Insect Innate Immune Response Research Articles

Systematic analysis of innate immune-related genes in the silkworm: Application to antiviral research.

The silkworm, a crucial model organism of the Lepidoptera, offers an excellent platform for investigating the molecular mechanisms underlying the innate immune response of insects toward pathogens. Over the years, researchers worldwide have identified numerous immune-related genes in silkworms. However, these identified silkworm immune genes are not well classified and not well known to the scientific community. With the availability of the latest genome data of silkworms and the extensive research on silkworm immunity, it has become imperative to systematically categorize the immune genes of silkworms with different database IDs. In this study, we present a meticulous organization of prevalent immune-related genes in the domestic silkworm, using the SilkDB 3.0 database as a reliable source for updated gene information. Furthermore, utilizing the available data, we classify the collected immune genes into distinct categories: pattern recognition receptors, classical immune pathways, effector genes and others. In-depth data analysis has enabled us to predict some potential antiviral genes. Subsequently, we performed antiviral experiments on selected genes, exploring their impact on Bombyx mori nucleopolyhedrovirus replication. The outcomes of this research furnish novel insights into the immune genes of the silkworm, consequently fostering advancements in the field of silkworm immunity research by establishing a comprehensive classification and functional understanding of immune-related genes in the silkworm. This study contributes to the broader understanding of insect immune responses and opens up new avenues for future investigations in the domain of host-pathogen interactions.

Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

Humoral immune response of Galleria mellonella after mono- and co-injection with Hypericum perforatum extract and Candida albicans.

Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co-injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co-injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono-injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono-injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

Cross-talk between immunity and behavior: insights from entomopathogenic fungi and their insect hosts.

Insects are one of the most successful animals in nature, and entomopathogenic fungi play a significant role in the natural epizootic control of insect populations in many ecosystems. The interaction between insects and entomopathogenic fungi has continuously coevolved over hundreds of millions of years. Many components of the insect innate immune responses against fungal infection are conserved across phyla. Additionally, behavioral responses, which include avoidance, grooming, and/or modulation of body temperature, have been recognized as important mechanisms for opposing fungal pathogens. In an effort to investigate possible cross-talk and mediating mechanisms between these fundamental biological processes, recent studies have integrated and/or explored immune and behavioral responses. Current information indicates that during discrete stages of fungal infection, several insect behavioral and immune responses are altered simultaneously, suggesting important connections between the two systems. This review synthesizes recent advances in our understanding of the physiological and molecular aspects influencing cross-talk between behavioral and innate immune antifungal reactions, including chemical perception and olfactory pathways.

Serpin-1a and serpin-6 regulate the Toll pathway immune homeostasis by synergistically inhibiting the Spätzle-processing enzyme CLIP2 in silkworm, Bombyx mori.

The Toll receptor signaling pathway is an important innate immune response of insects to pathogen infection; its extracellular signal transduction involves serine protease cascade activation. However, excessive or constitutive activation of the Toll pathway can be detrimental. Hence, the balance between activation and inhibition of the extracellular protease cascade must be tightly regulated to achieve favorable outcomes. Previous studies have shown that serpins-serine protease inhibitors-negatively regulate insect innate immunity by inhibiting extracellular protease cascade signaling. Although the roles of serpins in insect innate immunity are well described, the physiological mechanisms underlying their synergistic effects remain poorly understand. Here, we characterize the molecular mechanism by which serpin-1a and serpin-6 synergistically maintain immune homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Through in vitro biochemical assays and in vivo bioassays, we demonstrate that clip-domain serine protease 2 (CLIP2), as the Toll cascade-activating terminal protease, is responsible for processing proSpätzle1 to induce the expression of antimicrobial peptides. Further biochemical and genetic analyses indicate that constitutively expressed serpin-1a and inducible serpin-6 synergistically target CLIP2 to maintain homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Taken together, this study provides new insights into the precise regulation of Toll cascade activation signals in insect innate immune responses and highlights the importance and complexity of insect immune homeostasis regulation.

Microplitis bicoloratus Bracovirus Promotes Cyclophilin D-Acetylation at Lysine 125 That Correlates with Apoptosis during Insect Immunosuppression.

Cyclophilin D (CypD) is regulated during the innate immune response of insects. However, the mechanism by which CypD is activated under innate immunosuppression is not understood. Microplitis bicoloratus bracovirus (MbBV), a symbiotic virus in the parasitoid wasp, Microplitis bicoloratus, suppresses innate immunity in parasitized Spodoptera litura. Here, we demonstrate that MbBV promotes the CypD acetylation of S. litura, resulting in an immunosuppressive phenotype characterized by increased apoptosis of hemocytes and MbBV-infected cells. Under MbBV infection, the inhibition of CypD acetylation significantly rescued the apoptotic cells induced by MbBV, and the point-mutant fusion proteins of CypDK125R-V5 were deacetylated. The CypD-V5 fusion proteins were acetylated in MbBV-infected cells. Deacetylation of CypDK125R-V5 can also suppress the MbBV-induced increase in apoptosis. These results indicate that CypD is involved in the MbBV-suppressed innate immune response via the CypD-acetylation pathway and S. litura CypD is acetylated on K125.

A spätzle protein involved in the immune response of Neoseiulus barkeri (Acari: Phytoseiidae) against Beauveria bassiana

The predatory mite, Neoseiulus barkeri, is more tolerant to the entomopathogenic fungus, Beauveria bassiana, than small sap-feeding pests. Infection of insects by B. bassiana can induce insect innate immune response, and the spätzle protein (Spz), an important ligand of the insect Toll pathway, has been demonstrated to play an important role in insect innate immune defense system. Here, we investigated the infection process of B. bassiana on N. barkeri using scanning electron microscopy, and identified a Spz protein. The result showed that NbSpz-1 was expressed in all developmental stages of N. barkeri and its expression was up-regulated by B. bassiana infection. After knocking down the expression of NbSpz-1 by dsRNA, the tolerance of N. barkeri to B. bassiana was weakened, and the survival rate was reduced. Our results verified the potential role of the Spz protein in N. barkeri immune response against B. bassiana. This study enhanced our understanding on innate immune system of phytoseiid mite.

Spaetzle responds to the process of Neoseiulus barkeri resistance to Beauveria bassiana infection

The application of Beauveria bassiana is proved to be a feasible biological control measure against pest insects and mites (e.g. Liu et al. 2018; Al Khoury et al. 2020). But the complex and variable ecological environment can affect the efficiency of B. bassiana. Due to the powerful search and spread ability of predatory mites, they were proposed to carry and spread pathogenic microbial spores to places that are difficult to be covered by conventional spray methods (Chen et al. 2020; Hao et al. 2021). This biocontrol strategy of combining fungi with predator mites is predicted to play a synergistic role in pest control. Therefore, the tolerance of predator mites to B. bassiana and the underlying mechanism have been the subject of extensive research and remained to be explored further. The infection of insects by B. bassiana can induce insect innate immune response. Spaetzle protein (Spz), as an important ligand of insect Toll pathway, has been demonstrated to play an important role in insect innate immune defense system. 
  

Innate Immune Response of Galleria mellonella (Lepidoptera: Pyralidae) Larvae to Lemongrass Essential Oil and Citral

Abstract The innate immune response in insects relies on cellular responses. In most lepidopteran species, challenging the immune system causes an increase in hemocyte numbers and promotes encapsulation and melanization of invading objects, such as parasitoid eggs or artificial objects (e.g., nylon implants). We studied the effects of carbon particles (Sumi ink) on the immune response of Galleria mellonella L. (Lepidoptera: Pyralidae) last-instar larvae. Ink injections increased total hemocyte counts in a dose-dependent manner, with the 40 and 80% concentrations being most effective in eliciting hemocyte response. Hemocyte numbers increased significantly at 4 and 6 h after ink injection. Lemongrass essential oil (0.18, 0.36, and 0.72 µg/larva) and citral (0.12, 0.24, and 0.48 µg/larva) reversed the mobilization of hemocytes by the ink. Melanization of nylon larvae implants started as early as 15 min after implant insertion and reached maximum observed levels within 1 h. The degree of melanization also was higher in larvae challenged with ink 1 h prior to implant removal, which suggests initialization of the immune response. Citral injections inhibited implant melanization both in unchallenged and ink-challenged larvae. In the former case, citral concentrations needed for effective inhibition were less (0.3 µg/larva) than in ink-challenged larvae (1.2 and 2.4 µg/larva).

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis.

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.

Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species.

Innate immune systems are key defenses of animals and particularly important in species that lack the sophisticated adaptive immune systems as found in vertebrates. Here, we were interested to quantify variation in innate immune responses of insects in hosts that differ in their parasite susceptibility. To do this, we studied immune responses in honey bees, which can host a remarkable number of different parasites, which are major contributors of declining bee health and colony losses. The most significant parasite of honey bees is the mite Varroa destructor, which has infested the majority of global honey bee populations, and its control remains a major challenge for beekeepers. However, a number of nonmanaged honey bees seem able to control Varroa infections, for example, the Eastern honey bee Apis ceranacerana or the African honey bee Apis mellifera scutellata. These bees therefore make interesting study subjects to identify underlaying resistance traits, for example, by comparing them to more susceptible bee genotypes such as Western honey bees (A. melliferaligustica). We conducted a series of interlinked experiments and started with behavioral assays to compare the attractiveness of bee larvae to mites using different honey bee genotypes and castes. We found that 6-day-old larvae are always most attractive to mites, independently of genotype or castes. In a next step, we compared volatile profiles of the most attractive larvae to test whether they could be used by mites for host selection. We found that the abundance of volatile compounds differed between larval ages, but we also found significant differences between genotypes and castes. To further study the expected underlaying physiological differences between potentially resistant and susceptible host larvae, we compared the larval hemolymph proteomes of the three honey bee genotypes and two castes in response to mite exposure. We identified consistent upregulation of immune and stress-related genes in Varroa-exposed larvae, which differed between genotypes and castes. Tolerant honey bee castes and genotypes were characterized by stronger or more distinct immune esponses. In summary, we provide first insights into the complex involvement of the innate immune system of tolerant honey bees against mite infestations, which could be used for future breeding purposes.

Antimicrobial peptide expression in the cockroach gut during enterobacterial infection is specific and influenced by type III secretion.

ABSTRACTOmnivorous synanthropic cockroaches, such as the German cockroach (Blattella germanica), are reservoirs and vectors of enteric bacterial pathogens. A lifestyle conducive to frequent encounters with high loads of diverse bacteria may have led to the evolution of unique innate immune systems in these insects. The innate immune response of insects relies largely on generalized mechanisms to sense and eliminate foreign microbes. However, analyses of the genomes of common synanthropic cockroaches previously revealed a repertoire of pathogen associated molecular pattern (PAMP) receptors and antimicrobial peptides (AMPs) that is significantly expanded relative to most holometabolous insect models and vectors, supporting the intriguing possibility that cockroaches may encode enhanced recognition within their immune system and may possess an enhanced capacity to fine tune innate immune responses. Investigating how cockroaches respond to infection with enterobacteria provides the opportunity to expand our fundamental knowledge of the regulation of insect innate immunity in a context that is biologically and medically relevant. German cockroaches can harbor both Salmonella enterica serovar Typhimurium and Escherichia coli in their gut without experiencing pathogenesis. The former colonizes the gut and replicates while the latter persists only transiently. We hypothesized that differences in the innate immune response may contribute to or result from the difference in infection dynamics between the two enterobacteria. To test this hypothesis, we used qRT-PCR to analyze expression of five genes encoding representative AMPs (Attacins, Blattellicin, Defensins) in the gut of German cockroaches 1 and 24 h after ingestion of live or heat-killed enterobacteria. We found that robust AMP expression was induced in response to ingestion of a live wild-type strain of S. Typhimurium, but not in response to live E. coli, heat-killed S. Typhimurium, or a live mutant strain of S. Typhimurium lacking type III secretion systems. These results indicate that the cockroach immune system does not respond to stimulation with high levels of ingested bacterial PAMPs such as peptidoglycan. Rather, AMP expression in the gut appears to be induced by active bacterial colonization involving type III secretion. We speculate that this form of regulation may have evolved to prevent over activation of the immune system from frequent ingestion of innocuous, non-colonizing, or non-viable bacteria. While additional work is needed to delineate the molecular mechanisms underlying our observations, our findings provide significant novel insight into the immunological adaptation of cockroaches to life in septic environments as well as the factors that regulate bacterial pathogen transmission by these insects.

The Emerging Role of STING in Insect Innate Immune Responses and Pathogen Evasion Strategies.

Emerging evidence reveals that the stimulator of the interferon genes (STING) signaling pathway in insects and other animal cells helps them to sense and effectively respond to infection caused by numerous types of microbial pathogens. Recent studies have shown that genomic material from microbial pathogens induces the STING signaling pathway for the production of immune factors to attenuate infection. In contrast, microbial pathogens are equipped with various factors that assist them in evading the STING signaling cascade. Here we discuss the STING signaling pathway different animal groups compared to human and then focus on its crucial biological roles and application in the microbial infection of insects. In addition, we examine the negative and positive modulators of the STING signaling cascade. Finally, we describe the microbial pathogen strategies to evade this signaling cascade for successful invasion.

Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule

SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.

Peptidoglycan recognition protein-S1 (PGRP-S1) from Diaphania pyloalis (Walker) is involved in the agglutination and prophenoloxidase activation pathway.

Recognition of invading foreign exogenous pathogen is the first step to initiate the innate immune response of insects, which accomplished by the pattern recognition receptors (PRRs). Peptidoglycan recognition proteins (PGRPs) serve as an important type of PRRs, which activate immune response by detecting peptidoglycan of microbial cell wall. In this study, we have cloned the full-length cDNA of PGRP gene called PGRP-S1 from the Diaphania pyloalis (Walker). The open reading frame (ORF) of D. pyloalis PGRP-S1 encodes 211 amino acids which containing a secretion signal peptide and a canonical PGRP domain. Multisequence alignment revealed that PGRP-S1 possess the amino acid residues responsible for zinc binding and amidase activity. D. pyloalis PGRP-S1 exhibited the highest transcript level in fat body and followed in head. The mRNA concentration dramatically increased after an injection of Escherichia coli or Micrococcus luteus. Purified recombinant PGRP-S1 exhibit binding ability to peptidoglycans from Staphylococcus aureus or Bacillus subtilis and cause intensive agglutination of E. coli, M. luteus or S. aureus in the presence of zinc ions. Furthermore, phenoloxidase activity significantly increased when the plasma from larvae was incubated with recombinant PGPR-S1 and peptidoglycans from B. subtilis or M. luteus simultaneously. These results implied that PGRP-S1 was a member involving the prophenoloxidase activation pathway. Overall, our results indicated that D. pyloalis PGRP-S1 serve as a PRR to participate in the recognition of foreign pathogen and prophenoloxidase pathway stimulation.

Serine protease SP7 cleaves prophenoloxidase and is regulated by two serpins in Ostrinia furnacalis melanization

Melanization is an innate immune response in insects to defend against the invading pathogens and parasites. During melanization, prophenoloxidase (PPO) requires proteolytic activation by its upstream prophenoloxidase-activating protease (PAP). We here cloned a full-length cDNA for a serine protease, named as SP7, from Ostrinia furnacalis. The open reading frame of SP7 encodes 421-amino acid residue protein with a 19-residue signal peptide. qRT-PCR analysis showed that SP7 mRNA levels were significantly upregulated upon exposure to microbial infection. Recombinant SP7 zymogen was activated by serine protease SP2. The active SP7 could cleave O. furnacalis PPOs including PPO2, PPO1b and PPO3. Additionally, active SP7 could form covalent complexes with serine protease inhibitor serpin-3 and serpin-4. The activity of SP7 in cleaving a colorimetric substrate IEARpNA or O. furnacalis PPOs was efficiently blocked by either serpin-3 or serpin-4. Our work thus revealed that SP7 and SP2 partially constituted a PPO activation cascade in which SP7 was activated by SP2 and then likely worked as a PAP. SP7 was effectively regulated by serpin-3 and serpin-4. The results would allow further advances in the understanding of melanization mechanisms in O. furnacalis.

Physiological and Biochemical Effects of Klebsiella oxytoca Infection on Model Organism Galleria mellonella L.

Examination of physiological and biochemical effects on infection models is very important for the application of new treatments against human pathogens. It is used as an infection model because the innate immune responses of insects are similar to those of mammals. In this study, the physiological response of model insect Galleria mellonella to human pathogenic bacteria Klebsiella oxytoca infection was investigated. Changes in metabolic enzyme activities such as aspartate transferase (AST), alanine transferase (ALT), alkaline phosphatase (ALP), amylase (AMYL) and gamma glutamyl transferase (GGT) occurred due to K. oxytoca infection. In addition, amounts of non-enzymatic antioxidant such as uric acid (UA), bilirubin (BIL) and albumin (ALB) were altered in G. mellonella hemolymph due to infection. The results obtained from this study showed that G. mellonella can be used as a model organism in the evaluation of the physiological effects of K. oxytoca infection on the host.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Comparative response of Spodoptera litura challenged per os with Serratia marcescens strains differing in virulence

Host plays an important role in influencing virulence of a pathogen and efficacy of a biopesticide. The present study was aimed to characterize the possible factors present in Spodoptera litura that influenced pathogenecity of orally ingested S. marcescens strains, differing in their virulence. Fifth instar larvae of S. litura responded differently as challenged by two Serratia marcescens strains, SEN (virulent strain, LC50 7.02 103 cfu/ml) and ICC-4 (non-virulent strain, LC50 1.19 1012 cfu/ml). Considerable increase in activity of lytic enzymes protease and phospholipase was recorded in the gut and hemolymph of larvae fed on diet supplemented with S. marcescens strain ICC-4 as compared to the larvae treated with S. marcescens strain SEN. However, a significant up-regulation of antioxidative enzymes SOD (in foregut and midgut), CAT (in the midgut) and GST (in the foregut and hemolymph) was recorded in larvae fed on diet treated with the virulent S. marcescens strain SEN in comparison to larvae fed on diet treated with the non-virulent S. marcescens strain ICC-4. Activity of defense related enzymes lysozyme and phenoloxidase activity were also higher in the hemolymph of larvae fed with diet treated with S. marcescens strain SEN as compared to hemolymph of S. marcescens strain ICC-4 treated larvae. More number of over-expressed proteins was observed in the gut and hemolymph of S. marcescens strains ICC-4 and SEN treated larvae, respectively. Identification of the selected differentially expressed proteins indicated induction of proteins involved in insect innate immune response (Immunoglobulin I-set domain, Apolipophorin III, leucine rich repeat and Titin) in S. marcescens strain SEN treated larvae. Over-expression of two proteins, actin related protein and mt DNA helicase, were noted in S. marcescens treated larvae with very high levels observed in the non-virulent strain. Up-regulation of homeobox protein was noted only in S. marcescens strain ICC-4 challenged larvae. This study indicated that ingestion of non-virulent S. marcescens strain ICC-4 induced strong immune response in insect gut while there was weak response to the virulent S. marcescens strain SEN which probably resulted in difference in their virulence.

Peptides based on the reactive center loop of Manduca sexta serpin-3 block its protease inhibitory function

One innate immune response in insects is the proteolytic activation of hemolymph prophenoloxidase (proPO), regulated by protease inhibitors called serpins. In the inhibition reaction of serpins, a protease cleaves a peptide bond in a solvent-exposed reactive center loop (RCL) of the serpin, and the serpin undergoes a conformational change, incorporating the amino-terminal segment of the RCL into serpin β-sheet A as a new strand. This results in an irreversible inhibitory complex of the serpin with the protease. We synthesized four peptides with sequences from the hinge region in the RCL of Manduca sexta serpin-3 and found they were able to block serpin-3 inhibitory activity, resulting in suppression of inhibitory protease-serpin complex formation. An RCL-derived peptide with the sequence Ser-Val-Ala-Phe-Ser (SVAFS) displayed robust blocking activity against serpin-3. Addition of acetyl-SVAFS-amide to hemolymph led to unregulated proPO activation. Serpin-3 associated with Ac-SVAFS-COO− had an altered circular dichroism spectrum and enhanced thermal resistance to change in secondary structure, indicating that these two molecules formed a binary complex, most likely by insertion of the peptide into β-sheet A. The interference of RCL-derived peptides with serpin activity may lead to new possibilities of “silencing” arthropod serpins with unknown functions for investigation of their physiological roles.