Duan et al. Parasites Vectors (2021) 14:43 https://doi.org/10.1186/s13071-020-04536-x Parasites & Vectors RESEARCH Open Access Trematode diversity refecting the community structure of Danish freshwater systems: molecular clues Yajiao Duan*, Azmi Al‑Jubury, Per Walter Kania and Kurt Buchmann Abstract Background: Digenean trematodes are parasitic platyhelminths that use several hosts in their life cycles and are thereby embedded in various ecosystems afected by local environmental conditions. Their presence in a habitat will refect the presence of diferent host species and, as such, they can serve as ecological indicators. Only limited infor‑ mation on the occurrence of trematodes and their link to other trophic levels in the Danish freshwater ecosystems is currently available.Therefore, the main aim of the present study was to increase our knowledge in this feld. Methods: Snails were sampled from 21 freshwater lakes in Denmark, following which shedding procedures were performed, cercariae were recoved and the released parasites were identifed using molecular tools (PCR and sequencing). Results: A total of 5657 snail hosts belonging to ten species were identifed, revealing a highly diverse parasite fauna comprising 22 trematode species. The overall trematode prevalence was 12.6%, but large variations occurred between host species. The snail host Lymnaea stagnalis showed the highest prevalence and also exhibited the highest diversity, accounting for 47.6% of the species richness. Conclusions: This survey contributes updated information on parasite–host relations and compatibility and may assist in describing the ecological structure of the investigated Danish freshwater ecosystems. Keywords: Trematode, Snails, Diversity, Molecular identifcation, Freshwater lakes Background invertebrates and vertebrates may play a role as the sec- Trematodes, dominated by the digeneans, comprise a ond intermediate host, whereas the defnitive hosts are group of abundant and ubiquitous parasitic metazoans often vertebrates, such as various fshes, amphibians, rep- with high diversity and more than 18,000 nominal spe- tiles, birds and mammals [6]. Te trematode life cycles cies [1]. Tese metazoans utilize multi-host systems to involve intricate transmission patterns adapted to the complete their complex life cycles, often with a mollusc habitat, the abiotic and biotic conditions and especially as the frst intermediate host [2–4]. Details on the asso- the diferent host species present [7]. ciative life-cycle patterns between 1000 digenean species Due to the obligate relation to free-living organisms and their mollusc hosts have been recorded [5]. Other in the ecosystem, these parasites may serve as bioindi- cators and used to indicate presence of obligate hosts, environmental conditions and disease risks under vari- *Correspondence: [email protected] ous conditions (climate changes, anthropogenic impacts) Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University [2, 8–12]. Tis potential frames the importance of con- of Copenhagen, Via Stigbøjlen 7, 1870 Frederiksberg C, Copenhagen, ducting continuous surveys of trematode occurrence Denmark © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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However, of Denmark) and Jutland (Western part of Denmark). due to the characteristics of adult digeneans, often hid- Te precise locations of the lakes were determined den in their hosts and with uneven spatial distributions, using the global positioning system (Table 1), and sam- it is relatively difcult to detect and monitor that stage. In plings were conducted from March 2019 to September contrast, sampling of the mollusc host in a habitat creates 2019. Snails were collected by hand in shallow water a method for efcient biomonitoring of their larvae. Incu- areas, from stones and aquatic plants, placed in 1-l con- bation of an infected snail in the laboratory allows shed- tainers in cooled transport boxes and transferred (max- ding of cercariae and thereby detection of a parasite at imum 4 h transport time) to the university laboratory several trophic levels and hosts in the environment. Te (Frederiksberg, Copenhagen area) for subsequent shed- adults in the fnal hosts possess characteristic structures, ding procedures. In order to evaluate the efect of sea- but the larval stages related to the intermediate mollusc son (sampling date) on infection, three lakes (Bagsværd host (e.g. cercariae, rediae, sporocysts) and even metacer- sø, Furesø and Esrum sø) were sampled several times cariae in the second intermediate host are generally more between March and September 2019. difcult to identify [13]. With the advent of molecular tools, such as PCR and subsequent sequencing of selected Cercarial shedding from snails genes, it is now possible to elucidate the occurrence and Once in the laboratory, the sampled snails were rinsed in diversity of various trematodes species in freshwater de-chlorinated municipal water for removal of surface- systems simply by sampling the mollusc host, recover- adhering organisms (preventing interference of false ing the released cercariae, isolating DNA, performing positives), whereafter they were identifed to species by PCR and obtaining sequence information. Selected parts shell morphology [17]. Individual snails were placed at of the parasite genome, such as the nuclear ribosomal room temperature in 100-ml beakers containing 50 ml DNA regions comprising 18S, ITS1, 5.8S, ITS2, 28S and of 0.45-µm fltered lake water (Filtropur; Sarstedt, Nüm- the mitochondrial cytochrome c oxidase subunit genes brecht, Germany) [18] and incubated for 8 h in daylight. (cox1 and cox2) genes may be used to identify trematode Te beakers were subsequently examined under a ster- species across a broad taxonomic range [14]. Diagnosis eomicroscope (magnifcation ×7–40; Leica Microsystems can be done by performing BLAST analysis against well- GmbH, Wetzlar, Germany) to record presence/absence of described species in GenBank. cercariae. Infected snails were characterized as “positive” Almost one century ago, the limnologist Wesenberg- and non-infected snails as “negative”, following which the Lund investigated the occurrence of cercariae in snails cercarial type was determined [19–22]. Recovered cer- in a few Danish freshwater systems [15]. However, with cariae were preserved in 96% ethanol (Kemetyl A/S, Køge, a few exceptions, the link between the cercariae and Denmark) for subsequent molecular analysis [23]. other organisms in the environment was at that time poorly known. In addition, the identity of species of the Molecular identifcation of cercariae recovered cercariae and their connection to other hosts Individual ethanol-fxed cercariae were separated and in the ecosystem was in most cases not known. By using placed in Eppendorf tubes (Axygen®, Sorenson BioSci- a molecular approach it is now possible to supplement ence, Salt Lake City, UT, USA), following which the etha- our knowledge on the fauna of trematodes in Danish nol was evaporated (Eppendorf TermoMixer® Comfort; freshwater lakes [4, 16]. Te present study describes Eppendorf, Hamburg, Germany). DNA from each speci- the diversity of trematodes in 21 Danish freshwater men was then isolated following lysis (using a guanidine lakes (geographically representing a major part of the thiocyanate lysis bufer) (Tkach and Pawlowski [24]). national territory) by investigating ten species of mol- DNA was purifed from the PCR mixture using Illustra™ luscs and their released cercariae. By using current GFX™PCR and Gel band purifcation kit (VWR Inter- knowledge on transmission pathways, we obtained national, Søborg, Denmark). DNA concentration and information on second intermediate and defnitive host purity were measured using a Nanodrop 2000 spectro- occurrence and trophic interactions in the ecosystems. photometer (Saveen & Werner ApS, Jyllinge, Denmark). Te complete ITS1–5.8S–ITS2 regions of the rDNA were amplifed using forward primer BD1 (5′-GTC GTA Methods ACA AGG TTT CCG TA-3′) and reverse primer BD2 Field sampling (5′-TAT GCT TAA ATT CAG CGG GT-3′) [25] under Samples of snails were recovered from a total of 21 PCR cycling conditions as previously described [26]. selected freshwater lakes (sø) in Zealand (eastern part Each sequence obtained from the PCR conducted was Duan et al. Parasites Vectors (2021) 14:43 Page 3 of 15