Tick Artificial Membrane Feeding for Ixodes scapularis

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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 ful...

Dyskusje

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 envi...

Materiały

Name	Company	Catalog Number	Comments
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