Tusar T. Saha

bHLH-PAS heterodimer of methoprene-tolerant and Cycle mediates circadian expression of juvenile hormone-induced mosquito genes

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 Kr-h1 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.

Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression

Significance Mosquitoes transmit some of the most dangerous human diseases. Microarray analysis of developmental gene dynamics in the female Aedes aegypti fat body has demonstrated that 6,146 genes are differentially expressed during juvenile hormone-dependent posteclosion development. These genes show striking temporal and functional separation. A RNAi microarray screen revealed a differential action of Met in the down- and up-regulation of genes expressed during early and late PE, respectively. Thus, this study provides substantial evidence of a central role for insect-specific JH and its receptor Met in the regulation of female mosquito reproductive biology. 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, CACGC/TGA/GT/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi–down-regulated (LPE) genes but not in Met RNAi–up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.

High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections.

BackgroundInnate immunity is essential in defending against invading pathogens in invertebrates. The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most destructive lepidopteran pests, which causes enormous economic losses in agricultural production worldwide. The components of the immune system are largely unknown in this insect. The application of entomopathogens is considered as an alternative to the chemical insecticides for its control. However, few studies have focused on the molecular mechanisms of host-pathogen interactions between pest insects and their pathogens. Here, we investigated the immunotranscriptome of H. armigera larvae and examined gene expression changes after pathogen infections. This study provided insights into the potential immunity-related genes and pathways in H. armigera larvae.ResultsHere, we adopted a high throughput RNA-seq approach to determine the immunotranscriptome of H. armigera larvae injected with buffer, fungal pathogen Beauveria bassiana, or Gram-negative bacterium Enterobacter cloacae. Based on sequence similarity to those homologs known to participate in immune responses in other insects, we identified immunity-related genes encoding pattern recognition receptors, signal modulators, immune effectors, and nearly all members of the Toll, IMD and JAK/STAT pathways. The RNA-seq data indicated that some immunity-related genes were activated in fungus- and bacterium-challenged fat body while others were suppressed in B. bassiana challenged hemocytes, including the putative IMD and JAK-STAT pathway members. Bacterial infection elevated the expression of recognition and modulator genes in the fat body and signal pathway genes in hemocytes. Although fat body and hemocytes both are important organs involved in the immune response, our transcriptome analysis revealed that more immunity-related genes were induced in the fat body than that hemocytes. Furthermore, quantitative real-time PCR analysis confirmed that, consistent with the RNA-seq data, the transcript abundances of putative PGRP-SA1, Serpin1, Toll-14, and Spz2 genes were elevated in fat body upon B. bassiana infection, while the mRNA levels of defensin, moricin1, and gloverin1 were up-regulated in hemocytes.ConclusionsIn this study, a global survey of the host defense against fungal and bacterial infection was performed on the non-model lepidopteran pest species. The comprehensive sequence resource and expression profiles of the immunity-related genes in H. armigera are acquired. This study provided valuable information for future functional investigations as well as development of specific and effective agents to control this pest.

microRNA-309 targets the Homeobox gene SIX4 and controls ovarian development in the mosquito Aedes aegypti.

Significance We report here that microRNA-309 (miR-309) plays a critical role in ovarian development of female Aedes aegypti mosquitoes. The genetic disruption of miR-309 by CRISPR/Cas9 system displays a failure of ovarian primary follicle formation, and several pathways associated with ovarian development are down-regulated after miR-309 depletion. Comprehensive screening and functional identification have revealed that SIX homeobox 4 protein (SIX4) is a direct target of miR-309. miR-309–targeted degradation of SIX4 mRNA is required for appropriate commencement of the preparatory phase and initiation of the blood feeding-triggered phase of ovarian development. Thus, miR-309 serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA, allowing a shift from previtellogenic to postvitellogenic phases of ovarian development. Obligatory blood-triggered reproductive strategy is an evolutionary adaptation of mosquitoes for rapid egg development. It contributes to the vectorial capacity of these insects. Therefore, understanding the molecular mechanisms underlying reproductive processes is of particular importance. Here, we report that microRNA-309 (miR-309) plays a critical role in mosquito reproduction. A spatiotemporal expression profile of miR-309 displayed its blood feeding-dependent onset and ovary-specific manifestation in female Aedes aegypti mosquitoes. Antagomir silencing of miR-309 impaired ovarian development and resulted in nonsynchronized follicle growth. Furthermore, the genetic disruption of miR-309 by CRISPR/Cas9 system led to the developmental failure of primary follicle formation. Examination of genomic responses to miR-309 depletion revealed that several pathways associated with ovarian development are down-regulated. Comparative analysis of genes obtained from the high-throughput RNA sequencing of ovarian tissue from the miR-309 antagomir-silenced mosquitoes with those from the in silico computation target prediction identified that the gene-encoding SIX homeobox 4 protein (SIX4) is a putative target of miR-309. Reporter assay and RNA immunoprecipitation confirmed that SIX4 is a direct target of miR-309. RNA interference of SIX4 was able to rescue phenotypic manifestations caused by miR-309 depletion. Thus, miR-309 plays a critical role in mosquito reproduction by targeting SIX4 in the ovary and serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA. In turn, this microRNA (miRNA)-targeted degradation is required for appropriate initiation of a blood feeding-triggered phase of ovarian development, highlighting involvement of this miRNA in mosquito reproduction.

Temporal Coordination of Carbohydrate Metabolism during Mosquito Reproduction.

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.

Regulation of Gene Expression Patterns in Mosquito Reproduction

In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs) regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM). During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E) and the Ecdysone-Receptor (EcR). Between 36 h and 48 h, the third wave of gene activation—regulated mainly by HR3—occurs. Juvenile Hormone (JH) and its receptor Methoprene-Tolerant (Met) are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the previously unidentified large-scale effects of HR3 and JH/Met on transcriptional regulation during the termination of vitellogenesis and remodeling of the fat body.

Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression.

Significance Juvenile hormone is unique to arthropods and controls numerous essential functions in these organisms. Its receptor, Methoprene-tolerant, turns on genes by directly binding to E-box–like motifs in their regulatory regions. However, how juvenile hormone represses genes has remained unclear. Here, we used the Aedes aegypti female mosquito, in which juvenile hormone is necessary for reproductive maturation, to show that the repressor Hairy is required for the gene-repressive action of juvenile hormone and Methoprene-tolerant. Moreover, we demonstrate that a corepressor, Groucho, supports this Hairy function. Hence, Methoprene-tolerant recruits the evolutionarily conserved Hairy/Groucho molecular system to mediate the juvenile hormone gene-repressive function. The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box–like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

Hormone and receptor interplay in the regulation of mosquito lipid metabolism

Significance Mosquitoes require a blood meal to reproduce and by blood feeding transmit some of the most dangerous human diseases. Female mosquitoes have extremely high metabolism, and the elucidation of regulatory pathways coordinating reproductive and metabolic events is essential. RNAi of the juvenile hormone receptor Methoprene-tolerant (Met) promoted triacylglycerol (TAG) catabolism and β-oxidation and diminished TAG levels, whereas ecdysone receptor (EcR) RNAi had an opposite effect. Hepatocyte nuclear factor 4 (HNF4) directly regulated TAG catabolism and β-oxidation genes, and its RNAi silencing phenocopied EcR RNAi. The expression of the HNF4 gene was downregulated by Met and activated by EcR and Target of rapamycin. Thus, HNF4 mediates hormonal and nutritional signaling of lipid metabolism regulation in reproducing female Aedes aegypti mosquitoes. 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, HNF4 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.

Regulatory Pathways Controlling Female Insect Reproduction

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 and its receptor methoprene-tolerant promote ribosomal biogenesis and vitellogenesis in the Aedes aegypti mosquito

Juvenile hormone (JH) controls many biological activities in insects, including development, metamorphosis, and reproduction. In the Aedes aegypti mosquito, a vector of dengue, yellow fever, chikungunya, and zika viruses, the metabolic tissue (the fat body, which is an analogue of the vertebrate liver) produces yolk proteins for developing oocytes. JH is important for the fat body to acquire competence for yolk protein production. However, the molecular mechanisms of how JH promotes mosquito reproduction are not completely understood. In this study we show that stimulation of the JH receptor methoprene-tolerant (Met) activates expression of genes encoding the regulator of ribosome synthesis 1 (RRS1) and six ribosomal proteins (two ribosomal large subunit proteins, two ribosomal small subunit proteins, and two mitochondrial ribosomal proteins). Moreover, RNAi-mediated depletion of RRS1 decreased biosynthesis of the ribosomal protein L32 (RpL32). Depletion of Met, RRS1, or RpL32 led to retardation of ovarian growth and reduced mosquito fecundity, which may at least in part have resulted from decreased vitellogenin protein production in the fat body. In summary, our results indicate that JH is critical for inducing the expression of ribosomal protein genes and demonstrate that RRS1 mediates the JH signal to enhance both ribosomal biogenesis and vitellogenesis.