Ken Miura

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster, the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus, activating the Toll pathway, which in turn, induces AMP genes. Previous studies have shown the difference in immune systems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum. In T. castaneum, nine Toll and seven spaetzle (spz) genes were identified. To extend our understanding of AMP production by T. castaneum, we conducted functional assays of Toll and spaetzle genes related to Toll-pathway-dependent AMP gene expression in T. castaneum under challenge with bacteria or budding yeast. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll-pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll-pathway-dependent immune reactions, whereas spz7 plays a small part.

Insects rely only on their innate immune system to protect themselves from pathogens. Antimicrobial peptide (AMP) production is the main immune reaction in insects. In Drosophila melanogaster , the reaction is regulated mainly by the Toll and immune deficiency (IMD) pathways. Spaetzle proteins, activated by immune signals from upstream components, bind to Toll proteins, thus activating the Toll pathway, which in turn induces AMP genes. Previous studies have shown the difference immune ststems related to Toll and IMD pathways between D. melanogaster and Tribolium castaneum . In T. castaneum , 9 Toll and 7 spaetzle genes have been identified. To extend our understanding of AMP production by T. castaneum , we conducted functional assays of Toll and spaetzle genes related to Toll pathway-dependent AMP gene expression in T. castaneum under challenge with bacteria or budding yeast using RNA interference and qRT-PCR. The results revealed that Toll3 and Toll4 double-knockdown and spz7 knockdown strongly and moderately reduced the Toll pathway-dependent expression of AMP genes, respectively. Moreover, Toll3 and Toll4 double-knockdown pupae more rapidly succumbed to entomopathogenic bacteria than the control pupae, but spz7 knockdown pupae did not. The results suggest that Toll3 and Toll4 play a large role in Toll pathway-dependent immune reactions, whereas spz7 plays a small part.

In this study, the operation information and registration information of 2110 Elderly Housing with Supportive Services cases are used to analyze the corresponding situation for end-of-life care and severe dementia(hereinafter, “ECSD”) by hardware. The results are as follows:According to the different scale, living units and common facilities, there are obvious differences in the corresponding situation of ECSD. In addition, it is clear that among Elderly Housing with Supportive Services, the corresponding implementation rate of “unit type” to ECSD is higher, on the contrary, the implementation rate of “collective-house type” is lower.

Insects are solely dependent on an innate immune system. Antimicrobial peptide production is the main immune response of insects. The molecular mechanisms underlying this reaction in Drosophila melanogaster involves the induction of antimicrobial peptide genes, which is regulated by the Toll and IMD pathways. The Toll pathway is mainly activated by fungi or Gram-positive bacteria and the IMD pathway by Gram-negative bacteria. In terms of comparative immunology, we investigated the antimicrobial peptide production system in the beetle, Tribolium castaneum, which differs from that in D. melanogaster. To obtain a more detailed understanding, we examined whether Pelle and Tube, orthologues of which in D. melanogaster are the Toll pathway components, contributed to antimicrobial peptide production and immune reactions. These two genes were not induced by challenges from any type of microbe, which in this case were Gram-positive bacteria, -negative bacteria and an eukaryote. Using Pelle and Tube knockdown pupae, it was demonstrated that Pelle and Tube are involved in the induction of Cec2 as a representative Toll pathway-dependent gene in T. castaneum by Gram-positive and -negative bacteria and eukaryote challenges. Furthermore, neither Pelle nor Tube contributed to immune defences against two entomopathogenic bacteria. These results, taken together with our previous findings, led to the conclusion that the Toll pathway immune signaling reported in D. melanogaster indeed occurs in T. castaneum, and the gene sets involved in Toll signal transduction in T. castaneum did not differ significantly from those in D. melanogaster, but transduced immune signals to challenges from Gram-positive bacteria, -negative bacteria and an eukaryote, which differed from those in D. melanogaster.

We previously demonstrated that two immune signaling pathways, Toll and IMD, were concomitantly activated in the model beetle Tribolium castaneum by challenges to their immune system by several species of microbes, including Gram-positive and -negative bacteria as well as yeast. This contrasts with the Drosophila immune system in which more specific pathway activation depending on the type of microbe is well established. We suggest that the activation of an indiscriminate immune pathway in T. castaneum is due in part to an unselective recognition of pathogen-associated molecular patterns by the extracellular sensing modules of the two pathways. In order to obtain a more detailed understanding of the T. castaneum immune pathway, we investigated whether potential components of the T. castaneum IMD pathway, Caspar, DREDD and FADD, are involved in immune reactions triggered by microbial challenges. A sequence analysis of these three genes with the orthologues of other species, including insects, mouse and human, indicated that T. castaneum Caspar, DREDD and FADD functioned as immune signal transducers, which are usually induced by microbial challenges. However, these genes were not induced by microbial challenges. To establish whether these genes are involved in immune reactions, we used RNA interference-mediated knockdown of these genes to assess the microbial induction levels of the representative read-out antimicrobial peptide genes of the respective classes. The results indicated that these genes encode the canonical constituents of the IMD pathway of this beetle. DREDD and FADD influenced the induction of Toll-dependent antimicrobial peptide genes, providing novel crosstalk points between the two immune pathways, which appears to support indiscriminate pathway activation in T. castaneum. Furthermore, the phenotypes of DREDD or FADD knockdown pupae challenged by the two model bacterial pathogens correlated with AMP gene induction in the respective knockdowns, indicating that these intracellular factors contributed to antibacterial host defenses.

Coordinated regulation of apoptosis is critical for development, homeostasis, and immunity in larvae of Metazoa. We determined the full nucleotide sequence of an inhibitor of an apoptosis protein in a lepidopteran insect Mythimna separata (Walker) (MsIAP) and carried out functional analyses of the MsIAP. The full-length cDNA of MsIAP was 1642 bp, which encoded 379 amino acid residues with a calculated molecular mass of 41,834 Da, and two BIR domains and one RING domain revealed using amino acid sequence analysis. In addition, the sequences of these domains were similar to Drosophila IAP1 and those of some other lepidopteran insects. We carried out a functional analyses of MsIAP related to apoptosis regulation using RNA interference. The effects of MsIAP knockdown on adhering hemocytes and non-adhering hemocytes as controls were examined using Hoechst33342/propidium iodide staining, effector caspase activity and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. A significantly higher number of propidium iodide and TUNEL-positive cells was recorded in adhering hemocytes from MsIAP knockdown larvae than from control larvae, but these differences were not recorded for non-adhering hemocytes. However, higher effector caspase activity was detected in both adhering and non-adhering hemocytes from MsIAP knockdown larvae compared to that in control larvae. These results indicate that the knockdown of MsIAP induces apoptosis in larval adhering hemocytes, which MsIAP negatively and non-redundantly regulate apoptosis, and that IAP function is conserved in M. separata and other insect species including Drosophila and several lepidopteran insects.

Insect cellular immune reaction, which generally includes phagocytosis, encapsulation and nodule formation, is achieved by hemocytes circulating in insect haemolymph. The shift of hemocytes from the normal phase to the adhering phase is an important process in the cellular immune reaction, which includes the attachment of hemocytes to foreign surfaces or other hemocytes via adhesion factors. Neuroglian is one of the adhering factors associated with encapsulation in Manduca sexta and Drosophila melanogaster. Here we studied the localization of neuroglian (MsNrg) in Mythimna separata and its functional role in the cellular immune reaction. The distribution of MsNrg mRNA between hemocyte populations was determined using real-time quantitative reverse transcription PCR and in situ hybridization, which revealed that MsNrg was highly expressed in adhering hemocytes, especially in plasmatocytes. Unexpectedly, the transcript was observed as well in non-adhering hemocytes, implying neuroglian has a function in non-adhering hemocytes. Moreover, we showed that the amount of MsNrg mRNA was not changed by injections of either biotic or abiotic non-selves. Fewer latex beads were fully encapsulated by hemocytes in larvae treated with MsNrg double-stranded RNA than in control larvae, but their ability to achieve phagocytosis and nodule formation remained unchanged by the MsNrg knockdown. These results indicate that the function of neuroglian in the cellular immune reaction is conserved in D. melanogaster and lepidopteran species, and neuroglian in non-adhering hemocytes could possess unidentified function.