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.

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.

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.

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.

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.

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.