The Role of Mucus and Silk as Attachment and Sorption Sites in Streams Submitted by Chris Brereton for the degree of Doctor of Philosophy University College London 1998 ProQuest Number: U642856 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642856 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract This thesis is an examination of the characteristics of mucus and silk within freshwaters. The source of mucus was snail pedal mucus from Lymnaea peregra and Potamopyrgus jenkinsi, the source of silk was first instar silk threads of Simuliidae spp. Each chapter examines a different aspect, or role, of such materials within the habitat of snails and blackfly. In particular, the interactions of suspended particles, pollutants, sediments and biofilms are examined in relation to snail trail mucus (STM) and blackfly silk. The search for a particle type, suitable as a marker for STM is detailed. This was used to characterise STM, including the examination of the duration of STM integrity, the effect of water flow upon STM, the effect of disturbance and bacteria (airborne, waterborne and those included within the mucus trail). The advantage that different substrates offer in maintaining STM presence, as well as the physical dispersal of STM are aspects that are also examined. A comparison is made between the STM of two species of freshwater snail. The examination of the interactions of a range of pesticides to mucus and silk finds sorption to a degree previously unmeasured for any other natural substance. This presents many implications in terms of the bio-accumulation of pollutants within the freshwater ecosystem. The effect that STM has upon benthic sediment stability was found to be immeasurable. In looking at the role o f STM in the development of biofilm, it is found that STM can accelerate the accumulation of particulate matter as well as acting as a "seed-bed" for specific types of bacteria. This study provides evidence that organic materials, produced by freshwater macroinvertebrates are an understudied resource that have considerable impact on biological and chemical processes. They exist in considerable quantities and present a large surface area within the freshwater environment. In so doing, they act as a trap or sink for a wide variety of particulate material. Acknowledgements I would like to thank my three supervisors Dr.P.D.Armitage, Dr.W.A.House and Dr.R.S.Wotton for initiating this project and for each providing an invaluable source of information, advice and enthusiasm. In addition, I would also like to thank Dr.G.P.Irons for his patience and instruction regarding the operation of various software packages, as well as the commissioning of a computer. Mr.D.Orr and Mr.I.S.Farr provided a great deal of time in demonstrating the operation of various pieces of equipment, and Miss.F.M.Buchanan provided invaluable advice in the execution of those techniques described in Chapter 6. Mr.R.Clarke exhibited a great deal of patience in suggesting appropriate statistical tests. Dr.G.EImes kindly loaned his scanning electron microscope for which I am indebted to him, as I am to Mr.D.Hornby for his remote instruction regarding operation. I am also indebted to Miss F.M.Buchanan for performing the microbiological identifications, used in Table 6.1. I am also thankful to Dr.D.T. Chaloner for his ceaseless willingness to debate the finer points of experimental design and failure. Grateful thanks are also extended to those who have kindly offered their time to proof­ read this manuscript during its various stages o f completion, notably my three supervisors, and also Miss.F.M.Buchanan, Dr.S.Brown, Dr.M.Ladle, Miss C.Cannan, Dr.G.P.Irons and Mr. M. Bowes. I am grateful to the River Laboratory at East Stoke, at which I spent three years carrying out this work, for the provision of a friendly working environment as well as finest purpose built facilities imaginable for work o f this nature. A special acknowledgement is extended to Miss.F.M.Buchanan, without whose support and encouragement, this thesis would never have reached fruition. Finally I would like to thank EPSRC, who made this project possible. Table of Contents Page No. Title Page Abstract Acknowledgements Table of Contents List of Figures List of Tables 12 List of Plates 14 List of Abbreviations 15 Chapter 1 General Introduction 16 1.1 Introduction 16 1.2 The "re-cycling" of sites of attachment 17 1.3 Organisms chosen for use in this study 18 1.4 Review o f materials used: silk and STM 20 1.5 Characterisation of STM 22 1.6 Mucus and silk as sites o f attachment to DOM 23 1.7 Mucus as a potential agent of sediment consolidation 25 1.8 Sediment consolidation in aquatic environments 26 1.9 Consolidating properties of polysaccharide 27 1.10 Examination of estuarine consolidation 28 1.11 STM as a site of biofilm development 29 1.12 Formation of biofilm 31 1.13 Note on sorption 32 1.14 The progression o f study within this thesis 32 Chapter 2 Selection of a marker particle suitable for the observation of snail trail mucus 34 2.1 Introduction 34 2 . 1.1 Previous uses of marker particles in freshwater biology 34 2.1.2 Particle types available 35 2.1.3 Experimental objective 37 2.2 Materials and methods 37 2.2.1 Experimental protocol 37 2.2.2 Particles used in this trial 39 2.3 Results and discussion 42 2.3.1 Efficacy of particle types 42 2.3.2 Subjective approaches in the selection of an appropriate marker particle 50 2.3.3 Objective approaches in the selection o f an appropriate marker particle 51 2.4 Conclusion 52 Chapter 3 The physical behaviour of snail trail mucus 53 3.1 A background to the properties and functions of mucus 53 3.1.1 Experimental Objectives 56 3.2 Materials and Methods 56 3.2.1 Experimental protocols 60 3.2.2 Methods of counting and comparison by statistical analysis 62 3.3 Results and Discussion 65 3.3.1 W ithin treatment analysis - the uniform ity of 6 replicate response 65 3.3.2 Comparison of the snail trail mucus of two freshwater species 70 3.3.3 For how long does snail trail mucus remain sticky? 86 3.3.4 The effect of water speed upon snail mucus trail integrity 89 3.3.5 The effect o f sampling and snail action as disturbance events 91 3.3.6 The effects of ageing, water depth and airborne bacteria upon snail trail mucus longevity 97 3.3.7 The distribution mechanism of snail trail mucus and the effect upon it of waterborne consumers of mucus 100 3.3.8 Snail gut bacteria and mucus trails 102 3.3.9 Waterborne bacteria as consumers of snail trail mucus 103 3.3.10 The protection offered by surface relief in freshwater systems 106 3.3.11 Identified properties of the mucus trails of each species 107 3.4 Conclusions 109 Chapter 4 The affinity of pesticides to mucus and silk 110 4.1 Introduction 110 4.1.1 Parameters used to describe pesticide affinity for materials 110 4.1.2 Research developments into the role of pesticides in the environment: examining appropriate materials 111 4.1.3 Materials that have been overlooked by conventional techniques 113 4.1.4 Pesticide affinity for organic material produced by invertebrates 118 4.1.5 The aims of this study 118 4.2 Materials and Methods 119 7 4.2.1 Experimental Protocols - Mucus I 19 4.2.2 Estimation of mucus mass 121 4.2.3 Sample sizes - Mucus 121 4.2.4 Experimental Protocols - Silk 122 4.2.5 Sample'Sizes - Silk 124 4.2.6 Extraction and analysis of the pesticides 125 4.3 Results and discussion 126 4.3.1 Comparison with previously published pesticide affinity constants for various sediments and soils 139 4.3.2 Mucus sorption 141 4.3.3 The water content of mucus 142 4.3.4 Silk sorption 142 4.3.5 Individual pesticide sorption 143 4.3.6 Are certain pesticides sorbed to a greater extent than others? 144 4.3.7 Experimental constraints 145 4.3.8 Mucus and silk as water purifiers 146 4.3.9 Placing blackfly silk and snail mucus in the context of other materials studied 148 4.3.10 The importance of accounting for pesticide affinity to biological material 149 4.4 Conclusions 152 Chapter 5 Sediment stability 154 5.1 Introduction 154 5.1.1 The role of macroinvertebrates in sediment stabilisation and disturbance 155 5.1.2 Experimental objectives 156 5.2 Materials and Methods 157 5.2.1 Experimental Protocols 159 5.2.2 Equipment design 163 5.2.3 Calibration of light meter 163 5.2.4 Calibration of speed o f water in jars at each paddle setting 163 5.2.5 Sampling 164 5.2.6 Data manipulation 164 5.3 Results and Discussion 165 5.3.1 Water speed generated by paddle speed 165 5.3.2 Light meter readings at each paddle speed 165 5.3.3 Experimental improvements and suggestions 177 5.4 Conclusions 181 Chapter 6 Biofilm growth and the role of snail mucus 182 6.1 Introduction 182 6.1.1 The potential advantage offered by snail mucus to biofilm development 182 6.1.2 Experimental objectives 183 6.2 Materials and methods 184 6.2.1 Experimental protocols - SEM 186 6.2.2 Experimental protocols - M icrobiological analysis 186 6.2.3 M icrobial identification 188 6.3 Results and Discussion 191 6.3.1 Analysis