The function of juvenile-adult transition axis in female sexual receptivity of Drosophila melanogaster.

Female sexual receptivity is essential for reproduction of a species. Neuropeptides play the main role in regulating female receptivity. However, whether neuropeptides regulate female sexual receptivity during the neurodevelopment is unknown. Here, we found the peptide hormone prothoracicotropic hormone (PTTH), which belongs to the insect PG (prothoracic gland) axis, negatively regulated virgin female receptivity through ecdysone during neurodevelopment in Drosophila melanogaster. We identified PTTH neurons as doublesex-positive neurons, they regulated virgin female receptivity before the metamorphosis during the third-instar larval stage. PTTH deletion resulted in the increased EcR-A expression in the whole newly formed prepupae. Furthermore, the ecdysone receptor EcR-A in pC1 neurons positively regulated virgin female receptivity during metamorphosis. The decreased EcR-A in pC1 neurons induced abnormal morphological development of pC1 neurons without changing neural activity. Among all subtypes of pC1 neurons, the function of EcR-A in pC1b neurons was necessary for virgin female copulation rate. These suggested that the changes of synaptic connections between pC1b and other neurons decreased female copulation rate. Moreover, female receptivity significantly decreased when the expression of PTTH receptor Torso was reduced in pC1 neurons. This suggested that PTTH not only regulates female receptivity through ecdysone but also through affecting female receptivity associated neurons directly. The PG axis has similar functional strategy as the hypothalamic-pituitary-gonadal axis in mammals to trigger the juvenile-adult transition. Our work suggests a general mechanism underlying which the neurodevelopment during maturation regulates female sexual receptivity.

Modulation of response to braconid wasp venom by adipokinetic hormone in Drosophila melanogaster.

The minute wasp Habrobracon hebetor venom (HH venom) is a potent cocktail of toxins that paralyzes the victim's muscles and suppresses humoral and cellular immunity. This study examined the effect of HH venom on specific biochemical, physiological, and ultrastructural characteristics of the thoracic and nervous (CNS) tissues of Drosophila melanogaster under in vitro conditions. Venom treatment modulated the activities of superoxide dismutase (SOD) and catalase (CAT), endogenous Drome-AKH level, and affected the relative viability of the cells. Additionally, it reduced the expression of genes related to the immune system in the CNS, including Keap1, Relish, Nox, Eiger, Gadd45, and Domeless, as well as in the thoracic muscles, except for Nox. Besides, venom treatment led to deteriorative changes in the ultrastructure of muscle cells, particularly affecting the mitochondria. When venom and Drosophila melanogaster-adipokinetic hormone (Drome-AKH) were applied together, the effects of the venom alone were often modulated. The harmful effect of the venom on SOD activity was relatively reduced and the activity returned to a level similar to that of the control. In the CNS, the simultaneous application of venom and hormones abolished the suppression of previously reported immune-related genes (except for Gadd45), whereas in the muscles, this was only true for Eiger. Additionally, Drome-AKH restored cell structure to a level comparable to that of the control and lessened the harmful effects of HH venom on muscle mitochondria. These findings suggest a general body response of D. melanogaster to HH venom and a partial defensive role of Drome-AKH in this process.

Identification and Functional Analysis of Adipokinetic Hormone Receptor in Ostrinia furnacalis Guenée Larvae Parasitized by Macrocentrus cingulum.

As a typical G protein-coupled receptor, the adipokinetic hormone receptor (AKHR) has seven transmembrane domains (TMDs), and its structure and function are similar to the gonadotropin-releasing hormone receptor (GnRHR) in vertebrates. However, there is a dearth of information on other components of the AKHR signaling pathway and how it functions in the interaction between insect hosts and parasitoids. In this study, we cloned and analyzed the multifunctional Ostrinia furnacalis AKHR (OfAKHR) cDNA (GenBank accession number MF797868). OfAKHR has a 2206 bp full-length cDNA, which includes an open reading frame containing 1194 bp. OfAKHR contains the typical seven TMDs, and a "DRY" motif. OfAKHR has the highest relative expression in the fat body and the fifth instar larvae. The results revealed that ApoLpⅢ, PPO2, GS, TPS, Cecropin, and Moricin decreased the transcription levels from 48 to 72 h after the knockdown of OfAKHR expression by dsOfAKHR injection in the fourth instar O. furnacalis larvae. The parasitization of Macrocentrus cingulum selectively upregulated the expression levels of nutrition metabolism and immune-related genes in parasitized O. furnacalis larvae, stimulated lysozyme activity, and obviously raised the concentrations of triglyceride and trehalose in the hemolymph of O. furnacalis larvae. However, they inhibited the activities of PO and trehalase. This study is conducive to a deeper cognition of the roles of OfAKHR in nutrition and immune homeostasis, coevolution, and coexistence between parasitic wasps and hosts. It also sheds light on the potential as the target of pest control reagents.

Functional expression and characterization of CAPA receptor in the digestive tract and life stages of Drosophila suzukii, and differential activities with insect PRXamide peptides.

Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.

Expressions of sugar transporters/trehalases in relation to PTTH-stimulated ecdysteroidogenesis in the silkworm, Bombyx mori.

The prothoracic gland (PG) is the source of ecdysteoids in larval insects. Although numerous studies have been conducted on signaling networks involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in PGs, less is known about regulation of metabolism in PGs. In the present study, we investigated correlations between expressions of sugar transporter (St)/trehalase (Treh) genes and PTTH-stimulated ecdysteroidogenesis in Bombyx mori PGs. Our results showed that in vitro PTTH treatment stimulated expression of the St1 gene, but not other transporter genes. Expression of the Treh1 gene was also stimulated by PTTH treatment. An immunoblotting analysis showed that St1 protein levels in Bombyx PGs increased during the later stage of the last larval instar and were not affect by PTTH treatment. PTTH treatment enhanced Treh enzyme activity in a time-dependent manner. Blocking either extracellular signal-regulated kinase (ERK) signaling with U0126 or phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 decreased PTTH-stimulated Treh enzyme activity, indicating a link from the ERK and PI3K signaling pathways to Treh activity. Treatment with the Treh inhibitor, validamycin A, blocked PTTH-stimulated Treh enzyme activity and partially inhibited PTTH-stimulated ecdysteroidogenesis. Treatment with either a sugar transport inhibitor (cytochalasin B) or a specific glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) partially inhibited PTTH-stimulated ecdysteroidogenesis. Taken together, these results indicate that increased expressions of St1/Treh1 and Treh activity, which lie downstream of PTTH signaling, are involved in PTTH stimulation in B. mori PGs.

Transcriptome and proteome analyses reveal genes and signaling pathways involved in the response to two insect hormones in the insect-fungal pathogen Hirsutella satumaensis.

The insect hormones ecdysone (20E) and juvenile hormone III (JH) have been demonstrated to stimulate the secretion of conidia mucilage and pigments in Hirsutella satumaensis. However, the underlying mechanisms remain elusive. Here, comparative transcriptome and proteome analyses were performed to identify the fungal genes and proteins of H. satumaensis that are up- or downregulated in response to insect hormones. A total of 17,407 unigenes and 1,016 proteins in conidia mucilage were identified. The genes involved in response to the hormones were classified into four functional groups: (1) stress response-related genes that are required for the removal of reactive oxygen species (glutathione synthetase, c7144) and genes involved in the response to osmotic stress in the hemocoel, such as those encoding proteins involved in the G, mTOR, and MAPK signaling pathways (2); insect hormone metabolic genes, including genes encoding ecdysteroid UDP-glucosyltransferase, ecdysteroid-22-kinase, and a key aldehyde dehydrogenase in a juvenile hormone synthesis pathway (3); secretory proteins that share homology with those of the host Bombyx mori, including fibrohexamerin, sericin 1, metalloprotease 1 protein, and silk gum protein, which were revealed by the omics data; and (4) proteins related to amino sugar metabolism and oxidative phosphorylation that were specifically expressed in mucilage in response to 20E and JH, respectively. These findings revealed that H. satumaensis can mount effective responses by modulating the expression of genes involved in the detoxification, adaptation, and evasion of insect hormone-mediated immune responses, providing fresh insights into fungal pathogen-host insect interactions.IMPORTANCEInsect hormones are highly important for the regulation of insect growth, development, and immune system function. Thus, the expansion of entomopathogenic fungi (EPF) could be affected by these hormones when they inhabit the host hemocoel. However, the molecular basis of EPF in response to insect hormones has yet to be determined. Our results revealed that EPF are impacted by 20E and JH, both of which act as signals, as these hormones lead to changes in metabolic pathways of the fungus, thus demonstrating a direct relationship between the fungus and the hormones. Furthermore, adaptive strategies, such as the use of ecdysone-inactivating enzymes and secreted filamentous proteins in H. satumaensis, which strongly resemble those of the host insect, have been discovered, thus illustrating the importance of adaptation to insect hormones for a better understanding of the interaction between insects and EPF.

Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus (CLas),' the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

The allatotropin/orexin system as an example of immunomodulatory properties of neuropeptides.

The central nervous system (CNS) plays a critical role in signal integration in animals and allows the orchestration of life processes to maintain homeostasis. Current research clearly shows that inflammatory processes can also be modulated by the CNS via the neuroendocrine system. One of the neuropeptide families that participate in vertebrates in this process is orexins (OXs). Interestingly, our previous results suggested that a similar dependency may also exist between neuropeptides and immune system activity in insects. Due to the structural homology of orexin and allatotropin receptors and the functional similarity between these two neuropeptide families, the main aim of this research was to perform a complex analysis of the relationships between allatotropin (AT) and the insect immune response. Our results revealed functional similarities between vertebrate OXs and insect ATs. Similar effects were observed in the profile of the expression level of the gene encoding the AT precursor in the Tenebrio molitor nervous system and in the general action of Tenmo-AT on selected immune parameters of the tested beetles. Moreover, for the first time in insects, we confirmed the role of cytokines in the modulation of neuroendocrine system by determining the effect of Spätzle-like protein injection on the expression of genes encoding AT precursor and receptor. All these results are important for understanding the evolutionary basis of hormonal regulation of the immune response.

PTTH-Torso Signaling System Controls Developmental Timing, Body Size, and Reproduction through Regulating Ecdysone Homeostasis in the Brown Planthopper, Nilaparvata lugens.

In holometabolous insects, such as Drosophila and Bombyx, prothoracicotropic hormone (PTTH) is well established to be critical in controlling developmental transitions and metamorphosis by stimulating the biosynthesis of ecdysone in the prothoracic glands (PGs). However, the physiological role of PTTH and the receptor Torso in hemimetabolous insects remains largely unexplored. In this study, homozygous PTTH- and Torso-null mutants of the brown planthopper (BPH), Nilaparvata lugens, were successfully generated by employing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9). Further characterization showed that both NlPTTH-/- and NlTorso-/- mutants exhibited prolonged nymphal duration and increased final adult size. Enzyme-linked immunosorbent assay (ELISA) revealed that NlPTTH-/- and NlTorso-/- mutants exhibited a significant reduction in 20-hydroxyecdysone (20E) in fifth-instar nymphs at 48 h post-ecdysis compared to Wt controls. Furthermore, our results indicated that both NlPTTH-/- and NlTorso-/- mutants had shortened lifespan, reduced female fecundity, and reduced egg hatching rates in adults. These findings suggest a conserved role for the PTTH-Torso signaling system in the regulation of developmental transitions by stimulating ecdysone biosynthesis in hemimetabolous insects.

Compensatory enhancement of input maintains aversive dopaminergic reinforcement in hungry Drosophila.

Hungry animals need compensatory mechanisms to maintain flexible brain function, while modulation reconfigures circuits to prioritize resource seeking. In Drosophila, hunger inhibits aversively reinforcing dopaminergic neurons (DANs) to permit the expression of food-seeking memories. Multitasking the reinforcement system for motivation potentially undermines aversive learning. We find that chronic hunger mildly enhances aversive learning and that satiated-baseline and hunger-enhanced learning require endocrine adipokinetic hormone (AKH) signaling. Circulating AKH influences aversive learning via its receptor in four neurons in the ventral brain, two of which are octopaminergic. Connectomics revealed AKH receptor-expressing neurons to be upstream of several classes of ascending neurons, many of which are presynaptic to aversively reinforcing DANs. Octopaminergic modulation of and output from at least one of these ascending pathways is required for shock- and bitter-taste-reinforced aversive learning. We propose that coordinated enhancement of input compensates for hunger-directed inhibition of aversive DANs to preserve reinforcement when required.

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.

Elucidating the ecophysiology of soybean pod-sucking stinkbug Riptortus pedestris (Hemiptera: Alydidae) based on de novo genome assembly and transcriptome analysis.

Food security is important for the ever-growing global population. Soybean, Glycine max (L.) Merr., is cultivated worldwide providing a key source of food, protein and oil. Hence, it is imperative to maintain or to increase its yield under different conditions including challenges caused by abiotic and biotic stresses. In recent years, the soybean pod-sucking stinkbug Riptortus pedestris has emerged as an important agricultural insect pest in East, South and Southeast Asia. Here, we present a genomics resource for R. pedestris including its genome assembly, messenger RNA (mRNA) and microRNA (miRNA) transcriptomes at different developmental stages and from different organs. As insect hormone biosynthesis genes (genes involved in metamorphosis) and their regulators such as miRNAs are potential targets for pest control, we analyzed the sesquiterpenoid (juvenile) and ecdysteroid (molting) hormone biosynthesis pathway genes including their miRNAs and relevant neuropeptides. Temporal gene expression changes of these insect hormone biosynthesis pathways were observed at different developmental stages. Similarly, a diet-specific response in gene expression was also observed in both head and salivary glands. Furthermore, we observed that microRNAs (bantam, miR-14, miR-316, and miR-263) of R. pedestris fed with different types of soybeans were differentially expressed in the salivary glands indicating a diet-specific response. Interestingly, the opposite arms of miR-281 (-5p and -3p), a miRNA involved in regulating development, were predicted to target Hmgs genes of R. pedestris and soybean, respectively. These observations among others highlight stinkbug's responses as a function of its interaction with soybean. In brief, the results of this study not only present salient findings that could be of potential use in pest management and mitigation but also provide an invaluable resource for R. pedestris as an insect model to facilitate studies on plant-pest interactions.

Functional analysis of adipokinetic hormone signaling in reproductive diapause of Coccinella septempunctata.

BACKGROUND: The ladybeetle, Coccinella septempunctata, an important predator, is widely used to control aphids, whiteflies, mites, thrips, and lepidopteran pests. Diapause control technology is key to extending C. septempunctata shelf-life and commercialization. Lipid accumulation is a major feature of reproductive diapause, but the function of AKH signaling as a regulator of lipid mobilization in reproductive diapause remains unclear. This study aimed to identify and characterize AKH and AKHR genes, and clarify their functions in reproductive diapause. RESULTS: The relative expression levels of CsAKH and CsAKHR were the highest in the head and fat body, respectively, and were significantly decreased under diapause conditions, both in developmental stages and tissues (head, midgut, fat body, and ovary). Furthermore, CsAKH and CsAKHR expression was increased significantly after juvenile hormone (JH) injection, but CsMet silencing significantly inhibited CsAKH and CsAKHR expression, whereas CsMet knockdown blocked the induction effect of JH. CsAKH and CsAKHR knockdown significantly reduced water content, increased lipid storage, and promoted the expression of genes related to lipid synthesis, but significantly blocked ovarian development, and induced forkhead box O (FOXO) gene expression in C. septempunctata under reproduction conditions. By contrast, injection of AKH peptide significantly inhibited FOXO expression, reduced lipid storage, and increased water content in C. septempunctata under diapause conditions. CONCLUSION: These results indicate that CsAKH and CsAKHR are involved in the regulation of lipid accumulation and ovarian development during diapause in C. septempunctata, and provide a promising target for manipulating C. septempunctata diapause. © 2024 Society of Chemical Industry.

The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding.

Neuropeptides are the main regulators of physiological, developmental, and behavioural processes in insects. Three insect neuropeptide systems, the adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone/corazonin-related peptide (ACP), and their cognate receptors, are related to the vertebrate gonadotropin (GnRH) system and form the GnRH superfamily of peptides. In the current study, the two signalling systems, AKH and ACP, of the yellow fever mosquito, Aedes aegypti, were comparatively investigated with respect to ligand binding to their respective receptors. To achieve this, the solution structure of the hormones was determined by nuclear magnetic resonance distance restraint methodology. Atomic-scale models of the two G protein-coupled receptors were constructed with the help of homology modelling. Thereafter, the binding sites of the receptors were identified by blind docking of the ligands to the receptors, and models were derived for each hormone system showing how the ligands are bound to their receptors. Lastly, the two models were validated by comparing the computational results with experimentally derived data available from the literature. This mostly resulted in an acceptable agreement, proving the models to be largely correct and usable. The identification of an antagonist versus a true agonist may, however, require additional testing. The computational data also explains the exclusivity of the two systems that bind only the cognate ligand. This study forms the basis for further drug discovery studies.

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.

Authentication of a lophotrochozoan adipokinetic hormone receptor in a Gastropod, Aplysia californica.

Gonadotropin-releasing hormone (GnRH) superfamily comprises multiple families of signaling peptides in both protostomes and deuterostomes. Among this superfamily, vertebrate GnRH stimulates reproduction, but other GnRH superfamily members elicit diverse pleiotropic effects. Within the GnRH superfamily members, adipokinetic hormone (AKH) and its receptor are well described in ecdysozoans but understudied in other lineages. To fill this knowledge gap, we deorphanized a putative receptor for a lophotrochozoan AKH in a gastropod mollusk, Aplysia californica, and named it Aplca-AKHR. Phylogenetic analysis revealed an orthologous relationship of Aplca-AKHR with ecdysozoan AKHRs and other putative lophotrochozoan AKHRs. Aplca-AKHR bound specifically to the previously identified Aplca-AKH with high affinity and activated the inositol phosphate pathway. Aplca-AKHR was expressed widely among central and peripheral tissues, but most prominently in several central ganglia and the heart. The expression of Aplca-AKHR was downregulated by a hyposaline challenge, consistent with a role in volume and fluid regulation previously described for its ligand, Aplca-AKH. In summary, this is the first pairing of a lophotrochozoan AKH with its cognate receptor. Expression data further support diverse central and peripheral roles, including volume and fluid control, of this ligand/receptor pair.

Predicted novel hypertrehalosaemic peptides of cockroaches are verified by mass spectrometry.

Small neuropeptides from the corpora cardiaca are responsible in cockroaches for the mobilisation of trehalose from the fat body into the haemolymph. Such hypertrehalosaemic hormones (HrTHs) belong to the large family of insect adipokinetic hormones (AKHs); a few HrTHs were previously sequenced from cockroaches, and from genomic and/or transcriptomic information one may predict the genes encoding HrTHs from more species. Definite elucidation of the primary structure of the mature peptide with putative modifications needs analytical chemical methods. In the current study, we use high-resolution mass spectrometry coupled with liquid chromatography to identify unequivocally the HrTHs of 13 cockroach species. Either genomic/transcriptomic information was available for most of the species examined, or from related species. We confirm predicted novel sequences and find hydroxyproline modification for the majority of the peptides. The novel decapeptides are structurally close to Bladi-HrTH, which is found in all seven of the investigated blaberid subfamilies. Bladi-HrTH and all the novel peptides elicit a hypertrehalosaemic response in Periplaneta americana, a blattid cockroach.

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.

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.