Insect Innate Immune System Research Articles

Role of cuticular genes in the insect antimicrobial immune response.

Insects are established models for understanding host-pathogen interactions and innate immune mechanisms. The innate immune system in insects is highly efficient in recognizing and opposing pathogens that cause detrimental effects during infection. The cuticular layer which covers the superficial layer of the insect body participates in host defense and wound healing by inducing innate immune responses. Previous studies have started to address the involvement of cuticular genes in conferring resistance to insect pathogens, particularly those that infect by disrupting the insect cuticle. For example, the cuticular gene Transglutaminase (TG) in Drosophila melanogaster plays a structural role in cuticle formation and blood coagulation and also possesses immune properties against pathogenic infection. However, more information is becoming available about the immune function of other cuticular gene families in insects. In this review, we aim to highlight the recent advances in insect cuticular immunity and address the necessity of pursuing further research to fill the existing gaps in this important field of insect immunology. This information will lead to novel strategies for the efficient management of agricultural insect pests and vectors of plant and human disease.

Epigenetic remodeling in insect immune memory.

The innate immune system of insects can respond more swiftly and efficiently to pathogens based on previous experience of encountering antigens. The understanding of molecular mechanisms governing immune priming, a form of immune memory in insects, including its transgenerational inheritance, remains elusive. It is still unclear if the enhanced expression of immune genes observed in primed insects can persist and be regulated through changes in chromatin structure via epigenetic modifications of DNA or histones, mirroring observations in mammals. Increasing experimental evidence suggests that epigenetic changes at the level of DNA/RNA methylation and histone acetylation can modulate the activation of insects' immune responses to pathogen exposure. Moreover, transgenerational inheritance of certain epigenetic modifications in model insect hosts can influence the transmission of pre-programmed immune responses to the offspring, leading to the development of evolved resistance. Epigenetic research in model insect hosts is on the brink of significant progress in the mechanistic understanding of chromatin remodeling within innate immunity, particularly the direct relationships between immunological priming and epigenetic alterations. In this review, we discuss the latest discoveries concerning the involvement of DNA methylation and histone acetylation in shaping the development, maintenance, and inheritance of immune memory in insects, culminating in the evolution of resistance against pathogens.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Variation in Bombyx mori immune response against fungal pathogen Beauveria bassiana with variability in cell wall β-1,3-glucan.

Entomopathogenic fungi are protected by a cell wall with dynamic structure for adapting to various environmental conditions. β-1,3-Glucan recognition proteins activate the innate immune system of insects by recognizing surface molecules of fungi. However, the associations between pathogenicity and the different components of entomopathogenic fungal cell walls remain unclear. Three Beauveria bassiana strains were selected that have significantly differing virulence against Bombyx mori. The molecular mechanisms underlying the immune response in B. mori were investigated using RNA sequencing, which revealed differences in the immune response to different B. bassiana strains at 12h post-infection. Immunofluorescence assays revealed that β-1,3-glucan content had an opposite trend to that of fungal virulence. β-1,3-Glucan injection upregulated BmβGRP4 expression and significantly reduced the virulence of the high-virulence strain but not that of the medium-virulence or low-virulence strains. BmβGRP4 silencing in B. mori with RNA interference resulted in the opposite virulence pattern, indicating that the virulence of B. bassiana was affected by the cell walls' content of β-1,3-glucan, which could be recognized by BmβGRP4. Furthermore, interference with the gene CnA (calcineurin catalytic A subunit) involved in β-1,3-glucan synthesis eliminated differences in virulence between B. bassiana strains. These results indicate that strains of a single species of pathogenic fungi that have differing cell wall components are recognized differently by the innate immune system of B. mori.

Enhanced toxicity of entomopathogenic fungi Beauveria bassiana with bacteria expressing immune suppressive dsRNA in a leaf beetle

The entomopathogenic fungus is recognized as an ideal alternative to chemical pesticides, nonetheless, its efficacy is often limited by insect's innate immune system. The suppression of the host immunity may overcome the obstacle and promote the toxicity of the fungi. Here, by using an entomopathogenic fungus Beauveria bassiana and immune genes dsRNA-expressing bacteria, we explored the potentially synergistic toxicity of the two agents on a leaf beetle Plagiodera versicolora (Coleoptera: Chrysomelidae). We first determined the susceptibilities of P. versicolora to a B. bassiana 476 strain (hereafter referred to Bb476). And the immune genes were identified based on the transcriptome of Bb476 challenged beetles. Subsequently, five immune genes (PGRP1, Toll1, Domeless，SPN1，and Lysozyme) were targeted by feeding dsRNA-expressing bacteria, which produced a 71.4, 39.0, 72.0, 49.0, and 68.7% gene silencing effect, respectively. Furthermore, we found a significantly increased mortality of P. versicolora when combined the Bb476 and the immune suppressive dsRNAs. Taking together, this study highlights the importance of insect immunity in the defense of entomopathogens and also paves the way toward the development of a more efficient pest management strategy that integrates both entomopathogens and immune suppressive dsRNAs.

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.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity

BackgroundMosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated.MethodsLarvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR.ResultsPropagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF.ConclusionBy 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.Graphical

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. &#x0D;

Termicin silencing enhances the toxicity of Serratia marcescens Bizio (SM1) to Odontotermes formosanus (Shiraki)

Termites are often exposed to a variety of pathogens during their life cycle, which has led to the development of an innate immune system to resist these pathogens. Antimicrobial peptides (AMPs) play a crucial role in the innate immune system in insects. However, clear information on AMPs in termites has not been obtained. Therefore, exploring the function of AMPs in the subterranean termite Odontotermes formosanus (Shiraki) can lead to the development of novel termite control strategies that integrate RNA interference (RNAi) and pathogens. Here we first obtained two Oftermicins from O. formosanus and observed that the expression of these Oftermicin genes was significantly upregulated at the mRNA level after treatment with lipopolysaccharide (LPS) or Serratia marcescens Bizio (SM1). Interestingly, the expression of these Oftermicins increased not only in the donor termites but also in the recipient termites through transmission experiments. Bioassay experiments showed that the mortality of O. formosanus treated with SM1 after RNAi was significantly higher than that of other groups. In summary, dsOftermicins are important immunosuppressants for termite control and Oftermicins are optimal targets for termite control based on the combined use of RNAi and pathogens.

Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites.

The host defence of insects includes a combination of cellular and humoral responses. The cellular arm of the insect innate immune system includes mechanisms that are directly mediated by haemocytes (e.g., phagocytosis, nodulation and encapsulation). In addition, melanization accompanying coagulation, clot formation and wound healing, nodulation and encapsulation processes leads to the formation of cytotoxic redox-cycling melanin precursors and reactive oxygen and nitrogen species. However, demarcation between cellular and humoral immune reactions as two distinct categories is not straightforward. This is because many humoral factors affect haemocyte functions and haemocytes themselves are an important source of many humoral molecules. There is also a considerable overlap between cellular and humoral immune functions that span from recognition of foreign intruders to clot formation. Here, we review these immune reactions starting with the cellular mechanisms that limit haemolymph loss and participate in wound healing and clot formation and advancing to cellular functions that are critical in restricting pathogen movement and replication. This information is important because it highlights that insect cellular immunity is controlled by a multilayered system, different components of which are activated by different pathogens or during the different stages of the infection.

Immune response triggered by the ingestion of polyethylene microplastics in the dipteran larvae Chironomus riparius

The activation of insects’ immune system due to the ingestion of microplastics (MPs) has only been evidenced by the upregulation of specific genes. The activation of phenoloxidase (PO) system is one of the primary responses involved in insects' innate immunity when facing parasites and pathogens, and ingestion of MPs can trigger a similar process. This study aimed at addressing the activities of basal PO and total PO (PO+ prophenoloxidase – proPO), in Chironomus riparius larvae (a model species in ecotoxicology) exposed to sediments spiked with polyethylene microplastics (PE-MPs; size-range 32–63 µm; concentrations: 1.25; 5; to 20 g kg–1) for 48 h. The ingestion of PE-MPs by larvae triggered a significant increase of basal PO activity at 5 and 20 g PE-MPs kg–1, by 26% and 29%, respectively, whereas total PO increased significantly in the latter (+48%), suggesting de novo synthesis of proPO by organisms. Considering the particle size, the immune response's activation is probably linked to damage in the epithelial cells of the gut lumen. This research work provides the first evidence on the activation of the insect's innate immune system after ingestion of MPs and underlines the PO activity as a good indicator of the immune response induced by MPs' ingestion.

Cloning and functional characterization of two peptidoglycan recognition protein isoforms (PGRP-LC) in Bactrocera dorsalis (Diptera: Tephritidae)

The innate immune system of insects is the front line of self-defense against pathogen invasion. Peptidoglycan recognition proteins (PGRPs) are important components and play key roles in insect immune systems by recognizing peptidoglycan (PGN) in bacterial cell walls. We characterized two isoforms of the PGRP-LC gene, BdPGRP-LCa and BdPGRP-LCb, from Bactrocera dorsalis (Hendel), an important fruit and vegetable pest worldwide. These two isoforms contain an open reading frames of 1 668 bp and 1 731 bp, encoding a protein of 555 and 576 amino acids, respectively. Quantitative real-time PCR results showed that both transcripts were prominently expressed in midgut and fat body of B. dorsalis adult. Inoculation of pathogens showed that both isoforms actively responded to Escherichia coli PGN. We also observed a light response to Staphylococcus aureus PGN. Upon Beauveria bassiana inoculation, the expression of BdPGRP-LCa was enhanced, but the expression of BdPGRP-LCb was suppressed. Suppression of both transcripts by RNA interference led to increased mortality of flies challenged by E. coli, indicating that the two isoforms are involved in sensing Gram-negative bacterial infections.

Immunological Function of the Antibacterial Peptide Attacin-Like in the Chinese Oak Silkworm, Antheraea pernyi

Antibacterial peptides play important roles in the innate immune system of insects and are divided into four categories according to their structures. Although many antibacterial peptides have been reported in lepidopteran insects, the roles of an attacin-like gene in immune response of Antheraea pernyi remain unclear. In this study, the cloning and immunological functions of an attacin-like gene from Antheraea pernyi were investigated. The open reading frame of Ap-attacin-like gene was cloned by PCR using the specific primers and then was ligated to the pET-32a vector to construct the recombinant plasmids Ap-attacin- like-pET-32a. The recombinant Ap-attacin-like protein was expressed in E. coli (BL21 DE3) cells and purified by Ni-NTA affinity chromatography. The expression patterns of Ap-attacin-like in different tissues or under microorganism challenges were investigated by real-time PCR and western blotting. Finally, agar well diffusion assay was performed to determine the antimicrobial activity of the recombinant Ap-attacin-like proteins based on the inhibition rate. The expression level of Ap-attacin-like was highest in the fat body compared with the other examined tissues. The expression of Ap-attacin-like in the fat body was significantly elevated after E. coli, Beauveria bassiana, Micrococcus luteus or Nuclear Polyhedrosis Virus challenges. In addition, the recombinant Ap-attacin-like proteins had obvious antibacterial activity against E. coli. Ap-attacin-like was highly expressed in immune-related tissues and its expression level was significantly induced by different microorganism challenges, suggesting that Ap-attacin-like participated in the innate immunity of A. pernyi.

Identification, in silico characterization, and expression analysis of Tenebrio molitor Cecropin‐2

AbstractAntimicrobial peptides (AMPs) are considered to be candidate effectors for eliciting humoral immune responses against infectious pathogens in the host. Cecropins are α‐helical peptides of 30–40 amino acids, which are known to permeabilize bacterial membranes, and play authoritative roles in the innate immune system of insects. In the present study, we identified the full‐length open reading frame (ORF) encoding the Tenebrio molitor cecropin‐2 (TmCec2) gene using the Tribolium castaneum cecropin‐2 (TcCec2) gene to query a T. molitor Rnaseq database. Phylogenetic analysis revealed close identity of TmCec2 with TcCec2. TmCec2 was ubiquitously expressed in the insect during all developmental stages, with the highest expression observed in the adult. Tissue‐specific TmCec2 expression was highest in larval hemocytes and in the adult integument and hemocytes. Microbial challenge experiments revealed that TmCec2 was highly induced in response to gram‐positive and gram‐negative bacteria, and fungi. These data provide credible evidence for a putative role of TmCec2 in insect innate immunity against a plethora of pathogens.

Innate immune system function following systemic RNA-interference of the Fragile X Mental Retardation 1 gene in the cricket Acheta domesticus

Fragile X syndrome (FXS), caused by a mutation in the Fragile X Mental Retardation 1 (FMR1) gene, is a common form of inherited mental retardation. Mutation of the gene leads to a loss of the gene product Fragile X Mental Retardation Protein (FMRP). While a loss of FMRP has been primarily associated with neural and cognitive deficits, it has also been reported to lead to immune system dysfunction in both humans and flies. We used the Acheta domesticus transcriptome to identify a highly conserved cricket ortholog of FMR1 (adfmr1). We cloned a partial cDNA of adfmr1, used systemic RNA interference (RNAi) to knockdown adfmr1 expression, and examined the impact of this knockdown (KD) on the cellular and humoral responses of the insect innate immune system. Following RNAi, both male and female crickets exhibited an increase in the number of circulating hemocytes, a decrease in total hemolymph phenoloxidase (PO) activity, and an increase in fat body lysozyme expression. Despite similar changes in these immune parameters in both sexes, male and female crickets responded differently to an immune challenge. Most KD males failed to survive an intra-abdominal injection of bacterial lipopolysaccharide, while KD females were just as likely as control females to survive this challenge. Our results support that decreased fmr1 expression can alter the cellular and humoral defenses of the insect innate immune system, and may lead to a decrease in male, but not female, immunocompetence.

Predator-induced stress influences fall armyworm immune response to inoculating bacteria

The insect innateimmunesystem is assorted into two general categories, cellular and humoral immunity. Aside from direct challenge by invaders, predation risk can be perceived as odors, sounds or nearness. In this study, we evaluated influence of predation risk by the predatory bug Podisus maculiventris on immunity of an herbivore Spodoptera frugiperda. Under the predator-induced stress combined with Escherichia coli inoculation, several larval physiological parameters of S. frugiperda were studied, including body mass, nodulation, and phenoloxidase activity. Our findings offernew evidence that provides insight into the immunological mechanism of predator-induced stress effects on prey species.

The Absence of Nosema bombi in Bumblebees (Bombus spp.) on Farms in Michigan

The fungal pathogen Nosema bombi impacts bumblebee fitness and is a factor in population declines throughout North America. Pesticides are also thought to contribute to declines, because they harm the innate immune system of insects, making them more susceptible to disease. A total of 232 bumblebees were collected at nine different farms across Michigan (U.S.A.), including farms that used pesticides and those that did not, in summer 2017. The bees were identified and tested for Nosema bombi. All bees were found to be free from N. bombi infection, suggesting this microsporidian may not yet have a biologically important impact on bumblebee populations in Michigan.

Identification of a novel humoral antifungal defense molecule in the hemolymph of tasar silkworm Antheraea mylitta

Humoral defenses are the major components of insect innate immune system that include the production of several soluble effector molecules from fat body and hemocytes, and released in to the hemolymph upon microbial infection. Hemolymph was collected from the fungal immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, extracted with a mixture of solvent (methanol/glacial acetic acid/water) and fractionated through RP-HPLC. Several fractions were collected, lyophilized and their antifungal activity was tested against Candida albicans. Only the fraction showing strong antifungal activity was further purified via gel filtration chromatography and the purity of active compound was confirmed by thin layer chromatography which showed only single spot after staining with ninhydrin. The molecular mass of this purified compound was determined by high resolution mass spectrometry as 531 Da and analysis of 1H and 13C NMR spectral data along with mass fragmentation pattern indicated the probable structure of the isolated compound as symmetric bis-decanoate derivative. Scanning electron microscopic study revealed that the compound degraded fungal cell wall leading to its lysis and may be the major target for its antifungal activity. These results indicate that presence of this compound in the hemolymph of A.mylitta provides defense against fungal infection.

Preliminary analysis of PGRP-LC gene and structure characteristics in bumblebees

PGRP-LC is a significant pattern recognition receptor of the insect innate immune system that can recognize peptidoglycans and activate immune signaling pathways regulating the expression and release of antimicrobial peptides against infection. We for the first time analyzed the phylogenetic tree, purification and structure of bumblebee PGRP-LC. The results showed high conservation of bumblebee PGRP-LC among the 16 bumblebee species, and further phylogenetic analysis showed that the PGRP-LC phylogeny of different subgenera (Subterraneobombus, Megabombus, Melanobombus, Bombus) is consistent with that of the COI gene. Additionally, the phylogeny of PGRP-LCs among Bombus, Apis and the solitary bee Megachile rotundata coincides with the sociality evolution of bees. Moreover, bumblebee PGRP-LC (Bl-PGRP-LC) shares the Drosophila PGRP-LCx and PGRP-LCa topology, retaining conserved disulfide bonds and 80% binding residues involved in the interaction between TCT and PGRP-LCx. Therefore, Bl-PGRP-LC might share some similar binding characteristics with Drosophila PGRP-LCx. In addition, Bl-PGRP-LC has shorter β5 and β1 sheets, longer β2, β3, and β4 sheets and a shallow binding groove. To determine the characteristics of Bl-PGRP-LC, high-purity PGRP-LC inclusion bodies, soluble GST-tag Bl-PGRP-LC fusion protein and soluble pure Bl-PGRP-LC were obtained in vitro. The results will be helpful for further study of the function and structure of Bl-PGRP-LC.