Cloning And Functional Analysis Of Halloween Gene And Receptor In Diaphorina Citri Kuwayam (Hemiptera:Liviidae)

The citrus industry has been severely impacted by huanglongbing,and Diphorina citri is the disease’s main vector.Nowadays,chemical methods are the mainstay of D.citri control.Major environmental problems（including resistance and residue）have resulted from the extensive and extended use of chemical pesticides.Ecdysteroids（20-hydroxyecdysone,20E）play a crucial role in regulating insect growth and reproduction.One of the essential hormones for insects to function normally,allowing them to participate in physiological processes and control the growth of female ovaries.Ecdysteroids,which are generated by the Halloween gene family,require the binding of the ultraspiracle protein（USP）and the ecdysteroid receptor（Ec R）in order to have any effect.Moreover,there may be a connection between insect medication resistance and the genes for the Halloween gene family,ecdysone receptors,and ultraspiracle protein.In this study,four Halloween genes and the complete CDS sequences of the ecdysone receptor in the ecdysone synthesis pathway were successfully cloned by PCR and RACE techniques.The main functions of four Halloween genes and ecdysone receptors were investigated by RNA interference.The expression of Halloween and its receptor genes in different pesticides was clarified,and the susceptibility of D.citri to pesticides was investigated by knocking down the overexpressed genes.The main results are as follows:（1）Four D.citri Halloween genes’full c DNA sequences were cloned in this study,together with complete CDS sequences of Ec R and USP.The Dc CYP307A1 whole c DNA sequence measured 1748 bp,and its open reading frame（ORF）,which codes for466 amino acids,was 1491 bp long;the full length of Dc CYP302A1 was 2020 bp,the ORF was 1458 bp,encoding 485 amino acid residues;the Dc CYP315A1 gene’s c DNA sequence was 2220 base pairs long overall,with an ORF of 1497 base pairs that codes for 498 amino acids;the Dc CYP314A1 gene’s ORF,which was 1445 bp long and codes for 529 amino acids,had a full-length c DNA sequence of 1863 bp.The P450characteristic domains PFxx Gx Rx Cx G/A,P/G rich domains,Helix-C,Helix-I,Helix-K,and PERF were present in the four Halloween genes.The CDS sequence length of Dc Ec R was 1214 bp,the ORF was 930 bp,encoding 309 amino acids;the Dc USP gene’s ORF,which codes for 394 amino acids,had a length of 1185 bp and a CDs sequence length of 1355 bp.The usual DNA binding domain（DBD）and ligand binding domain（LBD）are present in both Dc Ec R and Dc USP.（2）To clarify the m RNA spatiotemporal and tissue specific expression profiles of the four key genes and receptors involved in the ecdysone synthesis pathway of D.citri.According to the q PCR data,as pest aged,their expression levels of Dc CYP307A1 and Dc CYP315A1 gradually increased;Dc CYP302A1 transcription peaked during the egg stage and the adult stage,respectively;when compared to Dc CYP307A1 and Dc CYP315A1,Dc CYP314A1 had the opposite transcription;the Dc Ec R transcription pattern revealed a general increasing trend,indicating that as D.citri continued to grow and mature,the transcription level of Dc Ec R gradually rose until it eventually peaked in the adult stage.The fifth instar nymph stage was when Dc USP’s transcription peaked,whereas the adult stage clearly exhibited downregulation.Moreover,mature female D.citri often expressed Halloween and its receptor genes at higher levels than males.According to tissue-specific expression,both the gene for Halloween and its receptor were expressed in the primary tissues of D.citri.In the main tissues,Dc CYP307A1 had a high transcription level in the male testis;The Dc CYP302A1 had a significantly high transcriptional level in female ovaries;The transcription level of Dc CYP315A1 in the ovary is the highest among all tissues,which was significantly higher than that in the male testis;The expression level of CYP314A1 in the intestine of D.citri were the highest,and the expression level in the male testis is significantly lower than that in the female ovary;The transcription level of receptor Dc Ec R was the highest in the head and the lowest in the wing;The specific expression pattern of Dc USP in various tissues is similar to that of Dc Ec R,with the highest expression level in the head,followed by the ovary.（3）The role of Halloween genes and nuclear receptors the in the growth and reproduction of D.citri was investigated using RNA interference technology.Any one of the four Halloween genes,when inhibited,significantly reduces the amount of 20E in nymphs.More seriously,D.citri development was negatively impacted by the reduction of the expression of Halloween and its receptor gene,and related measures,including the daily survival rate（Dc CYP307A1,Dc CYP302A1,Dc CYP315A1,Dc CYP314A1,Dc Ec R,and Dc USP were 27.43%,47.98%,49.02%,50.94%,43.28%,and 41.19%of the control,respectively）and the daily nymph eclosion rate（Dc CYP307A1,Dc CYP302A1,Dc CYP315A1,Dc CYP314A1,Dc Ec R,and Dc USP were 50.37%,33.74%,46.36%,53.28%,36.44%,and 47.17%of the control,respectively）,were drastically decreased.The adult ovary developed slowly,vitellogenin expression was down-regulated,and the number of eggs decreased after the expression of Halloween and its receptor gene was repressed,the male was largely unaffected.After injection of ds Ec R,wing deformities of adult D.citri were caused;the nymphs shrank and died when Dc USP expression was suppressed.（4）Halloween genes and nuclear receptor genes expression in response to thiamethoxam,cypermethrin,and pyriproxyfen exposure,as well as the impact of double-stranded RNA（ds RNA）in combination with the three different types of pesticides on D.citri survival rates.The outcomes demonstrated that the expression of Dc CYP315A1 and Dc CYP314A1 was overexpressed,whereas Dc CYP307A1,Dc CYP302A1,Dc Ec R,and Dc USP were suppressed when D.citri nymphs were subjected to three doses of thiamethoxam(LC₁₀,LC₃₀,and LC₅₀);cypermethrin can promote the overexpression of the Halloween genes and nuclear receptors at three concentrations;the nuclear receptors and Halloween genes expression were both dramatically decreased by pyriproxyfen;adults exposed to LC₃₀ doses of thiamethoxam only showed overexpression of Dc CYP315A1 and Dc CYP314A1,while genes expression were significantly inhibited at the other two doses;LC₁₀ doses of cypermethrin could stimulate the expression of Dc CYP315A1 and Dc USP,whereas under LC₃₀ and LC₅₀ conditions,Dc CYP314A1 would be overexpressed;D.citri’susceptibility to insecticides could be significantly increased by interfering with the expression of Dc CYP315A1,Dc CYP314A1,Dc Ec R,and Dc USP.This could result in a large rise in mortality.The four Halloween genes’full c DNA sequences,as well as the spatiotemporal and tissue-specific expression patterns of these four genes,ecdysone receptor,and ultraspiracle protein,were all obtained for this study.The roles of the Halloween gene and its receptor in the growth and reproduction of D.citri have been clarified.D.citri development would be impacted,and the normal operation of adult ovaries would be hampered by the inhibition of the expression of any gene.In addition,Dc CYP315A1,Dc CYP314A1,Dc Ec R,and Dc USP were upregulated to varying degrees in different pesticides.Knocking down the expression of these four genes would increase the susceptibility of D.citri to pesticides,suggesting that they may be involved in the detoxification and metabolism processes of insects and providing a theoretical basis for the prevention and control of D.citri.