Biochemical and Molecular Analysis of Juvenile Hormone Synthesis and its Regulation in the Mediterranean Fruit Fly (Ceratitis capitata)

Original Objectives and revisions: (1) "To determine the biosynthetic pathway of JHB3 in the adult C. capitata CA in order to establish parameters for the future choice and synthesis of suitable inhibitors". Modified: to determine the pattern of FR-7 biosynthesis during normal reproductive maturation, and identify enzymes potentially involved in its synthesis. (2) "To correlate allatal epoxidase activity to the biosynthesis of JHB3 at different stages of reproductive maturation/vitellogenesis and evaluate the hypothesis that a specific JH-epoxidase may be rate limiting". Modified: to study the effects of epoxidase inhibitors on the pattern of allatal JH biosynthesis in vitro and on female reproduction in vive. (3) "To probe and clone the gene homologous to ap from C. capitata, determine its exon-intron organization, sequence it and demonstrate its spatial and temporal expression in larvae, pupae and adults." The "Medfly" (Ceratitis capitata) is a serious polyphagous fruit pest, widely distributed in subtropical regions. Damage is caused by oviposition and subsequent development of larvae. JH's are dominant gonadotropic factors in insects. In the higher Diptera, to which the Medfly belongs, JHB3 is a major homolog. It comprises 95% of the total JH produced in vitro in D. melanogaster, with JH-III found as a minor component. The biosynthesis of both JH-III and JHB3 is dependent on epoxidation of double bonds in the JH molecule. The specificity of such epoxidases is unknown. The male accessory gland D. melanogaster produces a Sex Peptide, transferred to the female during copulation. SP reduces female receptivity while activating specific JH biosynthesis in vitro and inducing oviposition in vive. It also reduces pheromone production and activates CA of the moth Helicoverpa armigera. In a previous study, mutants of the apterous (ap) gene of D. melanogaster were analyzed. This gene induces previteilogenic arrest which can be rescued by external application of JH. Considerable progress has been made in recombinant DNA technology of the Medfly. When fully operative, it might be possible to effectively transfer D. melanogaster endocrine gene-lesions into the Medfly as a strategy for their genetic control. A marked heterogeneity in the pattern of JH homologs produced by Medfly CA was observed. Contrary to the anticipated biosynthesis of JHB;, significant amounts of an unknown JH-like compound, of unknown structure and provisionally termed FR-7, were produced, in addition to significant amounts of JH-III and JHB3. Inhibitors of monooxygenases, devised for their effects on ecdysteroid biosynthesis, affect Medfly JH biosynthesis but do not reduce egg deposition. FR-7 was isolated from incubation media of Medfly CA and examined by various MS procedures, but its structure is not yet resolved. MS analysis is being done in collaboration with Professor R.R.W. Rickards of the Australian National University in Canberra, Australia. A homologue of the ap gene of D. melanogaster exists in the Medfly. LIM domains and the homeo-domain, important for the function of the D. melanogaster ap gene, are conserved here too. Attempts to clone the complete gene were unsuccessful. Due to the complexity of JH homologs, presence of related FR-7 in the biosynthetic products of Medfly CA and lack of reduction in eggs deposited in the presence of monooxygenase inhibitors, inhibition of epoxidases is not a feasible alternative to control Medfly reproduction, and raises questions which cannot be resolved within the current dogma of hormonal control of reproduction in Diptera. The Medfly ap gene has similar domains to the D. melanogaster ap gene. Although mutant ap genes are involved in JH deficiency, ap is a questionable candidate for an endocrine lesion, especially since the D. melanogoster gene functions is a transcription factor.