| The endocrine regulation mechanism of insect diapause has always been a key issue in the field of entomology,and it is also the theoretical basis of pest control and successful insect reproduction.Previous studies have shown that the lack of juvenile hormone(JH)synthesis is a direct factor inducing reproductive diapause.However,in some insects with reproductive diapause characteristics,it was found the differences in 20-hydroxyecdysone(20E)titers between reproductive and diapausing individuals,suggesting the potential regulatory effect of 20 E on reproductive diapause.The studies have shown that the diapause preparation period is the key period for insects to enter diapause smoothly,which is important for the occurrence of diapause.At present,the molecular regulation mechanism of 20 E in the insect reproductive preparation is not clear.Therefore,the scientific problem to be solved in this study is to clarify the pathway and molecular mechanism of 20 E involved in the regulation of insect reproductive diapause preparation,so as to lay a foundation for solving the core scientific problem of how insects accumulate environmental information and start diapause procedure.Colaphellus bowringi Baly is a pest of cruciferous vegetables with characteristics of reproductive diapause.It has been proved that photoperiod and temperature are important factors to induce C.bowringi to enter reproductive diapause.C.bowringi diapause stages are obvious and easy to distinguish.It is an ideal model for the study of diapause regulation mechanism.The 25℃ and long-day(16L:8D)conditions induce females to enter reproductive diapause.The ovarian development is completely stagnant and the differentiation of ovariole is not obvious.A large amount of fat is accumulated in the fat body,which almost fills the whole abdominal cavity;whereas the 25℃ and short-day(12L:12D)conditions promote the development of ovaries and induce reproduction.In this study,C.bowringi was used as experimental material.We clarified the function of 20 E signaling in the reproductive diapause preparation of C.bowringi and explored the regulatory mechanism of 20 E signaling on the main physiological events in the diapause preparation period.The main results are as follows:1.Inhibition of 20 E signaling on reproductive diapause preparation of C.bowringiIn this study,we first identified and cloned the sequences of 20 E biosynthetic genes in C.bowringi,including spook(Spo),phantom(Phm),shadow(Sad)and shade(Shd).The 20 E titer and the expression pattern of 20 E biosynthetic genes under LD(diapause)and SD(non-diapause)conditions.The functions of 20 E signaling in the reproductive diapause preparation of C.bowringi was analyzed.The related results are as follows:(1)Phylogenetic tree construction showed that these 20 E biosynthetic genes were closely related to Leptinotarsa decemlineata.Multiple sequence alignment analysis showed that 4 20 E biosynthetic genes belonged to the cytochrome P450 superfamily and contained the classical characteristic motifs of CYP450.(2)20E titer was significantly different between LD-and SD-females,and showed a high level in the latter.Spo,Phm,Sad and Shd were specifically expressed in ovarian tissue.At the same time,Phm,Sad and Shd were highly expressed in the pre-oviposition stage of SD-induced females.(3)Exogenous injection of 20 E into LD-induced females resulted in an increase in ovarian size and yolk production,and a significant decrease in triglyceride content and total ester content.After silencing the 20 E receptor gene ecdysone receptor(Ec R)and 20 E biosynthetic genes Spo,Sad and Shd in SD females,it was found that ovarian development was seriously blocked and the lipid content increased significantly.The results showed that 20 E signaling could inhibit the reproductive diapause preparation of C.bowringi.(4)The fat bodies of LD-induced females treated with 20 E was analyzed by RNA-sequence.Combined with reports and screening of transcriptome database,the expression levels of lipolysis genes such as triacylglycerol lipase 1,Brummer and acyl Co A oxidase were significantly up-regulated after 20 E injection.The expression levels of lipogenesis genes such as fat acid synthase 2,transketolase 2,alcohol dehydrogenase NADP(+)decreased after 20 E treatment.2.20 E signaling suppressed reproductive diapause preparation of C.bowringi by regulating JH productionPrevious studies have shown that the lack of JH synthesis is the endocrine basis for insects to enter reproductive diapause.Therefore,this study analyzed the effects of 20 E signaling on JH production.The hormone rescue experiment determined that 20 E could participate in the regulation of reproductive diapause preparation through promoting JHsynthesis.The results are as follows:(1)We collected the heads of LD-females after 20 E injection for RNA-sequence and generated transcript profiles.KEGG analysis showed that the insect hormone biosynthesis pathway was significantly enriched.JH biosynthesis pathway and JH-inducible genes were identified and analyzed by heat map.The expression levels of 11 JH biosynthetic genes and 2 JH-inducible genes increased significantly after 20 E treatment.(2)Injection of 20 E could significantly induce the increase of JH synthesis in LD-females,and silencing Ec R gene of SD-females reduced JH production.The expression levels of biosynthetic genes and JH-inducible genes decreased significantly after silencing Spo,Sad and Shd in SD-females.(3)We first knocked down the JH receptor Methoprene-tolerant(Met)in LD females and applied 20 E.We found that 20E-induced ovarian development was completely blocked after Met depletion.However,the lipid content still decreased significantly.This suggested that there is a JH-independent unknown pathway for the regulation of 20 E on lipid accumulation.Furthermore,application of JHA on Ec R-depleted females resulted in induction of ovarian development,as well as an apparent suppression of lipid droplet accumulation.The above results showed that 20 E signaling could induce ovarian development and inhibit lipid accumulation by promoting JH production,and then participate in the reproductive diapause preparation of C.bowringi,while there is an unknown pathway independent of JH.3.The ecdysis-triggering hormone(ETH)acted as an important regulatory factor in the regulation of 20E-JH signaling on reproductive diapause preparation of C.bowringiETH could be used as an important intermediate regulator in the regulation of JH by 20 E signaling in the process of larval metamorphosis and adult reproduction.At present,its function in insect reproductive diapause has not been reported.In this study,the tissue and temporal expression patterns of ETH were measured,and the expression levels of ETH were detected after 20 E treatment and Ec R RNAi to clarify the effect of 20 E signaling on ETH.The effects of ETH signaling on JH synthesis and reproductive diapause related phenotypes were analyzed.The related results are as follows:(1)ETH has the conservative structural characteristics: signal peptide + mature peptide ETH1 +mature peptide ETH2.(2)ETH was expressed at a high level in the abdominal fat bodies attached with trachea,and transcripts of ETH in SD-females was significantly higher than that in LD-females.(3)20E signaling could induce the up-regulation of ETH.JH III titer,the expression levels of JH biosynthetic genes and JH-inducible genes were significantly decreased in ETH RNAi treatment group.(4)After silencing ETH,the ovarian development of female adults was significantly inhibited,indicating that ETH could induce the reproduction of C.bowringi by promoting JH production.(5)This study observed the development of ovary after injecting ETH mature peptide into LD-females,so as to further verify the function of ETH in reproductive diapause.Injection of different doses of ETH mature peptide did not significantly induce the ovarian development of LD-induced females.(6)The RNAi experiment of ETH receptor(ETHR)also led to the obstruction of ovarian development and the decrease of JH level in female adults.These results suggested that ETH could effectively activate JH signaling and promote ovarian development,but it is not a necessary condition for JH mediated reproduction.In conclusion,ETH signaling can be used as an intermediate regulatory factor to couple the regulatory effect of 20 E on JH synthesis,and then participate in the preparation of reproductive diapause of C.bowringi.4.20 E signaling regulated reproductive diapause preparation through DNA replication in C.bowringiIn this study,RNA-sequence technology and bioinformatics analysis were used to screen the other signaling pathways that may be involved in the occurrence of reproductive diapause and regulated by 20 E.The temporal expression patterns and functions of candidate genes were analyzed and verified.The relevant results are as follows:(1)Based on the transcriptome data of whole bodies during diapause preparation period and pr-oviposition period obtained previously,the KEGG database was used to enrich and analyze differentially expression genes(DEGs).It has been found that the DNA replication pathway was significantly enriched.Additionally,GO enrichment analysis in the transcriptome data of the fat bodies obtained from 20E-applied females showed that the DNA replication pathway can also be significantly enriched,indicating that 20 E signaling could regulate reproductive diapause preparation through DNAreplication.(2)This study identified and analyzed the sequence of the 14 DEGs enriched in the DNA replication pathway in the above two transcriptome databases.Phylogenetic tree analysis showed that these genes have high genetic relationship with those of Coleopteran insects.(3)Relative expression levels of 14 DNA replication genes were significantly different between LD-and SD-induced females,suggesting that the process of DNA replication has a potential regulatory effect on the occurrence of reproductive diapause.(4)RNAi experiments showed that silencing of 11 DNA replication genes could prevent the normal development of ovary of SD-females,and the expression levels of vitellogenin(Vg)1 and Vg2 also decreased significantly.(5)20E signaling promoted the up-regulation of DNA replication genes m RNA levels.In summary,20 E signaling could regulate the reproductive diapause preparation of C.bowringi through DNA replication.This study found that 20 E could respond to the photoperiodic cue in the stage of reproductive diapause preparation,and reveal the key role of 20 E signaling in the process of reproductive diapause preparation for the first time.It was also found that 20 E could determine the occurrence of reproductive diapause by regulating JH synthesis.ETH could be used as an intermediate regulator to couple the regulatory effect of 20 E on JH production,which defined the novel function of 20 E signaling as an upstream regulator of diapause.Among them,there is an unknown pathway independent of JH in the regulation of 20 E signal on fat accumulation.Finally,this study also revealed that 20 E signaling could also regulate the reproductive diapause preparation of C.bowringi through DNA replication.These results provide a new perspective for further understanding the mechanism of insect developmental plasticity,and provide a new idea for using insect growth regulators to regulate diapause and control pests. |
