Juvenile hormone signal transducer hairy inhibits Krüppel homolog1 expression.

Hairy and Krüppel homolog 1 (Kr-h1) are transcriptional repressors that act synergistically to mediate the gene-repressive action of juvenile hormone (JH). However, whether a regulatory relationship exists between Hairy and Kr-h1 remains unclear. In this study, an inhibitory effect of Hairy on Kr-h1 expression was found. Genetic studies in Drosophila have shown that the simultaneous overexpression of Hairy and Kr-h1 can rescue the defective phenotypes caused by the overexpression of a single factor. Reduced expression of Kr-h1 was observed in Hairy-overexpressing flies and cells, whereas the expression levels of Hairy were unaffected in cells with ectopic expression of Kr-h1. The inhibitory effect of Hairy on Kr-h1 expression was found to occur at the transcriptional level, as Hairy bound directly to the B-box within the Kr-h1 promoter via the bHLH motif and recruited the corepressors C-terminal binding protein (CtBP) and Groucho (Gro) through the PLSLV and WRPW motifs, respectively. Our findings revealed a regulatory relationship between two JH response factors, which advances our understanding of the molecular mechanism of JH signaling.

Juvenile hormone inhibits adult cuticle formation in Drosophila melanogaster through Kr-h1/Dnmt2-mediated DNA methylation of Acp65A promoter.

Differentiation of imaginal epidermal cells of Drosophila melanogaster to form adult cuticles occurs at approximately 40-93 h after puparium formation. Juvenile hormone (JH) given at pupariation results in formation of a second pupal cuticle in the abdomen instead of the adult cuticle. Although the adult cuticle gene Acp65A has been reported to be down-regulated following JH treatment, the regulatory mechanism remains unclear. Here, we found that the JH primary response gene Krüppel homologue 1 (Kr-h1) plays a vital role in the repression of adult cuticle formation through the mediation of JH action. Overexpression of Kr-h1 mimicked-while knocking down of Kr-h1 attenuated-the inhibitory action of JH on the formation of the adult abdominal cuticle. Further, we found that Kr-h1 inhibited the transcription of Acp65A by directly binding to the consensus Kr-h1 binding site (KBS) within the Acp65A promoter region. Moreover, the DNA methyltransferase Dnmt2 was shown to interact with Kr-h1, combined with the KBS to promote the DNA methylation of sequences around the KBS, in turn inhibiting the transcription of Acp65A. This study advances our understanding of the molecular basis of the "status quo" action of JH on the Drosophila adult metamorphosis.

Burkholderia gut symbiont induces insect host fecundity by modulating Kr-h1 gene expression.

Full-length cDNAs of the Broad-Complex (BR-C) from Riptortus pedestris were cloned. Moreover, Kr-h1 and BR-C expression levels in apo-symbiotic and symbiotic host insects were compared to verify whether they are modulated by Burkholderia gut symbionts. Interestingly, Kr-h1 expression level was significantly increased in symbiotic females. To determine how Kr-h1 affects fecundity in insects, the biosynthesis of two reproduction-associated proteins, hexamerin-α and vitellogenin, was investigated in R. pedestris females. Hexamerin-α and vitellogenin expression at the transcriptional and translational levels decreased in Kr-h1-suppressed symbiotic females, subsequently reduced egg production. These results suggest that Burkholderia gut symbiont modulates Kr-h1 expression to enhance ovarian development and egg production of R. pedestris by increasing the biosynthesis of the two proteins.

Histone deacetylase 1 suppresses Krüppel homolog 1 gene expression and influences juvenile hormone action in Tribolium castaneum.

Posttranslational modifications, including acetylation and deacetylation of histones and other proteins, modulate hormone action. In Tribolium castaneum TcA cells, Trichostatin A, a histone deacetylase (HDAC) inhibitor, mimics juvenile hormone (JH) in inducing JH response genes (e.g., Kr-h1), suggesting that HDACs may be involved in JH action. To test this hypothesis, we identified genes coding for HDACs in T. castaneum and studied their function. Knockdown of 12 HDAC genes showed variable phenotypes; the most severe phenotype was detected in insects injected with double-stranded RNA targeting HDAC1 (dsHDAC1). The dsHDAC1-injected insects showed arrested growth and development and eventually died. Application of JH analogs hydroprene to T. castaneum larvae and JH III to TcA cells suppressed HDAC1 expression. Sequencing of RNA isolated from control and dsHDAC1-injected larvae identified 1,720 differentially expressed genes, of which 1,664 were up-regulated in dsHDAC1-treated insects. The acetylation levels of core histones were increased in TcA cells exposed to dsHDAC1 or JH III. ChIP assays performed using histone H2BK5ac antibodies showed an increase in acetylation in the Kr-h1 promoter region of cells exposed to JH III or dsHDAC1. Overexpression or knockdown of HDAC1, SIN3, or both resulted in a decrease or increase in Kr-h1 mRNA levels and its promoter activity, respectively. Overexpression of the JH receptor Methoprene tolerant (Met) was unable to induce Kr-h1 in the presence of HDAC1 or SIN3. These data suggest that epigenetic modifications influence JH action by modulating acetylation levels of histones and by affecting the recruitment of proteins involved in the regulation of JH response genes.

Krüppel-homolog 1 exerts anti-metamorphic and vitellogenic functions in insects via phosphorylation-mediated recruitment of specific cofactors.

BACKGROUND: The zinc-finger transcription factor Krüppel-homolog 1 (Kr-h1) exerts a dual regulatory role during insect development by preventing precocious larval/nymphal metamorphosis and in stimulating aspects of adult reproduction such as vitellogenesis. However, how Kr-h1 functions both as a transcriptional repressor in juvenile metamorphosis and an activator in adult reproduction remains elusive. Here, we use the insect Locusta migratoria to dissect the molecular mechanism by which Kr-h1 functions as activator and repressor at these distinct developmental stages. RESULTS: We report that the kinase PKCα triggers Kr-h1 phosphorylation at the amino acid residue Ser154, a step essential for its dual functions. During juvenile stage, phosphorylated Kr-h1 recruits a corepressor, C-terminal binding protein (CtBP). The complex of phosphorylated Kr-h1 and CtBP represses the transcription of Ecdysone induced protein 93F (E93) and consequently prevents the juvenile-to-adult transition. In adult insects, phosphorylated Kr-h1 recruits a coactivator, CREB-binding protein (CBP), and promotes vitellogenesis by inducing the expression of Ribosomal protein L36. Furthermore, Kr-h1 phosphorylation with the concomitant inhibition of E93 transcription is evolutionarily conserved across insect orders. CONCLUSION: Our results suggest that Kr-h1 phosphorylation is indispensable for the recruitment of transcriptional cofactors, and for its anti-metamorphic and vitellogenic actions in insects. Our data shed new light on the understanding of Kr-h1 regulation and function in JH-regulated insect metamorphosis and reproduction.

20E-mediated regulation of BmKr-h1 by BmKRP promotes oocyte maturation.

BACKGROUND: Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori. RESULTS: Developmental and tissue-specific expression analysis revealed that BmKr-h1 was highly expressed in ovaries during the late pupal and adult stages and the expression was induced by 20E. RNA interference (RNAi)-mediated depletion of BmKr-h1 in female pupae severely repressed the transcription of vitellogenin receptor (VgR), resulting in the reduction in vitellogenin (Vg) deposition in oocytes. BmKr-h1 specifically bound the Kr-h1 binding site (KBS) between - 2818 and - 2805 nt upstream of BmVgR and enhanced the transcription of BmVgR. A 20E cis-regulatory element (CRE) was identified in the promoter of BmKr-h1 and functionally verified using luciferase reporter assay, EMSA and DNA-ChIP. Using pull-down assays, we identified a novel transcription factor B. mori Kr-h1 regulatory protein (BmKRP) that specifically bound the BmKr-h1 CRE and activated its transcription. CRISPR/Cas9-mediated knockout of BmKRP in female pupae suppressed the transcription of BmKr-h1 and BmVgR, resulting in arrested oogenesis. CONCLUSION: We identified BmKRP as a new transcription factor mediating 20E regulation of B. mori oogenesis. Our data suggests that induction of BmKRP by 20E regulates BmKr-h1 expression, which in turn induces BmVgR expression to facilitate Vg uptake and oogenesis.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (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 reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Vitellogenin expression in the ovaries of adult honeybee workers provides insights into the evolution of reproductive and social traits.

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin-signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non-reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co-opted to regulate behavioural differences between queens and workers.

Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes.

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

Krüppel homologue 1 interacts directly with Hairy and regulates ecdysis in the brown planthopper.

Juvenile hormone (JH) plays important roles in the growth and development of insects. JH and its receptor methoprene-tolerant (Met) regulate the expression of transcription factors to control the transcription of downstream genes. The expression of Hairy (Hry) and Krüppel homologue 1 (Kr-h1) is regulated by JH and JH receptors. Hry and Kr-h1 are both crucial in mediating JH signalling. However, whether they interact at the gene level in regulating metamorphosis and whether they interact physically at the protein level remain unknown. We used co-immunoprecipitation, glutathione S-transferase pull-down and RNA interference (RNAi) approaches to study the genetic and biochemical interactions of the two proteins Hry and Kr-h1. The results showed that brown planthopper (Nilaparvata lugens) Hry and Kr-h1 interact directly: Hry binds to the N-terminal of Kr-h1, which includes five zinc-finger domains. The RNAi experiment showed that downregulation of Hry reduced the ratio of ecdysis failure caused by knockdown of Kr-h1, indicating that the downregulation of Hry might mitigate ecdysis failure via the downregulation of Kr-h1. The expression of Hry increased significantly when Kr-h1 was downregulated, whereas it did not change significantly when both were downregulated. Our results suggest that the binding of Hry protein with Kr-h1 prevents the N-terminal five zinc-finger domains from binding with DNA, which in turn inactivates the transcription activator or inhibitor function of Kr-h1. Hry could possibly be used as a target for pesticide applications in the future.

MicroRNA miR-927 targets the juvenile hormone primary response gene Krüppel homolog1 to control Drosophila developmental growth.

Krüppel homolog1 (Kr-h1) is a juvenile hormone (JH) response transcriptional factor that transduces JH signalling to repress insect metamorphosis in both hemimetabolous and holometabolous insects. While few studies about microRNAs (miRNAs) downregulating Kr-h1 expression to mediate insect metamorphosis have been demonstrated in hemimetabolous insects, the miRNAs that target the Kr-h1 of holometabolous insects have not been reported. Here, we identified two miR-927 binding sites within the 3'UTR region of Kr-h1 in Drosophila melanogaster, and miR-927 was found to downregulate the expression of Kr-h1. The expression profiles of miR-927 and Kr-h1 displayed relatively opposite pattern during most of the larval development stages. Overexpression of miR-927 in the fat body significantly decreased the expression of Kr-h1 and resulted in reduced oviposition, increased mortality, delayed pupation, and reduced pupal size. Notably, the co-overexpression of Kr-h1 rescued the developmental and growth defects associated with miR-927 overexpression, indicating that Kr-h1 is a biologically relevant target of miR-927. Moreover, the expression of miR-927 was found to be repressed by JH and its receptor Met/gce, forming a positive regulatory loop of JH signalling. Overall, our studies support a conserved role for the JH/miRNA/Kr-h1 regulatory axis in growth control during insect development.

The Direct Interaction between E93 and Kr-h1 Mediated Their Antagonistic Effect on Ovary Development of the Brown Planthopper.

The juvenile hormone (JH) signalling and ecdysone signalling pathways are crucial endocrine signalling pathways that orchestrate the metamorphosis of insects. The metamorphic process, the morphological change from the immature to adult forms, is orchestrated by the dramatic reduction of JH and downstream transcription factors. The Krüppel-homologue 1 (Kr-h1), a downstream transcription factor of the JH signalling pathway, represses E93 expression with an anti-metamorphic effect. However, the biochemical interaction between Kr-h1 and E93 and how the interaction regulates ovary development, a sensitive readout for endocrine regulation, remain unknown. In brown planthopper, Nilaparvata lugens, we found that the downregulation of Kr-h1 partially recovered the deteriorating effect of E93 knock-down on metamorphosis. Dual knock down of E93 and Kr-h1 increased ovary development and the number of eggs laid when compared to the effects of the knock down of E93 alone, indicating that the knock down of Kr-h1 partially recovered the deteriorating effect of the E93 knock-down on ovary development. In summary, our results indicated that E93 and Kr-h1 have antagonistic effects on regulating metamorphosis and ovary development. We tested the biochemical interaction between these two proteins and found that these molecules interact directly. Kr-h1 V and E93 II undergo strong and specific interactions, indicating that the potential interacting domain may be located in these two regions. We inferred that the nuclear receptor interaction motif (NR-box) and helix-turn-helix DNA binding motifs of the pipsqueak family (RHF1) are candidate domains responsible for the protein-protein interaction between E93 and Kr-h1. Moreover, the HA-tagged E93 and FLAG-tagged Kr-h1 were co-localized in the nucleus, and the expression of E93 was increased when Kr-h1 was downregulated, supporting that these two proteins may interact antagonistically. JH and ecdysone signalling are critical for the control of ovary development and pest populations. Our result is important for understanding the interactions between E93 and related proteins, which makes it possible to identify potential targets and develop new pesticides for pest management.

Epigenetic modifications acetylation and deacetylation play important roles in juvenile hormone action.

BACKGROUND: Epigenetic modifications including DNA methylation and post-translational modifications of histones are known to regulate gene expression. Antagonistic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) mediate transcriptional reprogramming during insect development as shown in Drosophila melanogaster and other insects. Juvenile hormones (JH) play vital roles in the regulation of growth, development, metamorphosis, reproduction and other physiological processes. However, our current understanding of epigenetic regulation of JH action is still limited. Hence, we studied the role of CREB binding protein (CBP, contains HAT domain) and Trichostatin A (TSA, HDAC inhibitor) on JH action. RESULTS: Exposure of Tribolium castaneum cells (TcA cells) to JH or TSA caused an increase in expression of Kr-h1 (a known JH-response gene) and 31 or 698 other genes respectively. Knockdown of the gene coding for CBP caused a decrease in the expression of 456 genes including Kr-h1. Interestingly, the expression of several genes coding for transcription factors, nuclear receptors, P450 and fatty acid synthase family members that are known to mediate JH action were affected by CBP knockdown or TSA treatment. CONCLUSIONS: These data suggest that acetylation and deacetylation mediated by HATs and HDACs play an important role in JH action.

CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis.

BACKGROUND: CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-ß signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway. METHODS AND RESULTS: Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-ß, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-ß pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described here for the first time. CONCLUSIONS: Studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling. GENERAL SIGNIFICANCE: Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.

Multiple functions of CREB-binding protein during postembryonic development: identification of target genes.

BACKGROUND: Juvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect. RESULTS: CBP is ubiquitously expressed in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) mediated knockdown of CBP resulted in a decrease in JH induction of Kr-h1 gene expression in Tribolium larvae and led to a block in their development. Moreover, the injection of CBP double-stranded RNA (dsRNA) showed lethal phenotypes within 8 days of injection. RNA-seq and subsequent differential gene expression analysis identified CBP target genes in Tribolium. Knockdown of CBP caused a decrease in the expression of 1306 genes coding for transcription factors and other proteins associated with growth and development. Depletion of CBP impaired the expression of several JH response genes (e.g., Kr-h1, Hairy, early trypsin) and ecdysone response genes (EcR, E74, E75, and broad complex). Further, GO enrichment analyses of the downregulated genes showed enrichment in different functions including developmental processes, pigmentation, anatomical structure development, regulation of biological and cellular processes, etc. CONCLUSION: These data suggest diverse but crucial roles for CBP during postembryonic development in the coleopteran model insect, Tribolium. It can serve as a target for RNAi mediated pest management of this stored product pest.

Juvenile hormone suppresses sensory organ precursor determination to block Drosophila adult abdomen morphogenesis.

Juvenile hormone (JH) has a classic "status quo" action at both the pupal and adult molts when administrated exogenously. In Drosophila, treatment with JH at pupariation inhibits the formation of abdominal bristles, which are derived from the histoblasts. However, the mechanism via which JH exerts this effect remains poorly understood. In this study, we analyzed the effect of JH on histoblast proliferation, migration, and differentiation. Our results indicated that whereas the proliferation and migration of histoblasts remained unaffected following treatment with a JH mimic (JHM), their differentiation, particularly the specification of sensor organ precursor (SOP) cells, was inhibited. This effect was attributable to downregulated proneural genes achaete (ac) and Scute (sc) expression levels, which prevented the specification of SOP cells in proneural clusters. Moreover, Kr-h1 was found to mediate this effect of JHM. Histoblast-specific overexpression or knockdown of Kr-h1, respectively mimicked or attenuated the effects exerted by JHM on abdominal bristle formation, SOP determination, and transcriptional regulation of ac and sc. These results indicated that the defective SOP determination was responsible for the inhibition of abdominal bristle formation by JHM, which, in turn, was mainly mediated via the transducing action of Kr-h1.

Kruppel homolog 1 modulates ROS production and antimicrobial peptides expression in shrimp hemocytes during infection by the Vibrio parahaemolyticus strain that causes AHPND.

Shrimp aquaculture has been seriously affected by acute hepatopancreatic necrosis disease (AHPND), caused by a strain of Vibrio parahaemolyticus that carries the Pir toxin plasmids (V. parahaemolyticus (AHPND)). In this study, the transcription factor, Kruppel homolog 1-like of Peneaus vannamei (PvKr-h1), was significantly induced in shrimp hemocytes after V. parahaemolyticus (AHPND) challenge, suggesting that PvKr-h1 is involved in shrimp immune response. Knockdown of PvKr-h1 followed by V. parahaemolyticus (AHPND) challenge increased bacterial abundance in shrimp hemolymph coupled with high shrimp mortality. Moreover, transcriptome and immunofluorescence analyses revealed that PvKr-h1 silencing followed by V. parahaemolyticus (AHPND) challenge dysregulated the expression of several antioxidant-related enzyme genes, such as Cu-Zu SOD, GPX, and GST, and antimicrobial peptide genes, i.e., CRUs and PENs, and reduced ROS activity and nuclear translocation of Relish. These data reveal that PvKr-h1 regulates shrimps' immune response to V. parahaemolyticus (AHPND) infection by suppressing antioxidant-related enzymes, enhancing ROS production and promoting nuclei import of PvRelish to stimulate antimicrobial peptide genes expression.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Kr-h1, a Cornerstone Gene in Insect Life History.

Insect life cycle is coordinated by hormones and their downstream effectors. Krüppel homolog1 (Kr-h1) is one of the crucial effectors which mediates the actions of the two critical hormones of insects, the juvenile hormone (JH) and 20-hydroxyecdysone (20E). It is a transcription factor with a DNA-binding motif of eight C2H2 zinc fingers which is found to be conserved among insect orders. The expression of Kr-h1 is fluctuant during insect development with high abundance in juvenile instars and lower levels in the final instar and pupal stage, and reappearance in adults, which is governed by the coordination of JH, 20E, and miRNAs. The dynamic expression pattern of Kr-h1 is closely linked to its function in the entire life of insects. Over the past several years, accumulating studies have advanced our understanding of the role of Kr-h1 during insect development. It acts as a universal antimetamorphic factor in both hemimetabolous and holometabolous species by directly inhibiting the transcription of 20E signaling genes Broad-Complex (Br-C) and Ecdysone induced protein 93F (E93), and steroidogenic enzyme genes involved in ecdysone biosynthesis. Meanwhile, it promotes vitellogenesis and ovarian development in the majority of studied insects. In addition, Kr-h1 regulates insect behavioral plasticity and caste identity, neuronal morphogenesis, maturation of sexual behavior, as well as embryogenesis and metabolic homeostasis. Hence, Kr-h1 acts as a cornerstone regulator in insect life.

Identification of juvenile hormone-induced posttranslational modifications of methoprene tolerant and Krüppel homolog 1 in the yellow fever mosquito, Aedes aegypti.

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.