Parasites are mostly known for their negative effects on other organisms yet can also benefit ecosystem functioning (Gómez and Nichols, 2013, Stringer and Linklater, 2014, Wood and Vanhove, 2023). They can impact species community structure as well as genetic and phenotypic diversity, making them an invaluable component of healthy, diverse ecosystems. Increasingly, parasites are being viewed as conservation targets (Carlson et al., 2020, Kwak et al., 2020), with various theories suggesting that some parasite taxa have decreased significantly over the past decades (see Wood et al., 2023 for empirical evidence and references therein regarding theories). Trematode parasites stand out as a biodiverse yet neglected group of animals with an estimated 90% of species waiting to be discovered and formally described (Hechinger, 2023).

Trematode parasites of the superfamily Paramphistomoidea Fischoeder, 1901 − commonly referred to as ‘amphistomes’ or ‘stomach flukes’ − cause amphistomiasis or stomach fluke disease. Amphistomiasis is highly prevalent across many parts of the world and can be especially lethal to young animals (Toledo and Fried, 2014, Laidemitt et al., 2016, Pfukenyi and Mukaratirwa, 2018). Adult amphistomes are endoparasites of the digestive tract, found in a variety of vertebrate final hosts, from amphibians to hippos, while larval stages use freshwater snails as intermediate hosts (Toledo and Fried, 2014). Currently, no effective treatment against amphistomiasis exists (Mage et al., 2002). Despite a serious burden, significant gaps in our understanding of amphistomes persist, extending from host range, lifecycle biology and geographic distribution, to molecular characteristics (Laidemitt et al., 2016, Pfukenyi and Mukaratirwa, 2018, Toledo and Fried, 2014). To date, 254 amphistome species have been described (Sey, 1991). However, amphistome identification based on DNA-barcoding techniques remains severely constrained as genetic reference databases cover merely 37 species of which only 23 are represented by part of the COI barcoding region and a nuclear marker.

Morphological description and identification of amphistomes relies on median sagittal sections to investigate the morphology of the terminal genitalium, acetabulum and pharynx, complemented by Scanning Electron Microscopy (SEM) imaging of the tegumental surface to characterize the tegumental papillae (Sey, 1991). Both methods are destructive and hamper future analysis of the specimen, rendering them unsuitable for the analysis of museum or type specimens. Currently a non-destructive alternative is lacking, hampering the cataloguing of amphistome diversity.

Knowledge gaps in the field of amphistome biology could be effectively addressed through an integrative taxonomic approach combining the information on a parasite's lifecycle, its larval stages, intermediate and final hosts, along with an analysis of parasite morphology and genetics (Will et al., 2005, Schols et al., 2020). Moreover, by applying non-destructive morphological characterization methods, it would become feasible to responsibly examine valuable type and voucher specimens, both valorising museum collections and increasing the scientific accuracy of datasets.

Micro-computed tomography (micro-CT) is an imaging technique utilizing X-rays to produce a detailed three-dimensional representation of an object at a microscopic scale. The non-destructive characteristics of this technology facilitate the examination of both external and internal features of a sample without the need for dissection or any irreversible treatments, thereby preserving the original material. This is particularly advantageous when dealing with rare or precious specimens, as it maintains the physical integrity of the sample for subsequent analyses (Faulwetter et al., 2013). Additionally, the use of micro-CT does not alter the DNA of the specimen; molecular data can still be retrieved even post-imaging (Faulwetter et al., 2013). Crucially, the three-dimensional data obtained through micro-CT contain a wealth of information applicable in taxonomy and systematics and has the potential to substantially improve species identification. The investigation of specimens in their original, physical state, with characters in their natural positions within the organism, enables researchers not only to assess the specimens’ true shape but also to infer functionality from morphological structures and discover new diagnostic characters (Ziegler et al., 2010, Zimmermann et al., 2011, Faulwetter et al., 2013). The non-destructive nature of micro-CT imaging further allows the establishment of digital archives for specimens which provides continuous accessibility to type material for all potential users simultaneously.

Micro-CT imaging is an emerging technique in parasitology (O’Sullivan et al., 2018). It offers valuable insights in parasite location and migration in host tissues, allowing pathological and behavioural studies (O’Sullivan et al., 2018). For example, micro-CT imaging revealed vital insights in how the trematode Dicrocoelium dendriticum (Rudolphi, 1819) interacts with ant brain tissue to manipulate its host’s behaviour (Martín-Vega et al., 2018). Furthermore, this technology enabled Bulantová et al. (2016) to characterize larval migration of the neurotropic Trichobilharzia regenti Horák, Kolářová & Dvořák, 1998 in vertebrate hosts, explaining paralysis symptoms observed after incidental infection by this parasite. It has also been used to study trematode intramolluscan stages (Kremnev et al., 2020), trematode metacercariae in a 100 million years old lizard preserved in amber (Poinar et al., 2017), and encapsulated nematodes in snail shells (Falkingham and Rae, 2021). Micro-CT scanning clearly benefits parasitological research, yet no assessments have been made on the taxonomical value of this tool.

In this study, we test and optimize a non-destructive method for amphistome identification based on micro-CT imaging, using three parasites of Hippopotamus amphibius Linnaeus, 1758, the common hippopotamus, Gigantocotyle gigantocotyle (Brandes in Otto, 1896); Carmyerius aff. chabaudi van Strydonck, 1970; and Carmyerius aff. endopapillatus Dollfus, 1962. In doing so, we link each species to several molecular markers for future research endeavours and contribute to the limited knowledge on the unique trematode fauna of the common hippopotamus. Furthermore, the merits of micro-CT scanning for trematode species identification are revealed.