CHARACTERIZATION OF ANTI-MICROBIAL COMPOUNDS IN ORAL SECRECTIONS OF BEETLES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The carrion beetles (Coleoptera: Silphidae) consist of two subfamilies in North America. Members of the Silphinae arrive at carcasses during the mid-stage of decay and their larvae feed on developing maggots, while members of the Nicrophorinae or burying beetles bury and tend carcasses upon which their developing larvae feed. It has been hypothesized that the anal and/or oral secretions from these insects are antimicrobial, although quantification has not been made. My laboratory has been able to quantify the antimicrobial activity of the Nicrophorinae oral secretions with the Microtox Analyzer, which measures bioluminescence of the bacterium, Vibrio fischerii. The antimicrobial activity of the Nicrophorinae appears to be proteinaceous in nature. The future research of my laboratory is to identify these proteins. We plan to use a combination of techniques. One route is to identify all of the enzymatic activity in the oral secretions. The experimental techniques used for this research is colorimetric assays. Another route to identify the antimicrobial compounds is to compare the one species of Nicrophorinae, Nicrophorus carolinus, which does not exhibit antimicrobial activity to Nicrophorus marginatus, which does have antimicrobial activity in their oral secretion and is the most common burying beetle in Nebraska. We plan on using a subtractive comparison of gel electrophoresis banding patterns to help identify possible antimicrobial compounds. Once differences are identified the proteins can be isolated from gels and identified by sequencing these proteins. There is potential that the proteins responsible for the antimicrobial activity are small peptides. If the antimicrobial compounds seem to be peptides, we will use a combination of Tris-tricine gels, HPLC, and GC mass spec to separate and identify the compounds. By identifying these antimicrobial compounds we are hopeful that these proteins may be useful in treating antibiotic-resistant microorganisms. We have almost completed the characterization of the enzymes found in the oral secretion of N. marginatus. We have used colorimetric assays and have found the presence of alkaline phosphatase, peroxidase, and peptidase. We have further characterized the peptidases and find aspartic proteases and cysteine proteases present in the oral secretion. This is not surprising as these enzymes are commonly found in insect oral secretion. Since we have completed the characterization of the enzymes, we are now beginning fractionation of the secretion. After basic fractionation using microcon spin tubes, we find the active fraction in the solution left in the tube. We now believe that the antimicrobial compound could be a larger protein rather than a peptide as first hypothesized. Our next step is to fractionate samples further with the HPLC.