Expression and down-regulation of cytochrome P450 genes of the CYP4 family by ecdysteroid agonists in Spodoptera littoralis and Drosophila melanogaster

The function of CYP4 genes in insects is poorly understood. Some CYP genes are up-regulated by ecdysteroids and a number of Cyp4 genes in Drosophila melanogaster have been shown by microarray to be down-regulated when the ecdysteroid titre is high, suggesting hormonal regulation. Here, we report the utilization of certain cloned CYP4 cDNAs/fragments to probe their developmental/tissue expression in the Lepidopteran, Spodoptera littoralis, including the effects of ecdysteroid receptor agonists (bis-acyl hydrazines). CYP4L8 is expressed essentially throughout the final larval instar of S. littoralis and, together with CYP4M12, is down-regulated by agonist. Furthermore, expression of these genes occurs in midgut, but is undetectable in brain, fat body, and integument. Similarly, in D. melanogaster, Cyp4ac1, Cyp4ac3, Cyp4ad1 and Cyp4d1 gene expression is drastically down-regulated by ecdysteroid agonist. The significance of the results is discussed in relation to the plausible functions of the CYP4 genes in Lepidoptera and mechanisms of down-regulation.     

家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用,尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对,发现了家蚕直向同源基因(ortholog),其完全编码序列经RT-PCR检测和克隆、测序验证后,再以此为信息探针检索家蚕表达序列标签(expressed sequence tags,EST)数据库进行拼接延伸,获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列,验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988,P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp,编码541个氨基酸,含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域,其推定的分子量为61.67 kD,等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,5个内含子,外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测,显示出该基因的转录表达不仅具有时空特异性,而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。     

CYP306A1, a cytochrome P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic glands of Bombyx and Drosophila

Ecdysteroids mediate a wide variety of developmental and physiological events in insects. In the postembryonic development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although many studies have revealed the biochemical and physiological properties of the enzymes for ecdysteroid biosynthesis, most of the molecular identities of these enzymes have not been elucidated. Here we describe an uncharacterized cytochrome P450 gene, designated Cyp306a1, that is essential for ecdysteroid biosynthesis in the PGs of the silkworm Bombyx mori and fruit fly Drosophila melanogaster. Using the microarray technique for analyzing gene expression profiles in PG cells during Bombyx development, we identified two PG-specific P450 genes whose temporal expression patterns are correlated with changes in ecdysteroid titer during development. Amino acid sequence analysis showed that one of the Bombyx P450 genes belongs to the CYP306A1 subfamily. The temporal and spatial expression pattern of the Drosophila Cyp306a1 homolog is essentially the same as that of Bombyx Cyp306a1. We also found that Drosophila Cyp306a1 is disrupted in the phantom (phm) mutant, known also as the Halloween mutant. The morphological defects and decreased expression of ecdysone-inducible genes in phm suggest that this mutant cannot produce a high titer of ecdysone. Finally we demonstrate that S2 cells transfected with Cyp306a1 convert ketodiol to ketotriol via carbon 25 hydroxylation. These results strongly suggest that CYP306A1 functions as a carbon 25 hydroxylase and has an essential role in ecdysteroid biosynthesis during insect development.     

Ecdysteroid biosynthesis in workers of the European honeybee Apis mellifera L

We previously reported preferential expression of genes for ecdysteroid signaling in the mushroom bodies of honeybee workers, suggesting a role of ecdysteroid signaling in regulating honeybee behaviors. The organs that produce ecdysteroids in worker honeybees, however, remain unknown. We show here that the expression of neverland and Non-molting glossy/shroud, which are involved in early steps of ecdysteroid synthesis, was enhanced in the ovary, while the expression of CYP306A1 and CYP302A1, which are involved in later steps of ecdysone synthesis, was enhanced in the brain, and the expression of CYP314A1, which is involved in converting ecdysone into active 20-hydroxyecdysone (20E), was enhanced in the brain, fat body, and ovary. In in vitro organ culture, a significant amount of ecdysteroids was detected in the culture medium of the brain, fat body, and hypopharyngeal glands. The ecdysteroids detected in the culture medium of the fat body were identified as ecdysone and 20E. These findings suggest that, in worker honeybees, cholesterol is converted into intermediate ecdysteroids in the ovary, whereas ecdysone is synthesized and secreted mainly by the brain and converted into 20E in the brain and fat body.     

Ecdysteroid synthesis by the ring gland of Drosophila melanogaster during late-larval,prepupal and pupal development

Radioimmunoassay has been used to determine the characteristics of ecdysteroid synthesis by ring glands and brain-ring gland preparations from late 3rd-instar larvae of Drosophila melanogaster cultured in vitro. The rate of synthesis and secretion is linear for at least 4 hr in culture. Using a 4-hr culture period, variation in the rate of ecdysteroid synthesis by brain-ring gland preparations during larval, prepupal and pupal development has been examined. The rate of synthesis and secretion is highest in late 3rd-instar larvae and decreases after puparium formation. During pupal development, at a time when the endogenous ecdysteroid titre is again increasing, the rate of ecdysteroid synthesis by brain-ring gland preparations remains low and is only 10% of that prior to puparium formation. It is, therefore, likely that the ring gland is not a major source of ecdysteroids during this period.     

Haemolymph ecdysteroid in the housefly,Musca domestica,during oögenesis and its relationship with vitellogenin levels

Ecdysteroid titre in the haemolymph of the housefly, Musca domestica, cycled during oögenesis and peaked at ～50 pg/μl during stages 5, 6 and 7. Levels of 10–20 pg/μl were found in houseflies with pre- and post-vitellogenic ovaries. Removal of the corpus allatum and corpus cardiacum complex resulted in low ecdysteroid levels (10 pg/μl). Ovariectomized flies also had lower ecdysteroid levels than the controls at 2 days (5 pg/μl) after emergence but not at 6 days (22 pg/μl). It is possible that the ecdysteroid peak that occurred during stages 5, 6 and 7 was produced by the ovaries because ovaries secreted and synthesized ecdysteroid in vitro. Endogenous haemolymph ecdysteroid levels had a linear correlation with the amount of vitellogenin that held for hormone concentrations of 5–43 pg/μl. Furthermore, the injection of 20-hydroxyecdysone at doses of 10 ng?1.0 μg/fly increased the amount of vitellogenin from 6 h to 12 h after injection; by 24 h, the vitellogenin returned to control levels. When 20-hydroxyecdysone was injected into ovariectomized flies, it was rapidly degraded and 96% was cleared from the haemolymph within 1 h.     

A new cytochrome P450 from Drosophila melanogaster, CYP4G15, expressed in the nervous system

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed.     

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.     

Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.     

Molting cycle-dependent expression of CYP4C15, a cytochrome P450 enzyme putatively involved in ecdysteroidogenesis in the crayfish, Orconectes limosus.

A cytochrome P450 enzyme cDNA (CYP4C15) has been previously cloned from a cDNA library of crayfish steroidogenic glands (Y-organs). The conceptual translation of the CYP4C15 cDNA sequence was analyzed for regions of putative high antigenicity and a mixture of two synthetic peptides was chosen for the production of a specific polyclonal antibody. Western blot analysis on Y-organ subcellular fractions indicated an endoplasmic reticulum location of CYP4C15, in agreement with the structural feature of the predicted protein, i.e. the presence of a hydrophobic N-terminal segment.The protein is only expressed in Y-organs, thus showing a similar distribution to the corresponding mRNA. From this tissue specific expression, it has been postulated that CYP4C15 would play a role in ecdysteroid biosynthesis rather than detoxification and the variations of its expression during a molt cycle were carefully examined. CYP4C15 is not detectable in intermolt animals, expression levels are maximal during early premolt and decrease during late premolt. The results are discussed in relation to the variations of hemolymphatic ecdysteroid titers and steroidogenic capacities of the Y-organs during the molt cycle.     

Haemolymph ecdysteroid titres in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

The ecdysteroid titres of last-instar prediapausing, diapausing and nondiapausing larvae of Ostrinia nubilalis were determined by radioimmunoassay. In the nondiapause larvae a major peak of ecdysteroid activity preceded pupation by 24 h and continued through the pupal ecdysis. This peak was correlated with head and thorax critical periods as well as with changes in behaviour and physiology marking the transition from feeding larva to prepupa. Nondiapause larvae also displayed a rise in ecdysteroid titre during the feeding phase of development. This rise was approx one tenth that of the major peak and lasted 32 h. It was not correlated with any overt changes in larval physiology or behaviour. The diapause ecdysteroid profile was distinctive in that the levels measured were all lower than the lowest of the nondiapause curve. No peaks were observed in the diapause titres. Prepupal changes such as spinning and the cessation of feeding were not correlated with any increase in ecdysteroid levels. During diapause termination, under a long-day photoperiod, no increases in ecdysteroid titre were observed for the first 10 days. After 12 days individuals began to show ecdysteroids above the diapause levels. Pupation started after 16 long days.     

Metabolic imidacloprid resistance in the brown planthopper,Nilaparvata lugens,relies on multiple P450 enzymes

Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance.     

Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family,from cultured Coptis japonica cells

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.