Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles.

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

The Role of 20-Hydroxyecdysone in the Control of Carbohydrate Levels in Drosophila melanogaster under Short-Term Heat Stress.

It is known that 20-hydroxyecdysone is one of the most important hormonal regulators of development, reproduction and adaptation to unfavorable conditions in insects. Here, we show for the first time that exogenous 20-hydroxyecdysone increases the content of two main insect carbohydrates, trehalose and glucose, in Drosophila melanogaster females both in normal conditions and under short-term heat stress. It is found that the levels of both trehalose and glucose increase after 39 min of heat exposure and return to their original levels after 1.5 h. A scheme of hormonal regulation of carbohydrate content under heat stress, involving 20-hydroxyecdysone, juvenile hormone, and dopamine, is suggested.

Cell dynamics underlying oriented growth of the Drosophila wing imaginal disc.

Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.

Insulin and 20-hydroxyecdysone oppose each other in the regulation of phosphoinositide-dependent kinase-1 expression during insect pupation.

Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation.

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. We previously discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila However, the molecular events involved in this Mmp-mediated dissociation are unclear. Here we report that JH and 20E coordinately and precisely control the developmental timing of Mmp-induced fat body cell dissociation. We found that during the larval-prepupal transition, the anti-metamorphic factor Kr-h1 transduces JH signaling, which directly inhibited Mmp expression and activated expression of tissue inhibitor of metalloproteinases (timp) and thereby suppressed Mmp-induced fat body cell dissociation. We also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor ßftz-F1 Moreover, upon a decline in the 20E titer, ßftz-F1 expression was induced by the 20E early-late response gene DHR3, and then ßftz-F1 directly activated Mmp expression and inhibited timp expression, causing Mmp-induced fat body cell dissociation during 6-12 h after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmp-induced fat body cell dissociation. Our findings shed critical light on hormonal regulation of insect metamorphosis.

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.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis.

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, Bombyx mori.

Insect chitinases are hydrolytic enzymes required for the degradation of chitin. They are essential for insect moulting and metamorphosis. In this study, the regulation mechanism of a chitinase gene, Bombyx mori chitinase 5 (BmCHT5), was studied. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that BmCHT5 was up-regulated during the larval-larval and larval-pupa transitions and notably induced by 20-hydroxyecdysone (20E). Analysis of the BmCHT5 promoter revealed the presence of one Bombyx mori Broad-Complex Zinc-Finger Isoform 4 (BR-C Z4), two BR-C Z2 and two ecdysone-induced protein 74A (E74A) cis-regulatory elements (CREs) that are related to 20E. qRT-PCR showed that the expression of both BmBR-C Z4 and BmBR-C Z2 during metamorphosis, and when induced by 20E, was anastomotic with the variations in BmCHT5 mRNA level. In contrast, BmE74A did not follow this trend. An electrophoretic mobility shift assay did not retrieve a binding partner for the two BR-C Z2 CREs in the BmN cell line nuclear extract, whereas BR-C Z4 CRE specifically bound to BmBR-C Z4. Besides, luciferase activity analysis confirmed that BmBR-C Z4 could enhance the activity of the BmCHT5 promoter with BR-C Z4 CRE and could not enhance the promoter activity by mutating BR-C Z4 CRE. Taken together, these data suggest that the transcription factor BmBR-C Z4 enhances the expression of BmCHT5 during metamorphosis.

Insulin and 20-hydroxyecdysone action in Bombyx mori: Glycogen content and expression pattern of insulin and ecdysone receptors in fat body.

Insulin and ecdysone signaling play a critical role on the growth and development of insects including Bombyx mori. Our previous study showed that Bombyx larvae reached critical weight for metamorphosis between day 3.5 and 4 of the fifth larval instar. The present study showed that the effect of insulin on the accumulation of glycogen in fat body of Bombyx larvae depends on the critical growth period. When larvae are in active growth period (before reaching critical weight), insulin caused increased accumulation of glycogen, while its treatment in larvae at terminal growth period (after critical period) resulted in an increased mobilization of glycogen. During terminal growth period, insulin and 20-hydroxyecdysone (20E) showed an antagonistic effect on the accumulation of fat body glycogen in fed, food deprived and decapitated larvae as well as in isolated abdomens. Insulin treatment decreased the glycogen content, whereas, 20E increased it. Food deprivation and decapitation caused an increase in the transcript levels of insulin receptor (InR) and this increase in InR expression might be attributed to a decrease in synthesis/secretion of insulin-like peptides, as insulin treatment in these larvae showed a down-regulation in InR expression. However, insulin showed an up-regulation in InR in isolated abdomens and it suggests that in food deprived and decapitated larvae, the exogenous insulin may interact with some head and/or thoracic factors in modulating the expression of InR. Moreover, in fed larvae, insulin-mediated increase in InR expression indicates that its regulation by insulin-like peptides also depends on the nutritional status of the larvae. The treatment of 20E in fed larvae showed an antagonistic effect on the transcript levels since a down-regulation in InR expression was observed. 20E treatment also led to a decreased expression of InR in food deprived and decapitated larvae as well as in isolated abdomens. Insulin and 20E also modulated the expression level of ecdysone receptors (EcRB1 and USP1). 20E treatment showed an up-regulation in expression of ecdysone receptors, but only in fed larvae, whereas insulin treatment showed a down-regulation in the expression of EcRB1 and USP1 in all the experimental larvae studied. Further, the data indicates that an up-regulation of ecdysone receptors is associated with an increase in fat body glycogen content, whereas an up-regulation of insulin receptor expression causes glycogen mobilization. The study, therefore, suggests that the insulin and ecdysone signaling are linked to each other and that both insulin and ecdysone are involved in regulating the carbohydrate reserves in B. mori.

Sexual transfer of the steroid hormone 20E induces the postmating switch in Anopheles gambiae.

Female insects generally mate multiple times during their lives. A notable exception is the female malaria mosquito Anopheles gambiae, which after sex loses her susceptibility to further copulation. Sex in this species also renders females competent to lay eggs developed after blood feeding. Despite intense research efforts, the identity of the molecular triggers that cause the postmating switch in females, inducing a permanent refractoriness to further mating and triggering egg-laying, remains elusive. Here we show that the male-transferred steroid hormone 20-hydroxyecdysone (20E) is a key regulator of monandry and oviposition in An. gambiae. When sexual transfer of 20E is impaired by partial inactivation of the hormone and inhibition of its biosynthesis in males, oviposition and refractoriness to further mating in the female are strongly reduced. Conversely, mimicking sexual delivery by injecting 20E into virgin females switches them to an artificial mated status, triggering egg-laying and reducing susceptibility to copulation. Sexual transfer of 20E appears to incapacitate females physically from receiving seminal fluids by a second male. Comparative analysis of microarray data from females after mating and after 20E treatment indicates that 20E-regulated molecular pathways likely are implicated in the postmating switch, including cytoskeleton and musculature-associated genes that may render the atrium impenetrable to additional mates. By revealing signals and pathways shaping key processes in the An. gambiae reproductive biology, our data offer new opportunities for the control of natural populations of malaria vectors.

20-Hydroxyecdysone (20E) Primary Response Gene E93 Modulates 20E Signaling to Promote Bombyx Larval-Pupal Metamorphosis.

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis.

The steroid hormone 20-hydroxyecdysone via nongenomic pathway activates Ca2+/calmodulin-dependent protein kinase II to regulate gene expression.

The steroid hormone 20-hydroxyecdysone (20E) triggers calcium signaling pathway to regulate 20E response gene expression, but the mechanism underlying this process remains unclear. We propose that the 20E-induced phosphorylation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) serves an important function in 20E response gene transcription in the lepidopteran insect Helicoverpa armigera. CaMKII showed increased expression and phosphorylation during metamorphosis. 20E elevated CaMKII phosphorylation. However, the G protein-coupled receptor (GPCR) and ryanodine receptor inhibitor suramin, the phospholipase C inhibitor U73122, and the inositol 1,4,5-triphosphate receptor inhibitor xestospongin C suppressed 20E-induced CaMKII phosphorylation. Two ecdysone-responsible GPCRs and Gαq protein were involved in 20E-induced CaMKII phosphorylation by RNA interference analysis. 20E regulated CaMKII threonine phosphorylation at amino acid 290, thereby inducing CaMKII nuclear translocation. CaMKII knockdown by dsCaMKII injection into the larvae prevented the occurrence of larval-pupal transition and suppressed 20E response gene expression. CaMKII phosphorylation and nuclear translocation maintained USP1 lysine acetylation at amino acid 303 by inducing histone deacetylase 3 phosphorylation and nuclear export. The lysine acetylation of USP1 was necessary for the interaction of USP1 with EcRB1 and their binding to the ecdysone response element. Results suggest that 20E (via GPCR activation and calcium signaling) activates CaMKII phosphorylation and nuclear translocation, which regulate USP1 lysine acetylation to form an EcRB1-USP1 complex for 20E response gene transcription.

Juvenile hormone prevents 20-hydroxyecdysone-induced metamorphosis by regulating the phosphorylation of a newly identified broad protein.

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis. By contrast, juvenile hormone (JH) prevents metamorphosis. However, the mechanism by which JH inhibits metamorphosis remains unclear. In this study, we propose that JH induces the phosphorylation of Broad isoform Z7 (BrZ7), a newly identified protein, to inhibit 20E-mediated metamorphosis in the lepidopteran insect Helicoverpa armigera. The knockdown of BrZ7 in larvae inhibited metamorphosis by repressing the expression of the 20E response gene. BrZ7 was weakly expressed and phosphorylated during larval growth but highly expressed and non-phosphorylated during metamorphosis. JH regulated the rapid phosphorylation of BrZ7 via a G-protein-coupled receptor-, phospholipase C-, and protein kinase C-triggered pathway. The phosphorylated BrZ7 bound to the 5'-regulatory region of calponin to regulate its expression in the JH pathway. Exogenous JH induced BrZ7 phosphorylation to prevent metamorphosis by suppressing 20E-related gene transcription. JH promoted non-phosphorylated calponin interacting with ultraspiracle protein to activate the JH pathway and antagonize the 20E pathway. This study reveals one of the possible mechanisms by which JH counteracts 20E-regulated metamorphosis by inducing the phosphorylation of BrZ7.

The Drosophila nuclear receptors DHR3 and betaFTZ-F1 control overlapping developmental responses in late embryos.

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, betaFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, betaFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. In this study, we turn to embryonic development to address these two issues. We show that mid-embryonic expression of DHR3 and betaFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 and betaFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and betaFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of betaFTZ-F1. DHR3 and betaFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and betaFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade.

Fork head controls the timing and tissue selectivity of steroid-induced developmental cell death.

Cell death during Drosophila melanogaster metamorphosis is controlled by the steroid hormone 20-hydroxyecdysone (20E). Elements of the signaling pathway that triggers death are known, but it is not known why some tissues, and not others, die in response to a particular hormone pulse. We found that loss of the tissue-specific transcription factor Fork head (Fkh) is both required and sufficient to specify a death response to 20E in the larval salivary glands. Loss of fkh itself is a steroid-controlled event that is mediated by the 20E-induced BR-C gene, and that renders the key death regulators hid and reaper hormone responsive. These results implicate the D. melanogaster FOXA orthologue Fkh with a novel function as a competence factor for steroid-controlled cell death. They explain how a specific tissue is singled out for death, and why this tissue survives earlier hormone pulses. More generally, they suggest that cell identity factors like Fkh play a pivotal role in the normal control of developmental cell death.

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut.

Programmed cell death (PCD) plays an important role in insect midgut remodeling during metamorphosis. Insect midgut PCD is triggered by the steroid hormone 20-hydroxyecdysone (20E) and it is mediated by a series of genes. However, the mechanism by which 20E triggers midgut PCD is still unclear. Here, we report a protein phosphatase 6 (PP6) from Helicoverpa armigera playing roles in midgut PCD. PP6 was expressed in the midgut during larval growth and it is significantly increased during metamorphosis. The increase was proven to be regulated by 20E. The juvenile hormone analog methoprene has no effect on PP6 expression. RNA interference analysis suggests that 20E upregulated the PP6 transcript levels through the ecdysone receptor EcRB1. PP6 knockdown by larval feeding or PP6 dsRNA injection resulted in the repression of the midgut PCD during the metamorphic stage. The mechanism was demonstrated to be through the suppression of genes such as Broad (Br), E74a, E75b, HR3, E93, rpr, and caspase, which are involved in 20E signaling pathway or midgut PCD. These findings suggest that PP6 is involved in the 20E signal transduction pathway and participates in the PCD in midgut.

The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect.

Metamorphosis in holometabolous insects is mainly based on the destruction of larval tissues. Intensive research in Drosophila melanogaster, a model of holometabolan metamorphosis, has shown that the steroid hormone 20-hydroxyecdysone (20E) signals cell death of larval tissues during metamorphosis. However, D. melanogaster shows a highly derived type of development and the mechanisms regulating apoptosis may not be representative in the insect class context. Unfortunately, no functional studies have been carried out to address whether the mechanisms controlling cell death are present in more basal hemimetabolous species. To address this, we have analyzed the apoptosis of the prothoracic gland of the cockroach Blattella germanica, which undergoes stage-specific degeneration just after the imaginal molt. Here, we first show that B. germanica has two inhibitor of apoptosis (IAP) proteins and that one of them, BgIAP1, is continuously required to ensure tissue viability, including that of the prothoracic gland, during nymphal development. Moreover, we demonstrate that the degeneration of the prothoracic gland is controlled by a complex 20E-triggered hierarchy of nuclear receptors converging in the strong activation of the death-inducer Fushi tarazu-factor 1 (BgFTZ-F1) during the nymphal-adult transition. Finally, we have also shown that prothoracic gland degeneration is effectively prevented by the presence of juvenile hormone (JH). Given the relevance of cell death in the metamorphic process, the characterization of the molecular mechanisms regulating apoptosis in hemimetabolous insects would allow to help elucidate how metamorphosis has evolved from less to more derived insect species.

Increased levels of the cell cycle inhibitor protein, dacapo, accompany 20-hydroxyecdysone-induced G1 arrest in a mosquito cell line.

When treated with the steroid hormone 20-hydroxyecdysone (20E), C7-10 cells from the mosquito, Aedes albopictus, arrest in the G1 phase of the cell cycle. To explore whether 20E-mediated cell cycle arrest proceeds through increased levels of cell cycle inhibitor (CKI) proteins, we cloned the Ae. albopictus homolog of dacapo, the single member of the Cip/Kip family of CKI proteins known from Drosophila melanogaster. The Ae. albopictus dacapo cDNA encoded a 261-amino acid homolog of the Aedes aegypti protein XP_001651102.1, which is encoded by an ∼23 kb gene containing three exons. Like dacapo from D. melanogaster, the ∼27 kDa protein from Aedes and Culex mosquitoes contained several S/TXXE/D motifs corresponding to potential protein kinase CK2 phosphorylation sites, and a binding site for proliferating cell nuclear antigen (PCNA). When extracts from cells treated with 20E were analyzed by western blotting, using a primary antibody to synthetic peptides from the mosquito dacapo protein, up-regulation of an ∼27 kDa protein was observed within 24 h, and the abundance of the protein further increased by 48 h after hormone treatment. This is the first investigation of a cell cycle inhibitory protein in mosquitoes. The results reinforce growing evidence that 20E affects expression of proteins that regulate cell cycle progression.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Involvement of Leptinotarsa hormone receptor 38 in the larval-pupal transition.

In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata.