Introduction

Most parasitic organisms are in a co-evolutionary arms race with their host species [1,2]. In horizontally transmitted endoparasites this generally results in relatively low parasitic virulence for the parasites’ adult stages in the final host [3]. However, in punctuated events parasites may reach new host populations or get access to a new host species. The lack of co-evolved mechanisms between these parasites and the new host may potentially lead to elevated virulence resulting in increased host mortality thereby disturbing the equilibrium in the local ecological networks and communities [4–6], as it has been found for the Asian swim bladder nematode Anguillicoloides crassus colonizing the European eel (Anguilla anguilla) [6]. Therefore, it is important to detect and follow changes in parasite distributions. Records in historical literature are important to evaluate how uncommon such changes are, and such records are mostly based on morphological classification of the parasite species. As molecular methods (barcoding, next-generation sequencing) become more and more accessible to classify species [7,8], it becomes increasingly important to validate the reliability of the different classification methods and how the derived taxonomies relate to each other [9–12].

Acanthocephalans are a small phylum of obligatory endoparasites with limited variation in morphological structures that are common in aquatic ecosystems [13]. They require an arthropod intermediary host, and a vertebrate final host to which they may accrue considerable damage to the intestines [13,14]. Recent literature on Western-European acanthocephalans of the genus Pomphorhynchus has shown the importance of complementing morphological with molecular bar-coding methods in order to study recent changes in species distributions [4,15–17]. The current data suggests that Ponto-Caspian lineages of Pomphorhynchus laevis spread via the Danube-Main-Rhine canal into Western-Europe [18], and are effectively replacing Pomphorhynchus tereticollis populations in the Rhine [15]. Such a replacement could result in increased mortality of final host species as was shown for A. crassus [6]. The genus Pomphorhynchus include 28 species distributed over all continents and six of which have been described from the European continent (Retrieved 13 April 2020, from the Integrated Taxonomic Information System on-line database, http://www.itis.gov). They comprise about 2% of all acanthocephalans which in general are known to infect a wide range of intermediary and final host species to complete their complex life cycles [13,19]. When eggs are ingested by a suitable arthropod as intermediary host, the larva emerges and passes through the gut wall to reach the arthropod’s hemocoel where it further develops into a dormant larval stage, called cystacanth. Such infections are known to change the behaviour of the arthropod increasing the transmission probability of the cystacanth to the final host, usually through predation by a fish [20–22]. With the fish host, the young parasite emerges and uses its proboscis to penetrate the intestinal wall where it attaches, matures and reproduces dioeciously. Eggs produced by gravid females are excreted with the fish’s faeces. Fish occasionally are paratenic with the acanthocephalans remaining in a cystacanth stage in the body cavity [4,19]. The parasites has been shown to cause some inflammation and alteration of the intestinal wall, but this does not result in significant effects on mortality, growth or reproduction of their fish host [14].

Classical studies on this genus have differently addressed the morphological features of the proboscis to categorize and identify Pomphorhynchus species [23–25]. Although different forms of Pomphorhynchidae have been described in Western Europe, these forms were often combined under the "P. laevis species-complex" (Zoega in Müller, 1776) [23]. Genetic barcoding based on sequencing parts of relatively well conserved areas of mitochondrial (e.g. COI = cytochrome oxidase subunit I gene) and ribosomal (ITS = internal transcribed spacers) DNA has confirmed that at least one of the forms of the P. laevis species-complex, that was originally described on morphological characteristics as P. tereticollis (Rudolphi, 1809), should be considered a distinct species [26]. Dating techniques based on the molecular clock estimate that these two species started to diverge from the Late Miocene, about 8.4 million years ago [27]. Variation in mitochondrial COI and ribosomal ITS DNA further suggest that geographical populations of P. laevis diverged gradually since the late Miocene leading to genetically [16,28] and morphologically distinct lineages [16], whereas genetic variation in P. tereticollis is low, with populations starting to diverge from Middle Pleistocene [28].

The relation between the morphological and genetic classification of P. laevis and P. tereticollis has recently been questioned again, as molecular and morphological data seemingly pointed to a different species classification [29,30]. This has consequences for the interpretation of distribution patterns of the two species. For example, it was proposed that invasive intermediary and final hosts of Ponto-Caspian origin (e.g. the gammarid Dikerogammarus villosus and the round goby, Neogobius melanostomus), could carry around their parasites and thus spread non-native Pomphorhynchus species to new environments in the Rhine [4]. Identification based on morphological characteristics of parasites from invasive and local hosts, first led to the conclusion that P. tereticollis would replace endemic P. laevis [4]. However, a more recent molecular analysis of parasites taken from the invasive fish (N. melanostomus) indicated that these had ribosomal (ITS) sequences that were similar to those of P. laevis [29]. Moreover, the morphological characteristics (i.e. hooks of the bulbus) that were used to distinguish P. tereticollis from P. laevis [4,26], now also have been found in a subpopulation of these parasites in the Danube (i.e. P. bosniacus) that is genetically identical to P. laevis [16].

P. tereticollis was originally described on morphological characteristics from a specimen collected from European flounder (Platichthys flesus) from the Baltic coast [26]. P. tereticollis has since been verified using bar-coding methods in a wide range of gammarids, cyprinid and perciform hosts in lowland rivers in Europe [19,26]. Also gammarids captured from rhithral rivers in Europe have been shown to be infected with P. tereticollis, but the fish hosts was not identified in these rivers [31]. In rhithral rivers the dominant fish species are brown trout (Salmo trutta) and grayling (Thymallus thymallus). Trout and other salmonids are known to be suitable fish hosts for Pomphorhynchus species in the British Islands [24,32]. Although P. laevis and P. tereticollis have been collected from brown trout on the British Islands and Ireland [28], to the best of our knowledge salmonids have not been described as fish hosts for P. tereticollis in continental Western Europe.

As Ponto-Caspian lineages of P. laevis are spreading in the Rhine and Danube, it was questioned whether these parasites could have spread upstream in mountain rivers in Baden-Württemberg to infect Salmonidae. We expected P. tereticollis to be endemic as it has been commonly found in gammarids in the region in tributaries of the Rhine (in neighbouring Switzerland) [31]. The aims of this study, which was carried out in upstream tributaries of both Rhine and Danube in the federal state of Baden-Württemberg, were threefold: 1) to describe relationship between morphological and molecular data for the local Pomphorhynchus species; 2) to answer whether salmonids, e.g. brown trout and grayling, might serve as fish hosts for these parasites in Baden-Württemberg, and 3) to establish whether Ponto-Caspian lineages of P. laevis have expanded from the Rhine into rhithral rivers.

Material and methods

Results

Discussion

The framework of the study is an ongoing invasion of a Ponto-Caspian lineage of the acanthocephalan parasite Pomphorhynchus laevis in the Rhine and Danube [15,18]. It was hypothesized that this invasive lineage might migrate (via one of its intermediate or final hosts) further upstream in rhithral rivers in the Rhine and Danube areas, and thereby replaces the endemic Pomphorhynchus species which would alter existing parasite-host relationships. Using both morphological and molecular methods, the current study did not detect the invasive Ponto-Caspian lineage of P. laevis in the 120 samples obtained from rhithral rivers in Baden-Württemberg, Southern Germany. Most specimens found were of the endemic P. tereticollis and comparison with published sequences of DNA of the mitochondrial cytochrome oxidase subunit 1 (COI) and the ribosomal internal transcribed spacer (ITS) showed that all P. tereticollis and P. laevis had little genetic variation within the sampled area and were closely related to Western European lineages of the parasites.

In the current study species were initially classified based on differences in morphology using SEM, scientific drawing and microscopy (Figs 2 and 3). Most parasites that were collected in the study area showed bold hooks in middle rows on the proboscis and a characteristic projection on the top of the lower hooks on the proboscis. As these characteristics have been consistently attributed to specimens of P. tereticollis [4,15,26] most individuals were initially classified as this species. However, a recent study of Reier et al. [16] using both molecular and morphological methods, showed that Pomphorhynchus specimens captured in the Austrian part of the Danube with morphological characteristics of the proboscis that would classify them as P. tereticollis, had gene sequences (COI) that placed them closely related to P. laevis. In consequence, molecular evidence is necessary to certainly classify specimens: first a rapid qPCR method was developed to distinguish P. laevis from P. tereticollis for the populations of parasites in Baden-Württemberg based on 3 internal primers of the COI sequence for Pomphorhynchus. Two of these primers were specific for P. tereticollis and one for P. laevis. A large sample of the specimens collected confirmed the morphological classification with an accuracy of 85–99% (the lower threshold being caused by a single ambiguous outcome). A subset of these samples was Sanger sequenced (ITS and COI) and comparison with published sequences (Blastn, NCBI) confirmed the species identification.

The results show that fast molecular qPCR methods is effective for accurate molecular identification of local parasite species. Nevertheless, this cannot replace the first morphological assessment of the species. Moreover, a species classification based solely on measures of genetic similarity may fail to detect important variation in morphologic features that could be explanatory to delineate species [7], or may fail to detect life-history relevant variation in morphological features within species [49]. For example, the host environment might affect morphological features as has been shown for differences in hooks in a tapeworm species depending on being collected from perch or pike [49]. It therefore was concluded that both morphology and different molecular “barcoding” techniques (based on both ITS and COI gene sequences) are necessary to understand how morphological and genetic classification characteristics may be superimposed [16] in this intricate group of acanthocephalans.

Most studies on Pomphorhynchus species [4,15,18,19,26,27] have been carried out in larger lowland summer warm (potamal) rivers which are rich in fish species and dominated by cyprinid species. In these rivers both P. laevis and P. tereticollis can be found in a wide range of fish hosts in Central (including the Rhine) and in Ponto-Caspian (including the Danube) Europe [15,18,19,26], with cyprinid species like chub (Squalius cephalus) and barbel (Barbus barbus) as the most common hosts [19]. Here samples were taken from the more elevated, summer-cold and fast-flowing rhithral tributaries of the Danube and Rhine where salmonids are often the most abundant species. These samples clearly show that young brown trout and grayling are hosts for acanthocephalans in Baden-Württemberg. Almost all acanthocephalans that were found in brown trout and grayling in Baden-Württemberg resembled P. tereticollis. So far it was only shown for brown trout from the British Island to be a hosts for P. tereticollis [27], while grayling has never been described to be a host for P. tereticollis. The prevalence of P. tereticollis was higher in the Rhine area than in the Danube area, and was highest in tributaries of Lake Constance. It remains to be tested whether the Lake Constance plays a role in maintaining the parasite population at this higher level in tributaries of the lake.

No support was found for an invasion of the rhithral with Ponto-Caspian P. laevis. Only one brown trout in a tributary of the Danube was found infected with P. laevis. Molecular data indicate that this specimen originated from the Western European lineage of the species, and was clearly distinct of the invasive Ponto-Caspian lineage [18]. Also P. laevis sampled from chub in the river Kinzig was mostly related to the Western European lineage. There are two possible explanations for this deviating pattern found in “mountain rivers” (rhithral) from the ongoing invasion with Ponto-Caspian lineages of P. laevis in lowland rivers (potamal):

First, the invasion of P. laevis in the main lowland rivers connected to the Rhine and Danube is still ongoing, but they have not yet reached the upstream rhithral tributaries of these rivers that were sampled in the current study. P. laevis in the Rhine was rapidly expanding from 2003, reaching the High Rhine South of Baden-Württemberg already in 2004 [15,18], more than 10 years before the current study (see Fig 1). David et al. [18] postulated that P. laevis haplotypes that before were restricted to Eastern European lowland rivers, have expanded geographically via the Main-Danube canal to the Rhine as parasites of the round goby (Neogobius melanostomus). The round goby acts as a paratenic host for the parasite [4], but parasites can further develop and reproduce after predation of gobies by a piscivourous final hosts [4,29]. The front of the upstream migration of the round goby in the Rhine of Baden-Württemberg occurred already around 2011–2014 [50,51] and current census data of the round goby show the species is currently abundant in the Rhine and Neckar [37]. Indeed, Hohenadler et al. [15] has shown that P. laevis is in the process of replacing the endemic P. tereticollis in eels captured from the High Rhine. However, the gobies did not passed the Rhine Falls to reach the Bodensee area and have also not spread into the river Danube in Baden-Württemberg [37,52]. This is consistent with the front of the expansion of P. laevis over the Danube not reaching Austria yet in 2006 [18] and our finding that Graylings from the Danube River being infected with P. tereticollis rather than P. laevis. Therefore, the expansion of the round goby might explain the missing distribution of P. laevis in the Danube and Bodensee area, but cannot explain the missing distribution of P. laevis in the Rhine and Neckar area.

Second, the missing distribution of P. laevis in tributaries of the Rhine is suggesting that P. laevis might be less able to establish or maintain in the rhithral than P. tereticollis. The most likely explanation is that round gobies do not migrate from the Rhine into mountain rivers, thereby limiting further spatial spread of parasites it carries. However, cyprinid species like chub, barbel and nase (Chondrostoma nasus) that are final hosts for P. laevis [19,53], are known even to pass fast flowing barriers between potamal and rhithral rivers in order to reach upstream spawning areas [54–56]. Moreover, strains of brown trout are known to migrate between lowland and rhithral rivers, and hybrids between trout from the Danube and Rhine have been found in the upper Danube basin [57].Thus, both cyprinids and trout might carry the parasites upstream to the rhithral fish populations including the Rhine and Danube populations. Nevertheless mainly P. tereticollis was found in rhithral fish hosts (young brown trout and grayling) in this study.

Thus, P. tereticollis seems to own certain properties to successful adapt to the rhithral environment which in P. laevis are missing. A possible explanation for such a difference is the way these species manipulate their gammarid host in order to reach their final fish host. Recently Perrot-Minnot et al. [19] showed that in an area with sympatric occurrences of P. laevis and P. tereticollis, chub was more often infected with P. laevis while barbel was more often infected with P. tereticollis. They proposed this to be due to a weaker alteration in phototaxis and geotaxis in gammarids infected with P. tereticollis. As gammarids tent to drift downstream with current and thus have to actively migrate upstream [58], such an alteration in the behaviour of P. laevis infected gammarids would make them more vulnerable for effects of river current than P. tereticollis infected gammarids. A larger effect of drifting downstream would make it less likely for P. laevis infected gammarids to maintain themselves in the fast flowing rhithral zone and thereby to reach salmonids as final hosts. This explanation is consistent with Westram et al. [31] who found that P. tereticollis infected gammarids are abundant in rhithral rivers in Switzerland, while P. laevis infected gammarids were only found in one locality in Switzerland. It is becoming increasingly evident that environmental factors must be taken into account in understanding the biogeography of parasites or diseases [59]. The accumulated knowledge about the distribution of different Pomphorhynchus species, indicate geographical constraints might limit the spread of P. laevis, even when areas are connected by its intermediate and fish hosts. This implies that salmonids in rhithral rivers would not likely experience a substitution of Pomphorhynchus species as took place for eels in the river Rhine [15].

In summary, the current data shows that P. tereticollis, so far mostly associated with cyprinid fish in lowland (potamal) rivers, is generally able to infect brown trout and grayling, the most abundant salmonid fish species in the rhithral rivers of Baden-Württemberg. The parasite haplotype that was found in Baden-Württemberg Pt-L2/3, has been found in a wide range of fish hosts including the most common, i.e. the barbel and chub, but also in brown trout in the Otter river, UK [27]. To the best of our knowledge the parasite has not been shown yet to infect grayling and brown trout in continental Europe. Due to the possible differential behavioural effects of P. laevis and P. tereticollis on gammarids [19] in combination with the wandering of Salmonids, rhithral areas may provide a refuge for P. tereticollis when alien P. laevis lineages are expanding.

Supporting information

Acknowledgments

We thank the local fishing authorities and “Pächter” for their help with the collection of samples, and K. Blasel, J. Thonhofer, R. Haberbosch, B. Schmieder and their colleagues, and H.P. Billmann for their help with collecting the samples. Further we thank the LUBW, Viola Burkhardt-Gehbauer for providing the SEM pictures and Kirstin Zang for providing stereoscope pictures. Leonie Kneipp is thanked for drawing the scientific picture. The editor of PlosOne and two anonymous reviewers greatly helped improving the quality of the study and the manuscript.