Tick Blood Feeding through an Artificial Membrane on a 3D-Printed Chamber in Ixodid Tick Research

My research focuses on reducing the incidence of tick-borne diseases by understanding the microbial molecular factors that drive vector competence, as well as developing novel strategies for tick control. The existing artificial tick feeding systems typically require large volumes and complex setups. This protocol introduces a modular reusable system enabling small-scale customizable experiments.

To begin, using gloves, wash the parts of the feeding chambers to ensure there are no resin residues before assembly. If needed, gently sand the parts with fine 180 to 200 grit sandpaper and ensure that the bottom part is flat and smooth. Expose all dry pieces to ultraviolet light for 10 minutes to ensure complete resin polymerization.

Then glue the three millimeter by two millimeter round magnets into the slits on the top of the chambers and lid rings with clear epoxy glue and allow it to dry overnight. Ensure that the poles of the magnets face one direction to match the attraction of the poles on the magnets in the upper part of the lid ring. Assemble all pieces of the model to ensure that all components fit properly.

Place a 30 centimeter by 30 centimeter piece of plastic wrap on a clean surface. Stretch the plastic wrap on a flat surface to remove wrinkles and secure the edges with tape. Place four to five individual sheets of lens cleaning paper over the extended plastic wrap, spacing each sheet approximately five centimeters apart.

To prepare the silicone membrane, add 15 grams of silicone clear caulk, 4.5 grams of silicone oil, and 2.9 grams of hexane into a plastic weighing boat. Using a spatula, gently mix the ingredients until combined. Then place two to three grams of silicone mixture along the smaller edge of the lens cleaning paper to secure it to the plastic wrap.

Allow the silicone to seep through the lens paper for five to 10 seconds. Next, using a straight-edge instrument such as a lid, ruler, or spatula, evenly spread the silicone mixture across the lens cleaning paper. Ensure the tool covers the entire area of the lens cleaning paper in a single stroke to prevent membrane ruffling and edge ripples.

Return the spreading tool to the starting position to collect any remaining silicone mixture on the tool. Spread the collected silicone across the membrane to ensure all the mixture is used and to avoid gaps. Allow the membranes to air dry overnight at room temperature.

On the following day, measure the membrane thickness using a digital outside micrometer measuring tool. Select appropriate membrane thicknesses for different adult tick species, ranging from short mouth parts to longer mouth parts. Then remove all parts from the previously assembled chambers.

To apply silicone glue to the bottom rim of the chambers, fill a one milliliter syringe with silicone glue. Place a 200 microliter pipette tip onto the tip of the syringe. Screw the pipette tip securely onto the syringe end.

As with a needle tip, use this setup to enable fine application of glue to the bottom rim of each chamber. Place the chamber over the membrane and position a weighted object on top to ensure proper settling. Allow the assembly to sit overnight at room temperature.

Using curved scissors, cut away the excess membrane from around the chambers. Remove any remaining plastic wrap from the membrane before using the chamber for tick feeding. Assemble the chamber parts and include a 35 millimeter by 10 millimeter Petri dish at the bottom.

Invert the Petri dish lids and place them between the magnets to serve as chamber lids. Fill the bottom portion of the Petri dish with distilled water and allow the chamber membrane to soak in it overnight. Use a heated metal scalpel or a thin metal spatula to make two to three small breathing holes in the lids of each chamber.

Supplement the blood with gentamycin sulfate to a final concentration of 50 micrograms per milliliter. Store the supplemented blood at four degrees Celsius and use it within one week. Place two to three milliliters of blood into a clean 35 millimeter by 10 millimeter Petri dish.

Position the chamber on top of the Petri dish and adjust the chamber height by rotating the adjustment ring to increase or decrease the volume of blood used per chamber. Adjust the chamber to maintain a one to two millimeter gap between the membrane and the bottom of the Petri dish. Place unfed ticks into the chambers with equal numbers of males and females.

Ensure both sexes are included to support feeding. Then place the chambers in covered dry baths with a humidity source such as water cups under a 12-hour light and 12-hour dark photoperiod. Prepare two squirt bottles for rinsing solutions, one with 0.005%nystatin solution in distilled water and another with only distilled water.

After 12 to 16 hours, prepare new Petri dishes with fresh blood. Place the dishes in the dry bath at 37 degrees Celsius before positioning the chamber. In the meantime, remove the chamber from the used blood.

Briefly rinse the membrane underside with distilled water for three to five seconds, followed by a three to five second rinse in 0.005%nystatin to prevent fungal contamination, and finish with a final distilled water rinse to remove any residual nystatin. After rinsing, place the chamber into a new Petri dish containing fresh blood. Discard all used blood and rinsing water into a container containing 0.5%sodium hypochlorite solution according to institutional safety protocol.

Place the chambers in 0.5%sodium hypochlorite solution for up to 30 minutes to reuse them. Wash the chambers with dish soap after bleach treatment. Optimal feeding rates were achieved at membrane thicknesses of 100 to 200 micrometers for Amblyomma americanum and 100 to 250 micrometers for Amblyomma maculatum.

Rhipicephalus sanguineus showed the highest feeding rates at 70 to 100 micrometer membrane thickness. The highest feeding rates for Ixodes scapularis and Dermacentor variabilis were observed at a membrane thickness of less than 150 micrometers. Partially fed female Amblyomma americanum were classified into unfed, two to three days partially fed, four to five days partially fed, and engorged stages.

The engorged feeding stage included ticks at the onset of rapid engorgement with weights ranging from 48.9 milligrams to 109 milligrams, and fully engorged ticks with weights ranging from 272.7 milligrams to 348.7 milligrams. This protocol enables researchers to study tick feeding behavior, physiology, and molecular responses under controlled in vitro conditions. A key challenge is variable feeding success, requiring precise optimization of environmental conditions, membrane properties, and careful handling to maintain integrity and prevent contamination.

Future studies will optimize feeding across species, enable full lifecycle maintenance, and integrate molecular tools to study pathogen transmission and develop targeted control strategies.