Innovating stomach fluke identification: an integrative approach combining Micro-CT imaging and molecular tools

The rapid loss of biodiversity driven by anthropogenic pressures highlights the urgent need for improved species identification methods. Parasites, vital ecosystem regulators, are being lost at disproportionate rates, with amphistomes-a broadly distributed group of trematode parasites, infecting all major vertebrate groups-facing significant challenges. Many amphistome species remain undescribed, and reference sequences for known species are scarce, partly due to the reliance on labour-intensive identification methods, such as Scanning Electron Microscopy (SEM) and median sagittal sections. While sagittal sectioning is particularly informative for diagnostic traits, it is destructive, requires toxic chemicals, and demands specialized personnel. In this study, we evaluated micro-computed tomography (micro-CT) imaging as a non-destructive alternative for identifying three amphistome species, Gigantocotyle gigantocotyle (Brandes in Otto, 1896); Carmyerius aff. chabaudi van Strydonck, 1970; and Carmyerius aff. endopapillatus Dollfus, 1962, isolated from the common hippopotamus, Hippopotamus amphibius Linnaeus, 1758. By comparing micro-CT imaging with traditional sectioning, SEM and incorporating molecular barcoding, we reveal the need for a taxonomic revision of Carmyerius, focussed on identifying new diagnostic characters, to better reflect species boundaries. Moreover, the integrated taxonomic effort represented in this work uncovered evidence that C. aff. chabaudi is a new species record from the common hippopotamus. Additionally, we provide high-resolution images of the original type specimens of Carmyerius cruciformis (Leiper, 1910) and G. gigantocotyle and designate new lectotypes and paralectotypes. Our findings demonstrate that micro-CT imaging is a powerful, non-invasive tool for amphistome identification, facilitating access to fragile natural history collections and advancing integrative taxonomy.