Durham E-Theses Acclimatory response to environmental temperature in two species of marine crab: the stenothermal cancer pagurus and the eurythermal carcinus maenas Cuculescu, Mirela How to cite: Cuculescu, Mirela (1996) Acclimatory response to environmental temperature in two species of marine crab: the stenothermal cancer pagurus and the eurythermal carcinus maenas, Durham theses, Durham University. Available at Durham E-Theses Online: http://etheses.dur.ac.uk/5300/ Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in Durham E-Theses • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders. Please consult the full Durham E-Theses policy for further details. Academic Support Oce, Durham University, University Oce, Old Elvet, Durham DH1 3HP e-mail: [email protected] Tel: +44 0191 334 6107 http://etheses.dur.ac.uk 2 MIRELA CUCULESCU - Ph.D. 1995 ACCLIMATORY RESPONSES TO ENVIRONMENTAL TEMPERATURE IN TWO SPECIES OF MARINE CRAB: THE STENOTHERMAL CANCER PAGURUS AND THE EURYTHERMAL CARCINUS MAENAS ABSTRACT Temperature acclimation effects on leg muscle plasma membrane composition and biophysical state, neuromuscular function and whole organism thermal tolerance, and their dependence on the background seasonal acclimatization were investigated in the eurythermal Carcinus maenas and the stenothermal Cancer pagurus. Correlations between the changes observed at each of these levels are discussed. The average winter (5°C), annual (8°C), and summer (15°C) sea-water temperatures and a warmer summer temperature (22 °C) were chosen as acclimation temperatures for their ecological significance. Warm-acclimation (22°C) increased the thermal tolerance ·(CTMax) ·Jf both species and induced partial compensation of the neuromuscular function (assessed from leg nerve axonal conduction velocity and the amplitudes of excitatory junction potentials in dactylopodite closer muscle fibres). The time-course of acclimation showed it was complete after two weeks, but a longer exposure to 22°C reduced neuromuscular performance, an effect that may not be acclimation related. At plasma membrane level, the response to warm-acclimation depended on acclimation temperature. Acclimation to l5°C induced a significant increase in the S/U fatty acids ratio of PC and PE, but no increase in plasma membrane order. Acclimation to 22°C induced a significant increase in the Ch/PL molar ratio and in plasma membrane order, but no major changes in overall fatty acid composition of membrane phospholipids, compared to cold-acclimated crabs. A seasonal background, determined by complex factors, underlied these responses to temperature-acclimation. At all acclimation temperatures, crabs acclimated in autumn and winter had lower S/U fatty acid ratios and Ch/PL molar ratios than in spring, and the temperature dependence of plasma membrane fluidity was reduced in summer, compared to spring and autumn. The response to l5°C• acclimation was greater in winter than in other seasons. The CTMax and the magnitude or efficacy of the acclimation responses showed that the stenothermal species was more thermally sensitive than the eurythermal species. ACCLIMATORY RESPONSES TO ENVIRONMENTAL TEMPERATURE IN TWO SPECIES OF MARINE CRAB: THE STENOTHERMAL CANCER PAGURUS AND THE EURYTHERMAL CARCINUS MAENAS BY MIRELA CUCULESCU B.Sc., M.Sc. (University of Bucharest), M.Sc. (University of Manchester) The copyright of this thesis rests with the author. No quotation from it should be published without his prior written consent and information derived from it should be acknowledged. Being a Thesis presented in candidature for the Degree of Doctor in Philosophy University of Durham Department of Biological Sciences 1996 -~1996 TABLE OF CONTENTS Abstract Title Page ...................................................................................................................... .i Table of Contents ........................................................................................................... ii List of Tables ................................................................................................................ vi List of Figures ............................................................................................................... xi Declaration ............................................................................................................... xxiii Statement of Copyright ............................................................................................. xxiv Acknowledgements .................................................................................................... xxv Chemicals ................................................................................................................ xxvii Glossary ................................................................................................................... xxvii Chapter 1. General Introduction ................................................................................ ! Chapter 2. The Isolation of a Plasma Membrane Rich Fraction from the Leg Muscle of Two Species of Marine Crab, Carcinus maenas and Cancer pagurus .............................................................................. ~ ...................................... 18 2.1. Introduction ...................................................................................... 18 2.2. Materials and Methods ..................................................................... 2·1 2.2.1. Animals ............................................................................................ 21 2.2.2. Membrane Isolation Procedure ......................................................... 21 2.2.3. Enzymatic Assays ............................................................................ 22 2.3. Results ............................................................................................... 25 2.4. Discussion .......................................................................................... 26 Chapter 3. A Fluorescence Polarization Study of the Fluidity of Plasma Membranes of Cold and Warm-Acclimated Crabs ......................................... 30 3.1. Introduction ...................................................................................... 30 3.2. Materials and Methods ..................................................................... 36 3.2.1. Animals ............................................................................................ 36 3.2.2. Plasma Membranes .......................................................................... 37 3.2.3. Fluorescent Probes ........................................................................... 37 3.2.4. Experimental Set-Up ........................................................................ 38 3.2.5. Experimental Procedure ................................................................... 39 3.2.6. Homeoviscous Efficacy .................................................................... 40 ii 3.2.7. Statistical Analysis ........................................................................... 41 3.3. Results ............................................................................................... 41 3.4. Discussion .......................................................................................... 53 Chapter 4. A Comparative Study of the Plasma Membrane Lipid Composition of Cold- and Warm-Acclimated Crabs ............................................................ 64 4.1. Introduction ...................................................................................... 64 4.2. Materials and Methods ..................................................................... 69 4.2.1. Animals ............................................................................................ 69 4.2.2. Plasma Membranes .......................................................................... 69 4.2.3. Total Lipid Extraction ...................................................................... 69 4.2.4. Total Phospholipid Assay ................................................................. 71 4.2.5. Total Cholesterol Assay ................................................................... 71 4.2.6. Thin Layer Chromatography ............................................................ 72 4.2.7. Fatty Acid Composition Analysis ...................................................... 74 4.2.8. Statistical Analysis ........................................................................... 76 4.3. Results ............................................................................................... 7 6 4.3.1. Total Phospholipid, Cholesterol and CholesteroV Phospholipid Molar Ratio ...................................................................................... 7 6 4.3.2. Fatty Acid Composition ................................................................... 80 4.3.2.1. Total Phospholipid Fraction (TPL) ................................................ 82 4.3.2.2. Phosphatidylcholine (PC) .............................................................. 87 4.3.2.3. Phosphatidylethanolamine (PE) ....................................................