1. Preparing the tick membrane chamber
<ol>
	<li>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).</li>
	<li>Tape down 100% rayon lens cleaning paper to the prepared surface. Ensure it is flat and slightly taut with tape across all four side...

<ol>
	<li>Rosenberg, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+signs:+trends+in+reported+vectorborne+disease+cases+-+United+States+and+territories,+2004-2016.">Vital signs: trends in reported vectorborne disease cases - United States and territories, 2004-2016.</a> Morbidity and Mortality Weekly Report. 67 (17), 496-501 (2018).</li><li>Busch, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+movement+of+invasive+cattle+fever+ticks+(Rhipicephalus+microplus)+in+southern+Texas+leads+to+shared+local+infestations+on+cattle+and+deer.">Widespread movement of invasive cattle fever ticks (Rhipicephalus microplus) in southern Texas leads to shared local infestations on cattle and deer.</a> Parasites &amp; Vectors. 7, 188 (2014).</li><li>S&#252;ss, J., Klaus, C., Gerstengarbe, F. W., Werner, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+makes+ticks+tick?+Climate+change,+ticks,+and+tick-borne+diseases.">What makes ticks tick? Climate change, ticks, and tick-borne diseases.</a> Journal of Travel Medicine. 15 (1), 39-45 (2008).</li><li>Gray, J. S., Dautel, H., Estrada-Pe&#241;a, A., Kahl, O., Lindgren, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+climate+change+on+ticks+and+tick-borne+diseases+in+Europe.">Effects of climate change on ticks and tick-borne diseases in Europe.</a> Interdisciplinary Perspectives on Infectious Diseases. 2009, 593232 (2009).</li><li>Sonenshine, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Ticks. , (1991).</li><li>Schwan, T. G., Piesman, J., Golde, W. T., Dolan, M. C., Rosa, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+an+outer+surface+protein+on+Borrelia+burgdorferi+during+tick+feeding.">Induction of an outer surface protein on Borrelia burgdorferi during tick feeding.</a> Proceedings of the National Academy of Sciences. 92 (7), 2909-2913 (1995).</li><li>Massung, R. F., Priestley, R. A., Miller, N. J., Mather, T. N., Levin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inability+of+a+variant+strain+of+Anaplasma+phagocytophilum+to+infect+mice.">Inability of a variant strain of Anaplasma phagocytophilum to infect mice.</a> The Journal of Infectious Diseases. 188 (11), 1757-1763 (2003).</li><li>Koci, J., Bernard, Q., Yang, X., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borrelia+burgdorferi+surface+protein+Lmp1+facilitates+pathogen+dissemination+through+ticks+as+studied+by+an+artificial+membrane+feeding+system.">Borrelia burgdorferi surface protein Lmp1 facilitates pathogen dissemination through ticks as studied by an artificial membrane feeding system.</a> Scientific Reports. 8 (1), 1910 (2018).</li><li>Levin, M. L., Schumacher, L. B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+for+maintenance+of+multi-host+ixodid+ticks+in+the+laboratory.">Manual for maintenance of multi-host ixodid ticks in the laboratory.</a> Experimental and Applied Acarology. 70 (3), 343-367 (2016).</li><li>Hart, T., Yang, X., Pal, U., Lin, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Lyme+borreliae+proteins+promoting+vertebrate+host+blood-specific+spirochete+survival+in+Ixodes+scapularis+nymphs+using+artificial+feeding+chambers.">Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.</a> Ticks and Tick-Borne Diseases. 9 (5), 1057-1063 (2018).</li><li>Hart, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+tropism+determination+by+convergent+evolution+of+immunological+evasion+in+the+Lyme+disease+system.">Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.</a> PLoS Pathogens. 17 (7), (2021).</li><li>Bernard, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+in+early+immune+evasion+strategies+of+a+bacterial+pathogen.">Plasticity in early immune evasion strategies of a bacterial pathogen.</a> Proceedings of the National Academy of Sciences. 115 (16), 3788-3797 (2018).</li><li>Pierce, A. E., Pierce, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+note+on+the+cultivation+of+Boophilus+microplus+(Canestrini,+1887)+(Ixodidae:+Acarina)+on+the+embyonated+hen+egg.">A note on the cultivation of Boophilus microplus (Canestrini, 1887) (Ixodidae: Acarina) on the embyonated hen egg.</a> Australian Veterinary Journal. 32 (6), 144-146 (1956).</li><li>Doube, B. M., Kemp, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature,+relative+humidity+and+host+factors+on+the+attachment+and+survival+of+Boophilus+microplus+(Canestrini)+larvae+to+skin+slices.">The influence of temperature, relative humidity and host factors on the attachment and survival of Boophilus microplus (Canestrini) larvae to skin slices.</a> International Journal for Parasitology. 9 (5), 449-454 (1979).</li><li>Kr&#246;ber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+feeding+assay+to+test+acaricides+for+control+of+hard+ticks.">An in vitro feeding assay to test acaricides for control of hard ticks.</a> Pest Management Science. 63 (1), 17-22 (2007).</li><li>Kuhnert, F., Diehl, P. A., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life-cycle+of+the+bont+tick+Amblyomma+hebraeum+in+vitro.">The life-cycle of the bont tick Amblyomma hebraeum in vitro.</a> International Journal for Parasitology. 25 (8), 887-896 (1995).</li><li>Kr&#246;ber, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends in Parasitology. 23 (9), 445-449 (2007).</li><li>Andrade, J. J., Xu, G., Rich, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+silicone+membrane+for+in+vitro+feeding+of+Ixodes+scapularis+(Ixodida:+Ixodidae).">A silicone membrane for in vitro feeding of Ixodes scapularis (Ixodida: Ixodidae).</a> Journal of Medical Entomology. 51 (4), 878-879 (2014).</li><li>Oliver, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+immature+Ixodes+scapularis+(Acari:+Ixodidae)+by+membrane+feeding.">Infection of immature Ixodes scapularis (Acari: Ixodidae) by membrane feeding.</a> Journal of Medical Entomology. 53 (2), 409-415 (2016).</li><li>Oliver, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+dynamics+and+antibiotic+elimination+of+symbiotic+Rickettsia+buchneri+in+the+tick+Ixodes+scapularis+(Acari:+Ixodidae).">Growth dynamics and antibiotic elimination of symbiotic Rickettsia buchneri in the tick Ixodes scapularis (Acari: Ixodidae).</a> Applied and Environmental Microbiology. 87 (3), (2021).</li><li>Graham, E. E., Poland, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+Fluon+conditioning+for+capturing+cerambycid+beetles+in+different+trap+designs+and+persistence+on+panel+traps+over+time.">Efficacy of Fluon conditioning for capturing cerambycid beetles in different trap designs and persistence on panel traps over time.</a> Journal of Economic Entomology. 105 (2), 395-401 (2012).</li><li>Munderloh, U. G., Liu, Y., Wang, M., Chen, C., Kurtti, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment,+maintenance+and+description+of+cell+lines+from+the+tick+Ixodes+scapularis.">Establishment, maintenance and description of cell lines from the tick Ixodes scapularis.</a> Journal of Parasitology. 80 (4), 533-543 (1994).</li><li>Lehane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+single+and+coinfections+of+human+pathogens+in+Ixodes+ticks+from+five+geographical+regions+in+the+United+States,+2013-2019.">Prevalence of single and coinfections of human pathogens in Ixodes ticks from five geographical regions in the United States, 2013-2019.</a> Ticks and Tick-Borne Diseases. 12 (2), (2021).</li><li>Gonz&#225;lez, J., Bickerton, M., Toledo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+artificial+membrane+feeding+for+ixodid+ticks.">Applications of artificial membrane feeding for ixodid ticks.</a> Acta Tropica. 215, (2021).</li><li>Kr&#243;l, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+transmission+paths+for+three+different+Bartonella+spp.+in+Ixodes+ricinus+ticks+using+artificial+feeding.">Evaluating transmission paths for three different Bartonella spp. in Ixodes ricinus ticks using artificial feeding.</a> Microorganisms. 9 (5), 901 (2021).</li><li>Anderson, J. F., Magnarelli, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+ticks.">Biology of ticks.</a> Infectious Disease Clinics of North America. 22 (2), 195-215 (2008).</li></ol>

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...

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 diseases2,3. While there are likely numerous causes for the upward trend in case numbers, the changing climate is an important factor3,4. 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 transmission5,6. 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&#39; role by allowing infection or transmission to only occur on the tick&#39;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&#39; 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 host8,12.
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 1960s13,14. 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 impregnation15,16. Silicone was ideal as its physical properties mimic skin&#39;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. scapularis15,18. 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 antibiotics19,20. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>00-10 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Ecoflex 00-10</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>00-50 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Ecoflex 00-50</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>30 Hardness Silicone</td>
			<td>Smooth-On</td>
			<td>Mold Star 30</td>
			<td>Trial size from Smooth-On Store</td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Corning Incorporated</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine triphosphate (ATP)</td>
			<td>Millipore Sigma</td>
			<td>A1852-1VL</td>
			<td>Used to make an aqueous solution of 3 mM ATP that has been filter sterlized via 0.2 micometer filter</td>
		</tr>
		<tr>
			<td>Bovine blood</td>
			<td>HemoStat</td>
			<td>DBB500</td>
			<td>Mechanically defibrinated; 500 mL is usually sufficient for one experiment</td>
		</tr>
		<tr>
			<td>Clingwrap</td>
			<td>Fisherbrand</td>
			<td>22-305654&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Paper</td>
			<td>Fisherbrand</td>
			<td>09-790-2C</td>
			<td>Autoclave and let cool before using. Can use Fine quality instead of medium too</td>
		</tr>
		<tr>
			<td>Fluon (aqueous polytetrafluoroethylene)</td>
			<td>Bioquip</td>
			<td>2871</td>
			<td>Available from other sources such as https://canada-ant-colony.com/products/fluon-ptfe-10ml</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Millipore Sigma</td>
			<td>139386-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>Fisherbrand</td>
			<td>11-995</td>
			<td>100% rayon</td>
		</tr>
		<tr>
			<td>Nystatin &#160;</td>
			<td>Gold Biotechnology</td>
			<td>N-750-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisherbrand</td>
			<td>S37440&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/fungizone</td>
			<td>Gibco</td>
			<td>15240-096</td>
			<td>Or equivalent generic with concentration as follows (10,000 units/mL of penicillin, 10,000 &#181;g/mL of streptomycin, and 25 &#181;g/mL of Amphotericin B)</td>
		</tr>
		<tr>
			<td>Phagostimulant</td>
			<td>Made in House</td>
			<td></td>
			<td>Collected from prior tick feeds</td>
		</tr>
		<tr>
			<td>Polycarbonate Pipe</td>
			<td>McMaster-Carr</td>
			<td>8585K204&#160;</td>
			<td>Cut to 45 mm length, 1.25 inch outer diameter, 1 inch inner diameter. Cutting requires a chop saw grinding wheel.</td>
		</tr>
		<tr>
			<td>Rubber O-rings</td>
			<td>McMaster-Carr</td>
			<td>9452K38&#160;</td>
			<td>5 mm thick, 1.25 inch inner diameter</td>
		</tr>
		<tr>
			<td>Soft touch forceps</td>
			<td>VWR</td>
			<td>470315-238&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td></td>
			<td></td>
			<td>cyanoacrylate glue</td>
		</tr>
		<tr>
			<td>Unryu paper&#160;</td>
			<td>Art supply stores</td>
			<td></td>
			<td>mulberry fiber 10 g/m2. Purchased at Wet Paint art supply store, St. Paul, MN, USA</td>
		</tr>
	</tbody>
</table>

tick artificial membrane feeding for ixodes scapularis

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.

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...

Watch this Scientific Journal Video about Tick Artificial Membrane Feeding for Ixodes scapularis at JoVE.com

Tick Artificial Membrane Feeding for Ixodes scapularis

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.

Tick Artificial Membrane Feeding for Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

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.

tick-artificial-membrane-feeding-for-ixodes-scapularis

Research

JoVE Journal

Biology

2.8K vistas.  University of Minnesota. Si bien la alimentación por membrana artificial se ha realizado antes, nuestro protocolo es más simple de configurar y adaptable a otras especies de garrapatas con algunas modificaciones. La principal ventaja de la alimentación artificial por membrana es que permite la exposición a patógenos y tratamientos que pueden no ser factibles a través de la alimentación animal. Este sistema de alimentación por membrana artificial se puede aplicar a otras especies de artrópodos con algunas pru...