Our protocol expands the use of G.mellonella from a systemic infection model to a model that allows bacterial administration via the natural oral route. G.mellonella is a suitable replacement model for rodents in analyzing different hallmarks of innate micro-post interactions as the larvae can be used for human pathogenic bacteria. Begin by transferring the eggs laid by adult moths to two liter boxes containing wax moth substrate, at 30 degrees Celsius, in the dark.
After one to two weeks, transfer 25 grams of larva containing substrate into fresh substrate, when small and tiny larva become visible. Synchronize the larvae after two weeks according to their size, into groups of 30 to 40 larvae in new two liter containers on wax moth substrate. After two weeks, select larvae for the experiment by weight.
Using only pale, fast moving larvae with a mass of 180 to 200 milligrams. To force feed the larvae, load an insulin syringe with a blunt-ended needle for oral application, with one times 10 to the seventh bacteria per 10 microliters of DPBS per larva. Load the syringe into a microsyringe pump.
Wait for a larva to open its mouth parts before inserting the syringe carefully between the mandibles and delivering the bacteria. When all of the larva have been fed, incubate the animals in the dark at 37 degrees Celsius, between one to 24 hours. For larva processing, clean a safety hood with ethanol and spray reagent for preventing RNase contamination.
Snap freeze the living larva in liquid nitrogen. Homogenize each individual frozen larva using a mortar and pestle and a small volume of liquid nitrogen. When a powdered homogenate has been produced, transfer the homogenate to a disposable wait boat and wait for the liquid nitrogen to evaporate.
When all of the larva have been homogenized, mix the powdered homogenates with one milliliter of TRIzol in a two milliliter tube. Incubate the mixture at room temperature for one hour. At the end of the incubation, pellet the homogenate by centrifugation.
Transfer the supernatant into a new tube. Mix the supernatant with 200 microliters of 1-Bromo-3-chloropropane, or BCP. Vortex and incubate for five minutes at room temperature, followed by a 10 minute incubation on ice.
At the end of the second incubation, centrifuge the BCP added reaction and transfer the upper transparent layer into a new, two milliliter tube. Precipitate the RNA of the transferred upper layer with 500 microliters of isopropanol by mixing and inverting the tube for five minutes. Then collect the precipitate RNA by centrifugation.
Wash the precipitated RNA pellet with 500 microliters of 75%ethanol and dry the RNA for five to 10 minutes at room temperature. When the ethanol has evaporated, add 100 microliters of ribonuclease inhibitor in nuclease-free water to the dried RNA pellet, and carefully vortex the tube until the pellet is completely dissolved. Measure the RNA quality and quantity by standard protocols, ensuring that the 260 to 280 ratio is approximately two, and the 260 to 230 ratio is in the range of two to 2.2.
Then use five micrograms of the isolated RNA for DNase digestion and gene expression analysis, according to standard protocols. G.mellonella is able to clear the initial force fed bacterial load until no bacteria are detectable. With the 16s gene copy numbers of both B.vulgatus and E.coli substantially decreased within 24 hours.
Commensal administered a G.mellonella larvae induce RNA gene expression of various innate immunity marker genes. For example, the LPS recognition molecules, apolipophorin and hemolin are generally expressed more highly in the E.coli administered larvae compared to B.vulgatus administered larvae. Further, the production of reactive oxygen and nitrogen species are strongly upregulated upon E.coli force feeding compared to B.vulgatus, as well as antioxidative GST gene expression.
In addition, differential microbial peptide expression is induced more strongly after E.coli administration than in response to B.vulgatus force feeding. Remember to use only healthy, white, quickly moving larvae, to use patience during force feeding to avoid stressing the larvae and to include all of the necessary controls. The activation of G.mellonella immune markers can be determined directly by RUS or GST activity assays and the abundance of ANPs can be determined by bacterial growth inhibition assays.
This technique can be used as a screening tool for commensal and pathogenic bacteria and for exploring intestinal symbiomes and pathobiomes without sacrifice of many vertebrates. Always use the appropriate personal protective equipment when working with liquid nitrogen, BCP, or TRIzol substances.
