TAXONOMY AND ECOLOGY OF PREDATORY MARINE FLATWORMS (PLATYHELMINTHES: POLYCLADIDA) IN BOTANY BAY, NEW SOUTH WALES, AUSTRALIA

by

Ka-Man Lee

A thesis submitted in fulfilment of the requirements for the degree of Master of Science by research University of New South Wales

April 2006 ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed Ka-Man Lee April 2006

II ACKNOWLEDGEMENTS

Without the encouragement and enthusiasm of my supervisor, Dr. Emma Johnston, this thesis would not have been possible. Thank you for allowing me to pursue some innovative experiments and for your inspiration and criticism along the way. I thoroughly appreciated your patience and guidance.

I am eternally grateful to my co-supervisors, Assoc. Prof A. Michel Beal and Dr. Alistair Poore. Assoc. Prof Michel Beal has been incredibly supportive and generous with his time. I thoroughly enjoyed and appreciated your endless supply of patience and guidance. I enjoyed listening to your sharing. Thanks for visiting me in the dark room when I was doing the tedious observation work. I am also truly indebted to Dr. Alistair Poore who has helped me so much throughout my study. Thanks for telling me you are always there and willing to help whenever I need. Thanks for giving me the opportunity to gain the experience in demonstration. I really appreciate that.

I would especially like to acknowledge Professor John Hodgkiss for his professional advice and critical comment on the manuscripts and earlier drafts of my thesis. Thank you for your inspiration for scientific research. You are my constant source of encouragement and support.

Identification of the fabulous marine flatworms would not have been possible without the guidance from Dr. Leslie Newman at the Auckland Museum. Thanks for your endless support and advice in identifying and preserving the polyclads. I would also like to acknowledge the help of Gavin McKenzie at the Histology and Microscopy Unit at University of New South Wales for the preparation of whole mount specimens.

Many people from the Johnston and Poore Lab provided me with professional advice in the fields of experimental setup, statistical analysis and writing. I would like to thank the following in order of appearance: Graeme Clark, Richard Piola, Keyne Monro, Dave Roberts, Nicole Hill, Bronwyn Combo and Candida Barclay. In particular, thank you to Richard Piola for his assistance in the laboratory experimental setup on weekends.

I am grateful to the people from the workshop in the School of Biological, Earth and Environmental Sciences (School of BEES), John Matossian, Peter Boormian, and Ross Vickery, for their assistance in solving the technical problems. They are of great help in drilling holes and making windows on the experimental apparatuses whenever I need.

III I would like to acknowledge the help of Stephanine Poon, Titus Kwok and Chris Wong for their assistance in collecting seawater during weekends and holidays. Special thank is given to Carmen Lee who provided me with expert advice on the use of computer software and photo-taking.

Lee Ann Rollins, Candida Barclay, Nicole Hill, Keyne Monro, Richard Piola and Kelly Wright have provided me with valuable advice and endless care in the past two years. Thanks for your friendship and support. I am grateful to my friends who are miles away from me, but provide me constant support and love throughout my study. In particular, I would like to thank Dr. J-D Gu, Dr. Billy Hau, Jessie Lai, Jennifer Wong, Carmen Woo and Ida Yu. Thanks for encouraging me when my plates disappeared in the sea.

To my family, thank you for always encouraging me. Without your love and support, there is no way possible that I would have been able to undertake this project. I would like to thank my parents and grandparents who provide endless support and love even though I am away from home throughout my study. I sincerely thank for your encouragement and tolerance to my bad temper.

May all the glory and honour be unto God, the Creator of all things. Amen.

IV TABLE OF CONTENTS

Originality statement II Acknowledgements III Table of Contents V List of tables VIII List of figures IX Abstract XII

CHAPTER 1: GENERAL INTRODUCTION 1.1 Overview 1 1.2 Taxonomy of marine flatworms 1 1.3 Reproduction and parental care 3 1.4 Food and predation 4 1.5 Ecotoxicology 5 1.6 Research aims 7 1.6.1 Thesis structure 7

CHAPTER 2: DESCRIPTION OF A NEW PREDATORY FLATWORM (PLATYHELMINTHES, POLYCLADIDA) FROM BOTANY BAY, NEW SOUTH WALES, AUSTRALIA 2.1 Abstract 8 2.2 Introduction 9 2.3 Materials and methods 10 2.3.1 Specimen collection 10 2.3.2 Specimen processing 10 2.3.3 Predatory behaviour 12 2.4 Results 13 2.4.1 Systematics 13 2.4.2 Biology 18 2.4.3 Ecology: Predatory behaviour and feeding rate 18 2.5 Discussion 20

V CHAPTER 3: ROLE OF BROODING IN HATCHING SUCCESS OF ECHINOPLANA CELERRIMA AND STYLOCHUS PYGMAEUS (PLATYHELMINTHES: POLYCLADIDA) EGGS 3.1 Abstract 22 3.2 Introduction 23 3.3 Materials and methods 26 3.3.1 Study site 26 3.3.2 Specimen collection 26 3.3.3 Experimental design 27 3.3.4 Data analysis 32 3.4 Results 34 3.4.1 Interspecific differences in brooding behaviour 34 3.4.2 Effects of brooding on the hatching success of Echinoplana 34 celerrima and Stylochus pygmaeus eggs 3.4.3 Changes in the proportion of brooding time of Echinoplana 35 celerrima in the presence of potential flatworm egg predators 3.4.4 Significance of brooding to the hatching success of 35 Echinoplana celerrima eggs in the presence of potential flatworm egg predators 3.4.5 Size of flatworms 36 3.5 Discussion 43

CHAPTER 4: LOW LEVELS OF METAL POLLUTION AFFECT REPRODUCTIVE SUCCESS OF A MOBILE INVERTEBRATE 4.1 Abstract 47 4.2 Introduction 48 4.3 Materials and methods 52 4.3.1 Study site 52 4.3.2 Collection of flatworms and barnacles 52 4.3.3 Copper treatments 53 4.3.4 Experimental design 54 4.3.5 Data analysis 58 4.4 Results 59 4.4.1 Predation rate of Stylochus pygmaeus 59 4.4.2 Response of Stylochus pygmaeus to physical stimulation 59 VI 4.4.3 Reproductive success of flatworms 59 4.4.4 Feeding rate of Balanus variegatus 60 4.4.5 Size of barnacles and flatworms 60 4.4.6 Chemical analysis 61 4.5 Discussion 68 4.5.1 Predation rate of Stylochus pygmaeus 68 4.5.2 Reproductive success of Stylochus pygmaeus 70 4.5.3 Effects of Stylochus pygmaeus on the feeding rate of Balanus 71 variegatus 4.6 Conclusion 73

CHAPTER 5: SUMMARY AND IMPLICATIONS 5.1 Marine flatworm diversity 74 5.2 Reproductive behaviour of marine flatworms 75 5.3 Predatory behaviour of marine flatworms 76 5.4 Implications of sublethal effects of copper 77 5.5 Conclusion 77

CITED REFERENCES 78

APPENDIX: MARINE FLATWORM DIVERSITY AT KURNELL 95 PIER, BOTANY BAY, NEW SOUTH WALES, AUSTRALIA

VII LIST OF TABLES

Table 3.1 Summary of two-factor ANOVA of the hatching success and the 37 time taken for the Echinoplana celerrima eggs to finish hatching in the presence and absence of Morula marginalba, Bedeva hanleyi and Stylochus pygmaeus with and without the provision of parental care.

Table 3.2 Summary of one-factor ANOVA of the proportion of time spent 38 brooding that Echinoplana celerrima spent in the presence of (a) Morula marginalba and (b) Bedeva hanleyi and Stylochus pygmaeus. P-value in bold indicate significant difference at Į = 0.050.

Table 3.3 Summary of one factor ANOVA of the number of egg batches 39 that Echinoplana celerrima laid in the presence of (a) Morula marginalba and (b) Bedeva hanleyi and Stylochus pygmaeus.

Table 4.1 Summary of two-factor ANOVA and planned comparisons on the feeding rate of barnacles in the presence and absence of flatworms 62 and copper in Experiments 1 and 3. All planned comparisons were tested against the error term for the main test of Cu treatments. P-values in bold indicates significant differences at Į = 0.050.

Table 4.2 Nominal and measured copper concentrations (µg L-1) for copper treatments used in Experiment 1 & 2 indicate that the measured copper concentrations of the treatment solutions from the experiment 63 are close to their nominal values. Dashes represent copper concentrations not used in a particular experiment.

Table A1 Key to distinguish between the flatworm species at Botany Bay. 99

Table A2 Prevalent sessile species listed in the order of abundance on 107 settlement plates.

VIII LIST OF FIGURES

Figure 2.1 Living Imogine lateotentare sp. nov. from Kurnell Pier, Botany 16 Bay, New South Wales, Australia: (a) colour pattern on dorsal surface and (b) ventral view showing pharynx, gonopores and vas deferens. Scale bar: 1.4 mm.

Figure 2.2 Imogine lateotentare sp. nov. preserved: (a) Diagram of the 17 dorsal surface, (b) morphology of the ventral surface, (c) arrangement of the dorsal eyes, (d) diagrammatic reconstruction of the reproductive system (c - cerebral eyes, ce - cement glands, f - frontal eyes, fa - female antrum, go - gonopores, m - mouth, ma - male antrum, n - nuchal tentacle, p - penis papillae, ph - pharynx, pr - prostatic vesicle, s - seminal vesicle, va - vasa deferentia). Scale bars: 1.5 mm (a) and (b); 0.9 mm (c); 0.6 mm (d).

Figure 3.1 Experimental setup to for specimen collection. Attachment of 11 33 x 11 cm settlement plates on a 60 x 60 cm PVC backing plate. 16 settlement plates were attached to each backing plate.

Figure 3.2 (a) Proportion of time that Echinoplana celerrima and Stylochus 40 pygmaeus spent on brooding and (b) mean hatching success of E. celerrima and S. pygmaeus eggs in the presence and absence of parents. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

Figure 3.3 Proportion of time spent brooding by Echinoplana celerrima in 41 the presence and absence of (a) Morula marginalba and (b) Bedeva hanleyi, Stylochus pygmaeus. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

IX Figure 3.4 Mean hatching success of Echinoplana celerrima eggs in the 42 presence and absence of E. celerrima parents and (a) Morula marginalba and (b) Bedeva hanleyi, Stylochus pygmaeus. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

Figure 4.1 Effects of (a) 0, 25 and 50 μg L-1 and (b) 0, 10 and 25 μg L-1 Cu 64 treatments on the number of barnacles eaten by flatworm(s) in Experiment 1 and Experiment 3 respectively. Error bars represent mean (± 1 SE).

Figure 4.2 Effects of (a) 0, 25 and 50 μg L-1, (b) 0, 10 and 25 μg L-1 and (c) 65 0, 10 and 25 μg L-1 Cu treatments on flatworm response to physical stimulation in Experiments 1, 2 and 3 respectively. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

Figure 4.3 Effects of 0, 10 and 25 μg L-1 Cu treatment on the (a) number of 66 flatworm egg batches laid and (b) hatching success of flatworm eggs in Experiment 2. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

Figure 4.4 Effects of (a) 0, 25 and 50 μg L-1 and (b) 0, 10 and 25 μg L-1 Cu 67 treatments on the feeding rate of barnacles in presence and absence of flatworms in Experiment 1 and Experiment 3 respectively. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

Figure A1 Study site at Kurnell Pier, Botany Bay, New South Wales, 96 Australia.

Figure A2 Dorsal view of living Echinoplana celerrima brooding its eggs. 108 Scale bar: 0.9 mm.

Figure A3 Dorsal view of living Stylochus pygmaeus. Scale bar: 0.8 mm. 109 X Figure A4 Dorsal view of living Imogine lateotentare, sp. nov. Scale bar: 110 1.1 mm.

Figure A5 Dorsal view of living unidentified stylochid. 111

Figure A6 Dorsal view of living Cycloporus variegatus with (a) 112 cream-white dorsal surface and red branches, (b) cream-white dorsal surface covered in yellow spots with median white and intermittent purplish strip, (c) diagrammatic reconstruction of C. variegatus reproductive system (ce - cement glands, fa - female antrum, p - penis papillae, pr - prostatic vesicle, se - seminal vesicle) and (d) newly hatched C. variegatus larva. Scale bar: 0.8 mm (a), 0.7 mm (b), 0.1 mm (c) and 0.48 mm (d).

Figure A7 Dorsal view of living Eurylepta aurantiaca and (b) 113 Diagrammatic reconstruction of E. aurantiaca reproductive system (ce- cement glands, fa- female antrum, p- penis papillae, pr- prostatic vesicle, se- seminal vesicle). Scale bars: 0.07 mm.

Figure A8 Dorsal view of (a) anterior and (b) posterior of living 114 Thysanozoon sp.

XI Abstract

Abstract

Marine flatworms are important mobile predators on hard substrate, however little is known about their life history. I recorded seven species of flatworms in Botany Bay and described a new species of flatworm Imogine lateotentare sp. nov. It is distinguished from other species in the same genus by having small, transparent and inconspicuous tentacles and continuous bands of numerous frontal and cerebral eyes. This new flatworm species was found closely associated with the barnacle Balanus variegatus (Darwin, 1854) on which it fed.

Marine flatworms provide elaborate parental care to their offspring but its significance has not been experimentally confirmed. I provided quantitative measurements of the significance of parental care behaviour in Echinoplana celerrima and Stylochus pygmaeus under controlled laboratory conditions. I also examined the changes in reproductive behaviour of E. celerrima and the hatching success of their eggs when exposed to three putative flatworm egg predators. Brooding behaviour of neither species of flatworm enhanced the hatching success of their eggs and exposure of E. celerrima to the potential egg predators did not affect the timing of hatching or hatching success of its eggs. However, E. celerrima spent more time guarding their eggs when exposed to the potential egg predators. Brooding may be an innate behaviour in marine flatworms but it is not essential to their reproductive success.

Marine flatworms are closely associated with sessile organisms and these assemblages are common in bays and estuaries which are subject to anthropogenic inputs from various sources. Impacts of pollutants are known for many flatworm prey species but little is known about the effects on the flatworm themselves. I examined the influence of sublethal concentrations of copper ranging from 0 to 50 μg L-1 on the predatory and reproductive behaviour of Stylochus pygmaeus. These worms were more sensitive to low levels of copper pollution than their barnacle preys. Response of flatworms to physical stimulation, number of egg batches laid and hatching success were greatly reduced at higher copper concentrations. In areas polluted by heavy metals, flatworm populations will be affected at lower concentrations than their barnacle prey and which may alter sessile invertebrate community structure.

XII 1. General Introduction

CHAPTER 1: GENERAL INTRODUCTION

1.1 OVERVIEW

Predators exert a strong selection force and can influence population dynamics and community structure (Gurevitch et al. 2000; Osman and Whitlatch 2004; Sih 1987). In marine hard-substrate assemblages, predators create space when large numbers of prey are removed, and consequently, alter the community structure (Chase et al. 2002; Dayton 1971).

Some of the most common predators within sessile marine assemblages are flatworms.

Although simple animals, flatworms are highly successful mobile predators that prey on a wide range of organisms (Newman and Cannon 2003; Prudhoe 1985). Any factor that affects the abundance of marine flatworms may have ramifications throughout the trophic web.

Despite their prevalence in marine systems, there is a distinct lack of biological or ecological studies on marine flatworms. This may be due to the difficulty in collecting and preserving the delicate flatworm bodies. There have been so few well-preserved specimens in collections that the process of identification and description has been impeded (Newman and Cannon 2003; Prudhoe 1985). In the process of conducting this study, a new species of marine flatworm was identified and described. This study also represents the first behavioural and ecotoxicological experiments conducted on Australian marine flatworms.

1.2 TAXONOMY OF MARINE FLATWORMS

There are approximately 600 marine flatworms recorded in Australian temperate waters, however, many more species remain unidentified (Newman and Cannon 2003). 1 1. General Introduction

Marine flatworms are members of the phylum Platyhelminthes. This phylum includes a diverse group of early Metazoans. It contains more than 10,000 species having a great variety of body form, shape and size (Cannon and Newman 2003; Hyman 1951; MacGinitie and MacGinitie 1949; Prudhoe 1985; Ruppert and Barnes 1991). Recent research has divided the Platyhelminthes into two major groups: Neodermata (parasitic) and Turbellaria

(free-living) (Newman and Cannon 2003).

Marine flatworms are free-living turbellarians of the order Polycladida. They are known as ‘polyclads’ which means ‘many branches’ because they have a multi-branched digestive system to transport food to all parts of the body (Newman and Cannon 2003;

Prudhoe 1985). Conventionally, Polycladida is divided into two sub-orders, Cotylea and

Acotylea. Cotylea are flatworms with a muscular sucker on the ventral surface posterior to the female genital pore and tentacles, when present, are usually marginal. This suborder includes the colourful pseudocerotids and euryleptids. In contrast, Acotylea are flatworms without a ventral sucker; tentacles, when present, are usually nuchal type; and the copulatory complex is usually in the posterior body half. The voracious predators, the stylochids and leptoplanids, belong to the Acotylea (Faubel 1983; Hyman 1953; Newman and Cannon 2003; Prudhoe 1985; Prudhoe 1989).

Colour pattern cannot be reliably used to differentiate between species because marine flatworms may change their colour according to the pigment present in their prey (Newman and Cannon 1994). Instead, identification of marine flatworms requires study of the number and arrangement of the eyes and the reproductive structure (Newman and Cannon 1994;

Prudhoe 1985). The combined effects of colour photographs, field observations of the live animals and the new fixation method developed in recent years, facilitate the description of new species, and hence, improve our understanding on the biology of marine flatworms 2 1. General Introduction

(Newman and Cannon 1995).

1.3 REPRODUCTION AND PARENTAL CARE

Marine flatworms are hermaphrodites but they engage in mutual cross fertilization by intradermal hypodermic insemination or ‘penis fencing’ (Michiels and Newman 1998;

Prudhoe 1985). Most of the worms copulate by intradermal hypodermic insemination, which involves the posterior-to-posterior positioning of two worms with the posterior end of each worm being lift up from the substratum. One of them then stabs its penis through the skin of its partner and injects sperm. On the other hand, ‘penis fencing’ has only been observed in euryleptids and pseudocerotids. Two adult flatworms slowly rear up, lifting up the front part of their body. The muscles around the copulatory structures are tensed and the penis of the worm pushes out of the underside of the body and then directed to its partner.

Both of them try to stab its penis through the soft epidermis of its partner (Newman and

Cannon 2003).

Adult flatworms mainly breed between early and late summer and copulation usually occurs during late summer and autumn and they are able to lay more than one batch of eggs

(each batch consists of several hundreds to thousands of eggs) (Lytwyn and McDermott

1976; Pearse and Wharton 1938). Marine flatworms are considered as simple animals because they have neither coelom, definitive anus, circulatory, respiratory nor skeletal systems (Newman and Cannon 2003; Prudhoe 1985). However, they can maintain a high reproductive effort regardless of the abundance of food during the breeding season

(Chintala and Kennedy 1993) and appear to provide substantial parental care to their offspring.

3 1. General Introduction

Parental care increases the fitness of a parent’s offspring (Clutton-Brock 1991). It is a complex behavioural link between reproduction, ecology and evolution (Kutschera and

Wirtz 2001). The degree of parental care varies among and between marine invertebrate taxa (Fernandez and Brante 2003; Hoare and Hughes 2001). Marine flatworms deposit eggs in chains or plate-like masses covered with gelatinous mucus to ensure that the eggs are firmly attached on the substratum (Newman and Cannon 2003; Prudhoe 1985). It has been suggested that flatworms brood their offspring by covering the egg masses with their body in the first few days after the eggs are laid, or until all the eggs hatch (Rzhepishevskij 1979).

However, no previous study has examined the significance of brooding behaviour to the reproductive success of marine flatworms.

1.4 FOOD AND PREDATION

Marine flatworms are voracious predators and they can extensively reduce their prey populations and hence alter the community structure when they are at high densities

(Skerman 1960a; Skerman 1960b). Flatworms prey on a wide range of organisms, such as ascidians (Newman et al. 2000), bivalves (Ferrero et al. 1980; Galleni et al. 1980; O'Connor and Newman 2003), crustaceans (Hurley 1976; Rzhepishevskij 1979) and gastropods

(Phillips and Chiarappa 1980). However, it has been suggested that adult flatworms are unlikely to be subjected to predation themselves because of the highly toxic or distasteful chemical compounds, such as tetrodotoxin and staurosporine derivates in their epidermis

(Newman and Cannon 2003).

Previous research on predatory behaviour of marine flatworms revealed that they do not feed every day and they can endure protracted fasting (Galleni et al. 1980; Merory and

Newman 2005; O' Connor and Newman 2001). Marine flatworms identify the potential 4 1. General Introduction

prey item by detecting water movement generated from the prey and /or chemoreception

(Prudhoe 1985). Certain species cover the operculum of barnacles and bivalves with copious amounts of toxic mucus (Hurley 1976), rendering it unable to close its opercular valves (Merory and Newman 2005). The worm can then extend its pharynx between the opercular valves, allowing it to enter and consume its prey. They digest either the whole prey or just part of it at a time (Newman and Cannon 2003). When food enters the pharynx, it passes to the gut and nutrients are transported throughout the body through the intestinal branches. Digestion is partially extracellular and partially intracellular and the waste product is passed from the body via diffusion (Prudhoe 1985; O'Connor and Newman 2003).

Furthermore, after the flatworms have digested the flesh of barnacles and bivalves, they deposit eggs inside the empty shells of their prey (Murina et al. 1995; Pearse and Wharton

1938).

In recent years, increasing attention and research has been focused on acotylean flatworms of the family Stylochidae. The Stylochidae is one of the largest families of marine flatworms, with many species known as ‘oyster leeches’ as they prey predominantly on mussels, giant clams and barnacles (Ferrero et al. 1980; Jennings and Newman 1996b;

Newman et al. 1993). They have become a commercial pest to shellfish cultures throughout the world, particularly in the southeast Asia and the United States, and can cause huge economic losses (Friedman and Bell 2000; Littlewood and Marsbe 1990; Newell et al. 2000;

O'Connor and Newman 2003; Rho 1977).

1.5 ECOTOXICOLOGY

Australia’s population is concentrated in coastal areas (Apte et al. 1998). Industrial activity in these regions may increase the ambient level of heavy metals in the aquatic 5 1. General Introduction

environment (Cox and Preda 2005; Santos et al. 2000). Another major source of pollution in

Australian waterways is urban runoff (Stark 1998), which results in an increase in trace metals in bays and estuaries (Spooner et al. 2003). The sessile assemblage associated with marine flatworms occur in estuaries and shallow bays that are subjected to anthropogenic inputs from both industry and urbanisation (Battershill et al. 1998; Turner et al. 1997).

Exposure of these assemblages to heavy metal pollution may lead to a deterioration in the ecosystem’s health through direct effects, such as mortality (Hall et al. 1998; Johnston and

Keough 2002) and indirect effects, such as behavioural changes of exposed organisms and alterations in species interactions (Grue et al. 2002; Hamers and Krogh 1997, Johnston and

Keough 2003). However, no previous study has examined the effects of pollutants on predatory marine flatworms associated with sessile marine assemblages.

In order to investigate the effects of heavy metal pollution on the aquatic ecosystems, it is useful to conduct multispecies toxicity tests. Multispecies microcosms are the lowest level of an ecological organisation at which many processes or interactions can be observed

(Gillett 1989). Multispecies microcosm toxicity test provides information on the interactions between exposed species as well as the indirect effects of pollutants on exposed organisms (DeAngelis 1996; Pratt et al. 1987). Hence, the effects of pollutants on ecological processes at a community, such as population dynamics can be quantified (Gillett

1989; Hall et al. 1998; Kareiva et al. 1996). Anthropogenic impacts on coastal marine assemblages can be predicted through the multispecies microcosm toxicity test on abundant predatory marine flatworm Stylochus pygmaeus and its barnacle prey Balanus variegatus.

6 1. General Introduction

1.6 RESEARCH AIMS

The aims of this thesis were to describe the diversity of marine flatworms occurring at

Kurnell Pier in Botany Bay, to study the predatory and reproductive behaviour of two common flatworm species: Echinoplana celerrima, and Stylochus pygmaeus, and to assess the anthropogenic impacts on marine invertebrate assemblages using a multispecies toxicity test.

1.6.1 Thesis structure

Throughout my study, I collected and identified seven species of marine flatworm.

This constitutes the first observations on the diversity of marine flatworms in Botany Bay,

NSW (Appendix). One of these flatworms was identified as a new species and is described in chapter 2. Chapter 3 quantifies the effect of brooding on the reproductive success of two mostly found flatworm species as well as assessing the effect of parental care on hatching success in the presence and absence of three putative egg predators. In chapter 4, I investigate the sub-lethal effects of copper on the physical condition, reproductive success and predatory behaviour of the marine flatworm, Stylochus pygmaeus. Chapters 2, 3 and 4 of this thesis have been prepared in the form of a manuscript, and have either been submitted to or accepted by international journals. As a result, some aspects of methodology are repeated in each chapter.

7 2.Anewpredatorymarineflatworm

CHAPTER 2: A NEW PREDATORY FLATWORM (PLATYHELMINTHES,

POLYCLADIDA) FROM BOTANY BAY, NEW SOUTH WALES, AUSTRALIA

2.1 ABSTRACT

A new species of Stylochidae flatworm Imogine lateotentare is described from Botany

Bay, eastern New South Wales, Australia. This flatworm is distinguished from other species in the same genus by having small, transparent and inconspicuous tentacles, densely packed purplish pink flecks at the posterior of dorsal surface, distinctive purplish red colour gonopores and continuous bands of numerous frontal and cerebral eyes. Feeding and reproductive behaviour in the laboratory are described. This flatworm was found closely associated with the barnacle Balanus variegatus (Darwin, 1854) on which it fed, by extending its pharynx over the barnacle opercular and sucking out the flesh but ejecting the cirri. It consumed one B. variegatus in 14 d observation period and it was only observed feeding exclusively at night.

This paper has been accepted by Journal of Natural History on 21 October 2005. 8 2.Anewpredatorymarineflatworm

2.2 INTRODUCTION

Free living polyclad flatworms of the family Stylochidae, commonly referred to

‘oyster leeches’, are typically carnivores and are well known predators of bivalves, molluscs and barnacles all over the world (Jennings and Newman 1996b; Landers and

Rhodes 1970; Littlewood and Marsbe 1990; O' Connor and Newman 2001; Pearse and

Wharton 1938). Interest in the diversity, ecology, and especially the feeding behaviour of the family Stylochidae has been spurred by the recognition of stylochids as the cause of great economic loss of commercial bivalves (Chen et al. 1990; Newman et al. 1993; Pearse and Wharton 1938). Despite this, little is known regarding the biology and ecology of

Stylochidae in Australian waters, as only five Imogine spp.: Imogine lesteri (Jennings and

Newman 1996a), I. kimae, I. mcgrathi, I. meganae and I. pardalotus (Jennings and Newman

1996b) have been formally reported from the east coast of Australia.

Identification of marine flatworms remains difficult because of a lack of literature and reports containing detailed descriptions, as well as the rarity of well-preserved specimens for histological preparation (Newman and Cannon 2003). A new fixation method, developed in the last decade, preserves colour patterns of the flatworms with improved authenticity

(Newman and Cannon 1995) and the widespread use of this technique will no doubt lead to a rise in the number of polyclads described. The family Stylochidae has been revised and is subdivided into two major genera, Stylochus, which have a single-lobed seminal vesicle, and Imogine, with a tripartite seminal vesicle (Newman and Cannon 2003; Newman et al.

1993).

9 2.Anewpredatorymarineflatworm

2.3 MATERIALS AND METHODS

2.3.1 Specimen collection

The study site was located at Kurnell Pier, Botany Bay, New South Wales (33”59.92 S,

151”12.62’ E), 15 km south of Sydney. The pier extends 1.3 km from the southern shore of the bay and has restricted public access. Naturally occurring sessile assemblages on the pier include anthozoans, ascidians, bryozoans, hydrozoans, macroalgae, polychaetes and sponges (Clark and Johnston 2005). To assist in specimen collection, assemblages of sessile marine invertebrates were first allowed to develop on artificial substrata. Settlement plates consisted of 6 x 6 cm black Perspex tiles attached underneath two 60 x 60 cm PVC backing plate were deployed at Kurnell Pier on 4 May 2004. Backing plates were suspended horizontally at a depth of 3 m below the low water mark. After 12 weeks settlement plates were retrieved, and any flatworms found between settlement and backing plate were collected immediately using a small paintbrush and spatula. Settlement plates were retained and brought back to the laboratory in aerated water and placed in a dark constant-temperature room (23.5 oC), in a transparent plastic container (15 x 8 x 8 cm) with

1 L of field seawater. After 24 hours, any flatworms emerging from the assemblages were collected using a paintbrush. Collection of flatworms for taxonomic studies continued for a year.

2.3.2 Specimen processing

Collection and preservation of flatworms was made difficult by the mobility and extreme delicacy of the worms. The fixative solution used was 10% formalin in seawater.

The fixative solution was frozen and the polyclads were fixed by coaxing onto a filter paper 10 2.Anewpredatorymarineflatworm

and placed onto the frozen fixative. Cold fixative was added to just cover the flatworm in order to prevent the specimen being dried out. A soft brush was used to ensure the flatworm remained flat under the fixative and then the flatworm was left for 24 hours without disturbance. The fixative was replaced by 70% ethanol for long term preservation. This method ensures that the flatworms remained flat with the colour pattern preserved for histological preparation and microscopical examination (Newman and Cannon 1995;

Newman and Cannon 2003).

Whole mounts of two of the Imogine lateotentare were prepared by staining flatworms in Mayer’s haematoxylin for five minutes in order to obtain the best staining results.

Specimens were then dehydrated in graded alcohols, cleared in xylene and mounted in

Canada balsam. Longitudinal serial sections of the reproductive regions were prepared by embedding excised tissue in 56 oC Paraplast, cutting at 6 µm and staining with haematoxylin and eosin as described by Newman and Cannon (1995) and Newman and

Cannon (2003).

Drawings and measurements were made with the aid of a micro projector

(Ken-A-Vision, MFG, INC, USA). Measurements of the body were taken from live animals in a quiescent state and expressed as length (mm) x width (mm) for the type material only.

These measurements can only be used as a guide because of the plasticity of the polyclads.

Diagrammatic reconstructions of the reproductive systems are given. Descriptions of colours are based on the living animals and the colour descriptions were written in numbers referring to Pantone® Colour chart. All material was lodged at the Australian Museum: whole mounts are designated as WM, longitudinal serial section as LS and whole animal stored in 70% alcohol (S).

11 2.Anewpredatorymarineflatworm

2.3.3 Predatory behaviour

Imogine lateotentare were observed to prey on barnacles and the mechanism on how it consumed its prey was investigated in the laboratory. Size of barnacle opercular openings and length of flatworms were measured prior to the experiment. Barnacle size was tested using a one-factor analysis of variance (ANOVA) with the presence of flatworms as a fixed factor to ensure that the size of barnacle prey tested in the treatments and controls were consistent. Transparent plastic containers (15 x 8 x 8 cm) were used as experimental containers. Each container was filled with 1.5 L of field seawater, continuously aerated and changed once every 24 h. Of the 15 I. lateotentare collected during the study, six were used for a predatory behavioural study. One settlement plate with five Balanus variegatus recruits was placed in each predation treatment container. One flatworm was placed in each of the six predation treatment containers and a further six containers holding barnacles only were used as controls. All other organisms were scraped from the settlement plate.

Observations of flatworm predation on barnacles were taken once every four to six hours during both day and night for two weeks. A record was made of the number of barnacles consumed, and the activity and conditions of flatworms and barnacles.

12 2.Anewpredatorymarineflatworm

2.4 RESULTS

2.4.1 Systematics

Stylochidae Stimpson, 1857

Imogine Girard, 1853

Imogine lateotentare sp. nov.

(Figures 2.1-2.2)

Material examined

HOLOTYPE: W 29330, WM, 4 May 2004, Kurnell Pier, Botany Bay, New South

Wales, Australia.

PARATYPES: W 29331, WM, 16 July 2004, Kurnell Pier, Botany Bay, New South

Wales, Australia; W 29332, LS, same data; W 29333, S, same data.

Description

The size of flatworms measured live ranged from 9.5 x 4.7 mm to 19.2 x 8.0 (SE ± 0.7 x 0.2) mm (N = 15). Body rounded oval, thick and fleshy, blunt posterior, without marginal ruffles (Figure 2.2a). Background of dorsal surface cream-beige (721) with scattered brown

(731) mottling and light brown (722) flecking towards the margin. Purplish pink (507) flecks densely packed at the posterior of dorsal surface (Figure 2.1a). Gonopores purplish red (216) at posterior end on ventral surface (Figure 2.1b). Nuchal tentacles small and transparent, about 0.21 mm wide and 0.88 mm apart with 30 to 40 eyes aggregate at the tip of each nuchal tentacle. Four to five rows of scattered marginal eyes along the anterior margin, more densely packed anteriorly, reducing to two to three rows on both sides. 13 2.Anewpredatorymarineflatworm

Cerebral eyes numerous, embedded in the epidermis, aggregated in two bands lying between the tentacles, scattered to some distance to the back end of the tentacles, extending anteriorly into frontal eyes which are extremely numerous and scattered. Frontal eyes merge into anterior marginal eyes (Figure 2.2c). Pharynx long, narrow and ruffled, in middle of body, three quarters of body length, with about 22 complex pharyngeal folds. Mouth at two-third of pharynx (Figure 2.2b). Intestinal branches are non-anastomosing. Gonopores close but well separated posterior to pharynx. Female gonopore close and posterior to male pore. Vasa deferentia extend anteriorly from pores, originate laterally to pharynx, lying along the entire length of pharynx.

Testes scattered throughout body with tripartite globular seminal vesicle. Ventral, lateral and central lobes of seminal vesicle equally sized, about 0.5 mm long x 0.43 mm wide. Central lobe of seminal vesicle passes posteriorly, joins the prostatic duct at the proximal end of penis. Prostatic duct short joins dorsally to the mid-penis from prostatic organ. Prostatic organ large, muscular, about 0.78 mm long x 0.43 mm wide, horizontal to ejaculatory duct. Penis is simple, papilla small, within deep male antrum.

Ovaries scattered throughout the body. Vagina is long, muscular and narrow, with a shallow female antrum, accompanied by numerous cement glands (Figure 2.2d). Life history is indirect through Gotte’s larva.

Diagnosis

Relatively small size compared to other Imogine spp. Cream-beige and light brown with irregular pattern of dark brown flecks over the dorsal surface and densely packed medially. Purplish pink flecks densely packed at the posterior of the dorsal surface and 14 2.Anewpredatorymarineflatworm

purplish red gonopores at the posterior of ventral surface. Nuchal tentacles are not obvious.

Numerous and scattered cerebral and frontal eyes. Prostatic vesicle is relatively larger than seminal vesicle. Numerous cement glands.

Etymology

Named from the Latin, lateo = hidden, tentare = tentacles, for its inconspicuous and transparent nuchal tentacles.

Distribution

Imogine lateotentare were more common in spring and summer within emptied barnacle shells attached on the settlement plates deployed at Kurnell Pier. It also has been found associated with barnacles attached on the settlement plates at Port Kembla Harbour,

New South Wales, Australia.

15 2.Anewpredatorymarineflatworm

Figure 2.1. Living Imogine lateotentare sp. nov. from Kurnell Pier, Botany Bay, New South

Wales, Australia: (a) colour pattern on dorsal surface and (b) ventral view showing pharynx, gonopores and vas deferens. Scale bar: 1.4 mm.

16 2.Anewpredatorymarineflatworm

Figure 2.2. Imogine lateotentare sp. nov. preserved: (a) Diagram of the dorsal surface,

(b) morphology of the ventral surface, (c) arrangement of the dorsal eyes, (d) diagrammatic reconstruction of the reproductive system (c - cerebral eyes, ce - cement glands, f - frontal eyes, fa - female antrum, go - gonopores, m - mouth, ma - male antrum, n - nuchal tentacle, p - penis papillae, ph - pharynx, pr - prostatic vesicle, s – seminal vesicle, va - vasa deferentia). Scale bars: 1.5 mm (a) and (b); 0.9 mm (c); 0.6 mm (d).

17 2.Anewpredatorymarineflatworm

2.4.2 Biology

Egg deposition of Imogine lateotentare was observed in spring. Flatworms were observed to lay eggs within five days of solitary confinement in the laboratory (Lee and

Johnston unpublished data). Thousands of eggs were deposited, mainly within empty barnacle shells and also in the corners of the container. The egg mass was white and opaque when first deposited, laid in zig-zag chains, covered with sticky gelatinous substance fastening the eggs firmly on the substrata. The egg mass became yellowish brown after three to four days. Hatching began five to seven days after the eggs were laid. Gotte’s larva emerged, black in colour, with four ciliated lobes, anterior and posterior cilia tufts and were positively phototatic. Emptied egg capsules remained on the substrata after hatching.

2.4.3 Ecology: Predatory behaviour and feeding rate

Size of opercula of Balanus variegatus recruits ranged between 4.5 mm and 7.6 mm, with a mean size of 5.5 mm (SE ± 0.07). Length of flatworms ranged between 9.5 mm and

19.2 mm, with a mean length of 12.8 mm (SE ± 1.4) mm. Flatworms were observed to prey on B. variegatus exclusively at night. One B. variegatus was eaten in each of the six treatment replicates during the two week observation period. The flatworm was observed to glide across the barnacle to the opercular valves and to insert its pharynx between the tergum and scutum when the barnacle started to feed. The flatworm then stayed in that position for 15 to 30 minutes. Barnacle was observed trying to avoid the flatworm by closing or scraping its opercular valve around the edge of the opercular opening but that was not successful. The flatworm sucked out all the barnacle flesh using its pharynx, leaving the cirri untouched outside the shell. The flatworm then remained inside the dead barnacle shell for approximately two to three minutes. Dead barnacles eaten by Imogine 18 2.Anewpredatorymarineflatworm

lateotentare were recognized by the presence of open unmoving opercular plates. White food particles showed up clearly in the gut of the worm. After feeding, the flatworm emerged from the empty barnacle shell and moved to a position beneath the settlement plate in the container. It remained there stationary for about three hours presumably in the process of digesting the barnacle. Thereafter, the flatworm recommenced activities spending short periods of time moving around the container interspersed with longer periods of resting under the settlement plate.

The size of barnacle opercular openings did not differ between control and predation treatment containers (F1, 58 = 2.8, p = 0.10) and no barnacle died in the control containers. It is apparent that the replicate flatworms show similar behaviour by spending approximately

80% of time, hiding underneath the settlement plate with occasional exploration of the container. Feeding was observed at night between day 6 and 8 and the flatworms staying underneath the plate with flesh seen in the gut for few hours immediately after consumption.

Further exploratory behaviour was observed for intermittent periods (two to four days) after consumption of the barnacle.

19 2.Anewpredatorymarineflatworm

2.5 DISCUSSION

Stylochids are common voracious mobile predators on natural and artificial substrata, however, only few of them have been formally recorded from temperate Australian waters.

Identification of acotylea species based on their colour pattern only can be especially difficult as they may vary their colour pattern according to the colour of their prey items.

Imogine spp. are usually confused with Stylochus spp., necessitating the use of sectioning of the reproductive structures to distinguish between them. Imogine spp. differ from Stylochus spp. in having tripartite seminal vesicles (Jennings and Newman 1996a; Newman and

Cannon 2003). Imogine lateotentare is distinguished superficially from other Australian and worldwide Imogine spp. by having small and inconspicuous nuchal tentacles, reddish pink flecks densely packed at the posterior on its dorsal surface, distinctive purplish red gonopores clearly seen on its ventral surface, extremely numerous frontal and cerebral eyes, relatively larger prostatic vesicle compared to the seminal vesicle and numerous cement glands.

Particular attention has been paid to the feeding behaviour of stylochids since they are common predators of barnacles and mussels (for example, Chintala and Kennedy 1993;

Ferrero et al. 1980), and are a well known pest of commercial bivalves production throughout the world (Galleni et al. 1980; Jennings and Newman 1996b; Landers and

Rhodes 1970; Newman et al. 1993; Pearse and Wharton 1938). Our observations indicated that Imogine lateotentare only feed at night, and this has not been reported in other studies of feeding behaviour of stylochids (Rzhepishevskij 1979; Skerman 1960a). Such feeding behaviour may allow the flatworm to avoid potential visual predators, e.g. fish. I. lateotentare caused no physical damage to the shell of its prey, Balanus variegates.

However, the barnacle was unable to close its valves properly once the flatworm has 20 2.Anewpredatorymarineflatworm

inserted its pharynx. It has been suggested that the polyclads may paralyse the barnacles through the toxins in their tissues (Newman and Cannon 2003) or in the mucus (Hyman

1951).

Predation is an important factor influencing the characteristics of species, populations and communities, and is one of the most important factors to determine the distribution and abundance of organisms (Connell 1970; Menge 1995; Rand 1985; Sih et al. 1985).

Polyclads are one of the most common mobile predators found on artificial and natural hard substrata and are closely associated with sessile assemblages (Brown and Swearingen 1998;

Dean 1981; Newman 2002). Flatworms occurring at high densities, such as the typhloplanid flatworms which are important predators of mosquito larvae, are able to reduce their prey populations, and hence alter community structure (Blaustein and Dumont 1990). The predation rate of Imogine lateotentare in this study was relatively low when compared to other members of the family Stylochidae preying on other barnacle species in the field (for example, five to ten of barnacles, Balanus improvisus were consumed by one Stylochus tauricus in a month (Murina et al. 1995)). Possible explanations are the small size of I. lateotentare and the reduced energy expenditure of flatworms in well controlled laboratory conditions that are free of predators and other environmental stresses. I. lateotentare is a potential agent shaping assemblage structure if it occurs at high densities in the field.

Considerably more ecological studies of flatworms, such as I. lateotentare, are required before we understand the dynamics of sessile assemblages and their mobile predators.

21 3. Reproductive behaviour and parental care of marine flatworms

CHAPTER 3: ROLE OF BROODING BEHAVIOUR IN REPRODUCTIVE

SUCCESS OF TWO MARINE FLATWORMS

3.1 ABSTRACT

Marine flatworms spend significant amount of time brooding their eggs but the significance of this parental care has not been experimentally examined. Echinoplana celerrima and Stylochus pygmaeus are common free-living flatworms in Botany Bay, New

South Wales, Australia. They were observed using their body to cover their egg masses both in the field and in the laboratory. I provide quantitative measurements of the extent and significance of parental care behaviour in E. celerrima and S. pygmaeus under controlled laboratory conditions. S. pygmaeus spent significantly more time covering egg masses than

E. celerrima. Brooding behaviour of neither species of flatworm enhanced the hatching success of their eggs which was consistently high in both the presence and absence of parents (about 90% for both species). I also examined changes in the reproductive behaviour of E. celerrima and quantified the hatching success of eggs in the presence of three species of putative egg predators. E. celerrima spent less time brooding when exposed to putative egg predators because the flatworm parents devoted more time to guarding their eggs. However, exposure of E. celerrima to the putative flatworm egg predators did not affect hatching success or the time taken for eggs to hatch. Brooding may be an innate behaviour in marine flatworms however it is not essential to their reproductive success.

22 3. Reproductive behaviour and parental care of marine flatworms

3.2 INTRODUCTION

Mating and reproduction are the two most important acts in an animal’s life. Animals display a wide range of parental care behaviours to ensure their offspring’s survival, growth and breeding success (Foighil and Taylor 2000; Taborsky and Foertster 2004). ‘Parental care’ is a descriptive term referring to any form of parental behaviour that would increase the fitness of a parent’s offspring (Barnard 2004; Clutton-Brock 1991) and in some species it is required for egg hatching and egg development (St. Mary et al. 2004). In the broadest view, the establishment of territories, the production of yolked eggs and the protection of eggs and/or young both inside or outside the parental body against predation can be classified as parental care (Barnard 2004; Thiel 2003).

The contribution that parental care makes to offspring viability has been studied in a wide range of taxonomic groups (Charrassin et al. 1999; Eggert et al. 1998). Brooding is a parental care behaviour widely displayed in small invertebrates (Barnard 2004; Zworykin and Budaev 2000). It has been suggested that brooding is associated with small adult size.

This is because larger adults have a relatively greater capacity to produce offspring than their capacity to brood them following the allometric principles of morphological design

(Beekey and Hornbach 2004; Strathmann 1985; Strathmann and Strathmann 1982).

Brooding behaviour has been widely observed among small marine invertebrates, such as amphipods (Dick et al. 2002; Thiel 2000; Thiel et al. 1997), chitons (Creese 1986;

Eernisse 1988), crabs (Ruiz-Tagle et al. 2002), oysters (Foighil and Taylor 2000) and sea stars (Bosch and Slattery 1999; Byrne 2005; Strathmann et al. 1984). In general, among small marine invertebrates, brooding behaviour has been associated with adverse environmental conditions, such as low oxygen availability (Brante et al. 2003; Fernandez 23 3. Reproductive behaviour and parental care of marine flatworms

and Brante 2003; Lardies and Fernandez 2002). Protection of offspring from predation is also one of the important purported functions of brooding behaviour (Aoki and Kikuchi

1991; Kutschera and Wirtz 2001; Taborsky and Foertster 2004; Thiel 1997).

Although marine flatworms are among the simplest of animals, parental care has been observed in field and laboratory conditions (Faasse 2003; Murina et al. 1995; Pearse and

Wharton 1938; Prudhoe 1985). Care of fertilised eggs has been observed in marine flatworms. Adult flatworms lay eggs in chains or plate-like masses which are usually covered with a sticky gelatinous substance secreted from the glands lying in the ventral parenchyma and from the glands opening into the vagina. This ensures that the eggs are fastened firmly to the substratum (Prudhoe 1985). Flatworm parents also provide indirect protection to their offspring using empty barnacle shells as a cradle (Murina et al. 1995).

Moreover, it has been suggested that flatworms display brooding behaviour by covering the egg masses with their body for several days (Merory and Newman 2005; Pearse and

Wharton 1938). The significance of parental care to the reproductive success of flatworms has yet to be investigated.

Marine flatworms display diverse parental care behaviour that may also be useful in protecting their offspring from predators (Murina et al. 1995). Adult flatworms are unlikely to be subjected to predation because of the highly toxic or distasteful chemical compounds, such as tetrodotoxin and staurosporine derivates in their epidermis (Newman and Cannon

2003). Eggs, larvae and juvenile stages, however, may be susceptible to predation due to the weaker potency of epidermal secretions (Prudhoe 1985). Nevertheless, studies are yet to identify putative predators or to examine the role that parental care may play in protecting early life-history stage of the worms from predation.

24 3. Reproductive behaviour and parental care of marine flatworms

Echinoplana celerrima is the most abundant flatworm species in the eastern Australian waters (Newman and Cannon 2003). It is available throughout the year whereas Stylochus pygmaeus is mainly present during the warmer summer months (Lee and Johnston unpublished data, see also Merory and Newman 2005). In the present study, I investigated the contribution of apparent brooding behaviour by E. celerrima and S. pygmaeus to the hatching success of their eggs. I also determined whether reproductive behaviour of E. celerrima was altered by the presence of three putative flatworm egg predators: two predatory whelks, Morula marginalba (Blainville) and Bedeva hanleyi (Angas), and the predatory flatworm, S. pygmaeus. In the field, these three species of mobile predators were found inside shells of the barnacle, Balanus variegatus, together with E. celerrima eggs and they were identified as putative flatworm egg predators.

25 3. Reproductive behaviour and parental care of marine flatworms

3.3 MATERIALS AND METHODS

3.3.1 Study Site

The study site was located at Kurnell Pier (33”59.92’ S, 151”12.62’ E), on the southern margin of Botany Bay, New South Wales, Australia. The pier extends 1.3 km from the southern shore of the bay. It is a tidally flushed embayment and public access is restricted.

Naturally occurring sessile assemblages on the pier include anthozoans, ascidians, bryozoans, hydrozoans, macroalgae, polychaetes and sponges (Clark and Johnston 2005).

Seven flatworm species have been collected from the site, namely: Echinoplana celerrima,

Eurylepta aurantiaca, Cycloporus variegatus, Thysanozoon sp., Imogine lateotentare,

Stylochus pygmaeus and Stylochus sp. In the present study, I used the two most usually found flatworm species, E. celerrima (N = 92) and S. pygmaeus (N =12), collected at

Kurnell Pier during the 2004-2005 reproductive season.

3.3.2 Specimen collection

To assist in specimen collection, assemblages of sessile marine invertebrates were allowed to develop on settlement plates in the study site. Settlement plates consisting of 11 x 11 cm black Perspex tiles attached to the underside of a 60 x 60 cm PVC backing plate.

Five backing plates with a total of 80 settlement plates were suspended horizontally from the pilings of Kurnell Pier at a depth of 3 m below the low water mark in September 2005

(Figure 3.1).

After 13, 17 and 24 weeks (for Experiments 1, 2 (i) and 2 (ii) respectively), 30 settlement plates were retrieved from backing plates for each experiment. Flatworms found 26 3. Reproductive behaviour and parental care of marine flatworms

between the settlement and backing plates, or resting inside empty barnacle shells were collected immediately using a small paintbrush and spatula and placed in a sample jar filled with seawater collected from the study site. Settlement plates were retained and brought back to the laboratory and each of them was placed in a dark constant-temperature room (23 oC ± 0.5 oC) in a well-aerated transparent plastic container (15 x 8 x 8 cm) with 1 L of field seawater. After 24 hours, flatworms emerging from the assemblages were collected as described above. All flatworms were observed for 2 days prior to experiment to ensure that they had recovered from any collection and transport stress (Clesceri et al. 1998). In all experiments, the length (anterior-posterior axis, mm) and width (perpendicular to anterior-posterior axis, mm) of flatworms were measured when they were in a quiescent and relaxed state to ensure that the flatworm adults tested were of the same developmental stage.

No individuals were used in more than one experiment nor provided data more than once for any one experiment.

3.3.3 Experimental design

In all experiments, transparent plastic containers (15 x 8 x 8 cm) were used as experimental containers. Each container was filled with 1 L of filtered seawater. One 6 x 6 cm black Perspex tile was put into each of the containers to provide a resting place for the flatworms. All experiments were carried out in a constant-temperature room (23 oC ±ʳ0.5 oC) under a regular 11L: 13D photoperiod. Salinity, pH, temperature and dissolved oxygen were measured at the commencement of each experiment. Seawater collected from Kurnell Pier was filtered through a 0.2 µm filter and the filtered seawater in experimental containers was replaced once every 24 h.

The major prey of adult Stylochus pygmaeus flatworms are barnacles (Merory and 27 3. Reproductive behaviour and parental care of marine flatworms

Newman 2005). However, in order to ensure accurate assessment of hatching success of flatworm eggs, it is important not to have live barnacles in the experimental containers.

Flatworm larvae are planktonic immediately post-hatching and barnacles, as filter feeders, are potential predators of flatworm larvae. In pilot studies, both Echinoplana celerrima and

S. pygmaeus did not consume dead barnacles nor any similar crustacean food. Consequently, the flatworms were not fed for the duration of the experiments. E. celerrima and S. pygmaeus appeared to rely on the stored energy to produce and brood the eggs in the absence of available food. This is similar to S. ellipticus, which has been shown to maintain a relatively high reproductive effort despite a reduction in food supply (Chintala and

Kennedy 1993).

Experiment 1. Parental care: Brooding behaviour and hatching success of eggs

Twenty Echinoplana celerrima and twelve Stylochus pygmaeus flatworms were used in this experiment. A mating pair of similar sized flatworms were placed in an experimental container and allowed to lay eggs (10 pairs of E. celerrima and six pairs of S. pygmaeus).

The position of egg batches were marked on the wall of the containers with waterproof glass marker. The first batch of eggs laid was treated as the focal egg batch with subsequent egg batches being immediately and carefully removed with a spatula. The containers were randomly assigned to two treatments: containers holding eggs and a pair of flatworm parents and containers from which the flatworms were removed immediately after the focal egg batch had been laid. The number of replicates in the treatment and control was the same in each flatworm species (E. celerrima, n = 5 and S. pygmaeus, n = 3).

Photographs of egg batches were taken every 24 h under an Olympus compound microscope (SZE-ILLK200) connected to Pixelink (PL-A642) with a magnification of 40 x. 28 3. Reproductive behaviour and parental care of marine flatworms

The number of eggs in the focal egg batch were counted from the photographs using

Image-Pro Express 4.0.1 software. Behaviour of the flatworms, proportion of time that flatworms spent on brooding (i.e. the time when flatworm remained still and cover 50% or more of the focal egg batch) and the number of egg batches laid were observed and recorded once every four to six hours until the focal egg batch hatched. Flatworm larvae were collected daily by pouring the litre of seawater from the experimental container into a

1.25 L sample jar and preserving at 7% formalin. Once the focal batch had hatched, multiple solutions of preserved larvae from the same replicate container were collected by pouring through a 10 ͈m sieve. The larvae were then rinsed with Milli-Q® filtered water and resuspended in 500 mL sample solution. Triplicate estimations of flatworm larval numbers in each sample were made by counting the number of larvae in three 20 mL aliquots. Total number of flatworm larvae hatched in each container were estimated by multiplying the number of larvae present in 20 mL (mean of the three sub-samples) by twenty five.

Hatching success of flatworm eggs was then calculated as:

(Number of larvae/ Number of eggs) * 100%

Experiment 2. Brooding behaviour and hatching success in the presence of potential egg predators

The impacts of three potential flatworm egg predators, Morula marginalba, Bedeva hanleyi and Stylochus pygmaeus on the brooding behaviour of Echinoplana celerrima and on the hatching success of its eggs were investigated. E. celerrima was used as the subject in this experiment because it is the most common flatworm species in eastern Australian waters (Newman and Cannon 2003) and is readily available throughout the year at Kurnell

Pier (Lee and Johnston unpublished data). 29 3. Reproductive behaviour and parental care of marine flatworms

(i). Brooding behaviour and hatching success in the presence of Morula marginalba

Nine of the predatory whelks, Morula marginalba were collected and held individually for three days prior to the commencement of the experiment in well-aerated transparent plastic containers (15 x 8 x 8 cm) containing 1 L filtered seawater. Eighteen pairs of

Echinoplana celerrima were treated and observed in the same way as in Experiment 1 until the focal egg batch was laid. To assess the effect of the presence of M. marginalba on the hatching success of E. celerrima eggs, the 18 containers of E. celerrima were randomly assigned to one of the four treatments namely:

a. predator-parental care container (n = 5) parents retained and one M.

marginalba added immediately after the focal egg batch was laid;

b. parental-care container (n = 5) parents retained and left predator-free;

c. predator-egg treatment container (n = 4) parents removed and one M.

marginalba added immediately after the focal egg batch was laid;

d. egg hatching success control container (n = 4) parents removed, no predators

added. Containing the focal egg batch only.

The number of egg batches laid, the proportion of time that the flatworms spent covering the focal egg batch in the presence and absence of Morula marginalba and the hatching success in all containers were recorded and compared.

30 3. Reproductive behaviour and parental care of marine flatworms

(ii). Brooding behaviour and hatching success in the presence of Bedeva hanleyi or

Stylochus pygmaeus

Six Bedeva hanleyi and six Stylochus pygmaeus were collected and held as for Morula marginalba in Experiment 2(i). Eighteen pairs of Echinoplana celerrima were treated and observed as in Experiment 1 until the focal egg batch was laid. In order to assess the effect of the presence of B. hanleyi or S. pygmaeus on the hatching success of E. celerrima eggs, the 18 containers of E. celerrima were randomly assigned to one of the six treatments namely:

a. predator-parental care container (n = 3) parents retained and one B. hanleyi added

immediately after the focal egg batch was laid;

b. predator-parental care container (n = 3) parents retained and one S. pygmaeus

added immediately after the focal egg batch was laid;

c. parental-care container (n = 3) parents retained and left predator-free;

d. predator-egg treatment container (n = 3) parents removed and one B. hanleyi added

immediately after the focal egg batch was laid;

e. predator-egg treatment containers (n = 3) parents removed and one S. pygmaeus

added immediately after the focal egg batch was laid;

f. egg hatching success control containers (n = 3) parents removed and left predator

free.

Number of egg batches laid, proportion of time that the flatworms spent on covering the focal egg batch and the hatching success in all containers were recorded and compared.

31 3. Reproductive behaviour and parental care of marine flatworms

3.3.4 Data analysis

Flatworms produce eggs only when it reaches a certain size (Chintala and Kennedy

1993). In order to ensure that the flatworms used in each experiment were capable of producing eggs and providing similar levels of parental care, flatworm length in each experiment was tested using a one-factor analysis of variance (ANOVA). Differences in hatching success of Echinoplana celerrima and Stylochus pygmaeus eggs as a result of brooding were assessed using one-factor ANOVA with presence of flatworms as a fixed factor. The effect of the presence of each potential predator on the proportion of time spent on brooding by E. celerrima was tested separately using one-factor ANOVA with the presence of predator as a fixed factor. Hatching success and the time taken for the eggs to hatch with and without parental care were analysed separately and compared to that occurring when the potential flatworm egg predators were present and absent using two-factor ANOVA with the presence of predators and presence of parents as fixed factors.

Planned comparison tests were conducted on significant results to determine differences between specific predator-parental care treatments, with all planned comparisons tested against the error term for the main test of predator-parental care treatment (Quinn and

Keough 2002). Planned comparisons are conducted when specific post-hoc tests are required. They are usually more powerful than procedures that test every treatment combination (e.g. Tukeys). All statistical analysis was completed in Statistical Package for

Social Sciences (SPSS version 11.5), and plots of residuals versus means and descriptive statistics showed that the data satisfied assumptions of variance homogeneity and normality.

32 3. Reproductive behaviour and parental care of marine flatworms

3 m below low water mark

nylon rope bolt PVC backing plate settlement plate

about 1 m

brick weight for stability

Figure 3.1. Experimental setup to for specimen collection. Attachment of 11 x 11 cm settlement plates on a 60 x 60 cm PVC backing plate. 16 settlement plates were attached to each backing plate.

33 3. Reproductive behaviour and parental care of marine flatworms

3.4 RESULTS

3.4.1 Interspecific differences in brooding behaviour

There were interspecific differences in brooding behaviour between Echinoplana celerrima and Stylochus pygmaeus. Brooding behaviour in E. celerrima was observed only in the first three to six days after the egg masses were laid whereas S. pygmaeus brooded the eggs until the eggs started hatching. Both S. pygmaeus parents in the treatment containers were involved in brooding concurrently. In contrast, only one E. celerrima parent was involved in brooding at anyone time while the other parent moved around the container or rested underneath the Perspex tile. Furthermore, the focal egg batch of E. celerrima was covered by one of the parents for about 12 ± 2% of time, whereas, the focal egg batch of S. pygmaeus was covered by both parents for about 85 ± 8% of time until it hatched (F 1,6 =

123.2, p = 0.00; Figure 3.1a).

3.4.2 Effects of brooding on the hatching success of Echinoplana celerrima and

Stylochus pygmaeus eggs

Hatching success of Echinoplana celerrima and Stylochus pygmaeus eggs was not influenced by the presence or absence of parents (E. celerrima: F 1, 8 = 0.85, p = 0.38; S. pygmaeus: F 1, 4 = 0.07, p = 0.81, Figure 3.1b), nor was the time taken for the flatworm eggs to hatch influenced by the presence or absence of parents (E. celerrima: F 1, 8 = 0.2, p = 0.64;

S. pygmaeus: F 1, 4 = 0.1, p = 0.81). The time taken for S. pygmaeus eggs to hatch in the presence and absence of parents was about 12 d while those of E. celerrima hatched in about 14 d.

34 3. Reproductive behaviour and parental care of marine flatworms

3.4.3 Changes in the proportion of brooding time of Echinoplana celerrima in the presence of potential flatworm egg predators

Echinoplana celerrima spent less time brooding in the presence of Morula marginalba

(Table 3.2, Figure 3.2a), Bedeva hanleyi and Stylochus pygmaeus (Table 3.2, Figure 3.2b).

The presence of M. marginalba reduced brooding time by about 50% (Figure 3.2a). The presence of either B. hanleyi or S. pygmaeus had the same effect on E. celerrima reducing brooding time by about 80% (F 1, 4 = 0.8, p = 0.42, Figure 3.2b).

Echinoplana celerrima displayed different behaviour when confronted by different potential egg predators. Observations indicated that E. celerrima avoided contact with B. hanleyi or Stylochus pygmaeus while staying near, but not on top of, the focal egg batch. In contrast, E. celerrima spent about 21 ± 5 % of its time closely associated with Morula marginalba, with both parents simultaneously surrounding the whelk’s shell rendering it unable to move until the eggs hatched.

3.4.4 Significance of brooding to the hatching success of Echinoplana celerrima eggs in the presence of potential flatworm egg predators

The average hatching success of Echinoplana celerrima eggs in the presence and absence of the three potential egg predators were compared (Figure 3.3). Although E. celerrima parents guarded the eggs in the presence of potential egg predators, hatching success of E. celerrima eggs and the time taken for the eggs to hatch were not influenced by the presence of either parents or potential flatworm egg predators (Table 3.1, Figure 3.3). E. celerrima continued to lay batches of eggs regardless of the presence or absence of predators and the number of egg batches laid was not influenced by the presence of 35 3. Reproductive behaviour and parental care of marine flatworms

predators (Table 3.3). The time taken for E. celerrima eggs to hatch across treatments in

Experiment 2 was about 11 to 14 d.

3.4.5 Size of flatworms

There were no differences in the length of Echinoplana celerrima in the treatments and controls in Experiment 1 (F 1, 18 = 0.3, p = 0.60), Experiment 2 (i) (F 1, 34 = 0.3, p = 0.58) or

Experiment 2 (ii) (F 1, 34, = 0.1, p = 0.72). There were no differences in the length of

Stylochus pygmaeus across treatments in Experiment 1 (F1, 10 = 1.8, p = 0.21). The average length of S. pygmaeus used in Experiment 1 was 14.0 ± 0.4 mm and that of E. celerrima in

Experiments 1, 2 (i) and 2 (ii) was 17.5 ± 0.7 mm, 15.4 ± 0.4 mm and 13.8 ± 0.3 mm respectively. In each experiment, flatworms had a similar body size and thus were expected to show a uniform level of parental care for their eggs, consistent with the species (Chintala and Kennedy 1993).

36 3. Reproductive behaviour and parental care of marine flatworms

Table 3.1. Summary of two-factor ANOVA of the hatching success and the time taken for the Echinoplana celerrima eggs to finish hatching in the presence and absence of Morula marginalba, Bedeva hanleyi and Stylochus pygmaeus with and without the provision of parental care.

Independent variable df MS F P Dependent variable: Hatching success of the eggs Experiment 2(i) Presence of Morula marginalba 1 1.973 0.762 0.397 Presence of parents 1 0.221 0.085 0.775 Interaction (M. marginalba x parents) 1 0.005 0.002 0.965 Residual 14 2.589

Experiment 2 (ii) Presence of predator 2 1.508 1.155 0.348 Presence of parents 1 1.850 1.416 0.257 Interaction (predator x parents) 2 0.933 0.714 0.509 Residual 12 1.306

Dependent Variable: Time taken for the eggs to hatch Experiment 2(i) Presence of Morula marginalba 1 0.155 0.453 0.511 Presence of parents 1 0.251 0.734 0.405 Interaction (M. marginalba x parents) 1 0.155 0.453 0.511 Residual 14 0.342

Experiment 2 (ii) Presence of predator 2 3.389 2.179 0.156 Presence of parents 1 1.389 0.893 0.363 Interaction (predator x parents) 2 0.389 0.250 0.783 Residual 12 1.556

37 3. Reproductive behaviour and parental care of marine flatworms

Table 3.2. Summary of one-factor ANOVA of the proportion of time spent brooding that

Echinoplana celerrima spent in the presence of (a) Morula marginalba and (b) Bedeva hanleyi and Stylochus pygmaeus. P-value in bold indicate significant difference at Į =

0.050.

ʳ ʳ ʳ ʳ Time spent brooding Expert ʳ Source Treatment df MS F P 2 Main test Predator M. marginalba vs. control 1 0.007 6.756 0.032 Error 8 0.001

Main test Predator B. hanleyi vs. S. pygmaeus 2 0.016 17.502 0.003 vs. control Error 6 0.001

Planned Predator B. hanleyi vs. control 1 0.023 24.836 0.002 comparisons Error 4 0.001

Predator S. pygmaeus vs. control 1 0.025 27.596 0.002 ʳʳ Error ʳ 4 0.001 ʳʳ

38 3. Reproductive behaviour and parental care of marine flatworms

Table 3.3. Summary of one factor ANOVA of the number of egg batches that Echinoplana celerrima laid in the presence of (a) Morula marginalba and (b) Bedeva hanleyi and

Stylochus pygmaeus.

ʳ ʳ ʳ Number of egg batches Expert ʳ Source Treatment df MS F P 2 Main test Predator M. marginalba vs. control 1 0.400 0.889 0.373 Error 8 0.450

Main test Predator B. hanleyi vs. S. pygmaeus 2 5.778 0.800 0.492 vs. control Error 6 7.222 ʳʳʳʳ ʳʳʳʳ

39 3. Reproductive behaviour and parental care of marine flatworms

Figure 3.2. (a) Proportion of time that Echinoplana celerrima and Stylochus pygmaeus spent on brooding and (b) mean hatching success of E. celerrima and S. pygmaeus eggs in the presence and absence of parents. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

40 3. Reproductive behaviour and parental care of marine flatworms

Figure 3.3. Proportion of time spent brooding by Echinoplana celerrima in the presence and absence of (a) Morula marginalba and (b) Bedeva hanleyi, Stylochus pygmaeus. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

41 3. Reproductive behaviour and parental care of marine flatworms

Figure 3.4. Mean hatching success of Echinoplana celerrima eggs in the presence and absence of E. celerrima parents and (a) Morula marginalba and (b) Bedeva hanleyi,

Stylochus pygmaeus. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

42 3. Reproductive behaviour and parental care of marine flatworms

3.5 DISCUSSION

Echinoplana celerrima and Stylochus pygmaeus displayed active brooding behaviour in the field and in the laboratory. However, there were interspecific differences in this behaviour. S. pygmaeus spent significantly more time covering egg masses than E. celerrima. Differences in parental care patterns between species have been suggested to arise from interspecific differences in the parental costs and the benefits gained by the offspring (Clutton-Brock 1991). E. celerrima are generally more abundant and widespread than S. pygmaeus and recruit throughout the year (Newman and Cannon 2003) whereas S. pygmaeus only recruit during summer (Lee and Johnston unpublished data). The egg batches of E. celerrima contain a large number of eggs which, as a consequence of being arranged in plate-like masses, are well attached to the substratum. In contrast, the egg batches laid down by S. pygmaeus are made up of many egg capsules that each contains 12 embryos arranged in a sphere. This arrangement results in poor contact and loose attachment to the substratum. The reduced reproductive period of S. pygmaeus and the structural difference in egg batch construction may necessitate more diligent parental care, devoting more time to brooding to ensure the survival of eggs and production of larvae.

Echinoplana celerrima and Stylochus pygmaeus were observed covering egg masses with their body, however, consistently high hatching success of E. celerrima and S. pygmaeus was maintained regardless of the level of parental care provided. Protection of offspring is one of the most important functions of parental care behaviour (Clutton-Brock

1991). Morula marginalba, Bedeva hanleyi and S. pygmaeus are organisms usually found co-occurring with E. celerrima and are voracious mobile predators. M. marginalba and B. hanleyi are members of the family Muricidae, which are prevalent prosobranch gastropods in south-eastern Australia (Fairweather 1988; Fairweather et al. 1984; Jansen 2000; Jeffery 43 3. Reproductive behaviour and parental care of marine flatworms

2003; Moran et al. 1984; Troup et al. 2005). These three potential flatworm egg predators were observed not only preying on Balanus variegatus and but also staying inside the empty barnacle shells with the E. celerrima eggs (Lee and Johnston personal observations).

The presence of M. marginalba, B. hanleyi and S. pygmaeus, however, did not affect the hatching success of E. celerrima eggs in this study. Our putative egg predators were not observed consuming eggs in the presence or absence of flatworm parents. These co-occurring organisms may not be the major flatworm egg predators in the field.

Furthermore, egg batches may be protected by toxic chemicals secreted in the gelatinous substance surrounding the egg masses. The chemicals present in the gelatinous substance have not been examined. Nevertheless, changes in the brooding behaviour of E. celerrima were observed in the presence of putative flatworm egg predators.

Echinoplana celerrima spent less time covering the egg batch in the presence of other co-occurring organisms in this study. E. celerrima spent more of their time moving around the predator-parental care containers when any of the three potential egg predators were present, or staying beside the egg batch instead of covering it. E. celerrima parents even actively guarded its eggs in the presence of Morula marginalba. Both flatworm parents surrounded the M. marginalba’s shell and causing it to be unable to move. Guarding behaviour is an important function of parental attendance and is believed to be useful in protecting the egg masses from predators or to facilitate the embryonic development (Asoh and Yoshikawa 2001; Blumer 1986; Li and Jackson 2003; Markman et al. 1995).

Parental care is believed to improve offspring survivorship (Barnard 2004;

Clutton-Brock 1991) and in some species it is required for egg hatching and egg development (St. Mary et al. 2004). However, in the present study, presence of flatworm parents did not have beneficial effects on the hatching success of their eggs regardless of the 44 3. Reproductive behaviour and parental care of marine flatworms

presence or absence of putative egg predators. This brings up a question as to why the two flatworm species still invested time and energy in brooding and guarding their eggs.

The inability to demonstrate any changes in hatching success of flatworm eggs has two possible explanations. The first explanation stems from the benign nature of the physical and chemical environment maintained during experiments. Flatworms were held in well-controlled laboratory conditions with daily refreshment of filtered field seawater and a constant-temperature room. Flatworm eggs may not require parental care in order to hatch under these conditions. Attempts to imitate adverse environmental conditions such as strong currents by stirring the water at certain time intervals, and decreasing the oxygen level in the water by changing the seawater less frequently (every two days) were unsuccessful.

Under both of these conditions the flatworms did not lay eggs and mortality occurred (Lee and Johnston unpublished data). Other studies have shown that flatworms are sensitive to slight environmental changes and they have a low tolerance to physiological and physical stresses (Sagasti et al. 2001). They also tend to lay their eggs on stable substratum protected from strong currents (Merory and Newman 2005).

The second explanation for the lack of an effect of parental care is that the act of covering egg masses may not be ‘brooding’ behaviour as such and it may not be essential to egg hatching under any conditions. Likewise, ‘guarding’ may simply be a response to the presence of any other organisms, rather than specifically an attempt to protect eggs. Almost all eggs of Stylochus ellipticus hatched even though the eggs were completely unprotected by parents and subjected to two different temperatures (Chintala and Kennedy 1993).

Flatworms may primarily stay in safe environments, such as inside the empty barnacle shells in order to protect themselves against adverse conditions. Thereafter, flatworm parents lay eggs in that safe environment and spend time with the egg masses during the 45 3. Reproductive behaviour and parental care of marine flatworms

breeding season. The safe environment represents favourable conditions for both the parents and the eggs and consequently, this adaptive behaviour persists and passed on to offspring.

Parental care is a complex behavioural link between reproduction, evolution and ecology (Bickford 2004; Kutschera and Wirtz 2001). This study represents the first experimental work examining parental care in marine flatworms. More work is required to identify the flatworm egg predators and to confirm if the guarding behaviour of adult flatworms improves their reproductive success in the field. This would help to understand the evolution of parental care behaviour in marine flatworms and to address under what circumstances flatworm offspring will benefit from a close physical association with their parents.

46 4. Metal pollution affects reproductive success

CHAPTER 4: LOW LEVELS OF METAL POLLUTION AFFECT

REPRODUCTIVE SUCCESS OF A MOBILE INVERTEBRATE

4.1 ABSTRACT

Marine organisms that occur in urbanised bays are often subject to heavy metal pollution. Since many pollutants enter marine waters as low-level chronic toxicants, their harmful effects may not cause instant death, but result in sublethal changes to behaviour or reproduction. An ecotoxicological study determined the effects of low levels of copper on a subtidal predator-prey relationship. The effect of copper on the reproductive success of the predator was also quantified. The common free living flatworm Stylochus pygmaeus is a mobile predator of barnacles in Botany Bay, New South Wales, Australia. Flatworms and barnacles (Balanus variegatus) were exposed to low levels of copper pollution ranging from 0 to 50 μg L-1 in the laboratory. S. pygmaeus flatworms were more sensitive to copper than their barnacle prey. Copper reduced the reproductive success of S. pygmaeus and impaired their ability to respond to physical stimulus. Fewer egg batches were laid following exposure to copper and the hatching success of eggs was also reduced.

Barnacles were not affected by low levels of copper but exhibited avoidance behaviour

(feeding inhibition) in the presence of flatworm predators. In metal polluted areas, we predict that flatworm populations will be affected at lower concentrations than their barnacle prey. Field experiments are required to test the effects of reduced flatworm populations on sessile invertebrate community structure.

47 4. Metal pollution affects reproductive success

4.2 INTRODUCTION

Many near-shore marine ecosystems are strongly influenced by heavy metal pollution resulting from the discharge of domestic sewage, mining and industrial effluent (Jackson et al. 2005; Kertész and Fáncsi 2003; Kimball and Levin 1985). Since many pollutants enter marine waters as low-level chronic toxicants, their harmful effects may not result in obvious or instant mortality, however, they may disrupt the physiological or behavioural functions of the exposed populations (for example, Cebrian et al. 2003;

Moraitou-Apostolopoulou and Verriopoulos 1979).

Behavioural changes can be an early and sensitive indicator of stress (Lang et al.

1980) that may ultimately lead to deterioration in the health of the organisms and the affected ecosystems (DeAngelis 1996; McCahon and Pascoe 1990; Sayer et al. 1991). The sublethal effects of heavy metal pollution include changes in feeding and reproductive behaviour which may affect growth and reproductive success (Cebrian et al. 2003; Conradi and Depledge 1998; Sharp and Stearns 1997). The direct effects of heavy metals on survival rates of organisms are widely studied (for example, Blidberg 2004; Sharp and

Stearns 1997), however, sublethal effects of heavy metals on the behaviour of exposed organisms are not as well documented (Lefcort et al. 1999).

Ecotoxicological studies have largely focused on single species toxicity tests to determine the sublethal effects of heavy metals on organisms (Cairns 1983). However, ecological processes such as predation and competition, are a result of the interaction between two or more populations, and single species tests are unable to determine the effects of pollutants on these processes (Balczon and Pratt 1994; Hamers and Krogh 1997;

Luoma 1983). If a species is an important predator or competitor then the effects of 48 4. Metal pollution affects reproductive success

toxicants on this organism may result in a cascade of indirect effects throughout the community (Johnston and Keough 2003). A multispecies test approach using ‘meso’ or

‘microcosms’, can provide information on the effect of toxicants on the interactions between species (Hall et al. 1998). This will allow the community responses to heavy metal exposure to be predicted more accurately (DeAngelis 1996; Fairweather and

Underwood 1991; Kareiva et al. 1996).

Copper is an essential trace metal to all marine invertebrates but it becomes highly toxic at higher concentrations (Lewis and Cave 1982). Copper is a common pollutant in ports and estuaries and the harmful effects of copper on individual marine invertebrates has been widely studied and well documented (for reviews, see Cave and Lewis, 1982 and

Hall et al. 1998). However, there are fewer studies of the effects of copper on multi-species test systems.

In recent years, increasing attention and research has been focused on flatworms from the family Stylochidae as they are pests of commercial bivalves and can cause huge economic losses (Landers and Rhodes 1970; Littlewood and Marsbe 1990; Newman et al.

1993; O' Connor and Newman 2001). When free-living marine flatworms occur in high densities, they are able to reduce their prey populations extensively and hence alter community structure (Skerman 1960a; Skerman 1960b). Despite their important role as hard substrate predators preying on a wide range of mobile and sessile organisms (Galleni et al. 1980; Jennings and Newman 1996b; Newman 2002), few studies have examined the reproductive and predatory behaviour of marine flatworms.

Previous research has indicated that stylochids flatworms are also major predators of barnacles (Skerman 1960a). Field observations of the abundant local flatworm Stylochus 49 4. Metal pollution affects reproductive success

pygmaeus identified the barnacle Balanus variegatus as the major prey item, and the empty barnacle shells as a shelter and cradle for flatworm eggs and young (personal observation but see also Merory and Newman 2005). Barnacles are often the most abundant sessile organism recruiting throughout the year (Barbaro et al. 1978; Fairweather

1985; Ruelas-Inzunza and Páez-Osuna 1998; Underwood 1981) and B. variegatus was the most abundant barnacle species on hard substrates in this study. Despite the immobility of adult barnacles in a natural assemblage, they are not entirely unprotected from predators.

Predator-avoidance behaviour has been observed in some barnacle species through escape in size (Dayton 1971) or tolerance to desiccation which allows them to exploit areas higher on the shore and inaccessible to their predators (Connell 1961). Many species simply retreat into their hard calcium carbonate shell (Lively 1999); however, such behaviour may reduce their foraging time and may therefore affect their growth and reproductive rates

(Connell 1961).

The predatory marine flatworm Stylochus pygmaeus and its barnacle prey Balanus variegatus were identified as an important species pair that interact strongly within the local sessile invertebrate community. They also occur in estuarine and shallow bay communities that are subjected to inputs of heavy metals. I conducted an ecotoxicological study to determine the consequences of sublethal concentrations of copper on the predator-prey relationship between flatworms and barnacles. I also examined the reproductive behaviour and quantified the reproductive success of flatworms exposed to sublethal concentrations of copper. The following major hypotheses were tested: (i) exposure to sublethal concentrations of copper does not affect the predation rate of

Stylochus pygmaeus on Balanus variegatus (Experiment 1) (ii) exposure to sublethal concentrations of copper does not affect the reproductive success of Stylochus pygmaeus

(tested in the absence of food in Experiment 2, and the presence of food Experiment 3). In 50 4. Metal pollution affects reproductive success

addition, the following minor hypotheses were tested: i) exposure to sublethal concentrations of copper does not affect the ability of Stylochus pygmaeus to respond to physical stimulation (Experiments 1, 2 & 3), and ii) The feeding rate of the barnacle prey

(Balanus variegatus) does not change with exposure to copper and/or flatworm predators

(Experiments 1 and 3).

51 4. Metal pollution affects reproductive success

4.3 METHODS AND MATERIALS

4.3.1 Study Site

The study site was located at Kurnell Pier, Botany Bay, New South Wales, Australia

(33”59.92 S, 151”12.62’ E), 15 km south of Sydney. The pier extends 1.3 km from the southern shore close to the mouth of the bay. Naturally occurring sessile assemblages on the pier include anthozoans, ascidians, bryozoans, hydrozoans, macroalgae, polychaetes and sponges (Clark and Johnston 2005). Seven species of flatworm have been collected and identified from artificial substrata at Kurnell Pier, including: Echinoplana celerrima,

Eurylepta aurantiaca, Cycloporus variegatus, Thysanozoon sp., Imogine lateotentare,

Stylochus pygmaeus and Stylochus sp. (Lee and Johnston, unpublished data). Kurnell Pier is well flushed by oceanic waters and records less than 5 μg L-1 background concentrations of total copper (Piola and Johnston 2005). The underlying sandy sediment is also unlikely to retain metals.

4.3.2 Collection of flatworms and barnacles

To assist in the collection of cryptic mobile flatworms and sessile barnacles, assemblages of sessile marine invertebrates were first allowed to develop on artificial substrata. Settlement plates consisting of 6 x 6 cm black Perspex tiles attached to the underside of a 60 x 60 cm PVC backing plate. Six backing plates with a total of 294 settlement plates were suspended horizontally from the pier at a depth of 3 m below the low water mark in October 2005. After 21, 25 and 28 weeks (for Experiments 1, 2 and 3 respectively), about 50 settlement plates were retrieved for each experiment and any flatworms found between the settlement and backing plates, and inside empty barnacle 52 4. Metal pollution affects reproductive success

shells were collected immediately using a small paintbrush. Settlement plates were retained and brought back to the laboratory. Each settlement plate was placed in a well-aerated transparent plastic container (15 x 8 x 8 cm) with 1 L of filtered field seawater in a dark constant-temperature room (23.5 ̈́ 0.5 oC). After 24 h, any flatworms emerging from the assemblages were collected using a paintbrush. Sessile organisms other than

Balanus variegatus were then removed from the settlement plates. Flatworms and barnacles were observed for 2 d prior to exposure to allow them to recover from collection and transport stress they were not fed during this period (Clesceri et al. 1998).

4.3.3 Copper treatments

Analytical grade copper II chloride hydrous (CuCl2.2H2O) (Sigma-Aldrich USA) was used as the reference toxicant in all experiments. A 1000 mg L-1 primary copper stock

® solution was prepared by dissolving 2.6828g of CuCl2.2H2O in 1000 mL of Milli-Q filtered water. Stock solution was stored in the refrigerator at 4 oC to prevent the reduction of copper ions in the solution. A 1000 ͈g L-1 copper solution was prepared from this stock solution every day and diluted to obtain the sublethal experimental treatment solutions of

10, 25 and 50 ͈g L-1 copper. Earlier pilot studies indicated 100% flatworm mortality occurred following 24 h exposure to approximately 100 ͈g L-1 copper. Seawater collected from Kurnell Pier used as a dilution medium was filtered through a 0.2 ͈m filter to minimise the reaction of copper ions with organic particles which could reduce the amount of free copper ions in the treatment solutions. The added copper chloride would therefore have been present in seawater as dissolved (inorganic) carbonate and hydroxide complexes

+ (CuCO3 or CuOH /Cu(OH)2). Such complexes will readily dissociate to release copper ions (Cu 2+) that bind with organic matter causing toxicity. Treatment solutions were replaced every 24 h. The equipments used in all experiments were acid washed in 10% 53 4. Metal pollution affects reproductive success

nitric acid for 24 h and rinsed in Milli-Q® filtered water before use.

Samples of the experimental copper treatments were taken at the commencement of the experiments and were immediately acidified with analytical grade concentrated nitric acid according to Clesceri et al. (1998) and stored in a refrigerator at 4 oC. The concentrations of copper in the stock and experimental solutions were tested independently at the NATA accredited Australian Government National Measurement Institute using an inductively coupled plasma source mass spectrometer (ICP-MS) with a detection limit of 5

͈g L-1.

4.3.4 Experimental design

In all experiments, transparent plastic containers (15 x 8 x 8 cm) were used as the experimental containers. Each container was pre-soaked in the appropriate copper solution for 24 h before the start of the experiments to ensure minimal chelation of copper ions during the exposure period. Exposure to treatments was maintained for 10 d. Solutions were replaced once every 24 h and were prepared from the stock solution immediately before use. All experiments were conducted in a constant-temperature room (23 oC ̈́ 0.5 oC) subjected to 11:13 light: dark cycle. Salinity, pH, temperature and dissolved oxygen were measured at the commencement of all experiments. Following 10 d exposure to copper solutions organisms were placed in clean filtered field seawater for 2 d to assess latent mortality.

In all experiments, the length and width of flatworms were measured when they were in quiescent and relaxed state. The size of opercular opening of the barnacles was measured using a pair of callipers prior to exposure to ensure that the size range of 54 4. Metal pollution affects reproductive success

organisms in all treatments was consistent (Jenkins et al. 2001), and any changes in the response of the organisms during the experiment were not related to size differences. The response of flatworms to physical stimulation was tested using a mounting needle and recorded by counting the number of “pokes” that the flatworms experienced before moving. This was used as an indicator of the physical conditions of flatworms throughout the experiments. Flatworms exposed at 0 ͈g L-1 Cu were used as the control of the treatments.

Experiment 1: Predation rate

A settlement plate with three to five Balanus variegatus recruits (depending on the number of barnacles present on the settlement plate) was placed in each container. One

Stylochus pygmaeus was placed in each of the treatment containers. Containers holding only a settlement plate with barnacles were used as barnacle mortality controls at each copper concentration. The nominal copper concentrations used in this experiment were 0,

25 and 50 ͈g L-1, with four replicates for each treatment and three replicates for each barnacle mortality control. Each container was filled with 1L of treatment solution and was continuously aerated.

During the 10 d exposure period treatment solutions were replaced daily and barnacles were fed three times with 5 mL of hatched artemia cysts to ensure that any mortality of barnacles was not a result of starvation. Observations of the response of flatworms to physical stimulation (flatworms were gently poked with a stainless steel needle once every second until movement was observed), feeding rate of barnacles

(expressed as number of beats of cirri in one minute) and mortality of barnacles in all treatments were recorded every four to six hours for the entire 10 d observation period. A 55 4. Metal pollution affects reproductive success

reduced response to physical stimulation indicates a reduced ability to respond to potential threats such as encroaching predators or physical disturbance (e.g. from macroalgae brushing past). The consequences of this would likely be reduced survival. Pilot studies tested the behaviour of flatworms when physical stimulation was applied at the posterior, anterior and centre of the body. Worms moved in response to the stimulus regardless of where it was applied. We chose to apply the mechanical stimulation at the anterior end of the flatworm so as not to damage eye spots.

Experiment 2: Reproductive success in the absence of food

Brooding behaviour of adult flatworms and the subsequent hatching success of their eggs were observed with exposure to copper. The nominal copper concentrations used in this experiment were 0, 10 and 25 ͈g L-1 with seven replicates for each treatment. A pair of similar size flatworms was placed in each treatment container. Flatworm larvae are planktonic immediately post-hatching and barnacles, as filter feeders are potential predators of flatworm larvae. In order to collect the flatworm larvae and calculate the hatching success of the flatworm eggs it was important not to have live barnacles inside the experimental containers. In pilot studies flatworms were not observed to consume dead barnacles nor any similar crustacean food so flatworms were not provided with a food-source in this experiment.

Digital photographs of the flatworms and location of the egg batches in each container were taken every 24 h using Olympus compound microscope (SZX-ILLK200) connected to Pixelink (PL-A 642) with a magnification of 40 x. The number of eggs laid from each pair of flatworms was counted from the photographs using Image-Pro Express

4.0.1 software. Flatworm larvae were collected by pouring all the one-day old solutions 56 4. Metal pollution affects reproductive success

from each treatment container into a 1.25 L sample jar and preserved at 7% formalin. New copper solutions were then poured to each treatment container. All the preserved solutions from the same treatment were combined and poured through a 10 ͈m sieve, rinsed with

Milli-Q® filtered water and made up to a 500 mL sample solution. Flatworm larvae in each sample solution were counted by sub-sampling 20 mL of the sample solution five times.

Number of flatworm larvae present in each treatment was estimated by multiplying the average number of larvae present in the 20 mL sub-sampling solution by twenty five.

Hatching success of flatworm eggs was calculated as:

(Number of larvae/ Number of eggs) * 100%

Response of flatworms to physical stimulation, number of egg batches laid and the time taken for the flatworms to lay eggs were observed and recorded every four to six hours for 10 d.

Experiment 3: Reproductive success with food provided

In order to ensure that the response of flatworms and the timing of egg laying were not influenced by the absence of food, barnacles were placed in each treatment container as a food source in this experiment. A settlement plate with three to five barnacles and a pair of similar size flatworms was placed in each treatment container. Containers holding a settlement plate with three to five barnacle recruits only were used as barnacle mortality controls. The nominal copper concentrations used in this experiment were 0, 10 and 25 ͈g

L-1, with four replicates for each treatment and three replicates for each barnacle mortality control. Barnacles were fed with 5 mL hatched artemia cysts as described in Experiment 1.

The responses of flatworms to physical stimulation, time taken for flatworms to lay eggs 57 4. Metal pollution affects reproductive success

and feeding rate and mortality of barnacles in all treatments were recorded every four to

six hours for 10 d.

4.3.5 Data analysis

All statistical analysis was completed in Statistical Package for Social Sciences

(SPSS version 11.5). Plots of residuals versus means and descriptive statistics showed that

the data, except for the number of barnacles consumed by flatworms, satisfied assumptions

of variance homogeneity and normality. In each experiment, barnacle size was tested using

a one-factor ANOVA with the presence of flatworms as a fixed factor. Flatworm length

was tested using a one-factor ANOVA with copper treatments as the categorical factor.

Number of barnacles consumed over the 10 d period (between 0-5) was assessed using the

non-parametric Kruskal-Wallis test with copper treatment as the grouping factor. Response

of flatworms to physical stimulation (number of pokes required to elicit a response),

number of egg batches laid and the hatching success of flatworm eggs in all treatments

were tested separately using a one-factor ANOVA with copper treatment as the categorical

factor. The differences in the feeding rate of barnacles (cirri beats per minute averaged

over the 10 d period) in Experiments 1 and 3 were analysed using a two-factor ANOVA

with copper treatments and presence of flatworms as fixed factors. Planned comparison

tests were conducted on all significant results to determine the differences between

controls and individual copper treatments. Planned comparisons used the error term from

the main test of copper treatment (Quinn and Keough 2002). Planned comparisons are

conducted when specific post-hoc tests are required. They are usually more powerful than

procedures that test every treatment combination (e.g. Tukeys). In ecotoxicology studies

such as this, the tests of interest are usually a comparison of the control treatment against

individual toxicant concentrations. 58 4. Metal pollution affects reproductive success

4.4 RESULTS

4.4.1 Predation rate of Stylochus pygmaeus

No barnacles were observed to die in the mortality controls in Experiments 1 and 3 so any mortality of barnacle was therefore due to the presence of the flatworm predators. In

Experiment 1, exposure to 50 ͈g L-1 Cu appeared to reduce the predation rate of Stylochus pygmaeus on Balanus variegatus by more than half however the result was not significantly different (Kruskal-Wallis, p = 0.119; Figure 4.1a). Predation rate was not affected by exposure to 10 or 25 ͈g L-1 Cu in Experiment 3 (Kruskal-Wallis, p = 0.735; Figure 4.1b).

4.4.2 Response of Stylochus pygmaeus to physical stimulation

The sensitivity of flatworms to physical stimulation was reduced by exposure to copper in all three experiments (separate ANOVAs, p < 0.001, Figure 4.2). In Experiment 1, flatworms responded more slowly at 25 and 50 ͈g L-1 Cu when compared to the controls

(Planned comparisons, p < 0.001, Figure 4.2a). In Experiments 2 and 3 flatworms responded similarly in control and 10 ͈g L-1 Cu solutions (Planned comparisons, p > 0.554,

Figure 4.2b and 4.2c). They again took longer to respond when exposed to 25 ͈g L-1 Cu

(Planned comparisons, p < 0.001, Figure 4.2b and 4.2c).

4.4.3 Reproductive success of flatworms

Copper exposure reduced the reproductive success of flatworms. There were fewer egg batches laid by flatworms (ANOVA, p = 0.006, Figure 4.3a). Planned comparisons showed that this effect was evident at 25 ͈g L-1 Cu (p = 0.007) but not at 10 ͈g L-1 Cu (p =

59 4. Metal pollution affects reproductive success

0.763). Hatching success was highly consistent among replicates. In control treatments, about 92 ̈́ 0.4% of eggs hatched (Figure 4.3b). Hatching success of flatworm eggs was reduced by 10% at 10 ͈g L-1 Cu and 40% at 25 ͈g L-1 Cu compared to that in the controls

(ANOVA and Planned comparisons, p < 0.001, Figure 4.3b).

Flatworms were able to endure protracted fasting and fasting did not appear to affect

Stylochus pygmaeus reproductive behaviour. In Experiments 2 (flatworms unfed) and 3

(flatworms fed), there were no differences in the time taken for the flatworms to lay the first batch of eggs (five days) (Experiment 2: ANOVA, p= 0.303; Experiment 3: ANOVA, p = 0.142) and in both experiments flatworms were observed brooding the egg masses.

4.4.4 Feeding rate of Balanus variegatus

Feeding rate of barnacles was reduced by both the presence of flatworms and exposure to copper, however, the presence of flatworm predators had a much larger effect than copper exposure (Table 4.1, Figure 4.4). In both experiments, barnacle feeding rate was reduced by <10% when exposed to copper, but it was reduced by >30% by the presence of flatworm predators.

4.4.5 Size of barnacles and flatworms

Length of flatworm was used as an indicator of overall flatworm size (Chintala and

Kennedy 1993). The average length of flatworms collected and tested in Experiment 1 was

12.7 ̈́ 0.14 mm; Experiment 2 was 11.6 ̈́ 0.46 mm and Experiment 3 was 16.4 ̈́ 0.63 mm.

Flatworm length was similar across treatments within each experiment: Experiment 1

(ANOVA, p = 0.08); Experiment 2 (ANOVA, p = 0.27) and Experiment 3 (ANOVA, p = 60 4. Metal pollution affects reproductive success

0.06).

Operculum length was used as an indicator of overall barnacle size. Barnacle size was consistent between treatments in both Experiments 1 (ANOVA, P = 0.13) and 3 (ANOVA, p = 0.31). In Experiment 1, there were 3 ̈́ 0.2 barnacles (6.1 ̈́ 0.2 mm) in each treatment and 4 ̈́ 0.4 barnacles (5.7 ̈́ 0.16 mm) in each barnacle mortality control while in

Experiment 3, there were 4 ̈́ʳ0.3 barnacles (6.8 ̈́ 0.2 mm) in each treatment and 4 ̈́ 0.17 barnacles (6.5 ̈́ 0.25 mm) in each barnacle mortality control.

4.4.6 Chemical analysis

The nominal and measured copper concentrations taken at the commencement of

Experiments 1 and 2 are shown in Table 4.2. A lower range of copper concentration were used in Experiments 2 (0, 10 and 25 μg L-1) than experiment 1 (0, 25 and 50 μg L-1). All filtered field seawater samples contained <5 ͈g L-1 copper. Since all treatment solutions were prepared and changed every 24 h from a 1000 mg L-1 stock solution and the measured copper concentrations of the treatment solutions from the experiment are close to their nominal values, we have confidence in the accuracy of our treatment solutions.

61 4. Metal pollution affects reproductive success

Table 4.1. Summary of two-factor ANOVA and planned comparisons on the feeding rate of barnacles in the presence and absence of flatworms and copper in Experiments 1 and 3. All planned comparisons were tested against the error term for the main test of Cu treatments. P-values in bold indicates significant differences at Į = 0.050.

Cu treatments Main test Cu treatments Planned comparisons Expert Factors Condition (µg L-1) df MS F P (µg L-1) MS F P 1 Cu 0 vs. 25 vs. 50 2 56.983 33.321 <0.001 Presence of flatworms 0 vs. 25 5.014 2.932 0.107 Flatworms 1 5980.815 3497.174 <0.001 0 vs. 50 15.125 8.844 0.010 Flatworms x Cu 2 15.559 9.098 0.003 Error 15 1.710 Absence of flatworms 0 vs. 25 45.835 26.801 <0.001 0 vs. 50 112.67 65.880 <0.001

3 Cu 0 vs. 10 vs. 25 2 70.4460 123.375 <0.001 Presence of flatworms 0 vs. 10 45.125 79.030 <0.001 Flatworms 1 7458.8930 13063.142 <0.001 0 vs. 25 136.12 238.403 <0.001 Flatworms x Cu 2 7.6840 13.457 <0.001 Error 15 0.5710 Absence of flatworms 0 vs. 10 1.852 3.243 0.092 0 vs. 25 31.13 54.519 <0.001

62 4. Metal pollution affects behaviour of mobile invertebrate

Table 4.2. Nominal and measured copper concentrations (µg L-1) for copper treatments used in Experiment 1 & 2 indicate that the measured copper concentrations of the treatment solutions from the experiment are close to their nominal values. Dashes represent copper concentrations not used in a particular experiment.

Nominal copper concentration Measured copper concentration (µg L-1) (µg L-1) Experiment 1 Experiment 2 ʳ 0<5<5 ʳ 10 --ʳ 13.5 ʳ 25 26 22 ʳ 50 54 --

63 4. Metal pollution affects behaviour of mobile invertebrate

Figure 4.1. Effects of (a) 0, 25 and 50 µg L-1 and (b) 0, 10 and 25 µg L-1 Cu treatments on the number of barnacles eaten by flatworm(s) in Experiment 1 and Experiment 3 respectively. Error bars represent mean (± 1 SE).

64 4. Metal pollution affects behaviour of mobile invertebrate

Figure 4.2. Effects of (a) 0, 25 and 50 μg L-1, (b) 0, 10 and 25 μg L-1 and (c) 0, 10 and 25

µg L-1 Cu treatments on flatworm response to physical stimulation in Experiments 1, 2 and 3 respectively. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

65 4. Metal pollution affects behaviour of mobile invertebrate

Figure 4.3. Effects of 0, 10 and 25 μg L-1 Cu treatment on the (a) number of flatworm egg batches laid and (b) hatching success of flatworm eggs in Experiment 2. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

66 4. Metal pollution affects behaviour of mobile invertebrate

Figure 4.4. Effects of (a) 0, 25 and 50 µg L-1 and (b) 0, 10 and 25 μg L-1 Cu treatments on the feeding rate of barnacles in presence and absence of flatworms in Experiment 1 and Experiment 3 respectively. Error bars represent mean (± 1 SE). Letters indicate significant difference at Į = 0.050.

67 4. Metal pollution affects behaviour of mobile invertebrate

4.5 Discussion

Low levels of metal pollution had a major effect on the reproductive behaviour of the mobile flatworm, Stylochus pygmaeus. The reproductive success of the flatworm was reduced by exposure to sublethal concentrations of the toxicant. The flatworm was more sensitive to copper than its target prey species, the barnacle Balanus variegatus. The barnacle reduced its feeding rate in the presence of its predator but was barely affected by exposure to the pollutant. Copper concentrations used in this study fall into the lowest range of those typically used in laboratory toxicity tests and they reflect realistic concentrations experienced by marine assemblages in heavily polluted harbours (e.g.

Stauber et al. 2000) or close to ships painted with copper antifouling paints (Valkirs et al.

2003). The differential sensitivity of a predator and prey observed in this study warrants field experiments investigating the likely structural changes to the marine invertebrate community in areas of low level heavy metal pollution.

4.5.1 Predation rate of Stylochus pygmaeus

Predation is one of the important factors determining the distribution and abundance of organisms (Connell 1970; Menge 1995; Rand 1985; Sih et al. 1985).

Toxicant exposure can increase or decrease predation rates through effects on growth, reproduction and behaviour of the exposed predators and prey (Gómez et al. 1997;

Preston and Snell 2001). A pattern of reduced predation on barnacles was observed when flatworms were exposed to 50 μg L-1 copper, however this did not differ significantly from predation in the controls. While the predation rate of the mobile flatworm was largely unaffected, copper reduced the ability of the flatworm predator to respond to physical stimulation. In the real world this may leave the worm itself more vulnerable to 68 4. Metal pollution affects behaviour of mobile invertebrate

predation.

Previous research showed that high mortality and reduced predation rate of the flatworm, Stylochus ellipticus, could result from short hypoxic episodes that did not affect the barnacle prey Balanus improvisus (Sagasti et al. 2001). Flatworms have no shell to cover their fragile body, and they rely heavily on diffusion directly across the epidermis for the purposes of respiration and excretion of waste products. Hence, they may generally more susceptible to changes in water quality than their barnacle prey, which are protected by both a calcium carbonate external skeleton and a thick integument. The decrease in population size of flatworms due to slight changes in environmental conditions, such as low levels of copper or reduced levels of dissolved oxygen may create predation refuges for their prey populations.

The predation rate of Stylochus pygmaeus in this study was relatively low when compared to the field observations of other stylochids preying on barnacles (for example,

Stylochus tauricus consume five to ten Balanus improvisus in a month, Murina et al.

1995). This may be due to the relatively small size of S. pygmaeus compared to other stylochids. S. pygmaeus was named because of its small size (Merory and Newman 2005) and the maximum length of Stylochus pygmaeus collected in this study was 22 mm.

Other adult stylochids, such as S. inimicus and S. zebra commonly reach lengths of

40-50 mm (Lytwyn and McDermott 1976; Pearse and Wharton 1938). Low predation rates may also be a result of reduced energy expenditure of flatworms in well-controlled laboratory conditions. The predation rate of S. pygmaeus may be higher if measured in the field due to the need to compensate for energy costs associated with searching for food, hiding from predators, such as fish, and finding shelter from strong wave action.

69 4. Metal pollution affects behaviour of mobile invertebrate

The presence of a mating pair of Stylochus pygmaeus flatworms (Experiment 3) did not increase the predation rate relative to that of a single worm (Experiment 1). This might be because S. pygmaeus invested time in mating and brooding rather than foraging, when a mate was available. Chintala and Kennedy (1993) found that flatworms are able to maintain a high reproductive effort by using stored energy to produce eggs despite reductions in available food.

4.5.2 Reproductive success of Stylochus pygmaeus

Copper reduced the reproductive success of flatworms. There were fewer eggs laid when flatworms were exposed to low levels of copper, and the likelihood that any one of those eggs would hatch into a young larva was lowered. Copper was clearly reducing the capacity of flatworms to produce eggs, however, the mechanism by which the hatching success was affected is not clear in this study. The toxic effects of copper on adult health may have resulted in the laying of poor quality eggs, but eggs may have also been directly affected by copper exposure, and the newly hatched larvae may also have been directly affected. The early life stages of many organisms are more sensitive to pollutants and are often used in toxicity tests (Mora 2003). In a field situation, adults, eggs and larvae would all be exposed to pollution as they were in this study. The reproductive success of flatworms was the only test endpoint to be affected by the lowest copper concentration (10 μg L-1). Response to physical stimulation was only affected at 25 μg

L-1 copper and above. It therefore appears that reproductive success may be a more sensitive indicator of copper exposure than other sublethal endpoints.

It has been suggested that long term exposure of organisms to elevated copper concentrations might have serious consequences, such as local extinction of the exposed 70 4. Metal pollution affects behaviour of mobile invertebrate

species through decreased survival and growth rates (Conradi and Depledge 1998).

Decreased reproductive success in Stylochus pygmaeus at sublethal copper concentrations in this study might lead to a decline in flatworm population size. The combined effect of fewer egg batches and reduced rates of egg hatching resulted in declines in reproductive success of up to 80%. Within a couple of generations this may result in complete recruitment failure as the density of mature flatworms drops below a critical encounter threshold (Gascoigne and Lipcius 2004; Russell and Fowler 2002).

4.5.3 Effects of Stylochus pygmaeus on the feeding rate of Balanus variegatus

The trade-off between growth rate and consumption as a function of activity level is an important determinant of the life-style of organisms in many taxa (Gurevitch et al.

2000; Lima and Bednekoff 1999; Werner and Anholt 1993). Predator-avoidance behaviour has been widely studied in terrestrial (for example, Mondor et al. 2004;

Nelson et al. 2004) and aquatic ecosystems (for example, Hagen et al. 2002; Peacor

2002). Such behaviour has both direct and indirect effects upon the reacting prey such as reduction of survival and growth rates and disruption of the inter-and intra-specific relationships between the prey and its surrounding organisms (Nakaoka 2000).

Predator-avoidance behaviour (feeding inhibition) was clearly shown by Balanus variegatus in response to the presence of flatworms in this study. The presence of flatworms reduced the feeding rate of barnacles to a much greater extent than exposure to the toxicant copper. B. variegatus reduced its encounter rate with its flatworm predator by closing opercular valves. Such a defense mechanism was not noted in Hurley (1976) which examined the establishment of Balanus pacificus in the presence of its flatworm predators, Stylochus tripartitus. The mechanism by which B. variegatus identified the 71 4. Metal pollution affects behaviour of mobile invertebrate

presence of S. pygmaeus is not known, however, chemical cues may be involved (Hagen et al. 2002). This predator-avoidance behavioural response is presumably performed to reduce the predation risk but it comes at the cost of reduced foraging time and hence potentially reduced growth, reproduction and development of the prey species (Lefcort et al. 1999).

72 4. Metal pollution affects behaviour of mobile invertebrate

4.6 CONCLUSION

This study offers an important insight into the impacts of low level of heavy metal pollution on the reproductive success and the predator-prey interaction of marine invertebrates using a two-species microcosm approach. Copper had obvious detrimental effects on flatworms, reducing their reproductive success and their ability to respond to physical stimulus. It is likely that these changes would ultimately lead to a decrease in the population size of flatworms in polluted areas. Barnacles were less affected by copper exposure than by exposure to their flatworm predators. Patterns of population change in barnacles would need to be examined over a longer period and under field conditions in order to determine how the barnacle beds are replenished in the presence and absence of flatworms, and the importance of predator-avoidance behaviour of the barnacles within a sessile assemblage.

73 5. Summary and implications

CHAPTER 5: SUMMARY AND IMPLICATIONS

The aims of this thesis were to report on the diversity of marine flatworms at

Kurnell Pier in Botany Bay, to study the predatory and reproductive behaviour of the two most common flatworm species and to assess the anthropogenic impacts of low levels of pollution on a marine flatworm. This study offers important insights in understanding the reproductive biology of marine flatworms and the sublethal effects of pollutants on aquatic ecosystems mediated through behavioural changes to the predator-prey relationship between marine flatworms and their barnacle prey.

5.1 MARINE FLATWORM DIVERSITY

The awareness of the Australian marine flatworm fauna has been vastly increased over the few past decades and now nearly 600 species are recorded (Newman and

Cannon 2003). However, many more species remain unidentified and little is known about their life history. There is no literature on the biology and ecology of marine flatworms in New South Wales, Australia. Throughout my two years study period I recorded the relative abundance of each of the flatworm species present in the field.

This offers an insight in the spatial distribution pattern of the flatworm species at Botany

Bay. I recorded seven flatworm species and five of them, including Imogine lateotentare sp. nov. that I have described, were abundant at the study site (Chapter 2 and Appendix).

Marine flatworms were closely associated with sessile organisms, such as the barnacle, Balanus variegatus, the bryozoan, Watersipora substorquata and colonial ascidians, Botryllus schlosserii and Diplosoma listeranum in the study site (Appendix).

These assemblages are usually found in bays and estuaries which are subjected to 74 5. Summary and implications

anthropogenic inputs from various sources. Anthropogenic impacts on these assemblages can be determined through the multispecies microcosm tests.

5.2 REPRODUCTIVE BEHAVIOUR OF MARINE FLATWORMS

Marine flatworms are among the simplest of animals and yet they appear to provide parental care to their offspring. The worms take care of fertilised eggs by depositing the egg masses on safe and stable substratum with gelatinous mucus to ensure that the eggs are firmly attached on the substratum. Flatworms also cover their egg masses with their body in the first few days after the eggs are laid, or until all the eggs hatch. Understanding the diversity and ecology of marine flatworms has implications in other fields of biology, such as the evolution of the reproductive behaviour in hermaphrodites (Michiels and Newman 1998).

Echinoplana celerrima and Stylochus pygmaeus were usually found at my study site. I evaluated the influence of their brooding behaviour on the hatching success of their eggs under controlled laboratory conditions. I also examined the changes in reproductive behaviour of E. celerrima and quantified its reproductive success when exposed to each of three species of putative flatworm egg predators. Interestingly, the brooding behaviour of neither species of marine flatworm enhanced the hatching success of their eggs. Exposure of E. celerrima to the putative flatworm egg predators did not affect the timing of hatching or hatching success of its eggs. E. celerrima, however, devoted more time to guarding their eggs than to brooding when exposed to egg predators. Marine flatworms covered the egg masses with their body inherently and this behaviour was not essential to their reproductive success in the laboratory (Chapter

3). Attempts to investigate the significance of parental care behaviour of flatworms in the 75 5. Summary and implications

field failed due to the inability to observe the behaviour of flatworm parents under water.

Further work is needed to overcome the difficulties identifying flatworm egg predators and examining the benefits gained by the flatworm offspring from brooding in the field and, hence to understand the evolution of parental care behaviour in marine organisms.

5.3 PREDATORY BEHAVIOUR OF MARINE FLATWORMS

Marine flatworms are carnivores and prey upon a wide range of organisms. In recent decades, much interest has been focused on stylochids because they are voracious predators and they prey upon a wide range of organisms including barnacles and commercial bivalves. Modification in community structure and population dynamics will result from intense predation by stylochids on hard substrata (Newell et al. 2000).

Two common stylochids, Imogine lateotentare and Stylochus pygmaeus, are important mobile predators of young barnacles, Balanus variegatus, in an endemic sessile invertebrate community. The two species have same predation rates in controlled laboratory conditions, consuming one barnacle in a 14 d observation period. Their predation rate is presumed to be higher in the field due to reduced energy expenditure in the well-controlled laboratory conditions that were free of predators and other environmental stresses. However, the two species had different predatory behaviour. I. lateotentare were only observed feeding exclusively at night, whereas, S. pygmaeus were observed feeding both day and night. A white coloured spot clearly seen on the ventral surface of flatworm and ejected cirri found next to the empty barnacle shell are useful indicators of flatworm predation on barnacles (Chapters 2 and 4). There is a need for both field and laboratory research in understanding the predatory behaviour of stylochids and the implications for developing bivalve aquaculture industries. 76 5. Summary and implications

5.4 IMPLICATIONS OF SUBLETHAL EFFECTS OF COPPER

I was able to discern the toxic effects of copper on marine flatworms. Marine flatworms can be used as an indicator to low levels of heavy metal pollution since they are sensitive to slight elevation of toxicant concentration in the waters. Copper had obvious detrimental effects on marine flatworms, by reducing its reproductive success and ability to respond to physical stimulus. Barnacles, however, were less affected by copper exposure than by the presence of their flatworm predators (Chapter 4).

Long-term field experiments are required to address the effects of reduced flatworm populations on sessile invertebrate community structure under the exposure to heavy metals.

5.5 CONCLUSION

Marine flatworms have been overlooked for a long time despite their important role as hard substrate predators. This thesis represents first parental care and ecotoxicological studies on marine flatworms. This study also provided the first detailed descriptions on the flatworm species present in Botany Bay, New South Wales, Australia.

This thesis beings together knowledge relating to the biology and ecotoxicology of marine flatworms that may form the basis of understanding the evolution of reproductive behaviour of marine invertebrates and the anthropogenic impacts on marine hard substrata. It is hoped that this work encourages an increased interest in the study of marine flatworms and raises awareness of flatworm fauna in Australian waters.

77 Cited references

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94 Appendix. Marine flatworm diversity

APPENDIX: MARINE FLATWORM DIVERSITY AT KURNELL PIER,

BOTANY BAY, NEW SOUTH WALES, AUSTRALIA

Throughout my study, I collected and identified seven species of marine flatworms.

This constitutes the first observation on the diversity of marine flatworms in Botany Bay,

New South Wales, Australia. One of these flatworms was identified as a new species and is described in chapter 3. Previously only one species of marine flatworm has been reported from my study site, Kurnell Pier (Pollard and Pethebridge 2002). In this

Appendix, I summarise descriptions of the family and genus and provide initial observations on the relative abundance of each of the seven species.

Collection of marine flatworms

Assemblages of sessile marine invertebrates were allowed to develop on artificial substrata deployed at Kurnell Pier (33”59.92 S, 151”12.62’ E), southern side of Botany

Bay, New South Wales (Figure A1). Throughout my two years study period I deployed

16 Polyvinyl Chloride (PVC) backing plates (60 x 60 cm). There were two arrangements of settlement plates attached to the underside of the backing plate. 49 settlement plates consisted of 6 x 6 cm black Perspex tiles were attached to the underside of each of the eight PVC backing plates and a further 16 settlement plates consisted of 11 x 11 cm black Perspex tiles were attached on each of the other eight backing plates. Settlement surfaces were roughened with sand paper. The backing plates were suspended horizontally from pier pilings at a depth of 3 m below the low water mark. Marine flatworms within the assemblage and in between the settlement and backing plates were collected and placed separately into sample jars filled with seawater and brought back to the laboratory. Collection of marine flatworms from the settlement plates continued for two years. 95 Appendix. Marine flatworm diversity

Figure A1. Study site at Kurnell Pier, Botany Bay, New South Wales, Australia.

96 Appendix. Marine flatworm diversity

Flatworm specimen processing

Measurements of adult flatworm body size were taken from live animals when they were in a relaxed and quiescent state and expressed as length (mm) x width (mm).

Digital photographs of each flatworm species were taken using Olympus compound microscope (SZX-ILLK200) connected to Pixelink (PL-A 642) with magnification of a

40 x. Flatworms collected were preserved in the way suggested by Newman and

Cannon (1995) and Newman and Cannon (2003). Descriptions of colours are for living animals and the colour descriptions are identified by the number from Pantone® Colour chart. These numbers are given in parenthesis in the following descriptions.

Longitudinal serial sections of the reproductive regions were prepared when two or more of the same marine flatworm species were collected. Reconstruction of the reproductive anatomy of a paratype was done with the aid of a micro projector

(Ken-A-Vision, MFG, INC, USA) (details in fixation, processing of flatworm specimens and sectioning of reproductive structures of flatworms are described in Chapter 3).

Marine flatworm associated sessile assemblage composition

The most prevalent sessile species colonising settlement plates during my study were the barnacle, Balanus variegatus, the bryozoan, Watersipora substorquata and colonial ascidians, Botryllus schlosserii and Diplosoma listeranum (Table A2).

Diversity of marine flatworms

Photographs of living flatworm species collected at Kurnell Pier are shown in

Figures A2-8. Flatworms were identified to the lowest taxonomic level. Five marine flatworm species occurred at Kurnell Pier. In order of abundance they were:

Echinoplana celerrima, Stylochus pygmaeus, Imogine lateotentare sp. nov., Cycloporus variegatus, Eurylepta aurantiaca. Two variants of C. variegatus were found. 97 Appendix. Marine flatworm diversity

Longitudinal serial sections of copulatory structures of C. variegatus were prepared to confirm the species identification (Figure A6). Only one specimen of two further marine flatworms were collected and preserved. These were identified as Thysanozoon sp. and an unidentified stylochid. These specimens are being used as a reference collection by the Johnston marine laboratory who continues to do flatworm research. Key to distinguish between the flatworm species at Botany Bay is shown in Table A1.

98 Appendix. Marine flatworm diversity

Table A1. Key to distinguish between the flatworm species at Botany Bay.

Key to suborder A. Flatworms without a ventral sucker; tentacles, when present, are usually Acotylea (1) nuchal type; copulatory complex is usually in the posterior body half. B. Flatworms with a muscular sucker on the ventral surface posterior to the Cotylea (2) female genital pore and tentacles, when present, are usually marginal.

(1) Key to species of Acotylea at Botany Bay A. Cerebral eyes in two elongate clusters alongside cerebral organ; distinctive Echinoplana spiny cirrus-sac at the posterior end of the dorsal surface. celerrima

B. Dorsal surface with densely scattered dark and light brown mottling; three Stylochus to four rows of scattered marginal eyes along the anterior margin; 10–15 pygmaeus tentacular eyes each side; about 20 cerebral eyes.

C. Inconspicuous nuchal tentacles; numerous cerebral and frontal eyes; Imogine numerous cement glands. lateotentare

D. Marginal eyes present along the entire margin of the body; numerous Unidentified cerebral eyes present between nuchal tentacles. stylochid

(2) Key to species of Cotylea at Botany Bay A. Small marginal tentacles with eyes in their bases; cerebral eyes are in two Cycloporus distinct rectangular clusters; nine lateral intestinal branches variegatus

B. Pharynx is small, muscular and tubular; ventral sucker is prominent, in the Eurylepta mid third of body; a pair of large and conspicuous uterine vesicles. aurantiaca

C. Dorsal surface covered with long pointed yellowish brown papillae with Thysanozoon micro black dots at their tip; numerous pseudotentacular eyes occur along sp. and at the tips of pseudotentacles.

99 Appendix. Marine flatworm diversity

Diagnosis of flatworm species at Kurnell Pier

Suborder Acotylea

Echinoplana celerrima (Figure A2)

Stylochus pygmaeus (Figure A3)

Imogine lateotentare sp. nov. (Figure A4)

Unidentified stylochid (Figure A5)

Family Gnesiocerotidae

This family are mainly in elongate form with or without head tentacles. Cerebral and tentacular eyes, when present, are in two elongate clusters alongside cerebral organ.

Pharynx is highly folded and situated in the middle part of the body. Genital pores are well-separated and are posterior to the cirrus. Spiny cirrus-sac can be seen at the posterior end of the dorsal surface. Prostatic organ is interpolated between sperm ducts.

Vagina is simple and usually with Lang’s vesicle (Prudhoe 1985; Prudhoe 1989).

Genus Echinoplana

Animals are of elongate form with eyes in two elongate groups alongside cerebral organ. Pharynx is located in anterior half of body. Seminal vesicle is elongated with a muscular prostatic organ. Adults have thick musculature cirrus-sac which lined with spines gradually increasing in size towards posterior of the body (Prudhoe 1982;

Prudhoe 1989).

Species Echinoplana celerrima (Figure A2)

Echinoplana celerrima are elongate and flesh coloured (141). Eyes are in two elongate groups, disposed laterally to the cerebral organ, and tending to converge anteriorly. They have nuchal and cerebral eyes. The nuchal eyes are in the posterior 100 Appendix. Marine flatworm diversity

region of each group and are larger than the cerebral eyes. The pharynx is in the second quarter of the body. Distinct spiny cirrus can be clearly seen through the dorsal surface.

The male and female pores are well separated and are in the third quarter of the body.

They were always the most abundant flatworm species on settlement plates at Kurnell

Pier, Botany Bay and are common in the eastern Australian waters (Newman and

Cannon 2003). Detailed description of this species referred to Prudhoe (1982).

Family Stylochidae

Stylochids are thick and fleshy with a rounded or oval body varying in shape and size. Nuchal tentacles are often present in varying degrees of development. They have nuchal, cerebral, small marginal and frontal eyes. Intestinal branches seldom anastomose. Male and female genital pores are usually separated in posterior third of the body or near the posterior margin (Hyman 1953; Newman and Cannon 2003).

Genus Stylochus

Animals are in oval form with retractile nuchal tentacles. Marginal eyes are in bands of variable extent and cerebral eyes occur in single mass or two clusters, tentacular eyes are either within or at the bases of tentacles. They have a single-lobed seminal vesicle (Du Bois-Reymond Marcus and Marcus 1968; Hyman 1953; Prudhoe

1989).

Species Stylochus pygmaeus (Figure A3)

They are relatively small; body is oval to elongate, thick and fleshy and without marginal ruffles. Background cream-beige (127) with densely scattered dark and light brown mottling on the dorsal surface. Ventral surface is grey-white (453) without markings. There are three to four rows of scattered marginal eyes along the anterior 101 Appendix. Marine flatworm diversity

margin and the eyes are more densely packed anteriorly. Tentacular eyes are within the tentacles. The pharynx is central, about one-quarter the body length with complex ruffled folds. The mouth is in the middle of the pharynx. Gonopores are posterior to the pharynx and are near to the posterior margin (for detailed description and drawings of reproductive structures, see Merory and Newman (2005)). They were the second most abundant flatworm species on settlement plates in Botany Bay and were also found inside empty barnacle shells in Port Kembla Harbour, New South Wales, Australia.

Genus Imogine

Species of this genus have similar appearance to the Stylochus. Species identifications are mainly based on the arrangements of the eyes and details of the reproductive structures. Members of the genus Imogine have a seminal vesicle with three lobes (Jennings and Newman 1996a; Newman and Cannon 2003).

Species Imogine lateotentare sp. nov. (Figure A4)

The descriptive details of this new species are reported in Chapter 3 with its distinctive feature of having inconspicuous nuchal tentacles, numerous cerebral and frontal eyes and numerous cement glands. This species has been found inside the empty shells of the barnacle, Balanus variegatus, in Botany Bay and in Port Kembla Harbour,

New South Wales, Australia.

Unidentified stylochid (Figure A5)

This species is oval, thick and fleshy and without marginal ruffles. Dorsal surface brightly orange (158) mottled with darker orange (1595) and brown (167) flecks medially. Head tentacles are retractile with tentacular eyes concentrated at the tips of the tentacles. Marginal eyes present along the entire margin of the body and are more 102 Appendix. Marine flatworm diversity

densely packed at the anterior. Numerous cerebral eyes are present between nuchal tentacles. Pharynx is central, about one-third the body length. This species was found closely associated with colonial ascidians on the hard substrates and it was only recorded once throughout my study.

Suborder Cotylea

Cycloporus variegatus (Figure A6)

Eurylepta aurantiaca (Figure A7)

Thysanozoon sp. (Figure A8)

Family Euryleptidae

Euryleptids have elongate oval or round bodies, sometimes highly coloured.

Animals are of variable size, with a smooth or papillate dorsal surface. Marginal tentacles are of variable development. Distinctive tubular pharynx is directed anteriorly.

Ventral sucker is well developed in the middle third of the body, posterior to copulatory organs. Cerebral eyes are in two elongated clusters. Mouth situated at the anterior end of relatively short pharyngeal chamber. The male copulatory complex is located at anterior of body, posterior to pharynx while the female copulatory complex is located between male pore and ventral sucker. Vagina is short and simple, and is dorso-ventrally compressed. Detailed descriptions of reproductive structures of this family referred to

Newman and Cannon (2003) and Prudhoe (1985).

Genus Cycloporus

Members of this genus have an oval form with smooth or papillate dorsal surface of variable colour. Marginal tentacles are small with numerous eyes.

Cerebral eyes are in two closely associated elongate clusters. The intestinal trunk is 103 Appendix. Marine flatworm diversity

divided into three branches anteriorly with median branch passing between cerebral eye clusters while the side branches pass laterally to the eye clusters. They have six to ten pairs of lateral intestinal branches which terminate in small vesicles opening to the exterior on the side of the body (Newman and Cannon 2002).

Species Cycloporus variegatus (Figure A6)

Marine flatworms are small and rounded oval. The dorsal colour pattern is cream-white background with red branches (172). However, its colour pattern is variable, depending on the colour of the prey it has consumed (personal communication with Dr. Leslie Newman). A variant of Cycloporus variegatus with transparent cream dorsal surface, covered in yellow spots (102) with median white and intermittent purplish strip (261), was found. They have small marginal tentacles with eyes in their bases and ventral surfaces. The cerebral eyes lie over the cerebral organ in two distinct rectangular clusters. The lateral branches ramify and terminate in numerous elongate vesicles in the marginal region of the body. The vesicles open to the exterior through small pores in the peripheral epithelium. Detailed descriptions of this species are referred to Newman and Cannon (2002). They were the third most abundant flatworm species on the settlement plates that I have recorded throughout my study.

Genus Eurylepta

These animals have an oval body form with marginal tentacles containing many eyes. The dorsal surface is smooth with a ruffled margin. The ventral sucker is located in middle of the body. Marginal eyes form a distinct band anteriorly and cerebral eyes are in two rows alongside pharynx. Male copulatory complex is ventral to pharynx and posterior to male pore. Vas deferens opens into a muscular seminal vesicle. Female complex is posterior to female opening (Hyman 1953; Prudhoe 1985). 104 Appendix. Marine flatworm diversity

Species Eurylepta aurantiaca (Figure A7)

These animals are rounded, thick and fleshy, raised dorso-ventrally only over the area of the pharynx, with marginal ruffles and blunt posterior. Dorsal surface is usually orange (158). The marginal tentacles are short with many eyes. The cerebral eyes are small, in two rows alongside pharynx. Pharynx is small, muscular and tubular, directed anteriorly, mouth anterior. Ventral sucker is prominent, in the mid third of body, posterior to female pore. This species has a pair of large and conspicuous uterine vesicles. Gonopores are well separated. Vas deferentia unbranched, extending posteriorly along the intestine. Seminal vesicle is small (about 100 µm) and oval.

Ejaculatory duct is long, straight and muscular, prostate is oval with thick muscular walls and is larger than seminal vesicle (about 173 μm). This species has been found associated with colonial ascidians on hard substrate in Botany Bay and Port Kembla

Harbour, New South Wales, Australia. They were less abundant than Cycloporus variegatus on settlement plates at Botany Bay. Detailed description of this species referred to Hyman (1953).

Family Pseudocerotidae

This family comprises colourful elongate or oval marine flatworms, with a smooth or papillate dorsal surface and distinctive pseudotentacles. The pseudotentacles are folds of the anterior margin of the body. Pharynx is highly ruffled or smooth-margined and is located anterior to the middle of the body. Ventral sucker is situated at the middle of the body, posterior to the pharynx. The cerebral eyes are usually in a horseshoe shaped cluster, some may be in a single round cluster or two clusters. Genital pores are separated between mouth and ventral sucker. Members of this family have one or two male copulatory organs, which, when double, are arranged symmetrically. Vas deferentia extend on either side of intestinal trunk. Some genus may have multiple 105 Appendix. Marine flatworm diversity

female pores (such as Maiazoon). The female copulatory complex is closely posterior to male pore. Vagina is short and curved (Hyman 1953).

Genus Thysanozoon

Their dorsal surface bears numerous papillae. The pseudotentacles are pointed and ear-like with dorsal and ventral pseudotentacular eyes. Two male pores are close to the posterior of pharynx. Seminal vesicle is muscular and elongated and a relatively small prostatic organ. Members of this genus have one female copulatory complex (Hyman

1953; Prudhoe 1985).

Species Thysanozoon sp. (Figure A8)

Animal is large, oval, with few marginal ruffles. Background colour is translucent cream (600) mottled with light brown (110) and light yellow (106), and dark brown medially (161). The ventral surface is white with no markings. The dorsal surface covered with long pointed papillae which are yellowish brown (111) with micro black dots at their tip. The papillae are longer and more densely packed medially, declining in length and numbers laterally. Numerous pseudotentacular eyes occur along and at the tips of pseudotentacles. Two male pores are clearly seen on the ventral surface, posterior to the pharynx. This species was only recorded once throughout my study. It secreted more mucus under stress when compared to other flatworm species.

106 Appendix. Marine flatworm diversity

Table A2. Prevalent sessile species listed in the order of abundance on settlement plates.

Occurrences Sessile organisms Most common Balanus variegatus Watersipora substorquata Botryllus schlosserii Diplosoma listerianum Other colonial ascidians

Sometimes common Balanus trigonus Hydroides elegans Herdmamia momus

Uncommon Balanus amphitrite Tesseopra rosea Schizoporella errata Steyela plicata

107 Appendix. Marine flatworm diversity

egg masses

cerebral eyes

pharynx

spiny cirrus

Figure A2. Dorsal view of living Echinoplana celerrima brooding its eggs. Scale bar:

0.9 mm.

108 Appendix. Marine flatworm diversity

marginal eyes

nuchal tentacle tentacular eyes

pharynx

Figure A3. Dorsal view of living Stylochus pygmaeus. Scale bar: 0.8 mm.

109 Appendix. Marine flatworm diversity

marginal eyes

frontal eyes

pharynx gonopores

Figure A4. Dorsal view of living Imogine lateotentare, sp. nov. Scale bar: 1.1 mm.

Detailed description is in Chapter 3.

110 Appendix. Marine flatworm diversity

nuchal tentacle cerebral eyes

pharynx

marginal eyes

Figure A5. Dorsal view of living unidentified stylochid.

111 Appendix. Marine flatworm diversity

marginal tentacle tentacular eyes cerebral eyes pharynx

intestinal branch

Figure A6. Dorsal view of living Cycloporus variegatus with (a) cream-white dorsal surface and red branches, (b) cream-white dorsal surface covered in yellow spots with median white and intermittent purplish strip, (c) diagrammatic reconstruction of C. variegatus reproductive system (ce - cement glands, fa - female antrum, p - penis papillae, pr - prostatic vesicle, se - seminal vesicle) and (d) newly hatched C. variegatus larva. Scale bar: 0.8 mm (a), 0.7 mm (b), 0.1 mm (c) and 0.48 mm (d). 112 Appendix. Marine flatworm diversity

tentacle tentacular eyes cerebral eyes pharynx

uterine vesicle

Figure A7. (a) Dorsal view of living Eurylepta aurantiaca and (b) Diagrammatic reconstruction of E. aurantiaca reproductive system (ce- cement glands, fa- female antrum, p- penis papillae, pr- prostatic vesicle, se- seminal vesicle). Scale bars: 0.07 mm.

113 Appendix. Marine flatworm diversity

pseudotentacles

cerebral eyes

pharynx

papillae

Figure A8. Dorsal view of (a) anterior and (b) posterior of living Thysanozoon sp.

114