KNOCKDOWN OF CS‐SPOOK INDUCES DELAYED LARVAL MOLTING IN RICE STRIPED STEM BORER Chilo suppressalis

Abstract

Spook has essential roles in the biogenesis of the molting hormone 20-hydroxyecdysone (20-E). The function of spook in the rice striped stem borer (SSB) Chilo suppressalis remains unclear, prompting our hypothesis that it exerts actions similar to those reported for other insect species. Here we amplified the full-length transcript of spook (Cs-Spook) in SSB by 5' and 3' rapid amplification of cDNA ends. Cs-Spook has conserved P450 motifs such as Helix-C, Helix-I, Helix-K, and PERF motif (PxxFxPxRF). It was highly expressed in late instar larvae but less so in newly molted larvae. Cs-Spook was highly expressed in prothoracic glands. Cs-Spook was knocked down by dsRNA treatments. Compared with controls, the gene expression level was reduced to 9% at 24 h post injection (PI), 33% at 48 h PI, and 24% at 72 h PI. The ecdysteroid titer decreased significantly in the dsRNA-treated group (P < 0.05), resulting in delayed larval development. The delayed development in dsRNA-treatment group was rescued by treating with 20-E. Our work demonstrates that Cs-Spook participates in the biogenesis of 20-E and regulates the molt of SSB, as seen in other species.

Multiple miRNAs jointly regulate the biosynthesis of ecdysteroid in the holometabolous insects, Chilo suppressalis

“…Therefore, the timing of the appearance of ecdysteroids and JHs is important in determining developmental transition. The biosynthesis pathways of ecdysteroids and JH have been well characterized (Christiaens et al 2010;Yamazaki et al 2011;Shahzad et al 2015;Zhu et al 2017).…”

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

“…As Spot catalyses a relatively early step in 20E biosynthesis (presumably within the so‐called ‘black box’), we hypothesized that application of ecdysteroids (eg 20E) upstream of that step should be able to rescue the knockdown phenotype as demonstrated in other systems (Ono et al ., ; Jia et al ., ; Shahzad et al ., ). Topical application of 20E 24 h post‐ LhSpot dsRNA injection rescued adult eclosion (Fig.…”

“…). Although the consensus sequences for two additional P450 motifs, helix C (WxxxR) and helix I (A/GGxD/ETT/S), have diverged from that seen in the other Halloween proteins (Namiki et al ., ; Ono et al ., ; Iga & Smagghe, ; Marchal et al ., ; Zhou et al ., ; Cabrera et al ., ; Jia et al ., ; Shahzad et al ., ), the regions associated with these motifs are well conserved in the LhSpo‐like sequence and orthologues in other species (Fig. ).…”

“…Characteristic P450 domains/motifs (Pro/Gly rich region, helix K, Pro‐Glu‐Arg‐Phe (PERF) domain and heme‐binding domain) are indicated. The helix C and helix I motifs (dashed lines with grey font) do not meet conventional definitions for these regions (ie helix C = WXXXR; helix I = GxE/DTT/S) but correspond to regions defined as such in reports of insect CYP307A1 orthologues (Iga & Smagghe, ; Jia et al ., ; Shahzad et al ., ). Amino acid substitutions specific to Spot paralogues that are located near the putative helix C are indicated with closed ovals, those near helix I with open squares and miscellaneous spot specific changes are indicated with open ovals.…”

“…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 ., ; Ono et al ., ; Sztal et al ., ; Christiaens et al ., ; Iga & Smagghe ; Marchal et al ., ; Yamazaki et al ., ; Hentze et al ., ; Jia et al ., ,b; Luan et al ., ; Pondeville et al ., ; Zhou et al ., ; Shahzad et al ., ), as well as other arthropods (Rewitz & Gilbert, ; Cabrera et al ., ), 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 ., ; Sztal et al ., ; Rewitz & Gilbert, ).…”