The Role And Molecular Mechanism Of Juvenile Hormone Signaling Pathway Related Genes In Ovarian Development Of Pacific White Shrimp Litopenaeus Vannamei

Juvenile hormone(JH)signaling pathway plays a critical role in regulating the development and reproduction of arthropods.Methyl farnesoate(MF),a natural precursor of insect JH,has been identified as an innate JH of crustaceans.However,the molecular signaling of MF action in crustaceans is much less explored than JH signaling is in insects.A few reports have confirmed that MF can promote ovarian development in crustaceans,but its regulatory mechanism is still unclear.In this study,we perform a comprehensive transcriptomic analysis of various tissues at different ovary development stages,and screen the important regulatory pathways related to ovarian development such as juvenile hormone signaling pathway.In vitro incubation,RNA interference,yeast two hybrid and other molecular techniques are used to study the mechanisms for regulating juvenile hormone receptor expression.These results not only enrich our understanding of the molecular regulatory mechanism of juvenile hormone pathway in Litopenaeus vannamei,but also provide reference data for improving the basic theory of ovarian development in crustaceans.The main results are as follows:1.Deciphering ovarian development regulatory network of the L.vannamei through transcriptomic analysisIn this study,we performe a comprehensive transcriptomic analysis of five important organs related to ovary development,including the eyestalk ganglion,brain,thoracic ganglion,hepatopancreas,and ovary,at different ovary development stages of the Pacific white shrimp L.vannamei.A total of 48,722 unique transcripts(or unigenes)were obtained,of these 18,661 unigenes can be matched to homologous sequences in public databases.Pairwise comparisons revealed that a wide variety of genes were differentially expressed and several gene expression trends were significantly enriched in different organs during ovary development.Through weighted gene co-expression network analysis,six tissue-or stage-specific modules of co-expressed genes were identified.GO and KEGG analysis further disclosed that a rich set of genes,including those associated with the CHH family hormones,ecdysone signaling,juvenile hormone signaling,Insulin-PI3K-Akt signaling,EGFR signaling,and TGF? signaling pathways,were preferentially expressed in specific tissues,implying their crucial roles in the regulation of ovary development process.Taken together,the study reports a comprehensive analysis of transcriptomic modulation in major neuro-endocrine organs related to shrimp ovary maturation.The data obtained sheds light on the potential coordination between the nervous system and its target organs(i.e.ovary and hepatopancreas),and contributes to a deeper understanding of the regulatory network orchestrating ovary maturation process of shrimp.2.Cloning and functional analysis of juvenile hormone receptor gene(LvMet)in L.vannameiIn this study,we report the cloning and characterization ofMet from the shrimp L.vannamei.The open reading frame of LvMet was 3,165 bp in length encoding a deduced protein of 1,054 amino acids.Similar to other knownMets,the deduced protein of LvMet contained four typical domains of bHLH,PAS-A,PAS-B and PAC.Eight conserved amino acid residues related to JH or MF binding were found in PAS-B of LvMet.LvMet mRNA was widely expressed in various tissues,but the expression level was low in immature ovary and hepatopancreas and during ovarian development,its expression level was found to change significantly.In vitro experiment showed that LvMet,LvVg1 and LvVg2 mRNA in ovarian explants were significantly induced by appropriate concentrations of MF,but only induction of LvMet was observed in the hepatopancreas explants.However,differing from the in vitro experiment,injection of MF in in vivo experiment significantly stimulated the expressions of LvMet in ovary.When LvMet was silenced,the expression of LvVg1 in hepatopancreas and ovary was significantly increased and then decreased,similar to that of the LvMet in ovary.By contrast,LvVg2 in hepatopancreas was significantly decreased and then increased.Taken together,these results suggest that LvMet can potentially serve as a MF receptor,and may involve in the regulation of Vg in L.vannamei.3.Relationship between juvenile hormone receptor LvMet and retinoic acid X receptor LvRXR in ovarian development of L.vannameiThe 1,325 bp LvRXR cDNA sequence was obtained by PCR amplification.LvRXR mRNA was widely expressed in various tissues,but the expression level was lowest in immature ovary,eyestalk and hepatopancreas.The expression of LvRXR in the hepatopancreas and ovary gradually increased with the ovarian development,and reached the peak at the mature stage.Proper concentration of MF could effectively induce the expression of LvRXR,but inhibited the expression of LvRXR in ovarian explants.When LvRXR was silenced,both LvVg1 and LvVg2 were significantly upregulated and then down-regulated in both hepatopancreas and ovary.These results indicate that LvRXR expression is also regulated by MF and plays an important role in the regulation of Vg expression.When LvRXR was silenced,the trend of the relative expression of LvMet was similar to that of LvRXR.When LvMet was silenced,the expression of LvRXR in hepatopancreas was significantly up-regulated first,and then significantly down-regulated to a level lower than that of the control group(P<0.05),and the change trend of LvRXR expression in ovary was similar to that of LvMet.The yeast two-hybrid experiment further proved that there was a weak interaction between LvMet and LvRXR.These results suggest that MF may induce LvMet and LvRXR to form heterodimers,and participate in the Vg regulation of L.vannamei.4.The role of LvKr-h1,a downstream gene of juvenile hormone pathway,in the ovarian development of L.vannameiThe 2,021 bp LvKr-h1 cDNA sequence of was obtained,and the predicted ORF length was 1,833 bp,encoding 610 amino acids.Similar to the Kr-h1 s gene of other crustaceans,LvKr-h1 contained seven zinc finger structures(Zn2-Zn8).LvKr-h1 mRNA was widely distributed in various tissues,but its expression level is lowest in immature ovary,thoracic ganglia and hepatopancreas.In the intact eyestalk shrimp,the expression of LvKr-h1 gene in hepatopancreas and ovary fluctuated significantly with the ovarian development.After the eyestalk removed,the expression of LvKr-h1 in the hepatopancreas and ovary showed an overall upward trend,and reached the peak in the mature stage(UES ?),which was higher than that of the intact eyestalk shrimp(INT ?).High concentration of MF can significantly induce the expression of LvKr-h1 mRNA in hepatopancreas and ovary in vitro and in vivo(P<0.05).When LvKr-h1 was silenced,the expression of LvMet gene was significantly up-regulated,and LvRXR was significantly down-regulated at 48 h in hepatopancreas(P<0.05),while LvMet and LvRXR were significantly up-regulated and then down-regulated in ovary.In addition,LvVg1 and LvVg2 were significantly up-regulated.LvKr-h1 may be involved in the JH signaling pathway and Vg expression regulation in L.vannamei.When either LvMet or LvRXR gene was silenced,the expression of LvKr-h1 was significantly down-regulated(P<0.05).These results indicate that the transcription of LvKr-h1 is regulated by LvMet and LvRXR,and there may be exit a negative feedback regulation.These results would help to enhance the current understanding of the regulatory mechanism of MF signaling,and provide important resource for further research into the regulation of shrimp reproduction.