The synthesis of vitellogenin and its uptake by maturing oocytes during egg maturation are essential for successful female reproduction. These events are regulated by the juvenile hormones and ecdysteroids and by the nutritional signaling pathway regulated by neuropeptides. Juvenile hormones act as gonadotropins, regulating vitellogenesis in most insects, but ecdysteroids control this process in Diptera and some Hymenoptera and Lepidoptera. The complex crosstalk between the juvenile hormones, ecdysteroids, and nutritional signaling pathways differs distinctly depending on the reproductive strategies adopted by various insects. Molecular studies within the past decade have revealed much about the relationships among, and the role of, these pathways with respect to regulation of insect reproduction. Here, we review the role of juvenile hormones, ecdysteroids, and nutritional signaling, along with that of microRNAs, in regulating female insect reproduction at the molecular level.

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACG C

Mosquitoes transmit devastating human diseases because they need vertebrate blood for egg development. Metabolism in female mosquitoes is tightly coupled with blood meal-mediated reproduction, which requires an extremely high level of energy consumption. Functional analysis has shown that major genes encoding for enzymes involved in lipid metabolism (LM) in the mosquito fat bodies are down-regulated at the end of the juvenile hormone (JH)-controlled posteclosion (PE) phase but exhibit significant elevation in their transcript levels during the post-blood meal phase (PBM), which is regulated mainly by 20-hydroxyecdysone (20E). Reductions in the transcript levels of genes encoding triacylglycerol (TAG) catabolism and β-oxidation enzymes were observed to correlate with a dramatic accumulation of lipids in the PE phase; in contrast, these transcripts were elevated significantly and lipid stores were diminished during the PBM phase. The RNAi depletion of Methoprene-tolerant (Met) and ecdysone receptor (EcR), receptors for JH and 20E, respectively, reversed the LM gene expression and the levels of lipid stores and metabolites, demonstrating the critical roles of these hormones in LM regulation. Hepatocyte nuclear factor 4 (HNF4) RNAi-silenced mosquitoes exhibited down-regulation of the gene transcripts encoding TAG catabolism and β-oxidation enzymes and an inability to use lipids effectively, as manifested by TAG accumulation. The luciferase reporter assay showed direct regulation of LM-related genes by HNF4. Moreover, gene expression was down-regulated by Met and activated by EcR and Target of rapamycin, providing a link between nutritional and hormonal regulation of LM in female mosquitoes.

Hematophagous mosquitoes serve as vectors of multiple devastating human diseases, and many unique physiological features contribute to the incredible evolutionary success of these insects. These functions place high-energy demands on a reproducing female mosquito, and carbohydrate metabolism (CM) must be synchronized with these needs. Functional analysis of metabolic gene profiling showed that major CM pathways, including glycolysis, glycogen and sugar metabolism, and citrate cycle, are dramatically repressed at post eclosion (PE) stage in mosquito fat body followed by a sharply increase at post-blood meal (PBM) stage, which were also verified by Real-time RT-PCR. Consistent to the change of transcript and protein level of CM genes, the level of glycogen, glucose and trehalose and other secondary metabolites are also periodically accumulated and degraded during the reproductive cycle respectively. Levels of triacylglycerols (TAG), which represent another important energy storage form in the mosquito fat body, followed a similar tendency. On the other hand, ATP, which is generated by catabolism of these secondary metabolites, showed an opposite trend. Additionally, we used RNA interference studies for the juvenile hormone and ecdysone receptors, Met and EcR, coupled with transcriptomics and metabolomics analyses to show that these hormone receptors function as major regulatory switches coordinating CM with the differing energy requirements of the female mosquito throughout its reproductive cycle. Our study demonstrates how, by metabolic reprogramming, a multicellular organism adapts to drastic and rapid functional changes.

Juvenile hormone (JH) governs a great diversity of processes in insect development and reproduction. It plays a critical role in controlling the gonadotrophic cycles of female mosquitoes by preparing tissues for blood digestion and egg development. Here, we show that in female Aedes aegypti mosquitoes JH III control of gene expression is mediated by a heterodimer of two bHLH-PAS proteins-the JH receptor methoprene-tolerant (MET) and Cycle (CYC, AAEL002049). We identified Aedes CYC as a MET-interacting protein using yeast two-hybrid screening. Binding of CYC and MET required the presence of JH III. In newly eclosed female mosquitoes, the expression of two JH-responsive genes, Kr-h1 and Hairy, was dependent on both the ratio of light to dark periods and JH III. Their expression was compromised by in vivo RNA interference (RNAi) depletions of CYC, MET, and the steroid receptor coactivator SRC/FISC. Moreover, JH III was not effective in induction of Krh1 and Hairy gene expression in vitro in fat bodies of female mosquitoes with RNAi-depleted CYC, MET or SRC/FISC. A sequence containing an E-box-like motif from the Aedes Kr-h1 gene promoter specifically interacted with a protein complex, which included MET and CYC from the female mosquito fat body nuclear extract. These results indicate that a MET/CYC heterodimer mediates JH III activation of Kr-h1 and Hairy genes in the context of light-dependent circadian regulation in female mosquitoes during posteclosion development. This study provides an important insight into the understanding of the molecular basis of JH action.hormone receptor | insect hormone | transcriptional control M osquitoes serve as vectors of harmful human diseases because blood feeding is required for their egg development; disease pathogens use hematophagous female mosquitoes for obligatory stages of their life cycles. An insect-specific sesquiterpenoid juvenile hormone III (JH III) is essential for a newly eclosed female mosquito to reach a competence stage for blood feeding and egg maturation (1, 2). Although several aspects of the JH III-dependent development have been characterized (3-6), our understanding of molecular mechanisms underlying JH regulation of female mosquito posteclosion development is limited.Methoprene-tolerant (MET), which is a basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) protein, was discovered in Drosophila melanogaster (7), and its role in mediating a JH response has been established (8). MET binds to JH with a high affinity, suggesting that it is the JH receptor (9, 10). As a bHLH protein, MET requires either a homo-or heterodimer partner for its activity (11). Studies in Aedes aegypti, the beetle Tribolium castaneum, and the silkworm Bombyx mori have shown a bHLH-PAS domain-containing steroid receptor coactivator (SRC/FISC/ Taiman) to interact with MET (10,[12][13][14]. Whether, an additional bHLH transcription factor with DNA-binding properties is required as a Met partner remains to be established.Using yeast two-hybrid (Y2H) screening, we identified an Aedes ortholo...