C-type lectin-mediated microbial homeostasis is critical for Helicoverpa armigera larval growth and development.

The immune system of a host functions critically in shaping the composition of the microbiota, and some microbes are involved in regulating host endocrine system and development. However, whether the immune system acts on endocrine and development by shaping the composition of the microbiota remains unclear, and few molecular players or microbes involved in this process have been identified. In the current study, we found that RNA interference of a C-type lectin (HaCTL3) in the cotton bollworm Helicoverpa armigera suppresses ecdysone and juvenile hormone signaling, thus reducing larval body size and delaying pupation. Depletion of HaCTL3 also results in an increased abundance of Enterocuccus mundtii in the hemolymph, which may escape from the gut. Furthermore, HaCTL3 and its controlled antimicrobial peptides (attacin, lebocin, and gloverin) are involved in the clearance of E. mundtii from the hemolymph via phagocytosis or direct bactericidal activity. Injection of E. mundtii into larval hemocoel mimics HaCTL3-depleted phenotypes and suppresses ecdysone and juvenile hormone signaling. Taken together, we conclude that HaCTL3 maintains normal larval growth and development of H. armigera via suppressing the abundance of E. mundtii in the hemolymph. Our results provide the first evidence of an immune system acting on an endocrine system to modulate development via shaping the composition of microbiota in insect hemolymph. Thus, this study will deepen our understanding of the interaction between immunity and development.

Molecular characterization of immune responses of Helicoverpa armigera to infection with the mermithid nematode Ovomermis sinensis.

BACKGROUND: Mermithid nematodes, such as Ovomermis sinensis, display a broad host range including some lepidopteran pests. Infective juveniles penetrate their host through the cuticle, complete their growth within the hemocoel and eventually kill the host upon their emergence. Hence, mermithid nematodes are considered potential biological control agents of insect pests. Our previous data indicate that the infection rate of O. sinensis on cotton bollworm (Helicoverpa armigera) is low, which may be largely due to the strong immune system of the host. However, current knowledge on the interactions of mermithid nematodes with their hosts and the mechanisms employed by hosts to defend themselves against mermithid nematodes is limited. RESULTS: Here, we investigated the response of H. armigera to O. sinensis infection. Parasitism by O. sinensis caused a sharp decline in the survival rate of H. armigera. The hemocytic phagocytosis ability, antibacterial activity, and phenoloxidase (PO) activity in plasma of H. armigera increased at 1 d post parasitism (dpp) but decreased at 3 dpp. Further, we investigated gene expression in the fat body of parasitized and non-parasitized H. armigera larvae at 1, 3, and 5 dpp using a digital gene expression system. In total, 41, 60 and 68 immune-related differentially expressed genes were identified at 1, 3, and 5 dpp, respectively. These genes encoded pattern recognition receptors (PRRs), antimicrobial peptides (AMPs), serine proteases (SPs), SP inhibitors, mucins and other immune-related proteins. The expression of most PRRs, AMPs, SPs, and mucins was upregulated in the fat body of larvae at 1 dpp, downregulated at 3 dpp, and then again upregulated at 5 dpp by O. sinensis. The increased expression of SP inhibitors may contribute to the inhibited PO activity at 5 dpp. CONCLUSIONS: This study demonstrates that parasitism by O. sinensis modulates the immune reaction of the host H. armigera by altering the expression of immune-related genes. Our data provide a basis for future investigation of the molecular mechanisms employed by the mermithid nematode O. sinensis to modulate the immunity of the host H. armigera. These data will also likely facilitate the improvement of success in parasitism of H. armigera by O. sinensis.

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.

C-type lectin interacting with ß-integrin enhances hemocytic encapsulation in the cotton bollworm, Helicoverpa armigera.

The encapsulation reaction in invertebrates is analogous to granuloma formation in vertebrates, and this reaction is severely compromised when ecdysone signaling is blocked. However, the molecular mechanism underlying the encapsulation reaction and its regulation by ecdysone remains obscure. In our previous study, we found that the C-type lectin HaCTL3, from the cotton bollworm Helicoverpa armigera, is involved in anti-bacterial immune response, acting as a pattern recognition receptor (PRR). In the current study, we demonstrate that HaCTL3 is involved in defense against parasites and directly binds to the surface of nematodes. Our in vitro and in vivo studies indicate that HaCTL3 enhances hemocytic encapsulation and melanization, whereas H. armigera ß-integrin (Haß-integrin), located on the surface of hemocytes, participates in encapsulation. Additionally, co-immunoprecipitation experiments reveal HaCTL3 interacts with Haß-integrin, and knockdown of Haß-integrin leads to reduced encapsulation of HaCTL3-coated beads. These results indicate that Haß-integrin serves as a hemocytic receptor of HaCTL3 during the encapsulation reaction. Furthermore, we demonstrate that 20-hydroxyecdysone (20E) treatment dramatically induces the expression of HaCTL3, and knockdown of the 20E receptor (EcR)/ultraspiracle (USP), abrogates this response. Overall, this study provides the first evidence of the presence of a hemocytic receptor (Haß-integrin), that interacts with the PRR HaCTL3 to facilitate encapsulation reaction in insects and demonstrates the regulation of this process by the steroid hormone ecdysone.

WITHDRAWN: C-type lectin interacting with ß-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.