Rice stem borer (RSB, Chilo suppressalis) is an insect pest that causes huge economic losses every year. Control efforts rely heavily on chemical insecticides, which leads to serious problems such as insecticide resistance, environment pollution, and food safety issues. Therefore, developing alternative pest control methods is an important task. Here, we identified an insect-specific microRNA, miR-14, in RSB, which was predicted to target Spook (Spo) and Ecdysone receptor (EcR) in the ecdysone signalling network. In-vitro dual luciferase assays using HEK293T cells confirmed the interactions of Csu-miR-14 with CsSpo and with CsEcR. Csu-miR-14 exhibited high levels of expression at the end of each larval instar stage, and its expression was negatively correlated with the expression of its two target genes. Overexpression of Csu-miR-14 at the third day of the fifth instar stage led to high mortality and developmental defects in RSB individuals. We produced 35 rice transformants to express miR-14 and found that three lines had a single copy with highly abundant miR-14 mature transcripts. Feeding bioassays using both T and T generations of transgenic miR-14 rice indicated that at least one line (C#24) showed high resistance to RSB. These results indicated that the approach of miRNAs as targets has potential for improving pest control methods. Moreover, using insect-specific miRNAs rather than protein-encoding genes for pest control may prove benign to non-insect species, and thus is worthy of further exploration.

Insect chitinases participate in numerous physiological processes such as nutrition, parasitism, morphogenesis and immunity. These properties make chitinases good targets for pest control. Rice striped stem borer (SSB), Chilo suppressalis Walker, is one of the most destructive pests of rice causing huge yield losses. In our previous work, we reported the identification of 12 SSB chitinase (CsCht) genes, and studied the functions of CsCht1 to 4. Here, we have extended our study to investigate the expression patterns and functions of CsCht5 to 12. All eight chitinase genes displayed distinct temporospatial expression profiles. We looked at the effect of knocking down each gene at the developmental stage where highest expression was observed. Knocking down CsCht5, CsCht6 and CsCht8 resulted in high mortality and delayed development. Although silencing CsCht7, CsCht9, CsCht10, CsCht11 and CsCht12 had no apparent effect on development, knocking down CsCht10 in SSB individuals that were simultaneously treated with Beauveria bassiana (Bb84) led to higher mortality rates and quicker death, suggesting CsCht10 has an essential role in protecting SSB from exogenous microorganisms. In summary, we elucidated the functions of eight SSB chitinase genes and found that CsCht10 could be a good candidate for pest control.

The precise increase and decrease of hormone ecdysone are critical for accurate development in insects. Most previous works focus on transcriptional activation of ecdysone production; however, little is known about the mechanism of switching off ecdysone biosynthesis after ecdysis. Here, we showed that the precursor microRNA-14 (pre-miR-14) encodes two mature miRNAs in silkworm; both of these two mature miRNAs regulate various genes in the ecdysone-signalling pathway. Bmo-miR-14-5p targets on nine genes whereas Bmo-miR-14-3p targets on two genes in the same pathway. These two mature miRNAs increased immediately after the ecdysis, efficiently suppressing the 20-hydroxyecdysone (20E) biosynthesis, the upstream regulation, and the downstream response genes. Knocking down either of two mature miRNAs or both of them delays moult development, impairing development synchrony in antagomir-treated groups. In addition, overexpressing Bmo-miR-14-5p but not Bmo-miR-14-3p significantly affected the 20E titer and increased the moulting time variation, suggesting that Bmo-miR-14-5p, though it is less abundant, has more potent effects in development regulation than Bmo-miR-14-3p. In summary, we present evidence that a pre-miRNA encodes two mature miRNAs targeting on the same pathway, which significantly improves miRNA regulation efficiencies to programmatically switch off ecdysone biosynthesis.

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