The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide).

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Expression of brain-derived neurotrophic factor and formation of migrasome increases in the glioma cells induced by the adipokinetic hormone.

OBJECTIVE: It has been previously shown that brain-derived neurotrophic factor is linked with various types of cancer. Brain-derived neurotrophic factor is found to be highly expressed in multiple human cancers and associated with tumor growth, invasion, and metastasis. Adipokinetic hormones are functionally related to the vertebrate glucagon, as they have similar functionalities that manage the nutrient-dependent secretion of these two hormones. Migrasomes are new organelles that contain numerous small vesicles, which aid in transmitting signals between the migrating cells. Therefore, the aim of this study was to investigate the effects of Anax imperator adipokinetic hormone on brain-derived neurotrophic factor expression and ultrastructure of cells in the C6 glioma cell line. METHODS: The rat C6 glioma cells were treated with concentrations of 5 and 10 Anax imperator adipokinetic hormone for 24 h. The effects of the Anax imperator adipokinetic hormone on the migrasome formation and brain-derived neurotrophic factor expression were analyzed using immunocytochemistry and transmission electron microscope. RESULTS: The rat C6 glioma cells of the 5 and 10 µM Anax imperator adipokinetic hormone groups showed significantly high expressions of brain-derived neurotrophic factor and migrasomes numbers, compared with the control group. CONCLUSION: A positive correlation was found between the brain-derived neurotrophic factor expression level and the formation of migrasome, which indicates that the increased expression of brain-derived neurotrophic factor and the number of migrasomes may be involved to metastasis of the rat C6 glioma cell line induced by the Anax imperator adipokinetic hormone. Therefore, the expression of brain-derived neurotrophic factor and migrasome formation may be promising targets for preventing tumor proliferation, invasion, and metastasis in glioma.

Halloween genes are expressed with a circadian rhythm during development in prothoracic glands of the insect RHODNIUS PROLIXUS.

We analyse the developmental and circadian profiles of expression of the genes responsible for ecdysteroidogenesis (Halloween genes) in the PGs of Rhodnius prolixus throughout larval-adult development. Extensive use of in vitro techniques enabled multiple different parameters to be measured in individual PGs. Expression of disembodied and spook closely paralleled the ecdysteroid synthesis of the same PGs, and the ecdysteroid titre in vivo, but with functionally significant exceptions. Various tissues other than PGs expressed one, both or neither genes. Both gonads express both genes in pharate adults (larvae close to ecdysis). Both genes were expressed at low, but significant, levels in UF Rhodnius, raising questions concerning how developmental arrest is maintained in UF animals. IHC confirmed the subcellular localisation of the coded proteins. Gene knockdown suppressed transcription of both genes and ecdysteroid synthesis, with spook apparently regulating the downstream gene disembodied. Transcription of both genes occurred with a daily rhythm (with peaks at night) that was confirmed to be under circadian control using aperiodic conditions. The complex behaviour of the rhythm in LL implied two anatomically distinct oscillators regulate this transcription rhythm. First, the circadian clock in the PGs and second, the circadian rhythm of of Rhodnius PTTH which is released rhythmically from the brain under control of the circadian clock therein, both of which were described previously. We conclude ecdysteroidogenesis in Rhodnius PGs employs a similar pathway as other insects, but its control is complex, involving mechanisms both within and outside the PGs.

The proline effect and the tryptophan immonium ion assist in de novo sequencing of adipokinetic hormones.

Adipokinetic hormones (AKHs) in Arthopoda are characterized by special sequence features including limited choices of amino acid residues in certain positions, such as Trp in position 8. Over 100 different AKHs have been described, but de novo sequencing of novel peptide hormones can be a challenge. In a project of analyzing corpora cardiaca extracts from two fly species, two different moths, a termite and a beetle for their AKHs, we noted specific patterns in the fragmentation spectra of octapeptides in electrospray Q-TOF experiments resulting from the presence of Pro in position 6. The preference for cleavage N-terminal to Pro residues created an abundant y3″-ion, which, in conjunction with the two b-ions resulting from the fragmentation before and after Pro, provided a marker pattern. As Pro6 occurs in about 61% of known AKHs, this information is highly relevant for sequence elucidation. Moreover, the default presence of Trp8 allowed the use of its immonium ion for AKH candidate identification. In addition, we assembled the known AKH sequences from the literature and sequences of AKH-type format found in the Uniprot database in a single online resource. These efforts assisted in the analysis workflow and led to the assignment of two novel AKHs and evidence for the presence of Melme-CC and Dorpa-AKH in the corpus cardiacum of the scarab beetle Sinodendron cylindricum.

Signaling in cAMP-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

In the present study, we investigated downstream pathways of cyclic adenosine monophosphate (cAMP) signaling (which is related to prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis) in Bombyx mori prothoracic glands (PGs). Results showed that treatment with either dibutyryl cAMP (dbcAMP) or 1-methyl-3-isobutylxanthine (MIX) inhibited phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and activated phosphorylation of the translational repressor, 4E-binding protein (4E-BP), a marker of target of rapamycin (TOR) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside, AICAR) increased dbcAMP-inhibited AMPK phosphorylation and blocked dbcAMP-stimulated phosphorylation of 4E-BP, indicating that inhibition of AMPK phosphorylation lies upstream of dbcAMP-stimulated TOR signaling. Treatment of PGs with dbcAMP and MIX also stimulated phosphorylation of a 37-kDa protein, as recognized by a protein kinase C (PKC) substrate antibody, indicating that cAMP activates PKC signaling. Treatment with either LY294002 or AICAR did not affect dbcAMP-stimulated phosphorylation of the PKC-dependent 37-kDa protein, indicating that cAMP-stimulated PKC signaling is not related to phosphoinositide 3-kinase (PI3K) or AMPK. In addition, dbcAMP-stimulated ecdysteroidogenesis in PGs was partially inhibited by pretreatment with either LY294002, AICAR, or calphostin C. From these results, we concluded that AMPK/TOR/4E-BP and PKC pathways are involved in ecdysteroidogenesis of PGs stimulated by cAMP signaling in B. mori.

Comparative analysis of adipokinetic hormones and their receptors in Blattodea reveals novel patterns of gene evolution.

Adipokinetic hormone (AKH) is a neuropeptide produced in the insect corpora cardiaca that plays an essential role in mobilising carbohydrates and lipids from the fat body to the haemolymph. AKH acts by binding to a rhodopsin-like G protein-coupled receptor (GPCR), the adipokinetic hormone receptor (AKHR). In this study, we tackle AKH ligand and receptor gene evolution as well as the evolutionary origins of AKH gene paralogues from the order Blattodea (termites and cockroaches). Phylogenetic analyses of AKH precursor sequences point to an ancient AKH gene duplication event in the common ancestor of Blaberoidea, yielding a new group of putative decapeptides. In total, 16 different AKH peptides from 90 species were obtained. Two octapeptides and seven putatively novel decapeptides are predicted for the first time. AKH receptor sequences from 18 species, spanning solitary cockroaches and subsocial wood roaches as well as lower and higher termites, were subsequently acquired using classical molecular methods and in silico approaches employing transcriptomic data. Aligned AKHR open reading frames revealed 7 highly conserved transmembrane regions, a typical arrangement for GPCRs. Phylogenetic analyses based on AKHR sequences support accepted relationships among termite, subsocial (Cryptocercus spp.) and solitary cockroach lineages to a large extent, while putative post-translational modification sites do not greatly differ between solitary and subsocial roaches and social termites. Our study provides important information not only for AKH and AKHR functional research but also for further analyses interested in their development as potential candidates for biorational pest control agents against invasive termites and cockroaches.

The unique C-mannosylated hypertrehalosemic hormone of Carausius morosus: Identity, release, and biological activity.

Previous studies had shown that the corpora cardiaca (CC) of the Indian stick insect, Carausius morosus, synthesizes two hypertrehalosemic hormones (HrTHs)-decapeptides which differ in the way that the chromatographically less-hydrophobic form, code-named Carmo-HrTH-I, is modified by an unique C-mannosylated tryptophan residue at position 8. The availability of milligram amounts of this modified peptide in synthetic form now makes it possible to study physico-chemical and physiological properties. This study revealed that the synthetic peptide co-elutes with the natural peptide from the CC chromatographically, is heat stable for at least 30 min at 100°C, and causes hyperlipemia in acceptor locusts (a heterologous bioassay) and hypertrehalosemia in ligated stick insects (conspecific bioassay). In vitro incubation of Carmo-HrTH-I together with stick insect hemolymph (a natural source of peptidases) demonstrated clearly via chromatographic separation that the C-mannosylated Trp bond is stable and is not broken down to Carmo-HrTH-II (the more-hydrophobic decapeptide with an unmodified Trp residue). This notwithstanding, breakdown of Carmo-HrTH-I did take place, and the half-life of the compound was calculated as about 5 min. Finally, the natural peptide is releasable when CC are treated in vitro with a depolarizing saline (high potassium concentration) suggesting its role as true HrTHs in the stick insect. In conclusion, the results indicate that Carmo-HrTH-I which is synthesized in the CC is released into the hemolymph, binds to a HrTH receptor in the fat body, activates the carbohydrate metabolism pathway and is quickly inactivated in the hemolymph by (an) as yet unknown peptidase(s).

Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori.

The circadian clock plays an integral role in hormone biosynthesis and secretion. However, how the circadian clock precisely coordinates hormonal homeostasis to maintain normal animal development remains unclear. Here, we show that knocking out the core clock gene Cryptochrome 1 (Cry1) significantly delays the developmental time in Bombyx mori. This study focuses on the ecdysone and juvenile hormone signalling pathways of fifth instar larvae with the longest developmental time delay. We found that the mutant reduced prothoracicotropic hormone synthesis in the brain, and could not produce sufficient ecdysone in the prothoracic gland, resulting in a delayed peak of 20-hydroxyecdysone titre in the hemolymph of fifth instar larvae, prolonging developmental time. Moreover, further investigation revealed that the mutant enhanced juvenile hormone biosynthesis and signalling pathway and that this higher juvenile hormone titre also resulted in prolonged developmental time in fifth instar larvae. Our results provide insights into the molecular mechanisms by which the circadian clock regulates animal development by maintaining hormonal homeostasis.

Expression of tyrosine phosphatases in relation to PTTH-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori.

Protein tyrosine phosphorylation is a reversible, dynamic process regulated by the activities of tyrosine kinases and tyrosine phosphatases. Although the involvement of tyrosine kinases in the prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs) has been documented, few studies have been conducted on the involvement of protein tyrosine phosphatases (PTPs) in PTTH-stimulated ecdysteroidogenesis. In the present study, we investigated the correlation between PTPs and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that the basal PTP enzymatic activities exhibited development-specific changes during the last larval instar and pupation stage, with high activities being detected during the later stages of the last larval instar. PTP enzymatic activity was stimulated by PTTH treatment both in vitro and in vivo. Pretreatment with phenylarsine oxide (PAO) and benzylphosphonic acid (BPA), two chemical inhibitors of tyrosine phosphatase, reduced PTTH-stimulated enzymatic activity. Determination of ecdysteroid secretion showed that treatment with PAO and BPA did not affect basal ecdysteroid secretion, but greatly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated PTP activity is indeed involved in ecdysteroid secretion. PTTH-stimulated phosphorylation of the extracellular signal-regulated kinase (ERK) and 4E-binding protein (4E-BP) was partially inhibited by pretreatment with either PAO or BPA, indicating the potential link between PTPs and phosphorylation of ERK and 4E-BP. In addition, we also found that in vitro treatment with 20-hydroxyecdysone did not affect PTP enzymatic activity. We further investigated the expressions of two important PTPs (PTP 1B (PTP1B) and the phosphatase and tension homologue (PTEN)) in Bombyx PGs. Our immunoblotting analysis showed that B. mori PGs contained the proteins of PTP1B and PTEN, with PTP1B protein undergoing development-specific changes. Protein levels of PTP1B and PTEN were not affected by PTTH treatment. The gene expression levels of PTP1B and PTEN showed development-specific changes. From these results, we suggest that PTTH-regulated PTP signaling may crosstalk with ERK and target of rapamycin (TOR) signaling pathways and is a necessary component for stimulation of ecdysteroid secretion.

Functional insight and cell-specific expression of the adipokinetic hormone/corazonin-related peptide in the human disease vector mosquito, Aedes aegypti.

The adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic hormone (AKH). Unlike the AKH and CRZ signaling systems that are widely known for their roles in the mobilization of energy substrates and stress responses, respectively, the main role of ACP and its receptor (ACPR) remains unclear in most arthropods. The current study aimed to localize the distribution of ACP in the nervous system and provide insight into its physiological roles in the disease vector mosquito, Aedes aegypti. Immunohistochemical analysis and fluorescence in situ hybridization localized the ACP peptide and transcript within a number of cells in the central nervous system, including two pairs of laterally positioned neurons in the protocerebrum of the brain and a few ventrally localized neurons within the pro- and mesothoracic regions of the fused thoracic ganglia. Further, extensive ACP-immunoreactive axonal projections with prominent blebs and varicosities were observed traversing the abdominal ganglia. Given the prominent enrichment of ACPR expression within the abdominal ganglia of adult A. aegypti mosquitoes as determined previously, the current results indicate that ACP may function as a neurotransmitter and/or neuromodulator facilitating communication between the brain and posterior regions of the nervous system. In an effort to elucidate a functional role for ACP signaling, biochemical measurement of energy substrates in female mosquitoes revealed a reduction in abdominal fat body in response to ACP that matched the actions of AKH, but interestingly, a corresponding hypertrehalosaemic effect was only found in response to AKH since ACP did not influence circulating carbohydrate levels. Comparatively, both ACP and AKH led to a significant increase in haemolymph carbohydrate levels in male mosquitoes while both peptides had no influence on their glycogen stores. Neither ACP nor AKH influenced circulating or stored lipid levels in both male and female mosquitoes. Collectively, these results reveal ACP signaling in mosquitoes may have complex sex-specific actions, and future research should aim to expand knowledge on the role of this understudied neuropeptide.

The prothoracicotropic hormone (PTTH) of Rhodnius prolixus (Hemiptera) is noggin-like: Molecular characterisation, functional analysis and evolutionary implications.

Prothoracicotropic hormone (PTTH) is a central regulator of insect development that regulates the production of the steroid moulting hormones (ecdysteroids) from the prothoracic glands (PGs). Rhodnius PTTH was the first brain neurohormone discovered in any animal almost 100 years ago but has eluded identification and no homologue of Bombyx mori PTTH occurs in its genome. Here, we report Rhodnius PTTH is the first noggin-like PTTH found. It differs in important respects from known PTTHs and is the first PTTH from the Hemimetabola (Exopterygota) to be fully analysed. Recorded PTTHs are widespread in Holometabola but close to absent in hemimetabolous orders. We concluded Rhodnius PTTH likely differed substantially from the known ones. We identified one Rhodnius gene that coded a noggin-like protein (as defined by Molina et al., 2009) that had extensive similarities with known PTTHs but also had two additional cysteines. Sequence and structural analysis showed known PTTHs are closely related to noggin-like proteins, as both possess a growth factor cystine knot preceded by a potential cleavage site. The gene is significantly expressed only in the brain, in a few cells of the dorsal protocerebrum. We vector-expressed the sequence from the potential cleavage site to the C-terminus. This protein was strongly steroidogenic on PGs in vitro. An antiserum to the protein removed the steroidogenic protein released by the brain. RNAi performed on brains in vitro showed profound suppression of transcription of the gene and of production and release of PTTH and thus of ecdysteroid production by PGs. In vivo, the gene is expressed throughout development, in close synchrony with PTTH release, ecdysteroid production by PGs and the ecdysteroid titre. The Rhodnius PTTH monomer is 17kDa and immunoreactive to anti-PTTH of Bombyx mori (a holometabolan). Bombyx PTTH also mildly stimulated Rhodnius PGs. The two additional cysteines form a disulfide at the tip of finger 2, causing a loop of residues to protrude from the finger. A PTTH variant without this loop failed to stimulate PGs, showing the loop is essential for PTTH activity. It is considered that PTTHs of Holometabola evolved from a noggin-like protein in the ancestor of Holometabola and Hemiptera, c.400ma, explaining the absence of holometabolous-type PTTHs from hemimetabolous orders and the differences of Rhodnius PTTH from them. Noggin-like proteins studied from Hemiptera to Arachnida were homologous with Rhodnius PTTH and may be common as PTTHs or other hormones in lower insects.

Bombyxin-stimulated ecdysteroidogenesis in relation to sugar transporter/trehalase expressions in Bombyx prothoracic glands.

Our previous studies showed that bombyxin stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs) during a long-term incubation period in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. In the present study, we further investigated the downstream signaling cascade in bombyxin-stimulated PGs. Our results showed that upon treatment with bombyxin, expression levels of the sugar transport 1 (St1) and St4 genes and trehalase 1 (Treh1) gene, but not ecdysteroid biosynthesis genes were greatly enhanced compared to the controls. Treatment with LY294002 (an inhibitor of PI3K) reduced the enhanced St1 and Treh1 expression levels, clearly indicating the involvement of PI3K. Treatment with 1 mM of mpV(pic) (a potent inhibitor of protein phosphotyrosine phosphatase and activator of insulin receptor (InR) kinase) also stimulated expression levels of the St1 and Treh1 genes, thus further confirming the involvement of the InR. Determining Treh enzyme activity showed that bombyxin treatment stimulated Treh enzyme activity in time- and PI3K-dependent manners. Validamycin A (a Treh inhibitor) blocked bombyxin-stimulated Treh enzyme activity and partly decreased bombyxin-stimulated ecdysteroidogenesis. A specific sugar transport inhibitor (cytochalasin B) and a glycolysis inhibitor (2-deoxy-D-glucose (2-DG)) also reduced bombyxin-stimulated ecdysteroidogenesis. Taken together, these results indicated that increased expressions of Sts and Treh1 and enhanced Treh enzyme activity downstream of InR/PI3K are involved in bombyxin-stimulated ecdysteroidogenesis in B. mori PGs.

Time- and temperature-dependent dynamics of prothoracicotropic hormone and ecdysone sensitivity co-regulate pupal diapause in the green-veined white butterfly Pieris napi.

Diapause, a general shutdown of developmental pathways, is a vital adaptation allowing insects to adjust their life cycle to adverse environmental conditions such as winter. Diapause in the pupal stage is regulated by the major developmental hormones prothoracicotropic hormone (PTTH) and ecdysone. Termination of pupal diapause in the butterfly Pieris napi depends on low temperatures; therefore, we study the temperature-dependence of PTTH secretion and ecdysone sensitivity dynamics throughout diapause, with a focus on diapause termination. While PTTH is present throughout diapause in the cell bodies of two pairs of neurosecretory cells in the brain, it is absent in the axons, and the PTTH concentration in the haemolymph is significantly lower during diapause than during post diapause development, indicating that the PTTH signaling is reduced during diapause. The sensitivity of pupae to ecdysone injections is dependent on diapause stage. While pupae are sensitive to ecdysone during early diapause initiation, they gradually lose this sensitivity and become insensitive to non-lethal concentrations of ecdysone about 30 days into diapause. At low temperatures, reflecting natural overwintering conditions, diapause termination propensity after ecdysone injection is precocious compared to controls. In stark contrast, at high temperatures reflecting late summer and early autumn conditions, sensitivity to ecdysone does not return. Thus, here we show that PTTH secretion is reduced during diapause, and additionally, that the low ecdysone sensitivity of early diapause maintenance is lost during termination in a temperature dependent manner. The link between ecdysone sensitivity and low-temperature dependence reveals a putative mechanism of how diapause termination operates in insects that is in line with adaptive expectations for diapause.

Phytoecdysteroids: Distribution, Structural Diversity, Biosynthesis, Activity, and Crosstalk with Phytohormones.

Phytoecdysteroids (PEs) are naturally occurring polyhydroxylated compounds with a structure similar to that of insect molting hormone and the plant hormone brassinosteroids. PEs have a four-ringed skeleton composed of 27, 28, 29, or 30 carbon atoms (derived from plant sterols). The carbon skeleton of ecdysteroid is known as cyclopentanoperhydrophenanthrene and has a ß-sidechain on C-17. Plants produce PEs via the mevalonate pathway with the help of the precursor acetyl-CoA. PEs are found in algae, fungi, ferns, gymnosperms, and angiosperms; more than 500 different PEs are found in over 100 terrestrial plants. 20-hydroxyecdysone is the most common PE. PEs exhibit versatile biological roles in plants, invertebrates, and mammals. These compounds contribute to mitigating biotic and abiotic stresses. In plants, PEs play a potent role in enhancing tolerance against insects and nematodes via their allelochemical activity, which increases plant biological and metabolic responses. PEs promote enzymatic and non-enzymatic antioxidant defense systems, which decrease reactive oxygen species in the form of superoxide radicals and hydroxyl radicals and reduce malondialdehyde content. PEs also induce protein biosynthesis and modulate carbohydrate and lipid synthesis. In humans, PEs display biological, pharmacological, and medicinal properties, such as anti-diabetic, antioxidant, anti-microbial, hepatoprotective, hypoglycemic, anti-cancer, anti-inflammatory, antidepressant, and tissue differentiation activity.

Molecular and functional analysis of eclosion hormone-like gene involved in post-eclosion behavior in a beetle.

Eclosion hormone (EH) is a neurohormone that plays a key role in the regulation of insect pre-ecdysis behavior at the end of each molt. Previous research has reported more than one EH gene was found in certain insects, with their functions and mechanisms still unclear. Here, aside from the classical EH gene orthologous group, we characterized another novel orthologous cluster of eclosion hormone-like (EHL) genes in Arthropoda and investigated the roles of EHL during development in Tribolium castaneum. T. castaneum EHL (TcEHL) shows high expression levels during pupal - adult development, which also positively responded to 20-hydroxyecdysone (20E) treatment as well as RNA interference (RNAi) of ECR (20E nuclear receptor). Knockdown of TcEHL prevented the tanning of the adult cuticle and caused lethal phenotypes. Further analysis indicated that knockdown of TcEHL could upregulate expression levels of the classical TcEH, and downregulate the ecdysis behavior cascade genes, as well as tanning pathway enzymes. This suggests a critical role for TcEHL in adult eclosion and cuticle tanning. In addition, our data indicated that TcEHL is responsible for the female reproduction process. Taken together, these results suggest that TcEHL has specific roles in adult cuticle tanning during the post-eclosion process and female reproduction. They also suggest that EHL gene is the ancestral copy for the EH family and it is functionally shuffled by synfunctionalization.

Modulation of Metabolic Hormone Signaling via a Circadian Hormone and Biogenic Amine in Drosophila melanogaster.

In insects, adipokinetic hormone is the primary hormone responsible for the mobilization of stored energy. While a growing body of evidence has solidified the role of adipokinetic hormone (AKH) in modulating the physiological and behavioral responses to metabolic stress, little is known about the upstream endocrine circuit that directly regulates AKH release. We evaluated the AKH-producing cell (APC) transcriptome to identify potential regulatory elements controlling APC activity and found that a number of receptors showed consistent expression levels, including all known dopamine receptors and the pigment dispersing factor receptor (PDFR). We tested the consequences of targeted genetic knockdown and found that APC limited expression of RNAi elements corresponding to each dopamine receptor and caused a significant reduction in survival under starvation. In contrast, PDFR knockdown significantly extended lifespan under starvation, whereas expression of a tethered PDF in APCs resulted in significantly shorter lifespans. These manipulations caused various changes in locomotor activity under starvation. We used live-cell imaging to evaluate the acute effects of the ligands for these receptors on APC activation. Dopamine application led to a transient increase in intracellular calcium in a trehalose-dependent manner. Furthermore, coapplication of dopamine and ecdysone led to a complete loss of this response, suggesting that these two hormones act antagonistically. We also found that PDF application led to an increase in cAMP in APCs and that this response was dependent on expression of the PDFR in APCs. Together, these results suggest a complex circuit in which multiple hormones act on APCs to modulate metabolic state.

Molecular identification and lipid mobilization role of adipokinetic hormone receptor in Spodoptera litura (F.).

Energy homeostasis is essential for organisms to maintain fluctuation in energy accumulation, mobilization. Lipids as the main energy reserve in insects, their metabolism is under the control of many physiological program. This study aimed to determine whether the adipokinetic hormone receptor (AKHR) was involved in the lipid mobilization in the Spodoptera litura. A full-length cDNA encoding AKHR was isolated from S. litura. The SlAKHR protein has a conserved seven-transmembrane domain which is the character of a putative G protein receptor. Expression profile investigation revealed that SlAKHR mRNA was highly expressed in immatural stage and abundant in fat body in newly emerged female adults. Knockdown of SlAKHR expression was achieved through RNAi by injecting double-stranded RNA (dsRNA) into the 6th instar larvae. The content of triacylgycerol (TAG) in the fat body increased significantly after the SlAKHR gene was knockdown. And decrease of TAG releasing to hemolymph with increase of free fatty acid (FFA) in hemolymph were observed when the SlAKHR gene was knowned-down. In addition, lipid droplets increased in fat body was also found. These results suggested that SlAKHR is critical for insects to regulate lipids metabolism.

A nutrient-specific gut hormone arbitrates between courtship and feeding.

Animals must set behavioural priority in a context-dependent manner and switch from one behaviour to another at the appropriate moment1-3. Here we probe the molecular and neuronal mechanisms that orchestrate the transition from feeding to courtship in Drosophila melanogaster. We find that feeding is prioritized over courtship in starved males, and the consumption of protein-rich food rapidly reverses this order within a few minutes. At the molecular level, a gut-derived, nutrient-specific neuropeptide hormone-Diuretic hormone 31 (Dh31)-propels a switch from feeding to courtship. We further address the underlying kinetics with calcium imaging experiments. Amino acids from food acutely activate Dh31+ enteroendocrine cells in the gut, increasing Dh31 levels in the circulation. In addition, three-photon functional imaging of intact flies shows that optogenetic stimulation of Dh31+ enteroendocrine cells rapidly excites a subset of brain neurons that express Dh31 receptor (Dh31R). Gut-derived Dh31 excites the brain neurons through the circulatory system within a few minutes, in line with the speed of the feeding-courtship behavioural switch. At the circuit level, there are two distinct populations of Dh31R+ neurons in the brain, with one population inhibiting feeding through allatostatin-C and the other promoting courtship through corazonin. Together, our findings illustrate a mechanism by which the consumption of protein-rich food triggers the release of a gut hormone, which in turn prioritizes courtship over feeding through two parallel pathways.

The gut hormone Allatostatin C/Somatostatin regulates food intake and metabolic homeostasis under nutrient stress.

The intestine is a central regulator of metabolic homeostasis. Dietary inputs are absorbed through the gut, which senses their nutritional value and relays hormonal information to other organs to coordinate systemic energy balance. However, the gut-derived hormones affecting metabolic and behavioral responses are poorly defined. Here we show that the endocrine cells of the Drosophila gut sense nutrient stress through a mechanism that involves the TOR pathway and in response secrete the peptide hormone allatostatin C, a Drosophila somatostatin homolog. Gut-derived allatostatin C induces secretion of glucagon-like adipokinetic hormone to coordinate food intake and energy mobilization. Loss of gut Allatostatin C or its receptor in the adipokinetic-hormone-producing cells impairs lipid and sugar mobilization during fasting, leading to hypoglycemia. Our findings illustrate a nutrient-responsive endocrine mechanism that maintains energy homeostasis under nutrient-stress conditions, a function that is essential to health and whose failure can lead to metabolic disorders.

The effect of the brood and the queen on early gene expression in bumble bee workers' brains.

Worker reproduction in social insects is often regulated by the queen, but can be regulated by the brood and nestmates, who may use different mechanisms to induce the same outcomes in subordinates. Analysis of brain gene expression patterns in bumble bee workers (Bombus impatiens) in response to the presence of the queen, the brood, both or neither, identified 18 differentially expressed genes, 17 of them are regulated by the queen and none are regulated by the brood. Overall, brain gene expression differences in workers were driven by the queen's presence, despite recent studies showing that brood reduces worker egg laying and provides context to the queen pheromones. The queen affected important regulators of reproduction and brood care across insects, such as neuroparsin and vitellogenin, and a comparison with similar datasets in the honey bee and the clonal raider ant revealed that neuroparsin is differentially expressed in all species. These data emphasize the prominent role of the queen in regulating worker physiology and behavior. Genes that serve as key regulators of workers' reproduction are likely to play an important role in the evolution of sociality.