2021 Summer Undergraduate Research Program (SURP) Symposium
Characterization of the Intestinal Mucin Role in Colonization of the Spodoptera frugiperda Midgut
Location
Ballroom, Maucker Student Union, University of Northern Iowa
Presentation Type
Poster Presentation (Electronic Copy Not Available)
Document Type
poster
Abstract
The animal intestinal microbiome is a complex multispecies community which influences host development, physiology, and response to invasive pathogens. In lepidopteran (caterpillars) insects, intestinal mucins (IMs) are densely O-glycosylated proteins found in the midgut epithelium as either membrane-bound or gel-forming variants and possess chitin-binding domains which facilitate attachment to the protective mucus layer, the peritrophic matrix (PM) (Wang and Granados 1997). IMs are analogous to MUC2 the vertebrate mucin and major constituent in the mucosal layer of the mammalian gastrointestinal (G.I.) tract. In mice, it has been demonstrated that anaerobes secrete hydrolases that degrade mucin polysaccharides to mono- and disaccharides utilized by other intestinal residents (Mahowald et al. 2009). However, this mechanism of nutrient acquisition by stably colonized intestinal residents has not been elucidated. Using Spodoptera frugiperda (Fall armyworm) larvae, we attempted to clone and recombinantly express IMs with the goal of then using the purified mucins to supplement minimal growth media to test enteric bacteria for the ability to metabolize IM polysaccharides in vitro. Second, we attempted to quantify IM gene expression in each developmental instar by quantitative PCR to determine the optimal stage to induce gene silencing by RNA interference (RNAi). We anticipate that RNAi-mediated silencing of IM would significantly reduce the nutrient content in the mucosal layer, leading to a significant reduction in the number of stably colonized resident microorganisms. We have designed multiple primer sets that specifically target coding regions from S. frugiperda mucin transcripts (Legeai et al. 2014) and Aequorea victoria GFP to use as a control. We have also made complementary DNA (cDNA) from extracted RNAs of midgut epithelial tissue. With primers designed to specifically target S. frugiperda midgut IMs, we are closer to cloning the full-length transcript. Gradient polymerase chain reaction will be used in the next series of experiments to identify optimal amplification temperatures of S. Frugiperda intestinal mucin.
Start Date
30-7-2021 11:30 AM
End Date
30-7-2021 1:15 PM
Event Host
Summer Undergraduate Research Program, University of Northern Iowa
Faculty Advisor
Jerreme Jackson
Department
Department of Biology
Copyright
©2021 Noah Bond and Jerreme Jackson
File Format
application/pdf
Recommended Citation
Bond, Noah and Jackson, Jerreme Ph.D., "Characterization of the Intestinal Mucin Role in Colonization of the Spodoptera frugiperda Midgut" (2021). Summer Undergraduate Research Program (SURP) Symposium. 9.
https://scholarworks.uni.edu/surp/2021/all/9
Characterization of the Intestinal Mucin Role in Colonization of the Spodoptera frugiperda Midgut
Ballroom, Maucker Student Union, University of Northern Iowa
The animal intestinal microbiome is a complex multispecies community which influences host development, physiology, and response to invasive pathogens. In lepidopteran (caterpillars) insects, intestinal mucins (IMs) are densely O-glycosylated proteins found in the midgut epithelium as either membrane-bound or gel-forming variants and possess chitin-binding domains which facilitate attachment to the protective mucus layer, the peritrophic matrix (PM) (Wang and Granados 1997). IMs are analogous to MUC2 the vertebrate mucin and major constituent in the mucosal layer of the mammalian gastrointestinal (G.I.) tract. In mice, it has been demonstrated that anaerobes secrete hydrolases that degrade mucin polysaccharides to mono- and disaccharides utilized by other intestinal residents (Mahowald et al. 2009). However, this mechanism of nutrient acquisition by stably colonized intestinal residents has not been elucidated. Using Spodoptera frugiperda (Fall armyworm) larvae, we attempted to clone and recombinantly express IMs with the goal of then using the purified mucins to supplement minimal growth media to test enteric bacteria for the ability to metabolize IM polysaccharides in vitro. Second, we attempted to quantify IM gene expression in each developmental instar by quantitative PCR to determine the optimal stage to induce gene silencing by RNA interference (RNAi). We anticipate that RNAi-mediated silencing of IM would significantly reduce the nutrient content in the mucosal layer, leading to a significant reduction in the number of stably colonized resident microorganisms. We have designed multiple primer sets that specifically target coding regions from S. frugiperda mucin transcripts (Legeai et al. 2014) and Aequorea victoria GFP to use as a control. We have also made complementary DNA (cDNA) from extracted RNAs of midgut epithelial tissue. With primers designed to specifically target S. frugiperda midgut IMs, we are closer to cloning the full-length transcript. Gradient polymerase chain reaction will be used in the next series of experiments to identify optimal amplification temperatures of S. Frugiperda intestinal mucin.
