Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host-pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect-derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune-relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best-characterized immune-related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host-pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.

Comparative analysis of peptidoglycan recognition proteins in endoparasitoid wasp Microplitis mediator

Peptidoglycan recognition proteins （PGRPs） are a family of innate immune receptors that specifically recognize peptidoglycans （PGNs） on the surface of a number of pathogens. Here, we have identified and characterized six PGRPs from endoparasitoid wasp, Microplitis mediator （MmePGRPs）. To understand the roles of PGRPs in parasitoid wasps, we analyzed their evolutionary relationship and orthology, expression profiles during different developmental stages, and transcriptional expression following infection with Gram-positive and -negative bacteria and a fungus. MmePGRP-S1 was significantly induced in response to pathogenic infection. This prompted us to evaluate the effects of RNA interference mediated gene specific knockdown ofMmePGRP-S1. The knockdown of MmePGRP-S1 （iMmePGRP-S1） dramatically affected wasps＇ survival following challenge by Micrococcus luteus, indicating the involvement of this particular PGRP in immune responses against Gram-positive bacteria. This action is likely to be mediated by the Toll pathway, but the mechanism remains to be determined. MmePGRP-S 1 does not play a significant role in anti-fungal immunity as indicated by the survival rate of iMmePGRP-S wasps. This study provides a comprehensive characterization of PGRPs in the economically important hymenopteran species M. mediator.

Metabolic and immunological effects of gut microbiota in leaf beetles at the local and systemic levels

Insects’intestinal microbes have profound effects on the host’s physiological traits,which can impact their physiology at both the local(gut)and systemic(body)levels.Nevertheless,the molecular mechanisms underlying hostmicrobiota interactions,especially in non-model insects,remain elusive.Recently,tissue-specific transcriptomic analysis has been highlighted as a robust tool in studying host–microbe interactions.Plagiodera versicolora is a worldwide leaf-eating pest that primarily feeds on willows and poplar.The interplay between gut microflora and this host beetle has yet to be studied.Herein,we investigate the effects of the gut microbiota on the body mass of P.versicolora larvae,compare the nutrition status of larvae in absence and presence of gut microbiota,and profile gut bacterial loads throughout its developmental larval stages.We then perform comparative transcriptomic analysis of gut and body tissues in axenic and non-axenic larvae.Finally,we confirm the expression patterns of representative genes in nutritional metabolism and immunity.Results show that weight growth is retarded in conventional larvae,with a concomitant increase of total bacterial load by the 5^(th)development day,and germ-free larvae have a higher glucose content than conventional-reared larvae.Both nutritional and immunological analyses indicate that gut bacteria are a burden in the beetle’s larval development.These findings elucidate the impacts of gut microbiota on P.versicolora,and provide insight into tissue-specific responses to gut microflora in this pest at the genetic level,boosting our understanding of the molecular mechanisms underlying host–microbe interactions in leaf beetles and beyond.

Comparative analysis of the Monochamus alternatus immune system

The pine sawyer beetle, Monochamus alternatus, is regarded as a notorious for- est pest in Asia, vectoring an invasive pathogenic nematode, Bursaphelenchus xylophilus, which is known to cause pine wilt disease. However, little sequence information is available for this vector beetle. This hampered the research on its immune system. Based on the transcriptome of M. alternatus, we have identified and characterized 194 immunity-related genes in M. alternatus, and compared them with homologues molecules from other species known to exhibit immune responses against invading microbes. The lower number of puta- tive immunity-related genes in M. alternatus were attributed to fewer C-type lectin, serine protease （SP） and anti-microbial peptide （AMP） genes. Phylogenetic analysis revealed that M. alternatus had a unique recognition gene, galectin3, orthologues of which were not identified in Tribolium castaneum, Drosophila melanogastor, Anopheles gambiae and Apis mellifera. This suggested a lineage-specific gene evolution for coleopteran insects. Our study provides the comprehensive sequence resources of the immunity-related genes ofM. alternatus, presenting valuable information for better understanding of the molecular mechanism of innate immunity processes in M. alternatus against B. xylophilus.

Host-pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella.Infection with this fungus triggered systemic immune response in G.mellonella;nevertheless,the infection was lethal if spores entered the insect hemocel.We observed melanin deposition in the insect cuticle and walls of air bags,while the invading fungus interrupted tssue continuity.We have shown colonization of muscles,air bags,and finally colonization and complete destruction of the fat body-the main organ responsible for the synthesis of defense molecules in response to infection.This destruction was probably not caused by simple fungal growth,because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans.This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism.Finally,we were unable to reduce the extremely high virulence of B.bassiana against G.mellonella by priming of larvae with thermally inactivated fungal spores.

Characterization and mode of action analysis of black soldier fly(Hermetia illucens)larva-derived hemocytes

With the growing importance of the black soldier fly(Hermetia illucens)for both sustainable food production and waste management as well as for science,a great demand of understanding its immune system arises.Here,we present the first description of the circulating larval hemocytes with special emphasis on uptake of microorganisms and distinguishing hemocyte types.With histological,zymographic,and cytometric methods and with a set of hemocyte binding lectins and antibodies,the hemocytes of H.illucens are identified as plasmatocytes,crystal cells,and putative prohemocytes.Total hemocyte counts(THC)are determined,and methods for THC determination are compared.Approximately 1100 hemocytes per microliter hemolymph are present in naive animals,while hemocyte density decreases dramatically shortly after wounding,indicating a role of hemocytes in response to wounding(and immune response in general).The determination of the relative abundance of each hemocyte type(differential hemocyte count,DHC)revealed that plasmatocytes are highly abundant,whereas prohemocytes and crystal cells make up only a small percentage of the circulating cells.Plasmatocytes are not only the most abundant but also the professional phagocytes in H.illucens.They rapidly engulf and take up bacteria both in vivo and in vitro,indicating a very potent cellular defense against invading pathogens.Larger bioparticles such as yeasts are also removed from circulation by phagocytosis,but slower than bacteria.This is the first analysis of the potent cellular immune response in the black soldier fly,and a first toolbox that helps to identify hemocyte(types)is presented.