ADAPTIVE PLASICITY, EVOLUTION

AND SCORPION VENOM

Thesis submitted by Alexander N. Gangur in partial fulfilment of the requirements for

the degree of Bachelor of Science with honours in the School of Marine and Tropical
Biology of James Cook University, Cairns.

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I wish to dedicate this thesis to James; I couldnt have done it without you.

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Acknowledgments

To my many and varied supervisors Tobin Northfield, Michael Liddell, Jamie

Seymour, Mic Smout, and David Wilson I cannot thank you enough for your
infinite patience, invaluable guidance, and especially for the brutal criticism. And
thank you to Norelle Daly for helping to fund the project, and to whom I regretfully
owe a micropipette. I want to thank Sarah Kerr for helping to bring Frank to unlife;
Sandra Abell Davis for #52138 and all things fungi-related; and Alex Cheesman and
Will Edwards for suggesting the PCA analysis.

Sam Barnett thanks for showing me the ropes, and thank you to QTHA for
providing them, and to everyone there who kindly let me scour their brains for
advice. I also wish to thank Leanne, Jenni, and Jon for bearing with my boundless
capacity for forgetfulness and being such great sports about it. Thanks to all my
volunteers Chris, Dani, Dave, Eden, Martha (for the dodgy camera work), Nalisa,
Sky, and Tim for getting your hands dirty in the name of scorpion science.

And a special thanks to Mum and John, for keeping me smiling.

Copyright Acknowledgement

I, the undersigned, the author of this thesis, understand that James Cook University
will make it available for use within the University Library and, by microfilm or
other photographic means, allow access to users in other approved libraries. All users
consulting this thesis will have to sign the following statement: "In consulting this
thesis I agree not to copy or closely paraphrase it in whole or in part without the
written consent of the author; and to make proper written acknowledgment for any
assistance which I have obtained from it". Beyond this, I do not wish to place any
restriction on access to this thesis. Users of this thesis are advised that the policy for
preparation and acceptance of Honours theses does not guarantee that they are
entirely free of inappropriate analyses or conclusions. Users may direct enquiries
regarding particular theses to the relevant school head.

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19th Oct 2014

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(date)

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Declaration

I declare that this thesis is my own work and has not been submitted in any form for
another degree or diploma at any university or other institution of tertiary education.
Information derived from the published or unpublished work of others has been
acknowledged in the text and a list of references is given.

___________________
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19th Oct 2014

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Abstract

Organisms embedded within food webs typically need to balance arms races between
their predators and prey. Generally, studies of arms races have focused on pairwise
interactions and the co-evolution of prey-capture and anti-predator traits. However,
interactions between three trophic levels may be more representative of natural
systems. Theoretical investigations of arms races in three trophic-level systems have
revealed novel and often-unintuitive insights that are not evident from pairwise
studies of eco-evolutionary dynamics. There has been growing interest in the likely
importance of arms races in venom evolution, and the aim of this thesis is to evaluate
venom evolution in response to tritrophic interactions. Due to the putative high cost
of venom production, the venom optimisation hypothesis proposes that venomous
animals employ various strategies to use venom efficiently. Thus, an underlying
selection pressure for economical use of venom is believed to drive these arms races
with venomous predators and prey. The resulting escalation of venom toxicity
towards natural predators and prey and each trophic levels evolved venom resistance
may give rise to the extensive variation observed in venom use and composition.
Indeed, emerging evidence has strongly supported the role of diet in shaping the
evolution of venom, but the selection pressures and importance of venom in
defensive contexts is less well understood. I perform two general investigations of
predator-prey interactions in order to better understand the role of arms races, coevolution, and offensive and defensive selection pressures in venom production.

First, to develop general hypotheses to evaluate in an experiment, I investigated

selection pressures on offensive and defensive venom production in a venomous

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animal using a three trophic-level Lotka-Volterra model of eco-evolutionary

dynamics. I then tested these hypotheses in a microcosm experiment with a rainforest
scorpion. I identified rapid responses in scorpion venom profiles over the course of
six weeks, induced by simulated predator interactions. I manipulated selection for
offensive and defensive venoms by inducing venom-use via stinging, against a model
insect prey and a taxidermied vertebrate predator, respectively. Higher predator
pressure was expected to stimulate defensive venom production, and indeed we
found significant changes in venom composition. In contrast, a change from
predation to scavenging for dead prey did not alter scorpion venom composition. I
discuss the potential for induced defence mediated by venom composition for
stabilizing food webs. In the second investigation, I used a Lotka-Volterra three
trophic-level eco-evolutionary model to evaluate the evolutionary response of a
venomous consumer to co-evolution with its prey in the presence of an apex
generalist predator. I modified the model used in my first investigation to allow
venomous consumers to develop venoms associated with both prey capture and
predator defence, which may functionally overlap. Predator introduction promoted
investment in either defensive or offensive venoms. The investment type depended
on the effectiveness of the offensive venom when used in defensive contexts, and the
relative cost associated with investing in multiple venoms. Furthermore, the
evolutionary response of prey species to apex-predator-induced reductions in
consumer densities promoted the evolution of a novel defensive venom against the
predator. These dynamics suggest that interactions with other species can
substantially alter the venom complexity. Finally, I discuss how my findings may
provide valuable insights into the role of defensive venoms and predatory selection
pressure, co-evolution, and arms races in venomous animals.

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Table of Contents

INTRODUCTION TO THE THESIS ........................................................ 1

SPECIES INERACTIONS AND VENOM EVOLUTION ...................... 4

Abstract......................................................................................................... 5

1-1

Introduction .................................................................................................. 5

1-2

Ecological perspectives on venom an overview of venom ecology ........ 7

The venom optimisation hypothesis, a unifying theory of venoms.............8
The costs of chemical warfare .......................................................................9
Venom metering, the behavioural optimisation of venom expenditure ...10

1-3

Economy, arms races, and variation in offensive and defensive venoms .

..................................................................................................................... 11
Offensive venoms and the overkill controversy ........................................13
Defensive venoms time to reconsider? .....................................................15
Demographic variation in offensive and defensive venoms ........................19
Constraints upon venom evolution ..............................................................21

1-4

Venom biochemistry and the optimisation hypothesis ........................... 23

1-5

Future directions ........................................................................................ 26

INDUCED PLASTIC DEFENCE IN VENOM COMPOSITION ........ 28

Abstract....................................................................................................... 29

2-1

Introduction ................................................................................................ 29
Arms races in venomous animals ................................................................29
Theory and evidence for offensive and defensive venoms ..........................30
Adaptive plasticity in venoms ......................................................................32
Statement of aims and hypotheses ...............................................................33

2-2

Theoretical modelling ................................................................................ 34

Model description ........................................................................................34
Model analysis and results ...........................................................................38

2-3

Experimental materials and methods ...................................................... 41

Study organism ............................................................................................41
Experimental treatments ..............................................................................41
Collection and housing ................................................................................44
Choice of milking method ...........................................................................45
Venom milking ............................................................................................47
Fast protein liquid chromatographs (FPLC) venom analysis.......................47
Statistical analysis ........................................................................................48

2-4

Experimental results .................................................................................. 49

2-5

Discussion ................................................................................................... 52
Defensive venom .........................................................................................52
Offensive venom ..........................................................................................54
Implications of plasticity .............................................................................57
Applications and future directions ...............................................................58

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TRITROPHIC INTERACTIONS REVEAL NEW INSIGHTS INTO

VENOM EVOLUTION .......................................................................................... 60

Abstract ........................................................................................................ 61
3-1

Introduction ................................................................................................ 61
Balancing consumer-prey and consumer-predator arms races.....................61
Arms races in venomous animals ................................................................62
Offensive and defensive venoms and statement of aims .............................63

3-2

Model description ...................................................................................... 64

3-3

Analysis and results ................................................................................... 69

3-4

Discussion ................................................................................................... 73
Co-evolution with prey species may facilitate anti-predator venoms ..........73
Restricted co-evolution may give rise to overkill .....................................74
Conclusion and future directions .................................................................76

GENERAL DISCUSSION ........................................................................ 78

LITERATURE CITED ............................................................................. 81

APPENDIX I additional material relevant to Chapter 2 .................... 90

APPENDIX II additional material relevant to Chapter 3 .................. 95

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List of tables

Table 2-1

Description of parameters used in model describing change in

population densities for a prey-venomous consumer-predator system. .................... 36

Table 2-2

Description of functions used in model describing change in

population densities for a prey-venomous consumer-predator system. .................... 37

Table 2-3

MANOVA results, which demonstrated a significant overall difference

between the fraction loadings of predator and without-predator treatments............. 51

Table 2-4

ANOVA results, which demonstrated significantly different fraction

loadings between the predator treatments compared to the without-predator

treatments along both PC1 and PC2. ........................................................................ 51
Table 3-1

Description of parameters and functions used in model equations

describing change in population densities for a three trophic-level system involving a

prey, a venomous consumer, and a predator. ............................................................ 66
Table I-1

MANOVA results, which indicate there were no statistically

significent differences between fraction loadings of scorpions allocated to each

treatment prior to actually having been subjected to those treatments. .................... 93
Table I-2

MANOVA results, which indicate there were no statistically

significent differences between fraction loadings of treatments in the venom obtained

21 days after the cecession of experimental treatments. ........................................... 93
Table I-3

MANOVA results, which indicate there were no statistically

significent differences between fraction loadings of treatments in the venom obtained

21 days after the cecession of experimental treatments. ........................................... 93

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Table I-4

MANOVA results, which indicate there were no statistically

significent differences between fraction loadings of treatments in the venom obtained

21 days after the cecession of experimental treatments. ........................................... 94

List of figures and equations

Figure 1-1

Tree of venomous animals demonstrating the relationship between

animal lineages and the major ecological usage of venom, from Casewell et al.
(2013).

............................................................................................................ 18

Figure 2-1

4-panel figure of effects of offensive and defensive pressures on

equilibrium population densities and traits. .............................................................. 40

Figure 2-2

Comparison of reversed-phase venom profiles obtained via

electrostimulation and manual milking from the same individual. ........................... 46

Figure 2-3

Comparison of post-experiment mean venom profiles. ..................... 49

Figure 2-4

Sample values and priniciple component loadings for peaks 1-11

describing the venom profiles of the experimental scorpions. .................................. 50

Figure 3-1

4-panel figure of eco-evolutionary effects of an invading top predator

on densities and traits for a three-trophic-level food web where the prey species
either can or cannot co-evolve. ................................................................................. 71
Figure 3-2

8-panel figure of eco-evolutionary effects of an invading top predator

on densities and traits for a three-trophic-level food web for high and low M and g3,
where the prey species can always co-evolve. .......................................................... 72
Figure I-1

Comparison of venom profiles obtained prior to experimental

treatments.

............................................................................................................ 90

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Figure I-2

Comparison of venom profiles obtained 21 days after the conclusion of

the experiment, in the absence of treatment pressures. ............................................. 91

Figure I-3

11 fractions as determined by spline fitting, and elution volumes for

local minima. ............................................................................................................ 92

Figure II-1 Evolution of a defensive venom in the absence of co-evolution occurs
under high predatory pressure. .................................................................................. 95
Figure II-2 Effects of high and low M and g3 in the absence of co-evolution. ..... 96

Equations 2-1 Changes in population densities of the prey (Rt), venomous consumer
(Ct), and predator (Pt). ............................................................................................... 35
Equations 2-2 ....... Derivative equations showing changes in prey susceptibility (vR),
consumer prey-capture venom (v1), and consumer anti-predator venom (v2). .......... 37
Equations 3-1: Changes in population densities of the (Rt), venomous consumer
(Ct), and predator (Pt) in the expanded model. ......................................................... 65
Equations 3-2: Derivative equations showing changes prey susceptibility (vR),
consumer prey-capture venom (v1), and consumer anti-predator venom (v2) in the
expanded model. ....................................................................................................... 68

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General introduction to the thesis

While the role of diet in shaping the evolution and ecology of venoms in offensive
contexts is becoming increasingly understood, there is a general paucity of studies
investigating the selection pressures and relative importance of defensive venoms
(Casewell et al. 2013). Similarly, co-evolutionary arms races mediated by venom use
and counter-resistance have been recognized in the venom literature, but the role of
arms races in the evolution and ecology of venoms in offensive and defensive
contexts is poorly understood (Casewell et al. 2013). Thus, the overall aim of my
thesis was to investigate the role of arms races, co-evolution, and offensive and
defensive selection pressures in the evolution and ecology of venom use and
production. My thesis consists of three chapters, which fit within these overarching
themes, each of which will be submitted to a scientific journal.

Chapter 1 is a literature review of the underlying selection pressure for the

economical use of venom, which is believed to frame the evolution and ecology of
venoms, and how this fundamental pressure shapes venom in both offensive and
defensive contexts and may give rise to arms races. My intention is to publish this
chapter as a Toxicon review following the successful publication of the subsequent
two chapters, so that my results may be synthesised into the paper. Toxicon enforces
no strict formatting requirements or word limits for reviews, however I have used the
general article structure described by the Toxicon guide for authors. The broad,
general background to venom ecology, arms races, and offensive and defensive
venoms presented in Chapter 1 is not required, but will enhance an understanding of

the findings presented in Chapters 2 and 3. Chapter 1 concludes with a consideration

of future directions in venom research, segueing into in Chapter 2.

In Chapter 2 I use both theoretical and experimental approaches to investigate the

effects of offensive and defensive selection pressures on venom production. First, I
present theoretical modelling, which I used to generate hypotheses of the effects of
offensive and defensive selection pressures on venoms in a three trophic-level
context. I then present al microcosm experiment, in which I test these hypotheses by
attempting to provoke a plastic response in the venom composition of the rainforest
scorpion Liocheles waigiensis. I found evidence for an induced plastic defence in
venom composition; and to my knowledge, adaptive plasticity in venom composition
has not been previously reported. I intend to submit Chapter 2 as an American
Naturalist article, and have formatted the chapter accordingly. While there is no
strict page length, the average length for manuscripts is 21 (double-spaced) pages of
text.

Lastly, Chapter 3 continues from the investigation of balancing arms races and the
evidence for a novel defensive response presented in Chapter 2. I expand the coevolutionary model used in Chapter 2 in order to evaluate the effects of co-evolution
and a third trophic level (an apex predator) on offensive and defensive venoms. In
doing so, I find and discuss how co-evolutionary arms races with prey may influence
the evolution of defensive venoms, and also discuss novel insights provided by coevolution into a long standing controversy in venom ecology, the overkill
hypothesis (Reviewed in Chapter 1). My intention is to submit Chapter 3 as an
American Naturalist note. Chapter 3 therefore abides by the according formatting

requirements, including a limit of 3 figures/tables and 12 (double-spaced) pages of

text. When the abstract, figures, tables, and captions are excluded this chapter is
within the 12 page limit. I then conclude my thesis with a general discussion of my
findings.

1.

Target journal: Toxicon

Abstract

The production and use of venoms is inextricably tied to the ecology

of the animals that use them. The venom optimisation hypothesis attempts to present
a unifying theory of venom, proposing that the putative high cost of venom
production drives selection for economical venom use. This concept frames the
relationship between variation in venom use and toxicity and the ecology of venomusers. The hypothesis is corroborated by evidence of regulation of venom
expenditure and pressure on venoms for prey-capture and predator deterrence in
natural contexts. However, in general, venom has traditionally been considered to be
an offensive, rather than a defensive, adaptation. In light of emerging evidence
supporting the importance of defence as a selective pressure on venom evolution, we
review the production, use, and variation in venoms in both offensive and defensive
ecological and evolutionary contexts. However, the full extent to which ecology
shapes the immense biochemical variation in venoms remains unclear. We propose
coupling theoretical approaches to empirical work, which may help to inform future
research that attempts to improve both our understanding of defensive venoms and
the role and extent of arms races in venom evolution and ecology.

1.0 Introduction
An animals niche can be broadly defined as what it eats and where it lives, and the
threats that it faces there. The behavioural, morphological, and physiological traits an
animal uses to cope with these challenges, such as venom, can thereby benefit its
growth, reproduction, and survivability its fitness (Kearney et al. 2010). Venoms
generally perform three functions: prey capture, threat deterrence, and digestion,
which assists in prey capture and assimilation. Indeed, although a precise definition

has been contentious (Weinstein et al. 2013), the consensus view is essentially that
venom involves a complex mixture of chemicals variously used in the subjugation
and digestion of prey and in defence (Weinstein et al. 2012). The production and use
of venoms are therefore shaped by these functions in predator-prey interactions.
Offensive venoms (for prey capture) are typically biochemically complex and
variable in their physiological action and chemical composition (Fry et al. 2009). In
contrast, defensive venoms tend to be biochemically simpler and highly conserved in
their composition, producing the physiological effect of immediate, localised pain
(Church and Hodgson 2002; De Graaf et al. 2009; Peiren et al. 2005). Recent work
has challenged the characterisation of defensive venoms as comparatively simple
(Dutertre et al. 2014). Defence has traditionally been considered to be of secondary
importance to the use of venom in prey-capture (Casewell et al. 2013). Nonetheless,
because their use can thereby affect the vital rates growth, reproduction, and
survivability of a venom-user in different ways, offensive and defensive venoms
may be thought of as two different traits of venomous animals (Violle et al. 2007).

Together, offensive and defensive chemical warfare confers a substantial fitness

advantage by reducing the need to physically compete with and overcome potential
predators and prey (Morgenstern and King 2013). The broad taxonomic diversity of
animals which have independently evolved venoms attests to the significance of this
fitness advantage (Casewell et al. 2013). Venoms themselves are highly diverse,
exhibiting a huge variation in venom characteristics between phyla (Kordi and
Gubenek 2000), closely related species (Mackessy 2010), between populations of
the same species (Calvete et al. 2009; Glenn and Straight 1978), and even within the
lifespan of an individual in ontogenic variation (Andrade and Abe 1999) and

between sexes (Menezes et al. 2006). This literature review will first address the
underlying pressure for economical use of venom that frames the relationship
between venom evolution and the ecology of venom-users (Section 2). How
variation in venom use and production is shaped by predator-prey interactions in
light of this fundamental pressure will then be considered (Section 3). Finally, we
will highlight the paradox of the lavish biochemical complexity of venoms in spite of
the hypothesised selection for venom economy (Section 4), and consider future
directions in venom research (Section 5).

2.0 Ecological perspectives on venom an overview of venom ecology

Although there has been a traditional focus on human toxicity in venom research
(Fry et al. 2009), major advances in venom ecology have emerged in recent years.
Evidence explaining the geographic variation in venom composition in terms of diet
(Daltry et al. 1996) and for judicial behavioural regulation of venom-use (venom
metering) culminated in the proposition that venomous animals employ various
strategies to minimize venom expenditure (Morgenstern and King 2013; Wigger et
al. 2002). The potentially high costs underlying this pressure to use venom
economically have been subsequently elucidated (McCue 2006; Nisani et al. 2012;
Nisani et al. 2007; Pintor et al. 2010). In addition to venom metering, strategies for
minimising costs may include the differential secretion of venom components in
different ecological interactions (Inceoglu et al. 2003) and the evolution of toxicity
towards natural predators and prey (Barlow et al. 2009), which may drive coevolutionary arms races (Casewell et al. 2013). And while most attention has been
paid to the use of venom for prey-capture (Casewell et al. 2013), the recent discovery
of separate offensive and defensive venoms in cone snails may prompt a renewed

focus on understanding venom evolution and ecology in defensive contexts (Dutertre

et al. 2014). Objections have been raised to the hypothesised behavioural choice to
meter venom volumes in snakes (pressure-balance hypothesis (Young et al. 2002,
see section 2.3) and selection pressures driving economical use of venom snakes
(overkill hypothesis Mebs 2001, see section 3.1).

Nonetheless, the scope of our understanding still remains relatively narrow due to a
general research focus on pharmacologically important taxa. Indeed, venom research
has traditionally focused on snakes, and spiders and scorpions to lesser extent (Fry et
al. 2009). Relatively little attention has been afforded to other venomous taxa
including other arthropods and reptiles, cnidarians, molluscs, echinoderms, fish,
mammals, and amphibians (Casewell et al. 2013). Further, relatively little is
generally known about the ecology and natural history of venomous animals and
venom-use (Casewell et al. 2013). Improving our knowledge of venomous animal
ecology is critical to understanding the causes and processes underlying variation in
venoms, as well as critical for applied outcomes such as improving antivenoms and
human health outcomes (Richards et al. 2012).

2.1 The venom optimisation hypothesis, a unifying theory of venoms

The venom optimisation hypothesis is central to venom ecology, conceptually
framing the evolution and ecology of venom use and production. Wigger et al.
(2002) demonstrated a frugal choice in the volume of venom injected by a
polyphagous spider on the basis of prey type, and proposed that this behaviour
served to minimise waste of a valuable and expensive resource. Further evidence has
since emerged from a broader range of taxa and was synthesised by Morgernstern &

King (2013) in an attempt to generalize the hypothesis into a unifying theory of

venoms. It is argued that since venoms are rich in costly proteins and peptides (Fry
et al. 2009; Inceoglu et al. 2003), the expense of venom constrains the ability of
venomous animals to simply inject larger volumes in predator-prey interactions.
Instead, venomous animals evolve strategies to reduce venom expenditure
(Morgenstern and King 2013), including behavioural regulation (Section 2.3).
Although originally synonymous with venom metering only (Morgenstern and King
2013), selection for venom economy may also drive the evolution of venom toxicity
towards natural predators and prey (Section 3) (Casewell et al. 2013). Evidence for
the high cost of venom corroborates this hypothesised need for efficient venom
expenditure.

2.2 The costs of chemical warfare

It has been inferred from the secondary loss of venoms in a number of taxa that
venoms incur a considerable cost (King 2004; Li et al. 2005). Only four published
studies - limited to two species of snakes (McCue 2006; Pintor et al. 2010) and one
scorpion (Nisani et al. 2012; Nisani et al. 2007) - have investigated the metabolic
cost of venom regeneration to date. The nutritional costs of producing venom and the
maintenance of glands for venom production and storage have also been proposed as
potentially substantial costs (McCue 2006; Nisani et al. 2012), but remain uninvestigated. Though venom quantities are small relative to body mass (less than
0.5%) in snakes, spiders, and scorpions (Morgenstern and King 2013), increases in
metabolic rate ranging from 11% (McCue 2006) to 40% (Nisani et al. 2007) follow
the complete expulsion of venom (though see Pintor et al. 2010). This elevated
metabolic rate can be attributed to both the production of the biochemical

components of the venom itself, and the metabolic cost of maintaining the venomproducing and storage tissues (Morgenstern and King 2013). The time to fully
regenerate all components of a venom is not insignificant, requiring at least 28 days
in snakes (Oron and Bdolah 1973; Rotenberg et al. 1971), and from as few as 16
(Boev et al. 1995) to as many as 85 days in spiders (Perret 1977). Given that
elevated metabolic rates must be sustained in some degree to synthesise venom
components for such durations, the maintenance of a venom system is likely to be
metabolically expensive (Morgenstern and King 2013). However, further studies are
needed to confirm, generalize, and quantify the physiological and (though difficult to
estimate) direct reproductive fitness costs of venom to fully evaluate the evolutionary
implications for venom production.

2.3 Venom metering, the behavioural optimisation of venom expenditure

If chemical warfare is costly, then the optimisation hypothesis posits that regulatory
behaviour should ration this valuable resource (Wigger et al. 2002). Indeed, a
growing body of research has shown such venom metering in a range of venomous
taxa (Morgenstern and King 2013). Spiders, for instance, may evaluate venom
resistance in prey based on olfactory cues and use their venom accordingly
(Hostettler and Nentwig 2006). But the bulk of the research has historically come
from snakes, for which the evidence of metering has been disputed. Proponents of
the pressure-balance hypothesis maintain that variable secretion volumes are a
consequence of mechanical constraints and passive regulation upon contact of the
venom apparatus, not behavioural regulation prior to contact (Young and Kardong
2007). While more recent evidence has cast doubt upon this view (Morgenstern and
King 2013), there remain some valid criticisms. Notably, there is an inadequate

10

understanding of the sensory mechanisms by which venomous animals in general can

assess an interaction and utilize their venom accordingly (Morgenstern and King
2013; Young et al. 2002; Young et al. 2003).

In any case, the emerging evidence strongly supports behavioural metering of venom
to varying degrees in different taxa. A choice of whether or not to envenomate at all
or to simply physically subdue prey has been shown in both snakes and arachnids
based on the relative size and threat posed by the target (Edmunds and Sibly 2010;
Rein 1993). Once the decision to envenomate is made, a range of factors have been
shown to influence the delivery and volume of venom to minimize venom-use in
different trophically venomous taxa, including the relative sizes of the venom-user
and target (Edmunds and Sibly 2010; Hayes 1995; Malli et al. 1999), intensity of
struggling by the victim (Malli et al. 1999), venom-user hunger (Hayes 1993),
handling time (Edmunds and Sibly 2010), prey type (Edmunds and Sibly 2010;
Hayes 1992), and venom availability (Hostettler and Nentwig 2006). Thus, it appears
likely that venomous animals behaviourally regulate venom expenditure to minimize
the high cost of chemical warfare.

3.0 Economy, arms races, and variation in offensive and defensive venoms
Venom evolution, as well as behavioural strategies in the use of venom, is likely
driven by a pressure for economical use of venom (Casewell et al. 2013). Indeed,
selection for economy appears to drive the evolution of toxicity towards prey in
scorpion-eating saw-scaled vipers (Echis spp.), rather than kill-speed (Barlow et al.
2009). By increasing toxicity towards natural predators and prey, efficacy (i.e. in
subduing prey or deterring a threat) can be sustained with a smaller quantity of

11

venom. The resulting selection pressure on predators and prey to evolve venom
resistance to mitigate evolving toxicity may give rise to a reciprocal selection
pressure on venomous animals for yet higher toxicity and/or novel toxins (Casewell
et al. 2013). The net result is a Red Queen effect (Van Valen 1974), an escalating
co-evolutionary arms race. Venomous predator-prey systems demonstrate the basic
requirements for co-evolutionary arms races to occur, exhibiting specific toxicity
towards natural predators (Binford 2001; Dutertre et al. 2014) and prey (Barlow et al.
2009; Pekr et al. 2008), as well as venom resistance occurring in both natural
predators (Jansa and Voss 2011) and prey (Biardi and Coss 2011; Heatwole and
Poran 1995; Poran et al. 1987) of venomous animals.

There is a paucity of direct evidence of arms races between venomous animals and
their prey, and even more meagre evidence for defensive arms races with predators
(Casewell et al. 2013). Nonetheless, arms races and co-evolution may be key
processes driving the evolution of, and variation in, venoms across a mosaic of
different predator-prey communities, in both offensive and defensive contexts.
However, it is important to remember that this evolutionary adaptation is restricted
by the genetic constraints imposed by ancestral morphology and physiology.
Furthermore, other predator-prey systems show that arms races may not necessarily
escalate perpetually, but can be limited by settling to an equilibrium between preycapture and anti-predatory traits (Becks et al. 2010). In sum, selection for venom
economy in offensive and defensive contexts, which may give rise to arms races, are
likely major drivers of the inter- and intraspecific variation in the composition,
function and delivery of venoms.

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3.1 Offensive venoms and the overkill controversy

Selection for diet is a potent force which drives variation in venoms both between
and within venomous taxa (Casewell et al. 2013). But this view - and, more broadly,
the concept of venom optimisation - has been contentious. In snakes, there has
historically been inadequate evidence of adaptive venom composition (Chippaux et
al. 1991; Sasa 1999; Williams et al. 1988), and evidence of extreme venom toxicity
(Mebs 2001) and injection volumes (e.g. (Hayes et al. 2002; Young et al. 2002). The
overkill hypothesis cites such evidence of seemingly indiscriminate use of venom
and a lack of dietary pressures acting upon venom composition, and presents an
alternative neutral hypothesis of venom evolution (Gibbs et al. 2013; Mebs 2001).
Multiple studies of snake venom composition have failed to demonstrate a
correlation between venom composition and geographical differences in diet
(Chippaux et al. 1991; Williams et al. 1988). Williams et al. (1988) found that
variation in venom composition between populations of the tiger snakes Notechis
ater niger and N. scutatus was instead correlated with geographic distance, indicative
of neutral evolutionary processes, but noted that didnt exclude selection for genes
encoding particular venom components. Early evidence for a correlation between
diet and inter-population variation in the venom of the Malayan pit viper
Calloselasma rhodostoma was criticized for lacking toxicity data to demonstrate a
causal relationship between venom variation and toxicity towards prey (Mebs 2001;
Sasa 1999). Yet subsequent studies have since suggested that venom production is
generally subject to a strong island effect, giving rise to inter-population variation
driven by differences in diet (Zelanis et al. 2008).

13

Indeed, a wealth of evidence for prey-selection in venom has emerged from a broad
array of taxa, including spiders (Pekr et al. 2008), scorpions (Abdel-Rahman et al.
2009; Zhao et al. 2010), jellyfish (Kintner et al. 2005; McClounan and Seymour
2012), cone snails (Duda Jr et al. 2009; Elliger et al. 2011; Fainzilber et al. 1991;
Remigio and Duda Jr 2008), and also snakes (Barlow et al. 2009; Furry et al. 1991;
Jorge da Silva and Aird 2001). These studies have variously demonstrated adaptive
evolution for prey-specific toxicity extending from the whole venom to individual
toxins, such as the avian-specific denmotoxin in the mangrove snake Boiga
dendrophila (Pawlak et al. 2006). The secondary loss of venom toxicity in the seasnake Aipysurus eydouxii due to a dietary shift to an ovivorous diet corroborates the
importance of prey-selection, and provides compelling evidence for a strong positive
selection on venom production (Li et al. 2005). Thus, the emerging picture from
empirical evidence is in favour of selection upon venom composition for optimised
prey-capture and venom economy.

Nonetheless, remaining evidence for the overkill of prey via excessive venom
injection and toxicity by snakes is problematic. Sasa (1999) proposed that the
adaptive value of snake venoms may exhibit a continuum, where selective
advantages of venom might be evident in some taxa but absent in others. But such a
view is seemingly irreconcilable with the putative cost of venoms, the consequent
selection for venom economy, and the strong selective pressure that appears to act on
venom production. Thus, the evidence for wastefully large injection volumes and
excessive toxicity of some snake venoms must be addressed. Firstly, generalist
venomous predators, which can encounter prey exhibiting a broad range of
susceptibilities to their venom, may inject large quantities of venom as a form of bet

14

hedging to guarantee a meal (Hayes 1992). Investigations quantifying toxicity have

also frequently used model organisms, such as mice, which fail to exhibit the
physiological resistances to venom toxins arising in natural predators and prey
(Chippaux et al. 1991), potentially as a consequence of co-evolutionary arms races.
Secondly, laboratory studies have shown that snakes will inject venom volumes
hundreds of times greater than the lethal dose requirements (Mebs 2001). But studies
investigating metering by snakes upon different size classes of prey have indicated
that snakes do meter venom, but can only coarsely distinguish between smaller prey,
perhaps owing to the relatively insignificant venom volumes required or a limited
ability to finely meter their venom (Hayes et al. 2002; Young et al. 2002). In sum,
while minor points of contention may remain, empirical evidence strongly supports
the role of prey-capture and diet as a major force driving venom variation (Casewell
et al. 2013).

3.2 Defensive venoms time to reconsider?

Venoms are typically thought of as a foraging adaptation and, indeed, this has
historically been the main focus of study (Casewell et al. 2013). However, recent
evidence for the separate selection and production of a discrete defensive venom in
cone snails has led to the prediction that specialised defensive venoms may be more
widespread and important in other venomous taxa than previously recognized
(Dutertre et al. 2014). Histological evidence for different secretory units has been
found in sea anemones (Moran et al. 2013), spiders (Silva et al. 2008), snakes (Sakai
et al. 2012), centipedes (Nagpal and Kanwar 1981), and scorpions (Yahel-Niv and
Zlotkin 1979), suggesting that other venomous animals may be able to produce and
secrete separate venoms as in Conus. Scorpions can secrete a clear, chemically

15

cheap, pain-inducing prevenom to deter threats before resorting to their more

complex, protein-rich and chemically expensive main venom (Inceoglu et al. 2003).
Indeed, specialised pain-inducing toxins are known in snakes (Bohlen et al. 2011),
spiders (Siemens et al. 2006), ants (Schmidt 1990), centipedes (Undheim and King
2011), lizards (Strimple et al. 1997), and various fishes (Bohlen and Julius 2012) and
can effectively deter potential threats without causing significant tissue damage
(Bohlen and Julius 2012).

Though venom is employed primarily for defence by hymenopterans, lepidopterans,

echinoderms, stingrays, bony fish, amphibians, and eutherians, there is relatively
little empirical evidence to demonstrate defensive-related selection pressures acting
upon venom composition in any taxon (Casewell et al. 2013). The deleterious effects
of venom upon an aggressor in defensive contexts has been typically explained as
arising from conserved pharmacology between the predators and prey of venomusers, rather than from a separately-evolved defensive strategy (Dutertre et al. 2014;
Fry et al. 2009). Nonetheless, in addition to cone snails, defensive pressures have
been implicated in the evolution of defensive venoms in age-related (ontogenic)
shifts in ecology which heighten exposure to predators in bees (Owen and
Braidwood 1974) and spiders (Binford 2001; de Andrade et al. 1999).

Similarly, while many studies have supported the view that venomous animals can
behaviourally control venom expenditure in predatory contexts, few have
investigated metering in defensive situations. Those which do have been limited to
snakes (Hayes et al. 2008), ants (Haight 2006; Obin and Vander Meer 1985),
scorpions (Nisani and Hayes 2011), and spiders (Nelsen et al. 2014). High rates of

16

dry biting or stinging, in which no venom is actually delivered seems to be a

common mode of defensive venom-use (Morgenstern and King 2013), and snakes
exhibit highly variable injection volumes in threatening interactions (Hayes et al.
2002). Dry bites have been hypothesised, but not demonstrated, to deter predators,
perhaps with the threat of injection, while preserving valuable venom (Cooper et al.
2014). The threat of predation could pressure venomous animals to stockpile large
volumes of venom to ensure successful deterrence in a threatening encounter
(Cooper et al. 2014). Furthermore, there is limited evidence for defence-specific
morphological adaptations, such as specialized fangs to spit venom at an aggressor in
the elapid genera Naja and Hemachatus (Wster and Thorpe 1992). In sum, the
accumulated evidence appears to implicate prey selection as a stronger selective
force than defence in most venomous animals (Casewell et al. 2013), for which there
seems to be lower levels of adaptive evolution (Wster and Thorpe 1992) (Figure 1).

This supposedly generally weaker selection pressure on the defensive venoms seems
counter-intuitive. Survival is a compromise between resource acquisition, avoidance
of predation, and reproduction. But while the consequence of a failed foraging event
or mating event is simply a missed meal or opportunity to produce offspring, the
penalty for failing to avoid a predator is death, or at least injury. The life-dinner
principle describes this asymmetry in the price of failure for predators and prey
(Dawkins and Krebs 1979), and might superficially predict that selection for
investment in defensive venom-use should be stronger. However, winning a
defensive interaction only requires escape, while winning a predatory interaction
necessitates subduing and consuming prey. Defence is a major selective force for
cone snail venoms (Dutertre et al. 2014), but their limited mobility means that cone

17

Figure 1: Tree of venomous animals demonstrating the relationship between animal lineages and
the major ecological usage of venom, from Casewell et al. (2013). Coloured branches indicate
lineages which include venomous taxa. Red branches indicate a predominantly predatory role for
venom, blue a defensive role, and green a role in intraspecific competition. Note that for
Protostomes and reptiles, venom is predominantly used for prey-capture, while the use of venom by
other Deuterostomes is mainly for predator-deterrence.

snails possess few other means of effectively deterring threats, besides their shell.
Other venomous animals may be less dependent on their venom in defensive
contexts, given the putative high cost of venom production. Indeed, scorpions
generally prefer to retreat from threats (van der Meijden et al. 2013). Behavioural
responses in predatory contexts may thereby blur the relationships between
morphology, behaviour, and ecology, and decouple selection for maximum

18

performance in defensive contexts from ecology (van der Meijden et al. 2013). In
other words, venoms may appear to be relatively unimportant for defence because
venomous animals instead rely more heavily upon less costly behavioural antipredator strategies, such as retreat.

Lastly, there is relatively meagre evidence of co-evolutionary arms races between

venomous animals and their predators (Casewell et al. 2013). Sufficient reciprocal
selective pressure inducing the rapid evolution of defensive venom compounds is
more likely where the physiological and taxonomic diversity of predators do not
present a broad physiological target, and where the frequency of interactions with
predators are sufficiently high. Where predators are more generalist in their dietary
preferences there may be inadequate pressure to drive the co-evolution of venomresistance, whereas specialized predators are more likely to develop venom
resistance (Jansa and Voss 2011). In general, while the available evidence suggests
that the use and function of defensive venom may substantially differ to offensive
venoms, significantly more work is needed to understand the role of venom in
defensive contexts and the selection pressures acting on the evolution of defensive
venoms.

3.3 Demographic variation in offensive and defensive venoms

Ontogenetic niche shifts, whereby individuals exploit different resources, reduce
intraspecific competition between life stages and can thereby increase individual
fitness and the carrying capacity of the population (Werner and Gilliam 1984). When
this occurs, the venom optimisation hypothesis suggests that venom production and
use will change to mirror variation in the diet and threats faced by a venomous

19

animal across its lifespan, and sex-related differences in ecology. Indeed, the
avoidance of competition between adults and juveniles is regarded as the principal
ecological model to explain ontogenic shifts in venom composition (Underwood and
Seymour 2007).

There has been a long history of studies which correlate ontogenic shifts in prey
choice with concurrent shifts in venom use and composition, primarily in snakes
(Andrade and Abe 1999), spiders (Casper 1985), scorpions (Morgenstern and King
2013), and more recently also in jellyfish (McClounan and Seymour 2012;
Underwood and Seymour 2007). In offensive contexts, ontogenic shifts in venom are
thought to be associated with an increase in the energetic requirements of adults
(Hayes 1995; Zelanis et al. 2008). The resulting dietary change can be coupled with
changes in venom toxicity towards new prey (Underwood and Seymour 2007) and in
protease production to aid in the digestion of larger prey (Zelanis et al. 2008).

Upon reaching maturity, divergences may occur in venoms in tandem with the
emergence of different life histories between sexes. For example, male hobo spiders
(Tegenaria agrestis), which actively wander outside their burrows for mates, exhibit
selection for defence from the higher vertebrate toxicity of their venom compared to
that of more sedentary females, as a consequence of their higher exposure to
vertebrate predators (Binford 2001). Sexual dimorphism, which gives rise to
increased energy requirements in one sex, can also drive demographic variation in
offensive venom composition (Daltry et al. 1997; Furtado et al. 2006). And in the
burrowing scorpion Scopio maurus palmatus, the more complex chemical
composition of female venoms was attributed to a high frequency of sexual

20

cannibalism (Abdel-Rahman et al. 2009). Thus, evidence for variation in offensive

and defensive venoms precipitated by demographic differences highlights the need
for further research of the natural histories of venomous animals. Only knowledge of
the life histories of venom-users can elucidate this individual-level of variability
which may be invisible to geographic studies comparing venom composition
between populations.

3.4 Constraints upon venom evolution

While selection pressures drive evolution, constraints restrict and delimit the
pathways along which it can occur. Thus, it is also vital to consider the ancestral
morphological, physiological, and chemical innovations and limitations which
influence the present production, use, and delivery of venoms in offensive and
defensive contexts. Morphology is strongly genetically constrained, which limits the
evolution of delivery systems. The limited ability of cone snails to retreat from
threats may be responsible for the strong selection pressure for defensive venom (see
section 3.2). Similarly, spiders and scorpions have been shown to exhibit far more
sophisticated, highly judicial venom metering than snakes (Bub and Bowerman
1979; Malli et al. 1999; Nisani and Hayes 2011). Why might this be so? Spiders and
scorpions evaluate the need to continue injecting venom by assessing the struggling
of their grappled prey, injecting more venom as required (Morgenstern and King
2013). Snakes, conversely, tend to immediately release and then track their
envenomated prey by scent (Furry et al. 1991; Smith et al. 2000), avoiding retaliatory
injury and perhaps ultimately owing to a generally greater difficulty in maintaining a
grapple without limbs. Indeed, it has been suggested that necessary adaptations of
musculature and body conformation to suit constriction may generally be mutually

21

exclusive with the speed and agility necessary for venom-use in snakes (Shine and
Schwaner 1985), also constriction is known in some venomous snakes (Morgenstern
and King 2013; Shine and Schwaner 1985).

Similarly, phylogenetic constraints and ecological selection pressures may both

contribute to variation in the composition of venoms. Correlations between diet and
venom variation between congeners should be considered within a phylogenetic
context to pinpoint where evolutionary shifts in diet and venom composition have
occurred, to rule out similarities due to common ancestry (Barlow et al. 2009).
Toxins employed and adapted for prey- and predator-specificity by a venomous
animal are constrained by the venom components that have already been made
available for modification by ancestral evolution, which can vary between taxa.
Indeed, fundamental differences in toxin chemistry arises between major lineages of
venomous animals, shaping variation between taxa (Kordi and Gubenek 2000).

Toxic proteins generally evolve via recruitment and modification of proteins from
other physiological processes which satisfy a range of requirements (reviewed in Fry
et al. 2009) These proteins then typically undergo extensive gene duplication with
modification, generating closely-related toxin families (Casewell et al. 2013).
There is a high degree of convergence in the underlying biochemical structure and
pharmacology of toxic proteins between protein families (Fry et al. 2009). For
example: cone snails, scorpions, spiders, centipedes, bees, cnidarians, echinoderms,
and reptiles have all evolved different molecular strategies for targeting the same ion
channels (Fry et al. 2009). Further, there is a high frequency of independent
recruitment of the same proteins families in closely and distantly-related taxa

22

(Casewell et al. 2013). For example, the underlying chemical structure of

phospholipase A2 has been independently recruited four times into squamate reptiles,
but also in the venoms of cephalopods, cnidarians, insects, and scorpions (Casewell
et al. 2013). So long as they satisfy they satisfy the necessary biochemical criteria to
be eligible for recruitment as toxins, then is there any advantage to recruiting a given
protein over another? So long as they ultimately exhibit the same functional effects
of killing or immobilizing prey and deterring predators, the recruitment of a given
protein may simply be subject to chance and neutral processes. The relative extent to
which this underlying biochemical variation is related to adaptive significance is a
question that has yet to be seriously addressed.

4.0 Venom biochemistry and the optimisation hypothesis

The most confounding problem facing the optimisation hypothesis is the seemingly
lavish biochemical complexity of venoms (Morgenstern and King 2013). Non-toxic
proteins and peptides constitute a large proportion of venom, despite the substantial
reduction in metabolic cost that could be gained by even a small decrease in these
non-active proteinaceous components (Morgenstern and King 2013). Additionally,
the extraordinary degree of functional redundancy in the biochemical action
exhibited by different toxins in the same venom seems at odds with the putative
pressure for venom economy (Casewell et al. 2013).

The rapid evolution of seemingly redundant venom components may be a necessary

price to pay in order to continually generate novel toxins for use in predator-prey
interactions. When applied to venom evolution, the birth and death model of
evolution of multigene families (Nei and Rooney 2005) posits that extensive,

23

imperfect duplication of toxin genes to increase toxin production gives rise to

chemical redundancy due to minor mutations in the amino-acid sequence (Casewell
et al. 2013; Li et al. 2005). This process is hypothesized to provide the necessary
diversity of toxins for venomous animals to compete against evolving resistances in
co-evolutionary arms races with their predators and prey, and to target potentially
new predators and prey (Casewell et al. 2013). But though this model predicts that
redundant toxins, which confer no functional advantage, should degrade, this does
not always appear to be the case. For example, the venom of the scorpion Leiurus
quinquestriatus hebreus contains 14 toxins which bind to the same receptor in insects
(Morgenstern and King 2013). Some may truly be redundant and retained and
transcribed from the genome as a toll worth paying to maintain a large collection of
toxin encoding genes subjected to duplication and diversification (Weinberger et al.
2010).

To some extent, the lavish diversity of venom components can be explained by

synergistic effects, which improve the toxicity of the venom. These seemingly weak,
redundant, and even non-toxic compounds may cooperatively enhance the activity of
other venom components (Cohen et al. 2006). Indeed, the birth and death model
suggests that there is more benefit to maintaining large, multilocus toxin families
arising from gene duplication compared to optimising the potency of a single toxin,
due to the synergistic effects they could provide (Casewell et al. 2013). For example,
the nontoxic mutant scorpion peptide Bj-xtrIT-E15R induces a conformational
change in the physiological target of the alpha toxin LqhIT, enhancing its binding
and toxicity (Cohen et al. 2006). Similarly, cone-snails use synergistic pairs of toxins
called toxin-cabals to induce rapid paralysis in prey (Espiritu et al. 2001). Weak

24

and non-toxic venom components may provide other benefits, such as antimicrobial
activity to resist infection from prey ingestion (Kuhn-Nentwig 2003) or antiseptic
activity for honeycomb construction by bees (Baracchi et al. 2011).

Specific components from a broad armoury of available toxins may be selectively

produced or secreted in response to specific predator or prey interactions
(Morgenstern and King 2013). Similar biochemical modulation of venom has been
seen in the cheap and rapidly-regenerating prevenom of scorpions. Prevenom
contains pain-inducing potassium ions and fewer, simpler proteinaceous toxins, and
is preferentially used first in defensive interactions, preserving the more complex and
costly main venom (Inceoglu et al. 2003). Furthermore, prevenom can also be used
to immobilize arthropod prey, enabling scorpions to actively forage as well as deter
predators while main venom slowly regenerates (Inceoglu et al. 2003). The absence
of specific venom proteins in the initial stings of the scorpion Parabuthus
transvaalicus also suggests the selective use of certain proteins (Nisani and Hayes
2011). The potential for biochemical or physiological modulation in snakes (Cascardi
et al. 1999) and spiders (Boev et al. 1995) has been observed, but identification of
modulation in natural secretions was confounded in the latter by the use of
electrostimulation, which induces the complete evacuation of the venom glands.
Further research is needed in order to determine the extent to which biochemical
modulation in offensive and defensive encounters can explain the adaptive
significance of the venom complexity.

25

5.0 Future directions

The venom optimisation hypothesis lays a conceptual framework that relates venom
ecology and evolution to the high cost of venom production and a strong selection
pressure for venom economy. The underlying pressure to use venom economically
thereby frames the way in which venom and venom-use has evolved in response to
the predators and threats of venomous animals. While there has been growing
support for an association between variation in venoms and prey-capture (Casewell
et al. 2013), we still have a poor understanding of the pressures upon and role of
defence in venom evolution, likely due to the logistical difficulties in exposing
individuals to the often wide range of predators venomous animals might interact
with. Theoretical modelling can help to deal with these logistical difficulties, to
consider evolutionary questions that are difficult to empirically investigate due to the
time-frames involved, and to generate hypotheses that can be empirically tested. For
example, the recent discovery of separate offensive and defensive venoms in cone
snails has prompted a reconsideration of the role of defence in venom production,
and whether or not separate venoms might be more widespread (Dutertre et al. 2014).
Modelling can help elucidate the circumstances under which two separate venoms
for prey capture and predator deterrence might be more beneficial than using a single
venom that fulfils both functions.

Theoretical modelling has also been extensively used to investigate co-evolutionary

arms-races, due to difficulties in tracking the values of traits and their associated
fitness costs (Abrams 2000). Modelling may provide a rare opportunity for guided
empirical studies of arms races in venomous predator-prey systems. Theoretical

26

models may also be used to generalize the results found from simpler systems such
as toxin-producing bacteria (Kerr et al. 2002), where real-time evolutionary
experiments are more logistically feasible than for most venomous animals.

Further studies of the natural histories of venomous animals and venoms are vital,
especially in taxa that have traditionally been neglected, such as centipedes and
insects, fishes, mammals, and echinoderms. Indeed, the historical focus of study has
been on taxa for which venom is primarily an offensive adaptation. Broadening our
understanding of venom ecology in a wider range of venomous taxa, especially the
understudied organisms for which venoms are primarily a defensive adaptation, will
help to ensure the generality of the unifying theory of venoms, the optimisation
hypothesis. The optimisation hypothesis would further benefit from a more thorough
understanding of the precise nature of the costs of venom production, and from a
broader understanding from a wider range of taxa. In light of these costs, the
unexplained biochemical complexity and redundancy remains largely unexplained
and remains the biggest challenge to the optimisation hypothesis.

Finally, other strategies that may be employed by venomous animals to optimise the
use of their venom remain to be explored. Whether or not biochemical modulation of
venoms is consistent with the optimisation hypothesis should be a focus of future
studies (Morgenstern and King 2013). To our knowledge, the possibility for
phenotypic plasticity in venom biochemistry remains unexplored and may present
another important mechanism for managing the costs of chemical warfare in a
changing environment.

27

2.

Target journal: American Naturalist

28

Abstract

Animals generally balance the costs and benefits of prey-capture and

anti-predator traits in two simultaneous co-evolutionary arms races. We investigated

selection pressures on offensive and defensive venom production in a venomous
animal for prey-capture and for predator-defence using a co-evolutionary model
describing the dynamics of a 3-level trophic chain. We used the models to develop
hypotheses and tested them in a microcosm experiment with a rainforest scorpion.
We identified rapid responses in scorpion venom profiles over the course of six
weeks, induced by manipulated predator interactions. We manipulated selection for
offensive and defensive scorpion venoms by inducing stinging (venom use) against a
model insect prey and a stuffed model vertebrate predator, respectively. Higher
predation pressure was expected and confirmed to stimulate defensive venom
production. We discuss the potential for this induced defence for stabilizing food
webs.

Introduction
Arms races in venomous animals
Co-evolutionary arms races involving predatory and anti-predator traits are
ubiquitous in food webs (Wade 2007), and may be important in venom production
and counter-defence systems (Casewell et al. 2013). Perhaps as it is a costly resource
(McCue 2006; Nisani et al. 2012; Nisani et al. 2007; Pintor et al. 2010), venomous
animals employ various strategies to reduce the use of venom in both offensive and
defensive contexts (Morgenstern and King 2013). Behavioural strategies, such as the
metering of venom secretion volumes and assessment of the risk posed by a potential
predator, are employed to varying extents by venomous taxa (Morgernstern & King

29

2013). There is also growing evidence that venom economy drives selection for
lethality towards natural predators and prey (Barlow et al. 2009). In doing so, venom
can be used more economically to achieve the same efficacy. This gives rise to a
reciprocal selection pressure between predators and prey of the venomous animal,
which evolve a resistance to mitigate the increasing toxicity of the venom (Casewell
et al. 2013). The result, known as the Red Queen effect, is a co-evolutionary armsrace of escalating venom toxicity and venom resistance (Van Valen 1974). Although
venomous predator-prey systems demonstrate the basic requirement of a reciprocal
selection pressure for these arms races to arise, they have received little empirical
attention in offensive contexts and less-so for defensive contexts (Casewell et al.
2013). Nonetheless, evidence for the extraordinarily rapid evolution of venom toxins
compared to other Metazoan protein-coding genes (Chang and Duda 2012), for
toxin-specificity towards natural predators and prey (e.g. Dutertre et al. 2014), and
for a co-evolving resistance in both predators (e.g. Jansa & Voss 2011) and prey (e.g.
Coss et al. 1993) of venomous animals broadly supports the emergence of arms-races
in venomous animals. How these arms races influence the evolution of prey-capture
and anti-predator traits including venom may be understood by examining arms
races in other predator-prey systems, and using theoretical modelling of these
systems (Abrams 2000).

Theory and evidence for offensive and defensive venoms

The physiological, behavioural, and morphological traits with which an animal deals
with the challenges of capturing prey and deterring predators affect its vital rates
growth, reproduction, and survivorship and, therefore, fitness (Kearney et al. 2010).
Since the use of venom to facilitate feeding predominantly benefits growth and

30

reproduction, while its use for defence by the producing animal primarily benefits
survivorship, offensive and defensive venom efficacy could be thought of as two
traits of a venom-user (Violle et al. 2007). Further, they can be considered to be traits
which may overlap due to the potential for individual venom toxins to biochemically
target prey only, predators only, or both. Venoms consist of toxins which are both
broadly toxic and phyla-specific in their toxicity (Nicholson 2007), and different
toxins in a venom can separately target different taxa for maximal efficacy (Fry et al.
2009; Kordi and Gubenek 2000). Offensive and defensive venom-use may
consequently overlap to varying degrees, depending on the extent to which toxins are
specialized towards predator and prey physiology and biochemistry. For example,
Na+-channel blocking -toxins are a category of scorpion toxins containing a
subgroup of toxins only highly active only towards mammalian voltage-gated
sodium channels (VGSCs), another subgroup active only towards insect VGSCs, and
a third group of -like toxins toxic towards both insects and mammals (QuinteroHernndez et al. 2013). This capacity for different toxins to target separate
physiologies is seen in other venomous animals including jellyfish (McClounan and
Seymour 2012), centipedes (Yang et al. 2012), cone snails (Dutertre et al. 2014),
snakes (Zlotkin et al. 1975), and spiders (Nicholson 2007). In some venomous taxa
the same toxins may be used in both offensive and defensive contexts, resulting in no
distinctly different offensive and defensive toxins. For example, the diversity and
complexity of both phyla-specific and general toxins in spider venoms enable them
to prey upon specific groups of insects, though most are considered to be generalist
predators (Kuhn-Nentwig et al. 2011; Nicholson 2007). If there is sufficient selection
pressure and if the physiologies and biochemistries of predators and prey are

31

sufficiently different then the potential certainly exists for distinctly offensive and
defensive venom components to coexist in the same whole-venom mixture.

Adaptive plasticity in venoms

To our knowledge, an adaptive plastic response in underlying venom composition
arising from altered ecological interactions has not been previously reported.
Existing studies have found the that specific components of a whole-venom can be
differentially secreted in response to different interactions in a limited number of
venomous taxa including the spitting cobra Naja pallida (Cascardi et al. 1999), the
wandering spider Cupiennius salei (Boev et al. 1995), the dark scorpion Parabuthus
transvaalicus (Nisani and Hayes 2011). Cone snails can deploy separate offensive
and defensive venoms in prey and predator interactions, respectively (Dutertre et al.
2014). Scorpions can secrete a cheap pre-venom which can induce pain to deter
potential predators before resorting to a more costly main venom (Inceoglu et al.
2003), and which is preferentially used in low-threat encounters (Nisani and Hayes
2011). None of these studies have shown a plastic response in the composition of the
whole-venom itself. In sum, we know that different ingredients in the wholevenom recipe can be selectively used in a limited number of venomous animals, but
we dont know whether this recipe is fixed, or if it can plastically respond to a
changing environment. Previous studies in plant-herbivore and other predator-prey
systems have suggested that inducible defences should be most common when cues
for biological threats are reliably available, when biological attacks are
unpredictable, and when the fitness benefits exceed the costs of plasticity (Harvell
1990). Furthermore, plastic induced defences should also be favoured if defences are
costly and not always needed, as in the chemical warfare employed by both plants

32

and venomous animals (Karban 2011). Thus, the potential ability to mitigate the
costs of venom production by decreasing production or altering composition to
favour prey-capture and feeding would be consistent with the optimisation
hypothesis.

Statement of aims and hypotheses

In our broad investigation of the effects of predator-prey interactions upon venom
composition, the aims of this study were twofold, and combined theoretical and
experimental approaches. Our first aim was to understand how the offensive and
defensive traits of venoms might evolve when subjected to different ecological
interactions, using mathematical modelling of 3-trophic level predator-prey dynamics
and co-evolutionary trait-change. Although venomous animals are predators
themselves, for clarity we refer to the venomous animal in our model as a
consumer to avoid confusion with the apex generalist predator. The modelling was
used to investigate two hypotheses: 1) that applying a predatory pressure would
diminish the co-evolutionary arms race between the venomous consumer and prey by
decreasing their predatory and anti-predator trait and 2) increasing prey abundance
should enhance the arms race by increasing consumer and prey predatory and antipredator traits.

The second aim of the study was to evaluate the potential and direction for adaptive
plasticity in the venom profile of a venomous animal in response to manipulated
ecological interactions. Venoms are a complex mixture of molecules (Casewell et al.
2013), and this molecular diversity can be teased apart to varying extents and

33

captured using a range of analytical techniques such as chromatography and massspectrometry to obtain a venom profile (Shen and Noon 2004). We tested the null
hypothesis that manipulating ecological interactions would not give rise to changes
in venom profile compared to a control. Any observed change in the mature venom
profile in response to either the predatory or prey treatment would indicate a plastic
response. Theoretical studies suggest that plastic trait change is a good predictor of
the direction of evolutionary change (Draghi and Whitlock 2012), and we therefore
hypothesized that any plasticity we observed would occur in the same direction as
predicted by our eco-evolutionary model.

Theoretical modelling
Model description
Theoretical modelling of natural systems can be used to guide empirical work and to
investigate natural systems that are difficult to empirically study (Abrams 2000). In
our study, we investigate the evolution of prey-capture (offensive) and anti-predatory
(defensive) traits in a venomous animal embedded in a food web. The venomous
animal (e.g. a scorpion) feeds on a co-evolving prey (e.g. an insect), and is itself
preyed-upon by a generalist (non-co-evolving) predator (e.g. a small mammal).
Theoretical studies of 2 trophic-level predator-prey systems involving coevolutionary arms races suggest that reciprocal selection pressures give rise to an
escalating increase in offensive and defensive traits (Dieckmann et al. 1995). Studies
of co-evolution in tri-trophic-level systems involving plants, herbivores, and
herbivore-predators suggest that herbivores attempt to balance these arms races to
minimize their net costs (Nersesian et al. 2011). Thus, a consumer may adopt

34

intermediate trait values when arms races between their predators and prey need to
be balanced.

We used a three trophic-level, discrete-time Lotka-Volterra model of co-evolutionary

trait change to investigate the evolution of two traits offensive venom and
defensive venom in a venomous consumer. Our model is an extension of the 2
trophic-level model described by Northfield and Ives (2013), we also assume a type
1 functional response for simplicity. The changes in population densities of the prey
(Rt), venomous consumer (Ct), and predator (Pt) at time t are given by (Equations
2.1):

Rt

Rt erR ( r0 ,vR )

Rt q1 ( v1 ,vR )Ct

Ct

Ct ecq1 (v1 ,vR ) Rt

Pt

Pt e rP ( P )

m ( v1 ,v2 ) q2 ( v2 ) Pt

cP q2 ( v2 ) Ct mP

In the model we assume that consumer fitness is balanced by predation on the prey
species (where higher offensive venom investment increases consumer predation
rate), density-dependent mortality due to predation by the predator (where more
highly defensive venom reduces apex-predator predation rate), and mortality due to
other density-independent effects (which increases with investment in each venom).
The prey species may mitigate mortality due to consumer predation by investing in
venom resistance (modelled as venom susceptibility, where lower susceptibility
provides higher venom resistance), which is balanced by a cost (modelled as densityindependent mortality). Because we modelled the predator as a generalist predator,
its growth does not entirely depend on preying on the consumer species. Rather, it

35

has an alternative growth rate and carrying capacity. Since the predators fitness is
not tightly linked with the defensive venom of the consumer, we assume that it does
not co-evolve with the consumer. The parameters and general functions used in the
population density expressions are presented in Tables 1 and 2, respectively.

Table 1: Description of parameters used in model equations describing change in population densities
for a three trophic-level system involving a prey, a venomous consumer, and a predator.

Parameter
Rt
Ct
Pt
vR
v1
v2
vP

r0
f
m0
g1
g2
rP0
k
mP,0
gP
c
cP
a1
a2

Description
Prey species population density at time t.
Consumer species population density at time t.
Predator species population density at time t.
Prey susceptibility to the offensive venom of the venomous
consumer (higher values results in higher predation rate).
Offensive trait (venom) of the venomous consumer (higher values
results in higher predation rate).
Defensive trait (venom) of the venomous consumer (higher values
results in higher success rate by top predator).
Predator susceptibility to the offensive venom of the venomous
consumer (higher values results in higher predation rate of the
consumer).
Prey intrinsic rate of increase.
Fitness cost of venom susceptibility in the prey species (investing in
lower susceptibility to venom is more costly).
Venomous consumer base mortality rate (due to density-independent
effects).
Fitness cost of offensive venom in venomous consumer (investing
more in offensive venom is more costly).
Fitness cost of defensive venom in venomous consumer (investing
more in offensive venom is more costly).
Predator intrinsic rate of increase.
Carrying capacity of the predator population.
Predator base mortality rate (due to density-independent effects).
Fitness cost of venom susceptibility in the predator species (investing
in lower susceptibility to venom is more costly).
Base conversion rate of eaten prey into new venomous consumers.
Base conversion rate of eaten venomous consumers into new
predators.
Baseline attack rate of the venomous consumer, quantifying
searching efficiency for prey.
Baseline attack rate of the predator, quantifying searching efficiency
for venomous consumers.

36

Table 2: Description of functions used in model equations describing change in population densities
for a three trophic-level system involving a prey, a venomous consumer, and a predator.

Function
f
vR

rR(r0,vR) = r0

q1(v1, vR) = a1 (1 e

v1v R

m(v1, v2) =
m0

g 1 v1

g 2 v2

q2(v2) = a 2 Pe

v2 v P

rP(P) = rP 0 (1 kPt )

mP = mP , 0

gP
vP

Description
Population growth of the prey species. Large values
are given by high intrinsic rates of increase, high
susceptibility to venom, and small fitness cost of
venom susceptibility.
Predation rate of the prey species by the venomous
consumer. Large values are given when offensive
venom investment, prey susceptibility, and baseline
consumer attack rate are higher.
Density-independent consumer mortality. Increases
with base mortality rate, cost of offensive and
defensive venoms, and investment in offensive and
defensive venoms.
Predation rate of the consumer species by the
predator. Decreases when defensive venom
investment is higher, and predator susceptibility and
baseline predator attack rate are lower.
Population growth of the predator species on
alternative food sources. Higher values are driven by
higher predator intrinsic rate of increase and carrying
capacity.
Density independent predator mortality. Increases
with predator base mortality rate and susceptibility to
venom, and decreases with the increased cost of
venom susceptibility.

The changes in traits for the prey (i.e., prey susceptibility) and consumer (i.e.,
offensive and defensive venom investment) at each time step are then determined by
the populations genetic variance and mean fitness, as well as the derivative of mean
fitness with respect to the trait. If Fi is the mean fitness for species i, and VR, VC1 and
VC2 are additive genetic variance for prey susceptibility vC, consumer offensive
venom v1, and consumer defensive venom v2, respectively, then co-evolution of these
traits is given by the following derivatives (Equations 2.2):

vRt 1

vRt

1 FR
VR
FR vR

vRt

v1t 1

v1t

1 FC
VC1
FC vC1

v1t

(vRt ca1Rt e

vR

v1a1Ct e

v1vR

v1vR

)VR

g1 )VC1

37

v2t 1

v2t

1 FC
VC 2
FC vC 2

v2t

(vP q2 Pt

g 2 )VC 2

For our simulations predator venom susceptibility was considered a constant that
does not co-evolve. We made additional assumptions in our model as per the
assumptions presented by Northfield and Ives (2013). Firstly, although spatial
structure may influence predator-prey interactions and give rise to a geographic
mosaic of co-evolution, we assumed that each species was represented by a single,
panmictic population to focus on local adaptation. We used a quantitative genetics
approach and held the additive genetic variance of the traits constant, so that the rate
of evolution depended on the strength of selection. Although this assumption doesnt
hold true in the long term (without mutations to maintain genetic variation), under
very strong selection (resulting in loss of genetic variation, and for small populations
(which lack large initial genetic variation and are affected by genetic drift), we
believe this approach provides a reasonable starting point to investigate the shortterm (hundreds of generations) effects of changing ecological interactions.

Model analysis and results

Prey-driven arms races typically give rise to reciprocal co-evolution, while predatordriven arms races typically result in escalation whereby prey evolution is driven by a
predatory pressure, but predator evolution is not driven by a reciprocal pressure from
its prey (Dietl and Kelley 2002). To investigate the bottom-up and top-down effects
on venom of manipulating predator-prey interactions, we conducted two sets of
simulations. For each of these simulations, we assumed that all of the populations
began at eco-evolutionary equilibrium, with identical genetic variances for the coevolving prey and venomous consumer. In the first type of simulation, we changed

38

prey density by increasing prey intrinsic rate of increase r0. In the second type, we
changed top-predator density by increasing top-predator carrying capacity k. After
altering these ecological parameters, to find the equilibriums we simulated the
models for an additional 5000 generations to allow population densities and trait
values to stabilize (Figure 1). We tracked the trajectories of population densities and
traits as the parameters r0 and k increased. We ran the simulations with a range of
starting population density and trait values to evaluate the potential for alternative
stable states. Although alternative stable states were found for certain parameter
values, they did not qualitatively change the results.

When prey density is increased through increases in the intrinsic growth rate,
consumer and apex predator densities also subsequently increase (Figure 1A). The
increased consumer density enhances selection pressure on the prey, which leads to
reduced susceptibility to consumer venom. This prey trait change stimulates the
consumer species to invest more in venom production to compensate for the reduced
susceptibility. In response to higher densities of the apex predator, the venomous
consumer also increases investment in defensive venom to mitigate mortality due to
increasing top-predator density (Figure 1C).

39

Figure 1: Equilibrium population densities and traits. Equilibrium population densities (A, B) and
trait values (C, D) under changing bottom-up (prey growth rate) conditions (A, C) and top-down
(top-predator carrying-capacity) conditions (B, D). Red lines represent venomous consumer density
(A, B) and offensive and defensive venom investment (C, D. Solid and dashed lines, respectively).
The solid blue lines represent co-evolving prey density (A, B) and susceptibility to the offensive
venom trait (C, D). The dotted black line represents generalist, non-co-evolving top-predator density
(A, B). For changing prey growth rate, top-predator carrying-capacity k=0.015 was used. For
changing top-predator carrying capacity, the prey growth-rate r0 = 0.5 was used. For both
simulations, the parameters used were rP = 0.01, vR = 0.4, v1 = 0.3, v2 = 0.1, vR = 0.4, m0 = 0.05, mP0 =
0.01, a1 = 4, a2 = 3, c0 = cP = 0.25, f = 0.07, g1 = g2 = 0.02, gP = 0.005, Vn = V1 = V2 = VP = 1.

When the apex predator carrying capacity increases, higher apex predator densities
apply a stronger selection pressure for defence on the consumer and defensive venom

40

investment increases in the consumer species. Elevated mortality due to predation

reduces consumer density, resulting in a trophic cascade (Figure 1B). Consequently,
the prey experience less pressure from predation, and the co-evolutionary arms race
between the consumer and prey diminishes. The reduced consumer density results in
increased prey susceptibility, and reduced offensive venom in the consumer (Figure
1D).

Experimental materials and methods

Study organism
Loicheles waigienis is a rainforest scorpion found along the east coast of Australia,
South-East Asia, and the Pacific (Koch 1977). In the Wet Tropics of Far North
Queensland, L. waigiensis is a generalist predator of invertebrates, including crickets,
and is in turn preyed upon by a range of invertebrate and vertebrate (marsupial)
predators (Schneider 2011). Scorpions exhibit an inverse relationship between the
relative sizes of the pincers (chelae) and stinger (telson), and generally prefer to rely
upon the larger weapon for prey-capture and defence (van der Meijden et al. 2013).
L. waigiensis possesses relatively large, robust chelae and a small telson (Isbister et
al. 2004; Jeliffe 2010; Koch 1977). This ecomorphotype is consistent with other
members of Hemiscorpiidae (formerly Ischnuridae and Liochelidae) (van der
Meijden et al. 2013).

Experimental treatments
To subject scorpions to both offensive and defensive pressures (manipulated
ecological interactions) and elicit a plastic response, we used a 2 2 factorial design
with a prey treatment to provide an offensive pressure in one factor, and a predatory

41

treatment to provide an offensive pressure in the other. Each factor contained two
levels, which either attempted to provoke a sting (venom use) or not provoke a sting.
Thus, the treatments were live prey with predation (offensive and defensive
pressures), live prey without predation (offensive pressure), dead prey with predation
(defensive pressure), and dead prey without predation (control). Previous work has
shown that L. waigiensis produces different toxins that can target vertebrate and
invertebrate biochemistry (Schneider 2011). Because L. waigiensis preys upon many
insects, including crickets (Schneider 2011), and because they were readily available,
we used the common house cricket (Acheta domesticus) as a surrogate prey species.
Similarly, L waigiensis is preyed upon by a variety of marsupials including
bandicoots, white-tailed rats, and Antechinus sp. (Seymour, unpublished data). We
used a stuffed mouse (Mus musculus) to elicit anti-predator responses in a similar
fashion to the predator-on-a-stick method from Ramirez et al. (2010). Taxidermied
predators, including vertebrate predators, have been widely used in other studies of
prey behaviour (e.g. Digweed & Rendall 2009). Due to the small number of males (N
= 6), 4 males were picked at random and evenly dispersed between the four
treatments. The remaining 2 males were then randomly dispersed, to one treatment
each, for a total of 16 replicates per treatment. All other individuals in the experiment
were females (N = 58).

Previous studies investigating the venom regeneration in this species have shown that
L. waigiensis recovers a mature venom profile within 21 days of complete depletion
of the venom glands via electrostimulation (Jeliffe 2010; Schneider 2011). Thus, we
ran the treatments for 42 days so that the experiments lasted twice as long as the
venom regeneration time. By providing the scorpions with the full length of time

42

necessary to regenerate their venoms, we ensured that comparisons between

experimental treatments could detect differences in the complete, mature venom.
Although no ontogenic or sexual variation has been previously detected in the venom
profiles of L. waigiensis (Schneider 2011), we used mature scorpions only.

For the prey treatment, scorpions were each fed a cricket once per week (6 feedings
over 6 weeks). Live prey scorpions were given a cricket that was larger than their
chelae size, as larger prey are more likely to be stung during prey-capture (Edmunds
and Sibly 2010). Crickets for dead prey scorpions were frozen overnight before
feeding, to minimize loss of nutritional value. Cricket remains were removed the
following day to reduce fungal growth.

Our taxidermied mouse was used to provoke defensive stings from predation
scorpions three times a week (Monday, Wednesday, and Friday) for weeks 2-6, with
a one week acclimation period (15 treatments total per individual over 5 weeks).
Scorpion containers were placed within a 33 cm 17 cm 10 cm cardboard arena to
prevent animals from escaping, and animals were exposed without making direct
contact to avoid prematurely eliciting defensive behaviour. The mouse was then
continuously jabbed at the cephalothorax of the scorpions for 30 seconds, which
readily stimulated scorpion anti-predator responses, including alert and threat
postures (with chelae extended and open, and metasoma erect), grappling, pinching,
stinging, squirming, and retreat (van der Meijden et al. 2013; Warburg 1998).
Animals that escaped into the arena were immediately returned to their container
with forceps and the treatment resumed. The mouse was cleaned of dirt between each
treatment to prevent the spread of mould. Without predation scorpions were placed

43

in the arena and their containers opened for 30 seconds. This ensured that animals in
both treatments were handled and exposed to laboratory conditions outside their
growth chambers equally. Six weeks after commencement of the experiment,
scorpions were subjected to the control (dead prey without predation) treatment for
one week prior to re-milking to allow them time to recover a significant quantity of
mature venom.

Collection and housing

Individual Liocheles waigiensis, were collected from rainforest populations at
Crystal Cascades (1657'43.1"S 14540'46.9"E), Cairns, Queensland, Australia,
between 26th and 30th of March, 2014. Mature scorpions were collected, based on
cephalothorax shape and size, and sex was determined according to the presence
(males) or absence (females) of a distinctive notch in the chelae (Koch 1977).
Following Schneider (2011), we used this feature to identify the smallest mature
male in our collection and designated all smaller scorpions as juveniles (and
excluded them from the experiment) and larger scorpions without a notch as mature
females. Scorpions were then individually held in 170 110 50 mm 650 ml clear
plastic takeaway containers with one stone and moist organic soil (300 ml Searles
Premium Potting Mix brand potting mix) to provide a suitable microclimate for the
animal and to aid with moulting. These containers were randomly sorted and stacked
two high, in two Wisecube WGC-450 temperature and humidity chambers at 28 C
on a 14/10 light/dark cycle. Relative humidity was maintained at 70%, and after 3
weeks of treatments all scorpions were moved to new containers containing freshly
autoclaved soil to reduce fungal growth.

44

Choice of milking method

Repeated milking by electrostimulation can cause the venom apparatus to degrade in
a range of venomous taxa including some scorpions, compromising venom yield
(Cooper et al. 2014). However, previous work indicates that this is not the case for L.
waigiensis (Schneider 2011). It has also been proposed that electrostimulation may
contaminate venom samples with hemolymph or cellular contents (Oukkache et al.
2013). We found no evidence for this with L. waigiensis (Figure 2), and manual
milking produced inadequate volumes for chemical analysis of venom.
Electrostimulation is assumed to expel all venom material (Morgenstern and King
2013), and in scorpions may therefore elicit mixture of both prevenom and main
venom.

To evaluate whether or not milking method had an effect upon the venom profile
obtained from L. waigiensis we milked 4 scorpions with electrostimulation and 5
scorpions manually. Manual milking was carried out by dorsally poking the
scorpions to provoke stings to a triangular wire frame wrapped with Parafilm.
Droplets of venom were then collected and diluted in 10 L of deionised water.
Following Schneider (2011), electrostimulation was performed using an Arthur H.
Thomas Co. Z789 Square Wave Stimulator. Frequency, duration, pulse mode, and
output mode were 5.5 pulses/sec, 15 milliseconds, continuous, and + monophasic,
respectively. Scorpions were secured to a block of foam with a heavy-duty rubber
band, and a pair of electrodes wetted with saline solution were placed into contact
with opposing sides of the telson. A 20V stimulus was then applied, which was
gradually increased to up to 30V until venom was exuded. The venom was captured
with a micropipette tip slipped over the aculeus until no more venom was released

45

and then mixed with 10 L of deionised water. Following milking, the scorpions
were then fed a dead cricket, and subsequently fed each week for 3 weeks. They
were then each re-milked with the alternative milking method (i.e. manual, for
scorpions previously electrostimulated).

Figure 2: Reversed-phase venom profile obtained via electrostimulation (green chromatogram)

and manual milking (blue chromatogram) from the same individual. These venom profiles were
typical of the data we obtained to compare milking methods. Identical peaks aligned along the xaxis, indicating the presence of the same venom components. The elution time and relative heights
of peaks in the profiles closely matched with the exception of the very first peak, although
concentrations were lower due to a smaller volume of venom obtained via manual milking.

Venom samples were analysed using reversed-phase HPLC on a 1% gradient of 90%

acetonitrile/ 0.45% TFA at 0.4 mL/min using a Phenomonex C12 150 x 2 mm Jupiter
4m Proteo 90 column. We compared the venom profiles of individual animals
milked both manually and using electrostimulation and determined that the milking
method did not have a substantial effect upon the extracted venom composition.
Therefore, we used electrostimulation in order to maximize sample volumes for

46

optimal signal in the chemical analysis of venom composition in each of the

treatments.

Venom milking
Electrostimulation was used to obtain venom samples, which were then stored and
frozen at -80 C. After initial venom samples were collected and animals that did not
secrete venom excluded, individual scorpions were randomly, evenly assigned to one
of four treatments. Scorpions were first milked within 5 days of collection, and then
re-milked at the end of the experiment, 42 days later. All scorpions were then milked
a third time 21 days after the experimental treatments ceased to assess how it had
changed in the absence of offensive and defensive pressures.

Fast protein liquid chromatography (FPLC) venom analysis

Milked venoms were diluted in 150 L of degassed 50% phosphate buffered solution
(PBS), centrifuged for 10 minutes total at 32,000 RPM, and filtered through a
syringe-driven 0.22 m Millipore filter. Venom profiles were obtained using sizeexclusion fast protein liquid chromatograpy (FPLC) using a Superdex 75 10/300
(Tricorn) GL Column (13m, 10mm300mm) with PBS buffer at 0.50 ml/minute for
45 mL on an KTA FPLC. Venom contents were detected at a wavelength of 280
nm. Fractions were collected every 0.5 mL in 96 well plates and stored at 4C.
Venom profiles were then processed and exported using UNICORN 5.20 software
(2008, General Electric Company, General Healthcare Bio -Sciences AB, Sweden).

47

Statistical analysis

All analyses were performed in R (R Development Core Team 2014). In order to

compare between the venom profiles obtained in each treatment, we standardised
each FPLC chromatogram by dividing the intensity of each data point in a
chromatogram by the sum of all the data points for that chromatogram (the Riemann
sum), thereby normalizing every chromatogram so that the integral for each
chromatogram is equal to 1 (Sauve and Speed 2004). We then obtained the mean
chromatogram for each of the four treatments. This allowed us to compare relative
quantities of venom material in different parts of the venom profile, between
treatments. We removed four scorpions from the analysis, two of which gave rise to
chromatograms with a very poor signal-to-noise ratio, indicating low venom
concentrations. This may have been due to damage to the venom-producing glands of
these individuals, either as a result of electrostimulation or due to injury in the wild.
The other two scorpions were excluded due to highly atypical venom profiles,
suggesting that the venom samples were contaminated.

To delimit and evaluate the distinct fractions present in scorpion venom profiles, we
generated a mean chromatogram from all normalized chromatograms from the posttreatment milking and fitted a spline curve to the entire chromatogram using the
smooth.spline function in R, with the smoothing parameter, = 0.5 (R Development
Core Team 2014; Wehrens 2011). Fractions were then separated by local minima to
generate 11 fractions (Wehrens 2011), ignoring the first and final fractions as they
contained only noise (Appendix I, see Figure 3). Principle component analysis (PCA)
was then used to describe these 11 fractions across the data set (Wehrens 2011).

48

Finally, MANOVAs and ANOVAs were performed to evaluate treatment effects on

the principle components using type III sums of squares (Gotelli and Ellison 2004).

Experimental results
From the principle component analysis we obtained two major principal components,
PC1 and PC2, which explained 50% and 27% of the overall variability, respectively.
Venom profiles obtained from the mouse (defensive pressure, N=27) and non-mouse
(no defensive pressure, N=25) treatments were found to be significantly different
using a MANOVA to evaluate the treatments on the principal component weightings
(F1,48 = 0.237, p = 0.002; Figure 3; Figure 4; Table 2).

(n=14)
(n=13)

Normalised milliabsorbance (280 nm)

(n=11)
(n=14)

Elution volume (mL)

elution time

49

Figure 3: Comparison of post-experiment venom profiles. Because these represent the averaged,
normalized venom profiles for each treatment, the response variable is normalized milliabsorbance
units, and indicate relative quantity between any given portion of the mean of the venom profiles of
each treatment. Venom contents were detected at 280 nm. Defensive treatments are indicated in dark

10

0.2

-0.6 -0.4 -0.2

-0.15

-0.05

0.05
PC1 (50%)

0.15

0.0

PC2

0.05
0.00
-0.10

PC2 (27%)

0.4

0.6

0.10

0.15

and light blue, while the non-defensive treatments are shown in orange and red.

6
7

11
8
1

2
-0.6 -0.4 -0.2

0.0

0.2

0.4

0.6

0.8

PC1

Figure 4: Sample values (A) and priniciple component loadings for peaks 1-11 (B) describing the
venom profiles of the experimental scorpions. Defensive treatments are indicated in dark (live
prey) and light (dead prey) blue, while the non-defensive treatments are shown in red (live prey)
and orange (dead prey). The principle components were developed from the, 11 distinct fractions
fitted to the mean chromatogram obtained from all of the individual chromatograms. The two
main principal components (ANOVAs), PC1 and PC2, described 50% and 27% of the overall
variation, respectively.

Examination of the ordination graphs suggests clustering for the predator treatment
(Figure 4A). There were no interaction effects overall, nor any significant effects
from the live versus dead prey treatments. Scorpions for which anti-predator
stimulation was induced exhibited lower values of both PC1 (F1,48 = 6.42, p = 0.014)
and PC2 (F1,48 = 6.66, p = 0.013)(Table 3). Peak 10 strongly increased each principle
component (Figure 4B), where as peaks 6,7, and 9 reduced the values for both
(Figure 4B). Peaks 2 and 5 each had different effects on the two principle

50

components (Figure 4B). There were no interaction effects, nor any significant
effects from the live versus dead prey treatments. Furthermore, profiles obtained
after a 21 day recovery period following cession of treatments exhibited the same
patterns of difference between treatments as seen in Figure 3, but these differences
were not statistically significant (Appendix I). Lastly, the venom profiles obtained
from milking before the experimental treatments began were not significantly
different from each other (Appendix I).

Table 3: MANOVA results, which demonstrated a significant overall difference between the
fraction loadings of predator and without-predator treatments. There was no interaction effect.

Source
1. 2.
Prey
Predator
Predator
Prey
Residuals

d.f.

Pillai

approx F

Df den

1
1

0.028
0.236

0.698
7.395

2, 47
2, 47

0.503
0.002**

0.029

0.702

2, 47

0.501

48

Table 4: ANOVA results, which demonstrated significantly different fraction loadings between the
predator treatments compared to the without-predator treatments along both PC1 and PC2. There were
no interaction effects.

Source

d.f.

MSE

PC 1
Prey
Predator
Predator Prey
Residuals

1
1
1
48

0.008
0.038
0.001
0.006

1.385
6.522
0.203

0.245
0.014*
0.654

PC 2
Prey
Predator
Predator Prey
Residuals

1
1
1
48

0.000
0.021
0.004
0.003

0.124
6.626
1.336

0.727
0.013*
0.254

51

Discussion
In this study we have shown adaptive plasticity in the venom profile of a venomous
animal for the first time, to our knowledge. To understand and generate hypotheses
about how predator-prey interactions might influence offensive and defensive venom
production, we modelled eco-evolutionary dynamics in a three trophic-level
predator-prey system with a venomous consumer. In doing so, we found that
increasing predatory pressure would diminish a co-evolutionary arms race between
the consumer and its prey, while increasing defence. We also found that
independently increasing prey abundance would enhance the escalation of the arms
race. We then evaluated whether the direction of plastic change in offensive and
defensive venom components would be the same as expected from evolutionary
modelling. In doing so, we identified an induced plastic defence in a venomous
animal due to a predatory pressure that was consistent with our modelling.

Defensive venom
Our experimental results supported our modelling, which suggested that increasing
predatory pressure in the presence of consumer-prey co-evolution would diminish
the arms race between the consumer and prey. We have found evidence for adaptive
plasticity to predatory pressures in venom composition, whereby increasing pressure
for defence modifies venom composition to likely enhance venom components that
are associated with defensive interactions. This was consistent with our expectation
that a plastic response to this pressure would occur in the same direction as an
evolutionary response. Venom profiles obtained three weeks after the cessation of
experimental treatments sustained the general patterns of compositional change
observed in the profiles obtained at the end of the experiment. However, these

52

patterns had diminished and were no longer statistically significant in the absence of
a predatory pressure, suggesting that periods of relaxed selection pressure can return
scorpion venom to its original state.

A plastic response to predator exposure, which enhances toxicity towards that

predator, would be consistent with the venom optimisation hypothesis. The
optimisation hypothesis proposes that venomous animals must employ a range of
strategies to reduce the waste of their costly venom (Morgenstern and King 2013). A
plastic increase in vertebrate toxicity towards predators on a per-volume basis of
venom would improve the efficiency of venom-use, by demanding smaller injection
volumes in order to effectively deter a potential threat (Casewell et al. 2013). Indeed,
this same strategy is seen in venomous animals as an evolutionary response in preycapture contexts (Richards et al. 2012).

In addition to this strategy for optimising the use of defensive venom, L. waigiensis
is likely to partition its venom into at least two separate secretions a cost-effective
prevenom and a main venom. When milking L. waigiensis, we observed the same
succession of clear (prevenom) to milky (main venom) secretions, as has been
reported in other scorpion species (Gopalakrishnakone et al. 1995; Inceoglu et al.
2003; Nisani et al. 2012; Yahel-Niv and Zlotkin 1979). Studies of venom
regeneration in other scorpions have shown that prevenom toxins regenerate
substantially faster than main venom toxins (Nisani and Hayes 2011). Previous work
investigating regenerated venom profiles in L. waigiensis indicates that fractions 1
and 2 rapidly replenish only 1 day after milking, compared to 21 days for fractions 311, and are therefore likely to constitute the prevenom (Schneider 2011). Our results

53

suggest that both fractions 1 and 2 increased in response to the predation treatment,
in addition to fractions 7, 8, and 9, while fractions 4, 5, and 10 decreased. Thus, it
would appear that L. waigiensis reduces its relative investment in particular main
venom components in order to increase the relative production of both prevenom and
defensive main venom toxins in response to a predatory pressure. Prevenom in the
dark scorpion P. transvaalicus was found to be chemically cheaper to produce,
containing K+ ions and simpler, shorter peptide chains compared to the larger
neurotoxins in the main venom (Inceoglu et al. 2003; Nisani et al. 2012). Inceoglu et
al. (2003) concluded that prevenom is likely to be used as a rapidly-recovering
warning-shot to cheaply deter threats with immediate, localized pain, before
resorting to main venom, as well as acting as a less-effective back-up venom to
capture invertebrate prey when the main venom is depleted. Given its low cost and
rapid regeneration, induced investment in prevenom by L. waigiensis may be
indicative of a strategy to cope with a more uncertain, hostile environment.

Offensive venom
From our modelling results we hypothesized that the arms race between a venomous
consumer and its co-evolving prey would be enhanced. In contrast to our results for a
defence-inducing pressure, we did not observe a plastic increase in offensive venom
investment in response to our live prey treatment. Broadly speaking, the inability of
the live prey treatment to induce offensive-investment could have been due to
inadequate pressure to overcome the venom resistance of the model prey, or due to
an inability to plastically respond to prey resistance with an induced venom
investment. But given the plasticity observed from the defensive treatment, a
physiological inability to respond to any resistance pressure to venom resistance in

54

prey is unlikely. Instead, an inadequate (or entirely absent) pressure was likely
responsible, due to characteristics of both L. waigiensis and the model prey, which
could be taken into consideration for future studies.

Although theoretical studies have suggested that plastic phenotypic change should be
a good predictor of the direction of evolutionary change, empirical studies have also
shown that this is not necessarily always the case (Schaum and Collins 2014).
Furthermore, we did not model the eco-evolutionary effects of environmental
stochasticity to hypothesize the effects of increasing offensive pressure. When traits
respond to a selective pressure, evolution balances this response between optimising
the trait for the maximum fitness benefit, and over-investing in the trait to
compensate for the effect of environmental stochasticity to avoid extremely low
fitness (Rosenheim 2011). Such bet hedging strategies are ubiquitous (Beaumont et
al. 2009), and are also observed in venomous animals. For example, snakes will
overkill flying prey to ensure a meal, injecting quantities greatly in excess median
lethal doses (LD50 value) in order to guarantee that their prey cannot fly away
(Hayes 1992). Similarly, in times of prey scarcity where scavenging becomes an
alternative foraging strategy, or in the presence of an alternate prey which doesnt
require stinging to be subdued, it may be favourable to continue producing costly
venom even in the absence of live prey to ensure success in future opportunities to
catch a meal. Thus, bet hedging may have been responsible for a lack of plastic
response in venom composition due to the prey treatments.

We observed scorpions both stinging their prey and subduing the crickets with their
pincers alone. Thus, our venomous animal may have had a low preference for

55

stinging the prey used in our experiment. If this were the case then we might have
expected venom profiles from naive, freshly-captured scorpions to differ from those
after the treatments, where they were only fed crickets (and did not consistently need
to sting to subdue their prey). Our data appear consistent with this expectation. The
propensity to use venom in any given interaction is a property of both the venomous
animal and its prey (or threat), and is influenced by a wide range of factors of each
(for a review, see Morgernstern and King (2013). Previous studies have shown an
inverse relationship between pincer and stinger size in scorpions, and that scorpions
prefer to use their larger weapon in both offensive and defensive encounters
(Edmunds and Sibly 2010; van der Meijden et al. 2013). L. waigiensis has relatively
larger pincers than its metasoma and indeed, the scorpions were sometimes observed
catching and killing live crickets with their pincers alone. Although we anticipated
and attempted to mitigate this effect by feeding prey larger than the pincers, which
increases preference for envenomation to subdue prey in other scorpions (Edmunds
and Sibly 2010), it is possible that these crickets were still too small to reliably
provoke stinging (personal observation).

In sum, it remains unclear as to whether or not the lack of a plastic response in the
offensive venom of L. waigiensis was due to an inadequate pressure, due to a
preference by the scorpion to subdue its prey with pincers, or both. While the
availability of important ecological and toxicological information made L. waigiensis
a highly suitable model organism for our study, future work could better elucidate
the relationship between prey-capture and offensive venom by using a model
venomous animal which more readily relies upon venom to subdue prey, and a more
resistant model prey (such as cockroaches, e.g. Hostettler & Nentwig 2006) or even

56

natural prey. A longer study may be able to observe a delayed plastic response, and
the collection of milked venom volume data would enable the detection of any
changes in absolute volumes, whereby a decrease in absolute venom volumes would
indicate a plastic response to mitigate the costs of venom production.

Implications of plasticity
The capacity for plasticity in a given trait is itself a trait that is selected for, and
presents interesting ecological and evolutionary implications. Plasticity is favourable
in highly variable or cyclic environments where the fitness benefits of the phenotypic
flexibility it bestows outweigh the cost of maintaining this capacity for variability
(Svanbck et al. 2009). Plasticity would therefore enable a venomous animal to
minimize the production of costly venom between fluctuations in predator densities
or alternate prey with variable venom resistance. By comparing the lower-resolution
venom profile of L. waigiensis obtained from FPLC (Figure 3) to the higherresolution venom profile obtained from HPLC (Figure 2) it is clear that only a subset
of the peptides present in the venom have been increased or decreased in response to
a predatory pressure. Venomous animals evolve vast, complex armouries of peptides
and proteins in their venoms (Morgenstern and King 2013), and it would appear that
from its armoury of at least 40 different peptides L. waigiensis is able to modify the
production of small subset of these to suit a changing environment. Such a costeffective strategy would be consistent with the venom optimisation hypothesis, and
the magnitude of the pressure to minimize venom cost and the predatory pressure
may relate to how closely venom production tracks the rate of ecological dynamics.
Indeed, we found that the difference in venom profiles between predatory and non-

57

predatory treatments had noticeably diminished three weeks after the conclusion of
the experiment (Appendix I).

Thus, our findings suggest that L. waigiensis experiences sufficient variability in

predator density in its natural environment to select for venom plasticity. In addition
to the benefits for economical venom production, an induced plastic defence could
stabilize venomous animal populations against fluctuating predatory pressures,
stabilizing food webs (Cortez 2011). If more widespread in other venomous animals,
this could present a potent stabilizing force in ecological communities by
diminishing trophic cascades. Confronted with additional environmental pressures,
such as climate change and invasive species, adaptive plasticity can mitigate the
effects of these strong selective pressures on populations of venomous animals by
allowing more time for evolutionary adaptation and reducing the amount of
evolutionary change necessary to track a moving optimal trade-off between the costs
and benefits of venom production (Reed et al. 2011). Indeed, populations which
exhibit greater phenotypic plasticity can evolve more under global change and
thereby adapt to changing environments (Schaum and Collins 2014).

Applications and future directions

Venom research has historically been intently focused on human toxicity (Fry et al.
2009). The ecological and evolutionary perspectives that have been increasingly
explored, particularly in the last decade, offer critical insights that has improved
health outcomes as well as enriched our understanding of venom-use and production.
Indeed, if plastic responses are widespread in the other venomous animals,
antivenom production could be improved by accounting for this potential source of

58

variation (Richards et al. 2012). The potential role of venom in structuring and
stabilising ecological dynamics needs to be further explored and may be substantial.
For example, scorpions provide a major food resource in some ecological
communities (Polis 1990). Corroborating previous work (Casewell et al. 2013), our
modelling suggests that venomous animals and their predators and prey may provide
new insights into evolutionary arms races arising from both reciprocal co-evolution
and predator-driven escalation (Dietl and Kelley 2002). Finally, the evidence that
weve found for rapid trait change and adaptive plasticity in venomous animals may
present new opportunities for the empirical study of eco-evolutionary dynamics
using venomous animals as a model system.

59

3.

Target journal: American Naturalist

60

Abstract

Though pairwise co-evolution has received more attention due to

analytical simplicity, organisms embedded within multi-trophic food webs need to

balance two general arms races, between their predators and their prey. Here, we use
a simple co-evolutionary model to evaluate the evolutionary response of a venomous
consumer co-evolution of its prey and the presence of an apex generalist predator.
We assume that consumers can develop venoms for prey capture and predatordeterrence that can functionally overlap. Predator introduction promotes investment
in either defensive or offensive consumer venoms, or both. The investment type
depends on the effectiveness of offensive venom for predator-deterrence, and the
relative cost associated with investing in multiple venoms. Furthermore, the
evolutionary response of prey species to apex-predator-induced reductions in
consumer densities can promote evolution of a novel consumer anti-predator venom.
These dynamics suggest that interactions with other species can substantially alter
the venom complexity in predatory venomous animals.

Introduction
Balancing consumer-prey and consumer-predator arms races
Arms races between predatory and anti-predatory traits are likely to emerge when an
organism has an enemy that is capable of an evolutionary response (Dercole et al.
2010). Indeed, arms races may be of sufficiently widespread importance to shape the
evolution of consumer-prey interactions along all phylogenetic branches of the tree
of life (Dietl and Kelley 2002). Although consumer species are typically embedded
within food webs, there has been an historical emphasis on pairwise interactions in
theoretical and empirical studies of evolutionary arms races (Mougi and Nishimura
2009). Pairwise models offer the convenience of analytical simplicity (Mougi and

61

Nishimura 2009). However, paleontological and phylogenetic analyses have

accumulated evidence for the important role of three trophic-level interactions in coevolution (Currie et al. 2003; Dietl and Kelley 2002). Furthermore, the small but
growing body of studies of tri-trophic systems have shown that extending beyond
pairwise interactions can reveal novel processes and dynamics which are often not
intuitive (Abrams 1991; Dercole et al. 2010). Thus, there is a need for further
investigations of the evolutionary processes that govern arms races in a multi-trophic
level context (Dietl and Kelley 2002).

Arms races in venomous animals

Arms races involving predatory and anti-predator traits are ubiquitous in food webs
(Wade 2007), and are seen in venomous predator-prey systems (Casewell et al.
2013). Although studies have typically focused upon venom production as an
offensive adaptation for prey capture, venomous consumers may also co-evolve with
their predators (Casewell et al. 2013; Jansa and Voss 2011), and therefore need to
balance the costs and benefits of these offensive and defensive arms races.
Scorpions, for example, are prey for many species (Castilla et al. 2014; Polis 1991),
and may provide an important food prey in some habitats (van der Meijden et al.
2013). Indeed, venom provides venomous animals with one of the basic requirements
for the emergence of co-evolutionary arms races an effective defensive or offensive
weapon, which incurs a potentially steep allocation cost and therefore requires fitness
tradeoffs to produce (Dietl and Kelley 2002; McCue 2006; Nisani et al. 2012; Nisani
et al. 2007; Pintor et al. 2010). How consumers, including venomous consumers,
balance the costs and benefits associated with predatory and anti-predator traits in

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these arms races can be better understood by examining arms races in other predatorprey systems and by using theoretical modelling (Abrams 2000).

Offensive and defensive venoms and statement of aims

Venomous animals must balance arms races between predators and prey with toxins
that exhibit toxicity towards both predators and prey. Some traits which aid preycapture, such as crab claws, can also be used for defence (Abrams 1991), and the
same is true for venom toxins. Because some venom toxins act upon biochemical
processes which are highly conserved across all of Anamalia (Fry et al. 2009), the
same toxins can function both in offensive and defensive contexts. For example,
there are three sub-groups of scorpion -toxins. One exhibits activity towards insect
voltage-gated sodium channels (VGSCs) only, another towards mammalian VGSCs
only, and the third contains toxins that are active against both insects and mammals
(Quintero-Hernndez et al. 2013). Though most are considered to be generalist
predators, spiders can produce a suite of both general toxins and phyla-specific
toxins to predate upon specific groups of insects (Kuhn-Nentwig et al. 2011;
Nicholson 2007). Conversely, the evolution of discretely different offensive and
defensive venoms has been recently shown in Conus (Dutertre et al. 2014).
Scorpions secrete a chemically cheap prevenom for use in low-threat agonistic
interactions before resorting to the more chemically complex and costly main venom
(Inceoglu et al. 2003). Thus, venomous animals appear to vary in the extent to which
they can function with either two distinctly different offensive and defensive
venoms, or with a more functionally versatile single venom that can be used in both
prey-capture and predatory contexts.

63

Given the likely importance of arms races in venom evolution and the paucity of
studies addressing the potential need for venomous animals to balance prey and
predator arms races, we sought to investigate the effects of co-evolution on venom
production in a venomous consumer embedded in a tri-trophic chain. We use
theoretical models to evaluate how venomous animals may balance these two general
arms races with their predators and prey and to determine the circumstances under
which it would be preferable to evolve a separate defensive venom, rather than invest
more heavily in the venom used for prey capture.

Model Description
We use a trophic-level, discrete-time Lotka-Volterra model of co-evolutionary trait
change to investigate the evolution of two traits offensive venom and defensive
venom in a venomous consumer. The consumer feeds on a co-evolving prey, and is
itself preyed-upon by a generalist predator. Our model is an extension of the two
trophic-level model described by Northfield and Ives (2013). We re-parameterised
the model to suit venomous animals. Here, the venomous consumer has two traits
that correspond with two types of venom: offensive and defensive venom. Predation
of the prey by the consumer increases with the value of the offensive trait, whereas
predation of the consumer by the apex predator decreases with the value of the
defensive trait. Investing in each trait is associated with a cost administered to
consumer mortality.

We also include an additional fitness cost to produce two venoms, g3. This cost may
represent the additional cost associated with maintaining the physiological machinery
associated with producing two venom types, or super-additive metabolic costs

64

associated with producing two venom types. There have been relatively few studies
of venom costs (Morgenstern and King 2013). Investment in both offensive and
defensive venoms may be particularly costly due to the need to maintain specialised
structures and tissues to separately store or secrete two separate venoms, as in Conus
(Dutertre et al. 2014). We therefore considered the effects of including and excluding
g3 in our simulations. Venom toxins can exhibit general toxicity by attacking
biochemical targets that are conserved across Metazoans, or may be more specialized
(Dutertre et al. 2014). A prey-capture venom may also exhibit toxicity towards
predators. Furthermore, venom is typically considered to be driven by selection
pressures for offense (Casewell et al. 2013). Therefore we also include a measure of
the effectiveness of the prey-capture venom in deterring predators, M. We assume
the value of the parameter measuring this cross-effectiveness can range from 0 and 1,
with 0 representing no effectiveness of prey-capture venom at deterring predators,
and 1 representing the case where the offensive venom has equal effectiveness
against both prey and predators.

The changes in population densities of the prey (Rt), venomous consumer (Ct), and
predator (Pt) at time t are given by (Equations 3.1):

Rt

Rt erR ( r0 ,vR )

Rt q1 ( v1 ,vR )Ct

Ct

Ct ecq1 (v1 ,vR ) Rt

Pt

Pt erP ( P )

m ( v1 ,v2 ) q2 ( v1 ,v2 ) Pt

c P q 2 ( v1 , v 2 ) Ct m P

In the model we assume that consumer fitness is balanced by predation on the prey
species (where higher prey-capture venom investment increases predation rate),
density-dependent mortality due to predation by the predator (where higher anti-

65

predator venom reduces predation rates, as does higher prey-capture venom if M >
0), and mortality due to other density-independent effects (which increases with
investment in each venom). The prey species may mitigate mortality due to
consumer predation by investing in venom resistance (modelled as venom
susceptibility, where lower susceptibility provides higher venom resistance), which
is balanced by a cost (modelled as density-independent mortality). Because we
modelled the predator as a generalist predator, its growth does not entirely depend on
preying on the consumer species. Rather, it has an alternative growth rate and
carrying capacity, and since the predators fitness is not tightly linked with the
defensive venom of the consumer, we assume that it does not co-evolve with the
consumer. The parameters and general functions used in the population density
expressions are presented in Table 1.

Table 1: Description of parameters and functions used in model equations describing change in
population densities for a three trophic-level system involving a prey, a venomous consumer, and a
predator.

Parameter
Rt
Ct
Pt
vR
v1
v2
vP1

vP2

Description
Prey species population density at time t.
Consumer species population density at time t.
Predator species population density at time t.
Prey susceptibility to the prey-capture venom of the venomous
consumer (higher values results in higher predation rate).
Prey-capture trait (venom) of the venomous consumer (higher values
results in higher predation rate).
Anti-predator trait (venom) of the venomous consumer (higher values
results in higher predation rate by top predator).
Predator susceptibility to the anti-predator venom of the venomous
consumer (higher values results in higher predation rate of the
consumer).
Predator susceptibility to the prey-capture venom of the venomous
consumer (higher values results in higher predation rate of the
consumer, if M > 0).
Effectiveness of prey-capture venom for defence (ranges from 0 to 1,
with 0 representing no effectiveness of prey-capture venom at
deterring predators).

66

r0
f

Prey intrinsic rate of increase.

Fitness cost of venom susceptibility in the prey species (investing in
lower susceptibility to venom is more costly).
m0
Venomous consumer base mortality rate (due to density-independent
effects).
g1
Fitness cost of prey-capture venom in venomous consumer (investing
more in offensive venom is more costly).
g2
Fitness cost of anti-predator venom in venomous consumer (investing
more in offensive venom is more costly).
g3
Fitness cost of investing in two venoms in venomous consumer
(investing more in both venoms is more costly).
rP0
Predator intrinsic rate of increase.
k
Carrying capacity of the predator population.
mP,0
Predator base mortality rate (due to density-independent effects).
gP1
Fitness cost of venom susceptibility in the predator species to the
anti-predator venom (investing in lower susceptibility to venom is
more costly).
gP2
Fitness cost of venom susceptibility in the predator species to the
prey-capture venom (investing in lower susceptibility to venom is
more costly).
c
Base conversion rate of eaten prey into new venomous consumers.
cP
Base conversion rate of eaten venomous consumers into new
predators.
a1
Baseline attack rate of the venomous consumer, quantifying
searching efficiency for prey.
a2
Baseline attack rate of the predator, quantifying searching efficiency
for venomous consumers.
Function
Description
f
Population growth of the prey species. Large values are
rR(r0,vR) = r0 vR
given by high intrinsic rates of increase, high
susceptibility to venom, and small fitness cost of venom
susceptibility.
v1v R
Predation rate of the prey species by the venomous
)
q1(v1, vR) = a1 (1 e
consumer. Large values are given when prey-capture
venom investment, prey susceptibility, and baseline
consumer attack rate are higher.
m(v1, v2) =
Density-independent consumer mortality. Increases with
base mortality rate, cost of prey-capture, anti-predator,
m0 g1v1 g 2 v 2 g 3 v1v 2
and maintaining both venoms, and investment in preycapture and anti-predator venoms.
v 2 v P 1 Mv1v P 2
Predation rate of the consumer species by the predator.
q2(v2) = a 2 Pe
Decreases when anti-predator and prey-capture (if M >
0) venom investment is higher, and predator
susceptibility and baseline predator attack rate are
lower.
Population growth of the predator species from
rP(P) = rP 0 (1 kPt )
alternative food sources. Higher values are driven by
higher predator intrinsic rate of increase and carrying
capacity.

67

g P1
vP 1

mP = mP , 0

gP 2
vP 2

Density independent predator mortality. Increases with

predator base mortality rate and susceptibility to venom,
and decreases with the increased cost of venom
susceptibility.

The changes in traits for the prey (i.e., prey susceptibility) and consumer (i.e.,
offensive and defensive venom investment) at each time step are determined by the
populations genetic variance and mean fitness, as well as the derivative of mean
fitness with respect to the trait. If Fi is the mean fitness for species i, and VR, V1 and
V2 are additive genetic variance for prey susceptibility vR, consumer offensive venom
v1, and consumer defensive venom v2, respectively, then co-evolution of these traits
is given by the following derivatives (Equations 3.2):

vRt

vRt

1 FR
VR
FR vR

v1t 1

v1t

1 FC
V1
FC v1t

v2t 1

v2t

1 FC
V2
FC v2t

vRt

v1t

v2t

f
vR

v1a1Ct e

(vRt ca1Rt e

( g2

v1vR

v1v R

)VR

g1 (1 g3v2 ) MvP 2 q2 Pt )V1

g3v1 vP q2 Pt )V2

For our simulations predator venom susceptibility was considered a constant that
does not co-evolve. We made additional assumptions in our modelling. Firstly,
although spatial structure may influence predator-prey interactions and give rise to a
geographic mosaic of co-evolution, we assumed that each species was represented by
a single, panmictic population to focus on local adaptation. We used a quantitative
genetics approach and held the additive genetic variance of the traits constant, so that
the rate of evolution depended on the strength of selection. Although this assumption
doesnt hold true in the long term (without mutations to maintain genetic variation),
under very strong selection (resulting in loss of genetic variation, and for small
populations (which lack large initial genetic variation and are affected by genetic

68

drift), we believe this approach provides a reasonable starting point to investigate the
short-term (hundreds of generations) effects of changing ecological interactions
(Abrams 2001).

Analysis and Results

To understand the effects of co-evolution and the introduction of a third trophic level
on venom production, we performed simulations of an ecological invasion. Each of
these simulations began with a pairwise venomous consumer-prey system which was
able to reach eco-evolutionary equilibrium before the introduction of the generalist
predator after 1000 time steps. We performed two general types of simulations, and
set parameters to ensure non-zero equilibriums in both traits and densities. In the first
type, both the venomous consumer and prey species were assumed to have large
additive genetic variances (Vi = 1, for trait i) following the invasion of the predator,
promoting a high rate of evolution and thereby enabling the co-evolutionary arms
race to respond to this predatory pressure. In order to better understand the effects of
prey evolution on venom production in the consumer species, in the second
simulation the prey was assumed to have lost all additive genetic variance after
reaching equilibrium, but before predator introduction (at t = 1000). This second
scenario represents a stark contrast to the co-evolutionary scenario to better
understand the effects of evolution in the prey species on the final equilibrium
densities and consumer traits. We tracked the changes in population densities and
trait values as the simulation progressed and they reached equilibrium. To investigate
the effects of co-evolution and the invasion on the evolution of a novel antipredator venom, in both types of simulation we also investigated the effect of
additional costs associated with maintaining two venoms (g3 = 0 or g3 = 0.01) and the

69

effectiveness of the prey-capture venom as a defence against the predator (M = 0 vs.

M = 0.7). We ran these simulations with a range of starting population density and
trait values to evaluate the potential for alternative stable states.

Where the prey species can always evolve, elevated mortality due to the invasion of a
predator results in a decline in the consumer density (Figure 1A). Predatory pressure
on the prey species is thereby reduced, and allows the prey species to become more
susceptible to the consumer venom (Figure 1C). The arms race between consumer
and prey becomes diminished, enabling the consumer to divert investment to its antipredator trait (Figure 1C). The reduced arms-race cost and reduced consumer
pressure together increase prey densities (Figure 1A), and allow the consumer
species to invest less in offensive venom (Figure 1C).

In the absence of prey evolution after apex predator invasion, increased predation of
the consumer species does not stimulate reduced arms race investment by the prey
species (Figure 1D), unless predator carrying capacities are relatively high (data not
shown). There is no negative feedback loop mediated by prey trait change to reduce
the strength of the trophic cascade and the declines in consumer density are greater
than in the prey evolution case (Figure 1B). Because low venom susceptibility is
maintained in the prey species, the consumer may no longer decrease prey-capture
investment without compromising growth (Figure 1D). Thus, even when the
multiplicative cost of investing in two traits is low, and there is little overlap between
the functions of the prey-capture and anti-predator traits, the consumer does not
divert prey-capture to investing in a novel anti-predation trait for relatively low
predator carrying capacities. Instead, the predatory pressure induces a greater

70

investment in the prey-capture trait, taking advantage of its defensive properties even
when its effectiveness compared to the dedicated anti-predator trait is low (Figure
1D).

Figure 1: Eco-evolutionary effects of an invading top predator (at time = 1000) on the densities (A,B)
and traits (C,D) for a three-trophic-level food web where the prey species either can (A,C) or cannot
(B,D) co-evolve. Values for the prey, consumer, and predator species are shown in blue, black, and
red, respectively. Prey consumption increases with prey trait values (solid blue line) and consumer
traits associated with prey-capture (i.e., offensive venom investment; solid red line). Predator attack
rates decrease greatly with consumer anti-predator traits (i.e., offensive, and defensive venom
investment; dashed red line), and to a lesser extent the prey-capture trait. Addition of a predator
pressure when prey co-evolution becomes restricted gives rise to an overkill effect (D). The
parameters rP = 0.01, vR = 0.4, v1 = 0.3, v2 = 0.1, vP1 = 0.3, vP2 = 0.1, m0 = 0.05, mP0 = 0.01, a1 = 4, a2 =

71

3, c0 = cP = 0.25, f = 0.07, g1 = g2 = 0.02, g3 = 0, M = 0 gP1 = gP2 = 0.001, V1 = V2 = 1 were used for all
simulations. For the prey co-evolution scenario, VR = 1. For the inhibited co-evolution scenario, VR =
0.

Further evaluation of the no prey-evolution case suggests that investment in antipredator venom in response to the invasion only occurs under co-evolution, or when
predator carrying capacity is sufficiently large (Appendix II). In the case where the
prey species can evolve we find that evolution of a second, defensive venom is
discouraged by a high fitness cost g3 associated with producing multiple venoms and
the efficacy, and to a lesser degree by a low effectiveness of the prey-capture trait on
reducing attacks by the top predator (Figure 2).

Figure 2: Eco-evolutionary effects of an invading top predator (at time = 1000) on the densities (top
row) and traits (bottom row) for a three-trophic-level food web, where the prey species can always coevolve. Values for the prey, consumer, and predator species are shown in blue, black, and red,
respectively. Here we show the effect when g3 is set high (0.01, C,G,D,H) and low (0, columns
A,E,B,F), and M is also set high (0.7, columns B,F,D,H) and low (0, columns A,E,C,G). Investment in
the anti-predator venom is most strongly determined by the magnitude of g3, and is greater when g3 is
lower. Investment is affected to a lesser extent by M, to which it is also inversely proportional. The
outcome of the arms race between the venomous consumer and its prey is also affected by the values

72

of g3 and M. Low g3 and low M both result in higher prey-capture investment and higher susceptibility
in the prey species. The parameters rP = 0.01, vR = 0.4, v1 = 0.3, v2 = 0.1, vP1 = 0.3, vP2 = 0.1, m0 = 0.05,
mP0 = 0.01, a1 = 4, a2 = 3, c0 = cP = 0.25, f = 0.07, g1 = g2 = 0.02, gP1 = gP2 = 0.001, V1 = V2 = VR = 1
were used for all simulations.

Discussion
Co-evolution with prey species may facilitate anti-predator venoms
Our modelling suggests that a co-evolutionary interaction between a venomous
consumer with its prey can affect the consumers evolutionary response to a
generalised apex predator. Because it enables the trade-off between trait investments
via a reciprocal disarmament, a co-evolutionary arms race with its prey species can
facilitate the evolution of a novel anti-predator venom in a venomous consumer. This
finding brings new insight to an interesting paradox of the use of venoms in
defensive contexts. Currently, our understanding of the evolution and use of venoms
in defensive contexts is limited, and it is generally held that venoms are primarily an
offensive adaptation (Casewell et al. 2013). The life-dinner principle, proposed by
Dawkins and Krebs (1979), points out the inherent asymmetry of predator-prey
interactions. If a predator fails in a given interaction, it only loses a meal, but failure
by the prey results in death or injury. Given the life-dinner principle, it may seem
more intuitive that chemical warfare should be a more valuable mode of defence for
venomous animals to preserve their own lives, rather than as a weapon for preycapture. To some extent, behavioural responses may explain this apparent
discrepancy (van der Meijden et al. 2013).

There is a range of evidence suggesting that venom may be more important for
defence than previously thought. Recent identification of separate offensive and

73

defensive venoms in cone snails has suggested that venoms arising to deter predators
are more common than previously thought (Dutertre et al. (2014). Our findings
suggest that diets involving arms races with prey species are more likely to facilitate
the evolution of such defensive venoms, even at relatively low predator densities.
The use and evolution of defensive venom has been documented in other trophically
venomous taxa, including scorpion pre-venom and spitting cobras (Hayes et al. 2008;
Inceoglu et al. 2003). Furthermore, histological evidence suggests that a wider range
of venomous taxa may possess the necessary tissues for regionalized toxin
production and the deployment of separate offensive and defensive venoms (Moran
et al. 2013; Morgenstern and King 2013). In light of all this, and the putative
ubiquity of prey co-evolution in venomous animal systems, our findings may support
recent calls to reconsider the traditional view that defence is generally not a
significant selection-pressure or function of venoms (Dutertre et al. 2014).

Restricted co-evolution may give rise to overkill

Our results suggest that the ability to use prey-capture venom to deter predators,
paired limited co-evolution in prey, can lead to elevated investment in that venom in
response to increased predator exposure. In order to maximize the efficacy of the
prey-capture venom in defensive contexts, investment can elevate beyond that
necessary for prey capture. Consequently, when the pairwise consumer-prey
interaction is viewed in isolation of the predator, the decoupled arms race may
falsely suggest maladaptation by the venomous consumer due to overinvestment in
venom potency relative to its prey, reminiscent of the overkill hypothesis (Mebs
2001).

74

The view that adaptive venom composition, under selection for diet, gives rise to coevolutionary arms races between venomous consumers and their prey has not been
universally accepted (Casewell et al. 2013; Mebs 2001; Sasa 1999). The overkill
hypothesis instead proposes that neutral evolutionary processes are responsible for
the wide variability observed in the chemical composition, injection volumes, and
potency of snake venoms (Mebs 2001). A wealth of contrary evidence supporting a
strong selective pressure for diet has since emerged in the literature (Barlow et al.
2009; Gibbs and Mackessy 2009; Pawlak et al. 2009; Starkov et al. 2007). The
concept has been formalized by the venom optimisation hypothesis, which posits that
the high cost of venom production drives a strong selective pressure for strategies to
use it efficiently (Casewell et al. 2013; Morgenstern and King 2013). There is a
growing body of evidence for venom metering in a wide range of venomous taxa
which casts doubt over the assertion that injection volumes are highly variable
(Morgenstern and King 2013). Here we suggest that predator exposure may also
influence investment in prey-capture venoms, and these multi-trophic interactions
must be considered when developing estimations for optimal venom production.

Under what circumstances might co-evolution become restricted, potentially giving

rise to the overkill-like scenario? Co-evolution can become constrained due to
tradeoffs with other competing selection pressures (Dietl and Kelley 2002). Coevolution may also be limited or even entirely absent when the prey species is a
relatively rare species in a diverse community, or if the consumer feeds on a wide
range of prey species. According to the escalation hypothesis, these asymmetries in
predator-prey interactions can give rise to an escalation in the anti-predator traits of
prey species without a reciprocal elevation of prey-capture traits in the consumer,

75

which is characteristic of co-evolution (Vermeij 1994). Escalation may therefore be

a more common process in venomous consumer-prey interactions in venomous
animals that are dietary generalists. Thus, co-evolution may be limited or even absent
in generalist venomous consumers, satisfying what our results suggest to be the key
condition for the emergence of the overkill pattern in venom evolution.

Conclusion and future directions

In general, our investigation has shown that prey co-evolution has a marked effect on
the evolutionary response in the venom of a venomous consumer to the invasion of a
generalist apex predator. Abrams (1991) modelled a similar system, in which a
specialist (but non-co-evolving) predator was introduced to a co-evolutionary
consumer-prey arms race. When he assumed a negative correlation of prey-capture
and anti-predator traits in the consumer (as we did in our modelling using fitness
costs, such that investing in one trait requires a trade-off in the other) he also found
that consumer response to prey was diminished (Abrams 1991). Our results also
support earlier work proposing that additional interacting species will reduce the coevolutionary response in the members of an arms race (Vermeij 1982). Indeed,
empirical studies of tri-trophic chains in plant-herbivore systems have shown that
anti-herbivore defences are stronger in patches where there is less predation on
herbivores (Nersesian et al. 2011). The extent of a reciprocal disarmament between
venomous consumer and its prey species is strongest when the costs of possessing
multiple venoms are high and there is high functional overlap between these two
traits (i.e. when the prey-capture venom is effective in defensive contexts).

76

Venomous consumers offer fascinating opportunities as model systems for the study
of arms races, escalation, and co-evolution. For example, pit vipers and opossums
may be engaged in a co-evolutionary arms race involving a unique, biochemicallymediated process of role-switching between consumer and prey (Voss 2013). In our
investigation of the effects of predator-venomous consumer-prey interactions in a tritrophic chain we did not include evolution of the top predator, which can further
complicate the co-evolutionary arms races (e.g. giving rise to chaotic dynamics
Dercole et al. 2010). Nonetheless, our results suggest that considering venom
evolution in three-trophic levels can produce novel insights not readily apparent from
pairwise interactions. In sum, our study illustrates the importance of establishing a
theoretical framework for venom evolution, and that novel and unintuitive insights
can emerge by investigating more realistic, multi-trophic models of natural predator
prey systems.

77

4.

General discussion

In sum, from the induced defence observed in response to manipulated predator

interactions I found evidence for adaptive plasticity in venom composition for the
first time, to my knowledge. Additionally, I have made a number of findings using
theoretical modelling of arms races in venomous predator-prey systems. While an
offensive pressure enhances the arms race between a venomous animals and its prey,
a predatory pressure diminishes the arms race between a venomous animal and its
co-evolving prey and instead prompts investment in defensive venom. I have found
that co-evolution may enable the evolution of a novel defensive venom in response to
predatory pressures, and the absence of co-evolution may give rise to an overkill
effect in the presence of a defensive pressure.

There have been calls for a renewed interest for more research to understand the
evolution and ecology of defensive venoms (Casewell et al. 2013; Dutertre et al.
2014). My findings present several new insights into the importance of defensive
venoms and defensive pressures in venom evolution. The potential role for prey coevolution in facilitating the evolution of a separate defensive venom is of particular
interest in light of the recent discovery of a separate defensive venom in cone snails
(Dutertre et al. 2014). Studies of plant-herbivore systems have suggested that
induced plastic defences are generally favoured if defences are costly and are not
always needed (Karban 2011).Thus, induced plastic defence in the composition of
venom may present a new strategy for using venom economically, and adds further
evidence corroborating the venom optimisation hypothesis. The potential to provide
a vast armoury of toxins to plastically cope with different environmental conditions

78

may help to reconcile the seemingly lavish complexity of venoms can be reconciled
with the optimisation hypothesis (Morgenstern and King 2013).

Similarly, the likely importance of arms races in venom ecology and evolution has
been recognized, but studies of arms races in venomous animals are lacking
(Casewell et al. 2013). Furthermore, studies of predator-prey arms races in general
have traditionally focused on pairwise interactions (Mougi and Nishimura 2009), as
has the relatively meagre evidence for arms races in venomous animals (Casewell et
al. 2013). But multi-trophic models may be more representative of natural systems,
particularly three trophic-level models (Abrams 1991; Abrams 2000; Dercole et al.
2010). My results support the view that evaluating species-interactions in a three
trophic-level context can provide valuable new insights that are invisible to pairwise
studies. The accumulated, if limited, evidence for arms races in venomous animals
suggests that venomous animal systems may provide fertile ground as excellent
models for general studies of arms races in species interactions. For example, pit
vipers and opossums may be engaged in an entirely novel co-evolutionary arms race
involving a biochemically-mediated process of role-switching between predator and
prey (Voss 2013). Thus, further investigation of arms races in venomous animals is
essential.

There is also a growing acknowledgement of the need to investigate the interactions

between venomous animals in the broader context of their ecological communities
(Casewell et al. 2013), and of the potential role of venom in the structuring of food
webs and ecological communities (van der Meijden et al. 2013). Induced plastic
defences and co-evolution can stabilize predator-prey interactions and food webs

79

(Cortez 2011; Northfield and Ives 2013). Relationships between geographic variation
in venom composition and diet are becoming well established, but future studies
should consider how venom properties may also vary with respect to different
predators, different densities of predators and prey, as well as density-neutral
disturbances that can alter densities. Finally, a better understanding of the precise
costs of venom production is also essential, in order to better inform and improve the
modelling of fitness costs in future theoretical studies of venomous animal systems,
which in turn can guide and inform empirical work in this rapidly evolving and
exciting field of research.

80

5.

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6.

Appendix I additional material relevant to

Chapter 2

Normalised milliabsorbance (280 nm)

(n=14)
(n=15)
(n=15)
(n=15)

Elution volume (mL)

elution time

Figure 1: Comparison of venom profiles obtained prior to experimental treatments. Patterns of

difference between venom profiles were not statistically significant and arose from conditions in the
wild. Because these represent the averaged, normalized venom profiles for each treatment, the
response variable is normalized milliabsorbance units, and indicates relative quantity between any
given portion of the mean of the venom profiles of each treatment. Defensive treatments are indicated
in dark and light blue, while the non-defensive treatments are shown in orange and red.

90

Normalised milliabsorbance (280 nm)

(n=14)
(n=13)
(n=11)
(n=14)

Elution volume (mL)

elution time

Figure 2: Comparison of venom profiles obtained 21 days after the conclusion of the experiment, in
the absence of treatment pressures. Patterns of change between predator and without-predator
treatments in venom profiles obtained immediately after the conclusion of treatments were observed,
but were diminished and statistically insignificant. This lingering effect of the predatory pressure is
suggestive of an induced, plastic defence. Because these represent the averaged, normalized venom
profiles for each treatment, the response variable is normalized milliabsorbance units, and indicates
relative quantity between any given portion of the mean of the venom profiles of each treatment.
Defensive treatments are indicated in dark and light blue, while the non-defensive treatments are
shown in orange and red.

91

3 4

10

11

3e-04
0e+00

1e-04

2e-04

Absorbance

4e-04

5e-04

6e-04

10

15

20

25

30

35

40

45

Volume

Figure 3: We delimitated fractions by combining the normalized chromatograms of all individuals

into a single mean chromatogram, then fitted a spline curve ( = 0.5) and obtained 11 fractions by
separating at local minima. The correspinding elution volumes (mL) delimiting the fractions (from
smallest to highest) were as follows: 10.48, 13.75, 16.72, 17.93, 19.63, 22.70, 24.02, 28.73, 31.02,
34.66, 40.61, 44.10.

92

Table 1: MANOVA results, which indicated there were no statistically significent differences
between fraction loadings of scorpions allocated to each treatment prior to actually having been
subjected to those treatments (obtained from the first venom milking).

3.

Source
4.
Prey
Predator
Predator Prey
Residuals

d.f.

Pillai

approx F

Df den

1
1
1
55

0.017
0.033
0.006

0.483
0.927
0.165

2,54
2,54
2,54

0.619
0.402
0.849

Table 2: ANOVA results, which demonstrated significantly different fraction loadings between the
predator treatments compared to the without-predator treatments along both PC1 and PC2 for
scorpions prior to experimental treatments (obtained from the first venom milking). There were no
interaction effects.

Source

d.f.

MSE

PC 1
Prey
Predator
Predator Prey
Residuals

1
1
1
55

0.006
0.015
0.001
0.009

0.651
1.711
0.110

0.423
0.196
0.742

PC 2
Prey
Predator
Predator Prey
Residuals

1
1
1
55

0.002
0.001
0.001
0.005

0.334
0.175
0.225

0.565
0.678
0.637

Table 3: MANOVA results, which indicate dthere were no statistically significent differences
between fraction loadings of treatments in the venom obtained 21 days after the cecession of
experimental treatments (obtained from the final venom milking).

5.

Source
6.
Prey
Predator
Predator Prey
Residuals

d.f.

Pillai

approx F

Df den

1
1
1
49

0.042
0.025
0.012

1.076
0.619
0.299

2,48
2,48
2,48

0.349
0.543
0.743

93

Table 4: ANOVA results, which demonstrated significantly different fraction loadings between the
predator treatments compared to the without-predator treatments along both PC1 and PC2 for
scorpions milked 21 days after treatments ceased (obtained from the final venom milking). There were
no interaction effects.

Source

d.f.

MSE

PC 1
Prey
Predator
Predator Prey
Residuals

1
1
1
49

0.002
0.001
0.004
0.007

0.285
0.098
0.591

0.596
0.756
0.446

PC 2
Prey
Predator
Predator Prey
Residuals

1
1
1
49

0.008
0.005
0.000
0.004

1.899
1.170
0.018

0.174
0.285
0.895

94

6.

Appendix II additional material relevant to

Chapter 3

Figure 1: Under high predatory pressure (k=0.02 instead of 0.01, other parameters as per Figure 1), a
specialized defensive venom may evolve in the absence of co-evolution when both are true: the preycapture venom has no effect in defensive contexts (M=0), and there is no cost of maintaining two
venoms (g3=0).

95

Figure 2: High/low effectiveness of the prey-capture venom for defence, and high/low cost of
maintaining two venoms have a minimal effect on the population dynamics trait evolution in the
absence of co-evolution (Parameters as per Figure 2). Values for the prey, consumer, and predator
species are shown in blue, black, and red, respectively. Here we show the effect when g3 is set high
(0.01, C,G,D,H) and low (0, columns A,E,B,F), and M is also set high (0.7, columns B,F,D,H) and
low (0, columns A,E,C,G).

96