Juvenile Wild Lemon ( brevirostris) Health Assessment: Establishing a Baseline and Assessment Procedure for the Health of the South Caicos Juvenile Lemon Shark Population

Senior Thesis

Presented to The Faculty of the School of Arts and Sciences Brandeis University

Undergraduate Program in Biology Professor James Morris, Advisor

In partial fulfillment of the requirements for the degree of Bachelor of Science

By Mariah R. Beck April 2016

Copyright by Mariah R. Beck !ii

Abstract

Shark health is very important to the health of marine ecosystems, but it has been minimally studied in the past. In this study, a new health assessment procedure was established by combining traditional methods of overall health assessment, such as condition factor, with newer methods focused on parasitic parameters, such as fin rot infections and ectoparasites. This study indicates that condition factor may decrease and fin rot may increase with increased shark recaptures. Also, female may experience more intensive fin rot than males and the left side of the first dorsal fin may be more susceptible to fin rot as well. Dermophthirius nigrelli monogenean flatworms,

Stibarobdella macrothela leeches, and Rocinella signata isopods were found only on sharks exhibiting fin rot which may support the hypothesis that certain parasites increase shark susceptibility to other parasites and infections by decreasing immunity or acting as vectors. Analysis of these findings established a baseline for the juvenile lemon shark population in South Caicos, Turks and Caicos Islands. This population is in relatively good health and should be monitored for changes as coastal development continues on this island. It may act as good model for the effects of coastal development on shark populations and marine ecosystem health in the future. For continued monitoring of this population, condition factor measurements should be combined with fin rot analysis and ectoparasite observations as demonstrated in this study. !iii

Acknowledgments

A special thank you is extended to Professor James Morris for his continued and thoughtful support throughout my work on this project. I would also like to thank

Brandeis University and the Brandeis undergraduate Biology department for their support. The technical support provided by the School for Field Studies for this research is gratefully acknowledged. Special gratitude is also extended to Dr. Aaron Henderson and Travis Gomez-Phillips without whom this research would not have been possible.

Thanks is also extended to the students at the School for Field Studies for continued support during data collection and to the Turks and Caicos Islands government for allowing this research to take place. !iv

Table of Contents

Acknowledgments……………………………………………………………………..…………ii

List of Tables………………………………………………………………………………………v

List of Figures…………………………………………………………………………………….vi

I. Introduction………………………………………………………………………………..…..1

The Bell Sound Nature Reserve, South Caicos, Turks and Caicos Islands, British West

Indies…………………………………………………………………………………………..… 3

The Lemon Shark, Negaprion brevirostris………………………………………………..4

Lemon Shark Reproduction and Juvenile Behavior………………………………………7

Threats to Lemon Sharks………………………………………………………………….9

Health Impacts of Research Capture/Handling Procedures……………………………..10

Major Shark Capture Methods…………………………………………………………..13

Condition Factor…………………………………………………………………………15

Parasites: Fin Rot, Monogeneans, Isopods, & Leeches……………………..…………..16

Research Outline..….…………………………………………………………………….23

II. Materials & Methods…………………………………………………………………..……25

Study Area………………………………………………………………………………..25

Capture Method………………………………………………………………………….25

General Data Collection………………………………………………………………….26

Condition Factor: Data Collection and Analysis……………………………………..…26

Fin Rot: Data Collection and Analysis…………………………………………………..27

Ectoparasites: Data Collection and Analysis…………………………………………….28 !v

III. Results………………………………………………………………………………………29

No significant difference in condition factor was found between shark sexes and no

significant correlation was found between condition factor and shark size…………..…29

Condition factor may decrease with increased number of recaptures……………..…….30

The intensity of fin rot is low overall and no significant correlation was found between

condition factor and fin rot intensity……………………………………………………..31

Fin rot surface area may increase with the number of recaptures, female sharks may

experience higher intensity fin rot, and left fin surfaces may be more susceptible to fin

rot………………………………………………………………………………………..32

Certain parasites may increase the prevalence of other parasites, and some parasites are

found in specific regions of a shark………………………………………………..……34

IV. Discussion……………………………………………………………………………….….37

Condition Factor…………………………………………………………………………37

Fin Rot………………………………………………………………………………..…39

Ectoparasites……………………………………………………………………………..42

Conclusion……………………………………………………………………………………….45

References………………………………………………………………………………………..46 !vi

List of Tables

Table 1. List of data collected upon capture of a shark specimen in the field………………….26

Table 2. Prevalence, Mean Intensity, and Abundance of Parasites: Parameters for analyzing the infection rates and intensity of leeches (Stibarobdella macrothela), monogenean flatworms

(Dermophthirius nigrelli), and isopods (Rocinella signata)…………………………………… 35 !vii

List of Figures

Figure 1. Map of the Turks and Caicos Islands geographic region…………………..……..……3

Figure 2. Map of the Turks and Caicos Islands, highlighting South Caicos………………………3

Figure 3. Bell Sound Nature Reserve on South Caicos…………………………………………..3

Figure 4. Identifiable features of the lemon shark: (a) similarly sized dorsal fins (b) blunt, round

snout (c) yellow-brown hue…………………………..……………………..……………5

Figure 5. Global distribution of lemon sharks…………………..………………………………..6

Figure 6. Mangrove trees dominating a nursery habitat…………………………………………..7

Figure 7. Gill Net Capture Method…………………………………………..…………………..14

Figure 8. Long Line Capture Method…………………..………………………………………..14

Figure 9. Condition factor grouped frequency distribution shows variable health with a

potential tendency towards the lower end of the health range……..…..…………….…..29

Figure 10. Mean condition factor for sharks recaptured once, twice, three times, and four times

shows a possible trend of decreasing condition factor with increased number of

recaptures. Note: only one specimen was recaptured four times and the recorded

condition factor for that specimen was utilized in this graph……………………………30

Figure 11. Percent of captured sharks that experienced an increase, decrease, no change, or

inconsistent change in condition factor between recaptures over time shows the highest

percentage of sharks experienced decreased condition factor between captures. Note: N/A

represents sharks that were missing data essential for the condition factor

calculation………………………………………………………………………………..31 !viii

Figure 12. Median degree of change for sharks that experienced an increase (I) and decrease (D)

in condition factor between recaptures shows that condition factor increases were larger

on average than condition factor decreases between captures…………………………..31

Figure 13. Fin rot surface area ratio grouped frequency distribution shows a generally low degree

of fin rot in this population………………………………………………………………32

Figure 14. Percent of captured female and male sharks shows that more females were captured

than males………………………………………………………………………………..33

Figure 15. Mean fin rot surface area ratio (± SD) for female and male captured sharks shows that

females may generally experience more intense fin rot than males……………………..33

Figure 16. Specimen BSNR023 exhibiting highly variable fin rot between the two sides of

the same first dorsal fin…………………………………………………………………..34

Figure 17. Two Dermophthirius nigrelli monogenean parasites (BSNR023)……….………..…35

Figure 18. One Stibarobdella macrothela leech collected from BSNR024…………….….….…36

Figure 19. Front (A) and back (B) view of one Rocinella signata isopod collected from

BSNR013. Note: the distinctive half-moon pattern located towards the posterior end of

the specimen is distinct and helpful for species identification (C)……………………..36 !1

I. Introduction

Human beings have an incredibly unique relationship with sharks – Jaws or

Discovery Channel’s Shark Week might come to mind – but these are also one of the most important groups of animals for the health of the oceans worldwide, especially because they can greatly impact many species populations cascading down the food chain below them. Many people do not see themselves connected to marine ecosystems, but human survival is indeed linked to the health of the world’s oceans. Oceans provide human beings with air to breathe, food to eat, and a source of climate stability on Earth.

This vital resource is a system of diverse marine ecosystems, many of which are put at great risk when shark populations are threatened. This is because shark population changes have significant domino effects on the rest of the ecosystem’s food chain.

Sharks have been widely studied for this reason, but shark health specifically has been minimally explored, especially in wild shark populations. It is often difficult to study wild sharks in their natural environment because of their large size, free-ranging behavior, and the relative inaccessibility of the ocean for scientists (Sundstrom et al.

2000). Although wild shark health evaluation studies have been particularly infrequent, there is an important and increasing need for establishing reliable health assessment procedures for wild shark populations. Understanding and measuring wild shark health can support conservation goals by influencing development decisions or by creating increased understanding of shark health and pathology, which can help scientists better predict the effects of climate change on shark populations and subsequently, on the oceans and therefore on humans as well (Haman et al. 2012). !2

Currently, the relationships between different shark pathogens on each other and on shark health are very poorly understood. There is also disagreement among marine biologists regarding the health impacts of capture and handling procedures for this widely studied . Furthermore, the lack of wild shark health studies has left a procedural gap that must be filled with more specific and reliable methods for assessing shark health in wild populations.

The wild juvenile lemon shark (Negaprion brevirostris) population inhabiting the coastal waters of the Bell Sound Nature Reserve in South Caicos, an island in the Turks and Caicos Islands archipelago, has been studied for many years. However, like many other populations, there has been limited analysis regarding the health status of these sharks. My study focused on that well-known population in order to contribute to the development of a functional and reliable procedure for assessing health in wild juvenile shark populations. In doing so, this project will work towards (1) exploring the relationship between health and capture/handling research methods, (2) understanding elasmobranch disease and (3) establishing a current baseline health status of the individuals in this South Caicos population in order to support further health monitoring in the future as the island begins phases of coastal tourism development. In order to accomplish these goals, I combined traditional health assessment methods, such as condition factor analysis, with relatively new methods of assessing shark health through parasitic infection analysis, including pathogens such as bacteria, monogeneans (marine flatworms), isopods (small, parasitic ), and leeches. !3

The Bell Sound Nature Reserve, South Caicos, Turks and Caicos Islands, British West Indies

This study focused on the Turks and Caicos

Islands (TCI), which are located in the British West

Indies, southeast of the Bahamas (Fig. 1). The Turks and Caicos Islands archipelago includes about 42 islands with the major inhabited islands being

Providenciales, Grand Turk, East Caicos, Middle Figure 1. Map of the Turks and Caicos Islands Caicos, and South Caicos geographic region (WorldAtlas 2015)

(Fig. 2). South Caicos is located in the southeast area of the Turks and Caicos

Islands archipelago (Ministry of Environment and Home

Affairs 2014). The South Figure 2. Map of the Turks and Caicos Islands, highlighting South Caicos (Google Maps Caicos community is 2016) economically dependent on lobster and conch fisheries and this island has not yet been developed for tourism like many of the other islands in the TCI. Within and surrounding South

Caicos, a wide variety of ecologically important habitats can be found such as mangroves, seagrass beds, and coral reefs. These habitats foster a diverse array of marine life though food supply and nursery habitat, making this island a valuable location for marine conservation and research (Valiela et al. 2001; Orth et al. 2006; Rogers & Miller 2006). !4

The Bell Sound Nature Reserve (BSNR) is one of fourteen marine protected areas (MPAs) in the TCI network of MPAs (Fig. 3). The reserve is located on the northwestern coast of South Caicos and it spans a diverse habitat range including important mangrove, seagrass bed, and coal reef Figure 3. The Bell Sound Nature Reserve on South Caicos habitats (Sailrock 2015). Although the BSNR was originally created to protect bonefish spawning habitat on South Caicos, there has been minimal evidence suggesting that bonefish use this area for spawning. However, the

BSNR does protect a very important nursery habitat for lemon sharks and it is therefore of particular interest for this study (Carleton & Hambrey 2006). Currently, there are proposed plans to further develop the coastline surrounding this MPA, which would negatively impact the mangrove habitat upon which the juvenile lemon shark population depends. The current state and significance of this reserve for the future make it an ideal site for this study.

The Lemon Shark, Negaprion brevirostris

All sharks are categorized within the class and the

Elasmobranchii subclass. The Chondrichthyes class consists of cartilaginous fish and the

Elasmobranchii subclass is distinguished by cartilaginous fish with an unfused upper jaw and separated gill slit openings (Motta et al. 1997). Within the Elasmobranchii subclass, lemon sharks are in the Charcharhiniformes order (ground sharks) and the Carcharhinidae family. The !5

Carcharhinidae family is composed of requiem sharks, which are migratory, viviparous shark species. Viviparous shark species, such as the lemon shark, give birth to live offspring that are nourished by a placenta through an umbilical cord in utero. Oviparous species on the other hand, such as the , produce eggs that are left on the ocean floor to be nourished by the yolk sac within the egg. The lemon shark is further classified in the Negaprion genus and the species of interest in this study is the common lemon shark, Negaprion brevirostris (Elbert et al. 2013).

Lemon sharks can be easily identified by their unique dorsal fins, body color, and snout

(Fig. 4). This species of shark normally has similarly sized, large first and second dorsal fins.

They also typically have yellow-brown backs as well as blunt, rounded snouts (Humann &

DeLoach 2013). When they are born, lemon shark pups are about 60 to 65 cm (total length) and as adults they are considered to be moderately large with a typical length range of about 2.5 meters to 3.5 meters (total length) (Sundstrom et al. 2000).

a

c b

Figure 4. Identifiable features of the lemon shark: (a) similarly sized dorsal fins (b) blunt, round snout (c) yellow-brown hue (Nature’s Notebook 2013)

Lemon sharks are normally opportunistic feeders, meaning they will eat a wide variety of food depending on availability, but they have been observed to exhibit feeding selectivity as well. In general, juvenile lemon sharks prefer to eat mostly teleosts (bony fish), crustaceans, and !6 mollusks. With maturity and growth, the lemon shark diet shifts to include less mollusks and crustaceans and more teleosts and cartilaginous fish. In most ecosystems, the lemon shark is considered the apex predator, or the organism at the top of the food chain with few to no predators above it. Lemon sharks have been observed to be preyed upon by larger lemon sharks, but otherwise they do not have any typical documented predators (Cortés & Gruber 1990; Gruber

1982). Fluctuations in the populations of apex predators, such as the lemon shark, usually have very significant effects on ecosystem health, balance, and energy flow through the trophic cascade effect - a domino effect of population changes in an ecosystem in which one population change causes many other species populations to shift in numbers due to altered predator-prey interactions.

Lemon sharks are the apex predator in a variety of subtropical habitats including mangroves, seagrass, coral reefs, and river mouths (Compagno 1984). Their Figure 5. Global distribution of lemon sharks (Compagno 1984) normal geographic distribution includes the western Atlantic, from New Jersey to Brazil, the tropical eastern Pacific, from southern California to Ecuador, and sparse populations along the coast of western Africa (Fig. 5).

However, the number of lemon sharks in the eastern Pacific and Atlantic ranges are currently declining, especially in the coastal waters of Florida (MarineBio 2013; Compagno 1984). Within those ranges, lemon sharks normally inhabit shallow waters with depths of about 100 m, but they !7 periodically move to deeper waters during migration and to even shallower waters for pupping season (Humann & DeLoach 2013).

Lemon Shark Reproduction and Juvenile Behavior

Lemon sharks are generally considered juveniles for the first five to six years of their lives. They reach sexual maturity when they are six to seven years old, at which point they begin taking part in the reproductive cycle (Elbert et al. 2013). About 60% of all shark species are viviparous, including lemon sharks. In viviparous species, the shark fetus develops entirely within the body of the female parent for ten to twelve months until the fetus is fully developed, at which point it is released from the female parent during a process called ‘pupping’. Lemon sharks exhibit placental viviparity along with 10% of all other shark species. This means that the fetus develops within the female parent and it receives nourishment from a yolk sac that develops into a placental cord. Most female lemons sharks reproduce on a biennial reproductive cycle, pupping between four to eighteen juveniles per litter (Feldheim et al. 2002). After the pups are born, there is no direct parental care and the juveniles are forced to forage independently in the pupping area, which is also the juvenile nursery habitat.

The birthing females move inshore for pupping in specific nursery habitats for several reasons and the habitats are distinguished by a variety of characteristics (Gruber 1982; Cortes &

Gruber 1990). Shark nursery habitats have been classified as areas with higher concentrations of sharks (especially juveniles), higher site fidelity than other areas (sharks remain in the area for extended periods of time), and higher return rates than other areas (the area is used repeatedly over multiple years for pupping). The nursery areas are normally very warm, shallow areas that !8 are mainly dominated by mangrove habitats. Mangrove habitats are defined by the presence of mangrove trees, a group of saltwater- dwelling trees grouped in the

Rhizophoraceae family that are distinguished by their branching roots that Figure 6. Mangrove trees dominating a nursery habitat exist partly in the marine water and partly in the air (Fig. 6). These trees are also associated with higher quantities of food for marine organisms and lower quantities of predators for young species. The mangroves provide protection for the pregnant female during the pupping process and the newborns remain in the mangrove-dominated nursery areas for the first three years of their life because they provide protection from larger, full-grown predators and a reliable source of food for the juveniles

(Laegdsgaard & Johnson 2001; Morrissey & Gruber 1993). Many other species of fish and crustaceans use similar areas as nursery grounds and the juvenile lemon sharks commonly feed on those organisms (Huepel et al. 2007).

Although lemon sharks are often solitary, only aggregating in small groups from time to time, juvenile lemon sharks have been shown to actively and habitually prefer social living.

Some scientists hypothesize that the juvenile tendency towards social behavior is critical for the survival and development of the juveniles especially when they are in the nursery areas

(Guttridge et al. 2009).

The Bell Sound Nature Reserve in South Caicos has abundant mangrove habitats, making many areas in the reserve ideal nursery habitats for lemon sharks. The presence of those nursery habitats is largely responsible for the consistent population of juvenile lemon sharks in this study !9 area. The shallow nature of juvenile habitats also allows for easier capture and handling of juveniles for research, as opposed to adult lemon sharks in the open ocean.

Threats to Lemon Sharks

The United States , Caribbean, and eastern Pacific Ocean fisheries often target the lemon shark as a source of meat. In many Caribbean islands, the lemon shark is known as the tastiest of all shark species. Lemon sharks are also targets in the bottom longline fishery in the U.S.A. and they are often caught as by-catch in many other fisheries. In Asia, the fins of these animals are highly prized for use in traditional medicines and foods (FMNH 2015).

Furthermore, hunting of these animals is encouraged by the common misconception of sharks as human predators and enemies. However, from 1580 to present, there are only ten recorded lemon shark attacks and none of those attacks were fatal (ISAF 2015). Lemon sharks are categorized as ‘near threatened’ by the official International Union for Conservation of Nature and Natural Resources (IUCN) red list. They are considered an important conservation priority because they are being utilized by human industry and research, many populations are currently in decline, and they are a highly migratory species, which are more challenging to protect (IUCN

2015).

Lemon sharks are also at risk due to increased development of coastal areas within their distribution range. In many areas of the Caribbean and West Indies, there is a general trend of increased tourism and coastal development (Bryden 1973). In the TCI specifically, there is a significant level of concern and ongoing policy work in order to mitigate the effects of increased economic development on the TCI culture and natural environment (Siar et al. 2006). These !10 areas are important ranges for the lemon shark and many crucial nursery areas are located in the shallow coastal waters of these countries. The creation of coastal resorts and other development often leads to the dredging and destruction of mangroves which are extremely important for juvenile lemon sharks, and consequentially, adult lemon shark populations. Therefore, unregulated development of countries within the lemon shark distribution range is also of concern for this species.

Health Impacts of Research Capture/Handling Procedures

Lemon sharks are typically considered ideal research organisms because of their tendency to inhabit shallower waters, which makes them more accessible, and their relatively low level of aggression compared to other shark species. Most of the health-related research completed using these organisms has focused on exploring shark stress physiology and the health impacts of different shark capture and handling procedures.

During capture and handling procedures, many researchers often invert sharks for safer handling. This movement causes a state of tonic immobility (TI) in the animal, which is an induced, coma-like state triggered by dorsoventral inversion. It is a single stage of a large defensive cascade response experienced by sharks during stress. In sharks, TI is characterized by relaxed muscles, limp bodies, and deep rhythmical ventilations. Based on studies with blacktip reef sharks ( melanopterus), many believe that TI is not extremely harmful because sharks maintain continued, deep ventilation and a relatively stable heart rate and blood pressure

(Davie et al. 1993). However, Brooks et al. (2011) found that TI causes significant disruptions to physiological homeostasis in sharks over time, especially when combined with exhaustive !11 exercise and anaerobic ventilation (discussed further below). In that study, TI was observed to cause increased blood carbon dioxide, decreased blood pH, increased metabolic acidosis, and increased blood glucose. These physiological changes can sometimes affect the motility, dexterity, sensory perception, and/or short-term ventilation efficiency of the shark if it is weakened from prolonged disruptions. From these findings, the scientists concluded that TI is inherently stressful for sharks (Brooks et al. 2011).

Lemon sharks normally employ ram ventilation (a strategy in which the shark must move through the water for gas exchange to occur), but during capture they switch to a less efficient form of breathing called buccal pumping (gas exchange is completed by the buccal muscles in the cheeks pulling water over the gills, rather than moving through a current). This form of breathing is more susceptible to becoming anaerobic and most scientists agree that exhaustive anaerobic exercise intensifies the negative impacts of tonic immobility, such as increased respiratory stress (Brooks et al. 2011).

Buccal pumping is one of several ways that sharks cope with physiological changes such as metabolic acidosis. Other noted active compensatory mechanisms are blood vessel dilation and increased ion permeability for gas exchange and electrolyte balance. Although these mechanisms are meant to aid the organisms in coping with stress and acidosis, they can also lead to additional health repercussions. For example, increased permeability of blood vessels can also lead to an electrolyte imbalance. Furthermore, elasmobranchs normally maintain an internal environment hyper-osmotic to the surrounding environment by retaining nitrogenous organic compounds such as urea and trimethylamine oxide. Increased permeability often disrupts the !12 control of that delicate osmolarity balance and negatively impacts protein and membrane structure as well as lipid, ketone body, and amino acid metabolism (Richards et al. 2003).

Most studies agree that sharks do experience some level of stressful physiological perturbations during capture and handling procedures (Brooks et al. 2011; Hyatt et al. 2012).

However, there is some disagreement about whether the shark’s biological coping mechanisms compensate and eliminate significant handling stress, or whether capture and handling still causes significant stressful disruptions for the sharks that can impact them in a negative way.

Hyatt et al. (2012) completed a study in a Florida nursery ground using blood tests to explore the intensity of blood parameter changes that sharks experience during capture and handling stress. They found that every shark experienced metabolic and respiratory acidosis during capture and handling. Carcharhinid (requiem) sharks, including lemon sharks, were found to have a relatively low capacity for aerobic metabolism and they therefore switch to anaerobic metabolism quickly. When that happens, they often produce excess lactate and hydrogen ions that move into the bloodstream from muscle cells leading to a lower blood pH and eventually metabolic acidosis. Intensifying that change is the fact that most sharks are ram ventilators, meaning they must continuously swim to oxygenate their gills with water, as discussed above. During capture, Hyatt et al. (2012) observed that the sharks often reduce or terminate breathing. That causes an increase in carbon dioxide in the bloodstream, which further lowers blood pH by conversion to carbonic acid, leading to respiratory acidosis.

The intensity of this acidosis is dependent on several factors including the species’ sensitivity, capture method employed, duration of entrapment, degree of entanglement, and force of struggle experienced by the shark. In a one-hour gill net setting, a group of researchers !13 observed that lemon sharks did not exhibit compromised breathing nor did they have a

- significant drop in HCO3 (bicarbonate), indicating that they likely have a very sturdy compensatory buffering response for stressful experiences. This means that they are less sensitive compared to the other shark species, making them an ideal choice for a research organism.

For any shark species, Brooks et al. (2011) recommend using artificial ventilation throughout the handling process in order to decrease the intensity of the stress response experienced by the sharks. Artificial ventilation can be performed by simply moving the shark through the water for a period of time to allow more gas exchange to occur over the gills. This methodological step can greatly reduce physiological stress experienced, especially for ram ventilators such as the lemon shark. Artificial ventilation has been used in researching the population of lemon sharks in South Caicos for many years. This study began exploring whether shark condition factor, an evaluation of health, correlates with shark recaptures in order to eventually contribute to the discussion concerning whether the physiological response to stress experienced by sharks during capture and handling is associated with a decline in health for that organism.

Major Shark Capture Methods

The two major methods for catching wild sharks for research are gill netting and longlining. A gill net is a wall of netting with lead weights on one side and floats on the other. When these nets are deployed, they essentially create a wall of net in the water column of the research area (Fig. 7). Gill nets control species selectivity with variable !14

Figure 7. Gill Net Capture Method (AFMA) Figure 8. Longling Capture Method (AFMA) mesh sizes that target specific organisms. The animals swim into the net and when they attempt to swim backwards, the net holds the animal by sliding slightly under the gills.

Long lining involves a free floating device with a long, branched fishing line attached.

These lines can either be attached to additional buoys for industrial fishing purposes or they can hang from a single buoy to allow continued swimming for research purposes

(Fig. 8). Each branch of the line has an attached hook with fish bait. Species selectivity is accomplished in long lining by using variable hook sizes and bait types (NOAA 2014).

Many studies have explored which of these two methods incur greater health repercussions for the captured organism, but contradicting evidence remains inconclusive. Hyatt et al. concluded in 2012 that gill netting is more stressful for sharks.

However, Frick et al. supported the opposite conclusion after their study in 2010. Some research indicates that the entanglement experienced in gill nets prevents the shark from swimming, thereby minimizing the flow of water over their gills and affecting the respiratory efficiency of the shark. Longlines, in contrast, allow the shark to continue swimming throughout the capture, which could be especially beneficial for ram ventilating sharks, such as lemon sharks (Hyatt et al. 2012). On the other hand, longline !15 hooks can often cause flesh wounds and punctures on the shark and they leave the swimming shark as prey for larger sharks if they are not checked quickly. Gill netting does not have the problem of physical damage to the shark because it is a noninvasive method and predators do not inhabit the shallow waters where sharks are usually caught using gill nets.

Research does however suggest that lemon sharks specifically have a low sensitivity to gill net captures. In a one-hour gill net setting, some species experienced up to 31% mortality, but lemon sharks had a 0% mortality, indicating that they have a very sturdy compensatory buffering response for stressful experiences and a low sensitivity to gill net captures (Hyatt et al. 2012). In this study, gill netting was used to capture the juvenile lemon shark individuals for health parameter data collection.

Condition Factor

The traditional health assessment parameter that was used in this study is the condition factor (Dibattista et al. 2007). The condition factor is a quantitative measure of plumpness, robustness, or general fitness for animals based on the length and weight of an individual organism. It has been used in many studies to track the overall health of a variety of marine organisms such as fish, crustaceans, mollusks, and sharks (Dibattista et al. 2007; Adeogun et al.

2016; Fazli et al. 2012; Basusta et al. 2013; Sarkar et al. 2013). Although this measurement is an accepted assessment of the health of individuals in a population, it can be easily skewed by many factors, such as the most recent feeding, the size of the gonads, or the sex of the individual - any factor that can temporarily or permanently skew the weight of an organism. Due to the !16 amendable nature of the condition factor value, this study paired this established method of assessing health with relatively new methods of assessing health through observing bacterial infections and calculating parasitic infection intensity for the health of an organism (Dibattista et al. 2007).

Parasites: Fin Rot, Monogeneans, Isopods, & Leeches

There are limited published reviews of elasmobranch disease, but as sharks are increasingly kept in captivity and shark conservation gains more significance, it is becoming even more important for scientists to understand disease patterns and pathology in elasmobranchs. A recent study attempting to summarize elasmobranch disease found that infectious diseases were reported most often (33.5%) and, within that category, bacterial infections were the most commonly observed infectious diseases

(15%) (Garner 2013).

One common infection among wild sharks that is likely caused by bacterial agents is an ulcerative skin disease called ‘fin rot’, ‘tail and fin rot’, ‘gill rot’, or ‘black patch necrosis (BPN)’ (Devesa et al. 1989). In this study, this condition will be referred to as

‘fin rot’. In studies with fish, this infection is typically characterized by white discolored areas on the skin that eventually progress to a sloughing of skin cells which leads to hemorrhagic, ulcerative lesions on the skin. Similar symptoms are observed on sharks, but the infection is normally confined to the fins, specifically the first or second dorsal fins. In fish studies, fin rot has been associated with some increased mortality (ranging from 1% to 8%), increased respiratory distress, erratic swimming, or changes in appetite. !17

Some studies have noted that most fish die within 2-3 days of contracting this condition, but this observation has not been conclusively recorded in sharks at this point (Devesa et al. 1989).

There is also currently disagreement about whether or not this dermal infection causes systemic disease (Devesa et al. 1989). This question would normally be explored by examining internal organs for the presence of certain bacteria found in the skin lesions. However, there is currently no consensus regarding definitive agents for the fin rot infection therefore the assessment is hard to complete with confidence. Although there has been some progress, especially with fish species, the understanding of fin rot pathology in sharks is particularly uncertain.

Many studies are beginning to suggest that there are different bacterial agents for this disease in fish and elasmobranchs. Some of the major species being explored as causative agents in aquatic animal disease include Grimontia hollisae, Photobacterium damselae subsp. damselae, Vibrio alginolyticus, V. harveyi (V. carchariae), V. cholerae, V. fluvialis, V. furnissii, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus.

Nine out of these eleven suspected species are vibrio bacteria which supports the increasingly confident hypothesis that fin rot in sharks is caused by vibrios (Austin 2010).

Vibrios are often found in coastal and estuary habitats. They are gram-negative, rod-shaped, flagellated bacteria that require sodium chloride for normal growth. Thus far, vibrios have been associated with marine bioluminescence and they are now increasingly being associated with certain aquatic diseases and in some cases shark !18 mortality. Some studies have also linked vibrios to zoonotic infections, but that connection is fairly unexplored (Austin 2010; Grimes et al. 1984).

Of the aquatic animal disease agents listed above, V. parahaemolyticus, V. vulnificus, P. damselae subsp. damselae, and V. harveyi (V. carchariae) are considered potential candidates for fin rot in sharks because they have all shown some propensity to cause skin ulcers or lesions - all but one of these species are vibrio bacteria. V. parahaemolyticus is an accepted pathogen of invertebrates, but has recently been associated with external hemorrhages and tail rot leading to mortality in certain fish. V. vulnificus also creates hemorrhaging on the body surface and this species has been associated with systemic disease presented in the gills, GI tract, heart, liver, and spleen.

P. damselae subsp. damselae has been isolated from ulcers on fish fins and some shark species and it has been associated with intensification in warmer temperatures, a trend that may also be observed with fin rot intensity in sharks (Devesa et al. 1989). Lastly and most confidently, V. harveyi or V. carchariae, have been recorded in several species, including lemon sharks, on chronic skin ulcers and necrotic subdermal cysts that seem to cause subsequent lethargy, a change in appetite, and some disorientation (Austin 2010).

These symptoms have not been confirmed in cases of shark fin rot, but many hypothesize that the symptoms may exist based on observations made with other species of fish

(Devesa et al. 1989). Bertone et al. (1996) also found a correlation between Vibrio carchariae and chronic skin ulcers on sandbar sharks (Charcharhinus plumbeus) and another study associated Vibrio harveyi infections with fin and tail rot disease in several fish as well as chronic skin ulcers in sharks (Hashem & El-Barbary 2013). !19

It currently remains unclear whether fin rot is definitively associated with mortality in sharks. One study focused on damselfish did find increased mortality with fin rot, but sharks were not included in that study (Austin 2010). If fin rot is in fact caused by vibrio bacteria, then it is likely that it does increase shark mortality based on the finding that, after being injected with vibrio bacteria, a group of captive sharks died within eighteen hours of exposure (Grimes et al. 1984). Some believe that the possible increased mortality may be due to systemic infection induced by the bacterial agents of fin rot, while others believe that the bacterial fin rot infections simply make the sharks more vulnerable to other pathogens that can lead to increased mortality (Devesa et al.

1989; Grimes et al. 1985; Garner 2013). If scientists conclude that fin rot is caused by vibrio and that they can migrate internally causing systemic infection, then fin rot in wild sharks may be associated with increased mortality in the future. Due to limited repeated examinations of wild sharks, it is very difficult to track pathogenic progression.

Currently, fin rot has not been associated with wild shark mortality.

Although it is unclear whether fin rot is associated with shark mortality, the relationships between this disease and other environmental and pathogenic factors - such as size, water temperature, stress, capture/handling, and previous pathogenic infection - have been more clearly elucidated. Although it is often assumed that bigger sharks correlate with increased infection intensity, Dippenaar et al. (2008) found that larger surface area for attachment does not necessarily correlate with increased infection intensity. This may be important in explaining any correlations between parasitic !20 infection and condition factor because we may not assume that the size of the shark is necessarily a factor in parasitic abundance.

The other factors listed seem to be involved in a negative feedback effect on shark health. Several studies have agreed that increased water temperatures support increased fin rot frequency and intensity (Devesa et al. 1989). Some studies have also hypothesized that ulcerative skin diseases may be stress-induced. Dippenaar et al. (2008) suggest that capture and handling procedures for researching sharks may cause an increase in parasitic infection intensity because they generally disrupt proper water ventilation in sharks, causing respiratory stress in the animal. Some scientists believe that bacterial agents are opportunistic and take advantage of sharks weakened by any physiological stress (Garner 2013). That physiological stress may also lead to increases in other pathogens, which in turn have been connected to increased bacterial infections such as fin rot. One experiment infected lemon sharks with several vibrio bacteria and found that the infections were associated with increased mortality only when the shark was physiologically compromised before infection, either by stress or other parasites

(Grimes et al. 1985). Although the exact connections between increased stress, pathogen infection, and fin rot disease remain poorly understood, it is relatively clear that there are some feedback relationships between these factors, all of which may be affected by warmer water temperatures. If warmer water temperatures do impact the health of sharks in this way, then increased global water temperatures will likely have negative impacts on shark populations which, because sharks are important apex predators, can have effects !21 on whole ecosystems that support diverse marine life and significant human food resources.

One group of pathogens that may be involved in the feedback cycles described above are monogeneans. Monogeneans are one of several important ectoparasites, organisms that typically inhabit the skin surface of their host, that will be analyzed in this study. Monogeneans are flatworms that are commonly found on the gills, skin, or fins of aquatic animals (Reed et al. 2003). They have been shown to cause increased morbidity and increased skin lesions on sharks. Two species of monogeneans that have been specifically associated with lemon sharks are Neodermophthirius harkemai and

Dermophthirius nigrelli (Cheung & Ruggieri 1983). Dermophthirius nigrelli was first described by Cheung and Ruggieri in 1983 as a parasite with an ovoid/round body

(2.3-4.00 mm long, 2.1-3.5 mm wide) and genital complexes located in the anterior of the body (Cheung & Ruggieri 1983). Neodermophthirius harkemai individuals on the other hand generally exhibit lancoleate shape and their genital complexes are invisible to the unaided eye (Young et al. 2013). These species have only been recorded on the external surfaces of lemon sharks and have been associated with disease and death of captive lemon sharks in the past (Young et al. 2013). Young et al. (2013) also found that D. nigrelli infections were most common on the first dorsal fin and were never found outside of the two dorsal fins and the upper lobe of the caudal fin, the same locations that are common for fin rot infections. Based on their data collected from wild sharks, the authors also concluded that neonates, including the lemon shark, can become infected by these parasites almost immediately after parturition. They hypothesized that future !22 research may also find that the intensity of infection likely correlates with increased density of potential hosts (Young et al. 2013).

Monogenean infections on lemon sharks, such as that caused by D. nigrelli and N. harkemai, may actually facilitate increased skin lesions and other infections by viruses and/or bacteria such as vibrios (Young et al. 2013). One study confirmed an association between monogenean infections and skin lesions in wild blacktip sharks (Carcharhinus limbatus). In that study, the lesions were not associated with bacterial infections nor did they seem to be very detrimental to the organism’s health (Bullard et al. 2000). On the other hand, there have been some species of monogenean parasites, such as D. nigrelli, that have been associated with mortality in captive sharks (Young et al. 2013).

Lemon sharks can also be infected by certain marine-dwelling isopods (Bunkley-

Williams et al. 2006). Isopods are organisms within the subphylum Crustacea that are characterized by multiple similar legs. They can parasitize many organisms both on land, in freshwater, and in saltwater and the isopods that usually parasitize fish often feed on the blood or pus that oozes from wounds caused by the isopod’s attachment. There are also some marine isopods in the Caribbean, such as Rocinella signata, that can bite humans given the opportunity.

The last parasitic parameter analyzed in this study is the presence of marine leeches on the juvenile lemon sharks. Leeches, a group of hirudinean annelid worms, generally attach to the bodily surfaces of sharks, rays, and fish (Hutson 2010). They are usually found on the fins, cloaca, gill cavities, or spiracles, attached to the host by a sucker that ingests blood by making several small lesions on the host organism. !23

According to Marancik et al. (2012), leeches increase disease risks when attached to their elasmobranch hosts. In captivity, leech infestations have been associated with anemia, hypoproteinemia, anorexia, lethargy, and sometimes death. They have also been known to carry other infections such as trypanosomes, trypanoplasmas, viruses, bacteria, and/or fungi. One species of marine leech, Branchellion torpedinis specifically attaches to elasmobranchs and its biology may therefore be used to comparatively analyze leech-host interactions found in this study (Marancik et al. 2012).

Research Outline

Sharks are extremely important organisms for the healthy functioning of most marine ecosystems. However, many gaps remain in the scientific community regarding wild shark health and methods for measuring and assessing it. In this study, I developed a procedure for assessing the health of the juvenile lemon shark population in the coastal waters of South Caicos. A traditional overall health assessment method was combined with parasitic infection analysis in order to approach shark health assessment from a variety of angles, creating a fuller view of the animal’s health. General health was assessed using traditional condition factor analysis based on length and weight measurements. Parasitic infection analysis focused on two main parameters, fin rot and ectoparasites (monogeneans, isopods, and leeches). Fin rot was quantitatively assessed using photograph surface area analysis and ectoparasite infection intensity was analyzed using established methods that focus on parasite prevalence, mean intensity, and abundance (Margolis et al. 1982). The condition factor, fin rot, and ectoparasite data !24 collected will be used to establish a baseline health status for this population. The baseline will hopefully contribute to monitoring this population’s health in the future as coastal development continues by providing insight and protection for the ecosystem diversity on this island as well as the local economic relationship with that ecosystem. !25

II. Materials & Methods

Study Area

This study was conducted at a variety of sites within the Bell Sound Nature

Reserve on South Caicos in the Turks and Caicos Islands (Fig. 3). The Bell Sound

Nature Reserve spans 11.4 km2 of marine environment off the coast of South Caicos.

This area includes extensive mangrove habitat in addition to coral reef, seagrass, and pelagic (open ocean) habitats.

Capture Method

A 100-meter monofilament gill net was set on a rotating basis at seven sites throughout the

Bell Sound Nature Reserve including Man O’War Bush, West Wall, Horse Cay, Bell Sound West,

Bell Sound East, Bonefish Hole, and Sail Rock South (Fig. 3). The nets used in this study reach about two meters deep and have 6.5 cm2 mesh size. The nets were set between two to five times a week for about three hours at a time. After setting the nets, the GPS coordinates of the net start and end points were recorded as well as the start time for the net setting. Although captured sharks were generally noticed audibly and visually, researchers performed routine checks of the net every twenty minutes to remove bycatch and/or shark captures. For those net checks, one individual walked along the net with a flashlight checking for organisms. When an organism was captured in the net, it was removed promptly and delicately by hand from the net. The shark was then walked to shore for data collection while remaining in the water to prolong proper ventilation. Data collection generally took about five minutes, but if the procedure was !26 prolonged for any reason additional artificial ventilation was performed as needed during data collection. Once all of the data were collected, the shark was released by moving it through the water to ensure proper ventilation and then releasing the animal when signs of movement returned.

General Data Collection

Data relating to shark weight and length Data Collected have been collected for about three years General Data Collected GPS Location at Capture Location beginning in February 2012 and data regarding Time of Capture Depth of Capture parasites and fin rot have been continuously General Data Collected Length (Precaudal, Fork, On Shore Total) collected beginning in February 2015, with a Weight brief hiatus due to technical difficulties such as PIT Tag ID Tissue Sample (left pelvic storms. fin)

When an organism was captured in the Sex Health Parameter Data First Dorsal Fin Photos net, it was removed immediately while another Collected On Shore Ectoparasite Observations researcher recorded the GPS location, time, and (quantity and location)

Table 1. List of data collected upon capture of a shark specimen in depth of the capture in the water (Table 1). Once the field. on shore, measurements were taken by recording length to the nearest 0.1 cm (precaudal, fork, and total lengths) using a meter stick and weight to the nearest 0.01 kg using an electronic scale and mesh weight bag. The organism was scanned for a PIT tag to determine whether it had been previously captured and the PIT tag ID was recorded. If it was a new capture, a PIT tag was inserted, the number was recorded, and a small tissue sample was taken and stored in 70% !27 ethanol solution. The tissue sample was usually snipped from the left pelvic fin using scissors and forceps. Then, the sex of the animal was recorded.

Condition Factor: Data Collection and Analysis

Data collection for condition factor analysis included the measurement of shark length

(precaudal, fork, and total lengths) and weight as described above (Table 1). The condition factor for each of the captured sharks was calculated using the following formula (CF = W/Lb) where CF is condition factor, W is the total weight of the shark, L is the total length of the shark, and b is an exponent calculated from the equation of the best fit line for the length-weight curve

(King 1995). All statistical analyses were completed using JMP Pro (Version 10, SAS Institute

Inc.). The distributions of the condition factor and total length for all captured sharks were tested for normality using the Shapiro-Wilk test. The condition factor and total length were then assessed using the Spearman-rank correlation. The distribution of condition factor values among the number of recaptures for the sharks was also analyzed using the Kruskal-Wallis and

Spearman ranks correlation tests. Correlations between shark size, shark sex, and condition factor were analyzed using the two-tailed t-test and the Spearman-rank test.

Fin Rot: Data Collection and Analysis

The fin rot on the left and right side of the first dorsal fins were photographed using a

Nikon D5000 camera with a Tamron 18-270 mm lens or a Canon G9 housed in an underwater canon housing (camera equipment depended on the research team) (Table 1). Photos of the left and right side of the first dorsal fin, for every specimen collected beginning in February 2015, !28 were analyzed using ImageJ Software (1.49v, National Institutes of Health). A surface area ratio of fin area covered with fin rot to total fin area was calculated for every photo. For each side of each fin, the average surface area ratio was calculated based off of two, three, or four photos of each specimen. The distribution of fin rot intensity among the captured sharks was assessed for normality using the Shapiro-Wilk test. A possible correlation between condition factor and fin rot intensity was analyzed using the Pearson and Spearman-Rank tests. Finally, the two-tailed t- test was used to explore possible relationships between fin rot intensity and shark sex and fin rot intensity and the two sides of the fin.

Ectoparasites: Data Collection and Analysis

The final step of data collection was examining the skin of the shark for ectoparasites

(Table 1). A specimen of each type of parasite was collected for further analysis using a scalpel and forceps and species identification was completed in the lab.

The monogenean parasite samples were stored in 4% neutral buffered formalin solution, cleared in clove oil, and analyzed using a Leica Zoom 2000 dissection microscope. An isopod was also collected using a scalpel blade and forceps, stored in a 70% ethanol solution, then analyzed using an Omax stage microscope and ScopeImage 9.0 software was used for photographs. A leech sample was also collected and stored in a vial of seawater for species identification, then it was returned to the ocean. The infection rates of parasites were analyzed according to prevalence, mean intensity, and abundance (Margolis et al. 1982). !29

III. Results

This study was conducted using gill net capture methods in order to assess the health of the coastal juvenile lemon shark population in South Caicos, Turks and Caicos Islands. A new health assessment procedure was developed by pairing traditional condition factor measurements of overall health with specific parasitic parameters for assessing heath such as the surface area coverage of fin rot on these sharks and the infection intensity, prevalence, and abundance of ectoparasites.

No significant difference in condition factor was found between shark sexes and no significant correlation was found between condition factor and shark size.

In order to assess overall shark health using traditional methods, condition factor measurements were calculated based on length and weight measurement data. Before assessing any correlations between condition factor and other variables, the condition factor data set was tested for normality and found to be non-normally distributed on a histogram (Shapiro-Wilk, n =

204, p < 0.05). Overall, the condition factor was highly variable among this population of juvenile lemon sharks. In a grouped frequency distribution histogram, the values were distributed widely throughout the categories.

However, 92% of the condition factor Figure 9. Condition factor grouped frequency distribution shows values fell in the lower half of a variable health with a potential tendency towards the lower end of the health range. !30 grouped frequency diagram for this data set indicating that the majority of captured individuals were on the lower end of this population’s condition factor range (Fig. 9). In order to ensure that this measure of health is independent and therefore not skewed by shark size or shark sex, correlations between these two factors were assessed. Statistical analysis concluded that the condition factor measurement is independent of both shark sex and shark size. A two-tailed t-test found no significant difference in condition factor between shark sexes and a Spearman-rank test found no significant correlation between shark size and condition factor (two-tailed t-test, n =

18/22, p > 0.05; Spearman-rank, n = 204, p < 0.05).

Condition factor may decrease with increased number of recaptures.

In order to explore whether shark health in this population is being negatively impacted by capture and handling procedures for research, statistical analyses were used to explore whether the condition factor of an animal Mean Condition Factor v. Number of Recaptures decreased over time with increased 0.000480 number of capture experiences. A 0.000460 Spearman-rank correlation test found no

0.000440 significant correlation (n = 49, p < 0.05).

However, there was a slight decrease in 0.000420 Mean Condition Factor Condition Mean condition factor between groups of 0.000400 1 2 3 4 sharks that had been captured once, Number of Recaptures (#) twice, three times, and four times (Fig. Figure 10. Mean condition factor for sharks recaptured once (n=44), twice (n=43), three times (n=12), and four times (n=1) shows a possible trend of decreasing 10). Furthermore, nearly half (41%) of all condition factor with increased number of recaptures. Note: only one specimen was recaptured four times and the recorded condition factor for that specimen was utilized in this graph. !31

recaptured sharks experienced a decrease in Changes(in(Condi,on(Factor(Experienced(by(Sharks( Between(Subsequent(Captures( 2%$ 2%$ condition factor between subsequent captures 2%$ 12%$ 6%$ N/A$ 8%$ (Fig. 11). Among the other recaptured Increased$ Decreased$ Decreased,$Increased$ animals, 27% experienced an increase in 27%$ Increased,$Decreased$ Increased,$No$Change$ No$Change$ condition factor, 18% experienced No$Change,$Increase$ 41%$ inconsistent changes between captures, 12% Figure 11. Percent of captured sharks that experienced an increase, decrease, no change, or inconsistent change in condition were missing data to complete these factor between recaptures over time shows the highest percentage of sharks experienced decreased condition factor between calculations, and 2% of the captured sharks captures. Note: N/A represents sharks that were missing data essential for the condition factor calculation. experienced no change in condition factor between captures (Fig. 11). The sharks that experienced an increase in condition factor between captures changed by a greater degree than the sharks that experienced a decrease in condition factor (Fig. 12).

Notably, the only specimen recaptured four times

(SFS0058) had a decrease in condition factor after every

Figure 12. Median degree of change for sharks that subsequent capture. Overall, these descriptive statistics experienced an increase (I) and decrease (D) in condition factor between recaptures shows that condition factor increases were larger on average show a trend that may reveal a relationship between than condition factor decreases between captures. decreased condition factor and increased number of recaptures in the future as continued data collection increases statistical power over time.

The intensity of fin rot is low overall and no significant correlation was found between condition factor and fin rot intensity. !32

In order to explore whether fin rot has a negative impact on shark health, the relationship between condition factor and the surface area ratio of fin rot was analyzed. Before analyzing the data set, fin rot surface area ratios were tested for normality and found to be normally distributed

(Shapiro-Wilk, n = 40, p > 0.05). A grouped frequency distribution of the amount of fin rot in each shark specimen shows that many of the sharks in this population belong in the lower categories of fin rot intensity, with 68% of the specimens in the lowest intensity category (0.00 -

0.05) and another 23% occurring in the second lowest category (0.05 - 0.10) (Fig.

13). Condition factor was analyzed for a correlation with the degree of fin rot on the sharks and no significant correlation was found using parametric analyses first, then Figure 13. Fin rot surface area ratio grouped frequency distribution shows a generally low degree of fin rot in this non-parametric analyses second (Pearson, n = population.

40, p > 0.05; Spearman-Ranks, n = 40, p >

0.05).

Fin rot surface area may increase with the number of recaptures, female sharks may experience higher intensity fin rot, and left fin surfaces may be more susceptible to fin rot.

There are many gaps in the scientific understanding of fin rot disease in sharks.

Pathogenic patterns of this disease were explored in this study by analyzing possible correlations between fin rot and the number of recaptures in order to assess whether stressful experiences impact the degree of fin rot on the animal. Possible relationships between fin rot and shark sex !33 and fin rot and fin surface side were also analyzed in order to learn more about the pathogenic patterns of this disease.

In this round of data collection, six sharks were recaptured and every one of those sharks had at least a minimal level of fin rot. Although this sample size is not large enough for statistically significant results, qualitative assessment of this pattern indicates that fin rot may correlate positively with increased recapture experiences. Two of the six recaptured sharks in this round of data collection experienced increased rot between captures. Specimen BSNR005 was first captured with absent fin rot and upon its second capture it was observed with minimal fin rot. Specimen BSNR007 was originally observed with minimal fin rot and during its second capture it was recorded with moderate fin rot.

To further understand the pathogenicity of this disease, this study also analyzed whether fin rot patterns differ between shark sex. The data revealed that fin rot frequency did not vary greatly between sexes, but the intensity of fin rot may be more extreme in female sharks than in male sharks. Of the total captured sharks, 59% were female and 41% were male (Fig. 14). When it comes to fin rot, 90% of Female all captured males and 88% of all captured 59% females exhibited a degree of fin rot. The mean Male 41% surface area ratio of fin rot for females was higher than that of males. For female sharks, the mean fin rot was 0.069 ± 0.10 and for males it was 0.031 ± Figure 14. Percent of captured female and male sharks shows that more females were captured than males. 0.03 (Fig. 15). A two-tailed t test was used to !34

0.18 analyze the degree of fin rot in 0.135 males versus females and no

0.09 significant relationship was found

Ratio 0.045 (n = 40, p > 0.05). However, 0 future analysis may support these Mean Fin Rot Surface Areaa Areaa Surface Fin Rot Mean -0.045 preliminary trends with increased Female Male Figure 15. Mean fin rot surface area ratio (± SD) for female and male captured sharks shows that females may generally power from continuous collection of experience more intense fin rot than males. data.

Many sharks were observed with highly varied fin rot on either side of the same fin, as exhibited by specimen BSNR023 (Fig. 16). The mean fin rot surface area ratio on the left fin side in this population was 0.067 ± 0.10 and on the right side it was 0.037 ±

0.040. A two-tailed t-test was used to analyze the mean fin rot intensity on the right versus left side of the fin and no significant difference was found (n = 20, p > 0.05).

Left Right

Figure 16. Specimen BSNR023 exhibiting highly variable fin rot between the two sides of the same first dorsal fin.

Certain parasites may increase the prevalence of other parasites, and some parasites are found in specific regions of a shark. !35

The fins of all captured sharks were visually inspected for ectoparasites during this study. The data were analyzed for the prevalence, mean intensity, and abundance of each of the parasites found. Leeches were the most prevalent and most abundant parasite observed, but monogenean flatworms exhibited the highest mean intensity of infection

(Table 2).

Stibarobdella macrothela Dermophthirius nigrelli Rocinella signata

Prevalence (%) 24.1 13.8 3.45

Mean Intensity 1.14 1.25 1.00

Abundance 0.276 0.172 0.0345

Table 2. Prevalence, Mean Intensity, and Abundance of Parasites: Parameters for analyzing the infection rates and intensity of leeches (S. Macrothela), monogenean flatworms (D. nigrelli), and isopods (R. signata). The monogenean flatworm parasite infecting the individuals of this population was identified as Dermophthirius nigrelli (Fig. 17). D. nigrelli were found only on fins with visible fin rot and every one of the specimens were located specifically within the area of the fin experiencing the rot. It is also notable that the only shark specimen that was observed with more than one flatworm was also one of the two captured sharks experiencing highly extensive fin rot that had reached the phase of hemorrhagic ulcers on the Figure 17. Two Dermophthirius nigrelli monogenean parasites (BSNR023). skin (BSNR023).

The species of leech collected from this population was identified as

Stibarobdella macrothela (Fig. 18). Every recorded observation of leeches also occurred on shark specimens exhibiting at least minimal fin rot, although the leeches were always !36 observed on the anal fin rather than on the specific area of the first dorsal fin experiencing fin rot.

One isopod, Rocinella signata (Fig. 19), was collected from the cloaca of a captured shark experiencing moderate fin rot Figure 18. One Stibarobdella macrothela leech individual collected from BSNR024. (BSNR013). Several other isopods were A B observed, but they were unattached to the shark specimen and were therefore not recorded.

C

Figure 19. Front (A) and back (B) view of one Rocinella signata isopod collected from BSNR013. Note the distinctive half-moon pattern located towards the posterior end of the specimen which is helpful for species identification (C). !37

IV. Discussion

The goal of this study was to develop a procedure for assessing the health status of juvenile lemon sharks and to establish a baseline health status for the juvenile lemon shark population off the coast of South Caicos in the Turks and Caicos Islands. Gill nets were used to collect data regarding a variety of health parameters such as traditional condition factor, fin rot infections, and ectoparasites.

Condition Factor

Condition factor is a general assessment of an organism’s health based on length and weight measurements for that individual. Overall, I found that the condition factor of individuals in this population is variable. That variability may be attributed to the fact that condition factor values can be heavily influenced by the time elapsed since the shark’s last feeding (weight) and by the variability in length measurements caused by multiple research personnel taking the measurements. Otherwise, it is possible that the sharks in this population have a wide range of individual health. If that is the case, it would likely indicate that no environmental factor is currently impacting the health of this population in a major way, which would be possible for this geographic region because it has incurred minimal coastal development. Although the condition factor data showed variation, the majority of the values fell in the lower half of the overall range indicating that the individuals in this population may be leaning towards the lower end of the population’s health range (Fig. 9). It will be beneficial to continue observing this trend in the future as data collection continues. !38

In order to ensure that the condition factor measurement is a reliable method of health assessment, I analyzed shark length and sex in relation to the condition factor data set in order to confirm that the condition factor values were not skewed by independent variables. I found no significant correlation between the condition factor and shark size

(total length) thereby reducing the possibility that any discovered condition factor correlations may simply be due to growth over time. I also found no relationship between the sex of the shark and the measured condition factor indicating that this health assessment method is also not significantly skewed by shark sex. Therefore, I decided to retain the condition factor measurement as one piece of the overall health assessment procedure developed for this population.

There was also no significant correlation found between condition factor and the number of times a shark had been recaptured, suggesting that gill net capture and handling procedures do not negatively impact the shark post-release in this study. In addition to standard gill netting procedures, the methodology used in this study included artificial ventilation throughout the handling process, a method recommended by Brooks et al. (2011) to reduce handling stress. This methodological step may be partly responsible for the seemingly low impact on the shark’s health post-handling and capture.

Based on the results from this study, I recommend that shark handling studies in the future incorporate artificial ventilation.

Most physiological stress incurred by capture and handling is related to decreased blood pH as a result of stress which leads to respiratory and metabolic acidosis (Hyatt et al. 2012). Lemon sharks have been specifically identified as having low sensitivity to the !39 stresses of gill net capture and this study supports that claim (Hyatt et al. 2012). Lemon sharks seem to have a very robust compensatory response for acidosis, which may be the reason that they seem unaffected by the stress of gill net capture and research handling in this study. These findings suggest that lemon sharks are an ideal species choice for elasmobranch research involving gill nets in the future.

Although there was no significant statistical correlation between condition factor and recapture frequency, the data did show a slight decrease in mean condition factor between subsequent recaptures of sharks (Fig. 10). Furthermore, 41% of the captured sharks experienced a decrease in condition factor between captures and the only shark that was recaptured four times showed a decrease in condition factor between every subsequent capture (Fig. 11). These patterns may be due to chance or other variables, but further research with a larger sample size is recommended to elucidate any potentially emerging trends.

Fin Rot

I also chose to analyze the presence of fin rot disease on these sharks in order to establish a more complete perspective on the health of this population. To explore fin rot in this study, the first dorsal fin of each shark was photographed and the fin rot surface area coverage on the fin was measured using photo analysis software. Almost all of the individuals in this population are infected with some degree of fin rot, but the majority of the sharks do not have extensive fin rot with higher surface area ratios and/or hemorrhagic ulcers that would indicate an extensive progression of the disease (Fig. 11). !40

This fin rot baseline indicates that the population is in moderately good health, a finding that supports the conclusions drawn from condition factor analysis discussed above.

There have been contradicting findings regarding the impact of fin rot on the overall health of sharks. For example, Bullard et al. concluded in 2000 that blacktip reef sharks are unaffected by fin rot infections. However, Young et al. produced a contradicting study in 2012 claiming that the infection may become lethal. No significant correlation between general condition factor and the degree of fin rot was found in this study, indicating that fin rot may not have a noticeable impact on the shark’s overall health. However, there is inconclusive evidence regarding the reverse relationship which is whether fin rot will reflect other changes in the population’s health that are not characterized by shifts in weight or size. It is plausible that a change in fin rot prevalence and/or intensity would reflect other negative health changes in the future. Therefore, I believe it is beneficial to retain and utilize fin rot analysis in this health assessment procedure as a point of reference for the continued monitoring of this population’s health and as an additional evaluation of the overall health to ensure accuracy by comparison with condition factor results.

There is also disagreement among scientists regarding the disease-causing agent for elasmobranch fin rot. Previous studies regarding lemon shark health have associated fin rot with Vibrio spp. (V. harveyii and/or V. carchariae) bacterial infections and it is therefore likely that the rot observed in this study is also caused by the same bacteria

(Hashem & El-Barbary 2013). To confirm that hypothesis, further molecular analyses !41 would be required and may provide an area of research for future scientists studying this population.

As mentioned above, some studies have found fin rot to be harmless while others have concluded that it can be fatal (Bullard et al. 2000; Young et al. 2012). One study found V. carchariae, a probable fin rot causative agent, to be harmless on healthy lemon sharks, but when the infection was experienced by a physiologically weakened lemon shark the bacteria were lethal (Grimes et al. 2006). This means that it is possible that the physiological stress incurred by gill net capture and research handling could potentially contribute to increased V. carchariae infection intensity and therefore increased fin rot.

In this round of data collection, two out of six recaptured sharks exhibited increased fin rot between recaptures. One of those two specimens developed the first stages of fin rot after its first capture. The other specimen had a minimal amount of fin rot when it was first captured and when it was captured a second time it had significantly increased fin rot intensity. Although no statistically significant difference was found for these data, it is worth noting that the data may support the hypothesis that fin rot is intensified by the stress experienced by repeated captures. I intend to further explore this potential correlation in the future with a larger sample size of recaptured sharks with fin rot data.

The procedure for exploring this potential trend would be improved by adding the date and time of capture to the data collection step.

The findings from this study also suggest that female juvenile lemon sharks are more susceptible to increased fin rot intensity than male juvenile lemon sharks. The data revealed that the mean female fin rot surface area ratio was higher than that of males !42

(Fig. 15). However, fin rot was not necessarily observed more frequently on females than on males. It is possible that this finding may be skewed by the fact that slightly more females were captured than males in this study (59% of captures were female, 41% of captures were male) (Fig. 14). Therefore, it would be beneficial for future research to focus on whether there is a significant correlation between fin rot intensity and sex in lemon shark populations.

Another interesting fin rot pattern observed in this study was the variable intensity of fin rot on each side of a single fin (Fig. 16). One hypothesis is that the side with more extensive rot happened to also be infected by monogeneans, intensifying the rot on one side of the fin. Otherwise, it is possible that this study may elucidate an infection intensity trend in relation to the side of the shark. This observation has not been previously reported in other publications and it would therefore be very beneficial to further explore this emerging pattern. It would be interesting for future research to determine whether there is a specific side of the fin that is more commonly infected with more intensive fin rot.

Ectoparasites

Previous studies have reported that fin rot disease is likely intensified by concurrent monogenean infections (Grimes et al. 2006). Lemon sharks that were dually infected by D. nigrellii monogeneans and fin rot in this study provided evidence that D. nigrellii may increase fin rot intensity by acting as a vector for fin rot bacteria (likely

Vibrio spp.). If these monogeneans do not act as a vector, another hypothesis is that !43 ectoparasites or fin rot bacterial infections weaken the immune system of the shark, thereby facilitating the infection of the animal with other parasites. Observations made in this study, such as the repeated dual infection of monogeneans and fin rot on the sharks, support the existing hypothesis that D. nigrelli monogeneans facilitate fin rot disease caused by Vibrio spp. bacteria (Grimes et al. 2006). The fact that every monogenean was located within the fin area experiencing fin rot and the only record of multiple monogeneans on one specimen in this study was recorded on a shark with extensive fin rot further supports that claim, but further research would be essential to clarify the interconnected relationships between parasitic infections experienced by elasmobranchs.

Although the data are limited regarding other ectoparasites in this study, it is worth noting that all other ectoparasites recorded were also only located on lemon sharks experiencing some extent of fin rot. This observation further supports the interconnectedness of parasitic infections in elasmobranchs. Further research regarding potential correlations between fin rot, monogeneans, marine leeches, and isopods may provide further insight into the seemingly interconnected relationships between parasites and elasmobranch susceptibility as a host.

Many R. signata isopods were observed swarming non-target marine organisms, or bycatch, caught in the gill net as well as crawling over recently captured sharks. These ectoparasites were not recorded in the parasite counts because they were not attached to the shark’s body. However, this observation may indicate that the R. signata isopod exhibits micro-predatory behavior towards marine species, rather than purely parasitic !44 interactions. This would be an interesting finding for elasmobranch parasitology because that behavior is relatively unexplored in elasmobranchs and sharks in particular.

Overall, the infection rates of the parasites observed in this study were relatively low compared to several other studies reporting the same parameters for similar parasitic groups (Table 1) (Henderson & Dunne 2001; Henderson et al. 2002 ; McKiernan et al.

2004; Henderson et al. 2010). Within the study conducted by Henderson & Dunne

(2001), the parameters for infection on the first dorsal fin were established and generally much higher than that found in this study, indicating that this habitat is not optimal for these parasites or this population is not particularly susceptible to their infections.

Host-parasite interactions can be affected by increased anthropogenic pollutants, which can be a result of increased coastal development (Adlard et al. 2015). The low rates of infection intensity established in this study may play a vital role as a baseline for infection rates in this area for future studies as coastal development continues, potentially increasing anthropogenic pollutants in the area. !45

Conclusion

In this study, I developed a health assessment procedure that combines condition factor, fin rot, and ectoparasite health parameters to accomplish a more complete evaluation of wild shark health. I also observed several interesting pathogenic trends for fin rot infections and ectoparasites that will be further explored in the future. I successfully established a baseline health status for the juvenile lemon shark population in the coastal waters of South Caicos in the Turks and Caicos Islands and the baseline indicates that this population is relatively healthy overall. The newly developed procedure described in this study will be used to continuously monitor this population in the future in order to provide insight into the health of this population as South Caicos continues to develop economically.

Human beings cannot survive without a healthy ocean. Healthy shark populations are essential for healthy oceans. It is therefore our responsibility to take care of our oceans and the creatures living within them. Learning about the health of marine organisms is one important way to contribute because it can help guide conservation goals and public understanding of these animals. There are many other ways to do your part in maintaining the health of our oceans and it is within every individual’s power to do so.

It is my hope that this study will continue to contribute to the growing understanding of elasmobranch health and that my data will help inform future developments on South Caicos to motivate them towards sustainable plans that are compatible with shark health and ocean conservation. !46

References

Adeogun, A. O., Ibor, O. R., Onoja, A. B., & Arukwe, A. (2016). Fish condition factor, peroxisome proliferator activated receptors and biotransformation responses in Sarotherodon melanotheron from a contaminated freshwater dam (Awba Dam) in Ibadan, Nigeria. Mar Environ Res. doi:10.1016/j.marenvres.2016.02.002

Adeogun, A. O., Ibor, O. R., Regoli, F., & Arukwe, A. (2016). Peroxisome proliferator-activated receptors and biotransformation responses in relation to condition factor and contaminant burden in tilapia species from Ogun River, Nigeria. Comp Biochem Physiol C Toxicol Pharmacol, 183-184, 7-19. doi:10.1016/j.cbpc.2015.12.006

Adlard, R. D., Miller, T. L., & Smit, N. J. (2015). The butterfly effect: parasite diversity, environment and emerging disease in aquatic wildlife. Trends in Parasitology, 31(4), 160-166.

Affairs, M. o. E. a. H. (2014). National review (Turks and Caicos Islands) by the division for gender affairs of economic commission for latin america and the caribbean on way to Beijing. Retrieved from

Austin, B. (2010). Vibrios as causal agents of zoonoses. Vet Microbiol, 140(3-4), 310-317. doi: 10.1016/j.vetmic.2009.03.015

Australian Fisheries Management Authority (AFMA). Gillnets. Retrieved from http:// www.afma.gov.au/portfolio-item/gillnets/.

Basusta, A., Basusta, N., Calta, M., Ozer, E. I., & Girgin, H. (2013). Length-Weight Relationship and Condition Factor of Spiny Gurnard (Lepidotrigla dieuzeidei Blann and Hureau, 1973) Inhabiting Northeast Mediterranean Sea. Journal of Animal and Veterinary Advances, 12(2), 212-214.

Brooks, E. J., Mandelman, J. W., Sloman, K. A., Liss, S., Danylchuk, A. J., Cooke, S. J., . . . Suski, C. D. (2012). The physiological response of the Caribbean reef shark (Carcharhinus perezi) to longline capture. Comp Biochem Physiol A Mol Integr Physiol, 162(2), 94-100. doi:10.1016/j.cbpa.2011.04.012 !47

Brooks, E. J., Sloman, K. A., Liss, S., Hassan-Hassanein, L., Danylchuk, A. J., Cooke, S. J., . . . Suski, C. D. (2011). The stress physiology of extended duration tonic immobility in the juvenile lemon shark, Negaprion brevirostris (Poey 1868). Journal of Experimental Marine Biology and Ecology, 409(1-2), 351-360. doi:10.1016/j.jembe.2011.09.017

Bryden, J. M. (1973). Tourism and Development: A Case Study of the Commonwealth Caribbean. London: Cambridge University Press.

Bullard, S. T., Frasca, S., Jr., & Benz, G. W. (2000). Skin lesions caused by Dermophthirius penneri (Monogenea: Microbothriidae) on wild- caught blacktip sharks (Carcharhinus limbatus). Journal of Parasitology, 86, 618-622.

Bunkley-Williams, L., Williams, E. H., & Bashirullah, A. K. M. (2006). Isopods (Isopoda: Aegidae, Cymothoidae, Gnathiidae) associated with Venezuelan marine fishes (Elasmobranchii, Actinopterygii). Revista de Biologia Tropical, 54(3), 175-188.

Caira, J. (2001). Age of association between the , Ginglymostoma cirratum, and tapeworms of the genusPedibothrium (Tetraphyllidea: Onchobothriidae): implications from geography. Biological Journal of the Linnean Society, 72(4), 609-614. doi:10.1006/ bijl.2001.0521

Caira, J. N., Mega, J., & Ruhnke, T. R. (2005). An unusual blood sequestering tapeworm (Sanguilevator yearsleyi n. gen., n. sp.) from Borneo with description of Cathetocephalus resendezi n. sp. from Mexico and molecular support for the recognition of the order Cathetocephalidea (Platyhelminthes: Eucestoda). Int J Parasitol, 35(10), 1135-1152. doi: 10.1016/j.ijpara.2005.03.014

Camhi, M., Valenti, S., Fordham, S., Fowler, S., & Gibson, C. (2009). The conservation status of pelagic sharks and rays: Report of the IUCN shark specialist group pelagic shark red list workshop. IUCN Species Survival Commission Shark Specialist Group, 1-78.

Carleton, C., & J., H. (2006). Review and re-assessment of the TCI protected area system.

Cheung, P. T., & Ruggieri, G. D. (1983). Dermophthirius nigrelli n. sp. (Monogenea: Microbothriidae), an Ectoparasite from the Skin of the Lemon Shark, Negaprion brevirostris. Transactions of the American Microscopical Society, 102(2), 129-134. !48

Compagno, L. J. V. (1984). Sharks of the World. FAO Species Catalog, 4.

Coral Reefs of the United Kingdom Overseas Territories. (2013). (C. R. C. Sheppard Ed. Vol. 4): Springer.

Cortés, E., & Gruber, S. H. (1990). Diet, Feeding Habits and Estimates of Daily Ration of Young Lemon Sharks, Negaprion brevirostris (Poey). American Society of Ichthyologists and Herpetologists, 1990(1), 204-218.

Cresset, R. F. (1970). Copepods Parastic on Sharks from the West Coast of Florida (Vol. 38). Washingtong, D.C.: Smithsonian Institution Press.

Devesa, A., Barja, J. L., & Toranzo, A. E. (1989). Ulcerative skin and fin lesions in reared turbot, Scophthalmus maximus (L.). Journal of Fish Diseases, 12, 323-333.

Dibattista, J. D., Feldheim, K. A., Gruber, S. H., & Hendry, A. P. (2007). When bigger is not better: selection against large size, high condition and fast growth in juvenile lemon sharks. J Evol Biol, 20(1), 201-212. doi:10.1111/j.1420-9101.2006.01210.x

DiGirolamo, A. L., Gruber, S. H., Pomory, C., & Bennett, W. A. (2012). Diel temperature patterns of juvenile lemon sharks Negaprion brevirostris, in a shallow-water nursery. J Fish Biol, 80(5), 1436-1448. doi:10.1111/j.1095-8649.2012.03263.x

Dippenaar, S. M., van Tonder, R. C., & Wintner, S. P. (2008). Is there evidence of niche restriction in the spatial distribution of Kroyeria dispar Wilson, 1935, K. papillipes Wilson, 1932 and Eudactylina pusilla Cressey, 1967 (Copepoda: Siphonostomatoida) on the gill filaments of tiger sharks Galeocerdo cuvier off KwaZulu-Natal, South Africa? Hydrobiologia, 619(1), 89-101. doi:10.1007/s10750-008-9602-y

Fazli, H., Daryanabard, G., Salmanmahiny, A., Abdolmaleki, S., Bandani, G., & Afraei-Bandpei, M. A. (2012). Fingerling release program, biomass trend and evolution of the condition factor of Caspian Kutum during the 1991-2011 period. Cybium, 36(4), 545-550. !49

Feldheim, K. A., Gruber, S. H., & Ashley, M. V. (2002). The breeding biology of lemon sharks at a tropical nursery lagoon. Proc Biol Sci, 269(1501), 1655-1661. doi:10.1098/rspb. 2002.2051

Frick, L. H., Gallagher, A. J., Bushnell, P. G., Brill, R. W., & Mandelman, J. W. (2010). Blood gas, oxygen saturation, pH, and lactate values in elasmobranch blood measured with a commercially available portable clinical analyzer and standard laboratory instruments. Journal of Aquatic Animal Health, 22, 229-234.

Gallagher, A. J., Vianna, G. M. S., Papastamatiou, Y. P., Macdonald, C., Guttridge, T. L., & Hammerschlag, N. (2015). Biological effects, conservation potential, and research priorities of shark diving tourism. Biological Conservation, 184, 365-379. doi:10.1016/ j.biocon.2015.02.007

Gallucci, V. F., Taylor, I. G., & Erzini, K. (2006). Conservation and management of exploited shark populations based on reproductive value. Canadian Journal of Fisheries and Aquatic Sciences, 63, 931-942.

Garner, M. M. (2013). A retrospective study of disease in elasmobranchs. Vet Pathol, 50(3), 377-389. doi:10.1177/0300985813482147

Grimes, D. J., Gruber, S. H., & May, E. B. (1985). Experimental-Infection of Lemon Sharks, Negaprion-Brevirostris (Poey), with Vibrio Species. Journal of Fish Diseases, 8(2), 173-180. doi:DOI 10.1111/j.1365-2761.1985.tb01212.x

Grimes, D. J., Stemmler, J., Hada, H., May, E. B., Maneval, D., Hetrick, F. M., . . . Colwell, R. R. (1984). Vibrio Species Associated with Mortality of Sharks Held in Captivity. Microbial Ecology, 10(3), 271-282.

Gruber, S. H. (1982). Role of the lemon shark, Negaprion brevirostris (Poey) as a predator in the tropical marine environment: A multidisciplinary study. Florida Scientist, 45, 46-75.

Guttridge, T. L., Gruber, S. H., Gledhill, K. S., Croft, D. P., Sims, D. W., & Krause, J. (2009). Social preferences of juvenile lemon sharks, Negaprion brevirostris. Animal Behaviour, 78(2), 543-548. doi:10.1016/j.anbehav.2009.06.009 !50

Haman, K. H., Norton, T. M., Thomas, A. C., Dove, A. D., & Tseng, F. (2012). Baseline health parameters and species comparisons among free-ranging Atlantic sharpnose ( terraenovae), bonnethead (Sphyrna tiburo), and (Squalus acanthias) sharks in Georgia, Florida, and Washington, USA. J Wildl Dis, 48(2), 295-306. doi:10.7589/0090-3558-48.2.295

Harms, C. A. (1996). Treatments for Parasitic Diseases of Aquarium and Ornamental Fish. Seminars in Avian and Exotic Pet Medicine, 5(2), 54-63.

Haughton, M., Siar, S. V., & Tietze, U. (2006). Socio-economic indicators in integrated coastal zone and community-based fisheries management: case studies from the Caribbean (Vol. 491). Rome: Food and Agriculture Organization of the United Nations Fisheries Techinical Paper.

Henderson, A. C., & Dunne, J. (2001). The distribution of the microbothrid shark parasite Leptocotyle minor on its host, the lesser-spotted dogfish canicula. Biology and Environment, 101B(3), 251-253.

Henderson, A. C., Flannery, K., & Dunne, J. (2002). Parasites of the (Prionace glauca L.), in the north-east atlantic ocean. Journal of Natural History, 36, 1995-2004.

Henderson, A. C., McClellan, K., & Calosso, M. (2010). Preliminary assessment of a possible lemon shark nursery in the Turks & Caicos Islands, British West Indies. Caribbean Journal of Science, 46(1), 29-38.

History, F. M. o. N. (2016). Statistics on Attacking Species of Shark. International Shark Attack File. Retrieved from https://www.flmnh.ufl.edu/fish/isaf/contributing-factors/species- implicated-attacks/

Huepel, M. R., Carlson, J. K., & Simpfendorfer, C. A. (2007). Shark nursery areas: concepts, definition, characterization and assumptions. Marine Ecology Progress Series, 337, 287-297.

Humann, P., & DeLoach, N. (2014). Reef Fish Identification: Florida, Caribbean, Bahamas (4 ed.). Florida: New World Publications, Inc. !51

Hutson, K. S. (2010). Marine Leeches. In J. C. U. S. o. M. T. Biology (Ed.). Queensland, Australia: James Cook Univesity.

Hyatt, M. W., Anderson, P. A., O'Donnell, P. M., & Berzins, I. K. (2012). Assessment of acid- base derangements among bonnethead (Sphyrna tiburo), bull (Carcharhinus leucas), and lemon (Negaprion brevirostris) sharks from gillnet and longline capture and handling methods. Comp Biochem Physiol A Mol Integr Physiol, 162(2), 113-120. doi:10.1016/ j.cbpa.2011.05.004

IUCN. (2015). Negaprion brevirostris. Red List of Threatened Species. Retrieved from http:// www.iucnredlist.org/details/39380/0

Kensley, B., & Schotte, M. (1989). Guide to the Marine Isopod Crustaceans of the Caribbean. Washington, D.C.: Smithsonian Institution Press.

King, M. (1995). Fisheries biology, assessment and management. Oxford: Fishing News Books.

Kinney, M. J., & Simpfendorfer, C. A. (2009). Reassessing the value of nursery areas to shark conservation and management. Conservation Letters, 2(2), 53-60. doi:10.1111/j. 1755-263X.2008.00046.x

Laegdsgaard, P., & Johnson, C. (2001). Why do juvenile fish utilise mangrove habitats? Journal of Experimental Marine Biology and Ecology, 257, 229-253.

Hashem, M., & El-Barbary, M. (2013). Vibrio harveyi infection in Arabian surgeon fish (Acanthurus sohal) of red sea at Hurghada, Egypt. Egyptian Journal of Aquatic Research, 39(3), 199-203.

Marancik, D. P., Leary, J. H., Fast, M. M., Flajnik, M. F., & Camus, A. C. (2012). Humoral response of captive zebra sharks Stegostoma fasciatum to salivary gland proteins of the leech Branchellion torpedinis. Fish Shellfish Immunol, 33(4), 1000-1007. doi:10.1016/ j.fsi.2012.08.020 !52

Margolis, L., Esch, G. W., Holmes, J. C., Kuris, A. M., & Schad, G. A. (1982). The Use of Ecological Terms in Parasitology (Report of an Ad Hoc Committee of the American Society of Parasitologists). The American Society of Parasitologists, 68(1), 131-133.

MarineBio. (2015). Lemon Sharks. from MarineBio Conservation Society http://marinebio.org/ species.asp?id=490&arubalp=ce45767b-5400-4382-9956-08cf379adb

Mckiernan, J. P., Grutter, A. S., & Davies, A. J. (2004). Reproductive and feeding ecology of the parasitic gnathiid isopods of epaulette sharks ( ocellatum) with consideration of their role in the transmission of a haemogregarine. Int J Parasitol, 35(19-27).

Morrisey, J. F., & Gruber, S. H. (1993). Habitat selection by juvenile lemon sharks, Negaprion brevirostris. Environmental Biology Fishes, 38, 311-319.

Morgan, A. (2015). Lemon Shark. Retrieved from https://www.flmnh.ufl.edu/fish/discover/ species-profiles/negaprion-brevirostris/

Motta, P. J., Tricas, T. C., Hueter, R. E., & Summers, A. P. (1997). Feeding mechanism and function morphology of the jaws of the lemon shark, Negaprion brevirostris (Chondrichthyes, Carcharhinidae). The Journal of Experimental Biology, 200, 2765-2780.

National Oceanic and Atmospheric Administration (NOAA). (2014). Gillnets: Fishing Gear and Risks to Protected Species. Retrieved from http://www.nmfs.noaa.gov/pr/interactions/ gear/gillnet.htm

Olson, P. D., Caira, J. N., Jensen, K., Overstreet, R. M., Palm, H. W., & Beveridge, I. (2010). Evolution of the trypanorhynch tapeworms: parasite phylogeny supports independent lineages of sharks and rays. Int J Parasitol, 40(2), 223-242. doi:10.1016/j.ijpara. 2009.07.012

Orth, R. J., Carruthers, T. J. B., Dennison, W. C., Duarte, C. M., Fourqurean, J. W., Heck Jr., K. L., . . . Williams, S. L. (2006). A global crisis for seagrass ecosystems. Bioscience, 56(12), 987-996. !53

Pitman, F. W. (1917). The Development of the British West Indies, 1700-1763 (Vol. 4). New Haven: Yale University Press.

Reed, P., Francis-Floyd, R., & Klinger, R. E. (2003). Monogenean Parasites of Fish. University of Florida Institute of Food and Agricultural Sciences, 1-4.

Richards, J. G., Heigenhauser, G. J., & Wood, C. M. (2003). Exercise and recovery metabolism in the Pacific spiny dogfish (Squalus acanthias). J Comp Physiol B, 173(6), 463-474. doi: 10.1007/s00360-003-0354-8

Sailrock. (2015). National Parks Reserves. Retrieved from http://www.southcaicos.com/south- caicos/national-parks-reserves

Sarkar, U. K., Khan, G. E., Dabas, A., Pathak, A. K., Mir, J. I., Rebello, S. C., . . . Singh, S. P. (2013). Length weight relationship and condition factor of selected freshwater fish species found in River Ganga, Gomti and Rapti, India. Journal of Environmental Biology, 34, 951-956.

Sundström, L. F., Gruber, S. H., Clermont, S. M., Correia, J. P. S., C., d. M. J. R., Morrisey, J. F., . . . Oliveira, M. T. (2001). Review of elasmobranch behavioral studies using ultrasonic telemetry with special reference to the lemon shark, Negaprion brevirostris, around Bimini Islands, Bahamas. Environmental Biology, 60, 225-250.

SharkTrust. (2016). Longlining. Shark Conservation. Retrieved from http://www.sharktrust.org/ en/longlining.

Valiela, I., Bowen, J. L., & York, J. K. (2001). Mangrove forests: one of the world’s threatened major tropical environments. Bioscience, 51(10), 807-815.

Williams, E. H. Isopods as Parasites or Associates of Fishes.

WorldAtlas. (2015). Turks and Caicos. Retrieved from http://www.worldatlas.com/webimage/ countrys/namerica/caribb/tc.htm !54

Young, J. M., Frasca, S., Jr., Gruber, S. H., & Benz, G. W. (2013). Monogenoid infection of neonatal and older juvenile lemon sharks, Negaprion brevirostris (Carcharhinidae), in a shark nursery. J Parasitol, 99(6), 1151-1154. doi:10.1645/GE-3250.1