Vitellogenesis is pre-requisite to insect egg production and embryonic development after oviposition. During insect vitellogenesis, the yolk protein precursor vitellogenin (Vg) is mainly synthesized in the fat body, transported by the hemolymph through the intercellular spaces (known as patency) in the follicular epithelium to reach the membrane of maturing oocytes, and sequestered into the maturing oocytes via receptor-mediated endocytosis. Insect vitellogenesis is governed by two critical hormones, the sesquiterpenoid juvenile hormone (JH) and the ecdysteriod 20-hydroxyecdysone (20E). JH acts as the principal gonadotropic hormone to stimulate vitellogenesis in basal hemimetabolous and most holometabolous insects. 20E is critical for vitellogenesis in some hymenopterans, lepidopterans and dipterans. Furthermore, microRNA (miRNA) and nutritional (amino acid/Target of Rapamycin and insulin) pathways interplay with JH and 20E signaling cascades to control insect vitellogenesis. Revealing the regulatory mechanisms underlying insect vitellogenesis is critical for understanding insect reproduction and helpful for developing new strategies of insect pest control. Here, we outline the recent research progress in the molecular action of gonadotropic JH and 20E along with the role of miRNA and nutritional sensor in regulating insect vitellogenesis. We highlight the advancements in the regulatory mechanisms of insect vitellogenesis by the coordination of hormone, miRNA and nutritional signaling pathways.

Krüppel-homolog 1 (Kr-h1), a zinc finger transcription factor, transduces juvenile hormone (JH) signaling in inhibiting larval metamorphosis and promoting adult reproduction. While the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. By using the migratory locust, Locusta migratoria as a model system, we report here that microRNA let-7 and miR-278 bound to Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 agomiRs significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus discovers a previously unknown mechanism by which JH suppresses the expression of let-7 and miR-278, which, together with JH induction on Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. The results advance our understanding in the coordination of JH and miRNA regulation in insect development.

Vitellogenin receptor (VgR) plays a pivotal role in ovarian vitellogenin (Vg) uptake and vertical transmission of pathogenic microbes and Wolbachia symbionts. However, the regulatory mechanisms of VgR action as an endocytic receptor and translocation from oocyte cytoplasm to the membrane remain poorly understood. Here, by using the migratory locust Locusta migratoria as a model system, we report that juvenile hormone (JH) promotes VgR phosphorylation at Ser¹³⁶¹ in the second EGF-precursor homology domain. A signaling cascade including GPCR, PLC, extracellular calcium, and PKC-ι is involved in JH-stimulated VgR phosphorylation. This posttranslational regulation is a prerequisite for VgR binding to Vg on the external surface of the oocyte membrane and subsequent VgR/Vg endocytosis. Acidification, a condition in endosomes, induces VgR dephosphorylation along with the dissociation of Vg from VgR. Phosphorylation modification is also required for VgR recycling from oocyte cytoplasm to the membrane. Additionally, VgR phosphorylation and its requirement for Vg uptake and VgR recycling are evolutionarily conserved in other representative insects including the cockroach Periplaneta americana and the cotton bollworm Helicoverpa armigera. This study fills an important knowledge gap of low-density lipoprotein receptors in posttranslational regulation, endocytosis, and intracellular recycling. Significance During vitellogenesis of oviparous animals, vitellogenin receptor (VgR)–mediated endocytosis is crucial for the incorporation of yolk proteins into oocytes. VgR also serves as a gatekeeper for vertical transmission of pathogenic microbes and symbionts. Juvenile hormone (JH) acts as a gonadotrophic hormone and stimulates this process in many insect species, but the regulatory mechanisms remain elusive. We find that JH activates the GPCR-PLC-PKC-ι cascade and phosphorylates VgR in an EGF-precursor homology domain. Endosomal acidification leads to VgR dephosphorylation. Phosphorylation is essential for VgR to translocate from oocyte cytoplasm to the membrane to bind with vitellogenin on the membrane and to internalize vitellogenin in oocytes. Moreover, JH-promoted VgR phosphorylation for intracellular recycling and vitellogenin deposition is evolutionarily conserved across divergent insect orders.

Juvenile hormone (JH) represses insect metamorphosis and stimulates reproduction. JH titers are generally low in juveniles, drop to a nadir during metamorphosis, increase after eclosion and peak in vitellogenic phase. We found that Jhamt, a rate-limiting enzyme in JH biosynthesis, mirrors JH titer patterns in the migratory locust. Knocking down Jhamt reduced JH titers, led to precocious nymphal ecdysis, metamorphosis and impaired vitellogenesis. Jhamt is negatively regulated by miR-276 and positively by miR-182013-5p. miR-276 is abundant in late nymphal but low in adults, while miR-182013-5p shows the opposite pattern. In nymphs, miR-276 binds more to Jhamt, while in adults, miR-182013-5p dominates. Functionally, miR-276 reduced Jhamt and JH levels, shortening nymphal development and inhibiting Vg expression. Conversely, miR-182013-5p increased Jhamt and JH levels, prolonging nymphal development and enhancing Vg expression. Our findings identify miR-276 and miR-182013-5p as dual regulators in JH biosynthesis, acting as “brake” and “accelerator,” respectively. This study provides new insights into JH titer fluctuations and miRNA regulation in insect metamorphosis and reproduction.

Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JH-mediated polyploidization, vitellogenesis, and oogenesis.