Pollution-induced Immunomodulation in Biomphalaria glabrata; Implications for its Relationship with Obligate Parasite Schistosoma mansoni A thesis submitted for the degree of Doctor of Philosophy By Adam Lynch Institute for the Environment, Health and Societies, Brunel University, London 2015 Table of Contents DECLARATION .............................................................................................................. i ABSTRACT .....................................................................................................................ii ACKNOWLEDGMENTS ............................................................................................. iii LIST OF FIGURES ........................................................................................................ iv LIST OF TABLES ........................................................................................................... v Chapter 1: Introduction ................................................................................................... 1 1.1: Schistosomiasis .......................................................................................................... 2 1.1.1: Trematodes; parasites of man and molluscs .......................................................... 3 1.1.2: Species and distribution of Schistosomes ............................................................. 5 1.1.3: Evolutionary history of S.mansoni ........................................................................ 8 1.1.4: Schistosoma life-cycle .......................................................................................... 9 1.1.5: Biology of snail stages ....................................................................................... 11 1.1.5.1: Miracidia; contact and penetration .................................................................. 12 1.1.5.1.1: Epithelial cells ............................................................................................ 13 1.1.5.1.2: Papilla ........................................................................................................ 13 1.1.5.1.3: Adhesive glands ......................................................................................... 13 1.1.5.1.4: Penetration gland ........................................................................................ 13 1.1.5.2: Inside the snail ............................................................................................... 14 1.1.5.2.1: Development to mother sporocyst ............................................................... 14 1.1.5.2.2: The mother sporocyst ................................................................................. 15 1.1.5.2.3: Development of daughter sporocysts .......................................................... 16 1.1.5.2.4: Cercariae; development and release ............................................................ 16 1.2: Biomphalaria .......................................................................................................... 17 1.2.1: Biomphalaria glabrata ....................................................................................... 17 1.2.1.1: Evolutionary history and distribution .............................................................. 18 1.2.1.2: BBO2 .............................................................................................................. 21 1.2.1.3: Biology .......................................................................................................... 21 1.2.1.3.1: Reproduction ............................................................................................. 22 1.2.1.3.2: Respiration ................................................................................................. 23 1.2.2: Biomphalaria immune system ........................................................................... 25 1.2.2.1: Overview of the invertebrate/molluscan immune system ................................ 26 1.2.2.2: Hemocytes of B.glabrata ................................................................................ 29 1.2.2.3: Interaction with S.mansoni ............................................................................. 32 1.3: Does pollution affect the B.glabrata - S.mansoni relationship? ................................. 35 1.3.1: Transmission country pollution .......................................................................... 37 1.3.1.2: Ecotoxicology; pollutants in the environment ................................................. 41 1.3.1.2.1: Aquatic ecotoxicology ................................................................................ 43 1.3.1.2.2: Immunotoxicology ..................................................................................... 47 1.3.1.2.2.1: Immunotoxicology in Invertebrates ......................................................... 49 1.3.1.2.2.2: Immunotoxicology in molluscs …………………………………………..50 1.3.1.2.2.3: Immunotoxicology in commercial mollusc species ................................. 51 1.3.1.2.2.4: Parasitism and immunotoxicology in molluscs ......................................... 53 1.3.1.2.2.5: Molluscs and parasites as indicators of ecosystem health and beyond ...... 54 1.4: The hypothesis ......................................................................................................... 56 1.4.1: The importance of method development in testing the hypothesis ....................... 56 Chapter 2: Experimental Organisms; Husbandry and Biology .................................... 60 2.1: Biomphalaria Husbandry .......................................................................................... 61 2.1.1: Diet ..................................................................................................................... 61 2.1.2: Photoperiod ......................................................................................................... 62 2.1.2.1: Photoperiod for infected snails ........................................................................ 62 2.1.3: Pests.................................................................................................................... 63 2.1.4: Water .................................................................................................................. 63 2.1.5: Density................................................................................................................ 64 2.2: Biomphalaria cell culture ........................................................................................ 65 2.2.1: Biomphalaria cell buffers .................................................................................... 66 2.2.2: Hemolymph collection ........................................................................................ 67 2.2.2.1: Pooling ............................................................................................................ 69 2.2.2.2: Incubation with test chemicals ......................................................................... 72 2.3: Parasite culture ........................................................................................................ 73 2.3.1: Safety considerations .......................................................................................... 73 2.3.2: Mouse stage and egg collection ........................................................................... 74 2.3.3: Egg extraction and hatching ................................................................................ 75 2.3.4: Miracidia collection ............................................................................................ 76 2.3.4.1: Lighting .......................................................................................................... 76 2.3.4.2: Construction of the flask ................................................................................. 77 2.3.4.3: Chemical gradient ........................................................................................... 78 2.3.4.4: Pressure gradient ............................................................................................. 79 2.3.4.5: Addition of sample .......................................................................................... 79 2.3.4.6: Miracidia harvesting and transformation .......................................................... 80 2.3.4.7: Findings .......................................................................................................... 80 2.3.5: Miracidia transformation media........................................................................... 81 2.3.6: Infecting snails; background ................................................................................ 82 2.3.6.1: Number of miracidia ....................................................................................... 83 2.3.6.2: Age of snails ................................................................................................... 83 2.3.6.3: Snail infection protocol ................................................................................... 84 2.3.6.4: Shedding ......................................................................................................... 85 2.4: Alternative model: Marisa cornuarietis.................................................................... 88 2.4.1: Aim ..................................................................................................................... 88 2.4.2: Distribution ........................................................................................................