20E-response Genes Research Articles

A critical role for the nuclear protein Akirin in larval development in Henosepilachna vigintioctopunctata.

Akirin is a nuclear protein that controls development in vertebrates and invertebrates. The function of Akirin has not been assessed in any Coleopteran insects. We found that high levels of akirin transcripts in Henosepilachna vigintioctopunctata, a serious Coleopteran potato defoliator (hereafter Hvakirin), were present at prepupal, pupal and adult stages, especially in larval foregut and fat body. RNA interference (RNAi) targeting Hvakirin impaired larval development. The Hvakirin RNAi larvae arrested development at the final larval instar stage. They remained as stunted larvae, gradually blackened and finally died. Moreover, the remodelling of gut and fat body was inhibited in the Hvakirin depleted larvae. Two layers of cuticles, old and newly formed, were noted in the dsegfp-injected animals. In contrast, only a layer of cuticle was found in the dsakirin-injected beetles, indicating the arrest of larval development. Furthermore, the expression of three transforming growth factor-β cascade genes (Hvsmox, Hvmyo and Hvbabo), a 20-hydroxyecdysone (20E) receptor gene (HvEcR) and six 20E response genes (HvHR3, HvHR4, HvE75, HvBrC, HvE93 and Hvftz-f1) was significantly repressed, consistent with decreased 20E signalling. Conversely, the transcription of a juvenile hormone (JH) biosynthesis gene (Hvjhamt), a JH receptor gene (HvMet) and two JH response genes (HvKr-h1 and HvHairy) was greatly enhanced. Our findings suggest a critical role of Akirin in larval development in H. vigintioctopunctata.

Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half becamemalformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvβFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

Nanomaterial-encapsulated dsRNA of ecdysone-induced early gene E75, a potential RNAi-based SIT strategy for pest control against Bactrocera dorsalis

Bactrocera dorsalis is a notorious pest widely distributed across most Asian countries. With the rapid development of pesticide resistance, new pest control methods are urgently needed. RNAi-based sterile insect technique (SIT) is a species-specific pest management strategy for B. dorsalis control. It is of great significance to identify more target genes from B. dorsalis, and improve the RNAi efficiency. In this study, microinjection-based RNAi results showed that six 20E response genes were necessary for male fertility of B. dorsalis, of which E75 was identified as the key target according to the lowest egg-laying number and hatching rate after E75 knockdown. Three nanoparticles chitosan (CS), chitosan‑sodium tripolyphosphate (CS-TPP), and star polycation (SPc) were used to encapsulate dsE75 expressed by HT115 strain. Properties analysis of nanoparticle-dsRNA complexes showed that both CS-TPP-dsRNA and SPc-dsRNA exhibited better properties (smaller size and polydispersity index) than CS-dsRNA. Moreover, oral administration of CS-TPP-dsE75 and SPc-dsE75 by males resulted in more abnormal testis and significantly lower fertility than feeding naked dsE75. Semi-field trials further confirmed that the number of hatched larvae was dramatically reduced in these two groups. Our study not only provides more valuable targets for RNAi-based SIT, but also promotes the application of environment-friendly management against B. dorsalis in the field.

Interactions between 20-Hydroxyecdysone and Juvenile Hormone I, II, and III during the Developmental Stages of Spodoptera frugiperda

Juvenile hormones (JHs) play a crucial role in the development of the fall armyworm, Spodoptera frugiperda, with varying types and concentrations observed at different stages. However, the interplay between JHs and 20-hydroxyecdysone (20E) in co-ordinating the life cycle of S. frugiperda remains unknown. In this study, by using high-precision UPLC-MS/MS and qRT-PCR, we detected changes in JH and 20E levels and identified important 20E receptor and response genes. Our findings revealed that JH I antagonises JH II, whereas JH II promotes JH III synthesis. High JH I and JH II concentrations in the larval stage strongly affected moulting to the next instar. Furthermore, these hormones inhibit 20E synthesis and reduce its receptor expression, thereby affecting 20E signalling. During pupation, JH II plays a crucial role in stimulating 20E synthesis for larval–pupal transformation. JH I and JH II are essential for eclosion, precisely controlling emergence timing and subsequent reproductive organ maturation. These hormones likely regulate larval development, pupation, and adult reproduction in S. frugiperda. Further studies are warranted to explore the regulatory advantages of JH I and JH II over JH III.

Bruceine D Acts as a Potential Insecticide by Antagonizing 20E-EcR/USP Signal Transduction.

Bruceine D (BD) is an effective insecticidal compound found in the Chinese herb Brucea javanica (L.) Merr. BD inhibits the growth and metamorphosis of Plutella xylostella and Drosophila melanogaster; however, its target protein and the molecular mechanism of insecticidal activity remain unclear. In this study, proteins with high affinity for BD were screened using surface plasmon resonance and high-performance liquid chromatography coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, revealing the ecdysone receptor (EcR) is the main target of BD. In vivo results showed that BD inhibited insect growth and metamorphosis through inhibition of the expression of 20E response genes. In vitro dual luciferase and enhanced green fluorescent protein (EGFP) fluorescence experiments indicated that BD suppressed the transcriptional activation activity of EcR by blocking the ecdysone response element (EcRE)-triggered transcriptional cascade, suggesting that BD inhibits the formation of the 20E-EcR-USP-EcRE complex. Moreover, molecular docking demonstrated that BD bound well to EcR. Elucidating the insecticidal mechanism of BD will be helpful in the development of green pesticides to control pests.

Low GSK3β activity is required for insect diapause through responding to ROS/AKT signaling and down-regulation of Smad1/EcR/HR3 cascade

Glycogen synthase kinase 3β (GSK3β) plays important roles in gene transcription, metabolism, apoptosis, development, and signal transduction. However, its role in the regulation of pupal diapause remains unclear. In this paper, we find that low GSK3β activity in brains of diapause-destined pupae of Helicoverpa armigera is caused by elevated AKT activity. In response to ROS, AKT phosphorylates GSK3β to decrease its activity. In developing pupal brains, GSK3β can activate the transcription factor Smad1, which binds to the promoter region of the ecdysone receptor (EcR) gene and increases its expression. In the presence of 20-hydroxyecdysone (20E), EcR can bind to USP and increase the expression of 20E-response genes, including HR3, for pupal-adult development. In contrast, high levels of ROS in brains of diapause-destined pupae up-regulate p-AKT, which in turn decreases GSK3β activity. Low GSK3β activity causes low expression of EcR/HR3 via down-regulation of Smad1 activity, leading to diapause initiation. These results suggest that low GSK3β activity plays a key role in pupal diapause via ROS/AKT/GSK3β/Smad/EcR/HR3 signaling.

Smad on X is vital for larval-pupal transition in a herbivorous ladybird beetle

Insect development is regulated by a combination of juvenile hormone (JH) and 20-hydroxyecdysone (20E). Production of both JH and 20E is regulated by transforming growth factor-β (TGFβ) signaling. TGFβ can be classified into two branches, the Activin and Bone Morphogenetic Protein (BMP) pathways. In Drosophila melanogaster, BMP signaling is critical for JH synthesis, whereas Activin signal is required to generate the large pulse of 20E necessary for entering metamorphosis. However, to which extent the roles of these signals are conserved remains unknown. Here we studied the role of an Activin component Smad on X (Smox) in post-embryonic development in a defoliating ladybird Henosepilachna vigintioctopunctata. RNA interference (RNAi)-aided knockdown of Hvsmox inhibited larval growth, and impaired larval development. All Hvmyo RNAi larvae arrested at the fourth-instar larval stage. Moreover, knockdown of Hvsmox delayed gut and Malpighian tubules remodeling. Furthermore, the expression of a JH biosynthesis gene (Hvjhamt), a JH receptor gene HvMet and a JH response gene HvKr-h1 was greatly enhanced. Conversely, the expression levels of an ecdysteroidogenesis gene (Hvspo), a 20E receptor gene (HvEcR) and six 20E response genes (HvBrC, HvE74, HvE75, HvE93, HvHR3 and HvHR4) were significantly lowered. Knockdown of HvMet partially restored the negative phenotypes in the Hvsmox RNAi beetles. Our results suggest that Smox exerts regulative roles in JH production, ecdysteroidogenesis and organ remodeling, thus contributing to modulate the larva-pupa-adult transformation in H. vigintioctopunctata.

Dissecting the roles of FTZ-F1 in larval molting and pupation, and the sublethal effects of methoxyfenozide on Helicoverpa armigera.

In holometabolous insects, the major developmental transitions - larval molting and pupation - are triggered by a pulse of 20-hydroxyecdysone (20E) and coordinated by juvenile hormone. Methoxyfenozide (MF), an ecdysteroid agonist, represents a new class of insect growth regulators and is effective against lepidopteran pests. Fushi-tarazu factor 1 (FTZ-F1) is an ecdysone-inducible transcription factor. To date, the effect of MF on 20E-response genes remains unclear, and we speculate the involvement of FTZ-F1 in MF's growth regulating effect. MF at LC25 and LC10 caused severe ecdysis failure in Helicoverpa armigera, extended their larval duration, lowered their pupal weight, and reduced the respiratory, pupation and emergence rates. Furthermore, sublethal doses of MF inhibited ecdysteroidogenesis and lowered the intrinsic 20E titer, but showed an inductive effect on 20E-response genes including HaFTZ-F1. HaFTZ-F1, predominantly expressed in larval epidermis, was markedly upregulated before or right after larval ecdysis, and maintained a high level in prepupal stage. Knockdown of HaFTZ-F1 in 4th-instar larvae severely impaired larval ecdysis, whereas its knockdown in final-instar larvae caused abnormal pupation. Moreover, knocking down HaFTZ-F1 downregulated three critical ecdysteroidogenesis genes, lowered 20E titer, and suppressed the expression of 20E receptors and 20E-response genes. The introduction of 20E into HaFTZ-F1-RNAi larvae partly relieved the negative effects on the 20E-induced signaling cascade. Our findings reveal the adverse effects of sublethal doses of MF on the development of H. armigera and elucidate the resulting perturbations on the 20E-induced signaling cascade; we propose that HaFTZ-F1 regulates ecdysis and pupation by mediating 20E titer and its signaling pathway. © 2020 Society of Chemical Industry.

Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes.

Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action in Aedes aegypti mosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fed Ae. aegypti females but blocked the development of primary follicles at Christopher’s Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Krüppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, including HR3 and E75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism in Ae. aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF.

Molecular Characterization of Ca2+/Calmodulin-Dependent Protein Kinase II Isoforms in Three Rice Planthoppers-Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera.

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II (CaMKII) gene from Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20–81 amino acid “inserts” were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph−1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera, the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR, USP1, and HR3, in N. lugens. These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.

The CncC/keap1 pathway is activated in high temperature-induced metamorphosis and mediates the expression of Cyp450 genes in silkworm, Bombyx mori

Global warming is known to affect the growth, development and reproduction of insects. In this study, the larvae developmental process and endogenous hormone levels under high temperature (36 °C) stress were investigated in the lepidopteran model insect Bombyx mori (B. mori). After high temperature treatment, the duration of 5th instar larvae was shortened by 28 ± 2 h, the content of 20-hydroxyecdysone(20E) in hemolymph was significantly increased, and the transcription levels of the 20E response genes E93, Br–C, USP and E75 were up-regulated by 1.35-, 1.25-, 1.28-, and 1.27-fold, respectively. High temperature treatment also elevated the phosphorylation level of Akt and activated the downstream BmCncC/keap1 pathway, and the transcription levels of the 20E synthesis-related genes cyp302a1, cyp306a1, cyp314a1 and cyp315a1 were up-regulated by 1.12-, 1.51-, 2.17- and 1.23-fold, respectively. The transcription levels of cyp302a1 and cyp306a1 were significantly decreased in BmN cells after treatment with the double stranded RNA of BmCncC (dsBmCncC), whereas their transcription levels were significantly increased (2.15- and 1.31-fold, respectively) after treatment with the CncC agonist Curcumin. These results demonstrated that high temperature treatment promoted the metamorphosis and the BmCncC/keap1 pathway played a role in the metamorphosis of B. mori. Our results provided clues for understanding the CncC/keap1 pathway-mediated regulation of metamorphosis of Lepidopteran insects.

Cucurbitacin B acts a potential insect growth regulator by antagonizing 20-hydroxyecdysone activity.

20-Hydroxyecdysone (20E), a crucial insect steroid hormone, can bind to its cognate nuclear receptor composed of ecdysone receptor (EcR) and ultraspiracle (USP) to activate expression of 20E-response genes, enabling subsequent metamorphosis. In this study, we tried to find out which steroid-like compounds can block insect metamorphosis effectively and provide useful information for biopesticide study. For this purpose, we screened 126 steroid-like compounds for possible 20E antagonists using a dual-luciferase reporter assay with Drosophila melanogaster Kc and Bombyx mori Bm12 cells. Among 126 steroid-like compounds, three cucurbitacins (CucB, D and E) were identified as 20E antagonists in both Kc and Bm12 cells. Notably, CucB caused significant molting defects and mortality in both B. mori and D. melanogaster larvae, and dramatically hindered larval growth of Helicoverpa armigera by its anti-feeding activity. In vivo and in vitro experiments demonstrate that CucB acts as a potential insect growth regulator by antagonizing 20E activity and thus blocking molting and metamorphosis induced by 20E signaling. © 2017 Society of Chemical Industry.

Leptinotarsa hormone receptor 4 (HR4) tunes ecdysteroidogenesis and mediates 20-hydroxyecdysone signaling during larval-pupal metamorphosis

Hormone receptor 4 (HR4) is involved in the regulation of 20-hydroxyecdysone (20E) biosynthesis and the mediation of 20E signaling during larval-pupal transition in a holometabolan Drosophila melanogaster, whereas it acts as a repressor in 20E-responsive transcriptional cascade in a hemimetabolan, Blattella germanica. Here we characterized two HR4 splicing variants, LdHR4X1 and LdHR4X2, in a coleopteran Leptinotarsa decemlineata. LdHR4X1 was highly expressed in the prothoracic gland and epidermis while LdHR4X2 was abundantly transcribed in the nervous system. In vivo results showed that both prothoracicotropic hormone and 20E pathways transcriptionally regulated LdHR4, in an isoform-dependent pattern. RNA interference of LdHR4 at the final (fourth) larval instar, in contrast to the second- and third-instar periods, enhanced the expression of two ecdysteroidogenesis genes, increased 20E titer, upregulated transcription of five 20E-response genes, and reduced the mRNA level of Fushi tarazu-factor 1 (FTZ-F1). As a result, the fourth-instar LdHR4 RNAi larvae exhibited accelerated development and reduced body weight. Moreover, knockdown of LdHR4 at the fourth instar resulted in larval lethality and impaired pupation. Feeding of pyriproxyfen (a mimic of juvenile hormone) or silencing of a juvenile hormone degrading enzyme gene restored the normal course of ecdysteroidogenesis, duration of larval development, and body weight in fourth-instar LdHR4 RNAi larvae. The treatment partially suppressed the larval mortality but not the failure to pupate. The dual role of HR4 during larval-pupal metamorphosis appears to be evolutionarily conserved among holometabolans.

Leptinotarsa cap ‘n’ collar isoform C/Kelch-like ECH associated protein 1 signaling is critical for the regulation of ecdysteroidogenesis in the larvae

Drosophila cap ‘n’ collar isoform C (CncC) and Kelch-like ECH associated protein 1 (Keap1) regulate metamorphosis by transcriptional control of a subset of genes involved in ecdysteroidogenesis, 20-hydroxyecdysone (20E) signaling, and juvenile hormone (JH) degradation. In the present paper, we found that prothoracicotropic hormone signal was required for the activation of LdCncC and LdKeap1 in Leptinotarsa decemlineata. Moreover, RNA interference of LdCncC or LdKeap1 in the fourth-instar larvae delayed development. As a result, the treated larvae obtained heavier larval and pupal fresh weights and had larger body sizes than the controls. Furthermore, knockdown of LdCncC or LdKeap1 significantly reduced the mRNA levels of four ecdysone biosynthetic genes (Ldspo, Ldphm, Lddib and Ldsad), lowered 20E titer and decreased the transcript levels of five 20E response genes (LdEcR, LdUSP, LdE75, LdHR3 and LdFTZ-F1). However, the expression of two JH epoxide hydrolase genes and JH contents were not affected in the LdCncC and LdKeap1 RNAi larvae. Dietary supplementation with 20E shortened the developmental period to normal length, rescued the larval and pupal body mass rises, and recovered or even overcompensated the expression levels of the five 20E response genes in either LdCncC or LdKeap1 RNAi hypomorphs. Therefore, LdCncC/LdKeap1 signaling regulates several ecdysteroidogenesis genes, and consequently 20E pulse, to modulate the onset of metamorphosis in L. decemlineata.

Pupal diapause termination in Bactrocera minax: an insight on 20-hydroxyecdysone induced phenotypic and genotypic expressions

The Chinese citrus fruit fly, Bactrocera minax, is an economically important pest of citrus. It exhibits pupal diapause from November to May to combat harsh environmental conditions. Such a long pupal diapause is a barrier for laboratory rearing and development of control strategies against this pest. In the present study, 20-hydroxyecdysone (20E) was used to break pupal diapause of B. minax by topical application. After diapause termination by 20E treated, the pupal ontogenetic processes were observed along the temporal trajectory. The pupal response time to 20E was estimated by detecting the relative expression of 20E responsive genes at different times after 20E-treatment. Results revealed that 20E could effectively terminate the pupal diapause in a dose-dependent manner and significantly shorten the time for 50% adult emergence (Et50). 20E response genes, including ecr, broad and foxo, were up-regulated within 72h, indicating these genes are involved in pupal metamorphosis and diapause termination processes. Morphological changes showed the pupal metamorphosis began ~7 days after 20E-treatment at 22 °C. This study does not only pave the way for artificial rearing in the laboratory through manipulating of pupal diapause termination, but also deepens our understanding of the underlying pupal diapause termination mechanism of B. minax.

Knockdown of a putative insulin-like peptide gene LdILP2 in Leptinotarsa decemlineata by RNA interference impairs pupation and adult emergence

Five insulin-like peptide LdILP genes were identified in Leptinotarsa decemlineata. All of them contained three exons and two introns, with three genes tandemly arrayed and well separated from the other two. Phylogenetic analysis revealed that the three LdILPs from three tandemly-arrayed genes grouped with TcILP1, whereas the other two resembled with TcILP2 and TcILP4 from Tribolium castaneum. Thus, the five LdILP genes were provisionally named LdILP1a, LdILP1b, LdILP1c, LdILP2 and LdILP4. LdILP2 was widely expressed in several tissues such as the brain–corpora cardiaca–corpora allata (BR–CC–CA) complex, gut and fat body. In contrast, LdILP1a and LdILP1b were only transcribed in BR–CC–CA, LdILP4 was in ovaries, and LdILP1c was in both BR–CC–CA and ovaries. Ingestion of double-stranded RNAs (dsRNAs) targeting LdILP2 (dsLdILP2-1 and dsLdILP2-2) specifically knocked down LdILP2 and upregulated the transcription of both LdInR and Ld4EBP, indicating insulin/insulin-like growth factor signaling pathway (IIS) was inhibited. Approximately 50% of the LdILP2 RNAi larvae did not normally pupate and about 50% of the LdILP2 RNAi pupae did not emerge. Moreover, silencing LdILP2 reduced the expression of a juvenile hormone (JH) biosynthesis gene, lowered JH titer and disturbed JH signaling. Finally, knocking down LdILP2 inhibited an ecdysteroidogenesis gene, decreased 20-hydroxyecdysone (20E) titer, and repressed the expression of two 20E-response genes LdHR3 and LdFTZ-F1. Thus, the IIS pathway is involved in larval–pupal metamorphosis by modification of both JH and 20E signaling in L. decemlineata.

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

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.

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis.

RNA interference suppression of the receptor tyrosine kinase Torso gene impaired pupation and adult emergence in Leptinotarsa decemlineata

In Drosophila melanogaster prothoracic gland (PG) cells, Torso mediates prothoracicotropic hormone (PTTH)-triggered mitogen activated protein kinase (MAPK) pathway (consisting of four core components Ras, Raf, MEK and ERK) to stimulate ecdysteroidogenesis. In this study, LdTorso, LdRas, LdRaf and LdERK were cloned in Leptinotarsa decemlineata. The four genes were highly or moderately expressed in the larval prothoracic glands. At the first- to third-instar stages, their expression levels were higher just before and right after the molt, and were lower in the mid instars. At the fourth-instar stage, their transcript levels were higher before prepupal stage. RNA interference-mediated knockdown of LdTorso delayed larval development, increased pupal weight, and impaired pupation and adult emergence. Moreover, knockdown of LdTorso decreased the mRNA levels of LdRas, LdRaf and LdERK, repressed the transcription of two ecdysteroidogenesis genes (LdPHM and LdDIB), lowered 20E titer, and downregulated the expression of several 20E-response genes (LdEcR, LdUSP, LdHR3 and LdFTZ-F1). Furthermore, silencing of LdTorso induced the expression of a JH biosynthesis gene LdJHAMT, increased JH titer, and activated the transcription of a JH early-inducible gene LdKr-h1. Thus, our results suggest that Torso transduces PTTH-triggered MAPK signal to regulate ecdysteroidogenesis in the PGs in a non-drosophiline insect.

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

Our previous results revealed that RNA interference-aided knockdown of Leptinotarsa decemlineata FTZ-F1 (LdFTZ-F1) reduced 20E titer, and impaired pupation. In this study, we characterized a putative LdHR3 gene, an early-late 20E-response gene upstream of LdFTZ-F1. Within the first, second and third larval instars, three expression peaks of LdHR3 occurred just before the molt. In the fourth (final) larval instar 80 h after ecdysis and prepupal stage 3 days after burying into soil, two LdHR3 peaks occurred. The LdHR3 expression peaks coincide with the peaks of circulating 20E level. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdHR3 expression in the final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene Ldshd repressed the expression. Moreover, Hal rescued the transcript levels in the Ldshd-silenced larvae. Thus, 20E peaks activate the expression of LdHR3. Furthermore, ingesting dsRNA against LdHR3 successfully knocked down the target gene, and impaired pupation. Finally, knockdown of LdHR3 upregulated the transcription of three ecdysteroidogenesis genes (Ldphm, Lddib and Ldshd), increased 20E titer, and activated the expression of two 20E-response genes (LdEcR and LdFTZ-F1). Thus, LdHR3 functions in regulation of pupation in the Colorado potato beetle.