Balancing sample sizes in parasitology: A standardized experimental infection method using faecal parasite eggs and aquatic intermediate hosts

Balancing sample sizes between infected and uninfected hosts is a major challenge in ecological parasitology, particularly for intermediate hosts in complex life cycles. In natural populations, these hosts often accumulate parasites as they grow, making it difficult to obtain comparable numbers of infected and uninfected individuals across size classes. Here, we present a standardized and repeatable protocol to experimentally infect and re-infect intermediate hosts using parasite eggs obtained directly from fresh faeces of definitive hosts. We evaluated this approach in a marine host-parasite system involving the acanthocephalan Profilicollis altmani, the mole crab Emerita analoga, and the grey gull Leucophaeus modestus. Crabs were reared under controlled laboratory conditions from early juvenile stages to ensure they were parasite-free and were exposed to eggs through filtered faecal suspensions. Larval development was monitored across two inoculation events. The first larval stage (acanthellae) appeared six days post-inoculation and matured into cystacanths during the following week. After the first exposure, 83% (small) and 96% (large) of crabs were infected; following the second exposure, infection prevalence reached 100%. Larger crabs acquired more parasites than smaller individuals, likely due to higher filtration rates. This method closely mimics natural faecal-oral transmission and avoids the need to isolate or manipulate parasite eggs directly. It also addresses the common issue of parasite overdispersion in natural populations by enabling controlled infections across host size classes and experimental replicates. The technique is especially useful for species with opaque cuticle, where parasites cannot be visually detected in vivo, and can be potentially applied to other helminths (e.g. nematodes, cestodes, digeneans) whose eggs are shed in vertebrate faeces. Overall, this protocol provides an ecologically relevant and reliable approach for experimental parasitology in marine and freshwater systems.