Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

The gnotobiotic cockroach generation protocol facilitates testing of the impact of native gut microbial community removal and replacement on host health and microbiome assembly. This technique uses methods from multiple sources to form a cohesive, streamlined workflow for generating gnotobiotic cockroaches without the use of expensive specialty lab equipment. Considering the many different moving parts of this gnotobiotic protocol, visualization helps researchers more easily establish their own small-scale, low-cost, bench-top gnotobiotic facilities.

To facilitate collection of the appropriate number of females carrying oothecae for the planned experiments, use forceps to move the cardboard tubes containing gravid females in the stock tank. If a cardboard tube contains multiple insects in addition to the gravid female, shake the tube contents into an additional plastic container ringed with petroleum jelly and encourage the target insect to climb back into the cardboard tube alone. Upon collection, transfer each gravid female into the maternity ward.

Once the females have dropped their oothecae, return the females to the stock tank and use forceps to retrieve the oothecae from the litter. To clean the oothecae, place up to five egg cases in a five-milliliter centrifuge tube containing three milliliters of sodium dodecyl sulfate and vortex the oothecae for 10 seconds. After a second wash as just demonstrated, use a delicate task wipe to gently scrub the surface of each ootheca to remove any debris and place the cleaned ootheca into a weigh boat.

Before sterilization, fill two 1.5-milliliter centrifuge tubes per ootheca with one milliliter of sterile water per tube. Also add 10 microliters of 32%peracetic acid stock solution to 3.2 milliliters of double-distilled water in a five-milliliter centrifuge tube in a fume hood. Cap and invert several times to mix and place up to five cleaned oothecae in the freshly prepared 0.1%peracetic acid solution for five minutes, inverting the tube several times every 60 seconds.

Peracetic acid is a strong oxidizer an eye, skin, and respiratory irritant, and is flammable and corrosive. Always wear the proper PPE during handling and dispose of your waste appropriately. At end of the incubation, use sterile forceps and a laminar flow hood to transfer each ootheca to its own centrifuge tube of sterile rinse water.

Invert several times to mix before transferring the oothecae to the second set of tubes of rinse water. After the second rinse, use sterile forceps to transfer the oothecae to individual BHI slants. Place the slants into a sterilized secondary container and move the container into a humidified 30-degree-Celsius incubator for four to five weeks until hatched.

Check the slants regularly one to two times per week for fungal or bacterial growth until the fourth week, at which point the slants should be checked daily. When the nymphs begin to hatch, use sterile forceps and a laminar flow hood to aseptically transfer a pellet of sterilized rat chow to a prepared BHI flask. As a sterility check, place the flask in the secondary container in the 30-degree-Celsius incubator for 24 hours to confirm a lack of contaminating growth.

If the flask has remained sterile, shake the nymphs out of the slant, letting them fall into the flask in the laminar flow hood. Then water the nymphs with 300 microliters of sterile water once per week in the laminar flow hood. When nymph feces begin to cover the medium floor, transfer the nymphs to a new BHI flask containing sterilized rat chow added 24 hours in advance, as demonstrated.

Pregnant females can be identified by the ootheca attached to their posterior abdomens. The oothecae hatch an average of 34 days after sterilization. In cases of unsuccessful sterilization, growth can appear around the oothecae during the incubation, compromising the gnotobiotic status of the hatchlings.

Then nymphal growth rate can be tracked by measuring the insect body length. Restriction fragment length polymorphism of the 16S ribosomal DNA from a homogenized nymph can be used to confirm gnotobiotic status. Gnotobiotic cockroaches can be inoculated with synthetic or xenobiotic microbial communities.

While this particular workflow is new, previous studies using gnotobiotic cockroaches have generated insight into gut microbiome assembly and microbial roles in shaping social behavior, gut health, and immunity of their insect hosts.