Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in the vitellogenesis of insects. In this study, we cloned and characterized the two corresponding genes (TpVg and TpVgR) in an economically important insect, Thitarodes pui (Lepidoptera: Hepialidae), from the Tibetan plateau. The full length of TpVg is 5566 bp with a 5373 bp open reading frame (ORF) encoding 1,790 amino acids. Sequence alignment revealed that TpVg has three conserved domains: a Vitellogenin_N domain, a DUF1943 domain, and a von Willebrand factor type D domain (VWD). The full length of TpVgR is 5732 bp, with a 5397 bp ORF encoding 1798 amino acids. BLASTP showed that TpVgR belongs to the low-density lipoprotein receptor (LDLR) gene superfamily. Structural analysis revealed that TpVgR has a group of four structural domains: a ligand-binding domain (LBD), an epidermal growth factor (EGF)–precursor homology domain, a transmembrane (TM) domain, and a cytoplasmic domain. In addition, TpVgR has four cysteine-rich LDL repeats in the first ligand-binding site and seven in the second. Quantitative real-time polymerase chain reaction analysis revealed that the expression levels of TpVg and TpVgR are much higher in later pupa than in either the larval or adult stage, implying that the synthesis and uptake of Vg in T. pui occurs in the later pupal stage. These results will help us to understand the molecular mechanism of the reproductive capacity and will provide new insight into the mass rearing and utilization of T. pui.

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Drug-reinforced excessive operant responding is one fundamental feature of longlasting addiction-like behaviors and relapse in animals. However, the transcriptional regulatory mechanisms responsible for the persistent drug-specific (not natural rewards) operant behavior are not entirely clear. In this study, we demonstrate a key role for one of the de novo DNA methyltransferase, DNMT3a, in the acquisition of morphine self-administration (SA) in rats. The expression of DNMT3a in the hippocampal CA1 region but not in the nucleus accumbens shell was significantly up-regulated after 1-and 7-day morphine SA (0.3 mg/kg/infusion) but not after the yoked morphine injection. On the other hand, saccharin SA did not affect the expression of DNMT3a or DNMT3b. DNMT inhibitor 5-aza-2-deoxycytidine (5-aza) microinjected into the hippocampal CA1 significantly attenuated the acquisition of morphine SA. Knockdown of DNMT3a also impaired the ability to acquire the morphine SA. Overall, these findings suggest that DNMT3a in the hippocampus plays an important role in the acquisition of morphine SA and may be a valid target to prevent the development of morphine addiction.

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The development of opioid addiction involves DNA methylation. Accordingly, the DNA demethylation, induced by ten-eleven translocation (Tet) enzymes, may represent a novel approach to prevent opioid addiction. The present study examined the role of TET1 and TET3 in the development of morphine-seeking behavior in rats. We showed that 1 day of morphine self-administration (SA) training upregulated TET3 but not TET1 expression in the hippocampal CA1. With 7 days of morphine SA training, the expression of TET3 in the CA1 returned to the baseline level, while the TET1 expression was downregulated. No change of TET1 and TET3 in the nucleus accumbens shell was observed in morphine SA trained rats, or in the yoked morphine rats, or in rats trained for saccharin SA. Furthermore, we found that knocking down TET3 expression in the CA1 accelerated the acquisition of morphine SA, while overexpression of the catalytic domain of TET1 in the CA1 attenuated the acquisition. Together, these findings suggest that TET1 and TET3 in the CA1 are important epigenetic modulators involved in the morphine-seeking behavior and provide a new strategy in the treatment of opioid addiction.

Impairing reconsolidation may disrupt drug memories to prevent relapse, meanwhile long-term transcription regulations in the brain regions contribute to the occurrence of emotional memories. The basolateral amygdala (BLA) is involved in the drug-cue association, while the nucleus accumbens (NAc) responds to the drug reward. Here, we assessed whether DNA methyltransferases (Dnmts) in these two brain regions function identically in the reconsolidation of morphine reward memory. We show that Dnmts inhibition in the BLA but not in the NAc after memory retrieval impaired reconsolidation of a morphine reward memory. Moreover, the mRNA levels of Dnmt3a and Dnmt3b, rather than Dnmt1, in the BLA were continuously upregulated after retrieval. We further identified the differentially methylated regions (DMRs) in genes in the BLA after retrieval, and focused on the DMRs located in gene promoter regions. Among them were three genes (Gnas, Sox10, and Pik3r1) involved in memory modulation. Furthermore, Gnas promoter hypermethylation was confirmed to be inversely correlated with the downregulation of Gnas mRNA levels. The findings indicate that the specific transcription regulation mechanism in the BLA and NAc on reconsolidation of opiate-associated memories can be dissociable, and DNA hypermethylation of Gnas in the BLA is necessary for the reconsolidation of morphine reward memories.