Ecdysteroid Synthesis Research Articles

Effect of blocking transforming growth factor-β/Activin-Myostatin signaling on the expression of ecdysteroid metabolism and responsive genes in the crustacean molting gland (Y-organ).

Molting in decapod crustaceans is controlled by ecdysteroids synthesized and secreted by the molting gland, or Y-organ (YO). The YO undergoes phenotypic changes in ecdysteroid production that drive molt cycle stage transitions; these are the basal, activated, committed, and repressed states in the intermolt, early premolt, mid- and late premolt, and postmolt stages, respectively. Reduced secretion of molt-inhibiting hormone (MIH) by a neurosecretory center in the eyestalk ganglia activates the YO and the animal transitions to early premolt. During premolt, transforming growth factor-beta (TGFβ)/Activin-Myostatin (Mstn) signaling mediates the transition of the YO from the activated to the committed state, as SB431542 blocks this transition. In the blackback land crab, Gecarcinus lateralis, the YO expresses genes involved in ecdysteroid synthesis (Gl-NADK, Gl-ALAS and Halloween genes Gl-Nvd, Gl-Spo, Gl-Phm, Gl-Dib, and Gl-Sad) and catabolism (Gl-CYP18a1); ecdysteroid signaling (ecdysteroid responsive genes Gl-EcR, Gl-RXR, Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, and Gl-Ftz-f1); and Gl-FOXO. Intermolt adult G. lateralis were induced to molt by eyestalk ablation (ESA) and injected with either dimethyl sulfoxide (DMSO) vehicle (control) or SB431542 in DMSO (experimental) at Day 0. ESA increased hemolymph ecdysteroid titer at 1, 3, and 5 days post-ESA in both control and experimental groups, indicating that SB431542 had no effect on YO activation. Ecdysteroid titer did not increase further in the experimental group at 7 and 14 days post-ESA, indicating that SB431542 prevented transition of the YO to the committed state. ESA with or without SB431542 had no effect on the mRNA levels of the eight ecdysteroid metabolism genes, seven of the eight ecdysteroid responsive genes (the only exception was Gl-E74 at 1 day post-ESA), and Gl-FOXO at 1, 3, and 5 days post-ESA. Compared to the control group, SB431542 lowered the mRNA level of Gl-Nvd at 7 and 14 days post-ESA and mRNA levels of Gl-Spo, Gl-Phm, Gl-Dib, Gl-Sad, Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-EcR, Gl-RXR, Gl-Br-C, and Gl-FOXO at 14 days post-ESA. SB431542 had no effect on the mRNA levels of Gl-HR3 Gl-HR4, Gl-E74, Gl-E75 and Gl-Ftz-f1. These results suggest that TGFβ/Activin-Mstn signaling maintains the mRNA levels of genes needed for increased ecdysteroid synthesis and signaling in the committed YO during mid- and late premolt.

Ecdysteroids in Native Trees and Callus Cultures of the Genus Vitex L. of the Flora of Russia and Vietnam.

Vitex L. is a large genus of tropical and subtropical trees used in medicine of many nations. Some species are used in gynecology due to flavonoids, iridoids, and diterpenes. Other species were shown to contain ecdysteroids and are used as tonic remedies. This study aimed to investigate content and patterns of distribution of ecdysteroids in native trees and their accumulation in in vitro cell cultures as potential biotechnological agents of Vitex species growing in remote flora of Russia and Vietnam. Targeted UPLC-MS/MS was used for the quantitation of the ecdysteroids in samples of plants and callus cultures of Vitex species. Determination of nucleotide sequences was carried out on ABI PRISM 3730xl sequencer. Phylogenetic tree was constructed using Mega v.7.0. Samples of Vitex agnus-castus L. from Russian Black Sea coast of the Caucasus, as well as samples of nine species of Vitex from Vietnam were collected for ecdysteroids quantification. Relationships between the content of ecdysteroids and the systematic position of the Vitex species were shown. Higher levels of ecdysteroids were revealed in different plant parts of Vitex canescens Kurz, Vitex pinnata L., Vitex quinata (Lour.) F.N.Williams, and Vitex tripinnata (Lour.) Merr. It has been established that species rich in ecdysteroids, as well as those containing trace amount of ecdysteroids (V. agnus-castus L., Vitex negundo L., and Vitex rotundifolia L.f.) are grouped in certain clades isolated from each other on the molecular phylogenetic tree of the genus. The most promising raw materials are the young leaves of V. tripinnata (8562 μg/g of dry biomass), as well as the bark of V. canescens (16,520 μg/g) and V. quinata (19,130 μg/g). The bark of V. canescens could be a good source of turkesterone (11α-oxyecdysteroid) that has pronounced pharmacological effects. Its content reaches up to 1400 μg/g in the bark. Discovering ability of callus cultures for synthesis of ecdysteroids allows us to outline experimental ways to increase the level of ecdysteroid biosynthesis in biotechnological systems as alternative source of ecdysteroids.

Allatostatin-C signaling in the crab, Carcinus maenas is implicated in the ecdysis programme.

The allatostatin (AST) family of neuropeptides are widespread in arthropods. The multitude of structures and pleiotropic actions reflect the tremendous morphological, physiological and behavioral diversity of the phylum. Regarding the AST-C (with C-terminal PISCF motif) peptides, crustaceans commonly express three (AST-C, -CC, -CCC) that have likely arisen by gene duplication. However, we know little regarding their physiologically relevant actions. Here we functionally characterize the cognate receptor for AST-C and AST-CC, determine tissue expression and comprehensively examine the localization of AST mRNA and peptide. We also measured peptide release, circulating titers and performed bioassays to investigate possible roles. AST-C and -CC activate a single receptor (AST-CRd), but this, and other candidate receptors were not activated by AST-CCC. Whole mount in-situ hybridization (ISH) and hybridization chain reaction (HCR) FISH complemented neuropeptide immunolocalization strategies and revealed extensive expression of AST-Cs in the central nervous system. AST-C or -CCC expressing neurons were found in the cerebral ganglia, but AST-CC expression was never observed. Of note, we infer that AST-C and -CC are co-expressed in every neuron expressing crustacean cardioactive peptide (CCAP) and bursicon (BURS); all four peptides are released from the pericardial organs during a brief period coinciding with completion of emergence. In contrast to other studies, none of the AST-C peptides exhibited any effect on ecdysteroid synthesis or cardiac activity. However, expression of the AST-C receptor on hemocytes suggests a tantalizing glimpse of possible functions in immune modulation following ecdysis, at a time when crustaceans are vulnerable to pathogens.

Dietary cholesterol promotes the growth of mud crabs, Scylla paramamosain by regulating cholesterol metabolism, enhancing ecdysteroid synthesis and molting cascade response

A seven-week feeding trial was conducted to evaluate the effects of dietary cholesterol on growth, proximate composition, cholesterol metabolism, ecdysteroids synthesis and molting cascade response in mud crab (Scylla paramamosain). Six isonitrogenous and isolipidic diets (46% protein and 11% lipid) were formulated to contain graded levels of cholesterol, with the analyzed levels of cholesterol being 0.15%, 0.32%, 0.54%, 0.72%, 0.91% and 1.53%, respectively. A total of 180 crabs (initial weight 16.26 ± 0.02g) were separated into six treatments, each treatment dividing randomly into three replicates of 10 crabs. The results showed that crabs fed the diets with 0.54%-0.91% cholesterol displayed significantly higher FW, PWG, SGR and FE than those fed the diet with 0.15% cholesterol. The hepatopancreas lipid content of the crabs with dietary 1.53% cholesterol was significantly higher than that of the crabs without dietary cholesterol supplementation. Compared to crabs fed the diet with 0.15% cholesterol, significantly higher concentrations of ApoA, HDL-C, LDL-C, VLDL-C in serum and ApoA, HDL-C in hepatopancreas were observed at crabs fed the diet with 0.72% cholesterol. The highest concentrations of 20E and MF in serum were observed at crabs fed the diet with 0.72% cholesterol, whereas the concentration of MIH in serum was significantly lower in crabs fed the diets with 0.72% and 0.91% cholesterol than those fed the diets with 0.15% and 1.53% cholesterol. The expression levels of genes related to cholesterol metabolism (srb, ldlr, lrp2, cyp27a1 and start3) in hepatopancreas were significantly higher in crabs fed the diet with 0.54% cholesterol than those fed the diet with 0.15% cholesterol. The expression levels of molting-related genes (nvd, dib, rxr, br-c, e74b, e75, ftz-f1, met) were significantly higher in crabs fed the diet with 0.54% cholesterol than crabs fed the diet with 1.53% cholesterol. The highest expression level of ecr was in 0.72% and 0.91% cholesterol group. With dietary cholesterol increasing from 0.15% to 1.53%, the expression levels of mfe and jheh significantly decreased, whereas the expression levels of famet and hr38 significantly increased. In conclusion, 0.54%-0.72% cholesterol promoted cholesterol metabolism and activated expression levels of molting-related genes, leading the better growth performance of mud crabs. Based on PWG and the R-Square (R2) of the equations, two equations of the linear truncated model (Y = 45.98X + 57.562 and Y = − 33.312X + 112.31) were used to calculate the optimal cholesterol requirement of 0.68% for juvenile mud crabs.

Corazonin Stimulates Ecdysteroid Synthesis during the Molting Process of the Swimming Crab, Portunus trituberculatus.

The neuropeptide corazonin (Crz) exerts diverse physiological effects in insects, yet its role in crustaceans remains elusive. The abundant expression of Crz receptor (CrzR) in the Y-organs of several crustaceans suggests a potential involvement of Crz in regulating ecdysteroid synthesis. In this study, we examined the effects of PtCrz on ecdysteroid synthesis during the molting period of Portunus trituberculatus through PtCrz treatments and PtCrzR silencing. Our results showed that PtCrz peptide stimulates ecdysteroid levels and the gene expression involved in ecdysteroidogenesis both in vitro and in vivo, whereas dsPtCrzR treatments had opposite effects on ecdysteroid levels and associated gene expression. Thus, our study suggests that PtCrz may modulate ecdysteroid synthesis via Y-organ-expressed PtCrzR. Furthermore, we also discovered the involvement of PtCrz/PtCrzR signaling in regulating PtETH expression. Notably, the inhibitory effect of dsPtCrzR on ecdysteroid synthesis or PtETH expression can be reversed by PtCrz treatment, suggesting the potential existence of multiple receptors for PtCrz. This study provides new insights into the function of crustacean Crz and, for the first time, elucidates the presence of a neuropeptide that can stimulate ecdysteroid synthesis in crustaceans.

E93 gene in the swimming crab, Portunus trituberculatus: Responsiveness to 20-hydroxyecdysone and methyl farnesoate and role on regulating ecdysteroid synthesis

Ecdysone-induced protein 93 (E93) is a metamorphic determinant involved in crosstalk between 20-hydroxyecdysone (20E) and juvenile hormone (JH) during the insect molting process. The present study identified the E93 gene from the swimming crab, P. trituberculatus, and found it was widely distributed in adult tissues. PtE93 mRNA levels in Y-organ and epidermis fluctuated during the molt cycle, suggesting its involvement in juvenile molting. In vitro and in vivo treatments with 20E led to an induction of PtE93 expression in Y-organ and epidermis, while we found the opposite effect for methyl farnesoate (MF) treatments, a crustacean equivalent of insect JH. We also observed that two genes for ecdysteroid biosynthesis, Spook (Spo) and Shadow (Sad), were suppressed by 20E and induced by MF, showing a negative correlation between PtE93 and ecdysteroid biosynthesis. PtE93 RNA interference (RNAi) induced Spo and Sad expression levels, elevated ecdysteroid content in culture medium, and relieved the 20E inhibitory effect on ecdysteroid synthesis, indicating an inhibitory role of PtE93 on ecdysteroid synthesis. Overall, our results suggest that E93 may be involved in the crosstalk between 20E and MF during crustacean molting, and its presence in Y-organ is closely related to ecdysteroid synthesis.

Croquemort elicits activation of the immune deficiency pathway in ticks

The immune deficiency (IMD) pathway directs host defense in arthropods upon bacterial infection. In Pancrustacea, peptidoglycan recognition proteins sense microbial moieties and initiate nuclear factor-κB-driven immune responses. Proteins that elicit the IMD pathway in non-insect arthropods remain elusive. Here, we show that an Ixodes scapularis homolog of croquemort (Crq), a CD36-like protein, promotes activation of the tick IMD pathway. Crq exhibits plasma membrane localization and binds the lipid agonist 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. Crq regulates the IMD and jun N-terminal kinase signaling cascades and limits the acquisition of the Lyme disease spirochete B. burgdorferi. Additionally, nymphs silenced for crq display impaired feeding and delayed molting to adulthood due to a deficiency in ecdysteroid synthesis. Collectively, we establish a distinct mechanism for arthropod immunity outside of insects and crustaceans.

Effects of molting on the expression of ecdysteroid biosynthesis genes in the Y-organ of the blackback land crab, Gecarcinus lateralis

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid- and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.

The MicroRNA Ame-Bantam-3p Controls Larval Pupal Development by Targeting the Multiple Epidermal Growth Factor-like Domains 8 Gene (megf8) in the Honeybee, Apis mellifera.

20-Hydroxyecdysone (20E) plays an essential role in coordinating developmental transitions in insects through responsive protein-coding genes and microRNAs (miRNAs). However, the interplay between 20E and miRNAs during insect metamorphosis is unknown. In this study, using small RNA sequencing, a comparative miRNA transcriptomic analysis in different development stages, and 20E treatment, we identified ame-bantam-3p as a key candidate miRNA involved in honeybee metamorphosis. Target prediction and in vitro dual-luciferase assays confirmed that ame-bantam-3p interacts with the coding region of the megf8 gene and promotes its expression. Meanwhile, temporal expression analysis revealed that the expression of ame-bantam-3p is higher in the larval stage than in prepupal and pupal stages, and that this expression pattern is similar to that of megf8. In vivo, we found that the mRNA level of megf8 was significantly increased after the injection of ame-bantam-3p agomir. A 20E feeding assay showed that 20E downregulated the expression of both ame-bantam-3p and its target gene megf8 on larval days five, six, and seven. Meanwhile, the injection of ame-bantam-3p agomir also reduced the 20E titer, as well as the transcript levels of essential ecdysteroid synthesis genes, including Dib, Phm, Sad, and Nvd. The transcript levels of 20E cascade genes, including EcRA, ECRB1, USP, E75, E93, and Br-c, were also significantly decreased after ame-bantam-3p agomir injection. However, ame-bantam-3p antagomir injection and dsmegf8 injection showed the opposite effect to ame-bantam-3p agomir injection. Ame-bantam-3p agomir treatment ultimately led to mortality and the failure of larval pupation by inhibiting ecdysteroid synthesis and the 20E signaling pathway. However, the expression of 20E signaling-related genes was significantly increased after megf8 knockdown, and larvae injected with dsmegf8 showed early pupation. Combined, our results indicate that ame-bantam-3p is involved in the 20E signaling pathway through positively regulating its target gene megf8 and is indispensable for larval-pupal development in the honeybee. These findings may enhance our understanding of the relationship between 20E signaling and small RNAs during honeybee development.

Crosstalk between Nutrition, Insulin, Juvenile Hormone, and Ecdysteroid Signaling in the Classical Insect Model, Rhodnius prolixus

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.

The role of ecdysteroid in the regulation of ovarian growth and oocyte maturation in Rhodnius prolixus, a vector of Chagas disease.

Rhodnius prolixus is a blood-gorging insect that is medically important since it transmits Chagas disease via feces and urine that contain the parasite Trypanosoma cruzi. In adult females, ecdysteroid hormone (20-hydroxyecdysone, 20E) is involved in the growth of the ovary and development of eggs post-blood meal (PBM). Halloween genes are essential for ecdysteroid synthesis since they code for cytochrome P450 enzymes in the ecdysteroidogenic pathway. The ecdysone receptor (EcR/USP) binds 20E, resulting in activation of ecdysone-responsive genes. We have identified and characterized the Halloween genes, and the non-Halloween gene, neverland, in the R. prolixus ovary using transcriptomic data. We used BLAST to compare transcriptome sequences with other arthropod sequences to identify similar transcripts. Our results indicate that the Halloween genes, neverland and ecdysone receptor transcripts are present in the ovaries of R. prolixus. We have quantified, by qPCR, Halloween gene transcript expression in the ovary following a blood meal. Most of the Halloween genes are upregulated during the first 3 days PBM. Knockdown of EcR, USP and shade transcripts, using RNA interference, results in a significant reduction in the number of eggs produced and a severe reduction in egg laying and hatching rate. Furthermore, knockdown of the EcR or shade transcripts altered the expression of the chorion gene transcripts Rp30 and Rp45 at day 3 and 6 PBM. These results indicate that ecdysteroids play critical roles in reproduction of female R. prolixus.

Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Molecular cloning and functional characterization of sarco/endoplasmic reticulum Ca 2+ ‐ ATPase from Chinese mitten crab ( Eriocheir sinensis )

Ca2+ plays an important role in signal transduction and regulates various physiological functions of cells. The active transport of Ca2+ from the cytosol to the lumen of the sarcoplasmic or endoplasmic reticulum occurs via sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). However, the function of this protein during moulting cycles remains unclear in Chinese mitten crab (Eriocheir sinensis). In this study, we cloned a cDNA encoding a putative SERCA protein from the Y-organs (moulting glands) in E. sinensis. The cloned Es-SERCA cDNA (4552 bp) included an open reading frame of 3003 bp encoding a 1000-residue protein with a predicted molecular mass of 109.60 kDa. The predicted Es-SERCA protein was clustered with those from other arthropods in phylogenetic analysis. Further, qPCR revealed that Es-SERCA was widely expressed in in all tested tissues with the highest expression in the muscle, followed by the eyestalks, intestines, hepatopancreas, Y-organs and gills. The stage-specific fluctuations among different tissues indicated a functional role of SERCA in calcium homeostasis. The relative mRNA level of the Es-SERCA in Y-organs during the moulting cycle was similar to the level trend of ecdysteroid concentration in haemolymph, indicating that Ca2+ signalling stimulated ecdysteroid synthesis in Y-organs. Altogether, our study provides a reference for further research on the molecular mechanisms of the moulting in E. sinensis.

Understanding molt control switches: Transcriptomic and expression analysis of the genes involved in ecdysteroidogenesis and cholesterol uptake pathways in the Y-organ of the blue crab, Callinectes sapidus.

The crustacean molting process is regulated by an interplay of hormones produced by the eyestalk ganglia and Y-organs (YO). Molt-inhibiting hormone and crustacean hyperglycemic hormone released by the sinus gland of the eyestalk ganglia (EG) inhibit the synthesis and secretion of ecdysteroid by the YO, hence regulating hemolymph levels during the molt cycle. The purpose of this study is to investigate the ecdysteroidogenesis pathway, specifically genes linked to changes in ecdysteroid levels occurring at early premolt (ePM). To this end, a reference transcriptome based on YO, EG, and hepatopancreas was de novo assembled. Two genes (cholesterol 7-desaturase Neverland and cytochrome p450 307a1-like Spook) involved in ecdysteroidogenesis were identified from the YO transcriptome using sequence comparisons and transcript abundance. Two other candidates, Hormone receptor 4 and probable cytochrome p450 49a1 potentially involved in ecdysteroidogenesis were also identified. Since cholesterol is the ecdysteroid precursor, a putative cholesterol carrier (Apolipoprotein D-like) was also examined to understand if cholesterol uptake coincided with the increase in the ecdysteroid levels at the ePM stage. The expression level changes of the five candidate genes in the YO were compared between intermolt (IM) and induced ePM (iePM) stages using transcriptomic analysis. Expression analysis using qPCR were carried out at IM, iePM, and normal ePM. The increase in Spook and Neverland expression in the YO at the ePM was accompanied by a concomitant rise in ecdysteroid levels. The data obtained from iePM stage were congruent with those obtained from the normal ePM stage of intact control animals. The present findings support the role of Halloween genes in the ecdysteroidogenesis and molt cycle in the blue crab, Callinectes sapidus.

Role of Endocrine System in the Regulation of Female Insect Reproduction.

Simple SummaryThe abundance of insects indicates that they are one of the most adaptable forms of life on earth. Genetic, physiological, and biochemical plasticity and the extensive reproductive capacity of insects are some of the main reasons for such domination. The endocrine system has been known to regulate different stages of physiological and developmental processes such as metabolism, metamorphosis, growth, molting, and reproduction. However, in this review, we focus on those aspects of the endocrine system that regulate female insect reproduction. The proper understanding of the endocrine system will help us to better understand the insect reproductive system as well as to develop new strategies to control the insect pest population. The juvenile hormone analogs and molting hormone analogs have been widely used to control the insect pests. Such insect growth regulators are usually more specific and cause little harm to the beneficial organisms. Therefore, a proper understanding of these signaling pathways as well as their interaction with each other and other signaling pathways is very crucial. Further, the interaction of microbiome with the endocrine system is also discussed.The proper synthesis and functioning of ecdysteroids and juvenile hormones (JHs) are very important for the regulation of vitellogenesis and oogenesis. However, their role and function contrast among different orders, and even in the same insect order. For example, the JH is the main hormone that regulates vitellogenesis in hemimetabolous insect orders, which include Orthoptera, Blattodea, and Hemiptera, while ecdysteroids regulate the vitellogenesis among the insect orders of Diptera, some Hymenoptera and Lepidoptera. These endocrine hormones also regulate each other. Even at some specific stage of insect life, they positively regulate each other, while at other stages of insect life, they negatively control each other. Such positive and negative interaction of 20-hydroxyecdysone (20E) and JH is also discussed in this review article to better understand the role of these hormones in regulating the reproduction. Therefore, the purpose of the present review is to deeply understand the complex interaction of endocrine hormones with each other and with the insulin signaling pathway. The role of microbiomes in the regulation of the insect endocrine system is also reviewed, as the endocrine hormones are significantly affected by the compounds produced by the microbiota.

Signaling Pathways That Regulate the Crustacean Molting Gland.

A pair of Y-organs (YOs) are the molting glands of decapod crustaceans. They synthesize and secrete steroid molting hormones (ecdysteroids) and their activity is controlled by external and internal signals. The YO transitions through four physiological states over the molt cycle, which are mediated by molt-inhibiting hormone (MIH; basal state), mechanistic Target of Rapamycin Complex 1 (mTORC1; activated state), Transforming Growth Factor-β (TGFβ)/Activin (committed state), and ecdysteroid (repressed state) signaling pathways. MIH, produced in the eyestalk X-organ/sinus gland complex, inhibits the synthesis of ecdysteroids. A model for MIH signaling is organized into a cAMP/Ca2+-dependent triggering phase and a nitric oxide/cGMP-dependent summation phase, which maintains the YO in the basal state during intermolt. A reduction in MIH release triggers YO activation, which requires mTORC1-dependent protein synthesis, followed by mTORC1-dependent gene expression. TGFβ/Activin signaling is required for YO commitment in mid-premolt. The YO transcriptome has 878 unique contigs assigned to 23 KEGG signaling pathways, 478 of which are differentially expressed over the molt cycle. Ninety-nine contigs encode G protein-coupled receptors (GPCRs), 65 of which bind a variety of neuropeptides and biogenic amines. Among these are putative receptors for MIH/crustacean hyperglycemic hormone neuropeptides, corazonin, relaxin, serotonin, octopamine, dopamine, allatostatins, Bursicon, ecdysis-triggering hormone (ETH), CCHamide, FMRFamide, and proctolin. Contigs encoding receptor tyrosine kinase insulin-like receptor, epidermal growth factor (EGF) receptor, and fibroblast growth factor (FGF) receptor and ligands EGF and FGF suggest that the YO is positively regulated by insulin-like peptides and growth factors. Future research should focus on the interactions of signaling pathways that integrate physiological status with environmental cues for molt control.

Effects of Pergularia tomentosa active fraction on the oogenesis of the desert locust, Schistocerca gregaria: ovarian biochemical composition, and effects on the ecdysteroid pathway

AbstractFractionation of the methanolic extract of Pergularia tomentosa, using column chromatography, resulted in the separation of 10 fractions and only the active one (that gave mortality >70%) was kept and denoted the active fraction (AFPt). Adult female Schistocerca gregaria were treated with two concentrations of AFPt, 0.24% and 0.96%, to investigate the effect on the female reproductive potential. Both concentrations of AFPt significantly increased the duration between adult emergence and first oviposition, as well as affected fecundity and fertility. Furthermore, the effects of AFPt on vitellogenesis and ecdysteroid titres were examined. Biochemical analyses revealed a significant decrease in protein, lipid and carbohydrate contents in ovaries. This study also showed that AFPt induced a significant decrease in the ovarian ecdysteroids levels, as well as the haemolymph ecdysteroid titres. The transcript levels of the ecdysteroid synthesis enzymes Spook and Shadow were significantly decreased when treated with the highest concentration of AFPt, while transcript levels of the ecdysteroid synthesis enzymes Phantom and Disembodied increased significantly in animals treated with AFPt. Treatment with AFPt also resulted in a significantly higher transcript level of both the ecdysone receptor (EcR) and its partner ultraspiracle/retinoid‐X‐receptor (USP/RXR).

Discovery of Bioactive Compounds from Traditionally used Medicinal Plants: A Profile of Nilufar Z. Mamadalieva

Discovery of Bioactive Compounds from Traditionally used Medicinal Plants: A Profile of Nilufar Z. Mamadalieva

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world’s poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

De novo transcriptome assembly and functional annotation for Y-organs of the blue crab (Callinectes sapidus), and analysis of differentially expressed genes during pre-molt

Blue crabs (Callinectes sapidus) undergo incremental growth involving the shedding (molting) of the old exoskeleton, and subsequent expansion and re-calcification of the newly synthesized one. The cellular events that lead to molting are triggered by steroid hormones termed ecdysteroids released from Y-organs, paired endocrine glands located in the anterior cephalothorax. The regulatory pathways leading to increased synthesis and release of ecdysteroids are not fully understood, and no transcriptome has yet been published for blue crab Y-organs. Here we report de novo transcriptome assembly and annotation for adult blue crab Y-organs, and differential gene expression (DGE) analysis between Y-organs of intermolt and premolt crabs. After trimming and quality assessment, a total of 91,819,458 reads from four cDNA libraries were assembled using Trinity to form the reference transcriptome. Trinity produced a total of 171,530 contigs coding for 150,388 predicted genes with an average contig length of 613 and an N50 of 940. Of these, TransDecoder predicted 31,661 open reading frames (ORFs), and 10,210 produced non-redundant blastx results through Trinotate annotation. Genes involved in multiple cell signaling pathways, including Ca2+ signaling, cGMP signaling, cAMP signaling, and mTOR signaling were present in the annotated reference transcriptome. DGE analysis showed in premolt Y-organs up-regulated genes involved in energy production, cholesterol metabolism, and exocytosis. The results provide insights into the transcriptome of blue crab Y-organs during a natural (rather than experimentally induced) molting cycle, and constitute a step forward in understanding the cellular mechanisms that underlie stage-specific changes in the synthesis and secretion of ecdysteroids by Y-organs.