Synthesis of mitochondrial protein in the flight muscles of the Colorado beetle

This thesis deals with the biogenesis of flight-muscle mitochondria in the Colorado beetle ( Leptinotarsa decemlineata Say).In beetles, reared under conditions of long photoperiod, the activities of succinate dehydrogenase and cytochrome c oxidase and the contents of cytochromes aa3 , b , and c were found to increase roughly in parallel. This process starts around adult emergence and is completed after 8 days. The above enzymes provide an index of mitochondrial growth.Particular attention has been paid to the relative contributions of the two genetic systems concerned in mitochondriogenesis. In vivo, the incorporation of [ 14C]leucine was measured after administration of cycloheximide, a specific inhibitor of cytoplasmic protein synthesis. The incorporation into the cytosol protein of the flight muscles appeared to be inhibited by more than 99.5% with the antibiotic. However, a considerable amount of radioactivity was found in the protein of mitochondria. At the beginning of mitochondrial formation and in beetles with full-grown flight muscles the amino acid incorporation amounted to about 5% of the control value. During the period of rapid accumulation of mitochondrial enzymes, the percentage of cycloheximide-resistant incorporation increased to 14. This incorporation was sensitive to the inhibitor of mitochondrial protein synthesis, chloramphenicol.The incorporation activity of the isolated mitochondria in vitro was also examined. Attempts were made to elaborate a suitable incubation medium. It appeared, that proline was the most effective respiratory substrate in promoting the incorporation of [ 14C]leucine into protein. optimum incorporation furthermore required appropriate concentrations of KG, ADP, phosphate, Mg 2+- and H +-ions, and a synthetic amino acid mixture. Subsequently, the incorporation activity was estimated at various times during mitochondrial formation. Maximum rates of amino acid incorporation were recorded in the phase of rapid enzyme accumulation. Apparently, formation of mitochondria involves an enhanced contribution of the intrinsic translation system.While beetles reared at long photoperiods display locomotion and oviposition, treatment with a short-day régime induces diapause. In pre-diapause animals a development of the cytochrome system can be found after adult emergence, but the increase proceeds more slowly than in long-day beetles. This increase stops after one week and is then followed by a decline in cytochrome content. During deep diapause the cytochromes are hardly detectable in the flight muscles.Incorporation experiments with pre-diapause animals in vivo yielded similar results to those obtained with beetles reared under long-day conditions. Firstly, there was an increase in the percentage of cycloheximide-resistant [ 14C]leucine incorporation which later fell. Because of the similarities it is impossible to say which of the two proteinsynthesizing systems is responsible for the stagnation in mitochondrial growth at short photoperiods.The incorporation activities in vitro of flight-muscle mitochondria isolated from pre-diapause beetles were somewhat lower than those measured at corresponding times during normal flight-muscle development. Very low incorporation rates were recorded at the onset of diapause. Topical application of juvenile hormone not only broke diapause but also stimulated the incorporation activity of isolated mitochondria. No stimulation was found after direct addition of the hormone to the mitochondria in vitro. This indicates that the effect of juvenile hormone on amino acid incorporation is indirect.