Xu-Sheng Liu

Involvement of a pattern recognition receptor C-type lectin 7 in enhancing cellular encapsulation and melanization due to its carboxyl-terminal CRD domain in the cotton bollworm, Helicoverpa armigera.

C-type lectins play important roles in innate immunity as pattern recognition receptors (PRRs). We have previously reported a novel C-type lectin HaCTL7 from the cotton bollworm (Helicoverpa armigera) which contains two carbohydrate-recognition domains (CRDs), namely N-terminal CRD1 and C-terminal CRD2. Interestingly, there are four but not six of conserved cysteine residues in CRD2 of HaCTL7, which is different from that of other dual CRD C-type lectins. In the current study, we expressed and purified recombinant HaCTL7 (rHaCTL7) as well as rCRD1 and rCRD2, and demonstrated that both rHaCTL7 and rCRD2, but not rCRD1, owned the agglutinate ability against both Gram-negative and Gram-positive bacteria in a calcium dependent manner. In addition, both rHaCTL7 and rCRD2, but not rCRD1, could bind to various bacteria, and enhanced haemocytes mediated encapsulation and melanization processes. HaCTL7 secreted from fat bodies is able to bind to granulocytes, plasmatocytes and oenocytoids, but not to spherulocytes. Recombinant HaCTL7 and rCRD2 are capable of binding to both granulocytes and oenocytoids, while rCRD1 can only bind to granulocytes. Our data suggest that as a PRR HaCTL7 enhances encapsulation and melanization likely through its C-terminal CRD2, but not N-terminal CRD1, which imply that the characteristic four cysteine structure of CRD2 plays key roles in innate immunity.

Identification and characterization of two peptidoglycan recognition proteins with zinc-dependent antibacterial activity from the cotton bollworm, Helicoverpa armigera.

Peptidoglycan recognition proteins (PGRPs) specifically bind to peptidoglycan and play an important role in the innate immune responses as pattern recognition receptors (PRRs). Here we identified and characterized two PGRPs (HaPGRP-B and HaPGRP-C) from the cotton bollworm, Helicoverpa armigera. The comparative analysis indicated that five amino acids which are required for T7 lysozyme Zn(2+) binding and amidase activity are conserved in HaPGRP-B and HaPGRP-C, suggesting that the two PGRPs are members of the amidase-type PGRPs. HaPGRP-B and HaPGRP-C mRNA increased in both the fat bodies and the hemocytes after an injection of Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus. Recombinant HaPGRP-B and HaPGRP-C could agglutinate E. coli and S. aureus in a zinc-dependent manner. More importantly, both rHaPGRP-B and rHaPGRP strongly inhibited the growth of E. coli and S. aureus in the presence of Zn(2+). Moreover, the HaPGRP-B mRNA showed up-regulation post hormones (20E and methoprene) injection. Our results indicate that the two PGRPs of H. armigera may play an important role in defending against bacteria as amidase-type PGRPs and the hormones can function in regulating the expressions of PGRPs.

Expression profiles of six novel C-type lectins in response to bacterial and 20E injection in the cotton bollworm (Helicoverpa armigera).

C-type lectins can act as pattern recognition receptors (PRRs) and play an important role in innate immunity. Two C-type lectins (HaCTL1 and HaCTL2) have been previously identified in the cotton bollworm (Helicoverpa armigera). Here we isolate six C-type lectins from H. armigera (HaCTL3, 4, 5, 6, 7 and 8). All six new HaCTLs encode a signal peptide (or partial signal peptide) and complete tandem carbohydrate-recognition domains (CRDs). HaCTL4, 5, 6, 7 and 8 mRNA increased in the fat body after injection with both killed Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, whereas HaCTL3 mRNA was upregulated following E. coli injection only. Recombinant HaCTL3 exhibited agglutinating activity against both Gram-negative and Gram-positive bacteria in a calcium-dependent manner. Agglutination inhibitory analysis indicated that rHaCTL3 recognizes maltose, trehalose, peptidoglycan and lipopolysaccharides. HaCTL3 and HaCTL8 mRNA showed upregulation while HaCTL4, 5, and 6 mRNA downregulation post 20-Hydroxyecdysone (20E) injection. Our results indicate that the six novel C-type lectins of H. armigera may play important roles in defending against bacteria as PRRs and the hormone 20E can function in regulating immunity through lectins.

SRP gene is required for Helicoverpa armigera prophenoloxidase activation and nodulation response.

SRP gene was first identified from the fall webworm, Hyphantria cunea as one of genes up-regulated after bacteria injection. A rent study in Spodoptera litura showed that stress-induced elevation of SRP expression highly correlates with reduced feeding activities and growth retardation of larvae. In this study, we identified a SRP gene from the cotton bollworm, Helicoverpa armigera, namely Ha-SRP, and studied its precise roles in insect immunity. Expressions of Ha-SRP were upregulated in H. armigera larval hemocytes after injection of Escherichia coli. When the expression of Ha-SRP in H. armigera larval hemocytes was inhibited by dsHa-SRP injection, the transcription of prophenoloxidase genes in hemocytes was repressed, phenoloxidase activity in bacteria-challenged larval hemolymph was significantly decreased, and nodule formation in bacteria-injected larvae was reduced. More importantly, RNAi-treated insects infected with E. coli showed higher bacterial growth in hemolymph compared with infected controls. These results suggest that Ha-SRP gene plays importance roles in H. armigera innate immunity, possibly by mediating prophenoloxidase activation and nodulation response.

20‐hydroxyecdysone transcriptionally regulates humoral immunity in the fat body of Helicoverpa armigera

20‐hydroxyecdysone (20E) increases its titre level during the wandering stage and influences innate immunity in many holometabolous insects. However, the function of 20E as an immune‐activator or ‐suppressor needs to be determined. Here, the transcriptome of the peptidoglycan‐challenged fat body of the cotton bollworm, Helicoverpa armigera, was analysed using Illumina sequencing technology. Overall, 32 073 unigenes were assembled with a mean length of 643 nucleotides. Gene expression dynamics in the fat body during the wandering stage and of peptidoglycan‐challenged individuals were investigated by the digital gene expression system. Pattern recognition receptors [such as peptidoglycan recognition protein B (PGRP B), PGRP S2 precursor, C‐type lectin 5, hemolin and β‐1,3‐glucan recognition protein 2a] and antimicrobial peptides (namely attacin, gloverin, gloverin precursor, gloverin‐like, cecropin 2, cecropin D, cecropin D‐like and i‐type lysozyme) significantly increased their mRNA levels during the wandering stage. 20E treatment significantly induced the expression of these genes. Antibacterial activities were also enhanced during the wandering stage and after 20E injections. Bacillus subtilis peptidoglycan induced the expression of PGRP D, PGRP B, PGRP S2 precursor, gloverin, gloverin precursor, gloverin‐like, cecropin 2, cecropin D and lebocin‐like genes. These results demonstrate that 20E acts by enhancing humoral immunity in H. armigera.

Molecular characterization of a peptidoglycan recognition protein from the cotton bollworm, Helicoverpa armigera and its role in the prophenoloxidase activation pathway

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In this study, a PGRP (HaPGRP-A) from the cotton bollworm, Helicoverpa armigera was identified and characterized. Sequence analysis indicated that HaPGRP-A is not an amidase-type PGRP. Increased levels of HaPGRP-A mRNA were observed in the fat body and hemocytes of H. armigera larvae following the injection of microbes or Sephadex beads. Analysis using purified recombinant HaPGRP-A showed that it (i) could bind and agglutinate Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, (ii) enhanced prophenoloxidase activation in the presence of microbes, (iii) promoted the formation of melanotic nodules in vivo, and (iv) enhanced the melanization of Sephadex beads in vivo. RNA interference assays were performed to further confirm the function of HaPGRP-A. When the expression of HaPGRP-A in H. armigera larvae was inhibited by dsHaPGRP-A injection, the phenoloxidase activity in larval hemolymph was significantly decreased and RNAi-treated insects infected with bacteria showed higher bacterial growth in hemolymph compared with infected control larvae. These results indicated that HaPGRP-A acts as a pattern recognition receptor and binds to the invading organism to trigger the prophenoloxidase activation pathway of H. armigera, and the activated phenoloxidase may participate in the melanization process of nodulation and encapsulation responses.

Rab3 is involved in cellular immune responses of the cotton bollworm, Helicoverpa armigera

Rab3, a member of the Rab GTPase family, has been found to be involved in innate immunity. However, the precise function of this GTPase in innate immunity remains unknown. In this study, we identified a Rab3 gene (Ha-Rab3) from the cotton bollworm, Helicoverpa armigera and studied its roles in innate immune responses. Expression of Ha-Rab3 was upregulated in the hemocytes of H. armigera larvae after the injection of Escherichia coli or chromatography beads. The dsRNA-mediated knockdown of Ha-Rab3 gene in H. armigera larval hemocytes led to significant reduction in the phagocytosis and nodulation activities of hemocytes against E. coli, significant increase in the bacterial load in larval hemolymph, and significant reduction in the encapsulation activities of hemocytes toward invading chromatography beads. Furthermore, Ha-Rab3 knockdown significantly suppressed spreading of plasmatocytes. These results suggest that Ha-Rab3 plays important roles in H. armigera cellular immune responses, possibly by mediating spreading of hemocytes.

20-Hydroxyecdysone promotes release of GBP-binding protein from oenocytoids to suppress hemocytic encapsulation

Growth-blocking peptide (GBP) is an insect cytokine that stimulates plasmatocyte adhesion, thereby playing a critical role in encapsulation reaction. It has been previously demonstrated that GBP-binding protein (GBPB) is released upon oenocytoid lysis in response to GBP and is responsible for subsequent clearance of GBP from hemolymph. However, current knowledge about GBPB is limited and the mechanism by which insects increase GBPB levels to inactivate GBP remains largely unexplored. Here, we have identified one GBP precursor (HaGBP precursor) gene and two GBPB (namely HaGBPB1 and HaGBPB2) genes from the cotton bollworm, Helicoverpa armigera. The HaGBP precursor was found to be predominantly expressed in fat body, whereas HaGBPB1 and HaGBPB2 were mainly expressed in hemocytes. Immunological analyses indicated that both HaGBPB1 and HaGBPB2 are released from hemocytes into the plasma during the wandering stage. Additionally, 20-hydroxyecdysone (20E) treatment or bead challenge could promote the release of HaGBPB1 and HaGBPB2 at least partly from oenocytoids into the plasma. Furthermore, we demonstrate that the N-terminus of HaGBPB1 is responsible for binding to HaGBP and suppresses HaGBP-induced plasmatocyte spreading and encapsulation. Overall, this study helps to enrich our understanding of the molecular mechanism underlying 20E mediated regulation of plasmatocyte adhesion and encapsulation via GBP-GBPB interaction.

WITHDRAWN: C-type lectin interacting with β-integrin enhances hemocytic encapsulation in the cotton bollworm, Helicoverpa armigera

This article has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error. The article was subsequently accepted and published and can be viewed here: https://doi.org/10.1016/j.ibmb.2017.05.005 The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Identification and characterization of a gene involved in the encapsulation response of Helicoverpa armigera haemocytes: Identification of an encapsulation-related gene

Encapsulation is a kind of cellular immune response of insect haemocytes, which results in the formation of capsules around invading parasites. However, the molecular mechanism of this response is largely unknown. In this study, we identified a potential immune‐related gene in the cotton bollworm, Helicoverpa armigera, called defence protein 1 (Ha‐DFP1). A tissue distribution analysis revealed that Ha‐DFP1 protein was expressed in haemocytes and secreted into the haemolymph of Helic. armigera larvae. The Ha‐DFP1 mRNA transcript level in haemocytes and the concentration of the Ha‐DFP1 protein in haemolymph both increased after injecting chromatography beads. Purified recombinant Ha‐DFP1 bound to the surface of haemocytes and promoted haemocyte encapsulation on chromatography beads in vitro. The spreading ability of haemocytes was inhibited when Ha‐DFP1 expression in Helic. armigera larval haemocytes decreased in response to the injection of double‐stranded RNA specific to Ha‐DFP1, and the encapsulation ability of haemocytes was impaired. Based on these results, we speculate that Ha‐DFP1 plays an important role in the Helic. armigera encapsulation response, possibly by binding to the haemocyte surface and mediating spreading behaviour.