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1Microbiology and Pathogenesis Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 2Tick-Borne Diseases Activity, Bacterial Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention
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The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.
Patton, T. G., Dietrich, G., Brandt, K., Dolan, M. C., Piesman, J., Gilmore Jr., R. D. Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks. J. Vis. Exp. (60), e3894, doi:10.3791/3894 (2012).
Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.
1. Hemolymph collection for slide preparation
Note: When immobilizing the tick do not press too hard because this may disrupt the midgut or puncture the tick and contaminate the hemolymph.
Note: Do not cut the leg too close to the body because this may cause midgut contamination of the hemolymph.
2. Salivary gland removal
(Movies 2 & 3)
Note: Earlier in a feeding, the salivary glands are harder to locate because they are not as developed as compared to later on in the feeding.
Note: Wash the salivary gland clusters gently to reduce the loss and disruption of individual salivary glands.
3. Saliva collection
Note: Saliva acquisition can be stopped once enough saliva is collected for the study being performed.
4. Representative Results
Movie 1 demonstrates how to hold a partially fed I. scapularis nymph and amputate the legs to collect hemolymph onto a microscope slide. Once the leg or legs are amputated a clear fluid is secreted (figure 1A and 1B). If the midgut is ruptured the hemolymph appears cloudy as it is comes out of the amputated leg(s) (figure 1C and 1D).
The extraction of salivary glands after the nymph has been feeding for 48 or 72 hours is demonstrated in movies 2 and 3. After the tick is punctured there is generally a lot of debris (consisting of trachea, malpighian tubules, blood, connective tissue etc.), to prevent the loss or disruption of the salivary glands move the tick to a fresh pool of PBS. Figures 2A and 2B show where the salivary glands are located after the nymph has been cut open and figure 2C shows removed salivary gland clusters in a pool of PBS.
Saliva collection set up from I. scapularis adult females is shown in movie 4 and figure 3. A tick salivating into a capillary tube is observed in movie 5. This method of saliva collection used pilocarpine to stimulate salivation and can yield over 20 μl of saliva per adult female tick.
Figure 1. Labeled structures of an I. scapularis nymph.
Movie 1. Ixodes scapularis hemolymph collection. Click here to watch movie.
Figure 2. Uncontaminated (A & B) and contaminated (C & D) hemolymph exuding from the nymph's leg.
Movie 2. Salivary gland extraction from a 48 hour fed I. scapularis nymph.Click here to watch movie.
Movie 3. Salivary gland extraction from a 72 hour fed I. scapularis nymph. Click here to watch movie.
Figure 3. Ixodes scapularis nymph salivary glands. (A & B) Examples of salivary glands in a 72 hour fed nymph, prior to extraction. (C) Removed salivary gland cluster.
Movie 4. Saliva collection set up from an adult I. scapularis female tick. Click here to watch movie.
Figure 4. Saliva collection from adult I. scapularis female ticks. (A & B) Tick mounted on a slide with its hypostome in the pulled end of the capillary tube with the unpulled end of the capillary tube held by modeling clay. (C & D) Humidified chamber containing salivating adult I. scapularis female ticks.
Movie 5. Ixodes scapularis female tick salivating into a capillary tube. Click here to watch movie.
The collection of tick hemolymph, salivary glands, and saliva is important in the study of tick-borne pathogen transmission, prevalence, dissemination, proliferation, and persistence in both the tick and the host 6,11-13,20,23,29. There are several ways to dissect a tick30,31. However, when collecting salivary glands it is critical to dissect the tick properly so the salivary glands are not ruptured or lost in the tick's remains. Once the salivary glands are removed from the tick they need to be washed several times to remove midgut contamination and then can be fixed onto a slide for staining or ground in PBS to obtain salivary gland extract (SGE). SGE is easier to collect than saliva and possesses attributes similar to saliva; therefore, it can be used as a saliva alternative. However, SGE contains additional proteins originating from the salivary gland cells that are not present in tick saliva. The addition of extra proteins in SGE can be an advantage or a disadvantage depending on the study being performed, but is something a researcher needs to be aware of when working with SGE. SGE does have the advantage that pilocarpine is not used during the collection of salivary glands. Pilocarpine, a muscarinic cholinomimetic agent that acts as an agonist of salvation, has been shown to have a cytotoxic effect on B. burgdorferi during saliva collection 32,33. Dopamine is another agonist of salivation but is rapidly destroyed by the hemolymph and other tick fluids, as compared to pilocarpine that has a sustained effect33. Other stimulatory techniques have been explored for saliva collection and all were shown to affect the salivary composition 34.
Hemolymph collection can be difficult because the ticks are moving and it can be problematic to immobilize them without rupturing the midgut. Immobilizing the tick with double sided tape is an option, but the hemolymph can be lost onto the tape if the tick is not removed. Other studies have collected hemolymph from several ticks by cutting the tick's legs at the distal joints and using centrifugation to collect the hemolymph 16,35. Once hemolymph collection is mastered the hemolymph can be used to determine tick infectivity, tick to pathogen interactions, and the migration of pathogens from the midgut to the salivary glands.
Although our laboratory's focus is on B. burgdorferi and I. scapularis ticks, the techniques mentioned in this protocol can be used to study other tick-borne infectious agents and tick species. Furthermore, these techniques can also be used on nymphs or adults with relative ease. Using these techniques with larva could be challenging due to their size. Until the dissection technique is mastered it is suggested to use uninfected ticks. It is also important to exercise the proper biosafety measures when dissecting infected or field collected ticks. The methods in this video protocol can be used as guidelines on how to carry out tick dissections and what to look for when performing these techniques.
We have nothing to disclose.
The authors would like to thank the Division of Vector-Borne Diseases Animal Resources Branch, specifically Andrea Peterson, Lisa Massoudi, Verna O'Brien, and John Liddell for their care and maintenance of the mice and rabbits. We would also like to thank Amy Ullmann, Theresa Russell, and Barbara J. Johnson for their contributions toward this manuscript. Finally, we would like to acknowledge Alissa Eckert in the Office of the Associate Director for Communication at the CDC for producing the graphic illustrations and Judy Lavelle for directing all the legalities associated with the filming of this manuscript.
|Hydrogen peroxide||Fisher Scientific||H312-500|
|Dumont Fine forceps (3C)||Fisher Scientific||NC9906085|
|Silane treated microscope slides||Bioworld||42763007-1|
|Super frost plus microscope slides||Fisher Scientific||12-550-18|
|Protease inhibitor cocktail||Sigma-Aldrich||P2714|
|#11 disposable scalpel||Feather Safety Razor Co, Ltd.||2975#11|
|Nontoxic modeling clay||Fisher Scientific||S17307|
|Capillary tubes||Chase Scientific Glass, Inc.||40A502|
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