Insect Immune Response Research Articles

In Vivo Efficacy of a Nanoconjugated Glycopeptide Antibiotic in Silkworm Larvae Infected by Staphylococcus aureus

To contrast the rapid spread of antibiotic resistance in bacteria, new alternative therapeutic options are urgently needed. The use of nanoparticles as carriers for clinically relevant antibiotics represents a promising solution to potentiate their efficacy. In this study, we used Bombyx mori larvae for the first time as an animal model for testing a nanoconjugated glycopeptide antibiotic (teicoplanin) against Staphylococcus aureus infection. B. mori larvae might thus replace the use of mammalian models for preclinical tests, in agreement with the European Parliament Directive 2010/63/EU. The curative effect of teicoplanin (a last resort antibiotic against Gram-positive bacterial pathogens) conjugated to iron oxide nanoparticles was assessed by monitoring the survival rate of the larvae and some immunological markers (i.e., hemocyte viability, phenoloxidase system activation, and lysozyme activity). Human physiological conditions of infection were reproduced by performing the experiments at 37 °C. In this condition, nanoconjugated teicoplanin cured the bacterial infection at the same antibiotic concentration of the free counterpart, blocking the insect immune response without causing mortality of silkworm larvae. These results demonstrate the value and robustness of the silkworm as an infection model for testing the in vivo efficacy of nanoconjugated antimicrobial molecules.

Insect immunity in the Anthropocene.

Anthropogenic activities result in global change, including climate change, landscape degradation and pollution, that can alter insect physiology and immune defences. These changes may have contributed to global insect decline and the dynamics of insect-transmitted diseases. The ability of insects to mount immune responses upon infection is crucial for defence against pathogens and parasites. Suppressed immune defences reduce fitness by causing disease-driven mortality and elevated immune responses reduce energy available to invest in other fitness traits such as reproduction. Understanding the impact of anthropogenic factors on insect-pathogen interactions is therefore key to determining the contribution of anthropogenic global change to pathogen-driven global insect decline and the emergence and transmission of insect-borne diseases. Here, we synthesise evidence of the impact of anthropogenic factors on insect immunity. We found evidence that anthropogenic factors, such as insecticides and heavy metals, directly impacting insect immune responses by inhibiting immune activation pathways. Alternatively, factors such as global warming, heatwaves, elevated CO2 and landscape degradation can indirectly reduce insect immune responses via reducing the energy available for immune function. We further review how anthropogenic factors impact pathogen clearance and contribute to an increase in vector-borne diseases. We discuss the fitness cost of anthropogenic factors via pathogen-driven mortality and reduced reproductive output and how this can contribute to species extinction. We found that most research has determined the impact of a single anthropogenic factor on insect immune responses or pathogen resistance. We recommend studying the combined impact of multiple stressors on immune response and pathogen resistance to understand better how anthropogenic factors affect insect immunity. We conclude by highlighting the importance of initiatives to mitigate the impact of anthropogenic factors on insect immunity, to reduce the spread of vector-borne diseases, and to protect vulnerable ecosystems from emerging diseases.

Suppression of a transcriptional regulator, HexA, is essential for triggering the bacterial virulence of the entomopathogen, Xenorhabdus hominickii

A nematode-symbiotic bacterium, Xenorhabdus hominickii, exhibits two distinct lifestyles. Upon infection of its host nematode into a target insect, X. hominickii is released into the insect hemocoel and becomes pathogenic. This study examines the critical transformation in bacterial life forms concerning the activity of a transcriptional regulator, HexA. When X. hominickii was cultured in tryptic soy broth, HexA was expressed during the stationary phase of bacterial growth. Conversely, HexA was expressed in the early growth stage within the insect host, Spodoptera exigua, when infected with X. hominickii. The transient expression of HexA was succeeded by the expression of another transcriptional regulator, Lrp, which led to the production of bacterial virulent factors. Expression of HexA was manipulated by replacing its promoter with an inducible promoter controlled by the inducer, l-arabinose. In the absence of the inducer, the mutant bacteria expressed HexA at a low level, resulting in a bacterial culture broth that was more effective at suppressing insect immune responses than the wild type. When the inducer was added, HexA was expressed at high levels, rendering the culture broth ineffective in immunosuppression. Interestingly, expression of HexA inhibited the expression of another transcriptional regulator, Lrp, which in turn induced the expression of a non-ribosomal peptide synthetase, gxpS, leading to the production of an immunosuppressive metabolite, GXP. Suppression of HexA expression in mutant bacteria augmented GXP levels in secondary metabolites. This indicates that infection of X. hominickii into the insect host represses HexA expression and upregulates Lrp expression, leading to GXP production. The GXP metabolites inhibit insect immunity, thus protecting the bacteria-nematode complex. Therefore, the suppression of HexA expression in the insect hemocoel is crucial for the bacteria’s transition from a symbiotic to a pathogenic life form.

Unveiling the functions of the Lim-domain binding protein MaPtaB in Metarhizium acridum.

The Lim-domain binding protein PtaB, a homolog of Mfg1, governs conidiation and biofilm formation in several fungi. PtaB includes a conserved Lim-binding domain and two predicted nuclear localization sequences at its C terminus, and is co-regulated with the transcription factor Som1 downstream of the cyclic AMP-dependent protein kinase A (cAMP/PKA) pathway. However, the function of PtaB in entomopathogenic fungi remain poorly understood. Inactivation of PtaB in Metarhizium acridum resulted in delayed conidial germination, reduced conidial yield and increased sensitivities to cell wall disruptors, ultraviolet B irradiation and heat shock. In addition, the fungal virulence was significantly decreased after deletion of MaPtaB because of impairments in appressorium formation, cuticle penetration and evasion of insect immune responses in M. acridum. The MaPtaB-deletion and MaSom1-deletion strains showed similar phenotypes supporting that MaSom1/MaPtaB complex controls M. acridum normal conidiation and pathogenic progress. Upon loss of MaPtaB or MaSom1, the fungal sporulation mode in M. acridium shifted from microcycle conidiation to normal conidiation on SYA, a microcycle conidiation medium. Transcriptional analysis showed that more differentially expression genes were identified in MaSom1 RNA sequencing, and MaSom1 and MaPtaB may regulate the expression of genes for conidiation, nutrient metabolism and the cell cycle to control conidiation pattern shift. These data corroborate a complex control function for MaPtaB as an important central factor interacting with MaSom1 in the cAMP/PKA pathway, which links stress tolerance, conidiation and virulence in the entomopathogenic fungus M. acridum. © 2024 Society of Chemical Industry.

Morphological and functional characterization of circulating hemocytes in Tribolium castaneum larvae.

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye-staining properties were used to characterize hemocyte types from T.castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2h), then increased over time (at 4 and 6h) and eventually returned to the naive state by 24h post-injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T.castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph invivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes invitro, forming capsules to encapsulate and melanize nickel-nitrilotriacetic acid (Ni-NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T.castaneum larvae, offering valuable insights into cell-mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T.castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T.castaneum hemocytes in combating pathogens.

Identification of function modules in the co-expression protein–protein interaction network of Bombyx mori in response to Beauveria bassiana infection

Beauveria bassiana (B. bassiana) is a common fungal disease in sericulture. Previous research has primarily focused on investigating genes involved in innate immunity. However, the response of Bombyx mori (B. mori) to B. bassiana requires the coordination of other biological processes in addition to the immune system. We measured protein expression profile of B. mori after inoculating B. bassiana using iTRAQ technology in previous. Here we constructed a co-expression protein–protein interaction network of B. mori in response to B. bassiana infection. Subnetworks and modules were analyzed, and the functions of these modules were annotated. The results revealed the identification of numerous proteins associated with cellular immunity, including those involved in phagosomes, lysosomes, mTOR signaling, sugar metabolism, and the ubiquitin–proteasome pathway. Meanwhile, we observed that the pathways involved in protein synthesis were activated, including pyruvate and purine metabolism, RNA transport, ribosome, protein processing in endoplasmic reticulum, and protein export pathways, during B. bassiana infection. Based on this analysis, we selected six candidate genes (shock protein, ribosome, translocon, actin muscle-type A2, peptidoglycan recognition protein, and collagenase) that were found to be related to the response to B. bassiana. Further verification experiments demonstrated significant changes in their expression levels after inoculation with B. bassiana. These research findings provide new insights into the molecular mechanism of insect immune response to fungal infection.

The complexities of blood-feeding patterns in mosquitoes and sandflies and the burden of disease: A minireview.

Mosquitoes and sandflies exhibit a wide range of blood feeding patterns, targeting a wide range of vertebrate species, including birds, mammals, reptiles, and amphibians, for proteins vital for egg development. This broad host range increases the opportunity for them to acquire pathogens of numerous debilitating-and-fatal diseases from various animal reservoirs, playing a significant role in disease crossover between animals and humans, also known as zoonotic transmission. This review focuses on the intricate blood-feeding habits of these dipteran vectors, their sensory systems and the complex dance between host and pathogen during disease transmission. We delve into the influence of blood sources on pathogen spread by examining the insect immune response and its intricate interplay with pathogens. The remarkable sense of smell guiding them towards food sources and hosts is explored, highlighting the interplay of multiple sensory cues in their navigation. Finally, we examine the challenges in mosquito control strategies and explore innovations in this field, emphasizing the need for sustainable solutions to combat this global health threat. By understanding the biology and behaviour of these insects, we can develop more effective strategies to protect ourselves and mitigate the burden of vector-borne diseases.

Tomato Spotted Wilt Virus Suppresses the Antiviral Response of the Insect Vector, Frankliniella occidentalis, by Elevating an Immunosuppressive C18 Oxylipin Level Using Its Virulent Factor, NSs.

Orthotospovirus tomatomaculae (tomato spotted wilt virus, TSWV) is transmitted by the western flower thrips, Frankliniella occidentalis. Epoxyoctadecamonoenoic acids (EpOMEs) function as immune-suppressive factors, particularly in insects infected by viral pathogens. These oxylipins are produced by cytochrome P450 monooxygenases (CYPs) and are degraded by soluble epoxide hydrolase (sEH). In this study, we tested the hypothesis that TSWV modulates the EpOME level in the thrips to suppress antiviral responses and enhance its replication. TSWV infection significantly elevated both 9,10-EpOME and 12,13-EpOME levels. Following TSWV infection, the larvae displayed apoptosis in the midgut along with the upregulated expression of four caspase genes. However, the addition of EpOME to the viral treatment notably reduced apoptosis and downregulated caspase gene expressions, which led to a marked increase in TSWV titers. The CYP and sEH genes of F. occidentalis were identified, and their expression manipulation using RNA interference (RNAi) treatments led to significant alternations in the insect's immune responses and TSWV viral titers. To ascertain which viral factor influences the host EpOME levels, specialized RNAi treatments targeting genes encoded by TSWV were administered to larvae infected with TSWV. These treatments demonstrated that NSS expression is pivotal in manipulating the genes involved in EpOME metabolism. These results indicate that NSs of TSWV are crucially linked with the elevation of host insect EpOME levels and play a key role in suppressing the antiviral responses of F. occidentalis.

Exploring the role of RNASET2 in the immune response of black soldier fly larvae.

T2 RNases are transferase-type enzymes distributed across phyla, crucial for breaking down single-stranded RNA molecules. In addition to their canonical function, several T2 enzymes exhibit pleiotropic roles, contributing to various biological processes, such as the immune response in invertebrates and vertebrates. This study aims at characterizing RNASET2 in the larvae of black soldier fly (BSF), Hermetia illucens, which are used for organic waste reduction and the production of valuable insect biomolecules for feed formulation and other applications. Given the exposure of BSF larvae to pathogens present in the feeding substrate, it is likely that the mechanisms of their immune response have undergone significant evolution and increased complexity. After in silico characterization of HiRNASET2, demonstrating the high conservation of this T2 homolog, we investigated the expression pattern of the enzyme in the fat body and hemocytes, two districts mainly involved in the insect immune response, in larvae challenged with bacterial infection. While no variation in HiRNASET2 expression was observed in the fat body following infection, a significant upregulation of HiRNASET2 synthesis occurred in hemocytes shortly after the injection of bacteria in the larva. The intracellular localization of HiRNASET2 in lysosomes of plasmatocytes, its extracellular association with bacteria, and the presence of a putative antimicrobial domain in the molecule, suggest its potential role in RNA clean-up and as an alarm molecule promoting phagocytosis activation by hemocytes. These insights contribute to the characterization of the immune response ofHermetia illucens larvae and may facilitate the development of animal feedstuff enriched with highly valuable BSF bioactive compounds.

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Effects of two transglutaminases on innate immune responses in the oriental armyworm, Mythimna separata.

Transglutaminase (TGase) is a key enzyme that mediates hemolymph coagulation and is thought to contribute to the elimination of pathogenic microorganisms in invertebrates. The objective of this study was to elucidate the involvement of TGase in insect immune responses via functional analysis of this enzyme in the oriental armyworm, Mythimna separata,using recombinant proteins and RNA interference technique. We identified two TGase genes, mystgase1 and mystgase2, in Mythimna separata and found that both genes are expressed in all surveyed tissues in M. separata larvae. Significant changes were induced in hemocytes following Escherichia coli injection. Injection of Gram-positive bacteria (Micrococcus luteus) and Gram-negative bacteria (Escherichiacoli and Serratia marcescens) into larvae triggered a time-specific induction of both mystgase1 and mystgase2 in hemocytes. Recombinant MysTGase1 and MysTGase2 proteins bound to both E. coli and M. luteus, localizing within bacterial clusters and resulting in agglutination in a Ca2+-dependent manner. The hemocytes of larvae injected with recombinant MysTGase1 or MysTGase2 exhibited enhanced phagocytic ability against E. coli, improved in vivo bacterial clearance, and increased resistance to S. marcescens, decreasing larval mortality rate. Conversely, RNA interference targeting mystgase1 or mystgase2 significantly reduced hemocyte phagocytic capability, decreased bacterial clearance, and increased susceptibility to S. marcescens infection, thereby increasing larval mortality rate. The findings of this study are anticipated to expand our understanding of the function of TGases within insect immune responses and may contribute to developing new pest control strategies.

The allatotropin/orexin system as an example of immunomodulatory properties of neuropeptides

The central nervous system (CNS) plays a critical role in signal integration in animals and allows the orchestration of life processes to maintain homeostasis. Current research clearly shows that inflammatory processes can also be modulated by the CNS via the neuroendocrine system. One of the neuropeptide families that participate in vertebrates in this process is orexins (OXs). Interestingly, our previous results suggested that a similar dependency may also exist between neuropeptides and immune system activity in insects. Due to the structural homology of orexin and allatotropin receptors and the functional similarity between these two neuropeptide families, the main aim of this research was to perform a complex analysis of the relationships between allatotropin (AT) and the insect immune response. Our results revealed functional similarities between vertebrate OXs and insect ATs. Similar effects were observed in the profile of the expression level of the gene encoding the AT precursor in the Tenebrio molitor nervous system and in the general action of Tenmo-AT on selected immune parameters of the tested beetles. Moreover, for the first time in insects, we confirmed the role of cytokines in the modulation of neuroendocrine system by determining the effect of Spätzle-like protein injection on the expression of genes encoding AT precursor and receptor. All these results are important for understanding the evolutionary basis of hormonal regulation of the immune response.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

A C-type lectin with dual carbohydrate recognition domains functions in innate immune response in Asian corn borer, Ostrinia furnacalis.

C-type lectins (CTLs) act as pattern recognition receptors (PRRs) to initiate the innate immune response in insects. A CTL with dual carbohydrate recognition domains (CRDs) (named immulectin-4 [IML-4]) was selected from the Ostrinia furnacalis transcriptome dataset for functional studies. We cloned the full-length complementary DNA of O. furnacalis IML-4 (OfIML-4). It encodes a 328-residue protein with a Glu-Pro-Asn (EPN) and Gln-Pro-Asp (QPD) motifs in 2 CRDs, respectively. OfIML-4 messenger RNA levels increased significantly upon the bacterial and fungal infection. Recombinant OfIML-4 (rIML-4) and its individual CRDs (rCRD1 and rCRD2) exhibited the binding ability to various microorganisms including Escherichia coli, Micrococcus luteus, Pichia pastoris, and Beauveria bassiana, and the cell wall components including lipopolysaccharide from E. coli, peptidoglycan from M. luteus or Bacillus subtilis, and curdlan from Alcaligenes faecalis. The binding further induced the agglutination of E. coli, M. luteus, and B. bassiana in the presence of calcium, the phagocytosis of Staphylococcus aureus by the hemocytes, in vitro encapsulation and melanization of nickel-nitrilotriacetic acid beads, and a significant increase in phenoloxidase activity of plasma. In addition, rIML-4 significantly enhanced the phagocytosis, nodulation, and resistance of O. furnacalis to B. bassiana. Taken together, our results suggest that OfIML-4 potentially works as a PRR to recognize the invading microorganisms, and functions in the innate immune response in O. furnacalis.

A fungal pathogen secretes a cell wall-associated β-N-acetylhexosaminidase that is co-expressed with chitinases to contribute to infection of insects.

β-N-acetylhexosaminidases (HEXs) are widely distributed in fungi and involved in cell wall chitin metabolism and utilization of chitin-containing substrates. However, details of the fungal pathogens-derived HEXs in the interaction with their hosts remain limited. An insect nutrients-induced β-N-acetylhexosaminidase, BbHex1, was identified from the entomopathogenic fungus Beauveria bassiana, which was involved in cell wall modification and degradation of insect cuticle. BbHex1 was localized to cell wall and secreted, and displayed enzyme activity to degrade the chitinase-hydrolyzed product (GlcNAc)2. Disruption of BbHex1 resulted in a significant decrease in the level of cell wall chitin in the presence of insect nutrients and during infection of insects, with impaired ability to penetrate insect cuticle, accompanying downregulated cell wall metabolism-involved and cuticle-degrading chitinase genes. However, the opposite phenotypes were examined in the gene overexpression strain. Distinctly altered cell wall structures caused by BbHex1 mutation and overexpression led to the easy activation and evasion (respectively) of insect immune response during fungal infection. As a result, BbHex1 contributed to fungal virulence. Bioinformatics analysis revealed that promoters of some co-expressed chitinase genes with the BbHex1 promoter shared conserved transcription factors Skn7, Msn2 and Ste12, and CreA-binding motifs, implying co-regulation of those genes with BbHex1. These data support a mechanism that the fungal pathogen specifically expresses BbHex1, which is co-expressed with chitinases to modify cell wall for evasion of insect immune recognition and to degrade insect cuticle, and contributes to the fungal virulence against insects. © 2024 Society of Chemical Industry.

Hemolin increases the immune response of a caterpillar to NPV infection

Hemolin, a member of the immunoglobulin superfamily, plays a crucial role in the immune responses of insects against pathogens. However, the innate immune response of Hemolin to baculovirus infection varies among different insects, and the antiviral effects of Hemolin in Hyphantria cunea (HcHemolin) remain poorly understood. Our results showed that HcHemolin was expressed throughout all developmental stages, with higher expressions observed during pupal and adult stages of H. cunea. Additionally, HcHemolin was expressed in reproductive and digestive organs. The expression levels of the HcHemolin were induced significantly following H. cunea nucleopolyhedrovirus (HcNPV) infection. The susceptibility of H. cunea larvae to HcNPV decreased upon silencing of HcHemolin, resulting in a 40% reduction in median lifespan compared to the control group. The relative growth rate (RGR), the relative efficiency of consumption rate (RCR), the efficiency of the conversion of ingested food (ECI), and efficiency of the conversion of digested food (ECD) of silenced H. cunea larvae were significantly lower than those of the control group. Immune challenge assays showed that the median lifespan of treated H. cunea larvae was two-fold longer than the control group after HcNPV and HcHemolin protein co-injection. Therefore, we propose that HcHemolin plays a crucial role in regulating the growth, development, and food utilization of H. cunea, as well as in the antiviral immune response against HcNPV. These findings provide implications for the development of targeted nucleic acid pesticides and novel strategies for pollution-free biological control synergists for HcNPV.

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.

SfREPAT38, a pathogen response gene (REPAT), is involved in immune response of Spodoptera frugiperda larvae through mediating Toll signalling pathway.

REPAT (response to pathogen) is an immune-associated gene family that plays important roles in insect immune response to pathogens. Although nine REPAT genes have been identified in Spodoptera frugiperda (Lepidoptera: Noctuidae) currently, their functions and mechanisms in the immune response to pathogens still remain unclear. Therefore, SfREPAT38, a pathogen response gene (REPAT) of S. frugiperda, was characterised and its function was analysed. The results showed that SfREPAT38 contains a signal peptide and a transcription activator MBF2 (multi-protein bridging factor 2) domain. Quantitative real-time polymerase chain reaction analysis showed that SfREPAT38 was highly expressed in the sixth-instar larvae (L6) and was the highest in expression in the midgut of L6. We found that the expression of SfREPAT38 could be activated by challenge with four microbial pathogens (Bacillus thuringiensis, Metarhizium anisopliae, Spodoptera exigua nuclearpolyhedrosis and Escherichia coli), except 12 h after E. coli infection. Furthermore, the SfREPAT38 expression levels significantly decreased at 24, 48 and 72 h after SfREPAT38 dsRNA injection or feeding. Feeding with SfREPAT38 dsRNA significantly decreased the weight gain of S. frugiperda, and continuous feeding led to the death of S. frugiperda larvae from the fourth day. Moreover, SfREPAT38 dsRNA injection resulted in a significant decrease of weight gain on the fifth day. Silencing SfREPAT38 gene down-regulated the expression levels of immune genes belonging to the Toll pathway, including SPZ, Myd88, DIF, Cactus, Pell and Toll18W. After treatment with SfREPAT38 dsRNA, S. frugiperda became extremely sensitive to the B. thuringiensis infection, and the survival rate dramatically increased, with 100% mortality by the eighth day. The weight of S. frugiperda larvae was also significantly lower than that of the control groups from the second day onwards. In addition, the genes involved in the Toll signalling pathway and a few antibacterial peptide related genes were down-regulated after treatment. These results showed that SfREPAT38 is involved in the immune response of S. frugiperda larvae through mediating Toll signalling pathway.

The Diversity of Wolbachia and Other Bacterial Symbionts in Spodoptera frugiperda.

Bacterial symbionts associated with insects can be crucial in insect nutrition, metabolism, immune responses, development, and reproduction. However, the bacterial symbionts of the fall armyworm Spodoptera frugiperda remain unclear. S. frugiperda is an invasive polyphagous pest that severely damages many crops, particularly maize and wheat. Here, we investigated the infection, composition, abundance, and diversity of bacterial symbionts, especially Wolbachia, in different tissues of S. frugiperda female adults. The infection prevalence frequencies of Wolbachia in five provinces of China, namely Pu'er, Yunnan; Nanning, Guangxi; Sanya, Hainan; Yunfu, Guangdong; and Nanping, Fujian, were assessed. The results indicated that Proteobacteria, Firmicutes, and Bacteroidetes were the three most dominant bacterial phyla in S. frugiperda adults. At the genus level, the abundant microbiota, which included Enterobacter and Enterococcus, varied in abundance between tissues of S. frugiperda. Wolbachia was found in the ovaries and salivary glands of S. frugiperda adults, and was present in 33.33% of the Pu'er, Yunnan, 23.33% of the Nanning, Guangxi, and 13.33% of the Sanya, Hainan populations, but Wolbachia was absent in the Yunfu, Guangdong and Nanping, Fujian populations. Further phylogenetic analyses revealed that all of the Wolbachia strains from the different S. frugiperda populations belonged to the supergroup B and were named the wFru strain. Since there were Wolbachia strains inducing cytoplasmic incompatibility in supergroup B, these findings may provide a foundation for developing potential biocontrol techniques against S. frugiperda.