Predatory Behavior and Neuroanatomy of the Sessile Rotifer, Cupelopagis Vorax Elizabeth Preza University of Texas at El Paso, [email protected]

Predatory Behavior and Neuroanatomy of the Sessile Rotifer, Cupelopagis Vorax Elizabeth Preza University of Texas at El Paso, Epreza15@Gmail.Com

University of Texas at El Paso DigitalCommons@UTEP

Open Access Theses & Dissertations

2017-01-01 Predatory Behavior and Neuroanatomy of the Sessile Rotifer, Cupelopagis Vorax Elizabeth Preza University of Texas at El Paso, [email protected]

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Recommended Citation Preza, Elizabeth, "Predatory Behavior and Neuroanatomy of the Sessile Rotifer, Cupelopagis Vorax " (2017). Open Access Theses & Dissertations. 524. https://digitalcommons.utep.edu/open_etd/524

This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. PREDATORY BEHAVIOR AND NEUROANATOMY OF THE SESSILE ROTIFER, CUPELOPAGIS VORAX

ELIZABETH PREZA Master’s Program in Biological Sciences

APPROVED:

Elizabeth J. Walsh, Ph.D., Chair

Arshad M. Khan, Ph.D.

Rick Hochberg, Ph.D.

Charles Ambler, Ph.D. Dean of the Graduate School

by Elizabeth Preza 2017

Dedication

This thesis is dedicated to my parents, Blanca and Roberto Preza. PREDATORY BEHAVIOR AND NEUROANATOMY OF THE SESSILE ROTIFER, CUPELOPAGIS VORAX

ELIZABETH PREZA, B.S.

THESIS

Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Biological Sciences THE UNIVERSITY OF TEXAS AT EL PASO December 2017 Acknowledgements

I am blessed for having the support of many people. I wish to express my greatest gratitude and appreciation to Dr. Elizabeth J. Walsh for her continuous guidance throughout my academic career. She accepted me into her lab when I was an undergraduate and introduced me to the wonderful world of rotifers. Her mentorship provided me with the motivation and skills to excel as a scientist. I would also like to thank my committee members, Dr. Arshad M. Khan for giving me access to his lab and equipment, and Dr. Rick Hochberg for sharing his knowledge of rotifer neurobiology. The advice and support of my committee was essential for the completion of this work. A special thanks to Dr. Ellen Walker and Robert Walsmith for their invaluable help with the staining protocol and confocal microscope, Dr. Armando Varela for teaching me how to use the confocal microscope, Dr. Anais Martinez for generating the QTVR videos on Volocity 6.3 software, Dr. Robert Wallace for his input and for collecting the Wisconsin plankton samples, Dr. Scott Mills for providing the Logitech HD Pro Webcam C920 and AMCap Ver. 9.21 video software, Dr. Soyoung Jeon for her assistance with the statistical analyses, and Marcela Rivero Estens for contributing the prey size and swimming speed data included in this study. Rotifers from Minto Pond were collected under permit number OPRD-015-14 (E. Walsh). Thank you to the funding sources who made this research possible. This work was supported by NSF (DEB-1257068), the BBRC Cytometry, Screening, and Imaging and Biostatistics Core Facilities (NIH NIMH & HD 2G12MD007592-21), and the UTEP Graduate School (Dodson Research Grant).

I wish to extend my gratitude to my colleagues in Dr. Walsh’s lab for their help and for keeping me motivated. Finally, I am thankful for my mom, Blanca, my siblings, Yayis and Beto, my stepdad, Rito, and my boyfriend, Javier, for their unconditional love and encouragement.

They believed in me when I did not.

v Abstract

Predator-prey interactions contribute to the evolution of behavioral and morphological characteristics of species. However, research on zooplankton has mainly focused on planktonic species such Mesocyclops edax (Arthropoda, Cyclopoida) and Asplanchna spp. (Rotifera,

Ploima). Cupelopagis vorax (Rotifera, Collothecaceae) provides a unique model for studying the predatory behavior of a sessile species. Cupelopagis was chosen because it is the only rotifer known to exhibit rheotaxic behaviors in the presence of prey and because it undergoes indirect development where non-feeding, free-swimming larvae mature into feeding adults. The integration of behavioral techniques, immunohistochemistry, and confocal microscopy were used to provide insight as to how C. vorax feeds. Single prey and mixed prey feeding experiments were conducted using two prey species, the rotifer Lepadella triba and the gastrotrich

Lepidodermella squamata. The predatory behavior of Cupelopagis was classified into three categories: encounter, attack, and capture. When predator-prey interactions for both species were compared, there was no significant difference in the frequency of encounters (W = 80.5, p =

0.19). However, attacks were more frequent for gastrotrich prey (W = 44, p < 0.01) while capture events were higher for rotifer prey W = 189.5, p = 0.01). Ivlev’s electivity index (Ei) and Manly-

Chesson’s index (αi) were used to assess prey selectivity. Only mean αi values indicated active selection of gastrotrich prey by C. vorax; however, more evidence is needed to confirm if

Cupelopagis is a selective feeder. The feeding behavior of Cupelopagis is influenced by its ability to respond to its environment. Overall, these results provide additional evidence that

Cupelopagis uses mechanoreceptors to detect potential prey based on its reaction (attack and capture) when prey were within proximity of the infundibulum. It is also proposed that chemoreceptors may aid in the ingestion and rejection of prey given its tendency to prefer

vi gastrotrich prey. To gain insight into the neural basis of these behaviors, fluorescent labeling was employed to describe serotonin immunoreactivity (5HT-IR) in Cupelopagis larvae and adults.

The brain was dorsal to the mastax and bilaterally symmetrical. In larvae, paired lateral nerves innervated the corona, mastax, and foot. In adults they innervated the infundibulum and proventriculus. Both larvae and adults had four 5HT-IR perikarya in the central nervous system.

However, metamorphosis had an effect on the structure and position of 5HT-IR neurons in C. vorax. Larvae contained a single pair of anterior and posterior perikarya in the brain that were arranged in an X-shaped pattern. While in adults, the arc-shaped brain ganglion contained four single posterior perikarya. Perikarya in larvae were generally larger (1.7X) than those found in adults but their function was not discerned. In total, these results seem to indicate that the serotonergic nervous system of C. vorax is structured to meet the metabolic and physiological requirements of each developmental stage (i.e. locomotion in larvae and feeding in adults).

However, more research is needed to elucidate function from structure. The results of this study can help define the behavioral and sensory mechanisms involved in the evolution of feeding behavior within phylum Rotifera. It also increases our understanding of how the nervous system evolves and develops in invertebrate organisms.

vii Table of Contents

Acknowledgements ...... v

Abstract ...... vi

Table of Contents ...... viii

List of Tables ...... x

List of Figures ...... xi

Chapter 1: Introduction ...... 1

Chapter 2: Quantifying feeding behavior and prey selectivity ...... 9 2.1 Introduction ...... 9 2.2 Methods...... 10 2.2.1 Animals ...... 10 2.2.2 Feeding experiments ...... 11 2.2.3 Prey selectivity ...... 12 2.2.4 Statistical analyses ...... 13 2.3 Results ...... 13 2.3.1 Predatory behavior ...... 13 2.3.2 Prey selectivity ...... 17 2.4 Discussion ...... 21

Chapter 3: Describing the serotonergic nervous system of larvae and adults ...... 25 3.1 Introduction ...... 25 3.2 Methods...... 26 3.2.1 Fluorescent labeling ...... 26 3.2.2 Microscopy ...... 26 3.3 Results ...... 27 3.3.1 Larvae ...... 28 3.3.2 Adults ...... 28 3.4 Discussion ...... 33

viii Chapter 4: Conclusions ...... 38

References ...... 41

Appendices ...... 49 Appendix 1: Additional confocal images of serotonin immunoreactivity in larvae. A total of eight larvae were stained, image labels are described in section 3.3.1...... 49 Appendix 2: Additional confocal images of serotonin immunoreactivity in adults. A total of eight adults were stained, image labels are described in section 3.3.2. Circles indicate prey items or debris (*) in the proventriculus...... 53

Vita 57

ix List of Tables

Table 1.1. Electivity indices based on the proportions of prey type i in the diet (ri), in the environment (pi), and alive at the end of the experiment (wi)...... 3 Table 1.2. Distribution of serotonin in the central nervous system, peripheral nervous system, and mastax in rotifer taxa with diverse feeding behaviors (modified from Leasi et al. 2009)...... 8 Table 2.1. Morphology, size, and swimming speed of prey species. Prey types have a very distinct morphology. Lepadella triba was significantly larger and wider than Lepidodermella squamata (Mann–Whitney U test, p < 0.05)...... 10 Table 2.2. Feeding experiments testing the effect of two prey types, the rotifer Lepadella triba and the gastrotrich Lepidodermella squamata, on the behavior of a sessile predator, Cupelopagis vorax...... 11 Table 2.3. Summary of mean electivity values of C. vorax preying on rotifers and gastrotrich prey. Only mean αi values for gastrotrich prey reflected preference...... 17 Table 3.1. Reagents used for fluorescent labeling. Concentrations, incubation periods, and temperatures for anti-serotonin, phalloidin, and DAPI stains...... 27

x List of Figures

Figure 2.1. Images of predator and prey species. (Clockwise) Cupelopagis vorax adult, Cupelopagis larva, the rotifer Lepadella triba, and the gastrotrich Lepidodermella squamata. .. 12 Figure 2.2. Mean relative frequencies of behaviors exhibited by Cupelopagis vorax towards rotifer and gastrotrich prey in (A) single prey and (B) mixed prey trials. Mean relative frequencies were compared using the Mann–Whitney U test (*p < 0.05). Cupelopagis was more likely to attack gastrotrich prey but the frequency of capture events was higher for rotifers...... 15 Figure 2.3. Scatterplots showing the relationship between capture probability of prey and predator size in (A) single prey and (B) mixed prey trials. Fitted regression lines indicate capture probability (y) = β1*predator size. Capture probability was significantly higher for Lepadella (y = 0.0019x and y = 0.0011x, p < 0.001) than Lepidodermella (y = 0.0010x and y = 0.0007x, p < 0.001)...... 16 Figure 2.4. Electivity values for (A) Ivlev’s (Ei) and (B) Manly-Chesson’s (αi) indices when C. vorax preyed on both rotifers and gastrotrich prey. Values above the dashed line indicate preference, values on the line denote random feeding, and values below the line show avoidance. Gastrotrich prey were preferred 64.3% of the time in both cases...... 18 Figure 2.5. Relationship between αi values when C. vorax preyed on rotifers and gastrotrich prey. Preference for one prey resulted in the avoidance of the other (r = -1, p < 0.001)...... 19 Figure 2.6. Scatterplots showing the relationship between electivity values and predator size; (A) Ei and (B) αi values. Fitted regression lines indicate preference (y) = β1*predator size. Size was not a significant predictor of prey selectivity...... 20 Figure 3.1. Fluorescent labeling in C. vorax larva. Reagents successfully stained (A) nuclei, (B) musculature, and (C) serotonin immunoreactivity (5HT-IR). (D) A merged image of DAPI (blue), phalloidin (green), and anti-serotonin (red) shows colocalization of nuclei and 5HT-IR. Merged confocal stack of 88 slices. Scale bar = 50 µm...... 29 Figure 3.2. Labeled 5HT-IR in C. vorax larval specimen from Figure 3.1. (A) Anti-serotonin stain and (B) greyscale image are provided for contrast. Structures were labeled according to structure not function. BPa: anterior brain perikaryon; BPp: posterior brain perikaryon; BPaan: anterior neurite in BPa; BPapn: posterior neurite in BPa; BPpan: anterior neurite in BPp; np: neuropil; nc: lateral nerve cord. Merged confocal stack of 88 slices. Scale bar = 50 µm...... 30 Figure 3.3. Fluorescent labeling in a C. vorax adult. Reagents successfully stained (A) nuclei, (B) musculature, and (C) 5HT-IR. (D) A merged image of DAPI (blue), phalloidin (green), and anti- serotonin (red) shows colocalization of nuclei and 5HT-IR in the anterior end. Merged confocal stack of 140 slices. Scale bar = 50 µm...... 31 Figure 3.4. Labeled 5HT-IR in adult C. vorax specimen from Figure 3.3. (A) Anti-serotonin stain and (B) greyscale image. The asterisk indicates prey items and/or debris in the proventriculus. Adults do not have anterior brain perikarya. Merged confocal stack of 140 slices. Scale bar = 50 µm...... 32 Figure 3.5. Side by side comparison of a larva and adult C. vorax specimens. Brain neurons in the larva are arranged in an X-shaped pattern while neurons in the adult stage have an arc-shaped distribution. Scale bar = 30 µm...... 32

xi Chapter 1: Introduction

Aquatic animals are susceptible to predation at some stage in life (Mileikovsky 1974,

Beauchamp et al. 2007, Horning and Mellish 2012). Thus predator-prey interactions contribute to the evolution of behavioral and morphological characteristics of species (Lima and Dill 1990,

Ferrari et al. 2010). Aquatic prey have developed different strategies to reduce their vulnerability to predation (Greene 1983, Walls et al. 1990). Examples include the formation of spines in cladocerans and rotifers (Dodson 1989, Gilbert 2014), unpalatability in rotifers (Felix et al. 1995,

Walsh et al. 2006), changes in shell thickness in marine snails (Trussell and Smith 2000), and diel vertical migration in a variety of taxa (Lampert 1989, Pearre 2003). To counteract prey defenses, predators have also evolved specialized behaviors and sensory structures to capture prey (Cooper et al. 1985, Trites 2002, Kiørboe 2011a). Despite our knowledge of predator-prey interactions, more research is needed to understand the behavior of predators (Lima 2002,

Schmitz 2007, Peckarsky et al. 2008).

Predator-prey interactions are vital in structuring zooplankton communities (Lynch 1979,

Kerfoot and Sih 1987, Shurin 2001, Sailley et al. 2015). Zooplankton consist of three main groups: copepods, cladocerans, and rotifers. Because zooplankton live in a viscous, at low

Reynolds numbers, and dilute environment, finding and capturing food can be a challenge

(Purcell 1977, Kiørboe 2011b). Zooplankton feeding behavior is divided into two main strategies: active (filter and cruise feeding) and ambush feeding (Kiørboe 2011a). According to

Kiørboe (2011a), suspension feeders, or filter feeders, generate water currents and intercept prey using specialized mouthparts. Cruise feeders swim through the water and feed on encountered prey. Ambush feeders, or sit-and-wait predators, consume prey that swim within their capture

1 radius. Passive forms rely on the motility of the prey while active forms detect and attack their prey.

Predation is a series of interactions, encounter, attack, capture, and ingestion, that result in prey ingestion or prey escape (Gerritsen and Strickler 1977). Zooplankton mainly rely on mechanical disturbances to find prey in their vicinity but they also utilize tactile and/or chemical cues (Greene 1983, Kiørboe 2011a). Prey selectivity depends on encounter rates, handling ability, prey escape mechanisms, and predator behavior (Saha et al. 2010). Gilbert and

Williamson (1978) compared predator-prey interactions between a predatory copepod

(Mesocyclops edax), a predatory rotifer (Asplanchna girodi), and rotifer prey (Polyarthra vulgaris and Keratella cochlearis). They observed that P. vulgaris escaped predation from

Asplanchna, a slow-moving predator, by jumping away. The hard integument of K. cochlearis prevented attacks by M. edax and successful ingestion by A. girodi. In another study, Li and Li

(1979) studied predator-prey interactions between a copepod predator, Acanthocyclops vernalis, and cladoceran and rotifer prey. They demonstrated that prey size, shape, and swimming behavior played a role in capture success. Helenius and Saiz (2017) showed that larger, alternative prey were selected by nauplii of the calanoid copepod Paracartia grani even though such prey could not be completely ingested.

Optimal foraging theory predicts that predators will only attack prey that contribute to their optimal diet i.e. maximize the rate of net energy gain (Greene 1983, Parker and Smith 1990,

Watanabe et al. 2014). Electivity indices are used to compare the composition of a predator’s diet among available prey types in the environment (Table 1.1). Electivity, or preference, refers to a deviation from random sampling of available prey (Chesson 1978). Upon reviewing the statistical power of these electivity indices, Manly et al. (1993) determined that some indices

2 only reflect selection for a specific circumstance (w, E, and L) while others provide more biologically relevant estimates (α). Nonetheless, the former continue to be applied in aquatic research (Iyer and Rao 1996, Frau et al. 2016).

Table 1.1. Electivity indices based on the proportions of prey type i in the diet (ri), in the environment (pi), and alive at the end of the experiment (wi).

Index Reference

wi = ri/pi Forage ratio Ivlev (1961)

Ei = (ri – pi) / (ri + pi) Ivlev’s electivity index Ivlev (1961)

αi = ln(wi) / Ʃi ln(wi) Manly-Chesson index for Manly (1974) variable prey abundance

αi = (ri/pi) / Ʃi (ri/pi) Manly-Chesson index for Chesson (1978) constant prey abundance

Li = ri – pi Strauss’ linear index Strauss (1979)

The complexity of aquatic food webs can be better understood by studying the feeding behavior of species (Sih et al. 2004, Ings et al. 2009, Moya-Laraño 2011). Of all zooplankton, the feeding mechanisms of copepods have been most studied. Copepods are efficient predators; large species prey upon protozoans, rotifers, and small aquatic animals while smaller species tend to be plankton feeders (Fryer 1957, Hansen et al. 1994). Mechanoreceptors and chemoreceptors contribute to the detection of food by copepods (Strickler and Bal 1973, Buskey

1984). Furthermore, cyclopoid and calanoid copepods have different feeding strategies.

Cyclopoid copepods are slow, cruising feeders equipped with sensory setae in their first antennae

(Brandl 2005). Calanoid copepods are suspension feeders capable of redirecting food particles toward their mouthparts (Bundy and Vanderploeg 2002). Rotifers are often featured in the literature because they are the preferred food item of copepods (Brandl 2005). However, these

3 fascinating metazoans can have higher turnover rates and biomass than copepods thus making them important for trophic dynamics (Wallace et al. 2015).

Phylum Rotifera consists of three groups, Seisonidea, Bdelloidea, and Monogononta, comprising over 2,000 marine and freshwater species (Segers 2007). Rotifers are vital for connecting microbial life to higher trophic levels since they are prey to a variety of invertebrate predators (Wallace et al. 2006). Rotifers are characterized by a corona, a ciliated anterior end, the lorica, a thick body wall bearing variable appendages, and the mastax, a specialized pharyngeal organ with jaws, termed trophi. Their general life history is directly linked to their feeding behavior (Wallace et al. 2015). Most rotifer species are suspension feeders on microalgae, bacteria, or detritus, others are occasional or obligate predators, and a few are parasitic (Wallace et al. 2006). The majority of rotifers consume various prey items but some have highly specialized diets. Food is processed by the mastax where it is masticated by the trophi before being moved through the esophagus and into the stomach (Starkweather 1996, Wallace et al.

2015).

Class Monogononta consists of planktonic and sessile species with diverse lifestyles and morphologies. Sessile rotifers are found in three families of the superorder Gnesiotrocha,

Flosculariidae, Collothecidae, and Atrochidae. Sessile gnesiotrochans undergo indirect development where non-feeding, free-swimming larvae mature into feeding adults (Fontaneto et al. 2003). The juvenile motile stage is not a true larva since all the adult organs are present; however, the term larva is used since the corona is not fully developed in the young animal

(Wallace et al. 2015). Larvae do not feed but survive on limited food reserves and must find a suitable substrate before expending their energy (Wallace 1980). Because attachment is

4 permanent, substrate selection is a critical factor in the reproductive success and survival of adults (Butler 1983, Fontaneto et al. 2003).

Floscularid adults are filter feeders while collothecids are passive ambush feeders

(Wallace et al. 2015). A sessile lifestyle entails lower metabolic costs and predation risks; yet, a significant tradeoff is lower feeding efficiency (Kiørboe 2011a). An evolutionary adaptation to lower feeding efficiency may be the occurrence of larger, ornate corona in sessile rotifers in contrast to the smaller, ciliated corona of their planktonic counterparts. The morphological adaptations of sessile species are reviewed by Wallace et al. (2006). In collothecids and atrochids, the corona is modified and resembles a funnel or bowl and is known as the infundibulum. The infundibulum is a unique, apomorphic trait only found in gnesiotrochans

(Hochberg and Hochberg 2015). Collothecids, such as Stephanoceros fimbriatus, have long setae along the rim of the infundibulum that fold over to entrap food particles. Adult atrochids, such as

Cupelopagis vorax, lack setae or cilia. In this species, only larvae and males possess a ciliated corona that is only used for locomotion (Wallace et al. 2006).

Research has mainly focused on the predatory behavior of planktonic rotifers, more specifically on the monogonont Asplanchna. Asplanchna species are cruise feeders that only attack prey after physical interaction. An encounter occurs when prey come into direct contact with coronal chemoreceptors thus eliciting an attack (Gilbert and Williamson 1978). Asplanchna can be selective feeders, deciding whether to attack potential prey at the time of encounter

(Gilbert 1978, 1980). Chang et al. (2010) showed that the feeding behavior of Asplanchna is species-specific. The diet composition of A. herricki, A. priodonta, and A. girodi ranged among phytoplankton, protozoa, and zooplankton prey with A. girodi being the most carnivorous.

Asplanchna readily attack other rotifer species and engage in cannibalism (Gilbert 1980).

5 Asplanchna, as well as copepods, release biochemical cues that induce the development of spines in the next generation of rotifer prey (Wallace et al. 2006, Gilbert 2014). Other predatory rotifers include the bdelloid Abroctha carnivora (Ricci et al. 2001) and the monogononts Acyclus inquietus (Hochberg et al. 2010) and Cupelopagis vorax (Bevington et al. 1995).

Morphological and neurological traits develop concurrently to increase individual fitness

(Carvalho and Mirth 2015). As adults, sessile rotifers must overcome a limited scope of potential prey since they cannot actively search for food. Drastic ontogenetic changes in feeding behavior may result in modified neural structures associated with prey detection. Our knowledge of physiological functions and behaviors of rotifers can be enhanced by mapping specific neurotransmitters against sensory devices, muscles, and other organ systems (Hochberg 2009).

The rotifer nervous system consists of a brain, mastax ganglion, pedal ganglion, and paired nerve cords (Hochberg 2016). The brain is positioned dorsal to the mastax and connects to three types of sensory organs: mechano-, chemo-, and photoreceptors. Brain neurons comprise

20% of the total number of cells in the rotifer body (Kotikova et al. 2005). Neurons are bilaterally distributed with distinct species-specific patterns: x-shaped, arch-shaped, and ring- shaped (Kotikova 1998). Lateral or ventrolateral paired nerve cords originate from the brain and extend caudally where they fuse at the mastax ganglion, which innervates trophi muscles, and at the pedal ganglion, which innervates the foot (Leasi et al. 2009).

Histochemical and immunohistochemical techniques provide evidence for the presence of neurotransmitters in rotifer species. Leasi et al. (2009) summarized the presence of serotonin, catecholamines, acetylcholine, FMRFamide, and small cardioactive peptide b (SCPb) in the nervous system of bdelloid and monogonont rotifers. Serotonin is a primitive neurotransmitter found throughout Kingdom Animalia. It has been shown to regulate feeding behavior, ciliary

6 activity, development, and circadian rhythm in invertebrates (Hay-Schmidt 2000).

Immunohistochemical studies have shown that the serotonergic system is involved in the main centers that regulate feeding in insects (Falibene et al. 2012). Leasi and Ricci (2011) discovered that serotonin plays a role in the egg laying process and development of the bdelloid rotifer

Macrotrachela quadricornifera. Serotonergic neurons are widespread in rotifers and their distribution may have phylogenetic and functional significance (Hochberg 2009). An updated account of serotonin immunoreactivity across rotifer taxa is given in Table 1.2.

The purpose of this study was to investigate the feeding behavior and nervous system of a rotifer with a biphasic life history: planktonic larva and sessile adult. Cupelopagis vorax

(Rotifera, Collothecaceae, Atrochidae) provides a unique model since it is the only rotifer known to respond to vibrations produced by potential prey (Bevington et al. 1995). The predatory behavior of C. vorax has been categorized but the effect of prey type on capture efficiency and prey selectivity has not been assessed. Because C. vorax larvae do not feed, differences in the nervous system of larva and adult stages may reveal structures associated with feeding behavior.

The integration of behavioral techniques, immunohistochemistry, and confocal microscopy will increase our understanding of how C. vorax feeds and how the serotonergic system of larvae and adults is structured.

7 Table 1.2. Distribution of serotonin in the central nervous system, peripheral nervous system, and mastax in rotifer taxa with diverse feeding behaviors (modified from Leasi et al. 2009).

Taxa FeBeh Br PNS Ma Reference Bdelloidea Macrotrachela quadricornifera Filter 14N + - Leasi et al. (2009) Monogononta Ploima Asplanchna brightwellii Cruise 14N + - Hochberg (2009) Asplanchna herricki Cruise 4N + + Kotikova et al. (2005) Euchlanis dilatata Filter 4N + + Kotikova et al. (2005) Notommata copeus Cruise 10N + + Hochberg (2007) Platyias patulus Filter 6N + - Kotikova et al. (2005) Collothecacea Stephanoceros fimbriatus larva Filter 12N + - Hochberg and Hochberg (2015) Stephanoceros fimbriatus adult Filter 12N + - Hochberg and Hochberg (2015) Flosculariacea Conochilus coenobasis Filter 8N + - Hochberg (2006) Conochilus dossuarius Filter 8N + - Hochberg (2006) Sinantherina socialis Filter 3N(?) + - Hochberg and Lilley (2010)

FeBeh: Feeding behavior; Br: Brain; PNS: Peripheral nervous system; Ma: Mastax. N: number of neurons in the brain; (?): unknown. Serotonergic immunoreactivity was present (+) or absent (-).

8 Chapter 2: Quantifying feeding behavior and prey selectivity

2.1 Introduction

Cupelopagis vorax is a round, ambush predator (200–1,100 µm) that has a large hooded mouth (infundibulum) for prey capture (see Koste 1973 for detailed images). Larvae are vermiform and have a ciliated anterior end used for locomotion. Larvae settle on the underside of plants with broad, flat leaves or finely dissected leaves (e.g., Ceratophyllum, Elodea, and Potamogeton) (Edmondson 1949, Butler 1983) and metamorphose into adults. After metamorphosis, and unlike most rotifers, C. vorax uses its modified head (infundibulum) to detect and capture prey. The feeding behavior of adult Cupelopagis has been described and analyzed by Bevington et al. (1995). During an encounter, Cupelopagis rotates on its pedal stalk so that its infundibulum faces potential prey items (referred to as orientation). An encounter response seems to be elicited only by mechanical stimuli. An attack consists of two movements, in a swift motion Cupelopagis will extend its infundibulum over a prey item (lunge) and if successful, the infundibulum will contract thus reducing the opening of the infundibulum (coronal purse). Once a prey item is captured, it is pushed through the esophagus and stored in the proventriculus until the mastax macerates the prey, which is then transferred to the stomach (Wallace et al. 2015). Prey can escape from the coronal purse if the proventriculus is at full capacity (Bevington et al. 1995; personal observations). The proventriculus may play a role in food processing by temporarily storing food that requires further degradation by the trophi

(Hochberg et al. 2017). Cupelopagis has a diverse diet consisting of protists, cladocerans, ostracods, nematodes, gastrotrichs, and other rotifers. Bevington et al. (1995) determined that Cupelopagis had a higher capture efficiency for Paramecium bursaria (61.1%) in comparison to a small, unidentified flagellate (41.5%). This study considers the behavioral responses of C. vorax to two different prey types, the rotifer Lepadella triba and the gastrotrich Lepidodermella squamata. Prey characteristics are summarized in Table 2.1. The lorica of Lepadella and dorsal scales on Lepidodermella may offer protection from predators. The first objective of this study was to quantify the feeding 9 behavior of Cupelopagis. It was hypothesized that C. vorax would have a higher capture probability and show preference towards Lepadella triba instead of Lepidodermella squamata. Larger, faster prey, such as Lepadella, are more likely to be detected at a greater distance by ambush predators than smaller, slower prey (Kiørboe 2011a).

Table 2.1. Morphology, size, and swimming speed of prey species. Prey types have a very distinct morphology. Lepadella triba was significantly larger and wider than Lepidodermella squamata (Mann–Whitney U test, p < 0.05).

Prey species Prey characteristics Lepadella triba Lepidodermella squamata Morphology round flattened body Defense mechanisms thick body wall dorsal scales Length (µm) 157.2 ± 26.2a 115.0 ± 12.5b Width (µm) 124.6 ± 28.6a 29.3 ± 8.1b Swimming speed (µm s-1) 262.0 ± 0.07* 186.1 ± 0.4 Mean values ± SD; superscripts indicate a significant difference (Mann–Whitney U test, p < 0.05). *From Santos-Medrano et al. (2001).

2.2 Methods

2.2.1 Animals

Cupelopagis vorax populations were acquired from Minto Pond, Marion Co., OR (GPS coordinates: 44.9204 N, -123.06135 W) in May 2014 and Moon (Birch) Lake, Marquette Co., WI (43.802657 N, -89.369892 W) in September 2015. Rotifers were cultured under laboratory conditions in modified MBL medium (Stemberger 1981) and fed once a week with a mixture of Chlamydomonas reinhardtii (Culture Collection of Algae at The University of Texas at Austin

[UTEX] #90), Chlorella vulgaris (UTEX #30), Rhodomonas minuta, and two prey species, Lepadella triba (in culture, Walsh laboratory) and Lepidodermella squamata (Carolina Biological Supply). Prey were also cultured under laboratory conditions in MBL medium and a mix of algae.

10 2.2.2 Feeding experiments

Figure 2.1 illustrates the morphological differences between Cupelopagis vorax and the prey species used in the study. The experimental design for single and mixed prey experiments is summarized in Table 2.2. Round coverslips (12 mm ⌀) were placed in the culture dishes as a substrate for C. vorax larvae. Once larvae attached to a coverslip, rotifers were isolated in 2 ml of spring water (Carolina Biological Supply) to control for satiation for at least 24 h prior to observation. Spring water was used to control for metal ions. Individuals were transferred to an embryo dish with 1 ml of spring water and fed the rotifer Lepadella triba and/or the gastrotrich

Lepidodermella squamata. Prior to feeding, whole body and corona only length for Cupelopagis were measured using a micrometer. Feeding trials lasted 4 h and were recorded with a Leica Wild M10 stereomicroscope equipped with a Logitech HD Pro Webcam C920. Picture frames were captured every second with AMCap Ver. 9.21 video software. Behaviors were visualized in Windows Movie Maker Ver. 2012 and assigned a category (encounter, attack, or capture). The duration and frequency of each behavior was recorded. To control for prey availability, only trials with ≥ 2.3 predator-prey interactions h-1 were considered for statistical analysis.

Table 2.2. Feeding experiments testing the effect of two prey types, the rotifer Lepadella triba and the gastrotrich Lepidodermella squamata, on the behavior of a sessile predator, Cupelopagis vorax.

Experiment Treatment Type Trials (n) Prey type ind ml-1 Single prey 15 Rotifer ≥60 Gastrotrich 0 Single prey 15 Rotifer 0 Gastrotrich ≥60 Mixed prey 14 Rotifer 45 Gastrotrich 45 ind ml-1: prey density.

Figure 2.1. Images of predator and prey species. (Clockwise) Cupelopagis vorax adult, Cupelopagis larva, the rotifer Lepadella triba, and the gastrotrich Lepidodermella squamata.

2.2.3 Prey selectivity

Feeding selectivity was calculated using two indices: Ivlev’s and Manly-Chesson’s for variable prey abundance since prey items were not replaced after being eaten (Chipps and

Garvey 2006). Ivlev’s electivity index (1961) is defined as Ei = (ri–pi)/(ri+pi), where Ei is the relative abundance of prey type i in the diet (ri) in relation to its relative abundance in the environment (pi). The electivity index ranges from -1 to +1. When Ei = 0, the predator is feeding randomly, Ei > 0 indicates preference, and Ei < 0 indicates avoidance. Manly-Chesson’s index

(1974) was calculated as αi = ln(wi)/Ʃiln(wi) where αi estimates the relative abundance of prey type i left alive at the end of the experiment (wi). When αi = 1/m the predator is feeding randomly

(m = number of prey types considered), αi > 1/m indicates preference, and αi < 1/m indicates avoidance. Only the results from the mixed prey trials were used to assess preference.

12 2.2.4 Statistical analyses

Means and standard deviations for behavior categories (encounter, attack, and capture) were determined for single prey and mixed prey trials. Significant differences between behaviors exhibited towards each prey item were tested using the Mann–Whitney U test. A linear regression model was used to predict capture probability for each prey item based on predator size. The regression model used was y = β1x, where capture probability (y) equals the regression estimate (β1) multiplied by predator size (x). The {doBy} (Ver. 4.5-15) package was used to determine significant differences between linear slope estimates. The mean Ei and αi values obtained for each prey item was compared 0 and 0.5 (null hypothesis), respectively, using a one- sample t-test. Pearson’s product-moment correlation was used to test the association between paired samples of Ivlev’s and Manly-Chesson’s indices. A linear regression model was used to predict preference for each prey item based on predator size. The regression model used here was y = β1x, where preference (y) equals the regression estimate (β1) multiplied by predator size (x). All statistical analyses were done in RStudio Ver. 1.1.383. Graphs were generated in GraphPad Prism Ver. 7.03.

2.3 Results

2.3.1 Predatory behavior

Over 460 h of video were observed to categorize the predatory behavior of Cupelopagis vorax. However, only 44 experimental trials met conditions of high prey availability (≥ 2.3 predator-prey interactions h-1). Surveillance behavior was observed but not included in statistical analysis. Here, Cupelopagis appeared to scan its environment even when prey items were not within proximity. Cupelopagis exhibited encounter, attack, and capture behaviors in response to Lepadella triba and Lepidodermella squamata. The relative frequencies (mean ± SD) for each behavioral category are shown in Figure 2.2A for trials where Cupelopagis was fed only Lepadella (n = 15) or Lepidodermella (n = 15). There was no significant difference in the

13 frequency of encounters between rotifer and gastrotrich prey (W = 80.5, p = 0.19). However, Cupelopagis was significantly more likely to attack gastrotrichs (34.2% ± 0.17) than rotifers (15.0% ± 0.12; W = 44, p < 0.05). However, capture events were significantly more frequent for rotifer prey (59.0% ± 0.21) than for gastrotrichs (32.9% ± 0.16; W = 189.5, p < 0.01). Results for the mixed prey trials (n = 14) reflected those from the single prey trials (Figure 2.2B). There was no significant difference in the frequency of encounters between rotifer and gastrotrich prey (W = 72, p = 0.24). Cupelopagis was significantly more likely to attack gastrotrichs (38.6% ± 0.18) than rotifers (21.5% ± 0.16; W= 49, p = 0.03). Capture events were significantly more frequent for rotifer prey (43.2% ± 0.26) than gastrotrichs (21.7% ± 0.16; W = 149, p = 0.02). In two separate occasions, two rotifer prey escaped capture because the proventriculus was too packed. The impact of predator size on capture probability was considered (Figure 2.3). Because whole body and corona only length were correlated (Pearson’s product-moment correlation, r = 0.587, p < 0.001), whole body length was used to represent predator size. Cupelopagis size ranged from 262.5 to 362.5 µm (315 ± 37.9) in single prey trials with only Lepadella, 237.5 to

387.5 µm (308.7 ± 39.0) in single prey trials with only Lepidodermella, and 262.5 to 412.5 µm (329.8 ± 49.9) in the mixed prey trials. Predator size was a significant predictor of capture probability for rotifer prey in single prey trials when using a linear regression model (F = 99.42, p < 0.001, R2 = 0.88; y = 0.0019x, p < 0.001) and in mixed prey trials (F = 33.15, p < 0.001, R2 =

0.73; y = 0.0011x, p < 0.001). The probability of capturing gastrotrichs was also significantly influenced by predator size in single prey trials (y = 0.0010x, p < 0.001) and in mixed prey trials (y = 0.0007x, p < 0.001). Furthermore, there was a significant difference between the slope estimates of rotifer and gastrotrich prey in single prey (p < 0.001) and mixed prey trials (p = 0.05).

Figure 2.2. Mean relative frequencies of behaviors exhibited by Cupelopagis vorax towards rotifer and gastrotrich prey in (A) single prey and (B) mixed prey trials. Mean relative frequencies were compared using the Mann–Whitney U test (*p < 0.05). Cupelopagis was more likely to attack gastrotrich prey but the frequency of capture events was higher for rotifers.

Figure 2.3. Scatterplots showing the relationship between capture probability of prey and predator size in (A) single prey and (B) mixed prey trials. Fitted regression lines indicate capture probability (y) = β1*predator size. Capture probability was significantly higher for Lepadella (y = 0.0019x and y = 0.0011x, p < 0.001) than Lepidodermella (y = 0.0010x and y = 0.0007x, p < 0.001).

16 2.3.2 Prey selectivity

Only the mixed prey trials (n =14) were considered when assessing prey selectivity. Preference was consistent between the electivity indices used (Figure 2.4). Ivlev’s electivity index (Ei) and Manly-Chesson’s index (αi) indicated that Cupelopagis mostly preferred gastrotrich prey (64.3%) in comparison to rotifer prey (28.6%). There was only one case where

Cupelopagis fed randomly. There was a strong negative association between αi values for rotifer prey and αi values for gastrotrich prey (Pearson’s product-moment correlation, r = -1, p < 0.001) (Figure 2.5). Preference for one prey type resulted in avoidance of the other. Nonetheless, only mean αi values for gastrotrich prey indicated active selection (0.59 ± 0.34) (Table 2.3). Electivity values (mean ± SD) for rotifer prey (Ei: -0.23 ± 0.42, αi: 0.41 ± 0.34) and Ei values for gastrotrichs (-0.05 ± 0.46) were below the criteria for selective feeding. The impact of predator size was also considered for prey selectivity (Figure 2.6). Cupelopagis size ranged from 262.5 to 412.5 µm (mean ± SD: 329.8 ± 49.9). Predator size was

2 not a significant predictor of Ei values (F = 1.72, p > 0.05, R = 0.13; y = -0.0007x, p = 0.08) and

2 αi values (F = 15.34, p < 0.001, R = 0.74; y = -0.0026x, p = 0.20) for rotifer prey. Predator size was also not a significant predictor of Ei values (y = -0.0001x, p = 0.73) and αi values (y = 0.0026x, p = 0.20) for gastrotrich prey.

Table 2.3. Summary of mean electivity values of C. vorax preying on rotifers and gastrotrich prey. Only mean αi values for gastrotrich prey reflected preference.

Prey type Mean Ei Mean αi SD SE 95% CI Preference criteria Preference?

Rotifer -0.23 - 0.42 0.11 -0.47, 0.02 Ei > 0 No

- 0.41 0.34 0.09 0.21, 0.61 αi > 0.5 No

Gastrotrich -0.05 - 0.46 0.12 -0.31, 0.22 Ei > 0 No

- 0.59 0.34 0.09 0.39, 0.79 αi > 0.5 Yes

Figure 2.4. Electivity values for (A) Ivlev’s (Ei) and (B) Manly-Chesson’s (αi) indices when C. vorax preyed on both rotifers and gastrotrich prey. Values above the dashed line indicate preference, values on the line denote random feeding, and values below the line show avoidance. Gastrotrich prey were preferred 64.3% of the time in both cases.

Figure 2.5. Relationship between αi values when C. vorax preyed on rotifers and gastrotrich prey. Preference for one prey resulted in the avoidance of the other (r = -1, p < 0.001).

Figure 2.6. Scatterplots showing the relationship between electivity values and predator size; (A) Ei and (B) αi values. Fitted regression lines indicate preference (y) = β1*predator size. Size was not a significant predictor of prey selectivity.

20 2.4 Discussion

Zooplankton exhibit behavioral responses to hydromechanical signals generated by a potential mate, predator, or prey (Visser 2001). Setae within the antennae of copepods are highly sensitive to fluid motion thus allowing copepods to determine the position and velocity of other organisms (Strickler and Bal 1973, Yen et al. 1992, Kiørboe 2011b). Rheotaxic abilities are not uncommon in rotifers. Rheotaxic escape responses have been documented for Polyarthra remata, P. major, Filinia terminalis, and Keratella cochlearis when they become entrained in the feeding current of a predator (Williamson 1987, Kirk and Gilbert 1988). Nonetheless,

Cupelopagis vorax is the only rotifer known to exhibit rheotaxic behaviors in the presence of prey (Bevington et al. 1995). This study is the second to analyze predator-prey interactions between Cupelopagis and prey species. The behavioral responses towards rotifer and gastrotrich prey were very different. Cupelopagis attacked Lepidodermella squamata more frequently yet the number of successful captures was higher for Lepadella triba. Prey characteristics can help explain this discrepancy in behaviors. Lepidodermella have a flattened body that may make it more susceptible to attacks

(Strayer et al. 2010). In contrast, Lepadella possess a hard lorica that may deter predator attacks, similar to what has been observed for Keratella in the presence of the copepod Mesocyclops edax (Gilbert and Williamson 1978). The size and swimming behavior of Lepadella may have facilitated its detection and consequently its capture by Cupelopagis. Ambush predators are more likely to detect vibrations elicited by larger and faster prey (Kiørboe 2011a). Lepadella were significantly 1.4X longer and 4.3X wider than their gastrotrich counterparts (Mann– Whitney U tests, p < 0.05). Also, the swimming behavior of Lepadella was fast and erratic while

Lepidodermella tended to glide slowly across the surface (personal observations). Bevington et al. (1995) considered the impact of predator size on predator-prey interactions. They observed a positive correlation between encounter distance and Cupelopagis size, meaning that larger individuals could detect prey that were further away. In this study, Cupelopagis size was a significant predictor of capture probability (linear regression model, p <

21 0.05). As predator size increased so did the probability of capturing rotifer and gastrotrich prey. When considering mean predator size for single and mixed prey trials, capture probability for rotifer prey was 2X and 1.5X higher, respectively, than the estimates for gastrotrich prey. It was also observed that infundibulum size increased with body size (Pearson’s product-moment correlation, p < 0.001), however, the r coefficient was very low. This may indicate that a bigger infundibulum allows Cupelopagis to capture larger prey and detect prey from a longer distance. The behavioral responses of Cupelopagis provide evidence that lateral and coronal antennae are mechanosensitive (Vasisht and Dawar 1969). Cupelopagis engages in a surveillance behavior where it appears to scan the environment even when no prey are present (Bevington et al. 1995, personal observations). However, attack and capture behaviors are only exhibited when a prey item is within proximity of the infundibulum. Although chemodetection was not investigated here, it is unlikely that Cupelopagis uses chemical cues to detect prey. Any biological molecules, such as ammonia or pheromones, released by potential prey will spread by molecular advection or diffusion. In addition, chemical plumes form behind swimming prey and away from the predator (Kiørboe 2011a). Kiørboe (2011b) determined that chemical detection is restricted to ambush predators smaller than 50 µm, Cupelopagis size ranges from 200 to 1,100 µm. Generally, predator motility is required for the successful detection of prey via chemical cues (Svensen and Kiørboe 2000). Although chemoreceptors likely do not play a role in prey detection, they may aid in the ingestion or rejection of prey (Friedman and Strickler 1975, Wurdak et al. 1983, Vazquez Archdale and Anraku 2005). DeMott (1986) considered the role of taste in food selection by diverse zooplankton taxa. Copepod, cladocerans, and rotifer species fed selectively on flavored spheres (Diaptomus siciloides, Cyclops bicuspidatus, Bosmina longirostris, Eubosmina coregoni, and Filinia terminalis). DeMott (1986) hypothesized that zooplankton may use taste receptors to discriminate between high and low-quality food particles. Neuroethological studies have revealed that taste receptors in the mastax of rotifers relay information from the mastax ganglion to the brain (Hochberg et al. 2015). If prey items are acceptable, they are processed though the 22 digestive system of the rotifer. In this study, two rotifers escaped predation on two sperate occasions because the proventriculus of Cupelopagis was full to capacity; yet, Cupelopagis kept feeding. Our understanding of the function of taste receptors within phylum Rotifera is limited, therefore more studies are needed to elucidate the role of the nervous system in how prey is selected. Prey selectivity has been observed in Asplanchna species. Baumert (1998) determined that A. herricki preferred Keratella cochlearis, Brachionus patulus, and Pleosoma sp. despite polymorphic defense mechanisms (lorical spine development) in the former two species. Another study by Iyer and Rao (1996) showed that A. intermedia preferred Brachionus angularis, B. rubens, and Keratella tropica in contrast to B. budapestinensis, B. bidentus, and Polyarthra spp. when prey densities were alternated. Selectivity was influenced by the behavioral and morphological defenses of prey items rather than their size. Cupelopagis differentiated between prey types at least to some degree. Prey selectivity was measured using Ivlev’s electivity index (Ei) and Manly-Chesson’s index (αi) for variable prey abundance. These indices were selected because of their relevance in aquatic studies (Iyer and Rao 1996, Bernal et al. 2015, Frau et al. 2016). Only αi mean values reflected active selection of prey by C. vorax in this study. Cupelopagis preferred gastrotrich prey over rotifers when both prey items were present. Interestingly, there was a strong negative correlation between αi values, where preference for one prey type resulted in avoidance of the other in the predator-prey system tested here (Pearson’s product-moment correlation, p < 0.001). Selective feeding is an evolutionary trend meant to maximize energy intake, consequently selectivity should improve a predator’s growth and survival (Mayer and Wahl 1997). Prey selectivity generally seemed to decrease as Cupelopagis size increased; however, the results were not significant (linear regression model, p > 0.05). Because individual fitness is dependent on food abundance (Kent 1981, Gergs and Jager 2014), large sized Cupelopagis may opt for more generalized diets. When food is limited, some rotifer species respond by lowering their metabolic rates and curtailing reproduction (Kirk 2002). Zooplankton have the ability to switch 23 between prey items based on the quality of each prey item (Sailley et al. 2015). A life history study for Cupelopagis vorax showed that prey type has an impact on the lifespan and reproductive success of C. vorax when fed Lepadella triba, Lepidodermella squamata, and/or Paramecium multimicronucleatum. Cupelopagis had a longer life expectancy and higher reproductive success when fed Lepadella in comparison to a diet of L. squamata and P. multimicronucleatum. A mixed diet of L. triba and Lepidodermella, as well as a diet with all three prey types, resulted in longer life expectancy and higher survivorship than a diet of L. squamata and P. multimicronucleatum (Rivero Estens, unpublished data). More evidence is needed to confirm if Cupelopagis is a selective feeder. Feeding selectivity depends on factors such as predator hunger, prey distribution, and prey escape mechanisms (Chesson 1978). It has been documented that starvation will increase predation rates in zooplankton (Yen 1983, Salt 1987, Olsen et al. 2000). Williamson (1980) determined that the hunger state of a predator can influence prey preferences. Satiated Mesocyclops edax did not eat Bosmina, yet when the predator was starved, Bosmina became the second most preferred prey item. In this present study, animals were starved prior to feeding and trials were stopped at a set time (4 h) before satiation was observed in all experiments (i.e., prey were still being transferred into the proventriculus). This study provides insight on the predatory-prey interactions of Cupelopagis vorax and two prey species. The results supported the hypothesis that capture probabilities would be higher for Lepadella triba. However, Cupelopagis seemed to actively select Lepidodermella squamata when both prey types were present. Despite its sessile lifestyle, Cupelopagis is able to thrive in its environment. This fascinating organism is able to detect potential prey via mechanical cues. It is proposed that Cupelopagis may implement chemical cues in its decision to ingest specific prey types. Because the feeding behavior of Cupelopagis is influenced by its ability to respond to its environment, the nervous systems of larvae and adults are also considered (see next chapter).

24 Chapter 3: Describing the serotonergic nervous system of larvae and adults

3.1 Introduction

According to Vasisht and Dawar (1969), the nervous system of Cupelopagis vorax adults consists of a brain, three pairs of fine nerves, and eight pairs of lateral nerves. The fifth lateral nerve innervates two sensory organs: lateral and coronal antennae. Lateral antennae are located at the junction of the infundibulum and trunk while coronal antennae are found within the infundibulum. Mechanoreceptors within these sensory organs may aid Cupelopagis in detecting the position of potential prey (Bevington et al. 1995). The nervous system of Cupelopagis larvae has not been described and modern techniques have not been implemented to corroborate Vasisht and Dawar (1969)’s observations. The nervous system of rotifers at different life stages has only recently started to be described. Observations of planktonic larval stages allow us to observe the evolution of the serotonergic nervous system (Hay-Schmidt 2000). A study by Hochberg and Hochberg (2015) is the first to determine the distribution of serotonergic neurons in the larvae and adults of a rotifer species, the gnesiotrochan rotifer Stephanoceros fimbriatus. In the sessile, planktivore S. fimbriatus, both life stages had the same number of serotonergic neurons (12N); however, their central nervous system was structurally different. Larvae contained one pair of cerebral perikarya

(BPL) that was absent in adults; conversely, a large pair of perikarya (AP) could only be detected in adults (see Hochberg and Hochberg 2015 for detailed diagrams). The behavioral differences in the larval and adult stages of sessile rotifers may translate to their neuroanatomy since serotonin plays a role in the ciliary activity and feeding behavior of invertebrates (Hay-Schmidt 2000). The second objective of this study was to describe the serotonergic nervous system of Cupelopagis vorax larvae and adults. It was hypothesized that the nervous system of Cupelopagis would change as the animal matured.

25 3.2 Methods

3.2.1 Fluorescent labeling

Fluorescent staining was performed to visualize the serotonergic nervous system and complementary structures (i.e., muscles and nuclei) using methods modified from Hochberg (2009). The concentrations, incubation periods, and temperatures for all primary and secondary antibodies and fluorophores used are listed in Table 3.1. Antibodies were diluted to their desired concentrations using 0.5% PBT, a solution of 0.1M phosphate buffered saline (PBS; Bio-Rad Laboratories) and Triton X-100 (Bio-Rad Laboratories). All steps were conducted in 1.5 ml microcentrifuge tubes on an orbital shaker at 4ºC. Prior to staining, whole animals were relaxed in 0.5% bupivacaine, fixed in 4% paraformaldehyde for 2 h, and rinsed in 0.1M PBS for 1 h. Animals were placed in a blocking solution consisting of 1% bovine serum albumin (Sigma- Aldrich) and 0.1M PBS overnight. Animals were rinsed in 0.1M PBS for 1 h and transferred into primary antibody solution. Subsequently, animals were rinsed in 0.5% PBT for 24 h and incubated in secondary antibody solution. Following removal from the secondary solution, animals were rinsed in 0.5% PBT for 24 h and incubated in fluorophores. No primary controls were processed in the same manner but omitted from primary antibody incubation. To preserve fluorescence, microcentrifuge tubes were wrapped with foil during secondary and fluorophore incubations. Specimens were mounted in Fluoromount-G (Thermo Fisher Scientific) on glass slides and stored at 4ºC prior to imaging.

3.2.2 Microscopy

Fluorescent labeling was observed using a Zeiss LSM 700 confocal laser-scanning microscope. Confocal z-stacks were generated at 0.1 µm intervals and processed as .TIF files using ZEN 2.3 software. 3D images and QTVR videos were generated using Volocity 6.3 software. Serotonin immunoreactivity (5HT-IR) was labeled according to structure and topology rather than function (Richter et al. 2010). Labels were consistent with Hochberg and Hochberg

26 (2015). Measurements of serotonin-immunoreactive (5HT-IR) cell bodies (perikarya; mean ± SD) were performed on confocal images imported into ZEN 2.3 software.

Table 3.1. Reagents used for fluorescent labeling. Concentrations, incubation periods, and temperatures for anti-serotonin, phalloidin, and DAPI stains.

Reagent Antigen/Conjugate Host Type Source Dilution Incubation

Primary anti-serotonin Rb polyclonal Sigma- 1:2000 48 h, 4°C Aldrich Secondary anti-rabbit IgG Gt Alexa 546- Invitrogen 1:200 24 h, 4°C conjugated Conjugate phalloidin - Alexa 488- Invitrogen - 2 h, 4°C conjugated DAPI - - Invitrogen 1:4000 2 h, 4°C

Rb: Rabbit; Gt: Goat.

3.3 Results

The serotonergic nervous system, muscles, and nuclei of Cupelopagis larvae (n = 8) and adults (n = 8) was successfully visualized; the best results are shown in Figures 3.1 and 3.3. Serotonin immunoreactivity (5HT-IR) was present in the cerebral ganglion and paired nerve cords of larvae and adults. The cerebral ganglion was outlined by a large cluster of nuclei at the anterior end and verified by colocalization between 5HT-IR and DAPI staining. The brain was dorsal to the mastax and bilaterally symmetrical. In larvae, paired lateral nerves innervated the corona, mastax, and foot. In adults, they innervated the infundibulum and proventriculus. Not all cell bodies and neurites were labeled with equal intensity across specimens (see Appendix 1 and

2). Both larvae and adults contained four 5HT-IR perikarya in their central nervous system. However, in larvae, neurons were arranged in an X-shaped pattern while in the adults they followed an arc-shaped arrangement (Figure 3.5). In adult animals, visualization of 5HT-IR was complicated by the contraction of the infundibulum and background staining as a result of debris or prey items in the proventriculus.

27 3.3.1 Larvae

The larval brain contained two pairs of 5HT-IR perikarya. Anterior perikarya (BPa) were medium-sized (mean ±SD: 7.03 ± 1.47 µm long; 4.85 ± 1.06 µm wide) and bipolar with an anterior and posterior extended neurite (Figure 3.2). The anterior neurite (BPaan) projected toward the corona into an area of diffuse immunoreactivity. The posterior neurite (BPapn) bifurcated into the neuropil and nerve cords. The pretzel-shaped neuropil innervated the nerve cords and all 5HT-IR perikarya. The posteriorly located perikarya (BPp) were larger (9.72 ± 2.20

µm long; 6.62 ± 1.35 µm wide) and unipolar with an anterior neurite (BPpan) directed to the neuropil.

3.3.2 Adults

The adult brain consisted of a cluster of four, small perikarya (5.87 ± 1.06 µm long; 4.44

± 0.52 µm wide) on the posterior side of the brain (BPp1-4) (Figures 3.4). The two outermost perikarya (BPp1,4) were unipolar with a lateral neurite (BPpln) extending toward the neuropil. The two innermost perikarya (BPp2,3) were bipolar with anterior neurites bifurcating into the neuropil. Similar to the larvae, the pretzel-shaped neuropil innervated the nerve cords and all 5HT-IR perikarya.

Figure 3.1. Fluorescent labeling in C. vorax larva. Reagents successfully stained (A) nuclei, (B) musculature, and (C) serotonin immunoreactivity (5HT-IR). (D) A merged image of DAPI (blue), phalloidin (green), and anti-serotonin (red) shows colocalization of nuclei and 5HT-IR. Merged confocal stack of 88 slices. Scale bar = 50 µm.

Figure 3.2. Labeled 5HT-IR in C. vorax larval specimen from Figure 3.1. (A) Anti-serotonin stain and (B) greyscale image are provided for contrast. Structures were labeled according to structure not function. BPa: anterior brain perikaryon; BPp: posterior brain perikaryon; BPaan: anterior neurite in BPa; BPapn: posterior neurite in BPa; BPpan: anterior neurite in BPp; np: neuropil; nc: lateral nerve cord. Merged confocal stack of 88 slices. Scale bar = 50 µm.

Figure 3.3. Fluorescent labeling in a C. vorax adult. Reagents successfully stained (A) nuclei, (B) musculature, and (C) 5HT-IR. (D) A merged image of DAPI (blue), phalloidin (green), and anti-serotonin (red) shows colocalization of nuclei and 5HT-IR in the anterior end. Merged confocal stack of 140 slices. Scale bar = 50 µm.

Figure 3.4. Labeled 5HT-IR in adult C. vorax specimen from Figure 3.3. (A) Anti-serotonin stain and (B) greyscale image. The asterisk indicates prey items and/or debris in the proventriculus. Adults do not have anterior brain perikarya. Merged confocal stack of 140 slices. Scale bar = 50 µm.

Figure 3.5. Side by side comparison of a larva and adult C. vorax specimens. Brain neurons in the larva are arranged in an X-shaped pattern while neurons in the adult stage have an arc-shaped distribution. Scale bar = 30 µm.

32 3.4 Discussion

In many invertebrates, the activity and connectivity of the nervous system is reorganized during metamorphosis (Tissot and Stocker 2000, Carvalho and Mirth 2015, Kaul-Strehlow et al. 2015). Only sessile species in superorder Gnesiotrocha undergo indirect development. Gnesiotrochans in orders Collothecacea and Flosculariacea metamorphose from free-swimming, non-feeding larvae mature into sessile, feeding adults (Wallace 1980, Fontaneto et al. 2003). The larval and adult stage are morphologically distinct. Larvae are often vermiform while adults possess an ornate head that is used in feeding. Evidence suggests that this ornate head

(infundibulum) is not homologous to the larval corona since it develops with the larval digestive tract and not as an extension of the corona itself (Hochberg and Hochberg 2017). This may indicate that mechanoreceptors within the infundibulum are derived or connected to the digestive system of Cupelopagis. Morphological and neurological traits develop concurrently to increase the individual fitness of animals (Carvalho and Mirth 2015). This is evident in Cupelopagis vorax which uses its distinct bowl-shaped infundibulum to detect and capture prey. This is the second research effort to reveal serotonin immunoreactivity (5HT-IR) in a sessile rotifer. Contrary to the findings of Hochberg and Hochberg (2015), metamorphosis had an effect on the position of 5HT-IR neurons in C. vorax. In Stephanoceros fimbriatus, larvae contained one pair of cerebral perikarya (BPL) that was absent in adults; conversely, a large pair of perikarya (AP) could only be detected in adults. In Cupelopagis, larvae had one pair of anterior perikarya and one pair of posterior perikarya while adults only had four posterior perikarya. 5HT-IR perikarya were arranged in an X-shaped pattern in larvae and in an arc-shaped pattern in adults. In addition, perikarya in adults were 1.7X smaller than the largest perikarya in larvae (BPp). The fate of 5HT-IR neurons in larvae and adults is unknown, be it restructuring, neurogenesis, or apoptosis. Based on these findings, potential homologues between life stages cannot be ascertained.

The structural differences in the serotonergic nervous system of Cupelopagis is likely related to the alternate behavioral strategies employed by larvae and adults. Serotonin is known

33 to act as a modulator of ciliary activity and feeding behavior in invertebrates (Hay-Schmidt 2000). In larval specimens, paired lateral nerve cords innervated the corona, mastax, and foot. The ciliated corona aids in locomotion while the foot contains pedal glands that permit individuals to attach to a suitable substrate (Wallace et al. 2006). Because adults cannot detach and relocate to a better environment, substrate selection has major implications on the survival and reproductive success of adults (Wallace and Edmondson 1986). Substrate selection is likely guided by neurosensory behavioral mechanisms (Clément 1987). Wallace and Edmondson

(1986) determined that neurosensory processing in Collotheca larvae is limited by the presence of a substratum and the presence of Ca+2 in the water. Both cues assure that larvae choose plants that are actively photosynthesizing such as Elodea. Cupelopagis larvae often settle on the underside of plants with broad, flat leaves or finely dissected leaves (e.g. Ceratophyllum, Elodea, and Potamogeton) (Edmondson 1949, Butler 1983). However, the specific modalities of substrate selection have not been discerned for C. vorax larvae. The innervation of the mastax at the larval stage provides additional evidence about the development of this specialized pharyngeal organ and the trophi (jaws). In sessile taxa, the mastax is functional at the larval stage yet feeding is suppressed because larvae have a birefringent structure that theoretically serves as a food source (Wallace 1980, 1993). Furthermore, the size and shape of the trophi does not change for floscularid rotifers and C. vorax as they mature from and egg to an adult and from embryo to adult, respectively (Fontaneto et al. 2003, Fontaneto and Melone 2005). During the larval stage, sessile taxa are likely more concerned with finding a suitable substrate for settling as opposed to actively feeding. Conversely, Fontaneto and Melone (2005) observed significant trophi growth in Asplanchna priodonta. This growth may be indicative of the selective feeding behavior often displayed by Asplanchna spp. (Gilbert 1978, 1980). In Cupelopagis adults, paired lateral nerve cords innervated the infundibulum and proventriculus. Both structures are relevant in explaining the predatory behavior of Cupelopagis. Behavioral studies provide indirect evidence of the presence of mechanoreceptors within the 34 infundibulum. These mechanoreceptors are likely involved in the detection and capture of prey items (Bevington et al. 1995, present study). Cupelopagis initiates an attack by lunging forward on its foot. Once a prey item is captured, it is pushed through the esophagus and stored in the proventriculus until the mastax macerates the prey and transfers it into the stomach (Wallace et al. 2015). An area of interest in Cupelopagis neuroanatomy is the innervation of the foot. In larvae, the foot is located in the posterior end and is innervated by 5HT-IR nerve cords. However, as the animal metamorphoses, the foot shifts to a ventral position and is located directly below the proventriculus. Based on the results of this study, it is proposed that the neurites innervating the proventriculus form a circuit with the pedal ganglion thus informing the animal when to attack or not attack potential prey given the storage capacity of the proventriculus. Here, 5HT-IR nerve cords innervated the proventriculus of adult C. vorax (refer to Appendix 2). Nonetheless, more research is needed to elucidate the function of this and other 5HT-IR sensory structures. Fluorescent staining and microscopy techniques only revealed the location of the brain and posterior lateral nerves to be analogous with Vasisht and Dawar (1969)’s description of the adult nervous system. The scope of the present study is limited because it only considers the serotonergic components of the nervous system. Brain neurons comprise approximately 20% of the total number of cells in the rotifer body (Kotikova et al. 2005). Of these, 3-7% are catecholaminergic neurons (Kotikova 1998). Our understanding of how ontogeny restructures the nervous system of gnesiotrochan rotifers can be increased by implementing suites of neurotransmitters (Hochberg and Hochberg 2015). The distribution of catecholamines, acetylcholine, FMRFamide, and small cardioactive peptide b (SCPb) have been successfully investigated in other bdelloid and monogonont rotifers. Kotikova et al. (2005) noted a difference in the number and distribution of neurons when FMRFamide and serotonin were compared in Platyias patulus, Euchlanis dilatata, and Asplanchna herricki. The number of FMRFamide containing neurons generally outnumbered serotonergic neurons. In addition, Hochberg (2007) found anti-FMRFamide and -SCPb staining to be abundant in the cerebral ganglion, ventrolateral 35 nerve cords, and mastax of Notommata copeus. The application of additional neurotransmitters can be applied to obtain a more complete picture of the nervous system in both larvae and adults C. vorax. The nervous system can be an informative organ system for interpreting phylogenetic relationships (Kaul-Strehlow et al. 2015). Within phylum Rotifera, the distribution of 5HT-IR neurons is species-specific and does not seem to be constrained by lifestyle modalities or taxonomy (Table 1.2). Kotikova et al. (2005) demonstrated that two ploimate species,

Asplanchna herricki and Euchlanis dilatata both have four 5HT-IR neurons even though they employ different feeding strategies, cruise and filter feeding, respectively. However, Hochberg (2009) determined that Asplanchna brightwellii had fourteen 5HT-IR neurons indicating that A. brightwellii has a more elaborate nervous system than A. herricki. Within superorder Gnesiotrocha, the number of 5HT-IR neurons ranges from three to 12. The nervous system of Cupelopagis vorax is relatively simple (four 5HT-IR perikarya) when compared to Stephanoceros fimbriatus (twelve 5HT-IR perikarya), another sessile rotifer. Based on this finding, ambush feeding may be a derived behavioral trait. Most rotifer species are filter feeders on microalgae, bacteria, or detritus (Wallace et al. 2006), therefore ambush feeding may have evolved in gnesiotrochan rotifers. Studies on neuroanatomy may provide phenetic traits for phylogenetic studies (Wallace 2002). Nonetheless, Hochberg and Hochberg (2015) caution against using cell or neurite distribution to interpret phylogenetic relationships as they may not reveal clear evolutionary patterns within phylum Rotifera. Instead, neurophylogenetic research should focus on immunoreactivity in closely related species (e.g., Cupelopagis vorax and other sessile gnesiotrochan taxa).

This study provides insight on the serotonergic nervous system of a sessile predator. The results support the hypothesis that the serotonergic nervous system would change as the animal metamorphosed. The distribution of 5HT-IR neurons seems to indicate that the nervous system of C. vorax is structured to meet the metabolic and physiological requirements of each

36 developmental stage (i.e., locomotion in larvae and feeding in adults). However, more research is needed to elucidate the function of neurons across rotifer species.

37 Chapter 4: Conclusions

This investigation was conducted to elucidate the behavioral and neuroanatomical components involved in the predatory behavior of the sessile rotifer, Cupelopagis vorax. The behavioral study build upon the findings of Bevington et al. (1995). Attack and capture behaviors were significantly different between two distinct prey, Lepadella triba and Lepidodermella squamata. Similar to the findings of Bevington et al. (1995), Cupelopagis was more likely to capture larger prey items, with a higher capture probability for Lepadella instead of L. squamata.

The present study also showed that predator size had a positive effect on capture efficiency, with larger predators capturing more prey. The feeding strategies of zooplankton play a role in predator-prey interactions. Filter feeders have been shown to feed on smaller prey while raptorial species prey on larger animals (Hansen et al. 1994). Rotifers show size dependent feeding behavior (Salt 1987) and employ feeding strategies that are directly related to their general life history (Wallace et al. 2015).

Ivlev’s and Manly-Chesson’s indices were considered to assess prey selectivity. It was initially hypothesized that Cupelopagis showed active selection toward both prey species; however, further analysis determined that selection was only likely for gastrotrich prey. Prey selectivity has been confirmed in Asplanchna spp. (Gilbert and Williamson 1978, Iyer and Rao

1996, Baumert 1998). These studies determined that prey defenses played a role in whether prey were captured. It would be interesting to test how Cupelopagis responds to prey items with morphological defenses, such as spines in rotifers and helmets in cladocerans, and if these would be actively avoided. Future studies should consider the role of taste receptors in determining whether prey items are selected or not. Ultrastructural techniques and behavioral studies could inform if Cupelopagis feeds on unpalatable prey (Wurdak et al. 1983, Walsh et al. 2006).

38 It is only recently that the nervous system of sessile gnesiotrochans has started to be compared. Results of the present study integrating fluorescent labeling and confocal microscopy revealed that metamorphosis had an effect on the position of serotonin immunoreactive (5HT-IR) neurons in C. vorax. Contrary to the findings of Hochberg and Hochberg (2015) who determined no significant structural differences in the larvae and adults of Stephanoceros fimbriatus. The structural differences in the serotonergic nervous system of Cupelopagis is likely related to the alternate behavioral strategies employed by larvae and adults. In larvae, 5HT-IR nerve cords innervate the corona, mastax, and foot, structures that contribute to locomotion and substrate selection behavior. In adults, the nerve cords innervate the infundibulum and proventriculus, structures that help in the detection and capture of prey items.

There is much to learn about the behavior and neuroanatomy of C. vorax. Serotonin regulates behavioral responses, such as feeding and ciliary activity (Hay-Schmidt 2000), as well as physiological processes involved in energy balance (Tecott 2007) in invertebrates. It would be meaningful to elucidate the role of serotonin in the feeding behavior of Cupelopagis. Lent et al.

(1991) found significantly low levels of serotonin in the central nervous system of leeches that were satiated in comparison to hungry leeches. Multiphoton microscopy can be employed to quantify serotonergic distribution in live cells (Balaji et al. 2005). Future studies could utilize auto-fluorescence imaging to observe any changes in the brain of C. vorax after feeding on different prey types. Because Cupelopagis has a limited scope of potential prey, its nervous system may be wired to select prey with high energy content (DeMott 1986). However, in a situation where no palatable prey are available, consumption is likely to occur to meet minimum caloric demands (Mitra and Flynn 2006). Changes in serotonin levels when C. vorax is fed low quality versus high quality prey could be correlated with selective feeding behavior. Further

39 studies on energy balance could also observe the effects of exogenous serotonin in eliciting motor behaviors associated with feeding (i.e., attack and capture) (Tecott 2007).

Furthermore, electrophysiological techniques have been used to bridge the gap within structure and function. Joanidopoulos and Marwan (1998) observed that the stimulation of sensory receptors triggered specific male mating responses in Asplanchna sieboldi. The present study provides indirect evidence of the presence of mechanoreceptors within the infundibulum.

Our understanding of how Cupelopagis is able to respond to vibrations elicited by potential prey could be enhanced by measuring the electrical activity of 5HT-IR neurons. An application of an electrophysiological approach could also aid in elucidating central pattern generator (CPG) circuits within phylum Rotifera (Selverston 2010). Wallace (2002) outlined the challenges in characterizing rotifer neurobiology and proposed implementing a variety of techniques such as dye injection, cell labeling, photometry, and video imaging to aid in our understanding of the rotifer nervous system. In addition, integrative studies offer the opportunity to analyze aspects about the behavior and neurological responses in gnesiotrochans in a more holistic manner.

In conclusion, morphological and neurological adaptations allow Cupelopagis to thrive in its environment. Although in this study each component was analyzed separately, behavioral responses complement the distribution of neural structures and the distribution of neural structures can be used to help explain behavior. This research effort is the first to integrate behavioral and neuroanatomical techniques to investigate the feeding behavior of a sessile rotifer species. The results can help define evolutionary mechanisms involved in the development of feeding behavior within phylum Rotifera. In a more general context, it increases our understanding of the evolution of the invertebrate nervous system.

40 References

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48 Appendices

Appendix 1: Additional confocal images of serotonin immunoreactivity in larvae. A total of eight larvae were stained, image labels are described in section 3.3.1.

52 Appendix 2: Additional confocal images of serotonin immunoreactivity in adults. A total of eight adults were stained, image labels are described in section 3.3.2. Circles indicate prey items or debris (*) in the proventriculus.

56 Vita

Elizabeth Preza graduated from the University of Texas at El Paso with a Bachelor of Science in Biological Sciences in spring 2014. Her passion for research motivated her to continue her education in the Master’s Program in Biological Sciences. She worked as a Research Assistant funded by the National Science Foundation (DEB-1257068) in the Walsh laboratory from 2014- 2017. She was also a Teaching Assistant for Biol 1107 in fall 2016 and Biol 1108 in the spring 2017 and fall 2017 semesters. She presented posters for her research at the XIV International

Rotifer Symposium in České Budějovice, Czech Republic in 2016 and at the 2017 Evolution Meeting in Portland, OR. She was awarded Best Student Poster at the XIV International Rotifer Symposium. In 2016 she was awarded Outstanding Master’s Student in Ecology and Evolutionary Biology by the Department of Biological Sciences. She also received the Dodson Research Grant and the Graduate Student Research Assistant Summer Program award from the Graduate School. Elizabeth graduated with her Master of Science degree in December 2017.

Contact Information: [email protected]

This thesis was typed by Elizabeth Preza.