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2017

The context-dependency of predator-prey and competitive interactions between the invasive eastern mosquitofish, Gambusia holbrooki, and native Australian freshwater fauna

Laura Kate Lopez UnivFollowersity this of and Wollongong additional works at: https://ro.uow.edu.au/theses1

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Recommended Citation Lopez, Laura Kate, The context-dependency of predator-prey and competitive interactions between the invasive eastern mosquitofish, Gambusia holbrooki, and native Australian freshwater fauna, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2017. https://ro.uow.edu.au/ theses1/106

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School of Biological Sciences

The context-dependency of predator-prey and competitive interactions between the invasive eastern mosquitofish, Gambusia holbrooki, and native Australian freshwater

fauna

Laura Kate Lopez

This thesis is presented as part of the requirements for the award of the Degree of

Doctor of Philosophy of the University of Wollongong

June 2017

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iii PUBLICATIONS AND PRESENTATIONS

The following publications, presentations and workshop facilitation were completed during the course of my candidature:

Publications

Lopez L.K., Wong M.Y.L. and A.R. Davis (2016). The effects of diel cycle and the density of an invasive predator on predation risk and prey response. Animal Behaviour, 117:87-95.

Lopez, L.K., Couture P., Maher W.A., Krikowa F., Jolley D.F. and A.R. Davis. (2014).

Response of the hairy mussel Trichomya hirsuta to sediment-metal contamination in the presence of a bioturbator. Marine Pollution Bulletin, 88 (1-2): 180-187.

Conference and workshop presentations

Lopez L.K. (2016). Applications of behavioural ecology to aquatic conservation Endangered species research in Aquatic Ecosystems workshop. Wollongong, Australia.

Lopez L.K., Wong M.Y.L. and A.R. Davis (2016). Into the danger zone: temporal and density mediated effects of an invasive predator. Australasian Society for the Study of Animal

Behaviour (ASSAB).Annual Meeting, Katoomba, Australia.

Lopez L.K., Davis A.R. and M.Y.L. Wong. (2015). Hostile Waters: The effect of multiple stressors on aggression in freshwater fish. Animal Behaviour Society (ABS). Annual Meeting,

Anchorage, Alaska, U.S.A.

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Lopez L.K., Davis A.R. and M.Y.L. Wong. (2015). Antagonistic interactions between temperature and salinity mediate interference competition between invasive and native fish.

International Ethology Conference (EIC). Cairns, Australia.

Lopez L.K., Davis A.R. and M.Y.L. Wong. (2015). Negative behavioural interactions between the invasive eastern mosquitofish and Australian bass. Australian Society for Fish

Biology (ASFB). Annual meeting, Sydney, Australia.

Lopez L.K. (2015). Fish wars: the Bass strike back. University of Wollongong Postgraduate

Conference, Kioloa, Australia. (Winner Best Whole Organism Biology presentation).

Facilitated Workshops

Davis A.R., Lopez L.K. and M.Y.L. Wong. (2016). Endangered Species Research in Aquatic

Ecosystems. University of Wollongong.

v ABSTRACT

Invasive species can exert significant deleterious effects on native individuals, populations and communities. Freshwater ecosystems are especially vulnerable to invasion, due to their close association with anthropogenic activity and limited biogeographic connectivity.

Understanding the behavioural mechanisms which underlie predator-prey and competitive interactions between invasive and native species is critical when predicting the impacts of invasive species on freshwater habitats. However, due to the predominance of indirect experimental methods, these mechanisms are seldom identified. Furthermore, behavioural mechanisms, and subsequently, invasive-native species interactions may be mediated by environmental and individual-level context. Here I used both direct and indirect methods to determine whether and how predator-prey and competitive interactions between the invasive eastern mosquitofish, Gambusia holbrooki and native Australian freshwater fauna are mediated by environmental variables. Throughout, I also assessed how individual-level morphological variables, specifically size and sex, modulate behavioural interactions depending on environmental context. In the first section of this thesis, I investigated whether the consumptive and non-consumptive effects of predator-prey interactions between G. holbrooki and the glass shrimp, Paratya australiensis, are mediated by the invader’s density and the diel cycle. In the next section, I determined how the density of G. holbrooki influenced its competitive interactions with juvenile Australian bass, Macquaria novemaculeata, in the laboratory and field. Finally, I tested whether the intensity of interference competition between G. holbrooki and M. novemaculeata was dependent on a combination of temperature and salinity stress. While I found a positive relationship between

G. holbrooki density and the frequency of its direct interactions with P. australiensis and M. novemaculeata, this did not translate into reduced survivorship or growth of the native species. Temperature and salinity interacted in a non-additive manner on inter-specific

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aggression, highlighting the importance of considering multiple stressor effects. The responses of both P. australiensis and M. novemaculeata to predation and interference competition were modulated by interactions between body size and environmental context. In contrast, sex-specific aggression in G. holbrooki was shown to be dependent on its density.

These results demonstrate the importance of considering the complex interplay between environmental and individual-level variables when predicting the strength and outcome of interactions between G. holbrooki, native prey and competitors.

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ACKNOWLEDGEMENTS

There are many people to blame (ahem, thank) for helping me create this thesis (to quote

Andy Davis). First of all I thank Phil Byrne, Kris French, Sharon Robinson, Todd

Minchinton, Ben Gooden and James Wallman for giving me the opportunity to help teach their ecology courses. With the exception of 9 beautiful months in 2015 I have not have scholarship support and so I have relied a lot on teaching jobs, which have also been great fun. I also thank Tim Marchant and SMAH Research for their support and for helping me attend the ABS conference in Alaska. I am grateful to the NSW Department of Primary

Industries which supported my research with a Saltwater and Freshwater Trust Fund grant, as well as Boral Sand and Soil who provided a safe and friendly environment in which to conduct a field experiment. Many thanks to our resident fish gurus, Neil from Aquablue

Seafoods and Barry from BC aquarium, for helping me keep our fish happy and healthy.

I wish to thank the amazing volunteers and research support staff who took time out from what were undoubtedly more exciting ventures to help me clean tanks and collect fish-

Corinne de Mestre, Susan Rhind, Joy Williams, Stephen Poon, Matthew Chard, Driellie

Florencio de Melo, Thiego Hurbath da Silva, Carol Cordonis and Peter Garside. I am especially grateful to Daniel Swadling and Paul Gordon for their amazing work in the field- thanks for putting up with all the snakes and my episode of mild hypothermia!

I thank the many poor souls who have put up with me for the past few years. To my unofficial parents-in-law Jenny and Kevin King- thank you for welcoming me into your home and giving me an excuse to watch the Bachelorette. I also thank the other postgrad students (of whom there are too many to name), but particularly my sisters from other misters, Amy-Marie

Gilpin and Leesa Keough for your amazing support and friendship.

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Like any self-respecting millennial, I have adopted a semi-parasitic lifestyle and there is no way I would have been able to do this thesis without my parents, Maria and Michael. There are no words that can sufficiently express the gratitude for the financial and emotional support that you have provided. This PhD is a wonderful gift that you have given me and one day I will repay you by buying Dad a Porsche and Mum a French chateau (you will have to wait a really, really long time though). But seriously, thank you. I thank my beautiful grandmother

Josephina, your strength and attitude have always been a source of great inspiration to me. I also thank my wonderful cat Oliver for the company and cuddles, though I can do without the biting. To my fantastic and ridiculously good looking partner Tom, I thank you so much for your endless support. I truly admire your positive attitude and spirit, I am so lucky to have you in my life. I hope that I have finally proven to you that people from Dapto can read (we can also do some maths, but keep it simple, no calculus please).

Finally, thank you to the two best supervisors in the world, Andy Davis and Marian Wong. I have greatly enjoyed doing this PhD and that is really down to both of you. Thank you for providing me with so many opportunities. You have taught me everything I know- including that my spirit animal is the Chihuahua. Not only are you both amazing scientists, you’re also fantastic people and your students are so lucky to have you. I am really not articulate enough to fully express my gratitude, all I can say is thank you so, so much.

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DECLARATION

This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution to Laura Kate Lopez and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968.

The author acknowledges that copyright of published works contained within this thesis (as listed below) resides with the copyright holder(s) of those works. I also give permission for the digital version of my thesis to be made available on the web, via the University‘s digital research repository, the Library catalogue, the Australasian Digital Theses Program (ADTP) and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

This thesis contains four data chapters, each of which is written as a manuscript for publication, and is therefore intended to stand-alone. With this, some information may be redundant or repeated. While the candidate made substantial contributions to the manuscripts and is fully responsible for the work presented in this thesis, where the first person is used in the manuscripts it is used in the plural (‘we’) to reflect contributions from co-authors.

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Crowd: A witch! A witch! A witch! We've got a witch! A witch! Burn her! Burn her!

Bedemir: Quiet! There are ways of telling whether she is a witch.

Crowd: Are there? What are they?

Bedemir: Tell me, what do you do with witches?

Crowd: Burn, burn them up!

Bedemir: And what do you burn apart from witches?

Villager: Wood!

Bedemir: So, why do witches burn?

Villager: B--... 'cause they're made of wood...?

Bedemir: Good! So, how do we tell whether she is made of wood?

Villager: Build a bridge out of her.

Bedemir: But can you not also build bridges out of stone? Does wood sink in water?

Villager: It floats! It floats! Throw her into the pond!

Bedemir: What also floats in water?

Arthur: A duck.

Bedemir: Exactly! So, logically...,

Villager: If... she.. weighs the same as a duck, she's made of wood.

Bedemir: And therefore--?

Crowd: A witch!

Bedemir: We shall use my larger scales! Right, remove the supports!

- Monty Python’s The Holy Grail

The above describes a form of the scientific method which was NOT used during the development of this thesis.

iv

CONTENTS

1 General Introduction ...... 1

1.1 Biological invasions in freshwater ecosystems ...... 1

1.2 A mechanistic approach to understanding invasive species ...... 3

1.3 Who, what, when and where: the context-dependency of invasive-native species

interactions ...... 6

1.4 Context-dependent predation by invasive species...... 8

1.5 Context-dependent competition between invasive and native freshwater species 11

1.6 Multiple stressor effects ...... 14

1.7 The study species ...... 14

1.7.1 Eastern Mosquitofish, Gambusia holbrooki...... 14

1.7.2 Glass Shrimp (Paratya australiensis) ...... 17

1.7.3 Australian Bass (Macquaria novemaculeata) ...... 19

1.8 Thesis outline ...... 20

2 The effects of the diel cycle and the density of an invasive predator on predation risk and prey response ...... 22

2.1 Introduction ...... 23

2.2 Methods ...... 26

2.3 Results ...... 32

2.3.1 Consumptive effects ...... 32

2.3.2 Non-consumptive effects...... 33

2.4 Discussion ...... 38

3 Don’t be dense: the effect of density on interference competition between a highly invasive fish and its native competitor ...... 43

3.1 Introduction ...... 43

3.2 Methods ...... 47

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3.3 Results ...... 54

3.3.1 Aggression Given ...... 54

3.3.2 Submission Given ...... 59

3.4 Discussion ...... 64

4 Density dependent competition between the invasive eastern mosquitofish, Gambusia holbrooki and Australian bass, Macquaria novemaculeata ...... 70

4.1 Introduction ...... 70

4.2 Methods ...... 75

4.3 Results ...... 82

4.3.1 The survivorship and growth of Macquaria novemaculeata ...... 82

4.3.2 The survivorship and growth of Gambusia holbrooki ...... 83

4.4 Discussion ...... 87

5 Interference competition under multiple stressors: temperature and salinity mediate aggressive interactions between an invasive and native fish ...... 92

5.1 Introduction ...... 92

5.2 Methods ...... 96

5.3 Results ...... 100

5.4 Discussion ...... 105

6 General discussion ...... 110

6.1 Research framework ...... 110

6.2 The relationship between Gambusia holbrooki density, inter-specific interaction

strength and impact on native species ...... 111

6.3 The importance of intra-specific interactions ...... 113

6.4 Multiple stressor effects ...... 115

6.5 Interactions between population and individual-level variables ...... 116

6.6 Conclusion ...... 119 vi

LIST OF TABLES

Table 2.1Scaled P. australiensis size (P. australiensis carapace length/ G. holbrooki standard

length) in successful and unsuccessful predation attempts in low and high predator

density treatments. n = 12...... 33

Table 3.1 The densities of Gambusia holbrooki and Macquaria novemaculeata used in this

experiment investigating the effect of G. holbrooki density on inter- and intra-specific

interference competition, N = 10 replicates. The behaviours of focal fish (marked with *)

were recorded...... 49

Table 3.2 Ethogram of aggressive and submissive behaviours observed in Macquaria

novemaculeata and Gambusia holbrooki in this experiment...... 50

Table 4.1 Mean (± SD) water quality parameter recordings at the experimental site during the

course of the study, n = 10...... 76

Table 4.2 The densities of M. novemaculeata and G. holbrooki used in experimental

conditions of this study, n = 6...... 78

Table 5.1 Mean (± S.D.) temperature and salinity parameters used in this experiment...... 97

Table 5.2 Results of a generalised linear mixed model testing the effects of temperature and

salinity on inter-specific aggression from a) Macquaria novemaculeata and b) Gambusia

holbrooki. Non-significant terms were backward stepwise eliminated (p >0.016).

Significant terms are in bold. Pairwise post-hoc tests compared the means of control

temperature and control salinity (C), elevated temperature and control salinity (ET),

control temperature and elevated salinity (ES) and elevated temperature and elevated

salinity (ETS) treatments...... 101

Table 5.3 Results of a generalised linear mixed model testing the effects of species,

temperature and salinity on inter-specific aggression from Macquaria novemaculeata

and Gambusia holbrooki. Non-significant terms were backward stepwise eliminated.

Significant terms are in bold (p > 0.016). Pairwise post-hoc tests compared response of

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G. holbrooki (GH) and M. novemaculeata (MN) in control temperature and control

salinity (C), elevated temperature and control salinity (ET), control temperature and

elevated salinity (ES) and elevated temperature and elevated salinity (ETS) treatments.

...... 101

Table 5.4 Results of a generalised linear mixed model testing the effects of hetero-specific

size, temperature and salinity on aggression in a) Macquaria novemaculeata and b)

Gambusia holbrooki. Non-significant terms were backward stepwise eliminated.

Significant terms are in bold (p > 0.016). Pairwise post-hoc tests compared the responses

of individuals to small (S) and large (L) hetero-specifics in control temperature and

control salinity (C), elevated temperature and control salinity (ET), control temperature

and elevated salinity (ES) and elevated temperature and elevated salinity (ETS)

treatments...... 103

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LIST OF FIGURES

Figure 1.1 Generalised schematic view of the four stages of a biological invasion, detailing

barriers to progression and impacts upon native species, adapted from Colautti and

MacIsaac (2004)...... 4

Figure 1.2 The strength and outcome of competitive and predator-prey interactions is

dependent upon interactions between environmental variables, specific –specific

tolerances and individual level traits which modulate behavioural mechanisms...... 8

Figure 1.3 a) A female Gambusia holbrooki (source: www.ncfishes.com) and b) a pregnant

female G. holbrooki (top) and male G. holbrooki (bottom) (source:

www.guntherschmida.com.au)...... 16

Figure 1.4 Distribution of G. holbrooki in Australia as of 2008. Green dots mark the locations

of records and shaded areas catchments in which they occur (source: Rowe et al., 2008).

...... 17

Figure 1.5. Adult female gravid (berried) Paratya australiensis, tagged with an elastomer gel

(L.K. Lopez)...... 18

Figure 1.6 Macquaria novemaculeata fingerlings (source: scottgrayfishing.com.au)...... 19

Figure 2.1 Mean (± SE) number of predation events by G. holbrooki on P. australiensis at

low and high density treatments during AM (open bars) and PM (closed bars)

observation periods. n = 12...... 32

Figure 2.2 Mean (± SE) number of inter-specific interactions (the sum of approaches and

nips) received by P. australiensis from G. holbroooki at low and high density treatments

during AM (open bars) and PM (closed bars) observation periods. n = 12...... 34

Figure 2.3. The relationship between (a) carapace length and direct inter-specific interactions

from G. holbrooki to P. australiensis in low and high density controls (grey diamonds,

dashed line) and treatments (black squares, solid line) and (b) carapace length and shelter

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use for P. australiensis exposed to a low density (grey diamonds, dashed line) and high

density (black squares, solid line) treatments and controls...... 35

Figure 3.1 Mean (± SE) inter-specific aggression from G. holbrooki (open bars) and M.

novemaculeata (closed bars) at low and high density treatment conditions, n = 10...... 54

Figure 3.2 Mean (± SE) of inter- (solid line) and intra-specific (broken line) aggression from

large and small M. novemaculeata at low and high density conditions, n = 10...... 55

Figure 3.3 The average (± SE) frequency of aggressive behaviours performed by large and

small M. novemaculeata to male (open bars) and female (closed bars) G. holbrooki at

low and high density treatment conditions, n = 10...... 56

Figure 3.4 Mean (± SE) of inter- (solid line) and intra-specific (broken line) aggression from

male and female G. holbrooki at low and high density conditions, n = 10...... 57

Figure 3.5 Mean (± SE) frequency of aggressive behaviours performed by male and female G.

holbrooki to a) large (open bars) and small (closed bars) M. novemaculeata, b) large

(open bars) and small (closed bars) G. holbrooki and c) male (open bars) and female

(closed bars) G. holbrooki under low and high density conditions, n = 10...... 58

Figure 3.6 Mean (± SE) inter-specific submission given by G. holbrooki (open bars) and M.

novemaculeata (closed bars) at low and high density conditions, n = 10...... 59

Figure 3.7 Mean (± SE) of inter- (solid line) and intra-specific (broken line) submission given

by large and small M. novemaculeata at low and high density conditions, n = 10...... 60

Figure 3.8 The average (± SE) frequency of submissive retreats performed by large and small

M. novemaculeata to male (open bars) and female (closed bars) G. holbrooki at low and

high density treatment conditions, n = 10...... 61

Figure 3.9 Mean (± SE) of inter- (solid line) and intra-specific (broken line) submission given

by male and female G. holbrooki at low and high density conditions, n = 10...... 62

Figure 3.10 Mean (± SE) frequency of submissive retreats performed by male and female G.

holbrooki from a) large (open bars) and small (closed bars) M. novemaculeata, b) large x

(open bars) and small (closed bars) G. holbrooki and c) male (open bars) and female

(closed bars) G. holbrooki under low and high density conditions, n = 10...... 63

Figure 4.1 a) Macquaria novemaculeata fingerlings; b) measuring the standard length of a

Gambusia holbrooki; c) the experimental setup with floating aquaculture cages at

Dunmore Quarry and d) close up of the aquaculture cages...... 76

Figure 4.2 Mean (± S.E.) survivorship of M. novemaculeata fingerlings in low (open bars)

and high (closed bars) density intra-specific and mixed conditions, n = 6...... 82

Figure 4.3 Mean (± SE) standard lengths of M. novemaculeata at low (open bars) and high

(closed bars) density intra-specific and mixed conditions in T0 and T1 of the experiment,

n = 6 cages, sample sizes of individuals are above bars...... 83

Figure 4.4 Mean (± SE) survivorship of G. holbrooki in low (open bars) and high (closed

bars) density intra-specific and mixed conditions, n = 6...... 84

Figure 4.5 Mean (±SE) proportion of G. holbrooki males in low (open bars) and high (closed

bars) density intra-specific and mixed conditions at T0 and T1 of the experiment, n = 6.

...... 85

Figure 4.6 Mean (± SE) initial and final standard lengths for a) female and b) male G.

holbrooki in low (open bars) and high (closed bars) density intra-specific and mixed

conditions of T0 and T1 of the experiment, n = 6 cages, sample sizes for individuals are

above bars...... 86

Figure 5.1 Mean (± SE) number of interspecific aggressive acts (sum of approaches, nips and

chases) performed by a) Macquaria novemaculeata to Gambusia holbrooki, b) G.

holbrooki to M. novemaculeata, in four treatment combinations of 15 ppt and 35 ppt and

21 °C (open bars) and 28 °C (closed bars), n = 8. Means with the same letter indicate

statistically non-significant differences...... 102

Figure 5.2 The mean (± SE) proportion of total aggression directed from individual a) M.

novemaculeata and b) G. holbrooki to smaller (closed bars) and larger (open bars)

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hetero-specifics in four treatment combinations of 15 and 35 ppt and 21 and 28 °C, n = 8.

Letters above indicate statistically similar means of aggression following separate

analyses for each species...... 104

Figure 6.1 Conceptual framework depicting the outcomes of this thesis which can be applied

to studies examining behavioural interactions between invasive and native species. The

strength and outcome of inter-specific interactions is mediated by behavioural

mechanisms which are in turn dependent on species-specific, group and individual-level

responses to (multiple) abiotic and biotic variables...... 117

Figure 7.1 The mean (± SE) length of G. holbrooki (open bars) and M. novemaculeata (closed

bars) at T0 in low and high density mixed conditions in this study, sample sizes of

individuals are above bars...... 146

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LIST OF APPENDICES

Appendix 1: A comparison of Macquaria novemaculeata and Gambusia holbrooki size .... 146

Appendix 2: Additional Publication ...... 147

xiii

1 GENERAL INTRODUCTION

1.1 Biological invasions in freshwater ecosystems

Biological invasions are one of the ‘big five’ drivers of global biodiversity change, alongside

climate, land use, atmospheric CO2 and nitrogen disposition (Sala et al., 2000) and can have

major adverse effects on native populations and communities (Clavero and García-Berthou,

2005, Sato et al., 2010, Banks and Hughes, 2012, Simberloff, 2014). Generally an invasion is

defined as the introduction, naturalisation and spread of a species beyond its natural range via

an anthropogenic pathway (Elton, 1958; Richardson et al., 2000). In the last 200 years the

frequency of invasions has been exponentially increasing due to heightened anthropogenic

activity and movement, to the extent that in some areas of the world non-native species

comprise 20-50% of all species which are present (Simberloff, 2010). Of particular concern

is that other significant forms of global change may facilitate the rate of spread and overall

impact of invaders (Sato et al., 2010). However, the manner in which environmental context

influences biological invasions remains poorly understood (Rahel, 2002, Olden and Poff,

2003).

Freshwater ecosystems are considered to be particularly vulnerable to biological invasions,

and are among the most severely invaded habitats worldwide (Sala et al., 2000, Simon and

Townsend, 2003, Dudgeon et al., 2006). This is primarily due to two factors. Firstly,

freshwater environments are exposed to a wide range of anthropogenic activities revolving

around recreation, transportation as well as commercial and local resource development

(Dudgeon et al., 2006, Sato et al., 2010, Cucherousset and Olden, 2011). Deliberate

introductions of alien freshwater species typically derive from attempts to provide food for

human communities, to introduce a form of bio-control, or releases from hobby aquariums

(Mills et al., 1993, Lodge et al., 1998, García-Berthou et al., 2005, Gozlan, 2008). Accidental introductions are frequently due to escapes from aquaculture facilities and ballast water releases (García-Berthou et al., 2005, Gozlan, 2008, Gozlan et al., 2010, Maceda-Veiga et al.,

2013). Secondly, unlike many terrestrial and marine environments, freshwater bodies are effectively closed systems that act as biogeographic islands (Fenoglio et al., 2010, Gozlan et al., 2010, Sato et al., 2010). Since the connectivity between water bodies is limited, species develop in isolation and exist within a small geographic range (Gozlan et al., 2010, Olden et al., 2010, Sato et al., 2010, Strayer, 2010). Therefore, many native freshwater organisms are incapable of initiating rapid responses such as shifts in movement to mitigate the introduction of an invasive species (Rahel, 2002, Fenoglio et al., 2010).

The process of a biological invasion is generally separated into four distinct stages; introduction, establishment, naturalisation and expansion (see Figure 1.1). At each stage an alien species must overcome a range of abiotic (hydrological, chemical and climatological)

(Moyle and Light, 1996, Harrison, 1999, Fausch et al., 2001) and biotic (novel competitors and predators, pathogens) (Case, 1990, Levine, 2000) barriers to survive in a freshwater ecosystem. While it is possible that an invader may begin exerting impacts on native species and communities immediately following its introduction, most studies have highlighted the post-establishment period as the time when significant impacts emerge (an impact is defined here as a significant positive or negative change in a biological or ecological process or pattern) (Melbourne et al., 2007, Simberloff et al., 2013). Impacts are frequently measured as a change in nutrient dynamics, species diversity and absolute or relative abundance of native species (Sakai et al., 2001, Melbourne et al., 2007). The pathways by which an invasive freshwater species may exert impacts are multiple and often subtle, ranging from habitat alteration (Mack et al., 2000, Manchester and Bullock, 2000, Zedler and Kercher, 2004), hybridisation with native species (Capelli and Capelli, 1980, Bailey et al., 2007, D'Amato et

al., 2007), trophic web alteration (Khan and Panikkar, 2009, Nilsson et al., 2012, Gkenas et al., 2016), the introduction and proliferation of disease (Pinder et al., 2005, Gozlan et al.,

2006) and new or altered inter-specific interactions (Human and Gordon, 1996, Sakai et al.,

2001, Gurevitch and Padilla, 2004).

1.2 A mechanistic approach to understanding invasive species

Competition and predation between invasive and native freshwater species are major drivers behind changes in biodiversity following an invasion (Shea and Chesson, 2002, Dame and

Petren, 2006, Hasegawa, 2016). Predation by invasive species is seen to be a major factor in the decline and extinction of native freshwater fish (Kovalenko et al., 2010, Martin et al.,

2010, Strayer, 2010, Laske et al., 2012) (this chapter will focus on the effect of an invasive predators, rather than invasive species which become prey for native predators). A well- known example is the loss of 200 haplochromine cichlid species following the introduction of the Nile perch (Lates niloticus) into Lake Victoria, East Africa in 1954 (Fryer, 1960). In addition to predation, inter-specific competition is considered to play an important role in the displacement of autochthonous species (Peck et al., 2014, Grabowska et al., 2016).

Ultimately, where predator-prey and competitive interactions occur between invasive and native freshwater fish we may observe a homogenisation of taxa, whereby the species composition in locations becomes increasingly less distinct (Rahel, 2000, Rahel, 2002, Olden et al., 2004, Sato et al., 2010).

STAGE ONE: INTRODUCTION Overcomes stress due

to transportation The non-native species arrives in an area outside its natural range previously.

Overcomes stress due to STAGE TWO: ESTABLISHMENT exposure to novel abiotic (hydrological, The population overcomes abiotic climatological, chemical) and biotic barriers to its existence and biotic conditions though may depend upon repeated (competitors, predators, introductions. pathogens)

STAGE THREE: NATURALISATION Overcomes abiotic and Exerts negative or biotic barriers to The species develops a self- positive impacts on reproduction, dispersal native species via and recruitment perpetuating population without anthropogenic intervention. hybridisation, habitat alteration, trophic web alteration, disease and parasites, interspecific Overcomes resistance STAGE FOUR: EXPANSION interactions from native biota and

physical barriers The population undergoes dispersal, invading new areas.

Figure 1.1 Generalised schematic view of the four stages of a biological invasion, detailing barriers to progression and impacts upon native species, adapted from Colautti and MacIsaac (2004).

A central goal in the field of invasion ecology is determining how to detect and quantify the impacts of invasive predators and competitors (Sato et al., 2010, Thomsen et al., 2011,

Ricciardi et al., 2013). Thus far, indirect methods have predominantly been used to predict and measure changes in the survivorship, growth and fecundity of native species which can ensue following exposure to an invader (Holway et al., 2002, Almeida and Grossman, 2012).

For example, the gape size of an invasive predator can often provide an indication of the size range of vulnerable prey (Bence and Murdoch, 1986). In regard to competition, habitat and dietary overlap are typically used to predict the likelihood and strength of the interaction

(Gutierrez-Estrada et al., 1998, Encina et al., 2004). However, indirect methods only provide

a broad estimate of the likelihood of an interaction occurring and do not reveal the proximate mechanisms which underlie inter-specific interactions (Holway and Suarez, 1999, Almeida and Grossman, 2012). This is problematic since the dynamics of predator-prey and competitive interactions can often be traced back to mechanisms which arise from differences in individual and species level behaviours which are better identified using a direct approach

(Holway and Suarez, 1999, Dame and Petren, 2006).

In order to examine the impacts of an invasive predator, it is necessary to measure both its consumptive (CE) and non-consumptive effects (NCE) (Sih, 1986, Lima, 1998, Pintor and

Soluk, 2006, Salo et al., 2007, Sih et al., 2010). While a predator can directly reduce the survivorship of a prey population via consumption (CE), it may also force prey to make shifts in their key behaviours in order to reduce the risk of being consumed (NCE), but which may reduce their fitness overtime (Sih, 1986, Pintor and Soluk, 2006, Paterson et al., 2013).

Therefore, the outcome of predator-prey interactions is dependent on a range of behavioural mechanisms including foraging efficiency and prey preference of the predator as well as the prey’s response to predation risk (Ryer, 1988, Helfman, 1989, Ferrari et al., 2009, Ercit,

2014). While some studies have sought to use capture records of prey as an indication of

NCEs, (i.e. shelter use), direct observation of prey responses can provide more information on a range of behaviours which impact fitness, including reproductive activities and foraging

(Anholt et al., 2000, McArthur et al., 2014, Peck et al., 2014). Therefore, this approach facilitates a more comprehensive understanding of the impacts exerted by invasive freshwater predators on native prey.

Invasive competitors may interact aggressively, known as interference competition (Case and

Gilpin, 1974, Holway et al., 1998, Amarasekare, 2002, Almeida and Grossman, 2012, Capelle et al., 2015), or indirectly with native species, referred to as exploitation competition (Debach,

1966, Petren and Case, 1996, Dame and Petren, 2006), with native species. The mechanisms underlying exploitation and interference competition range from foraging efficiency, diet and habitat preferences to aggression and social dominance (Holway and Suarez, 1999, Dame and

Petren, 2006, Pintor et al., 2008). For many invasive and native competitors these mechanisms are seldom observed and are thus poorly documented. This is problematic since competition is often subtle and difficult to detect (Lodge et al., 1994, Almeida and Grossman,

2012, Peck et al., 2014). Furthermore, it becomes more challenging to distinguish and measure the relative magnitudes of exploitation and interference competition which gives an important insight into the processes behind biological invasions (Light, 2005, Almeida and

Grossman, 2012).

1.3 Who, what, when and where: the context-dependency of invasive-native species interactions

Alongside the growing recognition of the importance of behavioural mechanisms, there has been a realisation of the need to acknowledge the context in which freshwater invasive-native species interactions occur (Melbourne et al., 2007, Chamberlain et al., 2014, Maron et al.,

2014). Most studies have previously examined predator-prey and competitive interactions as though they were set in a homogenous environment (Skellam, 1951, Melbourne et al., 2007).

However, freshwater habitats, which range from ephemeral to permanent waterbodies, are subjected to temporally and spatially varying abiotic conditions (temperature, salinity, flow, turbidity and light) as well as biotic variables (organism density, habitat complexity)

(Fenoglio et al., 2010, White et al., 2015). Furthermore, they are becoming increasingly impacted by habitat degradation and climate change, both of which will bring about future shifts in abiotic and biotic variables (Fenoglio et al., 2010, Strayer, 2010, Simberloff et al.,

2013, Rehage et al., 2016).

The outcome and strength of inter-specific interactions has been demonstrated to change depending on the presence and level of abiotic and biotic conditions (Shea et al., 2005,

Chamberlain et al., 2014, Maron et al., 2014). A further layer of complexity is added by the morphological, physiological and behavioural individual level variation within a species, since the relative size, sex, personality and reproductive condition (among other traits) of an individual can influence behaviours which could mediate the outcome of an inter-specific interaction (Shine et al., 2000, Herrel et al., 2009, Magellan and García-Berthou, 2015).

Therefore, our ability to identify where and when a freshwater invader will exert the strongest impact is dependent on understanding how both population and individual level variables influence inter-specific interactions (Pintor and Sih, 2011, Ricciardi et al., 2013) (Figure 1.2).

I will briefly summarise the major findings of studies which explore the context-dependency of predatory and competitive invasive-native species interactions and identify potential areas for future investigation. This section will focus on interactions between freshwater finfish, and where possible place an emphasis on the underlying behavioural mechanisms.

Abiotic/ biotic Variable

Species -specific tolerance to Individual Level Variable (Sex, size, reproductive variable condition, personality)

Behavioural Mechanism

(Aggression, foraging)

Population Dynamics INTERACTION STRENGTH (Size, survivorship, AND OUTCOME age).

Figure 1.2 The strength and outcome of competitive and predator-prey interactions is dependent upon interactions between environmental variables, specific –specific tolerances and individual level traits which modulate behavioural mechanisms.

1.4 Context-dependent predation by invasive species

The Influence of Abiotic Variables

Thus far, studies which examine the role of abiotic variables as mediators of interactions

between invasive predators and native prey have almost exclusively focused on the effects of

turbidity, which can increase as a result of habitat degradation (Benfield and Minello, 1996,

Miner and Stein, 1996, Hazelton and Grossman, 2009a, Hazelton and Grossman, 2009b,

Ferrari et al., 2014). Heightened levels of particulate matter can limit both the ability of an invasive predator to detect prey, as well as prey responses to a predator (McMahon and

Holanov, 1995, Miner and Stein, 1996, Ferrari et al., 2014). For example, Miner and Stein

(1996) observed that the distance from which juveniles of the native bluegill, Lepomus macrochirus, reacted to the presence of the invasive largemouth bass, Micropterus salmoides, decreased with increasing turbidity. Even so, since the reaction time of L. macrochirus was consistently longer than that of M. salmoides, the predation rate was not affected (Miner and

Stein, 1996).

Besides turbidity, temperature and salinity may also mediate predator-prey interactions, since they affect metabolic rates and subsequently energetic requirements (Bystrom et al., 2006,

Zheng et al., 2008, Rohr and Palmer, 2013). Both prey and predators may increase their foraging rates under increased temperatures (Matassa and Trussell, 2015), while sub-lethal levels of salinity have been shown to interfere with anti-predator responses, including risk detection, in freshwater fish (Hoover et al., 2013).

The Influence of Biotic Variables

A loss of vegetation density and complexity is considered to be a problematic result of habitat degradation, since vegetation can act as a physical and visual refuge for native prey fish

(Savino and Stein, 1982, Takamura, 2007, Sutton et al., 2013, Ferrari et al., 2014, Belk et al.,

2016a). However, evidence pertaining to its role in reducing piscivory by invasive fish is currently mixed, most likely due to species-specific variation in predator foraging methods and prey responses (Segev et al., 2009, Westhoff et al., 2013, Belk et al., 2016b). For example, while predation upon bluegill, L. macrochirus, by largemouth bass, M. salmoides, decreased with increasing plant density, no such effect was observed for another prey species, the fathead minnow, Pimephales promelas (Savino and Stein, 1982). This was attributed to

the fact that while L. macrochirus responded to bass by remaining motionless, fathead minnows P. promelas displayed no such reaction (Savino and Stein, 1982).

Another important biotic variable may be the population density of the invasive predator, which may vary temporally and spatially potentially resulting in a heterogeneous predation risk (Hansen et al., 2013). Prey capture rates may increase with the rate of contact between predator and prey (Kaiser et al., 2013), however, an increase in competitive interactions between predators may reduce their predation rate, leading to mutual interference (Sih, 1986,

Mistri, 2003). Despite this, the density- impact relationship for invasive freshwater predators has yet to be examined.

The influence of Individual Level Variables

Invasive piscivores may be limited in their prey preferences by their gape size (Bence and

Murdoch, 1986), which can result in smaller size classes of a prey species being consumed while larger individuals are subject to competition or avoid interactions entirely (Mills et al.,

2004). In addition to size, predator-prey interactions may be mediated by sex (Norrdahl and

Korpimaki, 1998, Boukal et al., 2008). Male individuals are typically consumed more by predators since they often engage in risky behaviour associated with territoriality and reproduction (Burk, 1982, Boukal et al., 2008, Ercit, 2014). However, sexually dimorphic species may respond differently to abiotic stressors which influence metabolic rate since this is related to body size (Ruiz-Navarro et al., 2011, Ohlberger et al., 2012, O'Mara and Wong,

2015).

1.5 Context-dependent competition between invasive and native freshwater species

The influence of Abiotic Variables

Studies which examine condition-specific competition between invasive and native fish have predominantly focused on the effects of temperature, which can vary widely, both temporally and spatially, in freshwater habitats (Destaso and Rahel, 1994, Taniguchi and Nakano, 2000,

Rowe et al., 2007, Carmona-Catot et al., 2013a). Since fish are ectothermic, elevated temperature can promote aggression, food consumption and growth (Portner and Knust,

2007). Therefore, invasive species which are tolerant of elevated temperature have been reported to gain competitive dominance over native competitors, a phenomenon known as condition-specific competition (Rowe et al., 2007, Briffa et al., 2013, Carmona-Catot et al.,

2013a, Matthews and Wong, 2015)

In addition to temperature, there is growing evidence that salinity mediates competition between invasive and native competitors. Salinity stress can incur metabolic costs due to changes in energy costs related to osmoregulation, which may reduce activity levels and aggression (Swanson, 1998). Alcaraz et al. (2008) reported that aggressive behaviours from the eastern mosquitofish, Gambusia holbrooki, to the native Spanish toothcarp, Aphanius fasciatus, decreased with increasing salinity. Notably, A. fasciatus displayed no such change in behaviour, presumably due to a higher tolerance to elevated salinity (Alcaraz and García-

Berthou, 2007, Alcaraz et al., 2008).

Water velocity and turbidity have been reported to alter food capture rate, aggression and subsequently competitive dynamics, albeit to a lesser extent than temperature and salinity. For example, the invasive yellowfin shiner, Notropis lutippinis, was reported to aggressively dominate the native rosyside dace, Clinostomus funduloides, under low velocities, yet when

the flow rate increased C. funduloides were more successful at capturing prey and so at a competitive advantage (Hazelton and Grossman, 2009a). Considering that the number of studies which have examined the effects of water velocity and turbidity on competition between invasive and native species is currently limited, the role of these variables in mediating interactions needs to be further explored.

The influence of biotic variables

Increasing vegetation complexity and density may reduce the strength of competitive interactions between invasive and native fish (Magellan and García-Berthou, 2016). A number of native fish species have been observed to select denser plant cover in the presence of Gambusia spp. (Mills et al., 2004, Keller and Brown, 2008). Such a shift in habitat preference may provide a refuge by reducing their visibility and subsequent vulnerability to aggressive interactions (Keller and Brown, 2008). However, it could also reduce the native species’ fitness by restricting it to suboptimal habitats (Ayala et al., 2007).

The impacts of an invasive competitor are typically considered to worsen with its density since resource availability will decrease while rate of contact between invasive and native individuals will increase (Kaiser et al., 2013). However, the relationship between invader density, impact and competition strength is far from clear (Yokomizo et al., 2009, Jackson et al., 2015). While some studies do support the theory that invader impacts and inter-specific interactions increase with population density (Mills et al., 2004, Kaspersson et al., 2010), others have shown that the strength of competition decreases with density since intra-specific interactions within the invasive species may increase (Kornis et al., 2014). For example, growth rates of the invasive round goby, Neogobius melanostomus, were reduced at high population densities, due to a rise in competitive interactions within the species (Kornis et al.,

2014). Despite the likely importance of intra-specific competition, most studies focus solely

on inter-specific competition and do not report the relative magnitudes of both (Inouye, 2001,

Forrester et al., 2006).

The influence of individual level variables

The relative size of competing individuals is critical in determining the strength and outcome of both inter- and intra-specific competition (Shine et al., 2000, Stammler and Corkum, 2005,

Tatara and Berejikian, 2012). Typically larger individuals are more aggressive and therefore competitively dominant (Tatara and Berejikian, 2012, Little et al., 2013, Theis et al., 2015).

Therefore, where a native species is on average larger than an invasive competitor they may not be affected by aggressive inter-specific interactions (Garcia and Arroyo, 2002, Tatara and

Berejikian, 2012). Such a size advantage has been shown to effectively overrule temperature- dependent increases in competition strength. Specifically, despite being more aggressive in summer, G. holbrooki refrained from attacking the native Spanish toothcarp Valencia hispanica as it was much larger than the invader. Instead, V. hispanica was attacked more in autumn when the inter-specific size difference was not so pronounced (Rincon et al., 2002).

Besides body size, temperature-dependent patterns of aggression have been observed to differ between male and females (Carmona-Catot et al., 2013a). For example, although male G. holbrooki were more aggressive than female con-specifics regardless of temperature, males exhibited the greatest increase of aggression between 19 and 24° C, whilst females did so between 24 and 29°C (Carmona-Catot et al., 2013a). However, it is important to note that most studies which explore condition-specific competition between invasive and native freshwater fishes have not considered the effects of individual level variables (Magellan and

García-Berthou, 2015). Therefore, the manner in which individual level variations modulate responses to environmental variables and ultimately competition dynamics remains unclear.

1.6 Multiple stressor effects

It is worth noting that, with the exception of Ferrari et al. (2014) and Carter et al. (2010), who simultaneously manipulated turbidity and vegetation cover, studies thus far have examined the effects of only one abiotic or biotic variable on invasive-native interactions. However, multiple variables, in this context also referred to as stressors (Piggott et al., 2015), may interact, resulting in unexpected changes in the strength and outcome of inter-specific interactions (Townsend et al., 2008, Matthaei et al., 2010, White et al., 2015). Furthermore, some stressors typically occur in tandem (e.g. dissolved oxygen availability decreases with elevated temperature (Mallekh and Lagardere, 2002)) and so solely testing the effect of one in isolation is futile. Given that interactions between native and invasive freshwater species may occur in the presence of multiple variables, it is important to consider their effects in order to maximise ecological realism (Folt et al., 1999, Townsend et al., 2008, Piggott et al., 2015,

White et al., 2015).

1.7 The study species

1.7.1 Eastern Mosquitofish, Gambusia holbrooki

Gambusia holbrooki Girard, 1859, commonly known as the eastern mosquitofish or plague minnow, is a poeciliid fish (Order: Cyprinodontiformes) originating from the east coast of

North America, east of the Appalachian Mountains (Rosen and Bailey, 1963, Smith et al.,

1989). While some populations have been recorded together, it is typically allopatric from the closely related western mosquitofish, Gambusia affinis (Pyke, 2008). It is a sexually dimorphic species, with females being larger (maximum standard length of 60 mm) than males (maximum standard length of 35 mm) (Cadwallader and Backhouse, 1983) (Figure 1.3 a, b). Both sexes have an olive green or brown back, grey sides and a white belly

(Cadwallader and Backhouse, 1983). However, they can be differentiated by a dark brood

patch which is located above the vent in pregnant females and a modified anal fin in males which forms a gonopodium (Cadwallader and Backhouse, 1983) (Figure 1.3 a, b).

In the early twentieth century, G. holbrooki and affinis were transported around the world to act as biocontrols for mosquitoes and mosquito-vectored diseases (Pyke, 2005). Alongside G. affinis, G. holbrooki is now the most globally widespread species of freshwater fish (Pyke,

2008). While they do feed on mosquito larvae and pupae, studies have reported that this only makes up a small portion of their diet and in fact less than some endemic Australian fish

(Lloyd, 1986, Arthington and Lloyd, 1989). The first introduction of G. holbrooki in

Australian was in 1925 in the Sydney Botanic Gardens with fish from a population from

Georgia, U.S.A. (Wilson, 1960, Myers, 1965). G. holbrooki is now present in at least eight out of eleven main drainage basins on the Australian continent and in every state and territory

(Rowe et al., 2007, Pyke, 2008) (Figure 1.4). G. holbrooki is currently the only Gambusia spp. present in Australia (Pyke, 2008).

Populations of G. holbrooki have been recorded in habitats which range widely in salinity (0 –

41 ppt (Hubbs, 2000), pH (4.5 – 9) (Keup and Bayliss, 1964), temperature (0-45 °C) (Cherry et al., 1976) and dissolved oxygen levels (1-11mg l-1) (Cherry et al., 1976). Despite this, they are most commonly present in low lying swamps, streams and lakes which are shallow, slow moving, warm and which contain dense aquatic vegetation (Moyle and Nichols, 1973,

Arthington and Lloyd, 1989). The high tolerance of Gambusia spp. to extreme environmental conditions also assists this species to invade and persist in degraded habitats. Potentially this also gives the invader a competitive advantage over native species which may have been negatively impacted by other environmental stressors such as pollution (Rautenberg et al.,

2015, Magellan and Garcia-Berthou, 2016). G. holbrooki are frequently at a high density and often outnumber native fish (Kilby, 1955, Arthington and Milton, 1983), however, their

density does vary seasonally, peaking after the breeding season in early autumn and at its lowest in spring (Barney and Anson, 1921, Morton et al., 1988). For this reason, as well as its tolerance to a range of environmental variables, G. holbrooki is an ideal model species for which to examine the context-dependency of invasive and native species interactions.

a)

b)

Figure 1.3 a) A female Gambusia holbrooki (source: www.ncfishes.com) and b) a pregnant female G. holbrooki (top) and male G. holbrooki (bottom) (source: www.guntherschmida.com.au).

Although G. holbrooki has been implicated in the disappearance or decline of numerous species of invertebrates, amphibians and fish worldwide and in Australia (Hulbert et al., 1972,

Hurlbert and Mulla, 1981, Miura et al., 1984, Arthington, 1989, Courtney and Meffe, 1989,

Blaustein, 1991, Galat and Robertson, 1992, Lawler et al., 1999,), many reports are based on anecdotal evidence and don’t examine the underlying behavioural mechanisms (Pyke, 2008).

It is also becoming increasingly apparent that the impact of this invader on a native species is dependent on both environmental variables and the age and size class of all species (Mills et al., 2004, Alcaraz and García-Berthou, 2007, Carmona-Catot et al., 2013a). Therefore, there is a need to investigate, using a direct approach, the context-dependency of interactions between

G. holbrooki and native Australian species.

Figure 1.4 Distribution of G. holbrooki in Australia as of 2008. Green dots mark the locations of records and shaded areas catchments in which they occur (source: Rowe et al., 2008).

1.7.2 Glass Shrimp (Paratya australiensis)

The Glass Shrimp, Paratya australiensis, Kemp, 1917, is a native Australian crustacean widespread throughout freshwater and estuarine waterbodies on the east coast (Richardson et al., 2004, Cook et al., 2006, Bool et al., 2011). The species plays a significant role in nutrient cycling, since as a consumer it alters algal and invertebrate community composition (March et

al., 2002). In addition to this, it is known to be an important food source for native species

(Walsh and Mitchell, 1995, Walsh and Mitchell, 1998, Richardson et al., 2004).

Figure 1.5. Adult female gravid (berried) Paratya australiensis, tagged with an elastomer gel (L.K. Lopez).

Thus far, few studies have explored interactions between P. australiensis and G. holbrooki, although there is evidence that P.australiensis can learn to recognise and avoid the invader

(Bool et al., 2011). Despite this, there is ample evidence that Gambusia spp. consume and alter the behaviour of shrimp species outside of Australia (Leyse et al., 2004, Capps et al.,

2009, Carey et al., 2011). For example, the shrimp Halocaridina rubra which cohabits

Hawaiian anchialine pools with G. affinis displays a predominantly nocturnal activity pattern compared with H. rubra in fishless pools (Capps et al., 2009). Furthermore, G. affinis had altered the size distribution of H. rubra populations by selectively feeding on smaller individuals (Capps et al., 2009). Given this, among other studies, it is essential to quantify the consumptive and non-consumptive effects of G. holbrooki on P. australiensis.

1.7.3 Australian Bass (Macquaria novemaculeata)

The Australian bass, Macquaria novemaculeata, Steindachner, 1966, is carnivorous member of the Percichthydae family native to the eastern Australian drainage system (Harris, 1986).

The species is catadromous, with adults undergoing downstream migration from freshwater regions to estuaries to spawn between May and August (Harris, 1986, O'Mara and Wong,

2015). However, the construction of weirs and other barriers has prevented a number of populations from migrating which has resulted in their extinctions (NSW Fisheries 2003). In order to support isolated populations, each year hundreds of thousands of hatchery reared fingerlings (juveniles) are stocked into artificial and natural waterbodies (NSW Fisheries

2003).

Figure 1.6 Macquaria novemaculeata fingerlings (source: scottgrayfishing.com.au).

Adult M. novemaculeata have been observed to attack and consume G. holbrooki in captivity

(Grigaltchik et al., 2012). There is however, no knowledge of how fingerlings interact with the invader, which is problematic since the species are sympatric. Furthermore, there are a number of factors suggesting that G. holbrooki interacts aggressively with M. novemaculeata fingerlings. Firstly, being hatchery reared it is possible the fingerlings are naïve to the

aggressive behaviour displayed by the invader. Secondly, fingerlings are typically stocked when they are only 2- 3mm in length (L.K. Lopez, personal observation) which is equal to or smaller than adult G. hobrooki, thereby inferring the invader with a competitive advantage.

Finally, since G. holbrooki are already present in waterways when juvenile M. novemaculeata are stocked they have the advantage of prior residence which could lead to heightened aggression levels. Therefore, there is the possibility that G.holbrooki could significantly impede the conservation of M. novemaculeata by through aggressive interactions and resource domination.

1.8 Thesis outline

The overarching aim of this thesis was to investigate how abiotic and biotic variables, as well as size and sex, mediated predator-prey and competitive interactions between the invasive eastern mosquitofish, Gambusia holbrooki and native Australian freshwater fauna. This work predominantly quantified direct inter-specific behavioural interactions so as to identify and examine the significance of mechanisms behind the impact of G. holbrooki in the post- establishment biological invasion phase. Given the lack of consensus on how invader density modulates both predator-prey and competitive densities, much of this thesis examined the relationship between G. holbrooki and its potential impact on native species. In addition to this, it also examined competition in the presence of multiple abiotic variables.

The specific questions addressed by each data chapter were:

1. Does the density of G. holbrooki and the diel cycle mediate the consumptive (CEs) and

non-consumptive effects (NCEs) of this predator on a native prey species, the glass

shrimp, Paratya australiensis? (Chapter 2)

2. Does the density of G. holbrooki influence the intensity of interference competition

with juveniles of the native Australian bass, Macquaria novemaculeata? (Chapter 3)

3. Does the survivorship and growth of G. holbrooki and M. novemaculeata change

depending on the density of G. holbrooki? (Chapter 4)

4. How is the intensity of interference competition between G. holbrooki and M.

novemaculeata mediated by multiple abiotic stressors? (Chapter 5)

The final discussion section (Chapter 6) synthesizes the findings of this thesis and its implications for the management of G. holbrooki and invasive freshwater species in general.

2 THE EFFECTS OF THE DIEL CYCLE AND THE DENSITY OF AN INVASIVE PREDATOR ON PREDATION RISK AND PREY RESPONSE

A modified version of this chapter is published in Animal Behaviour:

Lopez L.K., Wong M.Y.L. and A.R. Davis (2016). The effects of diel cycle and the density of

an invasive predator on predation risk and prey response. Animal Behaviour, 117:87-95.

2.1 Introduction

To reduce the risk of being consumed by predators, prey often exhibit substantial behavioural changes including increased vigilance, refuge use, dispersal and changes in activity patterns

(Sih, 1986, Lima, 1998, Sih et al., 2010).Whilst frequently successful in reducing the rate of direct consumption (defined as Consumptive Effects, CEs), such behavioural changes may also negatively impact upon foraging and reproduction (defined as Non-Consumptive Effects,

NCEs), thus leading to a trade-off between minimising mortality from predation and maximising fitness (Sih, 1986, Paterson et al., 2013). Therefore, as posited by the threat- sensitive predator-avoidance hypothesis (Helfman, 1989), prey need to maximise their overall fitness by exhibiting anti-predator responses which are proportional to the level of predation risk to which they are exposed. If the avoidance behaviours exhibited by prey are in excess of the predation risk, then the strength of NCE’s on prey fitness will increase, yet if anti-predator behaviours are insufficient, the strength of CEs on prey should become greater (Sih, 1986,

Anholt et al., 2000, Ferrari et al., 2009).

Predator density can significantly influence perceived predation risk (Vucetich et al., 2002,

Foam et al., 2005, Ferrari et al., 2009). Density itself can be modulated if predators form aggregations in the environment, in turn generating a predation risk that is spatially heterogeneous (Butler, 1989, Navarrete and Menge, 1996, Mella et al., 2014). Currently, there is mixed support as to whether predator density and CEs positively correlate (Sih, 1986,

Vance-Chalcraft et al., 2004). Typically, higher predator densities have been related to an increased number of encounters between predators and prey, leading to a higher per capita kill rate, as observed in wolves and moose (Vucetich et al., 2002, Stier and White, 2014). Even so, negative correlations between predator density and prey capture and kill rates have been

reported, due to an increase in the frequency of competitive interactions amongst foraging predators, known as mutual interference (Sih, 1979, Abrams, 1993, Mistri, 2003).

With regards to NCEs, the relationship with predator density is also often complex. Flathead minnows (Pimephales promelas) show stronger anti-predator responses in the form of shoaling and flee behaviours when exposed to the odour cues of twelve pike compared to just two (Ferrari et al., 2006). Similarly, exposure to a high density of predatory mites led to greater dispersal by spider mites (Tetranychus kanzawai) (Bowler et al., 2013). However, a recent review by (Paterson et al., 2013) reported that crustacean prey from a number of taxa exhibited similar changes in activity levels and refuge use regardless of cue intensity or exposure time to fish predators. At this point it is unknown whether it may be advantageous for prey to display similar avoidance behaviours which may be an effective response to multiple predator densities, or if the intensity or combination of cue types used overpowered subtle indications of predator density (Paterson et al., 2013).

Levels of predation risk can also vary with diurnal changes in predator foraging behaviour

(Helfman, 1989, Griffin et al., 2005), effectively providing prey with an indication of predation risk (Clark et al., 2003). Typically, prey are more active in periods of ambient light levels in which predators are less active (Benfield and Minello, 1996). For example, buffalo, warthog and kudu are predominately active during the day in areas where nocturnal predators co-exist however, in their absence these prey species are also active during the night

(Tambling et al., 2015). There is also some evidence to suggest that the diel cycle can interact with other environmental variables such as depth, to alter predation risk and subsequently prey refuge use (Bollens and Frost, 1989, Bollens and Stearns, 1992, Clark et al., 2003). This has been observed in grass shrimp where mortality from predation by fish was found to be depth dependent during the day but not at night (Clark et al., 2003). Less well understood

however, is whether the diel cycle interacts with predator density to influence the magnitude of diurnal shifts in shelter use, swimming, foraging and other behaviours which have been observed in prey.

In this study, we investigated the effects of predator density and diel cycle on the

Consumptive and Non-Consumptive Effects of the invasive predator, the eastern mosquitofish, Gambusia holbrooki Girard, 1859, on the native Australian glass shrimp,

Paratya australiensis Kemp, 1917. P. australiensis is widespread in coastal Eastern Australia

(Cook et al., 2006) and plays a key role in nutrient cycling in freshwater and estuarine ecosystems as well as being a food source for native species (Walsh and Mitchell, 1995,

March et al., 2002, Richardson et al., 2004). The ubiquitous G. holbrooki has spread to eight of the eleven main drainage basins on the Australian continent since its introduction to

Sydney in 1925 (Pyke, 2008). G. holbrooki is an opportunistic omnivore which feeds on small decapod crustaceans, including P. australiensis (Arthington and Marshall, 1999).

Gambusia spp. are able to predate upon relatively large prey by nipping the body, tail and gills which results in immobilisation and death (Komak and Crossland, 2000, Segev et al.,

2009, Shulse and Semlitsch, 2014). Therefore Gambusia spp. are able to overcome the limitation imposed by their gape size on prey selectivity (Baber and Babbitt, 2003, Drake et al., 2014, Smith and Smith, 2015).The density of G. holbrooki is known to vary seasonally, with peak abundance in early autumn after the breeding season and the lowest abundance in spring (Barney and Anson, 1921, Morton et al., 1988). In addition, due to being a visual predator, the foraging behaviour of G. holbrooki is likely to be greater during the day than at night (Bool et al., 2011). Although it has been associated with a decline in populations of native fairy shrimp in California (Leyse et al., 2004), surprisingly few studies have quantified the behavioural interactions of G. holbrooki with native biota with the specific purposes of identifying the exact mechanisms behind its negative impacts on native species. Furthermore,

native prey may be especially vulnerable as they do not share an evolutionary history with the predator, possibly rendering them less adept at detecting risk and responding accordingly

(Bourdeau et al., 2013, Heavener et al., 2014).

Specifically, we determined if predator density and diel cycle affected the number of P. australiensis consumed by G. holbrooki. Direct behavioural interactions, in the form of approaches and nips by G. holbrooki to P. australiensis, were also recorded. Additionally, we assessed whether the behavioural responses exhibited by P. australiensis, namely shelter use, swimming and foraging behaviours co-varied with predator density and diel cycle. We hypothesised that 1) the number of predation events and direct interactions between G. holbrooki and P. australiensis would be greatest at the high predator density and during the day and 2) that in response to the greater predation risk, P. australiensis would exhibit greater behavioural changes in the presence of a high density of G. holbrooki and during the day.

2.2 Methods

Animal Collection and Aquaria Setup

Eastern mosquitofish, G. holbrooki (x̅ ± SE mass = 0.19 ± 0.14 g; x̅ ± SE standard length =

19.46 ± 0.46 mm; x̅ ± SE total length = 24.21 ± 0.53 mm) were collected from freshwater ponds located on the University of Wollongong campus (34° 24’ 19” S 150 ° 52’ 42” E) using a baited hand held landing net. Only adult females were collected so as to avoid the mating behaviours displayed by males which may have interfered with the predatory behaviours exhibited by females relevant to this study. Glass shrimp, P. australiensis, (x̅ ± SE mass =

0.07 ± 0.09 g; x̅ ± SE carapace length = 5.77 ± 0.1 mm) were acquired from the national supplier LiveFish.com. Berried P. australiensis were included in the study and a note of their condition was made. To conduct the experiment, 6 recirculating aquarium systems were used

at the University of Wollongong, each system containing 8 aquaria (37 × 22 × 27 cm) that were inter-connected and subjected to water conditions of 23 °C and 5 ppt salinity. Each aquarium was lined with 2 cm of natural river gravel and contained 3 black plastic tubes (7 ×

2 cm) positioned on the substratum to provide shelter for P. australiensis. The exterior sides of each tank were covered with black plastic to exclude visual cues from individuals in adjacent tanks. To acclimatise G. holbrooki and P. australiensis to laboratory conditions, G. holbrooki (N = 72 total fish) were placed into 12 aquaria spread equally across the 6 systems

(N = 12 fish per system). P. australiensis (N = 288 total individuals) were placed into 36 aquaria spread equally across the 6 systems (N = 48 individuals per system) and in separate systems from those housing G. holbrooki. All G. holbrooki and P. australiensis were maintained under these conditions for 7 days to ensure adequate acclimation to laboratory conditions. During this time, G. holbrooki were fed a commercial fish flake (New Life

Spectrum Thera formula) and P. australiensis were fed a shrimp granule (Fluval formula).

Water changes (< 20 %) were made once a fortnight and new water supplemented with Fluval bacterial and shrimp mineral additives. Due to the sensitivities of P. australiensis to traces of copper and other metals in the available tap water, deionised water was also treated with

CupriSorb (Seachem) to remove any traces of copper.

Experimental Design

To quantify the Consumptive Effects (CEs) and Non-Consumptive Effects (NCEs) of G. holbrooki on P. australiensis, we established two conditions: 1) Treatment condition, in which aquaria contained both G. holbrooki and P. australiensis (N = 24 aquaria) and 2)

Control condition, in which aquaria contained only P. australiensis (N = 24 aquaria). Within the Treatment condition aquaria (both species present), two density levels of G. holbrooki were established- i) low density (N = 1 individual/aquarium; N = 12 aquaria) and ii) high density (N = 5 individuals/aquarium; N = 12 aquaria), both with a constant density of P.

australiensis (N = 4 individuals/aquarium). Within the Control condition (P. australiensis only), the total number of P. australiensis was equal to the total number of P. australiensis and G. holbrooki in the equivalent low and high density treatment condition (N = 5 individuals in the low density; N = 12 aquaria, and N = 9 individuals in the high density; N =

12 aquaria). Treatment densities were based on estimates of seasonal low and high G. holbrooki densities in University of Wollongong campus ponds relative to the aquaria size used in this study (Lopez, personal observation). An alternative substitutive design (Inouye,

2001) was considered inappropriate as by changing the ratio of P. australiensis: G. holbrooki it would be impossible to determine whether any differences in the response variables were due to changes in the overall density of animals or species composition.

Behavioural Observations and Data Collection

Following the acclimatisation period, each P. australiensis was measured using hand-held callipers (mm carapace length (CL) ± 0.1 mm) and weighed with an electronic balance (grams

(g) ± 0.1 g). To enable the identification of individuals and control for any effect of body size, four P. australiensis (‘focal’ individuals) from each aquaria were tagged using a coloured fluorescent polymer elastomer (red, pink, orange or green) (Northwest Technologies Inc.) injected into the musculature of the telson.

Four P. australiensis were introduced into a test aquarium for twenty four hours prior to the addition of either one G. holbrooki (Treatment) or P. australiensis (Control) in the low density condition, or five G. holbrooki (Treatment) or P. australiensis (Control) in the high density condition. This order of residency was intended to simulate the natural temporal occurrence of species in the event of an invasion by G. holbrooki. Observations commenced after a 60 minute acclimation period followed by a 5 minute adjustment to the presence of the observer. Only one person (LKL) conducted observations to avoid potential issues with

observer bias. It was not possible to observe the animals blind due to the fact that the species composition and number differed between treatments and controls. Each tagged P. australiensis was observed twice, once during the day (0900- 1300 hrs under natural daylight and under red light) and once at night (1700- 2100 hrs under red light. Each observation period lasted for 10 minutes during which the time spent in shelter (seconds), time spent engaged in specific activities (swimming and foraging) (seconds), and direct behavioural interactions with G. holbrooki were recorded (approaches and nips). G. holbrooki and P. australiensis were therefore exposed to each other for a maximum of 12 hours after which they were separated and placed back into separate aquaria containing only con-specifics.

Data Analysis

P. australiensis was predated upon by G. holbrooki in the high density (N = 9 individuals) and low density (N = 6 individuals) treatments, and there was non-predation mortality in the low density control condition (N = 1 individual). Therefore, behavioural data collected during the experiment from these predated subjects has been excluded from subsequent analyses in order to use a repeated measures approach incorporating time as a repeated measures factor.

The effects of predator density (low or high) and time of day (AM or PM) on the number of predation events by G. holbrooki on P. australiensis was analysed using a two-way Analysis of Variance (ANOVA) with density (fixed), time (fixed) and tank ID (random) factors in the model (SPSS 21).

To assess whether the size difference between P. australiensis and G. holbrooki influenced whether a predation attempt (nipping by G. holbrooki) resulted in consumption of P. australiensis (successful outcome) or not (unsuccessful outcome), prey and predator size was scaled. This scaling was achieved by dividing the average carapace length of P. australiensis

in each low or high density treatment replicate tank by the average standard length of G. holbrooki in the same tank (Bence and Murdoch, 1986). Standard length of fish was used as it is linearly related to gape size in G. holbrooki (Bence and Murdoch, 1986). Scaled prey size was analysed using an ANOVA with density (fixed), outcome (fixed) and tank ID (random) as factors.

In order to assess the effects of predator density on the number of inter-specific interactions

(the sum of approaches and nips), a Generalised Linear Mixed Model (GLMM) with backward stepwise elimination was used, incorporating a negative binomial distribution and a log link function which is appropriate for analysing zero-inflated count data. The model consisted of density (fixed), time of day (repeated measure), carapace length (covariate) and tank ID (random) as factors (SPSS 21).

To assess the NCEs of G. holbrooki density and whether there was an effect of diel cycle, the amount of time in which focal P. australiensis engaged in shelter use, swimming and foraging

(seconds) was first converted into a percentage of total activity for each AM and PM observation period. For shelter use, data was categorised into whether individuals spent less than or equal to 50 % (0) or more than 50 % (1) of the observed time in shelters. Due to the small amount of time with which P. australiensis were observed swimming and foraging, the most suitable model was also found to use a binomial distribution, categorising the response variable depending on whether swimming and foraging was observed for an individual shrimp

(1 = yes, 0 = no). To investigate the effects of predator density and diel cycle on shelter use, swimming and foraging in P. australiensis, a GLMM with backward stepwise elimination, incorporating a binomial distribution and logit function was used, with density (fixed), species treatment (fixed), time of day (repeated measure), carapace length (covariate) and tank ID

(random factor) as factors in the model.

Ethical Note

As advised by Brennan et al. (2007), P. australiensis were not anaesthetised during the tagging procedure so as to avoid the risk of mortality as well as to reduce handling and recovery times. As a result, we did not observe mortality due to tagging. Upon completion of the experiment, G. holbrooki were euthanased using clove oil as it is illegal to release an invasive species under NSW legislation. P. australiensis were maintained in aquaria until their natural deaths. To minimise stress on both G. holbrooki and P. australiensis, G. holbrooki were not starved prior to exposure to P. australiensis. Shelters were provided to allow P. australiensis to hide from G. holbrooki when needed and tank sides were covered to prevent animals from being affected by the possible predation activities occurring in adjacent tanks. We limited behavioural trials to a maximum of 12 hours and separated P. australiensis and G. holbrooki immediately after this period. The methods used for animal capture, housing and tagging were approved by the Animal Ethics Committee of the University of Wollongong

(Animal Ethics Protocol No. 13/09) and adhered to the Scientific Collection guidelines

(permit No. P13/0011-1.3) of the NSW Department of Primary Industries.

2.3 Results

2.3.1 Consumptive effects

In this study G. holbrooki was observed to predate upon P. australiensis by nipping the telson and body until the prey was immobilised and deceased, upon which consumption would occur. G. holbrooki were not observed to consume the eggs of berried P. australiensis. In total, predation events of P. australiensis by G. holbrooki were observed in the low (N = 6 events) and high (N = 9 events) density treatments during the course of this experiment

(Figure 2.1). There was no significant effect of predator density (ANOVA: Density, F1, 44 =

0.37, p = 0.54), time of day (Time, F1, 44 = 3.31, p = 0.076) or an interaction between predator density and time of day (Density×Time, F1, 44 = 0.04, p = 0.84) on the number of P. australiensis predated upon.

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2 Average number Averagenumber predationof events

0.1

0 Low Density Treatment High Density Treatment Figure 2.1 Mean (± SE) number of predation events by G. holbrooki on P. australiensis at low and high density treatments during AM (open bars) and PM (closed bars) observation periods. n = 12.

There was no significant difference in scaled P. australiensis size between successful and unsuccessful predation attempts at either low or high density treatments (ANOVA: Density,

F1, 14 = 1.08, p = 0.31; Outcome, F1, 14 = 0.06, p = 0.82), and no significant interaction between density and outcome (Density×Outcome, F1, 14 = 0.78, p = 0.39) (Table 2.1).

Table 2.1Scaled P. australiensis size (P. australiensis carapace length/ G. holbrooki standard length) in successful and unsuccessful predation attempts in low and high predator density treatments. n = 12.

Predation Attempt Outcome Successful Unsuccessful

Low Density Treatment 0.21 ± 0.04 mm 0.25 ± 0.01 mm

High Density Treatment 0.29 ± 0.05 mm 0.25 ± 0.03 mm

2.3.2 Non-consumptive effects

Direct Inter-specific Interactions

P. australiensis exposed to a high predator density received significantly more direct behavioural interactions (nips and approaches) than P. australiensis in the low density treatment (GLMM Density, F1, 156 = 23.84, p = <0.0001) (Figure 2.2). In addition, P. australiensis experienced more interactions with G. holbrooki during the day than at night regardless of treatment (Time, F1, 156 = 22.22, p = <0.0001) (Figure 2.2). There was no significant interaction between density and time of day (Density ×Time, F1, 155 = 0.31, p =

0.58). Overall, smaller P. australiensis received more inter-specific interactions from G. holbrooki (Carapace Length, F1, 156 = 5.38, p = 0.02) although there was a significant interaction between density and shrimp size (Density×Carapace Length, F1, 156 = 6.80, p =

0.01). Therefore, smaller P. australiensis received more inter-specific interactions in the high density than low density treatments (Figure 2.3a).

4

3.5

specifc 3 -

2.5

2 interactions 1.5

1 Average number Averagenumber interof

0.5

0 Low Density Treatment High Density Treatment

Figure 2.2 Mean (± SE) number of inter-specific interactions (the sum of approaches and nips) received by P. australiensis from G. holbroooki at low and high density treatments during AM (open bars) and PM (closed bars) observation periods. n = 12.

30 a)

25

20

15

specific interactions - 10 5

Meaninter 0 0 2 4 6 8 10 12 Carapace Length (mm)

120 b)

100

80

60

Time Time (%) 40

20

0 0 2 4 6 8 10 12 Carapace length (mm) Figure 2.3. The relationship between (a) carapace length and direct inter-specific interactions from G. holbrooki to P. australiensis in low and high density controls (grey diamonds, dashed line) and treatments (black squares, solid line) and (b) carapace length and shelter use for P. australiensis exposed to a low density (grey diamonds, dashed line) and high density (black squares, solid line) treatments and controls.

Shelter Usage

P. australiensis spent significantly more time in shelters during the day compared to at night in both treatment and control conditions (GLMM Time, F1, 347 = 43.90, p <0.0001) (Figure

2.3a). Shelter use for P. australiensis was significantly greater in the treatments (both low and high density) than in the controls (both low and high density) during the day and night

(Species Treatment, F1, 347 = 17.61, p <0.0001). However, there was no effect of density on shelter usage (Density, F1, 346 = 2.73, p = 0.10) nor an interaction between species treatment

and density (Species Treatment ×Density, F1, 341 = 0.01, p = 0.91) (Figure 2.4a). Although animal density alone did not have an effect on the time spent in shelters, there was a significant interaction between body size and density, with larger P. australiensis spending more time in shelters under high density compared to low density control and treatment conditions (Density×Carapace Length, F2, 347 = 3.56, p = 0.03) (Figure 2.4b).

Swimming

P. australiensis exposed to both low and high densities of G. holbrooki spent significantly less time swimming than those where G. holbrooki was absent (GLMM Species Treatment,

F1, 350 = 10.12, p < 0.01) (Figure 2.3b). There was no significant effect of time, size or density on swimming behaviour (Time, F1, 345 = 1.24, p = 0.27; Density, F1, 343 = 0.08, p = 0.77;

Carapace length, F1, 343 = 0.73, p = 0.39).

Foraging

P. australiensis foraged for significantly longer periods of time during the night than the day in both treatment and control conditions (GLMM Time, F1, 349 = 33.67, p <0.0001) (Figure

2.3c). While there was no significant effect of G. holbrooki presence or absence (Condition,

F1, 348 = 0.07, p = 0.79), there was an effect of density, whereby, P. australiensis in the high density control and treatment conditions spent more time foraging than P. australiensis in low density control and treatment conditions (Density, F1, 349 = 6.37, p = 0.01) (Figure 2.3c). There was no significant effect of size on foraging behaviour (Carapace Length, F1, 343 = 0.38, p =

0.54).

80 a)

70 60 50

40

Time (%) 30 20 10

0 Low Density Control Low Density Treatment High Density Control High Density Treatment

6 b)

5

4

3

Time (%) 2

1

0 Low Density Control Low Density Treatment High Density Control High Density Treatment

35 c) 30

25

20

15 Time Time (%) 10

5 0 Low Density Control Low Density Treatment High Density Control High Density Treatment

Figure 2.4 Mean (± SE) time spent by P. australiensis engaging in a) shelter use, b) swimming, and c) foraging in the low and high density treatments and controls during AM (open bars) and PM (closed bars) observation periods. n = 12.

2.4 Discussion

The threat sensitivity predator avoidance hypothesis (Helfman, 1989) states that the magnitude of anti-predator behaviours exhibited by prey should be directly proportional to the level of predation risk to which they are exposed. In this study, we tested the effects of predator density and diel cycle on the Consumptive and Non-Consumptive Effects (CE and

NCEs) of predation by the invasive eastern mosquito fish, G. holbrooki, on the native

Australian glass shrimp, P. australiensis. As predicted, the frequency of direct behavioural interactions between predator and prey significantly increased with predator density and during the day when G. holbrooki is active, however, these factors did not significantly influence the number of P. australiensis actually consumed. P. australiensis displayed anti- predator behaviours in response to the presence of G. holbrooki, specifically an increase in shelter usage and a reduction in swimming. Yet the magnitude of these anti-predator behaviours did not differ depending on predator density or the time of day. In contrast, whether P. australiensis undertook foraging was primarily dependent on the total density of animals present and not species composition, with the number of P. australiensis foraging being greatest at a high density of both fish and shrimp. In addition, no interactions between predator density and diel cycle predicted any of these behaviours. Therefore, P. australiensis, by not altering its behaviours in relation to density or diel cycle, responded appropriately to the level of actual predation risk to which it was actually exposed.

Predator density has previously been either positively or negatively correlated with the frequency of direct interactions between predator and prey and the number of prey killed (Sih,

1979, Vucetich et al., 2002, Chang and Snyder, 2004, Gregory and Quijon, 2011). In the present study, the frequency of direct interactions, namely approaches and nips, which P. australiensis received from G. holbrooki was significantly greater at a higher predator

density. Additionally, significantly more direct interactions were recorded during the day than at night, regardless of G. holbrooki density. This concurs with previous studies which have reported a nocturnal decrease in interactions between G. holbrooki and P. australiensis most likely due to a decrease in the predators activity levels (Bool et al., 2011). However, despite the fact that predator density is commonly used as a proxy for predation risk (Ferrari et al.,

2009, Paterson et al., 2013), our results demonstrate that predator density and diel cycle did not alter the number of prey consumed, and therefore the level of predation risk. These findings are consistent with the occurrence of mutual interference, whereby aggressive and competitive behavioural interactions between predators can reduce foraging efficiency (Sih,

1979, Abrams, 1993, Mistri, 2003, Nilsson and Ruxton, 2004). This possibility would be an interesting avenue of future research.

Notably, smaller P. australiensis, particularly in the high density treatment, received significantly more nips and approaches from G. holbrooki, particularly when the predator’s density was high. However, since scaled prey size did not differ between successful or unsuccessful predation attempts in either low or high density treatments, G. holbrooki were apparently not limited to consuming smaller P. australiensis. This finding differs from that of

Capps et al. (2009), who reported selective predation by the Western Mosquitofish, Gambusia affinis, upon smaller individuals of theʻōpaeʻula shrimp, Halocaridina rubra. However, G. affinis has also been found to select medium and large sized prey and so does not follow the optimal diet theory since larger prey would impose a higher risk and effort during capture

(Pulliam, 1974, Bence and Murdoch, 1986, Metzke and Pederson, 2012). For example,

Metzke and Pederson (2012) reported that despite the gape size of G. affinis being smaller than the total length of large Daphnia spp., G. affinis did not reject large prey more than medium sized prey. It is also important to note that smaller P. australiensis in the current study spent less time in shelters when the density of G. holbrooki was high, increasing the

likelihood of an encounter with G. holbrooki. One possibility for this result was that smaller

P. australiensis were aggressively excluded from shelters by larger P. australiensis, as has previously been observed in crayfish and teleost fish (Mikheev et al., 1994, Figler et al., 1999,

Martin and Moore, 2007).

Increased refuge use and changes in activity are common behavioural responses observed in crustacean prey in response to predatory fish (Linden et al., 2003, Capps et al., 2009) and are likely to be a means of reducing visibility to a visually foraging predator (Meager et al., 2005,

Kunz et al., 2006, Lammers et al., 2009, Carey et al., 2011). In the present study P. australiensis reacted to the presence of G. holbrooki by significantly increasing shelter use and reducing swimming. However, there was no difference in the magnitude of change in these behaviours observed in P. australiensis exposed to low and high densities of G. holbrooki. Additionally, and in contrast to other studies (Clark et al., 2003, Capps et al., 2009,

Carey et al., 2011), there was no diurnal shift in shelter usage and swimming in P. australiensis, in response to G. holbrooki at either low or high density. Whilst P. australiensis spent more time in shelters during the day than at night, this was consistent across all treatments and densities. However, studies that have noted shifts in diurnal shelter usage and activities have predominately examined field populations of shrimp and fish predators and therefore such behavioural changes may occur over a time scale not possible in our study

(Weissburg et al., 2014).

While swimming and shelter use were predominantly affected by the presence of G. holbrooki, the frequency of foraging by P. australiensis was determined by the overall density of animals present. Specifically, P. australiensis in the high density control and treatment conditions foraged more than shrimp in the low density control and treatment conditions. In contrast to Bool et al. (2011), there was no shift from diurnal to nocturnal foraging upon

exposure to G. holbrooki. While P. australiensis exposed to a high density of G. holbrooki did forage less during the day and more at night than P. australiensis in the high density control condition (without G. holbrooki), the interaction between density and diel variation was not significant. As with shelter use and swimming, it is also possible that this is simply due to the length of time in which P. australiensis was exposed to G. holbrooki in the present study. However, as a generalist species, it is also possible that G. holbrooki competes with P. australiensis, as has been previously observed between larval killifish (Fundulus heteroclitus) and post-larval grass shrimp (Palaemontes pugio) whereby shrimp experienced reduced growth in the presence of fish (Cross and Stiven, 1997, Pyke, 2008).

The number of P. australiensis predated upon did not significantly differ between low and high density treatments. Therefore, P. australiensis was responding appropriately to the level of predation risk to which it was exposed, supporting the threat-sensitive predator-avoidance hypothesis (Helfman, 1989). Our results also suggest that P. australiensis was reacting to some form of alarm cue released by predated individuals as has been observed in some taxa

(Bouwma and Hazlett, 2001, Hazlett and McLay, 2005, Paterson et al., 2013), though this would require further testing. Proportional anti-predator responses have previously been reported for wood frog tadpoles, Rana spp. (Van Buskirk and Arioli, 2002, Schoeppner and

Relyea, 2008). For example, Schoeppner and Relyea (2008) manipulated predation risk by altering the number of predators which consumed a constant amount of prey and the number of prey consumed by a constant number of predators. Hiding and activity levels in Rana sylvatica tadpoles increased with the number of con-specifics consumed, but not with predator density (Schoeppner and Relyea, 2008). While a number of other studies have similar responses regardless of predator density and cue intensity in other crustaceans

(Paterson et al., 2013), there is also evidence in a number of crustacean taxa that some species are able to detect and respond appropriately to different levels of predation risk (Hill and

Weissburg, 2013, Arundell et al., 2014). For example, mud crabs decreased movement, foraging and distribution patterns in response to exposure to high biomass predator treatments of multiple small and single large blue crabs, both of which exert different forms of predation pressure. However, mud crabs did not respond to single small blue crabs (Hill and Weissburg,

2013). Therefore, there currently remains evidence both in support and opposition of the threat sensitivity predator avoidance hypothesis (Helfman, 1989) in regards to crustacean species.

In conclusion, this study demonstrates that the native crustacean P. australiensis is able to assess and respond appropriately to the level of predation risk posed by the invasive G. holbrooki in a relatively short period of time. This finding corresponds to other studies which have reported the innate or learned abilities of invertebrates to respond to the cues of both familiar and non-familiar predatory fish (Paterson et al., 2013). However, we also provide the first evidence that even at low densities G. holbrooki exerts multiple simultaneous negative interactions on P. australiensis via direct predation, significant behavioural shifts and potentially competition for resources. It would be valuable to further extend this study in the future to reflect the natural temporal variation in both P. australiensis and G. holbrooki densities as has been observed in the field (Walsh and Mitchell, 1998, Pyke, 2008). As such, the existence of multiple interactions may make co-existence between these species more challenging and suggests that traditional management techniques for invasive species, including reducing densities may prove to be ineffective (Mills et al., 2004, Kornis et al.,

2014).

3 DON’T BE DENSE: THE EFFECT OF DENSITY ON INTERFERENCE COMPETITION BETWEEN A HIGHLY INVASIVE FISH AND ITS NATIVE COMPETITOR

3.1 Introduction

Biological invasions can have a significant deleterious effect on global biodiversity and

ecosystem functioning (Holway and Suarez, 1999, Mack et al., 2000, Gallardo et al., 2016).

Often invaders engage in interference competition with native species which can include

contests, territoriality, nest vandalism and aggression (Case and Gilpin, 1974, Human and

Gordon, 1996, Amarasekare, 2002, Zhang et al., 2015). Such direct interactions have been a

key factor in the success of numerous invasive species, particularly since they are frequently

more aggressive than their native counterparts and so may gain competitive dominance

(Holway and Suarez, 1999, Pintor et al., 2008, Hudina et al., 2014). Therefore, the ultimate

effect of interference competition may be the displacement of a native species as its access to

food, shelter and reproductive partners becomes increasingly limited (Amarasekare, 2002).

The intensity and outcome of interference competition between invasive and native species is

frequently context dependent (Kaiser et al., 2013, Hudina et al., 2015, Jackson et al., 2015).

The abundance of many invaders is temporally and spatially heterogeneous, which in turn

suggests that the intensity of their aggressive interactions with natives is similarly variable

(Hansen et al 2013). Aggressive interactions have typically been thought to increase with the

population of an invader due to a higher rate of contact with native competitors as well as a

reduction in resources relative to population size (Kaiser et al., 2013). However, competition

within an invasive population (intra-specific) is also likely to increase with density, which

may lead to a reduction in inter-specific competition, in turn promoting co-existence with

native competitors (Britton et al., 2011, Kornis et al., 2014). Therefore, in order to predict the effects of an invader’s density on native competitors, it is necessary to examine the relative effects of inter-specific and intra-specific aggression which both species experience (Inouye,

2001).

According to reviews of classical competition theory, behavioural interactions with con- specifics should be stronger than those with hetero-specifics, particularly at high population densities (Connell, 1983, Munday et al., 2001, Forrester et al., 2006). This is because con- specifics share a higher degree of resource overlap and may be viewed as greater rivals than hetero-specifics (Connell, 1983, Munday, 2004, Forrester et al., 2006). For example, both the coral reef dwelling bridled goby (Coryphopterus glaucofraneum) and goldspot goby

(Gnatholepis thompsoni) were reported to be at least twice as aggressive towards con- as hetero-specifics (Forrester et al., 2006). However, support for competition theory is overwhelmingly provided by studies which have observed species in their natural ranges

(Connell, 1983, Goldberg and Barton, 1992, Mangla et al., 2011), neglecting the possibility that the behaviour of invaders may be inherently different to that of native species (Hansen et al., 2013). For example, invasive Argentine ant populations are known to display reduced intra-specific aggression between nests compared to populations in native ranges (Tsutsui et al., 2000). This gives some support to the theory that to reach high densities, it is advantageous for an invasive species to be more aggressive toward hetero- than con-specifics

(Pintor et al., 2008), or in accordance with the lottery model (Sale, 1977), equally aggressive to both. However, since the relationship between intra- and inter-specific aggression and population density remains poorly studied, there is no current consensus as to which competition model invaders, or the native species they interact with, may conform to.

In addition to population density, interference competition may be mediated by individual- level traits, including size and sex (Stammler and Corkum, 2005, Britton et al., 2011). Body size is a critical factor in the competitive potential of individuals as it is a reliable indicator of fighting ability (Tatara and Berejikian, 2012), with larger individuals typically being more aggressive and subsequently occupying dominant positions (Theis et al., 2015). Therefore, native species which are on average larger than their invasive competitors may be able to dominate aggressive encounters (Garcia and Arroyo, 2002, Tatara and Berejikian, 2012).

Alternatively, species-specific differences in aggression may render body size differences irrelevant in inter-specific competition (Sanches et al., 2012, Little et al., 2013). In regards to population density, it has been theorised that larger individuals lose their competitive advantage as group size increases, since smaller individuals may become more aggressive when the resource value is high (Pettersson et al., 1996, Tatara and Berejikian, 2012, Theis et al., 2015). Yet size-mediated shifts in aggression in relation to population density have rarely been examined, particularly in regards to inter-specific aggression.

Sex-based differences in aggression are frequently reported with males typically being more aggressive than females: a trait associated with the ability to successfully defend territory and offspring (Johnsson et al., 2001, Cain et al., 2011). It is also understood that sex-based intra- specific aggression can vary with the relative densities of the sexes (Bisazza and Marin, 1995,

Smith and Sargent, 2006, Dadda et al., 2008). Whether and how context dependent sex-based aggression is important in mediating inter-specific interference competition, however, has seldom been explored (Cain et al., 2011). This is problematic considering that in order to understand competitive interactions between invasive and native species it is necessary to quantify the role of individual-level variables which may be of further importance when devising targeted control strategies for invasive species (Balshine et al., 2005, Stammler and

Corkum, 2005, Fryxell et al., 2015).

Here we investigated how inter- and intra-specific interference competition between the native Australian Bass, Macquaria novemaculeata, (Steindachner, 1966) and the invasive eastern mosquitofish, Gambusia holbrooki were mediated by population density, as well as by

M. novemaculeata body size and G. holbrooki sex and size. Since its introduction into

Australia in the 1920s, G. holbrooki has spread to all Australian states and territories (Pyke,

2008). Its aggressive behaviour, including fin-nipping, is frequently cited as a key mechanism behind its success as an invader (Pyke, 2008, Carmona-Catot et al., 2013b, Sutton et al., 2013,

Westhoff et al., 2013). Populations of G. holbrooki are known to experience seasonal fluctuations, with densities being highest following breeding in early autumn and lowest in spring (Barney and Anson, 1921). Juveniles of the catadromous M. novemaculeata are stocked extensively throughout the East Australian drainage system to support populations no longer able to reach estuaries and breed due to habitat modification (Cameron et al., 2012).

Since juvenile M. novemaculeata are hatchery bred and therefore naïve to other species

(Brown and Laland, 2001), there is the possibility that the success of stocking for this species is negatively impacted by inter-specific interference competition with G. holbrooki.

Since invasive species are typically reported to be more aggressive than native species we predicted that 1) G. holbrooki will be more inter-specifically aggressive than M. novemaculeata. Since M. novemaculeata is a native species in the present context and in accordance with competition theory, we expected that 2) M. novemaculeata will be more aggressive towards con-specifics than hetero-specifics at a high density, while in contrast, the invasive G. holbrooki would 3) be more aggressive towards hetero- than con-specifics at a high density. Finally, we predicted that 4) small M. novemaculeata will be more aggressive in high density conditions and 5) that male G. holbrooki will be more aggressive than females at both low and high density conditions. We expected that the frequency of submissive

behaviours for both M. novemaculeata and G. holbrooki will show the inverse of patterns predicted for aggression (Drummond, 2006, Taborsky et al., 2012).

3.2 Methods

Study Species and Acclimation

Adult eastern mosquitofish, Gambusia holbrooki, (Male, x̅ ± SE mass = 0.14 ± 0.01 g; x̅ ± SE standard length = 20.43 ± 0.20 mm; Female, x̅ ± SE mass = 0.14 ± 0.01 g; x̅ ± SE standard length = 20.01 ± 0.27 mm), were collected from freshwater ponds located on the University of

Wollongong campus (34° 24’ 19” S 150 ° 52’ 42” E) using a baited hand held landing net.

Juvenile Australian bass, Macquaria novemaculeata (x̅ ± SE mass = 0.97 ± 0.03 g; x̅ ± SE standard length = 36.17 ± 0.35 mm) of mixed sex were obtained from a hatchery (Aquablue

Seafoods, Pindimar, NSW, Australia). M. novemaculeata were 6.9 times heavier and 1.78 times longer than both male and female G. holbrooki. M. novemaculeata were not sexed as this can only be done post-mortem, which was not possible in this study. To conduct the experiment, four recirculating aquarium systems at the University of Wollongong were set up in temperature controlled rooms. Each system contained 8 aquaria (60 × 30 × 30 cm) that were inter-connected and subjected to water conditions held constant at 21 °C and 15 ppt.

Each aquarium was aerated with a bubbler, lined with 2 cm of sand and contained 1 PVC pipe

(15 × 6 cm) to provide shelter. M. novemaculeata were placed into 24 aquaria (N = 130 total fish) spread across 4 separate systems (N = 33 or N = 32 fish per system). G. holbrooki were placed into 8 aquaria (N = 140 total fish) spread equally across 4 separate systems (N = 35 fish per system). All fish remained under these conditions for the next 7 days to ensure adequate acclimation. During this time, both species were maintained at a 12:12 light: dark cycle and fed an equal amount of a commercial fish pellet (New Life Spectrum Thera formula) and frozen bloodworms daily.

Experimental Design

To test for inter- and intra-specific competition in G. holbrooki and M. novemaculeata under low and high densities of G. holbrooki, we established 3 experimental conditions: 1) mixed, in which aquaria contained both G. holbrooki and M. novemaculeata (N = 20 aquaria), 2) M. novemaculeata intra-specific, in which aquaria contained only M. novemaculeata (N = 20 aquaria) and 3) G. holbrooki intra-specific where only G. holbrooki were present in aquaria

(N = 20 aquaria). Within the mixed treatment 2 density levels of G. holbrooki were established - low (N = 10 aquaria, n = 1 individual) and high (N = 10 aquaria, n = 4 individuals), while the density of M. novemaculeata was held constant (n =2 individuals)

(Table 3.1). In the intra-specific experimental conditions, the total number of M. novemaculeata and G. holbrooki was the same as the total number of both species in the equivalent low (N = 10 aquaria) or high density (N = 10 aquaria) mixed treatment (Table 3.1).

A substitutive design was considered inappropriate as by changing the ratio of G. holbrooki to M. novemaculeata it would be impossible to accurately determine whether any differences in the response variables were due to changes in the overall density of the individuals of both species or the density of one species (Inouye, 2001). Densities were based on estimates of seasonal low and high G. holbrooki densities in Wollongong water bodies relative to the aquaria size used in this study (L.K. Lopez personal observations).

Table 3.1 The densities of Gambusia holbrooki and Macquaria novemaculeata used in this experiment investigating the effect of G. holbrooki density on inter- and intra-specific interference competition, N = 10 replicates. The behaviours of focal fish (marked with *) were recorded.

Mixed M. novemaculeata G. holbrooki Intra-specific Intra-specific

Low Density 1 G. holbrooki* 1 M. novemaculeata 1 G. holbrooki* 2 M. novemaculeata* 2 M. novemaculeata* 2 G. holbrooki

High Density 4 G. holbrooki* 4 M. novemaculeata 4 G. holbrooki* 2 M. novemaculeata* 2 M. novemaculeata* 2 G. holbrooki

Behavioural Observations

Following the acclimation period, each M. novemaculeata and G. holbrooki were captured using hand nets, measured using hand- held callipers, (mm standard (SL) ± 0.1 mm) and weighed with an electronic balance (grams (g) ± 0.1 g). To enable the identification of individuals and control for any effect of sex, size and pregnancy, each fish was tagged with a polymer elastomer (yellow, green, red, pink and orange) (Northwest Industries Ltd) which was injected into the dorsal musculature (Malone et al., 1999).

In the mixed and G. holbrooki control conditions, 1 G. holbrooki (low density) or 4 G. holbrooki (high density) were introduced into a test aquarium 24 hours prior to the addition of

2 focal G. holbrooki or M. novemaculeata, respectively, heretofore referred to as ‘intruders’

(Table 3.1). In the M. novemaculeata control condition, 1 M. novemaculeata (low density condition) or 4 M. novemaculeata (high density condition) were introduced into a test aquarium 24 hours prior to the addition of 2 focal M. novemaculeata (Table 3.1). This order of residency was intended to simulate the temporal occurrence of species in the event of fish stocking whereby G. holbrooki is present prior to the introduction of M. novemaculeata.

Observations commenced after a 30 minute acclimation period followed by a 5 minute adjustment to the presence of the observer. Only one person (LKL) observed the fish to avoid potential issues with observer bias. Due to the fact that each test aquarium contained a different species composition or number of fish it was impossible to observe the animals blind. Each focal individual (see Table 3.1) was observed for 10 minutes during which time the number of behavioural interactions between other individuals was recorded (Matthews and Wong, 2015, O'Mara and Wong, 2015). Interactions were categorised into aggressive

(approach, chase, nip) and submissive (retreat) behaviours (Table 3.2). To account for diurnal variation in behaviour the same fish were observed once in the morning (0900-1200) and once in the afternoon (1300-1600). Therefore, the observed fish were exposed to each other for a maximum of 7 hours after which they were separated and placed back into separate aquaria.

Table 3.2 Ethogram of aggressive and submissive behaviours observed in Macquaria novemaculeata and Gambusia holbrooki in this experiment.

Type of Behaviour Behaviour Description Fish orients its self and swims toward another Aggressive Approach (Ap) fish.

Chase (Ch) Fish sustains an approach towards the fleeing fish.

Nip (Ni) Fish approaches and nips another fish.

Submissive Retreats (Re) Fish retreats and swims away from another fish.

Statistical Analysis

G. holbrooki were attacked and consumed by M. novemaculeata in the low (n = 1) and high

(n = 6) density mixed conditions and therefore behavioural data collected from these individuals was excluded from all subsequent analyses. All statistical analyses were performed using SPSS Statistics 21, IBM, Armonk, NY, U.S.A. No significant difference in aggression was detected for M. novemaculeata and G. holbrooki between the AM and PM observation times. Therefore the summed data from these periods was used in the analysis

(GLMM, M. novemaculeata, aggression; Time, F1,151 = 0.766, p = 0.383; submission; F1,151 =

0.000, p = 0.985; G. holbrooki, aggression; F1,170 = 0.079, p = 0.779; submission; F1,170 =

0.953, p = 0.330).

To determine the effects of individual body size on behavioural interactions in M. novemaculeata, the size ratio of each focal individual to its con-specific focal individual was calculated (SL M. novemaculeata 1/ SL M. novemaculeata 2, where SL was standard length).

The larger individual was categorised as ‘large’, while the remaining individual was categorised as ‘small’. The standard length of large individuals was 4.35 ± 0.52 mm (x̅ ± SE) greater than that of small con-specifics. To test the effects of condition, density and size on the frequency of inter- and intra-specific interactions for M. novemaculeata, a Generalised

Linear Mixed Model (GLMM) with a Poisson distribution and log-link function was used, which is effective for analysing count data (Bolker et al., 2009). The model also used a robust estimation which accounts for any violations of model assumptions (Cameron and Trivedi,

2009). The model included condition, density and size (fixed) and, to account for the non- independence of interacting groups of fish, Tank ID (random). Means were compared using simple contrast post-hoc tests. The dependent variables were aggression given and submission given. Where a significant three-way interaction between size and other fixed factors was found, separate GLMMs which included condition and density (fixed) and Tank ID (random)

as factors were performed for each size class. To determine if small and large M. novemaculeata responded differently to male and female G. holbrooki, a GLMM with a

Poisson distribution and log-link function was used which included density, size and G. holbrooki sex (fixed) and Tank ID (random) as factors and means were compared using a simple post-hoc test. Following application of the Bonferroni method, the alpha value was adjusted to 0.016.

To determine the effects of condition, density and sex on the frequency of inter- and intra- specific interactions for G. holbrooki, a GLMM with a Poisson distribution, log-link function and robust estimation was used. The model included condition, density and sex (fixed) and tank ID (random) as factors. The dependent variables were aggression given and submission given. Where a significant three-way interaction between condition, density and sex was found, separate GLMMs which included condition and density (fixed) and Tank ID (random) as factors were performed for each sex and means were compared using a simple contrast post-hoc test. To determine the effects of individual body size on behavioural interactions experienced by intruder G. holbrooki in control conditions, the size ratio of each individual to its con-specific focal individual was calculated (SL G. holbrooki 1/ SL G. holbrooki 2). From this metric intruder G. holbrooki were categorised as ‘large’ or ‘small’. The difference in standard length between large and small individuals was 2.26 ± 0.24 mm (x̅ ± SE). In order to determine if male and female G. holbrooki responded differently to M. novemaculeata based on size and other G. holbrooki based on sex and size, 3 GLMMs with Poisson distributions and loglink functions were used. The first model included density, G. holbrooki sex and M. novemaculeata size (fixed) and Tank ID (random) as factors. The second model consisted of density, G. holbrooki sex and intruder G. holbrooki size (fixed) and Tank ID (random) as factors and the third included density, G. holbrooki sex and intruder G. holbrooki sex (fixed)

and Tank ID (random) as factors and means were compared using simple post-hoc contrasts.

Following application of the Bonferroni method, the alpha value was adjusted to 0.01.

Ethical Note

At the conclusion of the experiment, G. holbrooki were euthanased using clove oil since it is illegal to release an invasive species under NSW law. M. novemaculeata were used in another experiment covered by a separate protocol. Animal collection and husbandry, observation data collection and euthanasia protocols were approved by the University of Wollongong

Animal Ethics Committee (protocol AE14/07) and adhered to the Scientific Collection guidelines (permit No. P13/0011-1.3) of the NSW Department of Primary Industries.

3.3 Results

3.3.1 Aggression Given

Comparison of Interspecific aggression by M. novemaculeata and G. holbrooki

M. novemaculeata displayed approximately 6 times more inter-specific aggression than G. holbrooki in the high density treatment. At a low density, M. novemaculeata and G. holbrooki were equally aggressive (GLMM, Density×Species, F1, 81 = 9.742, p = 0.002) (Figure 3.1).

10

9

8

7

6

5

4 AggressionGiven 3

2 1

0 Low Density High Density

Figure 3.1 Mean (± SE) inter-specific aggression from G. holbrooki (open bars) and M. novemaculeata (closed bars) at low and high density treatment conditions, n = 10.

The effect of size, density and treatment on aggression given by M. novemaculeata

Both large and small M. novemaculeata were more aggressive to G. holbrooki than to con- specifics at a high density, yet showed no such preference under low density conditions

(Large; Treatment×Density, F3, 36 = 24.686, p <0.0001; Small; Treatment×Density, F1,36 =

12.631, p = 0.001) (Figure 2). Small M. novemaculeata were twice as aggressive as large individuals, but only at a high density of G. holbrooki (GLMM, Treatment×Density×Size, F4,

72 = 12.230, p <0.0001) (Figure 3.2).

35

30

25

20 15

AggressionGiven 10

5

0 Low Density High Density Low Density High Density Large Small

Figure 3.2 Mean (± SE) of inter- (solid line) and intra-specific (broken line) aggression from large and small M. novemaculeata at low and high density conditions, n = 10.

Large and small M. novemaculeata were equally aggressive to male and female G. holbrooki regardless of density (Size×Density×Sex, F2,42 = 0.000, p = 1.000) (Figure 3.3).

60

50

40

30

Aggression Given Aggression 20

10

0 Large Small Large Small

Low Density High Density

Figure 3.3 The average (± SE) frequency of aggressive behaviours performed by large and small M. novemaculeata to male (open bars) and female (closed bars) G. holbrooki at low and high density treatment conditions, n = 10.

The effects of size, sex, density and treatment on aggression given by G. holbrooki

At a low density, male and female G. holbrooki were more aggressive toward con-specifics than M. novemaculeata. However, when present at a high density, equal aggression to con- specific and hetero-specific intruders was observed (Male; Treatment×Density, F1, 43 =

49.187, p <0.0001; Female; Treatment×Density, F1, 42 = 17.862, p <0.0001) (Figure 4). Male

G. holbrooki were approximately 3 times more aggressive than females, but only at a low density of con-specifics (Figure 3.4) (GLMM, Treatment×Density×Sex, F3, 85 = 14.899, p

<0.0001).

60

50 40

30

20

Given Aggression 10

0 Low Density High Density Low Density High Density Male Female

Figure 3.4 Mean (± SE) of inter- (solid line) and intra-specific (broken line) aggression from male and female G. holbrooki at low and high density conditions, n = 10.

Male and female G. holbrooki were equally aggressive toward large and small M. novemaculeata, regardless of density (GLMM, Aggressor Sex×Receiver Size×Density, F4, 84

= 0.540, p = 0.707; Aggressor Sex×Receiver Size, F2,84 = 0.023, p = 0.977) (Figure 3.5a). In regards to intra-specific aggression, however, male G. holbrooki were found to be more aggressive toward small than large G. holbrooki (Aggressor Sex×Receiver Size×Density, F5,

92 = 22.10, p <0.0001) (Figure 3.5b). There was also evidence of sex-based competitor preferences at the low density control condition whereby male G. holbrooki were approximately twice as aggressive to female compared to male con-specifics, while no difference existed in the high density control (Aggressor Sex×Receiver Sex×Density, F3,90 =

23.825, p <0.0001) (Figure 3.5c).

80 a) 70 60

50 40 30

Aggression Given Aggression 20 10 0 Male Female Male Female Low Density High Density 80 b) 70 60 50 40 30 20

Aggression Given Aggression 10 0 Male Female Male Female Low Density High Density 80 c) 70 60 50

40 30 20

Aggression Given Aggression 10 0 Male Female Male Female Low Density High Density Figure 3.5 Mean (± SE) frequency of aggressive behaviours performed by male and female G. holbrooki to a) large (open bars) and small (closed bars) M. novemaculeata, b) large (open bars) and small (closed bars) G. holbrooki and c) male (open bars) and female (closed bars) G. holbrooki under low and high density conditions, n = 10.

3.3.2 Submission Given

G. holbrooki performed more submissive retreats than M. novemaculeata from hetero- specifics at both low and high density treatments (GLMM, Species, F1, 79 = 81.596, p <

0.0001). M. novemaculeata were found to retreat more from a high than low density of G. holbrooki (Density×Species, F1, 79 = 14.052, p < 0.0001) (Figure 3.6).

12

10

8

6

Submission given 4

2

0 Low Density High Density

Figure 3.6 Mean (± SE) inter-specific submission given by G. holbrooki (open bars) and M. novemaculeata (closed bars) at low and high density conditions, n = 10.

The effect of size, density and treatment on submission given by Macquaria

novemaculeata

No difference in submission was observed between large and small M. novemaculeata regardless of experimental condition (Treatment×Density×Size, F4, 72 = 1.152, p = 0.339). M. novemaculeata retreated significantly more from a high density of G. holbrooki than from con-specifics, while the opposite pattern was observed under low density conditions

(Treatment×Density, F3, 76 = 13.72, p < 0.0001) (Figure 3.7), and both size classes were

equally submissive to male and female G. holbrooki (Size×Density×Sex, F1, 44 = 0.000, p =

0.999) (Figure 3.8).

6

5

4

3

2 Submission Given

1

0 Low Density High Density Low Density High Density Large Small Figure 3.7 Mean (± SE) of inter- (solid line) and intra-specific (broken line) submission given by large and small M. novemaculeata at low and high density conditions, n = 10.

4

3.5

3

2.5

2

1.5 Submission Submission Given 1

0.5

0 Large Small Large Small Low Density High Density Figure 3.8 The average (± SE) frequency of submissive retreats performed by large and small M. novemaculeata to male (open bars) and female (closed bars) G. holbrooki at low and high density treatment conditions, n = 10.

The effect of size, sex, density and treatment on submission given by Gambusia

holbrooki

Male and female G. holbrooki did not significantly differ in their patterns of submission

(GLMM, Treatment×Density×Sex, F6, 85 = 2.802, p = 0.015, Bonferroni correction). Both sexes performed significantly more retreats when exposed to M. novemaculeata than con- specifics, regardless of density (Treatment, F1, 91 = 32.51, p < 0.0001) (Figure 3.9).

12

10

Given 8

6

Submission 4

2

0 Low Density High Density Low Density High Density Male Female

Figure 3.9 Mean (± SE) of inter- (solid line) and intra-specific (broken line) submission given by male and female G. holbrooki at low and high density conditions, n = 10.

Both male and female G. holbrooki retreated more from small than from large M. novemaculeata, but only under high density conditions (GLMM, Density×M. novemaculeata

Size, F3,82 = 3.011, p = 0.035; Sex, F1, 81 = 2.042, p = 0.157) (Figure 3.10a) in regards to intra- specific submission, both sexes were more submissive to large con-specifics in the high density control condition (Density×G. holbrooki Size, F1, 94 = 8.52, p = 0.004) (Figure 3.10b).

Male G. holbrooki retreated significantly more from male con-specific intruders, yet only under low density conditions. Female fish displayed equal levels of submission to male and female intruders, regardless of density (Density×Submissive Sex×Aggressor sex, F4, 64 =

6.074, p < 0.0001) (Figure 3.10c).

20 18 a) 16 14 12

10 8

6 Submissive Submissive Given 4 2 0 Male Female Male Female Low Density High Density 20

18 b) 16 14 12 10 8

6 Submissive Submissive Given 4 2 0 Male Female Male Female

Low Density High Density 20 c) 18 16 14 12 10 8

6 Submissive Submissive Given 4 2 0 Male Female Male Female Low Density High Density

Figure 3.10 Mean (± SE) frequency of submissive retreats performed by male and female G. holbrooki from a) large (open bars) and small (closed bars) M. novemaculeata, b) large (open bars) and small (closed bars) G. holbrooki and c) male (open bars) and female (closed bars) G. holbrooki under low and high density conditions, n = 10.

3.4 Discussion

There is no current consensus as to how population density mediates the intensity of interference competition between invasive and native species (Yokomizo et al., 2009), even though aggression is key mechanism behind the success of many non-native species (Holway et al., 1998, Holway and Suarez, 1999, Tsutsui et al., 2000, Pintor et al., 2008). Furthermore, the role of size and sex in mediating aggression at different densities is poorly understood, despite the fact that identifying individual-level variation may assist in the design of more targeted management strategies (Knell, 2009). Here we show that the native Australian bass,

Macquaria novemaculeata is more inter-specifically aggressive than high densities of the invasive eastern mosquitofish, Gambusia holbrooki. In contrast to what was predicted, M. novemaculeata was more aggressive toward a high density of hetero- than con-specifics. Also unexpectedly, G. holbrooki displayed equal levels of inter- and intra-specific aggression at a high density, yet was more aggressive toward con- than hetero-specifics when at a low density. We also demonstrate that body size and sex interacted with density to modulate patterns of aggression and submission for both M. novemaculeata and G. holbrooki. These results emphasise the importance of considering both inter- and intra-specific interactions when exploring the influence of density upon interference competition between invasive and native species, as well considering individual-level variables.

In contrast to our first prediction, the native M. novemaculeata was more inter-specifically aggressive than G. holbrooki was in the high density treatment. This result was unexpected, given that previous studies have frequently reported aggression from G. holbrooki to exceed that of its native competitors (Howe et al., 1997, Rincon et al., 2002, Carmona-Catot et al.,

2013a). In cases of asymmetric competition, however, the larger species is often the more aggressive (Moretz, 2003, Little et al., 2013) and in the present study M. novemaculeata were

on average 1.67 times larger than G. holbrooki. Similarly, Garcia and Arroyo (2002) reported the hen harrier raptor (Circus cyaneus) to be both inter-specifically more aggressive and larger than the Montagu harrier (C. pygargus). It is likely that since M. novemaculeata were larger and also juveniles, they had a greater energetic requirement (Bystrom and García-

Berthou, 1999, Bystrom et al., 2006, Ohlberger et al., 2012), and therefore placed a higher resource value on food where G. holbrooki density was high. This result would suggest that the size difference between M. novemauleata and G. holbrooki is instrumental in determining competitive asymmetry.

In order to understand the effect of density dependent inter-specific aggression, it is also necessary to consider the strength of intra-specific interactions (Connell, 1983, Inouye, 2001,

Forrester et al., 2006). In opposition to our second prediction, the magnitude of inter-specific aggression and submission displayed by M. novemaculeata in high density conditions exceeded that of its intra-specific interactions. The intensity of interference competition is often predicted to increase with the invader’s density, due to the simultaneous increase in contact rate and a decrease in resource availability (Yokomizo et al., 2009, Britton et al.,

2011). However, this result contradicts competition theory which states that the strength of intra-specific interactions will be greater than inter-specific interactions at high competitor densities, due to conspecifics having a greater level of resource overlap (Connell, 1983,

Forrester et al., 2006). Even so, it is important to note that the majority of studies which support competition theory have observed interactions between species in their native ranges and thus do not account for invasive-native species dynamics. Indeed, inter-specific competition has been observed to be greater or equal to intra-specific competition for some native species competing with invaders (Tsutsui et al., 2000, Warnock and Rasmussen, 2013).

For example, Warnock and Rasmussen (2013) reported that the autochthonous bull trout

(Salvelinus confluentus) diverted more aggression toward a high density of the non-native

brook trout (S. fontinalis) than con-specifics. Therefore it is possible that the elevated levels of aggression often reported in invasive species may result in competition dynamics which do not conform to those commonly observed in autochthonous species. Our results illustrate the need for further research on density dependent interactions between native species and invaders which also consider the magnitude of intra-specific interactions.

Considering that the disparity in body size most likely mediated inter-specific aggression between M. novemaculeata and G. holbrooki, it is necessary to consider the role of size in determining intra-specific aggression and submission for M. novemaculeata. If an increase in resource value at high competitor densities led to increased inter-specific aggression in this study, a comparable or greater increase in intra-specific aggression may also be expected.

However, aggression is a costly behaviour and the risk of injury as a result of direct interactions typically increases for an individual with the relative body size of their competitor (Huntingford et al., 1990, Garcia and Arroyo, 2002, Herrel et al., 2009). In accordance with our third prediction, small M. novemaculeata were more aggressive under high density conditions, albeit only toward G. holbrooki and not con-specifics. Given that M. novemaculeata exposed to con-specifics did not have the size advantage of those exposed to

G. holbrooki, it is possible that their aggression was limited so as to lessen the risk of injury, despite the resource value still being high. Therefore, the effect of body size upon aggression in M. novemaculeata depended on density as well as competitor identity.

It has been hypothesised that invasive species display lower levels of intra- compared to inter- specific aggression at high densities, since this would reduce the likelihood of population self- regulation (Tsutsui et al., 2000, Pintor et al., 2009). In contrast to our fourth prediction, however, G. holbrooki were equally aggressive to a high density of M. novemaculeata and con-specifics, suggesting that both represented the same level of threat, as per the lottery

model (Sale, 1977). While this result contradicts studies which report stronger inter- than intra-specific aggression in invaders, (Tsutsui et al., 2000, Warnock and Rasmussen, 2013), there is also evidence demonstrating that invaders can be equally aggressive to con- and hetero-specifics (Kalinoski, 1975) or aggressive more so toward con- than hetero-specifics

(Sutton et al., 2013). Considering that M. novemaculeata may have been more aggressive towards hetero-specifics than was G. holbrooki because they were larger, it follows that G. holbrooki may have limited its aggressive behaviour toward M. novemaculeata in order to reduce the risk of injury or predation. Furthermore, neither aggression nor submission from G. holbrooki was influenced by M. novemaculeata size, which suggests that even small hetero- specifics had a significant size advantage. However, it is worth noting that inter-specific size differences do not always determine the relative strengths of inter- and intra-specific competition observed in invaders. For example, brook trout (S. fontinalis) were equal in size to bull trout (S. confluentus) yet more inter-specifically aggressive. In contrast, while house sparrows (Passer domesticus) were larger than house finches (Carpodacus mexicanus) yet equally aggressive to con- and hetero-specifics (Kalinoski, 1975). Therefore, while size was likely a key factor in the present study, when considered in the context of the literature our result also demonstrates that the outcomes of invasive-native interactions are frequently species-specific (Yokomizo et al., 2009).

Finally, male G. holbrooki were more aggressive than females in low density control conditions, and directed most of their aggression toward con-specifics which were smaller or female. Since female G. holbrooki were approximately 0.47 times smaller in length than males in low density conditions (Appendix A), this result suggests that male sexual aggression was a driver behind elevated intra-specific aggression at a low density. This theory is supported by the fact that male G. holbrooki in the low density control were predominantly submissive to other males, rather than females. Male sexual harassment in mosquito fish has

previously been shown to peak at low densities, since it is diluted in larger shoals of mosquitofish (Dadda et al., 2008). This is due to both male mating attempts being spread out over a higher number of females as well as suppression by a male hierarchy, though this is dependent on the operational sex ratio, which was not examined in the present study (Dadda et al., 2008). Therefore, a valuable avenue of future research would be to explore how sex ratio variation in conjunction with density influences intra-sexual aggression in this species.

Heightened sexual aggression has been shown to reduce the efficiency of important behaviours in female mosquitofish, including foraging (Pilastro et al., 2003). This result, along with the present study, suggests that male sexual aggression may be a limiting factor for mosquitofish populations at low densities. Therefore, the dilution of intra-sexual behaviour may in fact assist this species to exist in high abundance. However, it is important to note that male and female G. holbrooki did not significantly differ in submission behaviours. This is surprising since males are often considered to be less risk adverse than females (Johnsson et al., 2001, Piyapong et al., 2010). However, since males were only more aggressive than females in low density control conditions, which is likely sexually motivated, it follows that they would have been unlikely to flee an attempt to copulate with a female (Dadda et al.,

2008).

In conclusion, the present study indicates that M. novemaculeata juveniles will experience greater interference competition from a high than low density of G. holbrooki. Since M. novemaculeata were more aggressive than G. holbrooki at a high density, it is likely that the costs of such interactions, including injury and stress may in fact be lower for the native species than the invasive. However, M. novemaculeata may experience a decrease in fitness in the presence of a high density of G. holbrooki, as increased inter-specific aggression can also reduce time available for foraging or increase vulnerability to predation (Zhang et al.,

2015), which is less likely to occur in the presence of con-specifics only. This study also

demonstrates the importance of considering individual-level traits when examining mechanisms underlying inter- and intra-specific competition.

4 DENSITY DEPENDENT COMPETITION BETWEEN THE INVASIVE EASTERN MOSQUITOFISH, GAMBUSIA HOLBROOKI AND AUSTRALIAN BASS, MACQUARIA NOVEMACULEATA

4.1 Introduction

Invasive species pose a significant threat to global biodiversity and the preservation of natural

resources (Mack et al., 2000, Pintor and Sih, 2009, Strayer, 2010, Simberloff, 2014).

Freshwater systems are especially vulnerable to biological invasions due to their close

association with human societies and limited connectivity (Sato et al., 2010, Cucherousset and

Olden, 2011). Invasive species can exert significant negative impacts upon native individuals,

species and communities by engaging in competition for resources including food and habitat

(Human and Gordon, 1996, Bergstrom and Mensinger, 2009, Britton et al., 2009, Hentley et

al., 2016). In some instances, invaders are superior competitors to their native counterparts

(Hudina and Hock, 2012, Otturi et al., 2016), and may achieve competitive dominance by

having a higher relative abundance, exploiting resources more effectively or via direct

interference which can lead to asymmetrical competition (Ruetz et al., 2003, Britton et al.,

2011). This can result in significant reductions in the growth, fitness and survivorship of

native species, which may ultimately lead to their displacement (Strayer, 2010, Young et al.,

2010, Weis, 2011, Sowersby et al., 2016). Despite these implications, the ecological effects of

competition can be difficult to demonstrate, and the mechanisms underlying species

displacement are poorly understood (Holway and Suarez, 1999, Karlson et al., 2007,

Schumann et al., 2015).

Many invaders demonstrate significant temporal and spatial heterogeneity in abundance and so the strength of their competitive interactions may also vary (Hansen et al., 2013, Latzka et al., 2016). Therefore, it is necessary to consider the mediating role of population density

(Kaspersson et al., 2010, Jackson et al., 2015, Hasegawa, 2016). Commonly, the negative impacts of an invasive competitor are thought to increase with its density (Yokomizo et al.,

2009), as a function of both declining resource availability and an increased rate of contact with native species (Kaspersson et al., 2010, Kaiser et al., 2013, Hasegawa, 2016). However, the density-impact relationship for many invasive species remains to be quantified (Yokomizo et al., 2009). Furthermore, the few studies which have done so have reported conflicting evidence suggesting that for some invaders an increase in their density may in fact lower the strength of their competitive interactions with native species (Yokomizo et al., 2009, Kornis et al., 2012). This may be due to a rise in intra-specific competition, which could have a self- regulating effect on an invasive species’ population (Holway et al., 1998, Cross and Benke,

2002, Kornis et al., 2014). For example, Kornis et al. (2014) reported that growth rates of the invasive round goby, Neogobius melanostomus, were reduced at high population densities.

Notably, growth of the sympatric native species, the white sucker, Catostomus commersonii, and johnny darter, Etheostoma nigrum, did not differ between treatments containing a high density of N. melanostomus or an invader free control (Kornis et al., 2014). Therefore, increased competition within a population of an invader may reduce the strength of inter- specific competition, in turn promoting coexistence between invasive and native species

(Britton et al., 2011, Kornis et al., 2014).

As stated by the competition theory, the strength of intra-specific competition is typically thought to exceed that of inter-specific competition, particularly at high population densities

(Connell, 1983). This is due to the fact that con-specifics are expected to share a greater niche overlap than hetero-specifics, which would suggest that con-specifics are viewed as greater

rivals for resources (Connell, 1983, Forrester et al., 2006). While support for this dynamic is predominantly derived from studies which examine species within their native ranges, there is some evidence that competitive interactions for invaders also conform to this model. For example, growth of the invasive brown trout, Salmo trutta, was higher when exposed to Rio grande cutthroat trout, Oncorhynchus clarkii, than to con-specifics (Shemai et al., 2007).

In contrast, the lottery model predicts that the effects of inter- and intra-specific competition should be approximately equal if neither species is competitively dominant (Sale, 1977). This was reported for the non-native brook trout, Salvelinus fontinalis, the growth of which was equal in treatments containing con-specifics or the native bull trout S. confluentus (Gunckel et al., 2002). Alternatively, it is possible that invasive species will experience more significant effects of competition from hetero- than con-specifics (Pintor et al., 2008). While this dynamic is more compatible with the high population densities observed in some invaders as it would reduce self-regulating effects and promote competitive dominance over other species

(Pintor et al., 2008), little evidence in support of this model has been reported. Currently the lack of knowledge pertaining to which competition model is most frequently observed for both invasive and native competitors inhibits both the effective management of invasive species as well as the contribution of invasion ecology to fundamental science.

Given the important role that intra-specific interactions may have in mediating inter-specific competition, it is necessary to measure the relative magnitudes of both interactions (Inouye,

2001, Forrester et al., 2006, Rauschert and Shea, 2012, Barabas et al., 2016). However, most studies thus far, including those previously mentioned in this chapter, have used experimental designs which simultaneously manipulate the densities of con- and hetero-specifics (e.g. substitutive or additive designs). This prevents an independent assessment of the relative importance of intra- and inter-specific competitive interactions (Inouye, 2001, Damgaard,

2008). Therefore in the present study a form of the response surface design was employed which is an appropriate method to assess the relative magnitudes of intra- and inter-specific competition (Inouye, 2001).

In addition to population density, individual level traits including sex may play a key role in determining the intensity of both intra- and inter-specific competition. Many species display pronounced morphological and behavioural sexual dimorphism (Shine, 1989, Fryxell et al.,

2015). Specifically this can include differences in body size (Brose et al., 2006), feeding rates per unit body size (Brose et al., 2006, Fryxell et al., 2015), diet breadth (Bence and Murdoch,

1986), habitat preference (Alto et al., 2005), risk perception and aggression (Shine, 1989); all traits which could influence their ability to compete with hetero- and con-specifics (Pilastro et al., 2003, Arrington et al., 2009, Kishi et al., 2009). Therefore, sex ratio variation can significantly mediate the ecological impact of an invasive species (Fryxell et al., 2015).

Despite this, variation in sex ratio following exposure to inter- and intra-specific competition is seldom examined, which obscures the ability to better predict the population dynamics of invaders (Pilastro et al., 2003, Fryxell et al., 2015).

The aim of this study was to determine if population density mediates competition between the invasive eastern mosquitofish, Gambusia holbrooki and juveniles of the native Australian bass, Macquaria novemaculeata. G. holbrooki is a poeciliid species native to the south eastern

United States of America which was introduced into Sydney in the 1920’s and is now present in 8 out of 11 main catchments in Australia (Pyke, 2008). While G. holbrooki has been associated with the decline of numerous native fish species, its role as a competitor has seldom been examined in Australia (Pyke, 2008). G. holbrooki is typically observed in shallow, warm and slow-moving water bodies where it subsists on algae, detritus, zooplankton and invertebrates (Pyke, 2008). Juveniles, also known as fingerlings, of M.

novemaculeata are currently stocked extensively throughout the Australian eastern drainage system in artificial and natural waterbodies (Cameron et al., 2012). Many populations of this catadromous species are unable to reach the estuaries required for breeding as a result of habitat modification, and so their presence in some waterbodies is dependent on the annual stocking of fingerlings (Cameron et al., 2012). Juvenile M. novemaculeata are known to feed on insects, crustaceans and small fish and inhabit fast or slow moving water as shallow as 1 m in depth (Harris, 1986). Considering that M. novemaculeata shares its distribution with G. holbrooki, is similar in size to adult G. holbrooki and being hatchery bred is naïve to other species (Brown and Laland, 2001), it is possible that G. holbrooki limits the success of M. novemaculeata stocking programs.

For M. novemaculeata and G. holbrooki, the effect of density on the relative strengths of intra- and inter-specific competition were assessed by measuring growth and survivorship under experimentally altered densities. In addition to this, changes in the sex ratio of G. holbrooki and the growth of each sex were compared between treatments to determine if the sexes differed in their competitive abilities at different densities and species compositions. It was predicted that 1) as per competition theory, survivorship and growth of the native M. novemaculeata would be lowest when exposed to a high density of con-specifics. In contrast,

2) the survivorship and growth of the invasive G. holbrooki would be lowest when exposed to a high density of M. novemaculeata. Finally, since male G. holbrooki are typically more aggressive than females (Carmona-Catot et al., 2013a) and so may have more frequent direct contact with M. novemaculeata, it was predicted that 3) sex ratios of G. holbrooki exposed to

M. novemaculeata would become female biased.

4.2 Methods

Animal Collection, Experimental Set up and Data Collection

The experiment was conducted between the 21st of November and the 18th of December 2014.

Juvenile Australian bass, Macquaria novemaculeata (x̅ ± SE standard length = 22.30 ± 0.21 mm) of both sexes were acquired from a private hatchery (Aquablue Seafoods, Pindimar,

NSW Australia) (Figure 4.1a). Adult eastern mosquitofish, Gambusia holbrooki (female; x̅ ±

SE standard length = 21.53 ± 0.34 mm, male; x̅ ± SE standard length = 18.44 ± 0.0.21 mm) were collected from freshwater ponds located on site using a baited hand held landing net

(Figure 4.1b). Male G. holbrooki were identifiable by the presence of a gonopodium on the anal fin, while mature females have solely an anal fin (Pyke, 2008). Pregnant G. holbrooki, which have a dark brood patch on their abdomen (Pyke, 2008), were included in the study and a note of their condition was made. To house M. novemaculeata and G. holbrooki, 26 floating fish cages were set up in an artificial freshwater pond (average depth = 8 m) at Dunmore

Quarry, NSW, Australia (34° 36’ 32.15” S, 150° 50’ 38.99” E) (Figure 4.1c). The cages consisted of a PVC pipe frame (1 m² area by 1.5 m in height), foam floats and white nylon netting (2 mm) and were fitted with a mesh lid to prevent fish escaping (Figure 4.1d). Two

PVC pipes (15 × 7 cm) and algae (collected on site) were placed in each cage to provide shelter for both M. novemaculeata and G. holbrooki. During the experiment G. holbrooki and

M. novemaculeata were fed a commercial fish flake (New Life Spectrum Thera formula) and bloodworms ad libitum three times each week. Water parameters (temperature, salinity, pH, nitrates, nitrites and phosphates) were recorded three times a week (Table 4.1).

a) b)

c) d)

Figure 4.1 a) Macquaria novemaculeata fingerlings; b) measuring the standard length of a

Gambusia holbrooki; c) the experimental setup with floating aquaculture cages at Dunmore

Quarry and d) close up of the aquaculture cages.

Table 4.1 Mean (± SD) water quality parameter recordings at the experimental site during the course of the study, n = 10.

Temperature Salinity Nitrate Nitrite Ammonia pH (°) (ppt) (ppm) (ppm) (ppm)

25.3 ± 1.46 1.05 ± 1.8 7.72 ± 0.07 0 ± 0 0 ± 0 0.06 ± 0.11

Experimental Design and Data Collection

To quantify the effects of fish density and species composition on G. holbrooki and M. novemaculeata survivorship and growth, three experimental conditions were established: 1) mixed, in which cages (N = 12) contained both G. holbrooki and M. novemaculeata; 2) M. novemaculeata intra-specific, for which cages (N = 12) only contained M. novemaculeata and

3) G. holbrooki intra-specific, for which cages (N = 12) only contained G. holbrooki. Within the mixed condition, two density levels of G. holbrooki were established- low density (N = 6 cages, n = 10 individuals) and high density (N = 6 cages, n = 50) with a constant density of M. novemaculeata (n = 5 individuals per cage) (Table 4.2). Within the intra-specific conditions the total number of M. novemaculeata or G. holbrooki was equal to the total number of M. novemaculeata and G. holbrooki in the equivalent low and high density mixed condition

(Table 4.2). This design was used as it allowed the relative effects of inter- and intra-specific competition to be measured (Inouye 2001). Experimental conditions were randomly assigned to cages. Given that few studies have quantified G. holbrooki densities in Australian waterways and use varying methods to do so (Pyke, 2008), treatment densities in the present study were based upon estimates of low and high G. holbrooki abundances in local closed waterbodies (L.K. Lopez, personal observations).

Table 4.2 The densities of M. novemaculeata and G. holbrooki used in experimental conditions of this study, n = 6.

Mixed M. novemaculeata G. holbrooki Intra- Intra-specific specific

Low Density 10 G. holbrooki 10 M. novemaculeata 10 G. holbrooki 5 M. novemaculeata 5 M. novemaculeata 5 G. holbrooki

High Density 50 G. holbrooki 50 M. novemaculeata 50 G. holbrooki 5 M. novemaculeata 5 M. novemaculeata 5 G. holbrooki

At the beginning of the experimental period (T0), the standard lengths of G. holbrooki and M. novemaculeata was measured using hand-held callipers (Figure 4.1b). G. holbrooki were introduced into the treatment cages a week before M. novemaculeata. This order of residency was intended to simulate the temporal occurrence of species when M. novemaculeata is stocked into a water body. Since all M. novemaculeata were delivered at the same time from our supplier, this order of residency was unable to be replicated in M. novemaculeata control conditions. After a 2 week period (T1), M. novemaculeata and G. holbrooki were removed from low and high density treatment and control cages. The number of recovered M. novemaculeata and G. holbrooki were recorded and measured again. The experimental period was fourteen days, since M. novemaculeata are considered to be most vulnerable to G. holbrooki shortly after stocking since they are at their smallest size and naïve (NSW

Fisheries, 2003).

Statistical Analysis

All analyses were conducted using SPSS Statistics 21, IBM, Armonks, NY, U.SA. The normality of the data was checked visually using normal quantile plots. The residuals of the length data for both M. novemaculeata and G. holbrooki was plotted and found to conform to a normal distribution. The survivorship and sex ratio data was normalised using the arcsine transformation. One outlier was excluded from analysis of M. novemaculeata length data.

This datum did not change the results of the analysis pertaining to M. novemaculeata growth.

The mean survivorship rates for M. novemaculeata and G. holbrooki were calculated by dividing the final number of individuals retrieved at the end of the experimental period by the initial number placed in each cage (N1 / N0 × 100). An Analysis of Variance (ANOVA) was run to determine if density and species composition (treatment) had an effect on mean percent survivorship for M. novemaculeata or G. holbrooki. The model, run separately for each species, included density and treatment as fixed factors.

To determine if the sex ratio of G. holbrooki varied depending on fish density and species composition over time, the proportion of males was calculated in individual cages was calculated at T0 and T1 (number of males/total number of individuals). The male proportion of total individuals is commonly used to analyse the sex ratio (Wilson and Hardy, 2002).

Following this, an ANOVA with time (fixed), density (fixed) and treatment (fixed) as factors was used to determine if there was any change in the proportion of male G. holbrooki present.

The Bonferroni correction was applied to G. holbrooki survivorship data which adjusted the alpha value to 0.0025.

To test the whether the growth of M. novemaculeata was related to fish density and species composition, an ANOVA was used. The effects of time (fixed), density (fixed) and treatment

(fixed) on standard length was examined. For G. holbrooki, the additional effect of sex was investigated using an ANOVA with sex (fixed factor) and interactions between sex and time, density and treatment (fixed factors) on standard length. When a significant interaction between sex and another fixed factor was detected, separate ANOVAs for males and females were run to determine the effects of time (fixed), density (fixed) and treatment (fixed) for each sex individually. The Bonferroni correction was applied to the analyses of G. holbrooki length data which adjusted the alpha value to 0.0025.

During the first week of the experimental period a major storm event affected the study site.

Five aquaculture cages were observed to be sitting lower in the water column which may have enabled fish to escape. To determine if the survivorship of M. novemaculeata or G. holbrooki in these cages was affected by the storm, a separate t-test for each species was conducted comparing the final survivorship between affected and non-affected cages (Final number of individuals/Initial number of individuals ×100). The model contained state

(flooded/unflooded) (fixed) and cage ID (random) as factors. The survivorship of neither M. novemaculeata nor G. holbrooki did not differ in cages which were flooded compared to those which were not (t test; M. novemaculeata, State, t1,23 = 0.047, p = 0.830; G. holbrooki, t1,23 = 2.75, p = 0.115).

Ethical Note

At the conclusion of the experiment M. novemaculeata was released into the pond in which the cages had been held. As it is illegal to release G. holbrooki into water bodies in NSW, surviving G. holbrooki in this experiment were euthanased using clove oil. The methods used for animal capture, housing and euthanasia were approved by the Animal Ethics Committee of the University of Wollongong (Animal Ethics Protocol No. 14/18) and adhered to the

Scientific Collection guidelines (permit No. P13/0011-1.3) of the NSW Department of

Primary Industries.

4.3 Results

4.3.1 The survivorship and growth of Macquaria novemaculeata

The recapture rate for M. novemaculeata was 28 % lower in the high density mixed condition than in the other experimental conditions, however there was no significant effect of density, treatment or an interaction between them on survivorship (ANOVA, Density, F1,23 = 0.08, p =

0.42; Treatment, F1, 23 = 0.05, p = 0.59; Density×Treatment, F1,23 = 1.14, p = 0.27) (Figure

4.2).

100

90

80 70

60

50

40

Survivorship (%) Survivorship 30

20 10

0 IntraControl-specific InterMixed-specific Figure 4.2 Mean (± S.E.) survivorship of M. novemaculeata fingerlings in low (open bars) and high (closed bars) density intra-specific and mixed conditions, n = 6.

Overall, the average standard length of recaptured M. novemaculeata increased significantly

(by 8.12 ± 0.33 mm) over the course of the experiment (ANOVA; Time, F1, 157 = 16.59, p <

0.0001) (Figure 4.3). There was no significant effect of density or treatment on change in length over time (Time×Density, F1, 157 = 0.83, p = 0.41; Time×Treatment, F1, 157 = 1.47, p =

0.14; Time×Treatment×Density, F1, 157 = 1.11, p = 0.27) (Figure 4.4).

35

16 22 9 30 29

25 10 10 30 29

20

15

10

(mm) Length Standard

5

0 IntraControl-specific InterMixed-specific IntraControl-specific InterMixed-specific

T0 T1 Figure 4.3 Mean (± SE) standard lengths of M. novemaculeata at low (open bars) and high (closed bars) density intra-specific and mixed conditions in T0 and T1 of the experiment, n = 6 cages, sample sizes of individuals are above bars.

4.3.2 The survivorship and growth of Gambusia holbrooki

The recapture rate of G. holbrooki in the low density mixed condition was approximately 25

% higher than other experimental conditions (Figure 4.5). However, there was no significant effect of density, treatment or an interaction between them on recapture rate (ANOVA;

Density, F1, 23 = 1.72, p = 0.10; Treatment, F1, 23 = 1.36, p = 0.19; Density×Treatment, F1, 23 =

1.64, p = 0.12).

100 90

80 70 60 50

40

(%)Survivorship 30 20 10 0 IntraControl-specific InterMixed-specific

Figure 4.4 Mean (± SE) survivorship of G. holbrooki in low (open bars) and high (closed bars) density intra-specific and mixed conditions, n = 6.

Regardless of treatment and density, the proportion of males declined over time (ANOVA;

Time, F1, 47 = 3.81, p = 0.0005; Time×Density, F1, 47 = 0.53, p = 0.60; Time×Treatment, F1, 47

= 1.49, p = 0.14) (Figure 4.6).

1 0.9

0.8 0.7

0.6 0.5

0.4 0.3

ofmales Proportion 0.2 0.1

0 IntraControl-specific InterMixed-specific IntraControl-specific InterMixed-specific T0 T1

Figure 4.5 Mean (±SE) proportion of G. holbrooki males in low (open bars) and high (closed bars) density intra-specific and mixed conditions at T0 and T1 of the experiment, n = 6.

The average increase in length over time for female G. holbrooki across all conditions, 6.66 ±

0.69 mm, was more than twice that of males, 3.02 ± 0.16 mm (ANOVA; Sex×Time, F1, 936 =

3.80, p = 0.0002) (Figure 4.8a and b). The significant change in length for female G. holbrooki (Time, F7, 447 = 10.04, p < 0.0001) was not influenced by density (Time×Density,

F1,477 = 1.03, p = 0.30) or treatment (Time×Treatment, F1,477 = 1.47, p = 0.14) or an interaction between them (Time×Density×Treatment, F1, 477 = 0.29, p = 0.77), despite there being a trend toward fish being smaller in the high density control and mixed conditions at T0 than T1

(Figure 4.8a). Similarly, the increase in male G. holbrooki length over time (Time, F7, 458 =

5.70, p < 0.0001) was not dependent upon density (Time×Density, F1, 458 = 0.15, p = 0.88), treatment (Time×Treatment, F1, 458 = 0.30, p = 0.77) or an interaction between the factors

(Time×Density×Treatment, F1, 458 = 1.41, p = 0.16) (Figure 4.8b).

35 18 a) 33 30 24 41

13 30 25 158 40 20

15 Length(mm)

10

5

0 IntraControl-specific InterMixed-specific IntraControl-specific InterMixed-specific T0 T2

35 b)

30

16 25 7 8 182 12 50

20 29 155

15 Length(mm) 10

5

0

IntraControl-specific InterMixed-specific IntraControl-specific InterMixed-specific T0 T2

Figure 4.6 Mean (± SE) initial and final standard lengths for a) female and b) male G. holbrooki in low (open bars) and high (closed bars) density intra-specific and mixed conditions of T0 and T1 of the experiment, n = 6 cages, sample sizes for individuals are above bars.

4.4 Discussion

The effects of competition between invasive and native species are often expected to increase with invader density (Yokomizo et al., 2009), however, current evidence pertaining to the density-impact relationship for invasive species is mixed. Furthermore, few studies have simultaneously measured the relative effects of inter- and intra-specific competition for either native or invasive species, even though this provides a critical insight into the magnitude of inter-specific competition (Inouye, 2001, Forrester et al., 2006). In the present study, neither the survivorship nor growth of native Australian bass fingerlings, M. novemaculeata, were found to be dependent upon competitor identity and density. Similarly, the survivorship and growth of the invasive eastern mosquitofish, Gambusia holbrooki, were also independent of competitor identity or density. These results suggest that G. holbrooki has a negligible effect upon the growth and survivorship of stocked M. novemaculeata, regardless of density.

The lack of effect of density on the survivorship and growth of either species was unexpected and indicates that inter-specific competition was unrelated to G. holbrooki density. Further, it suggests that neither species experienced any effects of intra-specific competition at high densities. This result contradicts those of other studies which have reported both increased impacts of invasive species on natives (Mills et al., 2004, Kaspersson et al., 2010), as well as the enhanced effects of intra-specific competition with increasing density (Kornis et al.,

2014). One reason for this discrepancy could be that the high densities used in the present study were lower than for some other studies that have reported significant density-dependent effects of Gambusia spp on native competitors (i.e. 0.036 fish/L-1 in this study compared to

0.067 fish/L-1 and 0.074 fish/L-1 by Mills et al. (2004) and Belk and Lydeard (1994), respectively)). However, it is also known that density-impact relationships, particularly in regard to the threshold at which significant impacts emerge, vary significantly among species

(Yokomizo et al., 2009). Therefore, it is possible that at the same density G. holbrooki exerts effects depending on the identity of the native competitor. Since this is the first study to examine the competition between G. holbrooki and M. novemaculeata, and given that the densities used were based on observations of G. holbrooki populations in the field so as to maximise ecological relevancy, it is not possible to conclusively state that the density parameters employed were inappropriate. Even so, more research exploring density- dependent competition between G. holbrooki and native species over a wider range of densities would be valuable.

For native species, the relative effects of competition frequently conform to reviews of competition theory (Connell, 1983) whereby the strength and effects of intra-specific competition exceed that of inter-specific competition. Therefore, it was not expected that the survivorship and growth of the native M. novemaculeata did not differ following exposure to hetero- or con-specifics, regardless of G. holbrooki densities. It was anticipated that G. holbrooki, as an invasive species, would experience lower effects of intra-specific than inter- specific competition when at a high density, since this would limit population self-regulation

(Pintor et al., 2009). However, regardless of sex, the survivorship and growth of G. holbrooki did not differ between control and mixed conditions. These results suggest that in accordance with the lottery model (Sale, 1977) the costs of competing with hetero- and con-specifics are equivalent for both M. novemaculeata and G. holbrooki. Given that a lottery model competition dynamic is mostly thought to occur between species which have a high degree of resource overlap and ecological similarity (Kaplan and Denno, 2007), it was not anticipated that M. novemaculeata and G. holbrooki would conform to this dynamic, since they differ in regard to their habitat usage and diet breadth (Harris, 1986, Pyke, 2008). This dynamic would also suggest that competitive exclusion is unlikely to occur between these species, since the identity of the competitor had no effect (Cross and Benke, 2002). However, while

unexpected, there are a number of reasons as to why the effects of competition with con- and hetero-specifics did not differ for M. novemaculeata and G. holbrooki.

Firstly, it is important to consider the role of experimental design in determining the outcome of density-dependent competition studies. Most studies which explore the effect of invader density on a native competitor employ experimental designs that do not account for the relative effect of intra-specific competition (Inouye, 2001). This in turn may produce results which exaggerate the impact of invaders. For example, when Britton et al. (2011) added low, medium and high densities of the invasive topmouth gudgeon, Pseudorasbora parva, to a constant number of carp Cyprinus carpio, as per an additive design, it appeared that the growth of C. carpio became significantly reduced in the presence of P. parva. However, following a treatment which tested for the intra-specific effects of competition on C. carpio, it was revealed that when con-specifics were introduced at a similar biomass as P. parva, C. carpio experienced the same reduction in growth. This indicated that rather than being dependent on the presence of the invader, C. carpio growth rates were influenced by biomass alone (Britton et al., 2011). This result, as well as that of the present study, emphasises the value of comparing the effects of inter- and intra-specific competition when attempting to accurately assess the impact of invader density on native species.

Second, aggression from Gambusia spp. toward native competitors is frequently cited as a cause of higher mortality and injury to native species, particularly when the invader is present at a high density (Mills et al., 2004, Rowe et al., 2007, Pyke, 2008). However, the results of the present study suggest that there was no significant effect of inter-specific interference competition on either G. holbrooki or M. novemaculeata. Similarly, Schumann et al. (2015) reported that while G. affinis displayed aggression to the plains topminnow, Fundulus sciadicus, it did not compromise the native species’ growth or survivorship. In the present

study it is possible that M. novemaculeata may have been behaviourally dominant over G. holbrooki, since it has been observed to respond aggressively toward G. holbrooki, and in fact is more inter-specifically aggressive than the invader when it is present at a high density

(Chapter 3). Furthermore, the competing species which is larger is often also more aggressive and dominant (Garcia and Arroyo, 2002) and in this experiment M. novemaculeata fingerlings were on average 2 mm longer than G. holbrooki. However, this does not explain why G. holbrooki did not suffer reduced growth and survivorship following exposure to M. novemaculeata, unless the invader was able to effectively avoid interactions. While it is not possible to disentangle the effects of interference and exploitation competition in this study since interactions between individuals were not observed, our results do not support the hypothesis that G. holbrooki aggression has a greater deleterious effect than intra-specific aggression on M. novemaculeata fingerlings.

It was also predicted that male G. holbrooki survivorship would be reduced relative to females, which is based on the assumption that male G. holbrooki are more aggressive than females (Carmona-Catot et al., 2013a) and would engage in more direct aggressive interactions with M. novemaculeata. However, while the sex ratio of G. holbrooki did become female-biased, this did not differ between mixed and control conditions. Many Gambusia spp. populations commonly become female-biased over time due to females having a greater longevity than males (Haynes and Cashner, 1995, Fryxell et al., 2015). The results of the present study indicate that G. holbrooki conform to this pattern regardless of exposure to M. novemaculeata or overall fish density, and do not support the theory that the survivorship of males declines due to aggressive interactions.

Finally, we cannot disregard the possibility that a shift in habitat usage and activity levels may have occurred in mixed treatments which reduced the strength of competition between M.

novemaculeata and G. holbrooki. Shifts in diet or habitat use can promote coexistence between competing species (Mills et al., 2004). For example, least chub, Iotichthys phlegethontis, have been reported to use cooler and deeper habitats during the day rather than warm shallow waters so as to avoid western mosquitofish, G. affinis (Ayala et al., 2007). It would be beneficial in future studies to monitor foraging, refuge use and depth preference of both G. holbrooki and M. novemaculeata via remote underwater video (RUV) as well as to analyse gut contents to detect any changes in diet.

To conclude, the results of this study indicate the relative effects of intra- and inter-specific competition for the native M. novemaculeata and invasive G. holbrooki may be equal.

Therefore, G. holbrooki does not appear to have a negative effect on the survival or growth of stocked M. novemaculeata fingerlings. This study illustrates the importance of considering the relative importance of inter- and intra-specific competition when quantifying the magnitude of invader impacts.

5 INTERFERENCE COMPETITION UNDER MULTIPLE STRESSORS: TEMPERATURE AND SALINITY MEDIATE AGGRESSIVE INTERACTIONS BETWEEN AN INVASIVE AND NATIVE FISH

5.1 Introduction

The intensity and outcome of biotic interactions between invasive and native species is

critical to the success of an invasive species in an ecosystem (Amarasekare, 2002, Mills et al.,

2004, Shine, 2014, Gallardo et al., 2016). This includes interference competition, which refers

to the direct negative interactions, including aggression, which occurs between sympatric

species (Case and Gilpin, 1974, Human and Gordon, 1996, Zhang et al., 2015). Aggression

has been identified as an important factor in the competitive superiority of some invaders over

native species with aggressive interactions sometimes leading to the displacement of the

native species from food, habitat and reproductive partners (Pintor et al., 2008, Hudina et al.,

2014, Hudina et al., 2015).

In addition to invaders, many native species are subject to biotic and abiotic stressors, defined

as environmental conditions that exceed typical levels and cause shifts in physiology,

morphology and behaviour (Christensen et al., 2006, Alcaraz et al., 2008, Killen et al., 2013,

Piggott et al., 2015). Such changes may alter the intensity of aggressive interactions between

species (Barbieri et al., 2013, Killen et al., 2013, Barbieri et al., 2015) however, behavioural

responses are likely to be dependent on species-specific tolerances to stressors (Alcaraz et al.,

2008, Carmona-Catot et al., 2013a). For example, while exposure to a neurotoxin reduced

aggression in the Southern native ant (Monomorium antarcticum), the invasive Argentine ant

(Linepithema humile) behaved more aggressively toward its native competitor (Barbieri et al.,

2013). Therefore, by altering the intensity and outcome of inter-specific aggressive interactions, stressors can result in complex condition-specific distributions and abundances of native and invasive species (Dunson and Travis, 1991, Taniguchi and Nakano, 2000, White et al., 2015).

Empirical studies examining the interactions between abiotic stressors and interference competition between native and invasive species have predominantly examined the effects of single stressors (Destaso and Rahel, 1994, Alcaraz et al., 2008, Carmona-Catot et al., 2013a).

This is surprising, given that ecosystems may be subjected to multiple abiotic stressors that interact to produce additive and non-additive effects (Folt et al., 1999, Christensen et al.,

2006, Piggott et al., 2015). Whilst the effect of an additive interaction can be predicted from the sum of each stressor (Folt et al., 1999, Piggott et al., 2015), non-additive interactions occur when one stressor synergistically or antagonistically mediates the outcome of another, with the final effect being more than or less than what is expected under an additive model, respectively (Folt et al., 1999, Piggott et al., 2015). For example, when exposed to 31.5 °C and concentrations of CO2 of 420 and 530 µatm, the black anemonefish, Amphiprion melanopus, reduced its rate of foraging and food consumption. However, exposure to 31.5 °C and a higher concentration of CO2, 960 µatm, led to a synergistic increase in the frequencies of those behaviours (Nowicki et al., 2012) Therefore, the effects of non-additive interactions can deviate significantly from those expected under additive models, making them difficult to predict (Schram et al., 2014, Ferrari et al., 2015, Piggott et al., 2015).

While many habitats are exposed to multiple stressors, it is particularly pertinent to examine their effects on interactions between freshwater biota (Townsend et al., 2008, Matthaei et al.,

2010, White et al., 2015). This is because freshwater habitats are subjected to a wide range of anthropogenic activity, as well as being highly vulnerable to the effects of climate change

(Ficke et al., 2007, Fenoglio et al., 2010, Hobday and Lough, 2011), since conditions in freshwater ecosystems are closely linked to atmospheric variation (Ficke et al., 2007). Water temperatures are predicted to increase with rising air temperatures, which in south eastern

Australia could result in summer water temperatures of over 30 °C (Hobday and Lough, 2011,

Koehn et al., 2011, Pratchett et al., 2011, O'Mara and Wong, 2015). In addition to this, a reduction in average rainfall and runoff into freshwater habitats is likely to lead to higher salinity levels (Hobday and Lough, 2011, Koehn et al., 2011, Pratchett et al., 2011) while coastal waterbodies may experience an influx of seawater with rising sea levels (Hobday and

Lough, 2011, Koehn et al., 2011, Pratchett et al., 2011).

The effects of temperature and salinity on inter-specific interference competition between invasive and native freshwater species have previously been examined in isolation (Destaso and Rahel, 1994, Taniguchi et al., 1998, Alcaraz et al., 2008, Fobert et al., 2011, Carmona-

Catot et al., 2013a). Elevated temperature has been reported to increase the metabolic rate of fishes and promote heightened activity and aggression (Carmona-Catot et al., 2013; Rahel &

Olden, 2008). Conversely, elevated salinity can impose significant metabolic costs leading to a reduction in aggression between fish (Alcaraz et al., 2008, Uliano et al., 2010). While studies have reported an antagonistic interaction between temperature and salinity on the metabolic rate, osmoregulation and activity levels of freshwater fish (Wuenschel et al., 2005,

Uliano et al., 2010), whether and how these stressors mediate interference competition between native and invasive species has yet to be examined.

Here we tested the combined effects of temperature and salinity on interference competition between two freshwater fish, the native Australian bass, Macquaria novemaculeata and the invasive eastern mosquitofish, Gambusia holbrooki. M. novemaculeata and G. holbrooki are sympatric over parts of their ranges in south east Australia and inter-specific competition is

likely since G. holbrooki is known to aggressively displace native species (Pyke, 2008).

Furthermore, in order to support isolated populations, juvenile M. novemaculeata are stocked through the Australian Eastern Drainage System, therefore, interference competition with G. holbrooki may be a significant impediment to the success of M. novemaculeata stocking programs. Both species have wide thermal and salinity tolerance ranges and have been recorded in water bodies with temperatures of at least 0-30 °C and salinities of 0-35 ppt

(Langdon, 1987, Pyke, 2008).

We predicted that there would be an antagonistic interaction between elevated temperature, which would promote inter-specific aggression, and elevated salinity, which would reduce inter-specific aggression. Furthermore, since juvenile development of M. novemaculeata occurs in estuarine waters and hence M. novemaculeata are expected to be more tolerant of elevated salinities than G. holbrooki (Langdon, 1987), we predicted that M. novemaculeata would be more aggressive than G. holbrooki under elevated salinity. Finally, since inter- specific aggression is mediated by body size which may be influenced by physiological stress

(Poulos and McCormick, 2014, Poulos and McCormick, 2015), with individuals typically more aggressive to smaller hetero-specifics which pose a lesser threat (Moretz, 2003, Poulos and McCormick, 2014), we predicted that both M. novemaculeata and G. holbrooki would be more aggressive to smaller hetero-specifics under elevated salinity.

5.2 Methods

Study Species and Acclimation

Adult Gambusia holbrooki, (x̅ ± SE mass = 0.23 ± 0.02 g; x̅ ± SE total length = 22.72 ± 0.48 mm) were collected from freshwater ponds located on the University of Wollongong campus

(34° 24’ 19” S 150 ° 52’ 42” E ) using hand held nets. Only adult females were collected so as to avoid the mating behaviours displayed by males which may have interfered with competitive behaviours (Pyke, 2008). Juvenile Macquaria novemaculeata (x̅ ± SE mass =

0.74 ± 0.02 g; x̅ ± SE total length = 31.32 ± 0.29 mm) of both sexes were obtained from a hatchery (Aquablue Seafoods, Pindimar, NSW, Australia). To acclimatize the fish to laboratory conditions, 4 recirculating aquarium systems were set up with each system consisting of 8 interconnected aquaria (60 × 30 × 30 cm) subjected to constant conditions of

22 °C and 15 ppt. M. novemaculeata (N = 64 fish) were placed into 4 aquaria per system.

Similarly, G. holbrooki (N = 64 fish) were placed into 4 aquaria per system (but distinct from those housing M. novemaculeata). All fish were acclimated to these conditions for 7 days and fed an equal amount of a commercial fish pellet (New Life Spectrum Thera formula) and bloodworms daily.

After this period, G. holbrooki (N = 16 fish per treatment) and M. novemaculeata (N = 16 fish per treatment) were acclimated to four temperature and salinity condition treatments: 1) control salinity and control temperature (21°C and 15 ppt); 2) elevated temperature and control salinity (28 °C and 15 ppt); 3) control temperature and elevated salinity (21 °C and 35 ppt) and 4) elevated temperature and elevated salinity (28 °C and 35 ppt) (Table 5.1).

Temperature was gradually raised by 1°C every two days and salinity by 5 ppt every week.

These treatment conditions were maintained over the next 4 weeks after which behavioural observations were conducted.

Table 5.1 Mean (± S.D.) temperature and salinity parameters used in this experiment.

Treatment Temperature (°C) Salinity (ppt)

Control temperature control salinity 21.24 (± 0.38) 15.21 (± 2.47) Elevated temperature control salinity 28.12 (± 0.55) 15.1 (± 1.10) Control temperature elevated salinity 21.14 (± 0.28) 35 (± 0.47) Elevated temperature elevated salinity 28.19 (± 0.49) 35.1 (± 0.99)

Behavioural Observations and Data Collection

After the treatment acclimation period, all fish were captured, measured using callipers (mm standard length (SL) ± 0.1 mm) and uniquely tagged using a fluorescent elastomer (Northwest

Technologies Inc.) injected subcutaneously along the dorsal musculature (Malone et al.,

1999). To conduct the behavioural trial, G. holbrooki (n = 2) were introduced into a test aquarium followed by M. novemaculeata (n = 2) 24 hours later. G. holbrooki were added before M. novemaculeata to simulate the natural temporal occurrence of species when stocking a water body with M. novemaculeata. Water parameters within the test aquarium were the same temperature and salinity conditions that both species had been previously acclimated to (i.e. condition treatment 1, 2, 3 or 4). Focal observations of each fish commenced after a 30 minute acclimation period followed by a 5 minute adjustment to the presence of the observer (LKL). During a focal observation, each fish was observed for 10 minutes during which time the number of aggressive behavioural interactions (approaches, chases and nips) with its hetero-specifics was recorded following previous protocols (see

Table 3.2) (Matthews and Wong, 2015, O'Mara and Wong, 2015). To account for diurnal variation in behaviour the same fish were observed once in the morning (0900 - 1200) and once in the afternoon (1300 - 1600). This protocol was repeated until each fish had been observed under test conditions upon which hetero-specifics were separated.

Statistical Analysis

All statistical analyses were performed using SPSS Statistics 21, IBM, Armonk, NY, U.S.A.

Generalized Linear Mixed Models (GLMMs) with Poisson distributions and loglink functions were used. The model used a robust estimation which accounts for any violations of model assumptions (Cameron and Trivedi, 2009). No significant difference in aggression was detected for M. novemaculeata and G. holbrooki between the AM and PM observation times and so the summed data from these periods was used in the analysis (GLMM, M. novemaculeata, Time, F1,122 = 1.003, p = 0.319; G. holbrooki F1,114 = 0.041, p = 0.840). The initial model compared the frequency of inter-specific aggression in M. novemaculeata and G. holbrooki under elevated temperature and elevated salinity. Where a significant interaction between species and another fixed factor was found, separate GLMMs were conducted for each species to identify the effects of temperature and salinity. The models included species, temperature and salinity (fixed effects), body length (covariate) and tank ID (random) to account for the non-independence of interacting fish in each replicate. Interactions between factors were classified directionally as additive or positively or negatively synergistic or antagonistic (Piggott et al., 2015) and means were compared using pairwise post-hoc tests. A synergistic interaction occurred if we detected a significantly larger effect than what is predicted from the single stressors combined whereas an antagonistic interaction occurred if we detected an effect significantly lesser than what was predicted from each single stressor combined (Coors and De Meester, 2008). Using the framework detailed by Piggott et al

(2015), the magnitude of a non-additive interaction was also defined as negative or positive e.g. negative synergistic as an interaction which is less negative than predicted by an additive model and positive antagonistic as less positive than what would otherwise be predicted.

To determine how relative body size influenced the proportion of total aggression given by an individual to hetero-specifics under differing temperature and salinity conditions, the

proportion of aggression directed to larger and smaller hetero-specifics by each individual was calculated (i.e. number of aggressive acts toward each opponent/ sum of total aggression). This was related to the size ratio of each individual to its two hetero-specific opponents in each tank (SL G. holbrooki / SL M. novemaculeata; SL M. novemaculeata/ SL

G. holbrooki). From this metric, hetero-specifics were categorized as ‘larger’ or ‘smaller’.

The difference in standard length between large and small M. novemaculeata was 1.96 ± 0.29 mm (x̅ ± SE). The standard length of large G. holbrooki was 2.78 ± 0.44 mm (x̅ ± SE) greater than that of small con-specifics. Individuals of both species which displayed no aggressive behaviours throughout the observation period were excluded from this analysis since no proportion of size-based aggression could be calculated. For both M. novemaculeata and G. holbrooki, this proportional aggression data was normalised using an arcsine transformation.

While use of the arcsine transformation is controversial for binomially distributed proportional data (Warton and Hui, 2011), in the present instance, the data conformed to a sinusoidal distribution with few data at the upper and lower boundaries. GLMMs with a normal distribution were used to test the effect of hetero-specific size, temperature and salinity (fixed factors), and tank ID (random) on aggression for each species. The aggression received by each opponent (covariate) was included in the model to account for the non- independence of individual responses. Pairwise post-hoc tests were used to compare means.

The residuals were checked for homogeneity. Following the application of the Bonferroni correction the alpha value used was adjusted to 0.016.

Ethical Note

Upon completion of the experiment, G. holbrooki and M. novemaculeata were euthanased using clove oil. It is illegal to release an invasive species under NSW law and releasing M. novemaculeata following exposure to the experimental treatments was deemed excessively stressful. Observational data collection and euthanasia protocols were approved by the

University of Wollongong Animal Ethics Committee (protocol AE14/07) and adhered to the

Scientific Collection guidelines (permit No. P13/0011-1.3) of the NSW Department of

Primary Industries.

5.3 Results

The effect of temperature and salinity on interspecific aggression

Inter-specific aggression from M. novemaculeata increased following exposure to both elevated temperature and salinity compared to control conditions, yet remained lower than when M. novemaculeata were exposed to elevated temperature in isolation (Figure 5.1a).

Therefore, there was a significant positive antagonistic interaction between temperature and salinity on aggression from M. novemaculeata to G. holbrooki (Table 5.1a). For G. holbrooki, exposure to elevated temperature alone promoted a significant increase in inter-specific aggression (Figure 5.1b). Unlike M. novemaculeata, aggression in G. holbrooki was lower in the presence of both elevated temperature and elevated salinity compared to under the control condition, demonstrating a negative synergistic interaction between the stressors (Table 5.1b).

Species-specific responses to temperature and salinity

M. novemaculeata was more aggressive than G. holbrooki when exposed to elevated salinity, either as a single stressor or when combined with elevated temperature (Table 5.2, Figure 1a, b). When exposed to control conditions or elevated temperature alone, M. novemaculeata and

G. holbrooki were equally aggressive.

Table 5.2 Results of a generalised linear mixed model testing the effects of temperature and salinity on inter-specific aggression from a) Macquaria novemaculeata and b) Gambusia holbrooki. Non-significant terms were backward stepwise eliminated (p >0.016). Significant terms are in bold. Pairwise post-hoc tests compared the means of control temperature and control salinity (C), elevated temperature and control salinity (ET), control temperature and elevated salinity (ES) and elevated temperature and elevated salinity (ETS) treatments.

Comparison Post-hoc df1 df2 F p Group p value a) Macquaria novemaculeata Model 4 59 40.671 <0.0001 C vs ET < 0.0001 Temperature (T) 1 59 100.071 <0.0001 C vs ES 0.090 Salinity (S) 1 59 11.726 0.001 C vs ETS 0.010 Length 1 59 48.746 <0.0001 ET vs ES < 0.0001 T×S 1 59 31.102 <0.0001 ET vs ETS < 0.0001 Tank ID (random) 0.306 ES vs ETS 0.167

b) Gambusia holbrooki Model 4 56 40.769 < 0.0001 C vs ET 0.001 Temperature (T) 1 56 4.735 < 0.0001 C vs ES 0.002 Salinity (S) 1 56 32.855 0.034 C vs ETS 0.001 Length 1 56 62.145 < 0.0001 ET vs ES 0.001 T×S 1 56 8.006 0.006 ET vs ETS 0.001 Tank ID (random) 1.379 ES vs ETS 0.398

Table 5.3 Results of a generalised linear mixed model testing the effects of species, temperature and salinity on inter-specific aggression from Macquaria novemaculeata and Gambusia holbrooki. Non-significant terms were backward stepwise eliminated. Significant terms are in bold (p > 0.016). Pairwise post-hoc tests compared response of G. holbrooki (GH) and M. novemaculeata (MN) in control temperature and control salinity (C), elevated temperature and control salinity (ET), control temperature and elevated salinity (ES) and elevated temperature and elevated salinity (ETS) treatments.

Post-hoc df1 df2 F p Comparison Group p value

Model 5 119 52.389 < 0.0001 CGH vs CMN 0.848 Temperature (T) 1 119 35.998 <0.0001 ETGH vs ETMN 0.960 Salinity (S) 1 119 61.257 < 0.0001 ESGH vs ESMN < 0.0001 Species (Sp) 1 119 48.711 < 0.0001 ETSGH vs ETSMN < 0.0001 T×S 1 119 35.205 < 0.0001 T×Sp 1 118 0.15 0.700 S ×Sp 1 119 48.923 < 0.0001 T×S*Sp 1 117 0.727 0.396 Tank ID 0.259 (random)

20 a)

18 c 16

14

12

10 8 a

AggressionGiven 6 ab a 4

2

0 21 °C 28 °C

20 b) 18 c 16

14

12

10 8 AggressionGiven a 6

4 2 b b 0 21 °C 28 °C Figure 5.1 Mean (± SE) number of interspecific aggressive acts (sum of approaches, nips and chases) performed by a) Macquaria novemaculeata to Gambusia holbrooki, b) G. holbrooki to M. novemaculeata, in four treatment combinations of 15 ppt and 35 ppt and 21 °C (open bars) and 28 °C (closed bars), n = 8. Means with the same letter indicate statistically non- significant differences.

The effect of temperature and salinity on size-based interspecific aggression

M. novemaculeata elected to interact aggressively with large rather than small G. holbrooki under control salinity conditions and in combination with elevated temperature (Table 5.3a).

When exposed to elevated salinity, this pattern was reversed (Figure 5.2a). In contrast, G. holbrooki were equally aggressive to large or small M. novemaculeata in all temperature and salinity conditions (Table 5.3b, Figure 5.2b)

Table 5.4 Results of a generalised linear mixed model testing the effects of hetero-specific size, temperature and salinity on aggression in a) Macquaria novemaculeata and b) Gambusia holbrooki. Non-significant terms were backward stepwise eliminated. Significant terms are in bold (p > 0.016). Pairwise post-hoc tests compared the responses of individuals to small (S) and large (L) hetero-specifics in control temperature and control salinity (C), elevated temperature and control salinity (ET), control temperature and elevated salinity (ES) and elevated temperature and elevated salinity (ETS) treatments.

Comparison Post-hoc df1 df2 F p Group p value a) Macquaria novemaculeata Model 3 110 4.362 0.006 CS vs CL 0.029 Temperature (T) 1 106 0.004 0.949 ETS vs ETL 0.017 Salinity (S) 1 106 0.08 0.931 ESS vs ESL 0.022 Heterospecific size (H) 1 110 0.339 0.562 ETSS vs ETSL 0.524 T×S 1 106 0.151 0.699 T×H 2 108 0.326 0.722 S×H 3 110 4.362 0.006 T×S×H 2 106 0.219 0.804 Tank ID (random) < 0.0001

b) Gambusia holbrooki Model 3 66 0.884 0.454 Temperature (T) 1 62 0.152 0.698 Salinity (S) 1 62 0.181 0.672 Heterospecific size 1 64 0.117 0.733 (H) T×S 1 62 0.242 0.625 T×H 3 66 0.884 0.454 S×H 2 64 0.998 0.374 T×S×H 2 62 0.331 0.719 Tank ID (random) < 0.0001

1 a)

0.8 b b b a 0.6 a a a a

0.4

Proportion of total aggression totalof Proportion 0.2

0 21 °C 15ppt 28 °C 15 ppt 21 °C 35ppt 28 °C 35 ppt

1 b)

0.8 a a a a a a 0.6 a a 0.4

Proportion of total aggression totalof Proportion 0.2

0 21 °C 15ppt 28 °C 15 ppt 21 °C 35ppt 28 °C 35 ppt Figure 5.2 The mean (± SE) proportion of total aggression directed from individual a) M. novemaculeata and b) G. holbrooki to smaller (closed bars) and larger (open bars) hetero- specifics in four treatment combinations of 15 and 35 ppt and 21 and 28 °C, n = 8. Letters above indicate statistically similar means of aggression following separate analyses for each species.

5.4 Discussion

Understanding whether multiple environmental stressors interact in an additive, synergistic or antagonistic manner is critical when predicting the consequences of invasive-native species interactions. Multiple stressor effects are especially pertinent for interactions between freshwater biota which are predicted to experience greater exposure to temperature and salinity stress under future climate change. In the present study, elevated temperature promoted aggression in both the native Australian bass (Macquaria novemaculeata) and the invasive eastern mosquitofish (Gambusia holbrooki), yet this was partially or completely suppressed by elevated salinity, respectively. Therefore, in accordance with the first prediction, elevated temperature and elevated salinity interacted antagonistically on aggression for M. novemaculeata. Unexpectedly however, the stressors interacted synergistically on aggression in G. holbrooki. Notably, inter-specific aggression was modulated by species-specific and size-based responses whereby M. novemaculeata was more aggressive than G. holbrooki under elevated salinity and interacted more frequently with smaller hetero-specifics, while G. holbrooki showed no such change.

To date, few studies have investigated the influence of multiple stressors on interference competition, and as such, we know relatively little about the frequency of additive or non- additive interactions between stressors (Barbieri et al., 2013, Ferrari et al., 2015). This is surprising, given that non-additive interactions between temperature and other stressors, including CO2, moisture and pH have been observed to influence individual-level behaviours, including foraging (Nowicki et al., 2012, Barbieri et al., 2013), escape behaviours (Schram et al., 2014) and shelter use (Rohr and Palmer, 2013) in a range of ectothermic species.

Furthermore, recent evidence has emerged of non-additive interactions between temperature and CO2 altering predator-prey interactions in coral reef fish (Ferrari et al., 2015).

Specifically, elevated temperature and elevated CO2 interacted synergistically to increase the predation rate of the dottyback (Pseudochromis fuscus) upon two species of damselfish

(Pomacentrus amboinenses, P. nagasakiensis) (Ferrari et al., 2015). Notably, predator selectivity was mediated by an antagonistic interaction between these stressors resulting in P. fuscus showing no preference for either prey species (Ferrari et al., 2015). These results along with those of the present study emphasize the potential for multiple stressors to interact in unexpected ways.

Elevated temperature in isolation increased inter-specific aggression in both M. novemaculeata and G. holbooki, which is consistent with previous studies reporting an increase in aggression at high temperatures in a range of ectotherms (Pruitt et al., 2011, Briffa et al., 2013, Carmona-Catot et al., 2013a, Matthews and Wong, 2015). Metabolic rates typically increase with temperature (Briffa et al., 2013) which subsequently leads to increases in activity and aggression (Portner and Knust, 2007) provided sufficient food is available

(Portner and Knust, 2007). In contrast, the effect of elevated salinity on aggression is less well understood, with studies reporting negative or positive relationships (Alcaraz et al., 2008,

Schofield et al., 2009, Lorenz et al., 2016). Since osmoregulation may be affected by increases in salinity (Chervinski, 1983, Nordlie and Mirandi, 1996), a decrease in metabolic rate and activity levels may also be expected (Zheng et al., 2008, Uliano et al., 2010).While no change in aggression upon exposure to elevated salinity alone compared to the control was found for M. novemaculeata, G. holbrooki did reduce the frequency of its aggressive behaviour. In addition to this, M. novemaculeata was overall more aggressive under elevated salinity than G. holbrooki in accordance with the second prediction. While Gambusia spp. show physiological tolerance to elevated salinities (Chervinski, 1983, Pyke, 2008), M. novemaculeata is more likely to be tolerant of elevated salinity owing to its catadromous

lifestyle (Harris, 1986, Langdon, 1987), and as our results suggest, may become competitively dominant at elevated salinities.

Under elevated temperature and elevated salinity, M. novemaculeata was more aggressive than it was at control conditions, yet less aggressive than when exposed to elevated temperature alone. In contrast, G. holbrooki was less aggressive when exposed to both stressors than it was under control conditions. Therefore, temperature and salinity interacted in a positive antagonistic manner on aggression for M. novemaculeata and a negative synergistic manner on aggression for G. holbrooki. This suggests that salinity stress partially or entirely counteracted the increase in aggression due to elevated temperature in M. novemaculeata and G. holbrooki, respectively. Considering M. novemaculeata was more aggressive than G. holbrooki under elevated salinity, this stressor may be a key factor in determining whether the interaction was antagonistic or synergistic. Despite a paucity of studies examining the effect of temperature and salinity stress on inter-specific interactions, our results complement those of previous studies that have reported a significant interaction between temperature and salinity in altering metabolic rate, osmoregulation and activity levels in fishes (Imsland et al., 2001, Wuenschel et al., 2005, Uliano et al., 2010, Glover et al.,

2012). For example, the western mosquitofish, G. affinis, tolerated 35 ‰ salinity when acclimated to 20 °C yet reduced its oxygen consumption and activity levels when exposed to a combination of 35 ‰ and 27 °C (Uliano et al., 2010). By increasing metabolic rate, elevated temperatures increase ventilation rates and related ionic and osmotic losses, which in turn demands a greater energy input to sustain activity levels (Zheng et al., 2008). Considering this, it is possible that food availability may be a limiting factor in the current study. In the presence of unlimited food, individuals may have been more aggressive at the elevated temperature and elevated salinity treatment as this would have compensated for any energy

loss. An interesting avenue of future research would be to manipulate food availability to competing species during exposure to combinations of these stressors.

Body size is often an indicator of an opponent’s fighting ability and strength, with larger individuals typically displaying more aggression and subsequent dominance over smaller opponents (Moretz, 2003, Poulos and McCormick, 2014). In the present study, M. novemaculeata were more aggressive toward smaller, rather than similar-sized G. holbrooki under elevated salinity. Size-specific aggression could be a mechanism by which the metabolic costs of conflict are minimized under salinity stress, although further tests are required to explore this possibility. Similarly, the coral reef damselfish, Pomacentrus amboinenses, avoided engaging in conflict with similar-sized individuals of a hetero-specific

P. mollucensis upon exposure to elevated CO2, which they otherwise dominated under control conditions (McCormick et al., 2013). In contrast to M. novemaculeata, G. holbrooki did not preferentially attack hetero-specific opponents based on their relative size, regardless of temperature or salinity stress. Gambusia spp. are known to attack relatively large prey and competitors, therefore, it is possible that this species is less discriminant on the basis of body size (Komak and Crossland, 2000, Baber and Babbitt, 2004, Shulse and Semlitsch, 2014). In any case, our results highlight the fact that juvenile M. novemaculeata may not be able to obtain and maintain dominance over larger G. holbrooki under elevated salinity conditions

(McCormick et al., 2013).

To conclude, these results suggest that the negative outcomes of interference competition on

G. holbrooki and stocked juvenile M. novemaculeata, including reduced growth, body condition and greater mortality (Lehtonen et al., 2015, Zhang et al., 2015), are likely to increase in the future with water rising temperatures provided salinity levels remains relatively low. Ultimately this could have a profound impact on the distribution and

abundance of both species. This study demonstrates the value of considering the effects of multiple stressors as well as individual-level variables when investigating invasive-native species interactions.

6 GENERAL DISCUSSION

6.1 Research framework

Context-dependent inter-specific interactions are a commonly reported phenomenon for

species within their natural ranges (Shea et al., 2005, Melbourne et al., 2007, Chamberlain et

al., 2014). However, the manner in which predator-prey and competitive interactions between

invasive and native freshwater fauna are mediated by environmental and individual-level

variables remains poorly understood. Furthermore, since the majority of past studies have

employed indirect methods to examine invasive-native species interactions, information

pertaining to the behavioural mechanisms which underlie these interactions is often lacking

(Holway and Suarez, 1999, Almeida and Grossman, 2012, Peck et al., 2014). The absence of

these data poses an obstacle to the effective management of invasive freshwater species by

reducing our ability to account for spatial and temporal variation in the strength and impacts

of predation and competition (Knell, 2009, Thomsen et al., 2011, Hansen et al., 2013). This is

particularly true for interactions between native freshwater Australian fauna and invasive

species, for which the majority of information is anecdotal (Becker et al., 2005, Pyke, 2008) .

With the use of predominantly direct methods, I investigated how context, both at the

population and individual level, mediated predator-prey and competitive behavioural

interactions between the invasive eastern mosquitofish, Gambusia holbrooki, and two native

Australian species, the glass shrimp, Paratya australiensis, and Australian bass, Macquaria

novemaculeata. First, I examined how G. holbrooki’s density and the diel cycle influenced the

consumptive and non-consumptive effects of this predator on P. australiensis (Chapter 2).

Second, I determined how the sex and density of G. holbrooki and size of M. novemaculeata

mediated the intensity of intra- and inter-specific interference competition (Chapter 3). Third,

I tested whether the survivorship and growth of G. holbrooki and M. novemaculeata was dependent upon the invader’s density (Chapter 4). Finally, using a cross-factorial laboratory experiment, I measured the simultaneous effects of two stressors- temperature and salinity- on the intensity of interference competition between G. holbrooki and M. novemaculeata

(Chapter 5). Here I provide a synthesis of these findings and discuss avenues for future research.

6.2 The relationship between Gambusia holbrooki density, inter-specific interaction strength and impact on native species

A key aim of this thesis was to examine how the density of G. holbrooki influenced the strength and outcome of its interactions with native prey and competitors, since there is a lack of consensus on the relationship between population density and impact for invasive species

(Yokomizo et al., 2009, Kulhanek et al., 2011, Jackson et al., 2015). The negative impacts of an invasive predator or competitor on a native species are frequently expected to increase with its population density, since the rate of contact between individuals should theoretically rise while the availability of resources should decrease (Kaiser et al., 2013). This assumption is often at the core of management strategies which aim to reduce the densities of invasive species (Knell, 2009, Yokomizo et al., 2009).

This thesis does in fact support the hypothesis that the frequency of contact between G. holbrooki and native species increases with the invader’s density, yet it also suggests that this does not necessarily translate into reduced fitness for the native species. In Chapter 2, the prey species Paratya australiensis received a greater number of nips and approaches under a high compared to low density of G. holbrooki, yet the number of P. australiensis killed did not differ between density treatments, indicating that not every encounter between P. australiensis and G. holbrooki led to a predation event. This is consistent with mutual

interference, whereby intra-specific competition within G. holbrooki limited its predatory behaviours. However, since only 1 G. holbrooki was present in the low density treatment in this study, it is not possible to compare the frequency of intra-specific interactions with predation rate. Therefore, it is necessary to conduct further studies in order to definitively conclude whether mutual interference limits predation by G. holbrooki. Since predator behaviour is often neglected in the literature (Lima, 2002), I consider it to be highly valuable to further examine how the behaviour of invasive freshwater predators at different densities affects interactions with native prey.

Interference competition between G. holbrooki and M. novemaculeata was also found to be density dependent, whereby M. novemaculeata was more aggressive under a high rather than low density of G. holbrooki (Chapter 3). While G. holbrooki did not display an increase in inter-specific aggression with density, it was still expected that M. novemaculeata would experience lowered fitness when exposed to a high density of G. holbrooki. This is due to aggression being a costly behaviour which directs time and energy away from other activities and increases the risk of injury (Amarasekare, 2002, Capelle et al., 2015). However, the growth and survivorship of M. novemaculeata was not affected by G. holbrooki (Chapter 4).

Similarly, Schuman et al (2015) reported that interference competition between the invasive

G. affinis and the barrens topminnow and Sonoran topminnow did not alter the growth rates of the two native species. This result, along with that of the present study, would suggest that despite aggression being commonly cited as a key mechanism behind the impact of G. holbrooki, it may have a negligible effect on some native competitors.

It is however, critical to note that discrepancies between laboratory and field experiments examining inter-specific competition are not uncommon in the literature (Britton et al., 2011,

Kornis et al., 2014, Schumann et al., 2015). This is due to the presence of abiotic and biotic

variables in natural settings which cannot be replicated in the laboratory as well as the fact that the densities of invaders in some laboratory studies may be higher than those in the field

(Britton et al., 2011, Schumann et al., 2015). In the present study, while the high density level in Chapter 3 was 9 fish L-1, in Chapter 4 the high density equivalent was 0.036 fish L-1.

Therefore, it is possible that the disparity in results between Chapters 3 and 4 was due to a difference in the densities which were used. In addition to elevated densities, it is not possible to determine whether the discrepancy between the results of Chapters 3 and 4 were due to a shift in resource usage or because M. novemaculeata may have been behaviourally dominant over G. holbrooki. This could be determined by placing remote underwater video (RUV) units within enclosures as well as by measuring fin damage (which is indicative of aggression received) and gut contents (which provides an insight into resource usage) in the future.

Besides the above limitations, there is some evidence that the negative effects exerted by invasive species on native competitors peak at moderate densities (Yokomizo et al., 2009,

Kornis et al., 2014). Theoretically, at this stage, self-regulation due to intra-specific interactions within an invader’s population has not interfered with inter-specific competition

(Kornis et al., 2014). Given the possibility that there is a non-linear relationship between density and impact for G. holbrooki, it may be necessary to use a wider range of G. holbrooki densities in future experiments.

6.3 The importance of intra-specific interactions

Examining the relative effects of intra- and inter-specific interactions provides an insight into the dynamics of competitive interactions (Inouye, 2001, Forrester et al., 2006). Furthermore, it allows us to better predict the impact of an invasive species on native prey and competitors

(Britton et al., 2011). Despite this, intra-specific interactions are seldom considered in relation

to invasive-native competitive interactions which explore density-dependence. I set out to determine whether at a high density G. holbrooki and M. novemaculeata 1) engaged more in intra- than inter-specific competition, as per reviews of competition theory (Connell, 1983), 2) equal magnitudes of each type, as predicted by the lottery model (Sale, 1977) or 3) whether their inter-specific interactions exceeded the strength and outcome of their intra-specific competitive interactions (Pintor et al., 2009).

The patterns of interference competition displayed by M. novemaculeata and G. holbrooki did not conform to the competition model which I had expected in neither Chapters 3 nor 4. M. novemaculeata were more aggressive to G. holbrooki than con-specifics at a high density, which runs counter to the competition model dynamic commonly reported for species in their native ranges (Connell, 1983). Aggression from G. holbrooki was greater towards con- than hetero-specifics at a low density, yet otherwise did not differ at a high density. In Chapter 4, the competitive interactions of both species conformed to the lottery model, which would suggest that the costs of competing with con- or hetero-specifics were similar. As previously stated, a disparity between the results of laboratory and field experiments is not uncommon and is likely to be due to variations in population densities as well as the presence of variables which were not controlled in the laboratory (Britton et al., 2011, Schumann et al., 2015).

It is important to note that while the results of my thesis did not conform to what I initially predicted my expectations were based primarily on studies which examined species in their native ranges (Connell, 1983, Forrester et al., 2006). Furthermore, few studies which have examined density-dependent inter-specific competition between invasive and native freshwater species have employed experimental designs which allow an accurate assessment of the magnitudes of intra- and inter-specific competition. The substitutive design (where the overall density of individuals is constant but species composition is altered) and additive

design (where the density of one species is constant and the other varied) are effective at detecting the presence of inter-specific competition (Inouye, 2001). However, because these methods simultaneously alter the densities of con- and hetero-specifics present, it is not possible to accurately differentiate between the strength and outcomes of intra- or inter- specific competition (Forrester et al., 2006, Inouye, 2001). Furthermore, there is some evidence that, depending on the type of experimental design used, the effect of a freshwater invasive species upon a competitor can be exaggerated (Britton et al., 2011)

The present study employed a form of response surface design to measure the relative magnitudes of intra-and inter-specific competition in the laboratory and field. Therefore it is challenging to compare the results of this thesis to those of studies which have used a substitutive (Becker et al., 2005, Sutton et al., 2013) or additive design (Mills et al., 2004,

Kornis et al., 2014). Furthermore, density-impact relationships are likely to be species- specific (Yokomizo et al., 2009), and so the competitive dynamics displayed by G. holbrooki and M. novemaculeata in the present study may significantly differ from those of other competing invasive and native freshwater species. For these reasons, I consider it critical that there is a greater standardisation of experimental methods used by studies which investigate density-dependent competition for invasive freshwater species.

6.4 Multiple stressor effects

Despite their ubiquity in freshwater habitats (Christensen et al., 2006, Townsend et al., 2008,

White et al., 2015) little is known of how multiple stressor influence the strength and outcome of interactions between invasive and native species. This thesis demonstrates the value of considering the effects of more than one variable when examining interference competition

(Figure 6.1). I report that the frequency of aggressive behaviours displayed by M.

novemaculeata and G. holbrooki were determined by a positive antagonistic and negative synergistic interaction, respectively, between temperature and salinity. It is likely that the responses of these species fitted different forms of a non-additive interaction due to their tolerances to salinity stress, since M. novemaculeata were more aggressive than G. holbrooki under elevated salinity in isolation and elevated salinity plus elevated temperature. Therefore, examining how differing levels of tolerance to a single stressor between species, as well as between populations of single species, influences responses to multiple stressors would be highly valuable. Such an approach may assist in our ability to predict the occurrence of non- additive interactions (antagonistic and synergistic), which is currently exceedingly difficult to do (Christensen et al., 2006, Matthaei et al., 2010, Piggott et al., 2015).

6.5 Interactions between population and individual-level variables

Individual-level variables have an important influence on the strength and outcome of inter- specific interactions (Cain et al., 2011). Despite this, we still have a limited knowledge of how individuals respond to invasive competitors or predators according to environmental context (Bell, 2007). Where possible in this thesis, I sought to identify how individuals responded to competition and predation under different contexts, depending on their size and sex (see conceptual framework, Figure 6.1).

I observed that the size of P. australiensis influenced its interactions with the predatory G. holbrooki. Smaller P. australiensis were more frequently the recipients of nips and approaches from G. holbrooki than their larger conspecifics, particularly when predator density was high. Since G. holbrooki are not gape-size limited (Baber and Babbitt, 2003,

Smith and Smith, 2015), rather than this being a function of predator selectivity, it is likely due to intra-specific dominance hierarchies since smaller P. australiensis were also found to

occupy shelters less and prey size did not influence consumption. This result corresponds to other studies which suggest that subordinate individuals may be more vulnerable to predation than their more dominant con-specifics (Mappes et al., 1993, Halliday and Morris, 2013).

Abiotic/ biotic Interaction Abiotic/ biotic Variable 2 Variable 1

Species-specific Group Level Individual Level tolerances Context Variables (Sex ratio, social (Sex, size, reproductive structure) condition, personality)

Behavioural Mechanism (Aggression, foraging)

Population Dynamics INTERACTION STRENGTH (Growth, survivorship, AND OUTCOME reproductive output)

Figure 6.1 Conceptual framework depicting the outcomes of this thesis which can be applied to studies examining behavioural interactions between invasive and native species. The strength and outcome of inter-specific interactions is mediated by behavioural mechanisms which are in turn dependent on species-specific, group and individual-level responses to (multiple) abiotic and biotic variables.

In addition to predator-prey interactions, body size modulated the response of M. novemaculeata to interference competition. Notably, however, this varied with the density of con- and hetero-specifics to which the native was exposed (Chapter 3). Inter-specific aggression from small M. novemaculeata exceeded that of their larger con-specifics when exposed to a high density of G. holbrooki, which supports the hypothesis that smaller

individuals will become more aggressive as the resource value increases (Pettersson et al.,

1996, Tatara and Berejikian, 2012). A further interaction between an environmental and individual-level variable was observed in Chapter 5. Upon exposure to elevated salinity, both in isolation and combined with elevated temperature, M. novemaculeata elected to direct their aggression toward smaller G. holbrooki. This would suggest that under stressful conditions,

M. novemaculeata may attempt to minimise the risk of injury due to interference competition by interacting with smaller competitors.

In contrast to M. novemaculeata, aggression in G. holbrooki was not mediated by the size of hetero-specifics, regardless of environmental context. This result contradicts those of previous studies which report that Gambusia spp. are less aggressive toward large than small and medium sized hetero-specifics (Rincon et al., 2002, Magellan and García-Berthou, 2015).

However, this disparity could be due to species-specific variation, since G. holbrooki may respond differently to the juveniles of a predatory species such as M. novemaculeata, than to non-piscivorous species. I did however find evidence that interference competition was dependent on both density and sex for G. holbrooki. Male G. holbrooki were significantly more aggressive to female con-specifics at a low than high density. Notably, sexual aggression from male G. holbrooki has previously been shown to interfere with the foraging behaviour of female con-specifics (Pilastro et al., 2003). Therefore, the present study suggests that reduced intra-sexual aggression at high population densities may be a mechanism behind the ability of G. holbrooki to persist at high abundances.

In this thesis I considered individual-level variables to be independent of the social context.

However, individual-level responses can vary with social structure and sex ratio (Pettersson et al., 1996, Brown et al., 2009, Kaiser et al., 2013). Therefore, it would be beneficial for future studies to examine how environmental variables and social structure interact to modulate the

strength and outcome of inter-specific interactions for individuals (Figure 6.1). Furthermore, while I solely examined how morphological sources of individual-level variation mediated direct interactions, other sources of variation, including personality, can mediate predator- prey and competitive interactions, and should also be considered in conjunction with environmental variables (Webster et al., 2009, Brown and Irving, 2014, Belgrad and Griffen,

2016).

6.6 Conclusion

This thesis demonstrates that the strength and impact of predator-prey and competitive interactions between the invasive G. holbrooki and native Australian species depends on environmental and individual-level context. Notably, although the frequency of its direct interactions with native species may increase with density, my results suggest that the survivorship and growth of native prey and competitors is not dependent on G. holbrooki density. Therefore, management strategies which reduce the population densities of this invader without achieving complete eradication may be futile. Furthermore, despite not having been previously exposed to G. holbrooki and therefore possibly naïve, both P. australiensis and M. novemaculeata displayed avoidance and aggressive responses to the invader. This may be due to the native species having co-evolved with other predators and competitors (Banks and Dickman, 2007, Dunlop-Hayden and Rehage, 2011). My results also emphasise the importance of simultaneously measuring the effects of multiple variables

(stressors) on inter-specific interactions, as well body size and sex. Ultimately, the effective management of invasive species may require targeted management strategies which recognise the potential for complex interactions between environmental, group and individual level variables.

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APPENDIX 1: A COMPARISON OF MACQUARIA NOVEMACULEATA AND GAMBUSIA HOLBROOKI SIZE

I compared the standard lengths of Macquaria novemaculeata and Gambusia holbrooki which were exposed to one another in the low and high density mixed conditions of Chapter 3. The analysis was conducted using SPSS Statistics 21, IBM, Armonks, NY, U.S.A. The General

Linear Mixed Model included density and species (fixed) and cage ID (random) as factors.

The standard length of M. novemaculeata was significantly greater than that of G. holbrooki in both low and high density mixed conditions (ANOVA, Species, F1,125 = 1.295, p = 0.002)

(Figure 7.1).

45

40

35 5 5 30

25

20 (mm) Length 10 15 50

10

5

0 Low Density High Density

Figure 6.2 The mean (± SE) length of G. holbrooki (open bars) and M. novemaculeata (closed bars) at T0 in low and high density mixed conditions in this study, sample sizes of individuals are above bars.

APPENDIX 2: ADDITIONAL PUBLICATION

The following publication was prepared and published during the course of my PhD candidature. The data was collected during 2011 as part of my Honors thesis.