Methoprene-tolerant (Met) protein is a juvenile hormone (JH) receptor in insects. JH-bound Met forms a complex with the βFtz-F1-interacting steroid receptor coactivator (FISC) and together they regulate JH response genes in mosquitoes. Both proteins contain basic-helix-loop-helix (bHLH) and PAS motifs. Here we demonstrated that FISC is the obligatory partner of Met for binding to JH-response elements (JHREs). Met or FISC alone could not bind a previously characterized JHRE, while formation of the Met-FISC complex was necessary and sufficient to bind to the JHRE. This binding required participation of the DNA-binding domains of both Met and FISC. The optimal DNA sequence recognized by Met and FISC contained a core consensus sequence GCACGTG. While formation of the Met-FISC complex in mosquito cells was induced by JH, heterodimerization and DNA binding of bacterially expressed Met and FISC were JH-independent, implying that additional mosquito proteins were required to modulate formation of the receptor complex.
Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JHregulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membraneinitiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH.insect hormone | development | phospholipase C | protein kinase | transcription J uvenile hormones (JH) are a group of acyclic sesquiterpenoids produced in insects by the corpora allata, a pair of endocrine glands connected to the brain (1). They are important regulators in a wide variety of developmental and physiological events in insects, including development, reproduction, caste determination, behavior, diapauses, polyphenisms, and longevity (2-4).Many effects of JH are mediated by the methoprene-tolerant (MET) protein, an intracellular JH receptor (5). MET contains a basic helix-loop-helix (bHLH) DNA-recognition motif near the N terminus, followed by two tandem Per-ARNT-Sim (PAS) domains, PAS-A and PAS-B (6). In vitro studies have demonstrated that JH-III binds MET with relatively high affinity and have identified a putative JH-binding pocket in the PAS-B domain of MET (7,8). In the presence of JH, MET forms a heterodimer with a p160 steroid receptor coactivator (SRC), which also contains the bHLH-PAS domain (7, 9). The orthologs of SRC are called "Taiman" (TAI) in Drosophila melanogaster and "Ftz-F1-interacting steroid receptor coactivator" (FISC) in the yellow fever mosquito Aedes aegypti (10, 11). For simplicity, we will use a single name, Taiman, to describe all its orthologs in insects. TAI acts as the DNA-binding partner of MET; the MET-TAI complex recogni...
When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.
Krüppel homologue 1 (Kr-h1) is a zinc finger transcription factor that is upregulated in insects by juvenile hormone (JH) in metamorphosis and adult reproduction. The molecular function of Kr-h1 in reproduction remains largely unknown. Here we report that AaKr-h1 functions as an important transcription regulator in adult female Aedes aegypti mosquitoes. The amount of AaKr-h1 protein increases with rising JH levels after adult emergence, reaches its peak at 48 h after eclosion, then decreases gradually and disappears after blood feeding. RNA interference (RNAi)-mediated depletion of AaKr-h1 substantially reduced egg production after blood feeding. Using a chromatin immunoprecipitation cloning approach, we identified in vivo AaKr-h1 binding sites in previtellogenic female mosquitoes. Binding of AaKr-h1 to the target genes correlated with its protein abundance. Interestingly, RNAi experiments indicated that AaKr-h1 played distinct roles when it bound to individual target genes. For example, depletion of AaKr-h1 led to substantial upregulation of AAEL005545 and AAEL004444, but also significantly decreased the expression of AAEL005957 and AAEL013177 when compared with the control mosquitoes. In summary, AaKr-h1 directly binds to the regulatory regions of its target genes and acts as a transcriptional activator or a repressor in a promoter-specific manner.
Most animals employ aggressive behaviours to acquire resources such as food, territory and mates. Although mating is important for males, which typically exhibit competitive behaviours to gain mating opportunities, they generally tend to avoid conflict escalation; while extreme combat also occurs in some species and results in death. In this study, male-male lethal combat behaviour in Anastatus disparis was examined (Hymenoptera: Eupelmidae) by investigating the characteristics of fighting and the factors that influence fighting intensity in this species. Male fight intensity in A. disparis increased with both competitor density and female presence, while it was not influenced by the relatedness among male competitors. By comparing the frequency of received attacks between injured and non-injured males, we found that the former were more vulnerable to attack. In contrast to death due to lethal attack, death that occurs as a result of A. disparis combat may be the cumulative effect of injuries sustained over repeated competitive encounters. Combined with the biological characters of A. disparis, we discuss potential factors contributing to the evolution of fatal conflict in this species.
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