A free and user-friendly software protocol for the quantification of microfauna swimming behavior

Characterizing swimming behavior can provide a holistic assessment of the health, physiology and ecology of microfaunal species when done in conjunction with measuring other biological parameters. However, tracking and quantifying microfauna swimming behavior using existing automated tools is often difficult due to the animals' small size or transparency, or because of the high cost, expertise, or labor needed for the analysis. To address these issues, we created a cost-effective, user-friendly protocol for behavior analysis that employs the free software packages HitFilm and ToxTrac along with the R package ‘trajr’ and used the method to quantify the behavior of rotifers. This protocol can be used for other microfaunal species for which investigators may face similar issues in obtaining measurements of swimming behavior. Tracking microfauna behavior can be challenging due to their small size or transparency, or the cost and labor required. We created a user-friendly, cost-effective protocol to help quantify swimming behavior metrics for microfaunal studies. The protocol created allows users to add a semi-automated motion tracker to individuals of interest using HitFilm and to acquire tracking coordinates for each specimen without coding by using ToxTrac. Our approach allows quantification of swimming speed and acceleration, swimming efficiency, directional behavior, trajectory descriptors (distance and length) and movement time using the ‘trajr’ package in R. All code used for R, instructional information and tutorial videos for this protocol are provided to aid in the usability of the method. Characterizing and quantifying an organism's behavior can provide insights to its health, social interactions, psychology, ecology, life history and more, making behavioral studies relevant across a range of disciplines (including ecology, evolutionary biology, developmental biology, biomechanics and biomedicine). A plethora of software options for tracking animals are available, ranging from fully manual to completely automated, but many are labor-intensive, expensive, require a high degree of computational expertise, or are optimized for particular species. We created a protocol that uses free software to apply motion trackers to individuals (HitFilm), acquire tracking coordinates in each video frame for the targeted individuals (ToxTrac) and analyze the coordinates for various behavioral metrics (‘trajr’ package in R), to quantify and understand microfaunal behavior. Swimming behavior metrics that can be obtained from the protocol include speed and acceleration, swimming efficiency, directional behavior, trajectory descriptors and movement time. The protocol developed was proven to be efficient and effective when analyzing the behavior of rotifers supplemented either with the mitochondrial enhancer, elamipretide, or the mitochondrial inhibitor, rotenone. This protocol for analysis of microfaunal behavior can be applied to other microfaunal species and used for projects with limited funding, applied to videos taken with any imaging system and implemented without specialized coding expertise.