Characterisation of juvenile hormone esterase in Drosophila melanogaster

Abstract

Juvenile hormone (JH) is one of the main morphogenic hormones of insects and degradation of the hormone is believed to play an important role in its regulation in many insects. The two main routes for JH degradation are hydroy sis by JH esterase (JHE) and JH epoxide hydrolase (JHEH). • A major focus of research has been on juvenile hormone esterase (JHE) found in the haemolymph of final instar lepidopteran larvae, although considerable hydrolysis of JH occurs in other tissues and at other life stages. In contrast, no comprehensive study of JH degradation has been undertaken in any dipteran species. However, the available evidence suggests that very little JH degrading activity is found in the haemolymph of Diptera and several dipteran JHEs exhibit atypical patterns of sensitivity to inhibitors. These data suggested that both the expression pattern and the biochemical properties of dipteran JH degrading enzymes may differ from other insect orders, and therefore, that the role of JH degradation may be different.The aim of this project was to comprehensively characterise JH degradation in the dipteran, Drosophila melanogaster, with special emphasis on JHE. The first objective was to identify the JH degrading enzymes of D . melanogaster and the second was to deterrnine their expression patterns and biochemical properties in order to determine thei~ role in the regulation of JH titre. These data were compared with other data to deterrnine whether a general model could be developed for dipteran JH titre regulation. Finally, the roles of JH hydrolysis in the regulation of JH titre were compared among Diptera, Lepidoptera and other insects.Two approaches were taken to identify the JH degrading enzymes of D. melanogaster. The first was to investigate the properties of an esterase, denoted EST20, which can be visualised after native polyacrylarnide gel electrophoresis using artificial esterase substrates, a-and B-naphthylacetate. EST20 is shown to be a likely homologue of the p-esterase of Drosophila virilis, which has been claimed to be the JHE of this species. However, this study shows that JH hydrolytic activity is associated with neither EST20 in D. melanogaster nor the p-esterase in D . virilis.The second approach was to use a radiometric assay which identified two JH hydrolysing enzymes, JHE and JHEH. The developmental profile of JH hydrolytic activity in D. melanogaster correlates inversely with profiles of JH titre and production. Key features are prewandering and prepupal maxima of JH hydrolytic activity in whole body homogenates of final instar larvae. This developmental profile of expression is similar to the lepidopteran pattern suggesting that the role of JH hydrolysis is essentially similar during this period. In D. melanogaster, the prewandering maximum consists mainly of JHEH whereas the prepupal peak consists mainly of JHE. However, the relative contributions of JHE and JHEH at these times differs both among the Diptera and in other orders.JHE from the prepupal stage was purified to homogeneity by selectiv...

Evolutionary genetics ofDrosophila esterases

“…At least seven more can be resolved if a second dimension of electrophoresis involving isoelectric focussing is applied (Healy, Dumancic & Oakeshott, 1991;Campbell, Healy & Oakeshott, 1992). Most of these esterases are probably encoded by different genes.…”

“…If the aryl esterases of D. melanogaster behave in a similar way they would probably not have been detected under the conditions used to survey this species (Healy, Dumancic & Oakeshott, 1991, and references therein). Two carboxylesterases (esterase 6, or EST6, and juvenile hormone esterase, or JHE; White, Mane & Richmond, 1988;Campbell, Healy & Oakeshott, 1992) and two cholinesterases (ACHE and EST9;Fournier et al, 1988;Morton & Singh, 1985) have been substantially purified from D. melanogaster and a few others have been similarly characterised from other drosophilid (see below) and non-drosophilid insects (e.g., Devonshire, 1977;Kao, Motoyama & Dauterman, 1985;Ziegler et al, 1987;Mouchds et al, 1987;Abdel-Aal et al, 1988;Field et al, 1993). Like thei r mammalian Table 1.…”

Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle

“…A specific partition assay based on the inhibition of all of the JHE activity present in insect tissue extracts (Share and Roe, 1988) has been used to determine to which extent JHEH participates in JH degradation (Touhara and Prestwich, 1993;Wojtasek and Prestwich, 1996;Debernard et al, 1998). The assay reveals that JHEH is as critical as JHE in degrading JH in certain insects (Campbell et al, 1992;Halarnkar et al, 1993;Lassiter et al, 1995;Debernard et al, 1998). In the fourth larval instar of Culex quinquefasciatus, for example, JHEH activity is higher than JHE activity (Lassiter et al, 1995), implying its major function in JH degradation.…”

Endocrine regulation of reproduction and diapause in the boll weevil, Anthonomus grandis Boheman

“…Furthermore, we have characterized A. grandis hemolymph esterases using a series of inhibitor studies in which 5th instar Manduca sexta hemolymph was used as a positive control. We used four inhibitors: DFP (O,O-diisopropyl phosphorofluoridate) a general esterase inhibitor that typically does not inhibit JH-specific esterases (for exceptions, see Yu and Terriere, 1978;Peter et al, 1979;Campbell et al, 1992), and three compounds that inhibit JH-specific esterases in other systems. As expected, DFP was not a potent inhibitor of JH-specific esterase in M. sexta nor did it inhibit A. grandis JHE.…”