Hidden caveats in tick dissection: engorgement level and complex tracheal network architecture compromise internal...

Tick dissection is fundamental for isolating internal organs for histological, developmental, cell culture, microbiome, functional, and epidemiological studies. However, comparative anatomical details and methodological guidance for identification, and aseptic isolation of internal organs remain limited. The purpose of this study is to demonstrate the internal anatomy of hard ticks using Haemaphysalis longicornis Neumann, H. flava Neumann, and Amblyomma testudinarium Koch as the dissection materials and identify optimal dissection approaches for obtaining intact and contamination-free organs. Six groups of ticks, categorized by species, sex, life stage, and engorgement level, were dissected in aseptic conditions under a stereomicroscope. Internal morphoanatomical features were compared across groups, with particular emphasis on salivary glands, Malpighian tubules, gonads, tracheae, and associated organs. Given the risk of cross-organ contamination, the ease of tick handling, organ-specific separation techniques, difficulties, and immediate troubleshooting are documented. Dissection feasibility was found to vary by sexes, developmental stages, and engorgement states. A dense tracheal network was consistently observed across all groups, regardless of maturity. Organ isolation was most efficient in adult males owing to their simpler internal anatomy, reduced midgut blood volume, and less extensive tracheae. Engorgement level and tracheal complexity remained key limiting factors influencing the efficiency of dissection and ability to retrieve desired organs without cross-organ contamination, particularly due to fragmented tissues and midgut contents. Partly engorged, mid-sized adult ticks provide optimal conditions to master dissection techniques. This integrated approach has transformed the descriptive tick dissection into pictorial methodological guidance and offers comparative anatomical insights for tick dissection.