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.

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.

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.

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.

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.

A gene-driven recovery mechanism: Drosophila larvae increase feeding activity for post-stress weight recovery.

Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post-stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non-typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up-regulation of post-stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post-stress periods.

Heat stress hardening of oriental armyworms is induced by a transient elevation of reactive oxygen species during sublethal stress.

Pre-exposure to mild heat stress enhances the thermotolerance of insects. Stress hardening is a beneficial physiological plasticity, but the mechanism underlying it remains elusive. Here we report that reactive oxygen species (ROS) concentrations were quickly and transiently elevated in the armyworms, Mythimna separata, by exposing them to 40°C, but not other tested temperatures. Larvae exposed to 40°C had subsequently elevated antioxidant activity and the highest survival of all tested heating conditions. The elevation of ROS after lethal heating at 44°C for 1 h was approximately twofold compared to heating at 40°C. Injection of an optimal amount of hydrogen peroxide (H2 O2 ) similarly caused sequential elevation of ROS and antioxidant activity in the test larval hemolymph, which led to significantly enhanced survival after lethal heat stress. The H2 O2 -induced thermotolerance was abolished by coinjection of potent antioxidants such as ascorbic acid or N-acetylcysteine. Both preheating at 40°C and H2 O2 injection enhanced expression of genes encoding superoxide dismutase 1, catalase, and heat shock protein 70 in the fat body of test larvae, indicating the adequate heat stress induced a transient elevation of ROS, followed by upregulation of antioxidant activity. We infer that thermal stress hardening is induced by a small timely ROS elevation that triggers a reduction-oxidation signaling mechanism.

Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp.

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.

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.

Cloning and heterologous expression of the thioviridamide biosynthesis gene cluster from Streptomyces olivoviridis.

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.

Venom components of Asobara japonica impair cellular immune responses of host Drosophila melanogaster.

The endoparasitoid wasp Asobara japonica has highly poisonous venom: the host Drosophila larvae are killed by envenomation at a dose that is naturally injected by the female wasp at parasitism. This insecticidal venom is neutralized, however, because A. japonica introduces lateral oviduct components soon after venom injection at oviposition. Although the venom and lateral oviduct components of this parasitoid have been partially characterized, how the venom components favor successful development of wasp eggs and larvae in the host remains ambiguous. Here, we demonstrated that A. japonica venom did not affect host humoral immune responses, determined as expression of antimicrobial peptide (AMP) genes, but significantly diminished two cellular responses, spreading and phagocytosis, by host hemocytes. Moreover, venom components drastically elevated a serine protease-like activity 4 h after its injection. The lateral oviduct components did not negate the detrimental effects of the venom on host cellular immunities, but significantly reduced the venom-induced elevation of protease activity. Both active factors in venom and lateral oviduct components were roughly characterized as heat-labile substances with a molecular mass of at least 10 kDa. Finally, venom of A. japonica, with a wide host range, was found to be much more toxic than that of Asobara rossica, which has a limited host range. These results reveal that A. japonica venom toxicity allows exploitation of a broader range of host insects because it is essential to overcome cellular immune responses of the host for successful parasitism.

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1.

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

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.

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.

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.

Identification of a novel gene, anorexia, regulating feeding activity via insulin signaling in Drosophila melanogaster.

Feeding activities of animals, including insects, are influenced by various signals from the external environment, internal energy status, and physiological conditions. Full understanding of how such signals are integrated to regulate feeding activities has, however, been hampered by a lack of knowledge about the genes involved. Here, we identified an anorexic Drosophila melanogaster mutant (GS1189) in which the expression of a newly identified gene, Anorexia (Anox), is mutated. In Drosophila larvae, Anox encodes an acyl-CoA binding protein with an ankyrin repeat domain that is expressed in the cephalic chemosensory organs and various neurons in the central nervous system (CNS). Loss of its expression or disturbance of neural transmission in Anox-expressing cells decreased feeding activity. Conversely, overexpression of Anox in the CNS increased food intake. We further found that Anox regulates expression of the insulin receptor gene (dInR); overexpression and knockdown of Anox in the CNS, respectively, elevated and repressed dInR expression, which altered larval feeding activity in parallel with Anox expression levels. Anox mutant adults also showed significant repression of sugar-induced nerve responses and feeding potencies. Although Anox expression levels did not depend on the fasting and feeding states cycle, stressors such as high temperature and desiccation significantly repressed its expression levels. These results strongly suggest that Anox is essential for gustatory sensation and food intake of Drosophila through regulation of the insulin signaling activity that is directly regulated by internal nutrition status. Therefore, the mutant strain lacking Anox expression cannot enhance feeding potencies even under starvation.

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.

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster.

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.

A eukaryotic (insect) tricistronic mRNA encodes three proteins selected by context-dependent scanning.

Eukaryotic mRNAs are generally considered monocistronic and encode only one protein. Although dicistronic mRNAs encoding two proteins were found in fungi, plants, and animals, polycistronic mRNAs encoding more than two proteins have remained elusive so far in any eukaryote. Here we demonstrate that a single mRNA from silkworm encodes the precursor of an insect cytokine paralytic peptide (PP) and two new cytokine precursor-like proteins, uENF1 and uENF2. RT-PCR analysis showed that this mRNA is widely conserved in moths. Western blot analyses and reporter assays using its modified mRNAs, created by replacing each one of the three ORFs with the firefly luciferase ORF, showed that all three proteins were translated from this mRNA in cell lines, larval tissues, and cell-free systems. Insertion experiments using the Renilla luciferase ORF or a stem loop ruled out the possible involvement of internal ribosome entry site in the three protein translation. On the other hand, systematic mutation analysis of the translation initiation sequence of the 5'-proximal uENF1 ORF suggested that the context-dependent leaky-scanning mechanism is involved in translation of the downstream uENF2 and PP ORFs. In vitro, a synthetic peptide corresponding to the putative mature form of uENF1 stimulated spreading of hemocytes as did the synthetic PP, whereas that of uENF2 antagonized the stimulating activities of PP and the uENF1 peptide, suggesting that the three proteins control cellular immunity interactively. Thus, eukaryotes have a cellular tricistronic mRNA that encodes three functionally related proteins as in an operon.