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

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.

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.