Toward welfare indicators: leg condition and aversive stimuli modulate defensive behavior in Opilones

The history of invertebrate use in research and their growing use in managed settings highlight the need for validated behavioral indicators of welfare, yet these remain poorly understood across taxa. The arachnid group Opiliones (harvestmen or daddy long-legs) is an ideal group for identifying welfare indicators, given their well-characterized defensive behavior, including the common strategy of leg autotomy. The goal of this project was to identify candidate behavioral welfare indicators for Prionostemma sp. 1 in the Neotropical rainforest. We brought field-collected individuals with different leg conditions to the lab and briefly exposed them to an aversive stimulus (a gentle squeeze and release) paired with an assigned substrate (either rock or sand). A rewarding stimulus (food) was provided on the alternate substrate. We found no evidence of preference for roosting on the substrate associated with the rewarding stimulus, nor avoidance of the substrate associated with the aversive stimulus. Thus, evidence for associative learning was not supported. Additionally, substrate use did not differ based on leg condition (intact, missing 1-2 legs, or missing sensory legs). After an aversive stimulus, fleeing was consistently the most common response throughout 5 days. In contrast, the frequency of bobbing increased while the frequency of escaping decreased over trials. Individuals with all legs tended to escape more often than those missing legs, but neither the number nor the type of missing legs substantially improved predictions of behavioral responses. Overall, fleeing and bobbing may serve as behavioral indicators of negative welfare states, whereas the observed absence of exploratory tapping following the aversive stimulus suggests that this behavior may indicate neutral or positive welfare states. As one of the first investigations of welfare indicators in wild arachnids, this study provides novel insights into how defensive behaviors and physical condition may inform assessments of invertebrate welfare, laying the groundwork for future validation of behavioral indicators.