Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle

The gut bacteria can affect multiple physiological processes of insect hosts. Preparing and maintaining axenic larvae is a powerful tool to study gut bacteria functions. The main advantages of this protocol are that plant tissue culture is used to obtain germ-free leaves to rear axenic leaf eating insects derived form surface-sterilized eggs, and that this method is not restricted by artificial diets.

This method is convenient and increase visibility of maintaining axenic insects for future gut bacteria studies, especially for non-model insects without well-developed artificial diets. Maintain the P.versicolora population in a growth chamber at 27 degrees Celsius and 70%relative humidity, with a 16 hour light and eight hour dark photoperiod. Place the insects in perforated plastic boxes with tilted wet absorbing paper, and feed them fresh poplar branches.

Spray clean water on absorbing paper to maintain moisture, and change the branches every two days. Isolate adults for oviposition after pupation, and feed them tender leaves to obtain more eggs. Within 24 hours, collect newly laid eggs and place them on moist absorbing paper for 60 hours to prepare axenic larvae.

Prepare MS medium in one milligram per milliliter alpha-naphthalene acetic acid or NAA stock solutions. In a biosafety hood, add 50 microliters of the NAA stock solution to 500 milliliters of MS medium, and shake to mix well. Then pour approximately 50 milliliters per tissue culture container and wait for solidification.

Sterilize scalpels and forceps in an alcohol lamp flame in the biosafety hood. Cut three to four centimeter stem segments with apical buds or lateral buds from poplar seedlings at approximately one month of growth, and insert them into the culture medium, one or two stem segments per container. Incubate these stem segments in a growth chamber for approximately 30 days.

And use these germ-free leaves to feed the axenic larvae. Autoclave Petri dishes, paint brushes, filter paper, distilled water in a Petri dish containing LB agar medium. Place leaves with adherent eggs in a Petri dish.

Then carefully remove the eggs from the leaves using forceps and transfer them to another Petri dish. Wash these eggs with 75%ethanol for eight minutes. Then repeat the wash four times with sterile water.

Transfer the eggs onto LB agar medium to preserve moisture for hatching. Place the Petri dish in a growth chamber and wait for the eggs to hatch within 24 hours. In a biosafety hood, tile three pieces of wet filter paper in a Petri dish, and place germ-free poplar leaves on the paper.

Collect the larvae and place them on the leaves. Seal the Petri dish with Parafilm, and incubate it in a growth chamber. Change the leaves every two days.

For the conventionally reared groups, transfer the eggs from the leaves to a Petri dish containing moist filter paper, and feed these larva with germ-free poplar leaves. Randomly select three first, second and third instar larva from the germ-free and conventionally reared groups. Dissect the third instar larvae with sterile scissors and forceps under a stereo microscope, and collect their guts in microcentrifuge tubes.

Collect intact first and second instar larvae in tubes. Add 100 microliters of PBS and three steel balls to the 1.5 milliliter tubes, and grind the tissues into homogenates using a bead beating homogenizer. Add 100 microliters of the homogenate to LB agar medium and plate using a sterile glass spreading rod.

Incubate the plates at 37 degrees Celsius for 24 hours. Then observe the bacterial colonies. Extract the total DNA of the previously obtained tissues using a DNA extraction kit, and measure DNA concentration with a spectrophotometer.

Amplify the bacterial 16S rRNA gene using universal 16S rRNA primers with PCR, setting up the reaction as described in the text manuscript. Store the PCR products at four degrees Celsius until further analysis. Mix the PCR products with nucleic acid dye and analyze them using electrophoresis on a 1%agarose gel in 1X TAE buffer.

Use 10 microliters of a DNA marker as a reference. Observe the gel with a UV transilluminator, and look for the target fragment around 1, 500 base pairs. Although no bacterial colonies were observed in any germ-free group, they were observed in all conventionally reared groups, indicating that larvae from sterilized eggs that were fed tissue cultured poplar leaves contained no bacteria.

The 1, 500 base pair PCR bands appeared in all conventionally reared groups. In contrast, no band was observed in the germ-free groups or the negative control, implying that no gut bacteria existed in axenic larvae. For insect species with tiny eggs, the kinds of disinfectants and sterilization duration needed to be optimized.

In addition, endophytes in tissue-cultured plants should be notable. This protocol provides a new method to maintain germ-free insects, which is a handy tool to facilitate insect-gut bacteria interaction studies, especially for non-model insects without well-developed artificial diets.