title: Effects of azadirachtin on protein synthesis in specific tissues of the desert locust Schistocerca gregaria
Creators: name: Iqbal, Shagufta
Publisher: name: University of Glasgow
Year: 1999
Subjects: subject: Physiology, entomology.

description: Azadirachtin A, one of the most polar of the many terpenoids occurring in the the seeds of the neem tree (Azadirachta Indica) has long been known to be the most potent in terms of its toxic effects on insects. Although its exact primary mode of action remains unclear, much research has implied that it inhibits protein biosynthesis in various insect tissues. This then leads to many secondary effects, such as sterility, developmental failure and general loss of biological fitness. The aim of the work reported in this thesis was to choose two specific proteins, lysozyme and vitellogenin, which could be easily quantified, and synthesised mainly by the fat body. Previous work had suggested that the proteins synthesised by fat body were reduced by azadirachtin. It was hoped that the synthesis of both could be induced by appropriate chemical stimuli both in vivo and in vitro. The insect studied, the desert locust Schistocerca gregaria, is known to be sensitive to the effects of azadirachtin, and is sufficiently large to allow ease of tissue sampling. Preliminary studies with the uptake of [3H]-leucine into the proteins of fat body confirmed that aza reduced incorporation into the acid-preciptable proteins of the tissue and of the haemolymph by respectively 42% and 25% over a short time course. Injection of bacterial lipopolysaccharide (LPS) was found to double the amount of circulating lysozyme within 24 hours. Further results suggested that this originated in both the fat body and haemocytes. Subsequent studies concentrated on the fat body as it yielded more material. An incidental observation from this work was that lysozyme induction and release may be a general result of any mechanical or chemical insult to the locusts. Azadirachtin A (3mug/g body weight) largely eliminated the increase in lysozyme activity, reducing it to resting levels. Studies with isolated fat body confirmed that the effect of both LPS and azadirachtin could be shown to be direct effects on the fat body itself. In studying the production of vitellogenin, it was found necessary to estimate the protein by immunological means. Vitellin was purified from mature ovarian tissue, and was used to raise a polyclonal antiserum in rabbits. This antiserum was found to cross-react with vitellogenin, and was used as the basis for various immunological assays. An ELISA assay allowed quantitative assays of both vitellogenin in fat body and haemolymph, and of vitellin in ovaries. Over a period of a month after imaginal moult, the proportion of vitellogenin in the female haemolymph rose from undetectable below 8 days to a maximum of 70% of the total protein. Treatment of females at 15 and 19 days with azadirachtin reduced the amount of vitellogenin by 62% and that in ovaries by 73% by 48 hours after treatment. These effects in vivo could be largely reproduced in vitro with isolated fat-body, using methoprene, a Juvenile Hormone analogue, as the stimxilus. In conclusion, the results presented show that azadirachtin prevented at least part of the increased production, induced by appropriate factors, of two identified proteins. This is consistent with previous biological observations of the action of the terpenoid.