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Home , Hippocampus kuda, Seahorse, Tiger tail seahorse

Variation in sound production by the Pot-bellied , abdominalis,

during feeding

A thesis submitted to the

Graduate School of the University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in the Department of Biological Sciences

of the College of Arts and Sciences

by

Brittany A. Hutton

B.S. Miami University

August 2017

Committee Chair: Pete Scheifele, Ph.D LCDR USN (Ret)

Cincinnati, OH

ABSTRACT

The Pot-bellied seahorse, Hippocampus abdominalis, produces acoustic signals during feeding behavior. The acoustic signal, referred to as a “click”, is typically accompanied by a head-motion called a “snick”, and both occur during foraging by . I determined that clicks are usually associated with snicks (although not always) , supporting the “rub-knock” stridulation mechanism of sound production common in seahorses. I examined variation in peak frequency (Hz) as well as peak intensity (Watts/m2) of feeding clicks, but found no significant difference between or between adults and juveniles. Analyses of clicking in light and dark environments revealed that seahorses click much more in the light and exhibit an increased number of clicks in the presence of food. Female seahorses clicked significantly more often than males, suggesting a possible role for on acoustic signals in this -role reversed . A body condition index (BCI) of residual square root area (cm2) was inversely correlated with mean peak frequency (Hz) of feeding clicks, suggesting the acoustic signal may contain information on mate size and quality. These results suggest that the function of sound production by seahorses during feeding is related to conspecific communication.

ii

iii Acknowledgements:

Over the course of my education as a graduate student here at the University of

Cincinnati I have had the fortunate experience of working with and being supported by many advisors, educators and colleagues. I’d like to extend my gratitude to the Newport for allowing me to conduct the entirety of my study on site with the use of their seahorses. First and for most I would like to thank both of my advisors, Dr. Pete Scheifele and Dr. George Uetz for their support of my education and research in the Department of Biological Sciences. Dr. Pete

Scheifele not only encouraged my interest in bioacoustics and work in animal welfare but opened lines of communication to allow me to conduct the entirety of my research projects through the Newport Aquarium. Dr. Gerorge Uetz was crucial in the experimental design of my projects and was consistently a wonderful source for insight into animal behavior and scientific writing. I would like to also extend my heartfelt thanks to my other committee member Dr. John

Layne who took time to coach me through the sensory physiology of my model system. The graduate students of the Uetz Lab were all extremely supportive during my time here. I would especially like to thank Brent Stoffer, Alex Sweger, Rachel Gilbert, Tim Meyer, Emily Pickett,

Maddi Lallo and Trinity Waals for all their continued support and advice. To the many other graduate students in the Department of Biological Sciences I thank you for making my time here at the University of Cincinnati so enjoyable. I’d like to thank the FETCHLABTM and all of its members for not only support of my research but also providing the necessary equipment for me to conduct my studies. Finally, I would like to extend a personal thank you to my fiancé

Christian Romanchek for all his love and support. To my parents, sisters and brothers I’d like to say thank you for providing the necessary escape from school life and the always gentle reminder to not worry so much and just enjoy myself and my time in graduate school.

iv Table of Contents

Abstract ii

Acknowledgements iv

List of Tables & Figures vii

General Introduction 1

Study Organism 6

Objectives and Hypotheses 6

Chapter

I. Characterization of sound production by the Pot-bellied seahorse (Hippocampus

abdominalis) during feeding 13

Abstract 14

Introduction 15

Methods 16

Results 18

Discussion 20

References 23

Tables and Figures 25

II. Contexts of Sound Production 33

Abstract 34

Introduction 34

v Methods 36

Results 38

Discussion 38

References 40

Tables and Figures 44

III. Other Considerations- Function of Sound Production in Seahorses 47

Introduction 47

References 53

vi List of Figures & Tables:

Figure 1.1: Scatterplot of feeding clicks and snicks

Figure 1.2: Spectrogram

Figure 1.3: Power spectrum of the baseline noise level

Figure 1.4: Power spectrum for feeding click

Figure 1.5: Mean Peak Frequency (Hz) of feeding clicks

Figure 1.6: Mean Peak Intensity (Watts / m2) of feeding clicks

Figure 1.7: Mean RMS Level (Watts / m2) of feeding clicks

Figure 1.8: Mean number of feeding clicks between sexes, adults, juveniles

Figure 2.1: Bar graph of number of feeding clicks in Light and Dark within Food and No Food

Figure 2.2: Scatterplot of Seahorse Standard Length (Hz) and Mean Peak Frequency (Hz)

Figure 2.3: Peak Frequency (Hz) x Residual BCI (Body Condition Index)

vii GENERAL INTRODUCTION

Communication can be defined as an exchange of information between a sender and a receiver, wherein the receiver will process the information from the signal produced by the sender and then respond accordingly (Bradbury & Vehrencamp, 2011). Animal communication uses a multitude of signaling modalities which can be varied depending on both the context in which the signal is relayed for an intended receiver or the environment (Stevens, 2013). These signals can be very complex and include multiple modalities (Partan & Marler, 1999).

Communication has a variety of modes from which it can be transferred. Communication can take the form of vibrational or acoustic cues (vocalizations), visual cues (color displays) or chemical cues (pheromones) (Bradbury & Vehrencamp, 2011; Stevens, 2013; Partan & Marler,

1999). Visual cues may include a coloration or “body language” that sends a signal to another individual such as in between (Endler, 1987) and swordtail (Morris &

Hankison, 2001; Basolo, 1990). Chemical cues are also used in courtship such as the pheromones in silk (Pollard et.al, 2012). Acoustic cues are used in many contexts such as courtship, predator-prey interactions and territorial defense (Bradbury & Vehrencamp, 2011).

When one or more of these modes are used to convey information, it is called multi-modal communication (Partan & Marler, 1999) such as the use of visual and vibratory cues used in courtship by the spider, Schizocosa ocreata (Taylor et.al 2005, Hebets & Uetz, 1999).

Acoustic communication is among the best studied modes (Bradbury & Vehrencamp,

2011). Many use vocalizations or sound in communication. Many species of use songs in courtship or territorial defense (Thorpe, 1961). Crickets will also produce sound via stridulatory organs (file-scraper mechanism) (Gerhardt & Huber, 2002). Sound is defined as the vibration of molecules as a wave propagates through a medium. Sound waves are characterized

1 by direction, speed, amplitude (loudness) and frequency (pitch) (Gerhardt, 1992; Gerhardt,

1998). Sound waves can be digitized to visualize as a spectrogram or waveform for easier analysis. During communication, these properties of acoustic signals are perceived and analyzed by the receiver of the sound (Gerhardt, 1992).

Acoustic signals are received via mechanoreceptors. These receptors can sense vibration either airborne (sound) or through the substrate (seismic). Seismic-borne vibration can be received via specialized structures on the animals; for , these signals are received by the slit sensilla and lyriform organs (Barth, 2004). Airborne sound signals are received by the animal ear. The sound is transmitted into the ear and to hair-cells that will bend in response to the signal depending on the frequency of the sound (Davis, 1957). These hair-cells start a cascade of action potentials transmitting information so that the animal can assess the signal and respond accordingly (Davis, 1957). Acoustic signals that are transported through water are perceived by different mechanisms depending on the aquatic animal receiving the sound.

Sound waves behave very differently across terrestrial and aquatic environments. The speed of sound in air is 343 m/s while the speed of sound in water is 1482 m/s; due to the higher density of water as a medium. The increased speed of sound in water results in sound waves with a much higher wavelength, which can therefore travel much further a property used by many baleen whales during conspecific communication (Edds-Walton, 2012). Due to these differences in sound wave propagation, terrestrial and aquatic animals differ in the way they communicate acoustically. Terrestrial animals typically communicate acoustically by using vocal folds that vibrate with airflow (Ladich & Winkler, 2017). While some aquatic animals produce sound with modified vocal folds outfit for communication over long distances (cetaceans); fish produce and

2 receive sound via different mechanisms that are best for short-distance communication (Ladich

& Winkler, 2017).

The unique properties of sound waves in water influence acoustic communication by marine and freshwater animals. Aquatic species use various sound perception and production mechanisms. Odontocetes (Toothed whales) receive sound waves from the lower jaw, this signal is then conducted through mandibular fat bodies to the middle ear then auditory brain for processing (Ladich & Winkler, 2017). Fish are thought to receive sound via the otolith (Ladich

& Winkler, 2017), the lateral line (Coombs et.al, 2014) and in some cases the swim bladder

(Ladich & Winkler, 2017). There is controversy surrounding whether or not sound reception by the otolith and lateral line are always equivalent in their detection efforts or if they can also work independently in signal reception (Coombs et.al 2014). Both the inner ear and lateral line systems use hair cells in signal transduction. The lateral line has a distribution of spatial sensory organs designed to respond to differences in water flow. The hair cells in the system will respond to the changes in motion and transmit this information as an electrical signal via synapses

(Coombs et.al 2014).

The variety of mechanisms for sound reception across aquatic animal species are associated with the multiple contexts in which the acoustic signals are produced. Some fish use acoustic signals in conspecific communication; for example, the fish (Chaetodon multicinctus) uses fin slaps and tail smacks in territorial defense (Tricas et.al, 2006). Other fish produce sound in predator-prey interactions, e.g., the Longspine Squirrelfish (Holocentrus rufus), which uses short bursts of sound in alarm calling behavior (Smith, 1992). It is well known that cetaceans use a variety of mechanisms for sound production such as the echolocating clicks

3 of odontocetes, which are produced through the nasal passage, and the low-frequency vocal projections of mysticetes (Ladich & Winkler, 2017).

The contexts in which acoustic signals occur can convey specific information for the receiver. For example, the multimodal signaling used by male cichlids in courtship is accomplished with an acoustic call that augments the visual displays that are meant to attract potential mates (Smith & van Staaden, 2010). The role acoustic signals play in courtship is crucial and a research topic of intense focus. In the instance of sex-role reversal (males brood young), many species will exhibit courtship displays differently such as female ornamentation

(Berglund & Rosenqvist, 2003; Rosenqvist & Berglund, 2011). This is often associated with female-female competition, such as in the Bornean where females produce a higher rate of calls and group around males during courtship (Vallejos et.al 2017). In monogamous species, such as seahorses, sex-role reversal can influence aspects of communication.

SEAHORSES AS A MODEL FOR UNDERWATER ACOUSTIC SIGNALING

Seahorses (Family: ) can exhibit both conventional sex roles and sex-role reversal depending on population density (Wilson & Martin-Smith, 2007) but also develop monogamous relationships (Jones et.al. 1998; Kavarnemo et.al. 2000; Vincent & Sadler, 1995).

Seahorses use a variety of signals in communication such as visual ornaments and acoustic signals (Rosenqvist & Berglund, 2011). The acoustic signals produced by seahorses occur under a variety of contexts (i.e. courtship, foraging, stress). These traits make them an excellent model system for studying sexual selection and sound production in aquatic environments

4 Members of the family Syngnathidae produce sound in various contexts. In cases of stress, seahorses will produce a “growl”. The mechanism for the growl is debated as either drumming the swim bladder with the lateral trunk muscles (Oliveira et.al, 2014) or vibration originating in the cheek region (Lim & Chong, 2015).

Seahorses also produce a sound referred to as a “click”. This click typically occurs with a visual behavior described as a “snick” that involves elevation of the snout in a rapid upwards motion. This motion coincides with a stridulation of the supraoccipital bone and coronet, this rub-knock mechanism produces a transient sound (Lim & Chong, 2015). This sound production occurs under various contexts, the most common being feeding (Colson et.al, 1998; Lim &

Chong, 2015; Oliveira et.al, 2014), although, some species produce this sound when under stress, in male-male competition or in courtship (Anderson, 2009; Woods, 2000; Oliveira et.al, 2014).

While the mechanism for sound production by seahorses is well-understood, the function of this acoustic signal in feeding remains largely unknown.

The most popular and prevailing hypothesis for the function of the click behavior during feeding is intraspecific communication (Lim & Chong, 2015). Seahorses are known to be monogamous and develop strong bonds with mates, and acoustic signals produced in foraging could be a way to alert conspecifics or a nearby mate to the location of prey items. A second possibility is the prey-stunning hypothesis; a theory that suggests a predator will produce a cacophonous sound to disorient prey items. This hypothesis was recently considered and rejected in a study that muted seahorses and observed prey-capture success rates with the seahorse species Hippocampus erectus (Anderson, 2009).

Another possible hypothesis is that seahorses use the clicking acoustic signal to sense the environment and/or detect prey via echolocation. Toothed whales (odontocetes) use echolocation

5 for predation with ultrasonic pulses of sound. These short-wavelength signals bounce off objects and are received by the sender for processing the location, size, shape and environment mapping

(Møhl, 1988). Finally, the null hypothesis is that the acoustic signal is simply a byproduct of the snick head motion. The studies described here were designed to assess variation in acoustic behavior produced by seahorses during feeding, and to evaluate hypotheses for the function of sound production in this context.

STUDY ORGANISM

The Pot-Bellied seahorse, Hippocampus abdominalis, is the largest seahorse species and known for having plastic sex-roles in both the field and laboratory environments (Wilson &

Martin-Smith, 2007). Pot-Bellied seahorses inhabit sheltered costal bays and harbors in the

South-West Pacific Ocean near New Zealand and Australia. Little research on this species exists and may be critical for conservation efforts, needed to preserve this species’ habitats and reduce risk of becoming caught in nets of fisheries. The studies described here were designed to assess variation in acoustic behavior produced by H. abdominalis during feeding, and to evaluate hypotheses for the function of sound production by seahorses in this context.

OBJECTIVES

The research described here is focused on assessing variation of acoustic signals produced by H. abdominalis during foraging. Underwater monitoring of the acoustic environment with a hydrophone, coupled with quantitative analyses of behavior, was used to

6 investigate the function of sound production by seahorses during feeding. There are three objectives, as follows.

Obj. 1 Define and describe acoustic signals in males, females and juveniles (Chapter I)

Considering that there exists little previous research on H. abdominalis, it was necessary to develop a baseline behavior for analyses and comparisons. I first examined and analyzed variation in acoustic parameters of feeding clicks between adult males and adult females.

Second, I measured differences in sound production behavior between adults and juveniles. In addition, I assessed the relationship between acoustic parameters produced during feeding and the visual behavior associated with this signal.

Obj. 2 Assess variation in acoustic signals in different contexts (Chapter II)

Obj. 2a Presence/absence of food

I studied the role of the presence of prey on the production of sound by seahorses. I predicted that seahorses most commonly produce clicks in the context of foraging and would increase the number of clicks in the presence of food.

Obj. 2b Presence/absence of visual cues: Light vs. dark environment

I investigated how light and dark environments influence the number of clicks produced by seahorses. The dark environment eliminated visual cues to test the prediction that the amount of clicking will be reduced due to a decreased ability to sense prey items visually. Results of the light-dark experiment might also serve as a test of the hypothesis of seahorse use of echolocation in prey detection, which does not require visual information and should not be affected

7 Obj. 2c Body Size and Condition

By measuring seahorse standard length and developing a body condition index (BCI) I will investigate how the acoustic parameters of the click signal vary with body size and condition.

Obj. 3 Other Considerations: Echolocation & Communication (Chapter III)

Two hypotheses for describing the function of sound production by seahorses during foraging are echolocation and conspecific communication. I will comment on each of these hypotheses, show how they might be addressed within the limitations of this study.

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12 CHAPTER I

Characterization of sound production by the Pot-bellied seahorse (Hippocampus

abdominalis) during feeding

Brittany A. Hutton University of Cincinnati, 3202 Eden Ave., Cincinnati, Ohio USA 45267, [email protected]

Laurel Johnson Newport Aquarium, 1 Aquarium Way, Newport, KY 4107

George Uetz University of Cincinnati, Department of Biological Sciences. PO Box 210006, Cincinnati, OH 45221

Peter M. Scheifele University of Cincinnati, 3202 Eden Ave., Cincinnati, Ohio USA 45267, [email protected]

Department of Biological Sciences

University of Cincinnati

P.O. Box 210006

Cincinnati, OH 45221-0006

Formatted for submission to Journal of Acoustical Society of America- Express Letters

13 ABSTRACT

Sound production by the Pot-bellied Seahorse, Hippocampus abdominalis, during feeding was quantified at the Newport Aquarium (KY). Adults and juveniles were tested individually and observed with video and audio recordings. Spectra PLUS was used to evaluate the peak frequency, intensity and number of clicks (acoustic signal) present in the audio recordings. Video recording allowed for analysis of snick (visual behavior) occurrence. Clicks were found to be significantly positively correlated with snicks, supporting the “rub-knock” stridulation mechanism of sound production proposed for seahorses. Female seahorses clicked significantly more often than males. The mean peak frequency (Hz) of feeding clicks did not significantly differ between males and females or between adults and juveniles. The reduction in click behavior by males could be attributed to decreased foraging, increased stress behavior or an attribute of sex-role reversal.

14 INTRODUCTION

Seahorses produce a sound referred to as a “click” via stridulation of the supraoccipital bone and coronet (Lim & Chong, 2015). This sound production occurs under various contexts, the most common being feeding (Colson et.al, 1998; Lim & Chong, 2015; Oliveira et.al, 2014).

However, some species will also produce this sound when under stress, in male-male competition or in courtship (Anderson, 2009; Woods, 2000; Oliveira et.al, 2014). While the stridulation mechanism for sound production by seahorses is well-understood, the function of this acoustic signal in feeding remains unknown. The most popular and prevailing hypothesis for the function of the click behavior during feeding is intraspecific communication (Lim & Chong,

2015; Oliveira et.al, 2014) where seahorses could be indicating the presence of food to a nearby mate or an individual could use clicks to locate nearby mates. A second possibility is the prey- stunning hypothesis, which was recently considered in a study that muted seahorses and observed prey-capture success rates with the seahorse species Hippocampus erectus (Anderson,

2009). Additionally, there is the hypothesis that seahorse use the clicking acoustic signal to echolocate prey items. The null hypothesis for the function of the click during feeding is that the acoustic signal is a byproduct of the snick motion.

This click signal is produced by a stridulatory motion between the supraoccipital bone and cornet of the seahorse (Colson et.al, 1998; Lim & Chong, 2015; Oliveira et.al, 2014). Often these clicks are associated with feeding strikes, which is the most common context in which clicking is produced. A motion of the head referred to as a “snick” is produced prior to the feeding strike and is often associated with the click (the acoustic signal). (Lim & Chong, 2015;

Fish, 1958; Oliveira et.al, 2014).

15 The Pot-Bellied seahorse, Hippocampus abdominalis, inhabits sheltered costal bays and harbors in the South-West Pacific Ocean near New Zealand and Australia. Little research exists, but is critical to the conservation efforts needed to preserve its habitats and reduce risk of becoming a byproduct of fisheries. Our overarching goal is to determine the function of the seahorse click during feeding. We conducted experiments to examine how clicking behavior varies in occurrence, pattern and pitch between sexes and between adults and juveniles.

METHODS

Seahorses were kept in a community tank located at the Newport Aquarium (KY). The tank is approximately 350 gallons (pH 8.0+; salinity 34-36ppt; temp: 64-68 ̊C). In the community tank, approximately 50 individuals are grouped together. Seahorses are fed deceased mysis

(Order: ) and brine (Family: Artemiidae) shrimp twice a day.

For observational studies, an isolated acrylic tank (24in x 11in x 14in) of approximately

13 gallons was filled with filtered water with a pH of 8.0+, temperature of 64-68 ̊C and salinity was kept between 34-36ppt. Adult male (N = 13), adult female (N=19) and juvenile (N=6) seahorses were used for this study. Seahorse were tagged for identification with a small elastic band looped around the individual’s neck. Each band had a FloyTag number corresponding to the individual’s identity. Food was withheld from seahorses for at least 16 hours before testing.

Individuals were transferred from the community tank to the test tank, without air exposure, and allowed 1 hour to acclimate.

A video camera (Nikon Coolpix Full HD Waterproof Camera) was placed on a tripod in front of the tank to monitor behavior, and a hydrophone transducer (SQ-26, Research

16 Inc.) was attached to a recorder (M-Audio Microtrack II) for acoustic monitoring. The hydrophone was placed 12.7 cm deep at the center of the tank. Audio and video recordings began at the same time two minutes before food introduction. After two minutes of behavior monitoring an aliquot of were introduced. Feeding behavior by the individual was observed for ten minutes following the exposure to a food source. After the trial the individual was removed and returned to the community tanks, and the water in the experimental tank was siphoned clean for subsequent trials.

The behaviors of interest are the “snick”, a rapid upwards motion of the head prior to prey consumption, and the “click”, the stridulatory sound associated with this head motion. The snick was captured via the video recording and the clicking sound was observed with the hydrophone. I evaluated the number of snicks, the number of clicks, the time of onset for each click and snick as well as the frequency of each click. Acoustic recordings were analyzed with

Spectra Plus ® sound analysis software. I used spectral analyses, time-series analyses, power analyses and finally a hierarchical cluster analysis to quantify variance of acoustic signals during feeding behaviors of the seahorses in the previously described contexts.

17 RESULTS

The number of clicks was significantly positively correlated with the number of snick behaviors observed in linear fashion (Fig. 1.1). This linear regression was significant for females

(R2 = 0.6758), and males (R2 = 0.8455) as well as juveniles (R2 = 0.8959). However, this regression also shows a slope of less than one (0.5563 for females, 0.9385 for juveniles, and

0.9564 for males), suggesting that not all snick behaviors result in a click sound.

A spectrogram offered visualization of the acoustic parameters representing a feeding click from a seahorse (Fig. 1.2). An analysis of the background sound intensity (Fig.1.3) versus the click intensity revealed that at a click frequency of 486.49 Hz (average frequency for females) the intensity of the click was 6.208e-12 Watts/m2 or 58.7 dB re 1uPa (Fig. 1.4). At the same frequency, the baseline noise intensity is 1.901e-12 Watts/m2 or 53.56 dB re 1 uPa. The

SNR (AVG Signal to Noise Ratio) = 1.10.

To assess variation within and between individuals a multivariate hierarchical cluster analysis of click parameters (i.e. peak frequency (Hz) and peak intensity (Watts/m2) of clicks) was conducted. If signal parameters were clustered either by individual or other descriptor categories (sex, age), a high level of between-individual difference would be suggested. The analysis revealed that data did not cluster by individual, sex or age (suggesting a lack of consistent individual patterns, and subsequent analyses continued using aggregated means of acoustic properties of individuals for further analysis.

There was no significant difference among males, females and juveniles in mean peak frequency (Hz) (One-Way ANOVA: F2,35 = 0.0738, p = 0.9290) (Fig.1.5). There was no

2 significant difference in mean peak intensity (Watts/m ) of clicks (One-Way ANOVA: F2,34 = 1

18 .5702, p = 0.223) (Fig. 1.6) between males, females and juveniles. Likewise, an analysis of median peak frequency (Hz) of feeding clicks revealed no significant difference between adult females, adult males and juveniles (One-way ANOVA: F2,35 = 0.1843, p = 0.8325). The minimum frequency (females, 79.745 Hz; males, 117.135 Hz; juveniles, 174.516 Hz) did not differ between ages or sexes (One-way ANOVA: F2,35 = 1.5374, p = 0.2291). There was also no significant difference (One-way ANOVA: F2,35 = 0.2758, p = 0.7606) in maximum frequency

(females, 1281.073 Hz; males, 1299.445 Hz; juveniles, 1541.725 Hz).

Analyses of overall signal amplitude or intensity, measured as RMS (root-mean-square)

Level (Watts/m2) and collected as the area under the curve of the power spectrum (Fig. 1.2), revealed no significant difference between groups for adult males, adult females and juveniles

(One-way ANOVA: F2,29 = 1 .2499, p = 0.3015) (Fig. 1.7). There was a significant difference in the number of feeding clicks between males, females and juveniles (One-way ANOVA: F2,37 =

3.5378, p = 0.0393). A post-hoc test (Tukey HSD) revealed the difference to be between adult males and adult females, with female seahorses clicking more than males (Fig. 1.8).

19 DISCUSSION

Results show that sex of a seahorse likely determines the amount of snicking behavior and clicking signals during feeding, with females clicking and snicking more than males. We suspect this might be due to the likely stressful conditions of transport from the community tank to the experimental tank (Wright et.al, 2007). Seahorses are known for susceptibility to stressful conditions which in turn can often cause harmful consequences (Anderson, 2009; Matsunga &

Rahman, 1998). Common stress behaviors include piping, snout pulling and dwelling at the bottom of the enclosure. These behaviors were frequently observed when the seahorse was placed in the isolated experimental tank. The hour acclimation time was designated to allow the individuals time to recover from the stressful transport. When food was introduced to the tank the females would begin to feed but many males continued to exhibit stress behavior.

Male seahorses are unique for their sex roles (); perhaps this could be an underlying reason for male susceptibility to stress. Seahorse male pregnancy involves a large amount of hormone regulation. A key hormone in the process for seahorses is which functions in regulation of the , growth of embryos and osmoregulation in the brood pouch where the embryos are stored. Much of parental behavior has been showed to be tied to prolactin regulation. This could be one of the hormonal influences of behavior that leads to increased male stress after transport to the experimental tank. The influence of prolactin on secretion of testosterone and progesterone in male seahorses also implies variation in hormonal control that influences male behavior. (Stolting & Wilson, 2007)

20 The frequency range in H. abdominalis clicks varied from 100-1400 Hz. While the frequency (Hz) of the click fluctuated within and among individuals, results showed no difference in mean frequency between male and female H. abdominalis. Previous acoustic recordings of the click from other seahorse species, such as Hippocampus hudsonius were noted to peak at 4.8 kHz (Fish, 1953). Colson et.al (1998) observed Hippocampus zosterae clicking at a frequency range of 2.65 - 3.43 kHz and Hippocampus erectus at 1.96 - 2.35 kHz. Most recently a study noted to exhibit a range of click frequencies between 1- 4.8 kHz

(Chakrabortya et.al 2014). Given the large size of H. abdominalis, it is reasonable to assume that our results included a lower mean frequency of approximately 450Hz. This trend of decreased click frequency (Hz) with increased size was observed in comparisons of Hippocampus erectus with Hippocampus zosterae (Colson et.al, 1998).

These results also allow some insight to the function of sound production by seahorses.

The linear regression of behaviors and sounds shows that snick behaviors are usually followed by click sounds confirming a “rub-knock” stridulation mechanism as the method of sound production. The hypothesis of clicking as a mere by-product of head movement during feeding cannot be rejected entirely, although a slope of < 1.0 for “Number of Clicks” x “Number of

Snicks” regression in females is strongly suggestive. The fact that not all snick behaviors are associated with clicks in a one-to-one manner cannot be fully explained. Consequently, it is necessary to continue research into the various contexts (i.e. courtship, male competition and stress) in which this acoustic signal is produced. We propose further research into acoustic signal production by seahorses is necessary to determine the function of the click during feeding and other contexts.

21 ACKNOWLEDGEMENTS

The entirety of the experiment took place on site at the Newport Aquarium (KY), and we would like to thank them for allowing us to use their equipment and work with their Pot-Bellied

Seahorses. A special thanks is extended to Laurel Johnson for her assistance in the experimental design and tagging of seahorses. The support and advice from the members of the Uetz Lab in the Biological Sciences Department at the University of Cincinnati, Brent Stoffer, Alex Sweger,

Rachel Gilbert, Tim Meyer, Emily Pickett, Maddie Lallo and Trinity Waals was greatly appreciated. The FETCHLAB™ was a crucial component to the development and execution of this study. Thank you to all lab members for the assistance with this project.

22 REFERENCES

Anderson P.A. 2009. The functions of sound production in the lined seahorse, Hippocampus

erectus, and effects of loud ambient noise on its behavior and physiology in captive

environments. (Doctoral Dissertation). University of Florida

Chakrabortya B., Sarana A. K., Sinai Kuncolienkerb D., Sreepadaa R.A., Harisa K., Fernandes

W. 2014. Characterization of Hippocampus kuda (Bleeker, 1852) - yellow seahorse

feeding click sound signal in a laboratory environment: An application of probability

density function and power spectral density analyses. Bioacoustic. 23(1): 1-14.

Colson D.J, Patek S.N, Brainerd E.L, Lewis S.M. 1998. Sound Production during Feeding in

Hippocampus Seahorses (Syngnathidae). Environmental Biology of Fishes. 51: 221–229

Lim, A C O, and V C Chong. 2015. Sound Production in the Tiger-Tail Seahorse Hippocampus

comes : Insights into the Sound Producing Mechanisms. Journal of the Acoustical Society

of America. 138 (1): 404-412

Matsunga T. and Rahman A. 1998. What brough the adaptive immune system to vertebrates?-

The jaw hypothesis and the seahorse. Immunological Reviews. 166: 177-186.

Oliviera T.P.R., Ladich F., Abed-Navandi D., Souto A.S. Rosa I.L. 2014. Sounds produced

by the longsnout seahorse: a study of their structure and functions. Journal of

Zoology. 294: 114-121.

Stolting K.N & Wilson A.B. 2007. Male pregnancy in seahorses and pipefish: beyond the

mammalian model. BioEssays. 29: 884-896.

23 Woods C.M.C. 2000. Preliminary observations on breeding and rearing the seahorse

Hippocampus abdominalis (Teleostei: Syngnathidae) in captivity. New Zealand Journal

of Marine and Freshwater Research. 34:3, 475-485.

Wright K.A, Woods C. M. C. Gray B.E, Lokman P.M. 2007. Receovery from acute, chronic and

transport stress in the pot-bellied seahorse Hippocampus abdomialis. Journal of Fish

Biology. 70:1447-1457.

24 TABLES & FIGURES

60

50

40 Female Male 30 Juvenile 20 Linear (Female)

Number ofClicks Number 10 Linear (Male) Linear (Juvenile) 0 0 50 100 -10 Number of Snicks

Figure 1.1: Scatter plots of linear regression of the number of clicks and the number of snicks within females (slope = 0.5563), within males (slope = 0.9564) and within juveniles

(slope = 0.9385).

25

Figure 1.2: Spectrogram for visualization of a single feeding click from an adult female pot- bellied seahorse.

26

Figure 1.3: Power spectrum of the baseline noise level in the isolation tank without any seahorses present. Red line represents the average. Blue line represents the real-time relative amplitude and frequency.

27

Figure 1.4: Power spectrum for an adult female H. abdominalis showing an acoustic recording of a single feeding click. Red line represents the average. Blue line represents the real-time relative amplitude and frequency.

28 600

500

400

300

200

Mean Peak Frequency (Hz) Frequency Peak Mean 100

0 Adult Female Adult Male Juvenile

Figure 1.5: The mean peak frequency (± S.E.) of feeding clicks for males (n = 15; 463.41 ±

49.01), females (n = 19; 486.02 ± 40.951) and juveniles (n = 6, 486.73 ± 59.13). No significant difference in the peak frequency (Hz) was observed (One-way ANOVA: F2,35 = 0.0738, p =

0.9290).

29 1.2E-09

1E-09

8E-10 (dB re 1uPa) re (dB

6E-10

4E-10

Mean Click Intensity Click Mean 2E-10

0 Adult Female Adult Male Juvenile

Figure 1.6: Mean peak intensity (Watts/m2) (± S.E.) of feeding clicks for males (n = 13;

7.63E-10 ± 2.66E-10), females (n = 18; 4.38E-10 ± 1.23E-10) and juveniles (n = 6, 2.09E-10 ±

7.93E-11). No significant difference in intensity of clicks was observed (One-way ANOVA:

F2,34 = 1 .5702, p = 0.223) between males and females

30 0.16

0.14

) 0.12 2 0.1

0.08

0.06

RMS Level (Watts/m Level RMS 0.04

0.02

0 Adult Female Adult Male Juvenile

Figure 1.7: Mean RMS (Root Mean Square) Level (Watts/m2) (± S.E.) of feeding clicks for males (n = 12;), females (n = 14; 10) and juveniles (n = 6). No significant difference in intensity of clicks was observed (One-way ANOVA: F2,29 = 1 .2499, p = 0.3015) between males and females

31 35

30

25

20

15 Number ofClicks Number 10

5

0 Adult Female Adult Male Juvenile

Figure 1.8: Mean number of clicks (± S.E.) for males (n = 15; 13.47 ± 4.1792), females (n =

19; 26.11 ± 3.51) and juveniles (n = 6, 19.17 ± 5.41). A significant difference in the number of clicks was observed (One-way ANOVA: F2,37 = 3.5378, p = 0.0393). between males and females.

32 CHAPTER II

Contexts of Sound Production

Brittany A. Hutton, Laurel Johnson, George Uetz and Peter M. Scheifele

Department of Biological Sciences

University of Cincinnati

P.O. Box 210006

Cincinnati, OH 45221-0006

33 ABSTRACT

Sound production behavior of the Pot-bellied Seahorse, Hippocampus abdominalis was observed in light and dark environments in the presence and absence of prey items, and acoustic parameters were characterized. Seahorses produced clicks more often in the presence of food items and more often in light environments. Reduced clicking in the dark, along with calculations of transmission distance of acoustic signals within the confines of a small tank allowed rejection of the hypothesis of an echolocation function. Body size (measured as Standard

Length (SL) affected some aspects of acoustic signals. An inverse correlation between mean peak frequency (Hz) of feeding clicks and SL was found for females, but not males. A similar negative correlation between peak frequency (Hz) of feeding clicks and a residual body condition index (BCI) was found for both males and females, suggesting that acoustic signals may contain information regarding a mate’s quality.

INTRODUCTION

The effect of anthropogenic noise in natural aquatic environments as well as zoos and continues to be an area of concern, because many marine and freshwater animals use sound in conspecific communication, courtship and predator-prey interactions (Bradbury &

Vehrencamp, 2011). Underwater acoustic signaling by animals is especially interesting, because in aquatic environments, sound waves travel much faster and much farther than airborne sound in terrestrial environments. The contexts in which these sounds occur can influence the production and reception of the acoustic signal, and can sometimes provide insight to its function.

34 A common context of sound production by fish is in foraging or predator-prey interactions. For example, the Longspine Squirrel Fish, (Holocentrus rufus) produces a staccato alarm call in the presence of a predator, to warn conspecifics into hiding or initiate mobbing behavior (Smith, 1992). One of the most unique examples of the use of sound during predation is in echolocation, frequently produced by marine such as and whales

(Nachtigall, 1980). Echolocation works by a sender producing a sound as a series of high frequency transient sound (Møhl, 1988). The acoustic signal will reflect off objects and bounce back to the sender as an echo, allowing location of prey. However, the cod, Gadus morhua, is able to perceive the ultrasonic pulses produced by echolocating toothed whales (odontocetes) and thereby escape predation (Astrup & Mohl, 1993)

While many fish species use ornaments or visual behaviors in courtship, such as pipefish

(Berglund A. and Rosenqvist G. 2003; Berglund, 2000; Endler, 1987; Morris & Hankison, 2001;

Basolo, 1990), some species may also produce acoustic signals to seek out a mate and exhibit courtship behaviors to relay quality/fitness to a conspecific. Some species of seahorses have been known to use acoustic signals in the form of “clicks” in courtship settings (Anderson, 2009;

Woods, 2000).

Seahorses (Family: Syngnathidae) produce sound, particularly during foraging, courtship and when under stress (Anderson, 2009; Woods, 2000; Wright et.al. 2007). The acoustic signal, or click, is often associated with a visible head motion called a “snick”. The click signal is produced by a stridulatory motion between the supraoccipital bone and cornet of the seahorse

(Colson et.al, 1998; Lim & Chong, 2015; Oliveira et.al, 2014). In the case of Hippocampus comes it was determined that every click followed the snick head movement (Lim & Chong,

2015; Oliveira et.al, 2014). The Pot-Bellied seahorse, Hippocampus abdominalis, inhabit

35 sheltered costal bays and harbors in the South-West Pacific Ocean near New Zealand and

Australia, and are known to produce sound while feeding. As shown previously (Chapter I) these clicks are associated with snicks, although not always. This chapter elaborates on how the clicking behavior of H. abdominalis is influenced by environmental contexts (the presence of food; light and dark conditions) and individual size and body condition.

METHODS

Environment & Food Presence

All individuals were housed at the Newport Aquarium (KY) in a community tank (pH

8.0+; salinity 34-36ppt; temp: 64-68 ̊C). Approximately adult 50 seahorses of varying age were kept together in the community tank. Individuals were selected at random and tagged (Floy Tag) for observation and recording under light and dark conditions. Each seahorse was placed into an isolated experimental tank with water conditions approximately identical to the community tank.

A hydrophone transducer (SQ-26, Crustacean Research Inc.) was placed at the center of the tank. An audio recorder (M-Audio Microtrack II) was used to listen for clicks over the entire trial.

An ELIVE aquarium track light was used as the light source with three white LED’s

(approximately 80 lumens). The light source was turned on for the light portion of the trial and then turned off for the dark portion of the trial. The sides of the tank were covered in blackout curtains to eliminate light penetration and create the dark environment (approx. lumens).

Each seahorse was allowed one hour to acclimate to the new tank environment under either light or dark conditions. While each seahorse was recorded under both light and dark

36 conditions, the order of exposure to lighting environment was randomly alternated between trials

(Light then Dark or Dark then Light). After two minutes an aliquot of brine shrimp (Family:

Artemiidae) was introduced to the tank and the seahorse sound production behavior was recorded for five minutes. After five minutes the light fixture was turned on/off to introduce the opposite lighting condition, either light or completely dark. In either case, the seahorse was subsequently allowed to feed for five minutes (a total trial length of 12 minutes).

Body Size & Condition

Estimation of standard length (SL) for seahorses was determined with screenshots from video recordings and the use of a software program (Image J) to take pixel measurements from a known reference length. The specifications for acquiring SL measurements followed standard methodology (Lourie, 2003) with measurements taken of the head length (HL), trunk length

(TrL) and tail length (TaL). A rough estimate of Area (cm2) was also acquired with Image J to determine Body Condition Index (BCI).

The Standard Length (cm) was used to calculate a Residual Body Condition Index (BCI).

A linear regression of Linearized Area (√ Area) x Standard Length (SL) was used to derive residual values, which are commonly used as a body condition index (Jakob et al. 1996; Marshall et al. 1999). Residuals above the trendline were considered to represent seahorses with greater- area for a given standard length, indicating relative “fatness” or better condition. Residuals below the trendline were considered slimmer and smaller indicating lower condition. The estimates for BCI were derived from these residuals and plotted against the mean peak frequency

(Hz) of feeding clicks.

37

RESULTS

Environment & Food Presence

A two-way repeated measures analysis with both presence/absence of food and light/dark as treatments revealed significance for both Light/Dark (F1,48 = 6.0195, p = 0.0178) as well as

Food/No Food (F1,48 = 9.1449, p = 0.0040). The interaction for these main effects

Light/Dark*Food/No Food was also significant (F1,48 = 6.5925, p = 0.0134) The number of clicks was greatest in light in the presence of food (Fig. 2.1).

Body Size & Condition

Using standard length (SL) measurements, a regression analysis was used to compare mean peak frequency (Hz) of feeding clicks with size dimensions. A significant negative correlation between click frequency (Hz) and overall size was found for female seahorses (but not males or juveniles), where frequency decreased with increased SL (cm) (R2 = 0.1371) (Fig.

2.2). An additional regression analysis determined that mean peak frequency (Hz) was negatively correlated with residual BCI values (Fig. 2.3) for both females (F1,10 = 5.0977, p = 0.0475) and males (F1,7 = 16.5835, p = 0.0047).

DISCUSSION

The results of this study revealed that seahorses click more in the presence of food in light environments than in dark environments and/or the absence of food. These results suggest

38 that visual cues are important to foraging, which is the context where we encounter sound production in Pot-bellied seahorses most often. Because sound production by seahorses decreased in the absence of visual cues, these results also support rejection of the echolocation hypothesis as an explanation for the use of sound production by seahorses when foraging (see discussion in Chapter III below). Seahorses tend to be site specific and are often found in the field in close proximity to pair bond members (Vincent & Sadler, 1995). Therefore, individuals may signal more in the presence of food to indicate to nearby conspecifics that prey is close by.

The negative correlation between click frequency (Hz) and overall size found for female seahorses (but not males or juveniles) may reflect an overall trend for seahorses. The results from the previous study (Chapter I) suggested a frequency range for Hippocampus abdominalis to be

0.1 – 1.4 kHz. H. abdominalis is the largest seahorse species (up to 35 cm). Between species comparisons revealed that Pot-bellied Seahorses have a much lower frequency range than

Hippocampus zosterae (2.65 - 3.43 kHz), Hippocampus erectus (1.96 - 2.35 kHz) (Colson et.al

1998), and Hippocampus kuda (1- 4.8 kHz) (Chakrabortya et.al 2014). The sizes for H. zosterae,

H. erectus, and H. kuda are 5 cm, 12.7 cm, and 17 cm respectively. Considering the relationship between mean peak frequency (Hz) of feeding clicks to body condition in H. abdominalis perhaps frequency is influenced by size between and within species. More research is necessary to assess the relationship of size to acoustic parameters of clicks in other Syngnathidae species.

Seahorse body condition was negatively correlated with mean peak frequency (Hz) of feeding clicks. In other words, as the condition of the seahorse improved (higher BCI), or as a seahorse was larger/fatter the frequency (Hz) of the acoustic signal decreased. Members of the family Syngnathidae are known to prefer larger body sizes during mate selection (Berglund &

Rosenqvist, 2003; Rosenqvist & Berglund, 2011; Kavarnemo et.al. 2007; Mattle & Wilson,

39 2009). Therefore, there is a possibility that these signals might serve a function in intra-specific communication as condition/quality indicators to potential mates.

ACKNOWLEDGEMENTS

The entirety of this study took place at the Newport Aquarium (KY), and we would like to thank them for allowing us to use their equipment and work with their Pot-Bellied Seahorses.

We would like to extend a special thanks to Laurel Johnson for her assistance in the experimental design and tagging of seahorses. The support and advice from the members of the Uetz Lab in the Biological Sciences Department at the University of Cincinnati, Brent Stoffer, Alex Sweger,

Rachel Gilbert, Tim Meyer, Emily Pickett, Maddie Lallo and Trinity Walls was greatly appreciated. The FETCHLAB™ was a crucial component to the development and execution of this study. Thank you to all lab members for the assistance with this project.

REFERENCES

Anderson P.A. 2009. The functions of sound production in the lined seahorse, Hippocampus

erectus, and effects of loud ambient noise on its behavior and physiology in captive

environments. (Doctoral Dissertation). University of Florida

Astrup, J & Mohl B. 1993. Detection of intense ultrasound by the cod Gadus morhua. Journal of

Experimental Biology. 182: 71–80.

Basolo A.L. 1990. Female Preference Predates the Evolution of the Sword in Swordtail Fish.

Science. 250: 808-810.

40 Berglund A. 2000. Sex role reversal in pipefish: female ornaments as amplifying handicaps.

Annales Zoologica Fennici. 37: 1-13.

Berglund A. and Rosenqvist G. 2003. Sex role reversal in pipefish. Advances in the Study of

Behavior. 32: 131-167.

Bradbury J.W. and Vehrencamp S. 2011. Principles of Animal Communication. Sunderland,

MA: Sinauer Associates.

Chakrabortya B., Sarana A. K., Sinai Kuncolienkerb D., Sreepadaa R.A., Harisa K., Fernandes

W. 2014. Characterization of Hippocampus kuda (Bleeker, 1852) - yellow seahorse

feeding click sound signal in a laboratory environment: An application of probability

density function and power spectral density analyses. Bioacoustic. 23(1): 1-14.

Colson D.J, Patek S.N, Brainerd E.L, Lewis S.M. 1998. Sound Production during Feeding in

Hippocampus Seahorses (Syngnathidae). Environmental Biology of Fishes. 51: 221–229

Endler J.A. 1987. Predation, light intensity and courtship behaviour in Poecilia

reticulata (Pisces: Poeciliidae). Animal Behaviour.35(5): 1376 – 1385

Jakob, E. M., S.D. Marshall, Uetz, G.W.. 1996. Estimating fitness: a comparison of body

condition indices. Oikos 77: 61-67.

Kavernemo C., Moore G.I., Jones A.G. 2007. Sexually selected females in the monogamous

Western Australian seahorse. Proceedings of the Royal Society B. 274: 521-525.

Lim, A C O, and V C Chong. 2015. Sound Production in the Tiger-Tail Seahorse Hippocampus

comes : Insights into the Sound Producing Mechanisms. Journal of the Acoustical Society

41 of America. 138 (1): 404-412

Lourie S. 2003. Measuring Seahorses. Project Seahorse. Version One.

Marshall, S.D., E. M . Jakob, Uetz, G. W.. 1999. Re-estimating fitness: can scaling issues

confound condition indices? Oikos 87:401-402

Mattle B. and Wilson A.B. 2009. Body size preferences in the pot-bellied seahorse Hippocampus

abdominalis: choosy males and indiscriminate females. Behavioral Ecology and

Sociobiology. 63(10): 1403-1410.

Møhl B. 1988. Target Detection by Echolocating Bats. Animal Sonar. 156: 435-450.

Morris M.R. and Hankison S.J. 2001. Sexual selection and species recognition in the pygmy

swordtail, Xiphophorus pygmaeus: conflicting preferences. Behavioral Ecology and

Sociobiology. 51(2): 140-145.

Nachtigall P.E. 1980. Odontocete Echolocation Performance on Object Size, Shape and

Material. In: Busnel RG., Fish J.F. (eds) Animal Sonar Systems. NATO Advanced

Study Institutes Series (Series A: Life Sciences), vol 28. Springer, Boston, MA.

Oliveira T.P.R., Ladich F., Abed-Navandi D., Souto A.S. Rosa I.L. 2014. Sounds produced

by the longsnout seahorse: a study of their structure and functions. Journal of

Zoology. 294: 114-121.

Partan S., Marler P. 1999. Communication goes multimodal. Science. 283(5406):1272-3.

Rosenqvist G. and Berglund A. 2011. Sexual signals and mating patterns in Syngnathidae.

Journal of Fish Biology. 78: 1647-1661.

42 Smith R.J.F. 1992. Alarm Signals in Fish. Reviews in Fish Biology and Fisheries. 2:33-63.

Vincent A.C.J. and Sadler L.M. 1995. Faithful pair bonds in wild seahorses, Hippocampus

whitei. Animal Behavior. 50: 1557-1569.

Wilson A.B., Martin-Smith K.M. 2007. Genetic monogamy despite social promiscuity in the pot-

bellied seahorse (Hippocampus abdominalis). Molecular Ecology. 16: 2345-2352.

Woods C.M.C. 2000. Preliminary observations on breeding and rearing the seahorse

Hippocampus abdominalis (Teleostei: Syngnathidae) in captivity. New Zealand Journal

of Marine and Freshwater Research. 34:3, 475-485.

Wright K.A, Woods C. M. C. Gray B.E, Lokman P.M. 2007. Receovery from acute, chronic and

transport stress in the pot-bellied seahorse Hippocampus abdomialis. Journal of Fish

Biology. 70:1447-1457.

43 TABLES & FIGURES

Figure 2.1: Mean number of clicks (± S.E.) produced by seahorses recorded under different treatment conditions.

44 900 800 700 600 500 Female 400 Male 300 Juvenile 200 Linear (Female)

100 Mean Peak Frequency (Hz) Frequency Peak Mean 0 0 10 20 30 40 Standard Length (cm)

Figure 2.2: Scatterplot of linear regression of Standard Length (cm) against Mean Peak

Frequency (Hz). Regression analysis reveals a negative correlation between frequency and length. Decreased frequency is associated with increased standard length.

45

Figure 2.3: Linear regression of Mean Peak Frequency (Hz) of feeding clicks with a body condition index (BCI) derived from a regression using residuals of linearized area (√ Area) x Body Size (See text for details)

46 CHAPTER III

Other Considerations– Function of Sound Production in Seahorses

INTRODUCTION

There are four hypotheses for the function of sound production by seahorses during feeding: 1) prey-stunning; 2) echolocation; 3) by-product of head movement; 4) conspecific communication.

1) The prey-stunning hypothesis, which suggests a large cacophonous sound might be used by a predator to disorient prey items. This hypothesis was proposed as a function for seahorse clicks during foraging, but was then rejected when Anderson (2009) determined that muting seahorses did not impact prey-capture success rates.

2) Another theory to consider would be that seahorses use acoustic signals for echolocation or environment mapping. Toothed whales (odontocetes) will produce ultrasonic signals in environment mapping and prey localization. Echolocating for predation requires high frequency pulses of sound; these short-wavelength signals bounce off prey items and are received by the sender for processing the location, size, shape and even identity of the prey. The acoustic energy of an echolocation signal reflected from a target prey is referred to as target strength. The target strength depends on the ratio of the size of the prey and the wavelength of the sound produced by the predator (Møhl, 1988; Nachtigall, 1980). Therefore, a very short

47 wavelength i.e., a very high frequency sound, is necessary for localizing small prey items (e.g.

Dolphin echolocating pulse frequency ranges from 40 - 130 kHz) (Au, 2004).

3) The null hypothesis is that the click is a byproduct of the snick motion that occurs with the feeding strike. This hypothesis refers to the stridulatory mechanism between the supraoccipital bone and coronet of the seahorse (Colson et.al, 1998; Lim & Chong, 2015;

Oliveira et.al, 2014) that occurs with the snick and is often associated with the click signal

(Chapter I) in Hippocampus abdominalis.

4) The remaining hypothesis for the function of acoustic signal production during foraging is conspecific communication; which suggests that seahorses will use acoustic signals to communicate presence of prey items to nearby members of the same species. This chapter will discuss two of these hypotheses (echolocation and conspecific communication) in more depth, as well as how results from the previously described studies (Chapter I and Chapter II) relate to the others. These considerations are meant to speculate how these hypotheses could describe the function of seahorse sound production during feeding.

Possibility of Echolocation

An extensive test of the hypothesis of prey echolocation is beyond the capacity of this project, as it would require measurement of the production and reception of sound signals as they bounce off prey items or localize the environment. However, estimates of the capacity for echolocation can be made from data at hand. First, given the size of the container and the physical parameters of the environment-hydrophone recording, we can assume the tank is an acoustically dead space (little to no reverberation). This was determined with an RT-60 equation

48 (measurement of time it takes for any given sound to decay by 60 dB): RT-60 = k*(V/Sa), where k is a constant equal to 0.161 (if using metric units), V is the volume, and Sa represents the sum of all the surface areas in the tank multiplied by their respective absorption coefficients (units are

Sabins). The calculated RT-60 value was 0.01s, and therefore the tank is classified as an acoustically dead space. Additionally, the dimensions of the tank (24 x 12 x 14 in) represent a short distance, and the sound signal produced by H. abdominalis has an approximate wavelength of 3.3 m (given a frequency of 450 Hz and speed of sound in water 1500 m/s). Consequently, the seahorse will be producing sound within its own wavelength and so we suspect no attenuation in intensity based on the location of the seahorse relative to the hydrophone.

Second, the size of the prey item (Brine shrimp - Family: Artemiidae) is approximately

8mm and the average peak frequency of the click for Hippocampus abdominalis was determined to be around 450 Hz (Chapter I). An acoustic signal with such a long wavelength would be unable to bounce off a prey of that size, as objects smaller than the wavelength of the echolocating call will not be detected (Jacobs & Bastian, 2016). Clicks of H. abdominalis have an average frequency of 450 Hz, using λ= c/f, where λ is the wavelength =, c is the speed of sound in water (1500 m/s), and f is the frequency (Hz). Using this equation, the wavelength of the click can be calculated (3.3 m). Since brine shrimp are very small (8 mm) it is impossible for a sound of this wavelength to echolocate a prey item of that size. Therefore, the ability of a seahorse to localize the presence of prey with a click is highly unlikely. The characterization of average click frequency (Hz) from Chapter I, and the decrease in the number of clicks in the absence of visual cues (Chapter II) support rejection of the echolocation hypothesis as an explanation for the use of sound production by seahorses when foraging.

49 Possibility of Social Communication

Data presented here provide additional insight into the function of the clicking under various contexts, as seahorses did not appear to make more clicks without food present. This suggests the possibility of conspecific communication regarding the presence/absence of food.

However, signaling the presence of food to others might (at least at first) seem counter- intuitive or detrimental to individual fitness. Perante et.al. (2002) observed Hippocampus comes in a natural habitat and determined that seahorses were typically site specific (where a holdfast was available) and that pair bonded individuals were often in close proximity (<0.5m) to one another. Considering the small signal active space of the click (attenuates by approximately

20dB re 1uPa after 1 m), the signal is likely limited to seahorses nearby, such as pair bond members.

If seahorses click more in the presence of food, and if females click more often then perhaps these signals could be directed at a pair bond member to indicate the presence of prey items and recruit them to its source. To address the theory that the conspecific communication is specifically aimed at a mate, pair-bonded individuals should be observed. Answering the questions surrounding acoustic communication between monogamous pair-bonds in an aquatic environment could provide significant insight into this unique case of sex-role reversal. To further discern the information contained in clicks I suggest that the interactions between pair- bonds be explored further to evaluate the importance of acoustic traits in mate selection in

Syngnathidae.

Given the social structure of this species, it is also worthwhile to consider a possible function related to the context of sexual selection.

50 When considering the role conspecific communication plays in sexual selection, signals often contain information indicating partner quality (Andersson, 1994). While in most cases males may possess more sexually-selected traits, in sex-role reversed species the opposite is true

(Berglund & Rosenqvist, 2003). While many Syngnathids do exhibit sex role reversal (Berglund

& Rosenqvist, 2003; Berglund, 2000; Rosenqvist & Berglund, 2011); seahorses are typically monogamous (Jones et.al. 1998; Kavarnemo et.al. 2000; Vincent & Sadler, 1995) and are suggested to more often exhibit conventional sex roles despite the parental load on males

(Masonjones & Lewis, 2000; Vincent, 1994). There have been studies that have shown sexual selection can impact monogamous species and particularly can be shown in females, with male preference for larger female body size (Kavarnemo et.al. 2007).

The results of this study (Chapter II) showed that as the body condition of the seahorse improves (increased BCI), the mean peak frequency (Hz) of feeding clicks will decrease. This provides an insight into how seahorse condition may impact acoustic parameters of clicks.

Perhaps the acoustic signals produced by seahorses serve as indicators of mate quality or condition, a trait that is seen in other sex-role reversed species, (Vallejos et.al, 2017). It is well established that body size is an important quality in Syngnathidae mate selection (Berglund &

Rosenqvist, 2003; Rosenqvist & Berglund, 2011; Kavarnemo et.al. 2007; Mattle & Wilson,

2009), therefore we might conclude the click could contain information regarding body condition and play a role in mate selection.

The sex-roles of H. abdominalis are considered plastic; they exhibit monogamy and sex- role reversal (Bahr & Wilson, 2011; Wilson & Martin-Smith, 2007; Mattle & Wilson, 2009) in female dense populations where male-male competition is less apparent. Female-female competition in H. abdominalis has been noted to occur in laboratory and natural populations.

51 Previous findings (Chapter I), have shown that males click less than females, and while this might be attributed to stress it could also be related to the sex-role reversal, wherein males may click less than females because females use these signals to convey information of quality to males in cases of female-female competition. If clicks are intended as communicative signals to conspecifics, the response of the receiver is a necessary area of further research.

Many species of seahorse are classified as endangered or at risk of becoming such. A large contributor to this issue is habitat loss, but many seahorses are also lost to by-capture of fisheries. Anthropogenic noise is another issue; seahorses are known to be quite susceptible to stress. The large amount of noise in oceans (from military, commercial or transport vessels) can create a very loud environment. This not only can stress the seahorses but can also mask the signals they are trying to relay. Moreover, while in zoos and aquariums are sometimes useful in conservation, the presence of visitors and other activities within the captive environment creates noise as well. By understanding the way seahorses adjust their clicks and the contexts in which they produce them we can be better equipped to battle the ever-present conservation issues afflicting marine life.

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