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

Summary

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.

Abstract

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.

Introduction

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 health^1,2,3. 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 responses^5,6. 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.

Protocol

This study received ethical approval from the Pwani University Ethics Review Committee (Ref: ERC/EXT/002/2020E) and a research license from Kenya'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

1. 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.
2. 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.

2. Saliva collection

1. 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.
2. 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.
3. 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.
4. Inject 20 µL of a mixture consisting of 2% Pilocarpine HCl and 1× PBS (in a 1:1 ratio) directly behind coxa 4 using a 31 G syringe^14,15.
   NOTE: Take care not to inject too deeply as this will rupture the tick's body and affect the integrity of the sample. Alternatively, use 4-8 mm pen needle to control injection depth.
5. 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.
6. 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-µL tip over the hypostome of the tick. Using non-toxic modeling clay is optional.
7. Insert the 0.5-10-µL tip between the palps to ensure it covers the hypostome.
8. Wait for the tick to produce saliva or gently massage the hypostome by moving the tip in and out.
9. 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× PBS prewarmed to 38 °C at hand; pipetting a small amount (1 µL) 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.
10. Release the saliva from the tip into a sterile and properly labelled microtube and store at -20° C/-80 °C until further analysis.
    NOTE: It is possible to collect up to 10 µL of saliva from the tick. The saliva collection process is summarized in Figure 1.

3. Haemolymph collection

1. Continue with the same tick after saliva collection or use a new tick after surface sterilisation as mentioned above.
2. Place the tick on a new clean glass slide and cut off one of the tick's legs using a scalpel blade size no.11.
3. 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.
4. The haemolymph will be released as a drop from the leg, collect it with a pipette by placing a 0.5-10 µL 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.
5. 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 µL of haemolymph from the tick. The haemolymph collection method is summarized in Figure 2.

4. Tick dissection and tissue collection

1. Prepare the following:
   1. Melt the paraffin wax and pour it into a clean Petri dish.
   2. 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.
   3. Prepare separate slides/Petri dishes (one for each tissue type) by adding 50 µL of 1× PBS to each.
2. Heat the paraffin wax using a soldering iron to create a melted spot in the Petri dish where the tick will be placed.
3. 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.
4. Cut the dorsal edge of the body carefully using the scalpel blade and remove the dorsal cover of the tick body.
5. Extract the salivary glands, midgut, and other tissues using forceps and transfer each to a separate slide/Petri dish.
6. Using a pipette, place five drops (50 µL) of 1× PBS in separate slides/Petri dishes and thoroughly wash each of the extracted tissue types (salivary glands and midgut) using forceps multiple times.
7. Transfer each tissue type (salivary glands or midgut) to a sterile, labeled microtube and add 1× PBS (20 µL or more) until the tissue is fully submerged.
8. Store the tube containing the tissue for subsequent nucleic acid extraction.
   NOTE: The tick dissection and tissue collection method is outlined in Figure 3.

Results

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...

Discussion

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× PBS in a 1:1 ratio behind coxa 4 to stimulate saliva production^14,15, careful positioning of the pipette tip over the hypostome, and maintaining sterile technique throughout to minimise contamination. The use of prewarmed 1× PBS at...

Materials

List of materials used in this article

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