Ecdysteroid Receptor Research Articles

Serotonin induces ecdysteroidogenesis and methyl farnesoate synthesis in the mud crab, Scylla serrata

In the current study, we have examined the role of serotonin in regulating the levels of methyl farnesoate and ecdysteroids in the giant mud crab Scylla serrata and validated that serotonin indeed is a reproductive hormone. Administration of serotonin elevated circulatory levels of methyl farnesoate and ecdysteroids in crabs. Since methyl farnesoate and ecdysteroid act through retinoid X receptor (RXR) and ecdysteroid receptor (EcR) respectively and these receptors are involved in the regulation of reproduction in crustaceans, we have determined the mRNA levels of RXR and EcR in hepatopancreas and ovary after serotonin administration. The expression levels of both RXR and EcR increased significantly in the hepatopancreas and ovary of serotonin injected crabs when compared to the controls. In vitro organ culture studies revealed that incubation of Y-orgas and mandibular organ explants in the presence of serotonin resulted in a significant increase in the secretion of ecdysteroids by Y-organs, but without alterations in MF synthesis in mandibular organs. From the above studies it is evident that serotonin stimulates Y organs resulting in increased ecdysteroidogenesis. Though the circulatory levels methyl farnesoate elevated after serotonin administration, organ culture studies revealed serotonin mediated methyl farnesaote synthesis is indirect probably by inhibiting release of mandibular organ inhibiting hormone from eyestalks.

Dietary TiO2 particles modulate expression of hormone-related genes in Bombyx mori.

Silkworm (Bombyx mori) is an economically beneficial insect. Its growth and development are regulated by endogenous hormones. In the present study, we found that feeding titanium dioxide nanoparticles (TiO2 NP) caused a significant increase of body size. TiO2 NP stimulated the transcription of several genes, including the insulin-related hormone bombyxin, PI3K/Akt/TOR (where PI3K is phosphatidylinositol 3-kinase and TOR is target of rapamycin), and the adenosine 5'-monophosphateactivated protein kinase (AMPK)/target of rapamycin (TOR) pathways. Differentially expressed gene (DEG) analysis documented 26 developmental hormone signaling related genes that were differentially expressed following dietary TiO2 NP treatment. qPCR analysis confirmed the upregulation of insulin/ecdysteroid signaling genes, such as bombyxin B-1, bombyxin B-4, bombyxin B-7, MAPK, P70S6K, PI3k, eIF4E, E75, ecdysteroid receptor (EcR), and insulin-related peptide binding protein precursor 2 (IBP2). We infer from the upregulated expression of bombyxins and the signaling network that they act in bombyxin-stimulated ecdysteroidogenesis.

Characterization and expression patterns of key ecdysteroid biosynthesis and signaling genes in a spider mite (Panonychus citri)

Ecdysteroids play a crucial role in regulating molting in the phylum of Arthropoda and much is known with members of the subphylum of Hexapoda including the Insecta. However, this is still unclear in key pests as spider mites belonging to the subphylum of Chelicerata that originated earlier in the Cambrian period. In this study, we investigated 14 key genes of ecdysteroid biosynthesis and signaling and their expression over the different developmental stages in the citrus red mite, Panonychus citri (Acari: Stigmaeidae). P. citri is an economically important and widespread pest of citrus crops and it has five developmental stages of egg, larva, protonymph, deutonymph and adult. Typically, the expression of the ecdysteroid-synthesizing Halloween gene Spook (PcSpo) followed a positive zigzag-like pattern with a peak in the first half of each developmental stage and a drop in the second half prior to the molting to the next stage. Similar to PcSpo, PcDib, PcSad, PcRXR2, PcE75 and PcHR38 showed a positive zigzag-like expression pattern, while that of PcE78, PcHR3 and PcFTZ-F1 was opposite that we called a negative zigzag-like pattern. Silencing of the PcSpo gene by RNAi showed that molting was inhibited. Interestingly, we could rescue these RNAi effects by supplementing ponasterone A (PonA) and not by 20E, which is indicative that mites use PonA rather than 20E as ecdysteroid hormone. Modeling of the ecdysteroid receptor (PcEcR) hormone binding cavity also predicted binding of PonA, but showed a steric hindrance for 20E. We believe our data provide insight into the evolution and expression patterns of key ecdysteroid biosynthesis and signaling genes in a distant, non-insect species, and can become a foundation to develop new targets for controlling important agricultural pests such as spider mites.

Silencing of JHEH and EcR genes of Plutella xylostella (Lepidoptera: Plutellidae) through double stranded RNA oral delivery

Plutella xylostella (L.), Diamondback moth (DBM), is a notorious pest disrupting cruciferous crops across the world. Overuse of chemically synthesized insecticides as well as application of bio-pesticide like Bacillus thuringiensis has gained progressive resistance against them, resulting in DBM management failures. To overcome this, a more potential, non-chemical and safe technology like Ribonucleic acid interference (RNAi) is to be implemented. RNAi, is a reverse genetic tool with high sequence specificity, elicited by double-stranded RNA (dsRNA) for silencing the target genes. The objective of this study was to silence two candidate genes from DBM viz., Juvenile Hormone Epoxide hydrolase (PxJHEH) and Ecdysteroid receptor (PxEcR) involved in regulating metamorphosis and molting respectively. In this regard, we have custom designed the dsRNAs (500bp) by cloning and sequencing the candidate genes and delivered it orally (non-invasive mode). Further, we have assessed the extent of down regulation of target genes with five concentrations of dsRNA (1, 2, 5, 10 and 20μg/μl) at four time points (24, 48, 72 and 96h). The extent of gene silencing and mortality recorded for both genes were proportional to the dsRNA concentration. Our study projects that PxJHEH and PxEcR genes would be good target for dsRNA mediated gene silencing in management of insect pests such as DBM in near future.

Identification and expression patterns of 20-hydroxyecdysone-responsive genes from Procambarus clarkii

Ecdysteroids play an important role in the regulation of molting process in crustaceans. The suppression subtractive hybridization (SSH) technique was employed to search for 20-hydroxyecdysone-responsive genes in the hepatopancreas of the crayfish Procambarus clarkii. Ninety-three putative expressed sequence tags were identified by SSH, including three immune response-related genes, two cell cycle and apoptosis genes, four respiration and energy metabolism genes, four transport-related genes, six metabolism-related genes, two stress response genes, and eight transcription and translation regulation genes. The expression levels of the examined genes were regulated by 20-hydroxyecdysone and varied in the hepatopancreas during the molting stages; data were confirmed by real-time PCR. RNAi of the ecdysteroid receptor had significant effects on the expression levels of 20-hydroxyecdysone-responsive genes. These differentially expressed genes identified by SSH will provide insight into the ecdysteroid signaling pathway in P. clarkii.

Identification of ecdysteroid receptor-mediated signaling pathways in the hepatopancreas of the red swamp crayfish, Procambarus clarkii

The hepatopancreas of crustaceans plays an important role in lipid and carbohydrate metabolism, digestion of food, and biogenesis. In this study, the hepatopancreas transcriptome from the red crayfish Procambarus clarkii was characterized for the first time using high-throughput sequencing, producing approximately 41.4 million reads were obtained. After de novo assembly, 57,363 unigenes with an average length of 725bp were identified, Gene Ontology analysis categorized 22,580 as being involved in biological processes, among which metabolic process and cellular process groups were the most highly enriched. A total of 8034 unigenes were assigned to 223 metabolic pathways following mapping against the Kyoto encyclopedia of genes and genomes (KEGG) database. Ecdysteroid receptor (EcR)-mediated signaling pathways were investigated using digital gene expression (DGE) analysis following RNA interference targeting the EcR. A total of 529 differentially expressed genes (DEGs) were identified, including 322 downregulated and 207 upregulated unigenes. Of these, 445 (84.12%) were annotated successfully by alignment with known sequences, many of which were related to catalytic activity and binding functional categories. Using KEGG enrichment analysis, 183 DEGs were clustered into 78 pathways, and six significantly enriched pathways were predicted. The expression patterns of candidate genes identified by real-time PCR were consistent with the DGE results.

Effects of Elevated Temperature and Food Supply on the Termination of Over-Summering and Subsequent Development of the Calanoid Copepod Calanus sinicus: Morphology, Physiology and Gene Expression.

The copepod Calanus sinicus Brodsky dominates the zooplankton in the Yellow Sea, China, and undergoes over-summering within the Yellow Sea Cold Water Mass (YSCWM). Termination of over-summering and subsequent development are regarded as key processes in population recruitment, and are probably linked to environmental variations in the YSCWM. In this study, we examined the effects of temperature (9 and 18°C) and food conditions (0.1 μg C mL-1 and unfed) on metabolic rates, morphological characteristics, and relative gene expressions of six genes involved in molting, gonad development, lipid catabolism, and stress tolerance processes of C. sinicus during termination of over-summering and subsequent development. Both elevated temperature and external food supply rapidly ended over-summering of C. sinicus, accompanied by up-regulation of the ecdysteroid receptor (EcR) gene expression and increased metabolic rates. These environmental conditions resulted in irreversible termination of over-summering and ensure the success of molting. During subsequent development, the lipid reserve in oil sacs could permit only early gonad development. The food supply might be a trigger to activate the final maturity of gonad by up-regulating expression of the vitellogenin receptor (VgR) gene. Thus, food played an indispensable role in population recruitment after termination of over-summering, whereas the elevated temperature accelerated these physiological processes. This study revealed the first dynamic profiles of physiological processes involved in over-summering termination and the subsequent development of C. sinicus using morphological, physiological and molecular methods simultaneously, confirmed the quiescent state of over-summering C5 copepodites, detected the effects of environmental changes on over-summering termination and subsequent development, and provided a foundation for future investigations of the mechanisms involved in over-summering in YSCWM.

Mitochondria and the insect steroid hormone receptor (EcR): A complex relationship

The actions of the insect steroid molting hormones, ecdysteroids, on the genome of target cells has been well studied, but little is known of their extranuclear actions. We previously showed in Rhodnius prolixus that much of the ecdysteroid receptor (EcR) resides in the cytoplasm of various cell types and undergoes shuttling between nucleus and cytoplasm with circadian periodicity, possibly using microtubules as tracks for translocation to the nucleus. Here we report that cytoplasmic EcR appears to be also involved in extranuclear actions of ecdysteroids by association with the mitochondria. Western blots of subcellular fractions of brain lysates revealed that EcR is localized in the mitochondrial fraction, indicating an intimate association of EcR with mitochondria. Confocal laser microscopy and immunohistochemistry using anti-EcR revealed abundant co-localization of EcR with mitochondria in brain neurons and their axons, especially intense in the subplasmalemmal region, raising the possibility of EcR involvement in mitochondrial functions in subplasmalemmal microdomains. When mitochondria are dispersed by disruption of microtubules with colchicine, EcR remains associated with mitochondria showing strong receptor association with mitochondria. Treatment in vitro with ecdysteroids of brains of developmentally arrested R. prolixus (containing neither ecdysteroids nor EcR) induces EcR and abundant co-localization with mitochondria in neurons, concurrently with a sharp increase of the mitochondrial protein COX 1, suggesting involvement of EcR in mitochondrial function. These findings align EcR with various vertebrate steroid receptors, where actions of steroid receptors on mitochondria are widely known and suggest that steroid receptors across distant phyla share similar functional attributes.

Changes of exoskeleton surface roughness and expression of crucial participation genes for chitin formation and digestion in the mud crab (Macrophthalmus japonicus) following the antifouling biocide irgarol

Irgarol is a common antifoulant present in coastal sediment. The mud crab Macrophthalmus japonicus is one of the most abundant of the macrobenthos in the costal environment, and its exoskeleton has a protective function against various environmental threats. We evaluated the effects of irgarol toxicity on the exoskeleton of M. japonicus, which is the outer layer facing the environment. We analyzed transcriptional expression of exoskeleton, molting, and proteolysis-related genes in the gill and hepatopancreas of these exposed M. japonicus. In addition, changes in survival and exoskeleton surface characteristics were investigated. In the hepatopancreas, mRNA expression of chitinase 1 (Mj-chi1), chitinase 4 (Mj-chi4), and chitinase 5 (Mj-chi5) increased in M. japonicus exposed to all concentrations of irgarol. Mj-chi1 and Mj-chi4 expressions from 1 to 10μgL−1 were dose- and time-dependent. Ecdysteroid receptor (Mj-EcR), trypsin (Mj-Tryp), and serine proteinase (Mj-SP) in the hepatopancreas were upregulated in response to different exposure levels of irgarol at day 1, 4, or 7. In contrast, gill Mj-chi5, Mj-Tryp, and Mj-SP exhibited late upregulated responses to 10μgL−1 irgarol compared to the control at day 7. Mj-chi1 showed early upregulation upon exposure to 10μgL−1 irgarol and Mj-chi4 showed no changes in transcription in the gill. Gill Mj-EcR presented generally downregulated expression patterns. In addition, decreased survival and change of exoskeleton surface roughness were observed in M. japonicus exposed to the three concentrations of irgarol. These results suggest that exposure to irgarol induces changes in the exoskeleton, molting, and proteolysis metabolism of M. japonicus.

BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5–6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure.

QSAR ANALYSIS OF THE ECDYSTEROIDS’ AFFINITY BASED ON 2.5D-SIMPLEX REPRESENTATION OF THE MOLECULAR STRUCTURE

QSAR-studies of such classes of the organic compounds as steroids, are important because they can expand information about optimal ligands, research of the compounds analogical to existing steroids and understanding factors important for binding with receptors. In this study, we represent «2.5D»-SiRMS approach which is an expansion of the simplex representation of the molecular structure considering the labels of chirality. Steroid set of Kramer was used for test study, and set of ecdysteroids with affinity data to ecdysteroid receptor as the main target of the study. «2.5D»-SiRMS allowed to get satisfying QSAR models for both of those tasks. For Kramer set statistical parameters are: R2=0,84: Q2=0,79, RMSE=0,51. For ecdysteroids set statistical parameters are: R2=0,94: Q2=0,79, RMSE=0,44. Those results are comparable or higher than those of most of 3D-QSAR approaches. Functional and structural interpretations of the QSAR-model for ecdysteroid receptor affinity are also given in this study. There was shown that hydrophobic and electrostatic factors are the most important to the affinity. Analysis of structural fragments’ influence also allowed to distinguish some fragments, which provide activity more than other ones.

Differences in the physiological processes ofCalanus sinicusinside and outside the Yellow Sea Cold Water Mass

Calanus sinicus, the key zooplankton species in the Yellow Sea, China, over-summers in the Yellow Sea Cold Water Mass (YSCWM). Here, we compared the metabolic rates, morphological characteristics and relative expressions of seven genes associated with molting, gonad development, lipid catabolism and stress tolerance of C. sinicus captured both inside and outside the YSCWM in summer. With a large oil sac, low metabolic rate and suppressed molting development, the C5-stage copepods inside the YSCWM were probably quiescent. The gene expressions revealed differences in physiology between quiescent and active copepods in the two regions. When quiescent, the gene associated with molting [ecdysteroid receptor (EcR)] was down-regulated, while genes related to lipid catabolism (Hydroxyacyl CoA dehydrogenase) and stress tolerance (ferritin) were up-regulated. C5s at the margin of the YSCWM up-regulated EcR expression and this could be in response to the elevated Chl a concentration, suggesting that elevated food condition may serve as a trigger that terminated the over-summering of C. sinicus in the Yellow Sea. In conclusion, this study revealed the physiological processes of quiescent and active C. sinicus via morphological, physiological and molecular methods simultaneously, providing a foundation for future investigations of the mechanisms involved in over-summering in YSCWM.

Transcriptional, translational, and physiological signatures of undernourished honey bees (Apis mellifera) suggest a role for hormonal factors in hypopharyngeal gland degradation

Honey bee colonies function as a superorganism, where facultatively sterile female workers perform various tasks that support the hive. Nurse workers undergo numerous anatomical and physiological changes in preparation for brood rearing, including the growth of hypopharyngeal glands (HGs). These glands produce the major protein fraction of a protein- and lipid-rich jelly used to sustain developing larvae. Pollen intake is positively correlated with HG growth, but growth in the first three days is similar regardless of diet, suggesting that initial growth is a pre-determined process while later HG development depends on nutrient availability during a critical window in early adulthood (>3d). It is unclear whether the resultant size differences in nurse HG are simply due to growth arrest or active degradation of the tissue. To determine what processes cause such differences in HG size, we catalogued the differential expression of both gene transcripts and proteins in the HGs of 8d old bees that were fed diets containing pollen or no pollen. 3438 genes and 367 proteins were differentially regulated due to nutrition. Of the genes and proteins differentially expressed, undernourished bees exhibited more gene and protein up-regulation compared to well-nourished bees, with the affected processes including salivary gland apoptosis, oogenesis, and hormone signaling. Protein secretion was virtually the only process up-regulated in well-nourished bees. Further assays demonstrated that inhibition of ultraspiracle, one component of the ecdysteroid receptor, in the fat body caused larger HGs. Undernourished bees also had higher acid phosphatase activity, a physiological marker of cell death, compared to well-nourished bees. These results support a connection between poor nutrition, hormonal signaling, and HG degradation.

Induction of ecdysteroidogenesis, methyl farnesoate synthesis and expression of ecdysteroid receptor and retinoid X receptor in the hepatopancreas and ovary of the giant mud crab, Scylla serrata by melatonin

Melatonin, a chronobiotic molecule, is known to modulate several physiological functions in crustaceans including reproduction, molting and glucose homeostasis. In our earlier studies (Sainath and Reddy, 2010a), we observed hyperglycemia in crabs after melatonin administration and concluded that melatonin is another crustacean hyperglycemic hormone. In the current study, we have further examined the role of melatonin in regulating the levels of methyl farnesoate and ecdysteroid in the giant mud crab Scylla serrata and determined that melatonin indeed is a reproductive hormone. Further, we have determined partial nucleotide sequences of retinoid X receptor (RXR) and ecdysone receptor (EcR) in S. serrata and also studied the effect of melatonin on expression of these genes. Cloned RXR and EcR possess high sequence similarity with other Brachyuran genes. Administration of melatonin elevated circulatory methyl farnesoate (MF) and ecdysteroid levels in crabs. Since MF and ecdysteroid act through RXR and EcR respectively and these receptors are involved in the regulation of reproduction in crustaceans, we measured the expression levels of RXR and EcR in hepatopancreas and ovary after melatonin administration. The expression levels of both RXR and EcR increased significantly in the hepatopancreas and ovary of melatonin injected crabs when compared to the controls. In vitro culture of mandibular organ (MO) and Y-organ (YO) in the presence of melatonin resulted in a significant increase in the secretion of methyl farnesoate and ecdysteroid respectively. From the above studies it is clear that melatonin stimulates YO and MO, resulting in increased synthesis of ecdysteroids and methyl farnesoate, and thereby inducing reproduction in S. serrata.

Molecular cloning, characterization, and expression analysis of an ecdysone receptor homolog in Teleogryllus emma (Orthoptera: Gryllidae).

Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5′-untranslated region of 555 bp and a 3′-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods.

Identification and expression of an ecdysteroid-responsive amylase from red crayfish Procambarus clarkii

Amylases catalyze the hydrolysis of starch material and play important roles in the regulation of many physiological processes. Here, we report an amylase gene denoted Pc-Amy in the crayfish Procambarus clarkii (Decapoda: Cambaridae). The Pc-Amy gene encodes a polypeptide of 514 amino acids, and the deduced amylase protein is highly homologous to those from crustaceans. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis showed that Pc-Amy is highly expressed in hepatopancreas during the intermolt period. SDS-PAGE and western blot analysis demonstrated that a 58-kDa recombinant protein is successfully expressed in Escherichia coli cells. The expression of Pc-Amy was significantly downregulated in hepatopancreas at 48 h after ecdysteroid induction, as confirmed by qRT-PCR and western blot analyses. In addition, RNA interference with the ecdysteroid receptor had an impact on the expression of Pc-Amy. These results suggest that Pc-Amy is an ecdysteroid-responsive gene which plays a role in the developmental process of P. clarkii.

Characterization of a novel RXR receptor in the salmon louse (Lepeophtheirus salmonis, Copepoda) regulating growth and female reproduction.

BackgroundNuclear receptors have crucial roles in all metazoan animals as regulators of gene transcription. A wide range of studies have elucidated molecular and biological significance of nuclear receptors but there are still a large number of animals where the knowledge is very limited. In the present study we have identified an RXR type of nuclear receptor in the salmon louse (Lepeophtheirus salmonis) (i.e. LsRXR). RXR is one of the two partners of the Ecdysteroid receptor in arthropods, the receptor for the main molting hormone 20-hydroxyecdysone (E20) with a wide array of effects in arthropods.ResultsFive different LsRXR transcripts were identified by RACE showing large differences in domain structure. The largest isoforms contained complete DNA binding domain (DBD) and ligand binding domain (LBD), whereas some variants had incomplete or no DBD. LsRXR is transcribed in several tissues in the salmon louse including ovary, subcuticular tissue, intestine and glands. By using Q-PCR it is evident that the LsRXR mRNA levels vary throughout the L. salmonis life cycle. We also show that the truncated LsRXR transcript comprise about 50% in all examined samples. We used RNAi to knock-down the transcription in adult reproducing female lice. This resulted in close to zero viable offspring. We also assessed the LsRXR RNAi effects using a L. salmonis microarray and saw significant effects on transcription in the female lice. Transcription of the major yolk proteins was strongly reduced by knock-down of LsRXR. Genes involved in lipid metabolism and transport were also down regulated. Furthermore, different types of growth processes were up regulated and many cuticle proteins were present in this group.ConclusionsThe present study demonstrates the significance of LsRXR in adult female L. salmonis and discusses the functional aspects in relation to other arthropods. LsRXR has a unique structure that should be elucidated in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1277-y) contains supplementary material, which is available to authorized users.

Regulation of Drosophila circadian rhythms by miRNA let-7 is mediated by a regulatory cycle.

MicroRNA-mediated post-transcriptional regulations are increasingly recognized as important components of the circadian rhythm. Here we identify microRNA let-7, part of the Drosophila let-7-Complex, as a regulator of circadian rhythms mediated by a circadian regulatory cycle. Overexpression of let-7 in clock neurons lengthens circadian period and its deletion attenuates the morning activity peak as well as molecular oscillation. Let-7 regulates the circadian rhythm via repression of CLOCKWORK ORANGE (CWO). Conversely, upregulated cwo in cwo-expressing cells can rescue the phenotype of let-7-Complex overexpression. Moreover, circadian prothoracicotropic hormone (PTTH) and CLOCK-regulated 20-OH ecdysteroid signalling contribute to the circadian expression of let-7 through the 20-OH ecdysteroid receptor. Thus, we find a regulatory cycle involving PTTH, a direct target of CLOCK, and PTTH-driven miRNA let-7.

Significant fluctuations in ecdysteroid receptor gene (EcR) expression in relation to seasons of molt and reproduction in the grapsid crab, Metopograpsus messor (Brachyura: Decapoda)

Metopograpsus messor, a brachyuran crab inhabiting the estuaries of North Kerala (India), is a prolific breeder releasing approximately 14–16 broods a year. The present paper reports the sequence information on the DNA binding domain (C domain, DBD), linker (D domain) and ligand binding domain (E domain, LBD) of M. messor ecdysteroid receptor (MmEcR) gene, the first grapsid brachyuran crab EcR examined. We have also measured MmEcR transcript levels in the ovary and the hepatopancreas throughout the annual cycle, with special reference to seasons of molt and reproduction. MmEcR expression in both the tissues is found to be at its peak (P<0.05) in late premolt crabs (January/May, molt/reproduction season); the expression levels are lowest (P<0.05) during June/July, when the females would neither molt nor reproduce (season for molt/reproduction repose). Intermediate levels of expression were found during the breeding season (August/December). Interestingly, this pattern of gene expression is in concordance with the fluctuating ecdysteroid levels of the hemolymph and Y organ secretory activity. The significant levels of fluctuation in the ovarian expression of MmEcR strongly suggest the ovary as a potential target for ecdysteroid action. A season-wise comparison of the gene expression reveals that ovarian MmEcR transcript levels are higher in breeding crabs (August/December) than the non-breeding animals (June/July), implicating a possible ecdysteroid role in reproduction in M. messor.

Identification of potential markers and sensitive tissues for low or high salinity stress in an intertidal mud crab (Macrophthalmus japonicus)

Macrophthalmus japonicus is an intertidal mud crab is an ecologically important species in Korea, can tolerate a wide range of natural and anthropogenic stressors. Environmental changes especially salinity cause physiological stress to the marine habitats. Differential gene transcription of M. japonicus tissues provided information about tissue specific responses against salinity. Five potential genes were identified and their transcription levels were determined quantitatively comparison to seawater (SW: 31 ± 1 psu) in M. japonicus gills and hepatopancreas after exposed them to different salinities. Ecdysteroid receptor (Mj-EcR), trypsin (Mj-Tryp), arginine kinase (Mj-AK), lipopolysaccharide and β-1,3-glucan binding protein (Mj-LGBP) and peroxinectin (Mj-Prx) in hepatopancreas up-regulated against different salinities. In contrast, the gills, Mj-EcR, Mj-Tryp and Mj-AK showed late up-regulated responses to 40 psu compared to SW. All genes except Mj-LGBP showed up regulation in the gills as time dependent manner. These genes can be considered as potential markers to assess responses in salinity changes. This study suggests hepatopancreas is a suitable tissue for transcriptional, biochemical and physiological responses analysis on M. japonicus in low and high salinity stress.