Morphology of the Olfactory Apparatus in Leptocephalus

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MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS

LARVAE

MOLLY ANN WIGHTMAN

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 July 2018

MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS

LARVAE

A THESIS

MOLLY ANN WIGHTMAN

Approved as to style and content by:

Jonathan Shenker, Ph.D., Chairperson Ralph Turingan, Ph.D., Member Associate Professor Professor Department of Biological Sciences Department of Biological Sciences

Kelli Hunsucker, Ph.D., Member Richard Aronson, Ph.D. Research Assistant Professor Professor and Head Department of Ocean Engineering Department of Biological Science and Sciences

July 2018

ABSTRACT

MORPHOLOGY OF THE OLFACTORY APPARATUS IN LEPTOCEPHALUS

LARVAE

By Molly Ann Wightman, B.S., Florida Institute of Technology

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

Leptocephalus larvae are a unique larval form that unites the Elopomorph fishes. This larval form has morphological and cellular characteristics that differ from most other teleost fishes. The visual system of leptocephali rod-dominated retinas, leading to poor photopic vision and low visual acuity that would hinder feeding on planktonic prey. Recent studies indicate they feed marine snow, which is composed of mucilaginous materials, bacteria and plankton. Their low visual acuity raises the question of how these organisms find this gelatinous food source in the wild. I hypothesize that these larval fishes use chemoreception in order to find marine snow, and that their olfactory apparatus thus differs morphologically from other teleost fishes.

A variety of settlement-stage pelagic larvae were collected from Sebastian

Inlet State Park in Florida and Andros Island, Bahamas. Leptocephalus larvae were represented by tarpon (Megalops atlanticus), ladyfish (Elops saurus), bonefish

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(Albula vulpes), and speckled worm eels (Myrophis punctatus). Three species with non-leptocephalus larvae were examined: Atlantic menhaden (Brevoortia tyrannus), Atlantic croaker (Micropogonias undulatus), and pinfish (Lagodon rhomboides). Their external olfactory and visual systems were analyzed using

Scanning Electron Microscopy. Morphometric data were collected and compared to make comparisons between larval types regarding the dimensions and structures of these sensory organs. Developmental differences were also observed by comparing SEM images of larval and juvenile fishes.

Three out of the four species of leptocephalus larvae had large, exposed olfactory pits, while speckled worm eels and all non-leptocephalus larvae had their olfactory apparatus embedded under the skin of the head, with distinct anterior and posterior nostrils. Although there was no consistent difference among the groups in dimensions of the olfactory apparatus, eye size, or ratio between the sensory organs, the open olfactory pits of tarpon, ladyfish and bonefish larvae would directly expose sensory cells to chemical signals from marine snow. The enclosed olfactory apparatus of the non-leptocephalus species develops during embryogenesis, suggesting that exposed olfactory surfaces are not as important as vision in detecting motile plankton prey. Speckled worm eel larvae entering the estuary had already developed closed olfactory pits and nostrils, presumably in preparation for their rapid assumption of a life style where they bury in sediments during the day. Bonefish and ladyfish showed a similar development of closed olfactory pits and nostrils after metamorphosis within the estuarine habitat. iv

Studies of receptor densities, olfactory organ ontogeny, and testing the olfactory sensitivity of live larvae could help to further understand the life history of the Elopomorph fishes, and help with continued conservation and aquaculture efforts.

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

TABLE OF CONTENTS ...... vi

LIST OF FIGURES ...... viii

LIST OF TABLES ...... xi

ACKNOWLEDGEMENTS ...... xii

DEDICATION ...... xiii

INTRODUCTION ...... 1

LEPTOCEPHALUS LARVAE ...... 4

CHEMORECEPTION ...... 9

OLFACTION IN FISH ...... 11

RATIONALE AND HYPOTHESIS ...... 14

MATERIALS AND METHODS ...... 15

SAMPLE COLLECTION AND IDENTIFICATION ...... 15

EXTERNAL ASSESSMENT ...... 17

VISUAL ASSESSMENT ...... 17

SCANNING ELECTRON MICROSCOPY ...... 18

DATA COLLECTION AND ANALYSIS ...... 20

RESULTS ...... 22

MORPHOLOGY OF THE EYE AND OLFACTORY ORGANS...... 28

STANDARDIZATION WITH STANDARD LENGTH ...... 32 vi

PRE/POST METAMORPHIC ELOPOMORPHS ...... 35

DISCUSSION ...... 38

LITERATURE CITED ...... 50

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LIST OF FIGURES

Page

Figure 1 Chart of larval fish development (Northern anchovy, Engraulis mordax) from end of yolk-sac stage to juvenile stage (Moser and Watson, 2006) ...... 2

Figure 2 Picture of various distinctive features in leptocephalus larvae (Anibaldi et. al, 2016) ...... 6

Figure 3 Images of opsin immunohistochemistry of the retinas of ladyfish (A,C,E) and bonefish (B,D,F). 1 = dorsal; 2 = central; 3 = ventral retina regions. Antirod (magenta) and anticone (green, arrowheads) fluorescent stains show the distribution of rods and cones in the retinas of each species (Taylor et al. 2015) ...... 7

Figure 4 Schematic diagram of olfaction molecular mechanism (Buck and Axel, 1991) ...... 10

Figure 5 Schematic diagram of general morphology of fish olfactory area (Kasumyan, 2004) ...... 12

Figure 6 Imagery and drawings of various differences in morphology of fish olfactory area (a) Photo of preserved male anglerfish (Lophiiformes); (b) Electron micrograph from a goldfish (Carrasius auratus); (c) Electron micrograph from European eel (Anguilla anguilla); (ii) all drawings of rosette and lamellae for each corresponding species above (Cox, 2008) ...... 13

Figure 7 Images of morphometrics taken from an Atlantic menhaden (Brevoortia tyrannus) using Scandium program. (1) Total olfactory area; (2) Eye major and minor axis, posterier nostril major and minor axis, and anterior nostril major and minor axis (left to right); (3) Total eye area, posterior nostril area, anterior nostril area (left to right) ...... 21

Figure 8 Scanning Electron Micrograph image of an Atlantic croaker (Micropogonias undulatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril ...... 24

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Figure 9 Scanning Electron Micrograph image of an Atlantic menhaden (Brevoorita tyrannus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril ...... 24 Figure 10 Scanning Electron Micrograph image of a Pinfish (Lagodon rhomboides) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril ...... 25

Figure 11 Scanning Electron Micrograph image of a Speckled worm eel (Myrophis punctatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril ...... 25

Figure 12 Scanning Electron Micrograph image of a Ladyfish (Elops saurus) larva. Magnification = 50x. E = eye; Olf = olfactory pit ...... 26

Figure 13 Scanning Electron Micrograph image of a Bonefish (Albula vulpes) larva. Magnification = 50x. E = eye; Olf = olfactory pit . . 26

Figure 14 Scanning Electron Micrograph image of a Tarpon (Megalops atlanticus) larva. Magnification = 50x. E = eye; Olf = olfactory pit ...... 27

Figure 15 Mean eye area per species (+/- S.D.). Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae ...... 28

Figure 16 Mean olfactory area per species (+/- S.D.). Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae ...... 29

Figure 17 Mean (+/- S.D.) ratio of average olfactory area/average eye area in each species; Blue = leptocephalus larvae; Orange = Non- leptocephalus larvae ...... 30

Figure 18 Mean (+/- S.D.) of standardized eye diameter (% of SL); Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae ...... 33

Figure 19 Mean (+/- S.D.) of standardized olfactory pit length (% of SL); Blue = Leptocephalus larvae; Orange = Non- leptocephalus larvae ...... 34

Figure 20 Scanning Electron Micrograph image of a juvenile bonefish at 20x. SL = 40mm ...... 36

Figure 21 Scanning Electron Micrograph image of a juvenile ladyfish at 20x. 36 SL = 61mm ......

Figure 22 Scanning Electron Micrograph image of a juvenile pinfish at 20x. SL = 34mm ...... 37

Figure 23 Image of Muraenesox cinereus consuming squid paste (Mochioka et. al, 1993) ...... 39

Figure 24 Comparison of head versus body size of a leptocephalus larva versus a larval pinfish ...... 43

Figure 25 Image of various olfactory rosette types (Kasumyan, 2004) ...... 46

Figure 26 (A) Scanning Electron Micrograph image of a juvenile Bonefish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 40mm ...... 47

Figure 27 (A) Scanning Electron Micrograph image of a juvenile Ladyfish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 61mm ...... 48

LIST OF TABLES

Page

Table 1 Summary of settlement-stage larval fishes examined in this study ...... 23

Table 2 Matrix showing the results of the KW multiple comparison tests for total eye and olfactory areas. ** = significant at p<0.05. NS = not significant ...... 31

ACKNOWLEDGEMENTS

I would like to thank my academic advisor, Dr. Jon Shenker for his guidance and support throughout my master’s program. I would like to thank my remaining committee members, Dr. Turingan, Dr. Hunsucker, and Dr. Webbe for their patience and support. I am so grateful for my friends and labmates, Jake

Rennert, Tony Cianciotto, Alex Gering, Jamie Kelly, Mason Thurman, Louis

Penrod, and James King, who helped with all the sample collections and sorting.

This project would not have been possible without the help and patience of

Gayle Duncombe, who taught me all about various microscopy techniques crucial to this project. Thank you to Dee Dee for always keeping me on track and providing constant support. I would also like to thank Dr. Miller for his help and guidance with statistics, as well as Dr. Liz Wallace for providing the some of the samples for this study.

Lastly, I would like to thank my family, who have supported me and cheered me on throughout the entire process.

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DEDICATION

I would like to dedicate this to my parents. I love you both so much.

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INTRODUCTION

Many fish species have pelagic larvae that are called ichthyoplankton because they are relatively weak swimmers that are dispersed throughout the water column and transported by currents. The physical and ecological attributes of this pelagic environment influenced the evolution of larval morphology, behavior and physiology to enable larvae to find and ingest food, detect and avoid predators, and locate appropriate nursery habitats upon the completion of larval development.

Larvae not only had to evolve within the constraints of the pelagic realm, but they had to retain the capability to undergo an extensive series of ontogenetic changes to metamorphose into older life stages in varying habitats (Moser and Watson, 2006).

Early larvae of many species rely on endogenous nutrients contained within the remnants of their yolk sac, then transition to capturing food from the surrounding environment. This transition requires development of a functional gut and jaws and associated morphological changes in musculature, fins, and other organ systems (Figure 1). An increase in motility throughout larval development enhances prey capture and predator avoidance abilities. Congruent development of sensory systems is also crucial to their survival at the larval stage (Moser and

Watson, 2006).

Figure 1: Chart of larval fish development (Northern anchovy, Engraulis mordax) from end of yolk-sac stage to juvenile stage (Moser and Watson, 2006).

Most teleost fish larvae have very rudimentary sensory systems at hatching that quickly develop, enabling fish to begin detecting prey by the time they absorb the last of their yolk reserves (Figure 1). The three main sensory systems that begin development during this stage include mechanoreception, vision, and smell (Moser and Watson, 2006).

Mechanoreception is a crucial sensory system that enable fish to detect vibration in the water. Specialized hair cells called neuromasts are the foundation

3 for the development of the lateral line system in fish. As these mechanoreceptors are disturbed by pressure changes in the water, a signal is sent to the brain allowing the organism to detect movements and changes in water patterns around them

(Moser and Watson, 2006). These changes can alert them to predators or prey nearby. At the larval stage, these neuromasts are typically exposed on the surface of the skin and increase in abundance through early larval development. During the later larval period, neuromasts are encased within developing subdermal canals on the side of the body creating the lateral line system (Figure 1). This system enables detection of predators and mobile prey, but not for locating passive food sources that do not create physical disturbance in the water (Moser and Watson, 2006).

Vision or photoreception is another important sensory system used in the marine environment. Vision is the process of the eyes taking in a variety of wavelengths of light and creating an image of the environment around an organism.

This visual system is driven by specialized photoreceptor cells, which are housed in the posterior part of the retina. These photoreceptors fall into 2 main categories: rods and cones. Rods are extremely sensitive to light stimuli, and therefore are able to help organisms see in very low light level conditions, enabling nighttime

(scotopic) vision. Cones are less sensitive, needing more light to activate them, leading to photopic (daytime) vision (Pankhurst, 1994; Moser and Watson, 2006).

These cones are more densely arranged in the retina, allowing higher visual acuity, and are sensitive to different wavelengths of light that enable an organism to perceive color. In teleost fishes, development of the funtional visual system

4 typically occurs by the end of the yolk-sac stage, with retinas being densely packed with cone photoreceptors, providing larvae with the high visual acuity needed to detect and consume planktonic prey. Rod photoreceptors tend to develop later in larval or juvenile stages, when the need to navigate environments in lower light is higher (Pankhurst, 1994; Moser and Watson, 2006).

Olfaction or sense of smell is a third sensory modality important to fishes and other organisms. Specialized chemoreceptors detect different chemical cues in the environment, and send signals to the brain for processing (Buck and Axel,

1991). These odorants can have important effects on the behaviors of an organisms, including identifying conspecifics, detecting chemical attributes of spawning and nursery habitats, and sensing the presence of food and predators. (Døving, 1985;

Kasumyan, 2004).

LEPTOCEPHALUS LARVAE

Most studies dealing with larval development of teleost (bony) fishes and ontogenetic changes in sensory systems focus on the superorder of spiny-rayed fishes known as Acanthopterygii. This superorder, containing 13 orders and approximately 15,000 species is considered to be the most evolutionarily derived lineage within the teleosts (Nelson, 2006). The Elopomorpha is an older superorder, with a lineage dating back to the Jurassic period (Nelson 2006; Chen et al. 2014). Although the Elopomorpha consists of morphologically diverse fishes, their unusual leptocephalus (“slender head”) larvae are a synapomorphy that helps

5 define the superorder. This larval group is seen to be vastly different morphologically from those in the superorder Acanthopterygii or any other teleosts

(Nelson, 2006).

The Elopomorpha include widely divergent adult forms including eels

(Anguilliformes), ladyfish and tarpon (Elopiformes), bonefish (Albuliformes), and spiny eels (Notacanthiformes). Approximately 1000 extant species are currently known (Chen et al. 2014). The leptocephalus larvae of all of these group share unique characteristics including a laterally-compressed transparent body composed largely of acellular glycosaminoglycan (GAG) compounds (Miller, 2009), forward- pointing teeth, and a tubular gut (Figure 2). Each species can be differentiated through differences in overall body, head, and fin shape, gut length and morphology, pigmentation, and the number of muscle segments (myomeres).

Larvae of many coastal species have long developmental times, ranging from a mean of 22 days from hatching to metamorphosis in tarpon (Shenker et al. 2002),

55 days in bonefish (Mojica et al. 1995) and up to 200 days in American eels

(Wang and Tzeng, 2000), enabling long distance transport along continental shelves and pelagic areas by ocean currents (Miller, 2009). Deep sea taxa are commonly found at depths of 1000-5000 meters or more below the ocean surface

(Sutton et al., 2010), but little is known about their biology and development.

Figure 2: Picture of various distinctive features in leptocephalus larvae (Anibaldi et. al, 2016).

Along with their unique body morphology, leptocephali can be distinguished from Acanthopterygii larvae by their retinal structure. As previously stated, most teleost fishes have cone-dominated retinas at the larval stage, with rods developing closer to the juvenile stage (Moser and Watson, 2006). However, initial retinal structure and ontogeny is different in leptocephalus larvae. Taylor et. al

(2011, 2015) examined the visual systems of four species of larval Elopomorphs: tarpon (Megalops atlanticus), bonefish (Albula vulpes), ladyfish (Elops saurus), and speckled worm eels (Myrophis punctatus). This study showed that these larvae initially had rod dominated retinas, suggesting that their visual system was adapted for scotopic/nighttime vision, with low visual acuity and poor daytime vision

(Figure 3 ).

Figure 3: Images of opsin immunohistochemistry of the retinas of ladyfish (A,C,E) and bonefish (B,D,F). 1 = dorsal; 2 = central; 3 = ventral retina regions. Antirod (magenta) and anticone (green, arrowheads) fluorescent stains show the distribution of rods and cones in the retinas of each species (Taylor et al. 2015). Cones developed late in the larval period in these leptocephali. The number and proportion of rods and cones changed through ontogeny, resulting in development of photopic vision to accompany the scotopic vision (Taylor et. al,

2011:2015). This low visual acuity seen in leptocephalus larvae leads to the question of how these animals find and consume food in the wild at this crucial lifestage.

The feeding habits of leptocephalus larvae have long been a source of controversy. Identifiable prey items are not found in their guts, and their forward- pointing fang-like teeth are unlike those of other fish larvae, suggesting that these larvae do not capture and consume individual planktonic organisms. Recent studies have explored the carbon and nitrogen isotope ratios of these animals and suggest that leptocephali feed on a diet of gelatinous materials such as marine snow

(Miller, 2009; Miller et. al, 2013). Marine snow are mucilaginous aggregates that can include forms of detritius, inorganic materials, and various living organisms such as bacteria or phytoplankton. These aggregates can be created by mucus- based materials such as discarded larvacean houses and pteropod webs that collect various particles as they slowly fall through the water column. Because marine snow is able to support communities of bacteria and phytoplankton, it is an important site for nutrient recycling and production, making the marine snow a very nutricious food source for many organisms, including a variety of zooplankton and deep sea creatures (Alldredge and Silver, 1988).

Marine snow and gelatinous material in the plankton are generally transparent, which raise the question of how leptocephali with low visual acuity are able to locate food in pelagic environments. Based on their reliance on diffuse gelatinous food sources and and their retinal structure not being adapted for detecting transparent food, I hypothesize that chemoreception provides leptocephali with the ability to find and consume these gelatinous food sources.

CHEMORECEPTION

Chemoreception is the ability for animals to obtain information from chemical stimuli in the environment. Olfaction, or smell, is one crucial form of chemoreception that can help animals perceive their environment and affect their behavior. Odorant chemical stimuli in the environment impact a wide variety of fish behaviors, including identification of conspecific/prey/predator species, schooling behavior, migration, reproduction, defense mechanisms, and locating/orienting within habitats (Døving, 1985; Hara, 1986; Kasumyan, 2004;

Sola et. al, 1993).

Among the most intensively studied behaviors influenced by olfaction are migration and homing. Many species of salmon (Salmonidae) undergo ocean-wide migrations, returning to their natal stream to spawn, often dying after reproducing.

Various studies have shown that newly-hatched juvenile salmon imprint on stream- specific chemical stimuli, and use that olfactory signal to enter the same stream several years later to spawn (Cooper et. al, 1976; Graves et al 1968; Stabell, 1984).

In contrast with salmon who use olfaction to guide migration for reproduction, juvenile clownfish (Pomacentridae: Amphiprionae) frequently return to their natal range after a brief 9-12 day larval duration period. Jones et. al (2005) showed that one-third clownfish juveniles returned back to a natal area within a 2 hectare distance, with most settling less than 100 meters from their nest site (Jones et. al, 2005). Researchers believe that chemical cues are an important aspect in this

10 ability for coral-reef fishes to return to their natal area and locate suitable habitat

(Jones, 2005; Leis et. al, 2011; Gerlach and Artema, 2012).

The morphology of the olfactory apparatus that fish use to detect odorants and influence their behaviors can vary based on species. Despite these variations in morphology, however, the molecular mechanism remains constant. In all animals, odorants are detected by special receptors known as chemoreceptors. These chemoreceptors are specialized G-protein-coupled receptors within the olfactory epithelium that are activated by specific chemical stimuli (Figure 4; Buck and Axel,

1991).

Figure 4: Schematic diagram of olfaction molecular mechanism (Buck and Axel, 1991). As summarized in Figure 4, the G-protein itself is made up of 3 parts that include alpha, beta, and gamma subunits. As an odorant binds to the receptor within the olfactory epithelium, the protein goes through a conformational change, which allows the alpha subunit to break away from the beta and gamma subunits.

This alpha subunit and GTP then interact with the effector protein, which in the case of olfaction is adenylate cyclase (AC). This interaction stimulates the

11 adenylate cyclase, which elevates levels of cyclic AMP (cAMP). The build-up of cAMP in the cell opens a cyclic nucleotide gated channel and allowing ions such as sodium or calcium to flood into the cell (Buck and Axel, 1991). This flood of ions depolarizes the cell creating an action potential, which is passed along the olfactory receptor neurons to a glomerulus within the olfactory bulb. This glomerulus structure is a destination for multiple olfactory sensory cells to come together, and then pass the signal along to mitral or tufted cells, which take information to the brain for processing (Schoppa and Westbrook, 2001).

It was hypothesized by Buck and Axel (1991) that the olfactory system was made up of thousands of different odorant receptors. Kajiya et. al (2001) tested this hypothesis and examined if these olfactory receptors may overlap on the types of odorants they can recognize. Through cloning of particular receptors, they examined a variety of odorants including EG (Eugenol), EV (ethyl vanillin), and vanillin to obtain concentration data and intensity rates on different receptors. Their results showed that different receptors can detect overlapping odorants based on concentrations and change in molecular structure of the odorant. Through this research it was possible to see how different odorants are differentiated at a molecular level (Kajiya et. al, 2001).

OLFACTION IN FISH

The most important element in the mechanism of olfaction are the chemoreceptors. Without the stimulation of the receptors by an odorant, an action

12 potential cannot be created, and the stimuli cannot be changed into a form that can be processed by the brain. As with all vertebrate species, these receptors are contained within the olfactory epithelium.

Basic imaging of fish olfactory area shows that generally fish have an anterior and posterior nostril (Figure 5). The placement and distance apart of these nostrils, are unique for various species, however they are usually located between the eyes and snout of the fish. Water carrying odorants is brought in through the anterior nostril and down into the olfactory cavity or pit and back out through the posterior nostril.

Figure 5: Schematic diagram of general morphology of fish olfactory area (Kasumyan, 2004). As water moves through this olfactory pit, it is carried acorss the olfactory epithelium. The olfactory epithelium is specialized tissue that contains chemoreceptors to which odorants can bind. This specialized tissue containing receptor cells is folded into structures called lamellae, which arise from the bottom

13 of the pit and increase surface area within the pit. These lamellae then can come together forming a leaf-like structure called a rosette (Cox, 2008; Kasumyan,

2004). These rosette structures vary in shape, size, and number of folds or lamellae depending on species (Figure 6).

Figure 6: Imagery and drawings of various differences in morphology of fish olfactory area (a) Photo of preserved male anglerfish (Lophiiformes); (b) Electron micrograph from a goldfish (Carrasius auratus); (c) Electron micrograph from European eel (Anguilla anguilla) ; (ii) all drawings of rosette and lamellae for each corresponding species above (Cox, 2008). The development of these structures typically starts within a teleost fish embryo just a few days after fertilization. During the embryonic period, placodes on each side of the head start to elongate and increase in size. As this growth continues, the placodes invaginate and form the olfactory pit. As development of the organism continues, this structure will migrate to a place on the head that varies depending on species. Shortly after hatching, the anterior and posterior nostrils begin to open into the olfactory pit and expose the lamellae created during the

14 embryonic stage. As the fish begins the larval stage and continues to grow, the number and size of lamellae continue to increase, allowing various chemoreceptor types to develop (Noakes and Godin, 1988).

RATIONALE AND HYPOTHESIS

Leptocephalus larvae unite a group consisting of popular sportfish and eels.

As juveniles and adults, this group of fishes are important recreationally. For example, bonefish are a vital fishery in south Florida, contributing about 47 million dollars alone in 2009 (Fedler, 2009). Along with recreational importance, eels have a high level of commercial importance, and are a popular food source in many countries around the world. These different species are crucial to many different markets, and have prompted establishment of fishery regulations in many regions.

There is great interest in the development of aquaculture operations and produce fish for the consumptive market (Tanaka et. al, 2001; Kagawa et. al, 2005; Lago et. al, 2012). Despite this interest, one of the main problems for culturing the species is determining successful strategies for feeding leptocephalus larvae.

The unique leptocephalus larval stages have low visual acuity, yet they are able to navigate their environment and find gelatinous food sources. Determining if they have an olfactory capability to find food will help understand the ontogeny, feeding ecology and behavioral characteristics of these larvae. It will further help determine suitable feeding strategies for animals grown in aquaculture systems

15 devoted to producing food fish (eels) or for stock enhancement (bonefish and tarpon).

The overall goal of this study is to determine if chemoreception is a possible mechanism by which leptocephalus larvae can locate food in the wild. In order to achieve this, morphology of the olfactory system in leptocephalus larvae and other teleost larvae representatives will be characterized and compared. A variety of microscopy and histology techniques will be used to test the hypotheses:

H1: The olfactory apparatus of leptocephalus larvae is morphologically

different from other larval teleost fishes.

MATERIALS AND METHODS

SAMPLE COLLECTION AND IDENTIFICATION

The goal of this study is to compare leptocephalus larvae (tarpon, bonefish, eel, and ladyfish) with examples of other teleost fishes. For this comparison, samples of larvae were obtained from a variety of sources. I collected a diverse array of larvae by using a plankton net at Sebastian Inlet State Park in Florida during the months of October 2017 and February 2018. The plankton net was attached to the catwalk on the north side of the park and left floating in the water from 5-15 minutes during the incoming tide. A collection cup at the cod end of the net was used to collect any larvae washed into the net. After net retrieval, fish larvae were sorted from the other plankton and preserved in vials containing 10%

16 formalin in seawater. All samples were then brought back to the laboratory for further sorting and species identification.

In addition to obtaining samples from Sebastian Inlet, bonefish leptocephali were collected in late winter 2015 as they moved from the pelagic environment of the Tongue of the Ocean toward their juvenile nursery habitats in their way into the labyrinthine maze of creeks on South Andros Island, Bahamas. Staff from the

Forfar Field Station collected the larvae in a plankton net moored at the mouth of a creek, preserved the larvae 70% ethanol in seawater, and sent the samples to Dr.

Elizabeth Wallace at the Florida Fish and Wildlife Research Institute for genetic analysis. After confirming their identity as Albula vulpes, and not other species within the genus, Dr. Wallace provided 12 specimens for my analysis.

All leptocephalus larvae were identified on the basis of caudal fin structure, dorsal and anal fin placement and seasonality of collection. Round caudal fins indicated that the leptocephali were eels, while forked caudal fins designated the larvae as either bonefish, tarpon or ladyfish. Further identification to species was accomplished using morphological traits defined by Smith (1989).

Leptocephali in the February samples were dominated by speckled worm eels (Myrophis punctatus) and ladyfish (Elops saurus). The October samples also contained Atlantic tarpon (Megalops atlanticus).

After all leptocephalus specimens were sorted from the Sebastian Inlet samples, remaining larvae were sorted and identified for selection of teleost species to be used for comparison with the leptocephali. Richards (2005) was used for

17 identification of these larvae. Three taxa dominated the non-leptocephalus larvae in the collection, and were thus selected for analysis. Atlantic menhaden

(Brevoorita tyrannus) are planktivorous fishes that are members of the

Clupeomorpha, an early teleost superorder. Two species are members of the more recently-evolved Acanthopterygii: Micropogonias undulatus (Atlantic croaker) and Lagodon rhomboides (pinfish). Overall, this study thus used 4 different species to represent leptocephalus larvae and 3 to represent other teleost fishes.

EXTERNAL ASSESSMENT

Once specimens were obtained for each species, an external assessment of the olfactory region was completed using light microscopy and scanning electron microscopy (SEM).

VISUAL ASSESSMENT

After sample processing, an initial visual assessment was conducted for each species using light microscopy (Olympus CX31). Two larvae from each species were randomly chosen and photos of the head region were taken. This allowed for an initial assessment of the gross morphology of the fish without any type of imaging enhancement. These photos were not used for data collection or analysis, but instead were used as a guide to help develop and assess the quality of the scanning electron microscopy (SEM) preparations.

SCANNING ELECTRON MICROSCOPY

After these initial visual microscopy images were taken, another 14 larvae per species were chosen at random for morphometric analysis of the external olfactory apparatus via SEM. The process for obtaining these data included 6 steps: (1) Measuring standard length (2) Dehydrating sample, (3) Critical point drying, (4) Gold sputter coating, (5) SEM, and (6) Morphometric analysis. Before the start of this process, standard length (SL, from the tip of the notochord to the end of the caudal peduncle) was recorded, prior to any potential damage or shrinkage that could occur during the drying process. SL data were not available for bonefish larvae because their bodies had been removed for tissue for DNA identification of species; lengths of a subset of larvae taken from this Andros Island collection ranged from 42-63mm SL (mean = 54mm SL).

Extensive trials were performed on additional larvae to determine optimal specimen preparation procedures for use with the leptocephali and other larvae.

Presented here are the steps found to provide suitable specimens for analysis.

Specimen dehydration involved soaking samples in a series of dehydrating solutions: 20%, 50%, 75%, 90%, and 100% ethanol for 60 minutes each.

Following the ethanol series, the samples were then immersed in an intermediate fluid containing 50% ethanol and 50% amyl acetate for 60 minutes, before the final step which had the samples soak in 100% amyl acetate overnight. This process allowed for the slow replacement of water in the samples with amyl acetate.

After the samples were saturated with amyl acetate, they were placed a

Critical Point Dryer (Denton DCP-1) that initially replaced the amyl acetate with liquid CO2, which was then slowly volatilized from samples over a period of 35-45 minutes. Samples were placed in the dryer in specific groups in order to keep each species and class separate. After the samples were fully dehydrated, larvae were then adhered onto a SEM mount using small, black adhesive circles stuck directly onto the mount. A compound microscope was used to place the samples at the correct angle that allowed for the whole eye and olfactory apparatus to be seen. The mount was placed in the vacuum chamber of gold sputter coating instrument

(Denton Vacuum Desk III), and coated with a thin coating of gold. The coating time was dependent on the size of the larvae, but ranged from 2 -3.5 minutes in 60 or 90 second intervals. This layer of conductive metal reduces charging of specimens and helps give clearer imaging when in the scanning electron microscope.

After sample preparation was completed, the mounts were placed into the

SEM (JOEL, JSM-638OLV). The dorsal and lateral surface of the head of each larva was examined to visualize the olfactory pits, eyes and other head structures.

Images of the dorsal and lateral surface of the head of each larva were taken at magnifications of 30x, 50x, 75x, and 150x. All images were saved as TIFT files for subsequent morphometric measurement.

Suitable preparations that allowed SEM analysis were obtained for a total of

10 individual bonefish larvae and 12 individual larvae from the other taxa. The

20 process was also repeated for 1 juvenile bonefish sample and 1 juvenile ladyfish sample collected while seining in spring 2018 near a bridge close to Sebastian Inlet.

These extra samples were used to evaluate the ontogenetic changes that occurred after metamorphosis.

DATA COLLECTION AND ANALYSIS

After all images were taken, Scandium (Olympus Soft Imaging Solutions) was used to obtain the following general measurements: (1) Eye major/minor axis,

(2) Eye area, (3) Nostril major and minor axis, (4) Total nostril area, and (5) Total olfactory area (Figure 7).

Figure 7: Images of morphometrics taken from an Atlantic menaden (Brevoorita tyrannus) using Scandium program. (1) Total olfactory area; (2) Eye major and minor axis, posterier nostril major and minor axis, and anterior nostril major and minor axis (left to right); (3)Total eye area, posterior nostril area, anterior nostril area (left to right).

All measurements were taken from photos at 50x, allowing for consistency as well as a clear overall view of the eye and olfactory regions. Each picture was loaded into the program, the scale was set, and each measurement was obtained and saved onto an Excel sheet as well as in an image in case values needed to be double checked. After all measurements were taken, mean (+/- S.D.) data for each parameter was plotted per species and per group to characterize the structures.

Because larvae were of different sizes, measured parameters were also standardized relative to the SL of each specimen. To determine if morphometric parameters varied significantly among species, Kruskal-Wallis and KW Multiple Comparison tests (R Studio) were employed.

RESULTS

The morphology of the olfactory system and other larval features were examined in a total of 82 larvae compiled from 7 species of marine fishes (Table 1) that were collected in Sebastian Inlet, Florida, or Andros Island, Bahamas. Four of these species have leptocephalus larvae and 3 were representatives of other teleost superorders. All specimens were collected as they migrated from the pelagic ocean habitat into their juvenile nursery habitat. They can thus be considered as

“settlement-stage larvae,” and their sensory systems may begin to reflect the metamorphic changes as they shift to a new biological and ecological mode of existence.

A major difference between settlement-stage leptocephali and other teleost larvae is their length. Bonefish and ladyfish leptocephali were 3 to 5 times larger than croaker and pinfish, and twice the size of most menhaden larvae (Table 1).

The menhaden larvae and tarpon leptocephali were approximately equal in length, although menhaden grow continuously throughout their larval period while leptocephali shrink as they catabolize tissue while undergoing metamorphosis.

Table 1: Summary of settlement-stage larval fishes examined in this study.

Following initial species identification and sorting, fish larvae were prepared for SEM and images were taken for each species (Figures 8-14).

Figure 8: Scanning Electron Micrograph image of an Atlantic croaker (Micropogonias undulatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril

Figure 9: Scanning Electron Micrograph image of an Atlantic menhaden (Brevoorita tyrannus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril

Figure 10: Scanning Electron Micrograph image of a Pinfish (Lagodon rhomboides) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril

Figure 11: Scanning Electron Micrograph image of a Speckled worm eel (Myrophis punctatus) larva. Magnification = 50x. E = eye; A = Anterior nostril; P = Posterior nostril

Figure 12: Scanning Electron Micrograph image of a Ladyfish (Elops saurus) larva. Magnification = 50x. E = eye; Olf = olfactory pit

Figure 13: Scanning Electron Micrograph image of a Bonefish (Albula vulpes) larva. Magnification = 50x. E = eye; Olf = olfactory pit

Figure 14: Scanning Electron Micrograph image of a Tarpon (Megalops atlanticus) larva. Magnification = 50x. E = eye; Olf = olfactory pit The olfactory pits were easily observed in each species. When comparing the 7 different species, it was noted that the croaker, menhaden, pinfish, and eel all had very well developed anterior and posterior nostrils, and an enclosed olfactory pit (Figures 8-11). Although the size and shape were different, the croaker and menhaden seemed to have more elongated nostrils that were closer together (Figure

8 and 9) while the pinfish and eel (Figures 10 and 11) had more rounded and symmetric nostrils that were farther apart. The remaining 3 species, which include the ladyfish, bonefish, and tarpon seen in Figures 12-14 had large, open olfactory pits with no developed set of nostrils. None of these larvae had developed a visible olfactory rosette with associated lamellae.

MORPHOLOGY OF THE EYE AND OLFACTORY ORGANS

Following these initial morphological observations, various morphometric measurements were recorded from each SEM image. Because many organs grow in an isometric or allometric relationship with length, eyes would be expected to be larger in longer fish than in shorter fish. If body size alone influenced the size of sensory organs, the eyes and olfactory organs would be expected to be larger in the larger leptocephali than the other larvae. This simple size relationship was not observed in the eye area of the different species of larvae (Figure 15). While the eye area was the largest in the longest larvae (bonefish), the eye areas of the other leptocephali were smaller than the shorter non-leptocephalus larvae.

1.4

1.2 )

2 1 0.8 0.6

Eye Eye Area(mm 0.4 0.2 0

Species

Figure 15: Mean eye area per species (+/- S.D). Blue = Leptocephalus larvae; Orange = Non-leptocephalus larvae

Keeping in mind how organs grow in relation to legth, it would be expected that longer fish would have a larger olfactory pit. This size relationship was also not observed in the olfactory pit of these different species (Figure 16). While the largest olfactory area was observed in the ladyfish, areas of the other leptocephali were the same size or smaller than the shorter non-leptocephalus larvae.

0.16 0.14

0.12 )

2 0.1 0.08 0.06 0.04 0.02

Olfactory Olfactory Area(mm 0

Species

Figure 16: Mean olfactory area per species (+/-SD). Blue = Leptocephalus larvae; Orange = Non-leptocephalus larvae

To make these two measurements more comparable per species, average olfactory area was divided by average eye area for each species to compare the relative size of these sense organs (Figure 17).

0.6

0.5

0.4

0.3 Olf/Eye Olf/Eye Ratio 0.2

0.1

0 Bonefish Tarpon Ladyfish Eel Croaker Menhaden Pinfish Species

Figure 17: Mean (+/-SD) ratio of average olfactory area/average eye area in each species. Blue = Leptocephalus larvae; Orange = Non-leptocephalus larvae

This ratio of olfactory/eye area (Figure 17) is greatest in ladyfish, where the area of the olfactory apparatus is about 40% the size of the eye area. When compared to the non-leptocephalus larvae representatives, the ratio observed for the ladyfish was more than double for that of the non-leptocephalus respresentatives.

To determine if the differnce in sense organ size differed signficantly among species, both total eye area and total olfactory area were tested. After running Shapiro-Wilk normality tests on both variables, a p-value less than 0.05 was seen for both, concluding the data were not normal. Following this, a Bartlett’s test was used, which again producted a p-value less than 0.05 for both variables,

31 showing unequal variances. The lack of normal data and equal variances led determined the need for a non-parametric test to be used.

The Kruskal-wallis test was selected, and ran for both eye and olfacotry area. Both tests producted p-values less than 0.05, which showed a significant difference in these variables among species. In order to find which species were different from one another, KW multiple comparison tests were then used on the matrix of interspecific comparisons to determine which species were different from one another (Table 2).

Table 2: Matrix showing the results of the KW multiple comparison tests for total eye and olfactory areas. ** = significant at p <0.05. NS = not significant.

As shown in Table 2, there are indeed significant differences in sensory organ area among species. When looking at eye area, it was observed that bonefish were significantly different from the other leptocephalus larvae. This matches findings seen in Figure 15, which shows bonefish having an average eye area of

1.1mm2, compared to tarpon, ladyfish, and eels which all have averages less than

0.5mm2. With regards to olfactory area, there was a significant difference between

32 tarpon and the other leptocephalus representatives. This result corresponds to

Figure 16, which shows tarpon having the smallest olfactory area out of the leptocephalus group. Overall, it was seen that there were observable differences in sensory organ areas between species, however, there were no consistent differences that separated leptocephalus and non-leptocephalus larvae

Following analysis on sensory organ areas between species, a Shapiro-wilk normality test and a Bartlett’s test were also run for the olfactory/eye ratio data

(Figure 17). Both of these test concluded the data were again not normal and the variances were not equal, leading to the use of another Kruskal-wallis test. This

Kurskal-wallis test was used to see if there were differnces in this ratio among larval types. A p-value of less than 0.05 was produced, therefore showing that there was a significant difference in these ratios among leptocephalus and non- leptocephalus larvae.

STANDARDIZATION WITH STANDARD LENGTH

Although the KW multiple comparison tests presented above showed some differences sensory organ sizes among species, this raw data did not take into account the size differences between the fishes themselves.

Total eye area and olfactory area could not be directly standardized against standard length, so eye diameter and olfactory pit length were used. Eye diameter was initally measured during data collection for all fish, however olfactory pit length was not. For the bonefish, tarpon, and ladyfish, the exposed olfacotry pit was

33 initally measured. In the other 3 species, which had enclosed olfactory pits the length was caluculated by adding up the length of each nostril along with the distance between. After all the values were obtained, each was standardized against length by dividing the sensory variable by standard length for each fish and then multiplying it by 100. This turned the values into percentages of the total length of the fish, which were then compared between groups (Figures 18 and 19).

4 % Standard%Length

0 Bonefish Tarpon Ladyfish Eel Croaker Pinfish Menhaden Species

Figure 18: Mean (+/- S.D.) of standardized eye diameter (% of SL). Blue = Leptocephalus larvae; Orange = Non-leptocephalus larvae

When looking at eye diameter, a trend was seen between larval types, with the all leptocephalus larvae having a smaller mean of standardized eye diameter compared to that of the non-leptocephalus larvae (Figure 18). This same pattern was seen in Figure 19, with the leptocephalus larvae again havin a smaller mean of

34 standardized olfactory pit length compared to the non-leptocepahlus representatives.

4.5 4 3.5 3 2.5 2 1.5

%Standard Length 1 0.5 0

Species

Figure 19: Mean (+/- S.D.) of standardized olfactory pit length (% of SL). Blue = Leptocephalus larvae; Orange = Non-leptocephalus larvae

In order to confirm the difference between groups, a Kruskal-Wallis was used on both variables after the data were found be not be normal and variances were not equal. Both results of the Kruskal-Wallis tests produced a p-value less that

0.05, showing that the observable pattern was correct, and there were indeed signficant differnces between groups for both standardized eye diameter and olfactory pit length.

PRE/POST METAMORPHIC ELOPOMORPHS

The lack of anterior and posterior nostrils, and the open olfactory pits in the tarpon, ladyfish, and bonefish leptocephalus larvae is a significant difference from the development of other teleost fishes. Their olfactory pits are fully exposed on the surface of the larval heads, while the olfactory apparatus of settlement-stage eel leptocephali and other teleost fishes is embedded under the skin and exposed to odorants in water moving through an incurrent nostril. The metamorphosis of tarpon, bonefish and ladyfish leptocephali from pelagic larvae to coastal juveniles presumably includes transition of the olfactory apparatus to the juvenile/adult state.

Several juvenile bonefish and ladyfish were captured and examined to address this ontogenetic change in the olfactory apparatus. These juveniles were captured by seine net in the Indian River Lagoon in spring 2018. Imagery for one juvenile bonefish and ladyfish were then taken using the scanning electron microscope

(Figure 20 and 21).

Figure 20: Scanning Electron Micrograph image of a juvenile bonefish at 20x. SL = 40mm

Figure 21: Scanning Electron Micrograph image of a juvenile ladyfish at 20x. SL = 61mm

The SEM images of these juvenile ladyfish (61 mm SL) and bonefish (40 mm SL) show a dramatic change in olfactory morphology from the leptocephalus larval condition, with the olfactory epithelium embedded under the skin with clearly developed anterior and posterior nostrils. An olfactory rosette with epithelial lamellae is clearly visible within the nostrils. This olfactory structure is now morphologically similar to that of juvenile pinfish (Figure 22).

Figure 22: Scanning Electron Micrograph image of a juvenile Pinfish at 20x SL = 34mm

DISCUSSION

The superorder Elopomorpha is comprised of widely divergent adult fishes including eels (Anguilliformes), ladyfish and tarpon (Elopiformes), bonefish

(Albuliformes), and spiny eels (Notacanthiformes). This group is united by the unique leptocephalus (“slender head) larval stage, which has special morphological characteristics including a compressed, transparent body, forward-pointing teeth, and a tubular gut (Nelson, 2006). In addition to their distinctive morphological characteristics, leptocephali have cellular, physiological and behavioral differences from other teleost (bony fish) larvae. The visual system is an example of how larval groups differ. Most teleost larvae have retinas dominated by dense arrays of cone photoreceptors, allowing for high visual acuity and photopic (daytime) vision.

Leptocephalus larvae, however, have retinas dominated by rod photoreceptors, which are more functional for scotopic (low light) vision (Taylor, 2011; 2015).

Another difference between leptocephali and other teleost larvae is their feeding behavior. Most pelagic teleost larvae feed on zooplankton, and their retinal structure is adapted to detecting these small moving particles. As opposed to consuming active food sources, leptocephalus larvae eat marine snow (Miller,

2009; Miller et. al, 2013). Marine snow particles are aggregates derived from mucus and gelatinous zoopolankton that host bacterial colonies and adhere phytoplankton and other particulate matter (Alldredge and Silver, 1988).

Mochioka et. al (1993) demonstrated the feeding behavior of eel leptocephali when

39 fed on a diet of squid paste. The paste was dyed red in order to observe the digestion process and showed that the larvae used their fang-like teeth to carve pieces out of the artificial marine snow paste (Figure 23).

Figure 23: Image of Muraenesox cinereus consuming squid paste (Mochioka et. al, 1993).

This transparent and passive food source is hard to find in the water column. Previous research has shown that the leptocephali have rod-dominated retinas, meaning overall low visual acuity (Taylor et. al, 2011:2015). This lack of

40 emphasis on a visual feeding strategy is supported by the smaller eye diameter seen in this group.

The unique transparent nature of these larvae, along with their poor visual acuity creates the question of how these animals are able to find the gelatinous food sources in the pelagic environment. This study hypothesized that leptocephali use chemoreception to find the mucus aggregates of marine snow. This reliance on chemoreception was hypothesized to require an enhanced the olfactory apparatus of leptocephalus larvae compared to other teleost larval fishes. To test this hypothesis, the external olfactory apparatus of late larval stages of four species of leptocephali and three other teleost larvae was examined. Eye dimension data were also collected to allow analysis of intraspecific differences among species, and as an index of the relative development of visual and olfactory organs within each species.

These morphological analyses did indeed find variation in the structure of the olfactory apparatus between leptocephali and other teleost larvae. When comparing the 7 different species, the croaker, menhaden, pinfish, and eel larvae all had very well developed anterior and posterior nostrils, and an enclosed olfactory pit. The ladyfish, bonefish, and tarpon, however, had large, open olfactory pits with no developed set of nostrils. This unique open structure has not previously been described in any other fish larvae. In most teleost fishes, shortly after hatching, the anterior and posterior nostrils begin to open into the olfactory pit and expose the lamellae created during the embryonic stage (Noakes and Godin, 1988).

This opening of the nostrils can occur within the first couple days after hatching, making the delay seen in leptocephalus larvae unique.

The settlement-stage leptocephali with these exposed olfactory pits had odorant receptors directly exposed to surrounding seawater, presumably increasing their sensitivity to chemicals diffusing from their transparent mucus-based food source. All three of these species metamorphose into predatory, highly mobile species, and their olfactory apparatus apparently metamorphoses into the juvenile/adult state (Figure 20 and 21) concurrently with metamorphosis of the rest of the leptocephalus body into the juvenile body form.

Conversely, the speckled worm eel leptocephali had well-developed anterior and posterior nostrils. Leiby (1979) examined the ontogeny of the cranial osteology of this species throughout its larval period and metamorphosis. Although he did not focus on the olfactory apparatus, his drawings suggest the presence of a large open olfactory pit during early stages of larval development (from 13.9 to 49 mm Total Length [TL]). This open olfactory pit during early stages of development presumably also occurs in early developmental stages of tarpon, ladyfish and bonefish.

By the time the eel larvae began to metamorphose (65 mm TL), the open olfactory pit had been covered over by a developing nasal bridge, with openings restricted to anterior and posterior nostrils. This development of the closed olfactory pit and nostrils during metamorphosis is a preadaptation to the subsequent post-larval behavior of the species. Unlike mobile bonefish tarpon and ladyfish,

42 speckled worm eels burrow into the sediment and emerge only to forage (Able et. al, 2011). The early development of protected olfactory organs prior to metamorphosis enables the ability to quickly begin to bury in the sediment upon estuarine entrance. This early olfactory development could also account for the raised appearance of the anterior nostril as is the post-metamorphic fish could expose only their head to sense prey, while they have their bodies burrowed.

The morphology of the olfactory apparatus in leptocephalus larvae thus suggest that open exposed epithelial surfaces indeed help detect odorants in marine snow aggregates. The size of the olfactory and visual organs alone, however, do not serve as consistent predictors of the feeding behavior of larvae. Bonefish had the largest eyes of any of the larvae, while ladyfish had the largest olfactory pits

(Figures 15, 16 on pages 28, 29), although if sensor size was standardized to the length of the larvae, leptocephali had smaller sensors/larval length than did any of the other teleosts (Figures 18 and 19 on pages 33 and 34). One reason there could be no clear patterns in sensor dimensions could be the unique development patterns of the leptocephalus.

As compared to other larval teleost fishes, leptocephalus larvae go through a unique developmental change, which can be broken up into 2 phases. Phase 1 is classified as the premetamorphic interval, which aligns with a growth period where these larvae significantly expanding their body size in length and dorso-ventrally, while there is little growth in their head. Following phase 1 is the metamorphic interval or phase 2. In this phase the larvae begin to shrink and are no longer

43 laterally compressed, and start to lose some of the distinct leptocephalus characteristics. Once phase 2 is complete they are able to grow again and eventually enter the juvenile stage (Pfeiler, 1986).

A previously described by Pfeiler (1986), the head of leptocephalus grow significantly less than the body during the growth phase. This allometric growth is far more pronounced in leptocephali than in other teleost larvae (Figure 24).

Figure 24: Comparison of head versus body size of a leptocephalus larva versus a larval pinfish The larvae of other teleost fishes are not constrained by the “slender head” development pattern of leptocephali. Eye diameters, relative to the length of menhaden, pinfish and croaker (Figures 18 on page 33) were much larger than those in the leptocephalus larvae group. In these non-leptocephalus larvae, the eyes

44 are an important sensory system for larval fish to find food. The cone-dominated retina helps to find planktonic prey moving throughout the water column

(Pankhurst, 1994; Moser and Watson, 2006). Pinfish, croaker, and menhaden have a main diet of copepods at the larval stage, (June and Carlson, 1971; Darcy, 1985;

Govoni, 1986).

Although these three species with non-leptocephalus larvae use their visual systems to feed on zooplankton, other non-Elopomorph teleosts may rely on other sensory modalities to find and consume food. Newly hatched larvae of some other teleosts, such as freshwater cichlids and catfish, feed on detritus or other benthic materials. These animals presumably have well-developed olfactory systems, as well as chemoreceptors on barbels and epidermal surfaces, to help them obtain food, but the ontogeny of their olfactory sensors have not been studied (Mukai et. al, 2010).

Regardless of the type of larval development, metamorphosis from larval into juvenile form typically involves major changes in morphology, feeding and other behaviors. The olfactory systems of leptocephalus and non-leptocephalus larvae all develop into olfactory pits lined with the olfactory epithelium. This specialized tissue is folded into structures called lamellae, which are arranged into a leaf-like structure called a rosette. The rosette structures vary in shape, size, and number of lamellae depending on species. The increased number and size of the lamellae means there is more surface area for these chemoreceptors, and presumably greater olfactory sensitivity of the fish (Cox, 2008; Kasumyan, 2004).

The various shapes and folds in a rosette can vary among families (Figure

25). The eels (Anguilliformes) have a long rosette with many folds, with a large potential surface area for receptors. This is similar to what is seen in Figure 6 when looking at the rosette of the European eel in panel C. That rosette has a significantly longer shape with greater number of lamellae as compared to the anglerfish in panel A or the goldfish in panel B (Cox, 2008).

Figure 25: Image of various olfactory rosette types (Kasumyan, 2004).

The ontogenetic development of these rosettes was observed in several of the Elopomorph and other fishes examined in this study. Juvenile bonefish and ladyfish (Figure 20 and 21) are fish that have metamorphosed and adopted a

47 juvenile mode of life. They no longer feed on marine snow and mucus aggregates, instead feeding on various zooplankton and benthic invertebrates (Adams et.al,

2014). Their olfactory apparatus has also metamorphosed, with separate nostrils and a developing rosette taking on different biological roles than detecting marine snow (Figure 26 and 27).

Figure 26: (A) Scanning Electron Micrograph image of a juvenile Bonefish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 40mm

Figure 27: (A) Scanning Electron Micrograph image of a juvenile Ladyfish at 20x; (B) Scanning Electron Micrograph image of the olfactory apparatus at 75x; SL = 61mm By this life stage, the retinas have developed a balance between rods and cones, giving the organisms a higher visual acuity, which helps them become more visual feeders (Taylor et. al, 2015). The opposite result was seen in the pinfish development. These fish are already visual feeders and do not rely on olfaction as heavily in the larval stage. Like most teleost fishes, they start out with an olfactory apparatus that follows the basic schematic of having a covered olfactory pit with well-formed nostrils. Metamorphosis of these fish thus involves elaboration of their olfactory rosette and lamellae, rather than developing the entire nasal structure.

The results of this study supported the hypothesis that there is a significant difference in the morphology of the olfactory apparatus of leptocephalus larvae compared to other larval teleost fishes. It is important to understand how these

49 unique larvae perceive the world, with various challenges they face. Having a better understanding of these Elopomorphs at this stage can help give a more complete life history of these organisms, which could further conservation and aquaculture efforts. Further research needs to be conducted at the molecular level in order to better understand the importance of olfaction by looking at receptor densities and types present in these exposed, olfactory pits. Pairing this information with experiments testing larval sensitivity to smell would help to fully understand the importance olfaction in these larvae’s ability to find food.

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