Larval development of the New Zealand mussel Perna canaliculus and effects of cryopreservation Adam B. Rusk A thesis submitted to Auckland University of Technology in partial fulfilment of the requirements for the degree of Master of Science (MSc) 2012 School of Applied Science TABLE OF CONTENTS LIST OF FIGURES ............................................................................................. VI LIST OF TABLES ............................................................................................... XI ATTESTATION OF AUTHORSHIP ................................................................... XII ACKNOWLEDGEMENTS ................................................................................. XIII ABSTRACT ........................................................................................................ XV CHAPTER ONE: General Introduction .............................................................. 1 1.1 INTRODUCTION AND LITERATURE REVIEW ......................................... 2 1.1.1 Mussel biology & ecology ........................................................................ 2 1.1.2 Global & national aquaculture review .................................................... 10 1.1.3 Hatchery rearing of Perna canaliculus .................................................. 13 CHAPTER TWO: Larval Development ............................................................ 16 2.1 ABSTRACT ............................................................................................... 17 2.2 INTRODUCTION AND LITERATURE REVIEW ....................................... 19 2.2.1 General bivalve larval development ...................................................... 19 2.2.2 Bivalve larval shell morphology ............................................................. 24 2.2.3 Bivalve larval nervous system ............................................................... 26 2.2.4 Larval development of Perna canaliculus ............................................. 31 I 2.2.5 Significance of research on Perna canaliculus larvae .......................... 35 2.3 AIM & OBJECTIVES ................................................................................. 36 2.4 METHODS AND MATERIALS .................................................................. 36 2.4.1 General larval rearing ............................................................................ 36 2.4.2 Survivability ........................................................................................... 40 2.4.3 Shell length ............................................................................................ 40 2.4.4 Feeding consumption ............................................................................ 41 2.4.5 Light microscopy .................................................................................... 41 2.4.6 Scanning electron microscopy .............................................................. 42 2.4.7 Histology ................................................................................................ 43 2.4.8 Confocal microscopy ............................................................................. 44 2.4.9 Statistical analyses ................................................................................ 45 2.5 RESULTS ................................................................................................. 46 2.5.1 Survivability analysis ............................................................................. 46 2.5.2 Shell length analysis .............................................................................. 47 2.5.3 Feeding consumption analysis .............................................................. 48 2.5.4 Embryogenesis ...................................................................................... 49 2.5.5 Shell morphology ................................................................................... 52 2.5.6 Organogenesis ...................................................................................... 56 2.5.7 Neurogenesis ........................................................................................ 62 II 2.6 DISCUSSION............................................................................................ 70 2.6.1 Survivability ........................................................................................... 71 2.6.2 Shell morphology and growth ................................................................ 73 2.6.3 Feeding consumption ............................................................................ 76 2.6.4 Embryogenesis ...................................................................................... 78 2.6.5 Organogenesis ...................................................................................... 80 2.6.6 Neurogenesis ........................................................................................ 84 CHAPTER THREE: Cryopreservation ............................................................. 91 3.1 ABSTRACT ............................................................................................... 92 3.2 INTRODUCTION AND LITERATURE REVIEW ....................................... 93 3.2.1 General bivalve cryopreservation .......................................................... 93 3.2.2 Cryopreservation on Perna canaliculus ................................................ 96 3.2.3 Significance of cryopreserving Perna canaliculus larvae ...................... 98 3.3 AIM & OBJECTIVES ................................................................................. 98 3.4 METHODS AND MATERIALS .................................................................. 99 3.4.1 Experimental reagents .......................................................................... 99 3.4.2 Larval incubation and collection ............................................................ 99 3.4.3 Cryopreservation .................................................................................100 3.4.4 Larval rearing & sampling ....................................................................102 3.4.5 Statistical analyses ..............................................................................104 III 3.5 RESULTS ...............................................................................................104 3.5.1 Survivability .........................................................................................104 3.5.2 Shell length ..........................................................................................106 3.5.3 Feeding consumption ..........................................................................107 3.5.4 Shell morphology .................................................................................109 Control larvae .............................................................................................109 Cryopreserved at D-stage ..........................................................................109 Cryopreserved at trochophore stage .........................................................110 3.5.5 Organogenesis ....................................................................................117 Control larvae .............................................................................................117 Cryopreserved at trochophore ...................................................................123 Cryopreserved at D-stage ..........................................................................126 3.5.6 Neurogenesis ......................................................................................128 Control larvae .............................................................................................128 Cryopreserved at D-stage ..........................................................................134 Cryopreserved at trochophore ...................................................................138 3.6 DISCUSSION..........................................................................................141 3.6.1 Survivability .........................................................................................142 3.6.2 Shell length ..........................................................................................145 3.6.3 Feeding consumption ..........................................................................147 IV 3.6.4 Shell morphology .................................................................................148 3.6.5 Organogenesis ....................................................................................150 3.6.6 Neurogenesis ......................................................................................152 CHAPTER FOUR: General Discussion .........................................................156 4.1 DISCUSSION ............................................................................................157 REFERENCES .................................................................................................164 V LIST OF FIGURES Figure 1.1. Female green-lipped mussel releasing orange oocytes. .......................... 5 Figure 1.2. Male green-lipped mussel releasing white clouds of sperm. ................... 6 Figure 1.3. Unfertilised oocytes of Perna canaliculus.. ................................................ 7 Figure 1.4. Life cycle of mussel ..................................................................................... 8 Figure 2.1. General bivalve and freshwater bivalve life cycle ................................... 20 Figure 2.2. Diagram of