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

Abstract

The accurate rise and fall of active hormones is important for insect development. The ecdysteroids must be cleared in a timely manner. However, the mechanism of suppressing the ecdysteroid biosynthesis at the right time remains unclear. Here, we sequenced a small RNA library of Chilo suppressalis and identified 300 miRNAs in this notorious rice insect pest. Microarray analysis yielded 54 differentially expressed miRNAs during metamorphosis development. Target prediction and in vitro dualluciferase assays confirmed that seven miRNAs (two conserved and five novel miRNAs) jointly targeted three Halloween genes in the ecdysteroid biosynthesis pathway. Overexpression of these seven miRNAs reduced the titer of 20-hydroxyecdysone (20E), induced mortality, and retarded development, which could be rescued by treatment with 20E. Comparative analysis indicated that the miRNA regulation of metamorphosis development is a conserved process but that the miRNAs involved are highly divergent. In all, we present evidence that both conserved and lineage-specific miRNAs have crucial roles in regulating development in insects by controlling ecdysteroid biosynthesis, which is important for ensuring developmental convergence and evolutionary diversity.

Transgenic microRNA‐14 rice shows high resistance to rice stem borer

“…The expression of Csu-miR-14 negatively correlated with the expression of CsSpo and CsEcR Expression analysis indicated that the level of transcripts of Csu-miR-14 were high at the end of each instar and then declined sharply in the initial point of the following stage (Figure 2a). In our previous report (He et al, 2017;Sun et al, 2017), customized microarrays were designed to study the expression of small RNA and mRNA from the same batch of RSB samples including the stages of ageing larval, prepupal, early pupal, compound eye formation, pretarsal formation, pupal elongation and adult. This enabled us to calculate the co-expression patterns between Csu-miR-14 and its target genes.…”

“…Small RNA sequencing was performed as described in He et al . (). Purified RNA fragments were first separated by polyacrylamide gel electrophoresis (PAGE) and then ligated to two adaptors (5′/3′) for PCR primers binding.…”

“…(a) qRT-PCR analysis of Csu-miR-14 from the fifth day of the fourth-instar larvae (4L5D) to the third-day pupa (P3) stage, indicating that Csu-miR-14 is highly expressed at the end of each larval instar stage. (b) The changes in expression levels of Csu-miR-14, (c) CsSpo, and (d) CsEcR estimated by a previous microarray analysis at seven developmental stages of RSB (He et al, 2017;Sun et al, 2017) including aging larvae (aL), prepupae (pP), early pupae (eP), compound eye formation stage (cE), pupae elongation stage (Pe), and adult (Ad). The data are shown as means AE SE (n = 3).…”

“…Rice stem borer larvae were maintained on newly germinated rice seeds in glass bottles at 28 AE 1°C with a relative humidity of 70%-80% as described in He et al (2017). Nymphs of N. lugens and L. striatellus also were maintained on rice seedlings.…”

“…Total RNA was isolated from samples consisting of a mix of the eggs, larvae (nymphs), pupae and adults for each species (S. incertulas, N. lugens and L. striatellus) using TRIzol (Invitrogen, Carlsbad, CA) reagent following the instructions of the manufacturer. Small RNA sequencing was performed as described in He et al (2017). Purified RNA fragments were first separated by polyacrylamide gel electrophoresis (PAGE) and then ligated to two adaptors (5 0 /3 0 ) for PCR primers binding.…”