TY - BOOK

T1 - An analysis of the interaction between the gut microbiota and host gut immunity in Galleria mellonella during infection by Bacillus thuringiensis and Metarhizium robertsii

AU - Upfold, Jennifer Kate

PY - 2024

Y1 - 2024

N2 - Pathogens can severely affect insect fitness, often resulting in death and the further spread of the pathogen. This is particularly problematic in a mass rearing environment where there are large numbers of insects living in close proximity. Such environments are susceptible to the emergence of pathogens and vulnerable to the rapid proliferation of disease. At the same time, insects host mutually beneficial symbionts in their gut which are known to help mediate important functions such as nutrient assimilation or host resistance to pathogens when this microbial community is balanced. Changes to this community can occur following pathogen infection which can either contribute to- or decrease host resistance to pathogens. Due to this potential, more research is required to study the tripartite interaction of the gut microbiota, pathogen and host response. In this project the larvae of Galleria mellonella (wax moth) was used as a model insect host to study the role of the microbiota in the health and resistance against colonization by entomopathogens entering via oral or topical routes. Bacillus thuringiensis ssp. galleriae 69- 6 (Btg) and Metarhizium robertsii (Mr), were used to induce immune responses via an oral- and topical infectious route, respectively. Our strategy was to conduct fitness assays on Galleria mellonella larvae reared with a different microbiota status (sterile – “axenic” vs conventional larvae) and to investigate the host response following infection. We hypothesized that symbiotic bacterial isolates residing within the insect gut can enhance the host's immune response, aiding in the fight against pathogen infection. However, the efficiency of this symbiotically-boosted immune response may vary depending on the specific pathogen involved as well as its route of entry into the host organism. We observed that larvae without a microbiota exhibited more variation in their developmental time, but the overall fitness and ability to fulfill their lifecycle is not impeded.We then conducted survival assays on 250-300 mg larvae using halflethal doses of Btg and Mr, and found axenic larvae were more susceptible to an oral infection of Btg where the half-lethal dose for the conventional group led to 90-100% mortality in the axenic group. We however did not detect any difference in mortality rates between axenic and conventional larvae after the topical Mr infection. Through a dual-analysis experimental design, We determined the gut microbiota by 16S rRNA sequencing, and assessed the level of immunerelated gene expression (qRT-PCR) in the same group of larvae during infection by Btg and Mr. This analysis revealed that the microbiota of our conventionally reared population is dominated by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two AMPs, Gallerimycin and Gloverin, in the conventional larvae compared to the axenic. Furthermore, Enterococcus mundtii isolated from the gut of conventional larvae showed inhibition activity against Btg in vitro, and when axenic larvae were re-inoculated with this isolate, an improved resistance to Btg was observed by 96 hours post infection. These findings highlight the immune-modulatory role of the Enterococcusdominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

AB - Pathogens can severely affect insect fitness, often resulting in death and the further spread of the pathogen. This is particularly problematic in a mass rearing environment where there are large numbers of insects living in close proximity. Such environments are susceptible to the emergence of pathogens and vulnerable to the rapid proliferation of disease. At the same time, insects host mutually beneficial symbionts in their gut which are known to help mediate important functions such as nutrient assimilation or host resistance to pathogens when this microbial community is balanced. Changes to this community can occur following pathogen infection which can either contribute to- or decrease host resistance to pathogens. Due to this potential, more research is required to study the tripartite interaction of the gut microbiota, pathogen and host response. In this project the larvae of Galleria mellonella (wax moth) was used as a model insect host to study the role of the microbiota in the health and resistance against colonization by entomopathogens entering via oral or topical routes. Bacillus thuringiensis ssp. galleriae 69- 6 (Btg) and Metarhizium robertsii (Mr), were used to induce immune responses via an oral- and topical infectious route, respectively. Our strategy was to conduct fitness assays on Galleria mellonella larvae reared with a different microbiota status (sterile – “axenic” vs conventional larvae) and to investigate the host response following infection. We hypothesized that symbiotic bacterial isolates residing within the insect gut can enhance the host's immune response, aiding in the fight against pathogen infection. However, the efficiency of this symbiotically-boosted immune response may vary depending on the specific pathogen involved as well as its route of entry into the host organism. We observed that larvae without a microbiota exhibited more variation in their developmental time, but the overall fitness and ability to fulfill their lifecycle is not impeded.We then conducted survival assays on 250-300 mg larvae using halflethal doses of Btg and Mr, and found axenic larvae were more susceptible to an oral infection of Btg where the half-lethal dose for the conventional group led to 90-100% mortality in the axenic group. We however did not detect any difference in mortality rates between axenic and conventional larvae after the topical Mr infection. Through a dual-analysis experimental design, We determined the gut microbiota by 16S rRNA sequencing, and assessed the level of immunerelated gene expression (qRT-PCR) in the same group of larvae during infection by Btg and Mr. This analysis revealed that the microbiota of our conventionally reared population is dominated by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two AMPs, Gallerimycin and Gloverin, in the conventional larvae compared to the axenic. Furthermore, Enterococcus mundtii isolated from the gut of conventional larvae showed inhibition activity against Btg in vitro, and when axenic larvae were re-inoculated with this isolate, an improved resistance to Btg was observed by 96 hours post infection. These findings highlight the immune-modulatory role of the Enterococcusdominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections.

M3 - Ph.D. thesis

BT - An analysis of the interaction between the gut microbiota and host gut immunity in Galleria mellonella during infection by Bacillus thuringiensis and Metarhizium robertsii

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -