Drosophila cytokine GBP2 exerts immune responses and regulates GBP1 expression through GPCR receptor Mthl10.

Growth-blocking peptide (GBP), an insect cytokine, was first found in armyworm Mythimna separata. A functional analogue of GBP, stress-responsive peptide (SRP), was also identified in the same species. SRP gene expression has been demonstrated to be enhanced by GBP, indicating that both cytokines are organized within a hierarchical regulatory network. Although GBP1 (CG15917) and GBP2 (CG11395) have been identified in Drosophila melanogaster, immunological functions have only been characterized for GBP1. It is expected that the biological responses of two structurally similar peptides should be coordinated, but there is little information on this topic. Here, we demonstrate that GBP2 replicates the GBP1-mediated cellular immune response from Drosophila S2 cells. Moreover, the GBP2-induced response was silenced by pre-treatment with dsRNA targeting the GBP receptor gene, Mthl10. Furthermore, treatment of S2 cells with GBP2 enhanced GBP1 expression levels, but GBP1 did not affect GBP2 expression. GBP2 derived enhancement of GBP1 expression was not observed in the presence of GBP1, indicating that GBP2 is an upstream expressional regulator of a GBP1/GBP2 cytokine network. GBP2-induced enhancement of GBP1 expression was not observed in Mthl10 knockdown cells. Enhancement of GBP2 expression was observed in both Drosophila larvae and S2 cells under heat stress conditions; expressional enhancement of both GBP1 and GBP2 was eliminated in Mthl10 knockdown cells and larvae. Finally, Ca2+ mobilization assay in GCaMP3-expressing S2 cells demonstrated that GBP2 mobilizes Ca2+ upstream of Mthl10. Our finding revealed that Drosophila GBP1 and GBP2 control immune responses as well as their own expression levels through a hierarchical cytokine network, indicating that Drosophila GBP1/GBP2 system can be a simple model that is useful to investigate the detailed regulatory mechanism of related cytokine complexes.

Booklice Liposcelis bostrychophila are efficiently attracted by the combination of 2,3,5,6-tetramethylpyrazine and ultraviolet light.

BACKGROUND: Booklice Liposcelis bostrychophila are frequently found almost everywhere, including private houses and cleanrooms of factories and institutes. They often cause serious hygienic as well as agricultural problems, but a useful trap has not been developed so far. Therefore, an effective way to monitor and capture booklice is required. RESULTS: We here identified a new attractant, 2,3,5,6-tetramethylpyrazine (TMP), which efficiently captured booklice in combination with UV light. When booklice placed at both right and left edges of an assay tray were exposed to light stimulus from the center, test insects gathered at the center. The attraction was stronger with shorter wavelengths than longer ones: 365-nm ultraviolet (UV) light showed the strongest attraction of four tested light wavelengths. We found that cocoa powder attracted booklice weakly but significantly under total darkness. Furthermore, the cocoa smell was confirmed to enhance the attraction to light at all tested wavelengths irrespective of the difference between two brands of cocoa powders. Gas chromatography-mass spectrometry indicated that both cocoa products contain TMP as a major odor compound. Exposure of booklice to TMP significantly enhanced the attraction to UV light: the combined use with TMP almost doubled the attraction compared to the light only. By contrast, TMP homologs, pyrazine and dimethylpyrazines, showed strong repellent activities under UV light exposure. CONCLUSION: TMP enhanced the UV light attraction for booklice while pyrazine and dimethylpyrazines diminished it. Use of these attractant and repellent pyrazine derivatives together with UV light would enable us to develop a practical new way to monitor and capture booklice. © 2023 Society of Chemical Industry.

N-acetyloxfenicine strongly induces mitohormesis in mice as well as in insects.

Mitohormesis defines the increase in fitness induced by adaptive responses to mild mitochondrial stress. Here, we show that N-acetyloxfenicine (NAO) exerted higher thermotolerance than an endogenous mitohormesis inducer, N-acetyltyrosine (NAT). This activity was not observed in armyworm larvae injected with oxfenicine, suggesting the importance of N-acetylation. NAO-induced hormetic effect was triggered by transient perturbation of mitochondria, which causes a small increase in ROS production and leads to retrograde responses including enhanced expression of antioxidant enzyme genes via activation of FoxO transcription factors. Furthermore, pretreatment with NAO significantly repressed stress-induced peroxidation of lipids in mice and growth of colorectal cancer HCT116 cells that had been transplanted into nude mice. Taken together, NAO is a potent mitohormesis inducer that is similar to NAT in terms of structure and functions.

Prodigiosin R3, a new multicyclic prodigiosin formed by prodigiosin cyclization genes in the roseophilin producer.

The rph gene cluster for prodigiosin biosynthesis has been identified in Streptomyces griseoviridis 2464-S5, which produces cyclic prodigiosin derivatives including roseophilin (2), prodigiosin R1 (3) and prodigiosin R2 (4). A new cyclic prodigiosin, prodigiosin R3 (1), was produced by the redG redP double disruptant of Streptomyces coelicolor M511 expressing four cyclization gene candidates (rphG, rphG2, rphG3 and rphG4) in the rph cluster. The same compound was isolated from Streptomyces griseoviridis 2464-S5. The molecular formula of 1 was established as C27H33N3O by ESI and FAB mass spectrometry. The structure was determined to be a multicyclic prodigiosin with three alkyl linkages by NMR spectroscopic analysis. Prodigiosin R3 (1) showed cytotoxicity against HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells with IC50s of 2.1 µM and 3.2 µM, respectively.

13-Deoxo-13-iminodutomycin, a new neuroprotective compound from Streptomyces sp. RAP78.

A neuroprotective compound (2) was isolated from the culture broth of the dutomycin (1) producer Streptomyces sp. RAP78. The molecular formula of 2 was established as C44H55NO16 by high-resolution FAB-MS. The structure was determined to be a new dutomycin derivative possessing an acetimidoyl group in place of an acetyl group by NMR spectroscopic analysis. 13-Deoxo-13-iminodutomycin (2) but not dutomycin (1) protected C6 rat glioma cells and N18-RE-105 rat primary retina-mouse neuroblastoma hybrid cells from glutamate-induced toxicity with EC50s of 0.12 µM and 0.72 µM, respectively.

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.

N-acetyltyrosine-induced redox signaling in hormesis.

A suite of adaptations allows insects to survive in hostile terrestrial environments for long periods of time. Temperature represents a key environmental factor for most ectothermic insects, and they rapidly acclimate to high and low temperatures. Vast amounts of data in this research field support the idea that an insect's ability to tolerate fluctuating temperatures can be regarded as a biphasic hormetic dose response. Observation indicates that their thermal hormetic response represents a conservative estimate of their intrinsic capacity for rapid adaptation to environmental changes in nature because they naturally experience diel or seasonal temperature fluctuations. It is therefore reasonable to suppose that the hormetic response in insects reflects a surplus physiological capacity to deal with temperature changes that they would experience naturally. Although it has been unknown how thermal acclimation is induced, a stress-dependent increase in N-acetyltyrosine (NAT) was recently found to occur in insect larvae who had endured high temperatures. NAT treatment was demonstrated to induce thermotolerance in several tested insect species. NAT was also identified in the serum of humans as well as mice, and its concentration in mice was shown to be increased by heat and restraint stress, with NAT pretreatment lowering the concentrations of corticosterone and peroxidized lipids in stressed mice. These recent findings may give us some hints about how long a hormetic response lasts. Here, I will discuss recent findings underlying hormetic responses induced by an intrinsic factor, NAT, and how the hormetic response may begin and end.

The suppressive effect of bacterial-feeding nematodes on hemocyte spreading of Galleria mellonella.

Insect parasitic nematodes have developed a mechanism to escape from the cellular immunity of their insect hosts for successful parasitism. However, the detailed mechanism whereby they achieve this remains unclear. In our previous study, we demonstrated that non-parasitic nematodes such as Caenorhabditis elegans potentially have the ability to escape from the cellular immunity of the greater wax moth Galleria mellonella. Here we aimed to clarify the effect of non-parasitic and parasitic nematodes on the spreading of hemocytes-an essential cellular reaction for adhering to a foreign substance -from G. mellonella larvae. The hexane/methanol extract of C. elegans inhibited the spreading of hemocytes. Using 2D-TLC and reversed-phase HPLC, we detected a single peak that inhibited the spreading of hemocytes. In addition, the spreading of hemocytes recovered from C. elegans-injected insects was significantly delayed. Western blotting analysis showed that phosphorylated extracellular signal-regulated protein kinase (ERK) -an essential signaling component for spreading in hemocytes-was decreased by the injection of C. elegans, and that plasma from nematode-injected insects contained the factor that causes the decrease of phosphorylated ERK. We also observed this phenomenon using other non-parasitic and parasitic bacterial-feeding nematodes. These results suggest that the factors inhibiting hemocyte adhesion and delaying the spreading of hemocytes are conserved in bacterial-feeding nematodes and could be a pre-adaptation for parasitism.

Dunaimycin C3, a new GRP78 downregulator from Streptomyces sp. RAN389.

A new member of the dunaimycin family, dunaimycin C3 (2), was isolated from a fermented broth of Streptomyces sp. RAN389. The molecular formula of 2 was established as C42H70O10 by high-resolution FAB-MS, and the structure was elucidated by NMR spectroscopic analyses. Dunaimycin C3 inhibited the expression of the molecular chaperone GRP78 in HT1080 G-L cells in the presence of 10 mM of 2-deoxyglucose with an IC50 of 8.4 nM.

Neocurromycin A, a new GRP78 downregulator from Streptomyces sp. RAI364.

A new curromycin-related compound, neocurromycin A (2), was isolated from the fermented broth of Streptomyces sp. RAI364. The molecular formula of 2 was established as C35H44N4O7 by ESI-MS and the structure was elucidated by NMR spectroscopic analyses. Neocurromycin A showed selective cytotoxicity against MKN45 human gastric cancer cells in a nutrient-deprived medium with an IC50 of 380 nM and inhibited the expression of the molecular chaperone GRP78 in HT1080 G-L cells in the presence of 10 mM of 2-deoxyglucose with an IC50 of 1.7 µM.

N-acetyl-l-tyrosine is an intrinsic triggering factor of mitohormesis in stressed animals.

Under stress conditions, mitochondria release low levels of reactive oxygen species (ROS), which triggers a cytoprotective response, called "mitohormesis". It still remains unclear how mitochondria respond to stress-derived stimuli and release a low level of ROS. Here, we show that N-acetyl-l-tyrosine (NAT) functions as a plausible intrinsic factor responsible for these tasks in stressed animals. NAT is present in the blood or hemolymph of healthy animals, and its concentrations increase in response to heat stress. Pretreatment with NAT significantly increases the stress tolerance of tested insects and mice. Analyses using Drosophila larvae and cultured cells demonstrate that the hormetic effects are triggered by transient NAT-induced perturbation of mitochondria, which causes a small increase in ROS production and leads to sequential retrograde responses: NAT-dependent FoxO activation increases in the gene expression of antioxidant enzymes and Keap1. Moreover, we find that NAT represses tumor growth, possibly via the activation of Keap1. In sum, we propose that NAT is a vital endogenous molecule that could serve as a triggering factor for mitohormesis.

Bacterial feeding nematodes ingest haemocytes in the haemocoel of the insect Galleria mellonella.

Insect parasitic nematodes have acquired mechanisms to evade their host immune response for successful parasitism. Despite the importance of understanding of the evolution of evasion mechanisms from host immunity, insect immune response against non-parasitic nematodes has not been well studied. In our previous study, we demonstrated that a non-insect parasitic nematode Caenorhabditis elegans was not encapsulated by haemocytes in the larvae of the greater wax moth Galleria mellonella. To understand how nematodes influence insect haemocytes to escape encapsulation, we examined the effect of C. elegans on haemocytes in the haemocoel of G. mellonella larvae. Injection of nematodes resulted in the decrease of haemocyte density while mortality and spreading ability of haemocytes, the haematopoietic organs were not affected. In vitro co-incubation of haemocytes with nematodes resulted in a decrease of haemocyte density and we observed feeding on haemocytes by nematodes. Injection of C. elegans feeding-delay mutants into insects did not cause the decrease of haemocyte density. The decrease of haemocyte density was due to the nematode's ingestion of haemocytes. Furthermore, an entomopathogenic nematode and other bacterial feeding nematodes also showed similar feeding behaviour. The nematode's ability to feed on haemocytes may have played an important role in the evolution of nematode parasitism in bacterial-feeding nematodes.

Repeated phenotypic selection for cuticular blackness of armyworm larvae decreased stress resistance.

Armyworm Mythimna separata larvae show changes in cuticle darkening depending on population densities and are roughly categorized into two phenotypes, a pale brown solitary type and black-colored gregarious type. Although the color difference in both larval types is apparent, it remains ambiguous whether any change in physiological traits accompanies the cuticle darkening. To answer this query, we repeated genetic selection of the blackness phenotype over one hundred generations in our laboratory colony and produced a black-colored (BL) strain. Comparison between non-selected control (CTL) and BL strains revealed an increased fecundity and adult life span in the BL strain compared with the CTL strain. In contrast, BL strain larvae were found to be significantly more sensitive to heat stress than those in the CTL strain. Hemolymph reactive oxygen species (ROS) levels were higher in the BL strain than in the CTL strain under both non-stress and heat stress conditions. Antioxidant activities of the hemolymph were not significantly different between the two strains under non-stress condition, but the activities increased to higher levels in the CTL strain than those in the BL strain after heat stress. Activities and gene expression levels of antioxidant enzymes such as catalase and superoxide dismutase (SOD) in the fat body were significantly higher in CTL strain larvae than in BL strain larvae after heat treatment. Thermal stress tolerance of the offspring of crossings between the two strains showed a tolerance level almost equivalent to the maternal one: the cross between CTL females and BL males produced offspring with the higher tolerance compared with the oppositely crossed offspring. Expression levels of the antioxidant enzyme genes of the former offspring were found to be similar to those of CTL strain. These results indicate a trade-off between reproductive activity and stress resistance: the BL strain had acquired high reproductivity but had lost stress tolerance through repeated genetic selection. Furthermore, the present genetic analyses demonstrated that the phenotype of stress tolerance is derived from the maternal parent.

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.

Comments to Recent Studies Showing Systemic Mechanisms Enabling Drosophila Larvae to Recover From Stress-Induced Damages.

Compensational recovery from the damage created by stressors is important for all animals. However, how organisms recover from stress-induced negative impacts has been poorly understood. An 1-hour exposure to heat stress at 35°C led to reduced feeding activity of Drosophila melanogaster larvae, which caused reduction in body weight 2 hours after the stress, but not at other times. Such weight losses seem to be rescued by following enhanced feeding activities. We investigated the mechanisms underlying the accelerated feeding activity after the stress-induced reduction in feeding behavior. Our data showed increased expression of sweet taste gustatory receptor genes (Grs) and concomitant decreased expression of bitter taste Grs in the mouth parts 2 to 4 hours after the heat treatment for 1 hour. However, nontypical taste Gr expression was not changed. Furthermore, integration of both messenger RNA and protein expression analysis revealed that expression levels of tropomyosin and ATP (adenosine triphosphate) synthase ß subunit were significantly increased in their mouths 3 to 5 hours after the heat stress. The increased expression of these genes would contribute to accelerated muscular movement of the mouth hooks. This study indicated that Drosophila larvae possess an efficient systemic mechanism that enables them to recover from growth delay caused by stress conditions.

Pyroxazone, a new neuroprotective compound from Streptomyces sp. RAN54.

A neuroprotective compound designated pyroxazone (1) was isolated from the culture broth of Streptomyces sp. RAN54. The molecular formula of 1 was established as C18H14N2O5 by high-resolution FAB-MS. The structure was determined to be a new 2-amino-3H-phenoxazin-3-one derivative by NMR spectroscopic analysis. Pyroxazone (1) protected C6 rat glioma cells and N18-RE-105 rat primary retina-mouse neuroblastoma hybrid cells from glutamate-induced toxicity with EC50s of 8.2 µM and 1.7 µM, respectively.

Quinomycins H1 and H2, new cytotoxic antibiotics from Streptomyces sp. RAL404.

Two new cytotoxic antibiotics designated quinomycins H1 (2) and H2 (3) were isolated from the culture broth of Streptomyces sp. RAL404. The molecular formula of both compounds was established as C52H65N11O13S2 by electrospray ionization mass spectrometry (ESI-MS). Their structures were determined as echinomycin (1) derivatives containing a 3-hydoxyquinaldic acid molecule in place of one of the two quinoxaline-2-carboxylic acid chromophores. Quinomycins H1 (2) and H2 (3) showed selective cytotoxicity against RG-E1-4 cells bearing the adenovirus oncogenes with IC50s of 11 nM and 12 nM, respectively.

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.

Prodigiosin R2, a new prodigiosin from the roseophilin producer Streptomyces griseoviridis 2464-S5.

Roseophilin (2) is a unique prodigiosin-related compound produced by Streptomyces griseoviridis 2464-S5, and is characterized by a central furan ring and a bicyclic alkyl chain. During a search for biosynthetic intermediates of 2, a new metabolite designated prodigiosin R2 (1) was isolated from the culture of the roseophilin producer. The molecular formula of 1 was established as C27H35N3O by high-resolution FAB-MS. The structure of 1 was determined by NMR spectroscopic analyses as a prodigiosin derivative with the same bicyclic alkyl chain as 2. Prodigiosin R2 (1) showed potent cytotoxicity against HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells with IC50s of 0.41 and 0.82 µM, respectively.