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First published online February 15, 2006
Journal of Experimental Biology 209, 891-906 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02054

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Critical temperatures in the cephalopod Sepia officinalis investigated using in vivo ³¹P NMR spectroscopy

Frank Melzner^*, Christian Bock and Hans-O. Pörtner

Alfred-Wegener-Institute for Marine and Polar Research, Am Handelshafen 12, 27570 Bremerhaven, Germany

View larger version (39K): [in a new window]   Fig. 1. (A) Animal in chamber in front of the magnet, connected to a seawater perfusion system. Sliders restrict the space available to the animal to a minimum. A grey box indicates the position of the 31P surface coil below the animal's thick ventral mantle muscle. The pressure catheter (not visible) leaves the animal's mantle cavity and passes through the lid on the right towards a pressure transducer (see text). (B) Schematic illustration of the surface coil and its sensitive volume (grey semi-circle). Metabolite changes within radial and circular muscles of the mantle muscle organ can be recorded. Circular muscle consists of a central bulk of anaerobic fibres and two thin layers of outer, aerobic fibres. (C) Schematic illustration of intervals defined between the acquisition of successive in vivo NMR spectra. One complete 25 min interval is illustrated (n), which is divided into 12 segments (s1n–s12n). For each segment, swimming jet (SJ) pressure amplitudes and frequencies were determined, allowing us to calculate jet indices (JIs) for variable time intervals (by summing up segment JI within intervals a to l; e.g. interval g consists of JIs from segments s6n–s12n). For further explanations, see text.

View larger version (18K): [in a new window]   Fig. 2. Swimming jet (SJ) amplitude distribution and frequency. (A) Pressure amplitude (MMPA) distribution of spontaneously occurring high-pressure mantle cavity oscillations (SJs) of >0.2 kPa under control conditions (15°C). Amplitudes grouped into 1 kPa classes, frequencies expressed as percentage of total SJ frequency. The insert gives cumulative frequencies within selected intervals. (B) Frequency of SJs >0.2 kPa at all experimental temperatures, expressed in incidences per hour. N=5 animals per temperature. Error bars represent standard deviation. (C) SJ contribution to total mantle pressure generation in % of total mean mantle pressure (MMPtot) at all experimental temperatures. See text for calculations.

View larger version (24K): [in a new window]   Fig. 3. Metabolite changes due to spontaneous activity under control conditions (animal 4). In vivo 31P NMR spectra were acquired every 25 min. Each data point represents concentration information for the respective metabolite obtained from a single spectrum (acquisition time=3 min 40 s). Pi, inorganic phosphate; pHi, intracellular pH; PLA, phospho-L-arginine.

View larger version (24K): [in a new window]   Fig. 4. Metabolite changes under control conditions. Information from N=282 intervals of 25 min duration from all five animals. Intervals were grouped according to change in inorganic phosphate between successive NMR spectra. Groups were: [Pi]=0–0.99 µmol g–1 wet mass (N=88 cases), [Pi]=1–1.99 µmol g–1 wet mass (N=25 cases), [Pi]=2–2.99 µmol g–1 wet mass (N=9 cases) and [Pi]=>3 µmol g–1 wet mass (N=9 cases) on the positive side; [Pi]=0 to –0.99 µmol g–1 wet mass (N=90 cases), [Pi]=–1 to –1.99 µmol g–1 wet mass (N=31 cases), [Pi]=–2 to –2.99 µmol g–1 wet mass (N=18 cases) and [Pi]=<–3 µmol g–1 wet mass (N=9 cases) on the negative side. Number of cases per group (displayed in B) are a direct measure of the probability of occurrence, as all intervals were picked randomly. Linear regression equations: (A) pHi=7.444+0.022[Pi]; r2 =0.96, F1,5=121, P<0.001 (for the range <–3 to 3 µmol g–1 wet mass); (B) [PLA]i=–0.88[Pi]; r2 =0.99, F1,6=222, P<0.001; (C) H+i=–0.41–1.58[Pi]; r2=0.94, F1,5=88, P<0.001 (for the range <–3 to 3 µmol g–1 wet mass); (D) For ANOVA, see text. All concentrations in µmol g–1 wet mass (=wet mass–1).

View larger version (27K): [in a new window]   Fig. 5. Mantle organ metabolic status vs temperature. (A) A set of spectra obtained on animal 1 at different temperatures. Note increases in inorganic phosphate (Pi) peak area towards high and low temperatures. (B) ATP and Pi concentrations; (C) phospho-L-arginine (PLA) concentration; (D) intracellular pH (pHi). N=5 animals per temperature. Error bars represent standard deviation. Data derived from in vivo 31P NMR spectra. Concentrations of metabolites are proportional to the area under their respective peaks in the 31P NMR spectrum.

View larger version (27K): [in a new window]   Fig. 6. Mantle metabolic status and mean mantle pressure amplitude (MMPA) vs temperature. (A) MMPA maxima encountered in animals 1 and 5; (B) concomitant changes in [Pi] and free energy change of ATP hydrolysis (dG/d) in the radial muscle compartment, based on the assumption that observed changes in in vivo 31P NMR spectra solely represent the situation in working radial muscles (see text). Animal 1 had the lowest and animal 5 had the highest thermal tolerance of all animals investigated. Still, both show a tight correlation between stagnating and, eventually, decreasing, pressure amplitudes once |dG/d | decreases (the other three animals show similar patterns; data not shown).