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The activity of eight substituted imidazoles as inhibitors of juvenile hormone III synthesis in the cockroach, Diploptera punctata , was measured in a short-term in vitro radiochemical assay. The potency of these compounds (IC 50 of juvenile hormone III synthesis) ranged from 80 n M for KK-98 to 48 μ M for KK-71. For some compounds (KK-71, KK-51), inhibition of juvenile hormone III synthesis was not accompanied by an accumulation of the immediate precursor methyl farnesoate within the glands. For other compounds (KK-83, KK-96, KK-98) significant accumulation (i.e., more than 100 pmol/pair) of methyl farnesoate was observed. The results suggest that some substituted imidazole compounds are selective inhibitors of the corpus allatum methyl farnesoate epoxidase at low concentrations, but all inhibit methyl farnesoate synthesis at higher concentrations. Experiments with farnesol and farnesoic acid, two precursors of juvenile hormone III synthesis, suggested that the imidazole compounds may inhibit farnesoic acid O -methyl transferase in addition to their predicted inhibition of the epoxidase.

There are major changes in the sensitivity of corpora allata from the cockroach Diploptera punctata toward the allatostatic tridecapeptide APSGAQRLYGFGL-amide (ASAL) during the female reproductive cycle, as revealed by measurement of juvenile hormone (JH) biosynthesis in vitro. Glands from recently molted adult females show only 30%–40% inhibition at 10 nM ASAL, falling to a minimum of less than 10% on day 5 at the peak of spontaneous JH synthesis in vitro. The decline in JH synthesis observed in post-vitellogenic females is accompanied by a dramatic increase to ca. 90% ASAL sensitivity at 10 nM by day 6. Then sensitivity slowly wanes during subsequent ovulation and pregnancy to the levels typical of previtellogenic and virgin females. Full dose/response studies reveal a second level of response at ca. 1 μM, which resembles the pattern obtained from whole brain extracts. We conclude that physiological sensitivity to ASAL (IC50 ca. 0.1 nM) is correlated with the preparation for choriogenesis, and we suggest that 1000 times higher doses give a cross-reaction with related allatostatic receptor(s) that confer important sensitivity at other developmental stages.

Juvenile hormone III biosynthesis by corpora allata of adult female Leucophaea maderae was measured by an in vitro radiochemical assay. In fed females, JH III synthesis increases more than 20-fold after mating to a peak of 55 pmol/pair/h on day 9 and then rapidly declines. This increase in JH III synthesis concomitant with rapid oocyte growth in mated females is not observed in virgin females. The corpora allata from starved, virgin females appear to be inactive. The addition of 150 microM 2E,6E-farnesol (a) JH III precursor) to the incubation medium stimulates the corpora allata from starved, virgin females less than the corpora allata from starved, mated females. Both feeding and mating are necessary for the expression of a normal cycle of JH III synthesis in this cockroach.

The honeybee genome has substantially fewer protein coding genes (approximate to 11 000 genes) than Drosophila melanogaster (approximate to 13 500) and Anopheles gambiae (approximate to 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.

Background Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. Results Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76%) showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses). Conclusion This analysis of the host-polydnavirus interactions by a microarray approach indicates that the presence of HdIV induces, directly or indirectly, variations in transcript levels of specific host genes, changes that could be responsible in part for the alterations observed in the parasitized host physiology. Development of such global approaches will allow a better understanding of the strategies employed by parasites to manipulate their host physiology, and will permit the identification of potential targets of the immunosuppressive polydnaviruses.

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).

The structure and stereochemistry of nine steroid metabolites isolated in quantities ranging from 0.15 to 1.8 mg were determined using a variety of NMR techniques, including heteronuclear multiple bond correlation (HMBC) using broadband adiabatic 13C pulses and phase-sensitive data presentation. Testosterone, androstenedione and progesterone were oxidized with housefly cytochrome P450 6A1 enzyme reconstituted in vitro with housefly NADPH cytochrome P450 reductase and cytochrome b5. NMR analysis in CD3OD using a modified HMBC sequence as well as 2D heteronuclear single quantum correlation (HSQC), COSY and nuclear Overhauser and exchange spectroscopy (NOESY), combined with a detailed analysis of J couplings showed that hydroxylation occurs exclusively on the beta-face of the steroids, at positions 2, 12, and 15.

Transposons are well-known architects of genetic change but their role in insecticide resistance has, until recently, only been speculated upon. Transposon insertion, or transposon-mediated transposition, could alter either metabolic enzymes capable of degrading pesticides or could change the functionality of insecticide targets. The recent work of Aminetzach and coworkers suggests an exciting alternative, that transposon insertion can cause resistance by altering gene product function. This hypothesis is discussed in the light of other examples in which transposons have been implicated in insecticide resistance.

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

The effect of xenobiotics (phenobarbital and atrazine) on the expression of Drosophila melanogaster CYP genes encoding cytochromes P450, a gene family generally associated with detoxification, was analyzed by DNA microarray hybridization and verified by real-time RT-PCR in adults of both sexes. Only a small subset of the 86 CYP genes was significantly induced by the xenobiotics. Eleven CYP genes and three glutathione S-transferases (GST) genes were significantly induced by phenobarbital, seven CYP and one GST gene were induced by atrazine. Cyp6d5, Cyp6w1, Cyp12d1 and the ecdysone-inducible Cyp6a2 were induced by both chemicals. The constitutive expression of several of the inducible genes (Cyp6a2, Cyp6a8, Cyp6d5, Cyp12d1) was higher in males than in females, and the induced level similar in both sexes. Thus, the level of induction was consistently higher in females than in males. The female-specific and hormonally regulated yolk protein genes were significantly induced by phenobarbital in males and repressed by atrazine in females. Our results suggest that the numerous CYP genes of Drosophila respond selectively to xenobiotics, providing the fly with an adaptive response to chemically adverse environments. The xenobiotic inducibility of some CYP genes previously associated with insecticide resistance in laboratory-selected strains (Cyp6a2, Cyp6a8, Cyp12d1) suggests that deregulation of P450 gene expression may be a facile way to achieve resistance. Our study also suggests that xenobiotic-induced changes in P450 levels can affect insect fitness by interfering with hormonally regulated networks.

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14α-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14α-methyl-Δ8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14α-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.

A protein fragment of P450BM3 (residues 73-84) which participates in palmitoleate binding was subjected to scanning chimeragenesis. Amino acids 73-84, 73-78, 75-80, and 78-82 were replaced with the homologous fragments of the insect terpenoid hydroxylase CYP4C7. The four chimeric proteins, C(73-84), C(73-78), C(75-80), and C(78-82), were expressed, purified, and characterized. All the chimeric proteins contained all the cofactors and catalyzed monooxygenation of palmitate and of the sesquiterpene farnesol. Chimeragenesis altered substrate binding as shown by the changes in the amplitude of the palmitate-induced type I spectral shift. C(78-82) had monooxygenase activities close to those of P450BM3, while the rest of the chimeric proteins had monooxygenase activities that were inhibited relative to that of wild-type P450BM3. The extent of inhibition of the chimeric proteins varied depending on the substrate, and in the case of C(73-84), farnesol and palmitate oxidation was inhibited by 1 and 4 orders of magnitude, respectively. (1)H NMR spectroscopy and GC-MS were used to identify products of farnesol and palmitate oxidation. Wild-type P450BM3 and all chimeric proteins catalyzed oxidation of farnesol with formation of 9-hydroxyfarnesol and farnesol 10,11- and 2,3-epoxides. Three of the four chimeric proteins also formed a new compound, 5-hydroxyfarnesol, which was the major product in the case of C(73-78). In addition to hydroxylation of the C13-C15 atoms, the chimeric enzymes catalyze significant hydroxylation of the C10-C12 atoms of palmitate. In the case of C(78-82), the rates of formation of 11- and 12-hydroxypalmitates increased 7-fold compared to that of wild-type P450BM3 to 106 and 212 min(-)(1), respectively, while the rate of 10-hydroxypalmitate synthesis increased from zero to 106 min(-)(1). Thus, chimeragenesis of the region of residues 73-84 of the substrate binding site shifted the regiospecificity of substrate oxidation toward the center of the farnesol and palmitate molecules.

The molecular analysis of insect hormone biosynthesis has long been hampered by the minute size of the endocrine glands producing them. Expressed sequence tags from the corpora allata of the cockroach Diploptera punctata yielded a new cytochrome P450, CYP15A1. Its full-length cDNA encoded a 493-aa protein that has only 34% amino acid identity with CYP4C7, a terpenoid ω-hydroxylase previously cloned from this tissue. Heterologous expression of the cDNA in Escherichia coli produced >300 nmol of CYP15A1 per liter of culture. After purification, its catalytic activity was reconstituted by using phospholipids and house fly P450 reductase. CYP15A1 metabolizes methyl (2 E ,6 E )-3,7,11-trimethyl-2,6-dodecatrienoate (methyl farnesoate) to methyl (2 E ,6 E )-(10 R )-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate [juvenile hormone III, JH III] with a turnover of 3–5 nmol/min/nmol P450. The enzyme produces JH III with a ratio of ≈98:2 in favor of the natural (10 R )-epoxide enantiomer. This result is in contrast to other insect P450s, such as CYP6A1, that epoxidize methyl farnesoate with lower regio- and stereoselectivity. RT-PCR experiments show that the CYP15A1 gene is expressed selectively in the corpora allata of D. punctata , at the time of maximal JH production by the glands. We thus report the cloning and functional expression of a gene involved in an insect-specific step of juvenile hormone biosynthesis. Heterologously expressed CYP15A1 from D. punctata or its ortholog from economically important species may be useful in the design and screening of selective insect control agents.

Diflavin reductases are enzymes which emerged as a gene fusion of ferredoxin (flavodoxin) reductase and flavodoxin. The enzymes of this family tightly bind two flavin cofactors, FAD and FMN, and catalyze transfer of the reducing equivalents from the two-electron donor NADPH to a variety of one-electron acceptors. Cytochrome P450 reductase (P450R), a flavoprotein subunit of sulfite reductase (SiR), and flavoprotein domains of naturally occurring flavocytochrome fusion enzymes like nitric oxide synthases (NOS) and the fatty acid hydroxylase from Bacillus megaterium are some of the enzymes of this family. In this review the results of the last decade of research are summarized, and some earlier results are reevaluated as well. The kinetic mechanism of cytochrome c reduction is analyzed in light of other results on flavoprotein interactions with nucleotides and cytochromes. The roles of the binding sites of the isoalloxazine rings of the flavin cofactors and conformational changes of the protein in electron transfer are discussed. It is proposed that minor conformational changes during catalysis can potentiate properties of the redox centers during the catalytic turnover. A function of the aromatic residue that shields the isoalloxazine ring of the FAD is also proposed.

The synthesis of juvenile hormone III (JH III) by the isolated corpora allata (CA) of Aedes aegypti adult female was studied using an in vitro radiochemical assay. We dissected the corpora allata-corpora cardiaca (CA-CC) complex attached to a piece of aorta. The complex was left connected to the intact head capsule to facilitate the visualization and transfer of the glands. A linear increase in the cumulative amount of biosynthesized JH III was found for at least the first 6 h of incubation; approximately 45% of the synthesized JH III was present in the medium. There was a dependence of JH III synthesis on exogenous methionine supply. Using reversed phase high performance liquid chromatography two major labeled products biosynthesized by the CA were separated. They co-migrated with JH III and methyl farnesoate (MF). The identity of the biosynthesized JH III was confirmed by gas chromatography-mass spectrometry. JH III synthesis was only 2.0 fmol/pair gland/h immediately after adult emergence, but increased to 32.6 fmol/ pair gland/h 18 h later in sugar-fed females. Two days after emergence, the CA biosynthetic activity slowly started to decrease, and reached values of around 5.3 fmol/pair gland/h by one week after emergence. Synthesis of JH was similar from either sugar-fed females mated or unmated. A blood meal resulted in a decrease of JH III synthesis in CA from mated females by 12 h after feeding and from virgin females by 24 h after feeding. JH III biosynthesis remained low for at least 96 h in mated females, but was back to higher levels 72 h after feeding in virgin females. Rates of JH III biosynthesis closely reflected the hemolymph levels of JH III both after emergence and after a blood meal described by Shapiro et al. (1986). The activity of the CA in Aedes aegypti females seems to be regulated by developmental changes and nutritional signals, and to be independent of mating stimulus.

The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families— the carboxylesterases, glutathione transferases, and cytochrome P450s—that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae , the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development.

Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential roles in pollen maturation and dehiscence and wound-induced defence against biotic attacks. To better understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis knock-out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100-fold 1 h after wounding in wild-type plants, do not increase after wounding in the aos mutant. In addition, the mutant showed severe male sterility due to defects in anther and pollen development. The male-sterile phenotype was completely rescued by exogenous application of methyl jasomonate and by complementation with constitutive expression of the AOS gene. RT-PCR analysis showed that the induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be inducible by wound and jasmonate application in the wild-type, was absent in the aos mutant. In transgenic plants constitutively expressing AOS, wound-induced JA levels were 50-100% higher compared to wild-type plants. Taken together with JA deficiency in the aos mutant, our results show that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in unwounded plants.

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.

The interaction of recombinant house fly (Musca domestica) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one molecule of NADPH, whereas the rate of oxidation of a second molecule of NADPH is too slow to account for the observed rates of catalysis. This demonstrates that house fly P450 reductase does not require a priming reaction with NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectroscopy revealed that the enzyme forms two types of neutral FMN semiquinone. One serves as the catalytic intermediate of cytochrome c reduction, and another one is an ‘air-stable’ semiquinone, which reduces cytochrome c 3000 times more slowly. The results show that the reduction state of the house fly P450 reductase during catalysis cycles in a 0-2-1-0 sequence.

▪ Abstract The P450 enzymes (mixed function oxidases, cytochrome P450 monooxygenases), a diverse class of enzymes found in virtually all insect tissues, fulfill many important tasks, from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of foreign chemicals of natural or synthetic origin. This diversity in function is achieved by a diversity in structure, as insect genomes probably carry about 100 P450 genes, sometimes arranged in clusters, and each coding for a different P450 enzyme. Both microsomal and mitochondrial P450s are present in insects and are best studied by heterologous expression of their cDNA and reconstitution of purified enzymes. P450 genes are under complex regulation, with induction playing a central role in the adaptation to plant chemicals and regulatory mutations playing a central role in insecticide resistance. Polymorphisms in induction or constitutive expression allow insects to scan their P450 gene repertoire for the appropriate response to chemical insults, and these evolutionary pressures in turn maintain P450 diversity.

Eukaryotic P450 proteins are membrane proteins found predominantly in the endoplasmic reticulum. In vertebrates, several biosynthetic P450s are found in mitochondria as well. We cloned three putative insect mitochondrial P450s from larval house fly cDNA. These P450s are members of a new P450 family, CYP12. The CYP12 proteins are most closely related to the mammalian mitochondrial P450 of the CYP11, CYP24, and CYP27 families. The most abundant cDNA, CYP12A1, was expressed in Escherichia coli and purified. NADPH-dependent reduction of CYP12A1 was rapid and efficient with the bovine mitochondrial proteins adrenodoxin reductase and adrenodoxin as electron transfer partners. In contrast, house fly microsomal NADPH cytochrome P450 reductase reduced CYP12A1 only poorly. In a reconstituted system with the bovine mitochondrial electron donors, CYP12A1 metabolized a variety of insecticides and other xenobiotics, but did not metabolize ecdysteroids, juvenoids, or fatty acids. Subcellular localization of CYP12A1 by immunogold histochemistry established the mitochondrial nature of this protein. CYP12A1 mRNA levels are constitutively higher in an insecticide-resistant strain than in a susceptible strain, and this trait maps to chromosome II in the house fly, where the constitutive overexpression of the pesticide-metabolizing microsomal CYP6A1 also maps. Multiple mitochondrial P450s have evolved in insects and may play a role in the metabolism of xenobiotics in addition to their possibly ancestral functions in steroidogenesis.

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b 5 , and NADPH, the purified CYP4C7 metabolized (2 E ,6 E )-farnesol to a more polar product that was identified by GC-MS and by NMR as (10 E )-12-hydroxyfarnesol. CYP4C7 converted JH III to 12- trans -hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This ω-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

Allatostatins, peptides with an YXFGL-amide C-terminus, cause a rapid and reversible inhibition of the sesquiterpenoid juvenile hormone III biosynthesis by cockroach corpora allata in vitro. Inhibition is relieved by farnesol and by mevalonate, and neither HMG-CoA reductase nor HMG-CoA synthesis (HMG-CoA synthase and acetoacetyl-CoA lyase) are inhibited by allatostatin. The neuropeptide is more effective as an inhibitor of JH III synthesis in glucose or amino acid-driven glands than in acetate-driven glands. Incorporation of label from acetate into JH III is increased whereas incorporation of label from glucose is decreased by allatostatin. Citrate is a very effective precursor for HMG-CoA synthesis in homogenates of corpora allata. We propose that inhibition of JH III biosynthesis by the tridecapeptide APSGAQRLYGFGL-amide occurs at the first committed step(s) of JH III synthesis, i.e. the transfer of 2C units from mitochondria to the cytoplasm by the tricarboxylate carrier and/or the ATP-citrate lyase.

To the prevailing biochemical/physiological classification of mechanisms of organismal resistance to toxicants, an additional molecular dimension is proposed. Predictions are developed regarding the relative prevalence of different classes of mutations and are found to compare favorably with reports from the literature. In particular, point mutations in target loci were the dominant form of resistance for both lab and field selection. Amplifications of target loci were less common than structural mutations, and more common for lab-selected than for field-selected strains. Amplification was the most common mechanism of up-regulation of metabolizing enzymes. In comparison, only one mutation involving cis-regulation and several involving trans-acting regulation were found. Mutations involving gene disruption and down-regulation were uncommon, but were found in appropriate cases, i.e., when toxicants stimulated rather than inhibited target function and when metabolizing enzymes converted toxicants into more toxic metabolites. Additional phenomena of likely but uncertain importance are genetic "succession," recombinational limitation, and negative cross-resistance. More work on these phenomena and on quantification of fitness costs of resistance is recommended.

Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.

Publisher Summary This chapter focuses on the sampling of P450 diversity by cloning the polymerase chain reaction (PCR) products obtained with degenerate primers. Molecular cloning of P450 genes and their heterologous expression and reconstitution has so far provided the best method for the biochemical characterization of insect P450s. Molecular probes may also be needed to investigate a myriad of biological problems, such as pesticide resistance, induction by foreign chemicals, and hormonal or developmental control. One strategy is to use the conserved region around the conserved cysteine liganding the heme as a forward primer. Vector primers (for library screening) or oligo(dT) primers (for cDNA) are used as reverse primers. Another strategy is to use the region surrounding the conserved cysteine to design a reverse primer and to use as forward primer a sequence derived from another conserved region in the P450 protein. Thus, versatile methods are now available to clone P450 fragments from virtually any organism.

A cluster of genes of the CYP6 family was found in a series of overlapping lambda DASH clones from a genomic library of the house fly, Musca domestica. Four complete genes, CYP6A3, CYP6A4, CYP6A5, and CYP6C1, and fragments of two other genes, CYP6A6 and CYP6C2, were closely linked on a 24-kb segment of DNA. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified segments of two of the genes showed that the cluster is localized on chromosome V of the house fly. Each gene contained a short intron of 57 to 125 bp interrupting a conserved Glu codon, as in the previously described CYP6A1 gene. The gene fragment CYP6A6 consisted only of the coding region downstream from this intron, i.e., about one-third of the complete P450. The gene fragment CYP6C2 was missing a short amino-terminal part of the coding region, and may represent the two last exons of a larger gene. Gene duplication and chromosomal inversion events may explain the origin of this cluster. The P450 proteins deduced from the nucleotide sequences shared 39-71% amino acid identity with each other. This low identity and the lack of evidence of recent gene conversion events suggested that this cluster may be evolutionarily ancient and that homologous clusters may be found in other holometabolous insects. Evidence for transcription of the genes and for correct splicing of the introns was obtained by northern blotting and reverse transcription polymerase chain reaction (RT-PCR) experiments. No overexpression was observed in any of three insecticide-resistant house fly strains. RT-PCR and sequencing also revealed the existence of other genes or alleles closely related to the members of this cluster.

In order to grow, immature insects must periodically synthesize a new cuticle and shed the old one (a process of molting and ecdysis) until they have reached a stage permitting metamorphosis to the reproductive adult. Each molt is induced by a pulse of ecdysteroids, but the nature of the molt is determined by the juvenile hormones (JHs) [1]. These are sesquiterpenoids, synthesized from three isoprene units via the mevalonate pathway and decorated by an epoxide group on one end and a methyl ester on the other [1]. The JHs maintain larval characters, a status quo effect that is lifted when the level of JH drops. A threshold size must be attained for each molt and for metamorphosis to occur, and this size may relate to the limit of oxygen supply by the tracheal system [2], [3]. The number of molts varies among and sometimes within species; it is influenced by nutrition and by environmental and genetic signals. Yet, the mechanisms that “measure size” or “count the larval instars” are overridden by the experimental depletion of JHs. It has long been a tenet of insect endocrinology that removal of the corpora allata (CA), the endocrine glands that produce the JHs, causes precocious metamorphosis [1]. However, bringing the JH titer down experimentally is not as trivial as it may seem.

Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYVSEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several allatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.

The induction by dietary nicotine of a series of cytochrome P-450 enzyme activities was investigated in early fifth-instarManduca sexta larvae. At a low nicotine concentration in the diet (0.1 %), three of 12 midgut microsomal enzyme activities were significantly increased. At a higher concentration (0.75%) commonly found in plants of the genusNicotiana, nine of 12 activities were induced by 1.4- to 10.0-fold. Total cytochrome P-450, P-450 reductase activity, and midgut microsomal metabolism of nicotine were also increased by feeding 0.75% nicotine. Nicotine was metabolized by midgut microsomes to nicotine-1-N-oxide and cotinine-N-oxide. Fat body microsomal nicotine metabolism was low and unaffected by dietary nicotine. Isolated nerve cords were able to metabolize nicotine in vitro but this metabolism was not inducible by dietary nicotine. Nicotine-fed fifth-instarM. sexta larvae showed an increased tolerance to subsequent nicotine injection when compared to larvae fed a control diet. These results support the idea that induction of midgut cytochrome P-450-related metabolism is an adaptation ofManduca sexta to dietary nicotine.

Antisera to purified house fly NADPH-cytochrome P450 reductase were used to select cDNA clones from an expression library of abdomens of phenobarbital-treated house flies. A partial cDNA of 1841 bp containing a TAG termination codon, a consensus polyadenylation site and 269 bp of 3' untranslated sequence was obtained. Sequencing of a genomic clone coupled with mRNA sequencing yielded the complete coding sequence including the starting ATG. The resulting open reading frame of 2013 nucleotides codes for a protein of 671 residues. The native reductase apoprotein has a molecular weight of 76,366 and the deduced molecular weight of the holoenzyme (i.e. with 1 mol of FAD and FMN) is 77,608. The sequence of the house fly P450 reductase protein is highly similar to that of rabbit liver, the overall amino acid positional identity is 54.5% and the overall identity among eukaryotic P450 reductases is about 25%. The P450 reductase gene of 19-23 kb was located on chromosome III, as shown by comparison of RFLP-patterns of the P450 reductase gene in two house fly strains and their hybrids.

More than half the described species of eukaryotic organisms living on our planet are insects, and it is generally believed that a vast majority of insect species are still unknown. Among the known insect species are our best friends (pollinators of our crops) and our worst enemies (vectors of disease and avid consumers of our food). It is the chemical warfare against the latter that provided the framework for most of the early studies on insect cytochrome P450. The metabolism of insecticides, its inhibition by synergists such as piperonyl butoxide, and the resistance of insect pests to insecticides are only the most obvious reasons to study insect cytochrome P450. Insects are excellent model systems in which to study the induction of cytochrome P450 by dietary chemicals, or the molecular genetics of cytochrome P450. However, the study of cytochrome P450 has often been hampered by the small size of insects and the difficulty in obtaining enough tissue. Purification and reconstitution studies have been particularly difficulty (Hodgson 1985). Nonetheless, large numbers of insects obtained at low cost can sometimes compensate for small size, and the use of radiolabeled substrates has allowed enzymological studies of cytochrome P450 from tiny endocrine glands such as the corpora allata or the pro thoracic glands. This review is necessarily selective and will survey the known insect cytochromes P450 as well as some aspects of cytochrome P450 function in insects.

An octadecapeptide that inhibits juvenile hormone synthesis has been isolated by HPLC from brain-retrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this allatostatin has been elucidated by tandem mass spectrometry: Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu- NH2 (ASB2). The amidated three-residue C terminus of this type B allatostatin is identical to that of four known type A allatostatins, and the preceding three residues show close structural homology. ASB2 has over twice the activity of the type A tridecapeptide Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 (ASA1) in inhibiting juvenile hormone biosynthesis in corpora allata from females in early vitellogenesis (day 2), and its efficacy persists during pregnancy, but it is equally effective as ASA1 on glands from day-10 females (IC50 = 0.31 nM). The octadecapeptide is characterized by a potential dibasic cleavage site, Lys9-Arg10, the integrity of which is needed for high potency. The ASB2-(11-18)-octapeptide amide gives a full response at high concentrations at day 10 (IC50 = 48 nM), but the C-truncated (1-9)-, (1-11)-, and (1-17)-amide fragments of ASB2 are inactive. Thus, the endocrine message is located at the C terminus. N alpha-acetylation of the N-truncated (9-18), (10-18), and (11-18) fragments of ASB2 increases activity relative to the nonacetylated peptides. The site of action of type A and type B allatostatins is located before mevalonate kinase in the biosynthetic pathway for juvenile hormone.

Cytochrome P450 monooxygenases (P450) are a diverse and ubiquitous group of enzymes encoded by genes from a superfamily comprising at least 17 families, and probably many more (Nebert and Gonzalez, 1987; Nebert et al., 1989). Our present understanding of P450 enzymes comes mainly from studies of P450 in vertebrates, where they are involved in steroid biosynthesis and in the metabolism of drugs and chemical carcinogens, and in bacteria, with P450 cam from Pseudomonas putida being the only P450 enzyme for which a crystal structure is presently available (Poulos et al., 1987).

The control of growth, differentiation, metamorphosis, and reproduction in insects is a complex interplay of hormones, with the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone (JH) playing major roles. Molting is controlled by ecdysone, and the type of molt is controlled by JH. The presence of JH maintains the insect in the juvenile (larval) state, hence its name (1), and the relative absence of JH allows metamorphosis to proceed. With the discovery of insect JH came the idea that application of JH or its agonists at an inopportune time, when the insect required the absence of JH for metamorphosis—the “Achilles’ heel” of insect development—would disrupt this complex endocrine machinery. Analogs of JH, developed since the 1970s and euphemistically called “insect growth regulators,” are now among the most environmentally friendly types of insecticides. These chemicals, including methoprene, pyriproxyfen, and fenoxycarb, proved to be excellent mimics of the natural hormone, but, paradoxically, the molecular mode of action of JH has remained very much a mystery (2, 3). Now, studies on the insect’s response to these powerful chemicals—resistance—provide a direct avenue into the mode of action of JH itself. Ashok et al. (4) report in the current issue of the Proceedings the identity of the Methoprene-tolerant gene of Drosophila melanogaster as a member of the bHLH-PAS family of transcriptional regulators. This significant advance raises many questions, and its implications will be discussed here. Selection for methoprene-resistant mutants in D. melanogaster led to the definition of a genetic locus, Methoprene-tolerant or Met (5). Flies carrying the Met alleles are up to 100-fold resistant to structurally diverse JH analogs and to JH itself. The Met flies are not only resistant to the lethal effects of JH, but also to all the more subtle morphogenetic signs of hormone excess. For instance, treatment with methoprene …

Microarrays provide the means for the large-scale analysis of gene expression patterns in living organisms. My laboratory is part of three federally funded projects that are directed toward an understanding of the regulation of gene expression in higher plants.

After repetitive injections of moderate doses of ecdysone, ecdysterone or phenobarbital to young Vth (last) instar larvae of Locusta migratoria , the conversion rate of ecdysone to ecdysterone in vivo is significantly higher than in control insects. Similarly, 5 hr after injection of a low dose of ecdysone or ecdysterone, a strong ‘induction’ of ecdysone 20-monooxygenase activity occurs. This ‘inductive’ effect is blocked by cycloheximide. Simultaneous injections of ecdysone and ecdysterone show that hydroxylation of ecdysone is inhibited by the product of the reaction, ecdysterone. Removal of the prothoracic glands and X-ray treatment of the hemocytopoietic tissue do not affect ecdysone hydroxylation. The mechanism of induction and inhibition of ecdysone 20-monooxygenase shown in this study is probably responsible for the important variations of this key enzyme which have been reported from several insect species.

De novo biosynthesis of the sesquiterpenoid juvenile hormone III (JH- III , C 16 JH, methyl-10,11-epoxy-3,7,11-trimethyl -2E,6E- dodecadienoate ) by isolated corpora allata of the adult female cockroach Diploptera punctata has been studied in vitro using a radiochemical assay. Labelling from l -[ methyl - 3 H]methionine in the methyl ester moiety was used to quantify biosynthesized JH- III . When corpora allata were incubated in a medium lacking acetate but containing [1- 14 C]glucose, carbon from glucose labelled up to 30% of the sesquiterpenoid carbon chain. When the glands were incubated in a medium lacking glucose but containing [2- 14 C]acetate, dose-dependent utilization of labelled acetate was observed for the biosynthesis of juvenile hormone and its immediate precursor methyl farnesoate, reaching a plateau at stoichiometry, i.e. when 9 mol of acetate were incorporated for each mole of JH- III synthesized. This is a unique case of incorporation of exogenous, labelled acetate into an isoprenoid without significant dilution. The stoichiometric incorporation of exogenous [2- 14 C]acetate was utilized to measure the influence of farnesol and farnesoic acid (two sesquiterpenoid precursors of JH- III ) on the carbon flow through this biosynthetic pathway. Both compounds stimulated JH- III biosynthesis and inhibited [2- 14 C]acetate incorporation into juvenile hormone and methyl farnesoate in a dose-dependent fashion, resulting in a nearly total suppression of de novo JH- III biosynthesis.

Juvenile hormone esterase (JHE) activity and α-naphtyl acetate esterase (NacE) activity were studied in haemolymph of Diploptera punctata. The apparent Km value for JHE activity was 6.79 μM. Enzymatic activity of JHE and NAcE in females' haemolymph were measured during the first reproductive cycle. NAcE activity increased from emergence to oviposition and remained high during pregnancy. JHE activity was high at adult emergence and declined during the first three days. A sharp peak of JHE activity was observed on day 7 and high levels were maintained during pregnancy. JHE activity was not inhibited diisopropyfluorophosphate (DFP) whereas NAcEactivity was inhibited 90% at 1 mM. JHE activity profiles of virgin and allatectomized females did not show the peak on day 7 typical of normal mated females. Both oocyte growth and high JHE activity were restored in allatectomized females treated with hydroprene, a JH analogue, in a dose-dependent fashion. JH titre may regulate JHE activity at precise times during the reproductive cycle of D. punctata.

We compared the effects of three treatments (brain extracts, high K+, forskolin) which inhibit JH synthesis by corpora allata of the cockroach Diploptera punctata incubated in vitro. Corpora allata showed a similar developmental sensitivity to all three treatments, with a high level of inhibition in glands of low activity. Inhibition of JH III synthesis by forskolin, high K+ or brain extract was antagonized by mevalonate and farnesoic acid, thus implicating an early biosynthetic step as the target of inhibition. All three treatments were also antagonized by Mn2+, suggesting that the inhibitory mode of action may be dependent upon Ca2+. Inhibition for forskolin and by high K+ was not only reversible, but caused a stimulation of JH synthesis upon removal of the inhibitor, suggesting that more than one regulatory mechanism is affected by these treatments.

The synthesis of juvenile hormone-III by corpora allata of the cockroach Diploptera punctata is dependent under in vitro conditions upon a supply of exogenous methionine. Radiolabelled S-adenosylmethionine was identified by HPLC in extracts of corpora allata incubated with either [methyl-3H]methionine or [35S]methionine. Juvenile hormone (JH) synthesis by intact glands in vitro was inhibited by cycloleucine and selenomethionine, but this inhibition could be relieved by increasing the concentration of methionine. S-adenosylhomocysteine or sinefungin had little or no inhibitory effect on JH synthesis by intact glands, but 5′-deoxy-5′-methylthioadenosine was inhibitory. Adenosine and homocysteine synergistically inhibited JH synthesis. These results show that JH-III synthesis by intact glands can be inhibited by interfering with the S-adenosylmethionine-dependent transmethylation, and suggest that the product and inhibitor of that reaction, S-adenosyl-homocysteine, is rapidly hydrolyzed to adenosine and homocysteine in the corpora allata.

Two metyrapone analogues, 2-(l-imidazolyl)-2-methyl-l-phenyl-l-pro-panone (A-phenyl-B-imidazolyl-metyrapone; III) and 2-methyl-l-phenyl-2-{1,2,4-triazol-l-yl)-l -propanone (A-phenyl-B-triazolyl-meiyrapone; IV) as well as two cyclopropylamine derivatives. N-cyclopropyl-4-icrt-butylbenzylamine (V) and N-cyclopropyl-4-(3,7-dimethyl-7-methoxy-octyloxy)benzamide (cyclopropylamine acylated with a JH analogue acid of known structure; VI) were synthesized and evaluated in biological assays for JH biosynthesis on cockroach, Diploptera punctata corpora allata and egg growth in adult cockroach as well as for mixed function oxidase activities, i.e. epoxidation of aldrin to dieldrin and O-demethylation of 7-methoxy-4-methylcoumarin to 7-hydroxy-4-methylcoumarin on microsomes from housefly, Musca domestica, abdomen and from cockroach midgut. Compound VI was a good in-vitro inhibitor of JH biosynthesis, but it had significantly lower activities in the assays for inhibition of microsomal cytochrome P-450. Compound IV and metyrapone had moderate activity as inhibitors of oocyte growth. Compounds III, IV and V were more potent inhibitors of housefly aldrin epoxidation than metyrapone and they inhibited the enzyme activity by almost 100% at 02mM, while in cockroach midgut microsome assay metyrapone was more potent than these three compounds.

Hemolymph ecdysteroid titers during the last larval stage (lasting 20–21 days) of Diploptera punctata were determined by radioimmunoassay. A small peak (1 γM on day 10) preceded a larger peak (> 10 γM on day 17). Although ecdysone was the predominant circulating ecdysteroid in the beginning of the last larval stage, the ratio of 20-hydroxyecdysone to ecdysone increased to about 7 1 at the time of the large ecdysteroid peak. Ecdysone was converted to 20-hydroxyecdysone by fat body, midgut and Malpighian tubules incubated in vitro . Conditions for the measurement of ecdysone 20-monooxygenase activity were established for midgut homogenates. Maximal activity was found on days 5–9 and activity declined in the second half of the last larval stage. By using differential centrifugation and marker enzymes, most midgut ecdysone 20-monooxygenase activity was found to be associated with microsomes. Antibodies to NADPH-cytochrome P -450 reductase inhibited microsomal ecdysone 20-monooxygenase activity, thus confirming the cytochrome P -450 nature of this enzyme. The antibodies inhibited the ecdysone 20-monooxygenase activity associated with mitochondria as strongly as they inhibited the microsomal enzyme activity, thus showing that the mitochondrial activity was a microsomal contamination and that there was little or no intrinsically mitochondrial ecdysone 20-monooxygenase in the midgut.

The effect of 20-hydroxyecdysone upon the activity of corpora allata (CA) from female Diploptera punctata has been investigated. This ecdysteroid inhibits juvenile hormone (JH) biosynthesis by the CA, whether they have been implanted into a male, or remained in situ within the female. In the female, this inhibition is reflected in reduced oocyte growth and vitellin content. The allatostatic effect of 20-hydroxyecdysone becomes apparent in vivo within 24 hr. However, no inhibition was observed when the CA were maintained in vitro for 42 hr in medium containing up to 1·10−5 M 20-hydroxyecdysone. This suggests that the effect of the hormone upon the CA is indirect. These experiments raise the possibility that ecdysteroids play an allatostatic role during the normal gonotrophic cycle in Diploptera.

Ingested and injected radiolabeled α-ecdysone show quite different metabolic patterns in Locusta migratoria . Ingested α-ecdysone is rapidly taken up by the gut cells and converted to 3-dehydro-α-ecdysone and inactive conjugates, with hardly and β-ecdysone being produced. On the contrary, injected α-ecdysone is rapidly converted to β-ecdysone, this conversion being most efficient at the time of the endogenous hormone titre peak (day 7 of the last larval instar). Stage dependency is observed for the metabolic pattern and also for the distribution of the radiolabel after injection of α-ecdysone: when the hormone level is low, injected label passes rapidly into the gut and faeces. When the hormone level reaches its peak, β-ecdysone remains in tissues and hemolymph for a longer time and conjugation is delayed for several hours. In vitro experiments show that the fat body and the Malpighian tubules are the primary sites of ecdysone-20-hydroxylation while 3-dehydrogenation occurs in the digestive tract, and conjugation in the Malpighian tubules and hindgut.