Spectral Sensitivity of the Invasive Lionfish (Pterois

SPECTRAL SENSITIVITY OF THE INVASIVE LIONFISH (PTEROIS

SPECIES) RETINA AND ITS CAPACITY FOR CHANGE IN RESPONSE TO

LIGHTING CONDITION

MIRANDA ROSELAND CARROLL

B.S., Florida Institute of Technology

A thesis submitted to the Department of Biological Sciences of Florida Institute of Technology in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE in BIOLOGICAL SCIENCE

Melbourne, Florida May 2018

SPECTRAL SENSITIVITY OF THE INVASIVE LIONFISH (PTEROIS

SPECIES) RETINA AND ITS CAPACITY FOR CHANGE IN RESPONSE TO

LIGHTING CONDITION

MIRANDA ROSELAND CARROLL

Approved as to style and content by:

Jonathan Shenker, Ph.D., Chairperson Michael Grace, Ph.D., Member Associate Professor Professor Emeritus Department of Biological Sciences Department of Biological Sciences

Samantha Fowler, Ph.D., Member Lorian Schweikert, Ph.D., Member Assistant Professor Postdoctoral Scholar Department of Biological Sciences University of Florida

May 2018

ABSTRACT

SPECTRAL SENSITIVITY OF THE INVASIVE LIONFISH (PTEROIS

SPECIES) RETINA AND ITS CAPACITY FOR CHANGE IN RESPONSE TO

LIGHTING CONDITION

By Miranda Roseland Carroll, B.S., Florida Institute of Technology

Chairperson of Advisory Committee: Jonathan Shenker, Ph.D.

Since their first confirmed sighting in 1985 in the tropical western Atlantic, invasive lionfish of the genus Pterois have had a detrimental impact on marine systems in this region, including significant declines in native reef-fish populations.

The environmental tolerances of these predators are very broad, allowing them to inhabit a variety of environments: they can withstand large ranges of depths, salinities, and temperatures. Understanding their physiological and behavioral characteristics can provide insight for the success of these invaders. Adaptable visual capabilities is one biological characteristic that may assist their invasive success. I hypothesized that lionfish possess retinal plasticity and can adapt their spectral sensitivity in response to lighting conditions most prevalent in an invaded region, thus making them more effective predators. iii

Lionfish were collected alive along the East coast of Florida. To assess the adaptability of their visual systems, fish were housed for four months under either blue or red lighting conditions on a 12-hour-light/12-hour-dark cycle.

Electroretinography was used to assess the spectral sensitivity profile by measuring the initial retinal response of the fish to different wavelengths of light, from 350 to

650 nm in 50-nm increments, and again after two and four months under the experimental lighting conditions. Regardless of the lighting regime, lionfish were most sensitive to the green (500 – 550 nm) region of the spectrum, and very weakly sensitive to the red (600 – 650 nm) region of the spectrum. Neither lighting condition nor time of exposure had a significant influence on spectral sensitivity (2-

Way Repeated Measures ANCOVA and Anderson-Darling k-sample test). This lack of plasticity of spectral sensitivity may be due to the primarily crepuscular feeding habits of the species. Feeding under twilight and rapidly changing light conditions makes it more likely that their retinas are primarily adapted to photon capture in low light conditions, rendering color vision less essential. Further examination and characterization of the lionfish visual system is thus warranted to determine how lionfish use vision under changing and low light conditions. Such studies may help understand the biological traits that enable the success of this invader and help define additional habitats that are vulnerable to invasion.

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

TABLE OF CONTENTS ...... v

DEDICATION ...... vii

ACKNOWLEDGEMENTS ...... viii

LIST OF FIGURES ...... x

LIST OF TABLES ...... xiii

INTRODUCTION ...... 1

BIOLOGY...... 1

DISTRIBUTION AND GENETIC DIVERSITY...... 3

PHYSIOLOGICAL AND ECOLOGICAL VARIABILITY...... 6

THREATS TO INVADED COMMUNITIES...... 7

VISION AND LIGHT IN MARINE ENVIRONMENTS...... 8

VISION...... 8

LIGHT IN MARINE ENVIRONMENTS...... 13

ASSESSMENT OF VISUAL SYSTEMS OF FISH USING ELECTRORETINOGRAPHY ...... 16

RATIONALE AND RESEARCH OBJECTIVES ...... 22

MATERIALS AND METHODS ...... 24

LIONFISH COLLECTION AND HOUSING ...... 24

ELECTRORETINOGRAPHY...... 25

ANALYZING SPECTRAL SENSITIVITY ...... 28

RESULTS ...... 31

DISCUSSION...... 51

LITERATURE CITED...... 61

DEDICATION

I would like to dedicate this to my family, Jon, Deb, Matt, and Marc, who are all crazy. I love you all so much.

vii

ACKNOWLEDGEMENTS

I would like to thank my academic advisor, Dr. Jon Shenker for “adopting” me at the end of my master’s program and being incredibly supportive and thoughtful. I would like to thank my remaining committee members, Dr. Lorian

Schweikert, Dr. Samantha Fowler, and Dr. Michael Grace for crucial guidance with my project, providing me with connections, and challenging me as a scientist. I would like to specifically thank Lori, without whose guidance and support this work would have not been possible. Thank you to Dr. Grace and Dr. Emily Ralston for giving me the opportunities that allowed me to attend Florida Tech and ultimately complete this work and my degree. I’m very thankful to Dr. Glenn

Miller, who tirelessly answered all of my statistics questions and was always willing to assist me, and Dr. Rich Aronson for his support and reassurance throughout this process.

This project would not have been possible without the help of Emily Dark, who provided not only assistance in locating lionfish but also transportation throughout the Loxahatchee River and the Ft. Pierce inlet. Rena Trotter, Mark

Fusco, Jeff Beal, Gabor Tamasey, Dr. Mitchell Roffer, Roger and Vale Estrada, and Zack Jud were all also very helpful in the lionfish collection process.

I am immensely grateful to my friends and labmates, Molly Wightman,

Megan “Meggo” Setter, Aline “Weeny” Franqui, Jake “CB” Rennert, Tony

“Bologna” Cianciotto, Ann Wassick, Rachel Sales, Helen “Melon” Plylar, Nikia

viii

Rice, Kristin Kopperud, Brittan Steffel, and Aaron Martes for all of their love, encouragement, assistance with various aspects of this work, and frequent consoling. I would also like to thank my unofficial life coach, Dee Dee van Horn for her love and support, as well as Gayle Duncomb for her assistance during ERG testing.

I finally would like to thank my family. My parents, Jon and Deborah

Carroll, are the most wonderful and loving parents anyone could ask for and I love you both so much. I’d also like to thank my brothers, Matthew and Marc, for their support and for consistently keeping me in good spirits.

LIST OF FIGURES

Page Figure 1. A lionfish of the genus Pterois. (This Photo by Unknown Author is licensed under CC BY-NC-ND.)...... 3

Figure 2. Distribution of lionfish in their invaded region of the tropical western Atlantic (U.S Geological Survey, 2018) ...... 5

Figure 3. Figure 3: Global distribution of the lionfish (genus Pterois spp.), showing their native ranges (blue and green), the range of their invasion as of 2014 (red solid color) and predicted expansion of their invasion (red hatching) (U.S. Geological Survey, 2018)...... 5

Figure 4. Diagram of the eye of a teleostean fish (Ramel, 2018)...... 9

Figure 5. Location and Structure of the retina. This diagram shows the direction in which light travels through the retina to reach the photoreceptor cells located in the back of the eye. The general structure of the retina is, starting from the point of light detection, the photoreceptor cells, the horizontal cells, the bipolar cells, the amacrine cells, and the ganglion cells (DeRemur, 2016)...... 11

Figure 6. Molecular steps in photoactivation (Corneveaux, 2007)...... 12

Figure 7. How the visible spectrum attenuates through clear ocean water...... 14

Figure 8. Schematic representation of an electrical signal transmitted along the optic nerve as detected by electroretinography. The peaks and troughs of this wave indicate different points of the phototransduction cascade within the retina...... 17

Figure 9. Set-up of electroretinography with lionfish anesthetized (Photo by Miranda Carroll)...... 27

Figure 10. Spectral sensitivity curve for the red-light treated lionfish R1-1...... 34

Figure 11. Spectral sensitivity curve for the red-light treated lionfish R1-2...... 35

Figure 12. Spectral sensitivity curve for the red-light treated lionfish R1-3...... 35

Figure 13. Spectral sensitivity curve for the red-light treated lionfish R1-4. . 36

Figure 14. Spectral sensitivity curve for the red-light treated lionfish R1-5...... 36

Figure 15. Spectral sensitivity curve for the red-light treated lionfish R2-1...... 37

Figure 16. Spectral sensitivity curve for the red-light treated lionfish R2-2...... 37

Figure 17. Spectral sensitivity curve for the red-light treated lionfish R2-3...... 38

Figure 18. Spectral sensitivity curve for the red-light treated lionfish R2-4...... 38

Figure 19. Spectral sensitivity curve for the blue-light treated lionfish B1-1...... 39

Figure 20. Spectral sensitivity curve for the blue-light treated lionfish B1-2...... 39

Figure 21. Spectral sensitivity curve for the blue-light treated lionfish B1-3...... 40

Figure 22. Spectral sensitivity curve for the blue-light treated lionfish B1-4...... 40

Figure 23. Spectral sensitivity curve for the blue-light treated lionfish B2-1...... 41

Figure 24. Spectral sensitivity curve for the blue-light treated lionfish B2-2...... 41

Figure 25. Spectral sensitivity curve for the blue-light treated lionfish B2-3...... 42

Figure 26. Spectral sensitivity curve for the blue-light treated lionfish B2-4...... 42

Figure 27. The mean (+/- S.E.) of the initial spectral sensitivity of all lionfish (n=14 fish). The letters above the points indicate whether the spectral sensitivity between the tested wavelengths are significantly different from one another. If points have a letter in common, then they are not statistically different from one another...... 43

Figure 28. The mean (+/- S.E.) spectral sensitivity of lionfish after 2 months of either blue or red lighting treatment. The letters above or below the points indicate whether the spectral sensitivity between the tested wavelengths are significantly different from one another. The red letters correspond with the mean spectral sensitivity curve of the fish exposed to the red lighting condition, and the blue letters correspond with the mean spectral sensitivity curve of the fish exposed to the blue lighting condition ...... 45

Figure 29. The mean (+/- S.E.) spectral sensitivity of lionfish after 4 months of either blue or red lighting treatment. The letters above or below the points indicate whether the spectral sensitivity between the tested wavelengths are significantly different from one another. The red letters correspond with the mean spectral sensitivity curve of the fish exposed to the red lighting condition after 4 months, and the blue letters correspond with the mean spectral sensitivity curve of the fish exposed to the blue lighting condition after 4 months...... 47

xii

LIST OF TABLES

Page Table 1. Acquisition data for lionfish used for analysis of visual systems. Fish ID: R= fish acclimated to red light, B = fish acclimated to blue light. N/A = not available TL = Total Length; SL = Standard Length...... 31

Table 2. Table 2: Sample sizes of fish for each condition and time point. An X=no data available/fish perished before data could be collected; a ✓=data was collected from the individual fish at the corresponding time point...... 33

Table 3. Table 3: Results of 2-Way Repeated Measures ANCOVA...... 51

xiii

INTRODUCTION

Invasive lionfish of the genus Pterois pose a serious threat to the tropical western Atlantic marine environments and its inhabitants. Lionfish are very resilient and have broad tolerance ranges for depth, salinity, and temperature, which allow them to occupy a variety of habitats (Whitfield et al. 2002; Albins and Hixon,

2013; Jud et al. 2015). Because of their high environmental tolerances, venomous spines that inhibit potential natural predators, and highly efficient feeding capabilities, lionfish populations have increased exponentially since their introduction to the Atlantic (Whitfield et al. 2002; Morris et al. 2009). Studying the biological and ecological characteristics of lionfish can help determine the factors that contribute to the overwhelming success of this invasive species, predict limits to the extent of their invasion and impacts on native species, understand how ecosystems may ultimately adapt to lionfish populations, and suggest methods for mitigating their harmful effects.

Biology

Lionfish of the family Scorpaenidae are small (generally < 20 cm) but highly venomous fishes that are native to the Red Sea, Indian Ocean, and the western/central Pacific Ocean. Eleven species are currently recognized within the genus Pterois (fishbase.org, 2010). They are typically closely associated with coral reefs and other complex benthic habitats, where they primarily consume small benthic fish and invertebrates (Morris and Akins, 2009).

Two species of lionfish, Pterois volitans and P. miles, have been confirmed in the tropical western Atlantic. They are very similar morphologically and both are commonly referred to as “lionfish” (Figure 1; Schofield, 2009). Lionfish possess 18 long spines in their dorsal, anal and pectoral fins that have grooves that transmit venom from basal venom sacs. These spines are covered in skin that, when broken by contact, release a protein-based neurotoxin. These venomous spines inhibit predation from native Atlantic predators, contributing to the success of their invasion of Atlantic habitats (Morris and Whitfield, 2009; Cure et al. 2012). A sting from a lionfish to a human can cause a wide range of reactions, from mild irritation to extreme pain, swelling, and cardiovascular distress (Morris, 2009).

Researchers and fishery managers have speculated as to why lionfish are so successful invading habitats around the tropical western Atlantic. An initial factor is that lionfish have a very high rate of reproduction and fecundity, producing over

2 million eggs per year (Morris, 2009). Their pelagic larval stage lasts about one month, and larvae are dispersed via the Gulf Stream and other oceanic currents

(Hare et al. 2003). Following settlement into suitable benthic habitats, their success can be partially attributed to their generalist feeding behavior (Morris and Akins,

2009) and lack of predators (Morris and Whitfield, 2003). Lionfish are also most active during dawn and dusk (Myers, 1999; Randall, 2005) and this crepuscular feeding behavior may challenge prey that are visually adapted to greater or lesser levels of illumination (Helfman, 1986). Native prey may also lack predator avoidance behavior towards lionfish, commonly referred to as prey naiveté, due to

3 a lack of co-evolutionary history (Anton et al. 2016). This absence of coevolution between invasive lionfish and Atlantic prey and predatory species have helped enable lionfish to become much more abundant in their invaded range of the tropical western Atlantic than in their native range of the Indo-Pacific (Darling et al. 2011).

Figure 1: A lionfish of the genus Pterois. (This Photo by Unknown Author is licensed under CC BY-NC-ND.)

Distribution and Genetic Diversity

The first sighting of a lionfish in the tropical western Atlantic was in 1985 off the coast of Dania, FL. Lionfish continue to expand their range, moving farther north along the East Coast of the United States, as well as south along the eastern coast of South America (Figure 2). Kimball et al (2004) reported adult lionfish as far north as Cape Hatteras, North Carolina, however, Whitfield et al (2002)

4 reported juvenile lionfish as far north as Long Island, New York. It is predicted that their survival is inhibited farther north due to low temperatures in the winter months (Kimball et al. 2004). Lionfish have been documented throughout the Gulf of Mexico and the Caribbean Sea, in the Bahamas, Bermuda, Cuba, Turks and

Caicos, and as far south as the coasts of Venezuela (Semmens et al. 2004; Betancur et al. 2011). Ferriera et al (2015) reported the first lionfish to be found off the coast of Brazil, near Rio de Janeiro. Continuous updates of the distributions of lionfish are necessary to monitor their movements and determine where management practices should be implemented to attempt to mitigate their ecological impacts. A map of the global distribution of P. volitans and P. miles as of 2014 (Figure 3), indicated their expansion was expected along the coast of Brazil, which was confirmed the following year when Ferriera et al (2015) reported lionfish in Rio de

Janiero, Brazil.

Genetic analysis indicates that 93% of the Atlantic lionfish population is comprised of P. volitans that have very low genetic diversity. Courtenay (1995) and Semmens et al. (2004) suggest that lionfish introductions to the wild were perhaps the result of aquarium releases by negligent aquarists. More recently,

Schofield (2009) and Betancur et al. (2011) estimated that the invasion most likely originated from a small group of individuals composing of both P. volitans and P. miles during a single release event off the eastern coast of Florida.

Figure 2: Distribution of lionfish in their invaded region of the tropical western Atlantic (U.S Geological Survey, 2018).

Figure 3: Global distribution of the lionfish (genus Pterois spp.), showing their native ranges (blue and green), the range of their invasion as of 2014 (red solid color) and predicted expansion of their invasion (red hatching) (U.S. Geological Survey, 2018).

Physiological and Ecological Variability

The adaptability of lionfish to a wide variety of conditions contributes to their expansion throughout the tropical western Atlantic. They are found in a variety of habitats, including coral reefs, mangroves, artificial reefs or other man- made structures, and seagrass beds (Morris and Akins, 2009; Barbour et al. 2010).

All of these habitats are important nurseries for a variety of ecologically important fish species (Mumby et al. 2004). Claydon et al (2012) studied the movement of lionfish through various habitats and suggests that lionfish prefer shallow habitats but can move deeper, especially as they grow larger in size. Schultz (1985) found lionfish at depths of 0 to 80 m. However more recently fish have been observed at

300 m off Lyford Cay, Bahamas (McGuire & Hill, 2014; Harrell, 2017).

Being a tropical to subtropical species, lionfish prefer warmer conditions.

The minimum lethal temperature of lionfish is 10ºC, which allows their persistence along the eastern coast of the United States as far north as Cape Hatteras, NC, where the average bottom isotherm is 12ºC (Kimball et al. 2004). This tolerance to low temperatures also enables their movement to depths hundreds of meters below the surface. Gress et al (2017) predicted that temperature rather than light availability ultimately limits the maximum depth lionfish can inhabit. Jud et al

(2015) found that lionfish can survive extended periods of time in salinity as low as

7ppt, indicating their ability to inhabit brackish or estuarine waters in coastal regions. The light availability in these environments is significantly different in both intensity and spectral composition, suggesting that lionfish possess a plastic

7 retina that change in response to their environment. These characterizations and observations of lionfish in atypical habitats combined with their crepuscular feeding behavior suggest that lionfish have an adaptable retina that may greatly increase their fitness.

Threats to Invaded Communities

Lionfish threaten the diversity and abundance of native species through predation and competition. They can consume prey that is up to two-thirds of their body length, and currently 42 species of Atlantic fish have been identified from stomach-content analysis (Green et al. 2012). Lionfish are slow-moving and will guide and corner their prey with their extravagant pectoral fins, and perform suction feeding (Allen and Eschmeyer, 1973). Lionfish utilize a predatory technique in which they blow jets of water towards their prey to encourage a head- first orientation, which increases the chances of a successful capture (Albins and

Lyons, 2012). This behavior is believed to be unique to lionfish and has not been observed in other marine teleost species. Lionfish are crepuscular predators

(Randall, 2005) and are a threat particularly to fish that are not adapted to low light conditions (Helfman, 1986). Reefs where lionfish are now present are severely affected, with recruitment of reef fish reduced in some locations by 79% (Albins and Hixon, 2008) or 95% (Côté et al. 2013). Due to the presence of lionfish, these locations also often have smaller populations of ecologically similar predatory species like grouper and snapper (Côté et al. 2013). A study conducted by Green et al (2012) found a 44% reduction of native predators in coral reef communities of

New Providence, Bahamas. Because lionfish are such efficient predators, they are significantly outcompeting native piscivores such as grouper and snapper, which have critical ecological and commercial value.

The threats that lionfish populations in the tropical western Atlantic create, such as decreased biodiversity and native-fish biomass, are cause for great concern.

Lionfish are effective visual predators in a wide variety of environments, indicating that their visual system enables effective prey detection and predator avoidance over a broad range of lighting conditions. Water color and clarity vary tremendously among these habitats. Because lionfish are visually-guided predators

(Cure et al. 2012), understanding their visual system and its ability to adjust to different photic conditions may provide insight to their success as an invasive species.

Vision and Light in Marine Environments

Vision

Vision plays a significant role in organisms’ ability to interpret, respond, and interact with their environment. The first useful light detection in primitive animals apparently evolved approximately 530 million years ago, just before or during the Cambrian explosion (Land and Nilsson, 2012). It is possible that the evolution of visually guided predation contributed to the increase in animal size and evolution of macrofauna, fueling the diversity that arose during this time (Land and Nilsson, 2012). While all animals with opsin-based light sensitivity arose from

9 a common ancestor, photoreceptor structures that allow for spatial vision arose independently many times across many different phyla (Land and Nilsson, 2012).

Basic eye structure in vertebrates, including the teleost fishes that are the most recently evolved clade of bony fishes (Nelson, 2006), is diagrammed in Figure 4.

Light enters the eye through the pupil, an opening in the middle of the iris. Light then penetrates the lens, a transparent, spherical structure located posterior to the iris, where is it directed and focused onto the retina located in the back of the eye.

When light reaches the retina located in the most posterior part of the vertebrate eye, the photoreceptors begin the process to detect light.

Figure 4: Diagram of the eye of a teleostean fish (Ramel, 2018).

The process in which light received by a photoreceptor cell elicits an electrical signal within the vertebrate retina is known as the phototransduction

10 cascade. This cascade is a series of events initiated in the outer segment of photoreceptor cell, including the activation of light sensitive opsin proteins and hyperpolarization of the entire cell. Opsins are a class of proteins that are light- sensitive and change conformation when struck by a photon. These light sensitive proteins are found in the photoreceptor cells within the retina.

The two types of photoreceptors in the vertebrate retina include rods and cones (Figure 5). Rods are much more sensitive to light that cone cells, and are typically active in low light conditions because of their sensitivity (Ebrey and

Koutalos, 2001). These cells are typically concentrated at the edges of the retina and are also utilized in peripheral vision (Mannu, 2014). There are several types of cone cells, each sensitive to different wavelengths of light, which are perceived as different colors. Cones that differ in their spectral sensitivity have different photopigments (opsins) that are sensitive to distinct regions of the electromagnetic spectrum, so organisms that contain multiple cone types are able to discriminate multiple colors. Cones are less sensitive than rods but have a faster “reset” time, meaning that after the cell has been activated by light and has initiated the transduction of an electrical signal, it will be ready shortly after this first activation to receive more light. These cells are typically concentrated at the fovea, or the most central area of the retina where visual acuity is greatest (Mannu, 2014). Rods and cones are so named because the outer regions of both of these cells resemble these shapes. The outer region of both of these photoreceptors is located in the most posterior region of the retina and contain disc-like structures that possess

11 photosensitive, G-protein coupled receptor opsin proteins (Ebrey and Koutalos,

2001).

Figure 5: Location and Structure of the retina. This diagram shows the direction in which light travels through the retina to reach the photoreceptor cells located in the back of the eye. The general structure of the retina is, starting from the point of light detection, the photoreceptor cells, the horizontal cells, the bipolar cells, the amacrine cells, and the ganglion cells (DeRemur, 2016).

The phototransduction cascade in rod and cone cells is the same except for the type of opsin within the discs of the cells’ outer segments and therefore the wavelength of light that induces the cascade. Because cone cells are sensitive to a different wavelengths, there are different types of opsins that exist in the different

12 kinds of cone cells. These light sensitive pigments of cones are called photopsins.

In rod cells, phototransduction begins when a photon reaches the rhodopsin pigment, the opsin specific to rod cells. Photopsins and rhodopsins (labeled R* in

Figure 6) are G-protein coupled receptors with 7 transmembrane helical proteins, and contain the chromophore 11-cis retinal which photoisomerizes, or changes its structure upon photoexcitation to become all-trans retinal. All-trans retinal causes a conformational change in the opsin and leads to closure of cyclic guanosine monophosphate (cGMP)-gated channels via the activation of the regulatory protein transducin (labeled G in Figure 6). The nucleotide cGMP is a messenger molecule that is essential for mediating phototransduction. The guanosine diphosphate

(GDP) that is bound to transducin’s α-subunit is replaced by cytosolic guanosine triphosphate (GTP), allowing this α-subunit to dissociate from transducin’s β- and

γ-subunits. Activation of transducin leads to the reduction of cGMP. The unbound

α-subunit activates cGMP phosphodiesterase, which is an enzyme that that catalyzes the conversion of cGMP to 5’-GMP.

Figure 6: Molecular steps in photoactivation (Corneveaux, 2007).

In the dark, a large amount of cGMP is available and cGMP-gated channels remain open and allow the passage of cations, particularly Ca2+, between the membrane of the discs and the cytoplasm of the photoreceptor. When light is detected by the photoreceptors, the previously described cascade is activated, thus decreasing cGMP concentration, which in turn leads to the cGMP gated channels closing (Ebrey and Koutalos, 2001).

Light in Marine Environments

As light enters the water, it undergoes increasing attenuation with depth.

Attenuation, or the reduction of radiant flux, describes simultaneous absorption and scattering of light. The rapid absorption of red light with increasing depth is due to the fundamental vibration states of water molecules as well as the particulate matter present in water that absorbs and scatters these wavelengths. This combination of the properties of water molecules and the particulate matter in water prevents long- wavelength spectra from penetrating past certain depths. Figure 7 shows the attenuation of different wavelengths of the visible spectrum as depth in clear ocean water increases. The longer wavelengths in the “red” region of the spectrum attenuate rapidly with depth, while shorter wavelengths in the “blue” region of the spectrum are able to penetrate much greater depths. The same properties of water that attenuate red light as depth increases also act as a monochromator of blue light.

Shallow, marsh-like habitats contain tannin and decomposing plant matter that

14 causes absorption of all wavelengths of light and allows red to be the maximally transmitted region of the visible spectrum (Jerlov, 1968).

Figure 7: How the visible spectrum attenuates through clear ocean water.

The spectral composition of these aquatic habitats is extremely important for a fish’s ability to avoid predation, hunt or forage for food, find mates, and an overall awareness of surroundings. Teleost fish generally have excellent color vision, and have sensitivities that correspond with the light quality of their habitat and the time of day they are most active (McComb et al. 2010; McComb et al.

2013). Estuarine and shallow waters are highly variable with respect to light

15 quality; some species found in these habitats have high luminous sensitivity, which is the sensitivity to the presence of very little light, at the expense of resolution.

Species found in these shallow waters are typically sensitive to red because this wavelength has not attenuated at these depths as it does at greater depths. Turbidity and an increase in particulate matter can drastically influence the effectiveness of fish vision. Light intensity not only decreases with increasing depth, but also with turbidity and eutrophication (Omar and Matjafri, 2009). Portions of the visible electromagnetic spectrum are associated with specific marine locations or habitats.

Coastal species possess high sensitivity to green and blue, and deep and pelagic species possess high sensitivity to blue because these are the dominant wavelengths of these environments (Loew and Lythgoe, 1978).

In addition to water quality and depth, time of day also influences light intensity and spectral composition of marine habitats. Benthic species that are already adapted to low light conditions may experience little or no effects from changes to water and light quality. These species already possess other sensory mechanisms that compensate for conditions in which vision is rendered ineffectual

(Huber and Rylander, 1992). These species may also be relying more heavily on the functionality of their rod photoreceptors rather than their cone photoreceptors.

Crepuscular species, or species that are active during the hours of dawn and dusk, like lionfish (Cure et al. 2012), are also generally adapted to low light levels.

However, change in light quality may hinder a fish’s ability to distinguish fast

16 moving prey against a fast moving background. Coastal species that are green sensitive require enough light for optimal functionality, which in some areas are currently being altered anthropogenically (Horodysky et al. 2010). The variety of responses to changes in water quality makes it unclear as to whether lionfish will be influenced positively, negatively, or unaffected; however, they have already established themselves in highly turbid, variable estuarine and shallow habitats.

This could indicate that they have the potential to rapidly adapt to their environment, and would remain successful in changing water conditions.

Assessment of the Visual Systems of Fish using Electroretinography

A reliable method used to characterize vision and examine the functionality of the retina in live animals is a noninvasive electrophysiology technique called electroretinography (ERG). It has important clinical value in identifying retinal diseases and disorders, and allows investigation of the vision of live animals without surgical or harmful procedures. ERG measures the summed electrical response of the cells in the retina that is generated when light is absorbed by the visual pigments within photoreceptors. This neural response is measured by a recording electrode attached to the cornea, compared to a control or reference electrode attached elsewhere on the body, typically on the dermis next to the eye.

Various light intensities and wavelengths can be tested to determine the function of the different cell types within the retina. When a flash of light is administered to the eye, the response of the photoreceptors induces an electrical response which is

17 transmitted to ganglion cells, then to bipolar cells, and then the optic nerve. The optic nerve then transmits this information to the optic lobe of the brain. This response takes the form of a voltage wave that shows the summed action potentials generated in the activated photoreceptors (Figure 8). The amplitude of this wave represents the voltage that was generated by the retina over a given period. The higher the intensity of light, the greater the amplitude of the wave and the greater the voltage up to a threshold or maximum response is reached (Fishman, 1985).

The criterion response for studies of retinal sensitivity is a predetermined voltage that is induced in the retina from a certain intensity of light of a particular wavelength. It is important to note that light of different wavelengths emit different amounts of photons despite equal intensity, so light intensity is not kept constant for electroretinography because the response generated is dependent on the wavelength being administered (McComb et al. 2013).

b 20

Microvolts

-20 a

Time (s)

Figure 8: Schematic representation of an electrical signal transmitted along

the optic nerve as detected by electroretinography. The peaks and troughs of this wave indicate different points of the phototransduction cascade within the retina.

The ERG wave is composed of two basic elements. The “a wave” (Figure

8) represents hyperpolarization that occurs when light is introduced to a dark- adapted retina, causing a decrease of intracellular sodium ions in the discs of photoreceptors due to closing sodium gated channels of the plasma membrane. The

“b wave” peak represents depolarization of the photoreceptors because post- synaptic bipolar cells release potassium ions, resulting in extracellular accumulation of these cations. The amplitude of the wave, measured from the trough of the a wave to the peak of the b wave is a measure of the magnitude of the summed neural response to light stimulation (Fishman, 1985; Johnsen, 2012). The dashed lines on Figure 8 indicate a criterion response. If the predetermined criterion response for an individual fish was, for example, 40µV, then the difference between the peak of the b wave and the trough of the a wave shown in Figure 8 had to reach at least 40uV to be considered a functional response. The minimum intensity of light of a particular wavelength that generated the criterion response provides a measure of the sensitivity of the retina to that wavelength.

Electoretinography has been used successfully to explore the vision of various teleostean fishes. This sensory mechanism is important to explore as visual predators require a certain degree of water clarity to hunt their prey. The intensity of light being transmitted through water decreases as water depth increases. Deep

19 and open-ocean environments allow shorter wavelengths (blue) to penetrate, coastal waters allow intermediate wavelengths (green) to penetrate, and shallow environments allow long wavelengths (red) to penetrate.

Light quality varies greatly among marine habitats due to the different light intensities and spectral compositions they possess. Functional analyses of fish vision have consistently determined that these sensory systems strongly correlate to the lighting environments they inhabit. McComb et al (2013) utilized ERG to evaluate the spectral sensitivity of pinfish (Lagadon rhomboides), gray snapper

(Lutjanus griseus), and common snook (Centropomus undecimalis) collected from the Indian River Lagoon. They determined the presence of blue- and green- sensitive cones in the retinas of three fish species which are the prominent spectra in which they live. This indicates a visual adaptation that increases survival within their environment. Atlantic spadefish, tautog, and black sea bass possess cones that have specifically adapted to the light environments where they live, and accurately reflect their life histories (Horodysky et al. 2013). Spectral sensitivity of bonnethead (Sphyrna tiburo), scalloped hammerhead (Sphyrna lewini), and blacknose (Carcharhinus acronotus) sharks, which are generally nocturnal predators, exhibit sensitivities to spectra that exist during these times of low light levels and were maximally sensitive to 480 nm (McComb et al. 2010). Many fish species migrate to habitats of significantly different light quality during their ontogenetic development, which supports juveniles’ highly plastic retina that

20 readily adapts to photic change (Stenkamp, 2007; Taylor et al. 2015; Schweikert and Grace, 2017). Spectral sensitivity may also change in anticipation of habitat change, as demonstrated by tarpon (Megalops atlanticus) which reside in significantly different habitats between their juvenile and adult life stages. During their development, tarpon spectral sensitivity changes to match the dominant spectrum of their adult habitat before they actually migrate to these areas

(Schweikert and Grace, 2017). These studies support that vision can be highly adapted to a fish’s life history, and has the potential to shift during habitat change and through development.

Many crepuscular-feeding species do not exhibit color vision at all so they can maximize light absorption in rapidly-changing low light levels (Lythgoe,

1979). Lionfish are primarily benthic crepuscular feeders, staying close to the sea floor or to other substrate or structures. This lifestyle suggests that lionfish are highly sensitive to the presence of light, but because of the habitats varying so greatly in spectral composition, it is unclear how their color sensitivity will reflect their life history. During these times of low light, vision and perception of surroundings is dependent on an organism’s visual capabilities. Characteristics of vision such as temporal resolution, or the determination of objects over the course of sometimes fractions of seconds, and visual acuity, or the sharpness or ability to see details, can be analyzed electrophysiologically to determine and describe the vision of an organism.

The visual systems of fish of many different habitats and niches have been analyzed to correlate their vision to their behavior and life history. A characterization of the vision of crepuscular weakfish, Cynoscion regalis, found that while they exhibited relatively low acuities, they had very high temporal resolution. This indicates that crepuscular or nocturnal species are more adapted to seeing objects in low light quickly, rather than seeing the object clearly or with greater detail. This maximizes photon capture of the retina at the expense of seeing objects with acuity or in great detail (Horodysky et al. 2008). Deep-sea fish that reside in environments with almost no detectable light possess rods that are much longer than those of fish found in mesophotic habitats. This increases the chance of photon capture in an environment with extremely low light levels (Warrant, 2004).

Another method of detecting light in low levels is to decrease the speed at which light is integrated into the photoreceptor. At the expense of temporal resolution, slowing down the integration time of light improves the ability to see objects in low light, (Warrant, 1999). The nocturnal fourline cardinalfish, Ostorhinchus doederleini, is shown to have greater densities of rod cells than that of diurnal species that were examined in the same study. Through development, these cardinalfish also exhibited a decrease in cone density, indicating that color vision may only be influential during the juvenile stage of this marine fish (Shand, 1995).

The visual systems of fish are representative of their specific behaviors and accurately reflect their life history.

Rationale and Research Objectives

Marine fish visual systems have been investigated to understand the physiological basis for ecological interactions mediated by vision. Invasive lionfish are an excellent model organism for this study because they are visual predators, are ecologically detrimental to tropical western Atlantic ecosystems, and their vision and its capacity for change has not been previously examined. Many fish species exhibit retinal plasticity and can acclimatize to their environment in a manner that enhances their survival. Plasticity of the lionfish retina should allow for greater sensitivity to the wavelengths to which they are exposed. Analyzing the effect of lighting condition on lionfish vision using electroretinography (ERG) might determine the degree to which this plasticity is present in lionfish and potentially help explain their expansion into a variety of photic regimes.

The overall goals of this study are to determine: Do lionfish change their spectral sensitivity in response lighting condition? Do lionfish exhibit retinal plasticity and does it enable their invasion to diverse photic environments? Specific objectives to achieve these goals are:

1. To characterize the spectral sensitivity of lionfish.

2. To determine whether lionfish housed in specific, narrowband

wavelength lighting treatments will shift their spectral sensitivity.

3. To compare the spectral sensitivities of lionfish housed in different

narrowband wavelength lighting treatments over time.

These objectives will enable testing specific hypotheses:

H1: lionfish will exhibit similar spectral sensitivity upon capture from the wild,

H2: lionfish will shift their spectral sensitivities after extended exposure to narrow-banded lighting conditions,

H3: lionfish will shift their sensitivity over time during their exposure to narrow-banded light conditioning.

The methods of this study allow for quantitative comparisons of fish conditioned to two narrow-banded lighting conditions. This study may aid in the understanding of the mechanisms utilized by invasive lionfish to expand into a wide variety of habitats of significantly differing spectral composition.

MATERIALS AND METHODS

This investigation determines the initial spectral sensitivity of wild lionfish, and then assesses the potential retinal plasticity of fish that were conditioned to blue and red lighting conditions for up to 4 months. Non-invasive ERG techniques were performed on living animals to measure the mass electrical output of the retina in response to light stimuli. The “blue” treatment represented the dominant spectrum on deep water reefs, and the “red” treatment represented the dominant spectrum of tannin-stained coastal rivers and mangrove swamps (Jerlov, 1968).

Lionfish Collection and Housing

From April to September, 2017, I collected 17 adult lionfish by snorkeling in coastal waters, from SCUBA divers who donated the fish, and by purchasing fish from the Incredible Pets store in Melbourne, FL. All fish, including those purchased from the pet store, were collected from the Florida Keys to Sebastian,

FL. Fish size ranged from 10 to 23 cm Standard Length (SL). These fish were transported to the Aquaculture Laboratory at the Harris Center for Science and

Engineering at the Florida Institute of Technology in Melbourne, FL. Fish were acclimated in a clear, 160 L tank with a biofilter for at least 2 weeks under normal room lighting to ensure health before transfer to the experimental systems. For exposure to different light conditions, fish were then housed in four circular, black walled 380 L tanks with both a diameter and depth of about 1 m. The tanks were on recirculating systems with two tanks on one 150 L biofilter/sump and the other two

25 tanks on a 225 L biofilter/sump. Temperature was maintained at 22 ºC and a salinity of 32 ppt with periodic 15% water changes. Water quality, care and feeding were all recorded in an animal care log. All housing procedures and experimental protocols were conducted in accordance with Institutional Animal Care and Use

Committee (IACUC) protocol 2017-02.

Electroretinography

Following acclimation in standard full-spectrum room light for a period at least 2 weeks, initial ERG analyses were made. Then, fish were randomly assigned to blue (420 nm) or red (590 nm) light treatments, with 3 to 4 fish per 380 L tank, for a total of 7 fish per lighting condition. The other 3 fish died before they were exposed to any lighting condition. Lionfish were kept on a 12-hour-light/12-hour- dark cycle. Each tank was illuminated by a 1.3 m long LED light bar (BML

Horticulture, Austin, TX, USA). These lights provided 50 nm bandwidth spectra centered on 590 nm (red) and 420 nm (blue). Tank systems were enclosed within light-blocking curtains to ensure that fish received no light stimuli other than that provided by the LEDs. Light intensity measurements made with a radiometric probe (Ophir Photonics, North Andover, MA) 3 weeks into the conditioning period indicated that lights of the blue treatment were about 140% brighter than those of the red treatment. Aluminum mesh was wrapped around a portion of the blue light bars to decrease light intensity and match it to that of the red lights.

Spectral sensitivity of each fish was assessed after 2 months and 4 months of light conditioning under the blue or red light treatments (following McComb et

26 al., 2013 and Schweikert and Grace, 2018). For ERG measurements at the beginning of the project and at each of the assessment periods, individual fish were transported from the Harris Center for Science and Engineering to the Olin Life

Sciences building via a 40 L bucket that was fully covered in black, opaque contractor’s plastic sheeting to prevent exposure to daylight.

Each fish was sedated with tricaine methanesulfonate (MS-222; 0.5-1.0 g/L) for at least 10 min prior to testing, or until anesthesia was clearly achieved as evidenced by the lionfish no longer moving its operculum or gapping. The anesthetized fish was placed on its right side in shallow container, and propped with a wet washcloth to ensure both sets of gills remain open. The fish remained partially submerged for the duration of the ERG trial and was frequently kept wet using a squirt bottle full of MS-222-dosed water. A 6.4 mm PVC T-fitting fixed to a flexible tube connected an aquarium water pump was placed in the buccal cavity of the fish to allow MS-222 dosed water movement over its gills, at an estimated rate of 0.5 L/minute (Figure 9). A 1 mm, flexible, silver-wire electrode were held securely with a clamp and was gently but securely placed on the cornea of the left eye of the fish as the recording electrode, and another electrode was placed on the skin posterior to the left eye as a reference electrode using a clamp. A final electrode was gently wrapped around its body to serve as a ground electrode. After the fish was set-up for ERG testing, it was left in the dark for at least 30 minutes to dark-adapt the retina to ensure photoreceptors were reset and able to receive the light administered during ERG testing. The entire fish and the electrodes were kept

27 wet during the tests and dark adaption by pouring and squirting of MS-222 dosed water from a squirt bottle to ensure health of the fish and to maintain conductance of and adequacy of measurement from the electrodes.

Figure 9: Set-up of electroretinography with lionfish anesthetized (Photo by Miranda Carroll).

After dark adaption, stimulus light from a DC-powered, 150-W, quartz- tungsten halogen lamp was passed through an electronic, fast-acting shutter

(Uniblitz, Rochester, NY) and a grating monochromator (Oriel Instruments, Irvine,

CA, USA) for separation into the specified wavelengths. An aperture on the monochromator was manually adjusted using a slide bar to adjust light intensity. A

28 high-grade, fused-silica, fiber-optic bundle (Oriel) transmitted the stimulus light onto the eye. Light intensity for each wavelength was measured using a radiometric probe (Ophir Photonics, North Andover, MA). Starting with the shortest wavelength of 350 nm and ending with the longest wavelength of 650 nm, narrowband light was administered in 50-nm increments, and the retinal response was recorded to determine the spectral sensitivity. For each wavelength increment, 100- msec flashes were administered, with 2 min intervals between. Flashes of varying intensities were administered for each wavelength to obtain different microvolt responses for post-ERG trial determination of the criterion response. Flashes were limited to less than 4 for each wavelength to prevent photobleaching of the retina.

Fish sedation for ERG typically lasted for about an hour. After completion of the

ERG testing, the fish was revived in fresh saltwater and returned to its home tank containing normal saltwater. ERG testing took place during the 12 hour period in which the aquarium lights were on so that the measured sensitivity was that of daylight hours.

Analyzing Spectral Sensitivity

After collection of ERG data, the amount of light needed to generate the criterion responses for each individual fish at each wavelength was determined using the program Labview. The criterion response was generally set from 20 to 40

μV above baseline noise. When the criterion response was generated for a specific wavelength, the irradiance values (measured in μW s-1 cm-2) corresponding to that wavelength were calculated by taking the difference in voltage between the a- and

29 b-waves, or between baseline noise and the b-wave if electrical noise interfered with the recording. Irradiance was converted to photons using the following equation:

γ = W (λ) (5.05*1015) where γ is photons, W is irradiance in watts s-1 cm-2, λ is wavelength in nm, and the end term is Planck’s Constant (Johnsen, 2012). Data were converted to photons s-1 cm-2 for each wavelength. For comparisons to be made among fish and treatments, values were normalized on a zero to one scale by dividing each of the irradiance values for an individual fish by the greatest value for that trial. The reciprocals of normalized values were then calculated to present the data as measures of spectral sensitivity. Mean (+/- SD) spectral sensitivity curves were plotted for each treatment: initial fish before light treatments, as well as 2 month and 4 month trials for both blue and red light treated fish.

To statistically compare the mean spectral sensitivity curves, the data analysis software package “R” was used to conduct a 2-Way Repeated Measures

Analysis of Covariance (RMANCOVA) to determine whether the independent variables, lighting treatment and time, had an effect on the dependent variable, spectral sensitivity, while also taking into consideration the expected differing sensitivities to different wavelengths by using wavelength as a covariate. Although having normally-distributed data is an assumption of ANCOVAs, this analysis was still conducted because the variances were found to be equal between the variables tested. Normally-distributed data are often not representative of what happens in

30 nature, and having equal variances is much more important for maintaining the statistical power of this method (Sokal and Rohlf, 1995; Hilborn and Mangel, 1997;

Underwood, 1997; Underwood, 1981). Bartlett’s test for homogeneity of variances indicated equal variances among the independent variables, lighting condition and time. Mauchly’s sphericity test was performed after conducting a 2-Way

RMANOVA to ensure the variances between the levels of the independent variables are equal. A Games-Howell multiple comparisons test was conducted to determine if the sensitivities to each wavelength for each lighting treatment and time point were significantly different from one another. An Anderson-Darling k- sample test was also conducted to compare all possible combinations of the mean spectral sensitivity distributions between condition and through time to determine if any of these distributions were significantly different from one another.

RESULTS

A total of 17 lionfish were collected for this study from the Florida Keys,

Sebastian, Boynton Beach, and Ft. Pierce regions, from depths ranging from 3 to 27 m (Table 1). Lionfish were either collected through snorkeling, by SCUBA divers who donated them, or purchased from the Incredible Pets Store in Melbourne, FL.

The store purchased these fish from divers who collected the fish in the Florida

Keys. Fish were acquired from April through August 2017 and brought to the

Florida Institute of Technology. Collection data and identification numbers of each fish are provided in Table 1. Total (TL) and standard lengths (SL) of each fish are measured from the tip of the snout to the end of the tail, and from the tip of the snout to the end of the caudal peduncle, respectively.

Table 1: Acquisition data for lionfish used for analysis of visual systems. Fish ID: R= fish acclimated to red light, B = fish acclimated to blue light. N/A = not available TL = Total Length; SL = Standard Length.

Fish ID Location Collection Date of TL (cm) SL (cm) Depth (m) Acquisition R1-1 Florida Keys N/A 7/5/2017 16 10 R1-2 Florida Keys N/A 7/5/2017 16.5 10 R1-3 Ft. Pierce 3 4/28/2017 24 18 R1-4 Ft. Pierce 27 6/10/2017 18 15.5 R1-5 Ft. Pierce 3 4/28/2017 17 13 R2-1 Boynton 23 6/25/2017 20 16 R2-2 Sebastian 26 6/23/2017 23 18 R2-3 Florida Keys N/A 7/5/2017 13 8.5 R2-4 Ft. Pierce 3 4/28/2017 19.5 15 B1-1 Florida Keys N/A 8/10/2017 24.5 18 B1-2 Florida Keys N/A 7/5/2017 17 12 B1-3 Boynton 23 6/25/2017 28 23 B1-4 Florida Keys N/A 8/10/2017 23 18 B2-1 Ft. Pierce 4 5/12/2017 25 18 B2-2 Florida Keys N/A 7/5/2017 15 11.5 B2-3 Florida Keys N/A 7/5/2017 18 13 B2-4 Sebastian 26 6/23/2017 21.5 15

Following the initial acclimation to the culture tanks under normal lighting conditions, ERG analyses were conducted to assess the initial spectral sensitivity curves for each fish (Figures 10-26). Also presented in the figures are their responses after two and four months of exposure to the two light treatments. For three of the fish, I was not able to obtain initial curves prior to initiation of the light treatments, while four fish died during the experiment, preventing acquisition of either the two or four month (or both) spectral curves (Table 2). Due to Hurricane

Irma (September 2017) the fish were without a recirculating system for about 2 days, and as a result these four fish died after initial ERG analysis. Two other fish

33 that died during light conditioning refused to eat either for several days after ERG analysis, or for other unknown reasons (Table 2).

Table 2: Sample sizes of fish for each condition and time point. An X=no data available/fish perished before data could be collected; a ✓=data was collected from the individual fish at the corresponding time point.

Fish ID Initial Analysis 2-Month Analysis 4-Month Analysis R1-1 ✓ ✓ X R1-2 ✓ ✓ ✓ R1-3 ✓ ✓ ✓ R1-4 ✓ X X R1-5 ✓ X X R2-1 X ✓ ✓ R2-2 X ✓ ✓ R2-3 ✓ ✓ ✓ R2-4 ✓ X X B1-1 ✓ ✓ ✓ B1-2 ✓ ✓ ✓ B1-3 ✓ ✓ ✓ B1-4 ✓ X X B2-1 X ✓ X B2-2 ✓ ✓ ✓ B2-3 ✓ ✓ ✓ B2-4 ✓ ✓ ✓ Total 14 13 11

The y-axes of the spectral sensitivity curves (Figures 10-26) are the inversed normalized irradiances needed to generate the criterion response at each wavelength, essentially indicating the sensitivity to the corresponding wavelength.

The higher the value on this axis, the greater the sensitivity. The x-axes of these

34 curves are the seven different wavelengths tested for determination of spectral sensitivity.

Examination of the data collected from individual fish shown in Figures 10-

26 indicate that different fish had different sensitivity curves. For example, initial

ERGs of 4 of the fish showed highest sensitivities to 350-400 nm wavelengths, while 6 of the fish had very low sensitivity to those wavelengths. Calculating mean

(+/- S.E.) values for each wavelength for each treatment provided the ability to incorporate this variation into overall statistical analyses of how the experimental populations of fish responded to the treatments.

initial 1 2month

0.8 4month

0.6

0.4 Normalized Irradiance Normalized 1/(Photons/cm^2/s^1) 0.2