Efficient, Capsule-Free Nymphal Ixodid Tick Placement for Site-Specific Attachment to Assess Anti-Tick Host Behavior and Immune Responses.

Ixodid ticks are among the most widespread and permissive groups of arthropod disease vectors in the world. They transmit various viral, bacterial, protozoan, and even nematode pathogens infective for humans and animals. Ticks are essential to studying pathogen transmission and infection in a new host; however, exposing in vivo models to ticks is very challenging. Most current tick placement protocols rely on capsules to increase attachment rates at a specific location. However, capsules interfere with hosts' ability to engage with ticks by grooming/scratching. Conversely, available capsule-free tick infestation protocols, including free-roaming protocols, suffer from low and highly variable tick attachment efficacy. Here, we describe a highly efficient capsule-free nymphal tick placement protocol with tick attachment rates of ≥85% across various tick species and genera, offering the generation of highly reproducible data. This article also offers a set of protocols to the user primarily geared toward the study of itch-induced tick removal but are adaptable to other study settings. Published 2026. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Capsule-free, highly efficient multi-species nymphal tick placement for site-specific attachment Basic Protocol 2: Assessing efficacy of itch-induced tick removal by quantifying loss of ticks over time Basic Protocol 3: Assessment of health/ability of partially fed nymphal ticks to feed determined by scutal index Basic Protocol 4: Scratch bout quantification in guinea pigs Support Protocol: Development of a semi-supervised deep learning pipeline for automated detection and validation of scratching behavior Basic Protocol 5: Assessment of local skin inflammation due to immunity to tick bites using laser speckle contrast imaging.