Juvenile biology and captive rearing of the freshwater pearl mussel Margaritifera margaritifera Louise Lavictoire BSc. (Hons) October 2016 University of Lancaster This thesis is forsubmitted the degree in partial of Doctor fulfilment of Philosophy of the requirements ii Abstract Abstract Title: Juvenile biology and captive rearing of the freshwater pearl mussel Margaritifera margaritifera Author: Louise Lavictoire BSc. (Hons) Degree: Doctor of Philosophy Submission date: October 2016 Word count: 40,617 Captive breeding of the freshwater pearl mussel (Margaritifera margaritifera) is an important short-term strategy to conserve this critically endangered species. The aim of this thesis was to improve current knowledge of the factors affecting juvenile M. margaritifera in a captive setting, and to develop understanding of juvenile anatomy, ontogeny and the ecological requirements of juveniles in captivity. The substrate requirements of newly-excysted juveniles were investigated in an experimental flow-through system (Chapter 3) by analysing differences in survival and growth in two different substrate size clasts (0.25 - 1 mm or 1 - 2 mm), and cleaning regimes (weekly or monthly). Factors potentially affecting juvenile survival and growth were further investigated in Chapter 4. Results indicate that dissolved oxygen and flow were crucial for juveniles in this system. Investigations of juvenile anatomy and ontogeny (Chapter 5) using scanning electron microscopy have greatly improved our knowledge of the timing of key developmental stages, such as the onset of gill reflection. Analyses of gill ciliation suggest the species is capable of retaining very small particles (<2 µm diameter), offering a potential reason for why M. margaritifera is so sensitive to turbid and enriched conditions. Improving the efficiency and effectiveness of monitoring juveniles in captivity should be an objective for all rearing programmes. Batch marking of juveniles through immersion in calcein (Chapter 6) was shown to offer a quick and reliable method and has the potential to save rearing programmes time and money whilst improving juvenile monitoring. The findings of these investigations should inform other captive rearing programmes in order to improve juvenile survival. Rearing efforts should focus initially on ensuring sufficient flow and dissolved oxygen for post-excystment juveniles, before tailoring systems to ensure low-stress conditions for transforming juveniles. iii iv Contents Contents Abstract ..............................................................................................................................iii List of figures ......................................................................................................................xi List of tables ..................................................................................................................... xxi Acknowledgements ......................................................................................................... xxv Author’s declaration ...................................................................................................... xxvii Chapter 1: Introduction ...................................................................................................... 1 1.1. Background and context of freshwater bivalve conservation .................................... 2 1.2. The freshwater pearl mussel Margaritifera margaritifera ......................................... 3 1.2.1 Life history, distribution, habitat requirements and factors causing decline ..... 3 1.2.2 Taxonomy .......................................................................................................... 6 1.3. Captive rearing strategies ........................................................................................... 7 1.4. Thesis outline and objectives ................................................................................... 10 1.5. References ................................................................................................................ 13 Chapter 2: Freshwater Pearl Mussel Ark project and pilot studies ..................................... 21 2.1. Introduction .............................................................................................................. 22 2.1.1 Freshwater Pearl Mussel Ark project and the FBA captive rearing facility ...... 22 2.2. Pilot studies .............................................................................................................. 24 2.2.1 The effect of substrate size, depth and cleaning regime on growth and survival of juvenile mussels .................................................................................................... 25 2.2.2 Investigating the suitability of calcein immersion as a marking technique for juvenile mussels ........................................................................................................ 32 2.3. Conclusions .............................................................................................................. 40 2.4. References ................................................................................................................ 40 Chapter 3: Investigating the effects of substrate size and cleaning regime on growth and survival of juvenile freshwater pearl mussels Margaritifera margaritifera ....................... 43 3.1. Introduction .............................................................................................................. 44 3.1.1 Culture conditions affecting juvenile survival .................................................. 45 3.1.2 Objective of this study ..................................................................................... 47 3.2. Methods ................................................................................................................... 48 3.2.1 Experimental design ........................................................................................ 48 3.2.2 Data analysis ................................................................................................... 51 v Contents 3.3. Results ...................................................................................................................... 52 3.3.1 Survival ............................................................................................................ 52 3.3.2 Size .................................................................................................................. 57 3.3.3 Escaped mussels .............................................................................................. 63 3.4. Discussion ................................................................................................................. 64 3.4.1 Effect of substrate size ..................................................................................... 66 3.4.2 Effect of cleaning regime ................................................................................. 67 3.4.3 Effect of temperature ..................................................................................... 68 3.4.4 Conclusions and further research ................................................................... 70 3.5. References ................................................................................................................ 71 Chapter 4: Interstitial factors affecting growth and survival of juvenile freshwater pearl mussels Margaritifera margaritifera ................................................................................ 75 4.1. Introduction .............................................................................................................. 76 4.1.1 Interstitial conditions affecting juvenile mussels ............................................. 76 4.1.2 Objective of this study ..................................................................................... 78 4.2. Methods ................................................................................................................... 79 4.2.1 Experimental set-up ......................................................................................... 79 4.2.2 Chemical analyses ........................................................................................... 81 4.2.3 Flow and organic content analysis .................................................................. 83 4.2.4 Data analysis ................................................................................................... 83 4.3. Results ...................................................................................................................... 84 4.3.1 Survival ........................................................................................................... 86 4.3.2 Size .................................................................................................................. 87 4.3.3 Dissolved oxygen ............................................................................................. 91 4.3.4 Interstitial space and flow ............................................................................... 99 4.3.5 Substrate organic content and ion concentrations ........................................ 101 4.4. Discussion ............................................................................................................... 102 4.4.1 Effects on growth and survival – current investigation and comparisons over several years ..........................................................................................................