1) staining of retinal ganglion cells, to examine their distributional pattern, and (2) retrograde staining of retinal ganglion cells to determine morphology.
Thesis:
Thesis (M.A.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
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Mode of access: World Wide Web.
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by Amy L. Osmon.
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Title from PDF of title page.
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Document formatted into pages; contains 55 pages.

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aleph - 001478740
oclc - 56389394
notis - AJS2429
usfldc doi - E14-SFE0000381
usfldc handle - e14.381
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ABSTRACT: The goal of this project was to examine the visual system of the bonnethead shark (Sphyrna tiburo). The eyes of this shark are located at the extreme lateral ends of a broad, elongated cephalofoil. Better understanding of their visual system may aid in determining the adaptive benefits of their usual head shape. The proposed project examined one specific aspect of their visual system: the organization of retinal ganglion cells and identification of areas of increased resolution. Two experiments were conducted to realize these aims: (1) staining of retinal ganglion cells, to examine their distributional pattern, and (2) retrograde staining of retinal ganglion cells to determine morphology.
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The Organization of the Visual System in the Bonnethead Shark (Sphyrna tiburo) by Amy L. Osmon A thesis submitted in partial fulfillment Of the requirements for the degree of Cognitive and Neural Sciences Department of Psychology College of Arts and Sciences University of South Florida Major Professor: Toru Shimizu Sarah Partan, Ph.D. Robert Hueter, Ph.D. Cynthia Cimino, Ph.D. May 21, 2004 Keywords: Bonnethead, shar k, ganglion, vision, retina Copyright 2004, Amy L. Osmon

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iTable of Contents List of Tables ii List of Figures iii Abstract iv Overview 5 Organization of retinal ganglion ce lls in non-shark species 6 Illumination 9 Habitat 9 Behavior 10 Organization of retinal ganglion cells in sharks 12 Illumination 13 Habitat 14 Behavior 14 Possible retinal ganglion cell topography of the bonnethead shark 16 Methods 17 Topographic mapping of retinal ganglion cells 18 Retrograde labeling of retinal ganglion cells 19 Data Analysis 20 Results 22 Discussion 35 References 42 Appendix A 50

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iiList of Tables Table 1 Comparison of shark species from varied habitats 8 Table 2 Retinal ganglion cell counts and statistics 25

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iiiList of Figures Figure 1. Typical photographic image with retinal ganglion cells 22 Figure 2. Case 2627-1 26 Figure 3. Case 2626-4 27 Figure 4. Case 2626-5 28 Figure 5. Case 2626-8 29 Figure 6. Case IMF1 30 Figure 7. Case IMF2 31 Figure 8. Case 2625-2 32 Figure 9. Case 2626-6 33 Figure 10. Case 2627-1 50 Figure 11. Case 2626-4 51 Figure 12. Case 2626-5 51 Figure 13. Case 2626-8 52 Figure 14. Case IMF1 52 Figure 15. Case IMF2 53 Figure 16. Case 2626-5 53 Figure 17. Case 2626-6 54

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iv The Organization of the Visual System in the Bonnethead Shark ( Sphyrna tiburo ) Amy L. Osmon ABSTRACT The goal of this project was to examine the visual system of the bonnethead shark ( Sphyrna tiburo ). The eyes of this shark are located at the extreme lateral ends of a broad, elongated cephalofoil. Better understanding of their visual system may aid in determining the adaptive benefits of their usual head shape. The proposed project examined one specific aspect of their visual system: the organization of retinal gang lion cells and identification of areas of increased resolution. Two experiments were conducted to realize these aims: (1) staining of retinal ganglion cells, to examine their distributional pa ttern, and (2) retrograde staining of retinal ganglion cells to determine morphology.

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5Chapter One Introduction Elasmobranchs had long been thought to possess poor vision and utilize other sensory systems for navigation and detection of both prey and predators (Gruber, 1977). However, during the 1960’s and 1970’s the scientific community began to publish both anatomical and physiological research indicating that the visual systems of sharks and rays were capable of higher visual resolution than previously believe d (Ali and Anctil, 1974a; Gruber, 1977; Gruber, Gulley, and Brandon, 1975; Gruber, Hamasaki, a nd Bridges, 1963; Hamasaki and Gruber, 1965; Stell, 1972; Stell and Witkovsky, 1973). Concurre nt studies also attempted to test the visual acuity and learning ability of sharks by investiga ting visually mediated behaviors (Graeber, 1978; Tester and Kato, 1966; Wright and Jackson, 1964). These studies revealed complexity within sharks’ visual system and provided insight into the significance of the visual system in their daily existence. Research regarding retinal anatomy in sharks has shown variability in rod-to-cone ratios, the distribution of ganglion cells within the retina, as well as the presence of visual streaks (Bozzano and Collin, 2000; Gruber, 1977; Gruber et al., 1975; Gruber et al., 1963; Hamasaki and Gruber, 1965; Hueter, 1988; Peterson and Rowe, 19 80; Stell, 1972; Stell and Witkovsky, 1973). The implications of these retinal variations have not yet been thoroughly explored. However, several authors (Bonazzo and Collin, 2000; Gruber et al., 1975; Hueter, 1989; Hueter and Gruber, 1982) have related the variability of sharks’ vi sual systems to their feeding behaviors and habitats. This study examined the retinal anatom y of one shark species, the bonnethead shark. The bonnethead shark is one of nine species in the Sphyrnidae family, commonly referred to as “hammerhead” shar ks, possessing a broad and elongated head shape. The bonnethead shark inhabits clear to turbid inshore waters of the Gulf of Mexico, the Atlantic Ocean, and along the coasts of Central and South America (Cortes a nd Parsons, 1996; Hoese and Moore, 1958). Their

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6diet consists of a variety of swift-moving cr abs and cephalopods (Cortes, Manire, and Hueter, 1996; Motta and Wilga, 2000). As this shark speci es adjusts well to captivity (Cortes et al.,1996; Cortes and Parsons, 1996) and their ecology is well documented (Cortes et al., 1996; Cortes and Parsons, 1996; Hoese and Moore, 19 58; Myreberg and Gruber, 1974), it is an excellent subject for an investigation into the relationship betw een retinal anatomy and ecological niche. Theories regarding the function of Sphyrinid ae sharks’ unique cephalofoil focus on their head shape providing increased hydrodynamic lift, an area useful for capture of large prey items, and/or an enlarged area for electroreception a nd olfaction (Antcil and Ali, 1976; Compagno, 1984; Johnsen and Teeter, 1985; Kajiura and Holland, 2002; Martin, 1993; Nakaya, 1995; Strong, Gruber, and Snelson, 1990). Only two studies have examined the visual system of Sphyrinidae sharks (Anctil and Ali, 1974b; Gruber et al., 1963). Anctil and Ali (1974b) investigated the retinal morphology of the scalloped hammerhead shark ( Sphyrna lewini ) and designated some retinal ganglion cells as giant ganglion cells due to their large soma size. Gruber et al. (1963) reported similarities in the morphology of cones between the lemon shark ( Negaprion brevirostris ) and the great hammerhead shark ( Sphyrna mokarran) However, no research has been conducted regarding the visual system of the bonnethead shark. Information pertaining to the bonnethead shark’s visual system may lead to a better understanding of the true function and significance of this shark’s unusual head sh ape compared to other shark species. Organization of retinal ganglion cells in non-shark species How does retinal cell topography relate to th e diverse habitats of different vertebrate species? According to Hughes’ (1977) terrain theory, animals with a predominantly twodimensional horizon in their visual environment (e.g., the ocean with a sand-water boundary for a benthic aquatic species) gain an advantage from poss ession of a visual streak. A visual streak is an elongated area of increased ganglion cell dens ity relative to other areas within the retina

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7(Bozzano and Collin, 2000). The advantage of possessing a visual streak is higher resolving power in the visual fields corresponding to the area of increased cell density (Bozzano and Collin, 2000). A visual streak may also negate the necess ity of utilizing distinctive eye movements while an animal is gazing across an expansiv e horizon (Collin and Pettigrew, 1988b). Hughes’ theory (1977) has been tested a nd validated in many non-aquatic vertebrates including the fat-tailed dunnart ( Sminthopsis crassicaudata) (Arrese, Dunlop, Harman, Braekevelt, Ross, Shand, and Beazley, 1999), several ungulates (e.g. the pig, sheep, ox, dog, and horse) (Hebel, 1976), the tammar wallaby ( Macropus eugenii) (Wong,Wye-Dvorak, and Henry, 1986), and the African elephant ( Loxodonta africana) (Stone and Halasz, 1989). Visual streaks have also been found in teleosts occupying open areas with a distinct visual horizon such as the blue tuskfish ( Choerodon albigena ), red-throated emperor ( Lethrinus chrysostomas ), collared sea bream ( Gymnocranius bitorquatus) clown triggerfish ( Balistoides conspicillum) and painted flutemouth ( Aulostoma chinensis) (Collin and Pettigrew, 1988b). In addition, Hughes’ theory has been applied to other marine animals, including the loggerhead ( Caretta caretta) leatherback ( Dermochelys coriacea) and green ( Chelonia mydas) turtles (Oliver, Salmon, Wyneken, Hueter, and Cronin, 2000), and marine mammals including the sea otter ( Enhydra lutris) (Mass and Supin, 2000). However, there is scant inform ation regarding elasmobranch species (Bozzano and Collin, 2000; Hueter, 1989; Peterson and Rowe, 1980).

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8Table 1 : Comparison of elasmobranch species (table adapted from Bonazzo et al., 2000; Hueter 1991; Compagno, 1984; Motta, Tricas, Hueter, and Summer, 1997) There are multiple factors which appear to rela te to the width, length, and location of the visual streak (Bozzano and Collin, 2000; Hueter, 1991). These factors include: (1) the amount of light available to an animal, (2) the habitat of the animal (e.g., whether the horizon is completely open or partially obstructed), and (3) how an an imal utilizes its visual streak, which includes foraging and prey detection strategies. Environmental Factors Behavioral Factor Visual Streak Illumination Habitat and depth Foraging style Width Length Location Tiger shark Fair Pelagic 0-140m Biting/ bump and bite Narrow Not across entire retina Ventral to retinal meridian Epaulette shark Not recorded Benthic 0-50m N/A Broad Across most of retinal meridian Central Smallspotted dogfish shark Dim Benthic 50-400m N/A Narrow Across most of retinal meridian Just dorsal of the meridian Blackmouth dogfish shark Dim Benthopelagic 300-2,000m N/A Broad Across entire retinal meridian Central Velvetbelly shark Dim Mesopelagic 500-2,000m N/A Narrow Across entire retinal meridian Central Lemon shark Fair to Good Benthic 0-90m Ram-feederBroad Across most of retinal meridian Central Bigelow’s ray Not recorded Benthic 650-2200m Suction feeder Narrow Across most of retinal meridian Dorsal of the meridian

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9Illumination In regards to the variability of illumination, this factor appears to relate to the density of the ganglion cells within the visual streak and th e extent of the visual streak horizontally across the retina. This variability in the ganglion cell de nsity within the visual streak has been evident between nocturnal and diurnal species, including ungulates and primates (Hughes, 1977; Lima, Silveira, and Perry, 1996). These studies revealed th at the visual streak of nocturnal animals, if they possess a visual streak, generally contains lo wer cell densities than that of diurnal animals (Hughes, 1977; Lima et al., 1996). Within the realm of aquatic animals, Oliver et al. (2000) revealed that green sea turtles, inhabiting areas with clear water and a high level of illumination, have strong visual streaks (e.g. visual streaks with the highest number of retinal ganglion cells and longest horizontal extent). In contrast, the loggerhead and leatherback sea tur tles, both inhabiting areas with highly varied illumination, possess weaker visual streaks with lower cell densities and shorter horizontal extents. Habitat Referring to habitat, the visual streak gene rally extends the farthest horizontally in species living in open habitats with an unobstruc ted view of the horizon (Collin and Pettigrew, 1988b; Oliver et al., 2000). For instance, Co llin and Pettigrew (1988b) investigated several teleost species inhabiting coral reefs and found th at species with completely unhindered views of the horizon, such as the clown triggerfish and blue tuskfish, possess horizontal visual streaks extending across most of their retinal meridians. Whereas the Australian frogfish, inhabiting a more “closed environment” (e.g. with an obstruc ted view of the visual horizon) possess a weaker visual streak extending a much shorter distance acr oss the retinal meridian (Collin and Pettigrew, 1988a).

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10Behavior The third factor which may relate to an anim al’s visual streak concerns how an animal behaviorally utilizes its visual system. Collin a nd Pettigrew’s (1988b) study of coral reef fishes revealed that the differences in location of peak cell density within the visual streak and actual location of the visual streak may vary due to the way these fish species utilize them. More specifically, the topography of the visual streak may vary in association with the behavioral needs of the teleost (e.g. the visual streak may be mo re important for predatory behavior and/or for predator surveillance). For example, the blue tuskfish and painted flutemouth both possess a horizontal visual streak along their retinal meridians (Collin and Pettigrew, 1988b). However, the visual streak of these fish species differs in regards to whether a temporal area of increased ganglion cell density is separate from (blue tuskfish) or extends into (painted flutem outh) the visual streak. The variations in retinal topography possessed by these fish species may indicate whether their visual streak is useful primarily for predator surv eillance or for predatory behavior (Collin and Pettigrew, 1988b). The blue tuskfish forages by searching through and moving coral debris on the substrate in search of food. The authors believe that the visual streak of this fish species may be useful for predator detection, as possessing a visual streak congruent with the environmental horizon may help it to watch for predators while it forages (Collin and Pettigrew, 1988b). The temporal area of increased cell density in the blue tuskfish appears better suited to foraging for prey (Collin and Pettigrew, 1988b). The temporal ar ea subtends the visual region directly in front of the blue tuskfish, and should increase its resolv ing power in the area where the fish would be searching for invertebrates within the su bstrate (Collin and Pettigrew, 1988b). The painted flutemouth, however, shadows othe r fish species to approach its prey (swiftmoving fishes) by surprise (Collin and Pettigrew, 1988b) Possession of a visual streak correlated with the environmental horizon may be more usef ul to detect and approach unsuspecting prey,

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11rather than guard for predators in this fish species. The authors also state that this fish’s retinal topography (e.g. having the temporal retinal specia lization “extend” into a visual streak across the retinal meridian) is similar to the retinal t opography of other species where vision is more valuable for prey detection than predator surveillance (Collin and Pettigrew, 1988b). The study conducted by Oliver et al. (2000) also supports the idea that differences in feeding behaviors may be correlated with visual st reak length. Of the three species examined in the study, green turtles possessed the strongest and longest visual streak, likely due to their wellilluminated habitat containing an u nobstructed view of the visual horizon (Oliver et al., 2000). Both the loggerhead and leatherb ack turtles possess weaker visual streaks than the green turtle, with the leatherback turtle possessing the weakest visu al streak of the three (Oliver et al., 2000). Although the visual streak of the loggerhead tur tle was wider than that of the green turtle, its visual streak was less horizontally extensive a nd contained a lower ganglion cell density (Oliver et al., 2000). Loggerhead turtles forage for prey such as snails, sea anemones, and crustaceans contained within and around the mats of sea grasses or algae this turtle hides amongst (Oliver et al., 2000). The sea grasses and algae mats loggerh ead turtles feed within would hinder much of their vision, with the exception of objects located di rectly in front of them. Therefore, it is likely they would not need to possess a visual streak extending across their entire retinal meridian to provide them with increased sampling of visual ta rgets in their lateral/peripheral visual fields (Oliver et al., 2000). Of all three turtle species in the study, th e leatherback turtle po ssessed the weakest (least elongated across the retinal meridian and containi ng the lowest density of retinal cells) visual streak (Oliver et al., 2000). The leatherback tu rtle was also the only species in the study to possess an area centralis (e.g. a small area of increased retinal cell density) separate from their visual streak (Oliver et al., 2000). Leatherback turtles feed primaril y on jellyfish and other jellylike prey they capture via diving. The wea kness of the leatherback tu rtles’ visual streak

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12indicates that this type of visual adaptation may not be as beneficial to detect prey as their welldeveloped area centralis (Oliver et al., 2000). The lack of a lengthy and strong visual streak in the leatherback turtle could also be a result of their predilection for capturing prey in open water where the environmental horizon may be vague or even absent (Oliver et al., 2000). Other examples regarding how the location of th e visual streak may reveal its importance in a species daily survival, and how the visual str eak may relate to an animal’s behavior (e.g. foraging or predator surveillance be haviors) are the striped panchax ( Apolcheilus lineatus ) and Graham’s Hechtling ( Epiplatys grahami ), two freshwater fish species. Both the striped panchax and Graham’s Hechtling possess two “band-shaped ” thickenings extending across their retinal meridian. One band traverses the retinal meri dian, and the other “band” extends across the retina, just ventral to the retinal meridian (Collin and Pettigrew, 1988b). These band-shaped thickenings are the equivalent of visual streak s (Collin and Pettigrew, 1988b). These fish species feed on insects and other small prey items livi ng upon or just below the water surface where higher resolution power in the upper visual fields would aid these fish species in locating prey (Collin and Pettigrew, 1988b). Therefore, the aut hors believe that the ventral thickening is likely useful for detection of prey located just above the fish and the thickening of the central retinal meridian is congruent with the lateral visual fiel d of these fishes and useful for predator detection (Collin and Pettigrew, 1988b). Organization of retinal ganglion cells in sharks The visual surroundings of many shark species relate well to the terrain theory, as their environments are composed of a two-dimensiona l setting containing a sand-water boundary or a boundary containing a “horizontal gradation of light within the water column in the clear waters of the open ocean” (Bozzano and Co llin, 2000). Therefore, most shark species, especially those living in relatively well-illuminated benthic, pe lagic, or mesopelagic habitats, could possess a

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13visual streak. This appears to be true (see Tabl e 1) as shark species investigated thus far (e.g., the tiger, epaluette, black-mouthed, velvet-belly, lem on, small-spotted dogfish, and California horn shark) all possess a visual streak (Bozzano and Collin, 2000; Hueter, 1989; Hueter, 1991). Shark species that have been investigated also show species-specific variations in the width, length, and location of their visual streaks, similar to that found in teleosts and terrestrial vertebrates (Bozzano and Collin, 2000; Hu eter, 1991; Peterson and Rowe, 1980). These variations in visual streak organization appear to be associated with environmental factors as well as predatory or surveillance behavior, and not with phylogenetic relationships between shark species. Illumination In contrast to sea turtle hatchlings, there appears to be no apparent difference regarding overall ganglion cell density within the visual streak between deep-sea (low illumination) and shallow-water (higher illumination) shark speci es (Bozzano and Collin, 2000). However, there does appear to be a difference in the percentage of giant ganglion cells contained within the retina of shallow water versus deep-sea sharks (Bo zzano and Collin, 2000). Giant ganglion cells are characterized by a larger soma (e.g. two-to-three times the soma size of other ganglion cells) and are thought to possess larger receptive fields than the normal ganglion cells (Bozzano and Collin, 2000). Shallow water species, (e.g. the tiger and ep aulette sharks), regardless of whether they are benthic or pelagic, have the lowest percentage of giant ganglion cells, whereas deep-sea species (e.g. the blackmouth dogfish and velvet belly shar ks) have the highest percentage (Bozzano and Collin, 2000). This difference in the overall pe rcentage of giant ganglion cells may actually result from differences in the distinctiveness of the visual horizon between deep-sea and shallow water species (Bozzano and Collin, 2000).

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14Habitat Bozzano and Collin (2000) suggest that more pelagic than benthic species should possess a broad visual streak. This may be generally true, as the small-spotted dogfish and Bigelow’s ray are benthic and possess a narrow visual streak whereas the black-mouth dogfish shark is benthopelagic and possesses a broader visual str eak (Bozzano and Collin, 2000). However, the lemon shark, a shallow-water benthic species with a broad visual streak, may be an exception. The visual streak of the lemon shark forms a fairly wide horizontal band of increased cell density running along the retinal meridian (Hueter, 1989). This form of visual streak may aid the lemon shark in both prey and predator detection. Both diurnal and nocturnal in activity, the lemon shark pursues swift-moving crustaceans and fi sh (Hueter, 1991). It hunts via patrolling over a sandy substrate or sea grass flats sweeping its body from side-to-side (Hueter, 1991; Oliver et al., 2000). Possession of a broad and lengthy visual streak may allow this shark to detect movement of visual objects both directly in front of it and within the lateral periphery of this visual field (Hueter, 1991). Therefore, the visual streak of the lemon shark may play a role in detection of prey. Although hunters themselves, lemon sharks are also occasionally predated upon by larger sharks. The presence of a sizeable visual stimulus has been found to elicit a “rapid withdrawal response” in lemon sharks (Huete r, 1991). Therefore, it is also likely that the visual streak of the lemon shark may be useful for detection of predators as well. Behavior Whether a visual streak is broad or narrow ma y also be associated with whether detection of predators or prey is visually important to a shark species. The location of the visual streak either across the retinal meridian, or just dorsal or ventral to the retinal meridian, may vary in relation to the different types of predatory behaviors employed by shark species.

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15All shark species examined possessed rather cen trally located visual streaks with the exception of the tiger shark (Bozzano and Collin, 2000). The visual streak of this shark is located ventrally to the retinal meridian (Bozzano a nd Collin, 2000), and would subtend vision in the upper visual field. The tiger shark is a large pred ator generally found in shallow water, near the surface (Bozzano and Collin, 2000). This shark lik ely has the most varied diet of all shark species, as it feeds on bony fish, sea turtles, sea snakes, mollusks, and mammals, among other items (Compagno, 1984). As this shark generally attacks via a bump-and-bite or ambush-style predatory technique (Compagno, 1984), a visual streak allowing it to swim unnoticed underneath potential prey, such as one subtending the upper portion of the shark’s visual field, would be advantageous. In regards to predatory behavior and incr eases in ganglion cell density, an increased area of retinal ganglion cells was found in the center of the epaulette shark’s visual streak (Bozzano and Collin, 2000). This benthic shark inhabits re latively shallow waters and preys upon benthic invertebrates such as crustaceans and mollusks (C ompango, 1984). An increase in the resolving power within the central area of their frontal vi sual field may aid them in locating their prey (Bozzano and Collin, 2000). The benthopelagic blackmouth dogfish shark possesses two areas of increased cell density within the nasal and temporal areae of their visual streak (Bozzano and Collin, 2000). This shark consumes swift-moving prey such as bony fishes and cephalopods via sweeping its head and body from side-to-side (Bozzano and Collin, 2000). The two areas of increased cell density should increase the sampling of visual target s within the shark’s frontal and caudal visual fields and are likely useful for detection of prey (Bozzano and Collin, 2000). This sharks’ retinal topography is also congruent with the idea pr oposed by Collin and Pettigrew (1988b) that species possessing temporal areas of increased cell density which extend into a visual streak are more likely to utilize these areas for prey detection.

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16Possible retinal ganglion cell topography of the bonnethead shark The bonnethead shark is likely to possess a visual streak extending across the entire length of their retinal meridian, congruent with the vi sual horizon, due to its potentially well-illuminated shallow-water habitat which should possess a rather distinct visual horizon. Similar to the lemon shark, the vi sual streak of the bonnethead shark may be useful for both detection of predators and prey. However, the primary use of this shark’s visual streak should not be prey detection, as it would appear from the location of this shark’s eyes on its broad, elongated head, that this shark may lack a frontal visual field. This potential blind spot would negate the bonnethead shark’s ability to visua lly locate prey directly in front of it, though it may possess the ability to locate prey within its lateral visual fields. The sweeping side-to-side motion of this shark’s head while it patrols for pr ey may also aid it to detect prey within its peripheral visual fields. Even though pelagic sharks may, in genera l, possess wider visual streaks than benthic species, the bonnethead shark, like the lemon shark, may be an exception. Due to the location of this shark’s eyes within the extreme edges of its broad, shovel-shaped head, the bonnethead shark may also possess an elongated lateral visual field. This potential increase in the lateral visual field should allow the bonnethead shark to develop a broad horizontal visual streak extending across their entire retinal meridian. This type of visual streak should aid the bonnethead shark in avoiding predation by larger shark species, as it would provide the bonnethead shark with full visual access to the areas along its sides. Prey speci es of this shark are primarily crustaceans with the ability to change direction ra pidly, thus possessing a broad visual streak may also aid them in locating potential prey within their lateral visual fields.

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17Chapter Two Methods Eight retinas, from eight individual sharks, were utilized for this study. The sharks were obtained with help from Mote Marine Laboratory in Sarasota, Florida. Each shark was caught within the Tampa Bay region (Charlotte Harbo r) using gill nets. Measurements of length and weight were taken before the sharks were placed into a cooler containing ice. A preservative (4% paraformaldehyde solution in 0.5 M phosphate buffe r, 1.00 cc per eye) was injected intraocularly to prevent disturbing the retina within ten minut es of the sharks expiring to preserve the eyes. The eyes were then removed and immersed in a 4% paraformaldehyde solution in 0.1 M PB solution, Ph 7.4; Huxlin and G oodchild, 1997) in small containers and placed into a cooler for transport back to the lab. Excess tissue (e.g. c onnective tissue) was rem oved from the eye before the eye was placed into the preservative solution. The retinas were removed from the eyes and wholemounted within 24 hours of collection. Eight of the retinas were used for Nissl staining and two eyes (without the retina removed for the procedur e) were used for the retrograde tracing with DiI. Though eyes were to be counterbalanced betw een left and right for this project, seven were right eyes and one was from the left eye. This discrepancy was due to selection of the best wholemounts from the retinas available for topographic analysis of ganglion cells. Nissl Staining Before removal of the retina from the ey ecup, each eye was marked with a small indentation, using a # 11 scalpel (Hueter, 1988), to maintain the dorsal/ventral and anterior/posterior orientations of the eye. The eyes were then removed and placed in a deep petri dish containing the preservative solution (4% pa raformaldehyde solution in 0.1 M phosphate buffer). The preservative solution covered each eye to prevent them from becoming dehydrated. After fixation, an adaptation of Hueter’s (1988) retinal wholemount technique was utilized. To

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18remove the retina, the eye was placed in a petri dish filled with the 4 % paraformaldehyde solution. Using a scalpel, the eyes were cut ope n at the choroidal-scleral boundary to gain access to the retina. Cutting ceased when there was a small amount of tissue, forming a ‘lid’, left on the dorsal portion of the eyecup. The lens and vitr eous humor were then lifted from the eyecup and removed. Eye orientation (dorsal vs. ventral) was maintained while the retina was removed from the eyecup using small indentations made with a scalpel blade on the dorsal and ventral margins of the retina. Using a soft brush (camel hair, #0), the retina was then transferred unto a glass slide. The retina was then flattened against the glass s lide. If the retina did not lie flat against the glass slide, small incisions were made around the retina’s circumference to help it to lie flat. Any additional preservative solution and vitreous material was then carefully removed by touching filter paper to the coverslide to absorb them. Each retina was th en placed into a dustfree container for at least 12 hours to fully adhere to the slide before being taken out and gently washed with DH2O to prepare it for Nissl staining. Each retina was stained for 10-15 minutes in 0.05% Cresyl Violet. Each slide was then dehydrated, cleared, and coverslipped. Once the retinas were stained, the retinal ganglion cell layer was examined microscopically. Topographic Mapping Before taking pictures, each retina was traced into the Canvas7 computer software program using a computerized Wacom drawing ta blet, then divided into 1mm sections. A starting point for pictures was pinpointed, then th e coordinates for each 1mm section were labeled on the retinal drawings from readings taken from a Nikon microphot-FXA microscope using an X,Y grid system on its stage micrometer. A micro-photograph was taken of the lower right corner intersection between each 1 mm square with a Lucida camera attached to the Nikon

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19microscope. Retinal ganglion cell counts were taken from a 200 micron square area in the center of each microphotograph. Cell counts were not take n from the very edges of the retina, as retinal shrinkage (usually between 2 and 20%; Collin 1988; Oliver et al., 2000) can occur in these areas (Bozzano and Collin, 2000). Retinal ganglion ce ll counts were noted for each 1mm section of each retina. These raw counts were then conve rted to the number of cells per 1mm and topographic maps were composed to represent the visual topography. In the ganglion cell layer, ganglion cells were differentiated using morphological standards of Collin (1988). The author found retina l ganglion cells to be large, irregularly shaped with darkly stained somas (Collin, 1988). Ganglion cell morphology was to be assured by labeling ganglion cells within the re tina using DiI for retrograde tracing. Retrograde ganglion cell labeling In order to assure correct identif ication of ganglion cell morphology, four retinas were to be examined using retrograde labeli ng of ganglion cells. Two eyes from one bonnethead shark were used to test whether the crystal form of 1,1’ –dioctadecyl-3,3,3,’,3’ – tetramethylindocarbonocyanine perchlorate (DiI) would be suitable for retrograde labeling of ganglion cells. DiI is a lipophilic carbanocyann ine which attaches to and stains the plasma membrane. It is then is diffused laterally thro ugh the cell, eventually staining the entire cell. The optic nerve ending of the eyes utilized for retrograde labeling were cut to make the ends level and a small incision was made at into th e end of the optic nerve. A small crystal of DiI was then placed into the incision. As DiI is sensitive to light, this procedure was performed under minimal light conditions in the lab. Once the DiI crystal was securely placed into the incision at the end of the optic nerve, the ey e was returned to the container of preservative solution, with the optic nerve tip containing the DiI crystal supported above the preservative to keep it dry. In both cases, the eyes were placed into small containers filled with the 4% paraforamldehyde in 0.1M

PAGE 21

20PB buffer solution, Ph 7.4. The containers were then wrapped in aluminum foil to protect them from light contamination and placed in a dry, dark area. DiI was allowed to absorb into the cells of the eyes in one case for two weeks and for fo ur weeks for the second case. In both cases, after the allotted time for DiI absorption, the re tinas were removed and wholemounted using the aforementioned procedure with the exception of the retinas being removed under low light conditions. Once the retinas were wholemounted, several drops of glycerin were placed on the retinas and a coverslip applied. The retinas were then examined under a Nikon microscope under fluorescent lighting for evidence of DiI staining. Unfortunately, the DiI did not completely stain the entire ganglion cell bodies, making it impossible to use DiI to confirm ganglion cell morphology. In lieu of the DiI retrograde labe ling, morphology of ganglion cells was confirmed using the descriptions from Collin (1988) and Hueter (1991). Data Analysis Ganglion cells Wholemounted retinas were used to identify ganglion cells. The morphological criteria for identification of ganglion cells by Collin (1988) and Hueter (1991) was used. An average of 333 regions were sampled from each retina (see Table 1 in the results section for individual sampling data). A visual streak was to be defi ned in this study as any area of the retina where a significant increase in ganglion cell density was found. Nissl stains Eight retinas, from both male and female bonnethead sharks were whole mounted, stained with Nissl substance, and photographed. After completion of retinal counts, each retina was mapped. The resulting maps were divided into four quadrants (dorso -nasal and temporal and ventro-nasal and temporal) to aid the descriptive process. Retinal counts were divided into four

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21categories, low (under 25%), medium (26-50%), high (51-75%), and highest (over75%) number of retinal ganglion cells. These categories we re based on dividing the maximum and minimum counts averaged across the eight retinas into four equal quartiles and used to measure differences in retinal ganglion cell numbers across each retina. Expected results of these experiments The bonnethead shark was expected to possess a wide horiztonal visual streak (covering at least one-third of the longitudinal retinal diameter) across the entire length of the retinal meridian. This streak should mediate vision in their panoramic lateral visual field. If this visual streak was not found, then vision may not be important to the daily survival of this shark species. If retinal specializations were found (inc reased areas of peak retinal ganglion cell density) in the nasal region of the retina, then the bonnethead shark may use this area of increased resolution to detect predators coming from behind, and therefore, predator detection would likely be the primary function of their visual system, and its subsequent organization. If a retinal specialization was found within the temporal regi on of the bonnethead shark retina, then this shark species may utilize their visual sense to detect pr ey in front of or to the sides of the shark’s head. A visual streak in this retinal area w ould likely be aided by the head movements of the bonnethead shark, as they sweep their heads fro m side to side while patrolling for prey.

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22Chapter Three Results For the eight retinas utilized in this study pi ctures (0.5mm by 0.5mm each) were taken at 1mm intervals across the entire area of each retina. This cumulated in a total of 2,660 photographs. The number of pictures for each re tina varied between 266 (case # IMF2) and 413 (case # 2625-2). The average number of photographs taken per retina was 332.5. For each picture, retinal ganglion cells were identified and counted and this information was used to create topographic maps of ganglion-cell density across each individual retina (Figures 2 through 9). See Table 2 for individual data regarding the number of counted areas for each retina. A typical photographic image utilized for analysis is shown in Figure 1. Figure 1. Typical photographic image showing retinal ganglion cells Darkly stained irregularly shaped cells (solid arrows) were counted as ganglion cells. The smaller circular cells (open a rrow) were considered possible amacrine cells and not counted. In general, topographical maps revealed he terogeneous characteristics of retinal ganglion cell distribution. Therefore, some areas with in each retina showed hi gher ganglion cell density than the rest of the retina. In most cases, these areas of higher density formed a band-like shape

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23across the retinal meridian. The term “band” was used to describe them instead of “visual streak” for the results section. For the reasoning behind this terminology, see the discussion section. Of the eight retinas used for this study, six were from adult animals and two were from immature individuals that are referred to as IMF1 and IMF2. However, since no apparent differences regarding the cell distribution pattern were revealed between the adult and juvenile sharks, all results, including those from both imma ture and adult specimens, were combined and analyzed together. DiI crystals were placed into the optic nerve to stain and identify ganglion cells. However, the DiI substance did not fully stain the ganglion cells making it impossible to discern ganglion cell morphology. Descriptions of ganglion cell morphology from Hueter (1991) and and Collin (1988) were used to identify and count ganglion cells in lieu of DiI staining. General Results Cell Numbers/Density In all cases combined (from the 2,660 individual photographs), a total of 402,372 cells were identified as retinal ganglion cells. The minimum number of ganglion cells found was 127 cells per mm (case # 2625-2), whereas the maximu m number equaled 1571 cells per mm (case # 2626-8). The mean ganglion cell density was 693 cells per mm with a standard deviation of 43. Figures 2 through 9 show the frequency di stribution for each individual case. For the results section, cell numbers were cate gorized into quartiles in order to demarcate the general trend of retinal ganglion cell distri bution as well as utilize all data collected from the eight cases. The quartiles were obtained by averaging the minimum and maximum cell counts from each individual retina. These averaged mini mum and maximum counts were then used to establish the quartiles utilized for this study. These quartiles are defined as follows: Low (0-480 cells per mm), Medium (481-747 cells per mm) High (748-1010 cells per mm) and Highest

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24(1011+ cells per mm). Information for each individual case is found in Table 2 (counts are in cells per mm). High Density Band Out of the eight cases, two retinas revealed no clear heterogeneous pattern of ganglion cell distribution (cases 2625-2 and 2626-6). Howe ver, the remaining six cases (2627-1, 2626-4, 2626-5, 2626-8, IMF1 and IMF2) possessed th e area of increased ganglion cell density categorized as “higher density” along the retinal meridian (see Figures 2, 3, 4, 5, 6, and 7). These bands, though, varied in length between the individual cases. Four of the retinas (cases 2626-5, 2626-8, IMF1, and IMF2) contained areas of “higher” cell density running at least twothirds of the length of the entire retinal meri dian. In two cases (2626-4 and IMF2) the band extended across the entire length of the retinal meridian, and in one case (2627-1) the area of “higher” cell density covered at least half the length of the retinal meridian. Variations in the width of this “higher” de nsity band were also observed. In four cases (2626-4, 2626-5, IMF2 and 2626-8; see Figures 3, 4, 5, and 8), the “highe r” density band covered approximately one-third of the dorsal-to-ventral expa nse of the retina. The width of the “higher” density band was widest in one case, 2627-1(see Figure 2), where it covered at least half of the dorsal-to-ventral expanse of the retina. High density dorso-temporal area In addition to the band of “hi gher” density, there was a distinct area of increased ganglion cell density in the dorso-temporal retina in si x cases (2627-1, 2626-4, 26265, 2626-8, IMF1 and IMF2). Shape and density of this dorso-tem poral increase varied between all six cases (see Figures 2, 3, 4, 5, 6, and 7). Three cases, (262 7-1, 2626-5, and 2626-8) had fairly small dorsotemporal areas compared to the rest of the case s. In all five cases, density within the dorso-

PAGE 26

25temporal area was categorized as “High”. In three cases (2627-1, IMF1 and IMF2), this dorsotemporal higher-density area appeared to be an extension of the “higher” density band, whereas in the other four cases (2627-1, 2626-4, 2626-5, an d 2626-8) this area was not connected to the “higher” density band. Table 2 Retinal ganglion cell counts a nd statistics (all cell counts in cells/mm) Case Number 2627-1 2626-4 2626-5 2626-8IMF1 IMF22625-2 2626-6 Overall Mean Mean 702 742 634 751 720 801 575 620 693 Median 688 742 634 720 706 797 566 620 684 Minimum 136 285 127 353 208 371 127 140 218 Maximum 1317 1322 1036 1571 1249 1457 1118 1086 1270 Counted Areas 285 328 317 339 368 266 413 344 322.5

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26Individual Results Nasal Temporal Dorsal VentralOD Cells/mm Highest High Medium Low Figure 2. Topographic map of case 2627-1 Retina 2627-1 This retina came from the right eye of an a dult female shark. The band of “higher” density (outlined in figure), starting in the mid-nasal portion of the retina, was found running along most of the retinal meridian in this retina (Table 2, Figure 2). The dorso-nasal portion of the retina contained both low and medium cell counts. The dorso-temporal portion of the retina also contained mostly medium cell counts, with the exception of an area of high counts located just above the re tinal meridian in the extreme temporal edge of the retina. The band of “higher” cell density st arted just nasally of the optic disc (along the equator of the retina) and ran from this area across to the temporal portion of the retinal meridian

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27as well as down toward the ventro-central portion of the retina. The ventro-temporal portion of the retina contained mostly low and medium cell counts. 169 229 203 142 e 213 218 223 190 222 198 225 nc 205 e/c 161 222 292 182 179 154 176 200 e e 190 183 99 171 146 167 161 177 165 e 172 176 166 153 185 169 nc 129 170 175 156 180 222 209 173 170 180 174 161 134 174 156 169 e 170 183 182 165 149 155 155 216 198 195 244 198 203 183 189 179 172 nc e 209 141 139 180 166 203 192 223 218 270 202 e 164 184 159 166 187 nc 162 187 178 162 157 148 164 211 245 175 241 198 191 72 e/c 129 190 175 160 161 e 123 145 140 155 1510 188 218 174 169 198 195 173 238 162 154 143 164 e 124 168 158 125 157 158 141 201 258 217 205 175 229 187 190 194 135 156 151 157 131 116 1631 181 220 186 164 198 on 123 171 e e 150 161 151 155 175 218 169 168 127 162 e e 143 128 100 182 198 171 176 206 196 174 191 145 162 143 131 107 106 173 79 85 117 131 192 193 152 220 174 179 195 178 133 91 e 163 141 e 178 172 141 139 146 83 175 161 92 161 136 150 157 161 149 146 84 157 148 138 133 142 132 101 139 128 106 153 186 152 140 171 156 173 172 159 109 81 85 115 180 152 204 162 149 136 113 143 136 172 176 159 63 c 145 139 171 163 140 77 180 199 139 140 138 144 138 175 157 161 210 116 e 164 125 172 176 161 161 139 131 195 198 106 182 167 166 143 197 202 155 137 157 150 152 142 158 165 176 195 181 174 130 e 182 201 255 118 c 164 171 125 152 126 121 183 118 168 146 115 159 150 123 166 e 178 142 Temporal Nasal Dorsal ODCells/mm Highest High Medium Low Figure 3. Topographic map of case 2626-4 (areas marked with e= edge, nc= not countable) Retina 2626-4 This retina came from the right eye of an a dult female shark. A band of “higher” cell density was observed along part of the retinal meri dian, starting at the nasal edge of the retina and running across the entire retinal meridian (Table 2, Figure 3). The dorso-nasal portion of the retina contained mostly medium cell counts with a few randomly interspersed high counts. The dorso-tem poral portion of the retina also contained some medium and mostly high cell counts, as well as an area of High cell counts located just above the retinal meridian running from the mid-retina to th e extreme temporal edge of the retina. The ventro-nasal portion of the retina contained mos tly medium cell counts with a few low and high

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28counts at the extreme ventro-nasal edge. The ventro-temporal portion of the retina contained mostly high cell counts. 196173 195193 559377 e 11711293154 180228157 180 123 176 155 136 177 143131110139112 107 11812794120 91100 e 160 1781621541718040 59 141 171 239 148153132175131 15614786 147 115 e151 142 191250 192 14215317722595 63 180 134 173 147 144 246 244 256245203 243194207189 193 155116 165202 164 146124155157132 104 150 125 116107125114 e 127 nc nc nc nc nc nc 179 156 130 142 133 146 161 153 167 194 148 137 119 177 162 172 136 144 124 62 91 225 217 194 196 140 166 167 163 168 187 169 165 185 181 170 178 144 199 165 176 189 165 133 135 nc 181 181 51 193 171 163 126 107 133 102 124 168 156 131 127 142 80 186 170 158 58 181 142 159 161 153 182 147 134 185 209 166 208 217 171 154 168 186 147 171 132 109 165 210 207 209 171 142 164 171 147 168 170 118 124 156 176 198 148 170 172 140 137 169 159 89 113 111 175 168 207 147 164 149 89 157 64 158175 183 217 187 147 151 162 174 139 138 120 80 101 146 177 174 111 163 85 152 160 125 102 118 133 98 104 90 133 110 83 229 119 95 169 130 e 86126 118 99 185 182 147 238197 147 108 108 135 80 135 198 133 206 127 181 139114 144 129 74108142 129 175 143 119 159 16814215093 196173 187 206 165 175 114 153 152 229 139123 160 97 87 154 172 146 98 164 182 109 63 236 242 107131 1371909516271 159 158118 121 208 169 92139 155 209 1mm 0-480 481-747 748-1010 1011+ Dorsal Nasal Temporal Ventral Cells/mmHighest High Medium Low Figure 4. Topographic map of case 2626-5 (areas marked with e= edge, nc= not countable) Retina 2626-5 This retina came from the left eye of an adu lt female shark. A band of “higher” ganglion cell density (outlined in figure) began in the midnasal portion of the retina and ran across the rest of the retinal meridian (Table 2, Figure 4). The dorso-nasal portion of the retina contained mostly medium and low cell counts. The dorso-temporal portion of the retina contained mostly medium cell counts. The ventro-nasal portion of the retina contained mostly medium cell counts whereas the ventro-temporal portion of the retina contained mostly medium and some high ce ll counts in the most dorsal part of this area.

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29 1mm 170 138 124 nc 146 133 154 114 135 e 170 170 126 153 132 133 152 166 200 149 128 94 108 118 106 164 140 120 146 96 97 101 120 121 128 140 98 122 135 159 106 133 130 ck 131 134 140 153 139 118 nc 136 109 134 128 130 129 184 185 137 129 e 117 e 155 148 e 79 186 116 170 163 158 233 181 135 211 144 124 163 228 222 191 183 161 144 174 139 136 159 197 176 186 181 142 136 144 e 142 133 127 119 134 187 192 158 168 160 146 139 126 122 ne 107 101 95 138 163 123 180 163 167 156 125 131 128 121 nc 78 79 97 136 132 148 187 166 169 155 135 128 111 109 179 211200199224 257 170 220 ck 193 219 208 85146137 124135138 149123 193 228 238 264 253 248 281 nc 226219 176198 224 202181 174 144145 167 146 184 175 176226nc 213303222243274 266209210170 214205186176198203204 163 149 0-480 481-747 748-1010 1011+ nc nc nc nc ncnc nc nc nc nc nc nc nc nc 71 132 170 255 242 181 178 189 171 173 225249 216 244240250 263 233 222 218 215 173 165 184 172 158 152145 nc 292 264347264 230 180 159 173 179 142 199223 248 265 217 241 191 211 212 216 206 211 163 175 303 267 248 174 287 196 203 262 nc 186 171 176 168 140 162 nc nc 177 251 234 152 201 227 218 251 232 186 195 229 200 248 144 166 140 192 150 170 159 154 62 140 176 177 135 140 143 120 141 139 136 193 117 146 140 164 112 213 153 170 185 161 219 170 187 160 206 286 181 248 138 139 188 171 236 171 193 235 175 161 203 139 180 168 172 264 177 175 175 141 181 142 205 211 281 Dorsal Vent r al Nasal TemporalOD154 184 134 202 183 190 168 180 180 165 203 171 181 150 178 183 191 99 112 125 Cells/mmHighest High Medium Low Figure 5. Topographic map of case 2626-8 (areas marked with e= edge, nc= not countable) Retina 2626-8 This retina came from the right eye of an a dult female shark. A band of “higher” cell density (outlined) was located along the retinal meri dian. This band started in the nasal portion of the retina and ran across the majority of the retin al meridian as well slightly into the ventrotemporal portion of the retina (Table 2, Figure 5). The dorso-nasal portion of the retina contained mostly medium cell counts. The dorsotemporal portion of the retina contained mostly medium cell counts with a small area of high counts within the dorsal portion of this area. The ventro-nasal portion of the retina contained mostly medium cell counts interspersed with a few high counts. The ventro-temporal portion of

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30the retina contained mostly high cell counts with the very extreme ventral edges containing medium cell counts. 92 106 59 56 86 122 131 109 108 78 133 93 100 111 145 133 116 124 134 143 135 122 147 119 133 178 217 142 156 129 e 121 96 128 120 117 85 195 18 182 248 204 149 140 138 170 152 120 148 143 128 168 271 162 206 230 276 184 210 172 183 157 145 151 202 185 184 188 149 217 179 186 196 206 272 232 240 150 134 126 158 127 125 173 162 158 191 149 178 209 224 240 167 197 202 219 206 141 152 151 120 143 176 133 226 196 217 230 145 232 226 178 108 130 107 117 129 130 180 127 164 216 231 235 224 104 210 228 245 210 159 133 139 120 107 140 92 123 143 158 190 133 182 173 46 53 183 247 175 142 110 101 156 131 118 230 169 e 196 221 182 175 157 155 140 139 e 131 173 153 135 121 153 202 169 220 200 190 241 204 156 204 163 146 163 152 150 181 176 191 257 238 228 206 222 170 155 152 145 140 166 e 157 203 119 153 227 192 145 e 185 169 176 169 197 146 157 132 139 184 153 166 192 146 135 146 142 173 179 158 174 156 104 155 173 126 109 140 137 159 157 126 91 89 113 e 159 106 86 111 175 183 116 143 159 200 0-480 481-747 748-1010 1011+ 1mm Dorsal Ventral Temporal NasalCells/mm Highest High Medium Low Figure 6. Topographic map of case IMF1 Retina IMF1 This retina came from the right eye of an i mmature female. A band of “higher” cell density (outlined) was located along the retinal meridi an and started at the nasal edge of the retina and ran into both the dorso-temporal and ventro -nasal portions of the retina (Table 2, Figure 6). The dorso-nasal portion of this retina contained mostly medium cell counts with some a few high cell counts located at within the central portion of this area. The dorso-temporal portion of the retina contained high density cell counts within its mid-to-dorsal portion and medium and low counts at the extreme temporal edge of th e retina. The ventro-nasal portion of the retina

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31contained mostly high cell counts, whereas the ve ntro-temporal portion contained mostly medium cell counts with a small area of high counts in the most ventro-temporal portion of this area. 1mm 0-480 481-747 748-1010 1011+ 216 187 196 217 189 207 120 227 241 295 200 174 169 160 207 217 163 189 158 178 167 192 151 163 146 e 200 203 178 172 180 138 126 214 199 198 172 203 144 198 297 210 169 106 143 183 180 187 201 nc 200 167 196 218 224 186 nc e 203 264 199 193 204 186 185 176 137 119 147 194 205 201 194 176 195 175 226 e 223 182 127 163 103 195 158 160 182 251 222 187 e 202 179 117 170 224 e nc 219 190 200 195 201 164 139 182 197 226 231 e 206 215 263 196 238 211 168 e 108 192 175 157 178 e e 256 193 203 164 116 95 128 126 167 150 158 137 154 144 197 221 217 218 160 145 153 188 nc 175 167 234 236 195 123 138 171 205 135 185e 248 168 nc 197 231 240 170 199 113 136 128 203 154 138 168 e e nc 183 140 nc 130 166 173 211 224 278 190 200 139 164 127 124 175 107 179 196 141 111 130 159 175 178 175 186 228 273 215 169 96 92 207 175 201 172 183 190 187 199 161 172 170 169 169 171 177 182 221 260 183 177 99 nc 171 113 151 128 147 151 134 e 123 168 149 112 150 165 167 243 e 218 82 127 127 92 202 150 125 114 119 nc nc 132 135 148 168 166 188 e 296 e 154 e 164 e 217 nc nc nc 147 160 157 156 171 197 253 322 189 152 154 130 175 nc 232 137 164 174 173 154 228 221 220 237 182 157 130 nc 128 145 215 218 200 270 291 206 225 117 150 139 133 189 183 192 287 209 255 267 201 190 198 nc 180 nc44 45 Dorsal Temporal Ventral NasalCells/mmHighest High Medium Low Figure 7. Topographic map of case IMF2 (areas marked with e= edge, nc= not countable) Retina IMF2 This retina came from the right eye of an i mmature female. A narrow band of “higher” cell density, starting at the nasal edge of the retina and running across the entire retina, was located along the retinal meridian, just above the optic disc (Table 2, Figure 7). The dorso-nasal portion of the retina some medium cell counts with an area of high cell counts. The dorso-temporal portion of the retina contained mostly high cell counts. The ventronasal portion of the retina contained mostly medium and a scattering of high cell counts whereas

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32the ventro-temporal portion of the retina containe d a mixture of high and medium cell counts with no distinguishing pattern. 114 129 115 152 199 134 142 168 111 124 102 144 125 173 133 137 144 137 148 155 147 172 169 139 152 138 127 123 120 146 e 143 123 167 134 119 e 109 153 154 172 145 132 142 247 28 87 e 126 e/c e 58 171 51 146 198 137 153 108 101 134 109 126 110 65 150 94 136e 158 155 117 152 149 99 105 90 107 146 99 130 55 135 164 119 179 119 113 102 72 104 130 92 96 130 106 128 214 141 169 187 201 160 122 109 140 83 125 156 184 86 148 108 139 102 116 123109 111 110 111 155 164 159 168 129 116 e 121 108 114 108 132 101 117 106 e 126 171 74 192 92 134 141 108 139 156 12 127 107 113 143 82 139 e 146 89 130 86 154 102 138 102 136 62 137 51 164 132 100 136 157 122 82 193 207 191 223 e 185 170 100 132 130 106 98 e 138 121 62 79 139 87 159 180 138 173 173 152 132 156 116 127 133 172 85 121 121 125 127 104 105 116 134 91 148 116 145 105 101 82 151 68 72 184 145 153 165 136 122 148 155 116 177 135 175 111 138 109 113 92 129 112 139 108 162 108 116 122 184 143 127 193 159 85 135 157 119 151 148 155 188 149 e 122 122 107 131 120 122 102 110 97 127 94 156 144 137 173 123 nc 135 e 142 99 98 e 120 108 110 190 105 97 112 121 86 222 e 93 78 78 125 202 113 163 119 143 134 99 112 122 102 156 127 102 108 113 e 110 74 nc 135 105 127 e 94 126 125 121 128 113 140 102 124 e 114 e 139 e ncnc 103 Dorsal Ventral Temporal Nasal ON 1mmCells/mmHighest High Medium Low Figure 8. Topographic map of case 2625-2 (areas marked with e= edge, nc= not countable) Retina 2625-2 This retina came from the right eye of an a dult male. This retina did not contain any distinct pattern of cell distribution. However, there were scattered small areas containing high cell counts located in areas along the retinal meridian (Table 2, Figure 8). Both the dorso-nasal and dorso-temporal portio ns of the retina contained mostly medium and low cell counts without any definite pattern to their distribution. Both the ventro-nasal and

PAGE 34

33ventro-temporal portions of the retina containe d mostly medium cell counts. There were scattered areas of high counts along the retinal meridian, however these counts were not distributed in a way that would allow for the establishment of an observable cell distribution pattern. 195 163 183 167 156 160 170 128 125 138 175 164 141 208nc107 151 141 132 130 126 149 130 168 104 126 105 119 136 128 137 192 93 ck 142 131 133 106 115 138 131 143 154 109 111 168 145 170 140 ck 100 137 121 100 203 132 142 143 136nc136e140 137 132 136 121 127 116 119 129 171 159 141 137 140 133e nc163 160 170 138 149 132 123 140 91 155 160 139 163 154 131 132 159e182 178 177 162 138 137 110 103 122 160 158 131 190 146 168 166 151 153 126 130 119 131 112 140 129 147 175 175 178 146 201 199e145 153nc122 126 100 174 121 134 167 165 69 102 106 144 82 ck 174nc109 130 161 189 171 150 95 90 142e156 129 124 177 180 199 150 140 63 on 159e159 134 98 115 89 120 197 165 167 240 147 112 179 147 108 120 141 123 107 114 118 147 106 161 181 166 31 156 174 109 128 138 73 95 79 136 76 101 181 155 127 130 198 197 164 97 92 85 74 65 82 135 159 204 193nc135 145 160 123 101e90 57 53 93 142 182 218 198 107 73 82 50 41 67 92 124 152 173e54 67 68 112 127 161nc153 116 45nc nc nc134 163 115 110nc nc nc e132e111 36 40e nc e132 68nc nc nc nc83 84 129 57 36 95 125 116 109e nc131nc154 107 168e169nc 138 134 123 158 134 160 165 131nc154 107 168e177 170 162 137 135 173 138 145 152 188e180 167 177 108 136 132 176 141 182 154 173 167 207 154 137 168 134 172 171 178 131 142 145 158 162 128 149 218 163 167 161 134 130 140 147 163 142 173 107 136 136 168 153e85 117 94 141 153e127 115 92 120e128 106 129 135 1mm 0-480 481-747 748-1010 1011+Dorsal Nasal Temporal VentralCells/mmHighest High Medium Low Figure 9. Topographic map of case 2626-6 (areas marked with e= edge, nc= not countable) Retina 2626-6 This retina came from the right eye of an adult female shark. This retina showed absolutely no distinguishable pattern of cell di stribution along the retinal meridian (Table 2, Figure 9). The dorso-nasal and dorso-temporal portions of the retina contain mostly medium cell counts. Both the ventro-nasal and ventro-tem poral portions of the retina contained mostly

PAGE 35

34medium cell counts with some high and low c ounts interspersed across the retina in no distinguishable pattern.

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35Chapter Four Discussion The current results revealed that bonnethead sharks had some heterogeneity in retinal ganglion cell distribution. The results also showed that the bonnethead possessed a higherdensity “band” of ganglion cells traversing the cen tral potion of the retinal meridian. Finally, small areas of higher cell density in the dorso -temporal area were found in several cases. Although a higher density “band” of retinal ganglion cells was found within the retinal meridian of this species, the ratio between cell counts within the retinal meridian and other areas outside of the retinal meridian were not appreci ably different enough to warrant calling this area a visual streak. The term “band” was used in the present study, even though there was a high degree of individual variation in its overall shap e between cases, because of its fairly elongated shape in all cases. The results of this study may be explained by two factors (habitat openness and predatory behavior) that appear to be related to ganglion ce ll topography in sharks as well as other species. Although bonnethead sharks used in this study lived in fairly well-lit, shallow water habitat, their surroundings were likely obstructed, to some extent, due to particulate matter in the water. This factor may be related to the relatively short le ngth and low ratio between cell density within and outside of the “band” of higher cell density within their retinal meridian. The low ratio between cell counts within and outside of the “band” as well as the low overall density of ganglion cells within the bonnethead retina may also be expl ained by behavior. Though the diet of these animals is well-known, when they are most active during a 24-hour cycle is not. Therefore, the results from this study may also be explained by these sharks being predominately nocturnal in nature. If the bonnethead shark is a predominantly nocturnal predator, then a visual streak may not be necessary to aid them in prey detection. Sensitivity to light, rath er than visual acuity, would likely be more important to a nocturnal pred ator. Thus, if this species is predominately

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36nocturnal, this may explain their lack of a visual streak. The dorso-temporal area of increased ganglion cell density found in several cases c ould also be potentially associated with the predatory behavior of this species. Habitat openness Habitat openness may influence both the length of areas of higher ganglion cell density as well as the width of these areas. Hughes terrain th eory (1977) predicts that animals with a distinct visual horizon should possess a visual streak or areas of higher ganglion cell density along their retinal meridian. More specifically, species with unobstructed views of their surroundings generally possess lengthy visual str eaks which extend across the entire length of their retinal meridian (Collin and Pettigrew, 1988b). Species inhabiting areas with partially obstructed views of their habita t generally possess visual streaks that do not traverse the entire length of their retinal meridian (Oliver et al., 2001). Results of this study revealed that the “ba nd” of increased cell density in the bonnethead shark was rather narrow in width and did not trav erse the entire length of the retinal meridian. The increase in overall ganglion cell density across th e retinal meridian was expected because of the fairly-well illuminated, shallow water habitat of the bonnethead shark. Additionally, several other shark species (lemon, tiger, epaulette, sma ll-mouth dogfish, etc.) all possessed a visual streak. Although it does not meet the requirements of a visual streak, the existence of a short and weak “band” found in the present study is consistent with predictions from Hughes terrain theory. Why did the bonnethead sharks in the presen t study not possess a visual streak? One possibility may be their activity cycle. If this species is predominately nocturnal in nature, that could explain the lack of a strong visual streak as well as why their ganglion cell density is rather low considering their habitat. Nocturnal predator s require a visual system that is more sensitive to light (and would possess lower ganglion cell dens ities across their retinal meridians) than able

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37to resolve visual images with high acuity. Anot her possibility may be related to the water quality of their habitat. Sharks used in this study we re collected from the waters of Charlotte Harbor, which contains rather cloudy water (Humphrey s and Grantham, 1995; Tomasko, 2001). Because of murkiness of the water, the view of the visu al horizon was likely obstructed, to some extent, for this shark species, potentially making possession of a visual streak not useful to them. Therefore, sharks in this habitat may not have developed a visual streak, dependent on the amount of time they spend within this habitat during the year. A potential answer as to whether the water qua lity of the bonnethead sharks collected for this study or their behavioral patterns over a 24 hour-cycle affected the ganglion cell density within their retinas could be found from a comparis on of bonnethead sharks living in the waters of Charlotte Harbor to those inhabiting Florida Bay. Florida Bay is located near the Florida Keys and contains less particulate matter which can inte rfere with overall water clarity (Cortes et al., 1996; Cortes and Parsons, 1996). Preliminary results from mitochondrial DNA testing have also revealed that there is no significant diffe rence between the mitochondrial DNA of bonnethead sharks inhabiting either region (Lombardi-Carls on, Cortes, Parsons, and Manire, 2003). This comparison would be necessary to ascertain whether a relatively narrow and short area of increased ganglion cell density is found species-wide or is due to nocturnal behavior or even potentially regional differences in habitat. Behavior Behavior may also be an influential factor affecting retinal ganglion cell topography. This factor may influence both the width as well as the location of the visual streak and any associated specialized areas w ithin the retina (Bozzano and Collin, 2001; Collin and Pettigrew, 1988a and 1988b).

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38 In the bonnethead shark, their higher density “band” was found in a fairly central location within the retina. This higher density “band” di d not transverse the entire retinal meridian for the majority of cases. The higher density “band” lik ely subtends vision for this species visual horizon. Nonetheless, ganglion cell density may not be high enough, with in the higher density “band”, to provide this species with anything more than increased motion detection in this area of their visual environment. However, the areas of higher ganglion cell de nsity located within the temporal retina were not an expected finding, and could be related to the predatory behavior of this shark species. Species who have specialized methods of capturing prey may have specializations that provide them with a visual advantage in locating prey (Bozzano and Collin, 2001; Collin and Pettigrew, 1988a and 1988b; Oliver et al., 2001). Six of the bonnethead sharks from this study possessed a dorso-temporal area of increased ganglion cell density. According to Collin and Pettigrew (1988b), species possessing areas of increased ganglion cell density in thei r temporal retina likely use these areas for prey detection. Bonnethead sharks primarily predate upon swift moving blue crabs (Cortez et al., 1996). These crabs may be located moving along the substrate in front and slightly below the visual horizon of this shark. Therefore, an ar ea of higher ganglion cell density located within the dorso-temporal retina may be advantageous to th is shark species. An increased sensitivity to motion detection along the substrate would likely aid the bonnethead shark in locating potential prey items. Capture of these prey items would po ssibly then fall to other sensory systems, such as movement detected by the lateral line and el ectroreception of prey location detected through use of the ampullae of Lorenzini. Although results from the lone male retina utilized in the study did not reveal any topographic pattern, possibly due to methodological i ssues, the lack of a definitive pattern could also be related to sexually dimorphic differences in the head shape of th ese sharks between males

PAGE 40

39and females. Further investigation regarding the retinal topography of male bonnethead sharks is necessary to establish whethe r this is a possibility. Technical problems in the present study and possible solutions No definitive pattern of retinal ganglion cell distribution was found in two of the retinas (2625-2 and 2626-6) and considerable individual di fferences were observed in the rest of the retinas. This absence and variation of the retinal ganglion cell pattern may be due to methodological problems. In particular, in the field, it is possible that the eyes did not receive enough preservative to fix them properly or too mu ch time passed before preservative was added to the eyes to conserve them. If the eyes did not receive preservative in a timely matter, this could have resulted in ischemic damage which could explain the absence of a topographic pattern in two of the cases as well as the considerable i ndividual differences between the “band” of higher cell density as well as location and size of the dorso-temporal area between all cases. Retinal counts in this study may have also b een underestimated. The tracer DiI was to be utilized as a medium with which ganglion cell morphology in the bonnethead shark could be documented. However, DiI does not appear to be compatible with elasmobranch body chemistry and was unable to be used to document retinal ganglion cell morphology. Ganglion cell counts were then based on descriptions from both Hueter (1991) and Collin (1988). All ganglion cell counts in this study were based on darkness of the Nissl stain, cell-body shape, and presence of axon bodies. It is possible that the actual number of ganglion cells were undercounted. To correct for both problems, it may be bette r to watch the sharks after collection and inject preservative into them immediately after the shark expires, instead of attempting to inject preservative into the eyes within 15 minutes of the shark expiring. As of now, sacrificing the shark and immediately harvesting the eyes still appear s to be the most effective way to conduct a

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40study of retinal topography. It would also be he lpful to find a suitable tracer that works well with the body chemistry of elasmobranches. Future directions To further elaborate upon and better unders tand the significance of retinal ganglion cell topography in the bonnethead shark, an investigation of the visual threshold and visual capabilities of this shark species would be beneficial These types of studies should also take the differences in head shape between males and fema les into account. If this species is unable to detect targets or objects located in front and slightly below them, then vision is likely not as important to their daily survival as other sensory systems. Conducting an analysis of retinal ganglion cell topography on bonnethead sharks living in Florida Bay may also help to confirm the findings from this project. Findings from the same species of shark inhabiting a more illuminated and open habitat may shed light on whether or not the overall ganglion cell concentration as well as widt h and length of the higher density “band” is common to all bonnetheads living in Florida or vari es according to location (i.e. is influenced by environmental factors). If retinal topography betw een the two populations of bonnethead sharks is the same, then a study investigating whether or not these sharks are nocturnal in nature would also aid in better understanding of their retinal topography. Physically measuring and behaviorally testing the extent and limits of the visual field of the bonnethead shark (from either region) would also help to ascertain whether the re tinal topography of this shark could be related to its predatory behavior. Conclusions Even though a higher density “band” was reveal ed within the retinal meridian of this species, the ambiguity of the shape of this “ba nd” as well as the low ratio between the density of

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41this “band” may signify that vision is not as impor tant to this species as other sensory systems or that this shark could be more nocturnal than diur nal in its activity patterns. This region could also be left over from before the evolution of th is species unique head shape or could be used to lower the threshold for detection of disturban ces within the shark’s visual horizon. Further research regarding the visual capabilities of this sp ecies could reveal how, if at all, this area of increased cell density is utilized. Comparisons between this and other hammerhead species could also reveal whether or not retinal topography differs between the bonnethead shark and other hammerhead species. If there is a difference in re tinal ganglion cell topography within the other species of hammerhead sharks, it could signify that be havioral utility is the most influential factor behind retinal ganglion cell t opography in these sharks.

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42References Ali, M.A. and M. Anctil (1974a) Retinas of the electric ray ( Narcine brasiliensis ) and the freshwater stingray (Paratrygon motoro). Vision Research 14: 587-588. Ali, M.A. and M. Anctil (1974b) Giant ga nglion cells in the retina of hammerhead shark ( Sphyrna lewini ). Vision Research 14: 903-904. Ali, M.A. and M. Antcil, eds. (1976) Retinas of fishes: an atlas. Spriner-Verlag, Berlin. Arrese, C., Dunlop, S. A., Harman, A. M., Braekeve lt, C. R., Ross, W. M., Shand, J., and L. D. Beazley (1999) Retinal structure and visual acuity in a polyprotodont marsupial, the fat-tailed dunnart ( Sminthopsis crassicaudata ). Brain, Behavior, Evolution 53: 111-126. Bonazzo, and Collin, S.P. (2000) Retinal ga nglion cell topography in seven species of elasmobranch. Brain Behavior Evolution 55: 191-208. Collin, S.P. (1988) The retina of the shovel-nosed ray, Rhinobatos batillum ( Rhinobatidae ): morphology and quantitative analysis of the gang lion, amacrine, and bipolar cell populations. Experimental Biology 47: 195-207. Collin, S.P. and J. D. Pettigrew (1988a) Retinal topography in reef teleosts: some species with dell-developed area but poorly developed streaks. Brain, Behavior, and Evolution. 31:269-282.

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43Collin, S. P., and J. D. Pettigrew (1988b) Re tinal topography in teleosts: some species with prominent horizontal visual streaks and high-densit y areae. Brain, Behavior, Evolution. 31: 283295. Compango, L.V. J. (1984) Sharks of the world. Carcharhiniformes. FAO Fish Synops. Volume 4, Part2. Cortes, E. and G.R. Parsons (1996) Compar ative demography of two populations of the bonnethead shark ( Sphyrna tiburo ) in southwest florida. Canadian Journal of Fisheries and Aquatic Science 53: 709-718. Cortes, E., Manire, C.A., and R.E. Hueter, (1996) Diet, feeding habits, and diel feeding chronology of the bonnethead shark, Sphyrna tiburo in southwest florida. Bulletin of Marine Science 58: 353-367. Graeber, R.C. (1978) Behavioral studies correla ted with central nervous system integration of vision in sharks. Sensory Biology of Sharks Skates, and Rays. E.S. Hodgson and R.F. Mathewson, eds. Office of Naval Research, Arlington, Va. Pg. 11-105. Gruber, S.H. (1977) The visual system in sharks: adaptations and capability. American Zoologist. 17: 453-470. Gruber, S.H., Gulley, R.L., and J. Brandon (1975) Duplex retina in seven elasmobranch species. Bulletin of Marine Science. 25: 353-358.

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44Gruber, S.H., Hamasaki, D.I., and C.D. Bridges (1963) Cones in the retina of the lemon shark ( Negaprion brevirostris). Vision Research 3: 397-399. Hamasaki, D.I., and S.H. Gruber (1965) The photoreceptors of the nurse shark, Ginglymostoma cirratum and the stingray Dasyatis sayi Bulletin of Marine Science. 15: 1051-1059. Hebel, R. (1976) Distribution of retinal gang lion cells in five mammalian species (pig, sheep, ox, horse, dog). Anatomy and Embryology (Berl.). 150: 45-51. Hoese, H.D. and R.B. Moore (1958) Notes on the life history of the bonnetnose shark, Sphyrna tiburo The Texas Journal of Science 10: 69-71. Hueter, R.E. (1988) Retinal Topography and the retinotectal projection pattern in the juvenile lemon shark ( Negaprion brevirostris) Society of Neuroscience Abstracts. 14:1119. Hueter, R.E. (1989) The organization of spatial vision in the juvenile lemon shark ( Negaprion brevirostris ): retinotectal projections, retinal topography, and implications for the visual ecology of sharks. Dissertation Abstracts International Pa rt B: The Sciences and Engineering 50: 138. Hueter, R.E. (1991b) Adaptations for spatial vision in sharks. The Journal of Experimental Zoology Suppliment 5: 130-141. Hueter, R.E. and S.H. Gruber (1982) Recent Advances in studies of the visual system of the juvenile lemon shark ( Negaprion brevirostris ). Florida Scientist 45: 11-28.

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45Hughes, A. (1977) The topography of vision in mammals of contrasting lifestyle: comparative optics and retinal organization. Handbook of Sensory Physiology VII/5: The visual system in vertebrates. F. Crescitelli, ed. Springer-Verlag, Berlin. Pg. 613-756. Humphreys, J. and S.B. Grantham (1995) Char lotte Harbor. Fanthom Magazine, Winter edition. Huxlin K.R. and Goodchild A.K. (1997) Retin al ganglion cells in the albino rat: revised morphological classification. Journal of Co mparative Neurology 385: 309 323. Johnsen, P.B. and J.H. Teeter (1985) Behavioral responses of the bonnethead ( Sphyrna tiburo ) to controlled olfactory stimulation. Marine Behavior and Physiology 11:283-291. Kajiura, S.M. and K.N. Holland (2002) Elect roreception in juvenile scalloped hammerhead and sandbar sharks. Journal of Experi mental Biology 205: 3609-3624. Lima, S.M., Silveira, L.C., and V.H. Perry (19 96) Distribution of M ganglion cells in diurnal and nocturnal New World monkeys. Journal of Comparative Neurology 368: 538-52. Lombardi-Carlson, L.A., E. Cortes, G.R. Parsons and C.A. Manire. 2003. Latitudinal variation in life-history traits of bonnethead sharks, Sphyrna tiburo (Carcharhiniformes: Sphyrnidae) from the eastern Gulf of Mexico. Marine & Freshwater Research 54(7):875-884. Martin, A. (1993) Hammerhead shark origins. Nature 364: 494.

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46Mass, A. M. and A. Y. Supin (2000) Ganglion ce ll density and retinal resolution in the sea otter, Enhydra lutris Brain, Behavior, Evolution. 55: 111-119. Motta, P.J., Tricas, T.C., Hueter, R.E., and A.P. Summer (1997) Feeding mechanism and functional morphology of the jaws of the lemon shark, Negaprion brevirostri. Journal of Experimental Biology 200: 2765-2780. Motta, P.J. and C.D. Wilga (2000) Durophagy in sharks: feeding mechanics of the hammerhead Sphyrna Tiburo The Journal of Experimental Biology 203: 2781-2796. Myrberg and Gruber (1974) The behavior of the bonnethead shark. Copeia. 2: 358-374. Nakaya, K. (1995) Hydrodynamic function of the head in the hammerhead sharks (Elasmobranchii: Sphyrnidae). Copeia 1995: 330-336. Oliver, L.J., Salmon, M., Wyneken, J., Hueter, R. E., and T.W. Cronin (2000) Retinal anatomy of hatchling sea turtles: anatomical specializa tions and behavioral correlates. Marine and Freshwater Behavavior and Physiology 33: 233-248. Peterson, E.H., and M.H. Rowe (1980) Differe nt regional specializations of neurons in the ganglion cell layer and inner plexiform layer of the California horned shark, Heterodontus francisci Brain Research. 201: 195-201.

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47Shimizu, T., Cox, K., Karten, H., and L.R.G. Britto (1994) Cholera toxin mapping of retinal projections in pigeons ( Columbia livia ), with emphasis on retinohypothalamic connections. Visual Neuroscience 11: 441-446. Stell, W.K. (1972) The structure and morphologic relations of rods and cones in the retina of the spiny dogfish, Squalus Comparative Biochemistry and Physiology 42A: 141-151. Stell, W.K., and P. Witkovsky (1973) Retinal structure in the smooth dogfish, Mustelus canis : general description and light miscoscopy of giant ganglion cells. Journal of Comparative Neurology 148: 1-32. Stone, J. and P. Halasz (1989) Topography of the retina in the elephant Loxodonta africana Brain, Behavior, Evolution. 34: 84-95. Strong, W.R., Gruber, S.H., and F.F. Snelson (1 990) Hammerhead shark predation on stingrays: an observation of prey handling by Sphy rna mokkarran. Copeia. 1990: 386-340. Tester, A.L., and S. Kato (1966) Visual target discrimination in blacktip sharks ( Carcharrhinus melanopterus ) and grey sharks ( C. menisorrah ). Pacific Science 20: 461-471. Tomasko, D.A. (2001) Seagrass Restoration Varies in Sout hwest Florida's Estuaries. ERF Conference, Tradewinds Conference Center, St. Pete Beach, Florida November 4-8.

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48Wong, R. O., Wye-Dvorak, J., and G. H. Henry (1986) Morphology and distribution of neurons in the retinal ganglion cell layer of the adult tammar wallaby – Macropus eugenii Journal of Comparative Neurology. 253: 1-12. Wright, T. and R. Jackson (1964) Instrument al conditioning of young sharks. Copeia. 1964: 409-412.

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49 APPENDICES

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50Appendix A: Raw data reti nal counts (in cells per mm) Dorsal Ventral Temporal Nasal Figure 10. Raw data retina 2627-1

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51 Dorsal Nasal Ventral Temporal Figure 11. Raw data retina 2626-4 747 Dorsal Nasal Temporal Vent r al733 724 806 885785 Figure 12. Raw data retina 2626-5

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52 Nasal 1mm 770 783 625 1132 561 896 nc 797 661 552 602 566 697 507 516 448 611 865 e 828 770 806 770 679 570 819 693 774 598 919 602 747 688 815 752 815 905 761675 860 580 828 426 914 489 607 534 833 480 697 742Temporal634Ventral543Dorsal661 1182 435 955 439 928 457 643 543 819 548 638 580 792 634 792 435 801 611 1195 720 779 480 761 602 815 589 629 593 919 607 729 634 792 693 1064 629 874 534 774 nc 1068 616 774 493 851 607 629 580 625 589 1123 584 819 833 1204 838 933 620 724 584 847 e 770 530 991 e 729 702 838 670 770 e 693 358 964 842 507 525 742 770 634 738 661 715 530 1055 874 819 616 611 629 955 638 652 543 561 647 738 634 1032 611 1005 801 865 797 828 634 729 281 652 697 788 720 629 770 616 679 720 869 892 634 797 752 742 652 819 1123 643 905 616 1037 652 883 e 842 653 1050 602 1136 575 987 539 1028 607 910 847 720 869 1059 715 1136 761 801 724 nc 661 nc 629 733 570 634 552 761 ne 797 484 774 457 842 430 nc 625 1186 738 919 557 887 815 1299 738 788 756 1123 706 1209 566 1372 593 792 580 738 548 955 nc 933 353 978 358 960 439 955 616 865 598 1091 670 982 847 1200 752 1123 765 1010 702 901 611 643 580 810 503 783 493 720 815 810 1041 955 1195 905 1571 901 1195 1014 1322 1163 nc 770 656 996 688 ck 715 874 779 991 833 942 747 385 783 661 973 620 987 561 1005 611 1055 625 1191 675 1087 874 1105 1032 978 1077 1127 1195 1019 1145 774 1123 856 1272 806 nc 819 1023 1096 991 1154 1014 770 914 598 809 321 788 nc 652 nc 656 nc 756 nc 661 nc 833 nc 792 nc 797 nc 1023 nc nc nc 964 nc 1272 nc 1005 nc 1100 nc 1240 6751204 738 946924 951919 770896 969797 928842 Figure 13. Raw data retina 2626-8 Dorsal Ventral Temporal Nasal Figure 14. Raw data retina IMF1

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53 1mm 978 847 887 982 856 937 543 1028 1091 1336 905 788 765 878 724 937 982 738 856 715 806 756 869 684 738 661 e 905 919 806 779 815 625 570 969 901 896 779 919 652 896 1345 951 765 480 647 828 815 847 910 ne 905 756 887 987 1014 842 na e 919 1195 901 874 924 842 838 797 620 539 665 878 928 910 878 797 883 792 1023 e 1010 824 575 738 466 883 715 724 824 1136 1005 847 e 914 810 530 770 1014 e na 991 860 905 883 910 742 629 824 892 1023 1046 e 933 973 1191 887 1077 955 761 e 489 869 792 711 806 e e 1159874 919 742 525 430 580 570 756 679715 620 697 652 892 1001 982 987 724 656 693 851 na 792 756 1059 1068 883 557 625 774 928 611 838 1123 761 na 892 1046 1087 770 901 512 616 580 919 697 625 761 e e na 838 634 na 589 752 783 955 1014 1259 86 905 629 742 575 561 792 484 810 887 638 503 589 720 792 806 792 842 1032 1236 973 765 435 417 937 792 910 779 828 860 847 901 e 729 779 770 765 765 774 801 824 1001 1177 828 801 448 na 774 512 684 580 665 684 607e 403 557 761 675 507 679 747 756 1100 e 987 371 575 575 417 914 679 566 516 539 na na 598 611 670 761 752 851 e 1340 e 697 e 742 e 982 na nc nc 665 724 711 706 774 892 1145 1458 856 688 697 589 792 nc 1050 620 742 788 783 698 1032 1001 996 1073 824 711 589 nc 580656 973 987 905 1222 1317 933 1019 530 679 629 602 856 828 869 1299 946 1154 1209 910 860 896 nc 815 nc Dorsal Ventral Nasal Temporal Figure 15. Raw data retina IMF2 1mmDorsal Ventral Nasal Temporal

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54 Figure 16. Raw data retina 2625-2 1mm Dorsal Temporal Ventral Nasal Figure 17. Raw data retina 2626-6