Genome-wide identification of neuropeptides and their receptor genes in Bemisia tabaci and their transcript accumulation change in response to temperature stresses

Insect neuropeptides play an important role in regulating physiological functions such as growth,development,behavior and reproduction.We identified temperaturesensitive neuropeptides and receptor genes of the cotton whitefly,Bemisia tabaci.We identified 38 neuropeptide precursor genes and 35 neuropeptide receptors and constructed a phylogenetic tree using additional data from other insects.As temperature adaptability enables B.tabaci to colonize a diversity of habitats,we performed quantitative polymerase chain reaction with two temperature stresses(low=4℃ and high=40℃)to screen for temperature-sensitive neuropeptides.We found many neuropeptides and receptors that may be involved in the temperature adaptability of B.tabaci.This study is the first to identify B.tabaci neuropeptides and their receptors,and it will help to reveal the roles of neuropeptides in temperature adaptation of B.tabaci.     

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296–5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC₅₀, 1 × 10⁻⁹ M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC₅₀, 5 × 10⁻⁹ M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC₅₀, 7 × 10⁻⁸ M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10⁻⁶ M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.     

Neuropeptides and Neuropeptide Receptors: Drug Targets,and Peptide and Non‐Peptide Ligands: a Tribute to Prof. Dieter Seebach

The number of neuropeptides and their corresponding receptors has increased steadily over the last fourty years: initially, peptides were isolated from gut or brain (e.g., Substance P, somatostatin), then by targeted mining in specific regions (e.g., cortistatin, orexin in the brain), or by deorphanization of G‐protein‐coupled receptors (GPCRs; orexin, ghrelin receptors) and through the completion the Human Genome Project. Neuropeptides (and their receptors) have regionally restricted distributions in the central and peripheral nervous system. The neuropeptide signaling is somewhat more distinct spatially than signaling with classical, low‐molecular‐weight neurotransmitters that are more widely expressed, and, therefore, one assumes that drugs acting at neuropeptide receptors may have more selective pharmacological actions with possibly fewer side effects than drugs acting on glutamatergic, GABAergic, monoaminergic, or cholinergic systems. Neuropeptide receptors, which may have a few or multiple subtypes and splice variants, belong almost exclusively to the GPCR family also known as seven‐transmembrane receptors (7TM), a favorite class of drug targets in the pharmaceutical industry. Most neuropeptides are co‐stored and co‐released with classic neurotransmitters, albeit often only at higher frequencies of stimulation or at bursting activity, thus restricting the neuropeptide signaling to specific circumstances, another reason to assume that neuropeptide drug mimics may have less side effects. Neuropeptides possess a wide spectrum of functions from neurohormone, neurotransmitter to growth factor, but also as key inflammatory mediators. Neuropeptides become ‘active’ when the nervous system is challenged, e.g., by stress, injury, drug abuse, or neuropsychiatric disorders with genetic, epigenetic, and/or environmental components. The unsuspected number of true neuropeptides and their cognate receptors provides opportunities to identify novel targets for the treatment of both central and peripheral nervous system disorders. Both, receptor subtype‐selective antagonists and agonists are being developed, as illustrated by the success of somatostatin agonists, angiotensin, and endothelin antagonists, and the expected clinical applications of NK‐1/2/3 (substance P) receptor antagonists, CRF, vasopressin, NPY, neurotensin, orexin antagonists, or neuropeptide receptor modulators; such ligands have efficacy in preclinical or clinical models of pain and neuropsychiatric diseases, such as migraine, chronic/neuropathic pain, anxiety, sleep disorders, depression, and schizophrenia. In addition, both positive and negative allosteric modulators have been described with interesting in vivo activities (e.g., at galanin receptors). The field has become more complex now that an increasing number of heteromeric neuropeptide receptors are described, e.g., ghrelin receptors with 5‐HT_2C or dopamine D₁, D₂ receptors. At long last, structure‐based drug discovery can now be envisaged with confidence, since crystal or solution structure of GPCRs and GPCR? ligand complexes, including peptide receptors, are published almost on a monthly basis. Finally, although most compounds acting at peptide receptors are still peptidomimetics, the last decade has seen the emergence of low‐molecular‐weight nonpeptide ligands (e.g., for orexin, ghrelin, or neurokinin receptors), and surprising progress has been made with β‐ and γ‐peptides as very stable and potent mimetics of, e.g., somatostatin (SRIF), where the native SRIF has a half‐life limited to 2–3 min. This last point will be illustrated more specifically, as we have had a long‐standing collaboration with Prof. D. Seebach to whom this review is dedicated at the occasion of his 75th birthday.     

Neuropeptides in interneurons of the insect brain

A large number of neuropeptides has been identified in the brain of insects. At least 35 neuropeptide precursor genes have been characterized in Drosophila melanogaster, some of which encode multiple peptides. Additional neuropeptides have been found in other insect species. With a few notable exceptions, most of the neuropeptides have been demonstrated in brain interneurons of various types. The products of each neuropeptide precursor seem to be co-expressed, and each precursor displays a unique neuronal distribution pattern. Commonly, each type of neuropeptide is localized to a relatively small number of neurons. We describe the distribution of neuropeptides in brain interneurons of a few well-studied insect species. Emphasis has been placed upon interneurons innervating specific brain areas, such as the optic lobes, accessory medulla, antennal lobes, central body, and mushroom bodies. The functional roles of some neuropeptides and their receptors have been investigated in D. melanogaster by molecular genetics techniques. In addition, behavioral and electrophysiological assays have addressed neuropeptide functions in the cockroach Leucophaea maderae. Thus, the involvement of brain neuropeptides in circadian clock function, olfactory processing, various aspects of feeding behavior, and learning and memory are highlighted in this review. Studies so far indicate that neuropeptides can play a multitude of functional roles in the brain and that even single neuropeptides are likely to be multifunctional.The original research in the authors’ laboratories was supported by DFG grants HO 950/14 and 950/16 (U.H.) and Swedish Research Council grant VR 621-2004-3715 (D.R.N).     

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.     

G protein coupled receptors as targets for next generation pesticides

There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management.Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR.The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect.This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii.Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion hormone, ecdysis triggering hormone and crustacean cardioacceleratory peptide receptors) as well as the dopamine-2 like, latrophilin-like, starry night, frizzled-like, methuselah-like and the smoothened receptors may be suitable pesticide targets.From in vivo studies using native ligands and peptide analogues, receptors which appear to have a role in the regulation of feeding in the pea aphid, such as the PISCF-allatostatin and the various “kinin” receptors, are also potential targets.In Drosophila melanogaster various neuropeptides and their signalling pathways have been studied extensively. This may provide insights into potential pesticide targets that could be exploited in D. suzukii. Examples include the sex peptide receptor, which is involved in reproduction and host seeking behaviours, and those responsible for osmoregulation such as the diuretic hormone receptors.However the neuropeptides and their receptors in insects are often poorly characterized, especially in pest species. Although data from closely related species may be transferable (e.g. D. melanogaster to D. suzukii), peptides and receptors may have different roles in different insects, and hence a target in one insect may not be appropriate in another. Hence fundamental knowledge of the roles and functions of receptors is vital for development to proceed.     

Discovery of sea urchin NGFFFamide receptor unites a bilaterian neuropeptide family

Neuropeptides are ancient regulators of physiology and behaviour, but reconstruction of neuropeptide evolution is often difficult owing to lack of sequence conservation. Here, we report that the receptor for the neuropeptide NGFFFamide in the sea urchin Strongylocentrotus purpuratus (phylum Echinodermata) is an orthologue of vertebrate neuropeptide-S (NPS) receptors and crustacean cardioactive peptide (CCAP) receptors. Importantly, this has facilitated reconstruction of the evolution of two bilaterian neuropeptide signalling systems. Genes encoding the precursor of a vasopressin/oxytocin-type neuropeptide and its receptor duplicated in a common ancestor of the Bilateria. One copy of the precursor retained ancestral features, as seen in highly conserved vasopressin/oxytocin–neurophysin-type precursors. The other copy diverged, but this took different courses in protostomes and deuterostomes. In protostomes, the occurrence of a disulfide bridge in neuropeptide product(s) of the precursor was retained, as in CCAP, but with loss of the neurophysin domain. In deuterostomes, we see the opposite scenario—the neuropeptides lost the disulfide bridge, and neurophysin was retained (as in the NGFFFamide precursor) but was subsequently lost in vertebrate NPS precursors. Thus, the sea urchin NGFFFamide precursor and receptor are ‘missing links’ in the evolutionary history of neuropeptides that control ecdysis in arthropods (CCAP) and regulate anxiety in humans (NPS).     

The neurobiology of dopamine receptors: evolution from the dual concept to heterodimer complexes

Context: G protein-coupled receptors (GPCRs) have been classically thought to work as monomeric entities. The current view of their organization, however, assumes that they are part of highly organized molecular complexes, where different receptors and interacting proteins are clustered. These heteromers have peculiar pharmacological, signaling, and trafficking properties. GPCR heteromerization, raising different combinatorial possibilities, thus underlies an unexpected level of diversity within this receptor family.

Methods: In this paper, we summarize recent data, reported by different research groups, suggesting that the dopamine (DA) D1 receptor forms heteromers with receptors of the same family and with structurally and functionally divergent receptors.

Results and discussion: DA D1 and D3 receptors and glutamate NMDA receptors regulate rewarding mechanisms and motivated behavior, modulate emotional and cognitive processes and regulate locomotor activity by extensive cross-talk mechanisms. Co-localization of D1 and D3 receptors and D1 and NMDA receptors in specific neuronal populations in the striatum and nucleus accumbens, moreover, suggested that their cross-talk may involve direct interactions. By using different experimental approaches various groups have, in fact, demonstrated the existence of D1-NMDA and D1-D3 heteromers, in both transfected cell systems and in the straitum, with peculiar pharmacological, signaling, and functional properties. The putative role of the D1-D3 and D1-NMDA heteromers in the physiological regulation of striatal function and in the development of motor dysfunctions will be discussed.     

Mi‐mediated aphid resistance in tomato: tissue localization and impact on the feeding behavior of two potato aphid clones with differing levels of virulence

The Mi‐1.2 gene in tomato, Solanum lycopersicum L. (Solanaceae), confers resistance against several herbivores, including the potato aphid, Macrosiphum euphorbiae (Thomas) (Hemiptera: Sternorrhyncha: Aphididae) and the sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodidae). Previous studies on the tissue localization of resistance have given varying results; whitefly resistance was attributed to factors localized in the mesophyll or epidermis, whereas aphid resistance was attributed to factors localized in the phloem. Our study utilizes the direct current electrical penetration graph (DC‐EPG) technique to compare aphid feeding behavior on resistant (Mi‐1.2+) and susceptible (Mi‐1.2?) tomato plants. This study also compares the impact of resistance on the feeding behavior of two aphid clones that vary in their virulence, or their ability to survive and reproduce on resistant plants. Previous work had shown that the avirulent WU11 clone is almost completely inhibited by resistance, whereas the semi‐virulent WU12 clone can colonize resistant hosts. Here, DC‐EPG analysis shows that both aphid clones take longer to initiate cell sampling and to establish a confirmed sieve element phase on resistant plants than on susceptible hosts, and have shorter ingestion periods on resistant plants. However, the magnitude of these deterrent effects is far less for the semi‐virulent clone than for the avirulent aphids. In particular, the WU12 clone is less sensitive to factors that limit sieve element ingestion, showing shorter non‐probe duration and rapidly establishing sustained phloem ingestion on resistant plants when compared to the WU11 clone. We conclude that, in addition to previously described factors in the phloem that inhibit ingestion, Mi‐mediated aphid resistance also involves factors (possibly in the mesophyll and/or epidermis) that delay initiation of phloem salivation, and that act in the intercellular spaces to deter the first cell sampling. Furthermore, the relative effectiveness of these components of resistance differs among insect populations.     

Proctolin in the post-genomic era: new insights and challenges

Complete understanding of how neuropeptides operate as neuromodulators and neurohormones requires integration of knowledge obtained at different levels of biology, including molecular, biochemical, physiological and whole organism studies. Major advances have recently been made in the understanding of the molecular basis of neuropeptide action in invertebrates by analysis of data generated from sequencing the genomes of several insect species, especially that of Drosophila melanogaster. This approach has quickly led to the identification of genes encoding: (1) novel neuropeptide sequences, (2) neuropeptide receptors and (3) peptidases that might be responsible for the processing and inactivation of neuropeptides. In this article, we review our current knowledge of the biosynthesis, receptor interaction and metabolic inactivation of the arthropod neuropeptide, proctolin, and how the analysis and exploitation of genome sequencing projects has provided new insights.     

Galactose-NlGr11 inhibits AMPK phosphorylation by activating the PI3K-AKT-PKF-ATP signaling cascade via insulin receptor and Gβγ

As ligands of the sugar gustatory receptors, sugars have been known to activate the insulin/insulin-like growth factor signaling pathway; however, the precise pathways that are activated by the sugar-bound gustatory receptors in insects remain unclear. In this study, we aimed to investigate the signaling cascades activated by NlGr11, a sugar gustatory receptor in the brown planthopper Nilaparvata lugens (Stål), and its ligand. Galactose-bound NlGr11 (galactose-NlGr11) activated the -phosphatidylinositol 3-kinase (PI3K)-AKT signaling cascade via insulin receptor (InR) and Gβγ in vitro. In addition, galactose-NlGr11 inhibited the adenosine monophosphate-activated protein kinase (AMPK) phosphorylation by activating the AKT-phosphofructokinase (PFK)-ATP signaling cascade in vitro. Importantly, the InR-PI3K-AKT-PFK-AKT signaling cascade was activated and the AMPK phosphorylation was inhibited after feeding the brown planthoppers with galactose solution. Collectively, these findings confirm that NlGr11 can inhibit AMPK phosphorylation by activating the PI3K-AKT-PFK-ATP signaling cascades via both InR and Gβγ when bound to galactose. Thus, our study provides novel insights into the signaling pathways regulated by the sugar gustatory receptors in insects.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Incidence of Wolbachia in aquatic insects

Wolbachia is a genus of intracellular bacteria typically found within the reproductive systems of insects that manipulates those systems of their hosts. While current estimates of Wolbachia incidence suggest that it infects approximately half of all arthropod species, these estimates are based almost entirely on terrestrial insects. No systematic survey of Wolbachia in aquatic insects has been performed. To estimate Wolbachia incidence among aquatic insect species, we combined field‐collected samples from the Missouri River (251 samples from 58 species) with a global database from previously published surveys. The final database contained 5,598 samples of 2,687 total species (228 aquatic and 2,459 terrestrial). We estimate that 52% (95% CrIs: 44%–60%) of aquatic insect species carry Wolbachia, compared to 60% (58%–63%) of terrestrial insects. Among aquatic insects, infected orders included Odonata, Coleoptera, Trichoptera, Ephemeroptera, Diptera, Hemiptera, and Plecoptera. Incidence was highest within aquatic Diptera and Hemiptera (69%), Odonata (50%), and Coleoptera (53%), and was lowest within Ephemeroptera (13%). These results indicate that Wolbachia is common among aquatic insects, but incidence varies widely across orders and is especially uncertain in those orders with low sample sizes such as Ephemeroptera, Plecoptera, and Trichoptera.     

Structural and functional differences between pheromonotropic and melanotropic PK/PBAN receptors

Background

The pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions.

Methods

A sex pheromone gland receptor of the PK/PBAN family from Heliothis peltigera female moth and a Spodoptera littoralis larval receptor were cloned and stably expressed, and their structural models, electrostatic potentials and cellular functional properties were evaluated.

Results

Homology modeling indicated highly conserved amino-acid residues in appropriate structural positions as experimentally shown for class A G-protein coupled receptors. Structural differences could be proposed and electrostatic potentials of the two receptor models revealed net charge differences. Calcium mobilization assays demonstrated that both receptors were fully functional and could initiate extracellular calcium influx to start PK/PBAN signal transduction. Evaluation of the signaling response of both receptors to PBAN and diapause hormone (DH) revealed a highly sensitive, though differential response. Both receptors responded to PBAN whereas only Spl-PK/PBAN-R exhibited a high response toward DH.

Conclusions

The structural, electrostatic and cellular functional differences indicate that different PK/PBAN in vivo functions may be mediated by different PK/PBAN receptors and elicited by different peptide(s).

General significance

The results advance our understanding of the mode of action of the PK/PBAN family, and might help in exploring novel high-affinity receptor-specific antagonists that can serve as a basis for the development of new families of insect-control agents.     

Social behavior and the evolution of neuropeptide genes: lessons from the honeybee genome

Honeybees display a fascinating social behavior. The structural basis for this behavior, which made the bee a model organism for the study of communication, learning and memory formation, is the tiny insect brain. Neurons of the brain communicate via messenger molecules. Among these molecules, neuropeptides represent the structurally most‐diverse group and occupy a high hierarchic position in the modulation of behavior. A recent analysis of the honeybee genome revealed a considerable number of predicted (200) and confirmed (100) neuropeptides in this insect. 1 Is this quantity merely the result of advanced mass spectrometric techniques and bioinformatic tools or does it reflect the expression of more of these important messenger molecules, more than known from other insects studied so far? Our analysis of the data suggests that the social behavior is by no means correlated with a specific increase in the number of neuropeptides. Indeed, the honeybee genome is likely to contain fewer neuropeptide genes, neuropeptide paralogues and neuropeptide receptor genes than the solitary fruitfly Drosophila. BioEssays 29:416–421, 2007. © 2007 Wiley Periodicals, Inc.