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Summary

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

Abstract

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Introduction

Tick-borne diseases strongly impact the health of humans and animals across the world, being responsible for more than two-thirds of all vector-associated illness in the USA from 2004 to 2016¹. Additionally, case numbers have been growing in recent years, with more people and livestock being affected by ticks and their associated diseases^2,3. While there are likely numerous causes for the upward trend in case numbers, the changing climate is an important factor^3,4. The predicted ongoing increase in the number of tick-borne disease cases underlines the need to develop new tools to investigate the relationships between ticks and the pathogens they transmit.

It is known that ticks undergo changes in physiology and gene expression during feeding and that these changes play a role in pathogen transmission^5,6. It may be difficult to perform studies that examine the effects of full and partial feeding on pathogen transmission and acquisition using animal models, particularly in situations where rodent models are not susceptible to infection by a particular pathogen. For example, Anaplasma phagocytophilum Variant-1 strain is naturally transmitted between Ixodes scapularis and deer but is unable to infect mice, complicating tick infection in the lab⁷. Artificial feeding systems can also be applied to help study pathogens such as Borrelia burgdorferi via the use of transgenic mutants that have gene deletions that inhibit transmission or infection⁸. Using an artificial feeding system helps researchers isolate the genes' role by allowing infection or transmission to only occur on the tick's side, thereby isolating any host response that may confound such studies.

Similarly, some life stages of ticks involved in disease and animal transmission may not be induced to feed on common laboratory model species. Ixodes scapularis females, for example, must be fed on larger animals, typically rabbits⁹. While often accessible for laboratory experimentation, the administrative and husbandry requirements of using rabbits exceeds those of small rodents and may be prohibitive for some laboratories. Other tick species, particularly those of veterinary concern, must be fed on cattle or other large animals that are not practical to use in most laboratories. In vitro feeding and infection methods, such as artificial membrane feeding, provide alternatives to using large or exotic host animals.

Additionally, the use of an artificial feeding system allows certain analyses that may not be possible with traditional animal feeding methods. One such example is that, by separating the blood source from the feeding mechanism, examination of the role that different hosts' blood may have in B. burgdorferi transmission becomes possible¹⁰. This examination of host blood and the role blood itself plays in the absence of the host immune response is an important factor in being able to understand pathogen transmission cycles and one that artificial feeding systems are able to help answer¹¹. It also becomes possible to quantify the exact transmission numbers of a pathogen during a feed rather than just examining transmission success and establishment in a host^8,12.

Some of the first artificial feeding membranes made for hard ticks were made out of animal skins or animal-derived membranes in the 1950s and 1960s^13,14. Due to the biological nature of these membranes, there were problems with both the production of new membranes and shelf life. In the 1990s, fully artificial membranes were developed that utilized a backing of netting, paper, or fabric with silicone impregnation^15,16. Silicone was ideal as its physical properties mimic skin's stretchiness and slight tackiness, along with its bio-inherent nature. Building on this, Krober and Guerin, whose work this technique was based on, described a silicone-impregnated rayon membrane feeding technique for the artificial feeding of I. ricinus¹⁷.

Refinement of the methods for I. scapularis, a closely related species, has led to notable differences in the hardness of silicone used in membrane impregnation, the recipe for membrane production, dimensions of the chamber, and the attachment stimulant. While the refinements reported in this study have resulted in similar membrane characteristics as those reported by Andrade et al., who also developed a silicone-based membrane based on Krober and Guerin for use in I. scapularis, there is a difference in the silicone impregnation steps, which allows the flexibility to utilize this protocol for immature life stages of I. scapularis^15,18. This study also describes additions and technical alterations based on repeated use of this method, best practices that result in a successful feed, and troubleshooting of problems that may arise. This method has been used to feed all active life stages, infect ticks with pathogenic bacteria, and expose ticks to multiple dosages of antibiotics^19,20. While the artificial membrane feeding method shown is for I. scapularis, this method is readily adaptable to other species of ticks with minor modifications in membrane thickness.

Protocol

1. Preparing the tick membrane chamber

1. Prepare a flat, non-porous surface such as a plane of glass or ceramic-coated metal base of an arm stand by wiping it down with 70% ethanol and then cover it with a single layer of plastic wrap, making sure that the plastic wrap is flat and without bubbles or wrinkles (see Figure 1A).
2. 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. Two pieces of 4 in x 6 in lens paper can be made into membranes using the volumes described in step 1.3.
   NOTE: This is sufficient to make up to 12 feeding chambers (see Figure 1B). If the membrane is for larval ticks, use unryu paper instead of lens cleaning paper.
3. Prepare the 00-10 hardness silicone mixture by measuring out 5 mL of each part of the silicone kit into a disposable container, lightly mixing the two liquids before adding 1.5 mL of hexane. Continue mixing until the mixture is homogeneous and thoroughly mixed.
   NOTE: If making membranes for adult ticks, use 00-50 hardness silicone.
4. Use a small squeegee to distribute the silicone mix over the lens paper. Allow to stand for 1 min to ensure that the silicone has soaked into the lens paper. Steadily scrape excess silicone to the side with the squeegee using a small amount of downward pressure. Perform a second pass with the squeegee, exerting no downward pressure, to remove any lines of silicone and produce a smooth layer.
   NOTE: This step of the procedure may require some practice to produce membranes of optimal thickness for each life stage (adults: 150-200 µm; nymphs: 90-120 µm; larvae: 80-100 µm). A thicker membrane (within the feeding tolerances of the life stage) will usually produce better results by reducing condensation in the chamber and improving the self-healing characteristic of the membrane.
5. For larvae only, dip the top of the feeding chamber into Fluon fluoropolymer resin (PTFE-30) several times, allowing it to dry between applications to produce a consistent layer.
   NOTE: The application of Fluon will form a non-stick layer of fluoropolymer resin similar to a non-stick cooking pan. This will reduce the number of larvae that climb to the top of the chamber²¹.
6. Leave the membrane to cure for at least 24 h in a dust-free environment, such as a closed chemical or biosafety cabinet, before moving on to the next step. See Figure 1C for how the rayon lens paper looks after the silicone has been allowed to dry.
7. Prepare the 30-hardness silicone mixture to attach the chambers to the membrane by mixing equal parts of silicone mix A and B.
8. Dip the polycarbonate chambers about a quarter of an inch (6 mm) into the silicone mixture, and then place on the membrane sheet, taking care not to overlap any of the tape (Figure 1D).
9. Allow the attaching silicone to cure for at least 24 h before moving to the next steps.
10. 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 6-well plate without much scraping on the sides (Figure 1E). Allow sufficient material outside the chamber to maximize adhesion of the membrane.
    NOTE: If too much exterior material remains, repeated removal of the chamber from the 6-well plate can cause the membrane to partially detach from the chamber. Repairing detached membranes is described in section 5.
11. Cut a small piece of the leftover membrane from each of the corners and the center of the membrane sheet. Remove the plastic wrap from each piece. Measure the pieces with a micrometer to determine the average membrane thickness.
    NOTE: If the membrane thickness does not fall within the tolerance range for life stage (see step 1.4), the membranes should be discarded and remade. Unryu paper thickness is highly variable due to its thick strands of fiber and thin regions. While this characteristic allows larvae to find regions of optimal thickness, it makes it difficult to determine actual membrane thickness.
12. Place the feeding chambers along with one O-ring per chamber into a glass beaker to be autoclaved at 121 °C for at least 20 min to sterilize. Allow the chambers to cool before using them.

2. Setting up the tick feed

1. Prepare a lamp or room so that the lights are on for 16 h and then off for 8 h. If using a lamp, make sure that the water bath in the next step is in the dark for those 8 h.
2. Set up a water bath at 34 °C and with supports so that a 6-well plate can sit and float slightly in it. Use small glass beakers sunken in the water bath as supports. Use a transparent cover for the water bath to maintain a light-dark cycle for the ticks; if need be, use a T-stand and plastic wrap to make a cover. To reduce the risk of contamination, add 0.02% benzalkonium chloride to the water bath and set another water bath to 36 °C for warming chilled blood.
3. Take out the preprepared, autoclaved membrane chambers, placing the O-ring around the chamber (Figure 1F), and then fill each chamber with enough 70% ethanol to cover the membrane. Let sit in a 6-well plate well for 5 min and then look for any leaks between the chamber and membrane and in the membrane itself.
   NOTE: If the membrane is leaking, ethanol will accumulate in the base of the well. Leaky membranes should be discarded.
4. Empty out the ethanol from the chambers and then let it air dry inside a biosafety cabinet or laminar flow hood.
   NOTE: This may take several minutes depending on ambient humidity.
5. While waiting, heat-inactivate the complement in the blood by heating at 56 °C for 40 min. Supplement mechanically defibrinated bovine blood with 2 g/L glucose.
6. After the membrane is dry, apply ~20 µL of phagostimulant to the inside of the chamber and then spread it around the surface by tilting the chamber. See section 6 for instructions regarding preparing a phagostimulant from tick frass.
7. Wait until the phagostimulant is dry before adding the ticks to the chamber with either a brush or forceps. Work as quickly as possible when transferring the ticks to the chamber to prevent escape. Put the ticks in a tube on ice for 1-2 min before transferring them to slow down their ability to escape. After the ticks have been transferred, seal the top with parafilm; do not overstretch the parafilm, as the heat from the water bath can cause it to rip.
   NOTE: Forceps work best with nymphal and adult life stages and brushes work best with larval life stages.
8. Add 5 mL of the heat-inactivated blood per well/chamber into a conical tube and place in the water bath set at 36 °C. Bring chilled blood up to at least room temperature before exposing the ticks to it. Set aside four chambers per 6-well plate for easy handling of chambers. More chambers per plate should be avoided, however, depending on the size of the water bath, more plates can be used.
9. Add 5 µL of 3 mM ATP and 50 µL of 100x stock of penicillin/streptomycin/fungizone per 5 mL of blood to the tube.
10. Transfer 4.5 mL of the blood into a well of a 6-well plate, and then 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. Place the well into the blood at an angle to avoid air bubble formation between the blood and the membrane.
    NOTE: A completed plate is shown in Figure 2.
11. Place the lid of the 6-well plate on top of the feeding chambers; then, put the 6-well plate with chambers into the prepared 34 °C water bath and close the lid.
    ​NOTE: The lid on top will help stop condensed water from contacting the parafilm and diluting the blood in the wells.

3. Maintaining the feeding ticks by changing the blood every 12 h

1. Aliquot 5 mL of the heat-inactivated blood per well into a tube and warm it up in a 36 °C water bath. Thaw the ATP and penicillin/streptomycin/fungizone in the water bath at the same time.
2. Add 5 µL of 3 mM ATP and 50 µL of 100x stock of penicillin/streptomycin/fungizone per well to the blood tube. Transfer 4.5 mL of blood into a well of a fresh 6-well plate.
3. Take out the 6-well plate with the tick chamber from the water bath. Remove the chamber from the well and rinse the outside of the chamber and membrane with 10 mL of sterile 1x PBS to remove the blood.
4. Using autoclaved filter paper, gently dab dry the membrane and chamber to remove the excess PBS. Replace the parafilm on the top of the chamber. If there is a lot of condensation inside the chamber, dab dry the wet spots with autoclaved filter paper before sealing with fresh parafilm.
5. Place the tick chamber into the new 6-well plate with fresh blood, adjusting the O-ring height if needed. Repeat steps 3.3-3.5 for each chamber on the plate. Place the lid of the 6-well plate over the tops of the chambers. Move the new 6-well plate back into the 34 °C water bath.
6. Repeat steps 3.1-3.5 every 12 h until the conclusion of the tick feed. Wait for the ticks to detach from the membrane by themselves when engorged or gently remove them from the membrane with soft-touch tweezers while still attached to obtain partially engorged ticks.

4. Antifungal treatment

NOTE: Perform only when fungal growth is seen on the membrane. Fungus is likely to form on the blood side of the membrane if the feeding is of sufficient duration. The first indication of fungal contamination is small (1-3 mm) flakes of coagulated blood visible on the membrane. When fungal contamination is noted, antifungal treatments can prolong the duration of the experiment and improve engorgement success.

1. Dissolve 10,000 U nystatin per mL in distilled water, 5 mL per feeding chamber. Filter-sterilize with a 0.1 µm syringe filter.
2. Add 5 mL of nystatin solution per well of a new 6-well plate, one well for each feeding chamber.
3. Place PBS-rinsed chambers into the solution. Let sit for 10 min.
4. Rinse the treated membranes with PBS before putting back into fresh blood.
5. Repeat the antifungal treatment every 2-3 days (depending on the extent of fungal contamination) until the completion of the experiment.

5. Re-adhering detached membranes with cyanoacrylate glue

NOTE: Perform only when membrane detachment is noticed.

1. Membranes may partially detach from the feeding chambers, especially during removal from the 6-well plate. If this happens, rinse the membrane of blood with PBS.
2. Gently dry the affected area. It does not need to be perfectly dry, but the presence of moisture causes the glue to set faster.
   NOTE: Re-adhering the membrane limits the working time, however, depending on detachment size, a faster setting time may be desirable.
3. Squeeze a small amount of cyanoacrylate glue into the gap where the membrane has pulled away from the chamber. Hold in place for ~2 min to allow the glue to set.
4. Place the chamber back into the blood in the 6-well plate.

6. Making phagostimulant

NOTE: Perform at the end of a feed or before a membrane feed has started.

1. Collect tick feces from either a prior membrane feed or another source of feeding ticks. Store the feces at -20 °C prior to making the phagostimulant. Crush the pellets of tick feces and mix 0.1 g per 1 mL of water. Add 1 mM tripeptide reduced glutathione, dissolve, and mix thoroughly by vortexing.
2. Filter-sterilize with a 0.1 µm syringe filter.
3. Freeze at -20 °C until needed.

Representative Results

A successful feeding depends on whether a partial or full engorgement is desired. Successfully fed I. scapularis turn a shade of gunmetal grey for adults and detach on their own from the membrane. However, if they are at least pea-sized, they may be detached from the membrane when finishing up the feeding. For immature stages of I. scapularis, the size for fully engorged ticks varies, and because, unlike adults, they do not exhibit a color change, detachment is the best way to determine if a tick is fully engorged. See Figure 3 for an example of how nymphal I. scapularis may look during a feed. Adult I. scapularis ticks take 1 week or more from attachment until they start detaching from the membrane; nymphs often detach approximately 5 days after attachment, and larvae start detaching ~3-4 days after the first attachment. Feeds may continue for as long as one wants to maintain the twice-daily blood changes and as long as mold has not started to grow beyond what antifungal treatment can control. Partially engorged ticks have to be manually detached from the membrane.

Immature ticks (larvae and nymphs) that detach from the membrane on their own almost always successfully molt into their next life stage, and assuming that they have engorged sufficiently and are of a large enough size, even those that are detached manually from the membrane at the end of a feed molt. Female ticks that have turned a gunmetal grey during engorgement usually successfully lay eggs. However, unlike natural means of feeding, membrane-fed engorged females produce smaller egg masses and are of smaller size than their naturally animal-fed counterparts. Results from a recent experiment that fed I. scapularis larvae from eggs through nymphal engorgement and molting can be seen in Table 1. The resulting nymphs from the initial larval feed were fed 2 months post-molt. It is important to note, however, that this experiment had deer organ homogenate added to the blood, which may have had detrimental effects on the tick feeding success.

In another experiment, 60 female and 60 male I. scapularis ticks were placed in four feeding chambers (30 sex-matched ticks per chamber) and the female ticks were allowed to feed to repletion. These ticks originated as the offspring of engorged females collected from hunter-killed deer. The mean egg mass weight and range, in addition to engorgement success numbers (number of females who laid eggs post-engorgement and -detachment), can be seen in Table 1. Egg mass weights for I. scapularis are highly variable in the literature, but ticks previously collected from hunter-killed deer from Minnesota produced egg masses with a mean weight of 77 mg (19-147 mg)²². Tick mortality is low using the artificial membrane feeding system; ticks that do not attach and feed mostly survive the process, raising the possibility of attempting further membrane feeding experiments with the survivors.

This method has been used to infect nymphal I. scapularis with a variety of tick-borne pathogens, including A. phagocytophilum Variant-1, which cannot use traditional rodent infection methods⁷. Infection of the ticks by bacteria was confirmed with qPCR, and while all nymphal ticks that successfully fed were positive post-feeding, depending on the bacterial species, infection would or would not persist transstadially¹⁹. This method also has been utilized to feed female I. scapularis ciprofloxacin; the successfully fed ticks showed a comparable knockdown of bacterial symbiont levels to injected ticks²⁰.

Figure 1: The tick membrane chamber. (A) Ceramic-coated stand base covered with plastic wrap. (B) Lens paper taped on to plastic-wrapped base. The blue rectangle is the squeegee used in panel C. (C) Lens paper after being impregnated with silicone. (D) Chambers attached to the silicone-rayon membrane. Each 4 in x 6 in lens paper sheet can accommodate six chambers. (E) A completed chamber with the silicone-rayon membrane. A detached silicone-rayon membrane is shown on the right. (F) An assembled completed chamber, with an O-ring secured on it. Please click here to view a larger version of this figure.

Figure 2: Setting up the tick feed. An example of how a set of four chambers with adult I. scapularis will look once everything is set up and before it is placed in the water bath. Please click here to view a larger version of this figure.

Figure 3: An ongoing feed with partially engorged Ixodes scapularis nymphs. The black or brown dots around the attachment sites (see label A for example) are the frass that can be collected during and after the feed to make the phagostimulant. Partially engorged I. scapularis nymphs are seen attached on the membrane (see label B). A husk of the engorged larvae molt can be seen by label C. Please click here to view a larger version of this figure.

	Number of starting ticks	Number of successfully engorged ticks	Egg mass average mass in mg (range)
Larvae	2 hatched egg masses	150	N/A
Nymphs	150	24	N/A
Adult females	60	18	42.5 (5.3-77.8)

Table 1: Tick engorgement numbers and egg mass weights. Larval and nymphal engorgement experimental conditions had deer organ homogenate added to the blood in addition to the supplements used in this protocol. Adult female engorgement experimental conditions were as described in the protocol above. Successful engorgement was defined as either engorged ticks that successfully molted to the next life stage or engorged females that successfully laid eggs.

Discussion

Artificial membrane feeding of ticks provides a useful tool for a variety of experimental procedures, but is not likely to replace animal feeding for all applications. Maintaining large colonies of ticks at all life stages without animal feeding is generally untenable. Instead, the artificial feeding system is valuable for other purposes such as infecting ticks with pathogens not supported by model hosts, evaluating the impacts of controlled dosages of compounds or microorganisms on the ticks in a simplified feeding environment, or rearing small colonies of ticks that rely on host animals that are not readily available in the laboratory^{8,10,11,23,24,25}. Compared to traditional animal feeding methods, artificial membrane feeding is labor-intensive and presents challenges^15,24. It is for these reasons that artificial membrane feeding is not a replacement for traditional animal-based feeding methods and instead allows for experiments that cannot be done with traditional animal feeding.

Maintaining hygienic conditions both in the feeding chambers and the tick colony is vital to avoid fungal and bacterial contamination. However, when using artificial feeding to expose ticks to pathogens, penicillin/streptomycin/fungizone (P/S/F) supplements should be avoided from the blood to avoid killing the pathogens. Tick exposure to ciprofloxacin in the blood can completely eliminate Rickettsia buchneri, the ovarian endosymbiont of I. scapularis, from a subset of F1 progeny, while routine P/S/F addition to blood does not have this effect²⁰. Transovarial pathogen transmission studies were not performed using this system, but no changes were noted in the fecundity of engorged females in antibiotic-exposed versus control experiments. Rapid initiation of feeding and engorgement of ticks avoids many of the issues associated with fungal contamination; for this reason, the system tends to work more easily with larval and nymphal ticks, as they have shorter feeding times. Periodic treatment of contaminated membranes with antifungals such as nystatin can prolong the time available for engorgement by a few days. It is also important to use ticks that have been molted for long enough to be eager to feed. Larvae are ready 2 weeks after all larvae in the egg mass have completed emergence, but nymphs and adults fare better if used at least 10 weeks after molting.

This method has shown comparable tick feeding success to using laboratory animals, with typically 30%-50% of female and approximately 50% of nymphal I. scapularis completing engorgement, comparable to the engorgement success of adults fed on lab rabbits or nymphs fed on mice or hamsters¹⁹. Similarly, other studies using artificial feeding chambers have reported a 45% engorgement rate for I. scapularis females and 72% feeding rate for nymphs^10,18. Meanwhile, for Ixodes ricinus females and nymphs fed via an artificial membrane, engorgement rates were 71% and 58%, respectively²⁵. Therefore, results can be variable depending on the system in use and the tick species investigated.

Membrane thickness is an important factor in feeding success; thinner membranes are less able to seal around the tiny holes produced by tick probing and attachment. After feeding begins, more moisture gets into the chamber, which can lead to wet conditions, the liquification and subsequent redrying of frass, and mold. It is therefore important to produce membranes at the maximum thickness tolerable by the tick species and life stage's hypostome length that is to be fed (see protocol steps 1.1-1.4). Nymphs of I. scapularis and Dermacentor variabilis feed on membranes between 90 µm and 120 µm thickness, while adults feed on membranes up to thicknesses of nearly 200 µm. The adult I. scapularis hypostome is 500 µm in length, while nymphal and larval hypostomes are ~100 µm long, which proves sufficient to penetrate a slightly thicker membrane^15,26. Rayon lens paper on average is ~50 µm in thickness and membranes made from it have a minimum of 50 µm thickness; however, as stated above, thicker membranes produce better results. Larval feeding membranes work well at 80-100 µm in thickness using a very light unryu paper (10 g/m³). This irregular mulberry paper is laced with relatively thick fibers that provide support to a membrane that is very thin in places, allowing ample larvae attachment sites.

Based on the recommended membrane thickness and chamber sizes, it is best to limit the number of females in adult feeds to a maximum of 20 females (ticks are placed in chambers in protocol step 2.7). More females risk overcrowding the attachment sites, as the ticks tend to attach very closely together, which can compromise membrane integrity in the local area and increase the risk of a leak, as well as slow the feeding rate. For nymphal ticks, the maximum numbers are much higher and no significant detrimental effects are noted, even when feeding 50 nymphs per chamber. Since counting individual larvae is not reasonable and no overcrowding issues were noted, a one-half to a whole egg mass per chamber is recommended for ease of setup. Whole egg masses comprise of ~1,000 or more larvae, and over the course of a feed anywhere from dozens to ~200 engorged larvae may drop off from a single egg mass. Additionally, larval ticks that do not feed tend not to die and can potentially be rehoused for a later feeding.

Other plastics have been used to produce the feeding chambers, of which polycarbonate has been determined to be the most durable and non-reactive. Other plastics, such as acrylics, are sensitive to cleaning solutions, which can cause crazing and breakage, especially at the cut ends of the tubing. Polycarbonate holds up well to exposure to bleach, ethanol, autoclaving, and ultraviolet sterilization.

While ticks in a chamber may eventually bite and attach to the membrane due to the heat stimulus of the warmed blood underneath, additional stimuli in the form of chemical phagostimulants help speed up this process (for the recipe, see protocol step 6). For this study, the use of tick feces that have been dissolved in water is ideal due to their ability to be preserved at -20 °C for long periods. They are also easily renewable, as tick feces can be collected from subsequent feeds. Naturally, the first membrane feed may not have access to the suggested phagostimulant of tick feces extract and can be performed without it to help produce it for future feeds. Additionally, alternative phagostimulants may be used, with other studies having success with different chemical or mechanical stimuli such as hair or hair extracts^15,24.

Artificial membrane feeding provides additional tools for researchers working in tick and tick-borne pathogen biology. It is affordable and simple, but also labor-intensive, and will likely require some preliminary testing to optimize for their desired application and tick species/life stage.

Materials

00-10 Hardness Silicone	Smooth-On	Ecoflex 00-10	Trial size from Smooth-On Store
00-50 Hardness Silicone	Smooth-On	Ecoflex 00-50	Trial size from Smooth-On Store
30 Hardness Silicone	Smooth-On	Mold Star 30	Trial size from Smooth-On Store
6-well cell culture plates	Corning Incorporated	3516
Adenosine triphosphate (ATP)	Millipore Sigma	A1852-1VL	Used to make an aqueous solution of 3 mM ATP that has been filter sterlized via 0.2 micometer filter
Bovine blood	HemoStat	DBB500	Mechanically defibrinated; 500 mL is usually sufficient for one experiment
Clingwrap	Fisherbrand	22-305654
Filter Paper	Fisherbrand	09-790-2C	Autoclave and let cool before using. Can use Fine quality instead of medium too
Fluon (aqueous polytetrafluoroethylene)	Bioquip	2871	Available from other sources such as https://canada-ant-colony.com/products/fluon-ptfe-10ml
Glucose	Millipore Sigma	G8270-100G
Hexane	Millipore Sigma	139386-100ML
Lens paper	Fisherbrand	11-995	100% rayon
Nystatin	Gold Biotechnology	N-750-10
Parafilm	Fisherbrand	S37440
Penicillin/streptomycin/fungizone	Gibco	15240-096	Or equivalent generic with concentration as follows (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Amphotericin B)
Phagostimulant	Made in House		Collected from prior tick feeds
Polycarbonate Pipe	McMaster-Carr	8585K204	Cut to 45 mm length, 1.25 inch outer diameter, 1 inch inner diameter. Cutting requires a chop saw grinding wheel.
Rubber O-rings	McMaster-Carr	9452K38	5 mm thick, 1.25 inch inner diameter
Soft touch forceps	VWR	470315-238
Super glue			cyanoacrylate glue
Unryu paper	Art supply stores		mulberry fiber 10 g/m2. Purchased at Wet Paint art supply store, St. Paul, MN, USA