RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages

Abstract

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world’s poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

Silencing of transcription factor E93 inhibits adult morphogenesis and disrupts cuticle, wing and ovary development in Locusta migratoria

“…Our results showed that downregulation of LmE93 generate supernumerary nymphal instars. Similar phenotypes (supernumerary nymphal instar or second pupa) were found after E93 knockdown in other insects including D. melanogaster , T. castaneum , B. germanica , G. bimaculatus , C. lectularius , B. mori , S. gregaria and N. lugens (Ureña et al ., 2014; Liu et al ., 2015; Gujar & Palli, 2016; Li et al ., 2018; Ishimaru et al ., 2019; Gijbels et al ., 2020), suggesting that the function of E93 is conserved among insects. In our study, we also found that ds LmE93 ‐treated individuals remained in N4 and N5 nymphal stages for significantly shorter days.…”

“…Subsequently, it was found that E93 is highly expressed in the pupal stage and E93‐depleted larvae result in a failure in pupal–adult transition (Mou et al ., 2012; Ureña et al ., 2014, 2016). Several years later, functional studies confirmed E93 as a universal “adult specifier gene”, because knockdown of E93 induced reiteration of larval development to prevent pupal–adult or nymphal–adult and resulted in a supernumerary pupa or nymphal stage (Ureña et al ., 2014; Liu et al ., 2015; Gujar & Palli, 2016; Li et al ., 2018; Ishimaru et al ., 2019; Kamsoi & Belles, 2020; Gijbels et al ., 2020). Moreover, E93 has shown to be related to the JH signaling pathway.…”

Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle

“…At the penultimate instar stage in Tribolium castaneum , for instance, depletion of E93 perpetuates its juvenile identity by inducing the repetition of larval moults [ 13 ]. In immatures of the hemimetabolous Blattella germanica , Schistocerca gregaria , Locusta migratoria , Gryllus bimaculatus , Cimex lectularius and Nilaparvata lugens , depletion of E93 disallowed the nymphal-to-adult transformation, causing supernumerary juvenile instars [ 15 , 24 , 25 , 26 , 27 , 28 ].…”

Crucial Role of Juvenile Hormone Receptor Components Methoprene-Tolerant and Taiman in Sexual Maturation of Adult Male Desert Locusts

“…The significant reduction in Scg-Krh1 demonstrates that JH signaling was impaired upon knocking down Scg -Met ( Figure 2 A). E93 is an ecdysone inducible transcription factor, of which the expression is inhibited by Kr-h1, and vice versa [ 23 , 69 , 70 , 71 , 72 ]. In line with this, an increase in the relative mRNA levels of Scg -E93 was observed in the CA ( Figure 2 A).…”