Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

Abstract

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the “black box”, remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

RNA interference‐mediated knockdown of the Halloween gene Spookiest (CYP307B1) impedes adult eclosion in the western tarnished plant bug, Lygus hesperus

“…By contrast, Spo knockdown in Schistocerca gregaria and An. gambiae had no clear developmental phenotype despite reduced ecdysteroid production (Marchal et al ., ; Pondeville et al ., ). The incomplete development that we observed compared with the delayed development reported in other studies could be attributable to methodological variations in dsRNA delivery or biological compensatory effects.…”

“…gregaria (Marchal et al, 2011), Holcocerus hippophaecolus (Zhou et al, 2013), An. gambiae (Pondeville et al, 2013) and T. castaneum (Parthasarathy et al, 2010). RNAi-mediated knockdown of Spo and Phantom in An.…”

“…Given the indispensable role of ecdysteroids in regulating and coordinating the critical insect processes of growth, development and reproduction, the lack of functional redundancy amongst the Halloween suite of genes is surprising. Identification or prediction of Halloween genes in diverse insect species (Namiki et al, 2005;Ono et al, 2006Ono et al, , 2012Sztal et al, 2007;Christiaens et al, 2010;Marchal et al, 2010;Yamazaki et al, 2011;Hentze et al, 2013;Jia et al, 2013a,b;Luan et al, 2013;Pondeville et al, 2013;Zhou et al, 2013;Shahzad et al, 2015), as well as other arthropods (Rewitz & Gilbert, 2008;Cabrera et al, 2015), has revealed that each gene is more similar to its respective orthologues than to other P450 monoxygenases, suggesting the specific steps in ecdysteroid biosynthesis are catalysed by a single enzyme. Paralogues, however, have been identified for the CYP307 P450 monoxygenases, which have undergone lineage-specific duplications/losses, with three paralogues CYP307A1 (Spook, Spo), CYP307A2 (Spookier, Spok) and CYP307B1 (Spookiest, Spot) identified in various species, albeit with only two of the three present in any one species (Rewitz et al, 2007;Sztal et al, 2007;Rewitz & Gilbert, 2008).…”

The Anopheles gambiae transcriptome – a turning point for malaria control

“…To identify new enzymes associated with the activity of 20E, a small‐scale microarray analysis was performed to compare the expression of P450 enzymes in active and inactive steroidogenic tissues. The analysis revealed that only three genes encoding P450 enzymes, cyp4g16 , cyp6n1 and cyp307a1 , are up‐regulated in the active steroidogenic tissues of both male and female mosquitoes, but only cyp307a1 is involved in steroidogenesis (Pondeville et al ., ).…”

Calcium influx enhances neuropeptide activation of ecdysteroid hormone production by mosquito ovaries

“…Blood digestion provides amino acids to the fat body for the production of yolk proteins, which are then packaged into primary oocytes that develop into mature eggs. Studies conducted primarily with Aedes aegypti indicate that ecdysteroid hormone (ECD) produced by the ovaries is the primary factor that activates the fat body to synthesize yolk proteins (Attardo et al, 2005; Baldridge and Feyereisen, 1986; Gulia-Nuss et al, 2012; Gulia-Nuss et al, 2015; Pondeville et al, 2008; 2013; Roy et al, 2015). Prior studies further show that ovaries produce ECD in response to insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH), which are released from neurosecretory cells in the brain within a few hours after females consume a blood meal (Riehle and Brown, 1999; Riehle and Brown, 2002; Gulia-Nuss et al, 2011; Dhara et al, 2013).…”