Genome-wide CRISPR screening reveals key genes and pathways associated with 20-hydroxyecdysone signal transduction in the silkworm(Bombyx mori)

Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.

Simultaneous determination of six steroidal saponins and one ecdysone in Asparagus filicinus using high performance liquid chromatography coupled with evaporative light scattering detection

A high-performance liquid chromatography coupled with an evaporative light scattering detector(HPLC-ELSD)has been developed to evaluate the quality of Asparagus filicinus through a simultaneous determination of six steroidal saponins and one ecdysone,including aspafiliosides A,B,C,E,G,filiasparoside A and 20-hydroxyecdysone.With a C_(18) analytical column,the seven analytes were separated efficiently using acetonitrile-water as the mobile phase in a gradient program.The method limits of detection ranged 0.1250.225μg,and the method limits of quantitation ranged 0.408-0.720μg,respectively.The intra-and inter-day precisions of the method were evaluated and were all less than 3%.All the recoveries for the spiked analytes ranged 95.16%-100.61%.The proposed method was succesfully applied to quantify the seven components in thirteen samples from different localities in China.

MicroRNA targets the ecdysone signaling pathway E75 gene to control larval-pupal development in Bactrocera dorsalis

MicroRNAs (miRNAs) regulate various biological processes during insect developme nt;however, their role in larval-pupal development in oriental fruit fly, Bactrocera dorsalis (Hendel) remains unknown. In the current study, we address the biological function of a conserved miRNA, Bdo-Let-7 in the regulation of BdE75 gene, which belongs to the ecdysone signaling pathway and participates in the larval-pupal development in B. dorsalis. Using dual luciferase reporter assay in HEK293T cells we show that Bdo-Let-7 miRNA interacts with the 3' untranslated region of BdE75 gene and suppresses its expression. The Bdo-Let?7 and BdE75 are also co-expressed in the larval-pupal stages and in different tissues of B. dorsalis .In in vivo experiments, the injectio n of Bdo-Let-7 agomir and antagomir in third instar larvae down- and up-regulated the expression of BdE75、 respectively. The 20-hydroxyecdysone (20E) injection assay shows that 20E up-regulated the expression of Bdo-Let-7 on the 5th day of the larvae. Moreover, abnormal pupation and eclosion were observed after larval Bdo-Let-7 antagomir injection. Based on these results, we show that Bdo-Let-7 regulates the ecdysone signaling pathway through the exact dose of BdE75 gene, and is indispensable for normal larval-pupal development in B. dorsalis.

RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata

Henosepilachna vigintioctopunctata is a serious insect pest which attacks a large number of nightshades and cucurbits in Asian countries,Brazil and Australia.Prolonged application of traditional pesticides has caused environmental pollution and exerted deleterious effects on human health.Finding new approaches with high target specificity and low environmental contamination has become an urgent task.RNA interference(RNAi)induced by double-stranded RNA(dsRNA)is expected to be applicable to managing this pest.Here we evaluated the effects of Escherichia co/Z-expressed dsRNAs targeting ecdvsone receptor(EcR)gene via dietary delivery in laboratory and foliar spraying in a greenhouse.The target transcript was successfully knocked down when the 4th-instar larvae had fed on potatofoliage dipped with dsEcR in a laboratory bioassay.Around 85%of the HvEcR RNAi larvae remained as prepupae or became abnormal pupae,and failed to emerge into adults.Ingestion of ds£c7?-immersed foliage by the 3rd-instar larvae effectuated a comparable RNAi response and brought about more severe defects:all the resultant larvae arrested development,remained as prepupae and finally died.For assay in the greenhouse,a ds£c7?-contained E.coli suspension was directly sprayed to the foliage of greenhouse-growing potato plants and the 3rd-and 4th-instar larvae were transferred to the leaves.High RNAi efficacy was obtained and identical RNAi phenotypes were observed in treated larvae.In addition,spraying dsEcR reduced leaf damage.Our results indicate a possibility of practical application of dsEcR as an environmentally friendly RNA pesticide to control H.vigintioctopunctata larvae.

Multiple microRNAs control ecdysone signaling in the midgut of Spodoptera litura

Metamorphosis is one of the most important physiological processes in insects.It is regulated by a serial of ecdysone cascade genes.Recently,lots o f microRNAs(miRNAs)were investigated in insects;however,their function in metamorphosis is largely unknown.In the present study,the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis.A total of 101 miRNAs were identified,and 75 miRNAs were differentially expressed during the metamorphic process.The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs,and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets.Among them,miR-14-3p and its two targets(EcR and E75)were chosen for further validation.MiR-14-3p had higher expression level in the 6 th instar larvae as compared with either the prepupae or pupae,which was opposite to that of both EcR and E75,two ecdysone cascade genes.Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p.Interestingly,the 3'untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes,including EcR,USP,E75,E74,E78,E93,Hr3,Hr4,Hr39,K rhl and F tzfl.Thus,different transcript variants of one ecdysone cascade gene could be regulated by the same mi RNA.The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA.These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.

Ecdysone receptor isoforms play distinct roles in larval–pupal–adult transition in Leptinotarsa decemlineata

A heterodimer of two nuclear receptors,ecdysone receptor(EcR)and ultraspiracle,mediates 20‐hydroxyecdysone(20E)signaling to modulate many aspects in insect life,such as molting and metamorphosis,reproduction,diapause and innate immunity.In the present paper,we intended to determine the isoform‐specific roles of EcR during larval–pupal–adult transition in the Colorado potato beetle.Double‐stranded RNAs(dsRNAs)were prepared using the common(dsEcR)or isoform‐specific(dsEcRA,dsEcRB1)regions of EcR as templates.Ingestion of either dsEcR or dsEcRA,rather than dsEcRB1,by the penultimate(3rd)and final(4th)instar larvae caused failure of larval–pupal and pupal–adult ecdysis.The RNA interference(RNAi)larvae remained as prepupae,or became deformed pupae and adults.Determination of messenger RNA(mRNA)levels of EcR isoforms found that LdEcRA regulates the expression of LdEcRB1.Moreover,silencing the two EcR transcripts,LdEcRA or LdEcRB1 reduced the mRNA levels of Ldspo and Ldsad,and lowered 20E titer.In contrast,the expression levels of HR3,HR4,E74 and E75 were significantly decreased in the LdEcR or LdEcRA RNAi larvae,but not in LdEcRB1 depleted specimens.Dietary supplement with 20E did not restore the expression of five 20E signaling genes(USP,HR3,HR4,E74 and E75),and only partially alleviated the pupation defects in dsEcR‐or dsEcRA‐fed beetles.These data suggest that EcR plays isoform‐specific roles in the regulation of ecdysteroidogenesis and the transduction of 20E signal in L.decemlineata.

Dietary cholesterol promotes growth and ecdysone signalling pathway by modulating cholesterol transport in swimming crabs(Portunus trituberculatus)

Cholesterol,as an indispensable nutrient,regulates molting and growth in crustacean.As crustaceans are unable to biosynthesize cholesterol de novo,it is central to understand how dietary cholesterol affects molting in crustaceans.An 8-week feeding trial was conducted to evaluate the effects of dietary cholesterol level(0.12%,0.43%,0.79%,1.00%,1.30%and 2.50%)on growth,cholesterol metabolism and expression of genes related to lipid and ecdysone metabolism in female swimming crabs(Portunus trituberculatus).A total of 192 crabs(1.41±0.05 g)were randomly distributed into 192 aquaria.Each treatment had 4 replicates with each replicate containing 8 crabs.Crabs fed the 1.00%cholesterol diet showed best growth performance,and thus based on percent weight gain,the optimal dietary cholesterol requirement was calculated at 1.01%.Tissue cholesterol concentrations were positively correlated with dietary cholesterol level.The contents of functional fatty acids in hepatopancreas significantly increased as dietary cholesterol increased from 0.12%to 2.50%(P<0.05).The expression levels of genes related to lipogenesis pathway,lipid catabolism and fatty acid oxidation were significantly downregulated with increased dietary cholesterol level(P<0.05).The highest expression levels of cholesterol transport genes,low-density lipoprotein receptor(ldlr)and low-density lipoprotein receptorrelated protein 2(lrp2)occurred in crabs fed the 1.30%cholesterol diet.Moreover,hormones related to molting such as crustacean hyperglycemic hormone(CHH),methyl farnesoate(MF),molt-inhibiting hormone(MIH),and ecdysone in hemolymph were significantly influenced by dietary cholesterol level(P<0.05).The highest expression levels of ecdysone receptor(ecr)and chitinase 1(chi1)in eyestalk and hepatopancreas were found in crabs fed the diet containing 1.00%cholesterol(P<0.05).In conclusion,the optimal dietary level was beneficial to functional fatty acid accumulation,regulated lipid metabolism,promoted the ecdysone signalling pathway by improving the cholesterol transport,and improved the molting rate and growth of swimming crabs.

EcR/USP-1-mediated ecdysteroid signaling regulates wolf spider(Pardosa pseudoannulata) development and reproduction

Lycosidae females demonstrate meticulous maternal care of offspring by carrying egg sacs and juvenile spiderlings during the reproductive stage. Nuclear receptors(NRs), especially the ecdysone receptor(EcR) and ultraspiracle(USP), have attracted considerable attention in the regulation of arthropod development and reproduction due to their pivotal roles in ecdysteroid signaling cascades. In the present study, 23 NRs, including one EcR and two USPs, were identified in the genome of the predatory wolf spider Pardosa pseudoannulata. RNA interference(RNAi) targeting EcR and USP-1inhibited spiderling development and resulted in nonviable eggs in the egg sacs. EcR and USP-1responded to changes in ecdysteroid levels, and interference in ecdysteroid biosynthesis led to similar phenotypes as ds EcR and ds USP-1 treatments.These findings suggest that EcR/USP-1-mediated ecdysteroid signaling regulates P. pseudoannulata development and reproduction. The P.pseudoannulata females with suppressed ecdysteroid signaling proactively consumed their non-viable egg sacs, resulting in a 7.19 d shorter first reproductive cycle than the controls. Termination of the failed reproductive cycle enabled the spiders to produce a new egg sac more rapidly. This reproductive strategy may partially rescue the reduction in population growth due to non-viable eggs and compensate for the physiological expenditure of wasted maternal care, which would be beneficial for the conservation of P.pseudoannulata populations and their natural control of insect pests.

Brown planthopper E78 regulates moulting and ovarian development by interacting with E93

The brown planthopper(Nilaparvata lugens) is the main migratory pest in many rice growing areas in Asia. E78 is a member of the nuclear hormone receptor superfamily which plays an important role in egg development and maternal regulation of early embryogenesis. In this study, brown planthopper E78(NlE78) was cloned, and the predicted amino acid sequence showed that it contains two conserved domains: NR-LBD and DBD. qRT-PCR showed that the expression of NlE78 is high in the fifth instar nymphs and the ovaries of females. After downregulation of NlE78, the rate of moulting failure(33.2%) increased significantly, and ovarian development was delayed. However, when NlE78was downregulated together with NlE93, the emergence rate increased significantly(78.79%), and ovarian development was similar to that when NlE78 was downregulated but not delayed. A co-immunoprecipitation experiment showed that NlE78 interacts with NlE93, a crucial downstream transcription factor of the ecdysone signalling pathway. Cellular localization by immunofluorescence revealed that NlE78 and NlE93 are expressed in the nucleus. This study indicates that NlE78 regulates ovarian development and moulting, possibly through its interaction with NlE93. This study is of great significance for the development of new pesticides and control methods based on newly discovered targets.

Molecular characterization and functional analysis of USP-1 by RNA interference in the Asian gypsy moth Lymantria dispar

Ecdysteroids play an important role in regulating diverse physiological processes in arthropods,such as molting,metamorphosis,reproduction and diapause.Ecdysteroids mediate the response by binding to a heterodimeric complex of two nuclear receptors:the ecdysone receptor and the ultraspiracle(USP).To investigate the role of USP in development of the Asian gypsy moth(Lymantria dispar),a USP cDNA was obtained from the transcriptome of L.dispar and verified by PCR.In-depth profiling of transcript levels of L.dispar USP-1(LdUSP-1)at different developmental stages and over time in thirdinstar larvae and different tissues isolated during the thirdinstar stage of L.dispar was then carried out.Transcript levels of LdUSP-1 were relatively high before 72 h in the third-instar larvae after ecdysis and in the adult male.The function of LdUSP-1 in molting was analyzed by knockdown of LdUSP-1 in third instar larvae using RNA interference.Silencing of LdUSP-1 significantly downregulated the transcript level of E75,an ecdysone-inducible gene,and of Sad,a Halloween gene.In addition,the duration of the third-instar stage was slightly shortened and larval mortality increased after the LdUSP-1 knockdown.

Multiple endocrine factors regulate nutrient mobilization and storage in Aedes aegypti during a gonadotrophic cycle

Anautogenous mosquitoes must blood feed on a vertebrate host to produce eggs.Each gonadotrophic cycle is subdivided into a sugar-feeding previtellogenic phase that produces primary follicles and a blood meal-activated vitellogenic phase in which large numbers of eggs synchronously mature and are laid.Multiple endocrine factors including juvenile hormone(JH),insulin-like peptides(ILPs),ovary ecdysteroidogenic hormone(OEH),and 20-hydroxyecdysone(20E)coordinate each gonadotrophic cycle.Egg formation also requires nutrients from feeding that are stored in the fat body.Regulation of egg formation is best understood in Aedes aegypti but the role different endocrine factors play in regulating nutrient mobilization and storage remains unclear.In this study,we report that adult female Ae.aegypti maintained triacylglycerol(TAG)stores during the previtellogenic phase of the first gonadotrophic cycle while glycogen stores declined.In contrast,TAG and glycogen stores were rapidly mobilized during the vitellogenic phase and then replenishment.Several genes encoding enzymes with functions in TAG and glycogen metabolism were differentially expressed in the fat body,which suggested regulation was mediated in part at the transcriptional level.Gain of function assays indicated that stored nutrients were primarily mobilized by adipokinetic hormone(AKH)while juvenoids and OEH regulated replenishment.ILP3 further showed evidence of negatively regulating certain lipolytic enzymes.Loss of function assays indicated AKH depends on the AKH receptor(AKHR)for function.Altogether,our results indicate that the opposing activities of different hormones regulate nutrient stores during a gonadotrophic cycle in Ae.aegypti.

U1 small nuclear ribonucleoprotein is essential for early larval development in silkworm, Bombyx mori

U1 small nuclear ribonucleoproteins(U1 snRNP)associates with 5ʹsplice sites in the form of ribonucleoprotein particles and is highly conserved among species.The physiological functions of U1 snRNP in a lepidopteran model insect Bombyx mori is mostly unknown.Here,we showed that U1 snRNP plays an important role in the development of silkworm.Knockout of U1 snRNP in silkworm showed either delayed or stationary 1st instar larva development compared with the wild-type group.U1 snRNP deletion mutants exhibited abnormal cellular phenotypes with enlarged cell nucleus,scant cytoplasm and enlarged nuclei.RNA-seq analysis revealed that genes involved in metabolic pathway,biosynthesis of secondary metabolites and steroid hormone biosynthesis were significantly affected by U1 snRNP depletion.Taken together,our study suggests that U1 snRNP homeostasis plays an important role in silkworm development.

Molting process revealed by the detailed expression profiles of RXR1/RXR2 and mining the associated genes in a spider mite, Panonychus citri

Spider mites have one ecdysone receptor(EcR)and multiple retinoid X receptors(RXRs).However,the function of these RXRs in spider mite development is unknown.Here,we screened the expression dynamics of two PcRXR isoforms at 4 h intervals in the deutonymphal stage of Panonychus citri.The results showed that PcEcR had an expression pattern similar to that of PcRXR2.For PcRXR1,its expression remained at a certain high level,when there was a decrease of both PcEcR and PcRXR2.In situ hybridization showed that PcRXR2 was detected in the central nervous mass,while the ecdysteroid biosynthesis gene PcSpo was mainly expressed at the edge of the central nervous mass.RNAi-based silencing of PcRXR1 or PcRXR2 showed the same phenotype as in mites with that of silencing PcEcR.Furthermore,RNA-seq was used to mine the genes associated with the expression dynamics of PcRXR1 or PcRXR2,which revealed that the heterodimer of EcR-RXR2 in spider mites might be linked with the cell autophagy and tissue remodeling during apolysis,and RXR1 might be linked with new epicuticle and exocuticle secretion during ecdysis.Taken together,these results increase our understanding of the regulation mechanism of ecdysteroid signal pathway in spider mite development.

Insights into the mechanism of shortened developmental duration and accelerated weight gain associated with Wolbachia infection in Hylyphantes graminicola

Wolbachia infection is known to affect host reproduction and development.To date,however,the underlying mechanism related to the effects of Wolbachia on host development has not yet been reported.Here,we compared the developmental duration and body weight in different instars of Wolbachia-positive(W+)and Wolbachia-negative(W−)spiders(Hylyphantes graminicola)and detected the relative expression levels of 6 insulin-related genes and 3 ecdysone-related genes using reverse transcription qPCR.Results showed that the developmental duration was significantly shortened in W+spiders compared with W−spiders.Furthermore,W+spiders were significantly heavier thanW−spiders at the 3rd and 4th instars,although no significant differences in body weight were observed after maturity.We also found that the expression levels of insulin-like growth factor-2 mRNA-binding protein-1,insulin-degrading enzyme,and ecdysone-inducible protein-1 genes were significantly down-regulated in W+spiders compared with W−spiders,whereas the expression levels of insulin-like growth factor-binding protein-1,insulin-like peptide receptor,insulin receptor substrate 2-B,insulin-like,ecdysone-induced protein-2,and ecdysone receptor genes were significantly up-regulated inW+spiders.Our results suggest thatWolbachia may influence host development by affecting insulin and ecdysone signaling pathways.

Ontogeny of Drosophila melanogaster female sex-appeal and cuticular hydrocarbons

The chemical communication system in Drosophila melanogaster Meigen,1830 plays a major role in courtship and consists of the male-specific cis-Vaccenyl acetate and sex-specific contact pheromones,cuticular hydrocarbons(CHC),which build up during ontogeny(first 4 days).They replace longer CHCs,common to both sexes and present only after the imaginal eclosion.A detailed quantitative description of the evolution of cuticular unsaturated hydrocarbons with age is presented here for males and females of different D.melanogaster strains,which have been bred in well controlled environments.Monoenes appear in both sexes at around 12 h,before female dienes.The present paper argues that this is likely linked to the switching on of a new set of genes.Ecdysone,which is more abundant in females than in males during this critical period,might control this switch.Parallel behavioral studies show that whereas female of all ages trigger early mature male courtship steps like wing vibration,only females older than 1 day trigger late courtship steps like attempted copulation.This supports the hypothesis that late male courtship steps might be triggered by the CHCs,which build up after this age,especially female-specific(Z,Z)-7,11-and(Z,Z)-5,9-dienes.