Parasitism is one of the most diverse and common lifestyles on earth (Poulin and Morand, 2000), with parasites constituting a significant portion of the undescribed animal diversity (Okamura et al., 2018). The limited knowledge of extant parasite species and their distribution is particularly relevant in the context of current global biodiversity loss (Ceballos et al., 2015). Parasites are expected to be more vulnerable to extinction than non-parasitic species due to a combination of anthropogenic pressures on the definitive and intermediate hosts (e.g., habitat loss, overexploitation, and species invasion) as well as coextinction (Carlson et al., 2017, Cizauskas et al., 2017). Notwithstanding the general loss of diversity, several studies reported a concurrent increase in the abundance and spread of some parasites, often resulting in the emergence of infectious diseases in humans, livestock, and wildlife (Han et al., 2016, White and Razgour, 2020) with major impacts on ecosystems (Cizauskas et al., 2017, Okamura et al., 2018, Carlson et al., 2020). Thus, a detailed characterization of parasite diversity and distribution is an important prerequisite for the prediction of ongoing changes and their potential ecosystem-level consequences.

Diplostomidae (Trematoda: Digenea) is a group of parasitic platyhelminths that is ubiquitous in both marine and freshwater ecosystems and has a global distribution. It is a diverse taxonomic group with more than 25,000 estimated species, (Cribb et al., 2002; Carlson et al., 2020). However, due to the high level of cryptic diversity, the total species number is likely to be even greater (Poulin, 2011). Diplostomids have a complex life cycle that often involves lymnaeid snails and fish as intermediate hosts, whereas piscivorous birds frequently serve as definitive hosts (Esch, 2002). The larval stage found in the second intermediate host (metacercariae) is among the most frequently found parasite in fish communities (Karvonen and Marcogliese, 2020). Metacercariae usually establish in the eyes of fish where severe infections can lead to various detrimental effects such as cataracts that can reduce feeding efficiency, and predator avoidance by the host (Karvonen and Seppälä, 2008, Vivas Muñoz et al., 2017, Ubels et al., 2018). While their number per infected host can reach several hundred with coinfections of multiple species a common occurrence, metacercariae are extremely difficult to identify at the species level based on morphological characteristics alone, which renders the assessment of environmental factors and host-related characteristics that influence species diversity, composition, and coexistence in diplostomid communities a considerable challenge (Marcogliese et al., 2001, Faltýnková et al., 2011, Rellstab et al., 2011, Locke et al., 2013).

The application of molecular techniques and taxon-specific primers, especially for the cytochrome c oxidase subunit 1 (cox1), has proven to be highly advantageous for an effective identification of diplostomid species (Moszczynska et al., 2009, Chan et al., 2021). This approach has considerably improved our ability to assess diversity (Georgieva et al., 2013a, Georgieva et al., 2013b, Faltýnková et al., 2014, Otachi et al., 2015, Kudlai et al., 2017, Hoogendoorn et al., 2020), distribution (Locke et al., 2015, Ruehle et al., 2021), phylogenetic relationships (Achatz et al., 2022), and community assemblies of this species group (Désilets et al., 2013). However, individual parasite sampling and processing followed by Sanger sequencing are extremely labour- and time-consuming, which severely limits the ability to study complex diplostomid communities.

The development of new-generation sequencing, combined with the use of universal primers, has drastically changed how we assess biological diversity (Baird and Hajibabaei, 2012, Bik et al., 2012, Cristescu, 2014). Among these new approaches, the technique known as DNA metabarcoding allows the characterization of whole communities from complex samples which may include degraded DNA traces of the species (e.g., environmental DNA) or multiple individuals and species in a mixture (e.g., bulk samples) (Cristescu, 2014). DNA metabarcoding has been widely used to characterize high taxonomic ranks (e.g., eukaryotes or prokaryotes) (Yoccoz et al., 2012, Zaiko et al., 2015, Deiner et al., 2016, Petro et al., 2017) and to study more specific organism groups such as fish communities (Miya et al., 2015, Valentini et al., 2016, Bylemans et al., 2018). The ability to focus on just one target group is particularly relevant to studies of endoparasite communities because endoparasite sampling typically involves a great amount of host DNA (Bourret et al., 2021). To date, DNA metabarcoding has been successfully applied to survey the diversity of gastrointestinal nematodes in mammals (Avramenko et al., 2015, Aivelo et al., 2018), blood protozoan parasites in birds (Thomas et al., 2022), and monogenean ectoparasites in fish (Scheifler et al., 2022). However, few attempts have been made to apply DNA metabarcoding to diplostomid communities in teleosts. A notable exception is a recent study by Noreikiene et al. (2020) who used this approach to investigate diplostomids in Eurasian perch (Perca fluviatilis) and described how dissolved organic carbon (DOC) content together with a low water pH restricts diplostomids from humic lakes. Furthermore, they demonstrated that DNA metabarcoding can be used effectively to characterize intra-specific diversity in diplostomids. Yet, DNA metabarcoding also holds great promise for the assessment of abundance, richness, relatedness, and interactions of parasite communities at unprecedented resolutions and scales (Bass et al., 2015).

In this study, we further developed a cost-effective cox1 region-specific DNA metabarcoding approach to characterize parasitic diplostomid communities in lentic ecosystems. For this purpose, we screened the eyes of two common fish species (perch and roach) in seven temperate lakes and evaluated: (i) spatial intra- and inter-lake heterogeneities of eye fluke communities, (ii) the effects of host species and size on parasite communities, (iii) the potential role played by various environmental factors shaping eye fluke communities, (iv) the temporal variation of parasite communities, and (v) interspecific associations between different parasite species.