© 2011

Aabir Banerji

ALL RIGHTS RESERVED

DYNAMICS AND ADAPTIVE SIGNIFICANCE OF THE INDUCIBLE

TROPHIC POLYMORPHISM OF TETRAHYMENA VORAX

By

AABIR BANERJI

A Dissertation Submitted to:

Graduate School – New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements for the degree of:

Doctor of Philosophy

Graduate Program in Ecology & Evolution

Written under the direction of:

Dr. Peter J. Morin

and approved by:

_____________________________________

_____________________________________

_____________________________________

_____________________________________

_____________________________________

New Brunswick, New Jersey

October, 2011

ii

ABSTRACT OF THE DISSERTATION:

Dynamics and Adaptive Significance of the Inducible Trophic Polymorphism of

Tetrahymena vorax

By Aabir Banerji

Dissertation Director:

Dr. Peter J. Morin

This dissertation provides a four-part synopsis of the methodology, results, and

conclusions of an integrated set of experiments designed to explore the ecology of

inducible trophic polymorphisms (ITPs). These experiments were conducted using

isogenic populations of the polymorphic freshwater hymenostome ciliate, Tetrahymena

vorax Kidder, in combination with other protozoa, bacteria, algae, and micro-

invertebrates. Chapter 1 addresses the autecological significance of ITPs, revealing some

of their potential costs and benefits to individuals in the context of competition under

varying resource regimes. Chapter 2 demonstrates how ITPs can give rise to novel trait-

mediated indirect interactions that affect community structure and population dynamics.

Chapter 3 elucidates the extent to which the population dynamics of species exhibiting

ITPs are affected by the trophic complexity of their habitat. Chapter 4 presents a detailed

review of indirect offenses, a versatile adaptive strategy that ITPs can give rise to where

inducing agents are symbionts. The contents of these chapters provide unique insights

into the link between ITPs and related phenomena – such as cannibalism, intraguild

predation, omnivory, and expanded diet breadth – and open new avenues of inquiry

regarding the importance of the genetic and functional diversity of natural communities.

iii

ACKNOWLEDGEMENTS

First and foremost, I must thank Peter Morin, my academic advisor. I cannot

begin to describe the extent to which I am indebted to him for the countless ways in

which he has nurtured my personal growth and professional development since the day I

came to Rutgers. Rather than try to mold me into a re-creation of himself or persuade me

to become a mindless automaton to be exploited for his own agendas, Peter chose to

show respect for me both as his junior colleague and as an individual, which, I must add,

he does with all of his students, irrespective of where they are on the academic totem

pole. He did as much as he could to look out for me and was always willing to not only

hear me out, but also draw upon his vast, encyclopedic knowledge and years of

experience to help me achieve my goals. Time and again, I would come to him with

what I thought was an insurmountable obstacle, muttering profusely in my frustration

with it, only to have him take a quick look at it and systematically turn it into something

that I knew I could handle. Peter encouraged me when I needed it, straightened me out

when I needed it, left me to my own devices when I needed it, and did everything else

that was required to ensure that I would become a competent, self-respecting scientist

who was capable of finding his own way in the world. I could never have asked for a

better mentor, role-model, or friend than what Peter has been to me these past several

years.

Next, I must thank Marsha Morin, without whose intelligence, resourcefulness,

integrity, diligence, awareness, reliability, compassion, cat-herding ability, and sheer

tolerance for pain, my dissertation (along with our entire department) would have most

certainly burst into flames and/or collapsed into some kind of gravitational point

iv

singularity long ago. Her warmth and supportiveness have been a great source of

strength and joy for me from the very first day we met. To my gratitude for all of this, I

must also add my gratitude for the fact that she at some point forgave me for having

addressed her as “ma’am” for the first year or so of our acquaintance. Especially in light

of the fact that my parents had given both her and Peter express permission to beat me, I

consider this to have been most kind.

I thank my committee members (Tim Casey, Rebecca Jordan, Mike Pace, and

Cesar Rodriguez-Saona) for their always meaningful and prompt feedback on my various

drafts and progress reports, their willingness to work around their respective schedules to

accommodate my needs, their support of my research by way of resources and ideas, and

their having made every major milestone toward my degree fun and fulfilling for me.

I thank Martha Haviland, Doreen Glodowski, and Jana Curry (along with Jana’s

predecessor, Barbara Sena and the Busch MSLC’s administrative coordinator, Margie

Eickhorst) for having provided me with the opportunity and the means to take up

teaching, for having stood by me and come through for me whenever I needed help

solving problems and making things happen, for tolerating (and often even encouraging)

my various teaching-related shenanigans, and for being down-right awesome in general.

I thank the following people for helping me get started on the path to becoming a

full-fledged ecologist: Jim “Doc” Applegate, Barbara Goff, Colleen Hatfield, Bruce

Hamilton, Ted Stiles, Charles Harman, Fred Westcott, Ralph Caiazzo, Carol VanDalen,

Joyce Hartmann, and Kathy Parsell. Each of them in their own unique ways gave me

guidance, encouraged me to follow my heart, and enabled me to catch a glimpse of my

true potential. Their wisdom and their friendship will forever be a part of who I am.

v

I thank the following fellow participants in the 2005 Community Ecology course

(S211) at the Research School for Socio-Economic and Natural Sciences of the

Environment for the stimulating discussions and fun we had back then and the

stimulating discussions and fun we have had since: Anja Rubach, Karin Nilsson, Irene

van der Stap, Wade Ryberg, Tim Engelkes, Björn Christian Rall, Magnus Huss, Órla

McLaughlin, Euridice Leyequien, Geraldine Thiere, Sonia Kéfi, Martin Pedersen, Elly

Morrien, Jordie Netten, Maarten Eppinga, Roy van Grunsven, Mariska te Beest, Cleo

Gosling, Yuki Fujita, Ciska Veen, Marleen Riemens, Marleen Pierik, Tessa van der

Hammen, Charlotte Borvall, Johan Ahnström, Frans Kuenen, Ellen Weerman, and

Matthijs Schouten. Being together and working together with all of these fellow hard-

core community ecologists / citizens of the world will remain one of my fondest

memories in the years to come. Soon it will be our turn to do what our supervisors did

for us.

I thank my former lab mates (Lin Jiang, Christina Kaunzinger, Zac Long, Timon

McPhearson, Jen Price, Jenn Adams Krumins, Henry Stevens, Irene Karel, and Alex

Kulczycki) for showing me the ropes, helping me to imbibe the principle of K.I.S.S.

(Keep It Simple, Stupid!), and being a pleasure to hang out with.

I thank my current lab mates (Kevin Aagaard, Laura Chen, Jean Deo, Sean Hayes,

Ruchi Patel, Jack Siegrist, Maria Stanko, Holly Vuong, and Talia Young) for suffering

through my roughest drafts; putting up with my various quirks and ineptitudes within our

common, confined space; and sharing with me their ideas, enthusiasm, and friendship.

I thank the following faculty members and loose affiliates for profound, timely

advice and countless fascinating discussions (including everything from the need for

vi

applied ecology in modern medicine to the trouble with tribbles): Tamar Barkay, Mary

Cadenasso, John Dighton, David Drake, Siobain Duffy, David Ehrenfeld, Joan Ehrenfeld,

Nina Fefferman, Dina Fonseca, Jeremy Fox, Jason Grabosky, Ed Green, Steven Handel,

Brad Hillman, Claus Holzapfel, Henry John-Alder, Karl Kjer, Sharron Lawler, Julie

Lockwood, Bonnie McCay, Scott Meiners, Owen Petchey, Steward Pickett, Beth Ravit,

John Reinfelder, Peter Smouse, Lena Struwe, and Mike Sukhdeo.

While I owe every single one of my friends from Rutgers my gratitude and

acknowledgement for their empathy, encouragement, and collaboration, the following

deserve special recognition for having gone above and beyond the call of duty for me on

numerous occasions: Carrie Norin, Elena Tartaglia, Blake Mathys (and his lovely wife,

Dimitria), Wayne Rossiter (and his lovely wife, Melissa), Ai Wen (and her lovely

husband, Kenneth Elgersma), Dave Smith, Andrea Egizi, Ari Novy, Charlie Kontos,

Aspa Chatziefthimiou, Diana Johnson, Tricia Ramey-Balci, Kerri-Ann Norton, Rob

Hamilton IV, Kristen Ross, Em Stander, Jenni Momsen, Sharron Crane, Alison Seigel,

Alex Hernandez, Faye Benjamin, Wes Brooks, Brian Clough, Jeremy Feinberg, Zac

Freedman, Sona Mason, Christine Minerowicz, Mat Pompliano, and Stephanie

Baccarella.

Most of all, I thank my family. They have understood the formula.

vii

TABLE OF CONTENTS

Abstract of Dissertation …………………………….………………….… ii

Acknowledgements ……..……………………………………………..… iii

Table of Contents …………….………………………………………..… vii

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

Chapter 1: Phenotypic Plasticity, Intraguild Predation, and Anti-

Cannibal Defenses in an Enigmatic Polymorphic Ciliate …… 5

Chapter 2: Prey-Induced Gape Expansion Mediates Apparent

Competition …………………………………………………. 32

Chapter 3: Dynamics between Protists and Crustacean Zooplankton: Do

Copepods Show Preference for the Predatory Morph of

Tetrahymena vorax? ………………………………………… 52

Chapter 4: On the Strategy of Mounting an Indirect Offense: What If My

Enemy Has Allies, Too? …………………………………….. 71

Conclusion ………………………………………………………….….…. 97

Curriculum Vitae …………….….….…………………………………..… 98

1

INTRODUCTION

Phenotypic plasticity is the ability of an organism to alter its morphology or

physiology in response to environmental cues. It is distinct from evolutionary adaptation

in that it occurs at the individual level, rather than at the population level, and generally

involves no change in the organism’s genetic material (DeWitt and Scheiner 2004).

Many examples of phenotypic plasticity are conspicuous and familiar, such as the color

changes of chameleons (Stuart-Fox et al. 2008) and the phototropism of plants (Correll

and Kiss 2002). Others, including the various internal responses that allow for immunity

(Frost 1999) and homeostasis (Woods 2009), are more subtle and easy to overlook. In

population studies, phenotypic plasticity is fascinating not only as a versatile adaptive

trait (Via et al. 1995), but also as a potential driver of diversification and speciation

(Adams and Huntingford 2004, Pfennig et al. 2010).

Phenotypic plasticity has been shown to influence the dynamics and stability of

ecological communities (Agrawal 2001, Garay-Narváez and Ramos-Jiliberto 2009). A

particularly intriguing way that it may do so is by generating inducible trophic

polymorphisms. Inducible trophic polymorphisms (hereafter abbreviated as ITPs) occur

when some individuals in a population alter their morphology or physiology to expand

the number of trophic levels on which they feed. Induction of alternative morphs occurs

in response to environmental factors, such as specific chemical cues released by

competitors and/or potential prey. ITPs are functionally complementary to inducible

defenses among prey (Tollrian and Harvell 1999). As is true for inducible defenses, ITPs

may initially seem like unusual adaptations that occur infrequently, but are in reality

widely distributed across taxa.

2

The consequences of ITPs for long-term population and community dynamics are

likely significant, but difficult to assess. For most long-lived organisms, the amount of

time and effort required to monitor populations through two or more generations is

substantial, and opportunities for genetic recombination can make it nearly impossible to

distinguish induced variation from constitutive variation. To approach the challenge of

addressing these consequences, I have conducted my research using protists in

experimental microcosms. This approach has been shown to be valuable in testing

ecological and evolutionary theory and unveiling the mechanisms that underlie general

patterns (Cadotte et al. 2005, Holyoak and Lawler 2005). Experimental microcosms

make for highly controlled and replicable systems. The short generation times of protists,

together with their large population sizes, facilitate the study of population dynamics

(Morin and Lawler 1996, Price and Morin 2004). In the microcosms I made use of in my

research, the focal species was the freshwater hymenostome ciliate, Tetrahymena vorax

Kidder.

The natural history of T. vorax makes this a fascinating and powerful model

system for the study of ITPs. T. vorax exhibits a conspicuous ITP. Rapid morphological

differentiation allows different individuals in a single isogenic population to feed lower

(on bacteria) or higher (on ciliates that consume bacteria) in the same food chain (Kidder

et al. 1940, Ryals et al. 2002). The different morphs of T. vorax are easily distinguished

and counted via microscopy. Populations in the early logarithmic stage of growth

typically consist entirely of small (~ 45-75 m long), pear-shaped morphs, called

“microstomes,” which feed on bacteria. In response to known chemical cues that are

released by competing bacterivores, microstomes may transform into larger (~ 250 m

3

long), oval-shaped morphs, called “macrostomes,” which feed on other ciliates (Smith-

Somerville et al. 2000, Ryals et al. 2002). Once a microstome has transformed into a

macrostome, it can give rise to additional macrostomes, or it can revert into a microstome

at the time of cell division (~ every 6 hours).

My use of T. vorax for my experimental investigations has enabled me to explore

the autecological significance of ITPs, evaluate the potential of ITPs to generate complex

trait-mediated indirect effects, and elucidate the extent to which population and

community dynamics associated with ITPs are affected by trophic complexity. In so

doing, I have: (1) uncovered potential costs and benefits of different inducible

morphologies in the context of competition, (2) demonstrated how ITPs can potentially

mediate interactions such as “apparent competition” (Holt 1977), and (3) identified

possible roles of major groups of aquatic consumers in limiting the distribution of

polymorphic ciliates in nature. A portion of my work – Chapter 1 of this dissertation –

has already been published in the journal Functional Ecology (Banerji and Morin 2009).

References

Adams, C. E., and F. A. Huntingford. 2004. Incipient speciation driven by phenotypic

plasticity? Evidence from sympatric populations of Arctic charr. Biological

Journal of the Linnean Society 81(4): 611-618.

Agrawal, A. A. 2001. Phenotypic plasticity in the interaction and evolution of species.

Science 294: 321-326.

Banerji, A., and P. J. Morin. 2009. Phenotypic plasticity, intraguild predation and anti-

cannibal defences in an enigmatic polymorphic ciliate. Functional Ecology 23:

427-434.

Cadotte, M. W., J. A. Drake, and T. Fukami. 2005. Constructing nature: laboratory

models as necessary tools for investigating complex ecological communities.

Advances in Ecological Research 37: 333-353.

Correll, M. J., and J. Z. Kiss. 2002. Interactions between gravitropism and phototropism

in plants. Journal of Plant Growth Regulation 21(2): 89-101.

DeWitt, T. J., and S. M. Scheiner. 2004. Phenotypic variation from single genotypes: a

primer. Pages 1-9 in T. J. DeWitt and S. M. Scheiner, editors. Phenotypic

4

Plasticity: Functional and Conceptual Approaches. Oxford University Press,

Oxford.

Frost, S. D. W. 1999. The immune system as an inducible defense. Pages 104-126 in R.

Tollrian and C. D. Harvell, editors. The Ecology and Evolution of Inducible

Defenses. Princeton University Press, Princeton.

Garay-Narváez, L., and R. Ramos-Jiliberto. 2009. Induced defenses within food webs:

the role of community trade-offs, delayed responses, and defense specificity.

Ecological Complexity 6(3): 383-391.

Holyoak, M., and S. P. Lawler. 2005. The contribution of laboratory experiments on

protists to understand population and metapopulation dynamics. Advances in

Ecological Research 37: 245-271.

Kidder, G. W., D. M. Lilly, and S. L. Claff. 1940. Growth studies on ciliates. IV. The

influence of food on the structure and growth of Glaucoma vorax sp. nov.

Biological Bulletin 78: 9-23.

Morin, P. J. and S. P. Lawler. 1996. Effects of food chain length and omnivory on

population dynamics in experimental microcosms. Pages 218-230 in G. Polis and

K. Winemiller, editors. Food Webs: Integration of Patterns & Dynamics.

Chapman and Hall.

Pfennig, D. W., M. A. Wund, E. C. Snell-Rood, T. Cruickshank, C. D. Schlichting, and

A. P. Moczek. 2010. Phenotypic plasticity’s impacts on diversification and

speciation. Trends in Ecology and Evolution 25: 459-467.

Price, J. E., and P. J. Morin. 2004. Colonization history determines alternate community

states in a food web of intraguild predators. Ecology 85:1017-1028.

Ryals, P. E., H. E. Smith-Somerville, and H. E. Buhse Jr. 2002. Phenotype switching in

polymorphic Tetrahymena: a single-cell Jekyll and Hyde. International Review of

Cytology 212: 209-238.

Smith-Somerville, H. E., J. K. Hardman, R. Timkovich, W. J. Ray, K. E. Rose, P. E.

Ryals, S. H. Gibbons, and H. E. Buhse Jr. 2000. A complex of iron and nucleic

acid catabolites is a signal that triggers differentiation in a freshwater protozoan.

Proceedings of the National Academy of Sciences 97: 7325-7330.

Stuart-Fox, D., A. Moussalli, and M. J. Whiting. 2008. Predator-specific camouflage in

chameleons. Biology Letters 4(4): 326-329.

Tollrian, R., and C. D. Harvell, editors. 1999. The ecology and evolution of inducible

defenses. Princeton Univerity Press, Princeton.

Via, S., R. Gomulkiewicz, G. De Jong, S. M. Scheiner, C. D. Schlichting, and P. H. Van

Tienderen. 1995. Adaptive phenotypic plasticity: consensus and controversy.

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Woods, H. A. 2009. Evolution of homeostatic physiological systems. Pages 591-610 in

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Science Publishers, Plymouth.

5

CHAPTER 1

Phenotypic Plasticity, Intraguild Predation, and Anti-Cannibal Defenses in

an Enigmatic Polymorphic Ciliate

Summary

1. Inducible trophic polymorphisms are greatly underappreciated forms of

phenotypic plasticity that allow organisms to respond dynamically to

environmental variation by enabling them to change the trophic level upon

which they feed. Although inducible trophic polymorphisms occur in a

diverse array of organisms, their costs and benefits and their consequences for

long-term population and community dynamics are poorly understood.

2. I studied the inducible trophic polymorphism of the freshwater hymenostome

ciliate Tetrahymena vorax, whose isogenic populations can contain three

distinct morphs: pyriform, bacterivorous microstomes; larger, carnivorous

macrostomes; and elongate, “tailed” microstomes. I tested whether: (1) the

tailed microstome constitutes an inducible defense against macrostomes and

(2) the transformation of microstomes into macrostomes is size-dependent. I

also recorded the dynamics of the three morphs in the presence and absence of

an intraguild prey (Colpidium) across a gradient of growth medium

concentrations to infer potential tradeoffs in the success of different morphs at

different productivity levels.

3. Macrostomes do not discriminate between pyriform microstomes and readily-

consumed heterospecific prey (Colpidium) when provided only one of these

6

alternative prey items. Tailed microstomes display greatly reduced

susceptibility to consumption by macrostomes as compared to undefended,

pyriform microstomes. Morph dynamics are consistent with the hypothesis

that tailed microstomes serve as an inducible defense against cannibalism;

tailed microstomes and macrostomes appear simultaneously, in both the

presence and absence of Colpidium. At low productivity, T. vorax achieves

higher rates of growth when able to feed on Colpidium than when feeding on

bacteria alone. At higher productivity, this pattern is reversed, with growth

rates maximized in the absence of Colpidium.

4. The reduced consumption rate of tailed microstomes by cannibalistic

macrostomes, together with the simultaneous induction of tailed microstomes

and macrostomes, suggests that both morphs comprise a coordinated adaptive

response to the presence of intraguild prey.

Key-words: Induced offenses, Tetrahymena vorax, inducible trophic polymorphism,

omnivory

7

Introduction

Phenotypic plasticity can potentially have profound effects on the population

dynamics of interacting species. For instance, numerous examples of inducible defenses

have been shown to completely alter the predicted outcome of predator-prey interactions

(Tollrian and Harvell 1999). These inducible defenses can involve an array of

behavioral, physiological, metabolic, or life-history traits and can be direct or indirect

(Porter and Porter 1979; Abrahams 1993; Tollrian and Harvell 1999; Altwegg 2002).

While inducible defenses are the subject of extensive study (Tollrian and Harvell 1999),

their logical counterpart, inducible offenses (Padilla 2001), remain little-studied and

greatly underappreciated. Inducible offenses often involve inducible trophic

polymorphisms. In these situations, certain organisms alter the trophic level upon which

they feed via changes in morphology, physiology, or behavior, modifying the number and

types of interactions in their food web.

Inducible trophic polymorphisms take many forms. In some organisms,

reversible, morphological transformations provide flexible responses to environmental

variation within a single generation. In others, transformations constitute irreversible,

developmental trajectories that commit individuals to particular trophic roles. Some

inducible trophic polymorphisms produce changes in diet without changing the mode of

acquisition, such as where a predator turns into a higher-level predator or a cannibal

(Gilbert 1973, Wainwright et al. 1991). Others represent both a change in diet and in the

mode of acquisition, such as where an herbivore turns into a carnivore (Pfennig 1992,

Afik and Karasov 1995) or where an autotroph becomes a parasite (Baird and Riopel

1984).

8

Inducible trophic polymorphisms occur within a wide range of taxa that includes

birds (Afik and Karasov 1995), amphibians (Pfennig 1992), fish (Wainwright et al.

1991), gastropods (Padilla 2001), plants (Baird and Riopel 1984), fungi (Barron 1977,

Barron 1992, Kerry and Jaffee 1997), rotifers (Gilbert 1973), protists (Washburn 1988),

and bacteria (Berleman and Kirby 2007). The diversity and prevalence of inducible

trophic polymorphisms provide a compelling motivation to investigate what effects

inducible trophic polymorphisms have on long-term population dynamics. The study of

inducible trophic polymorphisms can also provide insights into related phenomena, such

as cannibalism (Fox 1975, Polis 1981), intraguild predation (Polis et al. 1989, Holt and

Polis 1997), omnivory (Polis 1991, Morin 1999, Diehl and Feissel 2000), variation in diet

breadth (Wainwright et al. 1991), and phenotypic plasticity in general (DeWitt and

Scheiner 2004). Inducible trophic polymorphisms may even shed light on certain

evolutionary processes, due to the fact that they potentially allow reciprocal induced

offenses and defenses to occur rapidly over short ecological time frames, changes that are

comparable to the shifting phenotypes seen over much longer time frames as a

consequence of reciprocal genetic changes in co-evolving species (Agrawal 2001).

The inducible trophic polymorphism of the freshwater protozoan Tetrahymena

vorax Kidder (Ciliatea: Hymenostomatida) offers five main advantages as a model

system for the experimental study of inducible offenses: (1) its small size (60-250 µm)

and ease of culture makes experimental studies relatively convenient; (2) its short

generation time (~ 6 hours) makes possible the collection of long-term population

dynamics data in a matter of days or weeks; (3) its clonal mode of reproduction (the cells

are amicronucleate and unable to conjugate) ensures that any short-term phenotypic

9

variation does not reflect underlying genetic variation; (4) its discrete polymorphism

facilitates distinguishing between morphs and quantifying the transformation; and (5) the

proximal cues triggering transformations are known and are easily manipulated (Ryals et

al. 2002).

Isogenic populations of T. vorax contain three distinct morphs: microstomes,

macrostomes, and tailed microstomes. Microstomes (~ 60 µm in length) are pyriform,

with a small cytostome that restricts their diet to bacteria. Macrostomes (~ 200 µm) are

ovoid, with a larger cytostome that allows them to prey on other smaller ciliates. Tailed

microstomes (~ 90 µm) also consume bacteria, but unlike pyriform microstomes, are

narrow, elongate cells with the posterior drawn out into a stiff, tail-like structure (Kidder

et al. 1941, Williams 1961). New T. vorax populations (at low density and high resource

levels) initially consist entirely of pyriform microstomes. As population density

increases, some of the pyriform microstomes transform into macrostomes and tailed

microstomes. Each of the three morphs can transform into any of the others (Fig. 1).

Microstomes transform into macrostomes when exposed to known cues, the

nucleic acid catabolites uracil and hypoxanthine in the presence of ferrous iron (Smith-

Somerville et al. 2000). The cues are released by other ciliates that are competitors for

bacteria, such as Colpidium striatum Stokes (Ciliatea: Hymenostomatida) and also by

conspecifics. In the absence of predators, Colpidium can reduce bacterial densities to a

greater extent than T. vorax (Kaunzinger and Morin 1998, Price and Morin 2004), which

suggests that microstomes might be competitively excluded by Colpidium. The ability of

T. vorax to mount an inducible offense against its competitors may provide a potentially

significant fitness advantage (Thingstad et al. 1996). However, as with any inducible

10

response, there may be constraints and / or costs that prevent this response from being

constitutive (Relyea 2002, DeWitt and Scheiner 2004). If, for example, the induced

macrostomes cannot discriminate between conspecifics and heterospecifics, the potential

advantage of being able to eat interspecific competitors could be offset by the costs of

cannibalism (Polis 1981).

Cannibalism can be adaptive if it allows a lineage to survive and reproduce during

severe resource limitation (Crump 1983, Van den Bosch et al. 1988, Hoffman and

Pfennig 1999). However, the observed rarity of cannibalism under less severe conditions

and among taxa in general suggests that it exacts significant fitness costs (Pfennig and

Collins 1993, Pfennig and Frankino 1997, Kusch 1999, Pfennig 1999, Pfennig 2000,

Michimae and Wakahara 2002). Therefore, where inducible trophic polymorphisms

create the potential for cannibalism, there is likely to be strong selection for mechanisms

that minimize cannibalism on close relatives, or that divert attacks to non-kin (Gilbert

1973, Pfennig and Collins 1993, Pfennig and Frankino 1997, Kusch 1999, Pfennig 1999).

Grønlien et al. (2002) have demonstrated that macrostomes capture heterospecific

prey in preference to conspecific microstomes and will engulf available microstomes

only after exhausting the supply of heterospecific prey. These results suggest that T.

vorax may indeed use kairomones or other cues to discriminate between kin and non-kin,

but they also indicate that this ability does not actually prevent macrostomes from

ultimately consuming smaller conspecifics. Nevertheless, T. vorax may still minimize

the costs of cannibalism through reciprocal, coordinated, morphological changes among

macrostomes and microstomes (Agrawal 2001, Kopp and Tollrian 2003a,b). My

preliminary observations indicated that tailed microstomes may be less susceptible to

11

cannibalism by macrostomes because elongate tailed microstomes cannot be completely

engulfed. If so, tailed microstomes may constitute an inducible defense against

cannibalism that would allow for a highly adaptive mixed foraging strategy, assuming

they are consistently present when macrostomes are present.

I speculated that the polymorphism of T. vorax could be the result of size-

dependent differentiation. Microstomes vary somewhat in size because of recent feeding

history, position in the cell cycle, and chance events. The successful transformation from

a microstome into a macrostome could depend on cell size at the time of induction, where

larger microstomes transform into macrostomes upon induction, while smaller ones

transform into tailed microstomes. Presumably, the proportion of larger microstomes in

the population is determined by the overall abundance of energy and nutrients (in the

form of bacteria) within the system. If macrostome production is energetically costly, or

depends on higher levels of productivity, then decreased bacterial abundances should lead

to a decreased frequency of macrostomes within the population. If resource limitation is

not a significant constraint on macrostome production, however, intraguild predation

theory predicts that T. vorax should switch from feeding on bacteria, the shared resource,

when bacteria are abundant, to feeding on Colpidium, the intraguild prey, when

Colpidium is abundant relative to bacteria. Increasing bacterial abundance by increasing

productivity would, in this case, reduce the relative frequency of macrostomes in the T.

vorax population and decrease intraguild predation of Colpidium by T. vorax (Křivan

2000, van Baalen et al. 2001).

This paper describes a series of studies designed to elucidate the costs and

benefits associated with each morph of T. vorax. I explore three questions arising from

12

the inducible trophic polymorphism. (1) Do tailed microstomes serve as an inducible

defense against cannibalism by conspecific macrostomes? (2) Is size-dependent

differentiation responsible for the mix of macrostomes and microstomes seen in isogenic

T. vorax populations? (3) Is the proportion of macrostomes in T. vorax populations more

indicative of the energetic and physiological requirements of transformation or of the

relative advantage to being a predator?

Methods

I obtained T. vorax from the American Type Culture Collection (strain 30421).

Isogenic populations of T. vorax were cultured in 240-ml glass jars containing 100 ml of

complex organic medium. Medium consisted of 0.37 g of Carolina Biological Supply

protozoan pellets (Carolina Biological Supply Company, Burlington, NC 27215, USA)

per L of filtered well water. Autoclaved medium was inoculated after cooling with three

species of bacteria (prey for the ciliates – Serratia marcescens, Bacillus subtilis, and

Bacillus cereus). I autoclaved all culture vessels before use. Screw caps on the jars

permitted some air circulation, while restricting evaporation and limiting contamination.

The experimental units used in this study contained detritus-based food-chains,

consisting of organic matter, bacteria, and ciliates. The organic matter in the growth

medium provided a conveniently manipulated energy source. Differences in the

concentration of the medium create differences in bacterial densities, which simulate

changes in the productivity of the system (Morin 1999).

Prey Selection by Macrostomes

To determine the potential effectiveness of the tailed microstome as an inducible

defense against cannibalism, I evaluated rates of prey consumption by macrostomes

13

feeding on different densities of pyriform microstomes, tailed microstomes, and

Colpidium. Feeding took place in 15-mm Petri dishes in 0.2 ml of medium, to confine all

cells (macrostomes and their prey) within the field of view of the microscope.

Each trial initially contained 30 macrostomes exposed to one of nine

combinations of prey type (pyriform microstomes, tailed microstomes, Colpidium) and

prey density (low ~ 201 cells / mL, medium ~ 403 cells / mL, and high ~ 599 cells /

mL). Prey densities varied to ensure that any differences found among the capture rates

for different prey items would be due to prey morphology, rather than to density-

dependent encounter rates. The response was the number of successful captures observed

within a 15-minute interval. I removed macrostomes that engulfed prey items using a

micropipette, to keep track of captures by macrostomes. Individual macrostomes were,

therefore, limited to completing a single capture per the 15-minute observation period.

There were three replicates for each combination of prey type and prey density.

I compared the number of prey captured per unit time with an analysis of

covariance (ANCOVA), focusing on differences among the three prey types (Colpidium,

microstomes, and tailed microstomes) using prey density as a covariate. Differences in

the slope of the prey-captured-vs.-prey-density relation among prey types would indicate

differences in susceptibility to macrostomes.

Phenology of the Tailed Microstome

I grew isogenic populations of T. vorax with and without Colpidium across a

gradient of 5 different nutrient concentrations to explore whether the phenology of tailed

microstomes is consistent with the possibility of their serving as an inducible defense

against cannibalism by macrostomes. The nutrient concentrations used were 50% (0.19

14

g/L), 75% (0.28 g/L), 100% (standard, 0.37 g/L), 125% (0.46 g/L), and 150% (0.56 g/L).

I recorded the population density for each morph and for Colpidium on 6 days (days 1, 2,

3, 4, 5, and 7) over a 7 day interval, sampling approximately 0.3 ml from each jar without

replacement. I first combined these observations of the densities of tailed microstomes

and macrostomes in a single correlation analysis to evaluate whether the changing

population density (cells/ml) of tailed microstomes was consistently correlated with the

changing population density of macrostomes over time. One microcosm became

contaminated with flagellates on Day 4 of the experiment. The data from this replicate

was subsequently discarded for Days 4, 5, and 7, yielding a total of 177 data points for

the analysis. This analysis assumes that population dynamics are sufficiently rapid that

observations made on different days in the same microcosm are effectively independent.

Given that T. vorax has a generation time of about 6 hours, this is a reasonable

assumption. A more conservative analysis evaluated the correlation between densities of

tailed microstomes and macrostomes separately for each of the sampling days (30

replicates on days 1, 2, 3; 29 replicates on days 4, 5, 7), treating each microcosm as an

independent observation on a given day. A positive correlation between the densities of

tailed microstomes and macrostomes would be consistent with the hypothesis that tailed

microstomes are more abundant when macrostomes are present, though it would not

indicate whether they arise in response to cues released by macrostomes or as a size-

dependent response to a common cue released by prey. Inclusion of populations grown

in the absence of Colpidium in this experiment ensured that the presence or absence of

Colpidium had no bearing on whether or not tailed microstomes are more abundant when

macrostomes are more abundant.

15

Effect of Microstome Cell Size on Transformation

Effects of cell size at the time of induction on macrostome formation were

determined by sorting microstomes into two size classes, small (< 60 µm) and large (≥ 60

µm), and culturing them within15-mm Petri dishes containing 2 ml of medium (standard

concentration) with or without Colpidium. I then monitored microstomes every 12 hr,

over a 48-hr period, and recorded the number of each morph within experimental groups.

Each experimental group consisted initially of 5 pyriform microstomal cells, and there

were 3 replicates for each group. I first compared differences in macrostome frequency

using a pooled two-way analysis of variance (ANOVA), after confirming that the effects

of time were not significant. I then conducted a more conservative analysis comparing

differences in macrostome frequency for each sampling time individually.

Population Dynamics in the Presence and Absence of an Interspecific Competitor

across a Productivity Gradient

Using the data collected from the phenology experiment, I compared the

individual morph population densities of each treatment to determine the effect of

productivity on the frequency of macrostome formation. In addition, I measured the

population growth rates of T. vorax as a whole, calculated as the natural log of the mean

number of cells per ml over time, with and without Colpidium across the productivity

gradient, to measure fitness.

Results

Prey Selection by Macrostomes

Capture rates by macrostomes were influenced by both prey type (ANCOVA,

F2,21 = 6.84, p = 0.0052) and prey density (ANCOVA, F1,21 = 106.44, p < 0.0001). Below

16

approximately 200 prey individuals / ml, prey capture rate by macrostomes was near 0,

irrespective of prey type (Fig. 2). There was a significant interaction between prey type

and prey density (ANCOVA, F2,21 = 23.61, p < 0.0001), which indicates that the slope of

the relation between prey density and the number of prey captured differed among prey

types. Macrostomes captured Colpidium and pyriform microstomes at approximately the

same rate, while few tailed microstomes were captured even at the highest prey density.

Phenology of the Tailed Microstome

The correlation analysis for which I had combined population densities across

time revealed that the population density of tailed microstomes was significantly

correlated with that of macrostomes (r = 0.687, p < 0.0001, n = 177). The more

conservative analysis yielded a significant positive correlation between the densities on

Day 2 (r = 0.770, p < 0.0001, n = 30), Day 3 (r = 0.764, p < 0.0001, n = 30), Day 4 (r =

0.739, p < 0.0001, n = 29), and Day 7 (r = 0.502, p = 0.0055, n = 29). On Day 1, as one

might expect, given that the cells would not have had time to differentiate, the correlation

between the densities tailed microstomes and macrostomes was not significant (r = 0.054,

p = 0.7763, n = 30). The correlation was also not significant on Day 5 (r = 0.251, p =

0.189, n = 29). Since, even in the conservative analysis, the correlation is positive on

four of the six days, the pattern appears robust. At each simulated productivity, in both

the presence and absence of Colpidium, macrostomes and tailed microstomes appeared

simultaneously. At standard productivity, with Colpidium present, pyriform microstomes

disappeared by the second day of population growth and did not reappear until after

Colpidium disappeared (Fig. 3).

Effect of Microstome Cell Size on Transformation

17

ANOVA revealed a significant interaction between microstome cell size and

Colpidium presence / absence on the induction of macrostomes (F1,56 = 29.57, p <

0.0001). This interaction was still observed when the analysis was conducted on each

sampling time separately (F1,8 = 15.21, p = 0.0045 for Hour 12; F1,8 = 147.05, p < 0.0001

for Hour 24; F1,8 = 201.79, p < 0.0001 for Hour 36; and F1,8 = 387.68, p < 0.0001 for

Hour 48). Microstomes less than 60 m long did not transform into macrostomes in the

presence of Colpidium. Although some pyriform cells transformed into tailed

microstomes, the majority remained microstomes. The majority of microstomes greater

than 60m transformed into macrostomes when induced by the presence of Colpidium

(Fig. 4).

Effects of Productivity and Intraguild Prey on Population Dynamics

Although the population density of macrostomes was significantly correlated with

both productivity (r = 0.296, p < .0001, n = 177) and the total population density of T.

vorax (r = 0.795, p < .0001, n = 177), the proportion of macrostomes within the T. vorax

population was not significantly correlated with these variables (r = 0.314, p = 0.0748, n

= 177 for productivity and r = 0.048, p = 0.5229, n = 177 for total population density).

Pyriform microstomes, which, at low productivity and in the presence of Colpidium

achieved the lowest densities of all the morphs, increased and achieved a higher density

than macrostomes at high productivity, in the absence of Colpidium. Tailed microstomes

achieved the highest densities of all the morphs in each treatment (Fig. 5). The overall

population growth rate of T. vorax was significantly influenced by productivity and the

presence or absence of Colpidium (ANCOVA, F4,28 = 3.40, p = 0.0295). At higher levels

18

of productivity, Colpidium decreased the growth rate of T. vorax. At low productivity,

the pattern was reversed, with Colpidium increasing T. vorax growth rates (Fig. 6).

Discussion

The results of my investigation strongly support the hypothesis that tailed

microstomes constitute a defense by the smaller size fraction of microstomes to potential

cannibalism by macrostomes. I recognize that I cannot separate whether the appearance

of the tailed microstome is directly induced by the appearance of macrostomes, or

whether tailed microstomes and macrostomes constitute different size-dependent

developmental responses to the same cue released by high densities of prey or by

conspecifics. Tailed microstomes clearly exhibit greatly reduced susceptibility to

consumption by macrostomes, relative to pyriform microstomes. Macrostomes do not

appear to discriminate between consuming conspecific and heterospecfic cells, as shown

by the observation that the capture rates of pyriform microstomes and Colpidium by

macrostomes did not differ. Furthermore, tailed microstomes appeared simultaneously

with macrostomes, even in the absence of Colpidium. The very low consumption rates of

tailed microstomes by macrostomes, together with the phenology of tailed microstome

morphs, enables the tailed microstome to serve as a coordinated, induced defense against

cannibalism when macrostomes appear in the population. Synchronous inductions of

offensive and defensive phenotypes such as this are unlikely to have evolved under

conditions favoring cannibalism, as such a defense would negate any selective advantage

of cannibalism. It is instead conceivable that the simultaneous induction may be part of a

mixed strategy to reduce intraspecific competition and to capitalize on multiple types of

available resources (Fisher 1958, Ehlinger 1990, Dudgeon and Buss 1996). In the case of

19

T. vorax, as the macrostomes feed on available ciliates, the tailed microstomes continue

to graze on bacteria, while remaining immune to the predation pressure that their

interspecific competitors face from macrostomes.

Differences in cell size at the time of induction provide one explanation for how

the polymorphism allowing for this potential mixed strategy arises. The significant effect

of cell size on the proportion of macrostomes induced within T. vorax populations

suggests that a size threshold exists for the successful transformation from microstome to

macrostome. Given that well-fed protozoa are larger than starved individuals (Kidder et

al. 1941), high productivities, where bacteria are abundant, should yield larger

proportions of macrostomes than low productivities, where bacteria are scarce. However,

increasing nutrient concentration did not significantly increase the proportion of

macrostomes, but instead merely increased the overall population density of T. vorax.

The fact that cell division was favored over transformation in a situation where

Colpidium was present at high density, but was neither as available nor as abundant as

bacteria, indicates that macrostome induction is not as much determined by the energetic

costs of transformation as it is by the relative advantage to being a predator.

The dominance of tailed microstomes over the other morphs at both high and low

productivity and in both the presence and absence of Colpidium may indicate that tailed

microstomes have a greater conversion efficiency of prey than either macrostomes or

pyriform microstomes. This, in combination with the resistance of the tailed microstome

to predation by cannibalistic macrostomes (and perhaps other gape-limited predators),

begs the question of why T. vorax populations contain pyriform microstomes at all. The

20

fact that the tailed microstome does not appear to be constitutive implies the existence of

either an unknown trade-off or a physiological constraint.

The reduced growth rate of T. vorax in the presence of Colpidium at the higher

productivity levels can be attributed to the inefficiency of energy transfer between trophic

levels. All else being equal, T. vorax, has less energy available to it when feeding on

Colpidium than it does when feeding directly upon the basal resources (the bacteria).

However, at low productivity, T. vorax had a higher growth rate in the presence of

Colpidium than it did when feeding on bacteria alone. This could suggest that, at higher

productivities, Colpidium is more difficult to capture and, therefore, yields a lower

energy return. The cell size of Colpidium increases with productivity or nutrient

concentration (Balciunas and Lawler 1995), and it is possible that Colpidium becomes

too large for macrostomes to consume at higher productivities.

Alternatively, this might indicate that intraguild predation can be particularly

advantageous at low resource levels. I can infer that Colpidium, in the absence of

predation, is a superior competitor to T. vorax microstomes, knowing that, in

monoculture, Colpidium reduces bacterial densities to a greater extent than T. vorax

(Kaunzinger and Morin 1998, Price and Morin 2004). At low productivity, where

bacteria are relatively sparse, T. vorax may gain greater net benefits from feeding on

Colpidium than from feeding on the bacteria. In addition, cells that do not transform into

macrostomes may still benefit from the presence of macrostomes as a result of

macrostomes reducing the competitive ability of Colpidium (Tilman 1982, Thingstad

1997, Fox 2002). Measuring the impact of the presence of Colpidium on microstome

growth rates to test this hypothesis would be virtually impossible because Colpidium

21

induces transformation in microstomes. However, future studies might examine the

impact of Colpidium on closely related Tetrahymena species that do not exhibit inducible

trophic polymorphism, such as an amicronucleate strain of T. pyriformis (Nanney et al.

1998). By comparing the population dynamics of T. pyriformis with and without

Colpidium over a range of productivity, I could indirectly evaluate the potential costs of

interactions with Colpidium to T. vorax microstomes and directly test the predictions of

competitive outcomes based on resource use.

Inducible trophic polymorphisms as spectacular as those exhibited by T. vorax

may be unusual. Even among protozoa, where the rapid reversibility of transformations

that take place within a single generation is far more commonplace than in larger, more

complex organisms with developmentally stable exo- or endoskeletons, the changes in

size that allow for shifts in trophic position are very seldom accompanied by drastic

reconstructions of morphology (Kidder et al. 1941, Agrawal 2001). However,

functionally similar trophic polymorphisms do occur in a diverse array of organisms and

habitats, and not necessarily through changes in morphology. Behavior can be an

extremely plastic trait in many higher-level organisms (DeWitt and Scheiner 2004) and

can greatly influence the diet and diet breadth of those organisms (Schluter and Grant

1984, Packer and Ruttan 1988, Dean et al. 1990). Investigating the mechanisms of how

inducible trophic polymorphisms occur may shed new light on our understanding of how

phenomena such as diet breadth (Polis 1991, Morin 1999, Diehl and Feissel 2000, Jiang

and Morin 2005), cannibalism (Polis 1981), and intraguild predation (Polis et al. 1989)

impact community structure and dynamics, and even long-term evolutionary processes.

Acknowledgements

22

I would like to thank Timothy Casey, Lin Jiang, Ben Baiser, Andrea Kornbluh,

Jean Deo, Jack Siegrist, Maria Stanko, Holly Vuong, and Jennifer Adams Krumins for

their helpful comments and invaluable insights. Support for this research came from the

Rutgers School of Environmental and Biological Sciences and the New Jersey

Agricultural Experiment Station.

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26

low density,

low competition

high density,

high competition

Colpidium:

interspecific

competitor

Microstome:

inducible

bacterivore

Macrostome:

induced

predator

Tailed

Microstome:

induced

bacterivore

Colpidium:

intraguild

prey

Resource = bacteriaResource = bacteria

60 μm

90 μm

< 3 μm

75 μm

75 μm

< 3 μm

200 μm

Fig. 1. Diagram of the inducible trophic polymorphism of T. vorax. Gray arrows

represent developmental pathways. Black arrows represent trophic links. Arrow size

represents relative interaction strength. Transformation of microstomes into

macrostomes is triggered by chemical cues released by conspecifics and by interspecific

competitors for bacteria, such as Colpidium (Kidder et al. 1941; Smith-Somerville 2000;

Ryals et al. 2002).

27

Fig. 2. Comparison of the differences in susceptibility of microstomes, tailed

microstomes, and Colpidium to consumption by macrostomes. Black squares mark the

regression line for microstomes; black triangles mark that of tailed microstomes; and gray

circles mark that of Colpidium.

28

Fig. 3. Morph dynamics of T. vorax at standard medium concentration in the presence

and absence of Colpidium during a 7-day period. Large unfilled black squares represent

macrostomes. Black triangles represent tailed microstomes. Small black squares

represent microstomes. Gray circles represent Colpidium.

29

Fig. 4. Effect of microstome cell size (small < 60 µm; large ≥ 60 µm) on the outcome of

induction. Symbols are as in Fig. 3.

30

Fig. 5. Morph dynamics in the presence and absence of Colpidium at low (50% of

standard) and high (150% of standard) medium concentrations. Symbols are as in Fig. 3.

31

Fig. 6. Interaction between the effects of productivity and the presence or absence of

Colpidium on the rate of growth of T. vorax, calculated as the natural log of the mean cell

density of T. vorax over time. Black diamonds represent growth rates with Colpidium

present. White diamonds represent growth rates with Colpidium absent.

32

CHAPTER 2

Prey-Induced Gape Expansion Mediates Apparent Competition

Summary

1. Phenotypic plasticity allows certain predators to increase their gape by feeding

on increasingly large prey. Where predators increase their gape enough to

feed on initially inedible species, a novel, trait-mediated indirect interaction

may occur – one that is akin to apparent competition.

2. I tested the hypothesis that feeding on Colpidium kleini, a large intraguild

prey, enables macrostomes of Tetrahymena vorax to increase their gape

enough to engulf Paramecium aurelia, a species too large for macrostomes to

engulf prior to such feeding.

3. My results were consistent with the hypothesis. Macrostomes allowed to feed

on C. kleini showed a significant increase in their mean cell diameter over

time. When exposed to P. aurelia, these enlarged macrostomes were able to

engulf P. aurelia, whereas macrostomes that had been cultured in the absence

of C. kleini were unable to engulf P. aurelia. The population dynamics of T.

vorax, C. kleini, and P. aurelia during a 22-day period revealed that the

occurrence and outcome of expanded diet breadth resulting from increased

gape in T. vorax depended on the recent history of community assembly.

Key-words: Chasmatectasis, phenotypic plasticity, priority effect, inducible trophic

polymorphism (ITP), Tetrahymena vorax, trait-mediated indirect interaction (TMII)

33

Introduction

Phenotypic plasticity is the ability of a single genotype to produce two or more

alternative phenotypes in response to differing environmental conditions (DeWitt and

Scheiner 2004). One intriguing consequence of phenotypic plasticity is the generation of

inducible trophic polymorphisms, wherein morphologically or physiologically different

individuals within a population come to occupy different trophic positions (Kopp and

Tollrian 2003a, Banerji and Morin 2009). Inducible trophic polymorphisms can result in

the utilization of a broader range of available resources than would be used by

monomorphic populations. In addition, induced individuals may be capable of

consuming some of their natural enemies (Washburn et al. 1988, Padilla 2001, Price and

Morin 2004). Since a major determinant of whether or not a predator is able to capture

and consume its prey is the disparity in size between the two (Maret and Collins 1996,

Urban 2007), it is not surprising that, in inducible trophic polymorphisms, a shift from a

lower trophic position to a higher one often simply entails an increase in size – either of

the body in its entirety or of specific internal and external feeding structures (Polis 1981,

Ricci et al. 1989, Wainwright et al. 1991, Maerz et al. 2006, Olsson et al. 2007).

More surprising is that certain species with inducible predatory morphs can

continue to increase their gape after the initial induction of their predatory morphology

by ingesting increasingly large prey. As I have not been able to find a precise term for

this phenomenon anywhere in the ecological literature, I have borrowed the pseudo-

clinical term used jokingly by a handful of medical students with whom I am acquainted

to describe the preferred training method of professional gurgitators (Levine et al. 2007,

O’Connor 2007): chasmatectasis (from the Greek words khasma, “gape,” and ektasis,

34

“stretching”). Chasmatectasis is an inducible offense (Padilla 2001) that has the potential

to significantly alter the shape and outcome of ecological dynamics (Miner et al. 2005,

Jeschke 2006, Mougi et al. 2010). It may, for example, provide a counter-measure

against the inducible defenses of prey (Kopp and Tollrian 2003b, Kishida et al. 2006) or

enhance the ability of predators to survive and recover from periods of starvation (Hewett

1987). There is anecdotal evidence of the occurrence of chasmatectasis in rotifers

(Gilbert 1973), cladocerans (Abrusán 2003), amphibians (Collins and Cheek 1983,

Pfennig 1990, Walls et al. 1993), fish (Wainwright et al. 1991, Persson et al. 2004), and

reptiles (Queral-Regil and King 1998). Among protists, and particularly among ciliates,

definite examples of chasmatectasis have been observed relatively frequently (Giese and

Alden 1938, Rickards and Lynn 1985, Hewett 1987, Ricci et al. 1989, Kopp and Tollrian

2003a).

An ecological aspect of chasmatectasis that has not previously been explored is its

potential to give rise to novel indirect interactions (Wootton 1994). In particular,

chasmatectasis might enable an induced predator to increase its gape enough to consume

a species that would have been too large for it to consume at the time of its initial

induction. The resulting exchange of negative indirect effects between the smaller and

larger prey species via their now shared predator would constitute apparent competition

(Holt 1977, Bonsall and Hassell 1997, Chaneton and Bonsall 2000). However, the

effects would be mediated via the predator’s gape rather than its population size, making

this a “trait-mediated” indirect interaction, instead of a “density-mediated” indirect

interaction (Peacor and Werner 2001). To explore this possibility, I made use of the

35

inducible trophic polymorphism of the freshwater protozoan Tetrahymena vorax Kidder

(Ciliatea: Hymenostomatida).

Isogenic populations of T. vorax may contain both “microstomes” and

“macrostomes.” Microstomes are small, pyriform individuals that feed on bacteria.

Macrostomes are larger, ovoid individuals that consume other ciliates. Microstomes can

reversibly transform into macrostomes in the presence of intraguild prey, which may

include conspecifics (Kidder et al. 1941, Williams 1961, Banerji and Morin 2009). My

preliminary observations of macrostomes exposed to varying sizes of intraguild prey

indicate that macrostomes are capable of some degree of chasmatectasis. Here I describe

my experimental approach to quantifying these observations and testing the hypothesis

that feeding on accessible prey, such as Colpidium kleini Foissner (Ciliatea:

Hymenostomatida), enables macrostomes to attain sizes large enough to engulf species

such as Paramecium aurelia Müller (Ciliatea: Hymenostomatida), which, prior to such

feeding, would exceed the limit of their gape (Fig. 1).

Methods

Culturing of Ciliates

The distribution of T. vorax in nature is poorly known, a situation that is typical

for protists and other microbes (Fenchel et al. 1997, Finlay 2002, Foissner 2009).

Fortuitously, many of the protists known to occur with T. vorax have cosmopolitan

distributions (Finlay 2002). Among these are species of the genus Colpidium, which

Kidder et al. (1941) were able to identify as intraguild prey of T. vorax almost

immediately upon its discovery. Members of the widely distributed Paramecium aurelia

complex are also likely to be found where T. vorax is present (Sonneborn 1975, Finlay

36

2002). I chose C. kleini and P. aurelia for use in my investigation for the ease with

which they could be obtained and cultured.

I purchased T. vorax (strain 30421) and P. aurelia (Item # 131546) from the

American Type Culture Collection and the Carolina Biological Supply Company,

respectively, and isolated C. kleini from the Adelphia Plant Science Research and

Extension Center (Freehold, NJ 07728, USA). I grew separate, isogenic lines of each of

these three ciliates in 240-mL glass jars containing 100 mL of complex organic growth

medium. The medium I used consisted of 0.37 g of Carolina Biological Supply

protozoan pellets per L of filtered well water. I sterilized and cooled the medium to room

temperature and then inoculated it with three different species of bacteria – Serratia

marcescens, Bacillus subtilis, and Bacillus cereus – to serve as a basal resource for the

ciliates. Loosely tightened screw caps on the jars permitted air circulation, while limiting

evaporation and contamination.

Measuring Chasmatectasis in T. vorax

To verify that macrostomes are able to increase their size and consume larger

prey, I performed an induction experiment. I inoculated six 15-mL Petri dishes,

containing 7 mL of growth medium, with T. vorax. I allowed three of these T. vorax

cultures to feed solely on bacteria (control replicates), while immediately inoculating the

other three with C. kleini (treatment replicates). After C. kleini had been extirpated from

the treatment replicates and all six replicates had entered into logarithmic growth (~ 4.5

days), I inoculated all six cultures with P. aurelia. I sampled approximately 0.3 mL from

the cultures every 12 hours over the course of 10 days to record average macrostome cell

size and the frequency of predation.

37

I measured average macrostome cell size as the mean cell diameter, in

micrometers, of macrostomes that were present within the sample. I made individual cell

diameter measurements by taking digital photomicrographs of the macrostomes in my

samples at 400X magnification. I used a Nikon Eclipse 80i microscope equipped with a

Nikon DXM1200C camera and ACT-1C imaging software. I analyzed the photos using

the NIH program, ImageJ (see Rasband 2007). I measured frequency of predation on P.

aurelia as the total number of observed incidences of a macrostome having engulfed P.

aurelia during my sampling interval (~ 5 minutes per dish).

Determining the Effect of Community Assembly on the Occurrence and Outcome of

Chasmatectasis in T. vorax

I performed a community assembly experiment to address: (1) whether feeding on

C. kleini prior to being exposed to P. aurelia is required for chasmatectasis in T. vorax

and (2) how chasmatectasis in T. vorax affects the relative fitness of all three species. My

experimental design involved four treatments, with five replicates per treatment. Each

replicate consisted of 100 ml of growth medium in a covered 240-mL glass jar that was

initially inoculated with T. vorax. C. kleini and P. aurelia were later added to the

replicates in different temporal sequences (Table 1).

I monitored the populations of all of the species present in each jar approximately

every 24 hours, over the course of 22 days, sampling approximately 0.3 mL without

replacement from each jar. To ensure that effects of the treatments were not confounded

by the build-up of toxins and metabolic wastes in the system, I removed and discarded

approximately 10% of medium by volume from each jar and replaced it with an

equivalent volume of fresh medium on Day 16. No measurements were recorded on this

38

day. Adhering to the same procedure, I constructed and monitored a pair of controls (5

replicates per control), each containing both C. kleini and P. aurelia, but without T.

vorax. This provided a baseline to assess any indirect effects among C. kleini and P.

aurelia mediated by T. vorax (Table 1).

Results

Measurement of Chasmatectasis in T. vorax

Macrostomes fed C. kleini showed a significant increase in size (ANOVA, F1,277 =

87.41, p < 0.0001) over the 10-day period of study (Fig. 2, Fig. 3). On Days 6-8, Day 9,

and Day 10, I recorded no average cell diameter of macrostomes in the control replicates

because no macrostomes were present in the control replicates during this period.

Macrostomes in the control replicates that had been cultured in the absence of C. kleini

appeared to have reverted to microstomes in the presence of P. aurelia. While I observed

8 separate incidences of macrostomes engulfing P. aurelia in the treatment replicates, I

did not observe any incidence of macrostomes doing so in the control replicates.

Effect of Community Assembly on the Occurrence and Outcome of Chasmatectasis in

T. vorax

The order in which T. vorax was exposed to C. kleini and P. aurelia had a

significant effect on the mean population densities of both C. kleini (ANOVA, F1,8 =

407.57, p < 0.0001) and P. aurelia (ANOVA, F1,8 = 3162.48, p < 0.0001) across time. It

also had a significant effect on the mean densities of macrostomes (ANOVA, F1,8 =

144.2, p < 0.0001) and microstomes (ANOVA, F1,8 = 179.98, p < 0.0001) in the T. vorax

population. In replicates where T. vorax was exposed to C. kleini first and to P. aurelia

second, P. aurelia was ultimately extirpated. In replicates where T. vorax was exposed to

39

P. aurelia before being exposed to C. kleini, however, P. aurelia was able to persist

throughout the experiment. In the controls, where C. kleini and P. aurelia were paired in

the absence of T. vorax, both species persisted throughout the experiment (Fig. 4). C.

kleini and P. aurelia were both able to persist at relatively high population density when

paired alone with T. vorax (Fig. 5). T. vorax macrostomes, in the presence of both prey,

attained higher densities and persisted longer when exposed to C. kleini first. In the

presence of C. kleini alone, T. vorax macrostomes attained higher population densities

and persisted longer than in the presence of P. aurelia alone (Fig. 6).

Discussion

My results strongly support the hypothesis that chasmatectasis can enable a

predator to expand its diet breadth to include species that were previously inaccessible

due to their large size relative to the predator’s initial gape. By feeding on C. kleini, a

relatively large but still initially consumable prey, T. vorax was able to expand its gape

enough to consume P. aurelia, a prey that T. vorax could not consume upon initial

transformation. Furthermore, I have demonstrated that the occurrence and ecological

consequences of chasmatectasis can be influenced by the history of community assembly.

Where T. vorax was exposed to C. kleini before being exposed to P. aurelia, T. vorax

underwent chasmatectasis and became able to feed on P. aurelia. Yet, where T. vorax

had been exposed to P. aurelia first, T. vorax not only failed to undergo chasmatectasis,

but also maintained a lower population density of macrostomes throughout the

experiment. It is conceivable that competition with P. aurelia for bacterial resources

might have prevented T. vorax from crossing the minimum size threshold for

transformation into macrostomes (Banerji and Morin 2009) upon the arrival of C. kleini

40

and that maintenance of the macrostome morphology requires ingestion of prey. In the

replicates in which T. vorax was able to undergo chasmatectasis and feed on P. aurelia,

P. aurelia was extirpated. Given the low rate of consumption of P. aurelia by T. vorax

indicated by the results of the induction experiment, the extirpation of P. aurelia in these

replicates cannot be attributed to predation alone. However, given the persistence of P.

aurelia in the replicates in which T. vorax had been unable to undergo chasmatectasis, it

appears that predation must still have played a significant role.

Chasmatectasis is an intriguing manifestation of phenotypic plasticity that

warrants further investigation in other taxa. The underlying genetic and physiological

basis for chasmatectasis has yet to be revealed, and its environmental basis in natural

systems remains uncertain. In the case of my system, for example, it is unclear whether

the ability of C. kleini and other large, but accessible prey to trigger chasmatectasis in T.

vorax is a function of the size of the prey, in and of itself, or of the composition – i.e.,

nutritional or energetic content – of the prey. Still to be determined, also, is the upper

size limit of prey that can be consumed by T. vorax via chasmatectasis. Future studies

might readily address these questions by repeating my experimental design using

different prey species of varying size and composition.

Chasmatectasis is not limited to ciliates. Though they differ from the examples

seen in ciliates in that they are generally irreversible, examples of chasmatectasis, as

previously mentioned, can be found in fish, amphibians, reptiles, and a range of

invertebrates. Dynamics similar to those I have uncovered in this investigation may very

well occur in these other systems. Such dynamics could have repercussions for the study

of colonization and succession. For example, given that overall body size has been

41

shown to play a significant role in determining the invasion success of predators (Persson

et al. 2004, Schröder et al. 2009), diversity of body sizes among incumbent prey may

facilitate invasion by predators capable of undergoing chasmatectasis.

Dynamics resulting from chasmatectasis might also have implications for

management practice, as shown by my demonstration of chasmatectasis mediating

apparent competition. Apparent competition has been shown to be an important

determinant of community structure (Jeffries and Lawton 1984, Settle and Wilson 1990,

Bonsall and Hassell 1997, Juliano 1998, Orrock et al. 2008), and classical apparent

competition theory has been applied in the contexts of biological control (Karban et al.

1994, van Veen et al. 2006) and restoration (Norbury 2001, Hoddle 2004).

Understanding the workings of chasmatectasis could enable us to determine if apparent

competition that is mediated by chasmatectasis has the same significance in ecological

communities as classical apparent competition and enable us to avoid the potential

pitfalls (Howarth 1991, Paynter et al. 2010) associated with its use in biological control

and restoration.

Acknowledgements

I gratefully acknowledge Rebecca Jordan, Mike Pace, Kevin Aagaard, Laura

Chen, Jean Deo, Sean Hayes, Ruchi Patel, Jack Siegrist, Maria Stanko, Holly Vuong, and

Talia Young for their worthwhile comments and suggestions. Support for this research

came from the School of Environmental and Biological Sciences and the New Jersey

Agricultural Experiment Station.

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45

==========================================================

Species Added

on Day 0

Species Added

on Day 3

Species Added

on Day 6

Treatment

1 T. vorax C. kleini P. aurelia

2 T. vorax P. aurelia C. kleini

3 T. vorax C. kleini C. kleini

4 T. vorax P. aurelia P. aurelia

Control

1 C. kleini P. aurelia P. aurelia

2 P. aurelia C. kleini C. kleini

============================================================

Table 1. Inoculation sequence for community assembly experiment. Treatment

replicates were initially inoculated with T. vorax and later inoculated with C. kleini, P.

aurelia, or both in different orders. Control replicates consisted of C. kleini and P.

aurelia without T. vorax.

46

Fig. 1. Chasmatectasis in T. vorax may create negative trait-mediated indirect effects

between C. kleini and P. aurelia. Black arrows represent trophic links, while gray arrows

represent developmental pathways.

47

Fig. 2. Mean macrostome cell diameter over a 10-day period. White diamonds represent

diameters observed in the presence of C. kleini; black diamonds represent diameters in

the absence of C. kleini. On Days 6-8, Day 9, and Day 10, no macrostomes were

observed in the absence of C. kleini. On Day 8.5, only 3 macrostomes were observed in

the absence of C. kleini, one of which being a low-end outlier in terms of its cell

diameter.

48

Fig. 3. Representative micrographs (differential image contrast scans) of T. vorax

macrostomes observed on the last day of my induction experiment (Day 10). Shown on

the left is a macrostome from a control replicate (monoculture with bacteria as the only

available prey). Shown on the right is a macrostome from a treatment replicate (initially

containing C. kleini as well as bacteria).

49

Fig. 4. Population dynamics of C. kleini and P. aurelia in replicates where they were

paired in the presence (top) and absence (bottom) of T. vorax. Gray circles represent the

abundance of C. kleini, and black crossed diamonds represent that of P. aurelia. Note the

log scales of the Y-axes.

50

Fig. 5. Population dynamics of C. kleini (left) and P. aurelia (right) alone with T. vorax.

Symbols are as in Fig. 4.

51

Fig. 6. Morph dynamics of T. vorax in the replicates shown at the top of Fig. 4 (top) and

shown in Fig. 5 (bottom). Large unfilled squares represent the abundance of

macrostomes. Small black squares represent that of microstomes. Note the log scales of

the Y-axes.

52

CHAPTER 3

Dynamics between Protists and Crustacean Zooplankton: Do Copepods

Show Preference for the Predatory Morph of Tetrahymena vorax?

Summary

1. Studies of protists in microcosms have made important contributions to

ecology, but some critics argue that the small spatial scale and trophic

simplicity of microcosms make them inappropriate as models of complex

natural systems. This concern is especially important to address in cases

where both the nature and strength of trophic links within the community are

density-dependent.

2. I used outdoor experimental mesocosms to assess whether increased trophic

complexity and variable abiotic conditions would alter the dynamics of a

simple freshwater protist community module that I had previously examined

in laboratory settings. This community contained the polymorphic ciliate

Tetrahymena vorax Kidder. I tested the following hypotheses: (1) that the

volume and ambient abiotic conditions of the mesocosm habitat would inhibit

the induction of predatory morphs of T. vorax, and (2) that predation by

crustacean zooplankton would result in the exclusion of T. vorax.

3. Differentiation did occur in the T. vorax population over the course of the

experiment. The proportion of T. vorax macrostomes was unaffected by the

presence or absence of crustacean zooplankton. T. vorax was not extirpated in

the presence of the zooplankton. However, the population densities of each of

53

the morphs of T. vorax, as well as that of Colpidium kleini (a competing

ciliate), were significantly reduced in the presence of zooplankton, relative to

what they were in the absence of zooplankton.

4. These results show that community patterns may change radically with the

scale and complexity of experimental systems, demonstrate the utility of

microcosms as a baseline for studies of larger systems, and offer insight into

possible reasons for the limited distribution of T. vorax in natural aquatic

systems.

Key-words: density-dependence, induced offenses, induced polymorphism, microcosm

experiments, predation, spatial scaling, Tetrahymena vorax, zooplankton

54

Introduction

The experimental tractability of protists in laboratory microcosms has made them

valuable tools for testing ecological and evolutionary principles and for uncovering the

mechanisms that underlie general patterns (Drake et al. 1996; Fraser and Keddy 1997;

Cadotte et al. 2005; Holyoak and Lawler 2005). The small size and self-contained

structure of microcosms allow for highly controlled experiments with adequate

replication (Jessup et al. 2004; Cadotte et al. 2005) – a feature that is sometimes lacking

and occasionally impossible in field experiments (Hurlbert 1984; Hargrove and Pickering

1992). These same convenient attributes can potentially make microcosms limited

models of larger, more complex systems (Carpenter 1996, 1999; Srivastava et al. 2004).

As noted by Schindler (1998) and Petersen and Hastings (2001), careful scaling

corrections must often be made even when the attempted extrapolation is simply from a

smaller habitat to a larger one of the same kind, located in the same geographic region,

and containing the same species. Seemingly contradictory results have been reported

even for investigations of community patterns thought to be scale-independent. For

instance, while productivity has been shown to be an important determinant of food chain

length in aquatic microbial communities (Kaunzinger and Morin 1998), it appears to be

unrelated to food chain length in lake systems (Post et al. 2000).

Recently, microcosm experiments with protists have been used to investigate the

phenomenon of inducible trophic polymorphisms, wherein phenotypic plasticity enables

organisms to alter their position in the food web and respond dynamically to changes in

resource availability and competitors (Kopp and Tolrian 2003; Price and Morin 2004;

Banerji and Morin 2009). In the case of the freshwater hymenostome ciliate

55

Tetrahymena vorax Kidder, exposure to chemical cues released by competing ciliates,

which include conspecifics, triggers a transformation from small (~ 60 µm), pyriform,

bacterivorous morphs, termed “microstomes,” to larger (~ 200 µm), ovoid, predatory

morphs, termed “macrostomes,” which can consume other ciliates (Kidder et al. 1941;

Smith-Somerville et al. 2000; Ryals et al. 2002). This ability to transform and

subsequently feed on competitors might not only enhance the fitness of T. vorax under a

variety of circumstances, but also give rise to trait-mediated indirect effects that would

influence the composition and stability of the community as a whole (Banerji and Morin

2009; Banerji and Morin in preparation). The fact that isogenic lines of T. vorax

differentiate into different morphs in response to high population densities of

conspecifics and/or heterospecifics indicates that transformation of microstomes into

macrostomes is a density-dependent response (Fig. 1).

The life-history of T. vorax in natural systems remains essentially unknown. All

lineages of T. vorax available in culture collections are descended from the collection

made by Kidder et al. (1940, see also Corliss 1952, 1953) from a small pond in the

vicinity of Providence, Rhode Island. T. vorax was also collected once from a stream in

Pennsylvania (Nanney et al. 1998). Other polymorphic Tetrahymena may enjoy less

restricted distributions (Ryals et al. 2002), but none appear to be as common or as widely

distributed as their smaller monomorphic congeners (Elliot 1973). Polymorphic

Tetrahymena species lack a drought-resistant resting stage and are consequently restricted

to permanent waters. Beyond this constraint, the reasons for the apparently limited

distribution of natural populations of T. vorax are unclear and raise fundamental

questions about the factors limiting the distribution of trophically plastic species.

56

Here I describe a study of T. vorax dynamics in outdoor experimental mesocosms

designed to uncover some of the possible causes of its limited distribution in nature. In

so doing, I also provide some insight as to whether or not the kinds of dynamics observed

in laboratory microcosms can be scaled up to larger, more complex, and more

realistically variable systems in the field.

Methods

Preparation of Protozoan and Bacterial Inoculants

I obtained T. vorax from the American Type Culture Collection (strain 30421)

and Colpidium kleini Foissner (Ciliatea: Hymenostomatida) from the Adelphia Plant

Science Research and Extension Center, Freehold, NJ 07728, USA. Species of the genus

Colpidium appear to have cosmopolitan distributions (Finlay 2002) and were found to be

naturally occurring prey / competitors of T. vorax in the original collections made by

Kidder et al. (1940). I cultured isogenic populations of T. vorax and C. kleini in 240-ml

glass jars containing 100 ml of complex organic medium. Loosely fastened screw caps

on the jars permitted some air circulation, while minimizing evaporation and

contamination. The complex organic culture medium used consisted of 0.37 g of

Carolina Biological Supply protozoan pellets (Carolina Biological Supply Company,

Burlington, NC 27215, USA) per L of filtered well water. After autoclaving and cooling,

I inoculated the medium with three species of bacteria (Serratia marcescens, Bacillus

subtilis, and Bacillus cereus) to standardize initial conditions and provide food for the

protozoa.

Construction of Outdoor Experimental Mesocosms

57

I modified twelve 19-L plastic buckets (Homer’s All-Purpose Bucket, The Home

Depot ®) to serve as my experimental units. I added 2 g of autoclaved dried alfalfa leaf,

14 L of well water, and 100 mL of bacterized alfalfa medium to each bucket. I covered

the buckets with 5-µm Nitex ® mesh glued to a removable plastic lid to prevent

contamination by larger aquatic organisms. The mesh size was small enough to exclude

the nauplii of cyclopoid copepods and enable me to maintain initial control over ciliate

species composition. The well water I used came from the same source used for the

medium of my laboratory cultures of T. vorax and C. kleini. The total volume in these

experimental units constituted a >140-fold increase from that of the jars used in my

microcosm experiments. The dilute alfalfa medium provided a nutrient source for the

bacteria, as well as some structural heterogeneity for the protozoa, that could be adjusted

to simulate the contributions of plant litter to natural pond systems. I placed the buckets

in a designated clearing located at the Hutcheson Memorial Forest of Rutgers University

(East Millstone, NJ, USA), arranging them with approximately 1 m of space between

them, with control and treatment replicates alternated to compensate for any potential

environmental gradients within the site (see Hurlbert 1984). Two days after filling and

positioning the buckets, I added a 2-day-old, 100-mL culture of T. vorax and a 2-day-old,

100-mL culture of C. kleini to each bucket.

Crustacean Zooplankton Treatment

There is an abundance of experimental and observational evidence implicating

cladocerans and copepods as important predators of the ciliates that persist in permanent

waters (Pace and Funke 1991; Burns and Gilbert 1993; Wickham 1995; Burns and

Schalenberg 1996; Adrian and Schneider-Olt 1999; Gaedke and Wickham 2004).

58

However, none of these studies address the very different problem of whether these

crustacean zooplankton actively exclude some ciliate taxa from otherwise favorable sites.

T. vorax in its macrostome state, for instance, could be more susceptible to grazing by

crustacean zooplankton due to its being more conspicuous than other, smaller co-

occurring ciliates. In addition, crustacean zooplankton could decrease the relative

abundance of macrostomes indirectly by reducing densities of all ciliates, and thereby

reducing the concentration of cues that induce macrostome formation in T. vorax.

After sampling to obtain baseline counts of the densities of T. vorax and C. kleini

in each bucket, I added 50 mL of unfiltered pond water from a local source to 6 of the 12

buckets (treatment replicates). The local source was a small permanent pond I had

chosen for the convenience of its proximity, containing cyclopoid copepods, cladocerans,

and other small aquatic organisms. To the remaining half of the buckets (controls), I

added pond water from the same source that had been filtered through 5-µm mesh to

remove the crustacean zooplankton, rotifers, and any large protozoa.

Sampling of Outdoor Experimental Mesocosms

I collected 7-mL samples from the surface and lower depths of each bucket (for a

combined total of 14 mL) in sterile Falcon® centrifuge tubes using a pair glass tubes that

I rinsed with hot water before and after each sampling interval. I counted copepods by

eye in the entire 14 mL sample, after confirming their identity via microscopy. I then

sub-sampled approximately 0.3 mL (measured by weight) from each of the 14 mL

samples using sterile glass pipettes to quantify the population densities of the protozoa. I

enumerated both macrostomes and microstomes of T. vorax and C. kleini. I also recorded

59

the presence (and, in some cases, density) of the other protozoa, algae, and macroscopic

zooplankton.

Results

Species Composition of Replicates after Initial Setup

The unfiltered pond water used for my treatment replicates introduced a number

of taxa to the treatment replicates. In addition, despite my use of the 5-µm mesh, all

replicates, including the control replicates, as of Day 26, were contaminated with large

densities of an alga of the genus Chlorella (mean average of 140.34 cells / mL) and a

species of microflagelate that I was unable to identify (mean average of 45.73 cells / mL).

As of Day 24, in treatment replicates Trt 1 and Trt 5 and in control replicate Con 4, I

observed hypotrichs of the genus Oxytricha (mean average of 10.84 cells / mL). During

the same period, in treatment replicate Trt 1 and control replicate Con 3, I observed

peritrichids of the genus Vorticella (mean average of 14.99 cells / mL).

Although I had observed species of Daphnia and other cladocerans in my source

pond, I did not observe any of these in my treatment replicates. The crustacean

zooplankton present in my treatment replicates consisted entirely of cyclopoid copepods,

the majority of which I could identify as being of the genus Acanthocyclops (either A.

vernalis or A. robustus), with two individuals (seen on Day 10 in Trt 6) possibly being of

the genus Diacyclops. One of the replicates with zooplankton added (Trt 1) also

contained eggs of the mosquito species Aedes albopictus (= Stegomyia albopicta Skuse),

which had presumably entered through my unfiltered pond water addition. These

emerged as larvae, developed into pupae, completed their metamorphosis into adults, and

then died between Days 17 and 26.

60

Effects of Zooplankton on Protist Community Dynamics

The population densities of T. vorax and C. kleini in treatment replicates were

significantly lower than in controls, which lacked large zooplankton (MANOVA, F3,8 =

10.83, p = 0.0034; Fig. 3). The appearance of the mosquitoes in Trt 1 did not appear to

change the overall patterns of abundance of T. vorax and C. kleini in that replicate, as

compared to the other treatment replicates. By Day 19, however, copepods were no

longer present in the replicate. In all of the treatment replicates, including Trt 1, protozoa

declined upon the addition of the unfiltered pond water containing macroscopic

zooplankton (Fig. 4). By contrast, without the zooplankton, T. vorax microstomes and C.

kleini maintained relatively constant population densities until the last three days of the

experiment (Fig. 5). Differences between the proportion of T. vorax macrostomes in the

presence and absence of crustacean zooplankton were not significant. In both cases, the

proportion of T. vorax macrostomes was significantly lower than what I had observed in

laboratory settings, across a range of productivities (ANOVA, F5,15 = 23.87, p < 0.0001;

see Banerji and Morin 2009). Follow-up contrast analysis of the means showed that

these differences were significant for each productivity-level compared to the outdoor

mesocosm settings.

Discussion

I am skeptical of there being a causal relationship between the presence of

mosquito larvae in Trt1 and the extirpation of copepods in that replicate. It is, for

example, highly unlikely that the copepods were consumed by the larvae. Apart from the

fact that I did not observe any such predation event in the course of my sampling,

copepods such as those I had observed prior to the emergence of the mosquito larvae

61

have been used as biological agents to kill mosquito larvae (Marten 1989, Marten and

Reid 2007). I can only speculate that other factors were responsible for the loss of

copepods in this replicate and that the timing was mainly coincidental. The steep decline

of the population densities of each of the protozoa in the control replicates during the last

three days of the experiment might be attributed to the fact that the water temperatures of

all of the replicates consistently exceeded 25 °C during this period.

Although T. vorax was not entirely extirpated from mesocosms in the presence of

crustacean zooplankton, the decline of T. vorax and C. kleini population densities

indicates that predation by crustacean zooplankton is still likely to be a factor in limiting

the distribution and abundance of T. vorax and other ciliates among natural aquatic

systems. This is not unexpected, as cladocerans and copepods have been shown to be

capable of affecting the abundance of protists in permanent ponds (Adrian and Schneider-

Olt 1999; Gaedke and Wickham 2004). Since the proportion of macrostomes in the T.

vorax population did not appear to differ between control and treatment replicates, it is

unlikely that crustacean zooplankton preferentially feed on macrostomes. It is uncertain

why the proportion of macrostomes in my outdoor mesocosms was smaller than what I

have observed in laboratory settings. The low density of ciliates and cues is a likely

cause, but I cannot rule out the possibility of the contents of the medium also having an

effect. It has been demonstrated, for example, that populations of T. vorax grown in

proteose peptone medium are unable to differentiate. Given that the dilute alfalfa

medium I used in this experiment resembled the environment of natural ponds more

closely than the protist pellet medium used for my laboratory cultures, it could be that the

62

expression of ITPs among protists raised in standard growth media is exaggerated,

relative to what it is in natural settings.

The results of my experiment show that community patterns may change

radically with the scale and complexity of experimental systems, but simultaneously

demonstrate the value of combining microcosm studies with field studies to uncover the

mechanisms underlying the dynamics of large, complex systems.

Acknowledgements

I thank Brian and Amy Clough for their aid in the setup of this experiment. I also

thank Kevin Aagaard, Jean Deo, Jack Siegrist, Maria Stanko, and Holly Vuong for their

helpful feedback. Support for this research came from the Rutgers School of

Environmental and Biological Sciences and the New Jersey Agricultural Experiment

Station, in the form of the Hutcheson Memorial Forest Summer Research Grant.

63

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66

low density,

low competition

high density,

high competition

Colpidium:

interspecific

competitor

Microstome:

inducible

bacterivore

Macrostome:

induced

predator

Tailed

Microstome:

induced

bacterivore

Colpidium:

intraguild

prey

Resource = bacteriaResource = bacteria

60 μm

90 μm

< 3 μm

75 μm

75 μm

< 3 μm

200 μm

Fig. 1. Diagram of the inducible trophic polymorphism of T. vorax (reproduced from

Banerji and Morin 2009). Gray arrows represent developmental pathways. Black arrows

represent trophic links. Arrow size represents relative interaction strength.

Transformation of microstomes into macrostomes occurs in the presence of chemical

cues released by competitors for bacteria.

67

Fig. 2. Semi-quantitative measurements of the abundances of macroscopic zooplankton

in treatment replicates. Black hexagons represent the abundance of copepods

(Acanthocyclops spp.). Dark gray triangles represent that of mosquitoes (Aedes

albopictus). Note that mosquitoes were detected as larvae on Days 19 and 21, as pupae

on Day 24, and as (dead) adults on Day 26.

68

Copepods Present

no yes

Mean

Den

sit

y +

1 S

.E.M

. (n

o. / m

L)

0

20

40

60

80

100

T. vorax macrostomes

T. vorax microstomes

C. kleini

Fig. 3. Effects of copepods on protozoa. Black columns represent the mean population

density of macrostomes of T. vorax. Light gray columns represent the mean population

density of microstomes of T. vorax. Dark gray columns represent the mean population

density of C. kleini.

69

Fig. 4. Population dynamics of protozoa in treatment replicates (replicates containing

crustacean zooplankton). Large unfilled black squares represent abundances of

macrostomes of T. vorax. Small black squares represent abundances of microstomes of

T. vorax. Gray circles represent abundances of C. kleini.

70

Fig. 5. Population dynamics of protozoa in control replicates (replicates lacking

crustacean zooplankton). Symbols are as in Fig. 4.

71

CHAPTER 4

On the Strategy of Mounting an Indirect Offense: What If My Enemy Has

Allies, Too?

Summary

An indirect offense is the use of symbionts or recruited associates by consumers

to increase their rate of acquisition of prey or hosts. This versatile adaptive strategy is

employed by a wide array of consumers in various aquatic and terrestrial habitats. Here I

present a review of indirect offenses, the purpose of which being to draw attention to the

phenomenon and elucidate its significance within ecology and evolution. I explain the

utility of recognizing indirect offenses as a unique category of species interaction, present

examples to illustrate and clarify the modes and diversity of indirect offenses, and

highlight promising directions for future research.

Key-words: Functional response; indirect defense; indirect offense; parasite; parasitoid;

predator; symbiosis.

72

Introduction

In recent years, the phenomenon of species relying on symbionts or recruited

associates for an “indirect defense” against their natural enemies has been of growing

interest to ecologists (Poelman et al. 2008, Degenhardt 2009). Best-known are the cases

of terrestrial plants deterring herbivores using fungus-derived compounds (Huitu et al.

2008, Afkhami and Rudgers 2009, Hartley and Gange 2009) or higher-level consumers

(Dicke and Sabelis 1988, Turlings et al. 1995, Thaler 2002, Hountondji et al. 2006), but

indirect defenses are also employed by a variety of other taxa (McAtee 1944, Owens and

Goss-Custard 1976, Wheelwright et al.1997, Machado and Vital 2001, Oliver et al. 2003,

Hay et al. 2004, Damiani 2005, Fenton et al. 2011). Studies have shown that this use of

symbionts or recruited associates can drastically reduce the rate of acquisition of prey or

hosts by consumers (Damiani 2000, Kessler and Baldwin 2001, Sabelis et al. 2001,

Turlings and Wäckers 2004) and significantly influence the composition of ecological

communities (Poelman et al. 2008, Utsumi et al. 2010). What these studies have

generally failed to take into account, however, is that the use of symbionts and recruited

associates is not unique to species under threat. In the same way that a species might

make use of another species to protect itself from natural enemies, a species might also

make use of another species to enhance its ability to feed on prey or hosts. In other

words, not only are there indirect defenses in ecological communities, but also “indirect

offenses.”

The Utility of the Concept of an Indirect Offense

Indirect offenses occur when consumers make use of symbionts or recruited

associates to increase their rate of acquisition of prey or hosts. Many kinds of symbiosis

73

and recruited association can give rise to indirect offenses, including both resource-

exchange mutualisms (Hoeksema and Schwartz 2003, Holland et al. 2005) and parasite-

host interactions (Poulin 1994, Lafferty 1999). Since the term “indirect offenses” has

never before been used to categorize these interactions, it seems appropriate to briefly

address the question of how it would be meaningful to apply the term currently. Simply

stated, the term provides us with a conceptual framework. It implies, as it is meant to,

that these interactions are the logical counterpart to indirect defenses. Recognizing the

existence and common features of these interactions enables us to generalize across taxa

and make predictions regarding their consequences and dynamics. In turn, our ability to

generalize and make predictions allows for new avenues of academic and applied

research, such as the development of integrated pest management strategies that target the

symbionts and recruited associates of invasive species or that manipulate the symbionts

and recruited associates of biological control agents.

Examples of Indirect Offenses

Theory predicts that, while foraging, a consumer’s rate of acquisition of prey or

hosts – also known as its “functional response” – is constrained by two separate factors:

“search time” and “handling time” (Abrams 1982, Jeschke et al. 2002). Search time is

the amount of time that the consumer spends locating its prey or hosts. Handling time is

the amount of time that the consumer spends subduing and assimilating its prey or hosts.

In an indirect offense, symbionts or recruited associates may benefit the consumer by

reducing one or the other of these limiting factors. In this section, I present some of the

ways in which they go about this.

Indirect Offenses That Reduce Search Time

74

… By Revealing the Presence of Prey or Hosts

Many prey species rely on the cover of darkness to avoid detection by visual

predators (Nelson and Vance 1979, Rydell and Speakman 1995, Wcislo et al. 2004). In

response, many nocturnal and deep-sea visual predators have evolved specialized

adaptations for foraging under low-light conditions (Healy and Guilford 1990, Collin and

Partridge 1996). The loose-jawed dragonfish, Malacosteus niger, has evolved an

especially unique adaptation. Using the green-sulfur bacteria residing in its suborbital

photophores, the dragonfish is able to project far-red light. All other known animals of

the deep sea are blind to this wavelength, which enables the dragonfish to illuminate its

prey without giving its own presence away (Douglas et al. 2000, Herring and Cope

2005).

… By Locating Prey or Hosts

Typically, if two species rely on the same food source, one would expect their

interaction with each another to be mostly competitive. Interspecific cooperative

foraging associations that involve guiding behavior allow for a fascinating exception to

this rule (Morse 1977, Giraldeau 1984, Berner and Grubb 1985). One of the most

striking examples is the guiding behavior of the coral grouper, Plectropomus

pessuliferus, which often leads its fellow predator, the giant moray eel, Gymnothorax

javanicus, to the location of prey that have taken refuge in narrow crevices within a reef.

The eel, due to the shape of its body, is able to enter these crevices, but the grouper is not.

It is the grouper that recruits the eel in this association. The grouper signals the

eel to follow it by approaching the eel at the eel’s den and performing a ritualized set of

visual displays. This entails the grouper rapidly shaking its head directly in front of the

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eel, a few centimeters away from the eel’s head. While doing so, the grouper keeps the

soft part of its dorsal fin erect and the bony part flat. Once the grouper has persuaded the

eel to leave the den and has led the grouper to the hiding place of the prey, the grouper

positions itself where it is best able to capture whatever prey the eel happens to flush out

without catching (Diamant and Shpigel 1985, Bshary et al. 2006).

… By Luring Prey or Hosts

For some consumers, the simplest way to reduce search time is to have their prey

or hosts come to them. The classic example of this kind of consumer is the bathypelagic

predatory female anglerfish, Melanocetus murrayi. The anglerfish uses the

bioluminescent, intracellular bacteria (Vibrionaceae) that reside in its light organ to lure

in its prey. It remains motionless with the exception of its light organ, which it wiggles

in order to mimic the movements of much smaller, bioluminescent organisms. Able to

discern only the wiggling light organ through the darkness, the anglerfish’s prey come

within range to investigate what they perceive as a potential meal. The moment they do,

the anglerfish is able to quickly swallow them whole (Hulet and Musil 1968).

Surprisingly, predators do not have to be larger than their prey to employ the

strategy of the anglerfish. In shallower waters, the squaloid shark, Isistius brasiliensis,

known also as the “cookiecutter shark,” uses its endosymbiotic bioluminescent bacteria to

camouflage all but a small portion of its body. Seeing it from below, larger pelagic

predators, such as tuna, mistake the shark for a very small, harmless prey. When these

larger predators attempt to consume the shark, the shark ambushes them and extracts

plugs of flesh from their bodies (Jones 1971, Widder 1998).

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Another manifestation of the use of symbionts to lure prey or hosts is the

attraction of definitive hosts by parasites with complex life-cycles. This phenomenon has

been termed “parasite-increased trophic transmission” (Lafferty 1999) and involves

parasites modifying the behavior or physiology of their intermediate hosts in such a way

as to make them more vulnerable to capture and consumption. In many cases, it can be

readily distinguished from mere artifacts of nutrient and energy loss because of the nature

of the changes in the host (Poulin 2000, Brown et al. 2001). A wide range of parasites

make use of this strategy, including the tapeworm, Schistocephalus solidus. This

tapeworm manipulates its intermediate host, the threespine stickleback, Gasterosteus

aculeatus, so as to be able to infect its definitive host, the piscivorous common

kingfisher, Alcedo atthis. It dramatically reduces the melanin content of the stickleback’s

scales and darkens the stickleback’s eyes, while causing the stickleback to move slowly

and be less responsive to danger. The stickleback thereby becomes significantly more

noticeable from above to its visual predator and significantly easier to catch (Ness and

Foster 1999, Milinski 2006).

Indirect Offenses That Reduce Handling Time

… By Concealing the Presence of the Consumer

Just as prey species can use symbionts to avoid detection by their predators

(Gradstein and Equihua 1995), predators can use symbionts to avoid detection by their

prey. The Hawaiian bobtail squid, Euprymna scolopes, for example, uses the

bioluminescent bacteria in its mantle to create disruptive color patterns that break up its

silhouette from beneath and eliminate its shadow. This enables the squid to approach

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prey such as red shrimp, Halocaridina rubra, without being seen (Travis 1996, Jones and

Nishiguchi 2004).

… By Enabling the Consumer to Metabolize Prey or Host Content

The presence of structural and chemical defenses can make feeding on prey or

hosts anything from unappealing to lethal for consumers (Simms and Fritz 1990, Jeschke

2006). Plants are especially well-equipped in terms of such defenses (Coley and Barone

1996). One of the ways in which herbivores and other consumers may respond to this is

by relying on specialized gut endosymbionts (Dyer 1989, Karban and Agrawal 2002).

The cigarette beetle, Lasioderma serricorne, for example, achieves this result using the

hydrolytic detoxifying enzymes of its symbiotic yeast (Dowd 1989, Dowd and Shen

1990, Dowd 1991).

It should be noted that a consumer’s inability to digest prey or host content is not

always a matter of the content being defensive in nature. In some cases, it is a matter of

the constraints set by the consumer’s own evolutionary history. The carnivorous plants,

Roridula gorgonias and Roridula dentata, for example, despite being fully capable of

trapping insects on their sticky leaves, lack the digestive enzymes necessary to

metabolize the insects. In lieu of the enzymes, these plants have developed a mutualistic

symbiosis with the predatory hemipteran, Pameridea roridulae. The hemipteran, which

is able to move freely across the plants’ sticky leaves, gains easy access to the already-

captured insects and enables the plants to assimilate nutrients from the insects via direct

absorption of its excrement (Anderson and Midgley 2003, Anderson 2005).

… By Poisoning Prey or Hosts

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Many consumers use venom to immobilize and/or kill their prey or hosts

(Whittaker and Feeny 1971). Some, however, are able to use pathogens or symbiont-

derived toxins to envenomate their prey or hosts, in lieu of endogenously produced

toxins. Soil-dwelling, predatory nematodes of the genus Heterorhabditis, for example,

make use of Photorhabdus luminescens, a bioluminescent bacterial symbiont they carry

within their guts. The tiny nematodes burrow inside of their larval insect prey and release

P. luminescens into the insects’ circulatory system, causing a “raging septicemia” which

kills the larvae within 24 hours. As P. luminescens begins to decompose the dead larvae

and rapidly proliferate, the nematodes complete their life cycle by laying eggs. Newly

hatched nematodes of the next generation will later feed on a portion of the P.

luminescens colony for 10 to 14 days and then assimilate the remainder as endosymbionts

(Mlot 1997, Fenton et al. 2000).

Larger-scale predators can also rely on microbial pathogens for venom. On land,

the Komodo dragon, Varanus komodoensis, compounds the lethal effects of its

venomous saliva with the use of up to 57 different species of virulent bacteria which it

obtains by feeding on carrion and houses in its gingival tissue (Montgomery et al. 2002,

Fry et al. 2006). In rocky intertidal zones, the blue-ringed octopus, Octopus maculosus,

paralyzes and kills its prey using the neurotoxins it obtains from 16 of the 22 bacterial

strains that reside in its posterior salivary glands and intestines (Sheumack et al. 1978,

Hwang et al. 1989, White 1998, Bonnet 1999).

Certain parasitoids use a similar strategy to subdue their hosts. The wasp,

Campoletis sonorensis (Hymenoptera: Ichneumonidae), for example, makes use of its co-

evolved endosymbiotic virus (Polydnaviridae). The virus replicates in the ovary of the

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wasp and is injected along with the wasp’s egg into a host caterpillar during oviposition.

There, it acts to suppress the caterpillar’s growth and encapsulating immune response to

the wasp’s egg, allowing the egg to successfully develop inside the caterpillar (Edson et

al. 1981, Theilmann and Summers 1988, Whitfield 1990, Shiga 2005).

… By Transporting the Consumers to Prey or Hosts

Phoresy is the symbiotic transportation of one organism by another (Binns 1982).

Consumers may make use of phoretic partners to acquire prey and hosts that are more

mobile than they are or are otherwise difficult to reach. One such consumer is the

mesostigmatid mite, Poecilochirus necrophori, which uses burying beetles of the genus

Necrophorus for transportation to the location of fly maggots and carrion on which to

feed. In return, the burying beetles may obtain the benefit of increased reproductive

success from the mite’s removal of their competitors (Springett 1968, Wilson 1983).

Pathogens commonly make use of phoretic partners, as well. In their case,

however, we refer to the partners as “vectors.” The mechanisms of recruitment of vectors

by pathogens are very similar to the attraction of primary hosts by parasites, in that they

often involve dramatic examples of host-manipulation. The fungus responsible for

Mummy-Berry Disease in blueberries and huckleberries, Monilinia vaccinii-corymbosi,

relies on pollinators for transportation from the leaves of its hosts to the flowers, where it

can sexually reproduce to make spores, which the pollinators then spread to other host

plants. To recruit the pollinators, the fungus causes the wilting leaves of its host to

secrete sugars, exude sweet odors, and reflect ultraviolet light in a manner similar to that

of nectar-rich flowers. While consuming the sugars, pollinators that have been attracted

80

to these imitation flowers take on some of the fungus and later deposit it into real flowers

as they make their normal feeding rounds (Ingvarsson and Lundberg 1993, Kaiser 2006).

Indirect Offenses versus Alternative Strategies for the Acquisition of Exogenous

Offensive Traits

Symbioses and recruited associations are favored where the benefits of

association outweigh the costs. Often included among the costs of association are

significant investments of nutrients, energy, and time (Dicke and Sabelis 1988, Thomas

et al. 1998, Siemens et al. 2002, van Rijn et al. 2002, Huntzinger et al. 2004, Wäckers

and Bonifay 2004). In some cases, the costs of association may also include conflicts

between partner-provided services and endogenous adaptations (Eisner et al. 1998) or

increases in the risk of attracting natural enemies and partners that are unsuitable

(Anderson and Midgley 2002, van Rijn et al. 2002, Horiuchi et al. 2003, Mouritsen and

Poulin 2003, Shenoy and Borges 2010). Given such costs, it would be reasonable to

assume that indirect offenses evolve when the alternative of developing and maintaining

endogenous offensive traits is still more costly. However, even in a situation where there

is strong selection for these traits and developing these traits endogenously is a

physiological impossibility, forming an indirect offense association is not the only

recourse. Besides indirect offenses, there are at least two other strategies for the

acquisition of exogenous offensive traits: opportunistic association and annexation. By

“opportunistic association,” I mean interspecific relationships such as scavengry (Tenney

1877, Farner and Kezer 1953), kleptoparasitism (Hamilton 2002), and aggressive

mimicry (Sazima 2002). By “annexation,” I mean the assimilation of exogenous traits

81

through collection (Levey et al. 2004) or consumption (Rothschild and Edgar 1978). In

this section, I expand on and clarify the distinctions between these alternative strategies.

Acquiring Exogenous Offensive Traits via Opportunistic Association

The ocellated antbird, Phaenostictus mcleannani, is an obligate associate of army

ants. It monitors the ants’ activity and follows them as they perform their raids, so that it

can capitalize on the swarms of hidden insect prey that the ants tend to flush out (Willis

and Oniki 1978, Swartz 2001, Chaves-Campos 2010). The antbird benefits from its

association with the ants in much the same way that other consumers benefit from

mounting an indirect offense. Yet, to obtain this benefit, the antbird neither has to recruit

the ants nor be recruited by the ants. It simply takes advantage of what the ants must do

irrespective of its presence or absence, scavenging off of them in a way that may actually

reduce their success rate in capturing prey (Wrege et al. 2005).

If being an opportunist is inherently less costly than initiating and maintaining an

indirect offense association, why, then, do species such as the coral grouper,

Plectropomus pessuliferus, invest in the latter (Diamant and Shpigel 1985, Bshary et al.

2006)? The likely scenario is that species such as the grouper, in reality, have more to

gain from recruiting and more to lose from scavenging. Unlike army ants, the moray eels

that the groupers rely on to flush out their hidden prey do not perform raids during the

grouper’s period of activity. Furthermore, if properly positioned, the eels are just as

capable as the grouper of chasing after the prey that flee vertically when flushed. As a

result, if the grouper were to simply follow the eel and scavenge off of it, rather than

recruit it, the grouper would be more likely to lose hidden prey to the eel when foraging

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with the eel, as well as miss the opportunity to capture prey during its own optimal

foraging period.

Acquiring Exogenous Offensive Traits via Annexation

To infiltrate a colony of aphids and avoid detection by the ants protecting this

colony, antlions, the predatory larvae of the green lacewing, Chrysopa slossonae,

camouflage themselves using the waxy remains of aphids they have already captured

(Caltagirone 1999). Similarly, predatory nudibranchs, such as Aeolidia papillosa, can

assimilate the stinging cells of their sea anemone prey and use them not only as a defense

against their own predators, but also as a tool for capturing and killing additional prey

(Greenwood and Mariscal 1984, Östman 1997, Edmunds 2009). Considering the

previously mentioned potential costs of association, what incentive would a species

capable of annexation have for mounting an indirect offense? There are at least two that

are apparent: (1) reduction of the handling time necessary to obtain the traits of another

species; and (2) renewability of the traits upon assimilation. Species that acquire

exogenous traits via annexation may be unable to replenish their supply of the traits when

necessary (Williams 2010), due to the absence or inaccessibility of their suppliers, for

which they themselves may be responsible. By contrast, species that form indirect

offense associations have better assurance of the presence of their suppliers, due to their

symbiosis and/or recruiting investments. In addition, species that form indirect offense

associations can, in some cases, also manipulate the level of production of the traits they

obtain from their associates to suit their short-term needs.

Future Research Questions

83

The paucity of attention given to indirect offenses thus far leaves many openings

for potential exploration and research. There is still much to be learned, for instance,

with regard to how indirect offenses have evolved and might continue to evolve, what

consequences indirect offenses give rise to, and what undocumented forms of indirect

offenses may be present in ecological communities. In this section, I highlight a few key

examples of these promising future directions.

Determining How Indirect Offenses That Involve Symbiosis Differ in Terms of Their

Evolution from Indirect Offenses That Involve Recruited Association

Interspecific associations that do not involve one partner living within, on top of,

or beside the other can often be so tightly co-evolved that researchers might still deem it

appropriate to refer to them as symbioses (Sapp 2004, Cimino et al. 2010). This is

certainly true for many of the cases of recruited association that give rise to indirect

offenses. Yet, differences between these types of associations and ones in which the

participants function all together as a single biological unit (Dyer 1989) can have

meaningful effects on how the associations persist. The former may be more easily

disrupted, for example, by cascading trait-mediation (Liere and Larson 2010) and

fluctuating abiotic conditions (Peterson 1995, Kersch and Fonseca 2005) than the latter,

even when the latter is highly exploitative rather than reciprocally beneficial (Law and

Dieckmann 1998, Kiers and van der Heijden 2006). Several explanations have been put

forth regarding why certain taxa have evolved intricate – and perhaps even obligate –

symbioses, while other taxa – even when closely related – have not (Keeler 1981, Daida

et al. 1996, Stadler and Dixon 1999, Kramer et al. 2009, Schemske et al. 2009). How

well any of these explanations apply to indirect offense associations remains to be seen.

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Exploring the Overlap between Indirect Offenses and Indirect Defenses

I refer earlier to indirect offenses as being the logical counterpart to indirect

defenses and depict them throughout this manuscript as essentially being analogous in

form, but diametrically opposed in purpose. This portrayal, though somewhat expressive

of how indirect offenses and defenses factor into consumer-resource dynamics, provides

little insight into the actual biological relatedness of these strategies. The relationship

between indirect offenses and defenses in general is often complex and can have

intriguing ecological and evolutionary significance. Indirect offenses can, for instance,

often be coupled with indirect defenses. Consider, for example, the case of the cassava

plant, Manihot esculenta. In response to herbivory by the green mite, Mononychellus

tanajoa, the plant releases a specific blend of volatile emissions that triggers infective

sporulation in the mite-pathogenic fungus, Neozygites tanajoae (Hountondji et al. 2006).

In this situation, the plant relies on the fungus for protection from the mite – meaning that

it is employing an indirect defense. Meanwhile, it simultaneously enhances the fungus’

ability to acquire new hosts – meaning that it is providing an indirect offense. Such

potential overlap between indirect offenses and indirect defenses leads to some

interesting questions. For example, how might we assess the relative importance of one

or the other in a situation like this?

Consider, also, the association between the red-billed oxpecker, Buphagus

erythrorhynchus, and the large mammals upon which it forages. This relationship is

obligate for the oxpecker and has, in classical literature, been cited as a mutualism – the

oxpecker reducing the mammals’ parasite loads by feeding. Tolerance of the plucking,

pecking, and scissoring behavior of the oxpecker foraging on their backs (Samish and

85

Rehacek 1999, Plantan et al. 2009), in other words, allows the mammals to obtain an

indirect defense against their ectoparasites. However, it has been demonstrated more

recently that the relationship between the oxpecker and the large mammals may be more

of a parasitism than a mutualism. Weeks (2000), for instance, reports that the exclusion

of oxpeckers from cattle in Zimbabwe has no significant effect on the adult tick load of

the cattle and that oxpeckers significantly prolong the healing time of wounds in cattle. If

open wounds in large mammals are attractive to other ectoparasites besides ticks, such as

flesh flies (Stevens et al. 2006), prolonging the healing time of wounds – and perhaps

even creating fresh wounds – could be an effective way for the oxpecker to acquire a

larger or steadier food supply. Employing this strategy would amount to exploiting the

traits of its mammalian hosts for an indirect offense. What factors might cause the

association to shift from being an indirect defense to being an indirect offense, or vice

versa?

Examining the Use of Symbionts and Recruited Associates in Interference Competition

Species in competition can make use of symbionts and recruited associates to

reduce the foraging ability, survival, or reproduction of their competitors. This

phenomenon constitutes a novel form of interference (Park 1962) and could also be

considered a “competitor” indirect offense. It has been observed in plants (Janzen 1966,

Janzen 1969, Schupp 1986, Agrawal 1998, Mattner and Parbery 2001, Mangla et al.

2008), insects (Yan and Stevens 1995, Yan et al. 1998, Currie et al. 2003, Little and

Currie 2007), free-living ciliates (Preer et al. 1953, Preer et al. 1974), and food-borne

microbial pathogens (Stecher et al. 2007, Brown et al. 2008). There is even some

evidence of its occurrence in mammals (Schmitz and Nudds 1994). What are the

86

consequences of this phenomenon in terms of population dynamics and community

structure? Do they fit the patterns we would expect to see with other trait-mediated

indirect interactions between competitors (Wootton 1994, Peacor and Werner 2001), such

as apparent competition (Holt 1977, Bonsall and Hassell 1997, Chaneton and Bonsall

2000)?

Conclusion

Indirect offenses are widespread, captivating, and diverse. Their occurrence

provides unique insights into the roles of symbiosis and solicited association in shaping

community structure and dynamics. There is still much to be learned with regard to how

indirect offenses have evolved and might continue to evolve, what consequences indirect

offenses give rise to, and what undiscovered forms of indirect offenses may occur in

natural systems. Although indirect offenses offer an enormous potential for promoting

interdisciplinary collaboration and fostering new ideas, their significance as a versatile

and general adaptive strategy has gone virtually unnoticed in the field of biology. It is

my hope that the contents of this review will draw attention to the phenomenon of

indirect offenses and stimulate further investigation into its mechanisms and potential

broader impacts in ecology and evolution.

Acknowledgements

I gratefully acknowledge Peter Morin, Jennifer Rudgers, Jeremy Fox, Nina

Fefferman, Michael Sukhdeo, Marilyn Scott, Robert Paine, Paul Craze, Mathew Leibold,

John Maron, Mark McPeek, Rebecca Jordan, Cesar Rodriguez-Saona, Michael Pace,

Timothy Casey, Peter Smouse, Jason Grabowsky, Deborah Goldberg, Robert Raguso,

James D. Thomson, John J. Wiens, Bradley Hillman, Claus Holzapfel, Michael May,

87

Zsofia Szendrei, Carolyn Norin, Ari Novy, Kristen Ross, my labmates (Kevin Aagaard,

Jean Deo, Jack Siegrist, Maria Stanko, and Holly Vuong), and a very long list of other

friends and colleagues for their helpful comments and invaluable insights. Support for

this review was provided by the Rutgers School of Environmental and Biological

Sciences and the New Jersey Agricultural Experiment Station.

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CONCLUSION

The research presented in this dissertation has uncovered many of the ecological

and evolutionary facets of inducible trophic polymorphisms (ITPs), as well as a good deal

of the fascinating life history of Tetrahymena vorax. My investigation of the potential

costs and benefits of ITPs allowed me to document the significance of the “tailed

microstome” morph of T. vorax as a reciprocal induced anti-cannibal defense. I was able

to demonstrate that the co-occurrence of the tailed microstome and the predatory

“macrostome” morph of T. vorax allows for a coordinated adaptive response to the

presence of intraguild prey. In exploring the potential of ITPs to give rise to novel trait-

mediated indirect interactions, I was able to draw attention to “chasmatectasis,” the

phenomenon of induced predators increasing their gape via the ingestion of increasingly

large prey. I demonstrated that chasmatectasis can mediate a previously undescribed

form of apparent competition. In assessing the differences between the expression of

ITPs in artificial and natural settings, I was able to show how community patterns may

change with the scale and trophic complexity of experimental systems, demonstrate the

utility of microcosms as a baseline for studies of larger systems, and offer insight into

possible reasons for the limited distribution of T. vorax in natural aquatic systems.

Finally, I also introduced the concept of an “indirect offense,” the use of symbionts or

recruited associates to increase the rate of acquisition of prey or hosts.

98

Curriculum Vitae

Aabir Banerji

Education

2004-2011 Ph.D.

Ecology & Evolution

GS-NB, Rutgers University

2000-2004 B.S.

Natural Resource Management

Cook College, Rutgers University

Employment

2004-2011 Teaching Assistant

Rutgers University

Publications

2009 Banerji, A., and P.J. Morin. Phenotypic plasticity, intraguild

predation and anti-cannibal defences in an enigmatic

polymorphic ciliate. Functional Ecology 23: 427-434.