Name: Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae) (Video) | JoVE Journal
Uploaded: 2025-10-31T13:20:00.000+00:00
Duration: 3 min 22 s
Description: Learn to extract saliva, haemolymph, salivary glands, and midgut from fed ticks using pilocarpine stimulation and micro-dissection. This protocol enables tissue-specific pathogen localization studies in Hyalomma and Rhipicephalus species.

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5 minutes

The authors sincerely acknowledge the financial support provided by the following organizations and agencies: the European Research Council (ERC) under the European Union&#39;s Horizon 2020 Research and Innovation Program (grant agreement No. 101000365, PREPARE4VBD); the Swedish International Development Cooperation Agency (Sida); the Swiss Agency for Development and Cooperation (SDC); the Australian Centre for International Agricultural Research (ACIAR); the Government of Norway; the German Federal Ministry for Economic Cooperation and Development (BMZ); and the Government of the Republic of Kenya. Rua Khogali gratefully acknowledges support from the German Academic Exchange Service (DAAD) through an icipe ARPPIS-DAAD scholarship, as well as a postgraduate bursary from the University of Pretoria. The views expressed in this publication are those of the authors and do not necessarily reflect those of the funding agencies.

This study received ethical approval from the Pwani University Ethics Review Committee (Ref: ERC/EXT/002/2020E) and a research license from Kenya&#39;s National Commission for Science, Technology and Innovation (NACOSTI) (Ref: NACOSTI/P/22/16467). This study used rabbits to maintain and rear ticks under controlled laboratory conditions at the International Centre of Insect Physiology and Ecology (icipe). All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) in accordance with its Standard Operating Procedures (2017). Animal handling was conducted by trained personnel following international animal welfare guidelines and Kenyan regulations to ensure humane care and minimise discomfort.
1. Application and scope
<ol>
	<li>Collect saliva, haemolymph, salivary glands, and midgut from a partially and fully engorged female or male. 
	NOTE: This protocol was applied at icipe to partially feed males and females of Hyalomma dromedarii, Hyalomma rufipes, Amblyomma gemma, and Rhipicephalus pulchellus collected from camels in Laikipia and Marsabit counties, Kenya13.</li>
	<li>Use isolated tissue samples in separate nucleic acid extractions for molecular analyses, including tissue-specific metagenomic sequencing. 
	NOTE: The required reagents and equipment are summarized in the Table of Materials.</li>
</ol>
2. Saliva collection
<ol>
	<li>Sterilize the tick exterior carefully by wiping the whole tick with a paper towel soaked in 1% bleach (sodium hypochlorite; NaOCl), followed by 70% ethanol for 30 s, and allow the tick to dry. 
	CAUTION: Sodium hypochlorite is corrosive and releases toxic fumes. Handle with gloves and protective eyewear in a chemical hood.</li>
	<li>Gently clean the mouth parts with the forceps by holding a small piece of paper towel soaked in 70% ethanol, and allow it to dry. 
	NOTE: This step will help to minimize the likelihood of contamination by tick surface microbes.</li>
	<li>Place the tick on a clean glass slide under the microscope with the ventral surface of the tick facing upward, and hold it securely between the finger and thumb before injecting the tick.</li>
	<li>Inject 20 &#181;L of a mixture consisting of 2% Pilocarpine HCl and 1&#215; PBS (in a 1:1 ratio) directly behind coxa 4 using a 31 G syringe14,15. 
	NOTE: Take care not to inject too deeply as this will rupture the tick&#39;s body and affect the integrity of the sample. Alternatively, use 4-8 mm pen needle to control injection depth.</li>
	<li>Rotate the tick so that the dorsal surface is exposed and fix it in place on one end of the slide by using transparent tape. 
	NOTE: Take care not to apply too much pressure when mounting the tick on the slide, as this may result in rupture and loss of samples.</li>
	<li>Put a small piece of non-toxic modeling clay at the opposite end of the slide to provide a fixed surface to support insertion of a 10-&#181;L tip over the hypostome of the tick. Using non-toxic modeling clay is optional.</li>
	<li>Insert the 0.5-10-&#181;L tip between the palps to ensure it covers the hypostome.</li>
	<li>Wait for the tick to produce saliva or gently massage the hypostome by moving the tip in and out.</li>
	<li>Draw the saliva into the pipette tip or create slight pressure for 5-10 s to stimulate its release. In case this does not work, always ensure to have 1&#215; PBS prewarmed to 38 &#176;C at hand; pipetting a small amount (1 &#181;L) of the prewarmed solution prior to fitting the tip over the hypostome and between the pedipalps assists with the release of saliva. 
	NOTE: Take care not to break/damage the hypostome as this will result in diffusion of haemolymph into the tips instead of saliva.</li>
	<li>Release the saliva from the tip into a sterile and properly labelled microtube and store at -20&#176; C/-80 &#176;C until further analysis. 
	NOTE: It is possible to collect up to 10 &#181;L of saliva from the tick. The saliva collection process is summarized in Figure 1.</li>
</ol>
3. Haemolymph collection
<ol>
	<li>Continue with the same tick after saliva collection or use a new tick after surface sterilisation as mentioned above.</li>
	<li>Place the tick on a new clean glass slide and cut off one of the tick&#39;s legs using a scalpel blade size no.11.</li>
	<li>Gently press the body, taking care not to apply too much pressure as this may rupture the tick or the internal organs, and release the midgut contents with the haemolymph.</li>
	<li>The haemolymph will be released as a drop from the leg, collect it with a pipette by placing a 0.5-10 &#181;L tip on the leg. 
	NOTE: Create a slight pressure using the pipette to facilitate the release of the haemolymph. Alternatively, cut several legs to obtain enough haemolymph.</li>
	<li>Dispense the haemolymph from the tip into a sterile, labeled microtube and store until further analysis. 
	NOTE: It is possible to collect up to 10 &#181;L of haemolymph from the tick. The haemolymph collection method is summarized in Figure 2.</li>
</ol>
4. Tick dissection and tissue collection
<ol>
	<li>Prepare the following:
	<ol>
		<li>Melt the paraffin wax and pour it into a clean Petri dish.</li>
		<li>Sterilize the surface of the tick by rinsing it three times in separate containers containing 1% bleach (sodium hypochlorite; NaOCl), followed by a final rinse in polymerase chain reaction (PCR)-grade water16.</li>
		<li>Prepare separate slides/Petri dishes (one for each tissue type) by adding 50 &#181;L of 1&#215; PBS to each.</li>
	</ol>
	</li>
	<li>Heat the paraffin wax using a soldering iron to create a melted spot in the Petri dish where the tick will be placed.</li>
	<li>Place the tick in the melted wax cavity, ensuring that the ventral part of the tick&#39;s body is submerged and held firmly in the wax.</li>
	<li>Cut the dorsal edge of the body carefully using the scalpel blade and remove the dorsal cover of the tick body.</li>
	<li>Extract the salivary glands, midgut, and other tissues using forceps and transfer each to a separate slide/Petri dish.</li>
	<li>Using a pipette, place five drops (50 &#181;L) of 1&#215; PBS in separate slides/Petri dishes and thoroughly wash each of the extracted tissue types (salivary glands and midgut) using forceps multiple times.</li>
	<li>Transfer each tissue type (salivary glands or midgut) to a sterile, labeled microtube and add 1&#215; PBS (20 &#181;L or more) until the tissue is fully submerged.</li>
	<li>Store the tube containing the tissue for subsequent nucleic acid extraction. 
	NOTE: The tick dissection and tissue collection method is outlined in Figure 3.</li>
</ol>

<ol><li>de la Fuente, J., Villar, M., Cabezas-Cruz, A., Estrada-Peña, A., Ayllón, N., Alberdi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tick-host-pathogen+interactions%3A+Conflict+and+cooperation%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Tick-host-pathogen interactions: Conflict and cooperation.</a> PLoS Pathog. 12 (4), e1005488(2016).</li>
<li>Michalik, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+of+Borrelia+burgdorferi+sensu+lato+species+in+Ixodes+ticks+%28Acari%3A+Ixodidae%29+associated+with+cave-dwelling+bats+from+Poland+and+Romania%5BTitle%5D)+AND+%22Ticks+Tick+Borne+Dis%22%5BJournal%5D)">Diversity of Borrelia burgdorferi sensu lato species in Ixodes ticks (Acari: Ixodidae) associated with cave-dwelling bats from Poland and Romania.</a> Ticks Tick Borne Dis. 11 (1), 101300(2020).</li>
<li>Lu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+survey+of+vector-borne+pathogens+in+ticks%2C+sheep+keds%2C+and+domestic+animals+from+Ngawa%2C+Southwest+China%5BTitle%5D)+AND+%22Pathogens%22%5BJournal%5D)">Molecular survey of vector-borne pathogens in ticks, sheep keds, and domestic animals from Ngawa, Southwest China.</a> Pathogens. 11 (5), 606(2022).</li>
<li>Boulanger, N., Boyer, P., Talagrand-Reboul, E., Hansmann, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ticks+and+tick-borne+diseases%5BTitle%5D)+AND+%22Med+Mal+Infect%22%5BJournal%5D)">Ticks and tick-borne diseases.</a> Med Mal Infect. 49 (2), 87-97 (2019).</li>
<li>Scholl, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunomodulatory+effects+of+tick+saliva+on+dermal+cells+exposed+to+Borrelia+burgdorferi%2C+the+agent+of+Lyme+disease%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Immunomodulatory effects of tick saliva on dermal cells exposed to Borrelia burgdorferi, the agent of Lyme disease.</a> Parasit Vectors. 9 (1), 394(2016).</li>
<li>Nuttall, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wonders+of+tick+saliva%5BTitle%5D)+AND+%22Ticks+Tick+Borne+Dis%22%5BJournal%5D)">Wonders of tick saliva.</a> Ticks Tick Borne Dis. 10 (2), 470-481 (2019).</li>
<li>Lejal, E., Moutailler, S., Šimo, L., Vayssier-Taussat, M., Pollet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tick-borne+pathogen+detection+in+midgut+and+salivary+glands+of+adult+Ixodes+ricinus%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Tick-borne pathogen detection in midgut and salivary glands of adult Ixodes ricinus.</a> Parasit Vectors. 12 (1), 152(2019).</li>
<li>Gudderra, N. P., Sonenshine, D. E., Apperson, C. S., Roe, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+distribution+and+characterization+of+predominant+hemolymph+carrier+proteins+from+Dermacentor+variabilis+and+Ornithodoros+parkeri%5BTitle%5D)+AND+%22J+Insect+Physiol%22%5BJournal%5D)">Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri.</a> J Insect Physiol. 48 (2), 161-170 (2002).</li>
<li>Liu, L., Yan, F., Zhang, L., Wu, Z. F., Duan, D. Y., Cheng, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+profiling+of+hemolymph+in+Haemaphysalis+flava+ticks%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Protein profiling of hemolymph in Haemaphysalis flava ticks.</a> Parasit Vectors. 15 (1), 179(2022).</li>
<li>Moreira, H. N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+deep+insight+into+the+whole+transcriptome+of+midguts%2C+ovaries+and+salivary+glands+of+the+Amblyomma+sculptum+tick%5BTitle%5D)+AND+%22Parasitol+Int%22%5BJournal%5D)">A deep insight into the whole transcriptome of midguts, ovaries and salivary glands of the Amblyomma sculptum tick.</a> Parasitol Int. 66 (2), 64-73 (2017).</li>
<li>Šimo, L., Kazimirova, M., Richardson, J., Bonnet, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+essential+role+of+tick+salivary+glands+and+saliva+in+tick+feeding+and+pathogen+transmission%5BTitle%5D)+AND+%22Front+Cell+Infect+Microbiol%22%5BJournal%5D)">The essential role of tick salivary glands and saliva in tick feeding and pathogen transmission.</a> Front Cell Infect Microbiol. 7, 281(2017).</li>
<li>Khanal, S., Taank, V., Anderson, J. F., Sultana, H., Neelakanta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rickettsial+pathogen+perturbs+tick+circadian+gene+to+infect+the+vertebrate+host%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Rickettsial pathogen perturbs tick circadian gene to infect the vertebrate host.</a> Int J Mol Sci. 23 (7), 3545(2022).</li>
<li>Khogali, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+localization+of+tick-borne+pathogens+in+ticks+collected+from+camels+in+Kenya%3A+Insights+into+vector+competence%5BTitle%5D)+AND+%22Front+Cell+Infect+Microbiol%22%5BJournal%5D)">Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: Insights into vector competence.</a> Front Cell Infect Microbiol. 14, 1382228(2024).</li>
<li>Patton, T. G., Dietrich, G., Brandt, K., Dolan, M. C., Piesman, J., Gilmore, R. D. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Saliva%2C+salivary+gland%2C+and+hemolymph+collection+from+Ixodes+scapularis+ticks%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks.</a> J Vis Exp. (60), e3894(2012).</li>
<li>Tirloni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Saliva+from+nymph+and+adult+females+of+Haemaphysalis+longicornis%3A+A+proteomic+study%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Saliva from nymph and adult females of Haemaphysalis longicornis: A proteomic study.</a> Parasit Vectors. 8, 338(2015).</li>
<li>Binetruy, F., Dupraz, M., Buysse, M., Duron, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+sterilization+methods+impact+measures+of+internal+microbial+diversity+in+ticks%5BTitle%5D)+AND+%22Parasit+Vectors%22%5BJournal%5D)">Surface sterilization methods impact measures of internal microbial diversity in ticks.</a> Parasit Vectors. 12 (1), 268(2019).</li>
<li>Oliveira, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteome+of+Rhipicephalus+sanguineus+tick+saliva+induced+by+the+secretagogues+pilocarpine+and+dopamine%5BTitle%5D)+AND+%22Ticks+Tick+Borne+Dis%22%5BJournal%5D)">Proteome of Rhipicephalus sanguineus tick saliva induced by the secretagogues pilocarpine and dopamine.</a> Ticks Tick Borne Dis. 4 (6), 469-477 (2013).</li>
<li>Edwards, K. T., Goddard, J., Varela-Stokes, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Examination+of+the+internal+morphology+of+the+ixodid+tick%2C+Amblyomma+maculatum+Koch+%28Acari%3A+Ixodidae%29%3A+A+%26%2334%3Bhow-to%26%2334%3B+pictorial+dissection+guide%5BTitle%5D)+AND+%22Midsouth+Entomol%22%5BJournal%5D)">Examination of the internal morphology of the ixodid tick, Amblyomma maculatum Koch (Acari: Ixodidae): A &#34;how-to&#34; pictorial dissection guide.</a> Midsouth Entomol. 2 (1), 28-39 (2009).</li>
<li>Khogali, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exploring+the+microbiomes+of+camel+ticks+to+infer+vector+competence%3A+Insights+from+tissue-level+symbiont-pathogen+relationships%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Exploring the microbiomes of camel ticks to infer vector competence: Insights from tissue-level symbiont-pathogen relationships.</a> Sci Rep. 15 (1), 5574(2025).</li>
</ol>

The authors have declared no conflict of interest.

This protocol builds upon prior efforts to isolate individual tick tissues for tissue-specific pathogen detection and microbiome profiling. Critical steps include precise injection of Pilocarpine HCl 2% solution mixed with 1&#215; PBS in a 1:1 ratio behind coxa 4 to stimulate saliva production14,15, careful positioning of the pipette tip over the hypostome, and maintaining sterile technique throughout to minimise contamination. The use of prewarmed 1&#215; PBS at...

Ticks are obligate blood ectoparasites that serve as vectors and reservoirs for many infectious agents such as viruses, bacteria, spirochetes, fungi, and helminths, which have a significant impact on human and animal health1,2,3. Ticks attach to their vertebrate hosts by piercing the skin with their hypostome and injecting saliva into the host to assist with feeding4. The saliva secreted by the salivary glands contains various bioactive molecules that control host responses5,6. Pathogen presence in saliva is an indicator that a pathogen has, following ingestion, successfully overcome several barriers (physiological and immunological) within the tick to reach the salivary glands; a key indicator of vector competence. Tick salivary glands and midgut are the primary organs involved in pathogen acquisition and transmission. The midgut is the first organ encountered by pathogens acquired during blood-feeding, while the salivary glands and their secretions play an important role in pathogen transmission to new hosts7. Indeed, tick-borne pathogens (TBPs) are transmitted via the saliva to hosts during bloodmeals. Tick haemolymph consists of the haemocytes and plasma8 and serves as a medium for nutrient and metabolite transport to and from all tick organs, while also providing protection against pathogens through the enzymes involved in innate immunity9. The tick ovaries play a crucial role in the transmission of pathogens from one generation to the next10.
Changes in the distribution of different tick species and the emergence of new TBPs have created an urgent need for more effective methods for tick control and risk assessment, and to unravel the tripartite interactions between specific tick species, the pathogens they transmit, and their vertebrate hosts11. However, most surveillance studies investigating these associations consider whole tick homogenates, which will include pathogens that may have simply been obtained during blood-feeding, which the tick may not readily transmit. It is therefore important to clarify the role of a tick species in pathogen transmission and distinguish between dead-end host and competent vector status. This will ensure that efforts directed at targeted interventions, such as transmission-blocking vaccines, are successful12.
Empirical demonstration of vector competence requires experimental infection studies, which are costly, time-consuming, and may not accurately reflect the vector-pathogen combinations in circulation in the field. Instead, we propose that investigating the microbiomes of specific tissues/fluids of field-caught ticks will identify microbes, including TBPs, that replicate within a specific tick species and can be transmitted to subsequent hosts. To achieve this, we established a detailed protocol to aseptically extract four tick tissue/organ and fluid components (saliva, haemolymph, salivary glands, and midgut) from individual ticks for microbiome sequencing. This article demonstrates a protocol for tissue-specific pathogen screening in ticks to improve studies on vector competence.

<table><tbody><tr><td>0.5&amp;ndash;10 &amp;micro;L pipette&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>3123000020</td><td></td></tr><tr><td>10-&amp;micro;L tips</td><td>Biologix</td><td>23-0010A</td><td></td></tr><tr><td>BD ultra-fine needle 1.0 mL 31 G, 12.7 mm</td><td>Becton Dickinson (BD) Biosciences SKU</td><td>328411</td><td></td></tr><tr><td>BioRender software</td><td>BioRender</td><td>https://biorender.com</td><td></td></tr><tr><td>Ethanol Absolute (AR)</td><td>Sigma-Aldrich Chemie GmbH&amp;nbsp;</td><td>107017</td><td></td></tr><tr><td>Fine forceps style # 5</td><td>Sigma Aldrich&amp;nbsp;</td><td>T4537</td><td></td></tr><tr><td>GraphPad Prism 8 software</td><td>GraphPad Software</td><td>https://www.graphpad.com</td><td></td></tr><tr><td>JIK Bleach Regular&amp;nbsp;</td><td>Reckitt Benckiser Ltd</td><td>Kikuubo Online</td><td></td></tr><tr><td>Microscope slides</td><td>Sigma Aldrich&amp;nbsp;</td><td>CLS294875X25</td><td></td></tr><tr><td>Microvolume spectrophotometer</td><td>Microvolume UV-Vis Spectrophotometer</td><td>Thermo Scientific NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer | Fisher Scientific</td><td></td></tr><tr><td>Paraplast Plus Paraffin Wax&amp;nbsp;</td><td>LEICA BIOSYSTEMS&amp;nbsp;</td><td>39602004</td><td></td></tr><tr><td>PCR water Ambion&amp;nbsp;</td><td>Ambion</td><td>AM9935</td><td></td></tr><tr><td>Petri dish, Glass, 100 mm &amp;acute; 15 mm</td><td>Duran</td><td>237554608</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma Aldrich&amp;nbsp;</td><td>79378</td><td></td></tr><tr><td>Plasticine (Non-toxic modelling clay)&amp;nbsp;</td><td>The Lab Warehouse&amp;nbsp;</td><td>SE180-45</td><td></td></tr><tr><td>Scalpel blades, No. 11</td><td>Sigma Aldrich&amp;nbsp;</td><td>S2771</td><td></td></tr><tr><td>Scalpel handle</td><td>Sigma Aldrich&amp;nbsp;</td><td>S2896</td><td></td></tr><tr><td>STEMI 2000-C STEREO MICROSCOPE</td><td>Zeiss, Oberkochen, Germany</td><td>455053-0000-000</td><td></td></tr><tr><td>Transperant tape</td><td>Scotch Magic</td><td>MID 70005189280</td><td></td></tr></tbody></table>

extraction of saliva, haemolymph, salivary glands, and midgut from individual ticks (acari: ixodidae)

Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae)

Ticks are blood-feeding ectoparasites recognised for their ability to transmit several infectious pathogens to humans and animals. Tick-borne pathogen (TBP) surveillance generally relies on detecting pathogens in homogenates of entire ticks, including those acquired during blood feeding, which may not be transmitted by the tick. To better understand the pathogen transmission mechanism, it is essential to investigate the dissemination and concentration of TBPs in various tick tissues, which is important for elucidating transmission mechanisms in ticks and determining their vector competence. This, in turn, rests on procedures for isolating saliva, haemolymph, salivary glands, and the midgut of individual ticks in order to examine the location of TBPs within these tissues and fluids. In this study, we describe a protocol for tick tissue collection, including a novel procedure for the collection of haemolymph. The results confirm that Rickettsia africae occurs at higher levels in the haemolymph of Amblyomma gemma, affirming the likely role of this tick species as a competent vector for R. africae. In contrast, Hyalomma dromedarii exhibited high rates of R. africae in the midgut, but the bacterium was absent in the haemolymph. The presence of TBPs in the haemolymph is therefore a valuable indicator of vector competence, and rests on the availability of a method that ensures ease of collection.

This protocol facilitated the successful collection of tick samples and biological tissues necessary for pathogen screening and other downstream analyses. Figure 1, Figure 2, and Figure 3 illustrate the collection of saliva, haemolymph, salivary glands, and midgut from partially and fully engorged female Hy. rufipes ticks. These ticks were obtained from a tick colony reared under controlled conditions on New Zealand rabbits...

Here, we present a protocol to collect saliva, haemolymph, salivary glands, and midgut, following the dissection of an individual fed tick, to study tissue-specific localization of tick-borne pathogens to advance understanding of vector competence and pathogen-microbiome interactions.

Ticks are blood-feeding ectoparasites recognised for their ability to transmit several infectious pathogens to humans and animals. Tick-borne pathogen ...

To begin collect specimens of Hyalomma dromedarii, Hyalomma rufipes, Amblyomma gemma, and Rhipicephalus pulchellus from camels. Sterilize the tick exterior carefully by wiping the whole tick with a bleach soaked paper towel and ethanol. When the tick has dried, gently clean the mouth parts with the forceps by holding a small piece of paper towel soaked in 70%ethanol, then dry again.Place the tick on a clean glass slide under the microscope with the ventral surface of the tick facing upward, and hold it securely between the thumb and index finger. Inject 20 microliters of a mixture, consisting of 2%pilocarpine hydrochloride and PBS in a 1:1 ratio directly behind coxa IV using a 31 gauge syringe Rotate the tick so that the dorsal surface is exposed and fix it in place on one end of the slide by using transparent tape. Place a small piece of non-toxic modeling clay at the opposite end of the slide to provide a fixed surface for support, wait for the tick to produce saliva.Insert the 0.5 to 10 microliter tip between the palps to ensure it covers the hypostome. Draw the saliva into the pipette tip. To collect the hemolymph cut off one of the tick's legs.Using a size 11 scalpel blade, gently press the body taking care not to apply too much pressure as this may rupture the tick or internal organs and release the midgut contents. Collect the hemolymph droplet using a pipette with a 0.5 to 10 microliter tip placed on the leg. To dissect the tick, first heat paraffin wax using a soldering iron to create a melted spot in the Petri dish where the tick will be placed.Place the tick in the melted wax cavity, ensuring that the ventral part of the tick's body is submerged and held firmly in the wax. Then cut the dorsal edge of the body carefully using the scalpel blade and remove the dorsal cover of the tick body. Extract the salivary glands, midgut, and other tissues using forceps.Thoroughly wash each of the extracted tissue types in five drops or 50 microliters of PBS in separate slides. DNA concentration was higher in hemolymph than in saliva across all four tick species. Among the four tick species, Rhipicephalus pulchellus hemolymph showed the highest DNA concentration compared to the others.

extraction-saliva-haemolymph-salivary-glands-midgut-from-individual

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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.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Lyme disease is the most commonly-reported vector-borne disease in North America.  The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks.  Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

This method outlines the development of an in vitro tick blood-feeding system incorporating a 3D-printed chamber and a laboratory-fabricated silicone membrane. The system enables artificial feeding of ticks, allowing for easy sampling at various feeding stages and utilizing low volumes of blood, providing an invaluable tool for studying ixodid ticks.

Tick Blood Feeding through an Artificial Membrane on a 3D-Printed Chamber in Ixodid Tick Research

Learn to extract saliva, haemolymph, salivary glands, and midgut from fed ticks using pilocarpine stimulation and micro-dissection. This protocol enables tissue-specific pathogen localization studies in Hyalomma and Rhipicephalus species.

Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae)

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