Insect Immunity Research Articles

Insect Antimicrobial Peptides as Guardians of Immunity and Beyond: A Review.

Antimicrobial peptides (AMPs), as immune effectors synthesized by a variety of organisms, not only constitute a robust defense mechanism against a broad spectrum of pathogens in the host but also show promising applications as effective antimicrobial agents. Notably, insects are significant reservoirs of natural AMPs. However, the complex array of variations in types, quantities, antimicrobial activities, and production pathways of AMPs, as well as evolution of AMPs across insect species, presents a significant challenge for immunity system understanding and AMP applications. This review covers insect AMP discoveries, classification, common properties, and mechanisms of action. Additionally, the types, quantities, and activities of immune-related AMPs in each model insect are also summarized. We conducted the first comprehensive investigation into the diversity, distribution, and evolution of 20 types of AMPs in model insects, employing phylogenetic analysis to describe their evolutionary relationships and shed light on conserved and distinctive AMP families. Furthermore, we summarize the regulatory pathways of AMP production through classical signaling pathways and additional pathways associated with Nitric Oxide, insulin-like signaling, and hormones. This review advances our understanding of AMPs as guardians in insect immunity systems and unlocks a gateway to insect AMP resources, facilitating the use of AMPs to address food safety concerns.

Insect immunity - adaptive mechanisms and survival strategies

Host-pathogen interactions play an important role in understanding the dynamics of the insect immune system. The analysis of environmental modulators, both biotic and abiotic, directs our attention to the impact of the surroundings on the effectiveness of immunological responses. This knowledge is essential for the comprehensive understanding of insect’s immune reaction after infection with a pathogen. This article discusses the role of the immune system in insect, with a special emphasis on greater wax moth Galleria mellonella and highlights its adaptive capabilities. The processes are not only extremely interesting area of scientific research but also indicate potential practical applications in the context of plan protection, pest population control and medicine.

Insect immune resolution with EpOME/DiHOME and its dysregulation by their analogs leading to pathogen hypersensitivity

Upon immune challenge, recognition signals trigger insect immunity to remove the pathogens through cellular and humoral responses. Various immune mediators propagate the immune signals to nearby tissues, in which polyunsaturated fatty acid (PUFA) derivatives play crucial roles. However, little was known on how the insects terminate the activated immune responses after pathogen neutralization. Interestingly, C20 PUFA was detected at the early infection stage and later C18 PUFAs were induced in a lepidopteran insect, Spodoptera exigua. This study showed the role of epoxyoctadecamonoenoic acids (EpOMEs) in the immune resolution at the late infection stage to quench the excessive and unnecessary immune responses. In contrast, dihydroxy-octadecamonoenoates (DiHOMEs) were the hydrolyzed and inactive forms of EpOMEs. The hydrolysis is catalyzed by soluble epoxide hydrolase (sEH). Inhibitors specific to sEH mimicked the immunosuppression induced by EpOMEs. Furthermore, the inhibitor treatments significantly enhanced the bacterial virulence of Bacillus thuringiensis against S. exigua. This study proposes a negative control of the immune responses using EpOME/DiHOME in insects.

Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect

Insects lack acquired immunity and were thought to have no immune memory, but recent studies reported a phenomenon called immune priming, wherein sublethal dose of pathogens or nonpathogenic microbes stimulates immunity and prevents subsequential pathogen infection. Although the evidence for insect immune priming is accumulating, the underlying mechanisms are still unclear. The bean bug Riptortus pedestris acquires its gut microbiota from ambient soil and spatially structures them into a multispecies and variable community in the anterior midgut and a specific, monospecies Caballeronia symbiont population in the posterior region. We demonstrate that a particular Burkholderia strain colonizing the anterior midgut stimulates systemic immunity by penetrating gut epithelia and migrating into the hemolymph. The activated immunity, consisting of a humoral and a cellular response, had no negative effect on the host fitness, but on the contrary protected the insect from subsequent infection by pathogenic bacteria. Interruption of contact between the Burkholderia strain and epithelia of the gut weakened the host immunity back to preinfection levels and made the insects more vulnerable to microbial infection, demonstrating that persistent acquisition of environmental bacteria is important to maintain an efficient immunity.

Current status of innate immune responses in Malpighian tubules of insects: A review focusing on the Toll signaling pathway

AbstractInnate immunity is the immediate and exclusive defense mechanism in invertebrates. It acts against foreign agents and pathogens. In insects, this defense mechanism involves a combination of humoral and cellular responses, orchestrated by specific recognition receptors and the activation of various signaling pathways. Remarkably, Toll and Toll‐related proteins play a pivotal role in insect immunity by inducing the production of antimicrobial peptides (AMPs) in response to pathogen attacks. Toll receptors, which are type I transmembrane proteins, significantly affect insect development and the innate immune response, primarily via the nuclear factor kappa‐B signaling pathway. The Malpighian tubules of insects play a crucial role in waste elimination, hemostasis maintenance, and immune defense. The Toll signaling pathway in Malpighian tubules is mainly involved in the production of AMPs. This review provides valuable insights into the mechanisms of the Toll signaling pathway and the morphological and physiological attributes of Malpighian tubules. Moreover, it discusses the classification and functionalities of AMPs synthesized in Malpighian tubules in response to pathogens, thereby contributing to our understanding of innate immunity in insects.

Thiourea as a polyphenol oxidase inhibitor enhances host infection by the entomopathogenic nematode, Heterorhabditis beicherriana

Entomopathogenic nematodes (EPN) are potent microbial biocontrol agents applied commercially against various economically important insect pests. The efficacy of EPN in the field may be partially reduced when compared to chemical insecticides due to its sensitivity to environmental stressors like temperature, desiccation, and UV light. Thus, researchers have developed methods to enhance the effectiveness of EPN, such as screening or breeding stress-resistant strains and creating adjuvants for EPN protection in the field. This study used another strategy to lower the host’s immunity so that the anti-infection ability of the pest wanes, and the virulence of EPN waxes accordingly. In addition to its use as a plant growth regulator, thiourea also functions as an inhibitor of polyphenol oxidase (PO) in insects. Thiourea can inhibit a key interaction in the humoral immunity of insects, so we utilized it to enhance the efficacy of EPN. To investigate the effects of thiourea on PO activity inhibition and EPN pathogenesis, we studied the effects of thiourea on survival, infectivity, reproduction, and host searching ability of the EPN, Heterorhabditis beicherriana, and monitored in vivo and in vitro polyphenol oxidase activity of the model insect host, Galleria mellonella. The survival of H. beicherriana infective juveniles (IJs) was barely affected after exposure to 0.5 mM and 1 mM thiourea solution, and the reproductive output of H. beicherriana IJs (after applying 50, 100 and 200 IJs per host) in 0.5 mM and 1 mM thiourea was not different compared to the control. However, the LD50 of G. mellonella larvae caused by H. beicherriana without thiourea was significantly higher than H. beicherriana IJs in thiourea. Moreover, thiourea inhibited in vivo and in vitro polyphenol oxidase activity of G. mellonella infected by EPNs at different concentrations. Thus, thiourea can improve the biological control efficacy of EPNs, and the enhanced environmentally friendly approach may apply to diverse cropping systems.

Insights into midgut cell types and their crucial role in antiviral immunity in the lepidopteran model Bombyx mori.

The midgut, a vital component of the digestive system in arthropods, serves as an interface between ingested food and the insect's physiology, playing a pivotal role in nutrient absorption and immune defense mechanisms. Distinct cell types, including columnar, enteroendocrine, goblet and regenerative cells, comprise the midgut in insects and contribute to its robust immune response. Enterocytes/columnar cells, the primary absorptive cells, facilitate the immune response through enzyme secretions, while regenerative cells play a crucial role in maintaining midgut integrity by continuously replenishing damaged cells and maintaining the continuity of the immune defense. The peritrophic membrane is vital to the insect's innate immunity, shielding the midgut from pathogens and abrasive food particles. Midgut juice, a mixture of digestive enzymes and antimicrobial factors, further contributes to the insect's immune defense, helping the insect to combat invading pathogens and regulate the midgut microbial community. The cutting-edge single-cell transcriptomics also unveiled previously unrecognized subpopulations within the insect midgut cells and elucidated the striking similarities between the gastrointestinal tracts of insects and higher mammals. Understanding the intricate interplay between midgut cell types provides valuable insights into insect immunity. This review provides a solid foundation for unraveling the complex roles of the midgut, not only in digestion but also in immunity. Moreover, this review will discuss the novel immune strategies led by the midgut employed by insects to combat invading pathogens, ultimately contributing to the broader understanding of insect physiology and defense mechanisms.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

Immunomodulatory Peptides as Vaccine Adjuvants and Antimicrobial Agents.

The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides.

The accumulation of modular serine protease mediated by a novel circRNA sponging miRNA increases Aedes aegypti immunity to fungus

BackgroundMosquitoes transmit many infectious diseases that affect human health. The fungus Beauveria bassiana is a biological pesticide that is pathogenic to mosquitoes but harmless to the environment.ResultsWe found a microRNA (miRNA) that can modulate the antifungal immunity of Aedes aegypti by inhibiting its cognate serine protease. Fungal infection can induce the expression of modular serine protease (ModSP), and ModSP knockdown mosquitoes were more sensitive to B. bassiana infection. The novel miRNA-novel-53 is linked to antifungal immune response and was greatly diminished in infected mosquitoes. The miRNA-novel-53 could bind to the coding sequences of ModSP and impede its expression. Double fluorescence in situ hybridization (FISH) showed that this inhibition occurred in the cytoplasm. The amount of miRNA-novel-53 increased after miRNA agomir injection. This resulted in a significant decrease in ModSP transcript and a significant increase in mortality after fungal infection. An opposite effect was produced after antagomir injection. The miRNA-novel-53 was also knocked out using CRISPR-Cas9, which increased mosquito resistance to the fungus B. bassiana. Moreover, mosquito novel-circ-930 can affect ModSP mRNA by interacting with miRNA-novel-53 during transfection with siRNA or overexpression plasmid.ConclusionsNovel-circ-930 affects the expression level of ModSP by a novel-circ-930/miRNA-novel-53/ModSP mechanism to modulate antifungal immunity, revealing new information on innate immunity in insects.

Destruxin A inhibits the hemocytin-mediated hemolymph immunity of host insects to facilitate Metarhizium infection

Insects have an effective innate immune system to protect themselves against fungal invasion. Metarhizium employs a toxin-based strategy using a nonribosomal peptide called destruxin A (DA) to counteract the host immune response. However, the mechanism by which DA inhibits insect immunity is still unclear. Here, we identified 48 DA-binding proteins in silkworm hemolymph, with the binding affinity (KD) ranging from 2 to 420μM. Among these proteins, hemocytin, an important immune factor, was determined to be the strongest DA-binding protein. DA binds to hemocytin and regulates its conformation in a multisite manner. Furthermore, DA exerts a significant inhibitory effect on hemocytin-mediated hemocyte aggregation. By disrupting the interaction between hemocytin, actin A3, and gelsolin, DA prevents the transformation of granules into vesicles in hemocytes. These vesicles are responsible for storing, maturing, and exocytosing hemocytin. Therefore, hemocytin secretion is reduced, and the formation of structures that promote aggregation in outer hemocytes is inhibited.

Growth and longevity modulation through larval environment mediate immunosenescence and immune strategy of Tenebrio molitor

BackgroundThe Disposable Soma Theory of aging suggests a trade-off between energy allocation for growth, reproduction and somatic maintenance, including immunity. While trade-offs between reproduction and immunity are well documented, those involving growth remain under-explored. Rapid growth might deplete resources, reducing investment in maintenance, potentially leading to earlier or faster senescence and a shorter lifespan. However, rapid growth could limit exposure to parasitism before reaching adulthood, decreasing immunity needs. The insect immunity’s components (cellular, enzymatic, and antibacterial) vary in cost, effectiveness, and duration. Despite overall immunity decline (immunosenescence), its components seem to age differently. We hypothesize that investment in these immune components is adjusted based on the resource cost of growth, longevity, and the associated risk of parasitism.ResultsWe tested this hypothesis using the mealworm beetle, Tenebrio molitor as our experimental subject. By manipulating the larval environment, including three different temperatures and three relative humidity levels, we achieved a wide range of growth durations and longevities. Our main focus was on the relationship between growth duration, longevity, and specific immune components: hemocyte count, phenoloxidase activity, and antibacterial activity. We measured these immune parameters both before and after exposing the individuals to a standard bacterial immune challenge, enabling us to assess immune responses. These measurements were taken in both young and older adult beetles. Upon altering growth duration and longevity by modifying larval temperature, we observed a more pronounced investment in cellular and antibacterial defenses among individuals with slow growth and extended lifespans. Intriguingly, slower-growing and long-lived beetles exhibited reduced enzymatic activity. Similar results were found when manipulating larval growth duration and adult longevity through variations in relative humidity, with a particular focus on antibacterial activity.ConclusionThe impact of growth manipulation on immune senescence varies by the specific immune parameter under consideration. Yet, in slow-growing T. molitor, a clear decline in cellular and antibacterial immune responses with age was observed. This decline can be linked to their initially stronger immune response in early life. Furthermore, our study suggests an immune strategy favoring enhanced antibacterial activity among slow-growing and long-lived T. molitor individuals.

Involvement of Sep38β in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38β) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38β was the highest in egg, followed by that in prepupa and pupa. Sep38β expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38β or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38β regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38β is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

The evolution of complex metarhizium-insect-plant interactions

Metarhizium species interact with plants, insects, and microbes within a diffuse co-evolutionary framework that benefits soil health, biodiversity, and plant growth. The insect host ranges of these fungi vary greatly. Specialization to a narrow host range usually occurs in the tropics with its stable insect populations, and is characterized by the rapid evolution of existing protein sequences, sexual recombination, and small genomes. Host-generalists are associated with temperate regions and ephemeral insect populations. Their mutualistic plant-colonizing lifestyle increases survival when insects are rare, while facultative entomopathogenicity feeds both the fungi and plants when insects are common. Host-generalists have lost meiosis and associated genome defense mechanisms, enabling gene duplications to diversify functions related to plant colonization and host exploitation. Horizontal gene transfer events via transposons have also contributed to host range changes, while parasexuality combines beneficial mutations within individual clones of host-generalists. There is also a lot of genetic variation in insect populations and an increasing understanding that both pathogen virulence and insect immunity are linked with stress responses. Thus, susceptibility to host-generalists can vary due to non-specific resistance to multiple stressors, multipurpose physical and chemical barriers, and heterogeneity in physiological and behavioral factors, such as sleep.

Discovery and Characterization of MaK: A Novel Knottin Antimicrobial Peptide from Monochamus alternatus.

Knottin-type antimicrobial peptides possess exceptional attributes, such as high efficacy, low vulnerability to drug resistance, minimal toxicity, and precise targeting of drug sites. These peptides play a crucial role in the innate immunity of insects, offering protection against bacteria, fungi, and parasites. Knottins have garnered considerable interest as promising contenders for drug development due to their ability to bridge the gap between small molecules and protein-based biopharmaceuticals, effectively addressing the therapeutic limitations of both modalities. This work presents the isolation and identification of a novel antimicrobial peptide derived from Monochamus alternatus. The cDNA encodes a 56-amino acid knottin propeptide, while the mature peptide comprises only 34 amino acids. We have labeled this knottin peptide as MaK. Using chemically synthesized MaK, we evaluated its hemolytic activity, thermal stability, antibacterial properties, and efficacy against nematodes. The results of this study indicate that MaK is an exceptionally effective knottin-type peptide. It demonstrates low toxicity, superior stability, potent antibacterial activity, and the ability to suppress pine wood nematodes. Consequently, these findings suggest that MaK has potential use in developing innovative therapeutic agents to prevent and manage pine wilt disease.

The roles of small RNAs in rice-brown planthopper interactions

Interactions between rice plants (Oryza sativa L.) and brown planthoppers (Nilaparvata lugens Stål, BPHs) are used as a model system to study the molecular mechanisms underlying plant-insect interactions. Small RNAs (sRNAs) regulate growth, development, immunity, and environmental responses in eukaryotic organisms, including plants and insects. Recent research suggests that sRNAs play significant roles in rice-BPH interactions by mediating post-transcriptional gene silencing. The focus of this review is to explore the roles of sRNAs in rice-BPH interactions and to highlight recent research progress in unraveling the mechanism of cross-kingdom RNA interference (ckRNAi) between host plants and insects and the application of ckRNAi in pest management of crops including rice. The research summarized here will aid in the development of safe and effective BPH control strategies.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Identification and immune analysis of antimicrobial peptides from the cigarette beetle (Lasioderma serricorne).

Antimicrobial peptides (AMPs) in insects are endogenous peptides that are effector components of the innate defense system of the insect. AMPs may serve as antimicrobial agents because of their small molecular weight and broad-spectrum antimicrobial activity. In this study, we performed transcriptome analysis of cigarette beetle (Lasioderma serricorne) larvae, parasitized by the ectoparasitic wasp, Anisopteromalus calandrae. Several AMP genes were significantly upregulated following A.calandrae parasitism, postulating the hypothesis that the parasitization enhanced the host's resistance against pathogenic microorganisms through the regulation of host AMP genes. Specifically, 3 AMP genes (LsDef1, LsDef2, and LsCole) were significantly upregulated and we studied their immune function in L.serricorne. Immune challenge and functional analysis showed that LsCole was responsible for the immune response against Gram-negative and Gram-positive bacteria, while LsDef1 and LsDef2 were involved in insect defense against Gram-positive bacteria. Purified recombinant LsCole exhibited antimicrobial activities against the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus. LsDef2 showed an antibacterial effect against S.aureus. LsCole and LsDef2 exhibited antibiofilm activity against S.aureus. The 2 AMPs disrupted cell membranes and caused leakage of S.aureus cell contents. The results indicated that the 3 AMPs in L.serricorne are involved in the innate immunity of this pest insect. These AMPs may have potential as antimicrobial agents for bacterial infection chemotherapy. Hence, data are discussed in relation to new control strategies with greater biosafety against pest insects with use of microbial biocontrol agents in combination with RNA interference against the insect's defensive AMP genes.

Silicon and Epichloë‐endophyte defences in a model temperate grass diminish feeding efficiency and immunity of an insect folivore

Abstract Plants deploy diverse anti‐herbivore defences which reduce feeding and performance of herbivores. Temperate grasses use silicon (Si) accumulation and Epichloë‐endophytes for physical and chemical (i.e. endophytic‐alkaloids) defence against insect herbivores. Recent studies suggest that Epichloë‐endophytes increase Si accumulation in their host grass. It is unknown, however, how this affects Si‐deposition on the leaf surface, their impacts on insect herbivore feeding efficiency and their immunity to potential infection/parasitism. To address this knowledge gap, we grew tall fescue (Festuca arundinacea) hydroponically with and without Si, in the absence or presence of the novel AR584 Epichloë‐strain. We exposed plants to Helicoverpa armigera (Lepidoptera: Noctuidae) in both in situ (intact leaves) and ex situ (excised leaves) feeding trials and determined the effects of Si and endophyte defences on herbivore feeding efficiency, growth rates and immunity against potential infection/parasitism. Endophytic plants supplied with Si showed 110% and 143% increases in leaf silica density and leaf Si concentrations, respectively, when exposed to herbivory, compared to non‐endophytic plants that were herbivore‐free. Despite the endophyte‐mediated increases in Si concentrations, H. armigera was only affected by Si supply; growth rates decreased by 87% and most feeding efficiency indices decreased by at least 30%. Si supply also increased mandibular wear by 16%, which was negatively correlated with H. armigera growth rates. Cellular and humoral immunity of H. armigera were negatively affected by both Si and endophytes. Endophytic‐loline alkaloid concentrations were unaffected by Si supply or herbivory, whereas herbivory increased peramine concentrations by 290%. To our knowledge, this is the first report of Si defences and Epichloë‐endophyte derived alkaloids compromising insect immunity via reduced melanisation response. Using tall fescue and H. armigera, our study suggests that deploying both physical (i.e. Si accumulation) and chemical (i.e. endophytic‐alkaloids) defences acting against multiple insect herbivore traits, including feeding efficiency, growth and immunity, may be a successful defence strategy in temperate grasses. This multi‐faceted defence may be particularly difficult for insect herbivores to overcome. Read the free Plain Language Summary for this article on the Journal blog.

Differential expression of immunity-related genes in larval Manduca sexta tissues in response to gut and systemic infection.

The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.