Regulation Mechanism Of Imidazole Derivative KK-42 On The Growth And Development Of Two Silkworms,Antheraea Pernyi And Bombyx Mori

The imidazole compound KK-42 is a synthetic hormone with the chemical structure of1-benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole(CAS: 96108-89-3),which can regulate the growth and development of insects.KK-42 can cause precocious metamorphosis of insect larvae,such as Bombyx mori,Antheraea pernyi and Locusta migratoria(e.g.,inducing fourth molting silkworms to trimolter),induce diapause release of eggs of Antheraea yamamai and Lymantria dispar,reduce the rate of B.mori diapause eggs,block the development of B.mori pupae,increase the ratio of diapause pupae of Helicoverpa armigera and Sarcophaga bullata,delay the release of pupae diapause of A.pernyi and H.armigera.KK-42 can enter the insect body through osmosis to exercise its activity,which makes KK-42 have a good prospect in controlling insect diapause or other stages of growth and development even the pest control.The mechanism of KK-42 regulating insect growth and development has been studied extensively in endocrinology.Previous studies have confirmed that the target organ of KK-42 is the prothoracic gland(the organ that syntheses ecdysone),which can temporarily inhibit ecdysone synthesis in the prothoracic gland to achieve the regulation of insect growth and development.However,it is not clear which link of the ecdysone synthesis pathway is inhibited by this process,and how to achieve the effect on ecdysone synthesis.especially the lack of molecular physiological evidence.In order to understand the mechanism of KK-42 regulating insect growth and development,we investigated the transcriptomic profile of KK-42 target organs,the in vitro binding of KK-42 to 20-hydroxyecdysone(20E),and the effects of KK-42 on the expression of three important hormone-related genes(ecdysone,[juvenile hormone,JH],[diapause hormone,DH]).In this study,the prothoracic gland(PG),the corpus allatum(CA)and the subpharyngeal ganglion(SG)of Antheraea pernyi(Lepidoptera: Saturniidae)and Bombyx mori(Lepidoptera: Bombycidae)were used as experimental materials combined transcriptome,endocrinology,and real-time quantitative PCR technology.The main results are as follows:1.In this study,we performed comparative transcriptome analysis of the PG,the target organ of KK-42,in order to explore the differences in gene expression between two different diapause types of insects A.pernyi(pupae diapause)and B.mori(egg diapause)larvae.The results showed that the gene expression profiles at the transcriptome level were highly similar in the prothymus of the two insect species.Almost all ecdysone biosynthesis related genes have relatively abundant expression levels in both two PGs.Moreover,two 20 E receptor genes,eleven JH related genes,and 13 genes involved in circadian clock were also represented in the two PGs.Quantitative real time-PCR(q RT-PCR)validated the expression of 8 juvenile hormone and 12 clock related genes in B.mori PG,with stage-specific expression patterns.This chapter confirms that different diapause types of A.pernyi and B.mori in the larvae PGs has the high similarity of gene expression and demonstrates the existence of a biological clock in the PG of insects at the molecular level.2.To explore the intrinsic link between PG and CA,we performed a comparative transcriptomic analysis of these two important endocrine organs in B.mori.The results showed that gene for Torso,the receptor of prothoracicotropic hormone(PTTH),is present in the PG,but not in the CA.Transcriptome comparison and q RT-PCR indicated that 11 genes related to JH biosynthesis and regulation and 6 genes for 20 E are expressed in both the CA and PG,suggesting that the two organs may cross talk with each other through these genes.The temporal expression profiles of the two genes for the multifunctional neurohormonal factor sericotropin precursor and the uncharacterized protein LOC114249572,the most abundant in the CA and PG transcriptomes respectively,suggested that they might play important roles in the JH and 20 E biosynthesis.This chapter shows the overall gene expression of the PG and the CA for the first time,and provids evidence that some 20 E and JH biosynthesis related genes are expressed in both two organs.Besides,this study provides the first transcriptome data for the isolated CA of insects.3.To investigate the physiological function of the PG during the diapause stage of insect pupae,we performed a comparative transcriptome analysis of the PG of the diapause pupae and post-diapause pupae of A.pernyi.The morphology of the PG of A.pernyi pupae did not change significantly during the whole diapause period(milky white with loose intercellular structure but still connected in clusters).After the diapause was ended,the volume of the PG gradually increased(milky white,but the cells were not in clusters)and finally dissolved and disappeared.For comparative transcriptome analysis,the overall genes expression had no significant difference in the PG between diapause and post-diapause pupae.KEGG pathway analysis showed that genes related to cell activity were obviously active during the transition from diapause to post-diapause development,which was consistent with the process of PG volume gradually increasing and then gradually dissociating and disappearing in morphological observation.With the transcriptomic sequencing and q RT-PCR analysis,the expression levels of five Halloween genes(nvd,spo,phm,dib and sad)began to increase after diapause termination of A.pernyi pupae,indicating that the PG cells were still active and maintained the function of synthesizing 20 E in the diapause stage of A.pernyi pupae.The expression levels of clock related genes(Period,Timeless,Vrille,HLF [hepatic leukemia factor])in the PG were also significantly up-regulated after diapause pupae of A.pernyi,suggesting that the PG may be a clock receptor to regulate the occurrence and dissolution of diapause of A.pernyi pupae.This chapter shows the overall gene expression of the PG in the diapause,and confirms that the PG still maintain the function of synthesizing 20 E during pupal diapause.4.In order to explore the inhibitory pathway of KK-42 on the synthesis of 20 E,we carried out the in vitro reaction test of KK-42 and 20 E,and then detected the titer change of 20 E in the hemolymph of A.pernyi pupae treated with KK-42.The in vitro test found that KK-42 and20 E can react directly,and they can combine with each other to offset substance content.The in vivo test found that the titer gradually decreased with the development of the pupae after KK-42 injection into the A.pernyi pupae.KK-42 treatment led to a significant decrease in the20 E titer in vivo and the phenotypically delayed the release of pupal diapause.Then 20 E titer increased with the pupal development.These results suggested that KK-42 may regulate the growth and development of A.pernyi by inactivating 20 E.This chapter confirms that KK-42 can react with 20 E directly in vitro for the first time,clarifies the metabolic kinetics of KK-42 in A.pernyi pupae,and shows the relationship between KK-42 and 20 E in vivo.5.To investigate the effect of KK-42 on the expression of 20 E related genes in the larval stage,this study compared the overall gene expression changes in B.mori larval PG between the KK-42 treatment group(trimolter silkworm)and the control group(fourth molting silkworm).Comparative transcriptome data showed that among the 17,335 genes annotated,3,650 differentially espressed genes were identified,of which 1,963 genes were up-regulated and 1,687 genes were down-regulated.KK-42 directly induced down-regulated expression of ecdysone biosynthesis related genes(nvd,spo,phm,dib,sad,Cyp6u1),induced 6 JH related genes up-regulation(Hydroxymethylglutaryl-Co A synthase,Hydroxymethylglutaryl-Co A reductase,Mevalonate kinase,Phosphomevalonate kinase,Diphosphomevalonate decarboxylase,Short-chain dehydrogenase)and 4 JH related genes down-regulated expression(Juvenile hormone epoxide hydrolase,Citrate(si)-synthase,ATP citrate lyase,Adenosine kinase).KK-42 also induced the upregulation expression of almost all circadian clock-related genes.This chapter confirms that KK-42 also directly inhibits the expression of20 E biosynthesis related genes.6.The study examined the effect of KK-42 on the development of A.pernyi eggs and compared the overall gene expression changes between the KK-42 treatment group and the control group,in order to explore the effect of KK-42 on the overall gene expression during embryonic development.The result showed that,2μg KK-42 applied to A.pernyi eggs during the appendage stage can induce embryo hatching 1 day earlier.Among 17,898 genes annotated by transcriptome sequencing,4,171 differentially expressed genes were detected,including 1,809 up-regulated genes and 2,362 down-regulated genes.However,neither transcriptome sequencing nor q RT-PCR detected the expression of 20 E biosynthesis related gene(Halloween gene).The results showed that KK-42-induced precocious metamorphosis of A.pernyi eggs by directly binding to ecdysone,rather than by inhibiting the expression of 20 E biosynthesis related genes.This chapter shows that KK-42 can accelerate the development of A.pernyi eggs,and the gene related to 20 E biosynthesis was not expressed or had a very low expression in A.pernyi eggs.7.In order to explore the link between KK-42 and the DH gene that controls B.mori egg diapause,the study examined the response expression of the DH gene in SG after KK-42 treatment.The expression level of the DH gene in the trimolter silkworm were significantly higher than that in normal silkworm(fourth molting silkworm)at the same period.In the B.mori and A.pernyi pupae the expression levels of the DH gene were also significantly higher than those in the control groups.These results suggested that KK-42 can induce up-regulation of the DH gene expression.This study is the first time to establish the link between KK-42 and the DH gene in insects.8.In this study,based on the findings mentioned above and observed in the previous studies,a new regulation mechanism of KK-42 on growth and development of insects was founded.When the exogenous KK-42 was applied to insects,(i)it can lower the titre of 20 E by directly reacting with 20 E at the hormone level,or(ii)it can lower the titre of 20 E by inhibiting the expression of 20 E biosynthesis related genes at the gene level.When the exogenous KK-42 was metabolized by the insects,the PG can recover the ability to the biosynthesis of 20 E that will influence the growth and development of insects.Meanwhile,(iii)KK-42 can directly or indirectly influence(up-regulate or downregulate)the expression of JH biosynthesis and regulation related genes and the DH gene in other endocrine glands and nerve tissue that will also influence the growth and development of insects.