The Molecular Mechanism Of Ecdysteroid Receptor Involved In Tick Salivary Gland Degeneration

Ticks are obligate hematophagous arthropods and live on the surface of the host.On a global scale,they represent the most important vectors of pathogens that affect animals,and are second only to mosquitoes where humans are concerned.Tick salivary glands are an important organ for ticks to suck blood and transmit pathogens.It is shown that tick salivary glands degenerate sharply in the short term after blood sucking and ecdysone(20E)plays a very important role in the degeneration of salivary glands.However,the molecular mechanism of how 20 E regulates the degeneration of female tick salivary glands is unknown.In this study,female ticks of Rhipicephalus haemaphysaloides were selected as the research object and ecdysone receptor as the research target.It focused on the process of female tick salivary gland degeneration.It could provide a theoretical basis for novel tick prevention and control strategies thatelucidating the molecular mechanism of ecdysone receptor in the process of salivary gland degeneration in female ticks.The main research results of this paper are as follows.1.Cloning and identification of ecdysone receptor gene in Rhipicephalus haemaphysaloidesA salivary gland transcriptome produced by our laboratory was used to identify the open reading frame(ORF)sequences of the three isoforms of Rh Ec R and the two isoforms of Rh USP.Subsequently,the ORF regions of three Rh Ec R isoforms and two Rh USP isoforms were cloned from the c DNA of the salivary gland from fully engorged female R.haemaphysaloides and named Rh Ec R1,Rh Ec R2,Rh Ec R3,Rh USP1,and Rh USP3,respectively.The ORFs of Rh Ec R1,Rh Ec R2,and Rh Ec R3 have 1680 bp,1362 bp,and 1260 bp,with deduced molecular weights(MW)and theoretical isoelectric points(PI)of 61.3k Da /8.39,51.2 k Da /7.80 and 47.2 k Da/8.48,respectively.The ORFs of Rh USP1 and Rh USP3 had 1308 bp and 1200 bp,with deduced molecular weights(MW)and the theoretical isoelectric points(PI)of 48.2k Da/8.93 and 44.4 k Da/8.60,respectively.The sequences had typical structural characteristics of nuclear receptors including a variable N-terminal domain(A/B),a highly conserved DBD(C),a variable hinge region(D),and a moderately conserved LBD(E).Rh Ec Rs and Rh USPs had a conserved DBD that had two conserved C4＿Zn F motifs characterized by four cysteine residues and one conserved LBD.Similar to other nuclear receptors,they also had one variable N-terminal A/B domain,a highly conserved DBD(C),a variable hinge region(D),one variable hinge region(E)and a moderately conserved LBD,but not the variable C-terminal region.They all had sequence similarity to various insect,other arthropods and were highly conserved in different tick species.2.Expression characteristics of ecdysone receptor and its effect on tick bloodsuckingTranscriptional expression of Rh Ec R/Rh USP was found in various tissues and organs of female ticks,such as salivary glands,intestine,ovary,adipose body and submandibular ganglion.After satiation,Rh Ec R/Rh USP transcriptional expression in salivary glands,intestines and ovaries was significantly higher than that in adipose bodies and submandibular ganglia.The transcriptional expression of Rh Ec R/Rh USP reaches its peak at the stage of high-speed degeneration,while the transcriptional expression of Rh Ec R/Rh USP in ovaries is always at a high level.The concentration of 20 E in salivary glands also increased rapidly in the early stage of rapid blood feeding until it reached the peak on the day of full blood feeding and decreased with the progress of degeneration.However,the concentration of 20 E in the hemolymph of the environment where the salivary glands are located remained at a high level from the early stage of rapid blood feeding until the salivary glands were degraded or the ovary was fully developed.Ecdysone receptors exist widely in various tissues and organs of female ticks,but their expression characteristics are different.Knockdown of Rh Ec R/Rh USP,Rh Ec R and Rh USP genes in vivo showed that both Rh Ec R and Rh USP genes could be knocked down well.There were similar phenotypes between the knockdown of Rh Ec R/Rh USP and Rh Ec R group.Bloodsucking of female ticks was inhibit,stagnation in the early stage of rapid bloodsucking made female ticks not to spawn and lay eggs.And knockdown of Rh USP did not affect the life of female ticks.There was no abnormal blood sucking phenomenon,and eventually the ticks could develop and reproduce normally.However,morphology about the knockdown of Rh Ec R group showed that the differentiation and remodeling of salivary gland tissue were inhibited,that is,the degeneration of salivary gland was blocked.3.The role of ecdysone receptor in tick salivary gland degenerationWhen the transcription level of Rh Ec R was significantly reduced by 54%,Caspases on the apoptosis pathway were also significantly reduced.It was displayed that the Rhcaspase7,8 and 9 transcriptional expression levels in the saliary glands were dropped down to 12.90%,34.17% and 8.70%with respect to the control group,respectively.Rhcaspases were significantly down-regulated and cell apoptosis process,which is caspase-dependent,was greatly affected.Because of the importance of programmed cell death in tissue remodeling degeneration,the salivary glands did not degenerate normally.In transcriptome and proteomics,the expression of related factors in apoptosis pathway,such as Rh Bc L2,Rh Cyt C and Rhcaspase7,was significantly inhibited when Rh Ec R was significantly knocked down in female ticks.At the same time,scanning transmission electron microscopy showed that the number of mitochondria and membrane-structured organelles in the salivary glands of Rh Ec R knockdown group were significantly reduced.All these indicated that Rh Ec R knockdown affected the internal apoptosis process in the process of salivary gland degeneration.4.Ecdysone receptor mediates salivary glands degeneration by regulating the transcription factor Rh FOXORh FOXO,a transcription factor regulating apoptosis,was also significantly reduced when Rh Ec R was knockdown significantly knocked down.The open reading frame of Rh FOXO was cloned and only one Rh FOXO was cloned according to the transcriptome database of the salivary glands of R.haemaphysaloides,which was different from the multiple FOXO genes in mammals but consistent with the theory that there is only one FOXO gene in arthropods.After the knockdown of Rh FOXO gene in vivo,blood sucking was slow,satiety time was significantly delayed,satiety weight was significantly increased,oviposition rate and hatching rate were significantly decreased.Morphology of the salivary glands showed that the differentiation and remodeling of salivary gland tissue were obviously inhibited,that is,the degeneration of salivary gland was blocked.Meanwhile,Rh PI3 K,Rh PDK1,Rh AKT,Rh Bc L2,Rh Cyt C and Rh Caspase7 decreased significantly in the early stage of degeneration.In normal degeneration stage,Rh Ec R,Rh USP,Rh PI3 K,Rh PDK1,Rh AKT and Rh Cyt C suddenly and significantly increased while Rh Caspase9 significantly decreased,suggesting that Rh Ec R regulates the internal apoptosis process by transcriptional regulation of Rh FOXO.However,due to the lack of tick-derived cell lines and relevant specific antibodies,Rh Ec R could only be inferred to regulate the internal apoptosis mediated by Rh FOXO transcription factor in salivary gland degeneration,but direct or indirect regulation is unknown.5.Interaction between Rh Ec Rs and Rh USPs in vitroDue to the lack of tick-derived cell lines and associated specific antibodies,Rh Ec Rs and Rh USPs interactions could not be verified in ticks.Based on other arthropod-related studies,the interaction between Rh Ec Rs and Rh USPs was verified in vitro in mammalian cell line HEK-293 T.It was found that the isomers of Rh Ec R could interact with the isomers of Rh USP.Single overexpression showed that the long-term expression of Rh Ec R isoforms could cause cell death,but the long-term expression of Rh USP isoforms could not cause cell death.However,co-overexpression results showed that the combination of Rh Ec R1 and Rh USP1 could promote cell death within 24 h after transfection through caspase-dependent apoptosis for a short time.In conclusion,five ecdysone receptor related molecules,including three Rh Ec R molecules and two Rh USP molecules,were cloned and identified in this study.Ecdysone receptors exist widely in various tissues and organs of female ticks,but their expression characteristics are different.Rh Ec R is necessary for the normal development and reproduction of female ticks.After the interference of Rh Ec R gene,female ticks could not satisfy blood and lay eggs.The interaction between Rh Ec R1 and Rh USP1 induced apoptosis.Rh Ec R affected salivary gland degeneration by regulating Rh FOXO-mediated internal apoptosis.These results elucidated the molecular mechanism of ecdysone receptor-mediated tick salivary gland degeneration and provided an important theoretical basis for novel prevention and treatment strategies.