Growth-blocking Peptide Research Articles

The growth-blocking peptide is a dual regulator of development and immunity with biocontrol potential in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Insect growth-blocking peptides (GBPs) are a family of cytokines found in several insect orders and are known for their roles in regulating development, paralysis, cell proliferation, and immune responses. Despite their diverse functions, the potential of GBPs as biocontrol targets against the pest Spodoptera frugiperda (Lepidoptera: Noctuidae) has not been fully explored. In this study, S. frugiperda GBP (SfGBP) was identified and functionally characterized. SfGBP is synthesized as a 146 amino acid proprotein with a 24 amino acid C-terminal active peptide (Glu123-Gly146). Predominant expression of SfGBP occurs in fourth to sixth instar larvae and in the larval fat body, with significant upregulation in response to pathogens and pathogen-associated molecular patterns. Injection of the synthetic active peptide into larvae induced growth retardation, delayed pupation, and increased survival against Beauveria bassiana infection. Conversely, RNA interference-mediated knockdown of SfGBP resulted in accelerated growth, earlier pupation, and decreased survival against B. bassiana infection. Further analysis revealed that SfGBP promoted SF9 cell proliferation and spreading, enhanced bacteriostatic activity of larval hemolymph, and directly inhibited germination of B. bassiana conidia. In addition, SfGBP enhanced humoral responses, such as upregulation of immunity-related genes and generation of reactive oxygen species, and cellular responses, such as nodulation, phagocytosis, and encapsulation. These results highlight the dual regulatory role of SfGBP in development and immune responses and establish it as a promising biocontrol target for the management of S. frugiperda.

Pleiotropic immunoregulation by growth-blocking peptide in Ostrinia furnacalis.

Insects rely on their innate immune system to eliminate pathogenic microbes. As a system component, cytokines transmit intercellular signals to control immune responses. Growth-blocking peptide (GBP) is a member of the stress-responsive peptide family of cytokines found in several orders of insects, including Drosophila. However, the physiological role of GBP in defence against pathogens is not thoroughly understood. In this study, we explored the functions of GBP in a lepidopteran pest, Ostrinia furnacalis. Injection of recombinant O. furnacalis GBP (OfGBP) precursor (proGBP) and chemically synthesised GBP significantly induced the transcription of antimicrobial peptides (AMPs) and other immunity-related genes including immune deficiency (IMD) and Dorsal. The level of OfGBP mRNA was upregulated after bacterial infection. Knockdown of OfGBP expression led to a decrease in IMD, Relish, MyD88 and Dorsal mRNA levels. OfGBP induced phenoloxidase activity and affected hemocyte behaviours in O. furnacalis larvae. In summary, GBP is a potent cytokine, effectively regulating AMP synthesis, melanization response and cellular immunity to eliminate invading pathogens.

Molecular cloning and characterization of a growth-blocking peptide from the honeybee Apis mellifera

• Bee ( Apis mellifera, A. cerana, Bombus terrestris, and B. ignitus ) growth-blocking peptides (GBPs) were cloned. • AmGBP was expressed at the larval stage. • RNAi-treatment of AmGBP affected larval development and larval-pupal transformation. Growth-blocking peptides (GBPs) are insect cytokines that have been characterized from several species. However, molecular studies on hymenopteran GBPs remain limited. In this study, we cloned four GBP cDNAs of honeybees and bumblebees and compared their sequences with those of other insect GBPs. Sequence analysis revealed that GBP from bees conserves a mature peptide with 21–25 amino acids, including two conserved cysteine residues. During Apis mellifera development, AmGBP was expressed at the larval stage but not at the pupal stage. AmGBP transcript reduction by RNAi affected larval growth and reduced body weight gain. AmGBP RNAi-treated larvae on day 4 were arrested to the larval stage and finally died. Our data suggested that AmGBP plays an essential role in larval growth and acts as a growth factor at the larval stage of A. mellifera .

Biocidal efficacy of tutin and its influence on immune cells and expression of growth-blocking and neuroglian peptides in Mythimna separata (Lepidoptera: Noctuidae).

Mythimna separata Walker (Lepidoptera: Noctuidae) is one of the major pests that can cause severe damage to grain crops. The development of low-toxicity and high-performance botanical insecticides is becoming the focus of new pesticide research to control M. separata. Tutin, a sesquiterpene lactone compound obtained from Coriaria sinica Maxim, a native Chinese poisonous plant, has antifeedant, absorption, and stomach poisoning against a variety of pests. To understand the toxic effect of tutin on M. separata larvae, we set out to determine their antifeedant, mortality, paralysis, weight change, and to examine the spreading of M. separata hemocytes under different concentrations of tutin treatment. Tissue distribution of the immune-associated gene growth-blocking peptide (GBP) and neuroglian peptide (Nrg) was detected by reverse transcription polymerase chain reaction (PCR). Furthermore, real-time quantitative PCR was carried out to determine the expression profiles of GBP and Nrg after different concentrations of tutin stimulation. Our results revealed that tutin exhibited significant antifeedant and insecticidal activities, paralysis, weight loss to M. separata. Besides, tutin significantly influenced on the morphology of hemocytes and enhanced the expression of GBP and Nrg in M. separata.

Stress-derived reactive oxygen species enable hemocytes to release activator of growth blocking peptide (GBP) processing enzyme

Insect cytokine growth blocking peptide (GBP) is synthesized as an inactive precursor, termed proGBP, that is normally present in a significant concentration in the hemolymph of non-stressed animals (Hayakawa, 1990, 1991). Under stress conditions, proGBP is instantly processed to active GBP by a serine protease and this is thought to be an important initial step for insects to cope with stress-induced adverse effects via GBP-induced physiological changes. However, the detailed mechanism underlying proteolytic processing of hemolymph proGBP in insects under stress conditions remains unknown. Here we demonstrated that proGBP processing requires ROS-induced release of a proteinaceous factor from hemocytes that activates the inactive proGBP processing enzyme. The release of the activator protein from hemocytes is initiated by an elevation of the cytoplasmic Ca2+ concentration induced by ROS. Therefore, we concluded that stress-induced activation of proGBP requires ROS-dependent stimulation of an intracellular calcium signaling pathway in hemocytes, followed by release of the hemocyte proteinaceous factor that specifically activates the proGBP processing enzyme.

Insect cytokine growth-blocking peptide may regulate density-dependent phase trait of cuticular melanization in the larval armyworm, Mythimna separata

Abstract Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities such as larval growth regulation, paralysis induction, cell proliferation and stimulation of immune cells. Density-dependent phase polyphenism is a phenomenon of phenotypic plasticity in which the expression of a variety of traits can be affected by local population density. In the present study, the armyworm Mythimna separata larvae with four rearing densities (1 larva/vial, 2 larvae/vial, 4 larvae/vial and 6 larvae/vial) were tested for cuticular melanization and body weight throughout the third-fifth instar, and the functional role of GBP in regulating the changes was investigated. The results indicated that when reared at high densities, the larvae exhibited less body weight and more degree of cuticular melanization than larvae reared at low densities. The gene expression of GBP in armyworm larvae showed an initial rise and then decline trend with increased rearing densities in the third to fifth instar. Compared with control, more degree of cuticular melanization was observed in GBP-injected larvae (500 ng/larva in volume 50 μL) than that in Ringer’s solution-injected counterparts. Furthermore, the gene expression level of dopa decarboxylase and prophenoloxidase increased significantly in GBP-injected fifth instar larvae from 6 h to 12 h after injection, suggesting the role of GBP in modulating density-dependent phase trait of armyworm cuticular melanization.

Functional Multiplicity of an Insect Cytokine Family Assists Defense Against Environmental Stress.

The widespread distribution of insects over many ecological niches owes much to evolution of multiple mechanisms to defend against environmental stress, especially because their ectothermic nature and small body size render them particularly susceptible to extremes in temperature and water availability. In this review, we will summarize the latest information describing a single, multifunctional cytokine family that is deployed by six orders of insect species to combat a diverse variety of environmental stresses. The originating member of this peptide family was identified in Mythimna (formerly called Pseudaletia) separata armyworm; the cytokine was named growth-blocking peptide (GBP), reflecting its actions in combating parasitic invasion. The peptide’s name has been retained, though the list of its regulatory activities has greatly expanded. All members of this family are small peptides, 19–25 amino acid residues, whose major source is fat body. They are now known to regulate embryonic morphogenesis, larval growth rates, feeding activities, immune responses, nutrition, and aging. In this review, we will describe recent developments in our understanding of the mechanisms of action of the GBP family, but we will also highlight remaining gaps in our knowledge.

Identification of a cytokine combination that protects insects from stress

Growth-blocking peptide (GBP) and stress-responsive peptide (SRP) are insect cytokines whose expression levels are elevated by various stressful conditions such as parasitization and high or low temperatures. Both GBP and SRP are synthesized as precursors and released into the hemolymph, where they are enzymatically processed to active peptides. Injection of active GBP or SRP into early last instar larvae elicits a reduction in feeding and consequent growth retardation in the armyworm Mythimna separata. Although such functions are thought to benefit insects under stressful conditions by affecting their physiologies and behaviors, the relationship between GBP and SRP remains elusive. Here we show that heat stress-induced reactive oxygen species (ROS) elevated hemolymph GBP, which activated SRP transcription and increased the SRP concentration in the hemolymph. Injection of both GBP and SRP elevated hemolymph antioxidant levels. We found that simultaneous increases in both active cytokines occurred in the larval hemolymph from 2 to 3 h after heat stress or H2O2 injection, suggesting a synergic action of the two factors. This speculation was confirmed by demonstrating that co-injection of GBP and SRP caused a more severe reduction in appetite and growth retardation than injection of an individual peptide alone. However, injection of GBP together with SRP did not elevate SRP expression at all, indicating the effect of negative feedback regulation. Furthermore, SRP RNAi larvae showed higher body weights compared to controls, and GBP-induced growth retardation was partially abrogated in SRP RNAi larvae. These results led us to conclude that GBP is an upstream cytokine in the regulation of SRP expression and that these cytokines synergistically retard larval growth by repressing feeding activities when insects are exposed to stress conditions.

The Drosophila cytokine, GBP: A model that illuminates the yin-yang of inflammation and longevity in humans?

Our laboratories have determined that the Drosophila cytokine, Growth-blocking peptide (GBP), mediates its biological effects through the Mthl10 G-protein coupled receptor. In this Cytokine Stimulus, we discuss the functional plasticity of the GBP/Mthl10 axis, and we propose that conserved components of this regulatory network may be relevant to human health.

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

20-Hydroxyecdysone promotes release of GBP-binding protein from oenocytoids to suppress hemocytic encapsulation

Growth-blocking peptide (GBP) is an insect cytokine that stimulates plasmatocyte adhesion, thereby playing a critical role in encapsulation reaction. It has been previously demonstrated that GBP-binding protein (GBPB) is released upon oenocytoid lysis in response to GBP and is responsible for subsequent clearance of GBP from hemolymph. However, current knowledge about GBPB is limited and the mechanism by which insects increase GBPB levels to inactivate GBP remains largely unexplored. Here, we have identified one GBP precursor (HaGBP precursor) gene and two GBPB (namely HaGBPB1 and HaGBPB2) genes from the cotton bollworm, Helicoverpa armigera. The HaGBP precursor was found to be predominantly expressed in fat body, whereas HaGBPB1 and HaGBPB2 were mainly expressed in hemocytes. Immunological analyses indicated that both HaGBPB1 and HaGBPB2 are released from hemocytes into the plasma during the wandering stage. Additionally, 20-hydroxyecdysone (20E) treatment or bead challenge could promote the release of HaGBPB1 and HaGBPB2 at least partly from oenocytoids into the plasma. Furthermore, we demonstrate that the N-terminus of HaGBPB1 is responsible for binding to HaGBP and suppresses HaGBP-induced plasmatocyte spreading and encapsulation. Overall, this study helps to enrich our understanding of the molecular mechanism underlying 20E mediated regulation of plasmatocyte adhesion and encapsulation via GBP-GBPB interaction.

Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation.

In Drosophila, the fat body, functionally equivalent to the mammalian liver and adipocytes, plays a central role in regulating systemic growth in response to nutrition. The fat body senses intracellular amino acids through Target of Rapamycin (TOR) signaling, and produces an unidentified humoral factor(s) to regulate insulin-like peptide (ILP) synthesis and/or secretion in the insulin-producing cells. Here, we find that two peptides, Growth-Blocking Peptide (GBP1) and CG11395 (GBP2), are produced in the fat body in response to amino acids and TOR signaling. Reducing the expression of GBP1 and GBP2 (GBPs) specifically in the fat body results in smaller body size due to reduced growth rate. In addition, we found that GBPs stimulate ILP secretion from the insulin-producing cells, either directly or indirectly, thereby increasing insulin and insulin-like growth factor signaling activity throughout the body. Our findings fill an important gap in our understanding of how the fat body transmits nutritional information to the insulin producing cells to control body size.

Enhancing the insecticidal activity of recombinant baculovirus by expressing a growth-blocking peptide from the beet armyworm Spodoptera exigua

Insect cytokine growth-blocking peptides (GBPs) play functional roles in growth regulation and the innate immune response. Moreover, high concentrations of GBPs significantly induce larval morphological abnormalities and lethality. In the present study, a recombinant AcNPV, AcNPV-SeGBPm, expressing a growth-blocking peptide from the beet armyworm Spodoptera exigua (SeGBP) under the control of a polyhedrin promoter was constructed. Western blot analysis indicated that the mature peptide of SeGBP (SeGBPm) is expressed as a 3-kDa peptide in recombinant baculovirus-infected Sf9 insect cells. SeGBPm expression in Sf9 insect cells decreased cell viability significantly and induced apoptotic cell death. In addition, the body weights of S. exigua larvae inoculated with AcNPV-SeGBPm decreased significantly compared to those of larvae inoculated with wild-type AcNPV. Moreover, AcNPV-SeGBPm improved the rate of larvae killing (LT50) by approximately 1day compared to wild-type AcNPV. Taken together, the obtained data demonstrate the great potential for future investigations of growth-blocking peptides for use as biopesticides.

Gain of long tonic immobility behavioral trait causes the red flour beetle to reduce anti-stress capacity

Tonic immobility (death-feigning) behavior of the red flour beetle, Tribolium castaneum, is a predator defense mechanism; it is a reflex elicited when a beetle is jarred with the substrate, often a result of the activities of a predator. We previously demonstrated that the frequency of predation by a jumping spider, Hasarius adansoni, was significantly lower among beetles with higher frequencies and longer durations of tonic immobility (L-type) than those with lower frequencies and shorter durations of tonic immobility (S-type). However, we found that the population of L-type beetles is much smaller than that of S-type beetles in their natural habitat. Here we demonstrated that L-type beetles are significantly more sensitive to environmental stressors such as mechanical vibration and high or low temperatures. We measured expression levels of stress-responsive genes such as heat shock proteins (Hsps) and antioxidant enzymes in both types of beetles. Among the genes we investigated, only catalase gene expression levels were significantly higher in S-type than in L-type beetles. Furthermore, a similar difference in the gene expression was observed in the T. castaneum ortholog of the insect cytokine growth-blocking peptide (GBP) gene. These results indicate the possibility that high expression of catalase and GBP in S-type beetles contributes to augmentation of their anti-stress capacity and expansion of their population in their natural habitat.

Morphological abnormalities and lethality in silkworm (Bombyx mori) larvae treated with high concentrations of insect growth-blocking peptide

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm (Bombyx mori) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.

Developmental regulation and antifungal activity of a growth-blocking peptide from the beet armyworm Spodoptera exigua

Insect cytokine growth-blocking peptides (GBPs) are involved in growth regulation and the innate immune response. However, the microbial binding and antimicrobial activities of GBPs remain unclear. Here, we investigate the developmental role and antifungal activity of a GBP from the beet armyworm Spodoptera exigua (SeGBP). Sequence analysis predicted that mature SeGBP consists of 24 amino acid residues, including 2 cysteine residues. During S. exigua development, SeGBP is constitutively expressed in the fat body during the larval and adult stages but not in pupae. SeGBP expression is up-regulated by 20-hydroxyecdysone and down-regulated by juvenile hormone analog. Recombinant SeGBP purified from baculovirus-infected insect cells retards the growth of S. exigua larvae. Additionally, SeGBP expression is acutely induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. Recombinant SeGBP can bind to B. bassiana but not to E. coli or B. thuringiensis. Consistent with these findings, SeGBP shows antifungal activity against B. bassiana. Therefore, these results provide insight into the role of SeGBP during the innate immune response following microbial infection, and furthermore, they suggest a novel function for SeGBP as a direct antifungal agent against entomopathogenic fungi, such as B. bassiana.

Toll recognition signal activates oenocytoid cell lysis via a crosstalk between plasmatocyte-spreading peptide and eicosanoids in response to a fungal infection

Plasmatocyte-spreading peptide (PSP) activates hemocyte-spreading behavior in response to various microbial pathogens. Its homolog, growth-blocking peptide, has several functions that activate immune cells and induce oenocytoid cell lysis (OCL). OCL is required for release of prophenoloxidase from oenocytoids in the beet armyworm, Spodoptera exigua. Injection of PSP to S. exigua larvae significantly induced in vivo OCL and resulted in significant increase of phenoloxidase (PO) activity. A fungal infection induced PSP expression and also significantly increased OCL. RNA interference (RNAi) of PSP expression significantly suppressed OCL induction and subsequently inhibited PO activation. Interestingly, an addition of dexamethasone (a specific phospholipase A2 inhibitor) inhibited the PSP activity to induce OCL. Toll signal pathway was associated with PSP action on inducing OCL because RNAi of Toll expression suppressed PSP expression and subsequent OCL induction. However, an addition of PSP to the larvae under RNAi of Toll expression rescued the progress of OCL.

Characteristics common to a cytokine family spanning five orders of insects

Growth-blocking peptide (GBP) is a member of an insect cytokine family with diverse functions including growth and immunity controls. Members of this cytokine family have been reported in 15 species of Lepidoptera, and we have recently identified GBP-like peptides in Diptera such as Lucilia cuprina and Drosophila melanogaster, indicating that this peptide family is not specific to Lepidoptera. In order to extend our knowledge of this peptide family, we purified the same family peptide from one of the tenebrionids, Zophobas atratus,11Zophobas atratus Zmc mRNA for cytokine, AB243070. isolated its cDNA, and sequenced it. The Z. atratus GBP sequence together with reported sequence data of peptides from the same family enabled us to perform BLAST searches against EST and genome databases of several insect species including Coleoptera, Diptera, Hymenoptera, and Hemiptera and identify homologous peptide genes. Here we report conserved structural features in these sequence data. They consist of 19–30 amino acid residues encoded at the C terminus of a 73-152 amino acid precursor and contain the motif C-x(2)-G-x(4,6)-G-x(1,2)-C-[KR], which shares a certain similarity with the motif in the mammalian EGF peptide family. These data indicate that these small cytokines belonging to one family are present in at least five insect orders.

Drosophila growth-blocking peptide-like factor mediates acute immune reactions during infectious and non-infectious stress

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevated expression of AMPs. GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential, indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.

Adaptor protein is essential for insect cytokine signaling in hemocytes

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin beta subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin beta subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens, indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.