Functions of nuclear receptor HR3 during larval-pupal molting in Leptinotarsa decemlineata (Say) revealed by in vivo RNA interference

20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development. In this study, three Leptinotarsa decemlineata Ecdysone-induced protein 75 (LdE75) cDNAs (LdE75A, B and C) were cloned from L. decemlineata. The three LdE75 isoforms were highly expressed just before or right after each moult. Within the fourth larval instar, they showed a small rise and a big peak 40 and 80 h after ecdysis. The expression peaks of the three LdE75s coincided with the peaks of circulating 20E levels. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdE75 expression in the day 1 final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene, Shade (LdSHD), repressed the expression of LdE75. Moreover, Hal upregulated the expression of the three LdE75s in LdSHD-silenced larvae. Thus, 20E pulses activate the transcription of LdE75s. Furthermore, ingesting dsE75-1 and dsE75-2 from a common fragment of the three isoforms successfully knocked down these LdE75s, and caused developmental arrest. Finally, knocking down LdE75s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered the 20E titre and affected the expression of two 20E-response genes. Silencing LdE75s also induced the expression of a JH biosynthesis gene, increased JH titre and activated the transcription of a JH early-inducible gene. Thus, Ld E75s are required for larval-pupal metamorphosis and act mainly by modulating 20E and JH titres and mediating their signalling pathways.

Section: Resultsmentioning

confidence: 97%

Nuclear receptor ecdysone‐induced protein 75 is required for larval–pupal metamorphosis in the Colorado potato beetle Leptinotarsa decemlineata (Say)

Guo

Liu

et al. 2015

“…Similarly, we individually knocked down genes coding for 20E signaling cascade components ecdysone receptor gene ( LdEcR ; Ogura et al ., ), hormone response protein 3 gene ( LdHR3 ), and ecdysone induced protein 75 ( LdE75 ; Ogura et al ., ; Liu et al ., ; Guo et al ., ; Guo et al ., ) by orally delivered dsRNA (Fig. S2), and discovered that inhibition of 20E signal in the LdEcR , LdHR3 , and LdE75 RNAi larvae significantly downregulated the expression of both LdCDA2a and LdCDA2b (Fig.…”

Section: Resultsmentioning

confidence: 99%

Silencing chitin deacetylase 2 impairs larval–pupal and pupal–adult molts in Leptinotarsa decemlineata

Chen

Wang

et al. 2018

Insect chitin deacetylases (CDAs) are carbohydrate esterases that catalyze N-deacetylation of chitin to generate chitosan, a process essential for chitin organization and compactness during the formation of extracellular chitinous structure. Here we identified two CDA2 splice variants (LdCDA2a and LdCDA2b) in Leptinotarsa decemlineata. Both splices were abundantly expressed in larval foregut, rectum, and epidermis; their levels peaked immediately before ecdysis within each instar. In vivo results revealed that the two isoforms transcriptionally responded, positively and negatively respectively, to 20-hydroxyecdysone and juvenile hormone signaling pathways. RNA interference (RNAi)-aided knockdown of the two LdCDA2 variants (hereafter LdCDA2) or LdCDA2b, rather than LdCDA2a, resulted in three negative effects. First, foliage consumption was significantly reduced, larval developing period was lengthened, and larval growth was retarded. Second, chitin contents were reduced, whereas glucose, trehalose, and glycogen contents were increased in the LdCDA2 and LdCDA2b RNAi larvae. Third, approximately 20% of LdCDA2 and LdCDA2b RNAi larvae were trapped within the exuviae and finally died. About 60% of the abnormal pupae died as pharate adults. Around 20% of the RNAi pupae emerged as deformed adults, with small size and wrinkled wings. These adults eventually died within 1 week after molting. Our results reveal that knockdown of CDA2 affects chitin accumulation. Consequently, LdCDA2 may be a potential target for control of L. decemlineata larvae.

“…To determine the effect of the LdUSP knockdown on the expression of ecdysone‐inducible genes, the mRNA levels of five known ecdysone‐inducible genes, EcR , HR3 , HR4 , E74 and E75 (Ogura et al , ; Guo et al , ; ; Xu et al , ; ), were quantified in treated larvae. Compared with control larvae having ingested ds egfp ( enhanced green fluorescent protein ), the expression levels of all transcripts tested were significantly decreased in LdUSP RNAi larvae (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The defects in LdUSP RNAi Leptinotarsa larvae are reminiscent of the phenotypes in 20E deficient (Kong et al , ) or 20E signalling repression (knockdown of LdHR3 , LdHR4 , LdE74 , LdE75 or LdFTZ‐F1 ) (Liu et al , ; Guo et al , ; ; Xu et al , ; ) L. decemlineata beetles; the resultant larvae remain as prepupae, or become deformed pupae and adults, partially or completely wrapped in old larval exuviae. In this paper, therefore, we determined that influence of LdUSP depletion on the ecdysteroidogenesis and the expression of representative 20E signalling genes.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

Deng

et al. 2019

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.