El-Wadawi, Rukaya A. (1996) Heat death and the development of thermotolerance in the blow fly calliphora viicina: a study of flight muscle mitochondrial function. Doctoral thesis, Durham University.
The LD(_50) of 10-day-old blowflies differed significantly in two different stocks, and were found to be 38.12 ± 0.07ºC for the Durham stock and 40.8 ± 0.18ºC for the Cambridge stock. A transitory increase in heat resistance occurred following the exposure of adult blowflies to a sublethal heat shock. This thermotolerance was apparent 1h after the application of heat shock, was maximal 2-3 h later and had disappeared after 6 h. Oxidative phosphorylation by flight muscle mitochondria from non-thermotolerant control flies was impaired by an LD(_50) dose in vivo. Respiration using glycerol-3- phosphate was more heat sensitive than that with pyruvate plus proline. State III respiration was markedly inhibited, acceptor control (RCI) was lost with (G 3P) as substrate and so ADP:0 ratios were not measurable; whereas with pyruvate + proline as substrates, although State III respiration was inhibited by 50% and acceptor control was significantly reduced, ADP:0 remained measurable. Uncoupling of oxidative phosphorylation was obvious only with pyruvate + proline where State IV was significantiy increased. The development of thermotolerance protected oxidative phosphorylation against heat damage. With G-3-P respiration State III was largely restored and acceptor control was not significantly different from controls, but ADP:0 remained lower. With pyruvate + proline as substrates State III respiration was inhibited, but State IV was also lower without evidence of uncoupling of oxidative phosphorylation. Acceptor control was restored to control levels but ADP:0 values were lower. The lower ADP:0 ratios indicate some impairment of mitochondrial function occurred. The effect of experimental temperature in vitro on respiratory performance of mitochondria from non-pretreated control and thermotolerant LD(_50) flies was also determined between 19 and 39ºC. State III respiration was markedly temperature- dependent in mitochondria from non-pretreated control flies with both substrates; it was maximal at 24-29ºC and fell progressively at higher measuring temperatures. In mitochondria from thermotolerant flies, State III respiration was less temperature dependent with both substrates, but this effect was more marked for G-3-P. The effect of experimental temperature on State IV respiration was similar in mitochondria from non- pretreated control and thermotolerant LD(_50) flies with the same substrate, but differed between the two substrates. With G 3P as substrate, respiration rate rose with temperature with a Q(_10) of approximately 1.5; however, with pyruvate + proline as substrate, the trend was for respiration rate to fall as experimental temperature rose. Differences in the temperature sensitivities of mitochondria from control and thermotolerant flies, in terms of acceptor control, were found. Using G-3-P, acceptor control was lost in mitochondria from control flies above 29ºC, but was still measurable at 34ºC in mitochondria from thermotolerant flies. With pyruvate + proline as substrate acceptor control was demonstrable in mitochondria from both non-pre-treated control and thermotolerant flies at all experimental temperatures. The thermal sensitivities of the respiratory complexes were studied using the inhibitors rotenone and antimycin A. In mitochondria from LD(_50) treated control flies respiration uncoupled with FCCP was not restored to State II levels. However, in LD(_50) treated mitochondria from thermotolerant flies respiration uncoupled with FCCP was not different from State III respiration. These data suggest that the reduction in State III respiration after heating is owing to an inhibition of oxidation rather than phosphorylation. Complex I, NADH coenzyme Q reductase, was shown to be the most temperature sensitive of the respiratory complexes.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||24 Oct 2012 15:06|