I'm working in a project where I need to test the enzyme activities of an insect's gut fluid. I've already tested it and found some good activity. Now I want to know whether the enzyme, showing high activity, is endogenous to the insect, or just produced by gut microbiota. There is no genome sequence or any other information available about that insect. How can I test it? How can I prepare enzyme solution, containing only endogenous enzyme of the insect?
I've read about the antibiotic feeding process. Anything else?
I found a paper where they incubated the insects for 24 hours, dissected, collected the gut fluid and then filtrated the gut fluid through 0.22 micrometer syringe filter, then demanded that this filtered solution contain no bacteria, neither their enzymes. But how can they demand there is no bacterial enzyme? If it is for starvation, then isn't it obvious for the insect's enzyme to reduce too? Can anyone provide any reference on support of this method, or concept? Please let me know.
I'm struggling in this issue and can't find any solution. I can't use the antibiotic method for a reason. Please help me out. Thank you.
If you can't kill off the bacteria in the insect gut by the use of antibiotics, can you somehow raise the insects in such a way that they do not have any gut bacteria, the way it is done with mice? This would involve first eliminating the gut bacteria of some parental insects with a combination of antibiotics, maintaining them in a sterile environment with sterile food, then breeding those insects to produce offspring that could be studied without antibiotic treatment. I don't know if anyone has ever done this.
Even if you had the entire genome sequence of the aseptic insect, it would not necessarily help you with your experiment if you are measuring the enzyme activity level with an enzyme assay, since that does not necessarily uniquely identify the enzyme(s) responsible for the activity. You would have to do a proteomics study to see what proteins were present in the gut fluid. You might be able to identify the enzymes that way by comparison with genomic sequences of related insects. You might also consider obtaining the full genome (or at least the full exome) sequence of your insect to inform your proteomics work. It's not that expensive nowadays.
You should also consider that there might be an interaction between the insect's gut lining cells and the gut microbiome that could affect the insect's secretion of enzymes. In other words, the insect and its microbiome may not be independent, so you would get a different amount and/or set of enzymes secreted by aseptic insects compared to insets with a normal microbiome.
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