We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.
"RNAi of integrin b1 significantly suppressed hemocyte encapsulation in M. sexta (Levin et al., 2005) and phagocytosis in C. capitata (Mamali et al., 2009). Cell-mediated responses of the innate immune system in insects involve a fast transformation from resting nonadherent states of hemocytes to their active adherent cells (Gillespie et al., 1997). "
[Show abstract][Hide abstract] ABSTRACT: Integrins are cell surface heterodimeric proteins interacting with the extracellular matrix and mediating environmental signals through cell membranes. A full-length cDNA sequence of the integrin β1 subunit gene (HaITGb1) was cloned from the Oriental tobacco budworm, Helicoverpa assulta, and analyzed for its physiological role in both immune response and development. HaITGb1 was expressed in all developmental stages from egg to adult and in all tested larval tissues of hemocytes, fat body, gut, and epidermis. Utilizing an RNA interference (RNAi) approach, injection of a specific double-stranded RNA (dsRNA) in larvae suppressed HaITGb1 transcript levels and significantly impaired hemocytes in their extracellular matrix adherence properties. Furthermore, the RNAi treatment significantly suppressed hemocyte nodule formation in response to bacterial challenge, which resulted in significantly enhanced susceptibility to both pathogenic and non-pathogenic bacteria. The RNAi treatment also interfered with H. assulta larval and pupal development. These results suggest that the extensive and constitutive expression of HaITGb1 is necessary for H. assulta to perform an efficient immune response against microbial pathogens and undergo normal immature development.
"Moreover, the integrin b subunit also participates in phagocytosis. The expression of an integrin b on the surface of medfly hemocytes could trigger the engulfment of pathogen (Mamali et al., 2009). In insects, another integrin b family, bn, may also regulate phagocytosis. "
[Show abstract][Hide abstract] ABSTRACT: When lepidopteran larvae are infected by a large quantity of pathogens or parasitized by nonadaptive parasitoids, hemocytes in the hemocoel will encapsulate these foreign invaders. Cellular encapsulation requires hemocytes, particularly plasmatocytes, to change their states from nonadhesive, spherical cells into adhesive, spreading cells. However, it is unclear how the changes of plasmatocytes are regulated. Here we report that the integrin β1 subunit from hemocytes of Ostrinia furnacalis (Ofint β1) plays an important role in regulating the spreading of plasmatocytes. The full length cDNA sequence (4477 bp) of Ofint β1 was cloned from hemocytes. Phylogenetic analysis showed that Ofint β1 belonged to the integrin βPS family of Drosophila melanogaster with highest sequence identity (78.7%) to the β-integrin of Pseudoplusia includens. Structural analysis of the deduced amino acid sequence indicated that Ofint β1 had similar functional domains to known β-integrins in other lepidopteran insects. RT-PCR, Northern blotting, Western blotting and immunohistochemical analyses showed that OfINT β1 was expressed mainly in hemocytes, especially in plasmatocytes, and weakly in fat body, Malpighian tubes and epidermis. After hemocytes had spread onto slides, fewer antibodies to OfINT β1 bound to the surface of plasmatocytes. Furthermore, anti-OfINT β1 serum clearly inhibited the spreading of plasmatocytes. Together these results indicate that OfINT β1 may play an important role in regulating the spreading of plasmatocytes.
[Show abstract][Hide abstract] ABSTRACT: Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.
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