the of ciliated protozoa in the dynamics ofresearchbank.rmit.edu.au/eserv/rmit:161821/watson.pdf ·...

Theroleofciliatedprotozoainthedynamics

ofmarineinvertebratesettlement

MatthewGrantWatson

B.Sc.(App.Aqua)

B.Sc(App.Biol.Hons)

Athesissubmittedinfulfilmentoftherequirementsforthedegreeof

DoctorofPhilosophy

SchoolofAppliedSciences

CollegeofScience,EngineeringandHealth

RMITUniversity

March,2015

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DeclarationofAuthenticity

I certify that except where due acknowledgement has been made, the work is that of the author

alone; the work has not been submitted previously, in whole or in part, to qualify for any other

academic award; the content of the thesis/project is the result of work which has been carried

out since the official commencement date of the approved research program; any editorial work,

paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and

guidelines have been followed.

Matthew Watson

Monday 30th March, 2015

School of Applied Science

RMIT University

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Acknowledgements

The success of any project is never limited to the individual who is undertaking the research.

Without the support, patience and guidance of the following people, this study would not have

been completed. It is to them that I owe my deepest gratitude.

First and foremost, I would like to thank my supervisor Dr. Jeff Shimeta. His willingness to

impart knowledge and commitment to the highest standards inspired and motivated me

throughout my time at RMIT. I am also very grateful to my associate supervisors, Dr. Liliana

Zalizniak and Dr. Andrew Scardino for their many insightful discussions and suggestions. I

could not have wished for a better, more knowledgeable or friendlier team of supervisors. It has

been my privilege to work with them and to have had the opportunity to learn from their wide

knowledge and experience. I am very grateful for their support and I will never forget their

integral contribution to my endeavours.

I would like to thank all members of the marine ecology laboratory for their support and for

making my candidature at RMIT an enjoyable experience. I would particularly like to recognise

the contributions made by Justin Cutajar and Thelma Vlamis for their pioneering work on larval

settlement which lead to the basis of this thesis, and Dr. Min Tang for her help with field

collections/rearing of invertebrates, insightful discussions and friendship during my final year of

study.

I would like to thank Mark Ciacic and Jim Dimas from the Defence Science & Technology

Organisation, for their assistance with panel preparation, deployment and sampling on many

cold Melbourne mornings.

I also gratefully acknowledge the financial support provided by the Australian Postgraduate

Award (APA), and a research grant from the Holsworth Wildlife Research Endowment.

Finally to my parents, Grant and Julie, and extended family who have always supported,

encouraged and believed in all my endeavours. This work is in no small way a reflection of your

input in my life and I will always be extremely grateful for that.

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Summary

Any natural or man-made structure immersed in the marine environment is rapidly colonised by

complex assemblages micro-organisms known as microbial biofilms. In most cases microbial

biofilms are mixed assemblages of bacteria, microalgae, protozoa and fungi. As the pioneering

organisms to colonise immersed substrates, microbial biofilms condition the substrate for

subsequent colonisation by macro-organisms. Settlement and metamorphosis of many marine

invertebrate species is well known to be mediated by cues originating from microbial biofilms.

In a highly selective process, larvae respond to a range of biofilm-associated signals,

interpreting variations in the physical and chemical cues presented by different substrates.

Despite the importance of physical and chemical cues originating from microbial biofilms, the

dynamics of protozoa within these complex assemblages remains largely unknown. The aim of

this thesis is to characterise the colonisation and succession of ciliated protozoa within

microbial biofilm assemblages, and define their subsequent influence on invertebrate settlement.

If the roles of ciliated protozoa in the dynamics of invertebrate settlement are to be understood,

the taxonomic differentiation of the assemblages that dwell within microbial biofilms must first

be characterised. Ciliate assemblage analyses were carried out on neutral substrates across

different seasons and on four recently developed fouling-release and biocide-releasing

antifoulant coatings (Chapters 2 and 6). The succession of ciliate assemblages colonising the

experimental substrates followed a similar pattern. The sessile peritrichs were among the first

ciliates to colonise the substrates and rapidly increased in abundance, quickly dominating the

assemblage in terms of abundance. Over time free-swimming vagile species, specifically

adapted for life on substrates, steadily increased in abundance and accounted for much of the

ciliate diversity within the assemblage. Once the biofilm had matured to the point that marine

invertebrates are induced to settle, the peritrichs were displaced as competition for substrate

space intensified leaving the free-swimming vagile species to dominate the assemblage. While

this pattern of colonisation was consistent, the rate of succession and species composition was

significantly influenced by the properties presented by the different antifoulants and a natural

disturbance event. This study was the first to characterise biofilm-dwelling ciliate assemblages

in temperate Australia.

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In the third chapter of this thesis a settlement assay protocol for Galeolaria caespitosa was

developed. Mature adults of G. caespitosa are fertile throughout the year, and their gregarious

settlement behaviour in the intertidal zone enables easy collection making them an ideal test

species. Static settlement assays are considered the standard tool for determining the settlement

preferences of marine invertebrates. Often used to assess and evaluate the properties of a given

substrate for colonization, the static assay format is technically simple, rapid, and inexpensive.

Unlike for other serpuid polychaetes a bacterial biofilm alone was not sufficient to consistently

induce the settlement of G. caespitosa. Instead, results showed that a conspecific cue was

required for reliable settlement under assay conditions. The static settlement assay protocol

developed in Chapter 3 provided a reproducible tool to define the impacts of ciliates on

invertebrate settlement investigated in the following chapters.

The influence of a mixed assemblage of ciliates on the settlement of several fouling

invertebrates had been previously described. Chapter 4 establishes the species-specificity of the

observed influence. Settlement assays were prepared with individual ciliate species belonging to

both vagile and planktonic niches and all common to natural assemblages (Chapter 2). The

presence of individual species was sufficient to significantly inhibit the settlement of G.

caespitosa and Mytilus galloprovincialis. Furthermore, the level of settlement inhibition caused

by the different ciliate species differed significantly, supporting the conclusion that there were

species specific factors mediating the extent of the settlement inhibition. The ciliate

assemblages that dwell within microbial biofilms are highly complex (Chapter 2). These results

again highlighted the importance of ciliates in the dynamics of invertebrate settlement, and

suggested that the differences in behaviour, morphology and grazing preferences expressed by

the different species present within an assemblage will have variable impacts on the settlement

response of invertebrate larvae.

Identifying the mechanisms of influence behind the settlement inhibition was the focus of

Chapters 4 and 5. Initially it was hypothesised that the mechanism of influence could be due to

the release of a chemical cue from the ciliates, which deters settlement, similar to negative cues

released by other members of the typical microbial biofilm assemblage. However, it was

determined that the physical presence of ciliates was required to significantly inhibit settlement.

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This lead to the hypothesis that the mechanism of influence could be based solely on physical

interaction between the larvae and ciliates. In Chapter 5 the exploration behaviours of G.

caespitosa larvae in the presence and absence of ciliates was investigated. It was found that the

presence of ciliates caused the natural behavioural progression of exploring larvae to reverse.

Upon making contact with a ciliate, crawling larvae would lift off the substrate, circle rapidly

and then resume typical substrate testing behaviour in another area of the dish, effectively

delaying settlement. This study successfully demonstrated a mechanism of influence that could

explain the settlement inhibition observed in previous chapters, and was the first to report a

behavioural change of invertebrate larvae in response to contact with a ciliate.

Overall, the research presented has produced substantial evidence supporting the hypothesis that

biofilm ciliates are an important factor influencing the variability of invertebrate recruitment

over time and space, and ultimately the structure and dynamics of natural invertebrate

assemblages. The recruitment and distribution of sessile marine invertebrates cannot be

understood without attempting to understand the ecology of the microbes, which condition the

immersed substrates for subsequent recruitment. The composition of the ciliate assemblage

present on a substrate, their influence on the structure of the associated microbial community,

and the degree to which these fouling taxa interact, could all contribute to the eventual

recruitment of sessile invertebrates. A greater understanding of these processes may ultimately

have valuable applications in aquaculture husbandry and the development of sustainable non-

toxic antifoulant technologies.

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Contents

Titlepage I

Declaration II

Acknowledgements III

Summary IV

Contents VII

ListofTables X

ListofFigures XI

Publications/Presentations XV

CHAPTER1‐Generalintroduction

1.1InvertebrateSettlementintheMarineEnvironment 1

1.2VariablesthatInfluenceInvertebrateSettlement 3

1.2.1PhysicalSettlementCues 4

1.2.1.1SurfaceCharacteristics 4

1.2.1.2Hydrodynamics 5

1.2.2ChemicalSettlementCues 6

1.2.2.1Conspecifics 7

1.2.2.2PreySpecies 8

1.2.2.3MicrobialBiofilms 8

1.3FoulingofHardSubstrata 10

1.3.1TheFoulingSequence 10

1.3.2BiochemicalConditioning 11

1.3.3PioneeringAttachment 12

1.3.4BiofilmMaturation 13

1.4TheMicrobialFoulingCommunity 14

1.4.1Bacteria 14

1.4.2Diatoms 15

1.4.3Protozoa 16

1.5DynamicsofProtozoainMicrobialBiofilms 18

1.5.1Protozoa&theMicrobialLoop 18

1.5.2ProtozoanGrazing 20

1.6Rationale&Aims 22

CHAPTER2‐Colonisationandsuccessionofmarinebiofilmdwellingciliatesinresponsetoenvironmentalvariation

2.1Introduction 23

2.2Methods 25

2.2.1StudySite&Sampling 25

2.2.2Identification&Enumeration 27

2.2.3DataAnalysis 28

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2.3Results 29

2.3.1TaxonomicComposition 29

2.3.2VariationBetweenSummer&WinterDeployments 32

2.3.3ChronologicalAssemblageSuccession 36

2.3.4Aspect 38

2.3.5ResponsetoDisturbance 39

2.4Discussion 41

CHAPTER3–OptimisingsettlementassaysfortheserpulidpolychaeteGaleolariacaespitosa

3.1Introduction 46

3.2Methods 51

3.2.1LarvalCulture 51

3.2.2BacterialCulture 53

3.2.3SettlementAssays 53

3.2.4SettlementInducingBacteria 54

3.2.5ConspecificInducers 54

3.2.5.1AdultWormHomogenate 54

3.2.5.2EmptyTubes 55

3.2.5.3ConspecificLiveWorms 55

3.2.6PharmacologicalInducerIBMX 56


3.3Results 56

3.3.1IBMXTrial 56

3.3.2BacterialInducers 57

3.3.3ConspecificInducers 58

3.4Discussion 60

CHAPTER4–Influenceofcommonbiofilm‐associatedciliatesonthesettlementofGaleolariacaespitosaandMytilusgalloprovincialis

4.1Introduction 64

4.2Methods 67

4.2.1Collection&CultureofCiliates 67


4.2.2.1Galeolariacaespitosa 67

4.2.2.2Mytilusgalloprovincialis 68

4.2.3PreparationofBioassayDishes 68

4.2.4SettlementAssays 70


4.3Results 71

4.3.1Galeolariacaespitosa 71

4.3.2Mytilusgalloprovincialis 73

4.3.3LarvalSettlement/CiliateAbundanceCorrelations 75

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4.3.4CiliateFiltrate 77

4.3.4.1Euplotesminuta 77

4.3.4.2Uronemamarinum 78

4.3.5BiofilmStructureAnalysis 79

4.4Discussion 80

CHAPTER5–BehaviouralresponseofGaleolariacaespitosalarvaetothepresenceofciliates

5.1Introduction 84

5.2Methods 86


5.2.2CiliateCulture 86

5.2.3PreparationofBioassayDishes 88

5.2.3.1AnalysisofSwimmingBehaviour 88

5.2.3.2AnalysisofBehaviourBefore&AfterDirectContact 88

5.2.4VideoRecordingofLarvalExploration 89


5.3Results 90

5.3.1SwimmingBehaviour 91

5.3.2SwimmingBehaviourBefore&AfterContact 92

5.4Discussion 94

CHAPTER6–Colonisationandsuccessionofmicrobialbiofilmassemblagesonantifoulingandfouling‐releasecoatings

6.1Introduction 99

6.2Methods 102

6.2.1StudySite&Sampling 102

6.2.2Identification&Enumeration 104


6.3Results 106

6.3.1BacteriaDensities 106

6.3.2DiatomDensities 107

6.3.3CiliateAssemblage 108

6.4Discussion 115

CHAPTER7–ConclusionsandFutureDirections

7.1NaturalCiliateAssemblages 122

7.2SpeciesSpecificityofCiliateInfluence 124

7.3MechanismsofCiliateInfluence 125

7.4FutureDirections 127

REFERENCES 130

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ListofTables

Table 2.1 Ciliate genera (by order) recorded in a total of 100 samples collected during winter (Jun and Jul 2012) and Summer (Feb and Mar 2013) seasons, including function group occupied (S: sessile, V: vagile, P: planktonic), peak abundance (±SE) and occurrence.

30

Table 2.2 Temporal variation of environmental parameters recorded at study site in Port Phillip Bay during summer (Feb‐March 2013) and winter (June‐July 2012) deployments (P values are comparisons of summer and winter means, significant difference at α = 0.05).

33

Table 2.3 SIMPER analysis displaying the contribution of the top 9 genera to the

average Bray‐Curtis dissimilarity in ciliate assemblages between seasons. 35

Table 4.1 Ciliate and bacterial densities in Galeolaria caespitosa settlement assays

(Mean ± SE cells cm‐2). Means with different superscripts were significantly

different by post hoc multiple comparison tests following a significant 1‐way

ANOVA (α=0.05).

72

Table 4.2 Ciliate and bacterial densities in Mytilus galloprovincialis settlement

assays (Mean ± SE cells cm‐2). Means with different superscripts were significantly

different by post hoc multiple comparison tests following a significant 1‐way

ANOVA (α=0.05).

75

Table 6.1 Peak abundances of ciliate genera and the functional group they occupy;

observed on each substrate treatment.

110

Table 6.2 Analysis of similarity (ANOSIM) evaluation of variation in ciliate assemblages between substrate treatments at weeks 3 and 10 (using Bray‐Curtis similarity matrix), computed from square root transformed ciliate genera abundances.

114

Table 6.3 SIMPER analysis evaluating the contribution of abundant ciliate genera to total dissimilarity (as percentages) between ciliate assemblages on control (CON), fouling‐release (FR) and biocide release (BR) coatings. Values in bold indicate highest contribution (%). Genera contributing < 2% of total dissimilarity are not shown.

115

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ListofFigures

Figure 1.1 Biofouling succession models. (a) The classical view of biofouling: a sequence of key growth stages based on causality between levels. (b) The seasonal or probabilistic model: biofouling communities are dynamic with all three fouling stages taking place concurrently, succession is based on the probability of substratum encounters (Adapted from: Clare et al. 1992; Terlizzi & Faimali 2009).

11

Figure 1.2 Basic ciliate morphology. (A) Example of the ciliate Parameciumshowing the nuclei and cilia common to this protozoan Phyla; (B) Structure of the pellicle highlighting the structures from which the cilia emerge. (Adapted from Hickman et al. 2001).

16

Figure 1.3 Examples of the diverse marine ciliates which colonise biofilms (Images adapted from Hickman et al. 2001; Lynn 2008).

17

Figure 1.4 Microbial Loop: DOC is produced (dashed arrows) or incorporated into the food chain (solid arrows) by bacteria, protozoa and metazoa. Small grazers provide the link between bacteria and larger grazers, incorporating DOC into the planktonic food web. Some small protozoa can directly utilise DOC from the system and are in competition with the heterotrophic bacteria. Larger grazers may short‐circuit the loop by directly grazing on heterotrophic bacteria (Sherr & Sherr 1987; Fenchel 1988; Sherr & Sherr 2002).

18

Figure 2.1 Map of study site Booth Pier, Hobsons Bay, Williamstown. 26

Figure 2.2 Panel design illustrating how each individual Petri‐dish slide was held in place by rails mounted on the PVC panel which was then attached to an aluminium frame and suspended vertically at 1 m underneath a raft.

27

Figure 2.3 Proportions of abundance (A, C) and cumulative genera number (B, D) of ciliate functional groups during summer (A, B) and winter (C, D).

31

Figure 2.4 Proportions of abundance (A, C) and cumulative genera number (B, D) of ciliate orders during summer (A, B) and winter (C, D).

32

Figure 2.5 Summer and winter assemblage comparison of total abundance and genera diversity (H’) (Mean ± SE).

34

Figure 2.6 Dendrograms of genera assemblages using group averages based on Bray‐Curtis similarity matrix of square root‐transformed genera abundances from summer (A) and winter (B) samplings.

35

Figure 2.7 Chronological variation of (A, C) relative abundance and (B, D) genera number of ciliate functional groups recorded during summer (left column) and winter (right column) deployments.

36

Figure 2.8 Chronological variation of (A, C) relative abundance and (B, D) genera number of ciliate assemblages at the level of order recorded during summer (left column) and winter (right column) deployments.

37

Figure 2.9 Multidimensional scaling (MDS) ordination of the 5 samples taken during summer and winter deployments based on Bray‐Curtis similarity matrix of square root transformed genera abundances.

38

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Figure 2.10 Temporal aspect comparisons of sessile (A, D), vagile (B, E) and planktonic (C, F) ciliates recorded during summer (A‐C) and winter (D‐F) (Mean ± SE).

39

Figure 2.11 Images of Petri‐dish slides in sequence displaying the sudden influx of debris adhering to the surface of the slides between days 10 and 14 following the disturbance event.

40

Figure 2.12Mulitdimensional scaling (MDS) ordination of the 10 aspect samples based on Bray‐Curtis similarity matrix, computed from square root transformed ciliate genera abundances recorded during (A) winter and (B) summer.

41

Figure 3.1 Natural aggregation of Galeolaria caespitosa on a rock in the intertidal

zone Williamstown, Port Phillip Bay, Australia (Photo M. Watson).

48

Figure 3.2 Competent Galeolaria caespitosa larva. 9‐12 days old displaying a

delineated head, forward growth of the mesodermal bands, and second eye spot

(Photo M. Watson; Sketch adapted from Marsden & Anderson 1981).

50

Figure 3.3 Spawning and rearing of Galeolaria caespitosa. (A) G. caespitosa intact

colony; (B) breaking adult worm from tube; (C) male specimen releasing sperm;

(D) female specimen releasing oocytes; (E) fertilised oocytes; (F) trochophore larva

(24 hours); (G) competent larva (9‐12 days); (H) recently settled larva undergoing

metamorphosis (9‐15 days) (Photos M. Watson).

52

Figure 3.4 Galeolaria caespitosa settlement rates in various concentrations of

IBMX. (Mean ±SE). Means with different subscripts indicate significant difference

at α = 0.05.

57

Figure 3.5 Galeolaria caespitosa settlement rates in the presence of a mixed

bacterial assemblage and a mono species (Pseudoalteromonas luteoviolacea)

biofilm (Mean ±SE). Means with different subscripts indicate significant difference

at α = 0.05.

58

Figure 3.6 Galeolaria caespitosa settlement rates in the presence of a mixed

bacterial biofilm and: (A) empty tubes of adult worms; (B) adult worm

homogenate; and (C) live pre‐settled conspecific worms. Bars show Mean ±SE,

different letters indicate significant difference at α = 0.05. (D) Example of a pre‐

settled specimen of G. caespitosa with competent larvae exploring the

surrounding substrate (Photo M. Watson).

59

Figure 4.1 Galeolaria caespitosa settlement rates in the presence/absence of

tested ciliate genera (Mean ± SE cells cm‐2). Means with different superscripts

within each time point were significantly different by post hoc multiple

comparison tests following a significant ANOVA (α=0.05).

72

Figure 4.2 Mytilus galloprovincialis settlement rates in the presence/absence of

tested ciliate genera. (Mean ± SE cells cm‐2). Means with different superscripts

within each time point were significantly different by post hoc multiple

comparison tests following a significant ANOVA (α=0.05).

73

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Figure 4.3 Correlations of ciliate densities against the proportion of Galeolaria

caespitosa (A, B, C) and Mytilus galloprovincialis (E, F, G) settlement, at 24 (A, D),

48 (B, E) and 72 (C, F) hours.

76

Figure 4.4 Galeolaria caespitosa settlement rates in the presence of (1) a bacterial

biofilm alone; (2) a bacterial biofilm and filtrate from a Euplotes minuta culture;

and (3) a bacterial biofilm and the physical presence of E. minuta (shown on

micrograph). (Mean ± SE). Means with different superscripts were significantly

different by post hoc multiple comparison tests following significant repeated

measures ANOVA at α = 0.05 (Photo M. Watson).

77

Figure 4.5 Galeolaria caespitosa settlement rates in the presence of (1) a bacterial

biofilm alone; (2) a bacterial biofilm and filtrate from a Uronema mariun culture;

and (3) a bacterial biofilm and the physical presence of U. marinum (shown on

micrograph) (Mean ± SE). Means with different superscripts were significantly

different by post hoc multiple comparison tests following significant repeated

measures ANOVA at α = 0.05 (Photo M. Watson).

78

Figure 4.6 Bacterial abundances and nearest neighbour distances (Mean ± SE),

Means with different superscripts within each analysis were significantly different

at α = 0.05.

79

Figure 4.7 Structure of Pseudoalteromonas luteoviolacea biofilms after 72 hours in

the absence (A) and presence (B) of Euplotes minuta.

80

Figure 5.1 (A) Protargol stain of Euplotes minuta highlighting the cirri and oral membranelles which have enabled this sub‐class of ciliate to thrive on substrates. (B) Sketch of crawling Euplotes sp. (adapted from Laurence & Snyder 1998). (C) Competent Galeolaria caespotisa larva in the presence of E. minuta, illustrating the difference in size.

87

Figure 5.2 Examples of recorded trajectories of larval movements in the absence (Control) and presence of the ciliate Euplotes minuta (Ciliate Treatment).

91

Figure 5.3 Comparison of mean values (A, B ,C) and coefficient of variation (D, E, F) in swimming parameters between control and ciliate treatments (Mean ± SE). Means with different subscripts indicate significant difference at α = 0.05.

92

Figure 5.4 Comparison of mean values (A ,B, C) and coefficient of variation (D, E, F) in swimming parameters 4 seconds before and after direct contact between larva and ciliate (Mean ± SE). Means with different subscripts indicate significant difference at α = 0.05.

93

Figure 5.5 Examples of larvae trajectories during instances of direct contact

between ciliate and larva. Heat maps and associated line graphs display the

significant increase in velocity following these events.

94

Figure 5.6 Mean change in angle distributions before and after direct larva/ciliate contact.

95

Figure 6.1 Map of study site Booth Pier, Hobsons Bay, Williamstown 102

Page | XIV

Figure 6.2 Panel design illustrating how each individual Petri‐dish slide was held in

place by rails mounted on the PVC panel which was then attached to an

aluminium frame and suspended vertically at 1 m underneath a raft.

104

Figure 6.3 Mean total bacteria abundance observed on each coating at 3 and 10

weeks (Mean ± SE). Means with different subscripts indicate significant difference

at α=0.05.

107

Figure 6.4 Examples of bacteria colonisation on control, fouling‐release and

biocide release coatings

107

Figure 6.5 Total diatom abundance observed on each coating at 3 and 10 weeks

(Mean ± SE). Means with different subscripts indicate significant difference at

α=0.05.

108

Figure 6.6 Total ciliate abundance observed on each coating at 3 and 10 weeks

(Mean ± SE). Means with different subscripts indicate significant difference at

α=0.05

109

Figure 6.7 Variation in the relative abundances (A, B) and genera number of ciliate functional groups between the different substrate treatments at week 3 (A, C) and week 10 (B, D).

111

Figure 6.8 Variation in the relative abundances (A, B) and genera number of ciliate functional groups between the different substrate treatments at week 3 (A, C) and week 10 (B, D).

112

Figure 6.9 Dendrogram of ciliate assemblage similarities based on Bray‐Curtis similarity matrix of square‐root transformed genera abundances on each coating at 3 (dashed lines) and 10 (solid lines) weeks.

113

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Publications/Presentations

Watson M.G., Scardino A.J., Zalizniak L. & Shimeta J. (2015) Colonisation and succession of

marine biofilm-dwelling ciliate assemblages on biocidal antifouling and fouling-release

coatings in temperate Australia. Biofouling. doi:10.1080/08927014.2015.1105221.

Watson M.G., Scardino A., Zalizniak L & Shimeta J. (2015) Influence of biofilm-associated

ciliates on the settlement of Galeolaria caespitosa and Mytilus galloprovincialis.

Presentation at the Australian Marine Sciences Association Annual Conference, Geelong,

Watson M.G., Scardino A.J., Zalizniak L. & Shimeta J. (2015) Colonisation and succession of

marine biofilm-dwelling ciliates in response to environmental variation. Aquatic Microbial

Ecology, 74, 95-105.

Watson M.G., Scardino A., Zalizniak L & Shimeta J. (2014) Influence of biofilm associated

ciliates on the settlement of Galeolaria caespitosa. Presentation at the Australian Marine

Sciences Association Annual Conference, Canberra, ACT.

Shimeta J., Watson M.G., Scardino A. & Zalizniak L. (2013) Roles of ciliated protozoa in

biofilm dynamics and invertebrate larval settlement. Presentation at the ANZPAC Worksop

on Biofouoling Management for Sustainable Shipping, Melbourne, VIC.

Watson M.G., Scardino A., Zalizniak L & Shimeta J. (2013) Colonization and succession of

marine biofilm dwelling ciliates in response to environmental variation. Presentation at the

Australian Marine Sciences Association Golden Jubilee Conference, Gold Coast, QLD.

Shimeta J., Cutajar J., Watson M. G. & Vlamis T. (2012) Influences of biofilm ciliates on

settlement rates of marine invertebrate larvae. Marine Ecology Progress Series, 449, 1-12.

1. General Introduction

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Chapter1 GeneralIntroduction

1.1 InvertebrateSettlementintheMarineEnvironment

The life cycle of many sessile marine invertebrates includes a free-living planktonic larval phase

which is morphologically and ecologically distinct from the adults. Upon release planktonic

larvae are widely dispersed to a variety of habitats primarily by wind patterns and water currents

(Rodrigues et al. 1993; Pineda et al. 2002; Shanks & Shearman 2009). The planktonic larval

stage is divided into a period of pre-competence, during which growth and development occurs;

and competence, in which development is completed and the larvae are ready to settle

(Rodrigues et al. 1993; Rittschof et al. 1998). Larval adaptations for planktonic life are,

however, retained until an adequate stimulus is found for settlement and metamorphosis into

sedentary juveniles (Rodrigues et al. 1993). When competent larvae encounter potential

attachment surfaces they actively explore them utilising sensory organs to analyse external

signals. The responses to specific physical and chemical cues at the surface are essential

determinants for final settlement and metamorphosis into adults (Holmström & Kjelleberd

1994; Hadfield & Paul 2001).

The settlement and recruitment processes of sessile marine invertebrates have long been

recognised as important. The health of marine ecosystems is greatly influenced by the

abundance and diversity of invertebrates (Hutchings 2003). They perform a variety of functions

in marine ecosystems such as nutrient recycling, the removal of detritus material, consumption

of algae, and as a food source for higher trophic level organisms (Hutchings 2003). In addition


Page | 2

to the ecological importance of marine invertebrates, many species are also commercially

exploited for human consumption. Recent market growth has accelerated the development of

mollusc aquaculture across the world with thriving abalone, muscle and oyster industries (Pillay

& Kutty 2005). A critical step in intensive mollusc culture is the settlement and metamorphosis

of larvae (Pawlik 1992; Prendergast 2009).

The importance of invertebrate settlement also extends to their role in biofouling. The

settlement and growth of both micro- (bacteria, diatoms, protozoa etc.) and macro- (barnacles,

tubeworms, bryozoans, algae etc.) organisms on a surface immersed in the aquatic environment

is known as ‘biofouling’ (Cooksey & Wigglesworth 1995). Biofouling is ubiquitous and may

form on any submerged surface (Fusetani 2004; Prendergast 2009). However, interest in the

fouling process mainly originates from its detrimental effects on man-made structures.

Biofouling increases frictional drag on ships, increases bulk of submerged structures, blocks

seawater pipelines, smothers monitoring equipment and promotes structural deterioration

(Fusetani 2004; Almeida et al. 2007).

The economic consequences of biofouling have led to the development of numerous fouling

control strategies, which are devised with the objective to either prevent surface colonization or

eliminate previously established fouling organisms on a surface (Prendergast 2009). The

development of these control measures requires an understanding of the variables that influence

the fouling process, such as invertebrate larvae settlement dynamics. Currently, the most

common approach to this problem is chemical control, which seeks to either limit the number of

viable organisms coming into contact with virgin surfaces or to remove the established fouling

organisms and their extracellular components (Cloete et al. 1998; Prendergast 2009). However,

growing environmental and health concerns on the unchecked usage of anti-microbial chemical

agents have led to more stringent regulatory requirements in the use of biocide-releasing

antifoulants, thus providing the impetus in recent times for the development of more


Page | 3

environmentally sustainable alternatives to biofouling control (Almeida et al. 2007; Marechal &

Hellio 2009).

This review aims to 1) summarise the specific substratum-associated cues, which present the

appropriate physical and chemical stimulus for settlement and metamorphosis of marine

invertebrates; and 2) to illustrate the importance of microbial fouling communities, with

particular reference to protozoan colonization and the subsequent implications for biofilm

community dynamics and invertebrate settlement. The information presented in this review

provides the background for the work described in this thesis, which takes a closer look at

ciliate communities associated with marine biofilms and the mechanisms by which they

influence the settlement of marine fouling invertebrates.

1.2 VariablesthatInfluenceInvertebrateSettlement

A range of terms, such as ‘adhesion’, ‘attachment’ and ‘settlement’, have been used in the

literature to describe the process of larvae encountering and attaching to a substrate. In this

thesis the term ‘settlement’ refers to the non-reversible attachment of larvae to the substrate,

including subsequent metamorphosis where applicable.

The transition from the planktonic to the sessile phase is a crucial stage in the life cycle of

sessile marine invertebrates. For many species, settlement on solid substrates is the prerequisite

for metamorphosis into sessile juveniles (Hadfield & Paul 2001). The selection of a suitable

substrate for settlement is part of a complex recruitment process. Competent larvae are highly

selective in their preferences for certain surfaces, exploring them utilising sensory organs to

analyse external signals, thereby determining the suitability of the substratum for settlement and

subsequent metamorphosis (Rodrigues et al. 1993). The settlement and metamorphosis of many

marine invertebrates is strongly influenced by both physical and chemical cues associated with a

substrate.


Page | 4

1.2.1PhysicalSettlementCues

Physical cues known to influence larval settlement include the surface characteristics (Raimondi

1988a; Holmes et al. 1997; Scardino et al. 2003; Herbert & Hawkins 2006), hydrodynamics

(Wethey 1986; Raimondi 1988b; Havenhand & Svane 1991), light and temperature (Rodrigues

et al. 1993), the presence of competitors/predators (Morse & Morse 1984) and the availability

of food (Butman 1989; Pawlik 1992; Rodrigues et al. 1993; Fusetani 2004).

1.2.1.1SurfaceCharacteristics

Settling invertebrate larvae have been shown to make choices on the suitability of a substrate

based on surface properties including wettability (Holmes et al. 1997; Dahlstrom et al. 2004),

surface composition (Raimondi 1988a; Holmes et al. 1997; Herbert & Hawkins 2006) and

surface contours, or microtopography (Wethey 1986; Berntsson et al. 2000; Scardino et al.

2003). Surface wettability influences the attachment strength of marine invertebrate larvae to a

substrate (Holmes et al. 1997). Reports on the settlement response of invertebrate larvae to

substrates of varying wettability have shown that the wettability of a substratum for settlement

is important for initial colonization, but not in determining the overall structure of the

invertebrate community on a surface (Roberts et al. 1991; Holmes et al. 1997). Larval

settlement behaviour of mussels (Aldred et al. 2005), barnacles (Dahlström et al. 2004) and

bryozoans (Holmes et al. 1997) has been shown to be affected by the wettability of a substrate

(Holmes et al. 1997; Dahlström et al. 2004). It is hypothesised that surface wettability

influences larval settlement by interacting with chemoreceptors when the larva is making

surface contact, or via a physical nature whereby surface contact is impeded by a repulsive

surface charge (Aldred et al. 2005; Dahlström et al. 2004; Di Fino et al. 2014).

The mineral composition of various rock substrates will determine their hardness and resistance

to weathering, and therefore influence surface topography and heterogeneity over time (Herbert

& Hawkins 2006). Studies investigating the effect of surface type on the settlement of


Page | 5

invertebrate larvae have often resulted in conflicting conclusions (Holmes et al. 1997; Herbert

& Hawkins 2006). Few studies have identified consistent patterns of settlement due to the

influence of surface composition alone. However, Herbert & Hawkins’s (2006) investigation of

numerous rock types identified relatively high post-recruitment mortality of the barnacle

Chthamalus montagui on chalk substrates. They concluded that the soft, flaky nature of chalk

and some limestones is likely to impede invertebrate colonisation as the rock surface is

constantly being weathered and broken away, leaving patchy assemblages.

The use of microtopography as a natural antifouling alternative to various toxic approaches has

been a growing subject of recent research (Wahl & Lafargue 1990; McKenzie & Grigolava,

1996; Wahl et al. 1998; Scardino et al. 2003). Scardino et al. (2003) found that the regular

ripple structure of the blue mussel Mytilus galloprovincialis outer shell significantly reduced

settlement of common fouling organisms, including barnacle larvae, tubeworms, bryozoans and

some species of algae. The common mechanism for attachment preferences of fouling

organisms is the number of available attachment points a surface provides (Schumacher et al.

2007; Scardino et al. 2008). When settling larvae cannot fit inside a surface feature, the number

of attachment points is reduced and settlement is generally decreased (Schumacher et al. 2007;

Scardino et al. 2008).

1.2.1.2Hydrodynamics

Hydrodynamic forces have influence over invertebrate larvae from the moment they are

released into the water column (reviewed by Koehl 2007). Water currents and eddies move

larvae away from the favourable habitat of their parents, and are widely dispersed to a variety of

new substrates (Shanks 1995). The importance of hydrodynamics continues once the larvae

reach competency and begin actively searching for favourable settlement substrates.

Hydrodynamic forces have been shown to influence the encounter rate between larvae and the

substratum (Hannan 1984; Wethey 1986; Havenhand & Svane 1991), subsequent settlement


Page | 6

rates (Jonsson et al. 2004), and post settlement growth and survival (Sanford et al. 1994; Judge

& Craig 1997) of many marine invertebrates.

Hydrodynamic forces impart a significant influence on the suitability of a substrate for initial

settlement and subsequent growth. Many invertebrate larvae cannot settle and attach above

certain flow rates. Nagaya et al. (2001), for example, found that a fluid velocity below

approximately 1 m s-1 was critical to attachment/detachment of the mussel Limnoperna fortunei

during the juvenile stage. Hydrodynamics can also influence the availability of food for many

suspension feeding invertebrates. Larsson & Jonsson (2006) showed that larvae of the barnacle

Balanus improvisus actively rejected high flow environments that were suboptimal for

suspension feeding. Hydrodynamic forces can also influence a larva’s direct contact with

surface-associated chemical cues, which is often necessary for the behavioural effect to be

triggered (Pawlik 1986; Raimondi 1988b). Numerous studies have shown that initial contact of

larvae with a surface is often due to water flow near that surface (Hannan 1984; Wethey 1986;

Havenhand & Svane 1991).

1.2.2ChemicalSettlementCues

There is considerable experimental evidence which suggests that chemical cues also play a

pivotal role in the settlement behaviours of fouling invertebrates (Wieczorek & Todd 1998;

Rittschof et al. 1998; Hadfield & Paul 2001). There are three main sources of stimulatory and

inhibitory chemical cues to which marine invertebrates respond: microbial biofilms (Bonar et al.

1990; Wieczorek & Todd 1998; Rittschof et al. 1998), conspecific adults (Jensen & Morse

1984; Pawlik 1986; Jensen et al. 1990) and prey species (Hadfield & Pennington 1990; Morse

1991).


Page | 7

1.2.2.1Conspecifics

Numerous laboratory-based studies of marine invertebrates have demonstrated settlement

induced by conspecific adults. These include common invertebrates such as polychaetes (Jensen

& Morse 1984; Pawlik 1986; Toonen & Pawlik 1996), abalone (Slattery 1992), ascidians

(Svane et al. 1987; Svane & Young 1989) and barnacles (Raimondi 1988b; Raimondi 1991).

Settlement induced by conspecifics is often observable in nature, accounting for the aggregated

distribution of many marine invertebrates. Settlement in large aggregations of conspecific adults

has many benefits, including selection of a habitat already shown to support the species (Pawlik

1986; Toonen & Pawlik 1994), increased probability of gamete fertilization (Pawlik 1986;

Rodrigues et al. 1993) and provides an effective defence against predators (Garnick 1978;

Toonen & Pawlik 1994). Research into the nature of conspecific signals has implicated adult-

associated compounds as being responsible for larval settlement responses into adult

aggregations. (Toonen & Pawlik 1994; Thiyagarajan 2010).

Wilson (1968) reported that marine polychaete worms of the family Sabellariidae are induced to

settle via a chemical cue produced in the tube cement of conspecific adults. In contrast, Toonen

& Pawlik (1996) discovered that the inductive compounds responsible for gregarious settlement

of the polychaete, Hydroides dianthus, are water soluble cues associated with the body of living

adults rather than the calcareous tubes they secrete. The Authors noted that while direct contact

with conspecific adults is not required to induce settlement, soluble settlement cues would be

diluted rapidly in turbulent flow. Therefore, it seems likely that settling larvae could only

perceive soluble cues released by conspecific adults at very short range (Toonen & Pawlik

1996). The chemical structure of conspecific settlement cues remain largely unknown, with few

compounds fully characterised (Fusetani 1997; Tsukamoto et al. 1995; Tsukamoto et al. 1999).

Further structural information is necessary as the ecological relevance and mechanism of

conspecific chemical inducers remains unclear (Hadfield & Paul 2001).


Page | 8

1.2.2.2PreySpecies

Chemicals released by prey species have also been identified as important settlement cues for

marine invertebrates. Morse & Morse (1984) identified settlement-inducing chemicals on the

surface of encrusting algae; the authors theorised that these chemicals could possibly be

released into the surrounding water column when the cells are grazed on by other organisms.

Many invertebrate larvae are induced to settle by the algae on which they feed. These include

invertebrates such as mussels (Soares et al. 2008; Yang et al. 2007), abalone (Morse & Morse

1984; Williams et al. 2008) and sea urchin (Swanson et al. 2006; Huggett et al. 2006). Abalone

larvae, for example, settle preferentially on crustose coralline red algae of the genera

Lithothamnion, Lithophyllum, and Hildenbrandia (Morse & Morse 1984; Daume et al. 1999).

The inducers have been partially characterized and appear to be associated with the

phycobiliprotiens of red algae (Morse et al. 1984).

Many prey species have also evolved the ability to synthesize chemical defences to inhibit

settlement of predators (Qian et al. 2010). These natural chemical defences are known as

biogenic agents, defined as chemical compounds that are synthesized by living organisms,

which if exceed certain concentrations cause damage or even death of other organisms by

chemical or physiochemical effects (Bhadury & Wright 2004). Biogenic agents are synthesized

in the secondary metabolism of the producer (de Nys & Steinberg 2002). The characterization

and development of these natural chemical defences as active ingredients in environmentally

friendly antifouling coatings has been of growing interest in recent decades (Maréchal & Hellio

2009; Qian et al. 2010).

1.2.2.3MicrobialBiofilms

Microbial biofilms have been identified as a source of chemical cues that can either induce or

inhibit settlement. Chemical cues released by the pioneering colonisers of biofilms are widely

considered to have the greatest influence on the settlement of macrofoulers (Wieczorek & Todd


Page | 9

1998; Rittschof et al. 1998), identified in numerous studies as important settlement cues for

polychaetes (Lau & Qian 1997), barnacles (Letourneux & Bourget 1988), oysters (Bonar et al.

1990), bryozoans (Hans et al. 2004) and many other invertebrate species. It is generally

accepted that the chemical cues are generated by the presence of extracellular polysaccharides

or glycoproteins attached to the bacterial wall (Kirchman et al. 1982, Hadfield 1986; Qian et al.

2010) or soluble compounds released by these films (Bonar et al. 1990; Qian et al. 2010).

Several studies have revealed that invertebrate settlement is somewhat mediated by the

composition of bacterial biofilms. For example, Lau et al. (2003) reported that biofilms

composed of Vibrio sp. and Roseobacter sp. inhibited the attachment of the barnacle Balanus

amphitrite. Khandeparker et al. (2003), in contrast, reported that bacteria isolated from

conspecific adult habitat significantly increased attachment of B. amphitrite when compared to

control assays. As the cues generated by bacterial films can either induce or inhibit settlement,

the bacterial composition of biofilms is, therefore, one of the predominant factors in whether

exploring larvae will accept or reject a substrate (Raimondi 1988a; Roberts et al. 1991; Werner

1967).

Recent research by Shikuma et al. (2014) identified another, previously unknown, mechanism

by which marine bacteria may induce settlement of invertebrates. It was discovered that arrays

of phage tail–like structures produced by the marine bacterium, Pseudoalteromonas

luteoviolacea, are responsible for triggering settlement and metamorphosis of Hydroides

elegans. Bacteria typically use the phage tail-like structures, also known as bactericins, to kill

other bacteria by puncturing their membrane, causing depolarization (Uratani & Hoshino 1984;

Michael-Briand & Baysse 2002). In fact, prior to the research presented in Shikuma et al.

(2014) no beneficial interaction had been linked to these structures. Similar arrays have also

been identified in the genomes of other marine bacteria (Persson et al. 2009), and suggest a

novel form of bacterium-animal interaction. However, how these arrays engaged with H.


Page | 10

elegans larvae remains unclear (Shikuma et al. 2014). Further research into how these phage

tail-like structures interact with larvae might yield new insights into the mechanisms

underpinning invertebrate settlement.

1.3 FoulingofHardSubstrata

1.3.1TheFoulingSequence

Biofouling of a submerged substrate occurs through a sequence of specific, but poorly

understood chemical and biological processes (Dobretsov et al. 2013). The classical view of

biofouling has been described as a sequence of key growth stages including an initial

accumulation of adsorbed organics known as biochemical conditioning, followed by the

settlement and growth of pioneering bacteria, and finally a succession of micro- and macro-

foulers (Wahl 1989). However, this simplified description of the biofouling sequence implies

some causality between the fouling stages and excludes acknowledgement of seasonal and

spatial variation in recruitment, growth, mortality and the influence of trophic interactions

between fouling organisms (Maki & Mitchell 2002).

While it is unrealistic to expect any model to incorporate all sources of variation present in the

fouling sequence, the seasonal or probabilistic model of biofouling is perhaps more applicable

to the fouling sequence as it occurs in the natural environment (Terlizzi & Faimali 2009). This

model suggests that all three fouling stages take place concurrently and form a complex web of

interactions between the substratum and the fouling organisms, and interspecifically between

fouling organisms (Maki & Mitchell 2002; Terlizzi & Faimali 2009). In this model, the

biofouling communities are dynamic both in their structure and function, continuously

developing by mechanisms, such as disturbance, facilitation, inhibition and tolerance (Wahl

1989; Maki & Mitchell 2002) (Fig. 1.1).


Page | 11

1.3.2BiochemicalConditioning

When a chemically inert substrate is first immersed in seawater, there is an almost immediate

accumulation of dissolved organic molecules onto the wetted surface. This initial ‘conditioning

film’ is generally composed of glycoproteins, humic acids, proteins, carbohydrates and amino

acids (Bauer et al. 1968; Abarzua & Jakubowksi 1995; Siboni et al. 2007). The formation of the

conditioning film on a substrate plays an important role in subsequent microbial adhesion by

altering the physical properties of the substrate. Conditioning films are known to influence

surface tension (Dexter 1979), interfacial free energy (Schneider 1996), hydrophobicity (Bakker

et al. 2004) and surface roughness (Bakker et al. 2004). In addition to altering the properties of

a substrate, conditioning films also provide attachment sites for microorganisms. Many of the

molecules (such as proteins) present in conditioning films are made up of long chains of

monomers which provide binding sites for microorganisms (Costerton et al. 1995).

Invertebrate Larvae

Unicellular Algae & Protozoa

Bacteria

Conditioning Film

Invertebrate Larvae

Algae & Protozoa

Bacteria

Conditioning Film

Particulates

? ?

?

?

A B

Figure 1.1 Biofouling succession models. (A) The classical view of biofouling: a sequence of key growth stages based on causality between levels. (B) The seasonal or probabilistic model: biofouling communities are dynamic with all three fouling stages taking place concurrently, succession is based on the probability of substratum encounters (Adapted from: Clare et al. 1992; Terlizzi & Faimali 2009).


Page | 12

Jain & Bhosle (2009) showed that in the absence of a conditioning film, the number of bacterial

cells able attach to a surface is significantly reduced. Furthermore, they discovered that rates of

successful attachment differed amongst species depending on the composition of the

conditioning film. For example, the concentration of carbohydrates in a marine conditioning

film was positively correlated to Pseudomonas species attachment, but negatively correlated to

Bacillus species attachment (Jain & Bhosle 2009). These differences are thought to be the result

of the differences in cell hydrophobicity between the different species of bacteria (Jain &

Bhosle 2009), or due to chemical attraction or repulsion (Chet et al. 1975; Gubner & Beech

2000).

1.3.3PioneeringAttachment

Following the formation of the conditioning film colonisation by micro- and macro- fouling

organisms can happen sequentially, overlap or occur in parallel (Clare et al. 1992; Terlizzi &

Faimali 2009) (Figure 1.1B). Although some marine invertebrates will settle on clean substrates,

settlement of many species is enhanced by the presence of microbial biofilms (Lau & Qian

1997; Wieczorek & Todd 1998; Rittschof et al. 1998). Hence some causality is often

demonstrated in pioneering attachment, i.e. colonisation by prokaryotes enhances settlement of

invertebrates in later phases of biofouling. Microorganisms that form biofilms can survive in

radically different environments, and are better equipped to attach onto substrata immediately

following biochemical conditioning (Costerton et al. 1995). Pioneering microorganisms secrete

extracellular polymeric substances (EPS), which envelop the microbial community and anchor

them to the substrate, providing a measure of protection from environmental variables and

predators (Marshall 1992; Hoagland et al. 1993; Cooksey & Wigglesworth 1995). Microbial

fouling is consequently rarely precluded during the initial stages of substrate colonisation.

Microbial biofilms in aquatic environments are very heterogenic and dynamic structures. The

composition of pioneering biofilms primarily depends on the presence of colonisers, the


Page | 13

conditions presented by the substratum and the environment in which it is immersed (Costerton

et al. 1995; Qian et al. 2007). Differences in biotic and abiotic environmental variables can

influence the composition of biofilms, biofilm density, productivity, architecture, succession

rate and the production of the compounds which influence invertebrate settlement (Qian et al.

2007; Dobrestov 2010). Overall, the rate of microbial biofilm development is generally

dependent on the nutrient status of the surrounding water. Biofilms can develop within serval

hours in nutrient rich coastal waters, whereas in the open ocean a microbial biofilm population

may take weeks to develop (Costerton et al. 1995; Lee et al. 2008). However, once established

on a substrate, microbial biofilms alter the surface chemistry, which then stimulates further

growth including the recruitment and settlement of macro-foulers.

1.3.4BiofilmMaturation

The fouling community develops into a mature stage through competitive or synergistic

interaction among the existing fouling organisms (Costerton et al. 1995; Moller et al. 1998;

Dang & Lovell 2000), and by recruitment of new fouling organisms (Wimpenny 1996). The

settlement of macro-foulers often follows in response to physical and chemical cues associated

with the substratum. The most notable macro-fouling invertebrate groups include Crustacea

(e.g. barnacles), Mollusca (e.g. mussels, oysters), Bryozoa, Annelida (e.g. tubeworms), Tunicata

(sea squirts), Cnidaria (anemones, hydroids) and Porifera (sponges) (Prendergast 2009).

Throughout the biofouling process communities are subject to constant dynamic changes (Wahl

1989; Jenkins & Martins 2010). Substrates immersed in the marine environment are subjected

to a range of physical (i.e. wave action, light exposure, storms) and biological (i.e. predation,

grazing) disturbance regimes (Sousa 1984; Jenkins & Martins 2010). The highly variable

disturbance regime to which fouled substrata are subjected ensures that composition of fouling

communities will show continual change through time.


Page | 14

1.4MicrobialFoulingCommunity

1.4.1Bacteria

Marine bacteria are often among the first organisms to colonize a surface after the biochemical

conditioning has taken place. The transition of bacteria from free-living planktonic cells to a

complex, surface-attached biofilm is a highly regulated process (Wahl 1989; Stoodley et al.

2002). In the first stage, reversible attachment is mainly mediated by physicochemical

interactions between the bacterial cell surface and the substratum (Wahl 1989). Interactions

between the bacterial cell wall and the substrate facilitate adhesion to a variety of inert surfaces

(van Pelt et al. 1985; Gilbert et al. 1991; Mueller et al. 1992). The second, irreversible,

attachment phase occurs following the production of extracellular polymers by the bacteria

(Marshall 1992). The polymers act as strong adhesives and guarantee that the pioneer

population of bacteria in a biofilm will be permanently attached to the surface, insulating that

surface from the water column (Marshall 1992).

Marine bacteria identified from biofilms include those from the genera Pseudomonas,

Alteromonas, Pseudoalteromonas and Oceanospirillum (Dang & Lovell 2002). Bacteria of the

α-proteobacteria (eg. Ochorbactrum anthropi, Paracoccus carotinifaciens) and the γ-

proteobacteria (e.g. Pseudomonas aeruginosa, Vibrio parahaemolyticus) were identified as

important primary colonising bacteria on surfaces exposed to seawater for 24-72 h (Dang &

Lovell 2000). The rate of bacterial biofilm development is dependent on the nutrient status of

the surrounding water. Biofilms can develop within days in nutrient rich coastal waters, whereas

in the open ocean a biofilm population may take weeks to develop (Costerton et al. 1995; Lee et

al. 2008). Once established on a substrate, bacterial biofilms alter the surface characteristics of

the substratum, rendering it suitable (or unsuitable) for subsequent colonization by secondary

micro- and macro-organisms (Characklis et al. 1990).


Page | 15

1.4.2Diatoms

Diatoms are usually the earliest eukaryotic colonizers of microbial biofilms, and are often a

dominant component when the substratum is situated within photic zones (Lam et al. 2005).

Similar to bacterial adhesion, diatoms attach to a surface through secretion of extracellular

polymers (EPS) (Hoagland et al. 1993; Cooksey & Wigglesworth 1995). Diatom attachment is a

two stage process, beginning with reversible adhesion (initial contact) and followed by

irreversible adhesion facilitated by the production of EPS (Wetherbee et al. 1998).

In marine ecosystems, raphid diatoms, such as Amphora sp., Navicula sp., and Nitzschia sp., are

the most abundant genera to colonise substratum in the early stages of biofilm establishment

(Wetherbee et al. 1998; Mitbavkar & Anil 2000). Colonisation by diatoms modifies the

properties of the underlying substratum due to the large quantities of EPS produced during

attachment (Wetherbee et al. 1998). The EPS improves the stability/attachment of the

developing biofilm, and has been shown to influence the efficiency of many antifoulant

treatments (Molino et al. 2009b; Dobretsov & Thomason 2011; Bressey & Lejars 2014).

Furthermore, diatoms also act as a settlement mediator for many fouling invertebrates such as

barnacles and polychaete worms (Letourneux & Bourget 1988; Harder et al. 2002; Lam et al.

2005; Patil & Anil 2005).

1.4.3Protozoa

Protozoa are unicellular eukaryotic microorganisms that range in size between 2 and 1000 µm.

For simplicity, they are generally divided according to the morphology of their locomotory

organelles: flagellates possessing flagella, ciliates possessing cilia and amoebae possessing

pseudopodia (Patterson 1996; Finlay & Esteban 1998). This simplified classification system is

utilised as a broad indicator of protozoan functional groups. It does not represent true

phylogenetic relationships of protozoa. Nevertheless, it is widely used in studies where such

information is not required (Finlay & Esteban 1998).


Page | 16

Ciliates are the most structurally complex and specialized of all protozoan groups (Patterson

1996). Classified into a single phylum, the Ciliophora, the nuclei rather than the cilia distinguish

ciliates from all other groups of protozoa (Patterson 1996; Finlay & Esteban 1998) (Figure

1.2A). Ciliates are dikaryotic organisms; the larger macronucleus regulates metabolism of the

cell, while the smaller micronucleus is primarily involved in the genetics and sexual

recombination (Finlay & Esteban 1998). The cilia are used for locomotion, to crawl on surfaces,

or to produce feeding currents (Parry 2004) (Figure 1.2B). Typically larger than other protozoan

groups, ranging in size between 10 µm and 2000 µm, ciliates are thought to be among the most

dominant grazers of microbes in aquatic benthic habitats (Finlay & Esteban 1998).

Ciliates often occur in very high abundances on marine biofilms and include both sessile filter

feeding forms (e.g. Vorticellia, Zoothamnium) permanently attached to the surface, specially

adapted free swimming motile predators of microorganisms (e.g. Euplotes, Stichotricha,

Dileptus) which browse directly on substrates, and planktonic species (e.g. Didinium, Uronema)

which typically swim in the surrounds of established biofilms occasionally exploiting transitory

patches of bacteria (Figure 1.3) (Patterson 1996; Arndt el al. 2003; Xu et al. 2009). Sessile

species rise above the substrate on stalks, and typically remove food (in the form of suspended

Figure 1.2 Basic ciliate morphology. (A) Example of

the ciliate Paramecium showing the nuclei and cilia

common to this protozoan Phyla; (B) Structure of the

pellicle highlighting the structures from which the

cilia emerge. (Adapted from Hickman et al. 2001).


Page | 17

particles) from the surrounding water by using their cilia to create a flow of fluid towards the

mouth (Patterson 1996). Many sessile species also have the ability to contract, a behaviour that

prevents damage from turbulence in the surrounding water, or from larger predators that may

swim over the submerged substratum (Patterson 1996; Gong et al. 2005). Ciliates from the

subclass Hypotrichida (e.g. Euplotes, Diophrys) are common examples of free-swimming

ciliates specifically adapted for life on substrates (Lynn 2008). Specialised somatic cilia have

been fused into cirri, which function as walking or crawling appendages. The oral ciliature of

this subclass is composed of a band of planar membranelles, which are located under the ventral

surface (Lawrence & Snyder 1998; Lynn 2008). The membranelles create feeding currents that

draw in particles, making these ciliates particularly effective at feeding from substrates (Lynn

2008).

1.5DynamicsofProtozoainBiofilms

1.5.1Protozoa&theMicrobialLoop

Microbial food webs are central to nutrient cycling and energy transfer in most aquatic

ecosystems (Fenchel 1988). The microbial loop consists of interacting bacteria, protozoa and

metazoa (Pernthaler 2005); consequently the quantity of carbon available to higher trophic

Figure 1.3 Examples of the diverse marine ciliates which colonise biofilms (Images adapted from

Hickman et al. 2001; Lynn 2008).


Page | 18

levels is influenced by the activities of all these organisms. Bacteria and protozoa can make up

half of the biomass in microbial food webs and are therefore responsible for the largest part of

energy flow in some aquatic ecosystems (Fenchel 1988). The basic microbial loop concept

describes how small protozoa act as a carbon-transfer bridge between heterotrophic bacteria and

larger predators, which cannot feed directly on bacteria (Fenchel 1988; Sherr & Sherr 2002;

Pernthaler 2005) (Fig. 1.2). Dissolved organic carbon (DOC) is released into the environment

by larger organisms through excretion, sloppy feeding, and the death and lysis of primary

producers (Sherr & Sherr 2002). Heterotrophic bacteria reincorporate the DOC into the food

chain by turning it into biomass (Sherr & Sherr 2002). The grazing activity of small grazers then

acts as a link that allows the transfer of carbon from this bacterial biomass into the food web

(Sherr & Sherr 2002; Pernthaler 2005).

Sho

rt-c

ircu

it

Dissolved Organic Carbon

Heterotrophic Bacteria &

Archaea (<1-2µm)

Bacterivorous Flagellates

& Ciliates

Zooplankton, Larger Ciliates

& Dinoflagellates

(feed on cells > 3-5µm)

Co

mp

etit

ion

Slop

py Feed

ing

Excretion

Death

& Lysis

Figure 1.4 Microbial Loop: DOC is produced (dashed arrows) or incorporated into the food chain (solid arrows) by bacteria, protozoa and metazoa. Small grazers provide the link between bacteria and larger grazers, incorporating DOC into the planktonic food web. Some small protozoa can directly utilise DOC from the system and are in competition with the heterotrophic bacteria. Larger grazers may short-circuit the loop by directly grazing on heterotrophic bacteria (Sherr & Sherr 1987; Fenchel 1988; Sherr & Sherr 2002).


Page | 19

In some situations, the basic microbial loop is modified, resulting in a reduction of the number

of trophic levels that carbon passes through before reaching metazoan grazers. For example,

mixotrophic protozoa can compete with heterotrophic bacteria for the DOC, incorporating it

directly into the food chain (Sherr & Sherr 1987). Also, some larger grazers, such as larger

ciliates, can short-circuit the loop by directly feeding on the heterotrophic bacteria (Sherr &

Sherr 1987). In all these scenarios, protozoa play an essential part by providing the trophic link

between prokaryotes and other eukaryotes (Sherr & Sherr 2002; Pernthaler 2005).

1.5.2ProtozoanGrazing

Protozoa ingest other microorganisms as food. This fundamental characteristic underpins the

ecological significance of protozoa in the natural environment. They are, in quantitative terms,

considered the most important grazers of microbes in aquatic environments (Berninger et al.

1991; Sherr & Sherr 1994; Corno & Jürgens 2006). There is a close link between protozoan

morphology (especially of the food-capturing organelles) and the way in which a protozoan

functions as a grazer (Finlay & Esteban 1998). Heterotrophic protozoa generally feed on smaller

microorganisms including prokaryotes, smaller protozoa, algae and fungi (Arndt et al. 2003;

Parry 2004). All three of the broad functional protozoan groups (flagellates, ciliates and

amoeba) may feed on the same type of microorganisms in the same place, but they differ in the

mechanics of food capture and in the food size spectrum that each can ingest. Amoeba for

example will typically engulf a much broader size range than a filter feeding ciliate of the same

size, the ciliate being adapted for filtering a narrow range of particle sizes (Iriberri et al. 1995;

Patterson 1996; Finlay & Esteban 1998).

The rate of bacterial consumption by bacterivorous protozoa depends on the type of grazer, the

size of the grazer and the nature and concentration of the prey present (Ayo et al. 2001; Boenigk

& Arndt 2002). Studies have shown that ciliates are able to consume up to 1,254 bacteria per

hour (Iriberri et al. 1995). Protozoa will often display a degree of feeding preferences when they


Page | 20

graze on bacteria. Preferences based on cell morphology (Hahn & Hofle 2001; Posch et al.

2001), motility (Matz & Jurgens 2005), metabolic state (del Giorgio et al. 1996) and prey

digestibility (Boenigk et al. 2001a; Weisse 2002) have been identified as important

characteristics that can determine whether a cell is grazed upon or not. The feeding behaviour is

also influenced by the nutritional status (Boenigk et al. 2001a), metabolic state (Boenigk et al.

2001b) and feeding strategy of the specific grazer (Finlay & Esteban 1998).

As the major consumers of bacteria in the environment, heterotrophic protozoa are one of the

most important selective forces that bacterial communities face (Hahn & Hofle 2001; Corno &

Jürgens 2006). Protozoan grazing has been shown to influence bacterial community structure

and composition in aquatic ecosystems (Lawrence & Snyder 1998; Pernthaler et al. 2005).

Lawrence & Snyder (1998) reported that intense localised feeding by the ciliate Euplotes on

attached bacteria resulted in increased spatial and temporal heterogeneity within the biofilm.

The authors suggested that these changes in structure could affect biofilm development by

creating areas of clearance and sloughing, which in turn could affect the overall activity of the

biofilm. Selective protozoan grazing has also been shown to exert a strong influence on the

composition of bacterial assemblages within biofilms (Hahn & Höfle 2001; Corno 2006).

Apart from the direct effect of predation, protozoan grazing can also affect bacterial biomass

indirectly by influencing nutrient availability (Parry 2004). Protozoan grazing has been found to

be important for nutrient recycling, and in some systems stimulate the bacteria involved in the

breakdown of detritus, returning carbon to the system (Sherr et al. 1982; Ribblet et al. 2005). In

addition to carbon cycling, protozoa play a crucial role in the cycling of nitrogen and

phosphorus (Andersson et al. 1985; Parry 2004). Protozoa re-mineralise the organic nitrogen

and phosphorus within their prey into soluble, inorganic nitrogen and phosphorus (Parry 2004).

These compounds are excreted as ammonium and orthophosphate respectively, and then utilised

by bacteria as an additional growth stimulant (Andersson et al. 1985). All this evidence


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indicates that there are many ways in which protozoan grazing can affect the bacterial

component of microbial biofilms, by either directly feeding on the bacteria or indirectly

contributing to their access to nutrients.

1.6Rationale&Aims

Despite extensive evidence that invertebrate larvae respond to a range of specific physical and

chemical cues that originate from microbial biofilms, the dynamics of protozoa in these

assemblages remains largely unknown (Arndt et al. 2003). Currently the study of free-living

protozoa is mostly limited to identification and taxonomic classification, determining their

diversity in different habitats, and understanding their grazing behaviour under different

conditions. Few studies have focused on investigating the influence of protozoa on marine

biofilm structure/function and the greater effects this may have on marine invertebrates that

colonise the biofilmed surfaces.

Any conclusions regarding the influence of ciliates on microbial biofilm dynamics and

subsequent impacts on invertebrate settlement will first require taxonomic differentiation of

ciliate assemblages. An understanding of the structure/function of ciliate assemblages in marine

microbial biofilms is central to interpret and predict the impacts of these assemblages on the

surrounding marine ecosystem. Investigations of succession on marine substrata have

contributed considerably to an understanding of the biofouling process. Given the impacts of

protozoan activities within microbial biofilms, their role in the fouling dynamics on antifoulant

coatings should also not be overlooked. The development of microbial biofilms can modify the

properties of the underlying antifoulant (Cassé & Swain 2006; Molino et al. 2009a). This

process is therefore an important determinant of the efficiency of antifoulant treatments.

Furthermore, the importance of physical and chemical cues, many of which derive from

microbial biofilms, on the settlement and metamorphosis of invertebrate larvae has been well


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documented (Wieczorek & Todd 1998; Rittschof et al. 1998; Hadfield & Paul 2001). It is,

therefore, reasonable to assume that biofilm dwelling protozoa could influence the settlement of

invertebrate larvae either indirectly by influencing the structure, abundance or community

composition of microbial biofilms, or directly through physical or chemical interactions with

larvae. The work described in this thesis is aimed at furthering our understanding of protozoan

dynamics within marine biofilms, with a focus on identifying the mechanisms by which they

influence the settlement of fouling invertebrates.

The specific aims are:

1. To characterise the community structure of ciliate populations associated with natural

marine biofilms.

2. To determine the extent to which settlement of biofouling invertebrates is influenced by

biofilm-dwelling ciliates, including identifying the species specificity of the observed

influence.

3. To investigate the direct or indirect mechanisms by which biofilm dwelling ciliates

influence the settlement of biofouling invertebrates.

4. To identify the impact of fouling-release and biocide-releasing antifoulant coatings,

commonly utilised by marine industry, on the development of ciliate assemblages and

associated biofilm-dwelling microbes.

2. Temporal succession of biofilm‐dwelling ciliates

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Chapter2

Colonisation and succession of marine biofilm-dwelling ciliates

in response to environmental variation

2.1Introduction

Substrates immersed in the ocean are rapidly colonised by complex assemblages of micro-

flora. These ‘microbial biofilms’ are primarily made up of bacteria, microalgae and protozoa

enmeshed within a layer of bacterial extracellular polymers (EPS), which anchor the

assemblages to the substrate (Arndt et al. 2003; Dobretsov 2013). The settlement and

metamorphosis of many marine invertebrates is strongly influenced by physical and

chemical cues associated with microbial biofilms (Rittschof et al. 1998; Qian et al. 2007).

Chemical cues released by the pioneering colonisers of biofilms in particular are widely

considered to have the greatest influence on the settlement of macrofoulers (Wieczorek &

Todd 1998; Rittschof et al. 1998; Hadfield & Paul 2001; Qian et al. 2007).

Protozoa, a ubiquitous component of microbial biofilms, are able to rapidly colonise new

substrata and over a short period of time reach high abundances within biofilms (Arndt et al.

2003; Gong et al. 2005; Xu et al. 2009). Heterotrophic protozoa are, in quantitative terms,

considered the most important grazers of microbes in aquatic environments (Berninger et al.

1991; Sherr & Sherr 1994; Corno & Jürgens 2006). There is increasing evidence that the

presence of protozoa can influence biofilm structure and population dynamics of microbial

biofilms (Jackson & Jones 1991; Lawrence & Snyder 1998, Kiørboe et al. 2003; Huws et al.

2005). The impact of biofilm dwelling protozoan assemblages, however, largely depends on

- Chapter 2 is adapted from: Watson M.G., Scardino A.J., Zaliziak L. & Shimeta J. (2015) Colonisation and succession of marine biofilm dwelling ciliates. Aquatic Microbial Ecology, 74(2), 95-105


Page | 24

their structure, both in terms of abundance as well as their taxonomical and functional

composition (Arndt et al. 2003).

Given the impacts of protozoan activities within biofilms, their influence could extend to the

settlement of invertebrate larvae either indirectly by influencing the structure, abundance or

community composition of microbial biofilms, or directly through physical or chemical

interactions with larvae. Shimeta et al. (2012) recently showed that the presence of a mixed

assemblage of ciliates had a range of impacts on the settlement and post-settlement mortality of

several invertebrate foulers. However, while the mixed assemblages of ciliates had strong

effects on invertebrate settlement, they appeared to be highly varied and species-specific

(Shimeta et al. 2012). These results point to a need for better description of species assemblages

and successional processes in marine biofilms.

Investigations of succession on marine hard substrata have contributed considerably to

understanding of the biofouling process. An array of direct and indirect species interactions,

stochastic recruitment events and environmental disturbance ultimately dictates the successional

trajectories of microbial biofilms (Henschel & Cook 1990; Stoodley et al. 2002; Jenkins &

Martins 2010). Succession driven by biological interactions is considered the primary

determinant of assemblage structure (Stoodley et al. 2002; Arndt el al. 2003; Qian et al. 2007).

As new substrates are immersed, they are gradually colonised by a number of species. These

early species may in turn facilitate or inhibit colonisation by later species (Sousa 1984; Arndt et

al. 2003; Jenkins & Martins 2010).

Currently the literature on biofilm-dwelling protozoa, while considerable in size, deals almost

exclusively with freshwater systems. Relatively little is known about community dynamics in

the marine environment. To understand the complex interactions of ciliates within microbial

biofilms, special attention must be paid to the taxonomic structure and successional dynamics of

the assemblages present. Although ubiquitous within biofilms, the density and species


Page | 25

assemblages of ciliate communities are known to vary seasonally (Gong et al. 2005; Xu et al.

2009). With their rapid growth and delicate external membranes, ciliate assemblages are

strongly influenced by environmental variation (Arndt el al. 2003; Gong et al. 2005).

Environmental factors including temperature, flow velocities, nutrient loadings and surface

composition have all been shown to significantly influence the structure of ciliate assemblages

(Franco et al. 1998; Gong et al. 2005; Norf et al. 2009; Risse-Buhl et al. 2009; Xu et al. 2009).

An understanding of the structure/function of ciliates in marine microbial biofilms is central to

interpret and predict the impacts of these assemblages on the surrounding marine ecosystem.

Here we report on the temporal succession of ciliate assemblages at genus level resolution in

Port Phillip Bay, Melbourne, Australia. The aims of this study were: (1) to document the

taxonomic structure and succession of ciliate assemblages in temperate Australian waters; (2) to

describe the successional patterns during two contrasting weather conditions, i.e. by studying

time-series in summer and in winter, including identifying the impact of north and south

orientations; and finally (3) a natural disturbance event, which occurred during the summer

deployment, provided a unique opportunity to investigate the response of established ciliate

assemblages to an environmental disturbance.

2.2Methods

2.2.1StudySite&Sampling

The study took place at the Defence Science and Technology Organisation (DSTO) Marine

Coatings exposure raft on Booth Pier, Williamstown, Victoria, Australia (37º51’41.40”S,

144º54’38.06”E) (Fig 2.1). The floating raft lies in Hobsons Bay, the northernmost part of

Port Phillip Bay which is a large inland bay covering 2000 km2 with a narrow opening into

Bass Straight. The site is approximately 6 m in depth with a tidal range of approximately 1

m. The assemblage analysis was conducted during June-July (Australian winter) 2012 and

again in February-March (Australian summer) 2013. The influence of aspect was


Page | 26

investigated by deploying the artificial substrates in different orientations (north/south

alignments), exposing the surfaces to different amounts of light.

Ciliate sampling was conducted using sealable plastic Petri-dish microscope slides

(AnalyslideTM, Pall Corp.) as artificial substrates for biofilm development. A total of 50 Petri-

dishes (25 on each aspect) were used to collect ciliates from a depth of 1 m below the water

surface. Each Petri-dish slide was secured vertically to a polyvinyl chloride (PVC) rack, which

was designed to attach to an aluminium frame suspended vertically beneath a raft (Fig. 2.2). A

total of 5 samplings were carried out over a 21 day period in each season. Assuming there

would be no significant differences between ciliate assemblages colonizing slides within the

same frame, five randomly selected replicate Petri-dish slides from each aspect were collected

on each sampling date. The slides were sealed with a cap under the water surface and

transferred into a cool box for transportation to the laboratory.

Figure 2.1 Map of study site Booth Pier, Hobsons Bay, Williamstown.


Page | 27

Temperature and luminosity (lux) were measured every 10 min throughout the deployment

period with data loggers (HOBO UA-002-64, Onset ltd.). Salinity, pH, turbidity and dissolved

oxygen were recorded in situ on each sampling date with a multi-parameter probe (Hydrolab

DS5X, Aqualab Scientific).

2.2.2Identification&Enumeration

Ciliates were initially observed live at 45 × magnification under a stereomicroscope (Leica

MZ9.5) to determine the classifications and abundances of sessile ciliate genera. Live

observations also gave insight into the behaviour, movement and ecological niches occupied by

vagile and planktonic ciliates in the samples. After live observation the samples were fixed in

2% glutaraldehyde in seawater solution within 1-2 h of sample collection. Identification and

enumeration were by quantitative protargol staining (QPS) techniques. The fixed samples were

first concentrated onto cellulose filters and embedded in agar, the filters then post-fixed in 10%

Figure 2.2 Panel design illustrating how each individual Petri‐dish slide was

held in place by rails mounted on the PVC panel which was then attached to

an aluminium frame and suspended vertically at 1 m underneath a raft.


Page | 28

Bouin’s solution prior to protargol impregnation. Staining followed the QPS method described

by Skibbe (1994).

Protargol impregnations were mounted in Permount and examined under a phase contrast

microscope (Leica DM2500) at 100–1250 × magnification to reveal details of kinetid patterns

and other morphological characteristics required for genus identification. Enumeration was

conducted at 200 × magnification, and 20 fields of view per slide were randomly selected for

counting. The ciliate abundances were determined based on the counts of all 5 replicate Petri-

dish slides collected at each sampling to confirm cell densities (cells/cm2). Specimens were also

photographed and catalogued for taxonomic classification using published keys by Carey

(1992), Lee et al. (2000) and Lynn (2008).

2.2.3DataAnalysis

Species diversity (H’) of samples was calculated as follows:

Where Pi = proportion of the total count arising from the ith species (Pielou 1966)

Abundance data were log10 (x + 1) transformed when required to reduce heterogeneity of

variances. A repeated-measures ANOVA (SYSTAT 13 Software, Inc.) was used to establish

whether there were significant differences in genera abundances/composition between time

points, aspect and season. Where one of these factors or an interaction was significant at α

=0.05, Tukey pairwise comparisons were run.

Multivariate analyses were performed with Primer 6.0 (Plymouth Marine Laboratory) software.

To prevent bias caused by highly abundant taxa, ciliate assemblage data were transformed prior

to analysis. Square-root transformations down-weighted the importance of the highly abundant

taxa, allowing rarer taxa to exert some influence in the diversity and similarity calculations.

Similarity percentage (SIMPER) analysis was performed to determine the percentage of


Page | 29

dissimilarity between ciliate genera within aspects/season and the percentage contribution of

each genus to the overall dissimilarity. Spatial and temporal patterns in community structure

were examined via hierarchal cluster analysis and non-metric multidimensional scaling (MDS)

ordinations also generated from this transformed data set.

2.3Results

2.3.1TaxonomicComposition

Ciliate abundance reached >350 cm-2 at its peak. The taxonomic composition of the ciliate

assemblages recorded during summer and winter deployments are summarized in Table 2.1. A

total of 16 genera representing eleven orders were recorded following examinations of 100

samples. Sessile ciliates were represented by species of the orders Sessilida and Heterotrichida.

Vagile forms belonged primarily to the orders Euplotida, Pleurostomatida and Stichotrichida.

Planktonic taxa were represented by the orders Philasterida, Pleuronematida and Strombidiida

(Table 2.1). The sessile ciliate Zoothamnium was the most abundant ciliate genus. The

combined sessile ciliates dominated the assemblage, accounting for 81.5% of the total ciliate

abundance at the point when the assemblage reached its peak abundance (Fig. 2.3). Vagile

ciliates had low abundance in comparison; however, they were the primary contributors to the

variation in genera diversity (Fig. 2.4). Euplotida and Pleurostomatida were the two orders

represented by the most genera, accounting for up to 25.8% and 13.3% of the total genera

present respectively (Fig. 2.4).


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Taxon Functional

Group

Summer Winter

Peak Abundance

(ciliates cm‐2)

Occurrence

(%)

Peak Abundance

(ciliates cm‐2)

Occurrence

(%)

Order: Sessilida

Zoothamnium S 224.62 ± 41.38 100 78.76 ± 13.96 100

Vorticella S 50.97 ± 10.13 100 4.36 ± 0.82 100

Order: Euplotida

Aspidisca V 6.92 ± 1.01 100 12.45 ± 1.50 100

Euplotes V 10.03 ± 0.65 100 5.11 ± 0.78 100

Uronychia V 0.36 ± 0.19 60 0.55 ± 0.19 40

Diophrys V 1.68 ± 0.21 60 0.00 ± 0.00 0

Order: Uronstylida

Holosticha V 0.66 ± 0.25 80 0.21 ± 0.13 60

Order: Stichotrichida

Sticotricha V 7.18 ± 0.67 100 3.05 ± 0.25 100

Order: Heterotrichida

Folliculina S 0.72 ± 0.19 100 0.00 ± 0.00 0

Order: Haptorida

Lacrymaria V 4.48 ± 0.55 100 1.02 ± 0.27 80

Order: Pleurostomatida

Amphileptus V 17.57 ± 1.06 100 17.25 ± 1.93 80

Litonotus V 1.68 ± 0.31 60 5.22 ± 0.55 80

Order: Dysteriida

Dysteria V 1.42 ± 0.33 20 0.74 ± 0.17 60

Order: Strombidiida

Strombidium P 1.02 ± 0.14 80 2.17 ± 0.24 100

Order: Philasterida

Uronema P 8.40 ± 0.77 100 3.31 ± 0.36 100

Order: Pleuronematida

Cyclidium P 5.80 ± 0.35 100 4.63 ± 0.64 100

Table 2.1 Ciliate genera (by order) recorded in a total of 100 samples collected during winter (Jun and Jul 2012)

and Summer (Feb and Mar 2013) seasons, including function group occupied (S: sessile, V: vagile, P:

planktonic), peak abundance (±SE) and occurrence.


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Figure 2.3 Proportions of abundance (A, C) and cumulative genera number (B, D) of ciliate functional groups during summer (A, B) and winter (C, D).


Page | 32

2.3.2VariationBetweenSummer&WinterDeployments

The ranges of environmental parameters recorded during summer and winter deployments are

summarised in Table 2.2. Among these variables, the mean values of temperature, salinity and

dissolved oxygen were significantly different between summer and winter deployments (Table

2.2). Correlations between the ciliate assemblages and environmental variables were weak as

the environmental parameters varied little during summer and winter samplings, and therefore

these data are not shown.

Figure 2.4 Proportions of abundance (A, C) and cumulative genera number (B, D) of ciliate orders during summer (A, B) and winter (C, D).


Page | 33

The ciliate assemblages varied significantly between summer and winter samplings.

Abundances ranged around mean values of 109.9 ± 64.9 and 39.2 ± 25.7 cm-2 in summer and

winter samplings respectively, with corresponding maximum abundances of 366.1 ± 50.1 and

138.7 ± 15.83 cm-2 (Fig. 2.5). The abundances were significantly different between summer and

winter samplings on days 3 (p < 0.001), 7 (p < 0.001), 10 (p < 0.001) and 21 (p = 0.001), which

had comparably higher abundances during summer (Fig. 2.5). A total of 16 genera were

identified during the summer deployment and 14 genera during the winter deployment (Table

2.1). The cumulative genera represented during both seasons showed little variation, with the

exception of Heterotrichida which was only identified in summer samples.

Table 2.2 Chronological variation of environmental parameters recorded at the study site in Port Phillip Bay

during summer (Feb‐March 2013) and winter (June‐July 2012) deployments (P values from t‐tests are

comparisons of summer and winter means; significant difference at α =0.05).

Paired sample t‐test: significant difference at the 0.05 level.


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The similarity percentage (SIMPER) analysis presented in Table 2.3 breaks down the

contribution of each ciliate genera to the observed dissimilarities between summer and winter

samplings. The sessile ciliate Zoothamnium was the primary contributor to the dissimilarity in

abundance/occurrence, due to the much higher abundances reached during the summer

deployment despite the fact they were ubiquitous during both seasons. The five most dominant

genera accounted for 62.7% of the total dissimilarity (Table 2.3). The vagile ciliates

Amphileptus, Aspidisca and Euplotes also represented high contributions to the dissimilarity due

to their high frequency of occurrence/abundance.

Figure 2.5 Summer and winter assemblage comparison of total abundance and genera diversity (H’) (Mean ± S.E).


Page | 35

Cluster analysis based on Bray-Curtis similarities of square-root transformed abundances

resulted in summer genera falling into three groups (I–III) at a 60% similarity level and winter

genera into four groups (I-IV) at 70% similarity (Fig. 2.6). Summer deployment groups I and II

represented by genera with high abundance and/or occurrence, whereas group III represented

assemblages of genera with low occurrence (Fig. 2.6). Similarly in the winter deployment

groupings, highly abundant genera are present in groups II and III, while the remaining groups

represented those rarer genera (Fig. 2.6).

Genus Av. Diss. Contrib. (%) Cum. %

Zoothamnium 10.80 24.44 24.44

Amphileptus 6.27 14.18 38.63

Aspidisca 4.55 10.29 48.92

Euplotes 3.05 6.91 55.84

Litonotus 3.05 6.91 62.74

Vorticella 2.85 6.46 69.21

Cyclidium 2.65 5.99 75.20

Uronema 2.38 5.39 80.59

Stichotricha 2.65 5.16 85.75

Figure 2.6 Dendrograms of ciliate assemblages using group averages based on Bray‐Curtis similarity matrix of square root‐transformed genera abundances from (A) summer and (B) winter samplings. Roman numeral indicate groups I to IV.

Table 2.3 SIMPER analysis displaying the contribution of the

top 9 genera to the average Bray‐Curtis dissimilarity in ciliate

assemblages between seasons.


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Figure 2.7 Chronological variation of (A, C) relative abundance and (B, D) genera number of ciliate functional groups recorded during summer (left column) and winter (right column) deployments.

2.3.3ChronologicalAssemblageSuccession

A clear pattern of temporal succession within ciliate assemblage structures in terms of both

genera number and abundance was observed during both seasons (Figs. 2.7 & 2.8). Sessile

ciliates dominated the ciliate assemblages in terms of abundance, particularly during summer,

where sessile ciliates accounted for up to 93.0% of the total assemblage abundance early in the

deployment (Fig. 2.7). Sessile and planktonic forms colonised the substrates early with few

additional recruits observed beyond seven days (Fig. 2.7). Vagile taxa conversely were

colonising the substrate throughout the deployment period. Ciliate genera from the orders

Euplotida and Pleurostomatida in particular exhibited high variability in occurrence and

abundance (Fig. 2.8).


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Multidimensional scaling (MDS) ordinations of the 5 samples taken during each season were

plotted from Bray–Curtis similarities on square root transformed genera abundances (Fig.

2.9). The analyses indicated that throughout the winter deployment ciliate assemblages

showed an uninterrupted trend of growth, with each day showing increased dissimilarity to

the next as the assemblage developed. The ciliate assemblages during the summer

deployment also initially followed this trend on days 3, 7 and 10. Colonisation however

occurred at a faster rate during summer with assemblages developing up to 7-10 days earlier

Figure 2.8 Chronological variation of (A, C) relative abundance and (B, D) genera number of ciliate assemblages at the level of order recorded during summer (left column) and winter (right column) deployments.


Page | 38

than the equivalent winter assemblage, with day 3 assemblages sharing high similarity to the

winter assemblages after 10 days of development (Fig. 2.9).

2.3.4Aspect

During the summer deployment the north aspect (187.3 ± 7.2 klx/day) was exposed to

significantly more light per day than the south facing aspect (96.2 ± 4.1 klx/day) (p < 0.001).

The different aspects were also exposed to significantly more average light per day during

summer (141.7 ± 15.5 klx/day) than winter deployments (61.6 ± 4.6 klx/day) (p < 0.001). The

ciliate assemblages between north and south aspects, however, were at no point significantly

different during either summer or winter deployments (p > 0.05). While no significant

difference was found in the assemblages between the north and south aspects, new ciliate

recruits tended to colonise the more exposed north aspect first. Figure 2.10 shows the temporal

variation of functional groups between aspects. Although the ciliate assemblages on both

aspects followed a similar trend of increasing abundance over time, the abundances on the south

aspect were frequently lower than on the north aspect (Fig. 2.10). It should be noted that

following the weather disturbance (see below) this trend changed, with higher abundances

recorded on the south aspect (Fig. 2.10).

Figure 2.9 Multidimensional scaling (MDS) ordination of the 5 samples taken during summer and winter deployments based on Bray‐Curtis similarity matrix of square root transformed genera abundances.


Page | 39

2.3.5ResponsetoDisturbance

A clear disturbance to succession occurred during the summer deployment in the samplings

following the tenth day (Fig. 2.5). Between sampling days 10 and 14, 38.4mm of rain fell on

site (Bureau of Meteorology 2013). The runoff and subsequent discharge from the nearby Yarra

River (Fig. 2.1) generated by this event resulted in a sudden increase in turbidity from 10.3 to

15.7 NTU (Table 2.2). An increase in the volume of debris adhering to the Petri dish slides

sampled on day 14 was clearly observed following this event (Fig. 2.11). This disturbance also

had a considerable impact on the abundance and/or occurrence of many established genera.

Figure 2.10 Temporal aspect comparisons of sessile (A, D), vagile (B, E) and planktonic (C, F) ciliates recorded during summer (A‐C) and winter (D‐F) (Mean ± SE).


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Subsequently, variability of the ciliate communities was distinctly higher during summer (Fig.

2.5).

Between sampling days 10 and 14, total abundance dropped from 366.1 ± 50.1 to 55.2 ± 12.2

ciliates cm-2 (Fig. 2.5). Figure 2.8A highlights the changes in the relative abundances of the

already established ciliate orders following the disturbance. The relative abundance of Sessilida

in particular was heavily reduced in response to the disturbance. In contrast, vagile and

planktonic ciliates from the orders Euplotida, Pleurostomatida and Philasterida were largely

unaffected, and subsequently increased in relative abundance (Fig. 2.8A). Despite the

considerable reduction in abundance the diversity of the assemblage increased following the

disturbance (Fig. 2.5).

MDS ordinations of the assemblages plotted from Bray–Curtis similarities on square-root

transformed genera abundances, show that post disturbance the ciliate assemblages reverted

back to a more similar composition as that recorded on day 7 (Fig. 2.9). Ordinations comparing

aspects also highlight the impact of the disturbance. The analysis shows that the similarities of

ciliate assemblages between aspects remained high throughout the winter and summer

deployments, with the exception of the south aspect following the disturbance during the

summer deployment (Fig. 2.12 D14/South).

Figure 2.11 Images of Petri‐dish slides in sequence displaying the sudden influx of debris adhering to the surface of the slides between days 10 and 14 following the disturbance event.


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2.4Discussion

The colonisation sequences observed here can be understood in terms of ecological niches of

the ciliates. The peritrichs Zoothamnium and Vorticella were among the first ciliates to colonise

the substrates and rapidly increased in abundance over the deployment period. The hypotrichs

Aspidisca and Euplotes, specifically adapted for life on substrates, were also ubiquitous in all

samples. Small planktonic ciliates Uronema and Strombidium, likely swimming in the

surroundings of the established biofilm exploiting transitory patches of bacteria (Fenchel 1980),

were more unpredictable in their colonisation dynamics but were generally present throughout

Figure 2.12Mulitdimensional scaling (MDS) ordination of the 10 aspect samples based on Bray‐Curtis similarity matrix, computed from square root transformed ciliate genera abundances recorded during (A) winter and (B) summer.

A

B


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the deployments. The remaining colonising genera were established in the assemblages for short

periods and thus may be considered transient or opportunistic in nature.

When comparing the taxonomic composition observed here with previous works on ciliates

colonising artificial substrates in marine environments, our results do not reveal large

differences in abundance, diversity or structure of the ciliate assemblages. Despite these

works being based on examinations over extended periods of time with substrates located at

various depths and sites, many genera were common to all observed assemblages (e.g.

Zoothamnium, Euplotes, Aspidisca, Amphileptus, Holosticha, and Litonotus) (Coppellotti &

Matarazzo 2000; Gong et al. 2005; Xu et al. 2009). This suggests that habitats providing

similar niches will hold comparable ciliate assemblages across a wide range of climates. This

seems particularly true for the dominance of sessile peritrich and vagile hypotrich ciliates

within marine biofilm habitats.

Previous works have shown that ciliate assemblages will eventually reach a state of

equilibrium, where few new species will colonise the substrate and abundances of

established ciliate groups remain more or less static (Coppellotti & Matarazzo 2000; Gong et

al. 2005). The time required for ciliate colonization to reach equilibrium is heavily

dependent on environmental factors, but typically takes between 1 and 4 weeks (Gong et al.

2005; Xu et al. 2009). In the present study, although the Petri-dish slides were exposed for

21 days, the ciliate assemblages did not reach this state of equilibrium. This may have been

due to low water temperatures during winter, and a significant environmental disturbance

during summer likely delaying the onset of equilibrium.

While equilibrium was not reached, the ciliate assemblages showed clear temporal succession

throughout colonisation during summer and winter deployments. Colonisation rates were

affected by differences between seasons with the development of ciliate assemblages being

much quicker during the summer sampling. While the cumulative genera represented during


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both seasons showed little variation, the abundances of individual genera varied significantly.

This was particularly evident with sessile ciliates Zoothamnium and Vorticella during the

summer sampling, which dominated the assemblage in terms of abundance, accounting for up to

93% of the relative abundance early in substrate colonisation (Fig. 2.6A). During the winter

sampling, when the lowest ciliate concentrations were recorded, the contribution of the different

ciliate genera was more evenly distributed. Consequently, the diversity of ciliate genera was

higher during winter despite there being fewer genera present overall (Fig. 2.4). It must be noted

that while our brief deployments revealed successional patterns with a fine scale of temporal

resolution, caution must be taken at the broader scale in attributing differences between the

summer and winter deployment solely to seasonality, as other vagaries of environmental factors

and successional stochasticity could have influenced results.

There is considerable evidence that environmental disturbance is a major source of temporal and

spatial heterogeneity in the structure and dynamics of natural biofilm assemblages (Underwood

1998; Sanz-Lazaro et al. 2011; Jones et al. 2013). Typically irregular events, the impacts of

disturbances can vary from negligible to extreme depending on the intensity of the disturbance

and the vulnerability of the different taxa within the assemblage (Sousa 1984; Turner et al.

1998; Jenkins & Martins 2010). The environmental disturbance experienced during the summer

deployment in this study distinctly influenced the colonization process, and the

structural/functional parameters of the already established ciliate assemblages.

Heavy rainfall and the subsequent increase of turbidity, the result of run off into the nearby

Yarra River, had a substantial impact on the abundance of many established ciliate genera.

Multivariate analysis highlighted the variations in assemblage structure over time, and revealed

how the environmental disturbance impacted the assemblage during summer. While both

summer and winter assemblages showed a clear trend of growth over time, following the

disturbance during summer (days 14-21) the ciliate assemblages reverted back to a more similar

composition as that recorded on day 7 (Fig. 2.8).


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The abundance of the sessile peritrichs in particular was heavily reduced in the samplings

following this event, whereas many of the vagile and planktonic species were more tolerant, and

after an initial decline in abundance showed sustained growth despite the influx of debris. This

is likely due to the sessile taxa being physically beaten or smothered by the sudden influx of

debris, whereas the motile vagile and planktonic taxa had the advantage of being able to avoid

the debris and adjust to the new substrate condition. Ciliate species from the orders Euplotida

and Pleurostomatida in particular seemed to benefit, taking advantage of the new substrate

condition and exerting a greater presence within the assemblage following the disturbance (Fig.

2.7).

Interestingly, while the abundances of many established ciliate genera were substantially

reduced, the diversity of the assemblage increased following the disturbance. Similar

patterns have been observed in freshwater ciliate assemblage studies (e.g. Cairns et al. 1971;

Eddison & Ollason 1978). In stable systems free from natural disturbances the relative

abundance of a small number of genera rise to dominate the ciliate assemblages. Whereas in

systems subject to frequent disturbances the relative abundances of the genera present are

comparatively lower than those in stable systems, and the domination of assemblages by a

small number of ciliate genera does not occur to the same degree, resulting in higher

assemblage diversity (Cairns et al. 1971; Eddison & Ollason 1978; Taylor 1983). The

changes in ciliate diversity observed here could be compared with the intermediate

disturbance hypothesis, as proposed by Connell (1978), which submits that species diversity

is maximised by moderately frequent environmental disturbances which keep the assemblage

in a non-equilibrium non-climactic state.

Therefore, while certainly dependent on the severity of the disturbance, it seems that the

diversity of an established assemblage can be maintained or even increase post disturbance

despite the abundances being subject to great fluctuation. This result also highlights that


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disturbances do not necessarily have a uniform effect on all ciliate taxa. In this case the

disturbance selectively reduced the abundance of the dominant sessile species, which were out-

competing the vagile and planktonic species through the course of succession. Various forms of

natural disturbance may result in a proportionally greater loss of certain taxa (Turner et al.

1998; Sousa 2001). Here we observed that an increase in debris selectively impacted the sessile

species. However, had the substrate been exposed to a hydrodynamic disturbance, such as wave

action or current flow, the vagile and planktonic species not attached to the surface may have

been at greater risk of removal from the assemblage.

Had the deployment period of this study extended further it is likely that new and/or the

remaining peritrichs would simply colonise on top of this new layer of debris. Indeed, the

subsequent assemblages of peritrichs might even benefit from the increase in ambient debris for

the increase in suspended bacteria present in the water column on which they feed (Arndt et al.

2003). It has been suggested that sessile filter feeders contribute to a tight coupling between the

water column and biofilm by channelling the organic carbon from the water column

(Augspurger et al. 2008). Hence, the recovery of the peritrichs may be important for the

eventual recuperation of the microbial community as a whole.

The recruitment and distribution of sessile marine invertebrates cannot be understood without

attempting to understand the ecology of the microbes which condition the immersed substrates

for subsequent recruitment. Studies that attempt to understand the influence of biofilms on the

recruitment and distribution of sessile marine invertebrates should take into account that ciliate

assemblages can vary in complex ways on relatively short time scales. How individual ciliate

species influence succession in the surrounding microbial community, and the degree to which

these influences interact, could all contribute to the eventual recruitment of sessile invertebrates.

Further study of protozoan assemblages across multiple temporal and spatial scales will build a

better understanding of these mechanisms within marine fouling assemblages.

3. Optimising Galeolaria caespitosa settlement assays

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Chapter3 Optimisingsettlementassaysfortheserpulidpolychaete

Galeolariacaespitosa

3.1Introduction

As settlement marks the transition from the planktonic to the sessile phase for many marine

invertebrates, it has become a focus of intense research (reviewed by Wieczorek & Todd 1998;

Hadfield 2011). Knowledge of such behaviour is not only important to understanding

recruitment dynamics, but also to the development of methods to interfere with or, in some

cases enhance this process, as applied in antifouling and aquaculture respectively (Qian et al.

2007). The settlement dynamics of marine invertebrates are a prime target for such management

strategies because it is both a key and vulnerable step in the colonisation process. Subsequently

there is a need for a better understanding of the phenomena which govern the colonisation of

surfaces by marine invertebrates (Wieczorek & Todd 1998; Qian et al. 2010).

Crucial to the development of new antifouling and aquaculture technologies is the establishment

of reliable, reproducible laboratory assays for the study of invertebrate settlement (Elbourne et

al. 2008). Laboratory based bioassays are commonly utilised to quantify the effects of

treatments and conditions on larval settlement. Among the many established assay methods

currently employed, static laboratory assays remain the primary research tool for studying

invertebrate settlement (Elbourne et al. 2008; Carl et al. 2011). Static assays are often

conducted in Petri dishes, and involve the enumeration of settlement after a period of incubation

under various manipulated conditions. Settlement assay protocols for various serpulid

polychaetes, particularly Hydroides elegans, are well established (Pawlik & Chia 1991; Bryan


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et al. 1997; Toonen & Pawlik 2001). However, to date there have been no established protocols

for standardised settlement assays of Galeolaria caespitosa.

Galeolaria caespitosa is a sessile, filter-feeding polychaete worm that inhabits mid to low

intertidal regions of exposed rocky shores of South-eastern Australia (Marsden & Anderson

1981). Where G. caespitosa occur in great densities, worms grow perpendicular to the

substratum and intertwine to form calcareous mats that can be greater than 4 cm thick

(Minchinton 1997) (Fig. 3.1). In Australia spawning is reported to occur primarily from late

September to early December, peaking during October and November (Marsden & Anderson

1981). However, mature adults of G. caespitosa are fertile throughout the year (Marsden &

Anderson 1981; personal observation, 2010-2014). Being fertile throughout the year is one of

the many factors that make G. caespitosa an ideal test species. Their gregarious settlement

behaviour in the intertidal zone also enables easy collection of large numbers of specimens. This

is particularly important in colder climates, where the rate of fouling on collection panels is

greatly diminished during the winter months impacting the reliable collection of alternative

fouling invertebrates for study. Furthermore, it is important to diversify the species utilised in

settlement assays to determine the true variability of settlement behaviour, and avoiding

generalisations of the settlement dynamics of serpulid polychaetes based on just a few species.


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Previous attempts to induce Galeolaria caespitosa larvae to settle under laboratory conditions

had been largely unsuccessful. Marsden & Anderson (1981) observed that in glass bowls a

small proportion of larvae would undergo the first steps in metamorphosis, including the

development of the neck, tentacle buds and the evagination of the collar (Fig. 3.2). However,

under this condition no larvae were observed to successfully complete metamorphosis and form

a tube (Marsden & Anderson 1981). Marsden & Anderson (1981) also attempted to induce

Figure 3.1 Natural aggregation of Galeolaria caespitosa on a rock in the intertidal

zone Williamstown, Port Phillip Bay, Australia (Photo M. Watson).


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settlement by exposing larvae to pieces of sandstone taken from the rocky shore on which the

adults were originally collected. Larvae were observed swarming around and over this

substratum, making temporary attachments and crawling into crevices on the surface but again

did not settle (Marsden & Anderson 1981). Permanent settlement was only achieved when

living pieces of G. caespitosa colony were added to the culture bowls (Marsden & Anderson

1981). Field observations support this finding, with settlement of G. caespitosa larvae almost

exclusively occurring on or nearby the tubes of adults of the same species (Minchinton 1997).

This indicates that contact made between larvae and resident conspecific adults stimulates their

settlement, and the larvae either attach within the mat of conspecific individuals or crawl to

nearby free space on the same substrate (Minchinton 1997).

Settlement in response to conspecific cues is a common phenomenon in marine invertebrate

larvae (Prendergast 2009). Numerous laboratory based studies of marine invertebrates have

demonstrated settlement induced by conspecific adults. These include common fouling

invertebrates such as molluscs (Slattery 1992), barnacles (Knight-Jones & Crisp 1953;

Raimondi 1991; Head et al. 2004) and polychaetes (Knight-Jones 1951; Wilson 1954; Pawlik

1986; Minchinton 1997). Despite the gregarious distribution of G. caespitosa in the field

(Marsden & Anderson 1981; Minchinton 1997), the conspecific effects on their larvae in

laboratory assays are unknown. To improve comparability of studies quantifying settlement, it

is important to establish conditions that optimise settlement. Inducing the settlement of

invertebrates that display conspecific attraction may involve ‘seeding’ the assay dishes with

appropriate conspecific cues. The presence of these conspecific cues then provides a stimulus

for the settlement of larvae which may not otherwise settle in typical assay conditions.

Another important consideration in optimising laboratory assays is the ability to consistently

identify larval competence. Determining the point at which a larva is competent to settle is often

based on morphological characteristics associated with different stages of larval development.

In Galeolaria caespitosa, larval competence is heralded by the forward growth of the


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mesodermal bands, formation of the second eye, development of sensory cilia and a profound

change in swimming behaviour (Marsden & Anderson 1981) (Fig. 3.2). At this point the larvae

have grown steadily over 9-12 days (at 25oC) and are now 250-300 µm long (personal

observation 2010-2014). Although larvae at this stage of development spend most of their time

near the bottom of the culture dish, they are still capable of effective swimming. Typically,

competent G. caespitosa larvae swim slowly close to the bottom of the culture dish, testing the

substrate with their apical tuft before accelerating in brief upward motions (personal

observation 2010-2014). Competent larvae will often make temporary attachments to the

culture dish or to a piece of detritus, and swimming individuals can often be seen trailing a

string of mucus (Marsden & Anderson 1981; personal observation 2010-2014).

Pharmacological approaches are also a valuable tool in determining the competence of larvae

(Pechenik & Qian 1998; Qian 1999). 3-isobutyl-1-methylxanthine (IBMX) is perhaps the most

commonly utilised compound in artificially inducing the settlement of invertebrate larvae.

IBMX is a phosphodiesterase inhibitor which causes an increase in cAMP levels (Qian 1999).

The exact mechanisms of this compound on inducing larval settlement remain unclear.

However, it has been suggested that the neurotransmitters enhance larval settlement by acting

Figure 3.2 Competent Galeolaria caespitosa larva. 9‐12 days old displaying a delineated head,

forward growth of the mesodermal bands, and second eye spot (Photo M. Watson; Sketch

adapted from Marsden & Anderson 1981).


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directly on the nervous systems of larvae, bypassing the chemoreceptors involved in natural

settlement processes (Qian & Pechenik 1998). Exploiting this mechanism, the competency of

larvae can be determined even in the absence of appropriate cues required for larvae to settle

naturally. This allows for the competency of larval cultures to be easily quantified in short 12 -

24 hour assays (Qian & Pechenik 1998).

This chapter reports on some of the trials conducted in establishing a protocol, which optimised

the settlement of Galeolaria caespitosa in laboratory assays. The aims of this study were: (1) to

determine the optimal concentration of IBMX for artificially inducing the settlement of

competent G. caespitosa larvae; (2) to investigate the inducing potential of bacterial biofilms on

the settlement of G. caespitosa; and (3) to examine the settlement response of G. caespitosa

larvae in the presence of various conspecific cues.

3.2Methods

3.2.1LarvalCulture

Adults of the serpulid polychaete Galeolaria caespitosa were collected from the intertidal zone

at Williamstown, Port Phillip Bay, Australia. Pieces of mature colony were chipped from the

rock with hammer and chisel. The animals were then transported back to the laboratory and held

in marine aquaria for no more than two weeks. The procedure for obtaining gametes and raising

the larvae followed that described in Marsden & Anderson (1981). Mature adults were isolated

into Petri dishes containing filtered seawater (FSW), and stimulated to release gametes by

gently removing them from their tubes (Fig. 3.3D). Oocytes collected from four to five

individual females were fertilized with a dilute sperm solution taken from two to three males

(Fig. 3.3C, E). After approximately 10 minutes, excess sperm was removed by passing fertilized

egg suspension through a 25 µm sieve. Fertilised eggs were then transferred into bowls

containing 450 mL of FSW. Cultures were maintained at 25oC on a 15:9 light-dark cycle

throughout larval development. Once hatched (approximately 20-24 hours) trochophore larvae


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were transferred to fresh FSW and fed the microalgae Isochrysis galbana (Australian National

Algae Culture Collection CS-177, cultured on F/2 medium) at 1 x 104 cells mL-1 on a daily basis

until competent (Fig. 3.3F, G, H).

Figure 3.3 Spawning and rearing of Galeolaria caespitosa. (A) G. caespitosa intact colony; (B) breaking

adult worm from tube; (C) male specimen releasing sperm; (D) female specimen releasing oocytes; (E)

fertilised oocytes; (F) trochophore larva (24 hours); (G) competent larva (9‐12 days); (H) recently

settled larva undergoing metamorphosis (9‐15 days) (Photos M. Watson).


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3.2.2BacterialCulture

Bacteria were cultured from natural biofilms that were grown in the intertidal zone at

Williamstown, Port Phillip Bay, Australia. A mixed assemblage of bacteria was isolated from

the rocks by scrapping off the biofilm, and placing samples into 15 mL capped test tubes for

immediate processing. In the lab samples were dispersed by sonicating for 5 s (Branson

Ultrasonics 200 W at 27% intensity), and passing the suspension through a 0.8 µm pore size

filter to remove debris and larger organisms. These bacteria were cultured in 0.2 µm filtered and

autoclaved sea water (FSW) with 0.005% yeast extract.

3.2.3SettlementAssays

Settlement assays were run in 55 mm diameter polystyrene Petri dishes. There were 6 replicates

of each treatment all of which included a monolayer bacterial biofilm, which was grown in each

dish prior to the commencement of the settlement assay. Petri dishes were placed into a large

tub containing 4 L of sterilised FSW ensuring that they were in a monolayer, the FSW baths

were aerated and a growth medium consisting of 0.001% yeast extract and 0.001% bacto-

peptone added to the bath. The bath was inoculated with a 5 mL suspension of the mixed

bacterial assemblage isolated from the field. The bacteria were allowed to incubate for 24 hours

at 25oC. After the 24-hour growth period the Petri dishes were gently rinsed with sterile FSW to

remove any unattached cells, Petri dishes were subsequently re-filled with 8 mL of sterilised

FSW.

Competent larvae (12-14 days post-fertilisation) were gently filtered through a 120 µm sieve to

remove algae and excess debris. Larvae retained in the sieves were then immediately transferred

into FSW ready to be inoculated into assay dishes using a pipette. Twenty competent larvae

were randomly added to each replicate dish. Throughout the 48 hour duration of the experiment

larvae were not fed and the testing solution was not changed. Bioassay dishes were kept in a

controlled environment at 25oC on a 15:9 light-dark cycle. Once the experiment had


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commenced, bioassay dishes were examined under dissection microscope every 24 hours.

During examinations larvae were classified as settled or unattached. The Galeolaria caespitosa

larvae were considered settled only once they had completed final metamorphosis, i.e. attached

to the bioassay dish, produced a tube, and grown tentacles.

3.2.4Settlement‐InducingBacteria

Typical static laboratory settlement assays rely upon bacterial biofilms as a natural settlement

inducer. Several species of bacteria that induce settlement have been identified, and many

settlement assay protocols utilise mono-species biofilms to naturally induce settlement (Bryan et

al. 1997; Hadfield et al. 2014). Pseudoalteromonas luteoviolacea is a bacterium shown to

strongly induce settlement in larvae of the serpulid polychaete Hydroides elegans in single-

species biofilms (Lau & Qian 1997; Hadfield et al. 2014). This trial was conducted to determine

whether the inducing properties of P. luteoviolacea would influence the settlement of

Galeolaria caespitosa. P. luteoviolacea (ATCC® culture #33492) mono-species biofilms were

grown in assay dishes prior to the assay as described above for the mixed assemblage biofilm.

The control treatment contained the mixed bacterial assemblage isolated from the field where

natural aggregations of G. caespitosa occur.

3.2.5ConspecificInducers

3.2.5.1AdultWormHomogenate

Adult worm homogenates were prepared by gently removing mature worms from their tubes

and gently rinsing them in FSW. Homogenates were prepared by crushing and sonicating

(Branson Ultrasonics 200 W at 27% intensity) the mature worms in Milli-Q water at a ratio of

0.2 mg worm mL-1, a concentration identified by Bryan et al. (1997) to induce the highest

percentage of metamorphosis of another serpulid polychaete, Hydroides elegans. The

homogenate was finally filtered through a Whatman grade 1 filter and stored at -20oC until use.


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Control dishes contained only the pre-prepared monolayer biofilm. Homogenate treatments

contained the monolayer biofilm and the clarified homogenate inoculated at 0.2 mg mL-1 FSW.

3.2.5.2EmptyTubes

Adult worms were gently removed from their tubes with the aim of keeping the tubes as intact

as possible. Empty tubes were gently rinsed in Milli-Q water and sterilised in an autoclave at

121oC for 20 minutes. Control dishes contained only the pre-prepared monolayer biofilm.

Empty tube treatments contained the monolayer biofilm and 0.5 g of sterilised empty tubes

scattered over the bottom of the dish.

3.2.5.3ConspecificLiveWorms

Marsden & Anderson (1981) induced the settlement of Galeolaria caespitosa in laboratory

conditions by introducing living pieces of G. casespitosa colony into their assay dishes.

However, repeating this method without inadvertently introducing the micro-fauna which also

inhabit the colonies (e.g. protozoa, microalgae algae etc.) would be very difficult. Here, in order

to avoid such contaminations, larvae from cultures were induced to settle in the assay dishes.

Following preparation of the mono-layer bacterial biofilm, approximately 15 competent larvae

were added to each dish in a 1 mM IBMX solution (as identified in IBMX trial) to induce

settlement. Once at least 10 larvae had successfully settled and completed metamorphosis in

each replicate dish (approx. 24 hours), excess settled and unattached worms were removed.

Dishes were then once again gently rinsed with sterile FSW to remove all trace of the IBMX

solution. Each replicate dish then contained the mono-layer bacteria biofilm and exactly 10 live

settled G. caespitosa which provided a natural conspecific cue for settlement. Control dishes

contained only the pre-prepared monolayer biofilm.


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3.2.6PharmacologicalInducerIBMX

IBMX was dissolved in sterilised FSW to give a stock solution of 0.1 M. Three test

concentrations (0.01 mM, 0.1 mM, 1 mM) were made immediately prior to the commencement

of the experiments by dilution of the stock with FSW, based on optimal concentrations

previously identified for Hydroides elegans (Qian & Pechenik 1998). Assays were carried out in

clean (no bacterial biofilm) 55 mm diameter polystyrene Petri dishes filled with 10 mL of each

test concentration. The control replicates only contained sterilised FSW.

3.2.7DataAnalysis

Settlement rates, converted to arc-sine square-root transformed proportions, were tested at each

time point by an a-priori test of main effects using the MSE from a repeated-measures ANOVA.

If the effect of the treatment was significant at α = 0.05, Tukey pairwise comparisons were run

among the treatments across time point using the MSE from the test of main effects. All

statistical tests were run on Systat v13.

3.3Results

3.3.1IBMXTrial

At 24 hours, settlement was significantly enhanced in the 1 mM treatment, at 81.6 % settlement,

compared with all other treatments (p < 0.01, Tukey’s test) (Fig. 3.4). Settlement in the 0.1 mM

and 0.01 mM treatments was not significantly different (p = 0.13), reaching 27.8 and 8.3 %

settlement respectivley (Fig. 3.4). No larvae had settled in the clean dishes. At 48 hours there

was little change in successful settlement and metamorphosis of the remaining larvae. The 1

mM treatment remained the only concentration to induce high levels of normal metamorphosis,

reaching 86.6 % settlement (Fig. 3.4) and remaining significantly enhanced compared to all

other treatments (p < 0.01, Tukey’s test). No larvae had settled in the clean dish treatment after

48 hours.


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3.3.2BacterialInducers

The presence of a mixed natural assemblage or mono-species biofilm alone failed to

consistently induce successful settlement and metamorphosis of Galeolaria caespitosa. After 24

hours settlement had only reached 2.5 ± 1.1 % and 3.3 ± 1.6 % in the mixed assemblage and

Pseudoalteromonas luteoviolacea treatments respectively (Fig. 3.5). Settlement had increased

after 48 hours, albeit almost exclusively in those dishes in which larvae had previously settled at

24 hours. Upon the conclusion of the assay, the proportion of settled larvae reached 5.8 ± 2.3 %

in the presence of the mixed assemblage, and 7.5 ± 2.8 % in the presence of a P. luteoviolacea

biofilm (Fig. 3.5). There was no significant difference between treatments at 24 (p = 0.84) or 48

(p = 0.78) hours. During the assay it was also observed that both biofilm treatments had induced

low levels of abnormal metamorphosis, where larvae had obviously begun but ultimately failed

to successfully complete metamorphosis.

Figure 3.4 Galeolaria caespitosa settlement rates in various concentrations of IBMX

(Mean ±SE). Means with different subscripts indicate significant difference at α = 0.05.


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3.3.3ConspecificInducers

The empty tubes and homogenate of adult worms had no significant impact on settlement

compared with the control, and both treatments showed high variance in successful settlement.

The presence of empty tubes induced 4.1 ± 2.0 % and 5.0 ± 1.8 % larvae to settle at 24 and 48

hours respectively (Fig. 3.6A). The settlement rates in the presence of empty tubes were not

significantly different to the controls at 24 (p = 0.56) or 48 (p = 0.30) hours (Fig. 3.6A). The

homogenate made from crushed adult worms induced similar proportions of larvae to settle. At

24 hours settlement reached 2.5 ± 1.7 %, while after 48 hours settlement had increased to 4.2 ±

2.4 % (Fig. 3.6B). The homogenate did not significantly induce more larvae to settle in

comparison with the control at 24 (p = 0.46) or 48 (p = 0.83) hours (Fig. 3.6B).

The presence of live conspecific worms was clearly the most successful method for naturally

inducing the settlement of G. caespitosa (Fig. 3.6D). After 24 hours settlement reached 32.0 ±

Figure 3.5 Galeolaria caespitosa settlement rates in the presence of a mixed bacterial

assemblage and a mono species (P. luteoviolacea) (Mean ±SE). Means with different

subscripts indicate significant difference at α = 0.05.


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3.3 % (Fig 3.6C). The biofilm alone induced a significantly smaller proportion of larvae to settle

in comparison (3.3 ± 0.6 %; p = < 0.001) (Fig 3.6C). At 48 hours the proportion of larvae to

have successfully settled increased to 44.3 ± 4.1 % and remained significantly higher than the

control (p = <0.001) (Fig 3.6C). On average the presence of live conspecific worms increased

settlement by 86.8 % compared to the presence of the mixed bacterial biofilm assemblage alone.

Figure 3.6 Galeolaria caespitosa settlement rates in the presence of a mixed bacterial biofilm and:

(A) empty tubes of adult worms; (B) adult worm homogenate; and (C) live pre‐settled conspecific

worms (Mean ± SE). Means with different subscripts indicate significant difference at α = 0.05. (D)

Example of a pre‐settled specimen of G. caespitosa with competent larvae exploring the

surrounding substrate (Photo M. Watson).


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3.4Discussion

The results of this study demonstrated that a conspecific cue is required for reliable and

reproducible laboratory settlement assays of Galeolaria caespitosa. The potent triggering of

metamorphosis in the presence of conspecific individuals, as observed here, is convincing

evidence of the importance of settlement inducing signals associated with living conspecific

individuals rather than the calcareous tubes they secrete. The gregarious behaviour displayed by

G. caespitosa and many other serpulid polychaetes has obvious benefits (Minchinton 1997;

Qian 1999). Larvae that settle within established aggregations benefit from choosing a habitat

likely to support post larval growth, and will ultimately share in the reproductive benefits of

being in close proximity with individuals of the same species (Pawlik 1992; Qian 1999).

Studies of gregarious settlement of invertebrate larvae have repeatedly implicated chemical cues

associated with adult conspecifics as being responsible for larval settlement responses

(Raimondi 1991; Toonen & Pawlik 1996; Head et al. 2003). However, in this study the

homogenate of crushed adult worms, presumably containing such compounds, did not induce a

significant settlement response compared with a bacterial biofilm alone (Fig. 3.6B). This could

suggest that the cue responsible for inducing the settlement of G. caespitosa was not a water-

soluble compound released by the conspecific individuals. Perhaps the larvae were instead

responding to touching the settled juveniles, rather than detecting a soluble cue. It must also be

noted that only one concentration of homogenate was trialled in this study, based on the optimal

concentration identified for the serpulid polychaete Hydroides elegans (Bryan et al. 1997). It is

possible that a higher concentration was required to initiate settlement induction in G.

caespitosa. Nonetheless, the presence of live settled juvenile worms was sufficient to induce

44.3% settlement of G. caespitosa after 48 hours (Fig. 3.6C).

Conversely, settlement on bacterial biofilms alone was minimal and highly inconsistent. Low

levels of abnormal metamorphosis, where larvae would begin but ultimately fail to complete


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metamorphosis, were frequently observed in treatments that did not contain living conspecific

individuals. The successful settlement recorded in the bacterial biofilm treatments was often

confined to a small number of replicate dishes. This suggests that a very small percentage of

larvae would successfully settle and complete metamorphosis in the absence of any conspecific

cue. Those larvae would then subsequently provide a conspecific cue leading to increased

settlement within those select replicate dishes. This phenomenon is has been observed among a

variety of gregarious invertebrate settlers (Toonen & Pawlik 1993; Toonen 2005). Individual

larvae of gregarious invertebrates will differ in their individual response to substrata. Upon

reaching competency a small proportion of larvae, so called ‘founders’, will accept a biofilm

alone as suitable habitat, whereas the majority, ‘aggregators’, require the presence of living

conspecific adults in order to settle and metamorphose (Toonen & Pawlik 1993, 2001). This

genetic variation undoubtedly plays a measurable and important part in the settlement of

gregarious invertebrates on previously uninhabited substrata in the field. However, the presence

of ‘founders’ cannot be reliably accounted for under laboratory assay conditions to induce

consistent settlement.

Furthermore, despite the varied settlement response, it cannot be assumed that the presence of a

bacterial biofilm does not play a part in the settlement of G. caespitosa. Chan & Walker (1998)

conducted similar experiments comparing settlement of the serpulid polychaete, Pomatoceros

lamarckii. Results showed that P. lamarckii settled preferentially on biofilmed substrates.

However, similar to the results presented here, the addition of conspecific adults was much

more conducive to larval settlement than a biofilm alone. It was concluded that settlement was

driven by two important factors acting together, a biofilm together with the ‘chemical influence’

of conspecific adults playing a significant sort range role in promoting final settlement and

metamorphosis. Competent larvae of G. caespitosa undoubtedly respond to a variety of cues

when assessing substratum suitability, including a suite of chemical and physical cues from

bacteria and other members of the fouling assemblage.


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IBMX at a concentration of 1 mM induced more than 80% of G. caespitosa larvae to settle. It

has been shown that IBMX at concentrations between 0.1 - 1 mM induces larval settlement and

metamorphosis of a range or marine invertebrates including abalone (Baxter & Morse 1987),

polychaetes (Qian & Pechenik 1998; Bryan et al. 1997), hydroids (Leitz 1997), and barnacles

(Clare et al. 1995). This observation suggests that IBMX may act on similar larval receptor

systems and/or affect the same or similar signal transduction pathways of many invertebrate

taxa (Qian & Pechenik 1998). Utilising the optimal concentration identified in this study it was

possible to develop modified IBMX rapid test for G. caespitosa. Subsamples of 10 larvae at 8-

10 days post-fertilization are placed into six-cell polystyrene microplate wells containing a 10

mL solution of 1 mM IBMX in seawater. The sub-sampled larvae are monitored for 24 hours.

Once at least 90% of larvae settled in response to the IBMX within a 24 hour period, larvae

cultures are considered competent to metamorphose. This method was used to quantify the

competency of G. caespitosa larval cultures prior to commencing the settlement assays

described in the following chapters.

Based on the results of this study it is recommended that in addition to the typical monolayer

bacterial biofilm, a number of pre-settled live juveniles are also included in the assay dishes to

provide a natural conspecific cue. This can be achieved via the following protocol: Following

preparation of the mono-layer bacterial biofilm, approximately 15 competent larvae are added to

each dish in a 1 mM IBMX solution to induce settlement. Once at least 10 larvae have

successfully settled and completed metamorphosis in each replicate dish (approx. 24 hours),

excess settled and unattached worms are removed. Dishes are then once again gently rinsed with

sterile FSW to remove all trace of the IBMX solution. Each replicate dish then contains the

mono-layer bacteria biofilm and exactly 10 live settled G. caespitosa. Following preparation of

the assay dishes with pre-settled juveniles the experimental variables may be incorporated into

the various replicate dishes.


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The static settlement assay protocol developed in this study provides a reproducible tool for the

study of the chemical and/or physical factors which influence the settlement of G. caespitosa,

and is the protocol utilised in the following chapters to investigate the influence of ciliates on

invertebrate settlement. Gaining an understanding of the dynamics of invertebrate settlement

will enhance knowledge in diverse areas such as developmental biology, marine benthic

community ecology, biofouling, and aquaculture (Wieczorek & Todd 1998; Qian et al. 2007).

4. Influence of biofilm‐associated ciliates on larval settlement

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Chapter4

Influence of common biofilm-associated ciliates on the settlement

of Galeolaria caespitosa and Mytilus galloprovincialis

4.1Introduction

Planktonic larvae of sessile invertebrates spend a variable period in the pelagic realm. During

this time they are dispersed to a wide variety of habitats, all the while maturing to the point of

attaining the competence to metamorphose (Marshall & Keough 2003; Marshall & Steinberg

2014). The transition from planktonic to sessile is a critical point in the life cycle of benthic

marine invertebrates; for many the settlement on solid substrates is the prerequisite for final

metamorphosis into sessile adults (Hadfield & Paul 2001). The choice of settlement site for

benthic invertebrates is critical, as the survival, growth and reproductive success is ultimately

dictated by this one act. To this end, larvae have developed complex patterns of behaviour and

finely tuned discriminatory abilities to ensure that settlement occurs in a habitat which will

maximise their expected fitness (Jenkins 2006; Burgess et al. 2009).

Numerous studies have shown that invertebrate larvae actively respond to both physical and

chemical cues associated with a substrate (e.g. Lau & Qian 1997; Kobak 2001; Hadfield &

Koehl 2004; Head et al. 2004). These cues include hydrodynamic forces (Havenhand & Svane

1991; Larsson & Jonsson 2006), surface composition and/or texture (Herbert & Hawkins 2006;

Scardino et al. 2003), the presence of competitors/predators (Morse & Morse 1984),

conspecifics (Jensen & Morse 1984; Pawlik 1986) and microbial biofilms (Hadfield & Huang

2003; Qian et al. 2007). Microbial biofilms are complex 3-dimensional structures composed of


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microorganisms including bacteria, diatoms, thraustochytrids, fungi, and protozoa enveloped

within a matrix of extracellular polymers that cover immersed substrata (Dobretsov 2010). The

bacteria and diatom assemblages within microbial biofilms have been identified as a major

source of both physical and chemical cues that can induce or inhibit invertebrate settlement

(Lau & Qian 1997; Wieczorek & Todd 1998; Rittschof et al. 1998). However, despite protozoa

being a ubiquitous component of microbial biofilms, their influence on invertebrate settlement

remains largely unknown.

Grazing of heterotrophic protozoa is one of the most important selective pressures that bacterial

communities face (Hahn & Hofle 2001; Corno & Jürgens 2006; Wey et al. 2008). Corno (2006)

demonstrated the effects of protozoan grazing on bacterial assemblage composition in a

simplified assay consisting of three competing bacterial strains and one protozoan predator.

Results showed that the bacterium Pseudomonas putida completely dominated in the predator-

free treatments, but with the addition of the flagellate grazer Ochromonas sp. it was completely

removed, allowing the other two bacterial strains to flourish. Selective grazing on particular

prey types via both mechanosensory and chemosensory cues has been observed in protozoa

(Ayo et al. 2001; Fenchel 1980). These mechanisms allow protozoa to select the size and

composition of food particles on which they feed (Ayo et al. 2001; Bernard & Rassoulzadegan

1990; Fenchel 1980; Gonzalez et al. 1990), and in turn allow protozoa to exert a strong

influence on the composition of bacterial assemblages within biofilms.

The feeding activities of protozoa have also been shown to have strong impacts on biofilm

architecture and sloughing dynamics (Jackson & Jones 1991; Lawrence & Snyder 1998; Huws et

al. 2005). Works conducted in simplified bioassays have shown that the grazing activities of

protozoa increase the spatial and temporal heterogeneity of bacterial biofilms, in extreme cases

creating areas of clearance or sloughing on the substrate (Lawrence & Snyder 1998; Jackson &

Jones 1991). Due to their high abundance, protozoa can also influence the three dimensional

structure of biofilms. Many protozoa have extracellular structures which persist within biofilms


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even after cell death (Arndt et al. 2003). As shown in Chapter 2, extensive colonies of peritrichs

(e.g. Zoothamnium sp., Vorticella sp.) were the most abundant ciliates in the assemblage. Reaching

276.3 cells cm-2, the colonies of peritrich ciliates had a considerable impact on the three

dimensional structure of the biofilm by rising above the substrate on stalks.

Given the impacts of protozoan activities within biofilms, we (Shimeta et al. 2012) recently

hypothesized that their influence could also extend to the settlement of invertebrate larvae,

whether indirectly by influencing other microbial species that make up the microbial biofilms,

or directly through physical and/or chemical interactions with larvae. Through laboratory based

assays it was determined that the presence of a mixed assemblage of ciliates had a range of

impacts on the settlement and post-settlement mortality of several invertebrate foulers (Shimeta

et al. 2012). Settlement of the serpulid polychaete, Galeolaria caespitosa, was significantly

reduced by up to 49% in the presence of a mixed assemblage of biofilm-dwelling ciliates.

Similarly the proportion of settled blue mussel larvae, Mytilus galloprovincialis, was

significantly reduced by 46% in the presence of a mixed ciliate assemblage, compared to

settlement on a pure bacterial biofilm (Shimeta et al. 2012). However, while these mixed

assemblages of ciliates had strong effects on invertebrate settlement, it was unknown whether

the impacts observed were species-specific or what the mechanism/s of influence were.

The ciliate assemblages that dwell within marine biofilms are highly varied (see Chapters 2 and

6). Differences in behaviour, morphology and grazing preferences of individual ciliate species

could have variable impacts on the settlement response of invertebrate larvae. In order to

interpret and predict the impacts of ciliate assemblages on invertebrate settlement in natural

systems, a taxonomic differentiation of the ciliate species that influence settlement is required.

Furthermore, investigation into the mechanism/s of ciliate influence on larval settlement might

provide new insights into the ecology of benthic communities, and may have valuable

applications in the development of sustainable antifoulant technologies. The aims of this study

were: (1) to identify the species specificity of our previously observed influence of ciliates on


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the settlement of Galeolaria caespitosa and Mytilus galloprovincialis; (2) to determine whether

the mechanism of influence is a chemical cue released by the ciliates, or whether their physical

presence is required to initiate the effect; and (3) to investigate the impact of the hypotrich

ciliate Euplotes minuta on the structure of the bacterial biofilms as a possible indirect

mechanism influencing larval settlement.

4.2Methods

4.2.1Collection&CultureofBiofilm‐DwellingCiliates

The ciliates were collected from plastic Petri dish microscope slides (AnalyslideTM, Pall Corp.)

which were deployed for two weeks underneath a pier at Williamstown, Port Phillip Bay. After

deployment, Petri dishes were capped and immediately transported to the laboratory. Dishes

were then analysed under a dissection microscope and motile ciliates isolated by micropipette

into monoclonal cultures. Isolated ciliates were cultured in tissue culture flasks (IWAKI)

containing 25 mL of 0.2 µm filtered sterilised fresh sea water (FSW), with 0.005% yeast extract

and a single grain of rice. Cultures were kept in a temperature controlled room at 25oC on a 15:9

light-dark cycle and transferred into fresh media fortnightly. Once cultures were well

established, ciliates were identified by first preserving in Bouin's fixative and then impregnating

with protargol as described by Skibbe (1992). Identifications were made following the keys in

Carey (1992), Lee et al. (2000) and Lynn (2008).

4.2.2LarvalCulture

4.2.2.1Galeolariacaespitosa

The procedure for obtaining gametes and raising the larvae followed those described in Marsden

& Anderson (1981) (See Chapter 3 for detailed description). The competency of the larvae was

determined based on morphological characteristics as described by Marsden & Anderson

(1981), and via the modified IBMX rapid test developed in Chapter 3. Subsamples of 5-10

larvae at 8-10 days post-fertilization were placed into six-cell polystyrene microplate wells

containing a 10 mL solution of 1 mM IBMX in seawater. The sub-sampled larvae were


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monitored for 24 hours. Once at least 90% of larvae settled in response to the IBMX within a 24

hour period, larvae cultures were considered competent to metamorphose.

4.2.2.2Mytilusgalloprovincialis

Pediveliger larvae of the mussel, M. galloprovincialis, were obtained in May 2013, at an age of

22 days from the Victorian Shellfish Hatchery, Dept. of Primary Industries, Queenscliff,

Victoria, Australia. The larvae were held at 20oC on a 15:9 light-dark cycle, and were

transferred into fresh FSW and fed equal parts Isochrysis galbana (CS-177), Pavlova lutherii

(CS-182) and Chaetoceros calcitrans (CS-176) at 5 x 104 cells mL-1 daily. The competency of

larvae was determined based on morphological characteristics. The settlement assay was begun

with the larvae at 24 days of age.

4.2.3PreparationofBioassayDishes

Settlement assays were run in 55 mm diameter polystyrene Petri dishes. All treatments included

a monolayer bacterial biofilm which was grown in each dish prior to the commencement of the

settlement assay. Petri dishes were placed into a large tub containing 4 L of sterilised FSW

ensuring that they were in a monolayer. The FSW baths were aerated and a growth medium

consisting of 0.001% yeast extract and 0.001% bacto-peptone was added to the bath. The bath

was inoculated with a 5 mL Pseudoalteromonas luteoviolacea suspension, a species common to

marine biofilms and well known to induce the settlement of another serpulid polychaete,

Hydroides elegans (Lau & Qian 2001; Hadfield et al. 2014). The bacteria were allowed to

incubate for 24 hours at 25oC. After the 24-hour growth period the Petri dishes were gently

rinsed with sterile FSW to remove any unattached cells, and Petri dishes were subsequently re-

filled with 8 mL of sterilized FSW.

As determined in Chapter 3, Galeolaria caespitosa larvae require a conspecific cue to settle

consistently within laboratory based settlement assays. This was achieved by inducing 10 larvae

to settle in each replicate dish prior to the assay. Following preparation of the mono-layer


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bacterial biofilm, approximately 15 competent larvae were added to each dish in a 1mM IBMX

solution to induce settlement. Once 10 larvae had successfully settled and completed

metamorphosis in each replicate dish (approx. 24 hours), dishes were once again gently rinsed

with sterile FSW to remove all trace of the IBMX solution. Each replicate dish then contained

the mono-layer bacterial biofilm and exactly 10 live settled G. caespitosa which provided a

natural cue for settlement.

The first set of settlement assays included five treatments and eight replicates of each treatment

dish. The control treatment dishes only contained the monospecies bacterial biofilm. The

remaining four treatments were inoculated with 10 cells cm-2 of a single species from the

following ciliate genera: (1) Euplotes minuta; (2) Amphisiella sp.; (3) Litonotus sp.; and (4)

Uronema marinum. The selected genera represented ciliates from both vagile and planktonic

niches, and were all highly abundant in natural marine assemblages as identified in Chapter 2.

Determining whether the observed inhibition was due to a chemical cue released by the ciliates

was achieved by utilising a filtrate from the ciliate cultures. The follow up assays were

conducted with Euplotes minuta and Uronema marinum. Selected as their presence significantly

inhibited the settlement of both G. caespitosa and M. galloprovincialis. Furthermore, as

identified in Chapter 2, ciliates from the genera Euplotes and Uronema were amongst the most

highly abundant ciliates identified within natural marine ciliate assemblages. The experiment

included three treatments with eight replicates of each treatment: (1) a control containing only

the Pseudoalteromonas luteoviolacea biofilm and 10 mL of FSW; (2) a ciliate filtrate treatment,

which contained the bacterial biofilm, 5 ml of media from the ciliate cultures passed through a

0.2 µm filter to remove all ciliates and bacteria, and an additional 5 mL of ciliate culture passed

through a 0.8 µm filter. This removed any ciliates, but allowed bacteria present within the ciliate

cultures, which would be present in the ‘ciliate treatment’, to pass through; and (3) a ciliate

treatment containing the bacterial biofilm, 5 mL of ciliate culture and 5 mL of FSW.


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4.2.4SettlementAssays

Competent larvae were gently filtered through a 90 µm sieve to remove algae and excess debris.

Larvae retained in the sieves were then immediately transferred into FSW ready to be

inoculated into assay dishes using a pipette. Twenty competent larvae were randomly added to

each treatment. Throughout the 72 hour duration of the experiment, larvae were not fed and the

testing solution was not changed. Bioassay dishes were kept in a controlled environment at 25oC

on a 15:9 light-dark cycle. Once the experiment had commenced, assay dishes were examined

under dissection microscope every 24 hours. During examinations, ciliate densities were

recorded and larvae were classified as settled or unattached.

Galeolaria caespitosa were considered settled only once they had completed final

metamorphosis, i.e. attached to the bioassay dish, produced a tube, and grown tentacles. Mytilus

galloprovincialis larvae were considered settled when they were motionless on the bottom, no

velum was visible through the shell, and they did not move when the dish was gently agitated.

Larvae that were unattached and swimming or crawling were considered unmetamorphosed and

classified as not settled.

After the final examination at 72 hours the contents of each dish were gently decanted and

replaced with 1% formalin in FSW to preserve the bacterial biofilms. Bacterial densities of

biofilms in each treatment were determined via epifluorescence microscopy. The formalin was

removed from the dishes and 50 µL of DAPI solution (0.02 mg mL-1) added to the dish, a 22 x

40 mm coverglass was randomly placed on top of the DAPI solution and bacteria cells counted

on an epifluorescence microscope (Leica DM2500) with UV excitation (340-380 nm). A

nearest-neighbour analysis was used to quantify the distribution of bacterial cells in the

presence/absence of E. minuta. Images of DAPI stained biofilms were imported into imageJ

v1.48 (National Institutes of Health, USA). An imageJ plugin made by Mao (2012) was utilised

for calculating the nearest neighbour distances of each cell within the image.


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4.2.5DataAnalysis

Settlement rates, converted to arc-sine square-root transformed proportions, were tested at each

time point by an a-priori test of main effects using the MSE from a repeated-measures ANOVA.

If the effect of the treatment was significant at = 0.05, Tukey pairwise comparisons were run

among the treatments at that time point using the MSE from the test of main effects. Bacterial

densities and nearest-neighbour distances were tested for differences among treatments by 1-

way ANOVA, followed by Tukey pairwise comparisons. All statistical tests were run on Systat

v13.

4.3Results

4.3.1Galeolariacaespitosa

After 24 hours, settlement differed significantly among treatments (p < 0.05). Compared to the

control, settlement was significantly reduced in the presence of Amphisiella sp. (p = 0.04),

Euplotes minuta (p = 0.03) and Uronema marinum (p = 0.04) (Fig. 4.1; 24 h). The Hypotrich

ciliate E. minuta had the greatest effect on settlement, causing a 34.3% reduction in comparison

with the control at 24 hours. Litonotus sp. was the only ciliate treatment not to significantly

reduce settlement in comparison with the control (p = 0.82). After 48 hours, settlement in the

control had increased by another 13.5%, while the average settlement in ciliate treatments rose

by only 5.2%. Litonotus sp. remained the only ciliate treatment not to reduce settlement

significantly in comparison with the control (p = 0.56). Few additional larvae settled in the E.

minuta and U. marinum treatments, which remained significantly different to control treatments

(E. minuta p = 0.01; U. marinum p = 0.02) (Fig. 4.1; 48 h.). At 72 h, Amphisiella sp., E. minuta

and U. marinum still caused a significant reduction in larval settlement compared to the control

(Amphisiella sp. p = 0.05; E. minuta p = 0.02; U. marinum p = 0.04) (Fig. 4.1; 72 h). Settlement

in these 3 ciliate treatments at 72 hours was on average reduced by 40.3% compared to the

control treatment.


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The densities of the ciliate genera differed significantly at all three time points (p < 0.001)

(Table 4.1). E. minuta and U.marinum had the highest densities of the ciliate treatments

reaching >35 cells cm-2, while the density within the Amphisiella sp. and Litonotus sp.

treatments remained comparatively low throughout the assay (Table 4.1). At the conclusion of

the assay (72 h), there was no significant difference in the abundance of biofilm bacteria

between the control and ciliate treatments (p = 0.94; Table 4.1).

Ciliate Density cm‐2 Bacteria Density

(×104 ) cm‐2

24 hours 48 hours 72 hours 72 hours

Control 0.00±0.00a 0.00±0.00a 0.00±0.00a 1.50±0.09a

Amphisiella sp. 11.40±0.43b 16.06±0.35b 18.40±0.31b 1.38±0.12a

Euplotes minuta 16.97±0.75c 31.52±0.90c 36.78±1.24c 1.38±0.14a

Uronema marinum 40.38±2.81d 42.23±2.28d 31.43±1.51c 1.14±0.24a

Litonotus sp. 12.10±0.74b 11.66±0.95e 8.66±0.58d 1.51±0.09a

Figure 4.1 Galeolaria caespitosa settlement rates in the presence/absence of tested ciliate genera

(Mean ± SE cells cm‐2). Means with different superscripts within each time point were significantly

different by post hoc multiple comparison tests following a significant ANOVA (α=0.05).

Table 4.1 Ciliate and bacterial densities in Galeolaria caespitosa settlement assays (Mean ± SE

cells cm‐2). Means with different superscripts within each time point were significantly

different by post hoc multiple comparison tests following a significant ANOVA (α=0.05).


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4.3.2Mytilusgalloprovincialis

At 24 hours, settlement was significantly reduced in Amphisiella sp. (p < 0.001), Euplotes

minuta (p = 0.003) and Uronema marinum (p = 0.003) treatments in comparrison with the

control treatment (Fig. 4.2; 24 h). In the Amphisiella sp. treatment no larval settlement was

observed at 24 hours. The presence of Litonotus sp. was the only ciliate treatment in which

settlement was not inhibited significantly when compared to the control treatment (p = 0.45)

(Fig. 4.2; 24 h.). At the remaining time points (48 and 72 h) settlement was significantly lower

in all ciliate treatments in comparrison with the control (Fig. 4.2; 48 h. 72 h). Settlement in the

Amphisiella sp. treatment increased up to 12% after 72 hours. However, this treatment remained

the most inhibiting to larval settlement. At 72 hours settlement in the presence of Amphisiella

sp. was reduced by 68.5% compared to the control treatment. The Litonotus sp. treatment had

the smallest impact on settlement, with settlement rates 36.8% lower than that recorded in the

contol at 72 h (Fig. 4.2; 72 h Litonotus).

Figure 4.2Mytilus galloprovincialis settlement rates in the presence/absence of tested ciliate

genera. (Mean ± SE cells cm‐2). Means with different superscripts within each time point were

significantly different by post hoc multiple comparison tests following a significant ANOVA (α=0.05).


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The densities of the ciliate genera differed significantly at all three time points (p < 0.001)

(Table 4.2). At 24 hours the densities of Amphisiella sp., E. minuta and U. marinum treatments

were not significantly different. The Hypotrich ciliate E. minuta subsequently multiplied at a

faster rate than the other ciliates and at 48 hours their densities were significantly higher than all

other ciliate treatments (p = 0.03). Upon the conclusion of the assay E. minuta and Amphisiella

sp. had the highest densities of the ciliate treatments reaching >35 cells cm-2, while the density

of Litonotus sp. treatments remained comparatively low throughout the assay (Table 4.2). At the

conclusion of the assay (72 h), there was no significant difference in the abundance of biofilm

bacteria between the control and ciliate sub-class treatments (p = 0.94; Table 4.2).

4.3.3LarvalSettlement/CiliateAbundanceCorrelations

At 24 hours the slope of the regression between the proportion of Galeolaria caespitosa

settlement and ciliate abundance was not significant (p = 0.58) (Fig. 4.3A). However, at 48 and

72 hours the impact of ciliate abundance had become more apparent with the regression slope

returning significant values (48 h p = 0.05; 72 h p = 0.02) (Fig. 4.3B, C). The impact of ciliate

abundance was evident earlier in the Mytilus galloprovincialis assay with the linear regression

returning a significant value after 24 hours (p = 0.04) (Fig. 4.3 D). However, at 72 hours the

slope of the regression was no longer significant (p = 0.09) (Fig. 4.3F). When significant, the

Ciliate Density cm‐2 Bacteria Density

(×104) cm‐2

24 hours 48 hours 72 hours 72 hours

Control 0.00±0.00a 0.00±0.00a 0.00±0.00a 1.42±0.08a

Amphisiella sp. 21.30±1.59b 28.40±1.66b 36.48±2.02b 1.29±0.06a

Euplotes sp. 26.24±2.90b 40.00±2.07c 36.48±2.03b 1.29±0.10a

Uronema sp. 22.20±0.56b 27.77±0.33b 21.60±0.63c 1.34±0.12a

Litonotus sp. 11.12±0.72c 15.06±0.85d 17.20±0.72d 1.48±0.10a

Table 4.2 Ciliate and bacterial densities in Mytilus galloprovincialis settlement assays

(Mean ± SE cells cm‐2). Means with different superscripts within each time point were

significantly different by post hoc multiple comparison tests following a significant


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slope of the regressions for both G. caespitosa and M. galloprovincialis were negative

indicating that increased ciliate abundance coincided with lower settlement rates.

The correlation coefficient of G. caespitosa settlement against ciliate abundance at 24 hours was

very weak at -0.13. At 48 and 72 hours the correlation coefficients were stronger but remained

relatively weak (48 h = -0.44; 72 h = -0.51) (Fig .4.3A, B, C). Correlation coefficients for M.

galloprovincialis were also very weak across all-time points. Although, in contrast to G.

caespitosa the coefficients for M. galloprovincialis remained relatively static throughout the

assay (24 h = -0.45; 48 h = -0.48; 72 h = -0.40) (Fig. 4.3D, E, F).


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Figure 4.3 Correlations of ciliate densities against the proportion of Galeolaria caespitosa (A, B, C) and

Mytilus galloprovincialis (E, F, G) settlement, at 24 (A, D), 48 (B, E) and 72 (C, F) hours.


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4.3.4CiliateFiltrate

4.3.4.1Euplotesminuta

After 24 hours larval settlement differed significantly among the treatments (p < 0.001; Fig.

4.4). Settlement was significantly inhibited in the physical presence of Euplotes minuta

compared to the pure bacterial biofilm (p < 0.001) and filtrate treatments (p = 0.002) (Fig. 4.4).

Settlement in the E. minuta treatment after 24 hours was on average 51.1% lower than that

observed in biofilm and filtrate treatments. At 48 and 72 hours, a similar pattern of settlement

persisted. The biofilm and filtrate treatments were at no point significantly different to each

other (p > 0.97). Settlement in the presence of E. minuta however, was still significantly

reduced compared to the biofilm and filtrate treatments at 48 (p < 0.001) and 72 hours (p <

0.001) (Fig. 4.4; 48 h and 72 h). Upon the conclusion of the assay, settlement was reduced by

36.8% in the E. minuta treatment compared to the biofilm and filtrate treatments.

Figure 4.4 Galeolaria caespitosa settlement rates in the presence of (1) a bacterial biofilm alone;

(2) a bacterial biofilm and filtrate from a Euplotes minuta culture; and (3) a bacterial biofilm and

the physical presence of E. minuta (shown on micrograph) (Mean ± SE). Means with different

superscripts were significantly different by post hoc multiple comparison tests following significant

repeated measures ANOVA at α = 0.05 (Photo M. Watson).


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4.3.4.2Uronemamarinum

The outcome of the the Uroema marinum filtrate assay was similar to Euplotes minuta After 24

hours settlement was significantly inhibited in the physical presence of U. marinum compared

to the pure bacterial biofilm (p < 0.001) and filtrate treatments (p < 0.001), but there was no

signifcant difference between the pure bacterial biofilm and filtrate treatments (p = 0.99) (Fig.

4.5). Settlement in the U. marinum treatment after 24 hours was on average 57.2% lower than

that observed in biofilm and filtrate treatments. Again at 48 and 72 hours, a similar pattern of

settlement persisted. Settlement in the presence of U. marinum was still significantly reduced

compared to the biofilm and filtrate treatments at 48 (p < 0.001) and 72 hours (p < 0.01) (Fig.

4.5; 48 h 72 h). The pure bacterial biofilm and filtrate treatments were at no point significantly

different to each other (p > 0.98). Upon the conclusion of the assay settlement was reduced by

32.3% in the U. marinum treatment compared to the biofilm and filtrate treatments.

Figure 4.5 Galeolaria caespitosa settlement rates in the presence of (1) a bacterial biofilm alone;

(2) a bacterial biofilm and filtrate from a Uronema marinum culture; and (3) a bacterial biofilm and

the physical presence of U. marinum (shown on micrograph) (Mean ± SE). Means with different

superscripts were significantly different by post hoc multiple comparison tests following significant

repeated measures ANOVA at α = 0.05 (Photo M. Watson).


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4.3.5BiofilmStructureAnalysis

Bacterial abundance prior to commencing the assay was 0.76 (±0.09) ×104 cells cm-2 (Fig 4.6).

At the conclusion of the assay (72 h), despite the introduction of Euplotes minuta into the ciliate

treatment, there was no significant difference in the abundance of biofilm bacteria between the

control (1.43 (±0.10) ×104 cells cm-2) and ciliate treatments (1.31 (±0.09) ×104 cells cm-2) (p =

0.57) (Fig. 4.6). In conjunction with an increase in bacterial abundance the nearest neighbour

distances between cells decreased in both treatments over time (Fig. 4.6). Furthermore, the

ciliate treatment had a significantly shorter nearest neighbour distance than the control (p <

0.001). The average nearest neighbour distance in the ciliate treatment was 1.51±0.20 µm,

compared with 2.46±0.32 µm in the control (Fig. 4.6). A shorter nearest neighbour distance is

evidence that the Pseudoalteromonas luteoviolacea biofilms in the presence of E. minuta had a

more clustered distribution. Figure 4.7 displays some examples of P. luteoviolacea biofilms in

the absence and presence of the ciliate E. minuta. The biofilm in the control treatment is

distributed relatively evenly over the substrate. In contrast the biofilm in the presence of E.

minuta show areas of clearance, with bacteria clustered together in tight groups.

Figure 4.6 Bacterial abundances and nearest neighbour distances

(Mean ± SE), Means with different superscripts within each

analysis were significantly different at α = 0.05.


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4.4Discussion

The presence of a single species of ciliate was enough to induce a significant reduction in the

settlement of Galeolaria caespitosa and Mytilus galloprovincialis. The ciliates in these

experiments included vagile species (Euplotes minuta, Amphisiella sp. & Litonotus sp.)

specifically adapted for life on substrates and a planktonic species (Uronema marinum) that

swims above the substratum periodically grazing upon attached bacteria. While no significant

difference was detected in the settlement response induced by ciliates from either ecological

niche, there were significant differences in the levels of settlement inhibition amongst the

different species (Figs. 4.1; 4.2). Despite weak correlations, the impact of ciliate abundance was

still apparent. The significant inverse relationship identified in the correlation analyses

illustrates density dependence and supports the hypothesis that some action of the ciliates is the

cause of the observed settlement inhibition.

Litonous sp. was the only ciliate species which did not significantly reduce settlement of both G.

caespitosa and M. galloprovincialis across all time points. A predatory species in the absence of

prey under assay conditions, the specimens of Litonotus sp. inoculated into the assay dishes did

not increase in abundance during the assays. Consequently it was the ciliate treatment with the

lowest density (Tables 4.1; 4.2). However, weak correlations between larval settlement and

ciliate abundance precludes the conclusion that abundance alone was the reason for the lack of

Figure 4.7 Epifluorescence micrographs of DAPI stained Pseudoalteromonas luteoviolacea biofilms after

72 hours in the absence (A) and presence (B) of Euplotes minuta.


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influence applied by Litonotus sp. on the settlement of G. caespitosa and M. galloprovincialis

larvae. This indicates that Litonotus sp. may have failed to significantly influence settlement for

some reason other than its abundance. Supporting the conclusion that there are species-specific

factors mediating the extent of settlement inhibition, and providing evidence that differences in

morphology and/or behaviour of the different ciliate species had a varied effect on larval

settlement.

It was hypothesised that the mechanism of influence could be due to the release of a chemical

cue from the ciliates, which deters settlement, similar to negative cues released by some bacteria

and algae (Lau & Qian 1997; Qian et al. 2007). However, while there was a pronounced effect

in the presence of ciliates, it was determined that filtrate from the ciliate cultures did not affect

settlement significantly (Figs. 4.3; 4.4). This result also argues against the possibility that ciliate

grazing induced a defensive response from the bacteria, releasing chemical cues that deterred

the larvae. It is well known that many species of biofilm bacteria can inhibit the settlement of

invertebrates (Qian et al. 2007). However, if such inhibitory chemicals were released by

bacteria in these assays they would have been present in the ciliate cultures, and subsequently

added to assays with the culture filtrate. Therefore, since filtrate alone did not inhibit

settlement, the inhibition observed here is unlikely to be due to bacterial chemical cues.

Furthermore, no such inhibitory chemical cues have been reported for Pseudoalteromonas

luteoviolacea. Rather, this species is frequently utilised in settlement assays of another serpulid

polychaete, Hydroides elegans, for which it is known to induce settlement (Lau & Qian 2001;

Hadfield et al. 2014).

As in our previous study (Shimeta et al. 2012), ciliate grazing had no significant effect on the

total bacterial abundance, so the settlement inhibition cannot be explained as a response to

bacterial density. However, upon further examination of the bacterial biofilms in a subsequent

assay, it was determined that the presence of Euplotes sp. had significantly altered the structure

of the biofilm (Fig. 4.6). Lawrence & Snyder (1998) also noted aggregates or clumps of bacteria


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formed in the presence of Euplotes sp. They repeatedly observed Euplotes sp. concentrating

feeding activity within certain zones on the experimental substrate, even in locations where

more abundant prey was available directly adjacent to these zones. It was hypothesised that the

concentrated feeding effort likely facilitates the physical removal of bacterial cells from the

surface. Furthermore, it is thought that repeated feeding in a targeted zone possibly increases the

likelihood of finding actively growing cells, which may be preferred over relatively starved

cells (Lawrence & Snyder 1998). Perhaps when a biofilm displays high heterogeneity, such as

areas of clearance or sloughing as observed here in the presence of E. minuta, the surface area

of substrate providing suitable stimulus for final attachment and metamorphosis is reduced,

leading to a subsequent reduction in settlement.

Recent studies have shown that physical contact with a bacterial biofilm is necessary for

metamorphosis to occur for many invertebrates (e.g. Matson et al. 2010; Penniman et al. 2013;

Hadfield et al. 2014). Hadfield et al. (2014) reported a significant increase (>90%) in settlement

and metamorphosis of another serpulid polychaete, Hydroides elegans, when physical contact

with a living biofilm was possible, against when contact was prevented with a screen.

Furthermore, it has been suggested that settling larvae actually employ the adhesive properties

of the EPS produced by bacteria to increase their own attachment strength (Zardus et al. 2008;

Hadfield 2011). In assessing attachment strength of H. elegans in turbulent flow, Zardus et al.

(2008) found that many of the primary tubes detached from clean surfaces during fixation.

Whereas those settling on biofilms remained attached and, at high magnification, showed an

intermingling of materials between the bacterial EPS and the tube matrix (Zardus et al. 2008).

Therefore, it must be conceivable that larvae are overlooking the zones of concentrated ciliate

grazing activities, due to the lack of bacteria and EPS required for stimulating a settlement

response.

Alternatively, the mechanism of influence could be solely based on physical interaction. The

response to specific physical cues at the surface are, in part, what determines the suitability of a


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site for final attachment and metamorphosis into adults (Holmström & Kjelleberg 1994;

Hadfield & Paul 2001). Conceivably, physical contact made between larvae and ciliates during

the exploration process is sufficient to impact the selection of a suitable site for final

metamorphosis. Determining the behavioural impact of direct interactions between ciliates and

larvae is the aim of the following chapter.

Whether ciliates are influencing settlement via direct and/or indirect mechanisms, these results

indicate that the recruitment of certain invertebrates onto an available patch of substratum can

depend on the ciliate assemblage present at that point in the succession of the biofilm

community. Further emphasising the importance of understanding the taxonomic structure and

successional dynamics of ciliate assemblages on immersed substrates. It is important to note

that the ciliates in this study only represent a fraction of the ciliates that grow on immersed

substrates, other species may influence settlement differently. Furthermore, biofilms in the field

are far more complex than those we created for our experiments (Arndt et al. 2003, Dobretsov

2010). Additional investigations are needed to determine how these interactions function in a

more complex, natural microbial biofilm community.

Overall, the results presented have demonstrated density dependant effects of ciliates on

invertebrate settlement. Supporting the hypothesis that biofilm-dwelling ciliates are an

important factor influencing the variability of invertebrate recruitment over time and space, and

ultimately the structure and dynamics of natural invertebrate assemblages. Increased knowledge

on the dynamics of ciliate assemblages, and the influence of each species present on a given

substrate, could have meaningful implications not only in understanding invertebrate

recruitment variation, but in improving the efficiency of mollusc aquaculture and the

development of sustainable antifouling technologies.

5. Tracking larval exploration behaviour

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Chapter5 BehaviouralresponseofGaleolariacaespitosalarvaetothe

presenceofciliates

5.1Introduction

Larvae settle in response to a variety of factors to which they respond behaviourally. Recent

observations have highlighted an array of larval sensory abilities and documented the

behavioural responses to a wide variety of physical and chemical cues presented by substrates.

Factors such as hydrodynamics (Jonsson et al. 2004; Koehl 2007), substratum complexity

(Scardino et al. 2003; Herbert & Hawkins 2006), conspecific cues (Toonen & Pawlik 1994),

bacterial metabolites (Hadfield & Koehl 2004; Qian et al. 2010), light and the presence of

competitors/predators (Morse & Morse 1984) have all been shown to have independent and

interactive effects on larval behaviour prior to settlement. The behavioural response to these

factors ultimately determines the success of settling in a habitat, which will maximise fitness.

Patterns of larval substrate exploration prior to permanent attachment and metamorphosis have

only been described in detail for a few species in still-water laboratory observations (e.g.

Balanus amphitrite (barnacle): Chaw et al. 2011; Maruzzo et al. 2011; Hydroides elegans

(polychaete tubeworm): Hadfield & Koehl 2004; Hadfield et al. 2014; Bugula neritina

(bryozoan): Burgess et al. 2009. Hadfield et al. (2014) identified that larvae of the serpulid

polychaete tube worm H. elegans, would engage in different types of behaviour when they

could touch biofilms versus when they could not. Larval movements were assigned to one of

four categories - straight swimming, turning, circling or crawling. Their results indicated that


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when larvae of H. elegans made physical contact with a biofilm, their swimming behaviour

shifted significantly. In effect, biofilm contact resulted in a switch from straight swimming and

circling to crawling, a behaviour which was significantly more prevalent in the experimental

conditions where larvae could make contact with a biofilm (Hadfield et al. 2014).

The importance of microbial biofilms, particularly the bacterial assemblage, on the settlement of

larvae from many invertebrate phyla has become increasingly apparent (Qian et al. 2010;

Hadfield 2011; Hadfield et al. 2014). The results presented by Hadfield et al. (2014) indicated

that the larvae of H. elegans must touch a biofilmed surface to detect the cues that stimulate a

settlement response. In a situation where settlement cues are surface bound rather than soluble,

behaviours involving physically touching potential settlement sites (e.g. crawling) would

naturally be more prevalent. Given the abundance of ciliates within the microbial biofilms,

which grow on immersed substrates (see Chapters 2 and 6), interactions between exploring

larvae and ciliates must be a frequent occurrence.

In Chapter 4 it was determined that the physical presence of ciliates is required to significantly

inhibit the settlement of G. caespitosa. Therefore, the mechanism of influence behind this

inhibition could be the result of direct interactions between a larva and a ciliate. Perhaps,

physical contact made between larvae and ciliates during the exploration process is sufficient to

impact the selection of a suitable site for final metamorphosis. Larval settlement behaviour is

generally characterised as responding to positive cues to stimulate settlement. However,

rejection of unsuitable substrata by settling invertebrates is an equally important mechanism in

ensuring that settlement occurs in an appropriate habitat (Berntsson et al. 2000). If a substratum

proves to be unsatisfactory, metamorphosis can usually be delayed, allowing the larva to locate

and test other substrates (Qian & Pechenik 1998; Prendergast 2009). Larvae will often make

repeated and active attempts to find a suitable place for development and growth (Berntsson et

al. 2000; Prendergast 2009).


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Experimental work on the influence of settlement cues has revealed much about the

discriminatory abilities of invertebrate larvae, and provided insight into understanding natural

settlement patterns. The present study sought to clarify whether the presence of ciliates impacts

larval pre-attachment exploration behaviours. The aims of this study were: (1) to determine

whether the presence of the Hypotrich ciliate, Euplotes minuta, elicits behavioural changes in

surface exploration of Galeolaria caespitosa larvae; and (2) to quantify any changes in

exploratory behaviour directly before and after contact is made.

5.2Methods

5.2.1LarvalCulture

The procedure for obtaining gametes and raising the larvae followed those described in Marsden

& Anderson (1981) (See Chapter 3 for detailed description). The competency of the larvae was

determined based on morphological characteristics as described by Marsden & Anderson

(1981), and via the modified IBMX rapid test developed in Chapter 3.

5.2.2CiliateCulture

The hypotrich, Euplotes minuta, was selected as the candidate for the assay based on results

presented in Chapter 4. The presence of E. minuta significantly inhibited the settlement of both

Galeolaria caespitosa and Mytilus galloprovincialis. Furthermore, as identified in Chapter 2,

ciliates from the genus Euplotes were amongst the most highly abundant ciliates identified

within natural ciliate assemblages. E. minuta is also a relatively large species of ciliate (approx.

50 µm), facilitating the observation of direct contact between larva and ciliate (Fig 5.1).

Belonging to the ciliate subclass Hypotrichida, these ciliates are specifically adapted for life on

substrates (Lynn 2008). Specialised somatic cilia have been fused into cirri, which function as

walking or crawling appendages (Fig 5.1). The oral ciliature of this genus is composed of a

band of planar membranelles, which are located under the ventral surface (Laurence & Snyder


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1998; Lynn 2008). The membranelles create feeding currents that draw in particles, making

these ciliates particularly effective at feeding from substrates (Lynn 2008).

Specimens of E. minuta were collected from plastic Petri dish microscope slides (AnalyslideTM,

Pall Corp.), which were deployed underneath a pier at Williamstown, Port Phillip Bay. After 7 days

of exposure Petri dishes were capped and immediately transported back to the laboratory. Petri-dish

slides were analysed under a dissection microscope. The motile ciliates present were isolated by

micropipette into monoclonal cultures. Isolated ciliates were cultured in 25 cm-2 tissue culture

flasks (IWAKI) containing 25 mL of 0.2 µm sterilised FSW, with 0.005% yeast extract and a single

grain of rice. Cultures were kept in a temperature controlled room at 25oC. Once cultures were well

established, ciliates were identified by first preserving in Bouin's fixative, and then impregnating

with protargol as described by Skibbe (1992). Identifications were made following the keys in Lee

et al. (2000), Carey (1992) and Lynn (2008).

Figure 5.1 (A) Protargol stain of Euplotes minuta highlighting the cirri and oral membranelles

which have enabled this sub‐class of ciliate to thrive on substrates. (B) Sketch of crawling

Euplotes sp. (adapted from Laurence & Snyder 1998). (C) Competent Galeolaria caespitosa

larva in the presence of E. minuta, illustrating the difference in size.

Oralmembranelles

Cirri “Walking legs”

B

100 µm

20 µm

CA

Cirri “walking legs”

Oral membranelles

Caudal cirri

Dorsal cilia


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5.2.3PreparationofBioassayDishes

Tracking assays were run in 35 mm diameter polystyrene Petri dishes. All assay dishes included

a monolayer bacterial biofilm which was grown in each dish prior to the commencement of the

settlement assay. Petri dishes were placed into a large tub containing 4 L of sterilised FSW

ensuring that they are in a monolayer, the FSW baths were aerated and a growth medium

consisting of 0.001% yeast extract and 0.001% bacto-peptone added to the bath. The bath was

inoculated with a 5 mL Pseudoalteromonas luteoviolacea suspension, a species common to

marine biofilms and known to induce the settlement of another serpulid polychaete, Hydroides

elegans. The bacterial suspension was allowed to incubate for 24 hours at 25oC. After the 24-

hour growth period the Petri dishes were gently rinsed with sterile FSW to remove any

unattached cells, Petri dishes were subsequently re-filled with 4 mL of sterilised FSW.

5.2.3.1AnalysisofSwimmingBehaviour

The first assay included 2 treatments: (1) control dishes without ciliates, and (2) treatment

dishes inoculated with the ciliate Euplotes minuta. A total of 20 replicates of each treatment

were prepared. Replicates randomly designated as ciliate treatments were inoculated with E.

minuta at 10 cells cm-2. Following assay dish preparation a single competent Galeolaria

caespitosa larva was placed into each replicate dish for examination. Larvae were allowed a

period of 5 minutes to acclimate to the conditions of the assay dishes before filming

commenced. Larvae in each replicate dish were recorded for 25 seconds at 24 frames per second

(fps). Video was recorded at 0.64 × magnification, ensuring that most of the assay dish was in

view during filming.

5.2.3.2AnalysisofBehaviourBefore&AfterDirectContact

Analysis of the impacts of direct contact was carried out in 4 replicate dishes that were

inoculated with Eupotes minuta at 10 cells cm-2. Following assay dish preparation 10 competent

Galeolaria caespitosa larvae were placed into each dish for examination. Video was recorded at


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6.0 × magnification to ensure instances of direct contact between ciliate and larva would be

clearly visible. Each replicate dish was then filmed for approximately 2 hours in these settings.

After recording, the video was analysed and instances of direct contact between larva and ciliate

identified and edited into shorter sequences for data analysis. The analysis of behaviour was

based on recorded swimming parameters 4 seconds directly before and after direct contact.

5.2.4VideoRecordingofLarvalExploration

The video recording of the larvae was conducted via stereomicroscope (Leica MZ9.5) and Leica

DFC 310 FX camera. Video clips were converted into image sequences with Adobe Media

Encoder v17.0.1 (Adobe Systems Incorporated). Image sequences were transferred into Image-

Pro Premier v9.1 (Media Cybernetics, Bethesda, MD, USA) for the quantification of motility.

Once imported, sequences were spatially calibrated based on the scale at which the video was

recorded. The spatial calibration of the image sequence allowed for various parameters of the

larval motility to be monitored. This was accomplished by relating the pixel size to physical

distances. Once spatial calibration was completed, 2D larval tracks were measured using the

automatic track objects operation in Image Pro Premier. Larvae tracks were determined on

image intensities with respect to the background of each image in sequence. Acceleration,

velocity, distance, turning angle and X/Y coordinates of each frame were recorded for each

replicate. Heat maps were generated via ImageJ v1.48 utilising the wiggle analysis plugin

(Preston et al. 2015).

5.2.5DataAnalysis

Based on X/Y coordinates of larvae positions at each frame, the velocity (v), acceleration (a)

and change in angle (Ɵ) were calculated as follows:

where d = distance between frames, t = time, v = velocity and Ɵ = angle.


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Swimming variability was quantified with the coefficient of variation, calculated as the ratio of

the standard deviation (σ) to the mean (µ):

Two sample t-tests (a = 0.05) were used to compare larval swimming parameters in control vs

treatment, as well as to compare swimming parameters 4 seconds before and after direct

contact. All statistics were performed with Systat 13.

5.3Results

5.3.1SwimmingBehaviour

Examples of the trajectories of competent Galeolaria caespitosa larvae in the presence/absence

of the Hypotrich ciliate Euplotes minuta are shown in Figure 5.2. In the absence of E. minuta,

most G. caespitosa larvae tended to swim close to the bottom, frequently pausing to touch their

apical tuft to the biofilm surface before lifting off again (Fig. 5.2). Less frequently, but still

typically, the larvae would directly crawl across the substrate, occasionally pausing before

moving on (Fig. 5.2C, F). While this same pattern of behaviour was often observed in the

presence of E. minuta, it was clear that the swimming behaviour of G. caespitosa was

significantly more erratic under this condition (Fig. 5.2). While engaged in the

touching/crawling behaviour the larvae would, on occasion, suddenly start to circle rapidly just

above the substrate (Fig. 5.2G). Following this event some larvae resumed the typical

touching/crawling exploration behaviours, while others simply swam away from the bottom of

the dish. While not examined, instances of larvae seemingly avoiding groups of grazing E.

mintua was also observed.

The mean change in angle between frames was significantly higher in the ciliate treatment

(39.22 ± 2.36o) compared to the larva in the control (31.04 ± 2.36o) (p = 0.02) (Fig. 5.3C). The

mean velocity recorded in the presence of E. minuta was also higher (5.26 ± 1.54 mm s-1) than


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that recorded in the control (4.22 ± 1.01 mm s-1), albeit not significantly different (p = 0.57)

(Fig. 5.3A). Similarly the mean acceleration was not significantly different (p = 0.72) in the

ciliate treatment (0.07 ± 0.10 mm s-2) compared with the control (0.01 ± 0.09 mm s-2) (Fig.

5.3B). There were no significant differences detected in the coefficient of variation values for

velocity and acceleration between treatments (p > 0.19) (Fig. 5.3). However, the coefficient of

variation in the change of angle while exploring the surface was significantly greater in the

control (2.17 ± 0.09) compared to the ciliate treatment (1.93 ± 0.08) (p = 0.03) (Fig. 5.3F).

Figure 5.2 Examples of recorded trajectories of larval movements in the absence (Control) and

presence of the ciliate Euplotes minuta (Ciliate Treatment).


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5.3.2SwimmingBehaviourBefore&AfterDirectContact

Examining the changes in behaviour at a finer scale of resolution immediately before and after

direct contact between larva and ciliate, both velocity and the change in angle were significantly

higher in the seconds following direct contact (Fig. 5.4A, C). The mean velocity prior to direct

contact was 2.75 ± 0.16 mm s-1, whereas following direct contact the mean velocity

significantly increased to 3.82 ± 0.32 mm s-1 (p = 0.02) (Fig. 5.4A). The mean change in angle

Figure 5.3 Comparison of mean values (A, B, C) and coefficient of variation (D, E, F) in swimming

parameters between control and ciliate treatments (Mean ± SE). Means with different subscripts

indicate significant difference at α = 0.05.


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also significantly increased from 34.22 ± 5.76o to 51.66 ± 6.41o (p = 0.05) following direct

contact with Euplotes mintua (Fig. 5.4C). Acceleration was higher following direct contact

although not significantly different (p = 0.57). There were no significant differences in the

coefficient of variation of swimming variables (Fig. 5.4D, E, F).

Figure 5.4 Comparison of mean values (A, B ,C) and coefficient of variation (D, E, F) in swimming parameters

4 seconds before and after direct contact between larva and ciliate (Mean ± SE). Means with different

subscripts indicate significant difference at α = 0.05.


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Example trajectories of competent G. caespitosa larvae before and after direct contact with

Hypotrich ciliate E. minuta are shown in Figure 5.5. Heat maps generated from larvae tracked

for this analysis highlight the increase in velocity following instances of direct contact. Turn

angle distributions of larvae observed before and after direct contact revealed a stronger

tendency to the smaller angles prior to contact (10o - 30o), indicative of larvae continuing in

more or less the same direction (Fig. 5.6). In contrast, following direct contact, larger angles

were more frequent (30o - 40o) signifying larger changes in direction (Fig. 5.6).

Figure 5.5 Examples of larvae trajectories during instances of direct contact between ciliate and larva. Heat

maps and associated line graphs display the significant increase in velocity following these events.


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5.4Discussion

The processes occurring immediately before the settlement of benthic invertebrate larvae are

fundamental to determining the distribution, abundance and dynamics of adult populations on

marine hard substrata. Complex behavioural mechanisms based on interaction with a range of

physical factors in part determine where settlement occurs and at what intensity. This study is

the first to experimentally show a behavioural response of invertebrate larvae in the presence of

ciliates. Through direct video observation of in situ surface exploration by Galeolaria

caespitosa larvae, it was possible to quantify a suite of behavioural swimming parameters.

The same touching and crawling behaviours observed here have been previously reported for G.

caespitosa larvae (Marsden & Anderson 1981). This type of behaviour is indicative of the

larvae ‘testing’ or ‘sampling’ the substrate. Larvae of polychaete worms possess an apical tuft

of elongate and somewhat stiff cilia. Pre-settlement behaviour typically includes swimming near

the substratum with their apical ends downward so that the apical tuft brushes or is pressed

against the substratum (Marsden & Anderson 1981; Hadfield et al. 2014). Thus the apical tuft,

Figure 5.6 Mean change in angle distributions before and after direct larva/ciliate contact.


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together with its underlying cells, has long been suspected to be the site of detection of

substrate-associated cues for settlement. Crawling behaviour is thought to begin once the larvae

have detected the stimuli associated with an optimal settlement site, and is the prelude to

permanent attachment and metamorphosis (Hadfield et al. 2014). This association was

substantiated by Hadfield et al. (2014), who identified that instances of Hydriodes elegans

larvae crawling were almost exclusively observed in settings where the larvae could contact a

biofilm known to possess settlement inducting stimuli.

Results clearly demonstrated that the presence of the Hypotrich ciliate, Euplotes minuta, can

significantly influence larval exploration behaviours. The analysis of swimming behaviour

revealed that in the presence of ciliates the larvae were making significantly larger changes in

angle during exploration (Fig. 5.3C). Interestingly the coefficient of variation in the change in

angle was significantly higher in the control treatment (Fig. 5.3F), perhaps suggesting that in the

absence of ciliates the larvae were less constrained in their behaviour, and so explored the

substrate with greater variability. When ciliates were present, however, the larvae were more

restricted in their exploration, avoiding groups of grazing ciliates and when the typical

touching/crawling behaviour was interrupted most larvae reacted by circling at consistent

angles. Upon examining the patterns of larval exploration before and after direct contact, it was

revealed that the rapid circling behaviour was almost exclusively initiated following contact

between ciliate and exploring larva. Following these events a clear disturbance to the typical

touching/crawling exploration behaviours was observed, with the larvae significantly increasing

their velocity and variation in angle in response to the contact (Fig. 5.4).

Hadfield et al. (2014) highlighted the importance of larvae making contact with biofilms, and

the behavioural transition from straight swimming and circling to crawling in response to this

contact. The findings of this study suggest that the presence of ciliates cause this natural

behavioural progression to reverse, i.e. the larvae lift off the substrate and resume the


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swimming/circling behaviours. Therefore, essentially the presence of ciliates is deterring the

larvae from making contact with the substrate. This disturbance inhibits the larvae’s ability to

detect surface bound cues from biofilm bacteria and conspecific adults required to initiate a

settlement response.

The consequences of disrupting the exploration process delaying settlement can have profound

impacts on settlement/metamorphic success and post settlement health. Larval settlement and

metamorphosis in many benthic invertebrates is often regarded as two distinct events, but in G.

caespitosa they are interlocked (Marsden & Anderson 1981). Metamorphosis begins before

settlement, but is not completed unless successful settlement is achieved. Larval metamorphosis

of G. caespitosa begins with the collapse of the prototroch cells (girdle of cilia) (Marsden &

Anderson 1981). This event robs the larva of its chief means of locomotion, and restricts them

to only weak swimming/crawling (Marsden & Anderson 1981). Importantly, this transition can

have serious implications on the selection of a settlement site. If the larvae are not in close

proximity to a suitable settlement site at this developmental stage, successful settlement and

metamorphosis is unlikely to occur.

The fate of invertebrates after successful metamorphosis is also determined, to a large degree,

by factors experienced during the larval exploration stage (Qian & Pechenik 1998; Qian 1999).

Rates of development, growth and survival are compromised for many marine invertebrates if

individuals have experienced a delay in their metamorphosis (Bhaud & Cha 1994; Qian &

Pechenik 1998; Qian 1999). Qian & Pechenik (1998) found that delaying the metamorphosis of

Hydroides elegans had a dramatic impact on juvenile growth and survival. Settled juveniles

resulting from larvae that were delayed for 3, 6 and 9 days were significantly smaller than

larvae in the controls allowed to metamorphose on day 0. Furthermore, mortality rates of

juveniles after attachment were higher the longer larval life was prolonged (Qian & Pechenik

1998).


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In this study the number of exogenous factors was minimised to focus on the impact of direct

interactions between larva and ciliate. Under laboratory conditions larvae are confined to a dish,

which differs from the reality in nature where larvae are free to explore other settlement sites.

Under natural conditions it is likely the larvae would simply continue searching until they

encounter an area of substrate relatively free from ciliates. This could explain spatial

distributions of invertebrates in nature, where natural aggregations of ciliates result in reduced

settlement in certain areas. Obviously in nature there are many additional factors that could

potentially influence larval behaviours prior to settlement. The patterns of pre-settlement

behaviour observed in future investigations would ideally examine the interaction under natural

field conditions. Nonetheless, this study represents an important first step in examining how

larval behaviour is influenced by the presence of ciliates.

In Chapter 4 it was determined that the physical presence of ciliates was required to inhibit

settlement of G. caespitosa, excluding possible mechanisms of influence linked to chemical

cues from ciliates and/or bacteria. In follow up, this study has successfully demonstrated a

mechanism of influence that could explain the settlement inhibition observed in Chapter 4. A

similar mechanism of swimming disruption observed here between G. caespitosa and E. minuta

could also conceivably extend to other invertebrate and ciliate species examined in Chapter 3.

Increasing our understanding of how pre-settlement behaviour impacts substrate selection will

accelerate the identification of surface cues that moderate or counteract settlement. Variation in

the behavioural response to external cues has implications for estimating dispersal profiles and

understanding recruitment variation, both of which are key processes in the dynamics of marine

populations. Furthermore, the development of methods focused on the substrate selection and

exploration stages of invertebrate settlement may have valuable applications in aquaculture

husbandry and the development of sustainable antifoulant coatings.

6. Colonisation and succession of microbial biofilm assemblages on antifoulants

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Chapter6 Colonisation and succession of microbial biofilm assemblages on

antifouling and fouling-release coatings

6.1Introduction

Biofouling is defined as the undesirable accumulation of organic material and a multitude of

other forms of life on man-made surfaces (Cooksey & Wigglesworth 1995). In the marine

environment, more than 4000 species are recognised as macro-fouling organisms (Almeida et

al. 2007). Biofouling is a worldwide problem affecting a multitude of industrial process,

including increasing frictional drag on ships, increasing bulk of submerged structures, blocking

seawater pipe-lines, smothering monitoring equipment and promoting structural deterioration

(Fusetani 2004; Almeida et al. 2007). The cumulative cost of marine biofouling may run into

billions of dollars per year worldwide, which explains why the development of cost effective

control measures has been a focus of intense research for many years (Yebra et al. 2004;

Almeida et al. 2007; Maréchal & Hellio 2009).

Due to the vast array of organisms involved in marine biofouling and the complexity of the

fouling process, development of an effective control measure to combat biofouling remains a

major technical challenge (Yebra et al. 2004). Historically, the most effective approach to

inhibit biofouling has been to utilise toxic compounds (biocides). Biocide-releasing paints (BR)

are the most widespread solution to fouling of structures submerged in the marine environment

(Alberte et al. 1992; Yebra et al. 2004; Almeida et al. 2007). The development of copper-based

antifouling paints during the 20th century provided the first efficient and long-lasting solution to


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marine fouling (Yebra et al. 2004). Although many different antifouling paints were developed

during this period, the tributyltin self-polishing paints (TBT) proved to be the most effective.

These coatings function by continuously leaching biocides, killing the marine fouling organisms

that come into contact with the surface. Although generally effective, some biocides, especially

TBT, have been found to cause significant environmental impacts on other non-target species

(Terlizzi et al. 2000; Gibbs et al. 1991).

Growing environmental and health concerns on the unchecked usage of these chemical agents

have led to more stringent regulatory requirements in the use of biocide releasing antifoulants,

thus providing the impetus in recent times for the development of more environmentally

sustainable alternatives to biofouling control (Almeida et al. 2007; Marechal & Hellio 2009). A

new generation of environmentally friendly, fouling-release (FR) coatings has been developed

as an alternative to biocidal paints. The mechanisms of action of FR coatings are generally

attributed to physical surface properties that interfere with the adhesion of fouling organisms.

Fouling-release coatings are mainly represented by 3 base materials: fluoropolymers, silicones

and more recently hydrogels (reviewed by Yebra et al. 2004). These materials produce a low

surface energy, often amphiphilic, surface which functions by minimising the strength of the

adhesive bond between the organism and the treated surface (Chambers et al. 2006). Any

attached fouling organisms are then simply dislodged once the treated surface is moving beyond

a critical velocity (typically 10-20 knots) (Terlizzi et al. 2000; Chambers et al. 2006), or

removed by water spray and light brushing (Townsin & Anderson 2009).

Marine biofouling is a multistage and complex process involving an interplay between the

substrate and the organisms that (actively or not) choose a substratum for settlement in what is

often a crucial step in their future development (Terlizzi & Faimali 2009; Dobretsov et al.

2013). The development of the primary microbial biofilm, complex assemblages of single celled

organisms including bacteria, diatoms, fungi and protozoa, can subsequently modify the


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properties of the underlying substratum (Bressey & Lejars 2014). This process is an important

determinant of the efficiency of antifoulant treatments, particularly FR coatings, which depend

on retaining an amphiphilic, low-surface-energy surface as the interface with potential fouling

organisms and their adhesives (Molino et al. 2009b; Dobretsov & Thomason 2011).

The development of microbial biofilm assemblages on several surfaces, including BR and FR

marine coatings, has been subject to numerous investigations that have focussed on the

characteristics of initial microbial settlement (Dempsey 1981; Cassé & Swain 2006; Molino et

al. 2009a; Molino et al. 2009b; Dobretsov & Thomason 2011). However, these studies did not

examine the colonisation dynamics of ciliate assemblages which, despite being ubiquitous

within microbial biofilms, remain largely unknown. As shown in Chapter 2, ciliates are able to

rapidly colonise new substrata and over a short period of time reach high abundances within

microbial biofilms. Investigations of succession on marine substrata have contributed

considerably to an understanding of the biofouling process. Given the impacts of protozoan

activities within microbial biofilms (see Chapters 4 and 5), their role in the fouling dynamics on

antifoulant coatings should not be overlooked.

This study utilised non-destructive sample analysis techniques to investigate the initial

colonisation by fouling microbes, with special attention paid to the ciliate assemblages, on four

currently employed BR and FR coatings. Intersleek 970® and Hempasil X3® are two globally

adopted non-biocidal FR coatings. Intersleek 970 utilises a fluoropolymer top coat which

applies amphiphilic properties designed to interfere with the adhesives secreted by marine

foulers. The Hempasil X3 coating alternatively utilises a silicone hydrogel top coat. Hydrogels

are complex polymer networks which have the ability to bind large amounts of water. The high

water content results in a soft and wet structure with high hydrophobicity. Intersmooth 360® and

Interspeed 5640® both rely on the release of biocides from the paint matrix. Intersmooth 360

utilises copper (I) and copper (II) oxide and zinc pyrithione as its active biocides. In contrast,


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Interspeed 5640 is considered as a copper-free system as it contains no cuprous or cupric oxide,

instead utilising zinc pyrithione and EconeaTM (2-(p-chlorophenyl)-3-cyano-4-bromo-5-

trifluoromethyl pyrrole) as biocides against fouling.

6.2Methods

6.2.1StudySite&Sampling

The study took place at the Defence Science and Technology Organisation (DSTO) Marine

Coatings and Corrosion Test Facility on Booth Pier, Port Phillip Bay, Williamstown (Fig. 6.1),

Victoria, Australia (37º51’41.40”S, 144º54’38.06”E). Sampling was conducted from a floating

raft which lies in Hobsons Bay, the northernmost part of Port Phillip Bay which is a large inland

bay covering 2000 km2 with a narrow opening into Bass Strait. The site is approximately 6 m in

depth with a tidal range of approximately 1 m. Sampling was conducted in May-June 2013

using sealable polystyrene Petri-dish microscope slides (AnalyslideTM, Pall Corp.) as artificial

substrates for biofilm development.

Control treatments were uncoated Petri dish slides, while treatment slides were painted by brush

with one of four coatings: (a) Intersleek 970® (FR coating, International Paint), (b) Hempasil

X3® (FR coating, Hempel), (c) Intersmooth 360® (BR coating, International Paint) and (d)

Interspeed 5640® (BR coating, International Paint). The Intersleek 970 coating scheme was

Figure 6.1 Map of study site Booth Pier, Hobsons Bay, Williamstown.


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applied as follows. One coat of Intersleek BXA737/738/739 (0.4:0.75:0.1 by volume) was

applied followed by one coat of Intersleek FXA972/980/981 (4.0:0.75:0.25 by volume). The

Hempasil X3 coating scheme required one coat Hempasil Nexus X-Tend 27500 (single

component) followed by one coat Hempasil X3 87509/98950 (17.8:2.2 by volume). The two BR

coatings, Intersmooth 360 and Interspeed 5640, required two coats of the primer Intershield

300® (International Paint) to be applied before the application of the top coat, Intersmooth 360

(single component) and Interspeed 5640 (single component). Each coat was applied in

accordance with the manufacturer’s specifications. To ensure coating thickness was in an

acceptable range, the thicknesses of dry films were cross checked on metallic substrates coated

at the same time as the Petri dishes. Differences in surface roughness and colour between

coatings were beyond our control and not accounted for. Once cured, sample slides were soaked

in distilled water for several days prior to deployment.

A total of 20 replicate Petri dish slides of each treatment were prepared. Each slide was then

randomly inserted vertically into a specially designed polyvinyl chloride (PVC) panel (Fig. 6.2).

Four panels were prepared in this manner, and then attached to aluminium frames and

suspended from a raft to a depth of 1 m below the water surface. Petri dish slides were sampled

at 3 and 10 weeks. On each sampling two PVC panels, holding 10 replicates of each treatment,

were collected. The slides were sealed with a cap under the water surface and placed into a cool

box for transportation to the laboratory. During the deployment the average water temperature

was 12.3 ± 0.9oC the salinity was 34.2 ± 1.3 ppt.


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6.2.2Identification&Enumeration

Five replicate slides of each treatment were randomly assigned for bacterial/diatom or ciliate

analyses. Slides intended for bacterial and diatom enumeration were preserved in 2%

glutaraldehyde and stored at 4°C until analysis. Bacterial and diatom densities were determined

by removing the glutaraldehyde, adding 50 µL of DAPI (4’6’-diamidino-2-phenylindole)

solution (0.02 mg mL-1) and a 22 × 40 mm cover glass over the top (Porter & Feig 1980). Cells

were counted at 1000 × magnification via oil immersion on an epifluorescence microscope

(Leica DM2500) with UV excitation 340-380 nm. At least 20 fields of view were randomly

selected and manually counted per replicate slide. To avoid error associated with edge effects,

care was taken to avoid the periphery of petri-dish slides during counting.

Ciliates were initially observed live at 6× magnification under a stereomicroscope (Leica

MZ9.5) to determine the classifications and abundances of sessile ciliate genera. Live

observations also gave insight into the behaviour, movement and ecological niches occupied by

vagile and planktonic ciliates in the samples. After live observation the samples were fixed in

2% glutaraldehyde solution within 1-2 h of sample collection. Identification and enumeration of

Figure 6.2 Panel design illustrating how each individual Petri dish slide was held in place by rails mounted

on the PVC panel which was then attached to an aluminium frame and suspended vertically at 1 m

underneath a raft.


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motile ciliates were made by quantitative protargol staining (QPS) techniques. The fixed

samples were gently rinsed from the dishes, and sub samples were concentrated onto cellulose

filters which were then embedded in agar. The filters were then post-fixed in 10% Bouin’s

solution prior to protargol impregnation. Staining followed the QPS method described by

Skibbe (1994).

Protargol impregnations were mounted in Permount and examined under a phase contrast

microscope (Leica DM2500) at 100–1250× magnification to reveal details of kinetid patterns and

other morphological characteristics required for genus level identification. Specimens were

photographed and catalogued for taxonomic classification using published keys by Lee et al.

(2000) and Lynn (2008). The ciliate abundances were determined based on the counts of sub

samples taken from the 5 replicates of each treatment to confirm cell densities (cells cm-2).

Enumeration was conducted at 200× magnification with the entirety of each slide carefully

examined for counts.

6.2.3DataAnalysis

Abundance data were log10 (x + 1) transformed when required to reduce heterogeneity of

variances. A repeated-measures ANOVA (SYSTAT 13 Software, Inc. 2009) was used to

establish whether there were significant differences in genera abundances/composition between

time points, and among substrate treatments. Where one of these factors or an interaction was

significant at α = 0.05, Tukey pairwise comparisons were run.

Multivariate analyses of ciliate assemblages observed on each treatment were performed with

Primer 6.0 (Plymouth Marine Laboratory) software. To prevent bias caused by highly abundant

taxa, ciliate assemblage data were transformed prior to analysis. Square-root transformations

down-weighted the importance of the highly abundant taxa, allowing rarer taxa to exert some

influence in the diversity and similarity calculations. Square-root transformed ciliate data


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normalized by Bray-Curtis similarity were used in an analysis of similarities (ANOSIM) to

identify significant differences of ciliate composition with regard to substrate treatment. Spatial

and temporal patterns in assemblage structure were examined via hierarchal cluster analysis and

non-metric multidimensional scaling (MDS) ordinations.

6.3 Results

6.3.1 Bacteria Densities

Differences in bacterial abundance among substrate treatments were statistically significant (p <

0.001, Fig. 6.3). The difference between the substrate treatments was also apparent in the

structures of the developing bacterial biofilms (Fig. 6.4). In the control, bacteria had attached

across most of the available surface in the Petri dishes, while colonisation of the FR coatings

seemed, at least initially, limited to certain patches on the treated surface (Fig. 6.4). Intersleek

970 exhibited the most rapid fouling by bacteria of the four fouling control coatings tested,

reaching (3.36 ± 0.39) × 104 cells cm-2 at its peak. Bacterial abundance at week 3 was

significantly reduced on BR coatings in comparison to control and FR treatments (Fig. 6.3). The

abundances on both FR coatings Intersleek 970 and Hempasil X3 were not significantly

different to controls at week 10; however, the BR paints Intersmooth 360 and Interspeed 5640

still exhibited significantly lower bacterial abundance.


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6.3.2DiatomDensities

Differences in diatom abundance between substrate treatments were statistically significant (p <

0.001, Fig. 6.5). Intersleek 970 exhibited the most rapid fouling by diatoms of the four

antifoulant coatings tested, reaching 219.6±58.6 cells cm-2 at its peak. Diatom abundance at

week 3 was significantly reduced on BR coatings (Intersmooth 360; Interspeed 5640) in

Figure 6.3Mean total bacteria abundance observed on each coating at 3 and 10 weeks (Mean ±

SE). Means with different subscripts indicate significant difference at α=0.05.

Figure 6.4 Examples of bacteria colonisation on control, fouling‐release and biocide release


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comparison to control and FR treatments (Fig. 6.5). The abundances on the FR coating

Intersleek 970 was not significantly different to controls at week 10; however the FR coating

Hempasil X3 and the BR paints Intersmooth 360 and Interspeed 5640 still exhibited

significantly lower diatom abundance.

6.3.3CiliateAssemblage

Differences in total ciliate abundance among substrate treatments were statistically significant

(p < 0.001, Fig. 6.6). The control exhibited significantly higher ciliate abundances than all

fouling control treatments at week 3. The FR coating Intersleek 970 exhibited the most rapid

fouling by ciliates of the four coatings, reaching 63.3 ± 5.9 cells cm-2 at week 3. In contrast the

BR coatings (Intersmooth 360; Interspeed 5640) proved highly effective against ciliate fouling,

restricting ciliate abundance to 0.02 ± 0.01 cells cm-2 at 3 weeks (Fig. 6.6). At 10 weeks ciliate

abundance had increased significantly on BR coatings (Intersmooth 360; Interspeed 5640).

Figure 6.5 Total diatom abundance observed on each coating at 3 and 10 weeks (Mean ± SE).

Means with different subscripts indicate significant difference at α=0.05.


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However, the abundance still remained significantly lower than that observed on the control and

FR treatments (p < 0.001). The abundances on the two FR coatings (Intersleek 970; Hempasil

X3) were no longer significantly different from each other after 10 weeks, as the abundance on

the Hempasil X3 coating increased (Fig. 6.6). Total ciliate abundance had decreased on the

control treatments, and the abundance on the FR treatments was no longer significantly different

to the control (p > 0.93).

A total of 15 genera from 10 orders were identified during the 10 week deployment (Table 6.1).

Sessile ciliates were represented by species of the orders Sessilida and Heterotrichida. Vagile

forms (free swimming ciliates adapted for life on substrates) were represented primarily by the

orders Euplotida and Pleurostomatida. Planktonic taxa (ciliates that primarily swim above the

substrate, occasionally feeding upon attached bacteria) were represented by the orders

Philasterida and Pleuronematida. All of the ciliate genera observed during this study were

present on Control and FR treatments at some point during the week 3 and 10 samplings.

Figure 6.6 Total ciliate abundance observed on each coating at 3 and 10 weeks (Mean ± SE). Means

with different subscripts indicate significant difference at α=0.05


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Ciliates from the genera Follicilina, Diophrys, Lacrymaria and Dysteria remained absent from

the assemblage on the BR coatings (Intersmooth 360; Interspeed 5640) throughout the

deployment period (Table 6.1).

At week 3 the combined sessile ciliates dominated the assemblage on the controls (68.4% of the

total ciliate abundance) and on the FR coatings (up to 54.0%) (Fig. 6.7A), this included ciliates

from the orders Sessilida and Heterotrichida (Fig. 6.8A). Vagile ciliates had low abundance in

comparison; however, they were the primary contributors to the variation in genera diversity

(Fig. 6.7C). Conversely, no ciliates were attached or even closely associated with the BR coated

surfaces after 3 weeks of immersion. Only a small number of planktonic ciliates, which only

intermittently come into contact with the substrate for grazing, were present on the BR coatings

at this time (Pleuronematida, Fig. 6.8A). At week 10 there was a shift in the assemblage as the

abundance of sessile ciliates was heavily reduced (Fig. 6.7B). The vagile ciliates, however, only

increased in abundance on controls and FR coatings (Intersleek 970; Hempasil X3), accounting

Table 6.1 Peak abundances of ciliate genera and the functional group they occupy; observed on each

substrate treatment.

S = Sessile; V = Vagile; P = Planktonic


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for approximately 60% of the total ciliate abundance (Fig. 6.7B). The assemblages on the BR

paints (Intersmooth 360; Interspeed 5640) at week 10 had begun to resemble those observed on

the controls and FR treatments at week 3, with sessile ciliates dominating the assemblage in

terms of abundance over vagile and planktonic forms (Fig. 6.7B).

Figure 6.7 Variation in the relative abundances (A, B) and genera number (C, D) of ciliate functional groups between the different substrate treatments at week 3 (A, C) and week 10 (B, D).


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A dendrogram (Figure 6.9) highlights the development of the ciliate assemblages on all

substrate treatments across both samplings. Control and FR treatments grouped together at 75%

similarity (Group II, Figure 6.9). Biocide-release coatings at week 3 remained highly dissimilar

to all other assemblages at all-time points (Group III, Figure 6.9). At week 10 the similarity had

increased as assemblages developed on the BR coatings; nevertheless, they still only reached

50% similarity to the assemblages present on the control and FR treatments (Group I, Figure

6.9).

Figure 6.8 Variation in the relative abundances of ciliate orders between the different substrate treatments at week 3 (A) and week 10 (B).


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Analysis of similarities (ANOSIM) revealed that the generic composition of the ciliate assemblages

were significantly different between substrate treatments at 0.1% significance. At week 3 there was

no significant difference in the structure of ciliate assemblages present on the two FR coatings and

similarly the two BR coatings. However, the control treatment displayed a significantly different

assemblage structure than all of the fouling control treatments (Table 6.2). At 10 weeks despite the

total ciliate abundance no longer being significantly different between control and FR treatments

(Fig. 6.6), the generic composition remained significantly different. The ciliate assemblages present

on the two FR and BR treatments at 10 weeks were still not significantly different to each other

(Table 6.2).

Figure 6.9 Dendrogram of ciliate assemblage similarities based on Bray‐Curtis similarity matrix of square‐root transformed genera abundances on each coating at 3 (dashed lines) and 10 (solid lines) weeks.


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The similarity percentage (SIMPER) analysis (Table 6.3) breaks down the contribution of the most

abundant ciliate genera to the observed dissimilarities between substrate treatments. At week 3 the

sessile ciliate Vorticella was the primary contributor to the dissimilarity in abundance/occurrence,

accounting for > 20% of the total dissimilarity between ciliate assemblages developed on the

control and antifoulant coatings (Table 6.3). The vagile ciliates Amphileptus, Aspidisca and

Euplotes also represented high contributions to the dissimilarity due to their high frequency of

occurrence/abundance on the control and FR treatments while being absent on the toxic BR

coatings (Table 6.3). At week 10 the dissimilarity between assemblages on each treatment had

reduced as ciliates continued to colonise the antifoulant coatings. Despite Vorticella remaining the

most abundant ciliate genus, it was no longer the primary contributor to the dissimilarity between

assemblages. The vagile genera Holosticha and Amphileptus alone accounted for 33.31% of the

total dissimilarity between assemblages on the control and FR coatings (Table 6.3). The

dissimilarities between the ciliate assemblages on the control and BR coatings were more evenly

spread as the assemblages were late to develop on the toxic BRF coatings. Ciliates from the genus

Dysteria were the primary contributors to the dissimilarity between the FR and BR coatings,

accounting for 10.98% of the total dissimilarity (Table 6.3).

Table 6.2 Analysis of similarity (ANOSIM) evaluation of variation in ciliate assemblages between substrate treatments at weeks 3 and 10 (using Bray‐Curtis similarity matrix), computed from square root transformed ciliate genera abundances.


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6.4Discussion

Microbial fouling is a process which begins immediately upon immersion in the ocean. Due to

their great abundance in the water column, various microbes will come into contact with

immersed substrates with high frequency. It is likely that individual cells from many species

colonise the substrates during this period. However, over the ensuing days, only those species

able to tolerate the properties presented by the particular substrate will proliferate. As observed

here, bacteria grew quickly on each antifoulant coating, forming a biofilm that subsequently

induced further recruitment of micro- and macro-foulers. Although biofilm colonisation in the

first 3 weeks was inhibited by both FR and BR coatings to varying extents, after an additional

seven weeks, bacterial, diatom and ciliate colonisation was only significantly inhibited by the

BR coatings.

Intersleek 970 exhibited the most rapid fouling of all four coatings. With the exception of ciliate

abundance at week 3, the rate of bacterial, diatom and ciliate colonisation on the Intersleek 970

Table 6.3 SIMPER analysis evaluating the contribution of abundant ciliate genera to total dissimilarity (as percentages) between ciliate assemblages on control (CON), fouling‐release (FR) and biocide release (BR) coatings. Values in bold indicate highest contribution (%). Genera contributing < 2% of total dissimilarity are not shown.


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coating was at no point significantly different to the control treatment. Comparing the

effectiveness of the two FR coatings, at week 3 bacteria and ciliate colonisation was

significantly reduced on the Hempasil X3 coating compared to the Intersleek 970 (Figs. 6.4 &

6.8). The antifouling mechanisms of hydrogel coatings are not well understood. Inspired by the

antifouling properties observed on soft body marine organisms, hydrogels are a form of

biomimetic antifouling treatment. Investigating the effectiveness of hydrogels on barnacle

settlement, Murosaki et al. (2011) suggested that the hydration of the polymer chain may

interrupt the adhesion of the cementing proteins, thereby affecting the settlement behaviours of

fouling invertebrates. Perhaps in a similar manner the adhesive properties of the EPS produced

by the pioneering bacteria were reduced, effectively delaying the development of a microbial

biofilm on the substrate. Despite the observed reduction in settlement at 3 weeks, after 10 weeks

of immersion there were no longer any significant differences in the total abundance of bacteria

or ciliates between the two FR coatings.

Overall despite an initial delay in colonisation, the amphiphilic properties of both FR coatings

tested here presented little barrier to ciliate colonisation and growth, with patterns of

colonisation sharing high similarity to control treatments (Fig. 6.9). Large colonies of the sessile

peritrichs Zoothamnium and Vorticella were among the first ciliates to colonise the substrates

and rapidly increase in abundance (Table 6.1; Fig. 6.7). During the early stages of assemblage

development, the combined sessile ciliates dominated the assemblage in terms of abundance

(Fig. 6.7A). As the biofilm assemblage matured, the dominance of the sessile ciliates was

reduced, allowing the vagile ciliates to exert a greater influence on the assemblage structure.

The hypotrichs, such as Aspidisca and Euplotes specifically adapted for life on substrates,

thrived and were the main contributor to the diversity of the ciliate assemblage (Fig. 7.8). This

pattern of ciliate colonisation is consistent with what was observed in Chapter 2, and other

ciliate assemblage analyses (Coppellotti & Matarazzo 2000; Gong et al. 2005; Xu et al. 2009).

Hence despite the amphiphilic properties of the FR coatings, they will hold comparable ciliate


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assemblages to those developing on polystyrene Petri dishes and glass (Gong et al. 2005; Xu et

al. 2009; Watson et al. 2015).

The main changes observed in the ciliate assemblages on the control and FR coating treatments

between weeks 3 and 10 were associated with the sessile Peritrich ciliates. The abundance of the

sessile Peritrich ciliates was heavily reduced between samplings (Fig. 6.7B). In contrast, the vagile

and planktonic genera present appeared to reach a state of equilibrium, with few new recruits and

little change in abundance of the already established genera. This phenomenon has been reported in

other ciliate assemblage studies (Gong et al. 2005; Xu et al. 2009). The time required for ciliate

colonisation to reach a state equilibrium is heavily dependent upon environmental variability, but

typically takes between 1 and 4 weeks (Gong et al. 2005; Xu et al. 2009). This considerable shift in

abundance is perhaps due to the sessile ciliates having to compete for space on the substrate with

colonising invertebrates, which are induced to settle as the biofilm matures. The motile vagile and

planktonic taxa conversely had the advantage of not being limited by substrate availability and thus

readily adjusted to the new substrate condition. It is also possible that the reduction of sessile

ciliates was due to an environmental disturbance similar to that observed in Chapter 2.

Environmental disturbances do not necessarily have a uniform effect on all ciliate taxa. Certain

types of disturbance, such as the heavy rainfall observed in Chapter 2, may have selectively

reduced the abundance of the dominant sessile ciliates.

The non-toxic nature of the FR coatings likely enabled a larger range of ambient species present

in the water column to be prospective colonisers should they contact the substratum. Fouling-

release coatings rely upon shear forces applied to the treated substrate to remove fouling

organisms. The static experimental conditions, aside from minimal currents generated from tidal

and wave action, meant that little to no shear force was applied to the developing microbial

biofilms during the deployment. Further studies conducted under variable flow conditions

would be required to comprehensively evaluate the efficacy of the Intersleek 970 and Hempasil


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X3 FR coatings. However, there are factors which may promote bacterial, diatom and ciliate

colonisation on FR coatings. Attractive hydrophobic interactions between the bacterial cell wall

and the substrate have been demonstrated to facilitate adhesion of marine bacteria (Mueller et

al. 1992; Bakker et al. 2004; Ista et al. 2004). Following the initial substrate contact, many

species begin producing extracellular polymers (EPS) of variable physical and chemical

properties. The EPS acts as a strong adhesive ensuring that the pioneering population of bacteria

are permanently attached to the surface (Marshall 1992). Once the initial bacterial biofilm is

formed, the amphiphilic properties of the FR coatings are likely reduced, allowing other

prospective colonisers to settle with little inhibition.

In contrast, the heavily inhibited bacterial, diatom and ciliate colonisation observed on the

biocide release coatings, compared with that recorded on the FR coatings, is likely the result of

the toxicity to potential colonisers (Trevors & Cotter 1990; Madoni & Romeo 2005; Molino et

al. 2009a). Consequently, the number of bacteria, diatom and ciliate species able to tolerate and

subsequently colonise the biocidal coatings will naturally be less than the number of species

able to colonise the non-toxic FR coatings. However, although biofilm colonisation was heavily

inhibited at the initial sampling, after several weeks bacterial, diatom and ciliate abundances on

the biocidal coatings had increased significantly, albeit still with significantly lower abundances

than that observed on control and FR coating treatments. Interestingly, although the toxicity of

the BR coatings to prospective colonisers was evident throughout the deployment, after 10

weeks of immersion the ciliate assemblage structure had begun to resemble that observed on the

control and FR coating treatments at 3 weeks (Fig. 5.7B; Fig. 5.8B). The sessile Peritrich

ciliates Vorticella and Zoothamnium dominated the assemblages in terms of abundance, while

vagile and planktonic genera accounted for much of the diversity.

The ability of fouling organisms to eventually tolerate and colonise biocidal antifoulant coatings

may be linked to the biosorptive properties of the pioneering microbial assemblage. The


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biosorption of metal ions into components of the biofilm, such as extracellular polymers, cell

membranes and cell walls, has been reported in numerous studies (Cooksey 1994; Langley &

Beveridge 1999; Nies 1999; Teitzel & Parsek 2003). The biosorptive components of microbial

biofilms sequester the toxic compounds released by the antifoulant, thereby providing an

opportunity for the successful colonisation of BR treated substrates. It has been suggested that

the two most important biosorptive components of microbial biofilms are dead cells and EPS

originating from the pioneering bacterial colonisers (Lewis 2001; Teitzel & Parsek 2003;

Harrison et al. 2007).

Dead cells are frequently ignored components of biofilms that are interspersed at varying ratios

with live cells in microbial biofilms (Harrison et al. 2007). Here we saw that the toxicity of both

BR coatings was very high during the first 3 weeks of colonisation (Fig. 6.4). Although the

staining protocol utilised does not provide information relating to the presence of dead cells, it

is reasonable to speculate that many of the pioneering microbial colonisers on these coatings

were killed following exposure to elevated concentrations of the active biocide present in each

coating. These dead cells then become chemically reactive biomasses that act as biosorptive

sites, protecting subsequent colonisers from the metal toxicity by sequestering or precipitating

the reactive metal ions released by the coating (Harrison et al. 2007). Similar biosorptive

properties have been linked to the EPS, specifically the polysaccharide components, produced

by many species of bacteria (Teitzel & Parsek 2003; Iyer et al. 2004). Species that are highly

resistant and/or tolerant to the biocides present in the BR coatings successfully colonise the

substrate and begin producing EPS, which envelopes the pioneering population and ensure

strong adhesion to the substrate. Heavy metal ions depleted from the antifoulant coating are then

partially bound within this EPS matrix. This effectively retards the diffusion of the active

biocides into the biofilm, allowing for the successful colonisation of less resistant fouling

microbe species.


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While the biosorption properties of the pioneering microbial assemblage may have eventually

enabled ciliate colonisation, the toxicity was still sufficient to generate selective pressures on

the structure of the ciliate assemblages. Ciliates from the genera Diophrys, Folliculina,

Lacrymaria and Dysteria, common on the control and FR treatments were absent from the

assemblage on the BR coatings (Table 6.1). Laboratory toxicity tests, based on ciliates, are

scarce and have primarily been carried out on freshwater species. Results obtained in these

toxicity studies suggest large variations in the tolerance to heavy metal exposure between

different ciliate species (Al-Rasheid & Sleigh 1994; Coppellotti 1994; Madoni 2000; Madoni &

Romeo 2006). However, despite the variation in tolerance, in all cases heavy metal ions have

been shown to be very harmful to ciliates even at low concentrations (Al-Rasheid & Sleigh

1994; Madoni & Romeo 2006). The tolerances of the aforementioned ciliate genera are

unknown, and there are no distinctive common physical or behavioural characteristics which

may explain their absence.

One of the most noteworthy observations of this study is that there were no significant

differences in microbial settlement between the two BR coatings tested. Intersmooth 360

utilising copper (I) / copper (II) oxide and zinc pyrithione as its active biocides. While

Interspeed 5640 is a copper free coating, utilising zinc pyrithione and EconeaTM as biocides

against fouling. This could have important implications for reducing the reliance on copper-

based biocidal paints. Adequate performance of copper-free systems may ultimately reduce

copper loadings in ports and harbours, ultimately enabling ship operators and the broader

maritime community to comply with increasingly stringent restrictions on the use of copper

based antifoulants.

The patterns of microbial fouling observed here parallels strongly with results of other

investigations of the microbial fouling of FR and BR coatings (Molino et al. 2009a; Molino et

al. 2009b; Dobretsov & Thomason 2011). For example, Molino et al. (2009a) found that within


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2-4 days after immersion, a FR coating (Intersleek 700®) displayed the quickest microbial

colonisation in comparison with biocidal coatings (Intersmooth 360 and Super Yacht 800).

Upon conclusion of the deployment, the percentage of bacterial cover on the FR coating was

1.3-2 times higher than on the biocidal coatings. Bacteria are accepted to be the initial, primary

colonising organisms on man-made surfaces in marine environments. Their ability to enable

colonisation of otherwise highly toxic or amphiphilic substrates is noteworthy (Cassé & Swain

2006; Dobretsov & Thomason 2011; Molino et al. 2009a). The results of this study suggest that

the presence of ciliates was highly correlated with successful bacterial colonisation, not only in

enabling colonisation, but as a source of prey on which the ciliates thrive.

In order to further develop alternative environmentally sustainable biofouling control measures

a better understanding of the variables which influence the fouling process, and the organisms

involved, is required. This is the first detailed study of the abundances and taxonomic

composition of the ciliate assemblages that colonise BR and FR coatings. The extent to which

ciliate assemblages provide selective pressures on the structure and species composition of

microbial biofilms, and their influence on the subsequent recruitment of fouling invertebrates

could play a pivotal role in shaping the fouling assemblage on an immersed substrate (see

Chapters 4 & 5). Further investigation into the influence of ciliates on important fouling

invertebrate species is warranted to better understand the processes behind this interaction.

Furthermore, a greater understanding of the colonisation dynamics of natural ciliate

assemblages, and the mechanisms by which they influence invertebrate settlement, may

accelerate the identification of novel surface cues that moderate or counteract the fouling

process.

7. Conclusions and future directions

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Chapter7

ConclusionsandFutureDirections

Interest in the settlement dynamics of marine invertebrates largely relates to its detrimental

effects on man-made structures and as a critical step in the intensive culture of invertebrates for

human consumption (Almeida et al. 2007; Pillay & Kutty 2005). The information to deliver

new surface-based technologies with applications for aquaculture and biofouling is in high

demand. Amidst the many investigations is an increasing recognition that the development of

such technologies to either promote or deter the settlement of marine invertebrates can only be

achieved through a greater understanding of the processes occurring within microbial biofilms.

The importance of physical and chemical cues originating from microbial biofilms on the

settlement and metamorphosis of invertebrate larvae has been well documented (Wieczorek &

Todd 1998; Hadfield & Paul 2001; Qian et al. 2010). However, the dynamics of protozoa in

these communities remains largely unknown (Arndt et al. 2003). The aim of this thesis was to

examine the importance of ciliated protozoa, an often overlooked but ubiquitous component of

microbial biofilms, and characterise their influence on the settlement of marine invertebrates.

The major outcomes of this study are:

1. Characterisation of the taxonomic structure and succession of natural ciliate

assemblages in temperate Australia, during two contrasting seasons on neutral

substrates and on four recently developed antifoulant coatings (Chapters 2 and 6).


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2. Development of a reliable and reproducible static settlement assay protocol for

Galeolaria caespitosa (Chapter 3).

3. Determining the species specificity of the influence that ciliated protozoa have on

invertebrate settlement (Chapter 4).

4. The identification and characterisation of a direct mechanism of influence ciliated

protozoa have on invertebrate settlement (Chapter 5).

7.1NaturalCiliate.Assemblages

Drawing any conclusions regarding the influence of ciliates on microbial biofilm dynamics and

subsequent impacts on invertebrate settlement first required the taxonomic differentiation of ciliate

assemblages within microbial biofilms. Ciliate assemblage analyses were carried out on neutral

substrates across different seasons and on four recently developed foul-release and biocide-release

antifoulant coatings. Despite these works being based on examinations over different seasons and

on substrates of differing properties, the succession of ciliate assemblages colonising the

experimental substrates followed a similar pattern. The sessile Peritrichs were among the first

ciliates to colonise the substrates and rapidly increased in abundance, quickly dominating the

assemblage in terms of abundance. Over time vagile species, particularly the Hypotrichs,

specifically adapted for life on substrates, steadily increased in abundance and accounted for much

of the ciliate diversity within the assemblage. Once the biofilm had matured to the point that

marine invertebrates were induced to settle, the Peritrichs were displaced as competition for

substrate space intensified, leaving the free-swimming vagile species to dominate the assemblage.

While this pattern of ciliate succession was consistent across experimental substrates, the

species composition and rate of succession was significantly influenced by natural disturbances

and the properties presented by the different antifoulant coatings. Heavy rainfall and a


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subsequent increase in turbidity selectively reduced the abundance of the dominant sessile

species, which were out-competing the vagile and planktonic species. This event abruptly

changed the typical course of succession, as the vagile species took advantage of the new

substrate condition, and subsequently exerted greater presence within the assemblage much

earlier in the development of the microbial biofilm.

Colonisation and succession were also influenced by the antifoulant coatings to varying extents.

The amphiphilic properties of the foul-release coatings presented little barrier to ciliate

colonisation and growth, only causing an initial delay in colonisation. In contrast, ciliate

colonisation was heavily inhibited by the biocide release coatings. The tolerances of marine

ciliates to heavy metal ions are largely unknown; however, results suggest that heavy metal ions

were very harmful to biofilm-dwelling ciliates even at low concentrations. The toxicity of these

substrate treatments had a significant impact upon the structure of the ciliate assemblages as

tolerances of individual genera varied. Certain genera common to the other non-toxic

experimental substrates were absent from the assemblage altogether on these coatings.

Despite being a ubiquitous component of microbial biofilms, very little was known about the

dynamics of natural ciliate assemblages in the marine environment. This study was the first to

characterise biofilm-dwelling ciliate assemblages in temperate Australian waters. Results

highlighted some of the factors involved in shaping a microbial biofilm assemblage, many of

which do not have a uniform effect on all taxa. In light of these findings, when evaluating

patterns of succession it is important to examine ciliate assemblages across a multitude of

environmental conditions and time scales. This approach would lead to a better understanding

of the processes generating the ever-shifting mosaic of marine fouling communities on hard

substrata. Through a greater understanding of the colonisation dynamics of natural ciliate

assemblages, and the mechanisms by which they influence invertebrate settlement, it may one


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day be possible to interpret and predict the recruitment and distribution of sessile marine

invertebrates in-part based on the local ciliate assemblage.

7.2SpeciesSpecificityofCiliateInfluence

We had previously established that a mixed assemblage of ciliates can significantly inhibit the

settlement of a range of fouling invertebrates (Shimeta et al. 2012). The species specificity of

this inhibition was examined in Chapter 4, with individual ciliate species chosen from those

abundant taxa identified in natural marine assemblages (Chapter 2). The selected genera

represented ciliates from both vagile and planktonic niches. However, while no differences were

detected in the settlement response of Galeolaria caespitosa and Mytilus galloprovincialis

induced by ciliates from either ecological niche, there were significant differences in the levels

of their settlement inhibition caused by the different ciliates species. Settlement rates were

correlated with ciliate abundance, revealing significant inverse relationships. This illustrated a

density-dependence and confirmed that some action of the ciliates was the cause of the

settlement inhibition. However, the correlation coefficients of this relationship were weak,

indicating that ciliate abundance alone cannot explain the differences in settlement inhibition.

These results support the conclusion that there were species-specific factors mediating the

extent of the settlement inhibition.

The results of this study further support the hypothesis that biofilm ciliates are an important

factor influencing the variability of invertebrate recruitment over time and space, and ultimately

the structure and dynamics of natural invertebrate assemblages. The ciliate assemblages that

dwell within marine biofilms are highly varied (Chapters 2 and 6). As such, ciliate assemblages

will display a variety of differences in behaviour, morphology and grazing preferences, all of

which may have variable impacts on the settlement response of invertebrate larvae. This

suggests that the recruitment of certain invertebrates onto an available patch of substratum can

depend on the ciliate fauna present at that point in the succession of the biofilm community, and


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further emphasises the importance of understanding the taxonomic structure and successional

dynamics of ciliate assemblages on immersed substrates.

Ultimately, increased knowledge on the dynamics of ciliate assemblages, and the influences of

each species present on the settlement of invertebrates could have meaningful implications not

only in understanding recruitment variation, but in improving the efficiency of mollusc

aquaculture and the development of sustainable antifouling technologies. The potential for

improvements to mollusc aquaculture husbandry is particularly apparent. To successfully settle

invertebrate larvae under culture conditions artificial substrates are prepared to induce larval

settlement. Substrates are exposed to seawater and sunlight to promote the establishment and

growth of biofilms (Hone et al. 1998). The introduction of free-swimming competent larvae into

settlement tanks is tightly linked to biofilm monitoring practices, with particular reverence

given to the growth of benthic diatoms (Hone et al. 1998). Incorporating techniques to

manipulate or control the presence of ciliates, which may influence settlement, could increase

the efficiency of larval settlement and subsequent metamorphosis under culture conditions. This

approach might further reduce the losses currently experienced by intensive culture operations

during this critical phase.

7.3MechanismsofCiliateInfluence

The previous chapters have examined the structure of natural ciliate assemblage and determined

the species specificity of their influence on larval settlement. Investigating the mechanism of

this influence, whether ciliates are affecting larval settlement indirectly by influencing the

structure, abundance or community composition of microbial biofilms, or directly through

physical or chemical interactions with larvae, is essential to build a better understanding of the

processes involved in invertebrate settlement. In this study two potential direct mechanisms of

influence were investigated. In Chapter 4 no evidence was found that a chemical cue from the

ciliates, which deters settlement, similar to negative cues released by some bacteria and algae


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(Lau & Qian 1997; Qian et al. 2007), was responsible for the observed settlement inhibition.

Only in the physical presence of the ciliates was settlement significantly inhibited. The fact that

the physical presence of ciliates was required to inhibit larval settlement pointed to the

possibility of direct interactions occurring between larvae and ciliates. This hypothesis formed

the basis of Chapter 5, where larval exploration swimming patterns in the presence/absence of

ciliates were quantified.

In the absence of ciliates, Galeolaria caespitosa larvae tended to swim close to the bottom,

frequently pausing to touch their apical tuft to the biofilm surface before lifting off again. This

touching behaviour is a form of substrate testing. Once the larvae have detected the stimuli

associated with an optimal settlement site the larvae transition into crawling across the

substrate, a behaviour which is thought to be the prelude to permanent attachment and

metamorphosis (Hadfield et al. 2014). Examining the patterns of larval exploration in the

presence of ciliates, it was revealed that direct contact with ciliates was causing this natural

behavioural progression to reverse. The net effect of which was to deter the larvae from making

contact with the substrate, subsequently inhibiting their ability to detect surface-bound cues

required to initiate a settlement response and thereby delaying settlement.

Results reported in Chapter 5 successfully demonstrated a mechanism of influence that could

explain the settlement inhibition observed in previous chapters, and represents the first time that

a behavioural response of invertebrate larvae caused by the presence of ciliates has been

reported. It is well known that pre-settlement larval behaviour can have profound implications

for larval dispersal and recruitment (Raimondi & Keough 1990; Morgan & Anastasia 2008;

Burgess et al. 2009). Increasing our understanding of how pre-settlement behaviour is impacted

by ciliates will accelerate the identification of surface cues that may enhance or counteract

settlement. Variation in the behavioural response to external cues has implications for


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estimating dispersal profiles and understanding recruitment variation, both of which are key

processes in the dynamics of marine fouling assemblages.

7.4FutureDirections

This thesis has revealed the complexities of the colonisation and succession of ciliates in

microbial biofilms, and the influence of these assemblages on invertebrate settlement. Based on

these outcomes various issues have emerged that are worthy of further investigation:

1. Quantitative characterisation and analysis of the impacts of sessile peritrich ciliates on

invertebrate settlement dynamics.

The sessile peritrich ciliates were by far the most abundant ciliates in natural assemblages

(Chapter 2). Unlike the vagile and planktonic ciliates analysed in this thesis, the interactions

between peritrichs and exploring larvae would be unique as the ciliates would be permanently

anchored to the substrate. Under these conditions the influence of ciliates may not only be based

on their presence, they may also be competing for available space on the substrate. In addition,

sessile peritrichs utilise their cilia to generate feeding currents, and it may be possible that

larvae swimming or sensory abilities are influenced by feeding currents generated by large

colonies of peritrichs on the substrate.

Several velocimetry methods have been applied to investigate flow dynamics induced by

microorganisms, including the highly abundant sessile ciliates identified in Chapter 2 from the

genera Vorticella and Zoothamnium (e.g. Vopel et al. 2002; Nagai et al. 2009). Utilising these

methods it would be possible to examine how the flow, generated by colonies of sessile ciliates,

influences the dispersion of dissolved settlement cues emanating from a substrate. In addition,

tracking larvae swimming patterns in the presence/absence of the turbulent flow generated by


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sessile ciliates would reveal the potential of another physical mechanism of influence ciliates

have on invertebrate settlement behaviour.

2. Determining the species specific influence of ciliates on additional invertebrates, including

other important fouling species and those cultured commercially.

In our previous study (Shimeta et al. 2012) a mixed assemblage of ciliates had a range of

impacts on different invertebrate species. The settlement of some invertebrates was enhanced

while others were not influenced by the presence of ciliates. Further investigation into the

influence of ciliates on other important fouling and aquaculture species is warranted to better

understand the mechanistic processes behind this interaction.

3. Observing and quantifying the impacts of larval/ciliate interactions during exploratory

behaviour under natural conditions.

In the artificial lab situation larvae are confined to a dish which differs from the reality in nature

where larvae are free to explore other settlement sites. Re-entering the water column would

allow the larvae to search over a greater area, with the possibility of encountering habitats with

varying, and perhaps more favourable, ciliate assemblages elsewhere. Furthermore, under

natural conditions there are many additional factors that could potentially influence larval

behaviours prior to settlement. Observations of this interaction under more natural conditions

and determining under what conditions protozoa will have a significant effect on invertebrate

recruitment in nature will further improve knowledge on the recruitment and distribution of

sessile marine invertebrates.

This could be achieved by observing settlement of larvae on substrates with increasing

complexity. The introduction of additional biotic (e.g. diatoms, fungi, mixed assemblages of

microbes) and abiotic (e.g. flow, temperature, substrate type) variables to laboratory settlement


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assays will better reflect the complex trophic interactions occurring in natural biofilms, and

reveal the extent to which ciliates influence invertebrate settlement in nature.

4. Identifying potential indirect mechanisms of influence with more natural bacterial biofilm

assemblages.

In Chapter 4 it was shown that concentrated grazing by Euplotes minuta was significantly

altering the biofilm architecture, creating areas of clearance on the substrate. Other studies have

also demonstrated that protozoan grazing can significantly alter bacterial assemblage

compositions (Corno 2006; Corno & Jürgens 2006; Wey et al. 2008). Further investigation with

mixed bacterial assemblages could measure the presence/absence of bacterial species, and their

relative abundances to determine the impact of ciliate grazing. In the event that the assemblages

are altered by ciliate grazing, the bacteria that were strongly altered by the protozoan presence

(either enhanced or reduced) could be isolated, and utilised in additional assays to determine

whether those species significantly induce or inhibit larval settlement.

Page | 130

References

Abarzua S. & Jakubowski S. (1995) Biotechnological investigation for the prevention of

biofouling. I. Biological and biochemical principles for the prevention of biofouling.

Marine Ecology Progress Series, 123, 301-312.

Alberte R.S., Snyder S. & Zahuranec B. (1992) Biofouling research needs for the United

States Navy: program history and goals. Biofouling, 6, 1-95.

Aldred N., Ista L.K., Callow M.E., Callow J.A., Lopez G.P. & Clare A.S. (2005) Mussel

(Mytilus edulis) byssus deposition in response to variations in surface wettability.

Journal of the Royal Society Interface, 22, 37-43.

Almeida E., Diamantino T.C. & Sousa O. (2007) Marine paints: The particular case of

antifouling paints. Progress in Organic Coatings, 59, 2-20.

Al-Rasheid K.A.S. & Sleigh M. (1994) The effects of heavy metals on the feeding rate

of Euplotes mutabilis (Tuffrau, 1960). European Journal of Protistology, 30,

270-279.

Andersson A., Lee C., Azam F. & Hagstrom A. (1985) Release of amino acids and

inorganic nutrients by heterotrophic marine microflagellates. Marine Ecology

Progress Series, 23, 99-106.

Arndt H., Schmidt-Denter K., Auer B. & Weitere M. (2003) Protozoans and biofilms. In:

Fossil and recent biofilms: a natural history of life on earth (eds. Krumbein W.E.,

Patterson D.M. & Zavarzin G.A.). Kluwer Academic Press, Norwell, MA, pp. 161-180.

Augspurger C., Gleixner G., Kramer C. & Kusel K. (2008) Tracking carbon flow in a 2-

week-old and 6-week-old stream biofilm food web. Limnology and Oceanography, 53,

642-650.

Page | 131

Ayo B., Santamarı´a E., Latatu A., Artolozaga I., Azu´a I. & Iriberri J. (2001) Grazing rates

of diverse morphotypes of bacterivorous ciliates feeding on four allochthonous bacteria.

Letters in Applied Microbiology, 33, 455-460.

Bakker D.P., Postmus B.R., Bisscher H.J. & van der Mei H.C. (2004) Bacteria strains

isolated from different niches can exhibit different patterns of adhesion to substrata.

Applied Environmental Microbiology, 70, 3758-3760.

Bauer R.E., Shafrin E.G. & Zisman W.A. (1968) Adhesion: Mechanisms that assist or

impede it. Science, 162, 1360-1368.

Baxter G. & Morse D.E. (1987) G protein and diaclyglicerol regulate metamorphosis of

planktonic molluscan larvae. Proceedings of the National Academy of Sciences, 84,

1867-1870.

Bernard C. & Rassoulzadegan F. (1990) Bacteria or microflagellates as a major food source

for marine ciliates: possible implications for the microzooplankton. Marine Ecology


Berninger U.G., Finlay B.J. & Kuuppo-Leinikki P. (1991) Protozoan control of bacterial

abundances in freshwater. Limnology and Oceanography, 36, 139-147.

Berntsson K.M., Jonsson P.R., Lejhall M. & Gatenholm P. (2000) Analysis of behavioural

rejection of micro-textured surfaces and implications for recruitment by the barnacle

Balanus improvisus. Journal of Experimental Marine Biology and Ecology, 251, 59-83.

Bhadury P. & Wright P.C. (2004) Exploitation of marine algae: biogenic compounds for

potential antifouling applications. Planta, 219, 561-578.

Bhaud M. & Cha J.H. (1994) Larvae-substrate relationships of Eupolymnia nebulosa

(Montagu), (Polychaeta, Terebellidae): An experimental analysis. Memoirs of the

Museum of Comparative Zoology, 162, 371-381.

Boenigk J. & Arndt H. (2002) Bacterivory by heterotrophic flagellates: community

structure and feeding strategies. Antonie Van Leeuwenhoek 81, 465-480.

Page | 132

Boenigk J., Matz C., Jurgens K. & Arndt H. (2001a) Confusing selective feeding with

differential digestion in bacterivorous nanoflagellates. Journal of Eukaryotic

Microbiology, 48, 425-432.

Boenigk J., Matz C., Jürgens K. & Arndt H. (2001b) The influence of preculture conditions

and food quality on the ingestion and digestion process of three species of heterotrophic

nanoflagellates. Microbial Ecology, 42, 168-176.

Bonar D.B., Coon S.L., Walch M., Weiner R.M. & Fitt W. (1990) Control of oyster

settlement and metamorphosis by endogenous and exogenous chemical cues. Bulletin of

Marine Science, 46, 484-498.

Bressy C. & Lejars M. (2014) Marine fouling: an overview. The Journal of Ocean

Technology, 9, 19-28.

Bryan P.J., Kreider L.J., Qian P.Y. & Chia F.S. (1997) Induction of settlement and

metamorphosis of the serpulid polychaete Hydroides elegans by conspecific associated

and pharmacological compounds. Marine Ecology Progress Series, 146, 81-90.

Burgess S.C., Hart S.P. & Marshall D.J. (2009) Pre-settlement behaviour in larval bryozoans:

the roles of larval age and size. Biological Bulletin, 216, 344-354.

Butman C.A. (1989). Sediment-trap experiments on the importance of hydrodynamical

processes in distributing settling invertebrate larvae in near-bottom waters. Journal of

Experimental Marine Biology and Ecology, 134, 37-88.

Cairns J., Dickson K.L. & Yonge W.H. (1971) The consequences of nonselective periodic

removal of portions of freshwater protozoa communities. Transactions of the American

Microscopical Society, 90, 71-80.

Carey P.G. (1992) Marine interstitial ciliates an illustrated key. Chapman & Hall, NY.

Carl C., Poole A.J., Vucko M.J., Williams M.R., Whalan S. & de Nys R. (2011) Optimising

settlement assays of pediveligers and plantigrades of Mytilus galloprovincialis.

Biofouling, 27, 859-868.

Page | 133

Casse F. & Swain G.W. (2006) The development of microfouling on four commercial

antifouling coatings under static and dynamic immersion. International

Biodeterioration & Biodegradation, 57, 179-185.

Chambers L.D., Stokes K.R., Walsh F.C. & Wood R.J.K. (2006). Modern approaches to

marine antifouling coatings. Surface & Coatings Technology, 201, 3642-3652.

Chan A.L.C. & Walker G. (1998) The settlement of Pomatoceros lamarckii larvae

(Polychaeta: Sabellida: Serpulidae): A laboratory study. Biofouling, 12, 71-80.

Characklis W.G., McFeters G.A. & Marshall K.C. (1990) Physiological ecology in biofilm

systems. In: Biofilms (eds. Characklis W.G. & Marshall K.C.). John Wiley & Sons,

New York, pp. 341-394.

Chaw K.C., Dickinson G.H., Ang K.Y., Deng Y. & Birch W.R. (2011) Surface exploration

of Amphibalanus amphitrite cyprids on microtextured surfaces. Biofouling, 27, 413-422.

Chet I., Asketh P. & Mitchell R. (1975) Repulsion of bacteria from marine surfaces. Applied


Clare A.S., Rittschof D., Gerhart D.J. & Maki J.S. (1992) Molecular approaches to non-toxic

antifouling. International Journal of Invertebrate Reproduction Development, 22, 67-

76.

Clare A.S., Thomas R.F. & Rittschof D. (1995). Evidence for the involvement of cyclic AMP

in the pheromonal modulation of barnacle settlement. Journal of Experimental Biology,

198, 655-664.

Cloete T.E., Jacobs L. & Brozel V.S. (1998) The chemical control of biofouling in industrial

water systems. Biodegradation, 9, 23-37.

Connell J.H. (1978) Diversity in tropical rain forests and coral reefs. Science, 199, 1302-

1310.

Cooksey D.A. (1994) Molecular mechanisms of copper resistance and accumulation in

bacteria. FEMS Microbiology Reviews, 14, 381-386.

Page | 134

Cooksey K.E. & Wigglesworth B. (1995) Adhesion of bacteria and diatoms to surfaces in the

sea. Aquatic Microbial Ecology, 9, 87-96.

Coppellotti O. (1994) Effects of cadmium on Uronema marinum (Ciliophora,

Scuticociliatida) from Antarctica. Acta Protozoologica, 33, 159-167.

Coppellotti O., Matarazzo P. (2000) Ciliates colonisation of artificial substrates in the

Lagoon of Venice. Journal of Marine Biological Association of the UK, 80, 419-427.

Corno G. & Jürgens K. (2006) Direct and indirect effects of protist predation on

population size structure of a bacterial strain with high phenotypic plasticity.

Applied Environmental Microbiology, 72, 78-86.

Corno G. (2006) Effects of nutrient availability and Ochromonas sp. predation on size and

composition of a simplified aquatic bacterial community. FEMS Microbiology and

Ecology, 58, 354-363.

Costerton J.W., Lewandowski D.E., Caldwell D.R. & Lappin-Scott H.M. (1995) Microbial

biofilms. Annual Review Microbiology, 49, 711-745.

Dahlstrom M., Jonsson H., Jonsson P.R. & Elwing H. (2004) Surface wettability as a

determinate in the settlement of the barnacle Balanus Improvisus (Darwin). Journal of


Dang H.Y. & Lovell C.R. (2000) Bacterial primary colonization and early succession on

surfaces in marine waters as determined by amplified rRNA gene restriction analysis

and sequence analysis of 16S rRNA genes. Applied Environmental Microbiology, 66,

467-475.

Daume S., Brand-Gardner S. & Woelkerling W.J. (1999) Settlement of abalone larvae

(Haliotis laevigata Donovan) in response to non-geniculate coralline red algae

(Corallinales, Rhodophyta). Journal of Experimental Marine Biology and Ecology, 234,

125-143.

de Nys R. & Steinberg P.D. (2002) Linking marine biology and biotechnology. Current

Opinion in Biotechnology, 13, 244-248.

Page | 135

Del-Giorgio P.A., Gasol J.M., Vaque D., Mura P., Agusti S. & Duarte C.M. (1996)

Bacterioplankton community structure: Protist control net production and the

proportion of active bacteria in a coastal marine community. Limnology and

Oceanography, 41, 1169-1179.

Dempsey M.J. (1981) Colonisation of antifouling paints by marine bacteria. Botanica

Marina, 24, 185-191.

Dexter S.C. (1979) Influence of substratum critical surface tension on bacterial adhesion –

in situ studies. Journal of Colloid and Interface Science, 70, 346-354.

Di Fino A., Petrone L., Aldred N., Ederth T., Liedberg B. & Clare A.S. (2014) Correlation

between surface chemistry and settlement behaviour in barnacle cyprids (Balanus

improvises). Biofouling, 30, 143-152.

Dobrestov S. & Thomason J.C. (2011) The development of marine biofilms on two

commercial non-biocidal coatings: a comparison between silicone and fluoropolymer

technologies. Biofouling, 27, 869-880.

Dobrestov S., Raeid M.M. & Teplitski M. (2013) Mini-review: inhibition of biofouling by

marine organisms. Biofouling, 29, 423-441.

Eddison J.C. & Ollason J.G. (1978) Diversity in constant and fluctuating environments.

Nature, 275, 309-310.

Elbourne P.D., Veater R.A. & Clare A.S. (2008) Interaction of conspecific cues in Balanus

amphitrite Darwin (Cirripedia) settlement assays: Continues argument for the single-

larva assay. Biofouling 24, 87-96.

Fenchel T. (1980) Suspension feeding in ciliated protozoa: functional response and particle

size selection. Microbial Ecology, 6, 1-11.

Fenchel T. (1988) Marine plankton food chains. Annual Review of Ecology, Evolution

and Systematics, 19, 19-38.

Page | 136

Finlay B.J. & Esteban G.F. (1998) Freshwater protozoa: biodiversity and ecological

function. Biodiversity and Conservation, 7, 1163-1186.

Franco C., Esteban G.F., Tellez C. (1998) Colonization and succession of ciliated protozoa

associated with submerged leaves in a river. Limnologica, 28, 275-283.

Fusetani N. (1997) Marine natural products influencing larval settlement and metamorphosis

of benthic invertebrates. Current Organic Chemistry, 1, 127-152.

Fusetani N. (2004) Biofouling and antifouling. Natural Product Reports, 21, 94-104.

Garnick E. (1978) Behavioural ecology of Strongylocentrotus droebachiensis (Muller)

(Echinodermata: Echinoidea). Aggregating behavior and chemotaxis. Oecologia, 37,

77-84.

Gibbs P.E., Bryan G.W. & Pascoe P.L. (1991) TBT-induced imposex in the dogwelk,

Nucella lapillus: geographical uniformity of the response and effects. Marine

Environment Research, 32, 79-87.

Gilbert P., Evans D.J., Evans E., Duguid I.G. & Brown M.R. (1991) Surface characteristics

and adhesion of Escherichia coli and Staphylococcus epidermidis. Journal of Applied.

Bacteriology, 71, 72-77.

Gong J., Song W.B. & Warren A. (2005) Periphytic ciliates colonization: annual cycle and

responses to environmental conditions. Aquatic Microbial Ecology, 39, 159-170.

Gonzalez J.M., Sherr E.B. & Sherr B.F. (1990) Size-selective grazing on bacteria by natural

assemblages of estuarine flagellates and ciliates. Applied Environmental Microbiology,

56, 583-589.

Gubner R. & Beech I.B. (2000) The effect of extracellular polymeric substances on the

attachment of Pseudomonas NCIMB 2021 to AISI 304 and 316 stainless steel.


Hadfield M.G. (1986) Settlement and recruitment of marine invertebrates: a perspective and

some proposals. Bulletin of Marine Science, 39, 418-425.

Page | 137

Hadfield M.G. (2011) Biofilms and marine invertebrate larvae: what bacteria produce that

larvae use to choose settlement sites. Annual Review of Marine Science, 3, 453-479.

Hadfield G. & Huang S. (2003) Composition and density of bacterial biofilms determine

larval settlement of the polychaete Hydroides elegans. Marine Ecology Progress Series,

260, 161-172.

Hadfield G.M. & Koehl M.R.R (2004) Rapid behavioural responses of an invertebrate larva

to dissolved settlement cue. Biological Bulletin, 207, 28-43.

Hadfield M.G., Nedved B.T., Wilbur S. & Koehl M.A.R. (2014) Biofilm cue for larval

settlement in Hydroides elegans (Polychaeta): is contact necessary? Marine Biology,

161, 2577-2587.

Hadfield M.G. & Paul V.J. (2001) Natural chemical cues for settlement and metamorphosis

of marine-invertebrate larvae. In: Marine Chemical Ecology (eds. McClintock J.B. &

Baker B.J.). CRC Press, Florida, pp. 431-461.

Hadfleld M.G. & Pennington J.T. (1990) Nature of the metamorphic signal and its internal

transduction in larvae of the nudibranch Phestilla sibogae. Bulletin of Marine Science,

46, 455-464.

Hahn M.W. & Hofle M.G. (2001) Grazing of protozoa and its effect on populations of

aquatic bacteria. Microbial & Ecology, 35, 113-121.

Hannan C.A. (1984) Planktonic larvae may act like passive particles in turbulent near-bottom

flows. Limnology and Oceanography, 29, 1108-1116.

Hans U., Dobretsov S. & Qian P.Y. (2004) The effect of bacterial and diatom biofilms on the

settlement of the byrozoan Bugula neritina. Journal of Experimental Marine Biology

and Ecology, 313, 191-209.

Harder T., Lam C. & Qian P.Y. (2002) Induction of larval settlement in the polychaete

Hydroides elegans by marine biofilms: an investigation of monospecific diatom films as

settlement cues. Marine Ecology Progess Series, 229, 105-112.

Page | 138

Harrison J.J., Ceri H. & Turner R.J. (2007) Multimetal resistence and tolerance in microbial

biofilms. Natural Reviews Microbiology, 5, 928-938.

Havenhand J.N. & Svane I. (1991) Roles of hydrodynamics and larval behaviour in

determining spatial aggregation in the tunicate Ciona intestinalis. Marine Ecology


Head R.M., Overbeke K., Klijnstra J., Biersteker R. & Thomason J.C. (2004) The effect of

gregariousness in cyprid settlement assays. Biofouling, 19, 269-278.

Henschel J.R. & Cook P.A. (1990) The development of a marine fouling community in

relation to the primary film of microorganisms. Biofouling, 2, 1-11.

Herbert R.J.H. & Hawkins S.J. (2006) Effect of rock type on the recruitment and early

mortality of the barnacle Chthamalus montagui. Journal of Experimental Marine

Biology and Ecology, 334, 96-108.

Hickman C.P., Roberts L.S. & Larson A. (2001). Integrated Principles of Zoology. 11th

edition. The McGraw-Hill Companies, New York, USA. pp. 231-239.

Hoagland K.D., Rosowski J.R., Gretz M.R. & Roemer S.C. (1993) Diatom extracellular

polymeric substances: function, fine structure, chemistry, and physiology. Journal of

Phycology, 29, 537-566.

Holmes S.P., Sturgess C.J. & Davies M. (1997) The effect of rock-type on the settlement of

Balanus balanoides (L.) cyprids. Biofouling, 11, 137-147.

Holmström C. & Kjelleberg S. (1994) The effect of external biological factors on

settlement of marine invertebrate and new antifouling technology. Biofouling, 8,

147-160.

Huggett M.J., Williamson J.E., de Nys R., Kjelleberg S. & Steinberg P.D. (2006)

Larvae settlement of the common Australian sea urchin Heliocodaris

erythrogramma in response to bacteria from the surface of coralline algae.

Oecologia, 149, 604-619.

Page | 139

Hutchings P.A. (2003) Threatened species management: out of its depth for marine

invertebrates. In: Hutchings P.A. & Lunney D. (eds). Conserving marine environments:

Out of sight out of mind. Royal Zoological Society, NSW, pp. 81-88.

Huws S.A., McBain A.J. & Gilbert P. (2005) Protozoan grazing and its impact upon

population dynamics in biofilm communities. Journal of Applied Microbiology, 98,

238-244.

Iriberri J., Ayo B., Santamaria E., Barcina I. & Egea L. (1995) Influence of bacterial

density and water temperature on the grazing activity of two freshwater ciliates.

Freshwater Biology, 33, 223-231.

Ista L.K., Callow M.E., Finlay J.A., Coleman S.A., Nolasco A.C., Simons R.H., Callow

J.A. & Lopez G.P. (2004) Effect of substratum surface chemistry and surface energy

on attachment of marine bacteria and algal spores. Applied Environmental


Iyer A., Mody K. & Bhavanath J. (2004) Accumulation of hexavalent chromium by an

exopolysaccharide producing marine Enterobacter cloaceae. Marine Pollution

Bulletin, 49, 974-977.

Jackson S.M. & Jones E.B.G. (1991) Interactions within biofilms: the disruption of biofilm

structure by protozoa. Kieler Meeresforschungen Sonderheft, 8, 264-268.

Jain A. & Bhosle N. (2009) Biochemical composition of the marine conditioning film:

implications for bacterial adhesion. Biofouling, 25, 13-19.

Jenkins S.R. (2006) Larval habitat selection, not larval supply, determines settlement patterns

and adult distribution in two chthamalid barnacles. Journal of Animal Ecology, 74, 893-

904.

Jenkins S.R. & Martins G.M. (2010) Succession on hard substrata. In: Biofouling (eds. Durr

S. & Thomason J.C.). Wiley-Blackwell, UK, pp. 60-69.

Page | 140

Jensen R.A. & Morse D.E. (1984) Intraspecific facilitation of larval recruitment: gregarious

settlement of the polychaete Phragmatopoma californica (Fewkes). Journal of


Jensen R.A., Morse D.E., Petty R.L. & Hooker N. (1990) Artificial induction of larval

metamorphosis by free fatty acids. Marine Ecology Progress Series, 67, 55-71.

Jones A.C., Liao T.S.V., Najar F.Z., Roe B.A., Hambright K.D. & Caron D.A. (2013)

Seasonality and disturbance: annual pattern and response of the bacterial and microbial

eukaryotic assemblages in a freshwater ecosystem. Environmental Microbiology, 9,

2557-2572.

Jonsson P.R., Berntsson K.M. & Larsson A.I. (2004) Linking larval supply to recruitment:

flow-mediated control of initial adhesion of barnacle larvae. Ecology, 85, 2850-2859.

Judge M.L. & Craig S.F. (1997) Positive flow dependence in the initial colonization of a

fouling community: results from in situ water current manipulations. Journal of


Jurgens K. & Gude H. (1994) The potential importance of grazing-resistant bacteria in

planktonic systems. Marine Ecology Progress Series, 112, 169-188.

Khandeparker L., Anil A.C. & Raghukumar S. (2003) Barnacle larval destination: piloting

possibilities by bacteria and lectin interaction. Journal of Experimental Marine Biology

and Ecology, 289, 1-13.

Kiørboe T., Tang K., Grossart H.P. & Ploug H. (2003) Dynamics of microbial communities

on marine snow aggregates: colonization, growth, detachment and grazing mortality of

attached bacteria. Applied Environmental Microbiology, 69, 3036-3047.

Kirchman D., Graham S., Reish D. & Mitchell R. (1982) Bacteria Induce settlement and

metamorphosis of Janua (Dexlosplra) brasiliensis Grube (Polychaeta: Spirorbidae).

Journal of Experimental Marine Biology and Ecology, 56, 153-163.

Knight-Jones E.W. (1951) Gregariousness and some other aspects of the settling behaviour

of Spirorbis. Journal of the Marine Biological Association UK, 30, 201-222.

Page | 141

Knight-Jones E.W. & Crisp D.J. (1953) Gregariousness in barnacles in relation to the fouling

of ships and to antifouling research. Nature, 171, 1109-1110.

Kobak J. (2001) Light, gravity and conspecifics as cues to site selection and attachment

behaviour of juvenile and adult Dreissena polymorpha Pallas. Journal of Molluscan

Studies, 67, 183-189.

Koehl M.R.A. (2007) Mini review: Hydrodynamics of larval settlement into fouling

communities. Biofouling, 23, 357-368.

Lam C., Harder T. & Qian P.Y. (2005) Induction of larval settlement in the polychaete

Hydroides elegans by extracellular polymers of benthic diatoms. Marine Ecology

Progess Series, 286, 145-154.

Langley S. & Beveridge T.J. (1999) Metal binding by Pseudomonas neruginosa is influenced

by growth of the cells as a biofilm. Canadian Journal of Microbiology, 45, 616-622.

Larsson A.I. & Jonsson P.R. (2006) Barnacle larvae actively select flow environments

supporting post-settlement growth and survival. Ecology, 87, 1960-1966.

Lau S.C.K. & Qian P.Y. (1997) Larval settlement in the serpulid polychaete Hydroides

elegans in response to bacterial films: an investigation of the natural of putative larval

settlement cue. Marine Biology, 138, 321-328.

Lau S.C.K., Thiyagarajan V. & Qian P.Y. (2003) The bioactivity of bacterial isolates in

Hong Kong waters for the inhibition of barnacle (Balanus amphitrite Darwin)

settlement. Journal of Experimental Marine Biology and Ecology, 8, 43-60.

Lawrence J.R. & Snyder R.A (1998) Feeding behaviour and grazing impacts of a

Euplotes sp. on attached bacteria. Canadian Journal of Microbiology, 44, 623-

629.

Lee J.J., Leedale G.F. & Bradbury P. (2000) An Illustrated Guide to the Protozoa. Second ed.

Society of Protozoologists. Allen Press, Kansas.

Page | 142

Lee J.W., Nam J.H., Kim Y.H., Lee K.H. & Lee D.H. (2008) Bacteria communities in the

initial stage of marine biofilm formation and artificial surfaces. The Journal of


Leitz T. (1997). Induction of settlement and metamorphosis of Cnidarian larvae: Signals and

signal transduction. Invertebrate Reproduction and Development, 31, 109-122.

Letourneux F. & Bourget E. (1988) The importance of physical and biological settlement

cues used at different spatial scales by the larvae of the barnacle, Semibalancus

balanoides. Marine Biology, 97, 57–66.

Lewis K.R. (2001) Riddle of biofilm resistance. Antimicrobial Agents and Chemotherapy,

45, 999-1007.

Lynn D.H. (2008) The ciliated protozoa. Characterization, classification and guide to the

literature, Third ed. Springer, Berlin.

Madoni P. (2000) The acute toxicity of nickel to freshwater ciliates. Environmental

Pollution, 109, 53-59.

Madoni P. & Romeo M.G. (2005) Acute toxicity of heavy metals towards freshwater

ciliated protists. Environmental Pollution, 141, 1-7.

Maki J.C. & Mitchell R. (2002) Biofouling in the marine environment. In: Bitton G. (ed)

Encyclopedia of environmental microbiology. John Wiley & Sons, New York, pp. 610-

619.

Marechal J.P. & Hellio C. (2009) Challenges for the development of new non-toxic

antifouling solutions. International Journal of Molecular Sciences, 10, 4623-4637.

Marsden J.R. & Anderson D.T. (1981) Larval development and metamorphosis of the

serpulid polychaete Galeolaria caespitosa Lamarck. Australian Journal of Marine and

Freshwater Research, 32, 667-680.

Page | 143

Marshall D.J. & Keough M.J. (2003) Variation in the dispersal potential of nonfeeding

invertebrate larvae: the desperate larva hypothesis and larval size. Marine Ecology


Marshall D.J. & Steinberg P.D. (2014) Larval size and age affect colonization in a marine

invertebrate. The Journal of Experimental Biology, 217, 3981-3987.

Marshall K.C. (1992) Biofilms: an overview of bacterial adhesion, activity, and control at

surfaces. ASM News, 58, 202-207.

Maruzzo D., Conlan S., Aldred N., Clare A.S. & Høeg J.T. (2011) Video observation of

surface exploration in cyprids of Balanus amphitrite: the movements of antennular

sensory setae. Biofouling, 27, 225-239.

Matson P.G., Steffen B.T. & Allen R.M. (2010) Settlement behavior of cyphonautes larvae

of the bryozoan Membranipora membranacea in response to two algal substrata.

Invertebrate Biology, 129, 277-283.

Matz C. & Jurgens K. (2005) High motility reduces grazing mortality of planktonic

bacteria. Applied Environmental Microbiology, 71, 921-929.

McKenzie J.D & Grigolava I.V. (1996) The echinoderm surface and its role in preventing

microfouling. Biofouling, 10, 261-272.

Michel-Briand Y. & Baysse C. (2002) The pyocins of Pesudomonas aeruginosa. Biochemie,

84, 499-510.

Minchinton T.E. (1997) Life on the edge: conspecific attraction and recruitment of

populations to disturbed habitats. Oecologia, 111, 45-52.

Mitbavkar S. & Anil C. (2000) Diatom colonization on stainless steel panels in estuarine

waters of Goa, west coast of India. Indian Journal of Marine Sciences, 29, 273-276.

Molino P.J., Childs S., Hubbard M.R.E., Carey J.M., Burgman M.A. & Wetherbee R.

(2009a) Development of the primary bacterial microfouling layer on antifouling and

Page | 144

foul release coatings in temperate and tropical environments in Eastern Australia.


Molino P.J., Campbell E., & Wetherbee R. (2009b) Development of the initial diatom

microfouling layer on antifouling and fouling-release surfaces in temperate and

tropical Australia. Biofouling, 25, 685-694.

Moller S., Sternberg C., Andersen J.B, Christensen B.B., Ramos J.L, Givskov M. & Molin S.

(1998) In situ gene expression in mixed-culture biofilms: evidence of metabolic

interactions between community members. Applied Environmental Microbiology, 64,

721-732.

Morgan S.G. & Anastasia J.R. (2008) Behavioural tradeoff conserves transport while

increasing the risk of predation across the ranges of marine species. Proceedings of the

National Academy of Sciences, 105, 222-227.

Morse A.N.C., Froyd C.A. & Morse D.E. (1984) Molecules from cyanobacteria and red

algae that induce larval settlement and metamorphosis in the mollusc Haliotis rufescens.

Marine Biology, 81, 293-298.

Morse A.N.C. & Morse D.E. (1984) Recuritment and metamorphosis of Haliotis larvae

induced by molecules uniquely available at the surface of crustose red algae. Journal of


Morse A.N.C. (1991) How do planktonic larvae know where to settle? American Scientist,

79, 154-167.

Mueller R.F., Characklis W.G, Jones W.L & Sears J.T. (1992) Characterization of initial

events in bacterial surface colonization by two Pseudomonas species using image

analysis. Biotechnology and Bioengineering, 39, 1161-1170.

Murosaki T., Ahmed N. & Gong J.P. (2011) Antifouling properties of hydrogels. Science and

Technology of Advanced Materials, 12, 1-7.

Nagai M., Oishi M., Oshima M., Asai H. & Fujita H. (2009) Three-dimensional two-

component velocity measurement of the flow field induced by the Vorticella picta

Page | 145

microorganism using a confocal microparticle image velocimetry technique.

Biomicrofluidics, 3, 014105.

Nagaya K., Matsui Y., Ohira H., Yuasa A., Yamamoto H., Ohkawa K. & Magara Y. (2001)

Attachment strength of an adhesive nuisance mussel, Limnoperna fortunei, against

water flow. Biofouling, 17, 263-274.

Nies D.H. (1999) Microbial heavy-metal resistance. Applied Microbiology and

Biotechnology, 51, 730-750.

Norf H., Arndt H. & Weitere M. (2009) Responses of biofilm dwelling ciliate communities

to planktonic and benthic resource enrichment. Microbial Ecology, 57, 687-700.

Parry J.D. (2004) Protozoan grazing of freshwater biofilms. Advances in Applied


Patil J.S. & Anil A.C. (2005) Influence of diatom exopolymers and biofilms on

metamorphosis in the barnacle Balanus amphitrite. Marine Ecology Progress Series,

301, 231-245.

Patterson D.J. (1996) Free-living freshwater protozoa, Manson Publishing Ltd, London

UK.

Pawlik J.R. (1986). Chemical induction of larval settlement and metamorphosis in the reef-

building tube worm Phragmatopoma californica (Sabellariidae Polychaeta). Marine

Biology 91, 59-68.

Pawlik J.R. (1992) Chemical ecology of the settlement of benthic marine invertebrates.

Oceanography and Marine Biology Annual Review, 30, 273-335.

Pawlik J.R. & Chia F.S. (1991) Larval settlement of Sabellaria cementarium Moore, and

comparisons with other species of sabellariid polychaetes. Canadian Journal of

Zoology, 69: 765-770.

Page | 146

Pechenik J.A. & Qian P.Y. (1998) Onset and maintenance of metamorphic competence in the

marine polychaete Hydroides elegans Haswell in response to three chemical cues.

Journal of Experimental Marine Biology and Ecology, 226, 51-74.

Penniman J.R., Doll M.K. & Pires A. (2013) Neural correlates of settlement in veliger larvae

of the gastropod, Crepidula fornicata. Invertebrate Biology, 132, 14-26.

Pernthaler J. (2005) Predation on prokaryotes in the water column and its ecological

implications. Nature Reviews Microbiology, 3, 537-546.

Persson O.P., Pinhassi J., Riemann L., Marklund B.I., Rhen M., Normark S., Gonźalez J.M.

& Hagström A. (2009) High abundance of virulence gene homologues in marine

bacteria. Environmental Microbiology, 11, 1348-1357.

Pielou E.C. (1966) The measurement of diversity in different types of biological collections.

Journal of Theoretical Biology, 13, 131-144.

Pillay R.V.T. & Kutty M.N. (2005) Aquaculture Principles and Practices (second edition).

Blackwell Publishing Ltd. Oxford.

Pineda J., Riebensahm D. & Medeiros-Bergen D. (2002) Semibalanus balanoides in winter

and spring: larval concentration, settlement, and substrate occupancy. Marine Biology,

140, 789-800.

Posch T., Jezbera J., Vrba J., Simek K., Pernthaler J., Andreatta S. & Sonntag B.

(2001) Size selective feeding in Cyclidium glaucoma (Ciliophora,

Scuticociliatida) and its effects on bacterial community structure: A study from a

continuous cultivation system. Microbial Ecology, 42, 217-227.

Prendergast G.S. (2009) Settlement and behaviour of marine fouling organisms. In:

Biofouling (eds. Durr S. & Thomason C.J). Blackwell Publishing, UK, pp 30-59.

Preston S., Jabbar A., Nowell C., Joachim A., Ruttkowski B., Baell J., Cardno T., Korhonen

P.K., Piedrafita D., Ansell B.R., Jex A.R., Hofmann A. & Gasser R.B. (2015) Low cost

whole-organism screening of compounds for anthelmintic activity. International

Journal of Parasitology, 45, 333-343.

Page | 147

Qian P.Y. (1999) Larval settlement of polychaetes. Hydrobiologia, 402, 239-253.

Qian P.Y. & Pechenik J.A. (1998) Effects of larval starvation and delayed metamorphosis on

juvenile survival and growth of the tube-dwelling polychaete Hydroides elegans

(Haswell). Journal of Experimental Marine Biology and Ecology, 227, 169-185.

Qian P.Y., Lau S.C.K., Dahms H.U., Dobrestov S. & Harder T. (2007) Marine biofilms as

mediators of colonization by marine macroorganisms: Implications for antifouling and

aquaculture. Marine Biotechnology, 9, 399-410.

Qian P.Y., Xu Y. & Fusetani N. (2010) Natural products as antifouling compounds: recent

progress and future perspectives. Biofouling, 26, 223-234.

Raimondi P.T. (1988a) Rock type affects settlement, recruitment and zonation of the

barnacle Chthamalus anisopoma Pilsbury. Journal of Experimental Marine Biology and

Ecology, 123, 253-267.

Raimondi P.T. (1988b) Settlement cues and determination of the vertical limit of an intertidal

barnacle. Ecology, 69, 400-407.

Raimondi P.T (1991) Settlement behaviour of Chthamalus anisopoma larvae largely

determines the adult distribution. Oecologia, 85, 349-360.

Raimondi P.T. & Keough M.J. (1990) Behavioural variability in marine larvae. Australian

Journal of Ecology, 15, 427-437.

Ribblett S.G., Palmer M.A. & Coats D.W. (2005) The importance of bacterivorous

protists in the decomposition of stream leaf litter. Freshwater Biology, 50, 516-

526.

Risse-Buhl U., Scherwass A., Schlussel A., Arndt H., Krower S. & Kusel K. (2009)

Detachment and motility of surface associated ciliates at increased flow velocities.

Aquatic Microbial Ecology, 55, 209-218.

Page | 148

Rittschof D., Forward R.B., Cannon G., Weich J.M., McClary M., Holm E.R., Clare A.S.,

Conova S., McKelvey L.M., Bryan P. & van Dover C.L. (1998) Cues and context:

Larval responses to physical and chemical cues. Biofouling, 12, 31-44.

Roberts D., Rittschof D., Holm E. & Schmldt A.R. (1991) Factors influencing initial larval

settlement: temporal, spatial and surface molecular components. Journal of


Rodrίguez S.R., Ojeda F.P. & Inestrosa N.C. (1993) Settlement of benthic marine

invertebrates. Marine Ecology Progress Series, 97, 193-207.

Sanford E., Bermudez D., Bertness M.D. & Gaines S.D. (1994) Flow, food supply and acorn

barnacle population dynamics. Marine Ecology Progress Series, 104, 49-62.

Sanz-Lazaro C., Navarrete-Mier F. & Arnaldo M. (2011) Biofilm responses to marine fish

farm wastes. Environmental Pollution, 159, 825-832.

Scardino A.J., de Nys R., Ison O., O’Connor W. & Steinberg P.D. (2003) Microtopography

and antifouling properties of shell surface of the bivalve molluscs Mytilus

galloprovincialis and Pinctada imbricate. Biofouling, 19, 221-230.

Scardino A.J., Guenther J. & de Nys R. (2008) Attachment point theory: diatom attachments

on microtextured polyimide biomimics. Biofouling, 24, 45-53.

Schumacher J.F., Carman M.L., Callow M.E., Finaly J.A., Clare A.S. & Brennan A.B.

(2007) Species specific engineered antifouling topographies: Correlations between the

settlement of algal zoospores and barnacles cyprids. Biofouling, 23, 307-317.

Shanks A.L. & Shearman R.K. (2009) Paradigm lost? Cross-shelf distributions of intertidal

invertebrate larvae are unaffected by upwelling or downwelling. Marine Ecology


Shanks A.L. (1995) Mechanisms of cross-shelf dispersal of larval invertebrates. In: Ecology

of marine invertebrate larvae (ed. McEdward L.R.). CRC Press, Florida, pp. 323-368.

Page | 149

Sherr B.F., Sherr E.B. & Berman T. (1982) Decomposition of organic detritus: A

selective role for microflagellate protozoa. Limnology and Oceanography, 27(4),

765-769.

Sherr E.B. & Sherr B.F. (1987) High rates of consumption of bacteria by pelagic

ciliates. Nature, 325, 710-711.

Sherr E.B. & Sherr B.F. (1994) Bacterivory and herbivory: key roles of phagotrophic

protists in pelagic food webs. Microbial Ecology, 28, 223-235.

Sherr E.B. & Sherr B.F. (2002) Significance of predation by protists in aquatic

microbial food webs. Antonie van Leeuwenhoek, 81, 293-308.

Shikuma N.J., Pilhofer M., Weiss G.L., Hadfield M.G., Jensen G.J. & Newman D.K. (2014)

Marine tube worm metamorphosis induced by arrays of bacterial phage tail-like

structures. Science, 343, 529-533.

Shimeta J., Cutajar J., Watson M.G. & Vlamis T. (2012) Influences of biofilm-associated

ciliates on the settlement of marine invertebrate larvae. Marine Ecology Progress

Series, 449, 1-12.

Siboni N., Lidor M., Kramarsky-Winter E. & Kushmaro A. (2007) Conditioning film and

initial biofilm formation on ceramics tiles in the marine environment. FEMS

Microbiology Letters, 274, 24-29.

Skibbe O. (1994) An improved quantitative protargol stain for ciliates and other planktonic

protists. Archiv fur Hydrobiologie, 130, 339-347.

Slattery M. (1992) Larval settlement and juvenile survival in the red abalone (Haliotis

rufescens): an examination of inductive cues and substrate selection. Aquaculture, 102,

143-153.

Soares A.R., Da Gama B.A.P., Da Cunha A.P., Teixeira V.L. & Pereira R.C. (2008)

Induction of attachment of the mussel Perna perna by natural products from the brown

seaweed Stypopodium zonale. Marine Biotechnology, 10, 158-165.

Page | 150

Sousa W.P. (1984) The role of disturbance in natural communities. Annual Review of

Ecological Systems, 15, 353-391.

Sousa W.P. (2001) Natural disturbance and the dynamics of marine benthic communities. In:

Bertness M., Gaines S. & Hay M. (eds) Marine Community Ecology. Sinauer

Associates, New York, pp. 85-130.

Stoodley P., Sauer K., Davies D.G. & Costerton J.W. (2002). Biofilms as complex

differentiated communities. Annual Review of Microbiology, 56, 187-209.

Swanson R.L., de Nys R., Huggett M.J., Green J.K. & Steinberg P.D. (2006) In situ

quantification of a natural settlement cue and recruitment of the Australian sea urchin

Holopneustes purpurascens. Marine Ecology Progress Series, 314, 1-14.

Svane I., Havenhand J.N. & Jorgensen A.J. (1987) Effects of tissue extract of adults on

metamorphosis in Ascidia mentula (O.F. Muller) and Ascidiella scabra (O.F. Muller).

Journal of Experimental Marine Biology Ecology, 110, 171-182.

Svane I. & Young C.M. (1989) The ecology and behaviour of ascidian larvae. Oceanography

and Marine Biology an Annual Review, 27, 45-90.

Taylor W.D. (1983) A comparative study of the sessile, filter-feeding ciliates of several small

streams. Hydrobiologia, 98, 125-133.

Teitzel G.M. & Parsek M.R. (2003) Heavy metal resistance of biofilm and planktonic

Pseudomonas aeruginosa. Applied Environmental Microbiology, 69, 2323-2320.

Terlizzi A. & Faimali M. (2009) Fouling on artificial substrata. In: Biofouling (eds. Durr S.

& Thomason C.J). Blackwell Publishing, UK, pp 30-59.

Terlizzi A., Cont E., Zupo V. & Mazzella L. (2000) Biological succession on silicone

fouling release surfaces: Long term exposure tests in the harbour of Ischia, Italy.


Page | 151

Thiyagarajan V. (2010) A review on the role of chemical cues in habitat selection by

barnacles: New insights from larval proteomics. Journal of Experimental Marine

Biology and Ecology, 392, 22-36.

Toonen R.J. & Pawlik J.R. (1993) For a marine tube worm, all larvae are not created equal.

American Zoologist 33, 118-128.

Toonen R.J. & Pawlik J.R. (1994) Foundations of gregariousness. Nature, 370, 511-512.

Toonen R.J. & Pawlik J.R. (1996) Settlement of the tube worm Hydroides dianthus

(Polychaeta: Serpulidae): cues for gregarious settlement. Marine Biology, 126, 725-733.

Toonen R.J. & Pawlik J.R. (2001) Settlement of the gregarious tube worm Hydroides

dianthus (Polychaeta: Serpulidae). I. Gregarious and nongregarious settlement. Marine

Ecology Progress Series, 224, 103-114.

Toonen R.J. & Pawlik J.R. (2001) Foundations of gregariousness: a dispersal polymorphism

among the planktonic larvae of a marine invertebrate. Evolution, 55, 2439-2454.

Toonen R.J. (2005) Foundations of gregariousness in barnacles. The Journal of Experimental

Biology, 208, 1773-1774.

Townsin R.L. & Anderson C.D. (2009) Fouling control coatings using low surface energy,

foul release technology. In: Advances in Marine Antifouling Coatings and

Technologies. (eds. Hellio C. & Yebra D.M.Y.). Woodhead Publishing Ltd., Oxford,

pp. 693-708.

Trevors J.T. & Cotter C.M. (1990) Copper toxicity and uptake in microoganisms. Journal

of Industrial Microbiology, 6, 77-84.

Tsukamoto S., Kato H., Hirota H. & Fusetani N. (1995) Pipecolate derivatives, anthosamines

A and B, inducers of larval metamorphosis in ascidians, from a marine sponge

Anthosigmella aff. Raromicrosclera. Tetrahedron, 51, 6687-6694.

Page | 152

Tsukamoto S., Kato H., Hirota H. & Fusetani N. (1999) Lumichrome: a larval

metamorphosis-inducing substance in the ascidian Halocynthia roretzi. European

Journal of Biochemistry, 264, 785-789.

Turner M.G., Baker W.L., Peterson C.J. & Peet R.K. (1998) Factors influencing succession:

lessons from large, infrequent natural disturbances. Ecosystems, 1, 511-523.

Underwood A.J. (1998) Grazing and disturbance: an experimental analysis of patchiness in

recovery from a severe storm by the intertidal alga Hormosira banksii on rocky shores

in New South Wales. Journal of Experimental Biology and Ecology, 231, 291-306.

Uratani Y. & Hoshino T. (1984) Pyocin R1 inhibits active transport in Pseudomnas

aeruginosa and depolarizes membrane potential. Journal of Bacteriology, 157, 632-636.

van Pelt A.W., Weerkamp A.H., Uyen M.H., Busscher H.J., de Jong H.P. & Arends J. (1985)

Adhesion of Streptococcus sanguis CH3 to polymers with different surface free

energies. Applied Environmental Microbiology, 49, 1270-1275.

Vopel K., Reik C.H., Arlt G., Pöhn M. & Ott A. (2002) Flow microenvironment of two

marine peritrich ciliates with ectobiotic chemoautotrophic bacteria. Aquatic Microbial

Ecology, 29, 19-28.

Wahl M. (1989) Marine epibiosis I: Fouling and antifouling, some basic aspects. Marine

Ecology Progress Series, 58, 175-189

Wahl M. & Lafargue F. (1990) Marine epibiosis II. Reduced fouling on Polysyncraton

lacazei (Didemnidae, Tunicata) and proposal of an antifouling potential index.

Oecologia, 82, 275-282

Wahl M., Kroger K. & Lenz M. (1998) Non-toxic protection against epibiosis. Biofouling,

12, 205-226.

Watson M.G., Scardino A.J., Zalizniak L. & Shimeta J. (2015) Colonisation and succession

of marine biofilm-dwelling ciliates in response to environmental variation. Aquatic

Microbial Ecology, 74, 95-105.

Weisse T. (2002) The significance of inter and intraspecific variation in bacterivorous and

herbivorous protists. Antonie van Leeuwenhoek, 81, 327-341.

Page | 153

Werner W.E. (1967) The distribution and ecology of the barnacle Balanus trigonus. Bulletin

of Marine Science, 17, 64-84.

Wetherbee R., Lind J.L., Burke J. & Quatrano R.S. (1998) The first kiss: establishment and

control of initial adhesion by raphid diatoms. Journal of Phycology, 34, 9-15.

Wethey D.S. (1986) Ranking of settlement cues by barnacle larvae: influence of surface

contour. Bulletin of Marine Science, 39, 393-400

Wey J.K., Scherwass A., Norf H., Arndt H. & Weitere M. (2008) Effects of protozoan

grazing within river biofilms under semi-natural conditions. Aquatic Microbial Ecology,

52, 283-296.

Wieczorek S.K. & Todd C.D. (1998) Biofilm cues and larval settlement. Biofouling, 12, 81–

118.

Williams E.A., Craigie A., Yeates A. & Degnan S.M. (2008) Articulated corralline algae of

the genus Amphiroa are highly effective natural inducers of settlement in the tropical

abalone Haliotis asinina. The Biological Bulletin, 215, 98-107.

Wilson D.P. (1954) The attractive factor in the settlement of Ophelia bicomis Savigny.

Journal of the Marine Biological Association UK, 33, 361-380.

Wimpenny J. (1996) Ecological determinants of biofilm formation. Biofouling, 10, 43-63.

Xu H., Min G.S., Choi J.K., Kim S.J., Jung J.H. & Lim B.J. (2009) Periphytic ciliate

colonization of an artificial substrate in Korean coastal waters. Protistology, 6, 55-65.

Yang J.L., Satuito C.G., Bao W.Y. & Kitamura H. (2007) Larval settlement and

metamorphosis of the mussel Mytilus galloprovincialis on different macroalgae. Marine

Biology, 152, 1121-1132.

Yebra D.M., Kiil S. & Dam-Johansen K. (2004) Antifouling technology - past, present and

future steps towards efficient and environmentally friendly antifouling coatings.

Progress in Organic Coatings, 50, 75-104

Page | 154

Zardus J.D., Nedved B.T., Huang Y., Tran C. & Hadfield M.G. (2008) Microbial biofilms

facilitate adhesion in biofouling invertebrates. The Biological Bulletin, 214, 91-98.

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