Noninvasive Methods Unveil the Trophic Transmission of the Tapeworm <scp><i>Ligula intestinalis</i></scp> in Gull‐Billed Terns

Recent developments in microscopic and molecular tools have allowed the implementation of new approaches for assessing parasitic infections in wildlife populations. This is particularly important for the noninvasive detection and quantification of endoparasites in live animals. Here, we combined copromicroscopic (Mini-FLOTAC) and molecular (qPCR) techniques to detect the infection of the macroparasite <i>Ligula intestinalis</i> (Cestoda, Pseudophyllidea) in fresh droppings of Gull-billed Terns <i>Gelochelidon nilotica</i> (Charadriiformes, Laridae) breeding in southwestern Spain. Additionally, we sequenced the cytochrome <i>b</i> gene in parasite isolates from Gull-billed Terns (definitive host) and Common Bleak <i>Alburnus alburnus</i> (second intermediate host) sampled around tern colonies to explore potential genetic differences between the isolates. The qPCR test showed a higher prevalence (18%; in 13/73 samples) than Mini-FLOTAC (9%; in 8/88 samples), indicating that qPCR was more sensitive for diagnostic purposes than fecal flotation alone. Although the agreement between both techniques was substantial (84.2%) mainly due to the large number of uninfected samples, only Mini-FLOTAC allowed us to quantify parasite shedding. When combining techniques, the prevalence of infection did not differ between adults and chicks, suggesting frequent trophic transmission from parents to their offspring via food provisioning. Phylogenetic analyses identified four haplotypes in the isolates from Gull-billed Terns and Bleak, all of which were placed within a European clade composed of tapeworms recovered exclusively from phylogenetically derived cyprinid fish. This, combined with the short lifespan of mature tapeworms, suggests that Gull-billed Terns became infected after consuming infected fish around their breeding colonies rather than on their West African wintering grounds. Altogether, our results represent the first record of <i>L. intestinalis</i> in Gull-billed Terns and the first molecular characterization of the parasite in the Iberian Peninsula. This integrative coprodiagnostic protocol can be applied to other host-parasite systems, allowing researchers to study helminth infections in wild populations using a noninvasive approach.