While artificial membrane feeding has been done before, our protocol is simpler to set up and adaptable to other tick species with some modifications. The main advantage of artificial membrane feeding is that it allows for exposure to pathogens and treatments that may not be feasible via animal feeding. This artificial membrane feeding system can be applied to other arthropod species with some testing for ideal phagostimulants and modifications on ideal membrane thickness.
Producing the correct membrane thickness requires some practice. Be sure to use a micrometer to measure the thickness at several locations on the membrane. Benjamin Cull, a postdoc in our laboratory will help me to demonstrate the procedure.
To begin, wipe down a flat, non-porous surface such as a pane of glass or ceramic coated metal base of an arm stand with 70%ethanol and then cover it with a single layer of plastic wrap. Make sure the plastic wrap is flat and without bubbles or wrinkles. Tape down 100%rayon lens cleaning paper to the prepared surface.
Ensure it is flat and slightly taut with tape across all four sides of the paper. Prepare the 0010 hardness silicone mixture by measuring out five milliliters of each part of the silicone kit into a disposable container. Lightly mix the two liquids and add 1.5 milliliters of hexane.
Continue mixing until the mixture is homogenous and thoroughly mixed. Use a small squeegee to distribute the silicone mix over the lens paper and allow it to stand for one minute to ensure that the silicone has soaked into the lens paper. Steadily, scrape the excess silicone to the side with the squeegee using a small amount of downward pressure.
Perform a second pass with the squeegee without exerting the downward pressure to remove any lines of silicone and produce a smooth layer. For larvae only, dip the top of the feeding chamber into Fluon fluoropolymer resin several times, allowing it to dry between applications to produce a consistent layer. Leave the membrane to cure for at least 24 hours in a dust-free environment such as a closed chemical or biosafety cabinet.
Next, mix equal parts of silicone mix A and B to prepare the 30 hardness silicone mixture to attach the chambers to the membrane. Dip the polycarbonate chambers about a quarter of an inch into the silicone mixture and then place them on the membrane sheet, taking care not to overlap any of the tapes. Allow the attaching silicone to cure for at least 24 hours.
Using a scalpel, carefully cut the membrane around each of the attached chambers, trimming down the edges so that it can fit smoothly into a well of the six well plate without much scraping on the sides. Allow sufficient material outside the chamber to maximize the addition of the membrane. Cut a small piece of the leftover membrane from each corner and the center of the membrane sheet.
Remove the plastic wrap from each piece and measure the pieces with a micrometer to determine the average membrane thickness. Place the feeding chambers along with one O-ring per chamber into a glass beaker to be autoclaved at 121 degrees Celsius for at least 20 minutes to sterilize. Allow the chambers to cool before using them.
After taking out the pre-prepared, autoclaved membrane chambers and placing the O-ring around them, fill each chamber with enough 70%ethanol to cover the membrane. Let it sit in a six well plate for five minutes and then look for any leaks between the chamber and membrane and in the membrane itself. Empty out the ethanol from the chambers and then let it air dry inside a biosafety cabinet or laminar flow hood.
To heat and activate the complement in the blood, heat it at 56 degrees Celsius for 40 minutes and supplement mechanically defibrillated bovine blood with two grams per liter of glucose. Once the membrane is dry, apply around 20 microliters of phagostimulant to the inside of the chamber and tilt the chamber to spread it around the surface. When the phagostimulant is dry, quickly add the ticks to the chamber with either a brush or forceps.
Seal the top with parafilm as the heat from the water bath can cause it to rip. Then add five milliliters of the heat and activated blood per well or chamber into a conical tube and place it in the water bath set at 36 degrees Celsius. Add five microliters of three millimolar ATP and 50 microliters of 100X stock of penicillin streptomycin fungizone per five milliliters of blood to the tube and transfer 4.5 milliliters of the blood into a well of a six well plate.
Gently place the chamber into the well, adjusting the O-ring height on the chamber so that the membrane sits in the blood, but the blood level around the side does not overtop the well. Then place the well into the blood at an angle to avoid air bubble formation between the blood and the membrane. Place the lid of the six well plate on top of the feeding chambers.
Put the six well plate with chambers into the prepared 34 degrees Celsius water bath and the lid. Add five microliters of three millimolar ATP and 50 microliters of 100X stock of penicillin streptomycin fungizone per well to the blood tube and transfer 4.5 milliliters of blood into a well of a fresh six well plate as shown earlier. Take out the six well plate with the tick chamber from the water bath and remove the chamber from the well.
Rinse the outside of the chamber and membrane with 10 milliliters of sterilized 1X PBS to remove the blood. Using autoclaved filter paper, gently dab dry the membrane and chamber to remove the excess PBS. If there is a lot of condensation inside the chamber, dab dry the wet spots with autoclaved filter paper before sealing it with fresh parafilm.
Then replace the parafilm on the top of the chamber. Place the tick chamber into the new six well plate with fresh blood, adjusting the O-ring height if needed. Repeat these steps for each chamber on the plate.
Finally, place the lid of the six well plate over the top of the chambers and move the new six well plate back into the 34 degrees Celsius water bath. An ongoing feed with partially engorged ixodes scapularis nymphs is shown here. The black or brown dots around the attachment sites are the frost that can be collected during and after the feed to make the phagostimulant.
Partially engorged ixodes scapularis nymphs are seen attached to the membrane. A husk of the engorged larvae mold can also be seen here. The tick engorgement numbers and egg mass weights are presented in this table.
Larval and nymphal engorgement experimental conditions had deer organ homogenate added to the blood in addition to the supplements used in this technique. Here, successful engorgement was defined as either engorged ticks that successfully molted to the next live stage or engorged females that successfully laid eggs. It takes practice to get acceptable thickness when applying silicone to the lens paper so it is always important to measure the thickness of the membrane before use.
Membrane feeding can expose ticks to pathogens or antibiotics. The effects of these on aspects of tick biology such as molting success, egg laying and survival can be assessed post feed.
