Neuropeptidergic control of the hindgut in the black-legged tick Ixodes scapularis

The hindgut, as a part of the tick excretory system, plays an important physiological role in maintaining homoeostases and waste elimination. Immunoreactive projections from the synganglion to the hindgut were found using antibodies against four different neuropeptides: FGLamide related allatostatin, myoinhibitory peptide, SIFamide, and orcokinin. The presence of FGLamide related allatostatin, myoinhibitory peptide and SIFamide in both synganglia (source) and hindgut (target organ) extracts was confirmed by MALDI-TOF. Tissue-specific PCR revealed the expression of four putative FGLamide related allatostatin receptors and an SIFamide receptor in the hindgut. An antibody against Ixodes scapularis SIFamide receptor detected immunoreactive spots in epithelial cells as well as the visceral muscles surrounding the rectal sac, while staining with the antibody against myoinhibitory peptide receptor 1 revealed that the immunoreactivity was only associated with the visceral muscles. In hindgut motility assays, SIFamide activated hindgut motility in a dose-dependent manner. None of other three neuropeptides (FGLamide related allatostatin, myoinhibitory peptide and orcokinin) activated hindgut motility when tested alone. Myoinhibitory peptide antagonised the SIFamide-stimulated hindgut mobility when it was tested in combination with SIFamide.     

Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing spreading behavior after eclosion. The cell death was accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy revealed that this cell death exhibited extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibited the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrated that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This was supported by evidence that wing cell death was inhibited by a mutation of rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeant analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induced precocious death. Conversely, cell death was inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion.     

Functional characterization of bursicon receptor and genome-wide analysis for identification of genes affected by bursicon receptor RNAi

Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action.     

Functional analysis of four neuropeptides, EH, ETH, CCAP and bursicon, and their receptors in adult ecdysis behavior of the red flour beetle, Tribolium castaneum

Ecdysis behavior in arthropods is driven by complex interactions among multiple neuropeptide signaling systems. To understand the roles of neuropeptides and their receptors in the red flour beetle, Tribolium castaneum, we performed systemic RNA interference (RNAi) experiments utilizing post-embryonic injections of double-stranded (ds) RNAs corresponding to ten gene products representing four different peptide signaling pathways: eclosion hormone (EH), ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP) and bursicon. Behavioral deficiencies and developmental arrests occurred as follows: RNAi of (1) eh or eth disrupted preecdysis behavior and prevented subsequent ecdysis behavior; (2) ccap interrupted ecdysis behavior; and (3) bursicon subunits resulted in wrinkled elytra due to incomplete wing expansion, but there was no effect on cuticle tanning or viability. RNAi of genes encoding receptors for those peptides produced phenocopies comparable to those of their respective cognate neuropeptides, except in those cases where more than one receptor was identified. The phenotypes resulting from neuropeptide RNAi in Tribolium differ substantially from phenotypes of the respective Drosophila mutants. Results from this study suggest that the functions of neuropeptidergic systems that drive innate ecdysis behavior have undergone significant changes during the evolution of arthropods.     

In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown.     

Molecular cloning and characterization of an allatostatin-like receptor in the cockroach Diploptera punctata

Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a lambda ZAP-cDNA library prepared from the corpora allata of the D. punctata. As an approach to testing the function of this receptor in vivo, the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.     

灰飞虱Bursicon基因的克隆、序列分析及在不同发育阶段的表达

Bursicon是通过G蛋白受体调节昆虫表皮硬化及展翅的功能蛋白, 它在昆虫蜕皮后的表皮硬化过程中起着关键作用。为探讨灰飞虱Laodelphax striatellus的 bursicon的功能, 利用RT-PCR和RACE技术克隆获得1 126 bp的bursicon α和761 bp的bursicon β全长序列, 将其分别命名为Lsburs-α和Lsburs-β。生物信息学分析表明： Lsburs-α开放阅读框长483 bp, 编码160个氨基酸, 该蛋白具有2个N-豆蔻酰化位点、 3个酪蛋白激酶Ⅱ磷酸化位点以及2个蛋白激酶C磷酸化位点。Lsburs-β开放阅读框长417 bp, 编码138个氨基酸, 该蛋白具有2个N-豆蔻酰化位点、 3个酪蛋白激酶Ⅱ磷酸化位点以及1个酪氨酸激酶磷酸化位点。qRT-PCR结果表明： Lsburs-α和Lsburs-β在灰飞虱各龄期均有转录表达, 并在若虫期随龄期增加呈上升趋势, 在羽化期达到峰值, 成虫期表达量逐渐降低。结果提示bursicon与灰飞虱蜕皮后的外表皮硬化关系密切。本文结果为深入研究bursicon的功能、受体调节和信号通路等奠定了基础。     

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.     

A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles

BACKGROUND: At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. RESULTS: To elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.     

Two peptide transmitters co-packaged in a single neurosecretory vesicle

Numerous neurosecretory cells are known to secrete more than one peptide, in both vertebrates and invertebrates. These co-expressed neuropeptides often originate from differential cleavage of a single large precursor, and are then usually sorted in the regulated pathway into different secretory vesicle classes to allow separable release dynamics. Here, we use immuno-gold electron microscopy to show that two very different neuropeptides (the nonapeptide crustacean cardioactive peptide (CCAP) and the 30 kDa heterodimeric bursicon) are co-packaged within the same dense core vesicles in neurosecretory neurons in the abdominal ganglia of Periplaneta americana. We suggest that this co-packaging serves a physiological function in which CCAP accelerates the distribution of bursicon to the epidermis after ecdysis to regulate sclerotization of the newly formed cuticle.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

Disrupting the Amblyomma americanum (L.) CD147 receptor homolog prevents ticks from feeding to repletion and blocks spontaneous detachment of ticks from their host

The CD147 receptor is a cell-surface glycoprotein in the IgG family that plays pivotal roles in intercellular interactions involved with numerous physiological and pathological processes such as extracellular matrix remodeling. We previously found an Amblyomma americanum (Aam) tick CD147 receptor homolog among genes that were up regulated in response to tick feeding stimuli. This study characterizes an AamCD147 receptor protein that is 72–83% conserved in other tick species and possess characteristic CD147 receptor sequence features: an extracellular (EC) region containing two IgG domains, a transmembrane and the cytoplasmic domains. Likewise, the AamCD147 EC domain folds into secondary structures that are consistent to the human homolog: an amino-terminus β-barrel that is linked to 2-carboxy-terminus β-sheets with consensus disulfide bonds conserved in each of the 2 domains. CD147 receptor signaling and regulatory mechanisms are putatively conserved in ticks as revealed by in silico analysis that show presence in the tick genome of CD147 receptor signaling protein homologs, cyclophilin (CyP) A and B, and chaperones that transport it to the plasma membrane, caveolin-1 and CyP60. The AamCD147 receptor has a dichotomous expression pattern of where it is up regulated in response to feeding in the salivary gland but remains constant at the midgut and ovary levels suggesting that it may regulate different functions in different tick organs. We speculate that biological functions of the AamCD147 receptor are essential to tick feeding success as revealed by RNAi-mediated silencing that caused ticks to obtain smaller blood meals, of which ～69% were below threshold to trigger spontaneous detachment of ticks from the host. These ticks showed unusual cuticle tenderness and assumed a reddish coloration, a phenomenon that has been attributed to tick midgut damage allowing red blood cells to leak into tick hemolymph. On the basis of the CD147 receptor being linked to tissue growth regulation in mammals, we speculate that silencing of the AamCD147 receptor blocked progression of the tick intermolt growth, a process that precedes tick engorgement and their spontaneous detachment of from the host to end feeding. The results are discussed in context of advances in tick molecular physiology.