Humoral immune response of Galleria mellonella after repeated infection with Bacillus thuringiensis.

The insect immune system relies on innate mechanisms only. However, there is an increasing number of data reporting that previous immune challenge with microbial elicitors or a low number of microorganisms can modulate susceptibility after subsequent lethal infection with the same or different pathogen. This phenomenon is called immune priming. Its biochemical and molecular mechanisms remain unravelled. Here we present that Galleria mellonella larvae that survived infection induced by intrahemocelic injection of a low dose of Bacillus thuringiensis were more resistant to re-injection of a lethal dose of the same bacteria but not other bacteria and fungi tested. This correlated with enhanced activity detected in full hemolymph as well as in separated hemolymph polypeptides. In addition, we observed differences in the hemolymph protein pattern between primed and non-primed larvae after infection with the lethal dose of B. thuringiensis. Expression of genes encoding inducible defence molecules was not enhanced in the primed larvae after the infection with the lethal dose of B. thuringiensis. It is likely that priming affects the turnover of immune related hemolymph proteins; hence, upon repeated contact, the immune response may be more ergonomic.

Short-term heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana.

We aimed to investigate how exposition of infected insects to short-term heat shock affects the biochemical and molecular aspects of their immune response. Galleria mellonella larvae were exposed to 43°C for 15min, at the seventy second hour after natural infection with entomopathogenic fungus Beauveria bassiana. As a result, both qualitative and quantitative changes in hemolymph protein profiles, and among them infection-induced changes in the amount of apolipophorin III (apoLp-III), were observed. Heat shock differently affects the expression of the tested immune-related genes. It transiently inhibits expression of antifungal peptides gallerimycin and galiomicin in both the fat body and hemocytes of infected larvae. The same, although to a lesser extent, concerned apoLp-III gene expression and was observed directly after heat shock. Nevertheless, in larvae that had recovered from heat shock, apoLp-III expression was higher in comparison to unshocked larvae in the fat body but not in hemocytes, which was consistent with the higher amount of this protein detected in the hemolymph of the infected, shocked larvae. Furthermore, lysozyme-type activity was higher directly after heat shock, while antifungal activity was significantly higher also in larvae that had recovered from heat shock, in comparison to the respective values in their non-shocked, infected counterparts. These results show how changes in the external temperature modulate the immune response of G. mellonella suffering from infection with its natural pathogen B. bassiana.

Changes in the hemolymph protein profiles in Galleria mellonella infected with Bacillus thuringiensis involve apolipophorin III. The effect of heat shock.

This report concerns the effect of heat shock on host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. We show enhanced activity against Gram-positive bacteria in the hemolymph of larvae pre-exposed to heat shock before infection with B. thuringiensis. Heat shock influenced the protein pattern in the hemolymph of infected larvae: more peptides with a molecular weight below 10 kDa were detected in comparison with nonshocked animals. Additionally, we noticed that the amount of apolipophorin III (apoLp-III) in the hemolymph decreased transiently following infection, which was considerably higher in larvae pre-exposed to heat shock. On the other hand, its expression in the fat body showed a consequent infection-induced decline, observed equally in shocked and nonshocked animals. This suggests that the amount of apoLp-III in the hemolymph of G. mellonella larvae is regulated at multiple levels. We also report that this protein is more resistant to degradation in the hemolymph of larvae pre-exposed to heat shock in comparison to nonshocked larvae. Two-dimensional analysis revealed the presence of three isoforms of apoLp-III, all susceptible to proteolytic degradation. However, one of them was the most abundant, both in the protease-treated and untreated hemolymph. Taking into consideration that, in general, apoLp-III has a stimulative effect on different immune-related hemolymph proteins and peptides, the reported findings bring us closer to understanding the effect of heat shock on the resistance of G. mellonella to infection.

Heat shock affects host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis.

We report that Galleria mellonella larvae exposed to heat shock was more resistant to infection with entomopathogenic bacteria Bacillus thuringiensis. The insects were exposed to a temperature of 40°C for 30 min directly before injection of vegetative bacterial cells. It appeared that the kinetics of the immune response was affected in heat-shocked animals. The infection-induced antimicrobial activity of larval hemolymph was stronger in shocked animals in comparison to the non-shocked ones. Hemolymph proteins of molecular weight below 10 kDa, corresponding to the size of antimicrobial peptides, were responsible for this activity. Furthermore, the transcription level of genes encoding antimicrobial peptides: cecropin, gallerimycin, and galiomycin was increased in the fat bodies of insects exposed to heat shock before infection. On the contrary, the heat-shock treatment did not enhance expression of the metalloproteinase inhibitor-IMPI in the infected animals. The difference in the amount of antimicrobial peptides and, consequently, in the defense activity of insect hemolymph, persisted after the action of bacterial metalloproteinases, which are well-known virulence factors. Furthermore, peptides with antimicrobial activity in the hemolymph of infected larvae pre-exposed to heat shock appeared to be more resistant to proteolytic degradation both in vitro and in vivo. Our results point to the mechanism of cross-protection of thermal stress toward innate immune response.