Biochemical Aspects of Juvenile Hormone Action in the Adult Locusta Migratoria by
The present study deals with the action of the juvenile hormone (JH) in the adult Locusta migratoria. It is divided into two parts. The first problem, discussed in chapter 3, is whether or not JH exerts a direct influence on respiratory metabolism. Many authors have tried to answer this question definitely by means of extirpation and/or implantation of the corpora allata (CA) in experiments in vivo. Only in vitro experiments can, however, provide a clue to this problem. Therefore, mitochondria isolated from flight muscles and fat body were used (section 3. 1.). No differences exist between the rates of oxygen consumption and oxidative phosphorylation of flight muscle mitochondria - with substrates -glycerophosphate and pyruvate/malate - from normal and allatectomiz ed Locusta. Homogenized active CA never have any effect on the oxygen consumption of both types of mitochondria. The CA have a stimulating effect on the oxidative phosphorylation of flight muscle mitochondria with -glycerophosphate and no effect with pyruvate/malate as a substrate. More evidence is given in support of the assumption that the phases of Locusta differ in their level of JH-activity. The solitary phase is more "juvenile". CA isolated from solitary locusts stimulate the oxidative phosphorylation in mitochondria from the gregarious and also from the allatectomized locusts; CA isolated from gregarious locusts only the mitochondria from allatectomized locusts. This applies to flight muscle- as well as to fat body mitochondria, incubated with -glycerophosphate. These data at the same time are a strong support for a specific action of the JH in these experiments (3.2.1.). The uncoupling agent 2.4.-dinitrophenol (DNP) stimulates oxygen consumption under non-phosphorylating conditions, but inhibits this process under phosphorylating conditions and uncouples oxidative phosphorylation of flight muscle mitochondria in both substrates. This effect of DNP has disappeared when it is applied at a concentration of 10-6 M. The CA depress the uncoupling effect of DNP at intermediate DNPconcentrations, at which the oxidative phosphorylation is not completely inhibited (10-4 - 10-5 M). The condition of the isolated mitochondria is controlled by a comparison of the Mg++- and DNP-induced adenosine triphosphatase activity. It appears that CA inhibit the DNP-induced ATP-ase activity of both flight muscle- and fat body mitochondria (3.2.2.). Cecropia oil, farnesol and farnesyl methyl-ether, added to flight muscle mitochondria, have no effects on oxygen consumption. Cecropia extract at 10-6 - 10-8 v/v enlarges the efficiency of the oxidative phosphorylation in contrast to the farnesol compounds (3.2.3.). We can conclude that there is no relation whatsoever between JH-action and oxygen consumption in vitro. However, a positive effect of JH on oxidative phosphorylation is evident. It is not possible to explain the present results against a background of data derived from insect endocrinology. The in vitro JH-effects are discussed in the following points: a. A comparison with thyroxine effects in rats; b. The possible stimulation of the so-called coupling factors, and c. The possible effect, via the ATP-ase activity, on the Na+/K+ ratio of the mitochondria (3.3.). After that, several aspects of the reproductive metabolism of normal and allatectomized adult Locusta have been compared (chapter 4). The relations between the different parts of the neuro-endocrine system which regulate reproduction and its underlying metabolic processes are very complicated. It is, therefore, difficult to establish, which part is played by various organs in the hormonal regulation of reproduction. Particularly the function of the CA is subject to much diversity of opinion. A study of extra-ovarian protein synthesis by the fat body yielded further useful information. For this purpose the haemolymph, as transporting medium of the proteins, can be used to convenience (4. 1.). At first quantitative analyses offat body, haemolymph and ovary have been carried out during the first 24 days of adult life. Dry weight and content of protein, RNA-phosphorus, free amino acids, fat and glycogen (only in the fat body) and trehalose (only in the haemolymph) have been determined and compared in normal and allatectomized Locusta. The relation with especially the first sexual cycle is clear in the normal females as well as in the males. There is no ovarian development in the allatectomized insects and the cyclicity of the above-mentioned constituents offat body, haemolymph and ovary completely disappears. The protein content in haemolymph, fat and glycogen in the fat body rises above normal (4.2.5.). The in vivo incorporation activity of 14C-labelled amino acids (as determined 24 hours after injection) into the proteins of fat body, haemolymph and ovary has been followed throughout the first 24 days of the adult stage. The normal Locusta exhibits a periodic activity, related to the sexual cycles, which disappears in the allatectomized insects. The protein synthesis is at its maximum at the 9th day, just at the beginning of the first cycle (4.2.6.). The in vitro incorporation activity of 14C-amino acids into the proteins of homogenized fat body shows only differences between normal and allatectomized Locusta at the time oocyte development ought to begin. In this respect mitochondria isolated from the fat body of both categories significantly differ, except for ageing locusts (4.2.7.). After separation of haemolymph proteins by agar-gel electrophoresis, 7-8 bands can be distinguished. Two "negative" proteins (6 and 7) are correlated with the sexual development of the locust. After allatectomy a "positive" protein 3 becomes the most important fraction (4.2.8.). Implantation of active CA in allatectomized female Locusta immediately brought the reproductive processes into action in more than 95 % of the cases, but only for the duration of a single cycle. By means of autoradiography of 14C-labelled haemolymph proteins a quick shift of the protein synthesis can be established (4.2.9.). Protein fraction c of the fat body is probably identical to haemolymph protein 3; fraction f to band 7 (4.2. 10.). The haemolymph protein patterns of allatectomized and NSCcauterized female Locusta bear a great similarity (4. 2 .11.). However, the haemolymph proteinpatterns of ovariectomized females bred in isolation are similar to normal ones, except for the accumulation of fraction 4. Haemolymph volume and protein content show an important increase in ovariectomized females bred with active males (4.2.12.) These results are in accord with the conception that the fat body produces a considerable fraction of the proteins required for oogenesis. We agree with the ideas of HIGHNAM et al. (1963) according to which the NSC activate protein synthesis in the fat body. We attach, however, a more independent rôle to the CA in this process. Our data are more in accord with the idea that JH exerts its activities in the presence of active protein synthesis and induces the formation of specific vitellogenic proteins. We possibly may explain the change of lipid and glycogen metabolism in the fat body in the same way (4.3.).