THE UNIVERSITY OF HULL The behavioural function of pheromones in crayfish being a Thesis submitted for the Degree of Doctor of Philosophy in the University of Hull by Fiona Catherine Berry (MRes University of York; BSc University of St Andrews) September 2008 Contents Acknowledgements 1 Abstract 2 Chapter 1 - Introduction 3 t.l Biological communication 4 1.1.2 Chemical communication 4 1.1.3 Aquatic chemical signalling 6 1.1.4 Aquatic chemical signalling - crustaceans 7 1.2 The chemical nature of pheromones 9 1.2.1 Pheromone identification 9 1.2.2 Bioassay development 10 1.2.3 Chemical analysis 11 1.3 The use of pheromones to control invasive species 11 1.4 Biological and ecology of crayfish (The Astacoidea) 12 1.4.1 Morphology and growth 13 1.4.2 Life history strategies 14 1.4.3 Crayfish in the UK and Europe 15 1.4.3.1 White-clawed crayfish in the UK 16 1.4.3.2 Non-indigenous crayfish 17 1.4.4 Controlling invasive crayfish species 19 1.5 Current knowledge of the role of chemical communication in crayfish, in the context of behaviour 21 1.5.1 Inter-specific chemical communication 22 1.5.2 Reproduction 22 1.5.3 Mate choice 25 1.5.4 Agonism 26 1.6 Aims and Objectives 27 Chapter 2 - General methodology 31 2.1 Animal maintenance 32 2.1.1 Collection 32 2.1.2 Legislation and holding 32 2.2 Experimental procedures 33 2.2.1 Identification 33 2.2.2 Isolation 33 2.2.3 Blindfolding 34 2.2.4 General experimental protocols 34 2.2.5 Checking for receptivity 34 2.2.6 Urine visualisation 35 2.2.7 Urine sampling technique 36 2.2.8 Conditioned water 36 2.2.9 Urine blocking 36 2.2.10 Behavioural analysis 37 2.2.11 Physiological measurements 37 2.2.11.1 Heartbeat analysis 3 7 2.2.11.2 Scaphognathite beat analysis 39 2.2.12 Statistical analysis 43 Chapter 3 - Urinary signalling in crayfish social interactions: miscommunication between females and males 44 3.1 Introduction 45 3.2 Materials and methods 47 3.2.1 Animals 47 3.2.2 Urinary signalling in social interactions 48 3.2.3 Urine visualisation procedure 48 3.2.4 Social interactions 48 3.2.5 Behavioural analysis 49 3.2.6 Agonistic behaviours 50 3.2.7 Reproductive behaviours 51 3.2.8 Urine release analysis 51 3.2.9 Statistical analysis 51 3.3 Results 52 3.3.1 Sex-specific differences in chemical signalling 52 3.3.2 Mating behaviour 53 3.3.3 Female urine release in sexual interactions 54 3.3.4 Agonistic behaviour 55 3.3.5 Urine release in agonistic interactions 55 3.4 Discussion 56 3.4.1 The role of female urinary cues in eliciting male courtship 56 iii 3.4.2 Absence of male urine release during courtship 57 3.4.3 Sexual conflict and urine signalling 57 3.4.4 Urinary signalling in agonistic interactions 58 3.4.5 The'dilemma'of urine signalling 58 3.4.5 Conclusions 59 Chapter 4 - Mate choice in crayfish, Pacifastacus leniusculus: Do females choose dominant partners when given a free choice? 60 4.1 Introduction 61 4.2 Materials and methods 63 4.2.1 Animal maintenance 63 4.2.2 Fight interactions 64 4.2.3 Mate choice experiments 64 4.2.4 Mate choice analysis 65 4.2.5 Statistical analysis 66 4.3 Results 66 4.4 Discussion 67 Chapter 5 - Ventilatory and heartbeat activity of crayfish, Pacifastacus leniusculus, during reproductive behaviours 70 5.1 Introduction 71 5.2 Materials and methods 72 5.2.1 Animal holding conditions 72 5.2.2 Mating interactions 73 5.2.3 Physiological recordings 73 5.2.4 Physiological analysis 74 5.2.5 Statistical analysis 74 5.3 Results 74 5.4 Discussion 77 Chapter 6 - Ventilatory measures in crayfish during social interactions: the effects of social dominance and urinary signalling 80 6.1 Introduction 81 6.2 Materials and methods 82 iv 6.2.1 Animal maintenance 82 6.2.2 Agonistic interactions 83 6.2.3 Ventilation rate recording 84 6.2.4 Urine visualisation procedure in P. clarkii 84 6.2.5 Behavioural analysis 85 6.2.6 Ventilation rate analysis 85 6.2.7 Statistical analysis 85 6.3 Results 86 6.4 Discussion 91 Chapter 7 - Development of behavioural and physiological assays to assess discrimination of male and female odours in crayfish, Pacifastacus lenimculus 94 7.1 Introduction 95 7.2 Materials and methods 97 7.2.1 Animal holding conditions 97 7.2.2 Urine and conditioned water collection 98 7.2.3 General bioassay procedure 98 7.2.4 Behavioural bioassay 99 7.2.5 Heartbeat assay 100 7.2.6 Ventilation assay 102 7.3 Results 102 7.3.1 Behavioural bioassay 102 7.3.2 Heartbeat assay 105 7.3.3 Ventilation assay 107 7.4 Discussion 108 7.4.1 Behavioural bioassay 109 7.4.2 Physiological assays 110 Chapter 8 - The effects of artificial urine release on the fight dynamics of crayfish, Pacifastacus leniusculm 113 8.1 Introduction 114 8.2 Materials and methods 117 8.2.1 Animal holding conditions 117 8.2.2 Urine blocking 117 8.2.3 General fight procedure 118 8.2.4 Stimulus collection and preparation 119 8.2.5 Stimulus introduction in fight two 119 8.2.6 Behavioural analysis 120 8.2.7 Statistical analysis 121 8.3 Results 121 8.4 Discussion 123 8.4.1 The effect of dominant urinary signals in fights 123 8.4.2 Candidate substances for crayfish status cues 124 Chapter 9 - Discussion 126 9.1 Chemical communication in crayfish 127 9.1.1 Chemical signalling during mating 127 9.1.2 Aggressive chemical signalling 129 9.1.3 Signal delivery 130 9.2 Future research 130 9.2.1 Determination of the chemical nature of crayfish pheromones 130 9.2.1.1 Sex pheromones 13 0 9.2.1.2 Dominance pheromones 132 9.2.2 Application of methodologies 132 9.2.3 Mate choice studies 133 9.3 Conclusion 134 References 135 VI Figures 1.1 Ventral views of the crayfish Procambarus clarkii (A) Form I male (B) Female 14 1.2 Distribution of white-clawed crayfish in the UK (A) 1979-1988(6) 1999-2008 16 1.3 Distribution of signal crayfish in the UK (A) 1969 - 1978 (B) 1979-1988 (C) 1989 - 1998 (D) 1999 - 2008 19 2.1 Blindfolded signal crayfish 34 2.2 Experimental equipment used to measure physiological parameters (heart rate, ventilation rate) of crayfish 38 2.3 Infra-red sensors used to measure (A) heartbeat activity (B) scaphognathite movement of crayfish 39 2.4 Ventilation rate recordings of male signal crayfish (A) right scaphognathite recording (B) left scaphognathite recording 41 2.5 Ventilation rate recording of an isolated male red swamp crayfish engaged in fighting (A) right scaphognathite recording (B) left scaphognathite recording 42 3.1 Urine release by dominant and subordinate crayfish in social interactions 53 3.2 Ethogram of a sexual interaction between signal crayfish 54 3.3 Urine release by female crayfish during sexual interactions 55 4.1 Experimental set up from mate choice experiments (A) acclimation period (B) experimental period 65 5.1 Ventilation rate of male and female crayfish during reproductive behaviours 75 5.2 Scaphognathite activity of (A) female and (B) male signal crayfish during reproductive behaviours 76 5.3 Heart rate of female crayfish during reproductive interactions 77 6.1 Scaphognathite activity of male red swamp crayfish during aggressive fighting 87 6.2 Ventilation rate of crayfish during agonistic behaviours (A) Procambarus clarkii (B) Pacifastacus leniusculus 88 6.3 Scaphognathite activity of male signal crayfish at the onset of aggressive fighting 89 vii 6.4 Ventilation rate of male red swamp crayfish during urinary signalling and aggressive fighting 90 6.5 Scaphognathite activity of male red swamp crayfish at the onset of urinary signalling 90 7.1 Number of male crayfish which mounted a female dummy following introduction of conspecific odours 103 7.2 Timed behavioural responses of male crayfish to a female dummy (A) mounting the female dummy (B) touching the female dummy with the chelae 104 7.3 Heart rate recording for male crayfish following the release of female urine 105 7.4 The number of male crayfish showing cardiac arrest to introduced stimuli 106 7.5 Standardised heart rate recorded by male crayfish following stimulus release 107 7.6 Number of male crayfish displaying ventilatory arrest to introduced stimulus 108 8.1 Fight dynamics of crayfish with unblocked nephropores on day one and blocked nephropores on day two (A) mean fight duration (B) mean fight intensity 122 Tables 1.1 The life history characteristics of three crayfish found in Europe 15 1.2 Native and non-indigenous crayfish of Europe 17 3.1 Agonistic and sexual behaviours displayed by signal crayfish in social interactions 50 3.2 Two-way repeated measures ANOVA results for urine release during social interactions 52 4.1 Mate choice and behaviour among female crayfish 67 5.1 Two-way repeated measures ANOVA results for ventilation rate of male and female crayfish during reproductive behaviours 75 6.1 Two-way repeated measures ANOVA results for the ventilation rate of male crayfish during agonistic behaviours (A) signal crayfish 86 (B) red swamp crayfish 87 8.1 Two-way repeated measures ANOVA results for (A) fight intensity (B) fight duration of second day fights where crayfish had blocked nephropores and were exposed to different experimental conditions 121 viii Acknowledgements I would firstly like to thank my supervisor, Thomas Breithaupt, for his enthusiasm towards my research and for the many hours spent discussing project ideas.