Copyright by Colt William Cook 2012

The Thesis Committee for Colt William Cook certifies that this is the approved version of the following thesis:

The early life history and reproductive biology of Cymothoa excisa,

a marine isopod parasitizing Atlantic croaker,

(Micropogonias undulatus), along the Texas coast

APPROVED BY SUPERVISING COMMITTEE:

Supervisor: Pablo Munguia

Ed Buskey

______Benjamin Walther

The early life history and reproductive biology of Cymothoa excisa,

a marine isopod parasitizing Atlantic croaker,

(Micropogonias undulatus), along the Texas coast

by

Colt William Cook, B.S. Bio.

Thesis Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

Master of Science in Marine Science

The University of Texas at Austin August 2012 Dedication

This thesis is dedicated to my mother and grandmother who have always been there to help me since I was young. Thank you for all of your support.

Acknowledgements

I would like to acknowledge everyone who has helped me throughout this process, including my mother, grandmother, sister, girlfriend and other family and friends. I would also like to thank my advisor Pablo Munguia, Zachary Darnell, Catalina Cuellar and Justin Crow for their help and input during field collections, experimentation and comments on the manuscript. I would like to thank the UTMSI statistics group for their help on experimental design and statistical analyses. I would also like to thank the

Houston Big Game Fishing Club for funding and Georgia Neblett for securing funding sources. Lastly, we would like to thank Stan Dignum and the crew of the R/V Katy for assisting with the sampling.

v Abstract

The early life history and reproductive biology of Cymothoa excisa, a marine isopod parasitizing Atlantic croaker, (Micropogonias undulatus), along the Texas coast

Colt William Cook, M.S.MarineSci The University of Texas at Austin, 2012

Supervisor: Pablo Munguia

Parasite population dynamics and the evolution of life history characteristics are strongly correlated with the processes of host infection, survival within a host and reproduction, with each process posing a challenge to the parasitic lifestyle.

Macroparasites living in marine environments have evolved extreme changes in physiology, morphology and life history traits to overcome these challenges. This study focused on the infective and reproductive stage of the parasitic isopod, Cymothoa excisa, a common parasite on Atlantic croaker, (Micropogonias undulatus), along the Texas coast. A two year survey identified infection rates and the relationship between fish density and size and parasite load, size and fecundity. Isopod morphology was quantified for each life stage, identifying shape transitions through ontogeny and sex change. Sex change in C. excisa was found to be driven by the absence of conspecific parasites within a host and sex change only occurred in the first individual to arrive. To understand the vi infective stage of C. excisa parasite energetics and host detection mechanisms were tested. Parasites with free-living life stages have a narrow window to infect a host and have evolved a number of mechanisms to detect and locate a host. I used a series of energetic experiments to determine an infection window for free-swimming larvae

(mancae) and behavioral response experiments testing both visual and chemical cues associated with host detection. Mancae were found to have a narrow infection window, where mancae began searching for a host as soon as they are born, but quickly switch to an ambush strategy to conserve energy. Mancae were also found to be responsive to both visual and chemical cues from its common fish host, as well as a non-host fish, indicating that chemical cues are used in host detection, but chemical specificity is not a mechanism that C. excisa uses to find its common host. The results from this study have implications to parasitic species and their hosts, as well as to other areas of study, including population and ecosystem dynamics.

vii Table of Contents

List of Tables ...... ix

List of Figures ...... x

INTRODUCTION...... 1

Chapter 1: Conspecific density-dependence drives sex change in a parasitic isopod ...... 4 Introduction ...... 4 Materials and Methods ...... 7 Results ...... 10 Discussion ...... 13

Chapter 2: Sensory cues associated with host detection in a marine parasitic isopod ...... 19 Introduction ...... 19 Materials and Methods ...... 23 Results ...... 29 Discussion ...... 31

CONCLUSION ...... 36

Tables ...... 39

Figures...... 41

Appendix 1 ...... 52

Appendix 2 ...... 53

Appendix 3 ...... 54

References ...... 55

Vita… ...... 71

viii List of Tables

Table 1: Isopod Morphology Discriminant Function Analysis (DFA) ...... 39

Table 2: Results from Chemical and Visual Cues ...... 40

ix List of Figures

Figure 1: Seasonal Infection Trends ...... 41

Figure 2: Isopod Location and Frequency ...... 42

Figure 3: Isopod Size by Fish Size ...... 43

Figure 4: Isopod Brood Size by Female Size ...... 44

Figure 5: Discriminant Function Analysis ...... 45

Figure 6: Isopod Morphology by Life Stage ...... 46

Figure 7: Isopod Gonopod Size by Isopod Size ...... 47

Figure 8: Mancae Energetics ...... 48

Figure 9: Mancae Behavior to Chemical Cues ...... 49

Figure 10: Mancae Taxis to Chemical Cues...... 50

Figure 11: Mancae Behavior to Chemical and Visual Cues...... 51

x Introduction

Parasitism is extremely common in nature and is one factor that can contribute significantly to ecosystem stability and diversity. Although most parasites are small in body size they have been found to account for a substantial amount of biomass within an ecosystem, which can lead to strong effects on community structure, composition, and energy flow. Due to the strong interaction of parasites within a community they can influence both their host population as well as other populations within the system.

Understanding how parasites influence populations within an ecosystem first requires understanding the population dynamics of the parasite. Both the evolution of parasite life history traits and parasite population dynamics are strongly affected by the ability to find and infect new hosts and reproduce within the host. The first step in identifying parasite population dynamics is understanding the reproductive system in play and the mechanisms by which parasites locate and infect a host.

Cymothoid parasites are a large family of ectoparasitic isopods that infect a diversity of fish from a wide range of climates. Although the family is widespread and has been found to infect a number of economically and ecologically important fish species, little is known about the life history characteristics of this family. This study focuses on Cymothoa excisa, a cymothoid parasite that infects Atlantic croaker,

(Micropogonias undulatus), along the Texas coast. Cymothoa excisa infects the buccal cavity of Atlantic croaker and utilizes the fish for growth and reproduction. Cymothoa excisa reproduces sexually within the host and bears free-swimming larvae (mancae) that

1 begin searching for a potential host immediately after birth. Previous research on cymothoid isopods has focused on cataloging seasonal infection rates, and identifying potential fish hosts, but little information exists on the mechanisms of host detection and the reproductive biology of cymothoid isopods.

Chapter one focuses on identifying the infection dynamics and the reproductive biology of C. excisa. A two year survey was used to identify infection rates and the relationship between fish density and size and parasite load, size and fecundity. Secondly, chapter one identifies morphological transitions as C. excisa undergoes sex change within its host. Chapter one highlights the importance of sex change, and discusses mating systems and mating strategies in parasitic isopods, which until now have received little attention.

Chapter two focuses on the metabolic constraints of searching for hosts of the cymothoid parasite, Cymothoa excisa, and the mechanisms associated with finding

Atlantic croaker, its common host. The infection window for mancae was determined by measuring metabolic activity and swimming ability as mancae age from the day they are born until they are inactive. Mancae could potentially experience and react to a number of cues associated with their host. To identify the response of mancae, both visual and chemical cues were used to explore how mancae respond to host proximity. Chapter two discusses the importance of host-finding mechanisms of C. excisa and the implications of host-detection in parasite population dynamics.

Parasitism has been largely ignored in the past, but in recent years the importance of parasitism in influencing ecosystem dynamics has come to light. The implications of 2 parasitism within ecosystems have initiated an influx of studies on the subject, but like any new area of study there are areas that require more attention. Parasitic species rely on their hosts for survival and locating, infecting and reproducing within a host are vital to the persistence of parasite populations. Understanding the processes that allow parasite populations to persist in different environments will allow us to predict how host and parasite populations fluctuate over time and how parasites influence the environment.

3 Chapter 1: Conspecific density-dependence drives sex change in a parasitic isopod

Introduction

The reproductive success of parasitic organisms is strongly influenced by their ability to infect a potential host. In order to infect a host, macroparasites have evolved life-history characteristics that allow parasites to successfully move between spatially and temporally separated patches of host resources (Viney and Cable 2011). Although transmission is a key component in the life history of parasites, parasites still face the challenge of surviving and reproducing within the host (Smith-Trail 1980; Viney and

Cable 2011). Macroparasites living in marine environments have evolved profound changes in physiology, morphology and life history traits to overcome these challenges

(Poulin 1995a). Given the above constraints, an important direction in the study of macroparasite ecology should focus on the relationship between ontogeny, morphology, and host-parasite population dynamics.

Macroparasites tend to have strong morphological transitions through their life cycle (Reitzel et al. 2006), often correlated with life history traits involved in infection and reproduction within a host (Fenton and Rands 2004). Some macroparasites have a free-living transmission stage as a juvenile and a sedentary stage as adults, in which the morphology of the parasite can dramatically shift when adapting to the different environments. For example, Trilles (2007) found that the parasitic isopod, Olencira praegustator, shows changes in morphology, including body size, mouthparts, eyes and tail when transitioning from juvenile to male to female. The most obvious morphological 4 change with ontogeny in parasites is the increase in body size from juveniles to mature adults. Reduced body size in juvenile parasites potentially increases the probability of infection by allowing the exploitation of more infection sites on a host (Price 1980;

Hanken and Wake 1993; Poulin 1995b; Tsai et al. 2001), are cheaper to produce (Vance

1973; Smith and Fretwell 1974; Marshall and Keough 2006), but can result in lower fecundity and reduction in mobility (Morand 1996; Iwasa an Wada 2006). To overcome this tradeoff, many parasitic species show distinct body sizes depending on the infection stage (i.e., host-searching juveniles and offspring-producing adults (Hechinger et al.

2012), and change sex to increase the probability of reproduction (Charnov 1982; Bull and Charnov 1988; Gusmao and McKinnon 2009).

Sex change is a common strategy in species that show patchy distributions or low mobility, and increases the probability of achieving sexual reproduction when the number of conspecific encounter rates is low (Ghiselin 1969; Warner 1975). For example, sequential hermaphroditism will be favored when an individual can reproduce more successfully as a member of one sex when that individual is small, but as a member of the opposite sex when that individual is large (Iwasa 1991; Brook et al. 1994; Tsai et al.

1999). Protogyny will be favored when there is selection for larger males, where small males are restricted from mating with females by larger males (Warner 1988; Brook et al.

1994). Conversely, protandry will be favored when there is selection for larger females because female size correlates with fecundity (Warner 1975; Warner 1988; Brook et al.

1994). Because sequential hermaphrodites change sex during their life cycle, an important component in understanding sex change is identifying the optimal time or size 5 at which individuals should change sex (Ghiselin 1969; Warner 1975; Charnov 1982,

1993; Iwasa 1991; Cadet et al. 2004). Previous studies have predicted that the optimal time to change sex depends on the existing sex ratio in the population and the relative fitness of each sex at that time (Charnov 1982; Proestou et al. 2008) however; social mating systems may also influence sex change (Hoagland 1978; Warner 1988; Warner and Swearer 1991; Warner et al. 1996; Proestou et al. 2008). While many macroparasites exhibit sex change strategies, limited information exists on morphological changes, reproductive output and the timing at which sex change becomes optimal (Brook et al.

1994; Poulin 1995b; Tsai 1999).

This study identifies morphological transitions as a parasitic isopod undergoes sex change within its host. Cymothoa excisa is a sequential hermaphrodite, changing sex from male to female during its life cycle as it infects the buccal cavity of its common host, the Atlantic croaker, (Micropogonias undulatus). Females fill the location of the tongue of the fish with one to five males attached to the gills or in the cavity between the gills and tongue of the fish. A two year survey identified infection rates and the relationship between fish density and size and parasite load, size and fecundity. Isopod morphology was quantified for each life stage, identifying shape transitions through ontogeny and sex change. The present study highlights the importance of sex change, and discusses mating systems and mating strategies in parasitic isopods, which until now have received little attention.

6 Materials and Methods

Field Collections

Collections occurred in Redfish Bay (N 27° 51’, W 97° 8’), a small shallow bay along the Texas coast, near Corpus Christi, Texas. The deeper water of the bay consists of mud and sand bottom, free from significant relief structure, with seagrass occurring in the shallow northern and southern portions of the bay. In this area, Atlantic croaker is a relatively common fish species and is frequently infected by C. excisa.

Fish samples were obtained from August 2010 to May 2012 using a 25 foot otter trawl for 15 minutes using University of Texas Marine Science Institute’s marine education vessel, the R/V Katy. Sampling occurred in the deeper portion of the bay (3 m depth), adjacent to the shallow areas that contain seagrass. Sampling trips were taken at least twice a month except when the vessel was out of commission for a period of 3 months each year (December-February). Trawls were done in the middle of the bay in the same location for every sampling event. All croaker collected were kept alive in a livewell with fresh flowing seawater and upon returning to the lab fish were transferred to holding tanks and each individual examined for infection by C. excisa. Infection was confirmed by the presence of an isopod and by the physical damage associated with infection. This second observation prevented scoring fish that were infected in transit as being counted as infected, but transfer of parasites between hosts is extremely low due to the limited mobility of the sedentary adult parasite (pers. obs.).

Morphological measurements were taken from 98 preserved isopods selected by their location (see below for the description of isopod location) in the buccal cavity of the 7 host, which is correlated with the different parasite life stages (per. obs.). Mancae

(juveniles) are born male and are free-swimming in search of a fish host. Mancae have an average half-life of 7 days and an average life span of approximately 11 days (Cook,

Chapter 2). Mancae display a severe reduction in swimming ability after their 7 day half- life (Cook, Chapter 2). If a manca locates a host, it attaches to the gill cavity of a fish, and shortly thereafter matures into a reproductive male. If the manca is the first to arrive to that host it quickly moves to the tongue and anchors itself, where it begins to change sex to a female; this stage is referred to as intermediate. Females are found anchored on the tongue and occur with 1 to 4 males. The courting male is found in the cavity between the tongue and the gill in close proximity to the female and if any other males are present they are found on the gill arches (tending males). Isopods selected for dissection consisted of 20 juveniles, 23 females, 24 intermediates and 31 males. Isopod body parts were photographed using a Canon G10 digital camera attached to a dissecting microscope and images were analyzed using ImageJ (Rasband 1997-2012); 19 measurements (to the nearest 0.1mm) (Appendix 1) were obtained from males, intermediates and females, and

5 from free-swimming juveniles.

Data analysis

To compare seasonal infection rates, sampling events were grouped into 5 categories based on seasonal fluctuations in temperature: winter, spring, early summer, late summer and fall. Winter consisted of the months of January through March. Spring was April through May. Early summer was June through July. Late summer was August to September. Fall was October through December. A one-way ANOVA tested whether 8 the proportion of infected fish varied among the seasons (winter was excluded because of few samples). Because fish grow with the seasons and larger fish (i.e., older fish) were observed to have more isopods, the median size of fish with parasite infections was calculated for each of the seasons. A Kruskal-Wallis nonparametric test was used to determine whether isopod frequency per fish varied among the four seasons (again winter was excluded because of few samples). A non-parametric test was used because data were not normally-distributed and it allowed for a test of ranks.

An ANCOVA was used to test whether parasite length varied between sex, taking into account fish length. Linear regression analysis was used to test whether brood size changed as a function of isopod length. When testing how parasite length varied as a function of fish size, only isopods that were found with two or more other isopods were used in the analysis. Isopods where labeled as female (on the tongue), courting male (in the gill-tongue cavity) or tending male (on the gills). Discriminant Function Analysis

(DFA) was used in two different ways to test whether adult individuals could be differentiated by either stage or location. Anderson’s CAP program (Anderson and

Robinson 2003; Anderson and Willis 2003) was used, where the number of eigenvectors was selected by the program and ran 9999 permutation tests for significance. Data were log(x+1) transformed and we used Bray-Curtis distances (Krebs 1999) in the DFA. The first DFA discriminated among males, intermediates and females to test whether the three stages could be discriminated using measured structures. The second DFA focused on males and intermediates, discriminating by location (gill, gill-tongue, and tongue) to test whether the structures could determine where an individual occurred. Using a DFA with 9 step-wise selection of variables (JMP, SAS Institute, Cary, NC) confirmed the six structures that differed the most between stages and locations were gonopod length, eye perimeter, first antenna length, uropod perimeter, pleotelson width and basis 7 width.

One-way ANOVAs were then used to test how these six structures differed between sexes. To assess how isopod sex (gonopod length) varied with location taking into account isopod length, an ANCOVA was used just using male and intermediate isopods.

Results

The 18-month survey produced 981 croaker individuals of which, 195 (19.88%) were infected by C. excisa (Appendix 2). Infection rate did not seem to change with season (F3, 21 = 1.45, P = 0.26). In spring 454 croaker were collected of which, 83

(18.28%) were infected (Appendix 2). In early summer 103 croaker were collected of which, 36(34.95%) were infected (Appendix 2). In late summer 338 croaker were collected of which, 62(18.34%) were infected (Appendix 2). In the fall 76 croaker were collected of which, 11(14.47%) were infected (Appendix 2). Winter was excluded because of few sampling trips. In spring parasite density was high with infection predominately on small fish (6-11cm) (Figure 1A), which contained a single isopod

(Figure 1B). By early summer isopod prevalence shifted to larger croaker (11-14cm)

(Figure 1A) and most of these fish were infected with 2 or more parasites (Figure 1B). In late summer larger fish (10-17cm) were infected (Figure 1A) in most cases with 2 or more parasites (Figure 1B). By fall only large fish (> 13cm) were collected (Figure 1A) and croaker density decreased from summer, but those fish that were infected were 10 hosting 2-3 parasites (Figure 1B). Only larger fish were collected in the winter (> 15cm)

(Figure 1A) with croaker density decreasing further, but fish showed infection by 2 isopods (Figure 1B). Parasite load varied among seasons (Kruskal-Wallis, χ² = 63.69, d.f.

= 1, P < 0.0001), where fish in early and late summer and fall had more isopods per fish than other seasons. This seasonal shift suggests that single parasites infect a small host early in the year and accumulate as fish age.

Location of parasites found within fish depends on the number of isopods present on a host (Figure 2). Hosts with one isopod almost always have the parasite on the tongue, with one exception of one isopod found on the gill. This lone individual was small and presumably had recently infected its host just prior to collection. When two parasites are present, one always occurs on the tongue while the other occurs in the cavity between the gills and tongue (Figure 2). When three or more parasites are found on a single host, one isopod is found always found on the tongue, one right behind the female in the cavity between the gills and tongue, and the rest are attached to the gill arches

(Figure 2).

Female isopods were larger than male isopods (F 2,239 = 1221.91, P < 0.0001), when taking into account fish length as a covariate (F 1,240 = 157.83, P < 0.0001) (Figure

3). However, there was an interaction between fish length and life stage, where tending males size was not influenced by fish length, but females and courting males were larger in larger fish (F 3,238 = 27.43, P < 0.0001). Brood size increased with female isopod size

(R² = 0.81, F= 81.92, P < 0.0001) (Figure 4).

11 Most of the 18 morphological structures varied with isopod length (Appendix 1).

The DFA was able to differentiate males, intermediates and females with only 14 misplaced assignments (Figure 5) (Table 1). Females were correctly assigned in 87% of the cases, while males were assigned correctly in 84% of the cases; intermediates showed the greatest uncertainty (75%) because of the transition between sexes. Morphological structures that were relevant and specific to male-female transitions were the gonopod length, eye, first antenna, uropod, pleotelson width and seventh basis width. These structures are likely the most important structures for identifying isopod sex when the isopods change sexes from male to female. Some structures were lost as males morphed into females, while some where gained. Structures that increased with ontogeny were:

th isopod size (length × width) (F3, 95 = 110.71, P < 0.0001), 7 basis width (F2, 76 = 21.81, P

< 0.0001) and pleotelson width (F 2, 76 = 34.93, P < 0.0001) (Figure 6). Structures that decreased with ontogeny were gonopod length (F 2, 76 = 133.51, P < 0.0001), eye perimeter (F 3, 95 = 59.11, P < 0.0001), uropod perimeter (F 2, 76 = 33.58, P < 0.0001) and first antenna length (F 3, 95 = 125.82, P < 0.0001) (Figure 6).

Males change morphology as they move from the gill to the tongue, and the DFA successfully differentiated males from different locations (Table 1). Tongue males showed the largest number of misclassifications because these males were transitioning into females. Here, structures that were driving differences in males found in the gill, gill- tongue and tongue were eye perimeter, gonopod length, uropod perimeter and first antenna length. These structures are likely the first structures lost as males move into the tongue position and start to morph into a female. Gonopod length was different in male 12 and intermediate isopods (F2, 53 = 19.22, P < 0.0001), but was strongly influenced by isopod length (F1, 54 = 18.44, P < 0.0001) (Figure 7). There was a significant interaction between isopod location and length (F 3, 52 = 3.54, P = 0.03) driven by males on the tongue that were transitioning to females.

Discussion

Sex change is a strategy among some organisms and sex allocation theory predicts that sex change should arise when reproductive success is distributed differently in males and females due to differences in size (Ghiselin 1969; Warner 1975, 1988; Charnov

1982; Warner et al. 1996). Sequential hermaphroditic species should change sex when a greater reproductive output is achieved by the rare sex (Warner 1988; Iwasa 1991;

Warner et al. 1996). Here, Cymothoa excisa was found to change sex as a function of parasite infection, where the absence of a female on the tongue induces sex change in the first parasite to infect a host. Frequency-dependent sex change has been found in other systems (e.g. Coe 1953; Robertson 1972; Hoagland 1978; Charnov1982; Soong & Chen

1991, 2003; Alonzo & Warner 2000a, b; Koeller et al. 2000; Chiba et al. 2003; Mackie

2003; Perry & Grober 2003). Thus, C. excisa shows sex change that is dependent on the presence of a female in a host with the first parasite to infect a host changing sex and becoming female and all subsequent parasites infecting the same host remaining male.

Parasites accumulated on infected fish as seasons progressed, suggesting a relationship between mating system and environmental conditions. As fish grow in size, so did the parasites, with female isopods being influenced the strongest by fish size because they 13 have been growing within their host the longest. Because sex change is related to the timing of infection and brood size is related to female size, it seems that parasites benefit the most by infecting early to increase body size as females and increase reproductive potential.

Drastic morphological changes through ontogeny are often related to transitions in habitat use (Rowe and Ludwig 1991; Peckarsky 2001; Svanback and Eklov 2002). For example, holometabolous insects, amphibians, polychaetes and crustaceans undergo metamorphosis when switching habitats (Pechenik and Cerulli 1991; Wolcott and

DeVries 1994; Denver et al. 1998; Arai et al. 1999; Peckarsky et al. 2001). Cymothoa excisa undergoes similar ontogenetic changes. One of the most notable changes in C. excisa is the dramatic increase in body size through ontogeny. Organisms capable of altering their sex during their lifetime do so because an increase in body size offers a fitness advantage (Ghiselin 1969; Warner 1975; Charnov 1982, 1993; Iwasa 1991; Cadet et al. 2004). In the first stage of ontogeny, free-swimming mancae have large uropods, eyes and antennae used in swimming and detecting environmental cues. The uropods in isopods have been shown to be used in swimming (Alexander and Chen 1990) and the antennae have been shown to be used in sensing chemical cues (Brusca 1981). Mancae are phototactic and large eyes help parasites detect shadows of potential hosts (Cook,

Chapter 2). Once an isopod attaches itself to an uninfected host, it undergoes a rapid transition as it moves to the tongue. It dramatically increases in size while its eyes, uropods and antennae are reduced, with the eyes eventually disappearing. The size reduction of these organs suggests that these sensory organs are no longer needed. 14 Gonopods are also reduced and presumably lose their function, yet the pleotelson becomes broader, and legs gain mass because they are structures that help anchor females to the host. However, if the parasite is not the first to infect a fish, it does not undergo these morphological changes, indicating that mating strategies may help determine parasite sex change, reproduction and fitness.

Some species change sex at specific stages, while others are more plastic and rely on external cues to change sex (Warner et al. 1996). Species that rely on external cues often respond to social situations where the rare sex often has the advantage when reproducing (Charnov 1982; Warner 1988; Warner et al. 1996). For example, in some tropical fishes large males monopolize multiple females in a harem, and sex change occurs when the male fish is removed from the group and the largest female becomes male (Robertson 1972; Warner et al. 1996). Cymothoa excisa seems to rely on cues from the female parasite within a host on whether to change sex. Brusca (1981) hypothesized that the absence of a resident female or the presence of a young male causes sex change in cymothoid isopods. This study confirms that the first parasite infecting a fish begins to change sex without other males present. Further sex change does not occur, suggesting that the female either deters other parasites from changing sex, there are no more resources available for the other parasites to change sex (i.e. only a single tongue is available), or a complex mating system is in place. Previous studies have shown that pheromones released by females of some parasitic crustaceans stimulate the neurosecretory system of conspecific males resulting in extended production of androgen in males and thus prolonging their masculinity (Legrand and Juchault, 1970; Raibaut and 15 Trilles 1993). By monopolizing the tongue, the female isopod controls resources and drastically increases in size, which translates to increased fecundity (Brook et al. 1994).

The presence of multiple males on a single fish is a strong indication that a mating system is in place where there is a process that is in play that determines which male gets access to the female. In protandrous species, males often compete for access to females

(Buston 2004). Why are multiple males present on a single host, and therefore which male gains access to the female isopod anchored on the tongue? Here I provide three hypotheses. First, multiple males may infect a single host simply because it is energetically costly to search for a new host. When C. excisa are juveniles they have limited energy reserves and a narrow infection window (Cook, Chapter 2), therefore it may be advantageous to remain on an infected host than to continue searching. This strategy would be reinforced if isopods change fish behavior to increase parasite load, a strategy observed in many other host-parasite systems. For example, parasites have been found to alter the behavior of fish (Lafferty and Morris 1996), snail (Levri 1999) and amphipod (Maynard et al. 1998) hosts to increase the probability of infection. Marine isopods are generally semelparous, dying shortly after releasing their brood (Shuster

1990; Warburg and Cohen 1991; Furota and Ito 1999) and limited female reproduction could create a mating chain. Males could mate in order of arrival, with the second parasite to arrive mating with the female, and once the female dies, the second parasite would become the new female and the next male the courting male. If this strategy was taking place, then it would be advantageous to be the first or second male to arrive as the reproduction potential is highest. This merry-go-round type of mating is found in other 16 species, where a mating group is composed of breeders and other individuals who either delay or completely forego their own reproduction until a later time (Buston 2004). For example, large females of the clown anemonefish, (Amphiprion percula), copulate with the largest male, and when the female dies the largest male becomes the reproductive male. Finally, while the first male of C. excisa becomes female, any male that arrives afterward may have to compete for access to the female. Males often compete for access to females (Bateman 1948; Williams 1966; Wade 1979; Clutton-Brock 1988; Shuster and

Wade 2003). In scenarios where male competition is strong, sexual selection favors male traits that increase fitness through access to females (Darwin 1874; Wade and Arnold

1980; Shuster and Arnold 2007). For example, Shuster and Arnold (2007) found that large male Paracerceis sculpta isopods displayed mate defense and acquisition. It is often found that male size is a sexually selected trait (Shuster and Wade 2003). In Cymothoa excisa the courting male is larger than the males found in the gills and could be guarding the female and preventing access to the remaining males (Jormalainen 1998).

This study is the first to show that sex change in a cymothoid isopod is driven by parasite density within the host, and the absence of other parasites within the host drives sex change. In C. excisa it is advantageous to be a large female as reproductive output increases with female size (Ghiselin 1969; Warner 1975, 1988; Charnov 1982; Hattori

2005). However, the presence of multiple males within a single host could be the result of an interaction between a complex mating system, environmental conditions and sex change. The evolution of sex change in parasites is likely an adaptive strategy evolved to

17 increase survival and reproduction within a host. Focusing on mechanisms behind sex change and parasitic mating systems will help resolve parasite evolution.

18 Chapter 2: Sensory cues associated with host detection in a marine parasitic isopod

Introduction

Both the evolution of parasite life history traits and parasite population dynamics are strongly affected by the ability to find and infect new hosts. Parasitic organisms are completely dependent on their host, and for parasites with free-living stages host detection is the most important stage for parasite survival (Sikkel et al. 2011). Due to this constraint parasites with free-living stages have evolved a number of sensory cues and behavioral responses to locate and infect potential hosts (Combes 1991, 2001; De Bruyn et al. 2011; Sikkel et al. 2011). These individuals have a narrow time window of days to a week to find a suitable host (Menzies et al. 1955; Sandifer and Kerby 1983; Fogelman and Grutter 2008). Therefore the mechanisms associated with host searching and any constraints during the free living phase are an important component behind parasite population dynamics.

Parasitic organisms display a vast spectrum of when and how they infect a host.

Parasites can search for a host either at a juvenile stage or as a mature adult. Parasitic species that search out hosts as adults do so mainly to use the host for reproduction

(Poulin 2009; Poulin 2011); this host searching trait is a major difference between parasitoids and parasites (Godfray 1994). For example, adult flesh fly parasitoids search out marine snails where fly larvae are deposited on the snail; the larvae feed on the host and eventually kill it in order to complete development (Pape et al. 2000). The outcome of host searching as an adult results in a larger window to find hosts because adult 19 parasites are free-living and are able to forage. Constraints associated with adult host searching involve a limited time budget for finding hosts and mates. Parasitic species that search out hosts as juveniles utilize hosts for shelter, energy resources and in some cases reproduction. Bopyrid parasites, for example, infect caridean shrimp at early life stages and grow within the host, ultimately reproducing within the shrimp and continuing their life cycle (Pike 1960; Truesdale and Mermilliod 1977; Beck 1980; Schuldt and Rodrigues

Capitulo 1985; Campos and Campos 1989; Oliveira and Masunari 1998). Host-searching juveniles are energy limited and have a narrow window of infection because they are not self-sustainable and must rely on energy reserves from birth to locate a host. Searching out a host as a juvenile is common strategy among most groups of ectoparasitic organisms (Poulin 2009; Poulin 2011). Yet, even if parasites infect hosts at different stages, all parasites must ultimately locate and infect a host.

Host searching strategies involve either ambushing hosts or actively searching for hosts. Passive parasites rely on the host’s behavior to induce the infection; this strategy has the greatest success when hosts are abundant or have high mobility (Campbell and

Lewis 2002; Fenton and Rands 2004). Alternatively, active parasites search out hosts and this strategy is considered to be best suited when hosts have patchy distributions or have limited mobility (Campbell and Lewis 2002; Fenton and Rands 2004). A trade-off exists between the two strategies, where searching consumes energy while increasing host encounter rates, and ambushing decreases energy expenditure while decreasing host encounter rate (Fenton and Rands 2004). An array of sensory abilities has evolved to curb this trade-off, and we can expect that host detection mechanisms should be highly 20 developed in active parasites when hosts occur at low densities to maximize infection probability.

These host detecting mechanisms also provide information on the identity of potential hosts, yet can be under different levels of selection. When parasites are able to reproduce on multiple host species, there is low selection pressure on host detection mechanisms giving rise to the generalist strategy. Alternatively, when parasite fitness is highest on a single host species, there is increased selective pressure on host detection mechanisms, which become specialized in detecting that single host and become maladapted towards other host species (Thompson et al. 2002). Often, parasites that rely on a single host species cause hosts to react and evolve avoidance mechanisms, giving rise to evolutionary “arms races” (Thompson et al. 2002). For example, the specialist copepod parasite, Lepeophtheirus salmonis, has evolved acute detection mechanisms to recognize specific chemosensory cues to locate only salmonid hosts and avoid cues from non-host species (Bailey et al. 2006). Conversely, the salmonid host has evolved specific physico-chemical characteristics in its skin mucus to prevent infection by the copepod

(Jones 2001). While some parasites might possess excellent host recognition ability, locating a suitable host in a low host environment presents a difficult task for free-living parasites, a problem that is compounded for specialist parasites.

Cymothoid isopods are a family of large ectoparasites that infest a wide diversity of fish from temperate to tropical climates and have a direct life cycle in which free- living juveniles (mancae) must find a potential host (Brusca 1981; Fogelman and Grutter

2008). During the crucial pre-settlement stage, free-swimming parasites have the best 21 opportunity to infect a host (Sikkel et al. 2011). Because cymothoid parasites reproduce sexually, juveniles need to find the correct host and ensure subsequent infection by at least one additional parasite. To successfully locate a host the mancae must isolate cues from the host and surrounding environment and then act upon them. Some cymothoid parasites have been found to be highly host and site-specific, thus limiting potential hosts

(Tsai et al. 1999; Fogelman and Grutter 2008). Due to high host specificity of some cymothoids, parasite populations are often affected by host population fluctuations. For example, a specialist cymothoid parasite in Florida shows high abundance levels corresponding to peaks in host fish population (Bakenhaster et al. 2006). While cymothoid parasite populations have been found to fluctuate with changes in host populations, the mechanisms by which cymothoid parasites find their hosts are unresolved. Recent research has examined the post-settlement stage, cataloged seasonal infection rates, and identified potential fish hosts (e.g. Segal 1987; Fogelman and Grutter

2008), but mechanisms for host detection in cymothoid isopods remain largely unknown.

This study focuses on the metabolic constraints of searching for hosts of the cymothoid parasite, Cymothoa excisa, and the mechanisms associated with finding

Atlantic croaker, Micropogonias undulatus, its common host. The infection window for free-swimming parasites was determined by measuring metabolic activity and swimming ability as mancae age from the day they emerge from the brood pouch until they are inactive (C. excisa broods their young and are direct developers). Due to the small size of the mancae, energy stores were predicted to be low and metabolic activity is likely to decrease rapidly. Mancae could experience several cues when searching for potential 22 hosts and cue redundancy could increase host detection at different spatial scales as observed with dispersing larvae (Kingsford et al. 2002). Here, both visual and chemical cues were used to explore how mancae respond to host proximity. If mancae show phototaxis, then this indicates that they have enough visual acuity to detect light.

Chemical cues would allow mancae to determine concentration gradients in the environment and short-distance proximity to host. Chemical cues are predicted to stimulate swimming and searching behavior, and allow the mancae to identify the correct host species. A series of laboratory experiments established phototaxis and behavioral response to chemical stimuli followed by a factorial experiment that tests the effect of shadow response in the presence of chemical cues. This study predicts that the mancae will use chemical cues secreted by the Atlantic croaker host as a fine-scale navigation tool, with sight as a secondary sensory system that can be used as a coarse-scale host finding mechanism.

Materials and Methods

Study System

Cymothoa excisa is a large ectoparasitic isopod where adult females reside in the buccal cavity above the tongue. Juveniles are born male and, when released by the mother, exit the buccal cavity and are free swimming until a new host is found. If a manca finds a host, it will settle on the tongue if no other parasite is present. If another parasite is present then the manca will settle just behind the tongue at the base of the gills

(pers. obs.). Any subsequent isopod that finds a parasitized fish will attach on the gills. 23 The first parasite that settles on the tongue will change sex from male to female, while other parasites attaching to other parts of the fish will remain male (Cook, Chapter1).

Cymothoa excisa is broadly distributed across the Gulf of Mexico. Along the Texas coasts it is commonly found in the Atlantic croaker, Micropogonias undulatus, one of the most widespread and abundant fishes in the Gulf of Mexico and the western Atlantic

(Welsh and Breder 1923; Bearden 1964; Anderson 1968; Franks et al. 1972; Chittenden and McEachran 1976; Diamond et al. 1999).

Atlantic croaker were collected from Redfish Bay, Texas (N 27° 51’, W 97° 8’) from June 2011 to May 2012 using a 25 foot otter trawl aboard the R/V Katy (working depth = 3m). Redfish Bay is a small shallow bay that consists of open mud and sand bottom with seagrass occurring in the northern portion of the bay and along the edges of the deeper water. All croaker collected were kept alive in a livewell with fresh, flowing seawater. If a fish contained a gravid female it would be separated and the gravid female kept in aerated tanks until release of mancae from the brood pouch. Once released, mancae were kept in individual containers containing 1500 mL of UV-sterilized seawater and used in the following experiments.

Parasite Energetics

To measure oxygen consumption rate mancae were carefully extracted from their holding tanks with a pipette and gently placed in 2mL tubes with 0.5mL of oxygen saturated water, with the remainder of the tube filled with air. Mancae activity was not restricted within the tube as mancae were seen swimming normally around in the tube

(personal obs.). To measure oxygen consumption, a fiber optic sensor (PreSens, 24 Germany) was inserted into each tube and sealed. 18 mancae were randomly selected from each brood right after they hatched. Oxygen concentration was measured every 15 seconds for 12 hrs using 9 individuals at a time, with a resting period of 36 hrs. Mancae were returned to their individual holding containers until the next set of trials. A sealed tube with 0.5mL of water but no manca was used to monitor oxygen levels in the absence of a manca during every run. Oxygen measurements were performed with the same individuals, unless an individual died, in which case it was replaced with another manca of the same brood and age. Oxygen consumption for each animal was standardized by oxygen loss in the blank tube and averaged across all individuals for a single day measurement. Daily swimming speed of individual manca was measured by placing the mancae in 2.5 cm wells containing 2ml of sterile seawater, and recorded for one minute with a digital camera. Videos were analyzed for maximum swim speed with ImageJ

(NIH, Bethesda, MD). The same mancae that were used at the beginning of the experiment were used each consecutive day until death. Thirty mancae were then randomly selected to monitor swimming time. Three mancae at a time were placed in a

3×1×5 cm (length × width × height) cuvette and allowed to adapt to the conditions of the cuvette for five minutes. Their behavior was recorded for one minute with a digital camera and then analyzed to determine the average percent time the mancae swam per trial. Manca survival, oxygen consumption, swimming speed and swimming time were measured as a function of age. A mixed-model ANOVA using batch as a random effect tested changes in oxygen consumption as a function of age. Regression models were used to show changes in swimming speed, swimming time, and survival. Half-life estimates 25 for each variable were estimated using the regression equations. These values were then averaged across all variables to estimate the age at which 50% of the mancae would be able to find a host.

Phototaxis and chemotaxis by free-swimming parasites

In order to test how mancae respond to light, individual manca were placed in an acrylic tank (20.5 × 3.5 × 10 cm) and their response to light was observed. Tanks were filled with sterile sea water to a depth of 3cm. Black sandpaper was applied to one side of the tank to prevent light from reflecting back in the tank. A fiber optic light source (53.52

μmol·photons·s-1·m-2) with a natural light filter was positioned at one end of the tank.

The tank was marked off into three sections: a section opposite the light source, a middle section, and a section directly adjacent to the light source. An individual manca was placed in the center of the tank and allowed to acclimate to total darkness for 3 minutes.

The light source was then turned on and the initial location of the manca in one of the three sections was recorded; after 3 minutes the location was recorded and the trial ended.

After each trial the orientation of the tank was switched to account for any potential bias towards either side of the tank. If the manca moved sections towards the light source a positive response was recorded, and if it moved away from the light a negative response was recorded. A χ2 test was used to determine positive or negative taxis to the light source using 31 trials. The response of mancae to fish chemical cues was measured using

3 treatments: a treatment with sterile water, a treatment with Atlantic croaker water and a treatment with pinfish (Lagodon rhomboides) water. Fish water was obtained by placing a fish in a one gallon aquarium for 24 hrs, allowing chemical cues from the fish to 26 saturate the water, and filtering the water to remove large particles. Individual manca were placed in sterile seawater for 1 minute to standardize each trial and then placed in separate one gallon aquaria containing 1500ml of water from one of the treatments. Each treatment lasted 16 minutes, and during each treatment parasite behavior was recorded with a camera (Canon G10) in five 1-min intervals spread throughout the 16 minute trial

(n = 52 individuals). Between treatments manca were again placed in fresh sterile seawater and then randomly assigned to a different treatment. The videos were visually analyzed using a chronometer and behaviors recorded to the nearest second. Behavior was categorized as swimming (individuals moving or suspended off the bottom) or resting (lying on the bottom of the aquaria without movement). A repeated-measures

ANOVA tested behavioral responses as a function of water type followed by a Tukey

HSD post-hoc test.

A Y-maze experiment was used to determine whether mancae show taxis toward or away from water saturated in fish chemical cues. The Y-maze (upper arms = 12cm, lower leg = 12cm) was built with one-way valves positioned at each of the upper arms to introduce a water current containing the respective water treatment (flow = 30ml min-1).

Clear demarcation of the flow down each arm and a mixing of the signal in the main leg of the apparatus were shown with food dyes prior to experimentation. Fish scented water was obtained as above. Orientation to light was controlled by flooding the maze with ambient white light from a 60W (52.68 μmol·photons·s-1·m-2) incandescent bulb placed directly above the maze. To avoid bias toward a particular arm of the maze, water treatments were switched between trials and allowed to run for a minimum of eight 27 minutes to clear arms of previous trial’s water. An individual manca was placed in sterile seawater for 1 minute then placed into the lower leg of the maze. Initial direction of a manca’s movement determined if there was a taxis toward a specific water type. Two conditions were run, sterile water versus croaker water (n = 127 individuals) and croaker water versus pinfish water (n = 40 individuals). A χ2 test was used to test preference between treatments for each of the conditions.

Planktonic organisms typically respond negatively to a shadow as a behavioral response to avoid a predator (Forward et al. 1996; Cohen and Forward 2003; Zeigler et al. 2010), so to test for the presence of a shadow response in mancae and the modification of this response by chemical stimuli, a laboratory experiment was conducted in an apparatus designed to mimic the natural angular light distribution (Forward et al. 1984).

An outer tank (50 × 50 × 25 cm) was filled with sterile seawater, and a 3 × 1 × 5 cm cuvette was filled with seawater and placed at the center of the large tank. The walls of the outer tank were painted flat black and the tank was lit from overhead by a 300 W

(42.28 μmol·photons·s-1·m-2) incandescent bulb. Behavior of mancae in the inner cuvette was recorded with a video camera (Panasonic WV-CP500). Pilot studies determined the parasite’s sensitivity to decreases in light intensity using 10 different filters that reduced the light intensity (Appendix 3), and the experiment ran with a filter that decreased 82% of light (Filter 3).

The experiment had a 2 × 2 factorial design, with the presence and absence of croaker scent crossed with presence and absence of a shadow. To create the chemical stimuli, water with croaker scent was made as above. In each experiment, the inner 28 cuvette was filled with either croaker water or sterile seawater (thus creating two odor treatments), and three mancae were placed in the cuvette. Isopods were placed underneath a 60 watt lamp (52.68 μmol·photons·s-1·m-2) for one hour before the experiment to standardize all of the mancae to full light conditions. At the time of testing, the mancae were subjected to a decrease in light intensity (shadow treatment) or no decrease (no shadow treatment). Behavior was monitored for 10 s prior to the shadow treatment and 1 minute after the shadow treatment was created. The recorded 10 s before the trial begun were used to identify the immediate response of the parasite to the shadow. Using ImageJ (NIH, Bethesda, MD), 3 variables were quantified: burst speed, change in time swimming and change in behavior. Burst speed was defined as the speed at which a manca makes its first vertical movement. Change in time swimming and number of behavioral changes was scored between the 10 s before and 10 s after the shadow occurred. The behaviors could be: sitting on bottom of tank, floating at the top of the tank or swimming normally in the water column. Behavioral change was scored as 1

(change) or 0 (no change) and averaged across the 3 manca for each trial. Behavioral change and change in swimming time identified the instantaneous response to a shadow.

Twenty mancae were used for each treatment totaling 80 parasites from two different cohorts. A mixed-model ANOVA tested each behavioral response as a function of water type and shadow presence and their interaction with cohort as a random effect using restricted maximum likelihood estimates (Fletcher and Underwood 2002). All analyses were performed in R (R Foundation for Statistical Computing, Vienna, Austria.) and JMP

(SAS Institute, Cary, NC). 29

Results

Oxygen consumption decreased as the mancae aged, with the largest decrease occurring between the day they emerged from the brood pouch and day 3 (O₂ consumed

= 1.12 - 0.04*Day, R² = 0.31, P = 0.03) (Figure 8A). Mancae decreased the amount of time that they swam as they aged (Swim Time = 71.37 - 5.09*Day, R² = 0.97, P =

0.0004) (Figure 8B). Swimming speed decreased as a function of age (Speed = 6.06 -

0.27*Day - 0.03*(Day-6.54)², R² = 0.92, P < 0.0001) (Figure 8C), with a sharp reduction in speed occurring after day 8 (-0.271*Day, P = 0.006). Mancae live for 10.69 ± 0.65 days (n = 77) but there is no sharp decline in survivorship with time (Survivorship =

103.84 - 5.19*Day, R² = 0.98, P < 0.0001) (Figure 8D). The reduction in the amount of time swimming after 3 days by the manca probably resulted in the reduction in oxygen use by the mancae. The ability of the mancae to maintain swimming response speed for over a week indicates that although they show a reduction in overall activity, they still have enough energy to move toward a host. Averaging the half-life for each of the four variables gives an overall half-life of 7.08 days.

When manca were stimulated with a light source, most individuals oriented positively to the light rather than away from the light (χ2 = 18.62, d.f. = 25, P < 0.0001) suggesting positive phototaxis. Twenty-four (77.42 %) of the mancae displayed a positive phototaxis. Two (6.45 %) mancae displayed a negative phototaxis and the remaining five showed no taxis (16.13%).

30 Mancae that where stimulated with water treated with croaker and pinfish scent swam significantly more than mancae in sterile water (Figure 9). However, there was no difference in swimming time between the two fish water treatments (F 2, 50 = 5.49, P =

0.002) (Figure 2). Mancae spent more time resting in sterile water relative to fish-scented water (F 2,50 = 4.80, P = 0 .008) (Figure 9). Mancae that were given a choice between croaker water and sterile water in a Y-maze, showed a positive response to croaker water

(χ2 = 9.33, d.f. = 83, p = 0.002) (Figure 10, Trial 1). But when mancae were given a choice between croaker water and pinfish water, there were no statistical differences in preference between the two scents (χ2 = 1.06, d.f. = 39, p = 0.3) (Figure 10, Trial 2), even if mancae moved more frequently towards croaker-scented water.

Mancae showed strong responses to shadows, often resulting in an increase in activity. As shown with previous results, manca are often swimming in search of a host or found resting at the bottom of tanks. When a shadow is presented, manca immediately burst vertically in search of a host. This bursting behavior was fastest when a shadow was presented in fish scented water (Figure 11A) relative to unscented water, and in the absence of fish scent burst speed was slower (Table 2). Mancae also swam more frequently in the presence of shadows (Figure11B), while scented water did not produce an effect (Table 2). Finally, manca were switching behaviors more frequently when presented a shadow relative to treatments without shadow (Figure11C) (Table 2); in the presence of a shadow scented water generated more behavioral changes than unscented water. This outcome could indicate that the shadow response could act as a cue for the mancae to attack, with the presence of fish chemical cue intensifying this effect. 31

Discussion

Parasites with free-living, infective stages have limited energy stores that are consumed when searching for a host (Croll 1972; Barrett 1976; Lee and Atkinson 1976;

Robinson et al. 1987; Fitters et al. 1997; Fenton and Rands 2004). The limited energy available in C. excisa creates a narrow window to locate and infect a host. Previous studies have shown the window to be as narrow as one week for cymothoid isopods

(Menzies et al. 1955; Sandifer and Kerby 1983; Fogelman and Grutter 2008). Results from this study show that the half-life of C. excisa is approximately 8 days and the average life span is 10 to 11 days, suggesting a rapid deterioration of energy stores after about a week. To prevent active searching when the chances of finding a host are low, parasites with free-living stages have evolved mechanisms to detect potential hosts

(Combes, 1991, 2001; De Bruyn 2011) and only search when they know a host is near.

Within the infection window, C. excisa could be modifying its searching strategy from active to passive strategy (Fenton and Rands 2004).

Hosts species represent an inherently patchy and dynamic resource that can vary spatially and temporally where parasites often have to overcome obstacles arising from such variation (Barrett et al. 2008). Cymothoa excisa has a limited energy supply and because of this constraint, individuals may switch between searching and resting while trying to locate a host. Mancae were frequently swimming the first few days after being born, but quickly became inactive, maintaining a constant and low metabolism. After the first three days of being born the mancae switched from a searching strategy to an 32 ambush strategy. However, individuals maintain a fast swim response within the first 8 days of their lives, but choose to wait for croaker to approach them. An example of parasites switching strategies has been found in the gut helminth parasite, Haemonchus contortus, where it begins searching actively for a host and switches to a passive behavior once it has reached an area that increases its chance of infecting a host (Fenton and Rands

2004). When manca are close to a potential host, both chemical cues and a shadow trigger an attack response. Chemical cues stimulate a systematic searching behavior as mancae constantly swim, while the presence of a shadow triggers mancae to prepare for an encounter with a host or swim vertically, attempting to attach to nearby hosts. This aggressive behavior is typical among parasites with free-living stages (e.g. copepods, gnathiids) (Poulin 2009), but uncommon among crustacean and echinoid larvae (Forward and Cronin 1978; Rumrill et al. 1985; Morgan 1995; Johnson and Shanks 2003; Metaxas and Burdett-Coutts 2006). Typically, planktonic larvae display a negative shadow response as a behavior to prevent predation (e.g. Forward et al. 1996; Cohen and Forward

2003; Zeigler et al. 2010). Mancae that search when no cues are encountered will likely spend more time in the plankton and fall victim to predation (Stepien and Brusca 1985), therefore searching for a host is only adaptive when specific host cues are present. To prevent searching when no host is present and to maximize its energy reserves, C. excisa has evolved specific cues to locate and successfully infect a potential host.

Along the Texas coastal bend, C. excisa is found exclusively on Atlantic croaker, suggesting that such high host specificity should be associated with well-honed detection mechanisms (Stevens 1990; Poulin 2007; De Bruyn 2011). Yet, in the laboratory mancae 33 initiate searching behaviors to both Atlantic croaker and pinfish, a species in which C. excisa has not been found. While waterborn cues did stimulate mancae to swim, the lack of species-specific chemical cues prevented mancae from distinguishing between croaker and pinfish water. Lack of pinfish infection by C. excisa in the field could be explained with three hypotheses. First, the fish scent is too generic and this study might have missed yet another cue (perhaps tactile) that helps determine whether manca have encountered an appropriate host or not. Second, host distribution, environmental constraints, or a combination of the two (Altizier et al. 2006) could prevent manca from successfully infecting pinfish. A parasite’s free-swimming stage could have environmental restrictions such as salinity levels or depth, constraining infection ability to those fish that occur within its environmental niche. For example the sea louse,

Lepeophtheirus salmonis, which infects salmonid species has reduced infection success in areas of low salinity, and changes its swimming behavior and orientation in order to avoid environments that reduce infection (Bricknell et al. 2006). Third, a pinfish could have evolved mechanisms to prevent infection by C. excisa, such as avoiding particular areas that have high parasite densities (Sorci et al. 1997) or have evolved behaviors to extrude the parasite if infected (e.g., Segal 1987). For example, when striped bass

(Morone saxatilis) and white perch (Morone Americana) are infected with the isopod parasite, Lironeca ovalis, the fish begin to employ behavioral strategies such as breaking the surface, swimming rapidly, thrashing their body, rubbing against the bottom or against objects on the bottom or dividing under sand in an attempt to remove the parasite

(Sandifer and Kerby 1983; Segal 1987). Fourth, there is the possibility that C. excisa 34 responds to a short-lived chemical cue from the Atlantic croaker that did not persist in the water treatment without the fish present. While the location of the host or the effect of environmental factors on infection determines if a parasite has the opportunity to infect a host, ultimately if the characteristics of the host are not suitable for growth and reproduction, the parasite will not be successful (Ward 1992). Potential hosts may be present to the parasite, but if the parasite is unable to successful reproduce on a host then the fitness of the parasite will be zero.

This study is the first to show how cymothoid isopods utilize both visual and chemical cues as host locating mechanisms. Results from this study show that C. excisa has a narrow window for infection and reduces activity a few days after being born. Once tired or energy stores are low, mancae only search when cues from a host are present, intensifying their search when multiple cues have been sensed. This type of behavior is thought to increase the chance of finding a host, while reducing energy costs (Fenton and

Rands 2004). Host detection, location and infection by parasites are the most important events in the life history of a parasite (Sikkel et al. 2011). By studying host detection mechanisms, we can begin to understand the complex population dynamics of parasitic species. The results from this study also will shed light on why some species of parasites are generalists and others specialists. Understanding the processes that allow parasite populations to persist in different environments will allow us to predict how host and parasite populations fluctuate over time.

35 Conclusion

While the study of parasitism is gaining interest there are significant areas within the field that require attention, especially marine parasites. This study aimed to resolve those areas where information about marine parasites was lacking, specifically focusing on an ectoparasitic crustacean with a free-living stage. Crustaceans comprise a large portion of marine ectoparasites, and infect a wide variety of marine organisms. The most important component in the life history of free-living parasites is infecting and reproducing within a host, and this study aimed to resolve these two components by studying, Cymothoa excisa, a marine, parasitic isopod in the common family cymothoidae.

Chapter one focused on identifying the infection trends and reproductive biology of C. excisa. The results are the first to show that sex change in a cymothoid isopod is driven by the absence of parasites within the host, where the absence of other parasites when the first parasite infects a host drives sex change. The results also show that infection rates in Atlantic croaker are constant throughout the year, where females are at an advantage to be large and the presence of multiple males indicate a complex mating system.

Chapter two focused on the early energetics and mechanisms of host detection involved in the searching behavior of C. excisa. The results from this chapter are the first to show that cymothoid isopods utilize both visual and chemical cues as host locating mechanisms. Results also show that C. excisa has a narrow window for infection and

36 reduces activity a few days after being born. Mancae were found to only search when cues from a host are present, intensifying their search when multiple cues have been sensed. This type of behavior is thought to increase the chance of finding a host, while reducing energy costs.

The results from this study have shed light on areas of parasitism that previously lacked detail. Understanding the mechanisms that marine ectoparasites use to locate and infect hosts is crucial in understanding parasite population dynamics. The reproductive biology of parasites also serves as a crucial component to understanding the evolution of mating systems in parasites as well as why parasites persist in multiple environments.

Although this study has brought to light new results and ideas on marine ectoparasites, areas of research are still lacking. First, we have shown that host searching mechanisms have evolved to conserve energy while maximizing host encounter rate. Like many other cymothoid isopods C. excisa is specialized on a single host, yet our results have shown that in laboratory conditions C. excisa begins searching for a host in the presence of non- host fish scent. Future research endeavors are needed to elucidate why C. excisa searches when non-host cues are present. By studying the specific chemical components of fish scent, future research will be able to identify why a specialist parasite responds to non- host cues, which will help elucidate why C. excisa is not found on other fish species. The current research has also created more questions involving the mating system of C. excisa. This study found that a single female exists within a host with multiple males present within the host. It is not known why the other males are present on the host or how/if they will mate with the female. Future research should focus on identifying why 37 multiple males exist on a single host and how the process of mating occurs in C. excisa.

These future research priorities will serve to further show why parasites are so successful in the marine environment and how parasite populations might change in the future.

38 Tables

Table 1. Isopod Morphology Discriminant Function Analysis (DFA). Classification results from the discriminant function analysis (DFA) using 18 morphological features identifying isopod by sex (top) and location (bottom). In the location DFA females were not included because the test focused on male morphological changes as individuals move from the gill to the tongue, just prior to changing sex.

Isopod Sex Female Intermediate Male Total %Correct Female 20 3 0 23 86.96% Intermediate 0 18 6 24 75.00% Male 0 5 26 31 83.87% Total correct = 64/78 = 82.05%

Isopod Location Gill Gill-Tongue Tongue Total %Correct Gill 13 2 1 16 81.25% Gill-Tongue 3 13 0 16 81.25% Tongue 6 3 14 23 60.87% Total correct = 40/55 = 72.73%

39 Table 2. Results from Chemical and Visual Cues. Statistical results from a laboratory experiment testing the effects of shadow and fish scent on mancae behavior using a mixed model. The scent treatment involved water infused with croaker scent or sterile water. The shadow treatment involved presenting mancae with a shadow, or no shadow.

Source effect degrees of freedom for burst speed = 106 and for change in swimming time and change in behavior = 139.

Burst speed SOURCE F P Scent 4.6429 0.0334* Shadow 30.9342 <0.0001* Scent x shadow 0.3524 0.5540 Change in swim time Scent 0.0508 0.8220 Shadow 27.1765 <0.0001* Scent x shadow 0.0006 0.9807 Change in behavior Scent 7.9754 0.0054* Shadow 195.5157 <.0001* Scent x shadow 11.8388 0.0008*

40 Figures

Figure 1. Seasonal Infection Trends. A) Seasonal frequency of isopods infecting fish as a function of fish size. Arrows indicate the median size of infected fish. B) Seasonal frequency of infected fish as a function of the number of isopods per infection.

A100 B 1000 Spring Spring 100 10 10

1 1 100 Early 1000 Early Summer Summer 100 10 10

1 1 100 Late 1000 Late Summer Summer 100 10

10 Number of Fish of Number 1 1 100 1000 Total Number of Isopods Total Fall Fall 100 10 10

1 1 100 1000 Winter Winter 100 10 10

1 1 0 5 10 15 20 0 1 2 3 4 5 6 Fish Size (cm) Number of Isopods

41 Figure 2. Isopod Location and Frequency. Location and frequency of isopod parasites occurring on Atlantic croaker, (Micropogonia undulatus) (n = 70). Locations were divided into three areas, the gills, the tongue and a cavity right behind the tongue next to the gills (T-G). Infected fish would have from 1(black bars) to 4 (white bars) parasites.

1.0 1 parasite

2 parasites 0.8 3 parasites

4 parasites 0.6

0.4

0.2

Fraction Fraction of Isopod Occurrence 0.0 Tongue T-G Gill Isopod Location

42 Figure 3. Isopod Size by Fish Size. Isopod size as a function of host fish size for female parasites (black circles, slope = 0.13), courting males (white triangles, slope = 0.064), and tending males (black triangles, slope = 0.029).

Female

Courting Male 2 Tending Male

1 Isopod(cm)Length

0 10 15 20 Fish Length (cm)

43 Figure 4. Isopod Brood Size by Female Size. Brood size of the isopod, C. excisa, in plotted against female isopod length (Brood Size = -286.91 + 515.72*Female Length, R² = 0.82, F = 81.92, P < 0.0001). Brood size includes total number of eggs, marsupiumites and mancae.

1200

800

Brood Brood Size 400

0 1 1.5 2 2.5 3 Isopod Length (cm)

44 Figure 5. Discriminant Function Analysis. First two dimensions of a discriminant function analysis (DFA) on morphological features of isopods. The DFA discriminated morphologies by sex (top) and location (bottom). To distinguish how morphological features changed as a result of location, only males and intermediates were used.

0.2 Female 0.15 Intermediate Male 0.1 0.05 0 -0.05 -0.1 -0.15 -0.2 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.2

Axis 2 Axis Gill 0.15 Gill-Tongue 0.1 Tongue 0.05 0 -0.05 -0.1 -0.15 -0.2 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 Axis 1

45 Figure 6. Isopod Morphology by Life Stage. (A) The average area of C. excisa separated by life stage (photos not to scale). The DFA was able to identify 6 traits that differed as C. excisa transitions from male to female. The six traits were converted into ratios by dividing the trait by the length of the isopod to standardize each isopod by size. The ratios of each trait are as follows: (B) Pleotelson width, (C) Basis 7 width [B7], (D) Gonopod length, (E) First antenna length, (F) Uropod perimeter, (G) Eye perimeter.

1.5 A

1

0.5 Isopod Area (cm²) Area Isopod

0 Juvenile Male Int Female 0.4 B E 0.4 0.3 0.3

0.2 0.2

0.1 Antenna 0.1 Pleotelson 0 0 C 0.16 F 0.8

0.12 0.6

0.08 0.4 B7

0.04 Uropod 0.2

0 0 D 0.2 G0.4 0.15 0.3

0.1 0.2 Eye

0.05 0.1 Gonopod 0 0 Male Int Female Juvenile Male Int Female

46 Figure 7. Isopod Gonopod Size by Isopod Size. Gonopod length of C. excisa males and intermediates as a function of isopod length. Symbols represent different locations in which individuals were found, the gill (black triangles, slope = 0.2), gill-tongue cavity (white triangles, slope = 0.13), and intermediate males found in the tongue (grey squares, slope = 0.04).

0.16 Gill

Gill-Tounge

0.12 Tongue

0.08

0.04 Gonopod Length (cm) Length Gonopod

0 0.3 0.6 0.9 Isopod Length (cm)

47 Figure 8. Mancae Energetics. (A) Average oxygen consumed by each batch of mancae of Cymothoa excisa from birth until their death (O₂ consumed = 1.12 - 0.04*Day, R² = 0.31, P = 0.03). (B) The average time mancae of Cymothoa excisa spent swimming as a function of age (Swim Time = 71.37 - 5.09*Day, R² = 0.97, P = 0.0004). (C) The average attack speed of Cymothoa excisa from birth until swimming halted (Speed = 6.06 - 0.27*Day - 0.03*(Day-6.54)², R² = 0.92, P < 0.0001). (D) The percent of mancae surviving as a function of age. The experiment lasted 20 days, but to compare against the other variables the figure stops at day 15 (Survivorship = 103.84 - 5.19*Day, R² = 0.98, P < 0.0001).

A 2.1 B 75

1.4 50 (mg/L)

₂ 0.7 25

O Time SwimTime (%) 0 0 0 5 10 15 0 5 10 15 C D 6 100

75 4 50 2

25

Surviving Surviving (%) Speed (cm/s) 0 0 0 5 10 15 0 5 10 15 Day Day

48 Figure 9. Mancae Behavior to Chemical Cues. Average time mancae spent swimming and resting in croaker (Micropogonias undulatus) water (dark gray bars), pinfish (Lagodon rhomboides) water (light gray bars) and sterile water (white bars). Swimming and resting were analyzed separately using a repeated measures ANOVA and letters above bars are representative of individual analyses.

250 a a 200 b b 150 a a Croaker

100 Pinfish Time (s) Time Sterile 50

0 Swimming Resting Behavior

49 Figure 10. Mancae Taxis to Chemical Cues. The percent of mancae of the isopod Cymothoa excisa positively orienting in a Y-maze to a specific water type. Trial 1 tested croaker water versus control water and Trial 2 tested croaker water versus pinfish water. A χ2 test was used to test for differences in manca orientation and an asterisk denotes a significant difference between the two treatments for each trial.

70 * 60 50 40 Croaker Sterile 30 Pinfish 20

10 Percent Orienting (%) Orienting Percent 0 Trial 1 Trial 2

50 Figure 11. Mancae Behavior to Chemical and Visual Cues. The percent of mancae of the isopod Cymothoa excisa that changed behavior in response to shadow and fish scent (a) The average first burst speed of the mancae, Cymothoa excisa. (b) The average percent time mancae of Cymothoa excisa spent in the water column 10 seconds after the trial began minus the average time spent in the water column 10 seconds prior to the trial. (c) The average number of mancae of Cymothoa excisa changing behavior 10 seconds prior to the start of each treatment compared to 10 seconds after each treatment started. For each behavior, different letters represent statistically different responses using a Tukey HSD post-hoc test (P < 0.05).

A 3 a ab Croaker 2.5 Sterile 2 bc 1.5 c 1

Speed Speed (cm/s) 0.5 0 B 25 a 20 a 15

10 Time Time 5 b b

Swimming(%) 0 -5 C 100 a 80 b 60

40 Behavior Behavior

Change Change (%) 20 c c 0 Shadow No Shadow Light Treatment

51

Appendix 1

Isopods were categorized by life stage (male, intermediate, female). The results show that male, intermediatemale,thatshowfemale).results The intermediate,(male, stage lifeby were categorizedIsopods with isopod length. length.isopodwith ( analysis.regressioneach for variable thedependentas used features 18 morphologicaltheofone each with variableindependenttheas was used (IL)lengthIsopodtheisopods. of features morphological 18 theof each 1 Appendix

Int

) and female eye perimeter, as well as intermediateas perimeter,welleyeas femaleand )

. .

Regression forMorphology.Isopod Analysis

gonopod

Table displaying the regression analyses for analysesTableregressionthe displaying length and female antenna length do not varydonot length antenna female and length

52 Appendix 2

Appendix 2. The seasonal prevalence of the isopod Cymothoa excisa on Atlantic croaker, (Micropogonias undulatus). Winter was excluded from data analysis due to few sampling trips. Error bars indicate the standard error for infection rates for each season.

0.5

0.4

0.3

of of Fish Infected 0.2

0.1 Fraction

0 Winter Spring E. Summer L. Summer Fall

53 Appendix 3

Appendix 3. Light curve. Light curve from the preliminary light intensity experiment to identify light intensity that the manca best responded (Light = 25.009e-0.442(Filter), R2 = 0.99). A series of filters were made by using one through nine pieces of window screen. A one-way ANOVA tested the number of manca swimming up as a function of filter number. Manca showed highest positive phototaxis to filter 3 (F = 8.25, d.f. = 35, P = 0.007, Tukey HSD post-hoc test, P < 0.05), which was used in the shadow response experiment.

30

25

/s) 2 20

15

10

Light Light (um photons/m 5

0 0 1 2 3 4 5 6 7 8 9 Number of Filters

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Vita

Colt William Cook was born in Wharton, Texas. After completing his work at Bay City

High School, Bay City, Texas, in 2003, he entered the University of Texas in Austin,

Texas. He received the degree of Bachelor of Science from the University of Texas at

Austin in 2007. During the following years he was employed as a research technician at the Mission-Aransas National Estuarine Research Reserve. In August, 2009, he entered

Graduate school at the University of Texas at Austin.

Permanent Address: [email protected]

This thesis was typed by the author.

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