A newly optimized protocol to extract high-quality hemolymph from the cattle tick Rhipicephalus microplus: Improving the old conditions

Ticks are hematophagous ectoparasites with importance to animal and human health. In recent years, the study of ticks has had significant development, including immune response, vector-host interactions, physiological and multi-omics approaches. However, one of the main impediments is obtaining a significant amount of high-quality hemolymph. For this reason, we developed a protocol that allows obtaining up to 100 μl of hemolymph free of host blood per engorged tick. The technique consists of continuous hipocuticular punctures of the tick dorsum and an anticoagulant buffer that impedes hemolymph coagulation, allowing constant extravasation and ensuring high yields. Additionally, the hemocytes recovered with this protocol are intact and can be used for further analysis. The high-quality hemolymph obtained using this protocol and its applications will help to better understand the processes involving the hemolymph and its components. Although there are other hemolymph extraction protocols, the method developed here is very well suited for Rhipicephalus microplus, and in our experience, results in better yields and high-quality samples.